Antibody-directed chemotherapy (ADC) offers an advantage over conventional chemotherapy because it provides antibody-directed targeting, with resultant improvement in therapeutic efficacy and reduced toxicity. Despite extensive research, with notable exceptions, broad clinical application of ADC remains elusive; major hurdles include the instability of antibody–chemotherapy linkers and reduced tumor toxicity of the chemotherapy when bound to the antibody. To address these challenges, we have developed a platform technology that utilizes the nab-paclitaxel formulation of paclitaxel, Abraxane, in which hydrophobic paclitaxel is suspended in 130-nm albumin nanoparticles and thus made water-soluble. We have developed a method to noncovalently coat the Abraxane nanoparticle with recombinant mAbs (anti-VEGF, bevacizumab) and guide Abraxane delivery into tumors in a preclinical model of human A375 melanoma. Here, we define the binding characteristics of bevacizumab and Abraxane, demonstrate that the chemotherapy agent retains its cytotoxic effect, while the antibody maintains the ability to bind its ligand when the two are present in a single nanoparticle (AB160), and show that the nanoparticle yields improved antitumor efficacy in a preclinical human melanoma xenograft model. Further data suggest that numerous therapeutic monoclonal IgG1 antibodies may be utilized in this platform, which has implications for many solid and hematologic malignancies. Cancer Res; 76(13); 3954–64. ©2016 AACR.

Systemic treatment of malignant disorders with chemotherapy remains a mainstay for systemic therapy for many types of cancer, including melanoma. Most chemotherapeutics are only slightly selective to the tumor cells, and toxicity to normal tissues can be high (1), often requiring chemotherapy dose reduction and even discontinuation of treatment. One approach to overcome chemotherapy toxicity, as well as improve therapeutic efficacy, is to target the chemotherapy drug to the tumor using antibodies that are specific for proteins selectively expressed (or overexpressed) by tumors cells (antibody-directed chemotherapy, ADC).

Conventional ADCs are designed with a cytotoxic agent (chemotherapeutic drug or potent toxin) linked to a targeting antibody via a synthetic protease-cleavable linker. The efficacy of such ADC therapy is dependent on the availability of receptors on the target cell that bind the antibody, ability of the linker to be cleaved, and the uptake of the toxic agent into the target cell (2).

Nearly 30 years ago, the first ADC, methotrexate, conjugated to an antihuman prostatic acid phosphatase (PAP), was tested in a mouse xenotransplant model of human prostate cancer using the cell line LNCaP (3). These experiments suggested that higher levels of the ADC penetrated the tumor relative to an isotype control antibody bound to methotrexate, which translated into reduced tumor growth in the ADC-treated mice. Since then, numerous ADCs have been tested both preclinically and in the clinical setting in phase I and II trials (2). However, despite significant development efforts, to date only three nonradioactive ADCs have received FDA approval: gemtuzumab ozogamicin for the treatment of acute myelogenous leukemia (4) in 2001, brentuximab vedotin for treatment of Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL) in 2011, and ado-trastuzumab vedotin for HER-2–positive breast cancer in 2013. Gemtuzumab ozogamicin, a CD33-targeted chemotherapy drug, had its FDA approval withdrawn when a confirmatory trial showed no benefit and greater toxicity compared with chemotherapy alone (5). Brentuximab vedotin, a CD30-targeted antimitotic agent, demonstrated an overall and progression-free survival of 40.5 and 9.3 months, respectively, in Hodgkin lymphoma patients (6). In a phase IIa clinical trial of ado-trastuzumab vedotin, a Her2-targeted taxane, the overall response rate was 33% in metastatic breast cancer patients previously treated with chemotherapy and 57% as first-line therapy with progression-free survival of 5.5 and 7.7 months, respectively (7). Thus, in clinical practice today, only two ADCs continue to demonstrate clinical efficacy, in the face of hundreds of tumor-specific mAbs and chemotherapy agents.

Nab-paclitaxel, Abraxane, is a recently developed paclitaxel formulation in which the hydrophobic paclitaxel is enveloped by albumin, to form a 130-nm nanoparticle. Unlike standard paclitaxel formulations, paclitaxel solubilized with Cremophor, Abraxane is water-soluble and may be delivered in higher doses and shorter infusion times, thereby altering the paclitaxel pharmacokinetics in the blood of the patient (8). Together, these allow the accumulation of paclitaxel in the tumor and increase chemotherapeutic efficacy (9). A phase II clinical trial demonstrated clinical efficacy of single-agent Abraxane in previously treated and chemotherapy naïve patients with metastatic melanoma (4), while a subsequent phase III clinical trial showed minimally improved clinical outcomes relative to dacarbazine (10).

It is commonly known that VEGF can be produced by tumors, resulting in high local VEGF concentration in the tumor microenvironment in melanoma (11). In addition, the clinical efficacy of the Abraxane is improved by administering Abraxane in combination with anti-VEGF antibody, bevacizumab (12, 13). Hence, we hypothesized whether bevacizumab could be noncovalently bound to the albumin scaffold of Abraxane forming a nano-immune conjugate the antibody may target the paclitaxel to the tumor increasing drug deposition in the tumor, thereby enhancing tumor regression and improving drug efficacy.

Here, we demonstrate that bevacizumab binds to the albumin scaffold of the Abraxane ex vivo to form a stable nano-ADC particle. We define binding characteristics, and show that the particle size can be customized. We present in vitro and in vivo efficacy of the 160 nm particles of ex vivo Abraxane-bound bevacizumab (AB160) relative to either Abraxane or bevacizumab individually. We also demonstrate that the albumin scaffold of Abraxane provides a platform to which other therapeutic antibodies can be bound. Unlike conventional ADC, the antibody is bound noncovalently to the toxic agent through the albumin scaffold. These results have broad implications in cancer therapeutics, beyond therapy of melanoma.

Nanoparticle preparation

Abraxane (10 mg) was suspended in bevacizumab (4 mg) in 1 mL of 0.9% saline at a final concentration of 10 mg/mL and 4 mg/mL of Abraxane and bevacizumab, respectively. The mixture was incubated for 30 minutes at room temperature to allow particle formation. For Mastersizer experiments to measure particle size of Abraxane:bevacizumab complexes, 10 mg of Abraxane was suspended in bevacizumab at concentrations of 0 to 25 mg/mL. Complexes of Abraxane with rituximab 0 to 10 mg/mL or trastuzumab 0 to 22 mg/mL were formed under similar conditions.

Flow cytometry

To determine binding of bevacizumab to Abraxane, visualization of AB160 was performed on an Accuri C6 flow cytometer (BD Franklin Lakes) and data analysis was done using Accuri C6 software. Biotinylated goat antimouse IgG (Abcam) was labeled with streptavidin phycoerythrin (PE; Abcam). The goat anti-mouse IgG was chosen to label AB160 because the Fab portion of the bevacizumab is mouse derived. Abraxane and AB160 were incubated with the PE-labeled goat anti-mouse IgG for 30 minutes at room temperature, washed, and visualized by flow cytometery.

Binding assay

Biotinylated bevacizumab, rituximab, or trastuzumab at 100 μg/mL, was bound to the streptavidin probe (ForteBio Corp.). The binding of Abraxane was measured by light absorbance on the BLItz system (ForteBio Corp.) at 1,000, 500, and 100 mg/mL. The association and dissociation constants were calculated using the BLItz software.

Mastersizer and nanosight

The particle size of Abraxane and antibody–Abraxane drug complexes were measured by dynamic light scattering on a Mastersizer 2000 (Malvern Instruments). To measure particle size, 2 mL (5 mg/mL) of Abraxane or complexes 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 using nanoparticle-tracking analysis (Malvern Instruments). The Abraxane or complex particles were diluted to the appropriate range to accurately measure particle sizes. Data were displayed by particle size distribution; however, the nanoparticle tracking analysis uses Brownian motion to determine particle size.

Electron microscopy

AB160 was diluted 1:5, and 5 μL of AB160 was loaded on nickel formvar–coated grid and dried. Samples were incubated for 1 hour in goat antimouse IgG (1:30 dilution) with 6-nm gold-conjugated particles (Electron Microscopy Sciences), washed 2× in PBS and 4× in water, and stained with a mixture of 2% methylcellulose and 4% UA (9:1) for 5 minutes and dried for 1 hour. Samples were incubated overnight in donkey anti-mouse IgG (1:25 dilution) with 6-nm gold-conjugated particles (Jackson ImmunoResearch), washed as above, stained with 1% phosphotungstic acid for 5 minutes, covered with 2% methylcellulose, and dried for 1 hour. The micrographs were taken on a JEOL1400 at operating at 80 kV.

In vitro toxicity

The A375 human melanoma cell line (ATCC) was cultured in DMEM with 1% penicillin, streptomycin, and glutamine (PSG) and 10% FBS. Cells were harvested and plated at 0.75 × 106 cells per well in 24-well plates. Cells were exposed to Abraxane or AB160 at paclitaxel concentrations from 0 to 200 μg/mL overnight at 37°C and 5% CO2. To measure proliferation, the Click-iT EdU kit (Molecular Probes) was utilized as per manufacturer's instructions.

VEGF ELISA

To determine whether bevacizumab could still bind its ligand, VEGF, when bound to Abraxane, we employed a standard VEGF ELISA (R&D Systems). AB160 was prepared and 2,000 pg/mL VEGF was added to the AB160 complex or Abraxane alone. The VEGF was incubated with the nanoparticles for 2 hours at room temperature. The suspension was spun at 6,000 rpm for 15 minutes, supernatants were collected, and VEGF content was measured by ELISA as per manufacturer's instructions. Color was developed with substrate (R&D Systems). Absorbance was measured at 450 nm using a Versamax ELISA plate reader (Molecular Devices). The concentration of unbound VEGF was determined with a standard curve ranging in concentration from 0 to 2,000 pg/mL.

Mouse model

To test tumor efficacy, female 5–6 week old mice were purchased from Harlan Sprague Dawley and injected with 1 × 106 A375 human melanoma cells in the right flank. When the tumors had reached about 700 mm3, the mice were randomized and treated by intravenous tail injection with PBS, Abraxane (30 mg/kg), bevacizumab (12 mg/kg), 12 mg/kg bevacizumab followed by 30 mg/kg Abraxane, or AB160, which contained 12 mg/kg bevacizumab and 30 mg/kg Abraxane. For the mouse experiments testing larger Abraxane:bevacizumab (AB) particles, the highest dose of bevacizumab (45 mg/kg) necessary to create the largest particles was used in the bevacizumab-only treatment group. Tumor size was monitored three times/week and tumor volume was calculated with the following equation: (length × width2)/2. Mice were sacrificed when the tumor size equaled about 10% of the mouse body weight. The day 7 and day 10 percent change from baseline was calculated as follows: [(tumor size on day 7 or 10−tumor size on day of treatment)/tumor size on day of treatment]×100.

Paclitaxel pharmacokinetics

The liquid chromatographic separation of paclitaxel was accomplished using an Agilent Poroshell 120 EC-C18 precolumn (2.1×5 mm, 2.7 μm, Chrom Tech) attached to an Agilent Poroshell 120 EC-C18 analytical column (2.1×100 mm, 2.7 μm Chrom Tech) at 40°C, under a gradient mobile phase [water with 0.1% formic acid (A)] and ACN with 0.1% formic acid (B) with a 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% to 85% for 4.5 minutes, held at 85% B for 0.2 minutes, and returned to initial conditions for 1.3 minutes. Auto-sampler temperature was 10°C and sample injection volume was 2 μL. Detection of paclitaxel was accomplished using the mass spectrometer in positive ESI mode with capillary voltage 1.75 kV, source temp 150°C, desolvation temperature 500°C, cone gas flow 150 L/hour, desolvation gas flow 1,000 L/hour, using multiple reaction monitoring (MRM) scan mode with a dwell time of 0.075 seconds.

Binding and function of AB160 in vitro

To understand the characteristics of the nanoparticles formed when binding bevacizumab to Abraxane, we determined the size of the Abraxane: bevacizumab complexes relative to Abraxane by light diffraction technology (Mastersizer 2000) after coincubating 10 mg/mL Abraxane with 4 mg/mL bevacizumab ex vivo. We found Abraxane: bevacizumab to be consistently larger (approximately 160 nm) than the 130 nm Abraxane alone (Fig. 1A). The size of the nanoparticle created directly correlated to the concentration of bevacizumab used with median sizes [d (0.5) μm] ranging from 0.157 to 2.166 μm. (Fig. 1A). The d (0.1) and d (0.9) represent the size of the 10th and 90th percentile size, respectively. With the goal of these studies being a phase I clinical trial, we chose to focus on the smallest Abraxane:bevacizumab particle (AB160) because it is the most similar to the 130-nm Abraxane.

Figure 1.

Demonstration of binding between abraxane and bevacizumab. Complex sizes were assessed by light scattering technology. Increasing concentrations of bevacizumab (0–25 mg) were added to 10 mg of Abraxane (ABX), and complex sizes determined. The average size of the complexes directly correlated to the concentration of bevacizumab (BEV) added (0.146–2.166 μmol/L; A). Data are of 5 drug preparations and are displayed as particle size distributions. Fluorescently labeled AB160 and Abraxane were analyzed by flow cytometry. The data are displayed as scatterplots, including Abraxane stained with secondary antibody only or goat anti-mouse IgG1-Fab plus secondary antibody and AB160 stained with secondary antibody only or goat anti-mouse IgG1-Fab plus secondary antibody (B). Electron microscopy was done after incubation of AB160 with gold particle–labeled anti-human IgG-Fc (C). AB160 was fractionated into particulate, proteins greater and less than 100 kDa, and analyzed for paclitaxel content by LC/MS; results are shown as a pie chart indicating the percentages of total paclitaxel (D) and Western blot analysis was performed with antibodies against mouse IgG Fab (bevacizumab) and paclitaxel to verify colocalization (E). Paclitaxel activity was confirmed in vitro with A375 human melanoma cells treated with Abraxane or AB160, and results are represented by the average (±SEM) proliferation index (F). These data represent three experiments and differences were not significant. A VEGF ELISA of supernatant after coincubation of VEGF with Abraxane and AB160 was utilized to determine binding of the ligand, VEGF. The results are shown as the average percentage (±SEM) of VEGF that was unbound by the two complexes (G). The data represents three experiments. **, P values < 0.005.

Figure 1.

Demonstration of binding between abraxane and bevacizumab. Complex sizes were assessed by light scattering technology. Increasing concentrations of bevacizumab (0–25 mg) were added to 10 mg of Abraxane (ABX), and complex sizes determined. The average size of the complexes directly correlated to the concentration of bevacizumab (BEV) added (0.146–2.166 μmol/L; A). Data are of 5 drug preparations and are displayed as particle size distributions. Fluorescently labeled AB160 and Abraxane were analyzed by flow cytometry. The data are displayed as scatterplots, including Abraxane stained with secondary antibody only or goat anti-mouse IgG1-Fab plus secondary antibody and AB160 stained with secondary antibody only or goat anti-mouse IgG1-Fab plus secondary antibody (B). Electron microscopy was done after incubation of AB160 with gold particle–labeled anti-human IgG-Fc (C). AB160 was fractionated into particulate, proteins greater and less than 100 kDa, and analyzed for paclitaxel content by LC/MS; results are shown as a pie chart indicating the percentages of total paclitaxel (D) and Western blot analysis was performed with antibodies against mouse IgG Fab (bevacizumab) and paclitaxel to verify colocalization (E). Paclitaxel activity was confirmed in vitro with A375 human melanoma cells treated with Abraxane or AB160, and results are represented by the average (±SEM) proliferation index (F). These data represent three experiments and differences were not significant. A VEGF ELISA of supernatant after coincubation of VEGF with Abraxane and AB160 was utilized to determine binding of the ligand, VEGF. The results are shown as the average percentage (±SEM) of VEGF that was unbound by the two complexes (G). The data represents three experiments. **, P values < 0.005.

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To visualize the AB160 particles, we utilized flow cytometry. Because the Fab portion of the IgG1 (bevacizumab) is of mouse origin, we selectively labeled particles containing bevacizumab with purified goat anti-mouse IgG followed by phycoerythrin (PE) conjugated anti-goat as a secondary antibody. As a negative control, we stained with the anti-goat PE alone. We demonstrated a bright signal of anti-mouse IgG1 binding to AB160 (41.2% positive) relative to Abraxane (6.7% positive) alone (Fig. 1B). To validate binding of bevacizumab to Abraxane, we labeled the bevacizumab with gold-labeled mouse anti-human IgG and visualized the particles with electron microscopy (Fig. 1C). The electron microscopy pictures suggest a monolayer of bevacizumab surrounding Abraxane nanoparticles, which is consistent with the 160-nm size of AB160.

To determine what protein (albumin or bevacizumab) the paclitaxel remains bound to when the complex dissociates, we made AB160 and collected fractions: the particulate, and proteins greater than 100 kDa and proteins less than 100 kDa. We measured paclitaxel in each fraction by LC/MS. We found that roughly 75% of the paclitaxel remained within the particulate, and the majority of the remaining paclitaxel was associated with the fraction containing proteins 100 kDa or greater, suggesting that when the particulate dissociates, the paclitaxel is bound to bevacizumab alone or a bevacizumab and albumin heterodimer (Fig. 1D). 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 Abraxane and AB160 (1 and 10 mg) particles, which were incubated in saline for 4 hours and overnight. Blots were probed with anti-mouse IgG Fab to detect bevacizumab and anti-paclitaxel, and results demonstrated that bevacizumab and paclitaxel colocalize at approximately 200 kDa, a size consistent with a paclitaxel–bevacizumab–albumin protein complex (Fig. 1E).

Finally, we wanted to confirm that the two key elements in the complexes, the antibody and the paclitaxel, retained their function when present in the complexes. AB160 has similar toxicity relative to Abraxane alone in an in vitro toxicity assay with the human melanoma cell line A375, suggesting that the paclitaxel functions equally as well in either formulation (Fig. 1F). To test the binding of VEGF to bevacizumab in the AB160 complex, we coincubated AB160 or Abraxane with VEGF, removed particulate, and measured VEGF content in the supernatant. The lack of VEGF in the supernatant measured from AB160 (<10% VEGF unbound) indicated that the VEGF was bound by the bevacizumab in the AB160 complex while it remained free when incubated with the Abraxane (>80% VEGF unbound) alone (Fig. 1G). Thus, AB160 retains the cytotoxic potential of Abraxane as well as the ligand-binding properties of bevacizumab.

Particle size and protein affinity

To assess the binding affinity of bevacizumab to Abraxane, we utilized Bio-Layer Interferometry (BLItz) technology. Biotinylated bevacizumab was bound to the streptavidin probe and exposed to Abraxane (1,000, 500, and 100 μg/mL). The dissociation constant (Kd) of bevacizumab and Abraxane is 2.2 × 10−8 mol/L at room temperature and pH 7 consistent with a strong noncovalent interaction (Fig. 2A). The binding affinity of bevacizumab and Abraxane is within the range of dissociation constants observed between albumin and natural or engineered albumin-binding domains of some bacterial proteins (14).

Figure 2.

Binding affinity of bevacizumab and Abraxane (ABX). A, binding affinity was determined by BLItz and dissociation constants (Kd) were calculated with BLItz software. Binding affinity was assessed at four pH levels and three temperatures. Data are representative of five experiments. Complex stability was determined by nanoparticle tracking analysis. Data are displayed as the number of particles/mg of Abraxane. AB160 was prepared at room temperature and pH 7 (AB16007) 58°C and pH 7 (AB1600758) 58°C and pH 5 (AB1600558) relative to Abraxane at each condition were compared. B, prepared particles were added to human AB serum for 15, 30, 45, and 60 minutes to determine stability in serum over time.

Figure 2.

Binding affinity of bevacizumab and Abraxane (ABX). A, binding affinity was determined by BLItz and dissociation constants (Kd) were calculated with BLItz software. Binding affinity was assessed at four pH levels and three temperatures. Data are representative of five experiments. Complex stability was determined by nanoparticle tracking analysis. Data are displayed as the number of particles/mg of Abraxane. AB160 was prepared at room temperature and pH 7 (AB16007) 58°C and pH 7 (AB1600758) 58°C and pH 5 (AB1600558) relative to Abraxane at each condition were compared. B, prepared particles were added to human AB serum for 15, 30, 45, and 60 minutes to determine stability in serum over time.

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We tested whether binding affinity was pH- and/or temperature-dependent by suspending the Abraxane in bevacizumab and diluting the mixture with saline at pH 3, 5, 7, or 9 before incubation at various temperatures (room temperature, 37°C, and 58°C) to allow particle formation. The binding affinity of bevacizumab and Abraxane is both pH- and temperature dependent, with the highest binding affinity observed when the particles are formed at pH 5 and 58°C (Fig. 2A).

To determine whether the higher affinity binding of bevacizumab and Abraxane at 58°C and pH 5 translated into stability of the complex, we compared various preparations (AB1600558) with AB160 prepared at room temperature and pH7 (AB16007) 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 with the Abraxane 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 prepared under higher affinity conditions (pH 7 and 58°C) were more stable as indicated by the number of particles present per mg Abraxane 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 Abraxane alone as determined by size and number of particles measured per mg Abraxane (Fig. 2B). This shows that the stability of the AB160 particles can be manipulated by adjusting temperature and pH under which the AB160 particles are formed.

In vivo efficacy of AB160

To test AB160 efficacy in vivo, we used a xenograft model of A375 human melanoma cells implanted into athymic nude mice. We tested AB160 relative to PBS, the single drugs alone, and the drugs administered sequentially. Mice treated with AB160 had significantly reduced tumor size relative to all other treatment groups (P = 0.0001–0.0089) at day 7 after treatment, relative to baseline (Fig. 3A). 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. 3B), with a survival of 7, 14, 14, 18, and 33 days for the PBS (P ≤ 0.0001), bevacizumab (P = 0.003), Abraxane (P = 0.0003), bevacizumab + Abraxane (P = 0.0006), and AB160 groups, respectively.

Figure 3.

In vivo testing of AB nanoparticles. A375 tumors were treated at about 700 mm3, the mice were treated with PBS, bevacizumab (BEV), Abraxane (ABX), bevacizumab + Abraxane, or AB160 in experiment 1 (A and B) or PBS, Abraxane, bevacizumab and AB160, AB580, and AB1130 in experiment 2 (C and D) or PBS, bevacizumab, Abraxane, AB160, or bevacizumab + AB160 in experiment 3 (E and F). Data are represented at day 7 after treatment (A and C) or day 10 after treatment (E) as the percent change in tumor size from baseline. The Student 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 two drugs given sequentially (A and C). The difference among the AB particles was not significant. The median survival difference between AB160 and bevacizumab+AB160 was significant (E). Kaplan–Meier curves were generated for median survival (B, D, and F) and median survival in days is presented in parentheses in the legend. The Mantle–Cox test was used to compare survival curves. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.

Figure 3.

In vivo testing of AB nanoparticles. A375 tumors were treated at about 700 mm3, the mice were treated with PBS, bevacizumab (BEV), Abraxane (ABX), bevacizumab + Abraxane, or AB160 in experiment 1 (A and B) or PBS, Abraxane, bevacizumab and AB160, AB580, and AB1130 in experiment 2 (C and D) or PBS, bevacizumab, Abraxane, AB160, or bevacizumab + AB160 in experiment 3 (E and F). Data are represented at day 7 after treatment (A and C) or day 10 after treatment (E) as the percent change in tumor size from baseline. The Student 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 two drugs given sequentially (A and C). The difference among the AB particles was not significant. The median survival difference between AB160 and bevacizumab+AB160 was significant (E). Kaplan–Meier curves were generated for median survival (B, D, and F) and median survival in days is presented in parentheses in the legend. The Mantle–Cox test was used to compare survival curves. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.

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Particles of increasing size were prepared using increasing bevacizumab: Abraxane ratios as shown in Fig. 1A. 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 (Fig. 3C and D).

VEGF is produced by tumors but is a soluble factor, as opposed to a membrane-bound protein, that can spill into the blood stream from the tumor microenvironment; therefore, we hypothesized that AB160 tumor targeting and efficacy may improve if a small dose of bevacizumab was given before AB160 administration. To test this hypothesis, we gave a 10% (1.2 mg/kg) dose of bevacizumab one day before AB160 and compared tumor response and median survival to the drugs individually and AB160 alone. The difference in percent change from baseline was significant between the bevacizumab + AB160 and the AB160 group at day 10 after treatment (P = 0.02; Fig. 3E). In addition, the median survival between bevacizumab + AB160 and AB160 groups was significantly different (P = 0.0085; Fig. 3F). The fact that AB160 efficacy improves after giving a dose of bevacizumab to clear circulating VEGF supports the idea that bevacizumab promotes tumor targeting of AB160.

Full toxicity studies were performed on the mice and no toxicities were noted (Supplementary Table S1).

Paclitaxel pharmacokinetics in mice

To compare the pharmacokinetics of AB160 and Abraxane, plasma paclitaxel concentrations were measured in mice administered with AB160 or Abraxane at 0, 1, 2, 4, and 8 hours. The AUC and maximum serum concentrations (Cmax) were calculated in A375 tumor-bearing mice with small or large tumors and non-tumor–bearing mice. The results are shown as concentrations of paclitaxel in the blood of 3 mice per group in log (Fig. 4A) and linear scale (Fig. 4B). In addition, Fig. 4C shows blood paclitaxel concentrations in bar graphs for the 0, 2, and 4-hour time points so the differences between groups can be seen. The results of this experiment demonstrated smaller AUC in tumor-bearing mice relative to non-tumor–bearing mice (Fig. 4D), with the lowest blood values of paclitaxel in mice with large tumors relative to the small tumor mice (80.4 ± 2.7, 48.4 ± 12.3, and 30.7 ± 5.2 μg/mL/hour) for Abraxane-treated non-tumor, small tumor, and large tumor-bearing mice, respectively; 66.1 ± 19.8, 44.4 ± 12.1, and 22.8 ± 6.9 μg/mL/hour for AB160-treated mice. Similarly, the Cmax (Fig. 4E) was lower in both treatment groups in mice with larger tumors (47.2, 28.9, and 19.7 μg/mL for Abraxane and 40.1, 26.9, and 15.3 μg/mL for AB160). The AUC and Cmax of paclitaxel in blood were lower in AB160-treated mice relative to Abraxane-treated mice. Although not statistically significant, these data are consistent with higher deposition of paclitaxel in the tumors treated with AB160.

Figure 4.

Mouse pharmacokinetics (PK). Blood and tumor samples were taken from mice (3/time point) at 0, 1, 2, 4, and 8 hours after intravenous injection with Abraxane (ABX) or AB160 and measured by LC/MS. Blood paclitaxel concentrations are shown in graphs displayed on log (A) and numeric (B) scales and a column graph of the 0, 2, and 4-hour time points (C). AUC (D) and Cmax (E) were calculated and displayed in bar graphs. Tumor paclitaxel concentrations were determined by LC/MS (F). Data are displayed as μg of paclitaxel/mg of tumor tissue. Significant differences of paclitaxel concentrations were seen at 4 and 8 hours in tumor tissue.

Figure 4.

Mouse pharmacokinetics (PK). Blood and tumor samples were taken from mice (3/time point) at 0, 1, 2, 4, and 8 hours after intravenous injection with Abraxane (ABX) or AB160 and measured by LC/MS. Blood paclitaxel concentrations are shown in graphs displayed on log (A) and numeric (B) scales and a column graph of the 0, 2, and 4-hour time points (C). AUC (D) and Cmax (E) were calculated and displayed in bar graphs. Tumor paclitaxel concentrations were determined by LC/MS (F). Data are displayed as μg of paclitaxel/mg of tumor tissue. Significant differences of paclitaxel concentrations were seen at 4 and 8 hours in tumor tissue.

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To directly test this hypothesis, we measured tumor paclitaxel concentrations by LC/MS. The tumor paclitaxel concentration was significantly higher in tumors treated with AB160 relative to Abraxane at the 4-hour (3,473 μg/mg ± 340 of tissue vs. 2,127 μg/mg ± 3.5 of tissue; P = 0.02) and 8-hour (3,005 ± 146 μg/mg of tissue vs. 1,688 μg/mg ± 146 of tissue; P = 0.01) time points (Fig. 4F), suggesting paclitaxel was retained in the tumor longer when targeted by the antibody. This is consistent with the pharmacokinetics and with redistribution of drug to tissues including the tumor.

Binding of other therapeutic antibodies to Abraxane

To determine whether other IgG therapeutic antibodies also exhibit binding to Abraxane when combined ex vivo, we tested binding of two other commercially available mAbs currently in active use in clinical practice: anti-human CD20 antibody (rituxamab) and anti-HER2/neu receptor antibody (trastuzumab) to Abraxane.

First, we measured particle size of the complexes with both rituxumab (AR) and trastuzumab (AT). The AB and AT particle sizes were very similar with average sizes ranging from 0.157 to 2.166 μmol/L (Fig. 2A) and 0.148 to 2.868 μmol/L (Fig. 5B), respectively. Particles formed with rituximab became much larger at lower antibody: Abraxane ratios, with particle sizes ranging from 0.159 to 8.286 μmol/L (Fig. 5A).

Figure 5.

Particle size measurements and affinity of nanoparticles made with rituximab (RIT) and trastuzumab (TRA). Abraxane (ABX; 10 mg/mL) was incubated with rituximab at 0 to 10 mg/mL (A) or trastuzumab at 0 to 22 mg/mL (B) 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. The tables show the sizes of the resulting particles at each concentration of antibody. Biolayer interferometry (BLItz) technology was utilized to determine the binding affinity of rituximab and trastuzumab to Abraxane at pH 3, 5, 7, and 9. C, the dissociation constants are displayed for each interaction.

Figure 5.

Particle size measurements and affinity of nanoparticles made with rituximab (RIT) and trastuzumab (TRA). Abraxane (ABX; 10 mg/mL) was incubated with rituximab at 0 to 10 mg/mL (A) or trastuzumab at 0 to 22 mg/mL (B) 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. The tables show the sizes of the resulting particles at each concentration of antibody. Biolayer interferometry (BLItz) technology was utilized to determine the binding affinity of rituximab and trastuzumab to Abraxane at pH 3, 5, 7, and 9. C, the dissociation constants are displayed for each interaction.

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The binding affinities of rituximab and trastuzumab with Abraxane were determined by BLItz under variable pH. Both antibodies bind with relatively high affinity in the picomolar range. The rituximab affinity to Abraxane decreased with higher pH, but trastuzumab affinity to Abraxane was unaffected by pH (Fig. 5C).

Therapeutic antibodies, including bevacizumab, can bind to the albumin portion of Abraxane to create a nano-immune conjugate that, unlike conventional ADCs, does not require a linker between the antibody and the toxic payload. Using BLItz technology, we demonstrated that bevacizumab binds to Abraxane with affinity in the picomolar range, indicating a strong noncovalent bond. Light scattering demonstrated a particle size distribution consistent with Abraxane surrounded by a monolayer of antibody molecules and the size of the particles created is dependent on the antibody concentration. Importantly, we showed by an in vitro toxicity assay and ELISA that the paclitaxel in AB160 retains its toxicity to tumor cells and the bound bevacizumab maintains the ability to bind its ligand, VEGF. We then tested AB160 in a human xenograft melanoma mouse model and demonstrated that the blood and tumor pharmacokinetics of paclitaxel are altered relative to Abraxane alone, which translated into enhanced tumor regression and longer median survival in AB160-treated mice. In addition, we showed that the albumin portion of the Abraxane provides a platform for other therapeutic antibodies to bind, including rituximab and trastuzumab.

Many natural and synthetic polymers as well as lipids have been tested as carriers in drug conjugates, including: micelles, liposomes, PEG, and albumin (15, 16). Albumin in particular has been utilized as a natural carrier (17). The fact that albumin is an excellent energy source for tumors and is able to accumulate in tissue makes albumin an attractive protein for tumor targeting of drugs (18). Albumin has proven to be very versatile in many clinical applications including diagnostics and therapeutics (19, 20).

The nab-paclitaxel formulation, Abraxane, is a nanoparticle containing an albumin shell around paclitaxel that has been shown to have clinical efficacy as a single agent and in combination with other drugs in breast (21) and pancreatic cancer (22), non–small cell lung carcinoma (23), and melanoma (12, 13). It has been suggested that the increased efficacy of Abraxane relative to Cremaphor-based paclitaxel is due at least in part to a change in paclitaxel pharmacokinetics of the two drugs and that Abraxane may accumulate in the tumor at higher concentrations than Cremaphor-based paclitaxel in mice (8) and humans (24). Our data suggest that noncovalently binding a tumor-targeting antibody to Abraxane, we alter the pharmacokinetics more dramatically than Abraxane alone, lowering the Cmax and AUC in the blood because of redistribution of AB160 to the tumor tissue. Our results from mouse blood paclitaxel pk (Fig. 4A–E) and tumor tissue levels of paclitaxel (Fig. 4F) in mice treated with AB160 relative to Abraxane alone suggest that antibody targeting of the Abraxane 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.

Conventional ADCs contain an antibody and a cytotoxic agent connected by a linker (25). Typically, ADCs require binding and internalization by the target cells. Upon internalization, the ADC must dissociate for the cytotoxic agent to effectively kill the tumor cell, which requires that the bond to the linker be cleaved (17). 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 (26). This has proven to be a major hurdle in developing effective drug conjugates (17, 26); therefore, an attractive feature of our nano-immune conjugate is that a chemical linker is not required. Our LC/MS and Western blot data (Fig. 1D and E) indicated that in systemic circulation 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.

Another issue with 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 higher concentration of the active agent in the tumor (3); however, this has not correlated in the treatment of human disease likely because human tumors are much more heterogeneous in permeability than mouse tumors (27). 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 cut-off size of vascular pores was permeable to liposomes up to 400 nm (28). 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 (29). We believe our nano-immune conjugate overcomes this issue by the fact that the large complex is less than 200 nm intact, which these studies show is amenable to tissue permeability. In addition, it is feasible that the complex partially dissociates into smaller functional units, which are 200 kDa in size and contain albumin, bevacizumab, and paclitaxel, as suggested by Western blot analysis (Fig. 1E). Furthermore, once the conjugate arrives at the tumor site, the smaller toxic payload can be released and only the toxic portion needs to be taken up by tumor cells.

In conclusion, we have demonstrated a simple way to construct a versatile nano-immune conjugate, which allows multiple proteins to be bound to a single albumin scaffold. In our mouse model, we demonstrate improved efficacy of the targeted drug relative to the single agents alone, which is at least in part due to altered pharmacokinetics of the antibody-targeted drug. We believe the versatility of our nano-immune conjugate that does not require a linker or target cell internalization will allow us to overcome the obstacles faced by other nanomedicines in translating results from mice to humans. A phase I clinical trial for AB160 is currently accruing. To ascertain its full clinical relevance, further investigation of this novel nano-immune conjugate is warranted.

W.K. Nevala and S.N. Markovic disclose that this technology has been licensed to Vavotar Life Sciences, LLC. No potential conflicts of interest were disclosed by the other authors.

Conception and design: W.K. Nevala, S.N. Markovic

Development of methodology: W.K. Nevala, J.M. Reid, E.A Atanasova, S.N. Markovic

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W.K. Nevala

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W.K. Nevala, S.A. Buhrow, D.J. Knauer, J.M. Reid, S.N. Markovic

Writing, review, and/or revision of the manuscript: W.K. Nevala, D.J. Knauer, J.M. Reid, S.N. Markovic

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.N. Markovic

Study supervision: S.N. Markovic

This work was supported by the Mayo Clinic Department of Development.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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