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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.

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TsAb3v1 and TsAb2v2 tumor cell proliferation inhibition. MDA-MB-175-VII and H322M cells were incubated for 5 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination of all four parental monospecific antibodies at the reported concentrations. Cell viability was measured in a Cell Titer Glo assay and calculated as growth inhibition versus untreated control.
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gzw037F4: TsAb3v1 and TsAb2v2 tumor cell proliferation inhibition. MDA-MB-175-VII and H322M cells were incubated for 5 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination of all four parental monospecific antibodies at the reported concentrations. Cell viability was measured in a Cell Titer Glo assay and calculated as growth inhibition versus untreated control.

Mentions: Inhibition of tumor cell growth was analyzed in MDA-MB-175-VII and H322M cells. Efficacy was evaluated in comparison to the combination of the four parental antibodies and two BsAbs, binding either HER3 and cMet or HER1 and IGF1R. Viability was assessed after 5 days of treatment with a concentration series of the antibodies. In MDA-MB-175-VII cells, TsAb2v2 and TsAb3v1 did not display any additional growth inhibitory effect compared to the BsAb cMet/HER3, while BsAb HER1/IGF1R was completely inactive (Fig. 4A). At the highest concentration growth inhibition reached 50%, and was slightly lower in comparison to the four monospecific antibodies sequestered simultaneously (60% growth inhibition; P < 0.01 and P < 0.001 significant difference versus TsAb2v2 and TsAb3v1 at 200 nM). Immunoblot data indicate strong activation of the HER3 receptor in MDA-MB-175-VII cells, presumably mediated by HER2 (Fig. 2).Fig. 4


TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations
TsAb3v1 and TsAb2v2 tumor cell proliferation inhibition. MDA-MB-175-VII and H322M cells were incubated for 5 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination of all four parental monospecific antibodies at the reported concentrations. Cell viability was measured in a Cell Titer Glo assay and calculated as growth inhibition versus untreated control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gzw037F4: TsAb3v1 and TsAb2v2 tumor cell proliferation inhibition. MDA-MB-175-VII and H322M cells were incubated for 5 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination of all four parental monospecific antibodies at the reported concentrations. Cell viability was measured in a Cell Titer Glo assay and calculated as growth inhibition versus untreated control.
Mentions: Inhibition of tumor cell growth was analyzed in MDA-MB-175-VII and H322M cells. Efficacy was evaluated in comparison to the combination of the four parental antibodies and two BsAbs, binding either HER3 and cMet or HER1 and IGF1R. Viability was assessed after 5 days of treatment with a concentration series of the antibodies. In MDA-MB-175-VII cells, TsAb2v2 and TsAb3v1 did not display any additional growth inhibitory effect compared to the BsAb cMet/HER3, while BsAb HER1/IGF1R was completely inactive (Fig. 4A). At the highest concentration growth inhibition reached 50%, and was slightly lower in comparison to the four monospecific antibodies sequestered simultaneously (60% growth inhibition; P < 0.01 and P < 0.001 significant difference versus TsAb2v2 and TsAb3v1 at 200 nM). Immunoblot data indicate strong activation of the HER3 receptor in MDA-MB-175-VII cells, presumably mediated by HER2 (Fig. 2).Fig. 4

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&nbsp;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