Composition composed of carrier and antibody, and methods of making and using the same

Abstract

To provide novel compositions composed of antibodies and carrier proteins and methods of making and using the same, in particular, as a cancer therapeutic.SOLUTION: In one aspect, provided herein are nanoparticle compositions comprising nanoparticles, wherein each of the nanoparticles comprises a carrier protein, between about 100 to about 1000 antibodies, and optionally at least one therapeutic agent, wherein the antibodies are arranged outward from the surface of the nanoparticles and wherein the nanoparticles are capable of binding to a predetermined epitope in vivo.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 060,484, filed October 6, 2014. No. 62 / 206,770, filed August 18, 2015. , claiming the benefit of the filing dates of Nos. 62 / 206,771 and 62 / 206,772. The disclosures of the above applications are incorporated by reference in their entirety.

[0002] The present disclosure relates to novel compositions of antibodies and carrier proteins, and in particular to therapeutic agents for cancer. and methods for their manufacture and use. [Background technology]

[0003] Chemotherapy remains the mainstay for the systemic treatment of many types of cancer, including melanoma. Most chemotherapy drugs are only weakly selective for tumor cells and do not affect healthy proliferating cells. (Allen TM. (2002) Cancer 2:750-763), and dose reduction may be required. and even interruption of treatment is often necessary. In theory, this would overcome the toxicity issues of chemotherapy. One way to achieve and enhance efficacy of a drug is to use a gene that is selectively expressed (or overexpressed) by tumor cells. Targeting chemotherapy drugs to tumors using antibodies specific to proteins expressed in tumors This allows the drug to be attracted to the tumor, thereby altering the biodistribution of chemotherapy drugs, resulting in more drug The goal is to deliver drugs to tumors and reduce their impact on healthy tissue. Nevertheless, specific targeting is rarely successful in therapeutic settings.

[0004] Conventional antibody-dependent chemotherapy (ADC) delivers antibodies via synthetic protease-cleavable linkers. The efficacy of such ADC therapeutics has been , the ability of the target cell to bind to the antibody, the linker being cleaved, and uptake of the toxic agent into the target cell. Schrama,D. et al. (2006) Nature reviews. Drug discovery 5:147 -159.

[0005] Antibody-targeted chemotherapy has the potential for targeting, multiple cytotoxic agents, and potentially low toxicity. It offers an improved combination of therapeutic capabilities that may offer advantages over conventional therapies. Despite extensive research, clinically effective antibody-targeted chemotherapy has not been achieved. The main obstacles are the instability of the linker between the antibody and the chemotherapeutic agent, The low tumor toxicity of chemotherapeutic agents when conjugated to antibodies, and the binding of the conjugates to tumor cells Furthermore, these treatments are limited by the inability to deliver the antibody-drug conjugates. The size could not be controlled.

[0006] The art has demonstrated that cytotoxicity is preserved for targeted drug delivery, while providing superior therapeutic efficacy compared to conventional therapeutic agents. There remains a need for antibody-based cancer therapeutics that provide more reliable and improved antitumor efficacy. It has been done.

[0007] It is also stable in terms of its physical, chemical and biological properties for all therapeutic applications. There remains a need for compositions which

[0008] Lyophilization or freeze-drying removes water from a composition. In this process, the dry The material to be dried is first frozen, and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients are included in the pre-lyophilized formulation to enhance stability during the lyophilization process. and / or may enhance the stability of the lyophilized product during storage. Pikal, M. Biopharm. 3( 9)26-30 (1990) and Arakawa et al., Pharm. Res. 8(3):285-291 (1991).

[0009] The protein may be lyophilized, however, the process of lyophilization and reconstitution may result in Proteins are larger and more efficient than traditional organic and inorganic drugs. Because they are complex (i.e., they have complex three-dimensional structures as well as multiple functional groups), The formulation of such proteins poses special problems. To achieve this, the formulation must preserve the structural integrity of at least the core sequence of amino acids of the protein. The protein must retain its molecular weight and at the same time protect multiple functional groups of the protein from degradation. Protein degradation pathways include chemical instability (i.e., catalysis by bond formation or cleavage). any process that produces new chemicals by modification of proteins) or physical anxiety Chemical instability may involve alterations in the structure of the protein (i.e., changes in the higher order structure of the protein). This may be due to deamidation, racemization, hydrolysis, oxidation, β-elimination or disulfide exchange. Physical instability can be due to, for example, denaturation, aggregation, precipitation or adsorption. The three most common Common protein degradation pathways are protein aggregation, deamidation and oxidation. and, et al.,Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993).

[0010] In the present invention, the composition comprises (a) a carrier protein, (b) an antibody, and (c) an optional therapeutic The antibodies are supported by weak hydrophobic interactions due to their nature. It is believed that the lyophilized and resuspended forms of such compositions are bound to the body proteins. The composition preserves not only the activity of the individual components but also their relative relationships in the nanoparticles. It must be.

[0011] The use of nanoparticles in therapeutics poses additional challenges.

[0012] For example, rearrangement of hydrophobic components in nanoparticles can be mitigated by covalent bonds between the components. However, such covalent attachment is not conducive to the therapeutic use of nanoparticles in cancer treatment. The antibodies, carrier proteins and further therapeutic agents typically They act at different locations and by different mechanisms. Non-covalent binding allows the nanoparticle components to target the tumor. Thus, covalent bonds can be advantageous for freeze-drying, but , which may be a drawback for therapeutic use.

[0013] The particle size and size distribution of the nanoparticles are also important. At larger particle sizes, nanoparticles or agglomerates of these particles may behave differently depending on the blood vessels. either of which may affect the performance and safety of the composition. be.

[0014] Finally, cryoprotectants and agents that aid in the freeze-drying process must be safe and therapeutically acceptable. It must be able to withstand practical use. Summary of the Invention [Means for solving the problem]

[0015] In one aspect, a nanoparticle composition comprises nanoparticles, each of which is coupled to a carrier protein. The present invention relates to a method for producing a medicament for the treatment of cancer, comprising administering to a patient a therapeutic agent comprising administering to said ... The antibodies are arranged outward from the surface of the nanoparticle, and the nanoparticles are capable of binding to a specific endogenous antibody in vivo. Provided herein are nanoparticle compositions capable of binding to a pitope. It will be offered.

[0016] When administered intravenously, large particles (e.g., >1 μm) become lodged in the pulmonary microvasculature. At the same time, larger particles are typically disliked because they can cause tumors or other abnormalities. The drug, called "TheraSphere," can accumulate in certain organs and nourish liver tumors. Glass particles of 20 to 60 microns, for example, used for injection into the hepatic artery to feed the liver (see Clinical Use for

[0017] Therefore, for intravenous administration, particles smaller than 1 μm are used. Particles larger than 1 μm are more Typically, administration is directly into the tumor ("direct injection") or into an artery that supplies the tumor site.

[0018] In another aspect, a nanoparticle composition comprising nanoparticles, each of the nanoparticles being an albumin. A carrier protein that is not an amino acid, about 100 to about 100 antibodies, preferably about 400 to about 800 00 antibodies, and optionally at least one therapeutic agent, wherein the antibodies are attached to the nanoparticles. The nanoparticles are arranged on the outer surface thereof and are capable of binding to a predetermined epitope in vivo. Provided herein are nanoparticle compositions in which the nanoparticles are multimerized. The number increases proportionally. For example, if a 160 nm nanoparticle contains 400 antibodies, 3 A 20 nm dimer contains approximately 800 antibodies.

[0019] In another aspect, a nanoparticle composition includes nanoparticles, each of the nanoparticles being supported by a carrier. At least about 400 to about 800 antibodies and any non-paclitaxel The antibody comprises a therapeutic agent, the antibody being arranged such that the binding portion of the antibody is directed away from the surface. The nanoparticles are arranged on the surface of the nanoparticles so as to bind to a specific epitope in vivo. Provided herein are nanoparticle compositions capable of binding.

[0020] In other embodiments, the nanoparticles are multimerized, e.g., dimerized. Weight or size multiples of a 160 nm particle are approximately 320 nm, 480 nm, 640 nm, etc. In some embodiments, multimerization may be observed when the 20 % of the population are multimers. In some embodiments, more than 80% of the population are multimers. .

[0021] In one embodiment, the carrier-bound drug:antibody (e.g., albumin-bound paclitaxel:bevacizumab) is In one embodiment, the weight ratio of carrier-bound drug:antibody (anti-coumab) is about 5:1 to about 1:1. In one embodiment, the antibody is present on the entire surface of the nanoparticle. It is essentially a monolayer of antibodies on the body or part. In one embodiment, the composition contains 0. Less than 0.1% of nanoparticles are greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, having a particle size selected from the group consisting of greater than 600 nm, greater than 700 nm, and greater than 800 nm. The larger particle sizes are the core and the entire or partial surface of each nanoparticle, respectively. This is believed to be the result of multimerization of some of the nanoparticles containing the antibody coating present.

[0022] The present invention further includes a freeze-dried composition, the freeze-dried composition being freshly prepared nanoparticles. The properties of the particles are not substantially different, i.e., the same. In particular, the lyophilized composition is When resuspended in aqueous solution, the particle size, particle size distribution, toxicity to cancer cells, antibody affinity and antibody activity were determined. The present invention relates to a freeze-dried nanoparticle formulation that is similar or identical to the new composition in terms of specificity. However, despite the presence of two different protein components in these particles, The surprising finding is that nanoparticles prepared in a situ retain their properties.

[0023] In one aspect, the present invention provides a nanoparticle comprising a carrier-bound drug core and a carrier-bound antibody core. It contains a quantity of antibodies arranged on the surface of the core such that the binding moieties are directed away from that surface. The antibodies retain their binding to the exterior surface of the nanoparticles upon reconstitution in aqueous solution. In one embodiment, the nanoparticle composition comprises a lyophilized nanoparticle. In one embodiment, the lyophilized composition is stable at room temperature for at least three months. The reconstituted nanoparticles retain the activity of the therapeutic agent and are capable of binding to targets in vivo. do.

[0024] In one embodiment, the average reconstituted nanoparticle size is about 130 nm to about 1 μm. In a preferred embodiment, the average reconstituted nanoparticle size is about 130 nm. m to about 200 nm, more preferably about 160 nm. The particle size of the nanoparticles produced is greater than 800 nm to approximately 3.5 μm, and the particle size of smaller nanoparticles is Multimers, for example, multimers of nanoparticles of 100-200 nm. In one embodiment, the core: The weight ratio of antibodies is greater than 1:1 to about 1:3.

[0025] In one aspect, the present disclosure provides a lyophilized nanoparticle composition comprising nanoparticles, the nanoparticles comprising: The nanoparticles consist of (a) an albumin-bound paclitaxel core, and (b) an antibody-bound The albumin-bound paclitaxel core is disposed on the surface thereof such that the moiety faces away from the surface. The antibody comprises about 400 to about 800 molecules of bevacizumab placed in the lyophilized The composition is stable for at least 3 months at about 20° C. to about 25° C., and the reconstituted nanoparticles As long as the molecules are capable of binding to VEGF in vivo, their corresponding molecules will bind to VEGF when reconstituted in aqueous solution. The present invention relates to a freeze-dried nanoparticle composition characterized in that the nanoparticles retain their surface association. do.

[0026] In another aspect, the present disclosure provides a lyophilized nanoparticle composition comprising nanoparticles, the nanoparticles comprising: The nanoparticles consist of (a) an albumin-bound paclitaxel core and (b) an antibody-bound core. on the surface of the albumin-bound paclitaxel core such that the binding moiety is directed away from the surface. and a quantity of bevacizumab disposed therein, the antibody being such that the lyophilized composition is about 20 The reconstituted nanoparticles are stable for at least 3 months at temperatures between 25°C and 50°C, and the reconstituted nanoparticles are stable for at least 3 months at temperatures between 25°C and 50°C. The surface of these nanoparticles when reconstituted in aqueous solution can bind to EGF. Furthermore, the average reconstituted nanoparticle size was approximately the same as that of the freshly prepared nanoparticles. A freeze-dried nanoparticle composition characterized in that the particle size of the freeze-dried nanoparticles is not substantially different from that of the freeze-dried nanoparticles In some embodiments, the particle size is 200 to 800 nm, for example 200, In other embodiments, the wavelength is 300, 400, 500, 600, 700 or 800 nm. The particles are larger, for example, greater than 800 nm to about 3.5 μm. In the present study, the particle is a polymer of nanoparticles.

[0027] In some embodiments, the weight ratio of albumin-bound paclitaxel:bevacizumab is: In another embodiment, the ratio of albumin-bound paclitaxel to bevacizumab is about 5:1 to about 1:1. In a further embodiment, the weight ratio of albumin-bound paclitaxel to ramucirumab is about 10:4. The weight ratio of xenobiotics:bevacizumab is greater than 1:1 to about 1:3.

[0028] In some embodiments, the core is albumin-bound paclitaxel, and the antibody have used antibodies that selectively recognize VEGF (e.g., bevacizumab / avastin), CD20 Antibodies that selectively recognize and antibodies that selectively recognize Her2 (trastuzumab / Herceptin). .

[0029] In some embodiments, at least one therapeutic agent is located within the nanoparticle. In other embodiments, the at least one therapeutic agent is located on the exterior surface of the nanoparticle. In some embodiments, the at least one therapeutic agent is present within and on the nanoparticle. Located on the exterior.

[0030] In some embodiments, the nanoparticles contain two or more therapeutic agents, e.g., a taxane and Platinum agents include, for example, paclitaxel and cisplatin.

[0031] In some embodiments, the antibody is ado-trastuzumab emtansine, arem Tuzumab, bevacizumab, cetuximab, denosumab, dinutuximab, ipilimumab, Nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pel In some cases, the drug is selected from the group consisting of tuzumab, rituximab, and trastuzumab. In embodiments, the antibody is present in a substantially monolayer on all or a portion of the surface of the nanoparticle. It is an antibody.

[0032] In a further embodiment, the antibody has a greater affinity than normally found in naturally occurring human antibodies. Such glycosylation may be effected, for example, by an expression system or by expression It may be affected by the presence of an existing glycosylation inhibitor. In the present case, the glycosylation state of an antibody is altered by enzymatic or chemical action.

[0033] In some embodiments, the at least one therapeutic agent is abiraterone, bendamustine , bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, da Satinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, Everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib , lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone , nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetidine Rexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine The drug is selected from the group consisting of cyclophosphamide and cyclophosphamide.

[0034] In some embodiments, the nanoparticles are not paclitaxel or bevacizumab. The composition further comprises at least one additional therapeutic agent.

[0035] In some embodiments, the antibody, carrier protein, and, if present, therapeutic agent are non-covalently linked. are bonded together by a bond.

[0036] In some embodiments, the carrier protein is gelatin, elastin, gliadin. From the group consisting of: legumin, zein, soy protein, milk protein and whey protein In other embodiments, the carrier protein is selected from albumin, e.g., human serum albumin. It is albumin.

[0037] In some embodiments, the composition is formulated for intravenous delivery. In the form, the composition is formulated for direct injection or perfusion into a tumor.

[0038] In some embodiments, the average nanoparticle size in the composition is greater than 800 nm to about 3.5 μm. It is.

[0039] In some embodiments, the nanoparticles are about 1×10 -11 M ~ approx. 1×10 -9 M It has a dissociation constant.

[0040] In another embodiment, the mixture is 5.0 to 7.0% by weight, under conditions and in ratios of components that allow for the formation of the desired nanoparticles. 0.5 and a temperature of about 5°C to about 60°C, about 23°C to about 60°C, or about 55°C to about 60°C. The carrier protein and any at least one therapeutic agent are combined with the antibody in a solution having a concentration of Provided herein is a method for producing a nanoparticle composition, comprising contacting a In an embodiment, the nanoparticles are produced at 55-60° C. and pH 7.0. (a) contacting the carrier protein and any at least one therapeutic agent to form a core; and (b) reacting the mixture under conditions and in ratios of components that allow for the formation of desired nanoparticles. A pH of 5.0 to about 7.5 and a temperature of about 5°C to about 60°C, about 23°C to about 60°C, or about 55°C to about contacting the core with the antibody in a solution having a temperature of about 60° C. Methods for making the compositions are provided herein.

[0041] To effect the formation of the desired nanoparticles, components (e.g., carrier proteins, antibodies, therapeutic agents, Compositions in which the amounts of the components are too dilute are described herein. In a preferred embodiment, the carrier protein:antibody does not form the nanoparticles described. The weight ratio is 10:4. In some embodiments, the amount of carrier protein is about 1 mg / m In some embodiments, the amount of antibody is about 1 mg / mL to about 100 mg / mL. For example, in some embodiments, the carrier protein:antibody:solvent ratio is about 30 mg / mL. The ratio of solutions is approximately 9 mg of carrier protein (e.g., albumin): 4 mg of antibody (e.g., BEV: 1 mL of a solution (e.g., saline). Sol) can also be added to the carrier protein.

[0042] In a further embodiment, the nanoparticles are prepared as described above and then lyophilized.

[0043] In another embodiment, a cancer cell is contacted with an effective amount of a nanoparticle composition disclosed herein. Provided herein are methods for treating cancer cells, comprising treating the cancer cells with

[0044] In another embodiment, a tumor cell is contacted with an effective amount of a nanoparticle composition disclosed herein. The present invention relates to a method for treating a tumor in a patient in need thereof, the method comprising the step of treating the tumor by administering the In some embodiments, the size of a tumor is reduced. In yet another embodiment, the nanoparticle composition is administered intravenously. The composition is administered by direct injection or perfusion into the tumor.

[0045] In some embodiments, the methods provided herein include: a) providing the nanoparticle composition; once a week for three weeks; b) administering the nanoparticle composition throughout the week. and c) repeating steps a) and b) as necessary to treat the tumor. Includes the process.

[0046] In a related embodiment, the treatment comprises administration of a targeting antibody prior to administration of the nanoparticles. In one embodiment, the nanoparticles are administered to the target tissue about 6 to 48 hours or 12 to 48 hours prior to administration. In another embodiment, the targeting antibody is administered 6 to 12 hours prior to administration of the nanoparticles. In yet another embodiment, the targeted antibody is administered 2 to 8 hours prior to administration of the nanoparticles. In yet another embodiment, the targeting antibody is administered to the body one week prior to administration of the nanoparticles. For example, a dose of BEV is administered 24 hours prior to administration of AB160. In this example, administration of rituximab precedes administration of the AR nanoparticles. The antibody to be administered is administered at 1 / 2, 1 / 10, or 1 / 20 of the amount normally considered to be therapeutic. Any subtherapeutic dose may be administered. Thus, in humans, the prevalence of BEV is Treatment consisted of administration of 1 mg / kg BEV, 1 / 10 the usual dose, followed by AB1 It may include 60 administrations.

[0047] In some embodiments, the therapeutically effective amount is about 75 mg / m 2 ~about 175mg / m 2 of The carrier protein (i.e., the patient's 2 (milligrams of carrier protein per milligram) In other embodiments, the therapeutically effective amount is about 75 mg / m 2 ~about 175mg / m 2 Treatment In another embodiment, the therapeutically effective amount is about 30 mg / m 2 ~about 70mg / m 2 In yet another embodiment, the therapeutically effective amount is , about 30mg / m 2 ~about 70mg / m 2 Including bevacizumab.

[0048] In one particular embodiment, the lyophilized composition has a concentration of about 75 mg / m 2 ~about 175mg / m 2 of carrier protein, preferably albumin, about 30 mg / m 2 ~about 70mg / m 2 of an antibody, preferably bevacizumab, and about 75 mg / m 2 ~about 175mg / m 2 of Contains paclitaxel.

[0049] The following figures are merely illustrative of the invention and are not intended to be limiting of the invention. In addition, the nanoparticles of the present invention using Abraxane® and bevacizumab are represented by the acronym " The numbers after AB, such as AB160, represent the average particle size of these nanoparticles (unit Similarly, if the antibody is rituximab, The acronym is "AR" and the numbers that follow have the same meaning. [Brief description of the drawings]

[0050] [Figure 1A] Flow cytometry scatter plots are shown including Abraxane® (ABX, commercially available from Celgene, Summit, NJ 07901) stained with secondary antibody only (top left panel), ABX stained with goat anti-mouse IgG1 Fab + secondary antibody (top right panel), AB160 (nanoparticles consisting of albumin-conjugated paclitaxel:bevacizumab in a ratio of approximately 10:4, with a mean particle size of 160 nm) stained with secondary antibody only (bottom left panel), or AB160 stained with goat anti-mouse IgG1 Fab + secondary antibody (bottom right panel). [Figure 1B] Representative electron micrographs are shown after incubation of AB160 with anti-human IgG Fc labeled with gold particles. [Figure 1C] Pie chart showing the percentage of total paclitaxel in the AB160 fraction (particulates, proteins >100 kD and proteins <100 kD) (top) and Western blot using mouse IgG Fab (BEV) and antibodies against paclitaxel to confirm colocalization (bottom). [Figure 1D] Figure 1 shows the activity of paclitaxel in an in vitro toxicity assay using A375 human melanoma cells compared to ABX alone. The results are expressed as the mean (+ / - SEM) proliferation index relative to the total proliferation rate of untreated cells. Data are representative of three experiments and the differences were not significant. [Figure 1E]Figure 1 shows the results of a VEGF ELISA of supernatants following incubation of VEGF with ABX and AB160 to determine ligand (VEGF) binding by the antibodies. Results are shown as the mean percentage of VEGF unbound by the two complexes + / - SEM. Data represent triplicate experiments (**P<0.005). [Figure 2A] Figure 1 shows the particle size (determined by light scattering techniques) of complexes formed upon addition of BEV (bevacizumab) to ABX under conditions that result in the formation of nanoparticles and larger particles. Increasing concentrations of BEV (0-25 mg) were added to 10 mg of ABX and the particle size of the complexes formed was determined. The mean particle size of the complexes (146 nm-2,166 nm) increased with increasing concentration of BEV. Data is presented as sample volume / particle size and the graph shows the particle size distribution. Data represents five separate drug formulations. In comparison, ABX by itself has a mean particle size of approximately 130 nm. [Figure 2B] The binding affinity of ABX and BEV, as determined by light absorption (BLItz) technique, is shown. Data are presented as dissociation constants (Kd). Binding affinity was evaluated for particles prepared at four pH levels (3, 5, 7, 9) and three temperatures (room temperature, 37°C, and 58°C), and data are representative of five experiments. [Figure 2C] Figure 2B shows the stability of the nanoparticle complex from Figure 2B in serum as determined by Nanoparticle Tracking Analysis (NTA) using a Nanosight 300 (NS300). Data is presented as number of particles per mg of ABX and compares AB160 prepared at room temperature and pH 7 (AB16007: particle size, pH), 58°C and pH 7 (AB1600758: particle size, pH, temperature), and 58°C and pH 5 (AB1600558: particle size, pH, temperature) to ABX alone under each condition. Once the particles were prepared, they were added to human AB serum for 15, 30 and 60 minutes to determine their stability over time in serum. [Figure 3A]In vivo testing of AB nanoparticles in athymic nude mice treated with PBS, 12 mg / kg BEV, 30 mg / kg ABX, 12 mg / kg BEV + 30 mg / kg ABX or AB160 (with approximately 12 mg / kg BEV and approximately 30 mg / kg ABX) at tumor sizes of approximately 600 mm3 to 900 mm3 after injection of 1 x 106 A375 human melanoma cells into the right flank. Data are expressed as percent change in tumor size from baseline (tumor size on the day of treatment) to day 7 after treatment. Significance was determined using Student's t-test. All p-values ​​for AB particles were significantly different from PBS, individual drugs alone and the two drugs administered sequentially. [Figure 3B] Kaplan-Meier curves generated for median survival of the mice analyzed in Figure 3A are shown. Significance was determined using the Mantel-Cox test comparing survival curves. [Figure 3C] Percent change from baseline is shown for mice treated when tumors were <700mm3 or >700mm3 to determine if tumor size influenced tumor response in the ABX alone and AB160 groups. Significance was determined using a Student's t-test, with the ABX alone group showing no significant difference based on tumor size (p=0.752) and the AB160 group being significantly different (p=0.0057). [Figure 3D]In vivo testing of AB nanoparticles in athymic nude mice treated with PBS, 30 mg / kg ABX or 45 mg / kg BEV+AB160, AB580 (nanoparticles consisting of albumin-bound paclitaxel:bevacizumab with a mean particle size of 580 nm) or AB1130 (nanoparticles consisting of albumin-bound paclitaxel:bevacizumab with a mean particle size of 1130 nm) at tumor sizes of approximately 600 mm3 to 900 mm3 with 1 × 106 A375 human melanoma cells injected into the right flank. Data are expressed as the percentage change in tumor size from baseline (tumor size on the day of treatment) 7 days after treatment. Significance was determined using Student's t-test. All p values ​​for AB particles were significantly different from PBS, individual drugs alone and the two drugs administered sequentially. Differences between AB particles with different particle sizes were not significant. [Figure 3E] Kaplan-Meier curves generated for median survival of the mice analyzed in Figure 3D are shown. Significance was determined using the Mantel-Cox test comparing survival curves. [Figure 4A] Blood paclitaxel concentrations displayed as a line graph with a logarithmic y-axis scale based on blood and tumor samples taken from tumor-free and tumor-bearing mice measured by LC-MS 0-24 hours after IV injection of 30 mg / kg paclitaxel associated with ABX or AB160. Mice were injected IV at time 0, blood samples were taken at time 0, 4, 8, 12 and 24 hours, and mice were sacrificed. There were at least 3 mice per time point. Student's t-test was used to determine whether any concentration differences between ABX and AB160 were significant. [Figure 4B] FIG. 4A shows blood paclitaxel concentrations displayed as a line graph with a numeric scale on the y-axis. [Figure 4C] Cmax, half-life and AUC values ​​calculated from the blood concentration data shown in Figures 4A and 4B are shown. [Figure 4D]FIG. 1 shows blood paclitaxel concentrations displayed as a line graph with a logarithmic y-axis scale from a second pharmacokinetic experiment using earlier time points (2-8 hours). [Figure 4E] FIG. 4D shows blood paclitaxel concentrations displayed as a line graph with a numeric scale on the y-axis. [Figure 4F] 1 shows blood paclitaxel concentrations in mice whose tumors were allowed to grow to a larger size before injection with ABX and AB160. [Figure 4G] Cmax and AUC calculated from the data in Figure 4F are shown. [Figure 4H] 1 shows the concentration of paclitaxel in tumors from a second mouse study as measured by LC-MS. Data are expressed as μg paclitaxel / mg tumor tissue. Student's t-test was used to determine whether differences were significant. [Figure 4I] Shows I-125 radioactivity levels in mice treated with AB160 compared to ABX alone. [Figure 4J] 4I depicts a graphical representation of the I-125 radioactivity levels shown in FIG. 4I. [Figure 5A] Particle size measurements and affinity of nanoparticles produced with rituximab. 10 mg / mL ABX was incubated with 0-10 mg / mL rituximab (RIT) and the particle size of the resulting particles was measured using light scattering techniques (Mastersizer 2000). Data are presented as volume fraction of particles at each particle size, and the curves represent the particle size distribution (top). The table (bottom) shows the particle size of the particles obtained at each concentration of antibody. [Figure 5B] Particle size measurements and affinity of nanoparticles prepared with trastuzumab. 10 mg / mL ABX was incubated with 0-22 mg / ml trastuzumab (HER) and the particle size of the resulting particles was measured using light scattering techniques (Mastersizer 2000). Data are presented as volume fraction of particles at each particle size, and the curves represent the particle size distribution (top). The table (bottom) shows the particle size of the particles obtained at each concentration of antibody. [Figure 5C] Binding affinities of Rituximab and Trastuzumab compared to ABX at pH 3, 5, 7 and 9 as determined by Biolayer Interferometry (BLItz) technology are shown. Dissociation constants are displayed for each interaction. [Figure 6A] Figure 1 shows the in vitro toxicity of AR160 tested using the CD20 positive Daudi human lymphoma cell line. Data are displayed as a graph of the proliferation index, which is the ratio of FITC positive cells in treated wells to FITC positive cells in untreated wells (highest level of proliferation). [Figure 6B] In vivo tumor efficacy in athymic nude mice injected with 5x106 Daudi human lymphoma cells in the right flank. Tumors were allowed to grow to 600mm3-900mm3 and mice were treated with PBS, 30mg / kg ABX, 12mg / kg rituximab, 12mg / kg rituximab + 30mg / kg ABX or AR160. On day 7 after treatment, tumor response was determined by the percent change in tumor size from day 1 of treatment. Significance was determined by Student's t-test and percent change from baseline was significantly different between mice treated with AR160 and all other groups (p<0.0001). [Figure 6C] Kaplan-Meier survival curves generated from the experiment shown in Figure 6B are shown. Median survival times for each treatment group are shown. Mantel-Cox tests were used to determine whether differences in survival curves were significant. [Figure 7A] Addition of another chemotherapeutic agent (cisplatin) to AB160 is shown. ABX (5 mg / ml) was incubated with cisplatin (0.5 mg / ml) for 30 min at room temperature and free cisplatin was measured by HPLC in the supernatant after removal of the ABX microparticles. The amount of free cisplatin was subtracted from the starting concentration to determine the amount of ABX-bound cisplatin. Data are displayed as a bar graph along with the starting concentration (cisplatin). [Figure 7B]Figure 1 shows the toxicity of cisplatin-conjugated ABX (AC) in a proliferation assay of A375 human melanoma cells. After 24 hours of drug exposure and EdU incorporation, the cells were fixed, permeabilized, and labeled with FITC-conjugated anti-EdU antibody. Data are displayed as a graph of the proliferation index, which is the percentage of FITC-positive cells in treated wells compared to FITC-positive cells in untreated wells (highest level of proliferation). [Figure 7C] In vivo tumor efficacy of AC (ABC complex, cisplatin-conjugated ABX) in athymic nude mice injected with 1 x 106 A375 human melanoma cells in the right flank. Tumors were allowed to grow to 600mm3-900mm3 and mice were treated with PBS, 30mg / kg ABX, 2mg / kg cisplatin, AB160, 2mg / kg cisplatin + AB160 or ABC160. On day 7 after treatment, tumor response was determined by the percentage change in tumor size from the day of treatment. Significance was determined by Student's t-test, and the percentage change from baseline was significantly different between ABC160-treated mice and PBS, cisplatin or ABX-treated mice (p<0.0001). No significant differences were observed between AB160, AB160 + cisplatin and ABC160-treated groups for the percentage change from baseline on day 7 after treatment. [Figure 7D] Kaplan-Meier survival curves generated from the experiment shown in Figure 7C are shown, with median survival times shown for each treatment group. Mantel-Cox tests were used to determine whether differences in survival curves were significant. [Figure 8A] FIG. 1 shows the particle size distribution of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted compared to fresh AB160 or ABX alone. [Figure 8B] 1 shows the ligand (VEGF) binding capacity of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted, compared to fresh AB160 or ABX alone. [Figure 8C] Figure 1 shows the in vitro cancer cytotoxicity of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted compared to fresh AB160 or ABX alone. [Figure 8D]FIG. 1 shows the particle size distribution of AB160 nanoparticles that were lyophilized, stored at room temperature for 10 months, and reconstituted compared to fresh AB160 or ABX alone. [Figure 8E] 1 shows the ligand (VEGF) binding capacity of AB160 nanoparticles that were lyophilized, stored at room temperature for 10 months, and reconstituted, compared to fresh AB160 or ABX alone. [Figure 8F] Figure 1 shows the in vitro cancer cytotoxicity of AB160 nanoparticles that were lyophilized, stored at room temperature for 10 months, and reconstituted compared to fresh AB160 or ABX alone. [Figure 9] Figures 9A-9C show the particle size distribution of ABX-BEV complexes (ABX final concentration: 5 mg / mL) under intravenous infusion conditions incubated in saline at room temperature for up to 24 hours (Figures 9A and 9B). There is some evidence of complex dissociation by ELISA by up to 4 hours at room temperature (20%, Figure 9C). [Figure 10] Shown is an ex vivo incubation of ABX (upper panel) or AB160 (lower panel) in saline or heparinized human plasma for 30 seconds at relative volume ratios of 9:1 or 1:1. [Figure 11A] In vivo study of athymic nude mice injected with 1x106 A375 human melanoma cells in the right flank and treated with PBS. Data are presented as tumor volume (in mm3) at 7 and 10 days after treatment. [Figure 11B] In vivo study of athymic nude mice injected with 1x106 A375 human melanoma cells in the right flank and treated with 12 mg / kg BEV. Data are presented as tumor volume (in mm3) at 7 and 10 days after treatment. [Figure 11C] Figure 1 shows an in vivo study of athymic nude mice injected with 1x106 A375 human melanoma cells in the right flank and treated with 30mg / kg ABX. Data are expressed as tumor volume (in mm3) at 7 and 10 days after treatment. [Figure 11D]Figure 1 shows an in vivo study of athymic nude mice injected with 1x106 A375 human melanoma cells in the right flank and treated with AB160. Data are presented as tumor volume (in mm3) at 7 and 10 days after treatment. [Figure 11E] In vivo study of athymic nude mice injected with 1x106 A375 human melanoma cells in the right flank and pre-treated with 1.2 mg / kg BEV and 24 hours later with AB160. Data are presented as tumor volume (in mm3) at 7 and 10 days after treatment. [Figure 11F] The data from Figures 11A-11E are summarized 7 days after treatment. [Figure 11G] The data from Figures 11A-11E are summarized 10 days after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] After reading this description, you may wish to learn how to implement the invention in various other embodiments and other applications. However, the various embodiments of the present invention are not limited to those described herein. It will be understood that the embodiments illustrated herein are presented by way of example only. Therefore, this detailed description of various alternative embodiments is not intended to be limiting of the present invention. The disclosure should not be construed as limiting the scope or breadth of the invention described below. do not have.

[0052] Before disclosing and describing the present invention, it is important to note that the embodiments described below are directed to certain compositions, such The present invention is not limited to the method of preparation of the composition or its use, which may, of course, vary. It should be understood that the terminology used herein is merely intended to describe particular embodiments. It should also be understood that these are merely illustrative and not limiting of the invention.

[0053] For the convenience of the reader only, the detailed description of the present invention has been divided into various sections. The disclosures contained herein may also be combined with the disclosures of other sections. Titles or subtitles may be used for convenience, but they are not intended to be limiting of the invention. It does not affect the range.

[0054] definition Unless otherwise defined, all technical and scientific terms used herein refer to the The term "compound" has the same meaning as commonly understood by a person skilled in the art. In this and the claims which follow, reference will be made to a number of terms which shall have the following meanings: This shall be the case.

[0055] The terminology used herein is merely for the purpose of describing particular embodiments. As used herein, the singular forms "a," "an," "an "An" and "the" are used unless the context clearly indicates otherwise. Unless otherwise specified, the plural forms are included.

[0056] "Optional" or "optionally" means that the event or circumstance described thereafter occurs. means that the event or circumstance may or may not occur, and the description represents This includes cases where this does not occur.

[0057] The term "about" may be used before any numerical designation that includes a range, e.g., temperature, time, amount, concentration, etc. If used, (+) or (-) 10%, 5%, 1% or anything in between. The ranges denote approximate values ​​that may vary by any subrange or subvalue. Preferably, the term "about" when used in relation to a dose means that the dose is + / - For example, "about 400 to about 800 antibodies" means that It is shown that the outer surface of the nanoparticles contains antibodies in the amount of 360 to 880.

[0058] "Comprising" or "comprise" means It is intended that the compositions and methods include the recited elements, but not exclude other elements. "Consisting essentially of" means a composition and when used to define a method, the combination for the stated purpose "The term 'commercial property' means the exclusion of any other element that is substantially significant to the existence of the commercial property." Thus, a composition consisting essentially of the elements defined herein is in accordance with the present invention as claimed. It does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the invention. "Consisting of" means that the substance contains more than trace amounts of other components and The embodiments defined by each of these transition terms are: is within the scope of the present invention.

[0059] As used herein, the term "nanoparticle" refers to a nanoparticle having at least one particle size smaller than 5 microns. In a preferred embodiment, the nanoparticles are, for example, for intravenous administration. The nanoparticles are less than 1 micron. For direct administration the nanoparticles are larger. Larger particles are expressly contemplated by the present invention.

[0060] In a group of particles, the particle sizes of the individual particles are distributed approximately evenly. The particle size of a population can also be expressed as a mean or percentile. D50 is the 5th percentile of the particles. The particle size that contains 0% or less of the particles. 10% of the particles are smaller than the D10 value and 90% of the particles are The particles are smaller than D90. If not clear, the "average" particle size is equal to D50. Therefore For example, AB160 refers to nanoparticles having an average particle size of 160 nanometers.

[0061] The term "nanoparticle" may also encompass discrete polymers of smaller nanoparticle units. For example, a 320 nm particle contains a dimer of a 160 nm nanoparticle unit. For 60 nm nanoparticles, the polymers are approximately 320 nm, 480 nm, 640 nm, and 800 nm. , 960nm, 1120nm, etc.

[0062] As used herein, the term "carrier protein" refers to a carrier protein that is capable of carrying an antibody and / or a therapeutic agent. The antibodies of the present disclosure are reversibly linked to the carrier protein. Exemplary carrier proteins are described in more detail below. .

[0063] As used herein, the term "core" refers to a carrier protein, carrier protein + therapeutic The core or interior of the nanoparticle may comprise a drug or other agent or combination of agents. In some embodiments, the hydrophobic portion of the antibody is incorporated into the core. Good too.

[0064] As used herein, the term "therapeutic agent" refers to an agent that is therapeutically useful, e.g., for treating a disease condition. for the treatment, amelioration or attenuation of, or the evaluation of, a condition, physiological state, symptom or etiological agent Therapeutic agents include chemotherapeutic agents, e.g., mitotic inhibitors, thrombin time inhibitors, Polyisomerase inhibitors, steroids, antitumor antibiotics, metabolic antagonists, alkylating agents, enzymes , a proteasome inhibitor, or any combination thereof.

[0065] As used herein, the term "antibody" refers to an immunoglobulin molecule and The immunologically active portion of a polypeptide molecule (i.e., an antigen-binding site that immunospecifically binds to an antigen) The term refers to a molecule that contains two immunoglobulin heavy chains and two immunoglobulin Antibodies consisting of light chains as well as various forms including full length antibodies and portions thereof, e.g., immunoglobulins Brine molecule, monoclonal antibody, chimeric antibody, CDR-grafted antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), bispecific antibody (diabody), multispecific antibody, bispecific antibody (dual specific antibody, anti-idiotypic antibody, bispecific antibody ), functionally active epitope-binding fragments thereof, bifunctional hybrid antibodies (e.g., Lanz et al. avecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chain (e.g., Huston et al. al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Scien ce 242, 423-426 (1988), which are incorporated herein by reference. In general, Hoodet al., Immunology, Benjamin, NY, 2ND ed. (1984), Harlow and Lane. , Antibodies. ALLaboratory Manual, Cold Spring Harbor Laboratory (1988), Hunkapi See, Eller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference. The antibodies may be of any type (e.g., IgG, IgA, IgM, Ig The antibody may be an IgG antibody (eg, IgE or IgD). Preferably, the antibody is an IgG. The antibody may be a non-human Human antibodies (e.g., mouse, goat, or any other animal origin), fully human antibodies, humanized antibodies The antibody may be an antibody specific for the human antibody or a chimeric antibody.

[0066] "K d The term "dissociation constant," also referred to as "dissociation constant," refers to the dissociation of a particular substance into its individual components (e.g., tandem dissociation). This refers to the amount that represents the degree to which a protein separates into its individual proteins (protein carriers, antibodies and any therapeutic agents).

[0067] As used herein, the terms "lyophilized," "freeze-dried," and the like refer to the material to be dried (e.g., For example, nanoparticles) are first frozen and then the ice or frozen solvent is sublimated in a vacuum environment. This refers to the process of removing excipients from previously lyophilized formulations during storage. In some embodiments, the nanoparticles enhance the stability of a lyophilized product of a therapeutic agent. The lyophilized components (carrier protein, antibody and any therapeutic agent) are formed into a lyophilized solution prior to use. In another embodiment, the carrier protein, antibody and optional therapeutic agent can be They are first combined into nanoparticles and then freeze-dried. The freeze-dried samples can be used without additional excipients. It may further include.

[0068] The term "bulking agent" includes agents that provide structure to the lyophilized product. Common examples used include mannitol, glycine, lactose and sucrose. In addition to providing a pharma- ceutical superior mass, bulking agents can also modify the collapse temperature. , providing freeze-thaw protection and enhancing protein stability during long-term storage. These agents can also provide useful qualities with respect to tonicity and It can also function as a filter.

[0069] The term "buffer" refers to an agent that maintains the pH of the solution within an acceptable range prior to lyophilization. Succinate (sodium or potassium), histidine, phosphate (sodium (tris(hydroxymethyl)aminomethane), diethanolamine Examples of the buffer solution of the present invention include amine, citrate (sodium), etc. The pH ranges from 5.5 to about 6.5, and preferably about 6.0. Examples of buffers that control the range include succinates (such as sodium succinate), gluconates, etc. Examples of suitable buffers include acid salt, histidine, citrate and other organic acid buffers.

[0070] The term "cryoprotectant" generally refers to a substance that is presumably preferentially excluded from protein surfaces. and an agent that confers stability to the protein against freezing-induced stress by: Protection can also be provided during primary and secondary drying and long term product storage. , polymers such as dextran and polyethylene glycol, sucrose, glucose , sugars such as trehalose and lactose, surfactants such as polysorbates, and Amino acids such as glycine, arginine and serine.

[0071] The term "lyoprotectant" probably refers to amorphous glass. providing a foamy matrix, binding the protein by hydrogen bonding, and The drying or "dehydration" process by replacing water molecules that are removed during the drying process. (primary and secondary drying cycles). Maintains protein structure, minimizes proteolysis during the lyophilization cycle, and provides long-term storage Helps improve product shelf life. Examples include polyols or sucrose and and trehalose.

[0072] The term "pharmaceutical formulation" refers to a form in which an active ingredient can be made effective and in which it is It refers to a formulation that does not contain additional components that are toxic to the subject to which the formulation is administered.

[0073] "Pharmaceutically acceptable" excipients (vehicles, additives) are used to deliver effective doses of the active ingredient. can be appropriately administered to a target mammal.

[0074] "Reconstitution time" is the time required to rehydrate a lyophilized formulation with a solution to produce a clear, particle-free solution. is the time required to

[0075] A "stable" formulation is one in which the protein therein maintains its physical stability and / or A formulation that essentially retains chemical stability and / or biological activity.

[0076] As used herein, the term "epitope" refers to the portion of an antigen that is recognized by an antibody. Epitopes include, but are not limited to, proteins (e.g., antibodies ) or a short amino acid sequence that allows for specific interaction with a ligand, i.e., a peptide (optionally glycosylated or otherwise modified). Examples For example, an epitope can be a part of a molecule that is bound by the antigen-binding site of an antibody.

[0077] The terms "treating" or "treatment" refer to a disease or disorder in a subject, such as a human. The present invention encompasses the treatment of a disease or disorder (e.g., cancer) and (i) inhibiting the disease or disorder, i.e., (ii) ameliorating the disease or disorder, i.e., causing its remission. (iii) slowing the progression of the disorder; and / or (iv) preventing or reducing the severity of the disease or This includes inhibiting, alleviating or slowing the progression of one or more symptoms of a disease or disorder. In embodiments, "treating" or "treatment" refers to killing cancer cells.

[0078] The term "killing" in the context of cancer treatment refers to at least one of the following: Regarding any kind of manipulation that leads to partial demise.

[0079] Additionally, some terms used herein are more specifically defined below. do.

[0080] Summary The present invention relates in part to a method for preparing nanoparticles, optionally lyophilized, that contain a carrier protein, an antibody, and a therapeutic agent. Surprising discovery that particles deliver targeted therapy to tumors while minimizing toxicity to patients Thus, the nanoparticles described herein have distinct advantages over conventional ADCs. It has been extensively improved.

[0081] For conventional ADCs to be effective, the linker must not dissociate during systemic circulation but must be able to reach the tumor site. It is important that the drug is stable enough to allow sufficient drug release at the site. ley, SC, et al. (2008) Bioconjug Chem 19:759-765. This is a useful tool to develop effective drug conjugates. It has been shown that MAbs 3: 467-478, Alley, SC, et al. (2008) Bioconjug Chem 19:759-765), thus nanoimmunization An attractive feature of nano-immuneconjugates is that no biochemical linker is required. This is the point.

[0082] Another drawback of current ADCs is that they result in substantially higher drug penetration into tumors in human tumors. Early testing of ADCs in mouse models has not been done using antibodies. It was suggested that the enhanced tumor targeting would deliver a higher concentration of the active agent into the tumor (Deguchi et al., 2013). , T. et al.(1986) Cancer Res 46: 3751-3755), but human tumors are probably more abundant than mouse tumors. This has no relevance in treating human diseases because the penetration is highly heterogeneous even in Jain,RK et al. (2010) Nat Rev Clin Oncol 7:653-664. Particle size is important for extravasation from the vasculature into the tumor. Human colon adenocarcinoma In mouse xenograft models, blood vessel pores were permeable to liposomes up to 400 nm in size. Yuan, F., et al. (1995) Cancer Res 55: 3752-3756. Another study of diameter and penetration showed that both properties were dependent on tumor location and growth status, and that regression It has been demonstrated that tumors and cranial tumors can be penetrated by particles smaller than 200 nm. Hobbs, SK, et al. (1998) Proc Natl Acad Sci USA 95:4607-4612. The nanoimmunoconjugates described in the paper are smaller than 200 nm in size, and the intact large complexes are distributed in the systemic circulation. It has been reported that the IL-10 receptor partially dissociates into smaller functional units that can easily penetrate tumor tissue. Moreover, once the complex reaches the tumor site, it is more This allows the release of a smaller toxic payload, so that only the toxic moiety, rather than the entire complex, hits the tumor. It must be taken up by the tumor cells.

[0083] Antibodies (i.e., Avastin), including therapeutic agents (i.e., Abraxane®) The advent of ALBUMIN coated nanoparticles has allowed the delivery of two or more therapeutic agents to the body. A new paradigm has been created for the targeted delivery of drugs to a specific site in the body. See Publication No. 2012 / 154861 ​​and International Publication No. 2014 / 055415. the entire disclosures of which are incorporated herein by reference.

[0084] A composition consisting of albumin and an antibody is mixed together in an aqueous solution at a specific concentration and ratio. When the antibody is bound to albumin, it spontaneously self-assembles into and onto albumin, forming multiple copies of the antibody. Without being limited to any theory, the following is believed to be the case: The antigen receptor portion of the antibody is oriented outward from the nanoparticle, and the hydrophobic tail is -It is thought to be incorporated into albumin via hydrophobic interactions.

[0085] Protein compositions containing single source proteins are generally stored in lyophilized form and Although such lyophilized compositions exhibit significant shelf life in the presence of hydrophobic-hydrophobic interactions, The study included self-assembled nanoparticles of two different proteins that were incorporated together via the interaction of Furthermore, the majority of the antibody binding moieties are exposed on the surface of the nanoparticles. Nanoparticle configurations allow for migration or reorganization under conditions that would otherwise be considered mild. For example, during lyophilization, the ionic charges on the protein are dehydrated, thereby The underlying charges are exposed, which alters the binding affinity of each protein with the others. This allows for charge-charge interactions between the two proteins that can be modified. As the medium (e.g., water) is removed, the concentration of the nanoparticles increases significantly. Such an increase in concentration can result in irreversible oligomerization. Merization reduces the biological properties of the oligomers compared to the monomeric form and sometimes It is a known property of proteins to increase the particle size of particles beyond Clon.

[0086] On the other hand, a stable form of the nanoparticle composition must have a shelf life of at least 3 months; and a shelf life of more than 6 or 9 months is preferred; necessary for clinical and / or commercial use Such a stable composition should be readily available for intravenous injection. The nanoparticles self-assemble when injected intravenously to target a desired site in the body. The drug must retain its integrated morphology and be able to prevent any ischemic events when delivered into the bloodstream. To avoid contamination, the particles must have a maximum particle size of less than 1 micron, and finally, The composition must be compatible with the aqueous composition used for injection.

[0087] compound As will be apparent to one of skill in the art upon reading this disclosure, the present disclosure provides methods for the preparation of carrier proteins, antibodies, and and optionally at least one therapeutic agent, and optionally lyophilized. do.

[0088] In some embodiments, the carrier protein is albumin, gelatin, elastin. (including topoelastin) or elastin-derived polypeptides elastin (e.g., α-elastin and elastin-like polypeptides (ELPs)), gliadin , legumin, zein, soy protein (e.g., soy protein isolate (SPI)), milk protein Proteins (e.g., β-lactoglobulin (BLG) and casein), or whey protein Proteins (e.g., whey protein concentrate (WPC) and whey protein isolate (WPI)) In a preferred embodiment, the carrier protein is albumin. In a preferred embodiment, the albumin is egg white (ovalbumin), bovine serum albumin (BSA In an even more preferred embodiment, the carrier protein is human serum albumin. In some embodiments, the carrier protein is generally a food It is considered a Gravely Approved (GRAS) excipient by the Food and Drug Administration (FDA).

[0089] In some embodiments, the antibody is ado-trastuzumab emtansine, arem Tuzumab, bevacizumab, cetuximab, denosumab, dinutuximab, ipilimumab, Nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pel In some cases, the drug is selected from the group consisting of tuzumab, rituximab, and trastuzumab. In embodiments, the antibody is present in a substantially monolayer on all or a portion of the surface of the nanoparticle. It is an antibody.

[0090] Table 1 shows a non-limiting list of antibodies. [Table 1]

[0091] In some embodiments, the at least one therapeutic agent is abiraterone, bendamustine , bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, da Satinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, Everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib , lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone , nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetidine Rexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine The drug is selected from the group consisting of cyclophosphamide and cyclophosphamide.

[0092] Table 2 provides a non-limiting list of cancer therapeutic agents. [Table 2] JPEG2025036633000004.jpg241161 JPEG2025036633000005.jpg242159 JPEG2025036633000006.jpg242161 JPEG2025036633000007.jpg243161 JPEG2025036633000008.jpg241159 JPEG2025036633000009.jpg241159 JPEG2025036633000010.jpg243160 JPEG2025036633000011.jpg152161

[0093] Of course, the therapeutic agent may be present inside the nanoparticle, on the exterior surface of the nanoparticle, or in any other suitable form. The nanoparticles may be located on both the nucleus and the nucleus. Contains a combination of three drugs, four drugs, five drugs or more Additionally, the nanoparticles may contain the same or different therapeutic agents on the interior and exterior of the nanoparticle. may also include

[0094] In some embodiments, any carrier protein, antibody, therapeutic agent, or any of the above. Any combination is expressly excluded. For example, in some embodiments, the composition comprises: Any carrier protein and chemotherapeutic agent other than Abraxane® and / or may include any targeting antibody other than bevacizumab.

[0095] In one embodiment, the nanoparticles have at least 10 non-covalently attached to the surface of the nanoparticles. In one embodiment, the nanoparticle comprises 0 antibodies non-covalently attached to the surface of the nanoparticle. In one embodiment, the nanoparticle comprises at least 200 antibodies. In one embodiment, the nanoparticle comprises at least 300 antibodies non-covalently attached to the The nanoparticle comprises at least 400 antibodies non-covalently attached to the surface thereof. The nanoparticles comprise at least 500 antibodies non-covalently attached to the surface of the nanoparticles. In one embodiment, the nanoparticles have at least 60 non-covalently attached to the surface of the nanoparticles. Contains 0 antibodies.

[0096] In one embodiment, the nanoparticles have about 100 to about 100 .mu.m non-covalently attached to the surface of the nanoparticles. In one embodiment, the nanoparticle comprises 000 antibodies non-covalently attached to the surface of the nanoparticle. In one embodiment, the nanoparticles comprise about 200 to about 1000 antibodies. In one embodiment, the nanoparticles include about 300 to about 1000 antibodies non-covalently bound to the surface of the nanoparticles. The nanoparticles contain about 400 to about 1000 antibodies non-covalently attached to the surface of the nanoparticles. In one embodiment, the nanoparticles have about 500 to about 1000 .mu.m non-covalently attached to the surface of the nanoparticles. In one embodiment, the nanoparticle comprises 000 antibodies non-covalently attached to the surface of the nanoparticle. In one embodiment, the nanoparticles comprise about 600 to about 1000 antibodies. In one embodiment, the nanoparticles include about 200 to about 800 antibodies non-covalently attached to the surface of the nanoparticles. The particles contain about 300 to about 800 antibodies non-covalently attached to the surface of the nanoparticles. In a preferred embodiment, the nanoparticles have about 400 .mu.m non-covalently attached to the surface of the nanoparticles. to about 800 antibodies. Values ​​contemplated include any of the recited ranges, including the endpoints. The present invention includes any value or subrange contained within any of the above.

[0097] In one embodiment, the average particle size in the nanoparticle composition is less than about 1 μm. The average particle size of the nanoparticle composition is about 130 nm to about 1 μm. The average particle size of the nanoparticle composition is about 130 nm to about 900 nm. In the present nanoparticle composition, the average particle size is about 130 nm to about 800 nm. In an embodiment, the average particle size of the nanoparticle composition is about 130 nm to about 700 nm. In one embodiment, the average particle size of the nanoparticle composition is about 130 nm to about 600 nm. In one embodiment, the average particle size of the nanoparticle composition is about 130 nm to about 500 nm. In one embodiment, the average particle size of the nanoparticle composition is about 130 nm to about 40 nm. In one embodiment, the average particle size in the nanoparticle composition is about 130 nm to about 300 nm. In one embodiment, the average particle size in the nanoparticle composition is about 130 nm. In a preferred embodiment, the average particle size in the nanoparticle composition is about 200 nm. In a particularly preferred embodiment, the nanoparticle composition has a particle size of about 150 nm to about 180 nm. The mean particle size in this study is approximately 160 nm. Expected values ​​include the listed endpoints. The present invention includes any value or subrange or range included in any of the ranges.

[0098] In one embodiment, the nanoparticle composition is formulated for intravenous injection. To avoid side effects, the nanoparticle composition formulated for intravenous injection has a diameter of about 1 μm. The nanoparticles must have an average particle size of less than 100 nm.

[0099] In one embodiment, the average particle size in the nanoparticle composition is greater than about 1 μm. The average particle size of the nanoparticle composition is about 1 μm to about 5 μm. The average particle size of the nanoparticle composition is about 1 μm to about 4 μm. The average particle size in the nanoparticle composition is about 1 μm to about 3 μm. In one embodiment, the nanoparticle composition has an average particle size of about 1 μm to about 2 μm. The average particle diameter in is about 1 μm to about 1.5 μm. Any value or subrange or range included in any of the listed ranges is included. Can be obtained.

[0100] In one embodiment, the nanoparticle composition is formulated for direct injection into a tumor. Injection includes injection into or near the tumor site and perfusion of the tumor. When formulated for direct injection into a tumor, the nanoparticles may be of any mean particle size. Without being bound by theory, larger particles (e.g., 500 nm and >1 μm) are more likely to be immobilized within the tumor and thus be beneficial Larger particles tend to accumulate in tumors or specific organs. For example, "TheraSphere (registered trademark)" (clinically used for liver cancer) It is used for injection into the hepatic artery, which feeds tumors in the liver called See the 20-60 micron glass particles used in the study. Therefore, for intravenous administration, Particles of less than 1 μm are typically used. Particles greater than 1 μm are more typically administered directly to the tumor. The drug is administered either by direct injection ("direct injection") or into an artery that supplies the tumor site.

[0101] In one embodiment, less than about 0.01% of the nanoparticles in the composition are greater than 200 nm, 300 nm, nm, 400 nm, 500 nm, 600 nm, 700 nm or 800 nm In one embodiment, less than about 0.001% of the nanoparticles in the composition have a particle size of 2 Over 00nm, over 300nm, over 400nm, over 500nm, over 600nm, over 700nm In a preferred embodiment, the composition has a particle size of about 0.01 nm or more. Less than % of the nanoparticles have a particle size greater than 800 nm. Less than about 0.001% of the nanoparticles in the composition have a particle size greater than 800 nm.

[0102] In preferred embodiments, the particle sizes and particle size ranges listed herein are reconstituted. The particle size of the freeze-dried nanoparticle composition is After resuspension in a solution (e.g., water, other pharma- ceutically acceptable excipients, buffers, etc.), the particles The size or average particle size is within the ranges recited herein.

[0103] In one embodiment, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96% , 97%, 98%, 99%, 99.5% or 99.9% of the nanoparticles are single nanoparticles present in the reconstituted composition as particles, i.e., less than about 50%, 40%, 30% The nanoparticles are dimerized or oligomerized.

[0104] In some embodiments, the particle size of the nanoparticles depends on the amount of carrier protein:antibody (e.g., For example, the particle size and particle size of the nanoparticles can be controlled by adjusting the ratio. The distribution is also important. The nanoparticles of the present invention behave differently depending on their particle size. With larger particle sizes, the agglomerates may block blood vessels. Therefore, the agglomerates of nanoparticles are On the other hand, larger particles may affect the performance and safety of the product under certain conditions. It may be more therapeutically effective under certain conditions (e.g., when not administered intravenously).

[0105] In one embodiment, the nanoparticle composition comprises at least one additional therapeutic agent. In some embodiments, at least one additional therapeutic agent is non-covalently attached to the exterior surface of the nanoparticle. In one embodiment, at least one additional therapeutic agent is present on the outer surface of the nanoparticle. In one embodiment, the at least one additional therapeutic agent is abiraterone. , bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, Lorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib , etoposide, everolimus, gemcitabine, gefitinib, idarubicin, imatinib hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, Trexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazo Panib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib , vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorel In one embodiment, the cyclophosphamide is selected from the group consisting of vin, vincristine and cyclophosphamide. In one embodiment, the at least one additional therapeutic agent is an anti-cancer antibody.

[0106] Method for producing nanoparticles In some aspects, the present invention provides a method for producing the nanoparticle compositions described herein. Regarding.

[0107] In one embodiment, the carrier protein particles or carrier protein- The carrier protein or carrier protein-therapeutic agent particles are coupled to the antibody in a ratio of therapeutic agent particle:antibody. The step of contacting with the body forms nanoparticles of the nanoparticle composition. In one embodiment, the ratio is from about 10:2 to about 10:25. In a preferred embodiment, the ratio is about 10:2 to about 10:6. In a preferred embodiment, the ratio is about 10:4. Any value, subrange or range included within any of the listed ranges is included.

[0108] In one embodiment, the solution or other liquid medium used to form the nanoparticles The amount of the carrier protein (or carrier protein-therapeutic agent) and the Nanoparticles will not form in an overly dilute solution of antibodies. In some embodiments, the solution used (e.g., sterile water, saline) The amount of solution (saline, phosphate buffered saline) is about 0.5 mL to about 20 mL of solution. In some embodiments, the amount of carrier protein is about 1 mg / mL to about 100 mg / mL. In some embodiments, the amount of antibody is about 1 mg / mL to about 30 mg / mL. For example, in some embodiments, the ratio of carrier protein:antibody:solution is about 9 mg of carrier. Somatic protein (e.g., albumin): 4 mg of antibody (e.g., BEV): 1 mL of solution (e.g., saline). A quantity of a therapeutic agent (e.g., taxol) is administered to the carrier tank. For example, 1 mg of taxol can be added to 9 mg of carrier protein. Add 4 mg of antibody to 1 mL of solution (10 mg carrier protein-therapeutic drug) For a typical IV bag, for example, about 1 liter of solution, 1 1000 times more carrier protein / carrier protein-therapeutic agent than used in mL Therefore, it is necessary to use antibodies and antibodies to form the nanoparticles in a standard IV bag. Moreover, adding these ingredients to a standard IV bag in the therapeutic amounts of the present invention is not possible. In this case, the components do not self-assemble to form nanoparticles.

[0109] In one embodiment, the carrier protein or carrier tag is in a solution having a pH of about 4 to about 8. The protein-therapeutic agent particles are contacted with the antibody. In one embodiment, the antibody is placed in a solution having a pH of about 4. The carrier protein or carrier protein-therapeutic agent particles are contacted with the antibody in a liquid. In one embodiment, the carrier protein or carrier protein is in a solution having a pH of about 5. - contacting therapeutic agent particles with the antibody. In one embodiment, the antibody is contacted in a solution having a pH of about 6. The carrier protein or carrier protein-therapeutic agent particle is contacted with the antibody. In one embodiment, the carrier protein or carrier protein-therapeutic agent is present in a solution having a pH of about 7. The particles are contacted with the antibody. In one embodiment, the carrier particles are contacted with the antibody in a solution having a pH of about 8. A protein or carrier protein-therapeutic drug particle is contacted with the antibody. In the present invention, the carrier protein or carrier protein-binding protein is bound to the carrier protein in a solution having a pH of about 5 to about 7. Therapeutic particles are contacted with the antibody.

[0110] In one embodiment, the carrier protein particles or carrier protein-therapeutic agent particles are heated to about 5° C. a temperature of up to about 60°C or any range, subrange, or value therein, including endpoints. In a preferred embodiment, the carrier protein is incubated with the antibody at 20° C. The carrier protein particles or carrier protein-therapeutic agent particles are then mixed with the antibody at a temperature of about 23° C. to about 60° C. Incubate.

[0111] Without being bound by theory, the stability of the nanoparticles in the nanoparticle composition at least one of the temperature and / or pH at which the nanoparticles are formed, and Dependent on the concentration of the components in solution (i.e., carrier protein, antibody and optionally therapeutic agent) In one embodiment, the K d is about 1 x 10 -11 M ~ approx. 2×10 -5 In one embodiment, the K of the nanoparticles is d is about 1 x 10 -11 M ~ approx. 2×10 -8 In one embodiment, the K of the nanoparticles is d is about 1 x 10 -11 M ~ approx. 7×10 -9 In a preferred embodiment, the K d is about 1 x 10 -11 M ~ approx. 3 ×10 -8 M. Possible values ​​are any of the ranges listed inclusively. The term "range" includes any value or subrange or range included in the range.

[0112] Freeze drying The present invention can be produced by standard freeze-drying techniques in the presence or absence of stabilizers, buffers, etc. Surprisingly, these conditions result in a relatively high concentration of the nanoparticles in a freeze-dried composition. Moreover, at least these nanoparticles can be freeze-dried without altering their relatively fragile structure. They retain their particle size distribution when resuspended and, more importantly, are substantially the same as when freshly prepared. Can be reconstituted for in vivo administration (e.g., intravenous delivery) in the same form and ratio .

[0113] formulation In one embodiment, the nanoparticle composition is formulated for direct injection into a tumor. Injection includes injection into or near the tumor site and perfusion of the tumor. The nanoparticle compositions are not administered systemically and are therefore formulated for direct injection into tumors. The nanoparticle composition may comprise any average particle size. Without being bound by theory, However, larger particles (e.g., >500 nm and >1 μm) were immobilized in the tumor. are more likely to be achieved, which may have beneficial effects.

[0114] In another embodiment, a compound as provided herein and at least one pharma- ceutically acceptable salt thereof are provided. Compositions comprising the excipient are provided herein.

[0115] In general, the compounds described herein are administered to a patient via any of the accepted modes of administration. Various formulations and drug delivery systems are known in the art. See, e.g., Gennaro, AR, ed. (1995) Remington's Pharm. See Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co. I want to.

[0116] Generally, the compounds described herein can be administered orally, systemically (e.g., via oral administration (e.g., intradermal, intranasal, or suppository) or parenteral (e.g., intramuscular, intravenous, or subcutaneous) It is administered by one of two routes:

[0117] The compositions generally comprise a compound of the present invention in combination with at least one pharma- ceutical acceptable excipient. Acceptable excipients are non-toxic, aid in administration, and are capable of carrying out the claimed compound. Such excipients are generally known to those skilled in the art and do not adversely affect the therapeutic efficacy of the compound being administered. Any available solid, liquid, semisolid or, in the case of an aerosol composition, gaseous excipient. It's fine.

[0118] Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, Sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate Sodium Stearate, Glyceryl Monostearate, Sodium Chloride and Dry skim milk, etc. Liquid and semi-solid excipients include glycerin, propylene glycol, glycol, water, ethanol and various oils, e.g. of petroleum, animal, vegetable or synthetic origin. Oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc. may be selected, particularly for injection solutions. Preferred liquid carriers for this purpose include water, saline, aqueous dextrose and glycols. Other suitable pharmaceutical excipients and their formulations are described in Remington's Pharmaceuticals. Maceutical Sciences (Remington's Pharmaceutical Sciences), edited by E. W. Martin (Mack Publishing (Fing Company, 18th ed., 1990).

[0119] The composition may, if desired, be presented in a pack or depot containing one or more unit dosage forms containing the active ingredient. Such a pack or device may be provided as a dispenser device. or plastic foil, e.g. blister packs or glass and vials The pack or dispenser device may contain a rubber stopper for administration. The present invention may be formulated in a compatible pharmaceutical carrier and may be accompanied by instructions for use in administering the composition to the patient. A composition containing the compound of formula (I) is prepared, placed in a suitable container, and labeled for treatment of the indicated condition. You may also attach a sticker.

[0120] Treatment method The nanoparticle compositions described herein can be used to treat cancer cells and / or tumors in a mammal. In a preferred embodiment, the mammal is a human (i.e., a human Preferably, the lyophilized nanoparticle composition is reconstituted (with an aqueous solution) prior to administration. (suspended in vehicle).

[0121] In one aspect, a cancer cell is contacted with an effective amount of a nanoparticle composition described herein to A method for treating cancer cells is provided, which comprises the step of treating the cancer cells. Including, but not limited to, the reduction of proliferation, the killing of cancer cells, and the prevention of metastasis of cancer cells. Some examples include:

[0122] In one aspect, a patient is administered a therapeutically effective amount of a nanoparticle composition described herein. The present invention provides a method for treating a tumor in a patient in need thereof, the method comprising the step of treating the tumor with In one embodiment, the tumor size is reduced. In one embodiment, the tumor size is reduced at least during treatment and and / or the tumor does not increase in size (i.e., progress) during the post-treatment period.

[0123] In one embodiment, the nanoparticle composition is administered intravenously. The composition is administered directly to the tumor. In one embodiment, the nanoparticle composition is administered by direct injection or administration to the tumor. The drug is administered by perfusion into the retina.

[0124] In one embodiment, the method comprises: a) administering the nanoparticle composition once a week for three weeks; b) ceasing administration of the nanoparticle composition for one week; and c) Optionally, repeat steps a) and b) as necessary to treat the tumor. Includes.

[0125] In one embodiment, a therapeutically effective amount of the nanoparticles described herein is about 50 mg / m 2 ~about 200mg / m 2 or carrier protein + therapeutic agent. In a preferred embodiment, the therapeutically effective amount is about 75 mg / m 2 ~about 175mg / m 2 Carrier Tan Includes protein or carrier protein + therapeutic agent. Possible values ​​include any enumerated list including endpoints. The term "range" includes any value or subrange or range included in any of the ranges listed.

[0126] In one embodiment, the therapeutically effective amount is about 20 mg / m 2 ~about 90mg / m 2 Contains antibodies to In a preferred embodiment, the therapeutically effective amount is 30 mg / m 2 ~about 70mg / m 2 The antibody Possible values ​​include any value in any of the listed ranges, including the endpoints. Any value or subrange or range may be included.

[0127] Cancers or tumors that can be treated by the compositions and methods described herein Cancers include, but are not limited to, biliary tract cancer, brain cancer (such as glioblastoma and medulloblastoma), breast cancer, Cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological tumors (acute lymphoma) myeloid and myeloid leukemia), multiple myeloma, AIDS-related leukemia and adult T-cell leukemia Lymphoma, intraepithelial neoplasia (such as Bowen's disease and Paget's disease), liver cancer (hepatocellular carcinoma), lung Cancer, lymphoma (Hodgkin's disease and lymphocytic lymphoma, etc.), neuroblastoma, oral cancer (tonsillectomy) squamous cell carcinoma, etc.), ovarian cancer (arising from epithelial cells, stromal cells, germ cells and mesenchymal cells) ), pancreatic cancer, prostate cancer, rectal cancer, sarcoma (leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma, etc.) ), testicular cancer (germinal tumor (seminoma, non-seminoma [teratoma, choriocarcinoma]), stromal and germ cell tumors), thyroid cancer (thyroid cancer adenocarcinoma and medullary carcinoma) and renal cancer (such as adenocarcinoma and Wilms tumor) In important embodiments, the cancer or tumor is breast cancer, lymphoma, or multiple myeloma. and melanoma.

[0128] In general, the compounds of the invention can be administered in any of the accepted modes of administration for agents that serve similar utilities. The compound of the present invention, i.e., the nanoparticles, is administered in a therapeutically effective amount by either method. The amount used will depend on the severity of the disease being treated, the age and relative health of the subject, the amount of compound used, and the dosage. The various factors that influence the composition of the present invention include the potency of the product, the route and form of administration, and other factors that are well known to those of skill in the art. It depends on the factors:

[0129] The effective amount of such an agent can be readily determined by routine experimentation and is most effective. The convenient route of administration and the most suitable formulation can also be determined. and drug delivery systems are available in the art. ed.(1995) Remington's Pharmaceutical Sciences, 18 th e Please refer to d.

[0130] An effective amount or therapeutically effective amount or dose of an agent, such as a compound of the invention, is a therapeutically effective amount or dose that is administered to a subject. "Amplification" refers to that amount of a drug or compound that results in amelioration of symptoms or prolongation of survival in a patient. The toxicity and therapeutic efficacy of such molecules can be measured, for example, by measuring the LD50 (lethal dose for 50% of the population) and and ED50 (therapeutic dose effective in 50% of the population) in cell cultures or experimental animals. Toxic effects: Therapeutic doses may be determined by standard pharmaceutical procedures. The ratio is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Drugs that are

[0131] An effective amount or a therapeutically effective amount may be sought by a researcher, veterinarian, physician or other clinician. Compounds that induce biological or medical responses in tissues, systems, animals or humans or the amount of pharmaceutical composition. The dosage amount depends on the dosage form used and / or the administration method utilized. It may vary within this range depending on the route. The exact formulation, route of administration, dosage and interval may vary. The interval should be selected according to methods known in the art, taking into account the nature of the subject's condition. It must be.

[0132] The plasma concentration of the active moiety sufficient to achieve the desired effect, i.e. the minimum effective concentration (MEC Dosage amount and interval can be adjusted individually to achieve the MEC. The MEC varies from individual to individual and can be estimated, for example, from in vitro data and animal studies. The dosage required to be achieved will depend on individual characteristics and the route of administration. In cases of selective uptake, the effective local concentration of the drug cannot be related to the plasma concentration. EXAMPLES

[0133] Albumin-bound paclitaxel (i.e., Abraxane®) or or cisplatin and the antibody bevacizumab (i.e., Avastin®) or using nanoparticles consisting of rituximab (i.e., Rituxan®), The present disclosure will now be described.

[0134] Those skilled in the art will appreciate that the methods for making and using the nanoparticles in the Examples are merely illustrative. It will be understood that the present disclosure is not limited to this example.

[0135] All abbreviations used herein have their usual scientific meaning unless otherwise specified. All temperatures are in °C. As used herein, the following terms have the following meanings unless otherwise defined: do. ABX = Abraxane® / (albumin-bound paclitaxel) AC = cisplatin-bound ABX ACN = acetonitrile ADC = antibody-dependent chemotherapy BEV = bevacizumab BSA = bovine serum albumin dH2O = distilled water DMEM = Dulbecco's modified Eagle's medium nM = nanomolar EdU = 5-ethynyl-2'-deoxyuridine EM = electron microscopy FCB = flow cytometry buffer FITC = fluorescein kD = kilodaltons K d = dissociation constant kg = kilograms KV = kilovolts L / hr = liters per hour LC-MS = Liquid Chromatography-Mass Spectrometry M = moles mCi = millicuries mg = milligrams ml or mL = milliliters m 2 = Square meters mm 3 = cubic millimeters μg = micrograms μl = microliter μm = micrometers / microns PBS = phosphate buffered saline pK = pharmacokinetics RT = room temperature rpm = revolutions per minute V = Volts ×g = ×gravitational acceleration

[0136] Example 1: Preparation of nanoparticles Abraxane (ABX) (10 mg) and bevacizumab (BEV) (unless otherwise stated) 4 mg [160 μl]) and 840 μl of 0.9% saline was added, and the mixture was incubated for 10 min. The mixture was incubated at room temperature (or The particles were formed by incubation at 4°C (indicated temperature) for 30 min. ABX:BEV complexes For the Mastersizer experiment to measure particle size, 10 mg of ABX was added to a sample of 0–25 mL. The antibodies were suspended in BEV at a concentration of 0.5 mg / ml. Under similar conditions, ABX and rituximab (0–1 0 mg / ml) or trastuzumab (0–22 mg / ml).

[0137] For human use, a number of 4 mL vials appropriate for a dose containing 25 mg / mL BEV were prepared. ABX was prepared by diluting each vial to 4 mg / mL according to the instructions below. ABX:BEV complexes may be prepared. Reconstituted to a final concentration of 10 mg / mL containing ABX nanoparticles. Prepare the appropriate number of vials containing 100 mg of ABX by composition. Using a sterile 3 mL syringe, inject 1.6 mL (40 mg) of bevacizumab ( 25 mg / mL) was withdrawn from each vial containing 100 mg of ABX for a minimum of 1 minute. The bevacizumab solution was poured slowly into the inner wall. Do not inject directly. Then, using a 12 mL sterile syringe, inject 8.4 mL of 0.9% Sodium Chloride Injection, USP, was withdrawn and 8.4 mL was mixed with 100 mg of ABX and Each vial containing 40 mg of BEV can be slowly injected into the inner wall over a minimum of 1 minute. 1.6 mL of BEV and 8.4 mL of 0.9% Sodium Chloride Injection, USP Once addition is complete, gently stir each vial until complete dissolution of any lumps / powder occurs. Rotate and / or gently invert for at least 2 minutes. At this point, the concentration of each vial is adjusted to 100 mg / 10 mL. The dose should be 40 mg / 10 mL of ABX and 40 mg / 10 mL of BEV. The vial containing the sucrose solution must be left for 60 minutes. Gently rotate the vial every 10 minutes. The complex should be mixed continuously by shaking and / or inversion. After 60 minutes, Withdraw the calculated dose volumes of BX and BEV from each vial and empty VIAFLEX points. The solution should be added slowly to the drop bag. Then an equal volume of 0.9% sodium chloride should be added. Add 50 mL of ABX (USP) to a final concentration of 5 mg / mL ABX and 2 mg / mL BEV. The bag is then gently rotated and / or slowly rotated for 1 minute. ABX:BEV nanoparticles should be mixed in the flask for up to 4 hours after final dilution. It can be stored at room temperature.

[0138] Example 2: Binding of ABX and BEV in vitro To determine whether ABX and BEV interact, the nanotubes formed in Example 1 were The particles were analyzed by flow cytometry and electron microscopy.

[0139] method [Flow Cytometry]: AB160 was generated as described in Example 1 above. To assess the binding of BEV to ABX, an Accuri C6 flow cytometer (BD Visualization of AB160 was performed at Accuri C (Franklin Lakes, NJ) and Data analysis was performed using software 6. 5 μg of biotinylated goat anti-mouse Ig G (Abcam, Cambridge, MA) was diluted with 5 μg of streptavidin- The Fab portion of BEV was labeled with PE (Abcam, Cambridge, Massachusetts). Goat anti-mouse IgG was used to label AB160 because the antibody is of mouse origin. ABX and AB160 were incubated with PE-labeled goat anti-mouse IgG for 30 min. The cells were incubated at room temperature for 1 h, washed, and visualized by flow cytometry.

[0140] [Electron microscopy]: 5 μl of ABX dissolved in PBS at 6 mg / ml was added to Parrodian. (parlodian)-Carbon coated 300 mesh copper grids were added and left for 1 minute. A pointed piece of filter paper was placed in contact with the droplet to remove excess liquid, leaving a thin film on the grid. The grid was then dried. To dissolve any remaining buffer crystals on the dried grid, The samples were washed three times in dH2O. 2) was added to the grid. The grid was then again placed in contact with the pointed piece of filter paper to remove excess. The liquid was removed, and a thin film was obtained on the grid and allowed to dry. 25 mg / ml BEV (Genentech) was diluted in PBS at a ratio of 1:10. 5 μl of BEV was placed on a nickel formvar-coated grid and incubated for 30 min to 1 h. For AB160, 10 mg / ml ABX and 10 mg / ml PBS were used. and 4 mg / ml BEV dissolved in 0.9% sodium chloride solution in a 2.5:1 ratio. The complex was further diluted 1:5 with PBS. 5 μl of the complex was added to nickel formaldehyde. The samples were then placed on a burr-coated grid and air dried for 30 minutes to an hour. 1 h in goat anti-mouse IgG with 100 μg of gold-conjugated particles (Electron Microscopy Sciences) Incubate, dilute 1:30 in 10% FCB / PBS, and repeat 6 times with PBS (respectively). 2 min), washed six times with dHO, then resuspended in 2% methylcellulose and 4% UA (9:1). The grids were stained with the mixture for 5 min. The stain was poured off using filter paper and the grids were air-dried for 1 h. Both samples were immunoblotted with 6 nm gold-conjugated particles (Jackson ImmunoResearch) in donkey anti-mouse IgG. Incubate overnight with 10% FCB / PBS, dilute 1:25 in PBS, and Wash 6 times (2 min each) with dH2O water, stain with 1% PTA for 5 min, and air dry. The plates were then covered with 2% methylcellulose and air-dried for 1 hour. Micrographs were taken with an L1400.

[0141] result In vitro, ABX (10 mg / ml) was incubated with 4 mg / ml BEV; We found that they formed 160 nm nanoparticles (herein referred to as AB160). Since the Fab portion of IgG1(BEV) is derived from mouse, particles containing BEV were Selective labeling was performed with purified goat anti-mouse IgG, followed by anti-goat PE as the secondary antibody. As a negative control, samples were stained with anti-goat PE alone. Visualization showed that anti-mouse IgG1 bound to AB160 compared to ABX alone (6.7% positive). The bright signal (41.2% positive) of BEV was demonstrated (Figure 1A). To confirm the binding of BEV to X, we labeled BEV with gold-conjugated mouse anti-human IgG and The particles were visualized using electron microscopy (Figure 1B). Remarkably, the EM images showed that ABX This suggests a monolayer of BEVs surrounding the nanoparticles.

[0142] When the complex dissociates, which protein (albumin or BEV) does paclitaxel bind to? To determine whether or not the AB160 remained bound, a fraction, i.e., fine particles (naphthalene), was prepared and (AB160), and recovered proteins above and below 100 kD. Liquid chromatography-mass spectrometry (LC-MS) was used to determine the amount of paclitaxel in each fraction. Approximately 75% of the paclitaxel remained in the microparticles, and the remaining paclitaxel Most of the protein was associated with a fraction containing proteins of 100 kD or more (Fig. 1C, top). When the microparticles were dissociated, paclitaxel was released into the BEV alone or into the BEV and albumin. This suggests that the dissociated complex is bound to a heterodimer of VEG These results indicate that the tumor microenvironment contains chemotherapeutic agents along with antibodies that still migrate to the tumor microenvironment. The findings were confirmed by Western blot analysis of supernatants from AB160, which showed BEV and paclitaxel bind to the paclitaxel-BEV-albumin protein complex. They were found to colocalize at a consistent size of approximately 200 kD (Fig. 1C , bottom).

[0143] Example 3: Function of AB160 in vitro The two main components of the complex, the antibody and paclitaxel, are present in the complex. We confirmed that these functions are maintained when the

[0144] method In vitro toxicity: A375 human melanoma cell line (ATCC, Manassas, VA) and and Daudi (B cell lymphoma line) (ATCC, Manassas, VA) were cultured at 1% P The cells were cultured in DMEM containing SG and 10% FBS. The cells were harvested and plated at 100 x g for 1 well. 0.75×10 6 The cells were seeded in a 24-well plate at 0–200 μg / mL. The mice were exposed to ABX or AB160 at a paclitaxel concentration of 1 ml overnight at 37°C and 5% CO2. To measure proliferation, Click-iT EdU (Molecular Probes, Eugene, OR) was used. Briefly, 10 mM EdU was added to the wells, and the cells and The cells were permeabilized with 1% saponin and incubated overnight with ABX or AB160. The FITC-positive cells from each treatment were treated with 5% ethanol and the intercalated EdU was labeled with FITC-conjugated antibody. The proliferation index was determined by dividing the maximum proliferation of untreated EdU-labeled cells by the maximum proliferation of untreated EdU-labeled cells.

[0145] [VEGF ELISA]: When BEV is bound to ABX, its ligand, VEG To determine whether the VEGF peptide was still capable of binding to F, a standard VEGF ELISA (R and D) was used. Systems, Minneapolis, MN). AB160 was prepared as described. 2000 pg / ml of VEGF was added to the AB160 complex or ABX alone. F was incubated with the nanoparticles at room temperature for 2 hours. The suspension was rotated at 6000 rpm for 1 After spinning for 5 min, the supernatant was collected and free VEGF was measured by ELISA. ELISA plates were coated with capture antibody overnight at 4°C. Plates were washed and blocked. After washing, the detection antibody was added and the plate was incubated with substrate (R an The absorbance was measured using a Versamax ELISA plate (D Systems, Minneapolis, MN). The 450 nm chromatogram was recorded using a chromatograph reader (Molecular Devices, Nieuvale, CA). The concentration of unbound VEGF was determined using a standard curve ranging from 0 to 2000 pg / ml. It was determined.

[0146] result AB160 exhibited anti-ABX activity in an in vitro toxicity assay using the human melanoma cell line A375. The toxicity of paclitaxel was similar in both formulations compared to rituximab alone. These results suggest that the IL-11 pathway may play a role in the regulation of IL-11 signaling (Fig. 1D).

[0147] To test the binding of VEGF to BEV in the AB160 complex, AB160 or ABX was incubated with VEGF, particulates were removed, and the supernatant was analyzed for VEGF content. The absence of VEGF in the supernatant measured from AB160 (less than 10%) Unbound VEGF) indicates that VEGF is bound by BEV in the AB160 complex. , which remains free when incubated with ABX alone (>80% unbound V EGF) (Figure 1E).

[0148] Importantly, these assays demonstrated that paclitaxel in AB160 binds to tumor cells. BEV retains its toxicity against VEGF and the ability of the bound BEV to bind to its ligand VEGF. It has been demonstrated that the

[0149] Example 4: Particle size and protein affinity To understand the properties of nanoparticles formed upon BEV binding to ABX, we investigated the mechanism of action of ABX: The particle size of the BEV complex was measured against ABX.

[0150] method [Mastersizer and Nanosight]: Mastersizer 2 000 (Malvern Instruments, Westborough, MA) for dynamic light scattering. The particle sizes of ABX and antibody-ABX drug conjugates were measured. Then, 2 ml (5 mg / ml) of Abraxane or conjugate was added to the sample chamber. Data was analyzed using ern software and the particle size distribution was expressed as a volume. Stability was subsequently evaluated using the NanoSight system (Malvern Instruments, West Virginia, MA). The particle size was confirmed by diluting the ABX or composite particles to an appropriate range. The data was expressed as particle size distribution. However, the nanoparticle tracking analysis showed that the particles were not Brownian. Particle size was measured using a flow meter.

[0151] [Binding assay]: Biotinylated BEV, rituximab or trastuzumab was incubated for 10 Streptavidin probe (ForteBio, Menlo Park, CA) at 0 μg / ml ABX binding was measured at 1000, 500 and 100 mg / ml, and BL Absorbance was measured using the Itz system (ForteBio, Menlo Park, CA, USA). Association and dissociation constants were calculated using BLItz software.

[0152] Binding affinity of BEV to ABX using Biolayer Interferometry (BLItz) technology The biotinylated BEV was bound to a streptavidin probe and ABX( The dissociation constants of BEV and ABX were (Kd) is 2.2 × 10 at room temperature and pH 7 -8 M, which is a strong non-covalent phase The binding affinities of BEV and ABX are consistent with their interaction with albumin and some cellular The dissociation constants observed between natural and artificial albumin-binding domains of bacterial proteins Within the range. Nilvebrant, J. et al. (2013) Comput Struct Biotechnol J 6:e2013030 09.

[0153] result The ABX:BEV nanoparticles were consistently larger than ABX alone at 130 nm (approximately 1 60 nm) (Figure 2A). The particle size of the nanoparticles formed correlated directly with the concentration of BEV used. The median diameters ranged from 0.157 to 2.166 μm (Figure 2A). The goal of their study is a Phase 1 clinical trial in which the smallest ABX:BEV particle (AB160) was We focused on the 30 nm ABX because it is the most similar. The particle size is consistent with the EM images of ABX + the surrounding monolayer of BEVs and particles. (See Figure 1B).

[0154] To determine whether intravenous administration conditions affect the size distribution of nanoparticles, Particle size distribution of AB160 (or ABX) incubated in saline for up to 24 hours The particle size distribution of AB160 did not change significantly for up to 24 hours (Figure 9A and Figure 9B). B). However, after 4 hours at room temperature, ELISA showed some degree of dissociation of AB160. There is evidence of this (Figure 9C).

[0155] To determine the stability of AB160 in plasma, ABX or AB160 were mixed at 9:1 or or incubated in saline or heparinized human plasma at a relative volume ratio of 1:1. Either ABX (Figure 10, upper panel) or AB160 (Figure 10, lower panel) When incubated in plasma at an equal volume (1:1), particles (0.01-1 μm) were detected. It is particularly noteworthy that this was not issued.

[0156] Western blots (data not shown) were performed in saline or heparinized human plasma. In the study, AB160 still contained tumor-targeting antibodies, albumin, and the cytotoxic drug paclitaxel. These protein complexes were found to be smaller than the tumor-associated protein complexes. Once delivered to the tumor site, it quickly dissolves, releasing its cytotoxic payload and killing the tumor. These nanoparticles can effectively initiate tumor regression without uptake of the entire nanoparticle by tumor cells. It holds the ability to.

[0157] Next, to test whether the binding affinity is pH and / or temperature dependent, BX was suspended in BEV, and the mixture was incubated at various temperatures (room temperature, 37°C, and 58°C). Prior to bating, particles were formed by dilution with saline at pH 3, 5, 7 or 9. The binding affinity of BX and BEV was pH- and temperature-dependent, with the particles binding at pH 5 and 58°C. The highest binding affinity was observed when the progeny was formed (Figure 2B).

[0158] Higher affinity binding of BEV and ABX at 58 °C and pH 5 contributes to the stability of the complex. Nanoparticle Tracking Analysis (NanoSight) was used to determine whether various products could be substituted. The agents were compared after incubation in human AB serum for 0, 15, 30 or 60 min. , 58°C and pH 5 (AB1600558), room temperature and pH 7 (AB16007) The stability of AB160 prepared at 58°C or 58°C and pH 7 (AB1600758) was examined using the same ABX0558, ABX07, and ABX0758 exposed to the conditions Compared.

[0159] As indicated by the number of particles present per mg of ABX after exposure to human AB serum Thus, particles prepared under higher affinity conditions (pH 7 and 58°C) were also more stable. AB160 particles exhibited increased stability in human serum that correlated with their binding affinity. In particular, AB16007 and AB1600558 were measured per mg of ABX. As determined by particle size and number of particles, saline and ABX alone were more effective. The AB160 particles were stable in both human serum and human serum (Fig. 2C and Table 3). By varying the conditions under which the AB160 particles are formed, the stability of the particles can be manipulated. This shows that. [Table 3]

[0160] These data demonstrate that BEV binds A with picomolar range affinity, indicating strong non-covalent binding. This is consistent with ABX being demonstrated to bind to BX and surrounded by a monolayer of antibody molecules. It was demonstrated that the particle size distribution, i.e. the size of the formed particles, depends on the antibody concentration. It was.

[0161] Example 5: Efficacy of AB160 in mice Using a xenograft model of A375 human melanoma cells implanted in athymic nude mice The efficacy of AB160 in vivo was tested.

[0162] method In vivo experiments were performed at least twice. Power analysis determined the number of mice required for this experiment. The tumors in the mice were measured 2-3 times a week, and the mice were euthanized when the tumor volume reached 10% by weight. Mice that completed the tumor response were monitored for 60-80 days after treatment. The endpoint was median survival time. Kaplan-Meier curves were generated and Mantel-C The ox test was performed to determine the significance of median survival times between treatment groups. Results are shown for at least five repeated experiments. Baseline differences were calculated using Student's t test. Statistical analysis of the in vitro and in vivo rate changes from the in experiments was performed.

[0163] [Mouse model]: 1 × 10 6 A375 human melanoma cells The cells were then transfected into athymic nude mice (Harlan Sprague Dawley, Indianapolis, IN). The tumor was transplanted into the right flank of a squirrel (Lucifer squirrel). 3When the size of The mice were then treated with PBS, ABX (30 mg / kg), BEV (12 mg / kg) (BEV followed by A BX) or the above concentrations of AB160. In the study, the highest dose required to form larger particles was observed in the BEV monotherapy group. A dose of BEV (45 mg / kg) was used. Tumor size was monitored three times a week, and tumor volume was Equation: (Length x Width 2 The tumor size was calculated using the formula: Approximately 2500 mm 3 Mice were sacrificed when the tumor size was equal to [(tumor size on day of treatment - 7 days Change from baseline to day 7 was calculated by [tumor size at day 1 (initial tumor size) / tumor size on treatment day] × 100. The conversion rate was calculated. 6 Daudi cells were cultured in athymic Nude mice were similar except that they were injected into the right flank.

[0164] result AB160 was tested against PBS, single agent alone and consecutively administered drugs. On day 1, mice treated with AB160 had a significantly higher AF than all other treatment groups compared to baseline. The tumor size was significantly reduced by AB (p=0.0001-0.0089) (Figure 3A). All mice treated with 160 experienced tumor regression by day 7, and this tumor response was consistent with that of all This was translated into a significant increase in median survival time in the AB160 group compared with the other groups ( Fig. 3B ). PBS (p<0.0001), BEV (p=0.003), ABX (p=0.0003) , 7, 14, and 14 in the BEV+ABX (p=0.0006) and AB160 groups, respectively. , 18 and 33 day survival.

[0165] Larger tumors are more likely to have higher local VEGF concentrations. (700mm 3 Less than and 700mm 3 Analyzing the data based on It was found that tumors with higher β-catenin had a greater response to AB160, which is consistent with the higher This suggests that tumor VEGF concentrations attract more BEV-targeted ABX to tumors. The difference in the rate of change from baseline was significant in the AB160 group (p=0.0 057). This observation was not observed in the ABX alone group (p=0.752), and ABX was not associated with targeted had no ability to do so (Figure 3C).

[0166] As shown in FIG. 2A, particles with increasing particle size with increasing BEV:ABX ratio Tumor regression and median survival time correlated positively with increasing particle size, which have shown that the biodistribution of larger particles may be altered compared to smaller particles. (Figures 3D and 3E). A full toxicity study was performed in mice and no toxicity was observed. Ta.

[0167] Example 6: Pharmacokinetics of Paclitaxel in Mice To compare the pharmacokinetics (pk) of AB160 and ABX, Plasma paclitaxel in mice administered X at 0, 4, 8, 12, and 24 hours The concentration was measured.

[0168] method [Pharmacokinetics of paclitaxel]: Agilent Poroshell 120 EC-C18 analytical column (2.1× The microscope was mounted on a 100 mm, 2.7 μm, Chrom Tech, Apple Valley, Minnesota Agilent Poroshell 120 EC-C18 precolumn (2.1 × 5 mm, 2.7 μm, Chrom Tech) (A) water containing 0.1% formic acid and (B) water containing 0.1% formic acid at 40°C using a 100% ethanol solution (Microfluidics, Inc., Apple Valley, MN). B) A constant flow rate of 0.5 ml / min using a gradient mobile phase consisting of ACN with 0.1% formic acid. Liquid chromatographic separation of paclitaxel and d5-paclitaxel was achieved by elution with Elution started with 60% A and 40% B for 0.5 min, then B for 4.5 min. The B concentration was increased linearly from 40% to 85% over 10 min, and 85% B was maintained for 0.2 min. The autosampler temperature was 10°C and the sample injection volume was 2μ. The mass spectrometer was used in the ESI positive mode, and the capillary voltage was 1.75 kV. voltage, source temperature of 150°C, desolvation temperature of 500°C, cone gas flow of 150 L / hr A desolvation gas flow rate of 1000 L / hr was used, with a data acquisition time of 0.075 seconds. A multiple reaction monitoring (MRM) scan mode was used to measure paclitaxel and intravenous Detection of standard d5-paclitaxel was achieved. MassLynx-Intellist The cone voltage and collision energy were measured using the art (v4.1) software. The energy ranged from 6 to 16 V and from 12 to 60 eV, respectively. For paclitaxel, it was 854.3> 105.2, and d5-paclitaxel had MRM precursor and m / z of 859.3 > 291.2. The product ions were monitored. Litaxel (1 mg / ml in EtOH) and d5 paclitaxel (1 mg / ml in EtOH) A primary stock solution of 100 µg / ml was prepared and stored at -20°C. Prepare working solutions by diluting the stock solution with ACN in cleaned glass vials and store at -20°C. 100 μl of plasma sample was diluted with d5 ​​paclitaxel (116.4 nM or 100 ng Add the solution to a 1.7 ml microcentrifuge tube containing 100 µl of PBS (100 µL / ml) and 300 µl of ACN, and vortex. Mix at room temperature for 10 minutes to precipitate proteins, then centrifuge for 3 minutes. Plasma samples were extracted by rotating at 14,000 rpm. The supernatant was then passed through an Agilent Captiv aND lipids The plates (ChromTech, Apple Valley, MN) were filtered and 96-well plated. The samples were collected on a well plate and dried using nitrogen gas. The plate was reconstituted and shaken for 5 minutes on a plate shaker (high speed). For this purpose, paclitaxel (0.59-5855 nM or 0.5-5000 ng / ml) Plasma standard curves containing d5 paclitaxel (116.4 nM) were generated daily. Tumors were thawed on ice, weighed, and diluted 2 parts (weight to volume) with 1× PBS. Then, a PRO200 tissue homogenizer using a serrated probe (5 mm x 75 mm) was used. Tumors were homogenized using ELISA kits. The tumor homogenates were then treated similarly to human plasma samples. Ta.

[0169] [Mouse Imaging]: Prepare Avastin and IgG control solutions and follow the protocol (Immunography) The antibodies were labeled with I-125 according to the protocol provided by Anis LifeSciences. 0.125 M Tris-HCl, pH 6.8, 0.15 M NaCl) and 5 mCi Na 125 I was diluted with iodination tubes (ThermoFischer Scientific, Waltham, MA). The iodide was activated and rotated at room temperature. The activated iodide was The mixture was mixed with the protein solution. 50 μl of capture buffer (10 mg / mL of chitin in PBS) was added. Tris / BSA buffer (pH 7.4) was added and incubated for 5 min. After addition and mixing, the sample was diluted in a 10K MWCO dialysis cassette with 4 mL of pre-chilled PBS. The mixture was dialyzed at 37°C for 30 minutes, 1 hour, 2 hours, and overnight. Radioactivity was measured using a gamma counter. The disintegrations per minute (DPM) and specific activity were then calculated. Abraxane-Avastin I-125, Abraxane-Human IgG I-125 or Abraxane alone. At 3, 10, 24, and 72 hours after administration, A SPECT-II / CT scanner (MILabs, Utrecht, The Netherlands) was used. Animals were imaged using SPECT-CT imaging. dered subsets by expectation maximization SPECT reconstruction was performed using the Feldkamp algorithm. The CT data were reconstructed during the algorithm. The simultaneously recorded images were processed using the PMOD software. Further rendering and analysis were performed using PMOD software (PMOD Technologies, Zurich, Switzerland). Animals were sacrificed and dissected 72 hours after injection. The organs and tissues were calibrated using a radioisotope dose calibrator (Capintec CRC-127R, Capintec). and measured.

[0170] result The results of the first pk experiment are shown in Figure 4A and Figure 4B. Area under the curve (AUC) and maximum serum concentration (C max In the first pk experiment, C max and AUC is AB160 and ABX was very similar in non-tumor bearing mice (63.3 + / - 39.4 vs. 65.5 + / - 14.4 and 129 vs. 133 μg / ml). In mice with C max and AUC were different (55.7+ / -21.2 vs. 63.3 + / - 17.3 and 112 vs. 128 μg / ml) (Figure 4C). Although this difference was not statistically significant, it does suggest superior targeting with AB160 compared to ABX. It matched.

[0171] A second study was performed using earlier time points and larger versus smaller tumor sizes. A pK experiment was performed (Figure 4D-F). The results of this experiment showed that compared with mice with small tumors, The lowest blood levels of paclitaxel in mice with large tumors (tumors treated with ABX) 80 in mice without tumors, mice with small tumors, and mice with large tumors, respectively. 4+ / -2.7, 48.4+ / -12.3 and 30.7+ / -5.2, cured with AB160 66.1+ / -19.8, 44.4+ / -12.1 and 22.8+ / -6 for those treated with cerebrospinal fluid. 9) showed that tumor-bearing mice showed smaller AU than non-tumor-bearing mice. C was demonstrated. Similarly, C max In mice with larger tumors, the tumor size was significantly higher in both treatment groups. The ABX group had a lower β-blocker concentration (47.2, 28.9, and 19.7 μg / ml, respectively, and the AB160 group had a lower β-blocker concentration (47.2, 28.9, and 19.7 μg / ml, respectively). The A of paclitaxel in blood was 40.1, 26.9, and 15.3 μg / ml (Figure 4G). U.C. and C. max was lower in AB160-treated mice compared to ABX-treated mice. Although not statistically significant, the data suggest that AB160-treated tumors This is further consistent with higher deposition of taxel.

[0172] To directly test this hypothesis, tumor paclitaxel concentrations were measured by LC-MS. Tumor paclitaxel concentrations were 3473 μg / mg + / - 340 in tissues vs. 212 in 4 h ( 7+ / -3.5μg / mg tissue, p=0.02) and 8 hours (3005μg / mg+ / -146 tissue vs. 1688 + / -146 μg / mg tissue, p=0.01). was significantly higher in tumors treated with AB160 compared with ABX, which was consistent with paclitaxel These results suggest that the IL-1 receptor remains in the tumor longer when targeted by the antibody. (Figure 4H). This describes the blood pK and coincides with the redistribution of the drug to tissues, including the tumor. It is being done.

[0173] I-125 labeled AB160 (Abx-AvtI125) and ABX-conjugated IgG In vivo live imaging of isotype (Abx-IgGI125) revealed that 3 days after injection Time (32.2uCi / g+ / -9.1 vs. 18.5uCi / g+ / -1.65, p=0. 06) and 10 hours (41.5uCi / g+ / -6.4 vs. 28.7uCi / g+ / -2 .66, p=0.03) in patients treated with AB160 compared with IgG-ABX. We confirmed the LC-MS results with higher levels of I-125 in the tumors of mice (Figure 4). I and Fig. 4J). Taken together, these data suggest that the binding of BEV to ABX mediates Blood pk changes and this change is related to the L of paclitaxel compared to isotype-matched IgG1. Subdistribution of drug to tumor tissue as demonstrated by both C-MS and I-125 labeling of BEV It is proven that the cause is fabric.

[0174] Without being bound by theory, it is believed that binding of a tumor-targeting antibody to ABX increases the pk to A The change was more dramatic than that of BX alone, and the redistribution of AB160 to the tumor tissue resulted in blood C max Oh The pk of paclitaxel in mouse blood, paclitaxel Tumor tissue levels of and I-1 in mice treated with AB160 compared to ABX alone These results of the 25 radioactivity levels suggest that elevated levels of paclitaxel reach the tumor. and retaining the ABX there for a longer period, thereby enhancing tumor regression. These results suggest that the biodistribution of paclitaxel may be altered by systemic targeting.

[0175] Example 7: Binding of other therapeutic antibodies Anti-human CD20 antibody (rituximab) and anti-HER2 / neu receptor antibody (trastuzumab) We tested the binding of ABX to other IgG therapeutic antibodies when combined in vitro. It was then determined whether or not ABX also exhibited binding to ABX.

[0176] method Nanoparticles containing rituximab or trastuzumab were prepared and prepared as described in the above examples. Tested as it is.

[0177] result 0.157-2.166 μm (Figure 2A) and 0.148-2.868 μm ( Complexes of BEV and trastuzumab (HER) with average particle sizes in the range of 0.1 to 1.0 mm (Figure 5B). The particle sizes of the complexes formed with rituximab were very similar. The induced particles were much larger at lower antibody:ABX ratios, with particle sizes reaching 0.1 The range was 59 to 8.286 μm (Figure 5A).

[0178] The binding affinity of rituximab and trastuzumab to ABX was investigated using BLI under variable pH conditions. Both antibodies bind with relatively high affinity in the picomolar range (Figure 5C). The affinity of rituximab for ABX decreases with increasing pH, whereas the affinity of trastuzumab for ABX decreases with increasing pH. The affinity of the mab for ABX was not affected by pH (Fig. 5C ).

[0179] The efficacy of 160 nm particles formulated with rituximab (AR160) was evaluated in vitro and in vivo. In vitro, increasing concentrations of α-aminobutyric acid (α-aminobutyric acid) were administered to Daudi (a B-cell lymphoma cell line) 0-200μg / ml) of paclitaxel, AR160, ABX or Rituximab Treatment was with umab alone. AR160(IC 50 = 10 μg / ml) is ABX (IC 50 > 200μg / ml) or rituximab (IC 50 >200μg / ml) alone When compared with 24 h treatment, it significantly inhibited the proliferation of Daudi cells (p=0.0 24) (Figure 6A).

[0180] In vivo, a xenograft model of Daudi cells was established in athymic nude mice. Once tumors were established, mice were treated with PBS, ABX, rituximab, or continuous ABX. + Rituximab or AR160. Tumors were measured 7 days after treatment and compared with baseline The percentage change in tumor size from baseline was calculated. Tumors treated with AR160 either regressed or remained stable. The tumors in all other treatment groups progressed (Figure 6B). The change in tumor size from baseline in the AR160 group was significant (p< 0.0001). Mice treated with AR160 had significantly higher pulmonary function than PBS (p<0.0001), ABX (p<0.0001) or rituximab (p=0.0002), respectively. significantly longer median survival time of over 60 days compared with 12, 16, and 12 days for the control group However, the difference in median survival time was smaller between the AR160 group and the continuous treatment group. The difference between the two groups was not significant (p=0.36), probably because rituximab was not able to bind to tumor cells. It remains bound to the cell surface, allowing subsequent administration of ABX to bind to soluble targets. Unlike BEV, which binds to IL-1 and does not bind to cell surface markers, upon entering the tumor site This is because it can bind to the antibody.

[0181] Example 8: Conjugation of other chemotherapeutic agents to AB160 The effectiveness of other chemotherapeutic agents to form functional nanoparticles was evaluated.

[0182] method Nanoparticles containing cisplatin were prepared and tested as described in the above examples. .

[0183] result To test whether other chemotherapeutic agents could be bound to the AB160 particles, Cisplatin was incubated with ABX and the amount of free cisplatin remaining in the supernatant was Approximately 60% of the cisplatin (i.e., only 40%) remained in the supernatant. ) bound to ABX (Fig. 7A).

[0184] Next, the tumor toxicity of AC compared with ABX and cisplatin alone was evaluated using A375 cells. The complex was centrifuged to remove unbound cisplatin, which is highly toxic, and Any additional toxicity of AC compared with ABX was solely attributable to cisplatin bound to ABX For parity, ABX alone was reconstituted in a similar manner. The cells were centrifuged by the AC (IC 50 = 90 μg / ml) to a greater extent than ABX alone Inhibited the proliferation of A375 cells (IC 50 >1000 μg / ml) (Figure 7B). Other toxicities The reduced toxicity in this experiment compared to the previous one is due to some loss of drug during the centrifugation step. However, the comparison between ABX and AC is still relevant.

[0185] To determine the tumor toxicity of the AB160 conjugate with cisplatin, AB160 was Incubation with cisplatin to form particles containing cisplatin (ABC complexes) The ABC complex was compared with each drug alone and AB160 in A375 melanoma xenograft models. AB160, sequential AB160 + cisplatin and ABC were tested. All tumors treated with the complex showed regression in tumor size 7 days after treatment (Figure 7C). The ABC complex had the longest median survival (24 and 26 days for AB160 and AB200, respectively). and AB160 + cisplatin for 35 days. The difference was not statistically significant. (p = 0.82 and 0.79) (Figure 7D), but the data do not support a long-term survival benefit. This is consistent with the benefits of ABC complexes.

[0186] These data suggest that the albumin moiety of ABX may be a novel therapeutic antibody that is able to effectively inhibit the effects of other therapeutic antibodies in vitro and in vivo. Rituximab and trastuzumab, which have similar efficacy to AB160 in demonstrated to provide a platform for conjugating other chemotherapeutic agents (e.g., cisplatin) to It was done.

[0187] Overall, these data suggest that multiple proteins or cytotoxic drugs can be linked to a single albumin. We present a simple method to construct versatile nanoimmunoconjugates that can be attached to scaffolds. The mouse model demonstrated high efficacy of the targeted drug compared to single agents alone. This is due, at least in part, to changes in the pK of antibody-targeted drugs. Without being bound by any of the above, the present disclosure does not require a linker or target cell uptake. The versatility of the nanoimmunoconjugates makes them a promising candidate for other nanomedicines when translating results from mice to humans. It is believed that the obstacles facing the product will be overcome.

[0188] Example 9: Lyophilization of AB160 8 mg (320 μl) of bevacizumab was added to 20 mg of Abraxane to obtain AB160 Then, 4 mg / ml of bevacizumab and 10 mg / ml of Abraxane were synthesized. Add 1.66 mL of 0.9% saline to a final volume of 2 mL for a final concentration of The mixture was incubated in a 15 ml polypropylene conical tube at room temperature for 30 minutes. .

[0189] After incubation at room temperature for 30 min, the mixture was diluted with 2 mg / ml bevacizumab and 5 mg / ml The Abraxane was diluted 1:2 in 0.9% saline to 20 mg / ml. are the concentrations of the two drugs when prepared by the pharmacy for administration to the patient. .

[0190] AB160 was placed in 20 1.5 ml polypropylene Eppendorf pipettes. Aliquots of 200 μl were separated and frozen at -80°C.

[0191] Once frozen, place the aliquots in a 3L Virtis tabletop freezer with a freezer section above it. The mixture was lyophilized overnight in a desiccator (SPScientific, Warminster, PA). A lyophilized formulation was produced.

[0192] The dried aliquot was placed in the same 1.5 ml polypropylene Eppendorf pipette and transferred to the chamber. These samples were easily reconstituted in room temperature saline for 30 min and then stored at room temperature for 20 min. The resulting sample was then centrifuged at 100 x g for 7 minutes. The resulting sample was then resuspended in an appropriate buffer, if necessary. It became suspended.

[0193] By comparison, samples dried with heat and speed vacuum could not be reconstituted.

[0194] Example 10: Testing of freeze-dried formulations Samples were reconstituted at different time points after lyophilization and were compared against ABX and freshly prepared AB160. Their physical properties were tested.

[0195] The particle size distribution was evaluated as described above.

[0196] The samples were incubated with VEGF for 2 hours at room temperature and centrifuged at 2000 x g for 7 minutes. VEGF binding was assessed by isolating the pellet (corresponding to the nanoparticles). The amount of VEGF bound to VEGF-binding proteins or remaining in the supernatant was measured by ELISA.

[0197] Paclitaxel activity was assessed by cytotoxicity against A375 cells in vitro.

[0198] Surprisingly, freeze-drying has been shown to be effective in inhibiting cancer cell proliferation. did not significantly affect particle size, VEGF binding, or activity of paclitaxel. The results were consistent with those obtained after storage for 1 month (Figures 8A to 8C) or 10 months (Figures 8D to 8F). This was also retained for the freeze-dried samples stored in a refrigerator.

[0199] Even more surprising is the lack of cryoprotectants or other drugs that may be detrimental to human therapeutic use. These results were observed when using freeze-dried nanoparticles without the use of an agent.

[0200] Example 11: Efficacy of AB160 in humans Safety of AB160 Administered to Patients with Metastatic Melanoma Who Have Failed Previous Therapies The study tested AB160 in a Phase 1 first-in-human clinical trial to test the efficacy and safety of AB160 in treating Using a typical 3+3 Phase 1 clinical trial design, three different doses of AB1 were administered in the following manner: Test 60. [Table 4]

[0201] These doses were selected relative to doses of Abraxane currently used in clinical practice AB160 was prepared prior to administration of each treatment. On days 1 and 8 of the 28-day treatment cycle Treatment was administered as a 30-minute intravenous infusion on days 10 and 15. No intolerable toxicity occurred. Treatment continued until failure, tumor progression, or patient refusal. Patients were evaluated for toxicity before each cycle and tumor assessments were performed once every two cycles (R ECIST).

[0202] This study included formal (inpatient) medications associated with cycles 1 and 2 of treatment administration 1. A physical status survey was also conducted at the same time.

[0203] 100 mg / m in 5 patients 2 of ABX and 40 mg / m 2 AB160 in BEV The study was conducted in 2013 and 2014, and four of them were analyzed. [Table 5]

[0204] PFS refers to progression-free survival, or the number of days of treatment before cancer recurs. There were no dose-limiting toxicities (DLTs). The findings were not associated with the dose of 60. Further details are shown in Table 6. [Table 6] [Table 7]

[0205] The mean PFS was 7.6 months, with a median of 7.0 months.

[0206] Comparison with other clinical trials The following table lists other published clinical trials of taxane therapy for metastatic melanoma. show. [Table 8]

[0207] In the current clinical trial, the dose of AB160 particles is 100 mg / m 2 Abraxane and 40 mg / m 2 The only study using BEV and ABX alone The study was conducted by Spitler, who used a higher dose of ABX. In addition, this study was conducted to determine the dose for the average patient (1.9 mg / kg). 2 surface area and quality of 90kg When adjusted for the BEV dose reported in previous studies (assuming Less than % was used.

[0208] Although the current study investigated patients who had failed previous treatment, Spilter Ineffective conventional treatments have reduced the time from expected PFS. Not only do they obtain cancer cells that are more resistant to treatment, but they also select cancer cells that are more resistant to treatment, typically resulting in a more favorable outcome for patients. Therefore, the “rescue” treatment (here AB16 0) has a lower PFS than the treatment-naïve population This is expected to have a positive effect on rescue and treatment-naïve patients with Abraxane alone. A Phase II clinical trial (Hersh et al., Cancer, January 2010, 116:155) investigated both In patients previously treated with Abraxane alone, PFS was 3.5 months. Hersh et al. Ann. Oncol 2015 (epub September 26, 2015) The PFS in treatment-naive patients treated with ABX alone was reported to be 4.8 months. [Table 9]

[0209] Thus, early results from a Phase 1 clinical trial with AB160 suggest that The study shows that PFS is increased in patients with advanced metastatic melanoma who received chemotherapy. and a higher dose of Abraxane and a nearly 12-fold higher dose of bevacizumab. This increase is particularly surprising given that it is higher than that of Spitler, who was administered The dose of BEV used in AB160 was much lower than in any other study. Therefore, the best comparison is to Hersh rather than Spitler.

[0210] Therefore, the ABX / BEV conjugate (AB160) is a novel anti-inflammatory drug that is effective in preventing and treating inflammatory bowel disease. was superior to ABX alone, and these excellent results were achieved using a very low effective dose of BEV. Thus, the data support the efficacy of BEV-mediated chemotherapeutic agents against tumors. This is consistent with AB160, which has improved targeting of albumin to tumors. This could help target BEV to tumors upon selective uptake. In addition, the presence of the BEV / ABX complex results in higher in vivo stability than Abraxane. It is also possible to show

[0211] Example 12: To investigate whether pretreatment with BEV improves targeting Follow-up study Following the general protocol described above, inoculate 1 x 10 mice into the right flank. 6 A 375 human melanoma cells were injected, followed by PBS, 12 mg / kg BEV, 30 mg / kg g of ABX, AB160, or 1.2 mg / kg BEV and 24 h later A The tumors were pretreated with B160. The data were the tumor volumes (unit: mm) on the 7th and 10th days after treatment. 3 11A-11E show the progression of tumor size over a 10-day period. Mice treated with AB160, with or without prior treatment with BEV, Only the group A and B showed a reduction in mean tumor volume. See also Figure 11F and Figure 11G.

[0212] Data summarized in Figure 11F at 7 days post-treatment showed that pretreatment with BEV was either a control or was statistically significantly greater than BEV alone (p ≤ 0.0001) or ABX alone (p ≤ 0.0001). These results show that treatment with 100 mg / kg / day of ...

[0213] Data from 10 days after treatment summarized in Figure 11G also showed that pretreatment with BEV improved efficacy compared with controls. Statistically, 100% of patients with ABX were not treated with BEV alone (p ≤ 0.0001) or ABX alone (p ≤ 0.0001). The results also show that the B Pretreatment with EVs was associated with a reduction in tumor volume compared with AB160 alone (p=0.02). Complete responses were observed in two mice.

[0214] In this study, BEV at a dose of 12 mg / kg had no therapeutic effect. The amount of BEV added was only 1.2 mg / kg, which is 1 / 10 of the usual dose in mice. However, pretreatment with subtherapeutic doses did not improve the efficacy of AB160 nanoparticles. The data appear to indicate improved efficacy of AB160 nanoparticles in tumor involvement. By allowing a higher relative concentration of VEGF to remain in the tumor, targeted VEGF targeting may be more effective. Therefore, pretreatment with subtherapeutic doses of BEV can eliminate systemic levels of VEGF. I support the idea that it is possible.

[0215] Example 13: Other means of delivering nanoparticles It is believed that the nanoparticles of the present invention can be delivered directly to tumors. For example, intra-arterial catheters Nanoparticles can be delivered via a catheter or by direct injection into the tumor. In such embodiments, large nanoparticles (e.g., It is believed that the optical fiber can deliver 580 nm or 1130 nm.

Claims

[Claim 1] The invention as depicted in the drawings.

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