Figures & data
© 2023 Affimed GmbH. Published with license by Taylor&Francis Group, LLC
Figure 1. FcRn-Ph-HPLC is capable of reliably assessing the FcRn dissociation pH of mAbs, which can be correlated with half-life established in vivo in humans.
(a) Bar graph depicting a single bar, with error bars and 15 individual data points, showing the FcRn dissociation pH of NIST mAb control antibodies. (b) Bar graph of FcRn dissociation pH values for cusatuzumab, cetuximab, margetuximab, durvalumab, rituximab, adalimumab, and briakinumab. (c) Bar graph showing differences in FcRn dissociation pH for cusatuzumab, cetuximab, margetuximab, durvalumab, rituximab, adalimumab, and briakinumab from physiological pH (7.4), with data points/ranges depicting the mean of the previously established, human half-life values in humans for each antibody, as reported in the prescribing information or a representative study.
Notes: (a) FcRn dissociation pH of NIST mAb control antibody as assessed by FcRn-pH-HPLC. Mean ± SD of pH values are shown from 15 independent experiments. (b) Means ± SD are depicted for each antibody, calculated from three independent experiments. (c) Bars: Mean difference in measured FcRn dissociation pH from pH of physiological IgG in vivo (pH 7.4) as established by FcRn-pH-HPLC. Values were calculated using the following: FcRn dissociation pH of physiological IgG (pH 7.4) ˗ mean FcRn dissociation pH of the indicated antibody. Mean ± SD are shown for each antibody. Data points/ranges (in green): In vivo half-lives in human of the indicated therapeutic mAbs, as established previously in clinical studies and presented in the prescribing information or a representative clinical study for each antibody.49-55
Figure 2. Examples of common Fc domain mutations used in the engineering of therapeutic IgGs do not significantly influence FcRn dissociation pH.
(a) Two IgG images describing the mutations introduced to the Fc domain to assess the influence of Fc modifications on FcRn dissociation pH. (b) Bar graph depicting the FcRn dissociation pH values observed for MOR208 IgG molecules with WT, Fc-silenced, Fc-enhanced, or KiH Fc domain modifications incorporated.
Notes: (a) Schematic IgG showing the location/position of Fc domain mutations, designed to silence effector function (FEAG, LALAPG, N297G), enhance effector function (S239D, I332E), or allow heterodimeric antibody assembly via KiH mutations. (b) FcRn dissociation pH of WT MOR208 mAb, and MOR208 mAbs with Fc-silencing, Fc-enhancing, or KiH mutations, as indicated. Mean ± SD is depicted for each antibody, calculated from three independent experiments.
Figure 3. Incorporating different antigen-binding domain sequences to a common antigen significantly influences the FcRn dissociation pH.
(a) Schematic depicting an IgG molecule with the CD16A effector antigen-binding domains highlighted, and text describing whether these had low, medium, or high affinity for CD16A, and that the Fc domains are identical. (b) Bar graph depicting significant differences in FcRn dissociation pH of IgGs incorporating either low, medium, or high affinity CD16A effector binding domains.
Notes: (a) Schematic showing standard IgG antibody format into which anti-CD16A effector antigen-binding domains with different sequences and affinities were incorporated. (b) FcRn dissociation pH values obtained for antibodies with low-, medium-, or high-affinity CD16A sequences incorporated in N-terminal Fab domains. Mean ± SD is presented for each antibody variant, calculated from two–three independent experiments. Statistical significance (p ≤ 0.05) was determined by unpaired T-test.
Figure 4. The number of antigen-binding domains presented in scFv or scDb significantly influences FcRn dissociation pH.
(a) Images of IgG, 2-Fab-Fc-2scFv, 2Fab-Fc-1scDb, and 2Fab-Fc-2scDb antibodies, with the numbers of C-terminal effector domains incorporated highlighted. Text highlights that all antibodies shown had identical Fab and Fc domains. (b and c) Two bar graphs depicting significant differences between the FcRn dissociation pH values of IgG, 2Fab-Fc-2scFv, and 2Fab-Fc-2scDb (left graph), and between 2Fab-Fc-1scDb and 2Fab-Fc-2scDb (right graph).
Notes: (a) Schematic figures depicting the two pairs of bsAbs used to investigate the effect of antigen-binding domain number on FcRn dissociation pH. (B and C) FcRn dissociation pH established by FcRn-pH-HPLC for (b) different numbers of C-terminally fused CD16A domains (2× CD16A binding moieties in 2× scFv vs. 4× CD16A binding moieties in 2× scDb) and (c) different numbers of C-terminally fused CD16A binding domains in scDb modules; mean ± SD are shown, calculated from three independent experiments. Statistical significance (p ≤ 0.05) was determined by unpaired T-test for each comparison. Note: the datasets for 2Fab-Fc-2scDb bsAbs in B and C were performed with two different versions of this antibody, incorporating two different target antigen‑binding domains. As such, these datasets cannot be consolidated into a single dataset.
Figure 5. Incorporation of different target domain sequences specific to different antigens within N-terminal Fab modules in bsAbs influences FcRn dissociation pH.
Schematic figure (a) depicts a 2Fab-Fc-2scFv bispecific antibody with the N-terminal target antigen-binding domains highlighted, and text describing these domains as being the independent variable in this experiment. Additional text denotes that the Fc and scFv parts of all antibodies tested in this experiment were identical. A correlation plot (B) presents FcRn dissociation pH plotted against the isoelectric point of the target antigen-binding domain incorporated.
Notes: (a) FcRn dissociation pH of bsAbs containing different N-terminal target domain sequences in Fab modules. (b) Isoelectric point/FcRn dissociation pH correlation plot.
Figure 6. Incorporation of stabilizing DSB within scFv modules of bsAb does not significantly influence FcRn dissociation pH.
(a) Two 2Fab-Fc-scFv bispecific antibody schematics with the C-terminal effector antigen-binding domains highlighted. The left antibody contains stabilizing disulfide bonds, the right antibody does not. Additional text highlights that the N-terminal target antigen-binding domains, and the Fc portions of the antibody are identical. (b) Bar graph depicting the FcRn dissociation pH of the 2Fab-Fc-2scFv antibodies with and without stabilizing disulfide bonds.
Notes: (a) Schematic depicting the tetravalent, bsAb formats (2Fab-Fc-2scFv) used to investigate the FcRn dissociation pH of antibodies with (rightleft) and without (leftright) stabilizing DSB within their C-terminal scFv modules. (b) FcRn dissociation pH values obtained for antibodies with and without stabilizing DSB incorporated in C-terminal scFv modules. Mean ± SD is presented for each antibody variant, calculated from three independent experiments. Statistical significance (p ≤ 0.05) was determined by unpaired T-test.
Figure 7. The structure in which antigen-binding domains are presented significantly influences FcRn dissociation pH.
(a) Schematics depicting three pairs of tetravalent, bispecific antibodies: Pair 1 = 2scFv-Fc-2scFv and 2Fab-Fc-2scFv; pair 2 = 1stgFab-1scDb-Fc and 2stgFab-Fc; pair 3 = 2Fab-Fc-2scFv and 2Fab-Fc-1scDb. Differences in how the target and effector antigen-binding domains are presented between the antibodies within each pair are highlighted; additional text depicts portions of the antibodies within each pair that are identical. (b) Bar graph depicting FcRn dissociation pH values observed for 2scFv-Fc-2scFv and 2Fab-Fc-2scFv. (c) Bar graph depicting FcRn dissociation pH values observed for 1stgFab-1scDb-Fc and 2stgFab-Fc. (d) Bar graph depicting FcRn dissociation pH values observed for 2Fab-Fc-2scFv and 2Fab-Fc-1scDb.
Notes: (a) Schematic figures depicting the three pairs of tetravalent, bsAbs used to investigate the effect of antigen-binding domain structure on FcRn dissociation pH. (B–D) FcRn dissociation pH established by FcRn-pH-HPLC for (b) different N-terminal fused target domain presenting modules (scFv or Fab), (c) different N-terminal fused CD16A effector domain presenting modules (scDb or stgFab), and (d) different CD16A C-terminal fused effector domain presenting modules (2× scFv or 1× scDb); mean ± SD are shown, calculated from three independent experiments. Statistical significance (p ≤ 0.05) was determined by unpaired T-test for each comparison.
Figure 8. The orientation of antigen-binding domains within a bsAb significantly influences FcRn dissociation pH.
Notes: (a and b) Schematic figures depicting the two pairs of tetravalent, bsAbs used to investigate the effect of antigen-binding domain orientation on FcRn dissociation pH. Black boxes indicate the modules which have been re-orientated within the two pairs of bsAbs. (c and d) FcRn dissociation pH established by FcRn-pH-HPLC for (c) swapped N- and C-terminally fused binding domains (d) re-oriented Fab and scDb domains; mean ± SD are shown, calculated from three independent experiments. Statistical significance (p ≤ 0.05) was determined by unpaired T-test for each comparison.
(a) Images depicting a pair of 2Fab-Fc-2scFv bispecific antibodies, with the N-terminal (orange) and C-terminal (blue) antigen-binding domains highlighted. The image on the left depicts the target domains on the N-terminus and the effector domains on the C-terminus (2FabTarget-Fc-2scFvEffector); the image on the right depicts the effector domains on the N-terminus and the target domains on the C-terminus (2FabEffector-Fc-2scFvTarget). (b) Images depicting a pair of antibodies. The left image depicts an antibody with a staggered Fab and scDb module on the N-terminus (1stgFab-1scDb-Fc); the image on the right has two Fab modules on the N-terminus, and a scDb module on the C-terminus (2Fab-Fc-1scDb). (c) Bar graph depicting FcRn dissociation pH values observed for 2FabTarget-Fc-2scFvEffector and 2FabEffector-Fc-2scFvTarget. (d) Bar graph depicting FcRn dissociation pH values observed for 1stgFab-1scDb-Fc and 2Fab-Fc-1scDb.
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