Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

Abstract

We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using ⁸⁹Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8⁺ T cells, skewing the CD8⁺:CD4⁺ ratio toward CD8⁺ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

Keywords: Antibody; CEA; CEACAM5; IL-2; IL2v; Immunocytokine.

Figure 1.

(A) IL2v/IL-2Rβγ complex structure. IL2v is shown yellow, the mutations in blue. The IL-2Rβ chain is colored in cyan and the γc chain in magenta. (B) Close-up view onto the loop region between helix A and helix B and the binding site of IL-2 with IL-2Rα. The picture shows an overlay of the IL2v-IL-2Rβγ (yellow) structure with 2B5I (gray representation). Mutations in the two hydrophobic patches with F42A, Y45A and L72G are highlighted in blue and circled. (C) Superposition of all atoms of IL2v-IL-2Rβγ with the quaternary complex IL-2, IL-2Rα, IL-2Rβ, γc (2erj). IL2v-IL-2Rβγ is colored as in Fig. 1A. 2B5I is shown in gray. (D) Schematic representation of CEA-IL2v and its key design features.

Figure 2.

(A–D) FACS analysis of STAT5 phosphorylation on (A) Treg cells (CD4+CD25+FOXP3+), (B) CD4⁺ T cells (CD4⁺CD25⁻), C) CD8⁺ T cells (CD3⁺CD8⁺) and (D) NK cells (CD3⁻CD56⁺) within freshly isolated PBMCs 20 min after treatment with CEA-IL2v, CEA-IL2wt and aldesleukin; (E–H) Fas-mediated apoptosis induction (E, F) and proliferation (G, H) of CD4⁺ and CD8⁺ T cells following treatment with CEA-IL2v and CEA-IL2wt after overnight pre-activation with PHA. Exemplary data from ≥ 3 independent experiments. Shown are means from triplicates and standard deviation.

Figure 3.

(A) Mean plasma concentration-time profiles of CEA antibody CH1A1A1–2F1, CEA-IL2v and CEA- IL2wt in CEA-transgenic C57BL/6 mice as determined by ELISA. (B) Organ biodistribution of ⁸⁹Zr-CEA-IL2v 3 d after injection at different doses in immunocompetent CEA-transgenic C57BL/6 mice bearing syngeneic CEA-positive MC38-CEA tumors and CEA-negative MC38 tumors on contra-lateral site. All values are expressed as %ID/g. Data represent mean value ± SD from at least four determinations. (C) Standardized Uptake Values (SUVs) of tumors derived from PET imaging of immunocompetent CEA-transgenic C57BL/6 mice bearing syngeneic CEA-positive MC38-CEA tumors after injection of ⁸⁹Zr-CEA-IL2v or ⁸⁹Zr-CEA-IL2wt. Data represent mean value ± SD from at least four determinations. (D) Biodistribution of ⁸⁹Zr-CEA-IL2v compared with ⁸⁹Zr-CEA-IL2wt 4 d after injection in immunocompetent CEA-transgenic C57BL/6 mice bearing syngeneic MC38-CE tumors. Data represent mean value ± SD from > 4 determinations.

Figure 4.

Immuno-pharmacodynamics in tumor-free and tumor-bearing C57BL/6 mice: (A) Peripheral T and NK cell expansion by CEA-IL2v. Shown are lymphocytes in blood 7 d after a single i.v. dose of CEA-IL2v. (B) Increase in the numbers of circulating (per μL blood) and intratumoral (per g tissue) CD8⁺ T cells, γδ T cells and NK cells in LLC1-CEA syngeneic tumor model 5 d after injection of 0.5 or 2 mg/kg muCEA-IL2v as determined by flow cytometry (upper panels). A skewing of the T cell compartment in favor of CD8⁺ T cells as shown by the ratio of CD8⁺ T to total CD4⁺ T and Treg in the blood and tumors of LLC1-CEA tumor-bearing mice (lower panels). (C) Increase of CD3 T cells in LLC1-CEA syngeneic tumor model 5 d after injection of 1.0 mg/kg CEA-IL2v as determined by immunohistochemistry.

Figure 5.

(A, B) Monotherapy efficacy: (A) Efficacy of 2 mg/kg CEA-IL2v (q1w3 starting on day 7 after tumor inocculation) as single agent in the syngeneic intrasplenic MC38-CEA model in CEA-transgenic C57/BL6 mice. (B) Efficacy of 0.5 mg/kg muCEA-IL2v (q1w3 starting on day 7 after tumor inocculation) as single agent in the syngeneic orthotopic PancO2-CEA model in CEA-transgenic C57/BL6 mice. (C, D) Combination of CEA-IL2v with PD-L1 checkpoint blockade in syngeneic models in CEA-transgenic C57BL/6 mice. (C) Efficacy of 0.25 mg/kg muCEA-IL2v (q1w5) combined with 10 mg/kg antibody 6E11 (q1w5) concomitantly starting on day 7 vs. vehicle and the respective monotherapies in the orthotopic PancO2-CEA pancreatic syngeneic model. (D) Efficacy of 0.25 mg/kg muCEA-IL2v or a matched dose of 0.2 mg/kg untargeted muDP47-IL2v control immunocytokine (q1w4) combined with 10 mg/kg antibody 6E11 (q1w4) concomitantly starting on day 7 vs. vehicle and the respective monotherapies. Pooled data from two independent studies are shown.

Figure 6.

(A–C) Combination of CEA-IL2v with ADCC competent/ADCC enhanced antibodies in human CD16-transgenic SCID mice: (A) Combination of CEA-IL2v (1 mg/kg, q1w3) with antibody trastuzumab in N87 (25 mg/kg, q1w3) (top) and KPL4 (10 mg/kg, q1w3) (bottom) xenograft models; (B) Combination of CEA-IL2v (1 mg/kg, q1w3) with antibody cetuxiumab (25 mg/kg, q1w3) in A549 (top) and Ls174t (bottom) xenograft models; (C) Combination of CEA-IL2v (1 mg/kg, q1w3) with glycoengineered antibody imgatuzumab (25 mg/kg, q1w3) in A549 (top) and LS174t (bottom) xenograft models.