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TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations

View Article: PubMed Central - PubMed

ABSTRACT

Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.

No MeSH data available.


TsAb3v1 and TsAb2v2 antibody generation. (A) Schematic representation of the two TsAb constructs. cMet and HER3 VH-(G4S)3-VL scFvs were fused to the C-terminus of ‘knob-into-hole’ heavy chains of an IgG1 antibody backbone by a (G4S)2 connector. N-terminal scFabs targeting HER1 and IGF1R were designed as VL-CL-(G4S)6GG-VH-CH1 in the TsAB2v2 construct. TsAb3v1 consists of an IGF1R targeting one arm scFab and a disulfide stabilized Fab binding to HER1. (B) SDS-PAGE analysis of purified TsAb3v1 and TsAb2v2. (C) Analytical SEC of purified TsAb3v1 and TsAb2v2. (D) SPR analysis of simultaneous binding of TsAb3v1 and TsAb2v2 to HER1, HER3, IGF1R and cMet. A color code of the injection order of the extracellular receptor domains of IGF1R, HER3 and cMet on the tetraspecific antibodies bound to a HER1-coated CM5 chip is depicted below the graphs.
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gzw037F1: TsAb3v1 and TsAb2v2 antibody generation. (A) Schematic representation of the two TsAb constructs. cMet and HER3 VH-(G4S)3-VL scFvs were fused to the C-terminus of ‘knob-into-hole’ heavy chains of an IgG1 antibody backbone by a (G4S)2 connector. N-terminal scFabs targeting HER1 and IGF1R were designed as VL-CL-(G4S)6GG-VH-CH1 in the TsAB2v2 construct. TsAb3v1 consists of an IGF1R targeting one arm scFab and a disulfide stabilized Fab binding to HER1. (B) SDS-PAGE analysis of purified TsAb3v1 and TsAb2v2. (C) Analytical SEC of purified TsAb3v1 and TsAb2v2. (D) SPR analysis of simultaneous binding of TsAb3v1 and TsAb2v2 to HER1, HER3, IGF1R and cMet. A color code of the injection order of the extracellular receptor domains of IGF1R, HER3 and cMet on the tetraspecific antibodies bound to a HER1-coated CM5 chip is depicted below the graphs.

Mentions: Two novel tetraspecific antibody formats TsAb2v2 and TsAb3v1 with antigen-binding specificities for HER1, IGF1R, HER3 and cMet were generated. A scFab format was employed to prevent light-chain mismatch of the IGF1R and HER1-binding Fab arms at the N-terminus of the human IgG1 backbone as described previously (Schanzer et al., 2014). Using either one or two scFab arms was the only difference between the two tetraspecific antibody formats. The antibody Fc region was further engineered by the knobs-into-holes technology to enable addition of two different scFv (Ridgway et al., 1996). HER3 and cMet-binding scFvs were joined by a G4S linker and fused at the C-terminus of the CH3 domain to generate tetraspecific antibodies (Fig. 1A).Fig. 1


TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations
TsAb3v1 and TsAb2v2 antibody generation. (A) Schematic representation of the two TsAb constructs. cMet and HER3 VH-(G4S)3-VL scFvs were fused to the C-terminus of ‘knob-into-hole’ heavy chains of an IgG1 antibody backbone by a (G4S)2 connector. N-terminal scFabs targeting HER1 and IGF1R were designed as VL-CL-(G4S)6GG-VH-CH1 in the TsAB2v2 construct. TsAb3v1 consists of an IGF1R targeting one arm scFab and a disulfide stabilized Fab binding to HER1. (B) SDS-PAGE analysis of purified TsAb3v1 and TsAb2v2. (C) Analytical SEC of purified TsAb3v1 and TsAb2v2. (D) SPR analysis of simultaneous binding of TsAb3v1 and TsAb2v2 to HER1, HER3, IGF1R and cMet. A color code of the injection order of the extracellular receptor domains of IGF1R, HER3 and cMet on the tetraspecific antibodies bound to a HER1-coated CM5 chip is depicted below the graphs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036864&req=5

gzw037F1: TsAb3v1 and TsAb2v2 antibody generation. (A) Schematic representation of the two TsAb constructs. cMet and HER3 VH-(G4S)3-VL scFvs were fused to the C-terminus of ‘knob-into-hole’ heavy chains of an IgG1 antibody backbone by a (G4S)2 connector. N-terminal scFabs targeting HER1 and IGF1R were designed as VL-CL-(G4S)6GG-VH-CH1 in the TsAB2v2 construct. TsAb3v1 consists of an IGF1R targeting one arm scFab and a disulfide stabilized Fab binding to HER1. (B) SDS-PAGE analysis of purified TsAb3v1 and TsAb2v2. (C) Analytical SEC of purified TsAb3v1 and TsAb2v2. (D) SPR analysis of simultaneous binding of TsAb3v1 and TsAb2v2 to HER1, HER3, IGF1R and cMet. A color code of the injection order of the extracellular receptor domains of IGF1R, HER3 and cMet on the tetraspecific antibodies bound to a HER1-coated CM5 chip is depicted below the graphs.
Mentions: Two novel tetraspecific antibody formats TsAb2v2 and TsAb3v1 with antigen-binding specificities for HER1, IGF1R, HER3 and cMet were generated. A scFab format was employed to prevent light-chain mismatch of the IGF1R and HER1-binding Fab arms at the N-terminus of the human IgG1 backbone as described previously (Schanzer et al., 2014). Using either one or two scFab arms was the only difference between the two tetraspecific antibody formats. The antibody Fc region was further engineered by the knobs-into-holes technology to enable addition of two different scFv (Ridgway et al., 1996). HER3 and cMet-binding scFvs were joined by a G4S linker and fused at the C-terminus of the CH3 domain to generate tetraspecific antibodies (Fig. 1A).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.

No MeSH data available.