Limits...
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.


Related in: MedlinePlus

Immunoblot analysis of TsAb3v1 and TsAb2v2 effect on signaling in BxPC3, H322M and MDA-MB-175-VII cells. Expression and phosphorylation status of HER1, HER3, cMet, IGF1R, AKT and MAPK in cells after a 30 minutes incubation with 0.07 µM TsAb3v1, TsAb2v2, BsAb HER1/IGF1R, BsAb cMet/HER3, and the combination of all four parental antibodies. Following the antibody incubation, cells were stimulated with the relative growth factors EGF, Heregulin, HGF and IGF1 for 10 minutes, lysed and subjected to immunoblotting. Tetra-, bi- and monospecific antibody molecules or combinations corresponding to expression and phosphorylation status of RTK are shown as ± symbols below the graph.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036864&req=5

gzw037F2: Immunoblot analysis of TsAb3v1 and TsAb2v2 effect on signaling in BxPC3, H322M and MDA-MB-175-VII cells. Expression and phosphorylation status of HER1, HER3, cMet, IGF1R, AKT and MAPK in cells after a 30 minutes incubation with 0.07 µM TsAb3v1, TsAb2v2, BsAb HER1/IGF1R, BsAb cMet/HER3, and the combination of all four parental antibodies. Following the antibody incubation, cells were stimulated with the relative growth factors EGF, Heregulin, HGF and IGF1 for 10 minutes, lysed and subjected to immunoblotting. Tetra-, bi- and monospecific antibody molecules or combinations corresponding to expression and phosphorylation status of RTK are shown as ± symbols below the graph.

Mentions: We initially addressed the presence and activation status of the four RTK in the tumor cell lines MDA-MB-175-VII, BxPC3 and H322M by immunoblotting. For in vitro receptor activation, the medium was supplemented with a mixture of the growth factors EGF, HRG, HGF and IGF to compensate for the absence of tumor–stroma interactions. Basal and ligand-dependent activation was determined by receptor phosphorylation status. Downstream signaling occurs predominantly via the MAPK and PI3K signaling pathways. BxPC3 and H322M displayed robust activation of all four RTKs as determined by receptor phosphorylation while MDA-MB-175-VII was only responding to IGF and HRG treatment by activation of the respective receptors (Fig. 2). MAPK as well as PI3K, as measured via phosphorylated AKT, were activated in all instances in which GF were added. We next asked the question if treatment with individual parental antibodies, a combination thereof or the tetraspecific antibodies (i) impairs this activation and if (ii) the tetraspecific antibody performs similar to the combination (combo) of all four parental antibodies. Indeed, the tetraspecific antibodies inhibited receptor activation as well as downstream signaling and showed similar activity as the combination of the parental antibodies (Fig. 2). Furthermore, we could not observe any agonistic activity.Fig. 2


TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations
Immunoblot analysis of TsAb3v1 and TsAb2v2 effect on signaling in BxPC3, H322M and MDA-MB-175-VII cells. Expression and phosphorylation status of HER1, HER3, cMet, IGF1R, AKT and MAPK in cells after a 30 minutes incubation with 0.07 µM TsAb3v1, TsAb2v2, BsAb HER1/IGF1R, BsAb cMet/HER3, and the combination of all four parental antibodies. Following the antibody incubation, cells were stimulated with the relative growth factors EGF, Heregulin, HGF and IGF1 for 10 minutes, lysed and subjected to immunoblotting. Tetra-, bi- and monospecific antibody molecules or combinations corresponding to expression and phosphorylation status of RTK are shown as ± symbols below the graph.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036864&req=5

gzw037F2: Immunoblot analysis of TsAb3v1 and TsAb2v2 effect on signaling in BxPC3, H322M and MDA-MB-175-VII cells. Expression and phosphorylation status of HER1, HER3, cMet, IGF1R, AKT and MAPK in cells after a 30 minutes incubation with 0.07 µM TsAb3v1, TsAb2v2, BsAb HER1/IGF1R, BsAb cMet/HER3, and the combination of all four parental antibodies. Following the antibody incubation, cells were stimulated with the relative growth factors EGF, Heregulin, HGF and IGF1 for 10 minutes, lysed and subjected to immunoblotting. Tetra-, bi- and monospecific antibody molecules or combinations corresponding to expression and phosphorylation status of RTK are shown as ± symbols below the graph.
Mentions: We initially addressed the presence and activation status of the four RTK in the tumor cell lines MDA-MB-175-VII, BxPC3 and H322M by immunoblotting. For in vitro receptor activation, the medium was supplemented with a mixture of the growth factors EGF, HRG, HGF and IGF to compensate for the absence of tumor–stroma interactions. Basal and ligand-dependent activation was determined by receptor phosphorylation status. Downstream signaling occurs predominantly via the MAPK and PI3K signaling pathways. BxPC3 and H322M displayed robust activation of all four RTKs as determined by receptor phosphorylation while MDA-MB-175-VII was only responding to IGF and HRG treatment by activation of the respective receptors (Fig. 2). MAPK as well as PI3K, as measured via phosphorylated AKT, were activated in all instances in which GF were added. We next asked the question if treatment with individual parental antibodies, a combination thereof or the tetraspecific antibodies (i) impairs this activation and if (ii) the tetraspecific antibody performs similar to the combination (combo) of all four parental antibodies. Indeed, the tetraspecific antibodies inhibited receptor activation as well as downstream signaling and showed similar activity as the combination of the parental antibodies (Fig. 2). Furthermore, we could not observe any agonistic activity.Fig. 2

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.


Related in: MedlinePlus