Patent Publication Number: US-11655301-B2

Title: Carrier-binding agent compositions and methods of making and using the same

Description:
RELATED APPLICATIONS 
     This application is a continuation of and claims priority to U.S. patent application Ser. No. 15/092,433 filed Apr. 6, 2016, now U.S. Pat. No. 10,618,969, the disclosure of which is incorporated herein by reference. The disclosures of PCT Application No. PCT/US15/54295, filed Oct. 6, 2015, U.S. Provisional Patent Application No. 62/060,484, filed Oct. 6, 2014, U.S. Provisional Patent Application Nos. 62/206,770; 62/206,771 filed Aug. 18, 2015, and U.S. Provisional Patent Application No. 62/206,772 filed Aug. 18, 2015 are incorporated by reference in their entireties. 
    
    
     FIELD OF THE INVENTION 
     This application relates to novel compositions of binding agents and carrier proteins and methods of making and using the same, in particular, as a cancer therapeutic. 
     STATE OF THE ART 
     Chemotherapy remains a mainstay for systemic therapy for many types of cancer, including melanoma. Most chemotherapeutics are only slightly selective to tumor cells, and toxicity to healthy proliferating cells can be high (Allen TM. (2002)  Cancer  2:750-763), often requiring dose reduction and even discontinuation of treatment. In theory, one way to overcome chemotherapy toxicity issues as well as improve drug efficacy is to target the chemotherapy drug to the tumor using antibodies that are specific for proteins selectively expressed (or overexpressed) by tumors cells to attract targeted drugs to the tumor, thereby altering the biodistribution of the chemotherapy and resulting in more drug going to the tumor and less affecting healthy tissue. Despite 30 years of research, however, specific targeting rarely succeeds in the therapeutic context. 
     Conventional antibody dependent chemotherapy (ADC) is designed with a toxic agent linked to a targeting antibody via a synthetic protease-cleavable linker. The efficacy of such ADC therapy is dependent on the ability of the target cell to bind to the antibody, the linker to be cleaved, and the uptake of the toxic agent into the target cell. Schrama, D. et al. (2006)  Nature reviews. Drug discovery  5:147-159. 
     Antibody-targeted chemotherapy promised advantages over conventional therapy because it provides combinations of targeting ability, multiple cytotoxic agents, and improved therapeutic capacity with potentially less toxicity. Despite extensive research, clinically effective antibody-targeted chemotherapy remains elusive: major hurdles include the instability of the linkers between the antibody and chemotherapy drug, reduced tumor toxicity of the chemotherapeutic agent when bound to the antibody, and the inability of the conjugate to bind and enter tumor cells. In addition, these therapies did not allow for control over the size of the antibody-drug conjugates. 
     There remains a need in the art for antibody-based cancer therapeutics that retain cytotoxic effect for targeted drug delivery to provide reliable and improved anti-tumor efficacy over prior therapeutics. 
     In addition, as to any therapeutic application, there also remains a need for the composition to be stable in its physical, chemical and biological properties. 
     Lyophilization, or freeze-drying, removes water from a composition. In the process, the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to enhance stability during the freeze-drying process and/or to improve stability of the lyophilized product upon storage. Pikal. M. Biopharm. 3(9)26-30 (1990) and Arakawa et al., Pharm. Res. 8(3):285-291 (1991). 
     While proteins may be lyophilized, the process of lyophilization and reconstitution may affect the properties of the protein. Because proteins are larger and more complex than traditional organic and inorganic drugs (i.e. possessing multiple functional groups in addition to complex three-dimensional structures), the formulation of such proteins poses special problems. For a protein to remain biologically active, a formulation must preserve intact the conformational integrity of at least a core sequence of the protein&#39;s amino acids while at the same time protecting the protein&#39;s multiple functional groups from degradation. Degradation pathways for proteins can involve chemical instability (i.e. any process which involves modification of the protein by bond formation or cleavage resulting in a new chemical entity) or physical instability (i.e. changes in the higher order structure of the protein). Chemical instability can result from deamidation, racemization, hydrolysis, oxidation, beta elimination or disulfide exchange. Physical instability can result from denaturation. aggregation, precipitation or adsorption, for example. The three most common protein degradation pathways are protein aggregation, deamidation and oxidation. Cleland, et al., Critical Reviews in Therapeutic Drug Carrier Systems 10(4): 307-377 (1993). 
     SUMMARY 
     In the present invention, the composition comprises nanoparticles which contain (a) carrier protein (b) a binding agent, and (c) optionally a therapeutic agent. The binding agent is believed to be bound to the carrier protein through hydrophobic interactions which, by their nature, are weak. Yet the activity of the individual components, as well as their relative relationship in the nanoparticle are preserved despite lyophilization and reconstitution of the composition. It is still further contemplated that binding to the carrier protein, e.g., complexation of the binding agent to the carrier protein, occurs through some or all of the hydrophobic portion of the binding agent, e.g., the Fc component, which results in all or part of the hydrophobic portion being integrated into the carrier protein core, while the target binding portions (regions) (e.g., an Fa and Fb portion) of the antibody remain outside of the carrier protein core, thereby retaining their target specific binding capabilities. In some embodiments, the binding agent is a non-therapeutic and non-endogenous human antibody, a fusion protein, e.g., fusion of an antibody Fc domain to a peptide that binds a target antigen, or an aptamer. 
     Further challenges are imposed because the nanoparticles are used in therapy. 
     While rearrangement of the hydrophobic components in the nanoparticle may be mitigated through covalent bonds between the components, such covalent bonds pose challenges for the therapeutic use of nanoparticles in cancer treatment. The binding agent, carrier protein, and additional therapeutic agent typically act at different locations in a tumor and through different mechanisms. Non-covalent bonds permit the components of the nanoparticle to dissociate at the tumor. Thus, while a covalent bond may be advantageous for lyophilization, it may be disadvantageous for therapeutic use. 
     The size of nanoparticles, and the distribution of the size, is also important. Nanoparticles may behave differently according to their size. At large sizes, nanoparticles or the agglomeration of the particles may block blood vessels either of which can affect the performance and safety of the composition. 
     Finally, cryoprotectants and agents that assist in the lyophilization process must be safe and tolerated for therapeutic use. 
     In the present invention, the inventive compositions comprise nanoparticles which nanoparticles contain (a) carrier protein (b) a binding agent and (c) optionally a therapeutic agent. Without wishing to be bound by theory, the binding agent is believed to be bound to the carrier protein through hydrophobic interactions which, by their nature, are weak. Yet, the activity of the individual components, and their relative relationship in the nanoparticle are still achieved despite lyophilization and reconstitution of the composition. 
     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 binding agents, and optionally at least one therapeutic agent, wherein the binding agents are arranged outward from the surface of the nanoparticles and wherein the nanoparticles are capable of binding to a predetermined epitope in vivo. 
     When administered intravenously, large particles (e.g. greater than 1 μm) are typically disfavored because they can become lodged in the microvasculature of the lungs. At the same time, larger particles can accumulate in the tumor or specific organs. See e.g. 20-60 micron glass particle that is used to inject into the hepatic artery feeding a tumor of the liver, called “TheraSphere” (in clinical use for liver cancer). 
     Therefore, for intravenous administration, particles under 1 μm are used. Particles over 1 μm are, more typically, administered directly into a tumor (“direct injection”) or into an artery feeding into the site of the tumor. 
     In another aspect, provided herein are nanoparticle compositions comprising nanoparticles wherein each of the nanoparticles comprises a carrier protein that is not albumin, between about 100 to about 1000 binding agents, preferably about 400 to about 800 binding agents, and optionally at least one therapeutic agent, wherein the binding agents are arranged on an outside surface of the nanoparticles and wherein the nanoparticles are capable of binding to a predetermined epitope in vivo. When nanoparticles multimerize, the number of binding agents is increased proportionally. For example, if a 160 nm nanoparticle contains 400 binding agents, a 320 nm dimer contains about 800 binding agents. 
     In another aspect, provided herein are nanoparticle compositions comprising nanoparticles, wherein each of the nanoparticles comprises carrier protein, between about 400 to about 800 binding agents, and optionally at least one therapeutic agent that is not paclitaxel, wherein the binding agents are arranged on a surface of the nanoparticles such that the binding portion of the binding agent is directed outward from that surface and wherein the nanoparticles are capable of binding to a predetermined epitope in vivo. 
     In other embodiments, the nanoparticles multimerize, e.g. dimerize. Multimerization may be observed as multiples of the weight or size of the unit molecule, e.g. 160 nm particles multimerize to about 320 nm, 480 nm, 640 nm, etc. In some embodiments, less than 20% of the nanoparticles in a population are multimers. In some embodiments, more than 80% of the nanoparticles in a population are multimers. 
     In one embodiment, the weight ratio of carrier-bound drug to binding agent (e.g. albumin-bound paclitaxel to bevacizumab) is between about 5:1 to about 1:1. In one embodiment, the weight ratio of carrier-bound drug to binding agent is about 10:4. In one embodiment, the binding agents are a substantially single layer on all or part of the surface of the nanoparticle. In one embodiment, less than 0.01% of nanoparticles in the composition have a size selected from the group consisting of greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm and greater than 800 nm. Larger sizes are believed to be the result of multimerization of several nanoparticles, each comprising a core and binding agent coating on all or part of the surface of each nanoparticle. 
     The invention further includes lyophilized compositions, and lyophilized compositions that do not materially differ from, or are the same as, the properties of freshly-prepared nanoparticles. In particular, the lypholized composition, upon resuspending in aqueous solution, is similar or identical to the fresh composition in terms of particle size, particle size distribution, toxicity for cancer cells, binding agent affinity, and binding agent specificity. The invention is directed to the surprising finding that lyophilized nanoparticles retain the properties of freshly-made nanoparticles after resuspension notwithstanding the presence of two different protein components in these particles. 
     In one aspect, this invention relates to a lyophilized nanoparticle composition comprising nanoparticles, wherein each of the nanoparticles comprises a carrier-bound drug core and an amount of binding agent arranged on a surface of the core such that the binding portion of the binding agent is directed outward from that surface, wherein the binding agents retain their association with the outside surface of the nanoparticle upon reconstitution with an aqueous solution. In one embodiment, the lyophilized composition is stable at room temperature for at least 3 months. In one embodiment, the reconstituted nanoparticles retain the activity of the therapeutic agent and are capable of binding to the target in vivo. 
     In one embodiment, the average reconstituted nanoparticle size is from about 130 nm to about 1 μm. In a preferred embodiment, the average reconstituted nanoparticle size is from about 130 nm to about 200 nm, and more preferably about 160 nm. In one embodiment, in the average reconstituted nanoparticle size is from greater than 800 nm to about 3.5 μm, comprising multimers of smaller nanoparticles, e.g. multimers of 100-200 nm nanoparticles. In one embodiment, the weight ratio of core to binding agent is from greater than 1:1 to about 1:3. In one embodiment, in the average reconstituted nanoparticle size is about 160 nm to about 225 nm. 
     In one aspect, this disclosure relates to a lyophilized nanoparticle composition comprising nanoparticles, wherein each of the nanoparticles comprises: (a) an albumin-bound paclitaxel core and (b) between about 400 to about 800 molecules of bevacizumab arranged on a surface of the albumin-bound paclitaxel core such that the binding portion of the binding agent is directed outward from that surface, wherein the binding agents retain their association with the surface of the nanoparticle upon reconstitution with an aqueous solution, provided that said lyophilized composition is stable at about 20° C. to about 25° C. for at least 3 months and the reconstituted nanoparticles are capable of binding to VEGF in vivo. 
     In other aspects, this disclosure relates to a lyophilized nanoparticle composition comprising nanoparticles, wherein each of the nanoparticles comprises: (a) an albumin-bound paclitaxel core and (b) an amount of bevacizumab arranged on a surface of the albumin-bound paclitaxel core such that the binding portion of the binding agent is directed outward from that surface, wherein the binding agents retain their association with the surface of the nanoparticle upon reconstitution with an aqueous solution, provided that said lyophilized composition is stable at about 20° C. to about 25° C. for at least 3 months and the reconstituted nanoparticles are capable of binding to VEGF in vivo, and further wherein the average reconstituted nanoparticle size is not substantially different from the particle size of the freshly prepared nanoparticles. In some embodiments, the average particle sizes are between 200 and 800 nm, including 200, 300, 400, 500, 600, 700 or 800 nm. In other embodiments, the average particles are larger, e.g. from greater than 800 nm to about 3.5 nm. In some embodiments, the particles are multimers of nanoparticles. In some embodiments the nanoparticles have average particle sizes of about 160 nm to about 225 nm either freshly made or after lyophilization and resuspension in an aqueous solution suitable for injection. 
     In some embodiments, the weight ratio of albumin-bound paclitaxel to bevacizumab is between about 5:1 to about 1:1. In other embodiments, the weight ratio of albumin-bound paclitaxel to bevacizumab is about 10:4. In further embodiments, the weight ratio of albumin-bound paclitaxel to bevacizumab is from greater than 1:1 to about 1:3. 
     In some embodiments, the core is albumin-bound paclitaxel, and the binding agents are selected from binding agents that selectively recognize VEGF (e.g. bevacizumab/Avastin), binding agents that selectively recognize CD20 (e.g. rituximab/Rituxin) and binding agents that selectively recognize Her2 (Trastuzumab/Herceptin). 
     In some embodiments, the at least one therapeutic agent is located inside the nanoparticle. In other embodiments, the at least one therapeutic agent is located on the outside surface of the nanoparticle. In yet other embodiments, the at least one therapeutic agent is located inside the nanoparticle and on the outside surface of the nanoparticle. 
     In some embodiments, the nanoparticle contains more than one type of therapeutic agent. For example, a taxane and a platinum drug, e.g. paclitaxel and cisplatin. 
     In some embodiments, the binding agents are selected from the group consisting of ado-trastuzumab emtansine, alemtuzumab, bevacizumab, cetuximab, denosumab, dinutuximab, ipilimumab, nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, and trastuzumab. In some embodiments, the binding agents are a substantially single layer of binding agents on all or part of the surface of the nanoparticle. 
     In further embodiments, the antibodies are less glycosylated than normally found in natural human antibodies. Such glycosylation can be influenced by e.g. the expression system, or the presence of glycosylation inhibitors during expression. In some embodiments, the glycosylation status of an antibody or other binding agent is altered through enzymatic or chemical action. 
     In some embodiments, the at least one therapeutic agent is selected from the group consisting of abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, and cyclophosphamide. 
     In some embodiments, the nanoparticle further comprises at least one additional therapeutic agent that is not paclitaxel or bevacizumab. 
     In some embodiments, the binding agents, carrier protein and, when present, therapeutic agent, are bound through non-covalent bonds. 
     In some embodiments, the carrier protein is selected from the group consisting of gelatin, elastin, gliadin, legumin, zein, a soy protein, a milk protein, and a whey protein. 
     In other embodiments, the carrier protein is albumin, for example, human serum albumin. 
     In some embodiments, the composition is formulated for intravenous delivery. In other embodiments, the composition is formulated for direct injection or perfusion into a tumor. 
     In some embodiments, the average nanoparticle size in the composition is from greater than 800 nm to about 3.5 μm. 
     In some embodiments, the nanoparticles have a dissociation constant between about 1×10 −11  M and about 1×10 −9  M. 
     In another aspect, provided herein are methods of making nanoparticle compositions, wherein said methods comprise contacting the carrier protein and the optionally at least one therapeutic agent with the antibodies in a solution having a pH of between 5.0 and 7.5 and a temperature between about 5° C. and about 60° C., between about 23° C. and about 60° C., or between about 55° C. and about 60° C. under conditions and ratios of components that will allow for formation of the desired nanoparticles. In one embodiment, the nanoparticle is made at 55-600C and pH 7.0. In another aspect, provided herein are methods of making the nanoparticle compositions, wherein said method comprises (a) contacting the carrier protein and optionally the at least one therapeutic agent to form a core and (b) contacting the core with the antibodies in a solution having a pH of about 5.0 to about 7.5 at a temperature between about 5° C. and about 60° C., between about 23° C. and about 60° C., or between about 55° C. and about 60° C. under conditions and ratios of components that will allow for formation of the desired nanoparticles. 
     The amount of components (e.g., carrier protein, antibodies, therapeutic agents, combinations thereof) is controlled in order to provide for formation of the desired nanoparticles. A composition wherein the amount of components is too dilute will not form the nanoparticles as described herein. In a preferred embodiment, weight ratio of carrier protein to binding agent is 10:4. In some embodiments, the amount of carrier protein is between about 1 mg/mL and about 100 mg/mL. In some embodiments, the amount of binding agent is between about 1 mg/mL and about 30 mg/mL. For example, in some embodiments, the ratio of carrier protein:binding agent:solution is approximately 9 mg of carrier protein (e.g., albumin) to 4 mg of binding agent (e.g., BEV) in 1 mL of solution (e.g., saline). An amount of therapeutic agent (e.g., paclitaxel) can also be added to the carrier protein. 
     In further embodiments, the nanoparticles are made as above, and then lyophilized. 
     In another aspect, provided herein are methods for treating a cancer cell, the method comprising contacting the cell with an effective amount of a nanoparticle composition disclosed herein to treat the cancer cell. 
     In another aspect, provided herein are methods for treating a tumor in a patient in need thereof, the method comprising contacting the cell with an effective amount of a nanoparticle composition disclosed herein to treat the tumor. In some embodiments, the size of the tumor is reduced. In other embodiments, the nanoparticle composition is administered intravenously. In yet other embodiments, the nanoparticle composition is administered by direct injection or perfusion into the tumor. 
     In some embodiments, the methods provided herein include the steps of: a) administering the nanoparticle composition once a week for three weeks; b) ceasing administration of the nanoparticle composition for one week; and c) repeating steps a) and b) as necessary to treat the tumor. 
     In related embodiments, the treatment comprises administration of the targeting binding agent prior to administration of the nanoparticles. In one embodiment, the targeting binding agent is administered between about 6 and 48, or 12 and 48 hours prior to administration of the nanoparticles. In another embodiment, the targeting binding agent is administered between 6 and 12 hours prior to administration of the nanoparticles. In yet another embodiment, the targeting binding agent is administered between 2 and 8 hours prior to administration of the nanoparticles. In still other embodiments, the targeting binding agent is administered a week prior to administration of the nanoparticles. For example, administration of a dose of BEV 24 hours prior to administration of AB 160. In another example, prior administration of rituximab prior to administering AR nanoparticles. The binding agent administered prior to the nanoparticle may be administered as a dose that is subtherapeutic, such as ½, 1/10th or 1/20 the amount normally considered therapeutic. Thus, in humans, pretreatment with BEV may comprise administration of 1 mg/kg BEV which is 1/10th the usual dose, followed by administration of AB160. 
     In some embodiments, the therapeutically effective amount comprises about 75 mg/m 2  to about 175 mg/m 2  of the carrier protein (i.e., milligrams carrier protein per m 2  of the patient). In other embodiments, the therapeutically effective amount comprises about 75 mg/m 2  to about 175 mg/m 2  of therapeutic agent (e.g., paclitaxel). In other embodiments, the therapeutically effective amount comprises about 30 mg/m 2  to about 70 mg/m 2  of the binding agent. In yet other embodiments, the therapeutically effective amount comprises about 30 mg/m 2  to about 70 mg/m 2  bevacizumab. 
     In one specific embodiment, the lypholized composition comprises from about 75 mg/m 2  to about 175 mg/m 2  of the carrier protein which is preferably albumin; from about 30 mg/m 2  to about 70 mg/m 2  of the binding agent which is preferably bevacizumab and from about 75 mg/m 2  to about 175 mg/m 2  of paclitaxel. 
     An embodiment of the invention includes a method for increasing the duration of tumor uptake of a chemotherapeutic agent by administering the chemotherapeutic agent in a nanoparticle comprising a carrier protein and the chemotherapeutic agent having surface complexation with an antibody, e.g., an antibody that specifically binds to an antigen on or shed by the tumor. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following figures are representative only of the invention and are not intended as a limitation. For the sake of consistency, the nanoparticles of this invention using ABRAXANE® and bevacizumab employ the acronym “AB” and the number after AB such as AB160 is meant to confer the average particle size of these nanoparticles (in nanometers). Likewise, when the binding agent is rituximab, the acronym is “AR” while the number thereafter remains the same. 
         FIG.  1 A  shows flow cytometry scatterplots including: ABRAXANE® (ABX—commercially available from Celgene Corporation, Summit, N.J. 07901) stained with secondary antibody only (top left panel), ABX stained with goat anti-mouse IgG1 Fab plus secondary antibody (top right panel), AB160 (which is a nanoparticle of albumin-bound paclitaxel to bevacizumab in a ratio of about 10:4 and has an average particle size of 160 nm) stained with secondary antibody only (bottom left panel), or AB160 stained with goat anti-mouse IgG1 Fab plus secondary antibody (bottom right panel). 
         FIG.  1 B  shows a representative electron micrograph after incubation of AB160 with gold particle-labeled anti-human IgG Fe. 
         FIG.  1 C  shows a pie chart (top) indicating the percentages of total paclitaxel in AB160 fractions (particulate, proteins greater than 100 kD and proteins less than 100 kD); and a Western blot with antibodies against mouse IgG Fab (BEV) and paclitaxel to verify co-localization (bottom). 
         FIG.  1 D  represents the activity of paclitaxel in an in vitro toxicity assay with A375 human melanoma cells, compared to ABX alone. The results are represented by the average (+/−SEM) proliferation index, which is the percentage of total proliferation of untreated cells. This data represents 3 experiments and differences were not significant. 
         FIG.  1 E  represents results from a VEGF ELISA of supernatant after co-incubation of VEGF with ABX and AB160 to determine binding of the ligand, VEGF, by the antibody. The results are shown as the average percentage+/−SEM of VEGF that was unbound by the 2 complexes. The data represents 3 experiments **P&lt;0.005. 
         FIG.  2 A  shows the size of the complexes (determined by light scattering technology) formed by adding BEV (bevacizumab) to ABX under conditions where nanoparticles and higher are formed. Increasing concentrations of BEV (0-25 mg) were added to 10 mg of ABX and the size of the complexes formed was determined. The average size of the complexes (146 nm to 2, 166 nm) increased as the concentration of BEV was increased. The data is displayed as volume of sample/size and graphs show the size distribution of the particles. This data is representative of 5 separate drug preparations. As a comparison, ABX, by itself, has an average particle size of about 130 nm. 
         FIG.  2 B  shows affinity of the binding of ABX and BEV (as determined by light absorption (BLItz) technology). The data is displayed as dissociation constant (Kd). The binding affinity of particles made at four pH levels (3, 5, 7, 9) and 3 temperatures (RT, 37° C. and 58° C.) was assessed, and the data are representative of 5 experiments. 
         FIG.  2 C  shows the stability of the nanoparticle complexes from  FIG.  2 B  in serum as determined by a nanoparticle tracking analysis (NTA) on Nanosight 300 (NS300). The data are displayed as the number of particles/mg of ABX and compares AB160 prepared at RT 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), relative to ABX alone under each condition. Once particles were prepared, they were added to human AB serum for 15, 30, and 60 minutes to determine stability in serum over time. 
         FIG.  3 A  shows in vivo testing of AB nanoparticles in athymic nude mice injected with 1×10 6  A375 human melanoma cells in the right flank and treated with PBS, 12 mg/kg BEV, 30 mg/kg ABX, 12 mg/kg BEV+30 mg/kg ABX, or AB160 (having about 12 mg/kg BEV and about 30 mg/kg ABX) at tumor size between approximately 600 mm 3  to 900 mm 3  Data is represented at day 7-post treatment as the percent change in tumor size from baseline (the size of the tumor on the day of treatment). Student&#39;s t-test was used to determine significance. The p-values for the AB particles were all significantly different than PBS, the individual drugs alone and the 2 drugs given sequentially. 
         FIG.  3 B  shows Kaplan-Meier curves generated for median survival of the mice analyzed in  FIG.  3 A . Significance was determined using the Mantle-Cox test comparing survival curves. 
         FIG.  3 C  shows the percent change from baseline for mice treated when tumors were less than or greater than 700 mm 3 , to ascertain whether the size of the tumor affected tumor response for the ABX only and AB160 groups. The Student&#39;s t-test was used to determine significance; the ABX only groups showed no significant difference (p=0.752) based on tumor size, while the AB160 groups were significantly different (p=0.0057). 
         FIG.  3 D  shows in vivo testing of AB nanoparticles in athymic nude mice injected with 1×10 6  A375 human melanoma cells in the right flank and treated with PBS, 30 mg/kg ABX, or 45 mg/kg BEV and AB160, AB580 (nanoparticle of albumin-bound paclitaxel to bevacizumab having an average particle size of 580 nm) or AB1130 (nanoparticle of albumin-bound paclitaxel to bevacizumab having an average particle size of 1130 nm) at tumor size between approximately 600 mm 3  to 900 mm 3 . Data is represented at day 7-post treatment as the percent change in tumor size from baseline (the size of the tumor on the day of treatment). Student&#39;s t-test was used to determine significance. The changes in tumor size after administration of the AB particles were all significantly different than PBS, the individual drugs alone and the 2 drugs given sequentially. The difference among the AB particles of different sizes was not significant. 
         FIG.  3 E  shows Kaplan-Meier curves generated for median survival of the mice analyzed in  FIG.  3 D . Significance was determined using the Mantle-Cox test comparing survival curves. 
         FIG.  4 A  demonstrates blood paclitaxel concentration displayed in line graph with y-axis in log scale, based on blood and tumor samples taken from non-tumor and tumor bearing mice at 0-24 hours after IV injection with 30 mg/kg of paclitaxel in the context of ABX or AB160 and measured by LC-MS. Mice were IV injected at time 0, with blood samples taken and the mice sacrificed at time points of 0, 4, 8, 12, and 24 hours. There were at least 3 mice per time point. Student&#39;s t-test was utilized to determine if any differences in concentrations between ABX and AB 160 were significant. 
         FIG.  4 B  demonstrates the blood paclitaxel concentration from  FIG.  4 A , displayed in line graph with y-axis in numeric scale. 
         FIG.  4 C  shows the C max , half-life and AUC values calculated from the blood concentration data provide in  FIGS.  4 A and  4 B . 
         FIG.  4 D  demonstrates blood paclitaxel concentration displayed in line graph with y-axis in log scale from a second pharmacokinetic experiment using earlier time points (2 to 8 hours). 
         FIG.  4 E  demonstrates the blood paclitaxel concentration from  FIG.  4 D , displayed in line graph with y-axis in numeric scale. 
         FIG.  4 F  shows blood paclitaxel concentration in mice in which the tumors were allowed to grow to a larger size before ABX and AB160 injections. 
         FIG.  4 G  shows the C max  and the AUC calculated from the data in  FIG.  4 F . 
         FIG.  4 H  shows paclitaxel concentrations in the tumors from the second mouse experiment as determined by LC-MS. Data are displayed as μg of paclitaxel/mg of tumor tissue. Student&#39;s t-test was utilized to determine if differences were significant. 
         FIG.  4 I  shows I-125 radioactivity levels in mice treated with AB160 relative to ABX alone. 
         FIG.  4 J  shows a graphical representation of the I-125 radioactivity levels shown in  FIG.  4 I . 
         FIG.  5 A  shows particle size measurements and affinity of nanoparticles made with rituximab. 10 mg/ml of ABX was incubated with rituximab (RIT) at 0-10 mg/ml and light scatter technology (Mastersizer 2000) was used to determine resulting particle sizes. Data are displayed as the percent volume of particles at each size and the curves represent particle size distributions (top). The table (bottom) shows the sizes of the resulting particles at each concentration of antibody. 
         FIG.  5 B  shows particle size measurements and affinity of nanoparticles made with trastuzumab. 10 mg/ml of ABX was incubated with trastuzumab (HER) at 0-22 mg/ml and light scatter technology (Mastersizer 2000) was used to determine resulting particle sizes. Data are displayed as the percent volume of particles at each size and the curves represent particle size distributions (top). The table (bottom) shows the sizes of the resulting particles at each concentration of antibody. 
         FIG.  5 C  shows the binding affinity of rituximab and trastuzumab as compared to ABX at pH 3, 5, 7 and 9, determined by biolayer interferometry (BLitz) technology. The dissociation constants are displayed for each interaction. 
         FIG.  6 A  shows in vitro toxicity of AR160 as tested with the CD20-positive Daudi human lymphoma cell line. The data are displayed in a graph of the proliferation index, which is the percent of FITC positive cells in treated wells relative to FITC positive cells in the untreated well (the highest level of proliferation). 
         FIG.  6 B  shows in vivo tumor efficacy in athymic nude mice injected with 5×10 6  Daudi human lymphoma cells in the right flank. The tumors were allowed to grow to 600 mm 3  to 900 mm 3  and the mice were treated with PBS, 30 mg/kg ABX, 12 mg/kg rituximab, 12 mg/kg rituximab+30 mg/kg ABX, or AR60. Tumor response was determined at day 7 post-treatment by the percent change in tumor size from the first day of treatment. Significance was determined by Student&#39;s t-test; the percent change from baseline was significantly different between the AR160 treated mice and all other groups (p&lt;0.0001). 
         FIG.  6 C  shows Kaplan-Meier survival curves generated from the experiment shown in  FIG.  6 B . Median survival for each treatment group is shown. A Mantle-Cox test was used to determine whether survival curve differences were significant. 
         FIG.  7 A  demonstrates addition of another chemotherapy drug (cisplatin) to AB160. ABX (5 mg/ml) was incubated with cisplatin (0.5 mg/ml) at room temperature for 30 minutes and free cisplatin was measured by HPLC in the supernatant after ABX particulate was removed. The quantity of free cisplatin was subtracted from the starting concentration to determine the quantity of cisplatin that bound to the ABX. The data are displayed in a column graph, along with the starting concentration (cisplatin). 
         FIG.  7 B  shows the toxicity of cisplatin-bound 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 a FITC conjugated anti-EdU antibody. The data is displayed in a graph of the proliferation index, which is the percent of FITC positive cells in treated wells compared to FITC positive cells in the untreated well (the highest level of proliferation). 
         FIG.  7 C  shows in vivo tumor efficacy of AC (ABC complex; cisplatin-bound ABX) in athymic nude mice injected with 1×10 6  A375 human melanoma cells in the right flank. The tumors were allowed to grow to 600 mm 3  to 900 mm 3  and the mice were treated with PBS, 30 mg/kg ABX, 2 mg/kg cisplatin, AB160, 2 mg/kg cisplatin+AB160 or ABC160. Tumor response was determined at day 7 post-treatment by the percent change in tumor size from the day of treatment. Significance was determined by Student&#39;s t-test; the percent change from baseline was significantly different between the ABC160 treated mice and PBS-, cisplatin-, or ABX-treated mice (p&lt;0.0001). There was no significant difference between the AB160, AB160+cisplatin, and ABC160 treated groups for day 7 post-treatment percent change from baseline. 
         FIG.  7 D  shows Kaplan-Meier survival curves generated based on the experiment shown in  FIG.  7 C  and median survival for each treatment group is shown. A Mantle-Cox test was used to determine whether survival curve differences were significant. 
         FIG.  8 A  shows the size distribution of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIG.  8 B  shows the ligand (VEGF) binding ability of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIG.  8 C  shows in vitro cancer cell toxicity of AB160 nanoparticles that were lyophilized, stored at room temperature for one month, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIG.  8 D  shows the size distribution of AB160 nanoparticles that were lyophilized, stored at room temperature for ten months, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIG.  8 E  shows the ligand (VEGF) binding ability of AB160 nanoparticles that were lyophilized, stored at room temperature for ten months, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIG.  8 F  shows in vitro cancer cell toxicity of AB160 nanoparticles that were lyophilized, stored at room temperature for ten months, and reconstituted, as compared to fresh AB160 or ABX alone. 
         FIGS.  9 A-C  show the size distributions of the ABX-BEV complexes at I.V. infusion conditions (ABX final concentration of 5 mg/mL) incubated in saline at room temperature for up to 24 hours ( FIGS.  9 A and  9 B ). By 4 hours at room temperature, there is some evidence of complex breakdown by ELISA (20%,  FIG.  9 C ). 
         FIG.  10    shows in vitro incubation for 30 seconds of ABX (top panel) or AB160 (bottom panel) in saline or heparinized human plasma at relative volume ratios of 9:1 or 1:1. 
         FIGS.  11 A-E  show in vivo testing of athymic nude mice injected with 1×10 6  A375 human melanoma cells in the right flank and treated with ( FIG.  11 A ) PBS, ( FIG.  11 B ) 12 mg/kg BEV, ( FIG.  11 C ) 30 mg/kg ABX, ( FIG.  11 D ) AB160, or ( FIG.  11 E ) pretreated with 01.2 mg/kg BEV and, 24 hr later, AB160. Data is represented at day 7-post and 10-day treatment as tumor volume in mm 3 . 
         FIG.  11 F  summarizes the day 7-post treatment data from  FIGS.  11 A-E . 
         FIG.  11 G  summarizes the day 10-post treatment data from  FIGS.  11 A-E . 
         FIG.  12    depicts the results of an experiment in which CD20 positive Daudi lymphoma cells were labeled with fluorescent tagged anti-human CD20 or isotype matched control in panels F and A, respectively, and analyzed by flow cytometry. In the other panels, the Daudi cells were pretreated with ABRAXANE® (ABX), AR160, AR160L, or Rituxan prior to CD20 labeling. As you can see, CD20 binding was specifically blocked by the AR particles and Rituxan, but not ABX alone suggesting AR160 and AR160L binds their CD20 ligand on these cells blocking binding of the fluorescent anti-CD20. 
         FIG.  13    is a histogram overlay of the scatterplots of  FIG.  12   . 
         FIGS.  14 A-B  depicts a particle size comparison of ABX alone relative to AR and AT freshly made and lyophilized.  FIG.  14 A  depicts a particle size comparison of ABX alone relative to AR freshly made and lyophilized.  FIG.  14 B  depicts a particle size comparison of ABX alone relative to AT freshly made and lyophilized. 
         FIG.  15    compares the toxicity of ABX and AR particles in a Daudi cell proliferation assay. 
         FIGS.  16 A-C  depicts the results obtained in mice treated with either labeled ABRAXANE®, labeled ABRAXANE® coated with non-specific antibodies (AB IgG), or labeled ABRAXANE® coated with Rituximab (AR160).  FIG.  16 A  depicts the fluorescence accumulation in regions of interest (ROI) in tumor (ROI 2, 3, and 4) and in background areas (ROI 1, 5, and 6). ROI 1, 5 and 6 serve as background references.  FIG.  16 B  is a bar graph of the average fluorescence per unit of tumor area of mice in all three treatment groups were determined to provide the gross tumor delivery.  FIG.  16    C is a bar graph of the average fluorescence per unit of tumor area normalized by background ROI to give proportion of drug delivered to tumor versus body. The data demonstrate that administration of AR160 nanoparticles results in an increased fluorescence as compared to ABRAXANE® alone or ABRAXANE® coated with non-specific antibodies. 
         FIG.  17    depicts the survival of the mice treated with a single dose of saline, BEV24 (24 mg/kg), ABX30 (30 mg/kg), AB160 (12 mg/kg BEV and 30 mg/kg ABX) and AB225 (24 mg/kg BEV and 30 mg/kg ABS). At 30 days post-administration the survival of mice treated with AB225 and with AB160 far exceeds the survival of mice treated with BEV alone of ABRAXANE® alone. 
     
    
    
     DETAILED DESCRIPTION 
     After reading this description it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below. 
     Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. 
     The detailed description of the invention is divided into various sections only for the reader&#39;s convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present invention. 
     Definitions 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: 
     The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. 
     “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. 
     The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by (+) or (−) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” when used with regard to a dose amount means that the dose may vary by +/−10%. For example, “about 400 to about 800 binding agents” indicates that an outside surface of a nanoparticles contain an amount of binding agent between 360 and 880 particles. 
     “Comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. 
     The term “nanoparticle” as used herein refers to particles having at least one dimension which is less than 5 microns. In preferred embodiments, such as for intravenous administration, the nanoparticle is less than 1 micron. For direct administration, the nanoparticle is larger. Even larger particles are expressly contemplated by the invention. 
     In a population of particles, the sizes of individual particles are distributed about a mean. Particle sizes for the population can therefore be represented by an average, and also by percentiles. D50 is the particle size below which 50% of the particles fall. 10% of particles are smaller than the DIO value and 90% of particles are smaller than D90. Where unclear, the “average” size is equivalent to D50. So, for example, AB160 and AR160 refer to nanoparticles having an average size of 160 nanometers. 
     The term “nanoparticle” may also encompass discrete multimers of smaller unit nanoparticles. For example, a 320 nm particle comprises a dimer of a unit 160 nm nanoparticle. For 160 nm nanoparticles, multimers would therefore be approximately 320 nm, 480 nm, 640 nm, 800 nm, 960 nm, 1120 nm, and so on. 
     The term “carrier protein” as used herein refers to proteins that function to transport binding agents and/or therapeutic agents. The binding agents of the present disclosure can reversibly bind to the carrier proteins. Examples of carrier proteins are discussed in more detail below. 
     The term “core” as used herein refers to central or inner portion of the nanoparticle which may be comprised of a carrier protein, a carrier protein and a therapeutic agent, or other agents or combination of agents. In some embodiments, a hydrophobic portion of the binding agent may be incorporated into the core. 
     The term “therapeutic agent” as used herein means an agent which is therapeutically useful, e.g., an agent for the treatment, remission or attenuation of a disease state, physiological condition, symptoms, or etiological factors, or for the evaluation or diagnosis thereof. A therapeutic agent may be a chemotherapeutic agent, for example, mitotic inhibitors, topoisomerase inhibitors, steroids, anti-tumor antibiotics, antimetabolites, alkylating agents, enzymes, proteasome inhibitors, or any combination thereof. 
     As used herein, the term, “binding agent”, “binding agent specific for”, or “binding agent that specifically binds” refers to an agent that binds to a target antigen and does not significantly bind to unrelated compounds. Examples of binding agents that can be effectively employed in the disclosed methods include, but are not limited to, lectins, proteins, and antibodies, such as monoclonal antibodies, e.g. humanized monoclonal antibodies, chimeric antibodies, or polyclonal antibodies, or antigen-binding fragments thereof, as well as aptamers, Fc domain fusion proteins, and aptamers having or fused to hydrophobic protein domain, e.g, Fc domain, etc. In an embodiment the binding agent is an exogenous antibody. An exogenous antibody is an antibody not naturally produced in a mammal, e.g. in a human, by the mammalian immune system. 
     The term “antibody” or “antibodies” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immuno-specifically bind an antigen). The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and portions thereof, including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies (e.g., Lanzavecchia et al.,  Eur. J Immunol.  17, 105 (1987)) and single chains (e.g., Huston et al.,  Proc. Natl. Acad. Sci. U.S.A.,  85, 5879-5883 (1988) and Bird et al.,  Science  242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al.,  Immunology , Benjamin. N.Y., 2ND ed. (1984); Harlow and Lane,  Antibodies. A Laboratory Manual , Cold Spring Harbor Laboratory (1988); Hunkapiller and Hood,  Nature,  323, 15-16 (1986), which are incorporated herein by reference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE or IgD). Preferably, the antibody is IgG. An antibody may be non-human (e.g., from mouse, goat, or any other animal), fully human, humanized, or chimeric. Antibody or antibodies include any biosimilar(s) of the antibodies disclosed herein. Biosimilars, as used herein, refers to a biopharmaceutical which is deemed to be comparable in quality, safety, and efficacy to a reference product marketed by an innovator company (Section 351(i) of the Public Health Service Act (42 U.S.C. 262(i)). 
     [The term “dissociation constant,” also referred to as “K d ,” refers to a quantity expressing the extent to which a particular substance separates into individual components (e.g., the protein carrier, antibody, and optional therapeutic agent). 
     The terms “lyophilized,” “lyophilization” and the like as used herein refer to a process by which the material (e.g., nanoparticles) to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient is optionally included in pre-lyophilized formulations to enhance stability of the lyophilized product upon storage. In some embodiments, the nanoparticles can be formed from lyophilized components (carrier protein, antibody and optional therapeutic) prior to use as a therapeutic. In other embodiments, the carrier protein, binding agent, e.g., antibody, and optional therapeutic agent are first combined into nanoparticles and then lyophilized. The lyophilized sample may further contain additional excipients. 
     The term “bulking agents” comprise agents that provide the structure of the freeze-dried product. Common examples used for bulking agents include mannitol, glycine, lactose and sucrose. In addition to providing a pharmaceutically elegant cake, bulking agents may also impart useful qualities in regard to modifying the collapse temperature, providing freeze-thaw protection, and enhancing the protein stability over long-term storage. These agents can also serve as tonicity modifiers. 
     The term “buffer” encompasses those agents which maintain the solution pH in an acceptable range prior to lyophilization and may include succinate (sodium or potassium), histidine, phosphate (sodium or potassium), Tris(tris(hydroxymethyl)aminomethane), diethanolamine, citrate (sodium) and the like. The buffer of this invention has a pH in the range from about 5.5 to about 6.5; and preferably has a pH of about 6.0. Examples of buffers that will control the pH in this range include succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers. 
     The term “cryoprotectants” generally includes agents which provide stability to the protein against freezing-induced stresses, presumably by being preferentially excluded from the protein surface. They may also offer protection during primary and secondary drying, and long-term product storage. Examples are polymers such as dextran and polyethylene glycol; sugars such as sucrose, glucose, trehalose, and lactose; surfactants such as polysorbates; and amino acids such as glycine, arginine, and serine. 
     The term “lyoprotectant” includes agents that provide stability to the protein during the drying or ‘dehydration’ process (primary and secondary drying cycles), presumably by providing an amorphous glassy matrix and by binding with the protein through hydrogen bonding, replacing the water molecules that are removed during the drying process. This helps to maintain the protein conformation, minimize protein degradation during the lyophilization cycle and improve the long-term products. Examples include polyols or sugars such as sucrose and trehalose. 
     The term “pharmaceutical formulation” refers to preparations which are in such form as to permit the active ingredients to be effective, and which contains no additional components that are toxic to the subjects to which the formulation would be administered. 
     “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. 
     “Reconstitution time” is the time that is required to rehydrate a lyophilized formulation into a solution. 
     A “stable” formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. 
     The term “epitope” as used herein refers to the portion of an antigen which is recognized by a binding agent, e.g., an antibody. Epitopes include, but are not limited to, a short amino acid sequence or peptide (optionally glycosylated or otherwise modified) enabling a specific interaction with a protein (e.g., an antibody) or ligand. For example, an epitope may be a part of a molecule to which the antigen-binding site of a binding agent attaches. 
     The term “treating” or “treatment” covers the treatment of a disease or disorder (e.g., cancer), in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments “treating” or “treatment” refers to the killing of cancer cells. 
     The term “kill” with respect to a cancer treatment is directed to include any type of manipulation that will lead to the death of that cancer cell or at least of portion of a population of cancer cells. 
     The term “aptamer” refers to a nucleic acid molecule that is capable of binding to a target molecule, such as a polypeptide. For example, an aptamer of the invention can specifically bind to e.g., CD20, CD38, CD52, PD-L1, Ly6E, HER2, HER3/EGFR DAF, ERBB-3 receptor, CSF-1R, STEAP1, CD3, CEA, CD40, OX40, Ang2-VEGF, and VEGF. The generation of antibodies with a particular binding specificity and the therapeutic use of aptamers are well established in the art. See. e.g., U.S. Pat. Nos. 5,475,096, 5,270,163, 5,582,981, 5,840,867, 6,011,020, 6,051,698, 6,147,204, 6,180,348 and 6,699,843, and the therapeutic efficacy of Macugen® (Eyetech, New York) for treating age-related macular degeneration. 
     Fc-fusion proteins are bioengineered polypeptides that join the crystallizable fragment (Fc) domain of an antibody with another biologically active agent, e.g., a protein domain, peptide, or nucleic acid or peptide aptamer to generate a molecule with desired structure-function properties and significant therapeutic potential. The gamma immunoglobulin (IgG) isotype is often used as the basis for generating Fc-fusion proteins because of favorable characteristics such as recruitment of effector function and increased plasma half-life. Given the range of aptamers, both peptide and nucleic acids, that can be used as fusion partners, Fc-fusion proteins have numerous biological and pharmaceutical applications. 
     Additionally, some terms used in this specification are more specifically defined below. 
     Overview 
     The current invention is predicated, in part, on the surprising discovery that optionally lyophilized nanoparticles comprising a carrier protein, a binding agent, e.g., an antibody, an aptamer, or a fusion protein having a hydrophobic domain and a binding domain, e.g., an Fc domain fused to an aptamer or the ligand of a cellular receptor, and a therapeutic agent provide targeted therapy to a tumor while minimizing toxicity to the patient. The nanoparticles as described herein are thus a significant improvement versus conventional ADCs. 
     For conventional ADCs to be effective, it is critical that the linker be stable enough not to dissociate in the systemic circulation but allow for sufficient drug release at the tumor site. Alley, S. C., et al. (2008)  Bioconjug Chem  19:759-765. This has proven to be a major hurdle in developing effective drug conjugate (Julien, D. C., et al. (2011) MAbs 3:467-478; Alley, S. C., et al. (2008)  Bioconjug Chem  19:759-765); therefore, an attractive feature of the nano-immune conjugate is that a biochemical linker is not required. 
     Another shortcoming of current ADCs is that higher drug penetration into the tumor has not been substantively proven in human tumors. Early testing of ADCs in mouse models suggested that tumor targeting with antibodies would result in a higher concentration of the active agent in the tumor (Deguchi, T. et al. (1986)  Cancer Res  46: 3751-3755); however, this has not correlated in the treatment of human disease, likely because human tumors are much more heterogeneous in permeability than mouse tumors. Jain, R. K. et al. (2010)  Nat Rev Clin Oncol  7:653-664. Also, the size of the nanoparticle is critical for extravasation from the vasculature into the tumor. In a mouse study using a human colon adenocarcinoma xenotransplant model, the vascular pores were permeable to liposomes up to 400 nm. Yuan, F., et al. (1995)  Cancer Res  55: 3752-3756. Another study of tumor pore size and permeability demonstrated that both characteristics were dependent on tumor location and growth status, with regressing tumors and cranial tumors permeable to particles less than 200 nm. Hobbs, S. K., et al. (1998)  Proc Natl Acad Sci USA  95:4607-4612. The nano-immune conjugate described herein overcomes this issue by the fact that the large complex, which is less than 200 nm intact, is partially dissociated in systemic circulation into smaller functional units that are easily able to permeate tumor tissue. Furthermore, once the conjugate arrives to the tumor site, the smaller toxic payload can be released and only the toxic portion needs to be taken up by tumor cells, not the entire conjugate. 
     The advent of antibody-(i.e. AVASTIN®) coated albumin nanoparticles containing a therapeutic agent (i.e., ABRAXANE®) has led to a new paradigm of directional delivery of two or more therapeutic agents to a predetermined site in vivo. See PCT Patent Publication Nos. WO 2012/154861 and WO 2014/055415, each of which is incorporated herein by reference in its entirety. 
     When compositions of albumin and an binding agent, e.g., antibody, are admixed together in an aqueous solution at specific concentrations and ratios, the binding agents useful in this invention spontaneously self-assemble into and onto the albumin to form nanoparticles having multiple copies of the binding agent (up to 500 or more). Without being limited to any theory, it is contemplated that the antigen (or ligand) receptor portion of the binding agent, e.g., the antibody or aptamer or Fc fusion molecule, is positioned outward from the nanoparticle while the hydrophobic tail of the binding agent in integrated into the albumin by hydrophobic-hydrophobic interactions. 
     While protein compositions comprising a single source protein are commonly stored in lyophilized form where they exhibit significant shelf-life, such lyophilized compositions do not contain a self-assembled nanoparticle of two different proteins integrated together by hydrophobic-hydrophobic interactions. Moreover, the nanoparticle configuration wherein a majority of the binding portions of the binding agent are exposed on the surface of the nanoparticles lends itself to being susceptible to dislodgement or reconfiguration by conditions which otherwise would be considered benign. For example, during lyophilization, ionic charges on the proteins are dehydrated thereby exposing the underlying charges. Exposed charges allow for charge-charge interactions between the two proteins which can alter the binding affinity of each protein to the other. In addition, the concentration of the nanoparticles increases significantly as the solvent (e.g., water) is removed. Such increased concentrations of nanoparticles could lead to irreversible oligomerization. Oligomerization is a known property of proteins that reduces the biological properties of the oligomer as compared to the monomeric form and increases the size of the particle sometimes beyond 1 micron. 
     On the other hand, a stable form of a nanoparticle composition is required for clinical and/or commercial use where a shelf-life of at least 3 months is required and shelf-lives of greater than 6 months or 9 months are preferred. Such a stable composition must be readily available for intravenous injection, must retain its self-assembled form upon intravenous injection so as to direct the nanoparticle to the predetermined site in vivo, must have a maximum size of less than 1 micron so as to avoid any ischemic event when delivered into the blood stream, and finally must be compatible with the aqueous composition used for injection. 
     Compounds 
     As will be apparent to the skilled artisan upon reading this disclosure, the present disclosure relates to compositions of nanoparticles containing a carrier protein, binding agents, and optionally at least one therapeutic agent, wherein said compositions are optionally lyophilized. 
     In some embodiments, the carrier protein can be albumin, gelatin, elastin (including topoelastin) or elastin-derived polypeptides (e.g., α-elastin and elastin-like polypeptides (ELPs)), gliadin, legumin, zein, soy protein (e.g., soy protein isolate (SPI)), milk protein (e.g., β-lactoglobulin (BLG) and casein), or whey protein (e.g., whey protein concentrates (WPC) and whey protein isolates (WPI)). In preferred embodiments, the carrier protein is albumin. In preferred embodiments, the albumin is egg white (ovalbumin), bovine serum albumin (BSA), or the like. In even more preferred embodiments, the carrier protein is human serum albumin (HSA). In some embodiments, the carrier protein is a generally regarded as safe (GRAS) excipient approved by the United States Food and Drug Administration (FDA). 
     In some embodiments, the binding agents are antibodies selected from the group consisting of ado-trastuzumab emtansine, alemtuzumab, bevacizumab, cetuximab, denosumab, dinutuximab, ipilimumab, nivolumab, obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, and trastuzumab. In some embodiments, the antibodies are a substantially single layer of antibodies on all or part of the surface of the nanoparticle. 
     Table 1 depicts a list of non-limiting list of antibodies. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Antibodies 
               
               
                 Antibodies 
               
            
           
           
               
               
               
            
               
                   
                 Biologic 
                 Treatment(s)/Taret(s) 
               
               
                   
               
               
                 Monoclonal 
                 Rituximab (Rituxan ®) 
                 Non-Hodgkin lymphoma 
               
               
                 antibodies 
                 Alemtuzumab (Campath ®) 
                 Chronic lymphocytic leukemia 
               
               
                 (MAbs)  
                   
                 (CLL) 
               
               
                 and antibodies 
                 Ipilimumab (Yervoy ®) 
                 Metastatic melanoma 
               
               
                 conjugates 
                 Bevaciztunab (Avastin ®) 
                 Colon cancer, lung cancer, renal 
               
               
                   
                   
                 cancer, ovarian cancer, 
               
               
                   
                   
                 glioblastoma multiforme 
               
               
                   
                 Cetuximab (Erbitux ®) 
                 Colorectal cancer, non-small cell 
               
               
                   
                   
                 lung cancer, head and neck 
               
               
                   
                   
                 cancer, cervical cancer, 
               
               
                   
                   
                 glioblastoma, ovarian epithelia, 
               
               
                   
                   
                 fallopian tube or primary 
               
               
                   
                   
                 peritoneal cancer, renal cell 
               
               
                   
                   
                 cancer 
               
               
                   
                 Panitumumab (Vectibix ®) 
                 Colorectal cancer 
               
               
                   
                 Trastuzumab (Herceptin ®) 
                 Breast cancer, Adenocarcinoma 
               
               
                   
                   90 Y-ibritumomab 
                 Non-Hodgkin lymphoma 
               
               
                   
                 tiuxetan (Zevalin ®) 
                   
               
               
                   
                 Ado-trastuzumab 
                 Breast cancer 
               
               
                   
                 emtansine (Kadycla ®, 
                   
               
               
                   
                 also called TDM-1) 
                   
               
               
                   
                 Brentuximab 
                 Hodgkin lymphoma, Anaplastic 
               
               
                   
                 vedotin 
                 large cell lymphoma 
               
               
                   
                 (Adcetris ®) 
                   
               
               
                   
                 Blinatumomab (Blincyto) 
                 Acute lymphocytic leukemia 
               
               
                   
                   
                 (ALL) 
               
               
                   
                 Pembrolizumab 
                 PD-1 (melanoma, non-small 
               
               
                   
                 (Keytruda ®) 
                 cell lung cancer) 
               
               
                   
                 Nivolumab (Opdivo ®)  
                 PD-1 (melanoma, non-small 
               
               
                   
                   
                 cell lung cancer) 
               
               
                   
                 Ofatumumab (Arzerra ®) 
                 Chronic lymphocytic leukemia 
               
               
                   
                   
                 (CLL) 
               
               
                   
                 Pertuzumab (Perieta ®) 
                 Breast cancer 
               
               
                   
                 Obinutuzumab (Gazyva ®) 
                 Lymphoma, diffuse large B-cell 
               
               
                   
                   
                 lymphoma (DLBCL), indolent NHL 
               
               
                   
                   
                 (1st-line) 
               
               
                   
                 Dinutuximab (Unituxjn ®)  
                 Neuroblastoma 
               
               
                   
                 Denosurnab (Prolia ®) 
                 Bone metastases, multiple 
               
               
                   
                   
                 myeloma, giant cell tumor of 
               
               
                   
                   
                 bone 
               
               
                   
                 RG6016 (LSD1 inhibitor) 
                 Acute myelogenous leukemia (AML) 
               
               
                   
                 mAB 
                   
               
               
                   
                 Small molecule according to 
                   
               
               
                   
                 BioCentury BCIQ 
                   
               
               
                   
                 RG7882 (antibody drug 
                 Pancreatic cancer, ovarian cancer 
               
               
                   
                 conjugate) 
                   
               
               
                   
                 Alternative Names: D-4064A; 
                   
               
               
                   
                 DMUC 4064A; RG7882 
                   
               
               
                   
                 Lifastuzumab vedotin 
                 Platinum-resistant ovarian cancer, 
               
               
                   
                 (antibody drug conjugate) 
                 NSCLC 
               
               
                   
                 Polatuzumab vedotin 
                 DLBCL, NHL 
               
               
                   
                 (antibody drug conjugate) 
                   
               
               
                   
                 Kadcyla ® (ado-trastuzumab 
                 HER2-positive breast cancer, NSCLC, 
               
               
                   
                 emtansine) 
                 gastric cancer 
               
               
                   
                 RG7446 (anti-PD-L1 mAb) 
                 bladder cancer. NSCLC, melanoma, breast, 
               
               
                   
                   
                 renal cell carcinoma, lymphoma 
               
               
                   
                 DLYE-5953A (anti-Ly6E 
                 Refractory solid tumors 
               
               
                   
                 mAB cytotoxic drug 
                   
               
               
                   
                 conjugate) 
                   
               
               
                   
                 Duligotuzumab (anti- 
                 Solid tumors with mutant KRAS 
               
               
                   
                 HER3/EGFR DAF mAb) 
                   
               
               
                   
                 RG7117 (ERBB-3 receptor 
                 Metastatic breast cancer 
               
               
                   
                 antagonist) 
                   
               
               
                   
                 RG7155 (CSF-1R antagonist) 
                 Solid tumors 
               
               
                   
                 RG7450 (anti-STEAP1 
                 Prostate cancer 
               
               
                   
                 antibody drug conjugate) 
                   
               
               
                   
                 RG7802 (CD3/CEA bispecific 
                 Solid tumors 
               
               
                   
                 antibody) 
                   
               
               
                   
                 RG7813 (CEA inhibitor) 
                 Solid tumors 
               
               
                   
                 RG7841 (antibody drug 
                 Solid tumors 
               
               
                   
                 conjugate) 
                   
               
               
                   
                 RG7876 (CD40 antigen 
                 Solid tumors 
               
               
                   
                 stimulant) 
                   
               
               
                   
                 RG7888 (anti-OX40 mAb) 
                 Solid tumors 
               
               
                   
                 RG7221 (Ang2-VEGF mAb) 
                 Metastatic colorectal cancer 
               
               
                   
                 RG7686 (glypican-3 mAb) 
                 Hepatocellular carcinoma 
               
               
                   
                 Perjeta ® pertuzumab 
                 HER3-positive breast cancer, gastric 
               
               
                   
                   
                 cancer 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the at least one therapeutic agent is selected from the group consisting of abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, and cyclophosphamide. 
     Table 2 depicts a list of non-limiting list of cancer therapeutic agents. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Cancer therapeutic agents 
               
               
                 Cancel Drugs 
               
            
           
           
               
               
            
               
                 Drug 
                 Target(s) 
               
               
                   
               
               
                 Abitrexate (Methotrexate) 
                 Acute lymphoblastic leukemia; breast 
               
               
                   
                 cancer; gestational trophoblastic disease, 
               
               
                   
                 head and neck cancer; lung cancer; mycosis 
               
               
                   
                 fungoides; non-Hodgkin lymphoma; 
               
               
                   
                 osteosarcoma 
               
               
                 ABRAXANE ® (Paclitaxel Albumin- 
                 Breast cancer; non-small cell lung 
               
               
                 stabilized Nanoparticle Formulation) 
                 cancer; pancreatic cancer 
               
               
                 ABVD (Adriamycin, bleomycin, vinblastine 
                 Hodgkin lymphoma 
               
               
                 sulfate, dacarbazine) 
                   
               
               
                 ABVE (Adriamycin, bleomycin, vincristine 
                 Hodgkin lymphoma (in children) 
               
               
                 sulfate, etoposide) 
                   
               
               
                 ABVE-PC (Adriamycin, bleomycin, vincristine 
                 Hodgkin lymphoma (in children) 
               
               
                 sulfate, etoposide, prednisone, 
                   
               
               
                 cyclophosphamide) 
                   
               
               
                 AC (Adriamycin cyclophosphamide) 
                 Breast cancer 
               
               
                 AC-T (Adriamycin, cylclophosphamide, 
                 Breast cancer 
               
               
                 Taxol) 
                   
               
               
                 Adcetris (Brentuximab Vedotin) 
                 Anaplastic large cell lymphoma; 
               
               
                   
                 Hodgkin lymphoma 
               
               
                 ADE (Cytarabine (Ara-C), Daunorubicin 
                 Acute myeloid leukemia (in children) 
               
               
                 Hydrochloride, Etoposide) 
                   
               
               
                 Ado-Trastuzumab Emtansine 
                 Breast cancer 
               
               
                 Adriamycin (Doxorubicin Hydrochloride) 
                 Acute lymphoblastic leukemia; acute myeloid 
               
               
                   
                 leukemia; breast cancer, gastric (stomach) 
               
               
                   
                 cancer; Hodgkin lymphoma; neuroblastoma; 
               
               
                   
                 non-Hodgkin lymphoma; ovarian cancer; 
               
               
                   
                 small cell lung cancer; soft tissue and bone 
               
               
                   
                 sarcomas; thyroid cancer; transitional cell 
               
               
                   
                 bladder cancer; Wilms tumor 
               
               
                 Adrucil (Fluorouracil) 
                 Basal cell carcinoma; breast cancer; 
               
               
                   
                 colorectal cancer; gastric (stomach) 
               
               
                   
                 adenocarcinoma; pancreatic cancer; squamous 
               
               
                   
                 cell carcinoma of the head and neck 
               
               
                 Afatinib Dimaleate 
                 Non-small cell lung cancer 
               
               
                 Afinitor (Everolimus) 
                 Breast cancer, pancreatic cancer; renal 
               
               
                   
                 cell carcinoma; subependymal giant cell 
               
               
                   
                 astrocytoma 
               
               
                 Alimta (Pemetrexed Disodium) 
                 Malignant pleural mesothelioma; non- 
               
               
                   
                 small cell lung cancer 
               
               
                 Ambochlorin (Chlorambucil) 
                 Chronic lymphocytic leukemia; 
               
               
                   
                 Hodgkin lymphoma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 Anastrozole 
                 Breast cancer 
               
               
                 Aredia (Pamidronate Disodium) 
                 Breast cancer; multiple myeloma 
               
               
                 Arimidex (Anastrozole) 
                 Breast cancer 
               
               
                 Aromasin (Exemestane) 
                 Advanced breast cancer; early-stage 
               
               
                   
                 breast cancer and estrogen receptor 
               
               
                   
                 positive 
               
               
                 Arranon (Nelarabine) 
                 T-cell acute lymphoblastic leukemia; T- 
               
               
                   
                 cell lymphoblastic lymphoma 
               
               
                 Azacitidine 
                 Myelodysplastic syndromes 
               
               
                 BEACOPP 
                 Hodgkin lymphoma 
               
               
                 Becenum (Carmustine) 
                 Brain tumors; Hodgkin lymphoma; 
               
               
                   
                 multiple myeloma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 Beleodaq (Belinostat) 
                 Peripheral T-cell lymphoma 
               
               
                 BEP 
                 Ovarian germ cell tumors; testicular germ 
               
               
                   
                 cell tumors 
               
               
                 Bicalutamide 
                 Prostate cancer 
               
               
                 BiCNU (Carmustine) 
                 Brain tumors; Hodgkin lymphoma; 
               
               
                   
                 multiple myeloma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 Bleomycin 
                 Hodgkin lymphoma; non-Hodgkin 
               
               
                   
                 lymphoma; penile cancer; squamous cell 
               
               
                   
                 carcinoma of the cervix; squamous cell 
               
               
                   
                 carcinoma of the head and neck; squamous 
               
               
                   
                 cell carcinoma of the vulva; testicular cancer 
               
               
                 Bosulif (Bosutinib) 
                 Chronic myelogenous leukemia 
               
               
                 Brentuximab Vedotin 
                 Anaplastic large cell lymphoma; 
               
               
                   
                 Hodgkin lymphoma 
               
               
                 Busulfan 
                 Chronic myelogenous leukemia 
               
               
                 Busulfex (Busulfan) 
                 Chronic myelogenous leukemia 
               
               
                 Cabozantinib-S-Malate 
                 Medullary thyroid cancer 
               
               
                 CAF 
                 Breast cancer 
               
               
                 Camptosar (Irinotecan Hydrochloride) 
                 Colorectal cancer 
               
               
                 CAPOX 
                 Colorectal cancer 
               
               
                 Carfilzomib 
                 Multiple myeloma 
               
               
                 Casodex (Bicalutamide) 
                 Prostate cancer 
               
               
                 CeeNU (Lomustine) 
                 Brain tumors; Hodgkin lymphoma 
               
               
                 Ceritinib 
                 Non-small cell lung cancer 
               
               
                 Cerubidine (Daunorubicin Hydrochloride) 
                 Acute lymphoblastic leukemia; acute myeloid 
               
               
                   
                 leukemia 
               
               
                 Chlorambucil 
                 Chronic lymphocytic leukemia; 
               
               
                   
                 Hodgkin lymphoma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 CHLORAMBUCIL-PREDNISONE 
                 Chronic lymphocytic leukemia 
               
               
                 CHOP 
                 Non-Hodgkin lymphoma 
               
               
                 Cisplatin 
                 Bladder cancer; cervical cancer; malignant 
               
               
                   
                 mesothelioma; non-small cell lung cancer; 
               
               
                   
                 ovarian cancer; squamous cell carcinoma of 
               
               
                   
                 the head and neck; testicular cancer 
               
               
                 Clafen (Cyclophosphamide) 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia; breast cancer; chronic 
               
               
                   
                 lymphocytic leukemia; chronic myelogenous 
               
               
                   
                 leukemia; Hodgkin lymphoma; multiple 
               
               
                   
                 myeloma; mycosis fungoides; 
               
               
                   
                 neuroblastoma; non- Hodgkin lymphoma; 
               
               
                   
                 ovarian cancer; retinoblastoma 
               
               
                 Clofarex (Clofarabine) 
                 Acute lymphoblastic leukemia 
               
               
                 CMF 
                 Breast cancer 
               
               
                 Cometriq (Cabozantinib-S-Malate) 
                 Medullary thyroid cancer 
               
               
                 COPP 
                 Hodgkin lymphoma; non-Hodgkin lymphoma 
               
               
                 COPP-ABV 
                 Hodgkin lymphoma 
               
               
                 Cosmegen (Dactinomycin) 
                 Ewing sarcoma; gestational trophoblastic 
               
               
                   
                 disease; rhabdomyosarcoma; solid 
               
               
                   
                 tumors; testicular cancer; Wilms tumor 
               
               
                 CVP 
                 Non-Hodgkin lymphoma; chronic 
               
               
                   
                 lymphocytic leukemia 
               
               
                 Cyclophosphamide 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia; breast cancer; chronic 
               
               
                   
                 lymphocytic leukemia; chronic myelogenous 
               
               
                   
                 leukemia; Hodgkin lymphoma; multiple 
               
               
                   
                 myeloma; mycosis fungoides; 
               
               
                   
                 neuroblastoma; non- Hodgkin lymphoma; 
               
               
                   
                 ovarian cancer; retinoblastoma. 
               
               
                 Cyfos (Ifosfamide) 
                 Testicular germ cell tumors 
               
               
                 Cyramza (Ramucirumab) 
                 Adenocarcinoma; colorectal cancer; non- 
               
               
                   
                 small cell lung cancer 
               
               
                 Cytarabine 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia; chronic myelogenous 
               
               
                   
                 leukemia; meningeal leukemia 
               
               
                 Cytosar-U (Cytarabine) 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia; chronic myelogenous 
               
               
                   
                 leukemia; meningeal leukemia 
               
               
                 Cytoxan (Cyclophosphamide) 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia; breast cancer; chronic 
               
               
                   
                 lymphocytic leukemia; chronic myelogenous 
               
               
                   
                 leukemia; 
               
               
                   
                 Hodgkin lymphoma; multiple 
               
               
                   
                 myeloma; mycosis fungoides; 
               
               
                   
                 neuroblastoma; non- Hodgkin 
               
               
                   
                 lymphoma; ovarian cancer; 
               
               
                   
                 retinoblastoma 
               
               
                 Dacarbazine 
                 Hodgkin lymphoma; melanoma 
               
               
                 Dacogen (Decitabine) 
                 Myelodysplastic syndromes 
               
               
                 Dactinomycin 
                 Ewing sarcoma; gestational trophoblastic 
               
               
                   
                 disease; rhabdomyosarcoma; solid 
               
               
                   
                 tumors; testicular cancer; Wilms tumor 
               
               
                 Daunorubicin Hydrochloride 
                 Acute lymphoblastic leukemia; acute 
               
               
                   
                 myeloid leukemia 
               
               
                 Degarelix 
                 Prostate cancer 
               
               
                 Denileukin Diftitox 
                 Cutaneous T-cell lymphoma 
               
               
                 Denosumab 
                 Giant cell tumor of the bone; breast 
               
               
                   
                 cancer, prostate cancer 
               
               
                 DepoCyt (Liposomal Cytarabine) 
                 Lymphomatous meningitis 
               
               
                 DepoFoam (Liposomal Cytarabine) 
                 Lymphomatous meningitis 
               
               
                 Docetaxel 
                 Breast cancer; adenocarcinoma of the 
               
               
                   
                 stomach or gastroesophageal junction; non- 
               
               
                   
                 small cell lung cancer; prostate cancer; 
               
               
                   
                 squamous cell carcinoma of the head and 
               
               
                   
                 neck 
               
               
                 Doxil (Doxorubicin Hydrochloride Liposome) 
                 AIDS-related Kaposi sarcoma; 
               
               
                   
                 multiple myeloma; ovarian cancer 
               
               
                 Doxorubicin Hydrochloride 
                 Acute lymphoblastic leukemia; acute myeloid 
               
               
                   
                 leukemia; breast cancer; gastric (stomach) 
               
               
                   
                 cancer; Hodgkin lymphoma; neuroblastoma; 
               
               
                   
                 non-Hodgkin lymphoma; ovarian cancer; 
               
               
                   
                 small cell lung cancer; soft tissue and bone 
               
               
                   
                 sarcomas; thyroid cancer; transitional cell 
               
               
                   
                 bladder cancer; Wilms tumor. 
               
               
                 Dox-SL (Doxorubicin 
                 AIDS-related Kaposi sarcoma; 
               
               
                 Hydrochloride Liposome) 
                 multiple myeloma; ovarian cancer 
               
               
                 DTIC-Dome (Dacarbazine) 
                 Hodgkin lymphoma; melanoma 
               
               
                 Efudex (Fluorouracil) 
                 Basal cell carcinoma; breast cancer; 
               
               
                   
                 colorectal cancer; gastric (stomach) 
               
               
                   
                 adenocarcinoma; pancreatic cancer; squamous 
               
               
                   
                 cell carcinoma of the head and neck 
               
               
                 Ellence (Epirubicin Hydrochloride) 
                 Breast cancer 
               
               
                 Eloxatin (Oxaliplatin) 
                 Colorectal cancer; stage III colon cancer 
               
               
                 Emend (Aprepitant) 
                 Nausea and vomiting caused by 
               
               
                   
                 chemotherapy and nausea and vomiting after 
               
               
                   
                 surgery 
               
               
                 Enzalutamide 
                 Prostate cancer 
               
               
                 Epirubicin Hydrochloride 
                 Breast cancer 
               
               
                 EPOCH 
                 Non-Hodgkin lymphoma 
               
               
                 Erbitux (Cetuximab) 
                 Colorectal cancer; squamous cell carcinoma of 
               
               
                   
                 the head and neck 
               
               
                 Eribulin Mesylate 
                 Breast cancer 
               
               
                 Erivedge (Vismodegib) 
                 Basal cell carcinoma 
               
               
                 Erlotinib Hydrochloride 
                 Non-small cell lung cancer; pancreatic cancer 
               
               
                 Erwinaze (Asparaginase 
                 Acute lymphoblastic leukemia 
               
               
                 Erwinia chrysanthemi) 
                   
               
               
                 Etopophos (Etoposide Phosphate) 
                 Small cell lung cancer; testicular cancer 
               
               
                 Evacet (Doxorubicin Hydrochloride Liposome) 
                 AIDS-related Kaposi sarcoma; 
               
               
                   
                 multiple myeloma; ovarian cancer 
               
               
                 Everolimus 
                 Breast cancer; pancreatic cancer; renal 
               
               
                   
                 cell carcinoma; subependymal giant cell 
               
               
                   
                 astrocytoma 
               
               
                 Evista (Raloxifene Hydrochloride) 
                 Breast cancer 
               
               
                 Exemestane 
                 Breast cancer 
               
               
                 Fareston (Toremifene) 
                 Breast cancer 
               
               
                 Farydak (Panobinostat) 
                 Multiple myeloma 
               
               
                 Faslodex (Fulvestrant) 
                 Breast cancer 
               
               
                 FEC 
                 Breast cancer 
               
               
                 Femara (Letrozole) 
                 Breast cancer 
               
               
                 Filgrastim 
                 Neutropenia 
               
               
                 Fludara (Fludarabine Phosphate) 
                 Chronic lymphocytic leukemia 
               
               
                 Fluoroplex (Fluorouracil) 
                 Basal cell carcinoma; breast cancer; 
               
               
                   
                 colorectal cancer; gastric (stomach) 
               
               
                   
                 adenocarcinoma; pancreatic cancer; squamous 
               
               
                   
                 cell carcinoma of the head and neck 
               
               
                 Folex (Methotrexate) 
                 Acute lymphoblastic leukemia; breast 
               
               
                   
                 cancer; gestational trophoblastic disease; 
               
               
                   
                 head and neck cancer; lung cancer; mycosis 
               
               
                   
                 fungoides; non-Hodgkin lymphoma; 
               
               
                   
                 osteosarcoma 
               
               
                 FOLFIRI 
                 Colorectal cancer 
               
               
                 FOLFIRI-BEVACIZUMAB 
                 Colorectal cancer 
               
               
                 FOLFIRI-CETUXIMAB 
                 Colorectal cancer 
               
               
                 FOLFIRINOX 
                 Pancreatic cancer 
               
               
                 FOLFOX 
                 Colorectal cancer 
               
               
                 Folotyn (Pralatrexate) 
                 Peripheral T-cell lymphoma 
               
               
                 FU-LV 
                 Colorectal cancer; esophageal cancer; 
               
               
                   
                 gastric cancer 
               
               
                 Fulvestrant 
                 Breast cancer 
               
               
                 Gefitinib 
                 Non-small cell lung cancer 
               
               
                 Gemcitabine Hydrochloride 
                 Breast cancer; non-small cell lung 
               
               
                   
                 cancer; ovarian cancer; pancreatic cancer 
               
               
                 GEMCITABINE-CISPLATIN 
                 Biliary tract cancer; bladder cancer; 
               
               
                   
                 cervical cancer; malignant mesothelioma; 
               
               
                   
                 non-small cell lung cancer; ovarian cancer; 
               
               
                   
                 pancreatic cancer 
               
               
                 GEIVICITABINE-OXALIPLATIN 
                 Pancreatic cancer 
               
               
                 Gemtuzumab Ozogamicin (antibody drug 
                 Acute myeloid leukemia 
               
               
                 conjugate) 
                   
               
               
                 Gemzar (Gemcitabine Hydrochloride) 
                 Breast cancer; non-small cell lung 
               
               
                   
                 cancer; ovarian cancer; pancreatic cancer 
               
               
                 Gilotrif (Afatinib Dimaleate) 
                 Non-small cell lung cancer 
               
               
                 Gleevec (Imatinib Mesylate) 
                 Acute lymphoblastic leukemia; chronic 
               
               
                   
                 eosinophilic leukemia or hypereosinophilic 
               
               
                   
                 syndrome; chronic myelogenous leukemia; 
               
               
                   
                 dermatofibrosarcoma protuberans; 
               
               
                   
                 gastrointestinal stromal tumor; 
               
               
                   
                 myelodysplastic/myeloproliferative neoplasms; 
               
               
                   
                 systemic mastocytosis. 
               
               
                 Gliadel (Carmustine Implant) 
                 Glioblastoma multiforme; malignant glioma 
               
               
                 Goserelin Acetate 
                 Breast cancer; prostate cancer 
               
               
                 Halaven (Eribulin Mesylate) 
                 Breast cancer 
               
               
                 Hycamtin (Topotecan Hydrochloride) 
                 Cervical cancer; ovarian cancer; small cell 
               
               
                   
                 lung cancer 
               
               
                 Hyper-CVAD 
                 Acute lymphoblastic leukemia; non- 
               
               
                   
                 Hodgkin lymphoma 
               
               
                 Ibrance (Palbociclib) 
                 Breast cancer 
               
               
                 Ibrutinib 
                 Chronic lymphocytic leukemia; mantel 
               
               
                   
                 cell lymphoma; 
               
               
                 ICE 
                 Hodgkin lymphoma; non-Hodgkin lymphoma 
               
               
                 Iclusig (Ponatinib Hydrochloride) 
                 Acute lymphoblastic leukemia; Chronic 
               
               
                   
                 myelogenous leukemia 
               
               
                 Idamycin (Idarubicin Hydrochloride) 
                 Acute myeloid leukemia 
               
               
                 Imatinib Mesylate 
                 Acute lymphoblastic leukemia; chronic 
               
               
                   
                 eosinophilic leukemia or hypereosinophilic 
               
               
                   
                 syndrome; chronic myelogenous leukemia; 
               
               
                   
                 dermatofibrosarcoma protuberans; 
               
               
                   
                 gastrointestinal stromal tumor; 
               
               
                   
                 myelodysplastic/myeloproliferative neoplasms; 
               
               
                   
                 systemic mastocytosis. 
               
               
                 Imbruvica (Ibrutinib) 
                 Chronic lymphocytic leukemia; mantle cell 
               
               
                   
                 lymphoma; Waldenstr6m 
               
               
                   
                 macroglo bulinemia 
               
               
                 Inlyta (Axitinib) 
                 Renal cell carcinoma 
               
               
                 Iressa (Gefitinib) 
                 Non-small cell lung cancer 
               
               
                 Irinotecan Hydrochloride 
                 Colorectal cancer 
               
               
                 Istodax (Romidepsin) 
                 Cutaneous T-cell lymphoma 
               
               
                 Ixempra (Ixabepilone) 
                 Breast cancer 
               
               
                 Jevtana (Cabazitaxel) 
                 Prostate cancer 
               
               
                 Keoxifene (Raloxifene Hydrochloride) 
                 Breast cancer 
               
               
                 Kyprolis (Carfilzomib) 
                 Multiple myeloma 
               
               
                 Lenvima (Lenvatinib Mesylate) 
                 Thyroid cancer 
               
               
                 Letrozole 
                 Breast cancer 
               
               
                 Leucovorin Calcium 
                 Colorectal cancer 
               
               
                 Leukeran (Chlorambucil) 
                 Chronic lymphocytic leukemia; 
               
               
                   
                 Hodgkin lymphoma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 Leuprolide Acetate 
                 Prostate cancer 
               
               
                 Linfolizin (Chlorambucil) 
                 Chronic lymphocytic leukemia; 
               
               
                   
                 Hodgkin lymphoma; non-Hodgkin 
               
               
                   
                 lymphoma 
               
               
                 LipoDox (Doxorubicin 
                 AIDS-related Kaposi sarcoma; 
               
               
                 Hydrochloride Liposome) 
                 multiple myeloma; ovarian cancer 
               
               
                 Lomustine 
                 Brain tumors; Hodgkin lymphoma 
               
               
                 Lupron (Leuprolide Acetate) 
                 Prostate cancer 
               
               
                 Lynparza (Olaparib) 
                 Ovarian cancer 
               
               
                 Margibo (Vincristine Sulfate Liposome) 
                 Acute lymphoblastic leukemia 
               
               
                 Matulane (Procarbazine Hydrochloride) 
                 Hodgkin lymphoma 
               
               
                 Mechlorethamine Hydrochloride 
                 Bronchogenic carcinoma; chronic 
               
               
                   
                 lymphocytic leukemia; chronic myelogenous 
               
               
                   
                 leukemia; Hodgkin lymphoma; malignant 
               
               
                   
                 pleural effusion, malignant pericardial 
               
               
                   
                 effusion, and malignant peritoneal effusion; 
               
               
                   
                 mycosis fungoides; non-Hodgkin lymphoma 
               
               
                 Megace (Megestrol Acetate) 
                 Breast cancer; endometrial cancer 
               
               
                 Mekinist (Trametinib) 
                 Melanoma 
               
               
                 Mercaptopurine 
                 Acute lymphoblastic leukemia 
               
               
                 Mesnex (Mesna) 
                 Hemorrhagic cystitis 
               
               
                 Methazolastone (Temozolomide) 
                 Anaplastic astrocytoma: 
               
               
                   
                 glioblastoma multiforme 
               
               
                 Mexate (Methotrexate) 
                 Acute lymphoblastic leukemia; breast 
               
               
                   
                 cancer; gestational trophoblastic disease; 
               
               
                   
                 head and neck cancer; lung cancer; mycosis 
               
               
                   
                 fungoides; non-Hodgkin lymphoma; 
               
               
                   
                 osteosarcoma 
               
               
                 Mexate-AQ (Methotrexate) 
                 Acute lymphoblastic leukemia; breast 
               
               
                   
                 cancer; gestational trophoblastic disease; 
               
               
                   
                 head and neck cancer; lung cancer; mycosis 
               
               
                   
                 fungoides; non-Hodgkin lymphoma; 
               
               
                   
                 osteosarcoma 
               
               
                 Mitoxantrone Hydrochloride 
                 Acute myeloid leukemia; prostate cancer 
               
               
                 Mitozytrex (Mitomycin C) 
                 Gastric (stomach) and 
               
               
                   
                 pancreatic adenocarcinoma 
               
               
                 MOPP 
                 Hodgkin lymphoma 
               
               
                 Mozobil (Plerixafor) 
                 Multiple myeloma; non-Hodgkin lymphoma 
               
               
                 Mustargen (Mechlorethamine Hydrochloride) 
                 Bronchogenic carcinoma; chronic 
               
               
                   
                 lymphocytic leukemia; chronic myelogenous 
               
               
                   
                 leukemia; Hodgkin lymphoma; malignant 
               
               
                   
                 pleural effusion, malignant pericardial 
               
               
                   
                 effusion, and malignant peritoneal effusion; 
               
               
                   
                 mycosis fungoides: non-Hodgkin lymphoma 
               
               
                 Myleran (Busulfan) 
                 Chronic myelogenous leukemia 
               
               
                 Mylotarg (Gemtuzumab Ozogamicin) 
                 Acute myeloid leukemia 
               
               
                 Nanoparticle Paclitaxel (Paclitaxel Albumin- 
                 Breast cancer; Non-small cell lung 
               
               
                 stabilized Nanoparticle Formulation) 
                 cancer; Pancreatic cancer 
               
               
                 Navelbine (Vinorelbine Tartrate) 
                 Non-small cell lung cancer 
               
               
                 Nelarabine 
                 T-cell acute lymphoblastic leukemia 
               
               
                 Neosar (Cyclophosphamide) 
                 Acute lymphoblastic leukemia; Acute 
               
               
                   
                 myeloid leukemia; Breast cancer; Chronic 
               
               
                   
                 lymphocytic leukemia; Chronic myelogenous 
               
               
                   
                 leukemia; Hodgkin lymphoma; Multiple 
               
               
                   
                 myeloma; Mycosis fungoides; 
               
               
                   
                 Neuroblastoma; Non- Hodgkin lymphoma; 
               
               
                   
                 Ovarian cancer; Retinoblastoma 
               
               
                 Nexavar (Sorafenib Tosylate) 
                 Hepatocellular carcinoma; Renal 
               
               
                   
                 cell carcinoma; Thyroid cancer 
               
               
                 Nilotinib 
                 Chronic myelogenous leukemia 
               
               
                 Nivolumab 
                 Melanoma; Squamous non-small cell 
               
               
                   
                 lung cancer 
               
               
                 Nolvadex (Tamoxifen Citrate) 
                 Breast cancer 
               
               
                 Odomzo (Sonidegib) 
                 Basal cell carcinoma 
               
               
                 OEPA 
                 Hodgkin lymphoma 
               
               
                 OFF 
                 Pancreatic cancer 
               
               
                 Olaparib 
                 Ovarian cancer 
               
               
                 Oncaspar (Pegaspargase) 
                 Acute lymphoblastic leukemia 
               
               
                 OPPA 
                 Hodgkin lymphoma 
               
               
                 Oxaliplatin 
                 Colorectal cancer; Stage III colon cancer 
               
               
                 Paclitaxel 
                 AIDS-related Kaposi sarcoma; Breast 
               
               
                   
                 cancer; Non-small cell lung cancer; Ovarian 
               
               
                   
                 cancer 
               
               
                 Paclitaxel Albumin-stabilized Nanoparticle 
                 Breast cancer; Non-small lung 
               
               
                 Formulation 
                 cancer; Pancreatic cancer 
               
               
                 PAD 
                 Multiple myeloma 
               
               
                 Palbociclib 
                 Breast cancer 
               
               
                 Pamidronate Disodium 
                 Breast cancer; Multiple myeloma 
               
               
                 Panitumumab 
                 Colorectal cancer 
               
               
                 Panobinostat 
                 Multiple myeloma 
               
               
                 Paraplat (Carboplatin) 
                 Non-small cell lung cancer; Ovarian cancer 
               
               
                 Paraplatin (Carboplatin) 
                 Non-small cell lung cancer; Ovarian cancer 
               
               
                 Pazopanib Hydrochloride 
                 Renal cell carcinoma; Soft tissue sarcoma 
               
               
                 Pegaspargase 
                 Acute lymphoblastic leukemia 
               
               
                 Pemetrexed Disodium 
                 Malignant pleural mesothelioma; Non- 
               
               
                   
                 small cell lung cancer 
               
               
                 Platinol (Cisplatin) 
                 Bladder cancer; Cervical cancer; Malignant 
               
               
                   
                 mesothelioma; Non-small cell lung cancer; 
               
               
                   
                 Ovarian cancer; Squamous cell carcinoma 
               
               
                   
                 of the head and neck; Testicular cancer 
               
               
                 Platinal-AQ (Cisplatin) 
                 Bladder cancer; Cervical cancer; Malignant 
               
               
                   
                 mesothelioma; Non-small cell lung cancer; 
               
               
                   
                 Ovarian cancer; Squamous cell carcinoma 
               
               
                   
                 of the head and neck; Testicular cancer 
               
               
                 Plerixafor 
                 Multiple myeloma; Non-Hodgkin lymphoma 
               
               
                 Pomalidomide 
                 Multiple myeloma 
               
               
                 Pomalyst (Pomalidomide) 
                 Multiple myeloma 
               
               
                 Pontinib Hydrochloride 
                 Acute lymphoblastic leukemia; 
               
               
                   
                 Chronic myelogenous leukemia 
               
               
                 Pralatrexate 
                 Peripheral T-cell lymphoma 
               
               
                 Prednisone 
                 Acute lymphoblastic leukemia; Chronic 
               
               
                   
                 lymphocytic leukemia; Hodgkin lymphoma; 
               
               
                   
                 Multiple myeloma; Non-Hodgkin 
               
               
                   
                 lymphoma; Prostate cancer; Thymoma and 
               
               
                   
                 thymic carcmoma 
               
               
                 Procarbazine Hydrochloride 
                 Hodgkin lymphoma 
               
               
                 Provenue (Sipuleucel-T) 
                 Prostate cancer 
               
               
                 Purinethol (Mercaptopurine) 
                 Acute lymphoblastic leukemia 
               
               
                 Radium 223 Dichloride 
                 Prostate cancer 
               
               
                 Raloxifene Hydrochloride 
                 Breast cancer 
               
               
                 R-CHOP 
                 Non-Hodgkin lymphoma 
               
               
                 R-CVP 
                 Non-Hodgkin lymphoma 
               
               
                 Regorafenib 
                 Colorectal cancer; Gastrointestinal 
               
               
                   
                 stromal tumor 
               
               
                 R-EPOCH 
                 B-cell non-Hodgkin lymphoma 
               
               
                 Revlimid (Lenalidomide) 
                 Mantle cell lymphoma; Multiple 
               
               
                   
                 myeloma; Anemia 
               
               
                 Rheumatrex (Methotrexate) 
                 Acute lymphoblastic leukemia; Breast 
               
               
                   
                 cancer; Gestational trophoblastic disease; 
               
               
                   
                 Head and neck cancer; Lung cancer; Non- 
               
               
                   
                 Hodgkin lymphoma; Osteosarcoma 
               
               
                 Romidepsin 
                 Cutaneous T-cell lymphoma 
               
               
                 Rubidomycin (Daunorubicin Hydrochloride) 
                 Acute lymphoblastic leukemia; Acute 
               
               
                   
                 myeloid leukemia 
               
               
                 Sipuleucel-T 
                 Prostate cancer 
               
               
                 Somatuline Depot (Lanreotide Acetate) 
                 Gastroenteropancreatic neuroendocrine tumors 
               
               
                 Sonidegib 
                 Basal cell carcinoma 
               
               
                 Sorafenib Tosylate 
                 Hepatocellular carcinoma; Renal 
               
               
                   
                 cell carcinoma; Thyroid cancer 
               
               
                 Sprycel (Dasatinib) 
                 Acute lymphoblastic leukemia; 
               
               
                   
                 Chronic myelogenous leukemia 
               
               
                 STANFORD V 
                 Hodgkin lymphoma 
               
               
                 Stivarga (Regorafenib) 
                 Colorectal cancer; Gastrointestinal 
               
               
                   
                 stromal tumor 
               
               
                 Sunitnib Malate 
                 Gastronintestinal stromal tumor; Pancreatic 
               
               
                   
                 cancer; Renal cell carcinoma 
               
               
                 Sutent (Sunitinib Malate) 
                 Gastronintestinal stromal tumor; 
               
               
                   
                 Pancreatic cancer; Renal cell carcinoma 
               
               
                 Synovir (Thalidomide) 
                 Multiple myeloma 
               
               
                 Synribo (Omacetaxine Mepesuccinate) 
                 Chronic myelogenous leukemia 
               
               
                 TAC 
                 Breast cancer 
               
               
                 Tafinlar (Dabrafenib) 
                 Melanoma 
               
               
                 Tamoxifen Citrate 
                 Breast cancer 
               
               
                 Tarabine PFS (Cytarabine) 
                 Acute lymphoblastic leukemia; Acute 
               
               
                   
                 myeloid leukemia; Chronic myelogenous 
               
               
                   
                 leukemia 
               
               
                 Tarceva (Erlotinib Hydrochloride) 
                 Non-small cell lung cancer; Pancreatic cancer 
               
               
                 Targretin (Bexarotene) 
                 Skin problems caused by cutaneous T- 
               
               
                   
                 cell lymphoma 
               
               
                 Tasina (Niltinib) 
                 Chronic myelogenous leukemia 
               
               
                 Taxol (Paclitaxel) 
                 AIDS-related Kaposi sarcoma; Breast 
               
               
                   
                 cancer; Non-small cell lung cancer; Ovarian 
               
               
                   
                 cancer 
               
               
                 Taxotere (Docetaxel) 
                 Breast cancer; Adenocarcinoma; Non- 
               
               
                   
                 small cell lung cancer; Prostate cancer; 
               
               
                   
                 Squamous cell carcinoma of the head and 
               
               
                   
                 neck 
               
               
                 Temodar (Temozolomide) 
                 Anaplastic astrocytoma; 
               
               
                   
                 Glioblastoma multiforme 
               
               
                 Temozolomide 
                 Anaplastic astrocytoma; 
               
               
                   
                 Glioblastoma multiforme 
               
               
                 Thiotepa 
                 Bladder cancer; Breast cancer; Malignant 
               
               
                   
                 pleural effusion, malignant pericardial 
               
               
                   
                 effusion, and malignant peritoneal 
               
               
                   
                 effusion; Ovarian cancer 
               
               
                 Toposar (Etoposide) 
                 Small cell lung cancer; Testicular cancer 
               
               
                 Topotecan Hydrochloride 
                 Cervical cancer; Ovarian cancer; Small 
               
               
                   
                 cell lung cancer 
               
               
                 Toremifene 
                 Breast cancer 
               
               
                 Torisel (Temsirolimus) 
                 Renal cell carcinoma 
               
               
                 TPF 
                 Squamous cell carcinoma of the head 
               
               
                   
                 and neck; Gastric (stomach) cancer 
               
               
                 Trastuzumab 
                 Adenocarcinoma; Breast cancer 
               
               
                 Treanda (Bendamustine Hydrochloride) 
                 B-cell non-Hodgkin lymphoma; 
               
               
                   
                 Chronic lymphocytic leukemia 
               
               
                 Trisenox (Arsenic Trioxide) 
                 Acute promyelocytic leukemia 
               
               
                 Tykerb (Lapatinib Ditosylate) 
                 Breast cancer 
               
               
                 Vandetabib 
                 Medullary thyroid cancer 
               
               
                 VAMP 
                 Hodgkin lymphoma 
               
               
                 VeIP 
                 Ovarian germ cell; Testicular cancer 
               
               
                 Velban (Vinblastine Sulfate) 
                 Breast cancer; Choriocarcinoma; 
               
               
                   
                 Hodgkin lymphoma; Kaposi sarcoma; 
               
               
                   
                 Mycosid fungoides; Non-Hodgkin 
               
               
                   
                 lymphoma; 
               
               
                   
                 Testicular cancer 
               
               
                 Veicade (Bortezomib) 
                 Mulitple myeloma; Mantle cell lymphoma 
               
               
                 Velsar (Vinblastine Sulfate) 
                 Breast cancer. Choriocarcinoma; 
               
               
                   
                 Hodgkin lymphoma; Kaposi sarcoma; 
               
               
                   
                 Mycosis fungoides; Non-Hodgkin 
               
               
                   
                 lymphoma; Testicular cancer 
               
               
                 VePesid (Etoposide) 
                 Small cell lung cancer; Testicular cancer 
               
               
                 Viadur (Leuprolide Acetate) 
                 Prostate cancer 
               
               
                 Vidaza (Azacitidine) 
                 Myelodysplastic syndromes 
               
               
                 Vincasar PFS (Vincristine Sulfate) 
                 Acute leukemia; Hodgkin lymphoma; 
               
               
                   
                 Neuroblastoma; Non-Hodgkin 
               
               
                   
                 lymphoma; Rhabdomyosarcoma; Wilms 
               
               
                   
                 tumor 
               
               
                 Vincristine Sulfate Liposome 
                 Acute lymphoblastic leukemia 
               
               
                 Vinorelbine Tartrate 
                 Non-small cell lung cancer 
               
               
                 VIP 
                 Testicular cancer 
               
               
                 Visbodegib 
                 Basal cell carcinoma 
               
               
                 Voraxaze (Glucarpidase) 
                 Toxic blood levels of the anticancer 
               
               
                   
                 drug methotrexate 
               
               
                 Votrient (Pazopanib Hydrochloride) 
                 Renal cell carcinoma; Soft tissue sarcoma 
               
               
                 Wellcovorin (Leucovorin Calcium) 
                 Colorectal cancer; Anemia 
               
               
                 Xalkori (Crizotinib) 
                 Non-small cell lung cancer 
               
               
                 Xeloda (Capecitabine) 
                 Breast cancer; Colorectal cancer 
               
               
                 XELIRI 
                 Colorectal cancer; Esophageal cancer; 
               
               
                   
                 Gastric (stomach) cancer 
               
               
                 XELOX 
                 Colorectal cancer 
               
               
                 Xofigo (Radium 223 Dichloride) 
                 Prostate cancer 
               
               
                 Xtandi (Enzalutamide) 
                 Prostate cancer 
               
               
                 Zaltrap (Ziv-Aflibercept) 
                 Colorectal cancer 
               
               
                 Zelboraf (Vemurafenib) 
                 Melanoma 
               
               
                 Ziv-Aflibercept 
                 Colorectal cancer 
               
               
                 Zoladex (Goserelin Acetate) 
                 Breast cancer; Prostate cancer 
               
               
                 Zolinza (Vorinostat) 
                 Cutaneous T-cell lymphoma 
               
               
                 Zometa (Zoledronic Acid) 
                 Multiple myeloma 
               
               
                 Zydelig (Idelalisib) 
                 Chronic lymphocytic leukemia; Non- 
               
               
                   
                 Hodgkin lymphoma (Follicula B-cell non 
               
               
                   
                 Hodgkin lymphoma and Small lymphocytic 
               
               
                   
                 lymphoma) 
               
               
                 Zykadia (Certinib) 
                 Non-small cell lung cancer 
               
               
                 Zytiga (Abiraterone Acetate) 
                 Prostate cancer 
               
               
                   
               
            
           
         
       
     
     It is to be understood that the therapeutic agent may be located inside the nanoparticle, on the outside surface of the nanoparticle, or both. The nanoparticle may contain more than one therapeutic agent, for example, two therapeutic agents, three therapeutic agents, four therapeutic agents, five therapeutic agents, or more. Furthermore, a nanoparticle may contain the same or different therapeutic agents inside and outside the nanoparticle. 
     In some embodiments of this invention the nanoparticles comprising ABRAXANE and bevacizumab are excluded. 
     In one aspect, the nanoparticle comprises at least 100 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 200 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 300 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 400 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 500 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises at least 600 binding agents non-covalently bound to the surface of the nanoparticle. 
     In one aspect, the nanoparticle comprises between about 100 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 200 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 300 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 400 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 500 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 600 and about 1000 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 200 and about 800 binding agents non-covalently bound to the surface of the nanoparticle. In one aspect, the nanoparticle comprises between about 300 and about 800 binding agents non-covalently bound to the surface of the nanoparticle. In preferred embodiments, the nanoparticle comprises between about 400 and about 800 binding agents non-covalently bound to the surface of the nanoparticle. Contemplated values include any value or subrange within any of the recited ranges, including endpoints. 
     In one aspect, the average particle size in the nanoparticle composition is less than about 1 μm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 1 μm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 900 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 800 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 700 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 600 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 500 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 400 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 300 nm. In one aspect, the average particle size in the nanoparticle composition is between about 130 nm and about 200 nm. In a preferred embodiment, the average particle size in the nanoparticle composition is between about 150 nm and about 180 nm. In an especially preferred embodiment, the mean particle size in the nanoparticle composition is about 160 nm. Contemplated values include any value, subrange, or range within any of the recited ranges, including endpoints. 
     In one aspect, the nanoparticle composition is formulated for intravenous injection. In order to avoid an ischemic event, the nanoparticle composition formulated for intravenous injection should comprise nanoparticles with an average particle size of less than about 1 μm. 
     In one aspect, the average particle size in the nanoparticle composition is greater than about 1 μm. In one aspect, the average particle size in the nanoparticle composition is between about 1 μm and about 5 μm. In one aspect, the average particle size in the nanoparticle composition is between about 1 μm and about 4 μm. In one aspect, the average particle size in the nanoparticle composition is between about 1 μm and about 3 μm. In one aspect, the average particle size in the nanoparticle composition is between about 1 μm and about 2 μm. In one aspect, the average particle size in the nanoparticle composition is between about 1 μm and about 1.5 μm. Contemplated values include any value, subrange, or range within any of the recited ranges, including endpoints. 
     In one aspect, the nanoparticle composition is formulated for direct injection into a tumor. Direct injection includes injection into or proximal to a tumor site, perfusion into a tumor, and the like. When formulated for direct injection into a tumor, the nanoparticle may comprise any average particle size. Without being bound by theory, it is believed that larger particles (e.g., greater than 500 nm, greater than 1 μm, and the like) are more likely to be immobilized within the tumor, thereby providing a beneficial effect. Larger particles can accumulate in the tumor or specific organs. See, e.g., 20-60 micron glass particle that is used to inject into the hepatic artery feeding a tumor of the liver, called “TheraSphere®” (in clinical use for liver cancer). Therefore, for intravenous administration, particles under 1 μm are typically used. Particles over 1 μm are, more typically, administered directly into a tumor (“direct injection”) or into an artery feeding into the site of the tumor. 
     In one aspect, less than about 0.01% of the nanoparticles within the composition have a particle size greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, or greater than 800 nm. In one aspect, less than about 0.001% of the nanoparticles within the composition have a particle size greater than 200 nm, greater than 300 nm, greater than 400 nm, greater than 500 nm, greater than 600 nm, greater than 700 nm, or greater than 800 nm. In a preferred embodiment, less than about 0.01% of the nanoparticles within the composition have a particle size greater than 800 nm. In a more preferred embodiment, less than about 0.001% of the nanoparticles within the composition have a particle size greater than 800 nm. 
     In a preferred aspect, the sizes and size ranges recited herein relate to particle sizes of the reconstituted lyophilized nanoparticle composition. That is, after the lyophilized nanoparticles are resuspended in an aqueous solution (e.g., water, other pharmaceutically acceptable excipient, buffer, etc.), the particle size or average particle size is within the range recited herein. 
     In one aspect, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the nanoparticles are present in the reconstituted composition as single nanoparticles. That is, fewer than about 50%, 40%, 30%, etc. of the nanoparticles are dimerized or multimerized (oligomerized). 
     In some embodiments, the size of the nanoparticle can be controlled by the adjusting the amount (e.g., ratio) of carrier protein to binding agent. The size of the nanoparticles, and the size distribution, is also important. The nanoparticles of the invention may behave differently according to their size. At large sizes, an agglomeration may block blood vessels. Therefore, agglomeration of nanoparticles can affect the performance and safety of the composition. On the other hand, larger particles may be more therapeutic under certain conditions (e.g., when not administered intravenously). 
     In one aspect, the nanoparticle composition comprises at least one additional therapeutic agent. In one embodiment, the at least one additional therapeutic agent is non-covalently bound to the outside surface of the nanoparticle. In one embodiment, the at least one additional therapeutic agent is arranged on the outside surface of the nanoparticle. In one embodiment, the at least one additional therapeutic agent is selected from the group consisting of abiraterone, bendamustine, bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gemcitabine, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib, oxaliplatin, pazopanib, pemetrexed, picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin, vinblastine, vinorelbine, vincristine, and cyclophosphamide. In one embodiment, the at least one additional therapeutic agent is an anti-cancer binding agent, e.g., an anti-cancer antibody. 
     Methods of Making Nanoparticles 
     In some aspects, the current invention relates to methods of making nanoparticle compositions as described herein. 
     In one aspect, the nanoparticles of the nanoparticle composition are formed by contacting the carrier protein or carrier protein-therapeutic agent particle with the binding agent at a ratio of about 10:1 to about 10:30 carrier protein particle or carrier protein-therapeutic agent particle to binding agent. In one embodiment, the ratio is about 10:2 to about 10:25. In one embodiment, the ratio is about 10:2 to about 1:1. In a preferred embodiment, the ratio is about 10:2 to about 10:6. In an especially preferred embodiment, the ratio is about 10:4. Contemplated ratios include any value, subrange, or range within any of the recited ranges, including endpoints. 
     In one embodiment, the amount of solution or other liquid medium employed to form the nanoparticles is particularly important. No nanoparticles are formed in an overly dilute solution of the carrier protein (or carrier protein-therapeutic agent) and the antibodies. An overly concentrated solution will result in unstructured aggregates. In some embodiments, the amount of solution (e.g., sterile water, saline, phosphate buffered saline) employed is between about 0.5 mL of solution to about 20 mL of solution. In some embodiments, the amount of carrier protein is between about 1 mg/mL and about 100 mg/mL. In some embodiments, the amount of binding agent is between about 1 mg/mL and about 30 mg/mL. For example, in some embodiments, the ratio of carrier protein:binding agent:solution is approximately 9 mg of carrier protein (e.g., albumin) to 4 mg of binding agent, e.g., antibody (e.g., BEV) in 1 mL of solution (e.g., saline). An amount of therapeutic agent (e.g., taxol) can also be added to the carrier protein. For example, 1 mg of taxol can be added 9 mg of carrier protein (10 mg carrier protein-therapeutic) and 4 mg of binding agent, e.g., antibody, Fc fusion molecule, or aptamer, in 1 mL of solution. When using a typical i.v. bag, for example, with the solution of approximately 1 liter one would need to use 1000× the amount of carrier protein/carrier protein-therapeutic agent and antibodies compared to that used in 1 mL. Thus, one cannot form the present nanoparticles in a standard i.v. bag. Furthermore, when the components are added to a standard i.v. bag in the therapeutic amounts of the present invention, the components do not self-assemble to form nanoparticles. 
     In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH between about 4 and about 8. In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH of about 4. In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH of about 5. In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH of about 6. In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH of about 7. In one embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH of about 8. In a preferred embodiment, the carrier protein or carrier protein-therapeutic agent particle is contacted with the binding agent in a solution having a pH between about 5 and about 7. 
     In one embodiment, the carrier protein particle or carrier protein-therapeutic agent particle is incubated with the binding agent at a temperature of about 5° C. to about 60° C., or any range, subrange, or value within that range including endpoints. In a preferred embodiment, the carrier protein particle or carrier protein-therapeutic agent particle is incubated with the binding agent at a temperature of about 23° C. to about 60° C. 
     Without being bound by theory, it is believed that the stability of the nanoparticles within the nanoparticle composition is, at least in part, dependent upon the temperature and/or pH at which the nanoparticles are formed, as well as the concentration of the components (i.e., carrier protein, binding agent, and optionally therapeutic agent) in the solution. In one embodiment, the K d  of the nanoparticles is between about 1×10 −11  M and about 2×10 −5 M. In one embodiment, the K d  of the nanoparticles is between about 1×10 −11  M and about 2×10 −8  M. In one embodiment, the K d  of the nanoparticles is between about 1×10 −11  M and about 7×10 −9 M. In a preferred embodiment, the K d  of the nanoparticles is between about 1×10 −11  M and about 3×10 −8  M. Contemplated values include any value, subrange, or range within any of the recited ranges, including endpoints. 
     Lyophilization 
     The lyophilized compositions of this invention are prepared by standard lyophilization techniques with or without the presence of stabilizers, buffers, etc. Surprisingly, these conditions do not alter the relatively fragile structure of the nanoparticles. Moreover, at best, these nanoparticles retain their size distribution upon lyophilization and, more importantly, can be reconstituted for in vivo administration (e.g., intravenous delivery) in substantially the same form and ratios as if freshly made. 
     Formulations 
     In one aspect, the nanoparticle composition is formulated for systemic delivery, e.g., intravenous administration. 
     In one aspect, the nanoparticle composition is formulated for direct injection into a tumor. Direct injection includes injection into or proximal to a tumor site, perfusion into a tumor, and the like. Because the nanoparticle composition is not administered systemically, a nanoparticle composition is formulated for direct injection into a tumor may comprise any average particle size. Without being bound by theory, it is believed that larger particles (e.g., greater than 500 nm, greater than 1 μm, and the like) are more likely to be immobilized within the tumor, thereby providing what is believed to be a better beneficial effect. 
     In another aspect, provided herein is a composition comprising a compound provided herein, and at least one pharmaceutically acceptable excipient. 
     In general, the compounds provided herein can be formulated for administration to a patient by any of the accepted modes of administration. Various formulations and drug delivery systems are available in the art. See, e.g., Gennaro, A. R., ed. (1995)  Remington&#39;s Pharmaceutical Sciences,  18th ed., Mack Publishing Co. 
     In general, compounds provided herein will be administered as pharmaceutical compositions by any one of the following routes: oral, systemic (e.g., transdermal, intranasal or by suppository), or parenteral (e.g., intramuscular, intravenous or subcutaneous) administration. 
     The compositions are comprised of, in general, a compound of the present invention in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the claimed compounds. Such excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art. 
     Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, aqueous dextrose, and glycols. Other suitable pharmaceutical excipients and their formulations are described in Remington&#39;s Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990). 
     The present compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient. Such a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass, and rubber stoppers such as in vials. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. 
     Treatment Methods 
     The nanoparticle compositions as described herein are useful in treating cancer cells and/or tumors in a mammal. In a preferred embodiment, the mammal is a human (i.e., a human patient). Preferably, the lyophilized nanoparticle composition is reconstituted (suspended in an aqueous excipient) prior to administration. 
     In one aspect is provided a method for treating a cancer cell, the method comprising contacting the cell with an effective amount of nanoparticle composition as described herein to treat the cancer cell. Treatment of a cancer cell includes, without limitation, reduction in proliferation, killing the cell, preventing metastasis of the cell, and the like. 
     In one aspect is provided a method for treating a tumor in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a nanoparticle composition as described herein to treat the tumor. In one embodiment, the size of the tumor is reduced. In one embodiment, the tumor size does not increase (i.e. progress) for at least a period of time during and/or after treatment. 
     In one embodiment, the nanoparticle composition is administered intravenously. In one embodiment, the nanoparticle composition is administered directly to the tumor. In one embodiment, the nanoparticle composition is administered by direct injection or perfusion into the tumor. 
     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 repeating steps a) and b) as necessary to treat the tumor.       

     In one embodiment, the therapeutically effective amount of the nanoparticles described herein comprises about 50 mg/m 2  to about 200 mg/m 2  carrier protein or carrier protein and therapeutic agent. In a preferred embodiment, the therapeutically effective amount comprises about 75 mg/m 2  to about 175 mg/m 2  carrier protein or carrier protein and therapeutic agent. Contemplated values include any value, subrange, or range within any of the recited ranges, including endpoints. 
     In one embodiment, the therapeutically effective amount comprises about 20 mg/m 2  to about 90 mg/m 2  binding agent, e.g., antibody, aptamer or Fc fusion. In a preferred embodiment, the therapeutically effective amount comprises 30 mg/m 2  to about 70 mg/m 2  binding agent, e.g., antibody, aptamer or Fc fusion. Contemplated values include any value, subrange, or range within any of the recited ranges, including endpoints. 
     Cancers or tumors that can be treated by the compositions and methods described herein include, but are not limited to: biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer, gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen&#39;s disease and Paget&#39;s disease; liver cancer (hepatocarcinoma); lung cancer; lymphomas, including Hodgkin&#39;s disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skin cancer, including melanoma, Kaposi&#39;s sarcoma, basocellular cancer and squamous cell cancer; testicular cancer, including germinal tumors (seminoma, non-seminoma[teratomas, choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer, including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor. In important embodiments, cancers or tumors include breast cancer, lymphoma, multiple myeloma, and melanoma. 
     In general, the compounds of this invention will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. The actual amount of the compound of this invention, i.e., the nanoparticles, will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors well known to the skilled artisan. 
     An effective amount of such agents can readily be determined by routine experimentation, as can the most effective and convenient route of administration, and the most appropriate formulation. Various formulations and drug delivery systems are available in the art. See, e.g., Gennaro, A. R., ed. (1995)  Remington&#39;s Pharmaceutical Sciences,  18th ed., Mack Publishing Co. 
     An effective amount or a therapeutically effective amount or dose of an agent, e.g., a compound of the invention, refers to that amount of the agent or compound that results in amelioration of symptoms or a prolongation of survival in a subject. Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Agents that exhibit high therapeutic indices are preferred. 
     The effective amount or therapeutically effective amount is the amount of the compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Dosages may vary within this range depending upon the dosage form employed and/or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be chosen according to methods known in the art, in view of the specifics of a subject&#39;s condition. 
     Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects; i.e., the minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from, for example, in vitro data and animal experiments. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. 
     EXAMPLES 
     The present disclosure is illustrated using nanoparticles composed of albumin-bound paclitaxel (i.e., ABRAXANE®) or cisplatin as core, and bevacizumab (i.e., Avastin®) or Rituximab (i.e., Rituxan®) as antibodies. 
     One skilled in the art would understand that making and using the nanoparticles of the Examples are for the sole purpose of illustration, and that the present disclosure is not limited by this illustration. 
     Any abbreviation used herein, has normal scientific meaning. All temperatures are ° C. unless otherwise stated. Herein, the following terms have the following meanings unless otherwise defined: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 ABX = 
                 ABRAXANE ®/(albumin-bound 
               
               
                   
                   
                 paclitaxel 
               
               
                   
                 AC = 
                 cisplatin-bound ABX 
               
               
                   
                 ACN = 
                 acetonitrile 
               
               
                   
                 ADC = 
                 antibody dependent chemotherapy 
               
               
                   
                 BEV = 
                 bevacizumab 
               
               
                   
                 BSA = 
                 bovine serum albumin 
               
               
                   
                 dH 2 O = 
                 distilled water 
               
               
                   
                 DMEM = 
                 Dulbecco&#39;s Modified Eagle&#39;s 
               
               
                   
                   
                 Medium 
               
               
                   
                 nM = 
                 nanomolar 
               
               
                   
                 EdU = 
                 5-ethynyl-2′-deoxyuridine 
               
               
                   
                 EM = 
                 electron microscopy 
               
               
                   
                 FCB = 
                 flow cytometry buffer 
               
               
                   
                 FITC = 
                 Fluorescein 
               
               
                   
                 kD = 
                 kilo-dalton 
               
               
                   
                 Kd = 
                 dissociation constant 
               
               
                   
                 kg = 
                 kilogram 
               
               
                   
                 KV = 
                 kilo-volts 
               
               
                   
                 L/hr = 
                 liter/hour 
               
               
                   
                 LC-MS = 
                 liquid chromatography-mass 
               
               
                   
                   
                 spectrometry 
               
               
                   
                 M = 
                 molar 
               
               
                   
                 mCi = 
                 millicuries 
               
               
                   
                 mg = 
                 milligram 
               
               
                   
                 ml or mL = 
                 milliliter 
               
               
                   
                 m 2  = 
                 square meters 
               
               
                   
                 mm 3    
                 cubic millimeter 
               
               
                   
                 μg = 
                 microgram 
               
               
                   
                 μl = 
                 microliter 
               
               
                   
                 μm = 
                 micrometer/micron 
               
               
                   
                 PBS = 
                 Phosphate buffered saline 
               
               
                   
                 pK = 
                 pharmacokinetics 
               
               
                   
                 RT = 
                 room temperate 
               
               
                   
                 rpm = 
                 rotations per minute 
               
               
                   
                 v = 
                 volts 
               
               
                   
                 x g = 
                 times gravity 
               
               
                   
                   
               
            
           
         
       
     
     Example 1: Nanoparticle Preparation 
     ABRAXANE® (ABX) (10 mg) was suspended in bevacizumab (BEV) (4 mg [160 μl] unless otherwise indicated), and 840 μl of 0.9% saline was added to give a final concentration of 10 mg/ml and 2 mg/ml of ABX and BEV, respectively. The mixture was incubated for 30 minutes at room temperature (or at the temperature indicated) to allow particle formation. For Mastersizer experiments to measure particle size of ABX:BEV complexes, 10 mg of ABX was suspended in BEV at concentrations of 0 to 25 mg/ml. Complexes of ABX with rituximab (0-10 mg/ml) or trastuzumab (0-22 mg/ml) were formed under similar conditions. 
     For use in humans, the ABX:BEV complexes may be prepared by obtaining the dose appropriate number of 4 mL vials of 25 mg/mL BEV and diluting each vial per the following directions to 4 mg/mL. The dose appropriate number of 100 mg vials of ABX can be prepared by reconstituting to a final concentration containing 10 mg/mL ABX nanoparticles. Using a sterile 3 mL syringe, 1.6 mL (40 mg) of bevacizumab (25 mg/mL) can be withdrawn and slowly injected, over a minimum of 1 minute, onto the inside wall of each of the vials containing 100 mg of ABX. The bevacizumab solution should not be injected directly onto the lyophilized cake as this will result in foaming. Then, using a sterile 12 mL sterile syringe, 8.4 mL 0.9% Sodium Chloride Injection, USP, can be withdrawn and slowly injected, over a minimum of 1 minute, 8.4 mL onto the inside wall of each vial containing ABX 100 mg and BEV 40 mg. Once the addition of BEV 1.6 mL and 0.9% Sodium Chloride Injection, USP 8.4 mL is completed, each vial can be gently swirled and/or inverted slowly for at least 2 minutes until complete dissolution of any cake/powder occurs. Generation of foam should be avoided. At this point, the concentration of each vial should be 100 mg/10 mL ABX and 40 mg/10 mL BEV. The vials containing the ABX and BEV should sit for 60 minutes. The vial(s) should be gently swirled and/or inverted every 10 minutes to continue to mix the complex. After 60 minutes has elapsed, the calculated dosing volume of ABX and BEV should be withdrawn from each vial and slowly added to an empty viaflex bag. An equal volume of 0.9% Sodium Chloride Injection, USP is then added to make the final concentration of ABX 5 mg/mL and BEV 2 mg/mL. The bag should then be gently swirled and/or inverted slowly for 1 minute to mix. The ABX:BEV nanoparticles can be stored for up to 4 hours at room temperature following final dilution. 
     Example 2: Binding of ABX and BEV In Vitro 
     To determine whether ABX and BEV interact, the nanoparticles formed in Example 1 were analyzed by flow cytometry and electron microscopy. 
     Methods 
     Flow Cytometry: 
     AB160 was produced as described in Example 1 above. To determine binding of BEV to ABX, visualization of AB160 was performed on an Accuri C6 flow cytometer (BD Franklin Lakes, N.J.) and data analysis was done using Accuri C6 software. Biotinylated (5 μg) goat anti-mouse IgG (Abeam, Cambridge, Mass.) was labeled with 5 μg of streptavidin PE (Abeam, Cambridge, Mass.). The goat anti-mouse IgG was chosen to label AB160 because the Fab portion of the BEV is mouse derived. ABX and AB160 were incubated with the PE-labeled goat anti-mouse IgG for 30 minutes at room temperature, washed and visualized by flow cytometry. 
     Electron Microscopy: 
     Five μl ABX, dissolved in PBS at 6 mg/ml, was added to a 300-mesh parlodian-carbon coated copper grid and allowed to sit for 1 minute. A pointed piece of filter paper was touched to the drop to remove excess liquid, leaving a thin film on the grid. The grids were allowed to dry. To dissolve the buffer crystals left on the dried grid, the sample was washed three times in dH 2 0. A small drop of 1% phosphotungstic acid (PTA), pH 7.2, was added to the grid. The grid was then again touched by a pointed piece of filter paper to remove excess liquid, leaving a thin film on the grid and allowed to dry. BEV (Genentech) at 25 mg/ml in 0.9% sodium chloride solution was diluted with PBS at 1:10 ratio. Five μl of BEV was loaded on nickel formvar-coated grid and allowed to air dry for 30 minutes to 1 hour. For the AB160, 10 mg/ml ABX, dissolved in PBS, and 4 mg/ml BEV, in 0.9% sodium chloride solution, were mixed at 2.5:1 ratio. The complex was further diluted with PBS at 1:5. Five μl of the complex was loaded on nickel formvar-coated grid and air dried for 30 minutes to 1 hour. Both samples were incubated for 1 hour in goat anti-mouse IgG with 6 nm gold-conjugated particles (Electron Microscopy Sciences), diluted 1:30 with 10% FCB/PBS, washed 6 times with PBS (each 2 minutes), 6 times with dH 2 0, then stained with the mixture of 2% methylcellulose and 4% UA (9:1) for 5 minutes. Filter paper was used to drain the stain and the grid was air dried for 1 hour. Both samples were incubated overnight in donkey anti-mouse IgG with 6 nm gold-conjugated particles (Jackson ImmunoResearch) diluted 1:25 with 10% FCB/PBS, washed 6 times with PBS (each 2 minutes), 6 times with dH 2 0 water, stained with 1% PTA for 5 minutes, air dried, covered with 2% methylcellulose, and air dried for 1 hour. The micrographs were taken on a JEOL 1400 at operating at 80 KV. 
     Results 
     ABX (10 mg/ml) was co-incubated with 4 mg/ml BEV in vitro and found that they formed 160 nm nanoparticles (referred to herein as AB160). Because the Fab portion of the IgGI (BEV) is of mouse origin, particles containing BEV were selectively labeled with purified goat anti-mouse IgG followed by anti-goat PE as a secondary antibody. As a negative control, samples were stained with the anti-goat PE only. Particles were visualized by flow cytometry and demonstrated a bright signal of anti-mouse IgGI binding to AB160 (41.2% positive) relative to ABX (6.7% positive) alone ( FIG.  1 A ). To validate binding of BEV to ABX, the BEV were labeled with gold-labeled mouse anti-human IgG and the particles were visualized with electron microscopy ( FIG.  1 B ). Surprisingly, the EM pictures suggest a monolayer of BEV surrounding ABX nanoparticles. 
     To determine what protein (albumin or BEV) the paclitaxel remains bound to when the complex breaks down, AB160 were made and collected fractions: the particulate (nanoAB160), proteins greater than 100 kD and proteins less than 100 kD. Paclitaxel was measured in each fraction by liquid chromatography-mass spectrometry (LC-MS). Roughly 75% of the paclitaxel remained within the particulate, and the majority of the remaining paclitaxel was associated with the fraction containing proteins 100 kD or greater ( FIG.  1    C, top), suggesting that when the particulate dissociates the paclitaxel is bound to BEV alone or a BEV and albumin heterodimer. This indicates that the dissociated complexes contain the chemotherapy drug with the antibody, which would still traffic to the high-VEGF tumor microenvironment. These findings were confirmed by Western blot analysis of the supernatants from AB160, which showed that BEV and paclitaxel co-localize at approximately 200 kD, a size consistent with a paclitaxel-BEV-albumin protein complex ( FIG.  1 C , bottom). 
     Example 3: Function of AB160 In Vitro 
     Confirmation that the two key elements in the complexes, the antibody and the paclitaxel, retained their function when present in the complexes was demonstrated. 
     Methods 
     In Vitro Toxicity: 
     The A375 human melanoma cell line (ATCC Manassas, Va.) and Daudi B-cell lymphoma line (ATCC Manassas, Va.) were cultured in DMEM with 1% PSG and 10% FBS. Cells were harvested and plated at 0.75×10 6  cells per well in 24 well plates. Cells were exposed to ABX or AB160 at paclitaxel concentrations from 0 to 200 μg/ml overnight at 37° C. and 5% CO 2 . To measure proliferation, the Click-iT EdU (Molecular Probes, Eugene, Oreg.) kit was utilized. Briefly, 10 mM EdU was added to the wells and incubated overnight with the cells and ABX or AB160. The cells were permeabilized with 1% saponin and intercalated EdU was labeled with a FITC-conjugated antibody. The proliferation index was determined by dividing the FITC positive cells from each treatment by the maximum proliferation of untreated EdU labeled cells. 
     VEGF ELISA: 
     To determine whether BEV can still bind its ligand, VEGF, when bound to ABX, a standard VEGF ELISA (R and D Systems, Minneapolis, Minn.) was employed. AB160 was prepared as described and 2000 μg/ml VEGF was added to the AB160 complex or ABX alone. The VEGF was incubated with the nanoparticles for 2 hours at room temperature. The suspension was spun at 6000 rpm for 15 minutes, supernatants were collected and free VEGF was measured by ELISA. Briefly, ELISA plates were coated with capture antibody overnight at 4° C. Plates were washed, blocked and standards and samples were added. After washing, detection antibody was added and plates were developed with substrate (R and D Systems, Minneapolis, Minn.). Absorbance was measured at 450 nm using a Versamax ELISA plate reader (Molecular Devices, Sunnyvale, Calif.). The concentration of unbound VEGF was determined with a standard curve from 0 to 2000 μg/ml. 
     Results 
     AB160 has similar toxicity relative to ABX alone in an in vitro toxicity assay with the human melanoma cell line, A375, suggesting that the paclitaxel functions equally in either formulation ( FIG.  1    D). 
     To test the binding of VEGF to BEV in the AB160 complex, AB160 or ABX was co-incubated with VEGF, the particulate removed, and the supernatant tested for VEGF content. The lack of VEGF in the supernatant measured from AB160 (&lt;10% VEGF unbound) indicated that the VEGF was bound by the BEV in the AB160 complex, while it remained free when incubated with the ABX (&gt;80% VEGF unbound) alone ( FIG.  1    E). 
     Importantly, these assays demonstrated that the paclitaxel in AB160 retains its toxicity to tumor cells and the bound BEV maintains the ability to bind its ligand, VEGF. 
     Example 4: Particle Size and Protein Affinity 
     To understand the characteristics of the nanoparticles formed when binding BEV to ABX, the size of the ABX:BEV complexes was determined relative to ABX. 
     Methods 
     Mastersizer and Nanosight: 
     The particle size of ABX and antibody-ABX drug complexes were measured by dynamic light scattering on a Mastersizer 2000 (Malvern Instruments, Westborough, Mass.). To measure particle size, 2 ml (5 mg/ml) of ABRAXANE® or complex was added to the sample chamber. Data were analyzed with Malvern software and particle size distributions were displayed by volume. The particle sizes and stability were later validated using the Nanosight System (Malvern Instruments, Westborough, Mass.). The ABX or complex particles were diluted to the appropriate range to accurately measure particle sizes. Data was displayed by particle size distribution; however, the nanoparticle tracking analysis uses Brownian motion to determine particle size. 
     Binding Assay: 
     Biotinylated BEV, rituximab or trastuzumab at 100 μg/ml was bound to the streptavidin probe (ForteBio Corp. MenloPark, CA). The binding of ABX was measured by light absorbance on the BLitz system (ForteBio Corp. MenloPark, CA) at 1000, 500 and 100 mg/ml. The association and dissociation constants were calculated using the BLItz software. 
     Bio-Layer Interferometry (BLItz) technology was utilized to assess the binding affinity of BEV to ABX. Biotinylated BEV was bound to the streptavidin probe and exposed to ABX (1000, 500, and 100 μg/ml). The dissociation constant (K d ) of BEV and ABX is 2.2×10 −8  M at room temperature and pH 7, consistent with a strong non-covalent interaction. The binding affinity of BEV and ABX is within the range of dissociation constants observed between albumin and natural or engineered albumin-binding domains of some bacterial proteins. Nilvebrant, J. et al. (2013)  Comput Struct Biotechnol J  6:e201303009. 
     Results 
     ABX:BEV nanoparticles were consistently larger (approximately 160 nm) than the 130 nm ABX alone ( FIG.  2 A ). The size of the nanoparticle created directly correlated to the concentration of BEV used, with median sizes ranging from 0.157 to 2.166 μm. ( FIG.  2 A ). With the goal of these studies being a Phase I clinical trial, the smallest ABX:BEV particle (AB160) were focused on because it is the most similar to the 130 nm ABX. The size of the AB160 particle was consistent with ABX plus a monolayer of BEV surrounding it and with the EM image of the particle (see  FIG.  1 B ). 
     To determine whether intravenous administration conditions affect nanoparticle size distributions, the particle size distributions of AB160 (or ABX) incubated in saline for up to 24 hours at room temperature were evaluated. AB160 size distribution does not significantly change for up to 24 hours ( FIGS.  9 A and  9 B ). However, by 4 hours at room temperature, there is some evidence of AB160 breakdown by ELISA ( FIG.  9 C ). 
     To determine the stability of AB160 in plasma, ABX or AB160 was incubated in saline or heparinized human plasma at relative volume ratios of 9:1 or 1:1. Notably, no particles (0.01 to 1 μm) were detected when either ABX ( FIG.  10   , top panel) or AB160 ( FIG.  10   , bottom panel) is incubated in plasma at equal volumes (1:1). 
     Western blot (data not shown) indicated that, in saline or heparinized human plasma, the AB160 dissociated into smaller protein conjugates that still contain the tumor-targeting antibody, albumin and the cytotoxic agent, paclitaxel. These protein conjugates retain their ability to target the tumor and, once at the tumor site, can quickly dissolve and release the cytotoxic payload to effectively initiate tumor regression without internalization of the entire nanoparticle by tumor cells. 
     Next, the ABX was suspended in BEV and the mixture diluted with saline at pH 3, 5, 7, or 9 prior to incubation at various temperatures (RT, 37° C. and 58° C.) to allow particle formation in order to test whether binding affinity was pH- and/or temperature-dependent. The binding affinity of ABX and BEV is both pH- and temperature-dependent, with the highest binding affinity observed when the particles are formed at pH 5 and 58° C. ( FIG.  2 B ). 
     To determine if the higher affinity binding of BEV and ABX at 58° C. and pH 5 translated into stability of the complex, various preparations were compared by nanoparticle tracking analysis (Nanosight). The stability of AB160 prepared at 58° C. and pH 5 (AB1600558), room temperature and pH 7 (AB16007), or 58° C. and pH 7 (AB1600758) was compared to ABX exposed to the same conditions (ABX0558, ABX07, and ABX0758, respectively) after incubation in human AB serum for 0, 15, 30, or 60 minutes. 
     The particles made under higher affinity conditions (pH 7 and 58° C.) were also more stable, as indicated by the number of particles present per mg ABX after exposure to human AB serum. The AB160 particles exhibited increased stability in human serum that correlated with their binding affinities. In particular, AB16007 and AB1600558 were more stable in both saline and human serum than ABX alone, as determined by size and number of particles measured per mg ABX ( FIG.  2 C  and Table 3). This shows that the stability of AB160 particles can be manipulated by changing the conditions under which the AB160 particles are formed. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Stability of AB160 and ABX in human AB serum 
               
            
           
           
               
               
               
            
               
                   
                   
                 Human AB Serum 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Saline 
                 0 min 
                 15 min 
                 30 min 
                 60 min 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 ABX07 
                 221.5 
                 54.4 
                 85.2 
                 84 
                 32.1 
               
               
                 AB16007 
                 2500 
                 516 
                 508 
                 756 
                 296 
               
               
                 ABX0758 
                 236 
                 182.4 
                 155.4 
                 54 
                 66 
               
               
                 AB1600758 
                 2460 
                 436 
                 236 
                 260 
                 176 
               
               
                 ABX0558 
                 348 
                 510 
                 86.8 
                 90 
                 64 
               
               
                 AB1600558 
                 7296 
                 2200 
                 1224 
                 1080 
                 960 
               
               
                   
               
            
           
         
       
     
     Particles per mg ABX×10 −8    
     These data demonstrated that BEV binds to ABX with affinity in the picomolar range, indicating a strong non-covalent bond, and demonstrated a particle size distribution consistent with ABX surrounded by a monolayer of antibody molecules; the size of the particles created is dependent on the antibody concentration. 
     Example 5: Efficacy of AB160 in Mice 
     A xenograft model of A375 human melanoma cells implanted into athymic nude mice was employed to test AB160 efficacy in vivo. 
     Methods 
     In vivo experiments were performed at least 2 times. The number of mice required for those experiments was determined by power analysis. Mouse tumors were measured 2-3 times/week and mice were sacrificed when the tumor was 10% by weight. Mice that had complete tumor responses were monitored for 60-80 days post-treatment. The end point of the mouse studies was median survival. Kaplan-Meier curves were generated and Mantle-Cox test was performed to determine significance of median survival between treatment groups. The in vitro results presented are representative of at least 5 repeated experiments. Statistical analyses of in vitro and in vivo percent change from baseline experiments were done using the Student&#39;s t-test. 
     Mouse Model: 
     To test tumor efficacy, 1×10 6  A375 human melanoma cells were implanted into the right flank of athymic nude mice (Harlan Sprague Dawley, Indianapolis, Ind.). When the tumors had reached a size of about 700 mm 3 , the mice were randomized and treated with PBS, ABX (30 mg/kg), BEV (12 mg/kg), BEV followed by ABX, or AB160 at the above concentrations. For the mouse experiments testing bigger AB particles, the highest dose of BEV (45 mg/kg) necessary to create the larger particles was used in the BEV-only treatment group. Tumor size was monitored 3 times/week and tumor volume was calculated with the following equation: (length×width 2 )/2. Mice were sacrificed when the tumor size equaled 10% of the mouse body weight or about 2500 mm 3 . The day 7 percent change from baseline was calculated as follows: [(tumor size on treatment day-tumor size on day 7)/tumor size on treatment day]×100. The in vivo testing of the AR160 was similar except 5×10 6  Daudi cells were injected into the right flank of athymic nude mice. 
     Results 
     AB160 was tested relative to PBS, the single drugs alone, and the drugs administered sequentially. Mice treated with AB160 had significantly reduced tumor size compared to all other treatment groups (p=0.0001 to 0.0089) at day 7 post-treatment, relative to baseline ( FIG.  3 A ). Tumors in all of the mice treated with AB160 had regressed at day 7, and this tumor response translated into significantly increased median survival of the AB160 group relative to all other groups ( FIG.  3 B ), with a survival of 7, 14, 14, 18 and 33 days for the PBS (p&lt;0.0001), BEV (p=0.003), ABX (p=0.0003), BEV+ABX (p=0.0006) and AB160 groups, respectively. 
     It is likely that larger tumors have a higher local VEGF concentration. When data were analyzed based on the size of the tumor on day of treatment (&lt;700 mm 3  and &gt;700 mm 3 ), the larger tumors were shown to have a greater response to AB160, suggesting that higher tumor VEGF concentration attracts more BEV-targeted ABX to the tumor. The difference in the percent change from baseline was significant for the AB160 groups (p=0.0057). This observation was not seen in the ABX only (p=0.752) group, where the ABX has no targeting capability ( FIG.  3 C ). 
     Particles of increasing size were prepared using increasing BEV:ABX ratios as shown in  FIG.  2 A . Tumor regression and median survival positively correlated with increasing particle size, indicating that biodistribution of larger particles may be altered relative to the smaller ones ( FIGS.  3 D and  3 E ). Full toxicity studies were performed on the mice and no toxicities were noted. 
     Example 6: Paclitaxel Pharmakokinetics in Mice 
     To compare the pharmacokinetics (pk) of AB160 and ABX, plasma paclitaxel concentrations were measured in mice administered AB160 or ABX at 0, 4, 8, 12 and 24 hours. 
     Methods 
     Paclitaxel Pharmacokinetics: 
     The liquid chromatographic separation of paclitaxel and d5 paclitaxel were accomplished using an Agilent Poroshell 120 EC-C18 precolumn (2.1×5 mm, 2.7 μm, Chrom Tech, Apple Valley, Minn.) attached to an Agilent Poroshell 120 EC-C18 analytical column (2.1×100 mm, 2.7 μm Chrom Tech, Apple Valley, Minn.) at 40° C., eluted with a gradient mobile phase composed of water with 0.1% formic acid (A) and ACN with 0.1% formic acid (B) with a constant flow rate of 0.5 ml/minute. The elution was initiated at 60% A and 40% B for 0.5 minutes, then B was linearly increased from 40-85% for 4.5 minutes, held at 85% B for 0.2 minutes, and returned to initial conditions for 1.3 minutes. Autosampler temperature was 10° C. and sample injection volume was 2 μl. Detection of paclitaxel and the internal standard d5-paclitaxel were accomplished using the mass spectrometer in positive ESI mode with capillary voltage 1.75 kV, source temp 150° C. desolvation temp 500° C., cone gas flow 150 L/hr, desolvation gas flow 1000 L/hr, using multiple reaction monitoring (MRM) scan mode with a dwell time of 0.075 seconds. The cone voltages and collision energies were determined by MassLynx-Intellistart, v4.1, software and varied between 6-16 V and 12-60 eV, respectively. The MRM precursor and product ions were monitored at m/z 854.3&gt;105.2 for paclitaxel and 859.3&gt;291.2 for d5 paclitaxel. The primary stock solutions of paclitaxel (1 mg/ml in EtOH) and d5 paclitaxel (1 mg/ml in EtOH) were prepared in 4 ml amber silanized glass vials and stored at −20° C. Working standards were prepared by dilution of the stock solution with ACN in 2 ml amber silanized glass vials and stored at −20° C. Plasma samples were extracted as follows, 100 μl plasma sample was added to a 1.7 ml microcentrifuge tube containing d5 paclitaxel (116.4 nM or 100 ng/ml) and 300 μl ACN, vortexed, incubated at room temperature for 10 minutes to precipitate proteins, and centrifuged (14,000 rpm) or 3 minutes. The supernatant was filtered on an Agilent Captiva ND lipids  plate (Chrom Tech, Apple Valley, Minn.), collected in a deep 96-well plate, and dried using nitrogen gas. The samples were reconstituted using 100 μl ACN and shaken on a plate shaker (high speed) for 5 minutes. Plasma standard curves were prepared daily containing paclitaxel (0.59-5855 nM or 0.5-5000 ng/ml) and d5 paclitaxel (116.4 nM) for paclitaxel quantitation. Mouse tumors were thawed on ice, weighed, and diluted 2 parts (weight to volume) in 1×PBS. Tumors were then homogenized using a PRO200 tissue homogenizer using the saw tooth probe (5 mm×75 mm). Tumor homogenate was than processed the same as the human plasma samples. 
     Mouse Imaging: 
     Avastin and IgG control solutions were prepared and 1-125 labeled per protocol (Imanis Life Sciences). Briefly, Tris Buffer (0.125 M Tris-HCl, pH 6.8, 0.15 M NaCl) and 5 mCi Na 125  I were added directly to iodination tubes (ThermoFischer Scientific, Waltham, Mass.). The iodide was allowed to activate and was swirled at room temperature. Activated iodide was mixed with the protein solution. 50 μl of Scavenging Buffer (10 mg tyrosine/mL in PBS. pH 7.4) was added and incubated for five minutes. After addition of Tris/BSA buffer and mixing, samples were applied in 10 K MWCO dialysis cassettes against pre-cooled PBS for 30 minutes, 1 hour, 2 hours, and overnight at 4° C. Radioactivity was determined by Gamma counter, then disintegrations per minute (DPM) and specific activity were calculated. Mice were injected in their tail vein with Avastin I-125, ABRAXANE®-AVASTIN® I-125, ABRAXANE®-human IgG I-125, or ABRAXANE® only. Animals were imaged at 3, 10, 24 and 72 hours post-administration via SPECT-CT imaging using the U-SPECT-II/CT scanner (MILabs, Utrecht, The Netherlands). SPECT reconstruction was performed using a POSEM (pixelated ordered subsets by expectation maximization) algorithm. CT data were reconstructed during the Feldkamp algorithm. Co-registered images were further rendered and visualized using PMOD software (PMOD Technologies, Zurich. Switzerland). Animals were sacrificed and dissected at 72 hours post-injection. Selected tissues and organs of interest were measured using radioisotope dose calibrator (Capintec CRC-127R, Capintec Inc.). 
     Results 
     Results of the first pk experiment are provided in  FIGS.  4 A and  4 B . The area under the curve (AUC) and maximum serum concentration (C max ) were calculated in A375 tumor bearing and non-tumor bearing mice. In the first pk experiment the C max  and AUC were very similar in the non-tumor bearing mice for AB160 and ABX (63.3+/−39.4 vs. 65.5+/−14.4 and 129 vs. 133 μg/ml, respectively). However, in the tumor bearing mice, the C max  and AUC for the treatment groups were different (55.7+/−21.2 vs 63.3+/−17.3 and 112 vs 128 μg/ml, respectively) ( FIG.  4 C ). Although this difference was not statistically significant, it is consistent with superior targeting by AB160, relative to ABX. 
     A second pk experiment was performed with additional early time points and large versus small tumor sizes ( FIGS.  4 D- 4 F ). The results of this experiment demonstrated smaller AUC in tumor bearing mice relative to non-tumor bearing mice, with the lowest blood values of paclitaxel in the large tumor mice relative to the small tumor mice (80.4+/−2.7, 48.4+/−12.3, and 30.7+/−5.2 for ABX-treated non-tumor, small tumor and large tumor bearing mice, respectively: 66.1+/−19.8, 44.4+/−12.1 and 22.8+/−6.9 for AB1160-treated). Similarly, the C max  dropped in both treatment groups in mice with larger tumors (47.2, 28.9 and 19.7 μg/ml for ABX and 40.1, 26.9 and 15.3 μg/ml for AB160) ( FIG.  4 G ). The AUC and C max  of paclitaxel in blood were lower in AB160-treated mice relative to ABX-treated mice. Although not statistically significant, this data is further consistent with higher deposition of paclitaxel in the tumors treated with AB160. 
     To directly test this hypothesis, tumor paclitaxel concentrations by LC-MS were measured. The tumor paclitaxel concentration was significantly higher in tumors treated with AB160 relative to ABX at the 4 hour (3473 μg/mg of tissue+/−340 vs 2127 μg/mg of tissue+/−3.5; p=0.02) and 8 hour (3005 μg/mg of tissue+/−146 vs 1688 μg/mg of tissue+/−146; p=0.01) time points, suggesting paclitaxel stays in the tumor longer when targeted by the antibody ( FIG.  4 H ). This explains the blood pk and is consistent with redistribution of drug to tissues including the tumor. 
     Live in vivo imaging of I-125 labeled AB160 (Abx-AvtI125) and IgG isotype bound ABX (Abx-IgGI125) confirmed the results of the LC-MS, with higher levels of I-125 in the tumor of mice treated with AB160 relative to IgG-ABX at 3 hours (32.2 uCi/g+/−9.1 vs 18.5 uCi/g+/−1.65; p=0.06) and 10 hours (41.5 uCi/g+/−6.4 vs 28.7 uCi/g+/−2.66: p=0.03) post injection ( FIGS.  4 I and  4 J ). Taken together, these data demonstrate that binding BEV to ABX alters blood pk, and this alteration is due to a redistribution of the drug to the tumor tissue as shown by both LC-MS of paclitaxel and I-125 labeling of BEV relative to an isotype matched IgGl. 
     Without being bound by theory, it is believed that by binding a tumor-targeted antibody to ABX, the pk is altered more dramatically than ABX alone, lowering the C max , and AUC in the blood because of redistribution of AB160 to the tumor tissue. These results from mouse blood paclitaxel pk, tumor tissue levels of paclitaxel, and I-125 radioactivity levels in mice treated with AB160 relative to ABX alone suggest that antibody targeting of the ABX alters biodistribution of paclitaxel such that increased levels reach the tumor and are retained there for a longer period of time, yielding enhanced tumor regression. 
     Example 7: Binding of Other Therapeutic Antibodies 
     The binding of the anti-human CD20 antibody (rituximab) and the anti-HER2/neu receptor antibody (trastuzumab) to ABX was tested to determine if other IgG therapeutic antibodies also exhibit binding to ABX when combined ex vivo. 
     Methods 
     Nanoparticles containing rituximab or trastuzumab were prepared and tested as described in the above examples. 
     Results 
     The particle size of the complexes with both BEV and trastuzumab (HER) were very similar, with average sizes ranging from 0.157 to 2.166 μm ( FIG.  2 A ) and 0.148 to 2.868 μm ( FIG.  5 B ), respectively. In contrast, particles formed with rituximab became much larger at lower antibody:ABX ratios, with particle sizes ranging from 0.159 to 8.286 μm ( FIG.  5 A ). 
     The binding affinities of rituximab and trastuzumab with ABX were determined by BLitz under variable pH. Both antibodies bind with relatively high affinity in the picomolar range ( FIG.  5 C ). The rituximab affinity to ABX decreased with higher pH, but trastuzumab affinity to ABX was unaffected by pH ( FIG.  5 C ). 
     The efficacy of the 160 nm particle made with rituximab (AR160) was tested in vitro and in vivo. In vitro, the B-cell lymphoma cell line Daudi was treated with AR160, ABX, or rituximab alone at increasing concentrations (0 to 200 μg/ml) of paclitaxel. AR160 (IC 50 =10 μg/ml) significantly inhibited proliferation of Daudi cells treated for 24 hours (p=0.024) compared to either ABX (IC 50 &gt;200 μg/ml) or rituximab (IC 50 &gt;200 μg/ml) alone ( FIG.  6 A ). 
     In vivo, a xenotransplant model of Daudi cells was established in athymic nude mice. Once tumors were established, mice were treated with PBS, ABX, rituximab, ABX and rituximab given sequentially, or AR160. On day 7 post treatment, tumors were measured and the percent change in tumor size from baseline was calculated. AR160-treated tumors regressed or remained stable, while tumors in all other treatment groups progressed ( FIG.  6 B ). The percent change from baseline tumor size in the AR160 group compared to all other groups was significant (p&lt;0.0001). The mice treated with AR160 had a significantly longer median survival of greater than 60 days compared to 12, 16, and 12 days for mice treated with PBS (p&lt;0.0001), ABX (p&lt;0.0001), or rituximab (p=0.0002), respectively ( FIG.  6 C ). However, the difference in median survival was not significant between AR160 and the sequentially treated groups (p=0.36). This may be because the rituximab binds to the tumor cells and remains on the cell surface, allowing the subsequently-administered ABX to bind to the antibody when it enters the tumor site, unlike BEV which binds a soluble target and not a cell surface marker. 
     Example 8: Binding of Other Chemotherapy Drugs to AB160 
     The efficacy of other chemotherapy drugs to form functional nanoparticles was evaluated. 
     Methods 
     Nanoparticles containing cisplatin were prepared and tested as described in the above examples. 
     Results 
     To test if another chemotherapy drug could bind to the AB160 particles, cisplatin and ABX were co-incubated and the amount of free cisplatin remaining in the supernatant was measured by HPLC. Approximately 60% (i.e., only 40% remains in the supernatant) of the cisplatin bound to the ABX ( FIG.  7 A ). 
     Next, tumor toxicity of AC relative to ABX and cisplatin alone was tested using A375 cells. The complexes were centrifuged to remove highly toxic unbound cisplatin, and reconstituted in media to ensure that any additional toxicity of AC relative to ABX is due only to ABX-bound cisplatin. For parity, the ABX only was centrifuged in a similar manner. AC (IC 50 =90 μg/ml) inhibited proliferation of A375 cells to a greater extent than ABX alone (IC 50 &gt;1000 μg/ml) ( FIG.  7 B ). The diminished toxicity in this experiment relative to other toxicity experiments is due to some loss of drug in the centrifugation step, but the comparison of ABX to AC remains relevant. 
     To determine the tumor toxicity of cisplatin-containing AB160 complexes, AB160 was co-incubated with cisplatin to form cisplatin containing particles (ABC complex). The ABC complex was tested in the A375 melanoma xenotransplant model relative to each drug alone and AB160. Tumors treated with AB160, AB160+cisplatin given sequentially, and the ABC complex all showed regression in tumor size at 7 days post treatment ( FIG.  7 C ), but the ABC complex conferred the longest median survival (35 days, relative to AB160 and AB160+cisplatin at 24 and 26 days, respectively). Although the difference was not statistically significant (p=0.82 and 0.79) ( FIG.  7 D ), the data is consistent with benefits of the ABC complex to long-term survival rates. 
     These data demonstrated that the albumin portion of the ABX provides a platform for other therapeutic antibodies to bind, such as rituximab and trastuzumab, as well as other chemotherapy agents (e.g., cisplatin), which all had similar efficacy in vitro and in vivo as AB160. 
     Together these data demonstrate a simple way to construct a versatile nano-immune conjugate, which allows multiple proteins or cytotoxic agents to be bound to a single albumin scaffold. Improved efficacy of the targeted drug relative to the single agents alone was demonstrated in the mouse model, which is at least in part due to altered pk of the antibody-targeted drug. Furthermore, without being bound by theory, it is believed that the versatility of the presently disclosed nano-immune conjugate that does not require a linker or target cell internalization will overcome the obstacles faced by other nanomedicines in translating results from mice to humans. 
     Example 9: Lyophilization of AB160 
     AB160 was synthesized by adding 8 mg (320 μl) of bevacizumab to 20 mg of ABRAXANE®. 1.66 ml of 0.9% saline was then added for a final volume of 2 ml for a final concentration of 4 mg/ml bevacizumab and 10 m g/ml ABRAXANE®, and the mixture was allowed to incubate at room temperature for 30 minutes in a 15 ml polypropylene conical tube. 
     After the 30 minute room temperature incubation, the mixture was diluted 1:2 in 0.9% saline to 2 mg/ml and 5 mg/ml bevacizumab and ABRAXANE®, respectively. These are the concentrations of the 2 drugs when prepared by the pharmacy for administration to patients. 
     AB160 was divided into twenty 200 μl 1 aliquots in 1.5 ml polypropylene eppendorfs and frozen at −80° C. 
     Once frozen, the aliquots were lyophilized overnight with the Virtis 3L benchtop lyophilizer (SP Scientific, Warmister, Pa.) with the refrigeration on. A lyophilized preparation was generated. 
     The dried aliquots were stored at room temperature in the same 1.5 ml polypropylene eppendorfs. These samples were readily reconstituted in saline at room temperature for 30 minutes, followed by centrifugation for 7 minutes at 2000×g. The resulting sample was then resuspended in the appropriate buffer, as needed. 
     By comparison, a sample that was dried with heat and a speed vacuum was impossible to reconstitute. 
     Example 10: Testing of Lyophilized Preparations 
     Samples were reconstituted at different time points after lyophilization and tested for their physical properties against ABX, and freshly made AB160. 
     Particle size distribution was evaluated as described above. 
     VEGF binding was evaluated by incubation of the sample with VEGF for 2 hours at room temperature, centrifuged at 2000×g for 7 minutes. The amount of VEGF bound to the pellet (corresponding to the nanoparticles) or remaining in the supernatant was measured with ELISA. 
     Paclitaxel activity was assessed by cytotoxicity against A375 cells in vitro. 
     Surprisingly, lyophilization did not significantly affect either the particle size, VEGF binding, or the activity of paclitaxel as shown by the ability to inhibit cancer cell proliferation. This result held for lyophilized samples stored for 1 month ( FIGS.  8 A- 8 C ) or 10 months ( FIGS.  8 D- 8 F ). 
     Further surprising is that these results were observed with nanoparticles lyophilized without the use of cryoprotectants or other agents that may adversely affect human therapeutic use. 
     Example 11: Efficacy of AB160 in Humans 
     AB160 was tested in a phase 1, first-in-man, clinical trial testing the safety of AB160 administered to patients with metastatic malignant melanoma that have failed prior therapies. The study utilizes a classical 3+3, phase 1 clinical trial design, testing 3 different doses of AB160 in the following schema: 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Dose 
                 AB-complex Both drugs MUST be reduced 
               
            
           
           
               
               
               
            
               
                 Level 
                 ABX dose 
                 Accompanying BEV dose 
               
               
                   
               
               
                 3 
                 175 mg/m 2   
                 70 mg/m 2   
               
               
                 7 
                 150 mg/m 2   
                 60 mg/m 2   
               
               
                  1* 
                 125 mg/m 2   
                 50 mg/m 2   
               
               
                 −1  
                 100 mg/m 2   
                 40 mg/m 2   
               
               
                 −2  
                  75 mg/m 2   
                 30 mg/m 2   
               
               
                   
               
               
                 *Dose level 1 refers to the starting dose. 
               
            
           
         
       
     
     The doses were selected relevant to doses of ABRAXANE® currently used in clinical practice. AB160 was made prior to each treatment dose. Treatments were administered as a 30 minute intravenous infusion on days 1, 8 and 15 of a 28-day treatment cycle. Treatments were continued until intolerable toxicity, tumor progression or patient refusal. Prior to every treatment cycle, patients were evaluated for toxicity; tumor evaluations were performed every other cycle (RECIST). 
     The study is accompanied by formal (in-patient) pharmacokinetic studies associated with dose 1 of cycles 1 and 2 of therapy. 
     Five patients have been administered AB160, at 100 mg/m 2  of ABX and 40 mg/m 2  of BEV, of which four have been analyzed. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Disease course in Phase I study* 
               
               
                 Disease Course: Dose Level 100 mg/m 2   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 off, 
                 follow- 
               
               
                   
                 number 
                   
                   
                 treatment 
                 up 
               
               
                 Patient 
                 of cycles 
                 response 
                 PFS time 
                 reasons 
                 time 
               
               
                   
               
               
                 1 
                 8 
                 stable 
                 238 
                 off, 
                 444+ 
               
               
                   
                   
                   
                   
                 progression 
                   
               
               
                 2 
                 6 
                 stable 
                  400+ 
                 off, toxicity 
                 400+ 
               
               
                 3 
                 1 
                 — 
                  182+ 
                 off, toxicity 
                 182+ 
               
               
                 4 
                 6 
                 stable 
                 181 
                 off, 
                 203+ 
               
               
                   
                   
                   
                   
                 progression 
               
               
                   
               
               
                 *Information as provided in application no. PCT/US2015/054295 filed Oct. 6, 2015. All patients are still living. 
               
            
           
         
       
     
     PFS refers to median progression free survival, i.e. the number of days of treatment before the cancer recurred. Adverse events are listed below. There was no dose limiting toxicity (DLT), i.e. the adverse events were not linked to the dose of AB160. More detail is provided in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Adverse events in Phase I study 
               
            
           
           
               
               
               
            
               
                 patient 
                 toxicity 
                 DLT 
               
               
                   
               
               
                 1 
                 grade 2 lymphopenia 
                 NO 
               
               
                 7 
                 grade 3 neutropenia and 
                 NO 
               
               
                   
                 leukopenia grade 2 
                   
               
               
                 3 
                 grade 2 colonic perforation, fatigue, and 
                 NO 
               
               
                   
                 blood bilirubin increase 
                   
               
               
                 4 
                 grade 2 neutropenia 
                 NO 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Treatment Course: Dose Level 100 m/m 2   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 number of 
                   
                   
                 number 
                 cycles 
                   
                   
               
               
                   
                 number of 
                 cycles 
                 cycles 
                 reasons 
                 of dose 
                 where dose 
                 reason 
                   
               
               
                   
                 cycles 
                 where day 
                 where day 
                 day 15 
                 reductions 
                 reduction 
                 for dose 
                   
               
               
                 patient 
                 administered 
                 15omitted 
                 15 omitted 
                 omitted 
                 taken 
                 taken 
                 reductions 
                 status 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 8 
                 0 
                   
                   
                 1 
                 4 
                 grd 2 sensory 
                 off, progression 
               
               
                   
                   
                   
                   
                   
                   
                   
                 neuropathy 
                   
               
               
                 2 
                 6 
                 3 
                 1, 2, 4 
                 grd 3 
                 2 
                 3, 5 
                 cycle 3: grade 
                 off toxicity 
               
               
                   
                   
                   
                   
                 neutropenia and 
                   
                   
                 3 neutropenia 
                 persistent grd 2 
               
               
                   
                   
                   
                   
                 leukopenia- all 
                   
                   
                 and leukopenia 
                 sensory 
               
               
                   
                   
                   
                   
                 3 cycles 
                   
                   
                 cycle 5: grade 3 
                 neuropathy 
               
               
                   
                   
                   
                   
                   
                   
                   
                 neutropenia, 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 leukopenia, and 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 fatigue and grd 2 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 sensory 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 neuropathy 
                   
               
               
                 3 
                 1 
                   
                   
                   
                   
                   
                   
                 off toxicity grd 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 2 colonic 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 perforation 
               
               
                 4 
                 6 
                   
                   
                   
                 2 
                 3, 5 
                 grd 2 sensory 
                 off, progression 
               
               
                   
                   
                   
                   
                   
                   
                   
                 neuropathy- both 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                 cycles 
               
               
                   
               
            
           
         
       
     
     The mean PFS was 7.6 months and the median was 7.0 months. 
     Comparison with Other Clinical Trials 
     The following table shows other published clinical studies for taxane therapy for metastatic melanoma. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Taxane therapy for metastatic melanoma 
               
            
           
           
               
               
               
               
               
            
               
                 Study or Author 
                 N 
                 Rx regimens 
                 PFS 
                 OS 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 HausChild 
                 135 
                 C = AUC 6 (q21) 
                 4.5 
                 10.5 
               
               
                   
                   
                 P = 225 mg/m2; D1 (q21) 
                   
                   
               
               
                 Flaherty 
                 411 
                 C = AUC 6 (q21) 
                 4.9 
                 11.3 
               
               
                   
                   
                 P = 225 mg/m2; D1 (q21) 
                   
                   
               
               
                 N057E 
                 41 
                 C = AUC2; D1, 8 15 (q28) 
                 4.5 
                 11.1 
               
               
                   
                 35 
                 A = 100 mg/m2; D1, 8, 15 (q28) 
                 4.1 
                 10.9 
               
               
                 N047A 
                 53 
                 C =AUC 6; D1 (q28) 
                 6.0 
                 12.0 
               
               
                   
                   
                 P = 80 mg/m2; D1, 8, 15 (q28) 
                   
                   
               
               
                   
                   
                 B = 10 mg/m2; D1, 15 (q28) 
                   
                   
               
               
                 BEAM 
                 71 
                 C = AUC5; D1 (q21) 
                 4.2 
                 8.6 
               
               
                   
                   
                 P = 175 mg/m2; D1 (q21) 
                   
                   
               
               
                   
                 143 
                 C = AUC5; D1 (q21) 
                 5.6 
                 12.3 
               
               
                   
                   
                 P = 175 mg/m2; D1 (q21) 
                   
                   
               
               
                   
                   
                 B = 15 mg/kg; 
                   
                   
               
               
                 N0775 
                 51 
                 C =AUC6 (5); D1 (q28) 
                 6.2 
                 13.9 
               
               
                   
                   
                 A = 100 (80) mg/m2; D1, 8, 15 
                   
                   
               
               
                   
                   
                 (q28) 
                   
                   
               
               
                 Spitler 
                 50 
                 A = 150 mg/m2; D1, 8, 15 (q28) 
                 7.6 
                 15.6 
               
               
                   
                   
                 B = 10g/kg; Dl, 15 (q28) 
               
               
                   
               
               
                 C = carboplatin, 
               
               
                 P = paclitaxel, 
               
               
                 A = nab-paclitaxel, 
               
               
                 B = bevacizumab 
               
               
                 References: 
               
               
                 Hauschild: Hauschild et al., (2009) J Clin Oneal .  27(17): 2823-30 
               
               
                 Flaherty: Flaherty et al., (2010) J Clin Oneal .  28: 15s (suppl; abstr 8511) 
               
               
                 N057E: Kottschade et al., (2010) Cancer 117(8): 1704-10 
               
               
                 N057A: Perez et al., (2009) Cancer 115(1): 119-27 
               
               
                 BEAM: Kim et al., (2012) J Clin Oneal .  30(1): 34-41 
               
               
                 N0775: Kottschade et al., (2013) Cancer 119(3): 586-92 
               
               
                 Spitler: Boasberg et al., (2011) J Clin Oneal .  29 (suppl; abstr 8543) 
               
            
           
         
       
     
     In the current trial, administration of AB160 particles is equivalent to a dose of 100 mg/m 2  of ABRAXANE®, and 40 mg/m 2  of bevacizumab. The only study that used BEV and ABX alone was Spitler. Spitler, however, used a higher dose of ABX. The present study also used less than 10% of the dose of BEV reported in previous studies, if the doses are adjusted to the average patient (assumed to have a surface area of 1.9 m 2  and a mass of 90 kg). 
     Spitler also examined patients who had not been previously treated, while the current study examined patients who had failed previous treatments. Ineffective prior treatment not only takes time from the expected PFS, but selects for cancer cells that are more resistant to treatment, and typically leaves a patient in poorer physical condition. Thus, the PFS for a population of patients on a “rescue” therapy (as here, with AB160) is expected to have a lower PFS than a naïve population. This can be seen in a Phase 2 clinical trial (Hersh et al.,  Cancer , January 2010, 116:155) that examined both rescue and naïve patients with ABRAXANE® alone. For previously treated patients with ABRAXANE® alone, the PFS was 3.5 months. Hersh et al.  Ann. Oncol  2015, (epub Sep. 26, 2015), reported a 4.8 month PFS for naïve patients treated with ABX alone. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Performance of AB160 in a limited study against published data 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 ABX dose in 
                 BEV dose in 
                   
               
               
                   
                 Prior  
                 average patient 
                 average patient 
                 PFS 
               
               
                 Study 
                 treatment 
                 (relative dose) 
                 (relative dose) 
                 (months) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 AB160 
                 Yes 
                 190 mg/patient 
                 76 mg/patient 
                 7.0 
               
               
                   
                   
                 (100 mg/m 2 ) 
                 (40 mg/m 2 ) 
                   
               
               
                 Spider 
                 No 
                 285 mg/patient 
                 900 mg/patient 
                 8.3 
               
               
                   
                   
                 (150 mg/m 2 ) 
                 (10 mg/kg) 
                   
               
               
                 Hersh 2010 
                 Yes 
                 190 mg/patient 
                 — 
                 3.5 
               
               
                   
                   
                 (100 mg/m 2 ) 
                   
                   
               
               
                 Hersh 2010 
                 No 
                 285 mg/patient 
                 — 
                 4.5 
               
               
                   
                   
                 (150 mg/m 2 ) 
                   
                   
               
               
                 Hersh 2015 
                 No 
                 285 mg/patient 
                 — 
                 4.8 
               
               
                   
                   
                 (150 mg/m 2 ) 
               
               
                   
               
            
           
         
       
     
     Thus, early results of the Phase I clinical trial with AB160 indicate an increase in PFS in late-stage metastatic malignant melanoma in previously treated patients. This increase is particularly surprising given that the PFS was greater than those in Spitler, who were chemotherapy naïve and were given a higher dose of ABRAXANE®, and an almost 12 fold higher dose of bevacizumab. The dose of BEV used in AB160 is far lower than any other study, so the best comparison is not Spitler, but Hersh. 
     Thus, the ABX/BEV complex (AB160) is superior to sequential administration of ABX and BEV, or ABX alone, and achieves this superior result with a very low effective dose of BEV. The data is therefore consistent with AB160 having improved targeting of the chemotherapeutics to the tumor, and that this targeting is mediated by BEV. It is possible that the ABX nanoparticle aids in targeting the BEV to the tumor, as albumin is selectively taken up by tumors. It is also possible that the existence of the BEVIABX complex shows greater stability in vivo than ABRAXANE®. 
     Example 12: Follow Up Study to Investigate Whether Pretreatment with BEV Improves Targeting 
     Following the general protocol above, athymic nude mice were injected with 1×10 6  A375 human melanoma cells in the right flank and then treated with PBS, 12 mg/kg BEV, 30 mg/kg ABX, AB160, or pretreated with 1.2 mg/kg BEV and, 24 hr later, AB160. Data is represented at day 7-post and day 10-post treatment as tumor volume in mm 3 .  FIGS.  11 A-E  track tumor size over 10 days. Only mice treated with AB160 (with or without pretreatment with BEV) showed a reduction in average tumor volume. See also  FIG.  11    F and  FIG.  11    G. 
     The day 7-post treatment data, as summarized in  FIG.  11 F , show that pretreatment with BEV was associated with a statistically significant reduction in tumor volume over control or BEV alone (p≤0.0001), or ABX alone (p≤0.0001). 
     The day 10-post treatment data, as summarized in  FIG.  11 G , again show that pretreatment with BEV was associated with a statistically significant reduction in tumor volume over control or BEV alone (p≤S0.0001), or ABX alone (p≤0.0001). Pretreatment with BEV before AB160 was also associated with a reduction in tumor volume over AB160 alone (p=0.02), with complete response in two mice. 
     In this experiment, a 12 mg/kg dose of BEV was not therapeutic. The amount of BEV added in the pretreatment group was only 1.2 mg/kg, which is 1/10 the usual dose in mice. Yet pretreatment with a subtherapeutic dose appears to show improved efficacy of the AB160 nanoparticle. This data support the idea that pretreatment with a subtherapeutic amount of BEV can clear systemic levels of VEGF, leaving a greater relative concentration at the tumor such that tumor-associated VEGF targeting by the AB160 nanoparticles is more effective. 
     Example 13: Alternative Means of Delivering Nanoparticles 
     It is contemplated that nanoparticles of this invention can be directly delivered to the tumor. For example, nanoparticles can be delivered via intra-arterial cannula or by direct injection into the tumor. In such embodiments, it is contemplated that large nanoparticles (e.g., 580 nm or 1130 nm) can be delivered by direct injection into or proximate to a tumor. 
     Example 14: Antigen Binding of Lyophilized AR160 
     CD20 positive Daudi lymphoma cells were labeled with fluorescent tagged anti-human CD20 or isotype matched control in panel F and A, respectively, and analyzed by flow cytometry. In the other panels, the Daudi cells were pretreated with ABX, AR160, AR160L (AR160 lyophilized and resuspended into a solution suitable for injection), or Rituxan prior to CD20 labeling.  FIG.  12    demonstrates that CD20 binding was specifically blocked by the AR particles and Rituxan, but not ABX alone. These results suggest that the AR binds to its CD20 ligand on these cells blocking binding of the fluorescent anti-CD20. 
       FIG.  13    is a histogram overlay of the same data presented in  FIG.  12   . 
       FIGS.  14 A and  14 B  depicts the particle size comparisons of ABX alone relative to AR ( FIG.  14 A ) and AT ( FIG.  14 B ) freshly made and lyophilized. 
       FIG.  15    presents the results of a Daudi proliferation assay comparing the toxicity of ABX and the AR particles. The data demonstrates the lyophilized and non-lyophilized nanoparticles have essentially the same toxicity in the Daudi assay. 
     Example 15: Fluorescent Analysis of Tumor Accumulation of AlexaFluor 750 Labeled Nanoparticles 
     Mice received intravenous (IV) injections of equal amounts of either labeled ABRAXANE®, labeled ABRAXANE® coated with non-specific antibodies (AB IgG), or labeled ABRAXANE® coated with Rituximab (AR160). Regions of interest (ROI) 2, 3, and 4 ( FIG.  16 A ) track tumor accumulation based on a fluorescence threshold, ROI 1, 5, and 6 ( FIG.  16 A ) serve as background references. Fluorescence was determined in the ROIs 24 hours post injection.  FIG.  16 B  is a bar graph of the average fluorescence per unit of tumor area of mice in all three treatment groups were determined to provide the gross tumor delivery.  FIG.  16    C is a bar graph of the average fluorescence per unit of tumor area normalized by background ROI to give proportion of drug delivered to tumor versus body. The data demonstrate that administration of AR160 nanoparticles results in an increased fluorescence as compared to ABRAXANE® alone or ABRAXANE® coated with non-specific antibodies. 
     Example 16: Nanoparticles Having a Size of 225 nm 
     To make a nanoparticle having a size of 225 nm, the particles were prepared in accordance with Example 1 but the ratio of BEV to ABRAXANE® was 4:5, i.e., 4 parts BEV and 5 parts ABRAXANE. This ratio produced nanoparticles having a size of 225 nm (AB225). The effect of AB225 was assayed in animals as set forth above.  FIG.  17    depicts the survival of the mice treated with a single dose of saline, BEV, ABX, AB160 and AB225 and with AB160 with a BEV pretreatment. At 30 days post-administration the survival of mice treated with AB225, and with AB160 with or without pretreatment with BEV far exceeds the survival of mice treated with BEV alone of ABRAXANE® alone.