Establishing in vitro in vivo correlations to screen monoclonal antibodies for physicochemical properties related to favorable human pharmacokinetics

Abstract

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

Keywords: AC-SINS; FcRn binding; IgG; clearance, in vitro assays; mAb; monoclonal antibody, neonatal Fc receptor; pharmacokinetics; polyreactivity.

Figure 1.

In vitro score range for assay controls. Assay negative control mAbs (mAb-01 and mAb-02) and positive control mAbs (mAb-03 and mAb-04) were tested in multiple replicates in the A) DNA binding assay (•), insulin binding assay (□), and AC-SINS assay (∆); B) hFcRn SPR assay (•); and C) immobilized hFcRn affinity column chromatography assay measuring relative retention time (rRT; •) and peak width (PW; □). Assay positive control mAb-05 (AC-SINS only) represents a distinct class of mAbs having high AC-SINS and low DNA and insulin scores. Error bars represent standard deviation.

Figure 2.

In vitro assay score range for mAb datasets. Assay control values are compared to the assay range observed for clinical mAb analog and study mAb datasets in assays A) DNA binding; B) insulin binding; C) AC-SINS; D) immobilized hFcRn SPR; and E-F) hFcRn column chromatography rRT and PW. Assay negative control mAbs (mAb-01 and mAb-02, green) and positive control mAbs (mAb-03 and mAb-04 for all assays + mAb-05 for AC-SINS, red) represent mean values of assay replicates. Clinical mAb analogs (blue) consist of the variable regions of commercially available marketed mAbs grafted onto human IgG1/kappa or lambda constant regions. Study mAbs (black) were chosen from research programs to represent a spectrum of in vitro scores or observed human CL.

Figure 3.

In vitro assay correlation with in vivo CL from mAbs administered to Tg32 mice and allometrically scaled to project human CL. The projected human CL is plotted against the measurements for each mAb from the in vitro assays: DNA binding, insulin binding, AC-SINS, hFcRn SPR, and hFcRn column chromatography rRT and PW. The threshold for identification of rapid CL is defined at ≥0.32 mL/hr/kg (dotted horizontal lines). The in vitro assay thresholds for the de-selection of mAbs based on CL is indicated by the dotted vertical lines, and defined as assay scores of ≥11 (DNA binding), ≥11 (insulin binding), ≥11 (AC-SINS), ≤451.7 nM (hFcRn SPR), ≥1.6 min (hFcRn column rRT), and ≥1.8 min (hFcRn column PW), based on ROC analysis (Fig. S4). Solid lines indicate the region where both in vitro assay scores and in vivo CL fall below threshold. Spearman correlation (ρ) is indicated for each in vitro assay with projected CL. All relationships show correlative significance at p<0.05. Circles and triangles, represent linear CL. Squares, apparent linear CL. •, study mAbs. Green ▴, negative control mAb-01. Red ▴, positive control mAbs-03 and -04. Control mAbs-02 and -05 do not have corresponding CL projected from Tg32 mice.

Figure 4.

In vitro assay parameter correlation with in vivo CL from mAbs administered to humans. The observed CL of mAbs administered to humans is plotted against the measurements for each mAb from each of the in vitro assays: DNA binding, insulin binding, AC-SINS, hFcRn SPR, and hFcRn column chromatography rRT and PW. Threshold values reflect those shown in Fig. 3, where rapid CL is defined at >0.32 mL/hr/kg (dotted horizontal lines) and in vitro parameter thresholds are shown as vertical dotted lines. Solid lines indicate the region where both in vitro assay scores and in vivo CL fall below threshold. Spearman correlation (ρ) are not depicted as sparse data leads to values <0.6. Correlative significance (p < 0.05) is observed for human CL relationship to the AC-SINS assay, hFcRn column rRT and PW results. Circles and triangles, linear CL. Squares, apparent linear CL. •, study mAbs. Green ▴, negative control mAb-01. Red ▴, positive control mAb-04. Control mAbs-02, -03 and -05 do not have corresponding CL in human.

Figure 5.

Retrospective comparison of in vitro parameters and categories to human CL with triage assessment. Data from the set of study mAbs are rank-ordered by descending projected human CL from Tg32 mouse data. In vitro assay data and CL are scored as below threshold (green) or above threshold (red). Triage category: proposed decisions for each molecule based on the combined in vitro assay data are as follows: advance mAb candidate (green; accept mAbs scoring below threshold in 3/3 assay categories); characterize further (yellow; mAbs scoring above threshold in 1/3 or 2/3 assay categories); or do not advance mAb candidate (red; mAbs scoring above threshold in 3/3 assay categories). Key: Assay negative control (mAb-01) and positive controls (mAb-03 and -04) denoted in mAb listing. n/a indicates data not generated.

Figure 6.

Screening paradigm for PK risk mitigation during mAb discovery and lead selection. High-throughput assays (DNA and insulin binding; AC-SINS) are implemented when hundreds of mAbs are available for screening. MAbs scoring below threshold in both categories would be accepted. Following additional screens including biological activity, expression, stability, etc., mAb panels of 10–50 would be screened by matrix-immobilized hFcRn binding interactions. MAbs with below-threshold scores in this category would be accepted and only those with 1/3 or 2/3 categories above threshold would require further characterization for de-risking PK.