Structural Characterization of a Monoclonal Antibody–Maytansinoid Immunoconjugate

Structural characterization was performed on an antibody–drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solvent-exposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0–8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the C_H2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the C_H2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the C_H2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody

Chemical Modification by glycation does not enhance the response of PBMC in the IVCIA assay.

<p>Three representative mAbs (Avastin, mAb1, and Humira), that were treated by glycation with different sugars (galactose, glucose and mannose, and a non-glycating sugar, sorbitol), were tested in the IVCIA assay at the early (20 h) (n = 11 donors for most samples) and late (7 day) phases (n = 6 donors). Avastin, mAb1, and Humira have low, medium, and high rates of clinical or predicted immunogenicity, respectively. Heatmaps depict the percentage of donors that responded to the glycated mAbs above the original forms of each molecule (mAb before glycation treatment) and were at least two fold above the background, to highlight the differential response that might be due to glycated mAbs. The percentage of donors with increased secretion of signature cytokines in adherent monocytes at the early phase (red), and in PBMC at the early (yellow) and late (green) phases is highlighted. The percentage of donors with an increase in the number of IFN-γ secreting cells is shown on the far right (blue). The grey boxes show low level responses that were observed in less than or equal to 20% of donors, in contrast to colored boxes (red, yellow, green and blue) that show responses in a greater number of donors (40–100%).</p

High numbers of aggregates of a wide array of antibodies can be detected by the IVCIA assay.

<p>10 mAbs (with known or predicted rates of clinical immunogenicity) were aggregated by stirring stress and then evaluated in the IVCIA assay in a population of 50 heathy human donors. The percentage of donors that responded to the original mAb (<i>solid bars</i>) and the aggregated mAb (<i>striped bars</i>) by A) positive T-cell proliferative responses ([³H]-thymidine uptake) or B) an increase in the number (No.) of IL-2 secreting cells (Elispot) over the course of the entire study (5–8 days) are displayed. The percentage of donors that showed C) either a positive T-cell proliferation response or an increase in the concentration of IL-2 secreted (multiplex cytokine analysis) or D) a positive T-cell proliferation response and an increase in the number of IL-2 secreting cells are depicted. The y-axes of graphs in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.g004" target="_blank">4</a> are on different scales. Not all donors were tested for IL-2 concentration for some samples (<i>grey circles</i>). A response was considered positive if the SI ≥ 2.0 (<i>p</i><0.05) for proliferation or number of IL-2 secreting cells, or the SI ≥ 1.9 for IL-2 concentration (above the background response). Borderline responses were also included in some cases (SI≥1.9, <i>p</i><0.05), and are shown with one asterisk. mAbs are in the same order as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.g001" target="_blank">Fig 1</a>. The scale bars at the top of each graph show the highest incidence of immunogenicity reported for each mAb in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.t001" target="_blank">Table 1</a>. All rates are associated with diverse disease indications and assay testing platforms with variable sensitivity. <i>Black circles</i> show the responses in duplicate experiments in different sets of 50 donors.</p

Particle counts, size distribution, and morphology of stirring induced aggregates of a broad array of antibodies.

<p>10 mAbs (with known or predicted rates of clinical immunogenicity) were aggregated by stirring stress and then examined for their subvisible and visible aggregate content. A) Aggregates were quantitated by HIAC to determine the number and size range of particles present. Bar height represents the differential particle counts per ml in each size range (average of 3 runs). B) Aggregates images were captured on a Micro-flow Imaging System to evaluate the morphology of particles present. Representative images of the largest particles detected are shown. The size threshold indicates the lower size limit of the particles that were used for comparison. The aggregate content of the original mAbs (before stirring stress) is also shown and highlights that only a few small sized aggregates were detected.</p

Biotherapeutics with minor differences in sequence can be distinguished by the IVCIA assay.

<p>Several sequence variants of FP1 varying by one (mutant-1 and mutant-2) or two (mutant-3) amino acids, as well as FP1 lacking the Fc domain (FP1 (no Fc)) were tested in the in the IVCIA assay using 50 healthy human donors over 5–8 days. FP1 is a biotherapeutic fusion protein of an enzyme fused to the Fc domain of a monoclonal antibody. The percentage of donors with either A) a positive T-cell proliferative response ([3H]-thymidine uptake) or B) an increase in the number (No.) of IL-2 secreting cells (Elispot), or C) both positive T-cell proliferative responses and an increase in the number of IL-2 secreting cells are shown. A response was considered positive if the SI≥2.0 (<i>p</i><0.05) above the background response.</p

Stages of biotherapeutic development where the IVCIA assay can be used for risk assessment related to attributes.

<p><i>In silico</i> or algorithm based assessments rank order and identify lead candidates based on the least sequence based risk. <i>In vitro</i> assessments identify non-sequence attributes and any immune mediated target based risk at preclinical stage prior to first in human (FIH). Pharmacogenomic assessments for HLA can be introduced in long-term clinical studies (Phase 1b/2) to evaluate associations of HLA with clinical immunogenicity. Immunogenicity assessments measured in serum as ADA span the breadth of clinical trials and disease indications (FIH to Launch/post launch). <i>In vitro</i> assays also provide attribute related risk assessment of manufactured lots, lot-to-lot comparability, and risk post packaging due to attributes related to storage, shipping, handling, and device-related leachates.</p

The response of CD4⁺ T-cells in the IVCIA assay agrees with the rate of clinical immunogenicity for biotherapeutic mAbs.

<p>10 mAbs, with known rates of clinical immunogenicity, were evaluated in the IVCIA assay in a population of 50 healthy human donors over 5–8 days. mAb1 has not been tested in the clinic. Donors that responded by multiple readouts were evaluated for the most effective relative risk ranking. The percentage of donors that showed A) a positive T-cell proliferative response ([³H]-thymidine uptake) or B) an increase in the number (No.) of IL-2 secreting cells (Elispot) over the course of the entire study are displayed. Results were also combined to illustrate the percentage of donors that showed C) either a positive T-cell proliferation response or an increase in the concentration of IL-2 secreted (multiplex cytokine analysis) or D) a positive T-cell proliferation response and an increase in the number of IL-2 secreting cells are shown. Not all donors were tested for IL-2 for some samples (<i>grey circles</i>). A response was considered positive if the SI ≥ 2.0 (<i>p</i><0.05) for proliferation or number of IL-2 secreting cells or the SI ≥ 1.9 for IL-2 concentration (above the background response). mAbs are ordered approximately within each graph from the lowest to the highest response in the IVCIA assay. The scale bars at the top of each graph show the highest incidence of immunogenicity reported for each mAb in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159328#pone.0159328.t001" target="_blank">Table 1</a>. All rates are associated with diverse disease indications and assay testing platforms with variable sensitivity. <i>Black circles</i> represent duplicate experiments in different sets of 50 donors.</p

The IVCIA assay can be used to compare mAb lots from both the same manufacturer and from different manufacturers (biosimilars).

<p>A) Several different lots of Erbitux (2 lots), Remicade (2 lots) and Humira (5 lots) were tested in the IVCIA assay for a response at the early (20 h) phase (n = 12 donors), at 40 μg/mL. The concentration (pg/mL) of signature cytokines that were secreted 20 hours after stimulation is shown. B) Biosimilars, Humira and ABP 501, from two different manufacturers were compared in the IVCIA assay for the secretion of signature cytokines at the early (20 h) and late (7 day) phases (n = 4 donors), at 100 μg/mL. C) Multiple lots of another set of biosimilars, Herceptin and ABP 980, from two different manufacturers (which are both associated with a low rate of clinical immunogenicity), were assessed in the IVCIA assay at 40 μg/mL for T-cell proliferative responses on Day 7 only. No statistically different responses in the assay were observed between different lots of Herceptin and mAb5-B (p = 0.12). In all assays, the average response across the population tested was similar, although slightly different responses in specific donors could be observed. In all panels, <i>bars in shades of white and grey</i> show the average level of cytokine secretion at the early and late phases, and <i>purple bars</i> depict the average level of T-cell proliferation, across the population. <i>Colored and black circles</i> represent the response of individual donors and show the variability of the population tested.</p

Use of <i>In Vitro</i> Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics

<div><p>An <i>In Vitro</i> Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed.</p></div