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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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


(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.
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f0001: (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.

Mentions: To confirm that the mutations in IL2v indeed only interfere with CD25 binding, and do not affect the interaction with IL-2Rβγ, we determined the co-crystal structure of the IL2v-IL-2Rβγ ternary complex at a resolution of 2.3 Å (Fig. 1A, Table S1). The structure contains one ternary complex molecule in the asymmetric unit and consists of the β-chain residues 6–208, γ-chain residues 33–225 and IL2v residues 4–30, 37–133. Although the protein was treated with PNGase F before crystallization, electron density for 5 N-glycosylated Asn residues on the receptor chains was observed (data not shown). In addition, we identified two covalently attached mannose moieties at W194 and W197 within the class I cytokine receptor WSXWS motif of the IL-2Rβ chain.36 The overall structure of the IL2v-IL-2Rβγ complex is essentially identical to the wild-type quaternary complex (pdb code 2b5i) with only minor changes observed within the βγ interface and within IL-2 (superposition of all atoms with a root mean square deviation (rmsd) of 1.02 Å) (Fig. 1B). Binding of IL-2 to CD25 is mediated by two hydrophobic patches around F42, Y45 and L72 on the IL-2 surface.34,35 F42 and Y45 are located in the loop AB, whereas L72 is part of helix B. The mutations F42A, Y45A and L72G remove a large part of the Van’ der Waal interaction surface and thereby prevent binding of IL2v to CD25 without affecting the IL-2 structure (Fig. 1C) or the interaction with IL-2Rβγ.Figure 1.


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
(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.
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f0001: (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.
Mentions: To confirm that the mutations in IL2v indeed only interfere with CD25 binding, and do not affect the interaction with IL-2Rβγ, we determined the co-crystal structure of the IL2v-IL-2Rβγ ternary complex at a resolution of 2.3 Å (Fig. 1A, Table S1). The structure contains one ternary complex molecule in the asymmetric unit and consists of the β-chain residues 6–208, γ-chain residues 33–225 and IL2v residues 4–30, 37–133. Although the protein was treated with PNGase F before crystallization, electron density for 5 N-glycosylated Asn residues on the receptor chains was observed (data not shown). In addition, we identified two covalently attached mannose moieties at W194 and W197 within the class I cytokine receptor WSXWS motif of the IL-2Rβ chain.36 The overall structure of the IL2v-IL-2Rβγ complex is essentially identical to the wild-type quaternary complex (pdb code 2b5i) with only minor changes observed within the βγ interface and within IL-2 (superposition of all atoms with a root mean square deviation (rmsd) of 1.02 Å) (Fig. 1B). Binding of IL-2 to CD25 is mediated by two hydrophobic patches around F42, Y45 and L72 on the IL-2 surface.34,35 F42 and Y45 are located in the loop AB, whereas L72 is part of helix B. The mutations F42A, Y45A and L72G remove a large part of the Van’ der Waal interaction surface and thereby prevent binding of IL2v to CD25 without affecting the IL-2 structure (Fig. 1C) or the interaction with IL-2Rβγ.Figure 1.

View Article: PubMed Central - PubMed

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.

No MeSH data available.