Pharm Res DOI 10.1007/s11095-014-1541-x
RESEARCH PAPER
S m a l l A m o u n t s o f S u b -V i s i b l e A g g r e g a t e s E n h a n c e the Immunogenic Potential of Monoclonal Antibody Therapeutics Maryam Ahmadi & Christine J. Bryson & Edward A. Cloake & Katie Welch & Vasco Filipe & Stefan Romeijn & Andrea Hawe & Wim Jiskoot & Matthew P. Baker & Mark H. Fogg
# Springer Science+Business Media New York 2014
ABSTRACT Purpose Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. Methods Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (10 μm were present. Results were calculated as mean value of three measurements of 0.3 ml and referred to a sample volume of 1.0 ml.
Fluorescent Labelling of Monomeric and Aggregated Antibody For preparation of fluorescent-labelled rituximab aggregates for MoDC uptake experiments, Alexa Fluor 488 (A488) carboxylic acid, N-hydroxysuccinimide ester was obtained from Invitrogen (Merelbeke, Belgium). The fluorescent labelling of rituximab (rituximab-A488) was performed according to the manufacturer’s instructions, using an IgG concentration of 10 mg/ml and a molar ratio of 4:1 (dye: IgG). A pH of 7.4 was chosen for the labelling buffer, in order to achieve selective labelling of the amine termini. The rituximab-A488 was dialyzed back to PBS using a Float-A-Lyzer® G2 (Spectrum, Rancho Dominguez, CA, USA) with a 100 kDa molecular weight cut-off membrane to remove excess dye, and the labelling ratio achieved was about two A488 labels per rituximab molecule.
Preparation of Peripheral Blood Mononuclear Cells (PBMC) PBMC were isolated from healthy community donor buffy coats (from blood drawn within 24 h) obtained from the UK National Blood Transfusion Service (Addenbrooke’s Hospital, Cambridge, UK) and according to approval granted by Cambridgeshire Local Research Ethics Committee. PBMC were isolated from buffy coats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation and CD8+ T cells depleted using CD8+ RosetteSep™ (StemCell Technologies Inc., London, UK). PBMC were then frozen and stored in liquid nitrogen until required.
In Vitro T Cell Assays Where indicated healthy donors were selected based on broad representation of HLA-DR allotypes (40 donors for rituximab and 25 donors for trastuzumab). After donor selection, PBMC were thawed, counted, viability assessed and suspended in AIM-V® culture medium (Invitrogen, Paisley, UK) at 4–6 × 106 PBMC/ml. Bulk cultures were established for each donor where 1 ml of cell suspension was added to the wells of a 24well plate (Corning Life Science, Amsterdam, NL) along with 1 ml of antibody in AIM-V® culture medium. Proliferation of CD4+ T cells within the culture was measured on days 5, 6, 7 and 8 post-stimulation by gently re-suspending the cells in the bulk cultures followed by removal of 3 × 100 μl samples, which were transferred to a round bottomed 96-well plate for pulsing with 1 μCi/well tritiated thymidine (Perkin Elmer, Buckingham, UK). After 18 h the pulsed cultures were harvested onto filter mats (Perkin Elmer) using a TomTec Mach III cell harvester and counts per minute (cpm) for each well determined by Meltilex™ (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, low background counting mode.
Generation of Monocyte-Derived Dendritic Cells Monocytes were extracted from PBMC by negative selection with monocyte isolation kit II MACS beads (Miltenyi Biotech, Bisley, UK) according to the manufacturer’s instructions. The CD14+ monocytes were cultured at 1 × 106 cells/ml with 1000 IU/ml of IL-4 and GM-CSF (Peprotech, London, UK) in AIM-V® for 72 h to produce MoDC, which were stimulated with antibodies for a further 24 h and harvested before measuring cytokine secretion via cytometric bead array (CBA), protein uptake by phagocytosis (laser scanning confocal microscopy) or cell surface phenotype (flow cytometry).
Cytometric Bead Array Supernatants were collected from cultured cells and analyzed for cytokine production using BD CBA Enhanced Sensitivity Flex Set System (BD Biosciences, Oxford, UK), according to the manufacturer’s instructions. Briefly, thawed culture supernatant and serially diluted standards were incubated with premixed capture beads for 2 h followed by additional 2 h with detection reagents. The sensitivity of detection was enhanced following a 1 h incubation with enhanced detection reagent. Cytokines were acquired by a BD Accuri C6 instrument (BD Biosciences). Data were analyzed using FCAP v3.01 software (Softflow, Inc, Minnesota USA).
Ahmadi et al.
Rituximab Aggregate Uptake by MoDC Coverslips coated with MoDC (MoDC were initially grown on round coverslips) were transferred to 1 ml fresh AIM-V® medium and rituximab-A488 was added followed by incubation at 37°C or on ice (for control). At 0.5 h and 3 h the cells were washed three times with PBS followed by 15 min incubation on ice with BD Bioscience Cell Fix™. Cells were stained with HLA-DR PE (BD Bioscience) on ice for 30 min. Coverslips were inverted onto microscope slides with Vectashield (Vector Labs, Peterborough, UK) mounting medium. The stained cells were imaged with a Zeiss LSM 510 META laser scanning confocal microscope using a 40_NA 1.3 oil immersion objective. Single confocal slices were acquired to generate the data images. Three-dimensional reconstructions of confocal slices were made using Volocity software (Improvision, Perkin Elmer). Using image J software (http:// rsbweb.nih.gov/ij/) the intensity across the width of the images was plotted for each wavelength (grey scale).
Data Analysis For proliferation assays, a stimulation index (SI) was calculated as described previously [3]. Briefly, the counts/min for each donor treated with aggregated antibodies was divided by the background response. An SI≥1.9 was considered positive, based on a statistically derived cut point for the population tested for an adaptive response.
Statistical Analysis Statistical analysis was carried out using GraphPad Prism Software version 6 (GraphPad Software Inc, California, USA). Two sample paired t-test or one way repeated measure ANOVA were carried out (corrected for multiple tests where appropriate).
RESULTS In this study, therapeutic mAbs, stressed by a variety of pharmaceutically relevant methods to induce small amounts of aggregates, were used to investigate the effects of different aggregation conditions on in vitro T cell responses. Moreover, the effects of differently stressed IgG on protein uptake and activation of MoDC was studied. We chose two frequently used therapeutics for these studies; a chimeric monoclonal IgG, rituximab, and a humanized monoclonal IgG, trastuzumab.
Analytical Characterisation of Aggregated Monoclonal Antibodies To test the effect of aggregate-containing therapeutics on T cell responses, monomeric antibody was exposed to stir, heat, or freeze/thaw stress conditions (Table I). The stress conditions were fine-tuned to ensure that for post-stress samples hardly any monomer was lost and the aggregates that were induced involved only a minor fraction of the total protein in the stressed samples. Therefore, these samples may reflect products that are administered to patients, e.g. after long-term or inadequate storage conditions or handling. Indeed, for all samples, hardly any loss of monomer was found after stress treatment, as determined by HP-SEC (Table I). For both antibodies, mechanical stress through stirring generated the highest level of micron-sized aggregates, as evidenced by light obscuration (increased levels of particles with a size of 1–10 μm) and by DLS (increased Z-average diameter/PDI score), indicating a very broad size distribution of aggregates in the nano- and low micron-size range. Heat-stressed aggregates for both rituximab and trastuzumab were mainly in the low nanometre-size range according to DLS (Table I). These aggregates were not detected by HP-SEC, although there was some monomer loss (Table I), indicating that they were too large or too sticky to pass through the HP-SEC column. Finally, freeze/thawing stress resulted in the formation of small (1 μm
>10 μm
>25 μm
Unstressed Stir Heat Freeze/thaw Unstressed Stir
100.0 (0.08) 103.1 (0.46) 98.7 (0.02) 100.5 (0.11) 100.0 (0.20) 96.7 (0.24)
99.1 99.1 99.3 98.9 97.5 97.7
10.2±0.3 1540±134 19.7±0.4 10.1±0.1 10.3±0.1 2203±857
0.09±0.02 1.00±0.00 0.38±0.14 0.18±0.01 0.14±0.02 0.77±0.24
2,989±287 98,978±2,125 34,836±1,314 4,511±520 13,554±904 896,544±33,073
117±58 60±10 133±29 28±19 17±0 33,073±67
22±0 0±0 11±19 0±0 6±10 0±0
Heat Freeze/thaw
96.9 (0.08) 95.6 (0.17)
97.6 96.8
28.7±6.2 53.3±30.1
0.11±0.01 0.14±0.03
12,982±1,021 80,925±10,404
6±10 278±67
antibody (Fig. 1), although this was not statistically significant. In contrast, rituximab stressed by freeze/thawing did not induce any increase in proliferation, consistent with the absence of any measurable aggregate formation after stressing the rituximab material (Table I). Interestingly, CD4+ T cell proliferation was significantly enhanced in the presence of all three stressed trastuzumab samples compared to the nonstressed sample (Fig. 1). Overall, these data suggest that minute amount of aggregates generated by different stress conditions can induce activation of T cells in vitro. This effect was significant for trastuzumab but not rituximab, most likely because of the intrinsic immunogenic potential of unstressed rituximab which is not significantly enhanced by its aggregation.
0±0 0
Cytokine Production in CD8+-Depleted PBMC Cultures Stimulated With Aggregated Humanized Antibody Proliferation data indicated different magnitudes of CD4+ T cell responses to stressed rituximab and trastuzumab. To examine whether such differences are reflected in cytokine production by PBMC, culture supernatants taken at day 4 of the in vitro T cell proliferation assays were assessed for the presence of inflammatory and anti-inflammatory cytokines (Fig. 2). Unstressed rituximab significantly increased TNF-α levels compared to the monomeric trastuzumab, confirming that rituximab is associated with increased immunogenicity compared to trastuzumab in its monomeric form (Fig. 2). In-keeping with no significant increase in CD4+ T cell proliferation, there was no significant difference in cytokines, including IL-2 and IFN-γ, produced in response to rituximab in the non-stressed form compared to the stressed conditions, although there was a trend towards increased TNF-α levels for the stressed samples. In contrast, all three stressed forms of trastuzumab induced significantly increased levels of IL-2 and IL-10 compared to non-stressed antibody. Although, incubation with stressed trastuzumab did not lead to increased levels of IL-21 in the entire cohort, 3 donors with increased CD4+ T cell proliferation and IL-2 production in response to trastuzumab, also showed increased levels of IL-21. Consequently these individuals had a strong correlation between IL21 secretion and increased IL-2 production. Phenotype of Dendritic Cells Stimulated With Aggregated Antibody
Fig. 1 Proliferation of CD4+ Tcells cultured in the presence of non-stressed or stressed rituximab and trastuzumab. Proliferation was measured by 3Hthymidine incorporation and is expressed as maximum stimulation index (SI) from days 5–8. F/T – freeze thaw. Statistical significance was determined by Student’s paired t-test by comparison to non-stressed antibody conditions; ** p
RESEARCH PAPER
S m a l l A m o u n t s o f S u b -V i s i b l e A g g r e g a t e s E n h a n c e the Immunogenic Potential of Monoclonal Antibody Therapeutics Maryam Ahmadi & Christine J. Bryson & Edward A. Cloake & Katie Welch & Vasco Filipe & Stefan Romeijn & Andrea Hawe & Wim Jiskoot & Matthew P. Baker & Mark H. Fogg
# Springer Science+Business Media New York 2014
ABSTRACT Purpose Determine the effect of minute quantities of sub-visible aggregates on the in vitro immunogenicity of clinically relevant protein therapeutics. Methods Monoclonal chimeric (rituximab) and humanized (trastuzumab) antibodies were subjected to fine-tuned stress conditions to achieve low levels (10 μm were present. Results were calculated as mean value of three measurements of 0.3 ml and referred to a sample volume of 1.0 ml.
Fluorescent Labelling of Monomeric and Aggregated Antibody For preparation of fluorescent-labelled rituximab aggregates for MoDC uptake experiments, Alexa Fluor 488 (A488) carboxylic acid, N-hydroxysuccinimide ester was obtained from Invitrogen (Merelbeke, Belgium). The fluorescent labelling of rituximab (rituximab-A488) was performed according to the manufacturer’s instructions, using an IgG concentration of 10 mg/ml and a molar ratio of 4:1 (dye: IgG). A pH of 7.4 was chosen for the labelling buffer, in order to achieve selective labelling of the amine termini. The rituximab-A488 was dialyzed back to PBS using a Float-A-Lyzer® G2 (Spectrum, Rancho Dominguez, CA, USA) with a 100 kDa molecular weight cut-off membrane to remove excess dye, and the labelling ratio achieved was about two A488 labels per rituximab molecule.
Preparation of Peripheral Blood Mononuclear Cells (PBMC) PBMC were isolated from healthy community donor buffy coats (from blood drawn within 24 h) obtained from the UK National Blood Transfusion Service (Addenbrooke’s Hospital, Cambridge, UK) and according to approval granted by Cambridgeshire Local Research Ethics Committee. PBMC were isolated from buffy coats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation and CD8+ T cells depleted using CD8+ RosetteSep™ (StemCell Technologies Inc., London, UK). PBMC were then frozen and stored in liquid nitrogen until required.
In Vitro T Cell Assays Where indicated healthy donors were selected based on broad representation of HLA-DR allotypes (40 donors for rituximab and 25 donors for trastuzumab). After donor selection, PBMC were thawed, counted, viability assessed and suspended in AIM-V® culture medium (Invitrogen, Paisley, UK) at 4–6 × 106 PBMC/ml. Bulk cultures were established for each donor where 1 ml of cell suspension was added to the wells of a 24well plate (Corning Life Science, Amsterdam, NL) along with 1 ml of antibody in AIM-V® culture medium. Proliferation of CD4+ T cells within the culture was measured on days 5, 6, 7 and 8 post-stimulation by gently re-suspending the cells in the bulk cultures followed by removal of 3 × 100 μl samples, which were transferred to a round bottomed 96-well plate for pulsing with 1 μCi/well tritiated thymidine (Perkin Elmer, Buckingham, UK). After 18 h the pulsed cultures were harvested onto filter mats (Perkin Elmer) using a TomTec Mach III cell harvester and counts per minute (cpm) for each well determined by Meltilex™ (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, low background counting mode.
Generation of Monocyte-Derived Dendritic Cells Monocytes were extracted from PBMC by negative selection with monocyte isolation kit II MACS beads (Miltenyi Biotech, Bisley, UK) according to the manufacturer’s instructions. The CD14+ monocytes were cultured at 1 × 106 cells/ml with 1000 IU/ml of IL-4 and GM-CSF (Peprotech, London, UK) in AIM-V® for 72 h to produce MoDC, which were stimulated with antibodies for a further 24 h and harvested before measuring cytokine secretion via cytometric bead array (CBA), protein uptake by phagocytosis (laser scanning confocal microscopy) or cell surface phenotype (flow cytometry).
Cytometric Bead Array Supernatants were collected from cultured cells and analyzed for cytokine production using BD CBA Enhanced Sensitivity Flex Set System (BD Biosciences, Oxford, UK), according to the manufacturer’s instructions. Briefly, thawed culture supernatant and serially diluted standards were incubated with premixed capture beads for 2 h followed by additional 2 h with detection reagents. The sensitivity of detection was enhanced following a 1 h incubation with enhanced detection reagent. Cytokines were acquired by a BD Accuri C6 instrument (BD Biosciences). Data were analyzed using FCAP v3.01 software (Softflow, Inc, Minnesota USA).
Ahmadi et al.
Rituximab Aggregate Uptake by MoDC Coverslips coated with MoDC (MoDC were initially grown on round coverslips) were transferred to 1 ml fresh AIM-V® medium and rituximab-A488 was added followed by incubation at 37°C or on ice (for control). At 0.5 h and 3 h the cells were washed three times with PBS followed by 15 min incubation on ice with BD Bioscience Cell Fix™. Cells were stained with HLA-DR PE (BD Bioscience) on ice for 30 min. Coverslips were inverted onto microscope slides with Vectashield (Vector Labs, Peterborough, UK) mounting medium. The stained cells were imaged with a Zeiss LSM 510 META laser scanning confocal microscope using a 40_NA 1.3 oil immersion objective. Single confocal slices were acquired to generate the data images. Three-dimensional reconstructions of confocal slices were made using Volocity software (Improvision, Perkin Elmer). Using image J software (http:// rsbweb.nih.gov/ij/) the intensity across the width of the images was plotted for each wavelength (grey scale).
Data Analysis For proliferation assays, a stimulation index (SI) was calculated as described previously [3]. Briefly, the counts/min for each donor treated with aggregated antibodies was divided by the background response. An SI≥1.9 was considered positive, based on a statistically derived cut point for the population tested for an adaptive response.
Statistical Analysis Statistical analysis was carried out using GraphPad Prism Software version 6 (GraphPad Software Inc, California, USA). Two sample paired t-test or one way repeated measure ANOVA were carried out (corrected for multiple tests where appropriate).
RESULTS In this study, therapeutic mAbs, stressed by a variety of pharmaceutically relevant methods to induce small amounts of aggregates, were used to investigate the effects of different aggregation conditions on in vitro T cell responses. Moreover, the effects of differently stressed IgG on protein uptake and activation of MoDC was studied. We chose two frequently used therapeutics for these studies; a chimeric monoclonal IgG, rituximab, and a humanized monoclonal IgG, trastuzumab.
Analytical Characterisation of Aggregated Monoclonal Antibodies To test the effect of aggregate-containing therapeutics on T cell responses, monomeric antibody was exposed to stir, heat, or freeze/thaw stress conditions (Table I). The stress conditions were fine-tuned to ensure that for post-stress samples hardly any monomer was lost and the aggregates that were induced involved only a minor fraction of the total protein in the stressed samples. Therefore, these samples may reflect products that are administered to patients, e.g. after long-term or inadequate storage conditions or handling. Indeed, for all samples, hardly any loss of monomer was found after stress treatment, as determined by HP-SEC (Table I). For both antibodies, mechanical stress through stirring generated the highest level of micron-sized aggregates, as evidenced by light obscuration (increased levels of particles with a size of 1–10 μm) and by DLS (increased Z-average diameter/PDI score), indicating a very broad size distribution of aggregates in the nano- and low micron-size range. Heat-stressed aggregates for both rituximab and trastuzumab were mainly in the low nanometre-size range according to DLS (Table I). These aggregates were not detected by HP-SEC, although there was some monomer loss (Table I), indicating that they were too large or too sticky to pass through the HP-SEC column. Finally, freeze/thawing stress resulted in the formation of small (1 μm
>10 μm
>25 μm
Unstressed Stir Heat Freeze/thaw Unstressed Stir
100.0 (0.08) 103.1 (0.46) 98.7 (0.02) 100.5 (0.11) 100.0 (0.20) 96.7 (0.24)
99.1 99.1 99.3 98.9 97.5 97.7
10.2±0.3 1540±134 19.7±0.4 10.1±0.1 10.3±0.1 2203±857
0.09±0.02 1.00±0.00 0.38±0.14 0.18±0.01 0.14±0.02 0.77±0.24
2,989±287 98,978±2,125 34,836±1,314 4,511±520 13,554±904 896,544±33,073
117±58 60±10 133±29 28±19 17±0 33,073±67
22±0 0±0 11±19 0±0 6±10 0±0
Heat Freeze/thaw
96.9 (0.08) 95.6 (0.17)
97.6 96.8
28.7±6.2 53.3±30.1
0.11±0.01 0.14±0.03
12,982±1,021 80,925±10,404
6±10 278±67
antibody (Fig. 1), although this was not statistically significant. In contrast, rituximab stressed by freeze/thawing did not induce any increase in proliferation, consistent with the absence of any measurable aggregate formation after stressing the rituximab material (Table I). Interestingly, CD4+ T cell proliferation was significantly enhanced in the presence of all three stressed trastuzumab samples compared to the nonstressed sample (Fig. 1). Overall, these data suggest that minute amount of aggregates generated by different stress conditions can induce activation of T cells in vitro. This effect was significant for trastuzumab but not rituximab, most likely because of the intrinsic immunogenic potential of unstressed rituximab which is not significantly enhanced by its aggregation.
0±0 0
Cytokine Production in CD8+-Depleted PBMC Cultures Stimulated With Aggregated Humanized Antibody Proliferation data indicated different magnitudes of CD4+ T cell responses to stressed rituximab and trastuzumab. To examine whether such differences are reflected in cytokine production by PBMC, culture supernatants taken at day 4 of the in vitro T cell proliferation assays were assessed for the presence of inflammatory and anti-inflammatory cytokines (Fig. 2). Unstressed rituximab significantly increased TNF-α levels compared to the monomeric trastuzumab, confirming that rituximab is associated with increased immunogenicity compared to trastuzumab in its monomeric form (Fig. 2). In-keeping with no significant increase in CD4+ T cell proliferation, there was no significant difference in cytokines, including IL-2 and IFN-γ, produced in response to rituximab in the non-stressed form compared to the stressed conditions, although there was a trend towards increased TNF-α levels for the stressed samples. In contrast, all three stressed forms of trastuzumab induced significantly increased levels of IL-2 and IL-10 compared to non-stressed antibody. Although, incubation with stressed trastuzumab did not lead to increased levels of IL-21 in the entire cohort, 3 donors with increased CD4+ T cell proliferation and IL-2 production in response to trastuzumab, also showed increased levels of IL-21. Consequently these individuals had a strong correlation between IL21 secretion and increased IL-2 production. Phenotype of Dendritic Cells Stimulated With Aggregated Antibody
Fig. 1 Proliferation of CD4+ Tcells cultured in the presence of non-stressed or stressed rituximab and trastuzumab. Proliferation was measured by 3Hthymidine incorporation and is expressed as maximum stimulation index (SI) from days 5–8. F/T – freeze thaw. Statistical significance was determined by Student’s paired t-test by comparison to non-stressed antibody conditions; ** p
