Abstract
Antibody responses during infection and vaccination typically undergo affinity maturation to achieve high-affinity binding for efficient neutralization of pathogens1,2. Similarly, high affinity is routinely the goal for therapeutic antibody generation. However, in contrast to naturally occurring or direct-targeting therapeutic antibodies, immunomodulatory antibodies, which are designed to modulate receptor signalling, have not been widely examined for their affinity–function relationship. Here we examine three separate immunologically important receptors spanning two receptor superfamilies: CD40, 4-1BB and PD-1. We show that low rather than high affinity delivers greater activity through increased clustering. This approach delivered higher immune cell activation, in vivo T cell expansion and antitumour activity in the case of CD40. Moreover, an inert anti-4-1BB monoclonal antibody was transformed into an agonist. Low-affinity variants of the clinically important antagonistic anti-PD-1 monoclonal antibody nivolumab also mediated more potent signalling and affected T cell activation. These findings reveal a new paradigm for augmenting agonism across diverse receptor families and shed light on the mechanism of antibody-mediated receptor signalling. Such affinity engineering offers a rational, efficient and highly tuneable solution to deliver antibody-mediated receptor activity across a range of potencies suitable for translation to the treatment of human disease.
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Data availability
Original raw data will be provided upon request to include all supporting information. Models of Fab–receptor complexes were obtained from the PDB with the following accession codes: 6FAX for the ChiLob 7/4–CD40 complex; 6MI2 for the utomilumab–4-1BB complex; and 5WT9 for the nivolumab–PD-1 complex. Source data are provided with this paper.
References
Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000).
Viant, C. et al. Antibody affinity shapes the choice between memory and germinal center B cell fates. Cell 183, 1298–1311.e11 (2020).
Forthal, D. N. Functions of antibodies. Microbiol. Spectr. 2, 1–17 (2014).
Singh, S. et al. Monoclonal antibodies: a review. Curr. Clin. Pharmacol. 13, 85–99 (2018).
Mullard, A. FDA approves 100th monoclonal antibody product. Nat. Rev. Drug Discov. 20, 491–495 (2021).
Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).
Clackson, T., Hoogenboom, H. R., Griffiths, A. D. & Winter, G. Making antibody fragments using phage display libraries. Nature 352, 624–628 (1991).
Pedrioli, A. & Oxenius, A. Single B cell technologies for monoclonal antibody discovery. Trends Immunol. 42, 1143–1158 (2021).
Mayes, P. A., Hance, K. W. & Hoos, A. The promise and challenges of immune agonist antibody development in cancer. Nat. Rev. Drug Discov. 17, 509–527 (2018).
White, A. L. et al. Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody. J. Immunol. 187, 1754–1763 (2011).
Yu, X. et al. Complex interplay between epitope specificity and isotype dictates the biological activity of anti-human CD40 antibodies. Cancer Cell 33, 664–675.e4 (2018).
White, A. L. et al. Conformation of the human immunoglobulin G2 hinge imparts superagonistic properties to immunostimulatory anticancer antibodies. Cancer Cell 27, 138–148 (2015).
Ahonen, C. et al. The CD40–TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells. Nat. Immunol. 3, 451–456 (2002).
Dahan, R. et al. Therapeutic activity of agonistic, human anti-CD40 monoclonal antibodies requires selective FcγR engagement. Cancer Cell 29, 820–831 (2016).
Yu, X. et al. Isotype switching converts anti-CD40 antagonism to agonism to elicit potent antitumor activity. Cancer Cell 37, 850–866.e7 (2020).
Ma, D. Y. & Clark, E. A. The role of CD40 and CD154/CD40L in dendritic cells. Semin. Immunol. 21, 265–272 (2009).
Yu, X. et al. TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity. Commun. Biol. 4, 772 (2021).
Chester, C., Sanmamed, M. F., Wang, J. & Melero, I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 131, 49–57 (2018).
Chin, S. M. et al. Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab. Nat. Commun. 9, 4679 (2018).
Gong, J., Chehrazi-Raffle, A., Reddi, S. & Salgia, R. Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. J. Immunother. Cancer 6, 8 (2018).
Paluch, C., Santos, A. M., Anzilotti, C., Cornall, R. J. & Davis, S. J. Immune checkpoints as therapeutic targets in autoimmunity. Front. Immunol. 9, 2306 (2018).
Curnock, A. P. et al. Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors. JCI Insight 6, e152468 (2021).
Bryan, C. M. et al. Computational design of a synthetic PD-1 agonist. Proc. Natl Acad. Sci. USA 118, e2102164118 (2021).
Lee, J. Y. et al. Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy. Nat. Commun. 7, 13354 (2016).
Bardhan, K. et al. Phosphorylation of PD-1-Y248 is a marker of PD-1-mediated inhibitory function in human T cells. Sci. Rep. 9, 17252 (2019).
Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H. & Riley, J. L. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J. Immunol. 173, 945–954 (2004).
Patsoukis, N., Wang, Q., Strauss, L. & Boussiotis, V. A. Revisiting the PD-1 pathway. Sci. Adv. 6, eabd2712 (2020).
Jones, B., Tite, J. P. & Janeway, C. A. Jr Different phenotypic variants of the mouse B cell tumor A20/2J are selected by antigen- and mitogen-triggered cytotoxicity of L3T4-positive, I-A-restricted T cell clones. J. Immunol. 136, 348–356 (1986).
Lu, R. M. et al. Development of therapeutic antibodies for the treatment of diseases. J. Biomed. Sci. 27, 1 (2020).
Tabasinezhad, M. et al. Trends in therapeutic antibody affinity maturation: from in-vitro towards next-generation sequencing approaches. Immunol. Lett. 212, 106–113 (2019).
Chodorge, M. et al. A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ. 19, 1187–1195 (2012).
Segal, N. H. et al. Phase I study of single-agent utomilumab (PF-05082566), a 4-1BB/CD137 agonist, in patients with advanced cancer. Clin. Cancer Res. 24, 1816–1823 (2018).
Rudnick, S. I. et al. Influence of affinity and antigen internalization on the uptake and penetration of anti-HER2 antibodies in solid tumors. Cancer Res. 71, 2250–2259 (2011).
Wajant, H. Principles of antibody-mediated TNF receptor activation. Cell Death Differ. 22, 1727–1741 (2015).
Roghanian, A. et al. Antagonistic human FcγRIIB (CD32B) antibodies have anti-tumor activity and overcome resistance to antibody therapy in vivo. Cancer Cell 27, 473–488 (2015).
Krissinel, E. Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids Res. 43, W314–W319 (2015).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Davis, C. B. et al. Combination of a PD-1 antagonist and a 4-1BB agonist for treating cancer. International patent publication number WO 2015/119923 A1 (2015).
Korman, A. J. et al. Human monoclonal antibodies to programmed death 1 (PD-1) and methods of treating cancer using anti-PD01 antibodies alone or in combination with other immunotherapeutics. International patent publication number WO 2006/121168 A1 (2006).
Keler, T et al. Antibodies that bind human CD27 and uses thereof. US patent 9,169,325 (2015).
Hanke, T et al. Nucleic acids encoding superagonistic anti-CD28 antibodies. US patent 7,585,960 (2009).
Arakawa, F. et al. Cloning and sequencing of the VH and Vκ genes of an anti-CD3 monoclonal antibody, and construction of a mouse/human chimeric antibody. J. Biochem. 120, 657–662 (1996).
Meyer, L. et al. A simplified workflow for monoclonal antibody sequencing. PLoS ONE 14, e0218717 (2019).
Sallusto, F. & Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118 (1994).
Gate, D. et al. Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease. Nature 577, 399–404 (2020).
Fernandes, R. A. et al. Immune receptor inhibition through enforced phosphatase recruitment. Nature 586, 779–784 (2020).
Austin, C. D. et al. Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol. Biol. Cell 15, 5268–5282 (2004).
Sopp, J. M. et al. On-target IgG hexamerisation driven by a C-terminal IgM tail-piece fusion variant confers augmented complement activation. Commun. Biol. 4, 1031 (2021).
Acknowledgements
We would like to thank the preclinical unit staff for animal husbandry; D. Johnston from the Biomedical Imaging Unit, Southampton General Hospital, Southampton, UK, for assistance with confocal microscopy; D. Kavanagh from the University of Oxford for sharing the dSTORM buffer recipe; and J. Felce from ONI, Oxford, UK, for advice on dSTORM data analysis. The dSTORM microscopy experiments were made possible through the funding of an ONI Nanoimager by the Mark Benevolent Fund. Funding was provided by CRUK grants A20537, A25139, A25169 and DRCDDRPGM-Apr2020\100005. X.Y. is funded by a Careertrack Fellowship provided by the Faculty of Medicine in conjunction with the Cancer Immunology Talent fund.
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X.Y. designed and performed the experiments, analysed and interpreted data and wrote the manuscript. C.M.O., H.T.C.C., S.J., C.A.P., J.K., K.L.C., T.I. and C.I.M. generated or provided key reagents or performed and analysed the experiments. J.W.E. and I.T. supported acquisition of funding and contributed to the conception of the work and approach. M.J.G. designed the study, discussed and interpreted data. M.S.C. designed the study, supervised data collection, discussed and interpreted data and wrote the manuscript with X.Y. All authors commented on and approved the final manuscript.
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M.S.C. acts as a consultant for a number of biotechnology companies, being retained as a consultant for BioInvent and has received research funding from BioInvent, GSK, UCB, iTeos and Roche, and receives institutional payments and royalties from patents and licences relating to antibody immunotherapy. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Characterization of anti-CD40 mIgG1 mAb ChiLob 7/4 affinity mutants.
a, SPR of various ChiLob 7/4 m1 affinity mutants injected at 250, 50, 10, 2, 0.4, and 0 nM binding to CD40ECD. Data representative of 3 independent experiments. b, ChiLob 7/4 m1 affinity mutants were evaluated for their binding affinity for CD40ECD by SPR as indicated in a, with affinity constants (ka, kd and KD) calculated. Fold change indicates affinity change compared with WT ChiLob 7/4 m1. c, Ramos cells were incubated with 0.5 μg/mL of AF647-labelled ChiLob 7/4 h1 and various concentrations of competing ChiLob 7/4 m1 affinity mutants as indicated and then washed and bound AF647-labelled ChiLob 7/4 h1 detected. Means ± SEM, n = 3, data representative of 3 independent experiments.
Extended Data Fig. 2 Low affinity anti-CD40 mIgG1 mAb exhibit potent agonism.
a, Purified hCD40Tg mouse B cells were incubated with ChiLob 7/4 m1 mutants for 2 days and then stained for surface expression of CD23. Left plot, exemplar raw data. b, Same experiment as (a). Surface expression of CD86. Left plot, exemplar raw data. c, Purified hCD40Tg mouse B cells were incubated with ChiLob 7/4 m1 mutants for 3 days and then 3H-thymidine was added for 18 h to measure proliferation. Inset cell culture images were taken on day 2. Scale bar, 0.5 mm. For (a—c), Means ± SEM, n = 3, data representative of 3 independent experiments. Rightmost plots illustrate the CD23 expression, CD86 expression or proliferation as a function of the on-rate (ka) or off-rate (kd). d, hCD40Tg mice were inoculated with EG7 cells. 7 days later, mice received OTI cells and the next day were treated with ChiLob 7/4 m1 mutants as indicated. Tumor growth curves are shown with numbers as proportion of tumour free mice at experiment end inset. n = 13–17, data pooled from two to three independent experiments.
Extended Data Fig. 3 Characterization of anti-CD40 hIgG2 mAb ChiLob 7/4 affinity mutants.
a, SPR of various ChiLob 7/4 h2 affinity mutants injected at 250, 50, 10, 2, 0.4, and 0 nM binding to CD40ECD. Data representative of 3 independent experiments. b, ChiLob 7/4 h2 affinity mutants were evaluated for their binding affinity for CD40ECD by SPR as indicated in a, with affinity constants (ka, kd and KD) calculated. Fold change indicates affinity change compared with WT ChiLob 7/4 h2. c, Ramos cells were incubated with 0.5 μg/mL of AF647-labelled ChiLob 7/4 h1 and various concentrations of competing ChiLob 7/4 h2 affinity mutants as indicated and then washed and bound AF647-labelled ChiLob 7/4 h1 detected. Means ± SEM, n = 3, data representative of 3 independent experiments. d, e, Purified hCD40Tg mouse B cells were incubated with ChiLob 7/4 h2 affinity mutants as indicated for 2 days and then stained for surface expression of CD23 (d, left plot, exemplar raw data) and CD86 (d, right plot, exemplar raw data). B cell proliferation was assessed by 3H-thymidine incorporation (e). Plots show affinity (KD) vs maximum CD23 MFI, maximum CD86 MFI or maximum proliferation. Means ± SEM, n = 3, data representative of 3 independent experiments. f, OTI cells were adoptively transferred into hCD40Tg mice 1 day before treatment with ChiLob 7/4 h2 mutants with peripheral SIINFEKL+ CD8 cells identified by flow cytometry on day 4. Mean ± SEM, n = 7, data pooled from two independent experiments. Each dot represents one mouse. Two-tailed, non-paired Student’s t test, the p values for WT h2 vs FW-12 h2, vs FW-22 h2, vs FW-16 h2 are (from left to right) 0.0023, 0.0023 and 0.0023. g, Purified human B cells were incubated with ChiLob 7/4 h2 affinity mutants for 2 days and then stained for surface expression of CD23 (left plot, exemplar raw data) and CD86 (right plot, exemplar raw data). h, Purified human B cells were incubated with ChiLob 7/4 h2 mutants as indicated for 3 days and then 3H-thymidine was added for 18 h to measure proliferation. Inset cell culture images were taken on day 2. For g, h, Means ± SEM, n = 3, data representative of 3 independent experiments. i, Purified human B cells were incubated with ChiLob 7/4 h2 affinity mutants as indicated for 2 days. B cell proliferation was assessed by 3H-thymidine incorporation. Plots show affinity (KD) vs maximum CD23 MFI, maximum CD86 MFI or maximum proliferation. Means ± SEM, n = 3, data representative of 3 independent experiments.
Extended Data Fig. 4 Low affinity anti-CD40 mAb exhibit potent agonism in human systems.
a, Human DCs were stimulated with various ChiLob 7/4 h2 affinity mutants for 2 days and then evaluated for CD86 expression. The ranking of CD86 MFI was plotted against KD. Means ± SEM, each dot represents an average value from 9 donors. b, Gating strategy and representative histograms for a. c, Human DCs were pre-treated with ChiLob 7/4 h2 affinity mutants for 2 days and co-cultured with allogeneic CD4+ T cells for 5 days. CD4+ T cell proliferation was measured by 3H-thymidine incorporation. Ranking of CD4+ T cell proliferation was plotted against affinity (KD). Means ± SEM, each dot represents the average from 7 donors. d, Human monocyte-derived DCs were pre-treated with ChiLob 7/4 h2 affinity mutants for 2 days and then co-cultured with allogeneic CD4+ T cells at different ratios for 5 days. CD4+ T cell proliferation was measured by 3H-thymidine incorporation. The ranking of CD4+ T cell proliferation was plotted against affinity (KD). Means ± SEM, each dot represents the average value from 7 healthy donors. e, CFSE-labelled PBMCs were stimulated with antigenic peptides and ChiLob 7/4 h2 affinity mutants for 5 days and proliferating CD8+ T cells evaluated for surface expression of CD25. Ranking of CD25 MFI plotted against KD. Means ± SEM, each dot represents the average from 8 donors. f, Gating strategy and representative histograms for CD25 expression of proliferating CD8+ T cells in PBMCs stimulated with CEFX Ultra SuperStim Pool and ChiLob 7/4 h2 affinity mutants. Data representative of 8 donors.
Extended Data Fig. 5 Low affinity anti-CD40 mIgG1 mAb induce agonism independent of FcγR.
a, Jurkat-NFκB-GFP-CD40 reporter cells were incubated with various ChiLob 7/4 m1 affinity mutants for 6 h and the level of NFκB activation (GFP) assessed. Means ± SEM, n = 3, data representative of 3 independent experiments. Representative flow data shown in left panels. Subsequent plots reflect the dose-response for the different mAb followed by cumulative plots of the data as a function of affinity parameters; ka (top) and kd (bottom), respectively. b—d, Purified hCD40Tg/FcγRnull mouse B cells were incubated with ChiLob 7/4 m1 mutants for 2 days and then stained for surface expression of (b) CD23 and (c) CD86. Representative flow data shown in left panels. Subsequent plots reflect the dose-response for the different mAb followed by cumulative plots of the data as a function of affinity parameters; ka (top) and kd (bottom), respectively.(d) On day 3 3H-thymidine was added for 18 h to assess B cell proliferation. Inset cell culture images were taken on day 2. Means ± SEM, n = 3, data representative of 3 independent experiments. Scale bar, 0.5 mm. Plots reflect the dose-response for the different mAb followed by cumulative plots of the data as a function of affinity parameters; ka (top) and kd (bottom), respectively.
Extended Data Fig. 6 Low affinity anti-CD40 hIgG2 mAb induce agonism independent of FcγR.
a—c, Purified hCD40Tg/FcγRnull mouse B cells were incubated with ChiLob 7/4 h2 mutants for 2 days and then stained for surface expression of (a) CD23 and (b) CD86. Representative flow data shown in left panels. Subsequent plots reflect the dose-response for the different mAb followed by cumulative plots of the data as a function of affinity (KD). (c) On day 3 3H-thymidine was added for 18 h to assess B cell proliferation. Inset cell culture images were taken on day 2. Means ± SEM, n = 3, data representative of 3 independent experiments. Scale bar, 0.5 mm. Plots reflect the dose-response for the different mAb followed by cumulative plots of the data as a function of affinity (KD).
Extended Data Fig. 7 Low affinity anti-CD40 mAb induce agonism through receptor clustering with minimal receptor internalization.
a, Schematic of the method for calculating clustering index. Confocal images through the centre of cells were opened in LAS X software and fluorescence intensity measurements taken for regions of interest at the cell:cell junctions (red) or at the periphery of the cells (blue). b, Jurkat-CD40-GFP cells were incubated with ChiLob 7/4 m1 affinity mutants as indicated for 3 h at 37 °C and then imaged by confocal. Green: CD40-GFP. Scale bar: 4 μm. Image representative of at least fifteen images taken from 3 independent experiments. c, Same experiment as (b) Left panel: cell circularity was measured by ImageJ for five confocal images per treatment and the results of three independent experiments were pooled. Each dot represents one cell. Right panel: plot showing cumulative circularity data as a function of B cell proliferation. Means ± SEM. d, Binding of Fab fragments of ChiLob 7/4 m1 affinity mutants to Ramos cells. Data representative of 3 independent experiments. e, Jurkat-NFκB-GFP-CD40 reporter cells were incubated with various ChiLob 7/4 m1 affinity mutant IgG versus Fab pairs for 6 h and the level of NFκB activation (GFP) assessed. Means ± SEM, n = 3, data representative of 3 independent experiments. f, Same experiment as (b) evaluating the clustering potential of 4 different ChiLob 7/4 m1affinity mutant Fab versus an IgG positive control. Clustering index calculated as indicated in (a). Means ± SEM, n = 5, data representative of 3 independent experiments. g, Ramos cells were treated with AF488-labelled ChiLob 7/4 m1 affinity mutants for 10, 30, 60, 120 or 180 min (left to right) at 37 °C or 4 °C as indicated. Cells were then washed and half the cells treated with anti-AF488 mAb at 4 °C to quench cell surface associated AF488 fluorescence. Remaining cell surface-bound CD40 was expressed as % Total expression. Data representative of 3 independent experiments. h, Jurkat-CD40-GFP cells were incubated with ChiLob 7/4 m1 affinity mutants as indicated for 3 h at 37 °C and then fixed with PFA, counterstained with DAPI and imaged by confocal. Orthogonal images shown. Blue: nucleus; Green: CD40-GFP. Image representative of at least ten images taken from 3 independent experiments.
Extended Data Fig. 8 Super-resolution dSTORM analysis of receptor clusters induced by low affinity anti-CD40 mAb or CD40L.
a, Jurkat-CD40-GFP cells were incubated with various CD40 agonists as indicated for 1 h, and then CD40-GFP detected with AF647-conjugated anti-GFP nanobody and visualised by wide field fluorescence microscopy and dSTORM. A region of interest was drawn around the cell:cell junctions and clustering analysis was performed using HDBSCAN, example results of subclusters are shown. Scale bars; 10 μm (wide field), 5 μm (dSTORM), 1 μm (HDBSCAN) b, Subcluster density (number of localisation per unit area). One-way ANOVA followed by Kruskal-Wallis test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n= number of subclusters, y=number of cell:cell junctions examined: Untreated, n = 281, y = 28; ChiLob 7/4 h2, n = 664, y = 26; CD40L, n = 427, y = 25; WT m1, n = 138, y = 23; FW-16 m1, n = 642, y = 29; FW-32 m1, n = 151, y = 30. c, Plot of subcluster area versus density. Results shown are representative of 3 independent experiments.
Extended Data Fig. 9 Characterization of low affinity utomilumab and nivolumab variants.
a, Utomilumab affinity mutants were evaluated for their binding affinity for 4-1BBECD by SPR, with affinity constants (ka, kd and KD) calculated as well as fold change which indicates affinity change compared with WT utomilumab. b, Jurkat-4-1BB-GFP cells were incubated with utomilumab affinity mutants and then imaged by confocal. Green: 4-1BB-GFP. Scale bar: 4 μm. Images representative of at least fifteen images taken from 3 independent experiments. c, Same experiment as (b) Left panel: cell circularity was measured by ImageJ for five confocal images per treatment and the results of three independent experiments were pooled. Each dot represents one cell. Right panel: plot showing cumulative circularity data as a function of NFκB activation. Means ± SEM. d, The expression level of 4-1BB on Ramos-4-1BB cells was analysed by flow cytometry. e, The expression level of 4-1BB on IIA1.6-4-1BB cells was analysed by flow cytometry. f, SPR of various nivolumab affinity mutants injected at 250, 50, 10, 2, 0.4, and 0 nM binding to PD-1ECD. Plots display sensorgram data representative of 3 independent experiments. g, Nivolumab affinity mutants were evaluated for their binding affinity for PD-1ECD by SPR as indicated in f, with affinity constants (ka, kd and KD) calculated. Fold change indicates affinity change compared with WT nivolumab. h, PD-1-transfected Jurkat cells were incubated with various nivolumab affinity mutants as indicated and then washed and bound hIgG detected. Means ± SEM, n = 3, data representative of 3 independent experiments.
Extended Data Fig. 10 Low affinity nivolumab variants induce PD-1 agonism through receptor clustering.
a, Schematic of assays investigating the antagonistic and agonistic properties of anti-PD-1 affinity mutants. Left panel: Assay to evaluate the ability of mAb to block PD-L1-mediated T cell suppression. Middle panel: Assay to evaluate the ability of mAb to induce PD-1 signalling. Right panel: Assay to evaluate the ability of mAb to suppress anti-CD3 mAb-mediated T cell activation. b, Histogram showing CHO-SB2H2-scFv-CD8α cells binding to various nivolumab affinity mutants, data representative of 3 independent experiments. c, CHO-SB2H2-scFv-CD8α cells were opsonized with OKT3 and nivolumab affinity mutants as indicated and then co-cultured with Jurkat-NFAT-Luc-PD-1 for 6 hours. NFAT signalling activity was then assessed. Plots showing relative luciferase units (RLU) vs ka and RLU vs kd. Means ± SEM, n = 3, data representative of 3 independent experiments. d, Same experiment as (c). Plots showing CD69 MFI vs ka and RLU vs CD69 MFI. e, IIA1.6-PD-1-GFP cells were incubated with nivolumab affinity mutants for 3 h and then imaged using confocal. Green: PD-1-GFP. Scale bar: 4 μm. Image representative of at least 15 images taken from 3 independent experiments. f, IIA1.6-PD-1-GFP cells were incubated with nivolumab affinity mutants as indicated for 3 h and then fixed, counterstained with DAPI and imaged using confocal. Z-stack projections shown. Blue: nucleus; Green: PD-1-GFP. Scale bar: 4 μm. Image representative of at least ten images taken from two independent experiments. g, Jurkat-NFκB-GFP-PD-1 reporter cells were incubated with various nivolumab affinity mutants as indicated for 30 min then, Left panel: washed with the level of mAb remaining bound after various periods quantified or Right panel: the level of NFκB activation (GFP) assessed after various periods. Means ± SEM, n = 3, data representative of 3 independent experiments. h, Same experiment as (e) Left panel: cell circularity was measured by ImageJ for five confocal images per treatment and the results of three independent experiments were pooled. Each dot represents one cell. Right panel: plot showing cumulative circularity data as a function of CD69 MFI, Means ± SEM, n = 34, 32, 39, 39, 36, 38, 33, 36, 33, 37 (from left to right).
Extended Data Fig. 11 Low affinity mutants retain target-specific binding and exhibit agonism at multiple receptor densities.
a, Heat map showing MFI values of ChiLob 7/4 m1 affinity mutants binding to Jurkat-NFκB-GFP-TNFRII cells versus a positive control anti-TNFRII mAb. b, Heat map showing SPR response of ChiLob 7/4 m1 affinity mutants binding to soluble TNFRII immobilized onto a CM5 chip versus a positive control anti-TNFRII mAb. c, Heat map showing MFI values of utomilumab affinity mutants binding to Jurkat-NFκB-GFP-CD27 cells versus a positive control anti-CD27 mAb. d, Heat map showing SPR response of utomilumab affinity mutants binding to soluble CD27 immobilized onto a CM5 chip versus a positive control anti-CD27 mAb. e, Heat map showing MFI values of nivolumab affinity mutants binding to Jurkat-CD28-GFP cells versus a positive control anti-CD28 mAb. f, Heat map showing SPR response of nivolumab affinity mutants binding to soluble CD28 immobilized onto a CM5 chip versus a positive control anti-CD28 mAb. g, Representative z plane images covering the entire z-axis of Jurkat-CD40-GFP cells treated with anti-CD40 mAb. Blue: nucleus; Green: PD-1-GFP. Scale bar: 4 μm. Data representative of 3 independent experiments. h, Representative z plane images covering the entire z-axis of Jurkat-4-1BB-GFP cells treated with anti-4-1BB mAb. Blue: nucleus; Green: PD-1-GFP. Scale bar: 4 μm. Data representative of 3 independent experiments. i, Representative z plane images covering the entire z-axis of IIA1.6-PD-1-GFP cells treated with anti-PD-1 mAb. Blue: nucleus; Green: PD-1-GFP. Scale bar: 4 μm. Data representative of 3 independent experiments. j, Jurkat-NFκB-GFP reporter cells expressing low, medium or high levels of CD40 were incubated with various ChiLob 7/4 m1 affinity mutants for 6 h and the level of NFκB activation (GFP) assessed. k, Quantification of CD40 receptor number (as expressed by Molecules of Equivalent Soluble Fluorochrome, MESF) on various cell lines and primary cells as indicated. l, Jurkat-NFκB-GFP reporter cells expressing low, medium or high levels of PD-1 were incubated with various nivolumab affinity mutants for 6 h and the level of NFκB activation (GFP) assessed. m, Quantification of PD-1 receptor number (MESF) on various cell lines and primary cells as indicated. n, Jurkat-NFκB-GFP reporter cells expressing low, medium or high levels of 4-1BB were incubated with various utomilumab affinity mutants for 6 h and the level of NFκB activation (GFP) assessed. o, Quantification of 4-1BB receptor number (MESF) on various cell lines and primary cells as indicated. p, mAb (black) or soluble ligands (red) were injected at 250, 50, 10, 2, 0.4, and 0 nM to evaluate binding by SPR to their cognate soluble receptor CD40, 4-1BB or PD-1, as indicated. Values indicate the equilibrium affinity KD of the ligands for their cognate receptor. Means ± SEM, n = 3, data representative of 3 independent experiments.
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Yu, X., Orr, C.M., Chan, H.T.C. et al. Reducing affinity as a strategy to boost immunomodulatory antibody agonism. Nature 614, 539–547 (2023). https://doi.org/10.1038/s41586-022-05673-2
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DOI: https://doi.org/10.1038/s41586-022-05673-2
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Cancer Immunology, Immunotherapy (2024)
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