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. 2019 May 20;10(1):2141.
doi: 10.1038/s41467-019-10088-1.

Optimization of 4-1BB Antibody for Cancer Immunotherapy by Balancing Agonistic Strength With FcγR Affinity

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Free PMC article

Optimization of 4-1BB Antibody for Cancer Immunotherapy by Balancing Agonistic Strength With FcγR Affinity

Xinyue Qi et al. Nat Commun. .
Free PMC article

Abstract

Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab). Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG. While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB. All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB+ cells. This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance. As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.

Conflict of interest statement

The research was funded in part by Lyvgen Biopharma. Y.W. and J.W. are employees of Lyvgen Biopharma. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Distinct activity and toxicity profiles of anti-4-1BB Abs. a WT Balb/c (n = 4–5/group) or b B6 mice (n = 5/group) were injected subcutaneously with 1 × 106 (a) CT-26 (b) B16-OVA cells, then 200 μg of anti-4-1BB (3H3 or LOB12.3) or control IgG was administered on days 9, 16, and 23. Tumor growth was measured twice a week. c, d Twenty-one days after the first treatment, serum ALT levels in a, b were measured. e Naive Balb/c (n = 4/group) or f B6 mice (n = 4/group) were treated with 200 μg of anti-4-1BB (3H3 or LOB12.3) or control IgG on days 0, 7, and 14, and serum ALT levels were measured on day 21. g Same as in e, f liver tissue was stained by HE. Mean ± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated. Scale bar: 50 µm
Fig. 2
Fig. 2
FcγRs-crosslinking enhanced the activity of anti-4-1BB Abs. a Total splenocytes or b purified CD8+ T cells from B6 mice were stimulated with anti-CD3 and indicated anti-4-1BB Abs, 2 days later, the IFN-γ in culture medium was analyzed by CBA. c, d Purified CD8+ T cells from B6 mice were stimulated with anti-CD3 and indicated anti-4-1BB Abs when indicated single mouse FcγR-expressing B16-OVA cells were present, 2 days later, the IFN-γ in culture medium was analyzed by CBA. Mean ± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated
Fig. 3
Fig. 3
Activating FcγRs compromise anti-4-1BB Abs’ activity in vivo. a Fcgr2b−/− (n = 6–9/group) and b Fcgr3−/− mice (n = 5/group) were injected subcutaneously with 1 × 106 B16-OVA cells, then 200 μg of indicated control IgG or anti-4-1BB Abs were administered on days 9, 16, and 23. Tumor growth was measured twice a week. ce Fcgr2b−/− (n = 6–7/group), Fcer1g−/− (n = 7/group) and Fcgr3−/− mice (n = 5–6/group) were injected subcutaneously with 1 × 106 B16-OVA cells, then 200 μg of indicated control IgG or chimeric anti-4-1BB Abs were administered on days 9, 16, and 23. Tumor growth was measured twice a week. f, g WT Balb/c mice (n = 5/group) were injected subcutaneously with 1 × 106 CT-26 cells, then 200 μg of indicated chimeric anti-4-1BB Abs or control IgG was administered on days 9, 16, and 23. Tumor growth was measured twice a week. h Same as in g, splenocytes were analyzed by flow cytometry. i, j Splenocytes from Rag1−/− mice were co-cultured with EL4-4-1BB+ CFSEhigh and EL4-4-1BB- CFSElow cells in the presence of indicated anti-4-1BB Abs. One day later, the ratio of EL4-4-1BB+/4-1BB was analyzed by flow cytometry. Mean ± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated
Fig. 4
Fig. 4
Fab and Fc co-determine liver toxicity by anti-4-1BB Abs. a After anti-4-1BB Ab treatment, liver leukocytes (n = 3–4/group) were analyzed by flow cytometry directly or stimulated with PMA/Ionomycin/BFA for 1 h, followed by intracellular staining for IFN-γ. b WT B6 mice (n = 4–5/group) were treated with 200 μg of anti-4-1BB Abs on days 0, 7, and 14, with 200 μg of anti-CD8 administered on days 0 and 7. c, d Fcgr2b−/− (n = 4/group) and Fcgr3−/− mice (n = 4/group) were treated with 200 μg of anti-4-1BB rat Abs on days 0, 7, and 14. eg Fcgr3−/− (n = 3–6/group), Fcer1g−/− (n = 4–7/group), and Fcgr2b−/− mice (n = 4–7/group) were treated with 200 μg of anti-4-1BB chimeric Abs on days 0, 7, and 14. hi WT Balb/c mice (n = 6/group) were treated with indicated anti-4-1BB chimeric Abs on days 0, 7, and 14. bi All serum ALT levels were analyzed on day 21 after first antibody treatment. Mean ± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated
Fig. 5
Fig. 5
Activation and toxicity profile of human anti-4-1BB Abs. a Human PBMC or b purified T cells from human PBMC were stimulated with anti-CD3 and indicated anti-4-1BB Abs. Two days later, the IFN-γ in culture medium was analyzed by CBA assay. c Purified T cells from h-4-1BB KI B6 mice were stimulated with anti-CD3 and indicated anti-human 4-1BB Abs in the presence of single human FcγR-expressing 3T3 cells. Two days later, the IFN-γ in culture medium was analyzed by CBA assay. d Human 4-1BB KI BMC mice (n = 5/group) were treated with 200 μg of indicated anti-4-1BB Abs on days 0, 7, and 14. Serum ALT levels were analyzed on day 21. e Same as in d, liver tissue was stained by HE and IHC. Mean ± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated. Scale bar: 50 µm
Fig. 6
Fig. 6
Engineering potent anti-h4-1BB Ab with limited toxicity. a GS-H2/4-1BB reporter cell line was stimulated with LVGN6051 anti-4-1BB Ab in the presence of single human FcγR-expressing cells. Two days later, reporter activity was analyzed. b Purified T cells from human PBMC were stimulated with anti-CD3 and indicated anti-4-1BB Abs in the presence of human FcγRIIB-expressing cells. Two days later, the IFN-γ in culture medium was analyzed by CBA assay. c Human 4-1BB KI mice (n = 5–6/group) were treated with 200 μg of anti-4-1BB Ab LVGN6051. Tumor growth was measured twice a week. d Same as in c, Serum ALT levels were analyzed on day 21. e Human 4-1BB KI mice (n = 7/group) were treated with 200 μg of in-house Urelumab. Tumor growth was measured twice a week. f Same as in e, Serum ALT levels were analyzed on day 21 after first antibody treatment. Mean± SEM are shown. *p < 0.05, **p < 0.01 compared with control group or as indicated
Fig. 7
Fig. 7
Optimization strategies for different anti-4-1BB Abs. Strong agonistic anti-4-1BB Abs can co-stimulate T cell when FcγRs are absent. Weak agonistic anti-4-1BB Abs require FcγR-mediated crosslinking for activity. When anti-4-1BB Abs are crosslinked by activating FcγRs, ADCC will comprise the co-stimulation ability. When anti-4-1BB Abs are crosslinked by FcγRIIB, it will not cause ADCC and induce strong T cell activation. When strong agonistic anti-4-1BB are engineered with low A/I Fc, it can cause super-activation of T cells and induce liver toxicity. For optimization of strong agonistic anti-4-1BB Abs, it requires fine tuning the Fc within a narrow range to balancing efficacy and toxicity. For optimization of weak agonistic anti-4-1BB Abs, if equipped with FcγRIIB selective Fc, it shows potent anti-tumor activity with desired liver safety profile

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