Impact of a Heat Shock Protein Impurity on the Immunogenicity of Biotherapeutic Monoclonal Antibodies
Anti-drug antibodies can impair the efficacy of therapeutic proteins and, in some circumstances, induce adverse health effects. Immunogenicity can be promoted by aggregation; here we examined the ability of recombinant mouse heat shock protein 70 (rmHSP70) - a common host cell impurity - to modulate the immune responses to aggregates of two therapeutic mAbs in mice.
Heat and shaking stress methods were used to generate aggregates in the sub-micron size range from two human mAbs, and immunogenicity assessed by intraperitoneal exposure in BALB/c mice.
rmHSP70 was shown to bind preferentially to aggregates of both mAbs, but not to the native, monomeric proteins. Aggregates supplemented with 0.1% rmHSP70 induced significantly enhanced IgG2a antibody responses compared with aggregates alone but the effect was not observed for monomeric mAbs. Dendritic cells pulsed with mAb aggregate showed enhanced IFNγ production on co-culture with T cells in the presence of rmHSP70.
The results indicate a Th1-skewing of the immune response by aggregates and show that murine rmHSP70 selectively modulates the immune response to mAb aggregates, but not monomer. These data suggest that heat shock protein impurities can selectively accumulate by binding to mAb aggregates and thus influence immunogenic responses to therapeutic proteins.
KEY WORDSadaptive immunity aggregation host cell impurity immunogenicity monoclonal antibody
Asymmetric flow field flow fractionation
Antigen presenting cells
Bone marrow-derived dendritic cells
Bovine serum albumin
Chinese hamster ovary
Dynamic light scattering
Host cell protein
Normal mouse serum
Phosphate buffered saline
Raster image correlation spectroscopy
Recombinant mouse heat shock protein 70
Region of interest
Single chain variable fragment
Monoclonal antibodies (mAbs) are the fastest growing sector of the global pharmaceutical industry, particularly for the treatment of cancer and inflammatory diseases [1,2]. Unwanted immunogenicity and the formation of anti-drug antibodies (ADAs) is a significant challenge for the use of biotherapeutics, even for those that have been humanized , impacting safety and efficacy. A variety of factors can influence immunogenicity: these include product-related factors such as protein conformation or impurities, patient-related variables such as immune and genetic background, disease status and treatment-related factors such as route and duration of exposure [4, 5]. Aggregation is an important factor which has been implicated in immunogenicity and its likelihood is increased with the use of mAbs at high concentrations [6, 7, 8]. The link between aggregation and enhanced immunogenic responses is well established in mouse models (including transgenic animals). For example, aggregate percentage and the extent of denaturation of interferon beta-1a (IFNβ-1a) have been shown to influence the ability of aggregates to break tolerance in transgenic mice . Only aggregates that retained native epitopes were able to stimulate a transient immune response and their removal prevented the breakdown of tolerance . Aggregation is thought to be initiated by association of partially unfolded conformational states which can occur at any stage of bioprocessing and storage. They range in size and dimensions in the 0.1-10 μm range have been identified as being the most immunogenic . Characteristics of aggregates which may contribute to immunogenic potential include the formation of neo-epitopes, multiple valency, post-translational modifications, concentration and size [11, 12, 13]. Despite the wealth of evidence demonstrating that aggregation results in enhanced immunogenicity, the underlying mechanisms are incompletely understood .
The removal of host cell protein (HCP) impurities is an important problem in the isolation of biologics for clinical use . HCPs originate from the expression host, most commonly Chinese Hamster Ovary (CHO) cells for monoclonal antibodies. Measurement of total HCP content is commonly conducted by ELISA: null cell line isolates are used to immunize mice and obtain polyclonal antisera. This method is limited, however, in that it is dependent on the total average response to a crude mixture of HCPs which will, individually, have differential abilities to stimulate antibody production . HCPs have been shown to influence immunogenicity as antigens or adjuvants [16,17], through HCP-induced protease activity  or direct biological activity , highlighting the need for HCP identification and individual quantification.
Heat shock proteins (HSPs) are a common HCP impurity ; they participate in housekeeping functions and are part of responses to environmental stresses [20,21]. Their chaperone activity in macromolecular complex assembly, protein transport and degradation acts to stabilize and correct the folding of nascent polypeptides de novo, dissociating aggregates, and re-folding stress-denatured proteins . Stresses can exacerbate protein conformational problems, exceeding the capacity of chaperone systems to prevent aggregation. In addition, the combined use of HSPs has been explored as a strategy for enhancing vaccine potency . Studies have shown, for example, that HSP-peptide complexes successfully elicited MHC class I restricted cytotoxic T lymphocyte responses, whereas HSP or peptide alone were not immunogenic, establishing the tumor-derived HSP gp96 as the first adjuvant of mammalian origin . HSP-based cancer vaccines, such as artificially reconstituted HSP peptide antigen complexes, have been widely exploited . In addition, the adjuvant property of mycobacterial Heat Shock Protein 70 fusion protein has been demonstrated using a variety of model antigens . Our laboratory has recently shown that low levels of an E. coli HSP, DnaK, were able to enhance the immunogenicity of a recombinant 25 kDa human single chain variable fragment (scFv) following immunization of BALB/c strain mice . HSPs therefore have the potential to function as adjuvants.
The principal aim of the current investigation was to establish whether this adjuvant-like effect could also be observed with aggregated human biotherapeutic mAbs and a cognate mammalian HSP, similar to that found in CHO cells. To this end, we used recombinant mouse HSP70 (rmHSP70), an ortholog of E. coli DnaK which is 98% identical to CHO HSP70 . We show that rmHSP70 binds preferentially to aggregates and is able to exert an adjuvant-like effect on immune responses in a BALB/c mouse model. The implications for the contribution of HCPs to the immunogenicity of therapeutic protein aggregates are discussed.
Materials and Methods
Female BALB/c strain mice (8–12 weeks old) were used for these experiments (Envigo, Bicester, UK). Mice were housed on sterilized wood bedding with materials provided for environmental enrichment. Food (Beekay Rat and Mouse Diet No1 pellets; B&K Universal, Hull, UK) and water were available ad libitum. The ambient temperature was maintained at 21 ± 2°C and relative humidity was 55 ± 10% with a 12 h light/dark cycle. All procedures were carried out in accordance with the Animals (Scientific Procedures) Act 1986, and approved by Home Office licence.
Two human monoclonal antibodies were used for the current study, hereafter referred to as mAb1 and mAb2. mAb1 has a theoretical molecular mass of 148 kDa and an experimentally measured pI of 7.6. mAb2 (a bispecific antibody) has a theoretical molecular mass of 204 kDa and an experimentally measured pI of 9.1. Both the mAbs were provided by MedImmune (Cambridge, UK).
Aggregate Formation and Spiking with rmHSP70
Purified mAbs were diluted into 1 mg/mL in Dulbecco’s phosphate buffered saline (DPBS) without Ca+2 or Mg+2 (Sigma-Aldrich, St Louis, Missouri). In order to form aggregates of mAb1 by thermal stress, it was treated at 60°C for 25 min. To generate mAb1 aggregates using shaking stress, the solution at 1 mg/mL was shaken in a bench top shaker at 3000 rpm for 12 h at 22°C. mAb2 aggregates were formed by shaking stress in the same way, but at 1500 rpm for 4 h at 22°C. rmHSP70-aggregate complex samples were prepared by addition of rmHSP70 (Enzo Life Sciences, UK) to 0.1% by mass into the mAb aggregate within 5 min of mAb aggregation. The aggregates formed were stable and did not dissociate into monomers when the temperature was subsequently decreased by refrigeration, or after storage at −80°C.
Dynamic Light Scattering (DLS)
Measurements of DLS were performed using a Malvern Zetasizer Nano ZS ZEN3600 (Malvern, Herrenberg, Germany), equipped with a 633 nm laser. Each sample (70 μL) was measured in a Suprasil® quartz cuvette (Hellma GmbH, Muellheim, Germany) with a path length of 3 mm and 200–2500 nm spectral range. Monomeric and stressed samples at 1 mg/mL were measured at 25°C to determine the volume-based average protein particle diameter in solution.
Raster Image Correlation Spectroscopy (RICS)
SYPRO® Red (Molecular Probes, Oregon) was prepared as a 50x stock solution in pre-filtered histidine-sucrose buffer and diluted to a final working concentration of 2.5x for fluorescence studies immediately prior to use (all solutions were prepared on the day of use) . SYPRO® Red was added 15 min prior to visualization with confocal microscopy. A Zeiss 510 Confocor 2 (Zeiss, Jena, Germany) confocal microscope equipped with a c-Apochromat 40×/1.2NA water-immersion objective was used for image acquisition. Imaging was carried out by exciting the dye with a Helium-Neon laser at 543 nm and the emitted fluorescence collected above 585 nm (LP585 filter set). A confocal image time series of 1024 × 1024 pixel resolution was captured over 100 frames with a corresponding pixel dwell time of 6.4 μs. In-house RICS software (ManICS) was applied to analysis of images acquired using confocal microscopy. A full description of the RICS algorithm has been described elsewhere [28,29]. The image time series were sub-divided into 32 × 32 pixel sub-regions and the diffusion coefficients (D) of each region of interest (ROI) was generated. The method is described in greater detail previously .
Asymmetric Flow Field Flow Fractionation (AF4)
Asymmetric flow field-flow fractionation (AF4) is used to measure aggregate content and molecular mass distribution, as an orthogonal method to size exclusion chromatography or analytical ultracentrifugation. In the current study, aggregates of mAb1 (generated by thermal stress) and mAb2 (generated by shaking stress) in the presence and absence of rnHSP70 were prepared as described above. Separation was achieved by a liquid cross-flow which takes place in a narrow, ribbon-like channel of trapezoidal geometry, which is built up by a spacer, between a porous and a nonporous plate. The porous plate is covered by a membrane, which acts as accumulation wall and allows the eluent to pass the membrane, while the particles/macromolecules are retained . Water was used as solvent for the method at 25°C, with 30 min analysis time and 0.294 s sampling time. The total area of the peaks and the molecular mass of the rmHSP70-aggregates were measured. Corresponding monomer mAbs (with and without rmHSP70) were used for comparison. The ASTRA chromatography software (Wyatt Technology) was used to analyse the data.
rmHSP70 (low endotoxin) was purchased and was obtained commercially labelled with BODIPY FL by Life Technologies, USA. BODIPY FL labelled rmHSP70 (0.1%) was added to the thermal stressed mAb1 and shaking stressed mAb2 aggregates. 2.5x SYPRO® Red dye was added to the aggregated mAb immediately before analyzing the sample using a Zeiss 510 Confocor 2 microscope.
Protein samples were diluted in SDS-PAGE sample buffer (Bio-Rad, Berkley, CA, USA) containing 1% (v/v) 2-mercaptoethanol and heated for 5 min at 90°C. Samples were resolved on a pre-cast NuPAGE 4–12% Bis-Tris Protein gels (Invitrogen™) and stained using InstantBlue™ Coomassie protein stain (Expedeon, Swavesey, UK).
Protein samples were resolved on pre-cast 4–12% acrylamide gel at various concentrations (0.1, 0.01, 0.001, 0.005 and 0.05 μg/mL in PBS), transferred onto nitrocellulose membrane and detected using anti-HSP70 horseradish peroxidase (HRP) conjugated antibody (StressMarq Bioscience Inc., Victoria, British Columbia, Canada) diluted at 1:2000. Proteins on the blots were visualized using enhanced chemiluminescence reagents (Thermo Scientific).
Monomer and aggregate samples were prepared as described previously. rmHSP70 was added at 1:1000 ratio by mass to monomer and aggregated mAbs. For monomeric samples, where pellets were not observed, 30 μl PBS was added to the tube to dissolve any sedimented material. Protein samples were resolved on a pre-cast 4–12% acrylamide gel and transferred onto a nitrocellulose membrane. The presence of rmHSP70 was detected using anti-HSP70 horseradish peroxidase (HRP) conjugated antibody and blots were visualized using enhanced chemiluminescence reagents, as described above.
Mice (n = 3 or 5 per group) were immunized by intraperitoneal (I.P.) injection on days 0, 7 and 14 and were exsanguinated on day 21. Animals were immunized with 250 μL of 1 mg/mL of mAb1 and 150 μL of 1 mg/mL of mAb2 (monomeric or aggregated) in PBS with or without rmHSP70 at a ratio of 1 in 1000 (0.1% by mass) relative to the immunizing mAb immediately after aggregate formation. Individual serum samples were prepared and stored at −80°C until analysis.
ELISA for Analysis of Serum from mAb Immunized Mice
To analyse the serum from mAb immunized mice, plastic Maxisorb® plates (Nunc, Copenhagen, Denmark) were coated with 0.1 mg/mL monomer or 0.05 mg/mL aggregate mAbs in PBS overnight at 4°C. Protein-coated plates were blocked with 2% (w/v) bovine serum albumin (BSA)/PBS (Sigma Aldrich) at 37°C for 30 min. Doubling dilutions of serum samples were added (starting dilution 1:140 for IgG, 1:1120 for IgG1, 1:35 for IgG2a and 1:70 for IgM) prepared in 1% BSA/PBS and plates incubated for 3 h at 4°C. Negative control naïve mouse serum (NMS) or PBS sham control mouse serum samples were analyzed concurrently. Plates were incubated for 2 h at 4°C with HRP labelled sheep anti-mouse IgG diluted 1:4000, sheep anti-mouse IgG1 diluted 1:2000 (both Bio-Rad) or goat anti-mouse IgM diluted 1: 6000 (Invitrogen, Paisley, UK), diluted in 1% BSA/ PBS. For IgG2a ELISAs, MCA1588P rat anti mouse IgG2a HRP-heavy chain antibody (Bio-Rad) was used for mAb1 and STAR133P goat anti mouse IgG2a HRP antibody (Bio-Rad) was used for mAb2 (both at 1:1000 dilution). Plates were washed between incubations with 0.05% Tween 20 in PBS. For color development, plates were incubated with substrate (1.6 mg/mL o-phenylenediamine and 0.4 mg/mL urea hydrogen peroxide in 0.5 M citrate phosphate buffer (pH 5)), 100 μL/well, for 15 min in the dark and reactions were stopped with 50 μL/well of 0.5 M citric acid. Absorbance was read at 450 nm using an automated reader (ELx800; BioTek Instruments, Inc., Winooski, US), using the Gen 5 1.10 software. Data are displayed with respect to antibody titre (log2) calculated as the lowest serum dilution at which a 3x serum blank OD450nm reading was reached.
[3H]Thymidine Splenocyte Proliferation Assay
A single cell suspension of splenocytes from immunized mice was prepared using mechanical disaggregation. Splenocytes were stimulated in vitro with respective protein samples for 7 days. 5 × 104 cells/well splenocytes were co-cultured with 5 × 103 bone marrow derived dendritic cells (BMDCs) pulsed with the protein immunogen or no protein (for control) in quadruplicate in round-bottom 96-well plates . [3H]thymidine incorporation proliferation assay method is described elsewhere . Cultures were incubated for approximately 60 h at 37°C, 5% CO2 and [3H]thymidine (3HTdR) (PerkinElmer, Waltham, MA, USA) was added at 37 kBq/well for the final 18 h, plates were harvested onto glass fibre filter mats with a multichannel semi-automated harvesting device (Titertek, Skatron AS, Lierbyen, Norway) and quantified with β scintillation counting. Results are presented as counts per min (cpm) as means of quadruplicates as described previously .
Measurement of IFNγ Secretion Using ELISpot Assay
Splenocytes from immunized mice were cultured ex vivo using mAb1 or mAb2. BMDCs prepared as described previously were used as Antigen Presenting Cells (APCs) . ELISpot assays were performed according to the manufacturer’s protocol (Mabtec, Nacka Strand, Sweden). Aliquots of 5 × 104 cells/well were assayed in triplicate. The plates were developed after 48 h with BCIP (5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt) and NBT (p-nitro blue tetrazolium chloride) color development solution (Bio-Rad) for 30–45 min and plates were rinsed with tap water. Spots were quantitated with an ELISpot reader (Cellular Technology Limited, Bonn, Germany). An animal was scored as positive when the response in the peptide-containing well was at least twice that of control wells, as described previously .
Statistical analyses were performed using Graphpad Prism 7. Analysis of variance (ANOVA) was used to determine statistical significance of differences between groups. Experiments were analyzed by non-parametric one way or two way ANOVA followed by Tukey’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).
Characterization of Therapeutic mAbs in Native and Aggregated States
Binding of rmHSP70 to mAb1 and mAb2 Aggregates
Asymmetric Flow Field Flow Fractionation (AF4)1
Elution time (min)
Effect of rmHSP70 on Immune Responses to mAb1 and mAb2 in BALB/c Model
In vivo approaches have been usefully adopted to compare immune responses elicited by aggregated proteins and their monomeric counterparts. A common approach is to measure antibody responses provoked in mice by immunizations with the monomeric or aggregated protein [33,34]. A BALB/c mouse model was therefore used in the current study to assess the immune responses generated following intraperitoneal (I.P.) immunization with monomer, rmHSP70-monomer, aggregated or rmHSP70-aggregated human mAbs .
In order to investigate whether these observations were particular to mAb1, mAb2 was investigated in a similar manner. Preliminary studies performed using mAb2 indicated that lower doses were needed to obtain measurable serum antibody responses in immunized mice (data not shown), and a dose of 150 μg mAb2 was used for the ensuing experiments. Mice immunized with shaking stress-induced mAb2 aggregates demonstrated increased IgG, IgG1, and IgG2a responses compared to immunization with monomer (Fig. 5c) and, as recorded for mAb1, rmHSP70 further enhanced IgG2a antibody production. A specific anti-mAb2 IgM antibody response was recorded for aggregate-treated mice, but not monomer. Similar patterns of antibody responses were observed in ELISAs for both monomer and aggregate-coated plates of mAb1 and mAb2.
Protein aggregation can be described as the self-association of monomers in their native or partially unfolded forms [35,36], and is a common phenomenon observed in biopharmaceutical preparations. There is evidence that aggregates can stimulate an anti-drug immune response which may impair drug efficacy. However, the mechanisms through which immunogenicity is enhanced or conferred on proteins are only poorly understood . Recently we reported that IgG2a antibody responses to an aggregated model scFv were stimulated in a mouse model by addition of a low (0.1%) level of a bacterial HSP, DnaK . This observation led us to investigate whether this effect was also observed with human mAbs and a murine HSP. Aggregates that may be present in protein products can range from dimers to subvisible or visible particles. They can be formed during different stages of production, transport or delivery to the patient, in response to diverse stresses [35,38]. Aggregates formed in biotherapeutic monoclonal antibodies under the influence of various stresses have been characterized by various techniques on the basis of their sizes, ranging from nm to micron dimensions [39, 40, 41, 42]. The mAb aggregates employed in this study fall within this range and can therefore be regarded as typical, at least in terms of size, compared with those studied previously.
It is well established that HSP family members recognize exposed hydrophobic residues of misfolded or denatured proteins which are retained for ubiquitination and subsequent targeting to the proteasome for degradation [20,43]. Using co-sedimentation and western blotting, we have shown that rmHSP70 bound to aggregates of both mAbs. Similar phenomena were observed when aggregates were analyzed using AF4 where the presence of rmHSP70 had an effect on the migration profile of each mAb. Supporting these observations, microscopy demonstrated that fluorescently labelled rmHSP70 bound to the aggregated mAbs. Although the level of rmHSP70 used in these studies is high by industry standards (1:1000), our results provide evidence that HSP70 impurities can be selectively accumulated by adhesion to aggregates. This opens up the possibility that HSPs in general can selectively accumulate by adhesion to aggregated material, even though the overall levels of HSPs are low.
Our observations on IgG subclass specificity are consistent with those made previously by Ratanji et al. , which showed a stimulation of IgG2a levels characteristic of Th1-skewing of the immune response (Fig. 5). Interestingly, no increases in IgM antibody levels above PBS controls were detected for the immunizations of mAb1. This observation could be due to the immunogenic potential of mAb1, the serum IgM repertoire and the lifespan of IgM in the in vivo system . Additional evidence for the stimulatory effects of rmHSP70 was obtained from cellular proliferation assays, using splenocytes cultured with antigen-primed DCs. Splenocyte proliferation was elevated in the group of mice immunized with rmHSP70-aggregated protein, compared with those immunized with aggregate alone, demonstrating an enhanced CD4-mediated T cell response. Stimulation of IFNγ secretion was particularly pronounced for mAb1 aggregates in splenocytes from the mice which received rmHSP70-aggregate, compared with aggregate alone. We think that the enhancement of mAb immunogenicity by rmHSP70 which we observe is unlikely to be attributable to its immunogenicity, for several reasons. First, the levels of rmHSP70 used were very low (0.1% of total protein inoculated). It is remarkable that such a low level of impurity can drive significant responses to IgG2a (Fig. 5) and splenocyte proliferation (Fig. 6a). Second, the Cricetulus griseus (Chinese Hamster) HSP70 is 98% identical to the mouse HSP70 sequence. Third, we do not observe an enhancement of IgG2a levels for monomer compared with rmHSP70-monomer, only for aggregate compared with rmHSP70-aggregate (Fig. 5).
Previous studies using mouse models have shown that HSP70 can be used as an adjuvant in cancer vaccine development strategies [45,46]. HSPs have therefore been harnessed as adjuvants of vaccines against cancers and infectious diseases : examples include Phase I clinical trials for Glioblastoma (HSP70), colon carcinoma, Phase I-II studies for Non-small cell lung carcinoma (HSP70-activated NK cells) and a Phase I HIV vaccine study . The adjuvant-like activity of HSP70 has been demonstrated in vitro by coupling to superparamagnetic iron oxide nanoparticles (SPIONs) to form HSP70-SPION conjugates. HSP70-SPIONs have shown effective delivery of immunogenic peptides from tumor lysates to DCs, stimulating a tumor-specific, CD8+ cytotoxic T cell response in experimental glioma models . It is also known that HSP–peptide complexes can act as typical tumor-specific foreign antigens, chaperokines and adjuvants that facilitate uptake, processing, and presentation for tumor-specific antigens which are cross-presented by APCs to T lymphocytes . The influence of HSPs on the induction of immunogenicity therefore appears to be a general phenomenon, rather than being confined to specific HSP-immunogen pairs.
HSP70 is released from cells in response to stress conditions ; more generally, intracellular proteins will be released by damaged, necrotic cells in a passive manner . It is therefore not surprising that HSP family members have been recorded as HCP impurities in purified mAbs [15,52]. HSP70 is listed as a persistent HCP impurity in 29 mAb preparations following cross-interaction or Protein A affinity chromatography . Accepted limits of HCP impurities are generally between 1 and 100 ppm ; ostensibly this would appear to be a low level but it does not take account of selective adsorption to aggregates, which would increase the apparent local concentration. Doses of therapeutic mAbs are well above 100 mg [55,56], so an impurity level of 0.1% of a particular HCP would result in administration of 100 μg, a level which could influence immunogenic responses to aggregates.
It has been reported that HSPs interact with and activate the immune system via Toll-like receptors on antigen-presenting cells [57,58]. It is also possible that aggregation itself enhances antigen uptake, and the presence of DnaK within the complex somehow enhances processing and presentation once inside the antigen-presenting cell. It is interesting to note that elevated levels of HSPs have been reported in the plasma of patients with certain illnesses such as dyslipidaemia , prostate cancer , and neurodegenerative diseases .
These observations confirm and extend our previous work on DnaK/scFv , and provide additional evidence that the adjuvant-like effects of HSPs are general, rather than specific, to particular HSP/immunogen pairs. The precise mechanism(s) for the adjuvant effects of HSPs are currently a matter of debate, however. It is therefore difficult to predict which HSPs, or indeed other categories of HCPs, might act in this way and, if so, at what levels. Here we have used HSP70 as an exemplar HCP but our observations open up the possibility that a complex mixture of multiple HCPs may have synergistic effects on the immunogenicity of biotherapeutic mAbs. The implications for the immunogenicity of therapeutic mAbs in humans are harder to discern. We would argue that further work is needed to investigate the nature of this effect and determine the extent to which it contributes to the immunogenicity of aggregates of biopharmaceutical drugs.
ACKNOWLEDGMENTS AND DISCLOSURES
We thank Lorna Beresford for her valuable support with animal work and Angela Thistlethwaite for her help with gel electrophoresis. We would also like to thank the Faculty Biomolecular Core Facility for DLS for instrument support and expertise. S. Rane was employed on a grant from MedImmune to carry out this research project. J. Derrick and the University of Manchester have received research grants from MedImmune. The other authors declare that they have no other relevant conflicts of interest.
This work was funded by MedImmune.
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