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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 mediated tumor cell death. H322M cells were incubated for 2 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination as well as all four parental monospecific antibodies individually. Apoptosis induction was measured after cell lysis in a Caspase Glo assay and reported as luminescence units. Staurosporine was used as positive control. Statistical significance was calculated versus a negative control (cells treated with an unspecific huIgG antibody); **P < 0.01; ***P < 0.001.
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gzw037F3: TsAb3v1 and TsAb2v2 mediated tumor cell death. H322M cells were incubated for 2 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination as well as all four parental monospecific antibodies individually. Apoptosis induction was measured after cell lysis in a Caspase Glo assay and reported as luminescence units. Staurosporine was used as positive control. Statistical significance was calculated versus a negative control (cells treated with an unspecific huIgG antibody); **P < 0.01; ***P < 0.001.

Mentions: We next asked if treatment with the inhibitory antibodies results in increased apoptosis in tumor cells. Apoptosis induction was followed by measuring Caspase 3/7 activity in H322M cells. As expected, only addition of antibody combinations elicited a significant increase of apoptosis after 2 days of incubation. Both tetraspecific antibodies significantly induced apoptosis at a concentration of 200 nM in this assay, to a similar degree as the combination treatment, which was almost equally potent as incubation with a 10 µM solution of the multi-kinase inhibitor staurosporine (Fig. 3).Fig. 3


TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations
TsAb3v1 and TsAb2v2 mediated tumor cell death. H322M cells were incubated for 2 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination as well as all four parental monospecific antibodies individually. Apoptosis induction was measured after cell lysis in a Caspase Glo assay and reported as luminescence units. Staurosporine was used as positive control. Statistical significance was calculated versus a negative control (cells treated with an unspecific huIgG antibody); **P < 0.01; ***P < 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gzw037F3: TsAb3v1 and TsAb2v2 mediated tumor cell death. H322M cells were incubated for 2 days with TsAb3v1, TsAb2v2, the BsAbs HER1/IGF1R and cMetHER3 and the combination as well as all four parental monospecific antibodies individually. Apoptosis induction was measured after cell lysis in a Caspase Glo assay and reported as luminescence units. Staurosporine was used as positive control. Statistical significance was calculated versus a negative control (cells treated with an unspecific huIgG antibody); **P < 0.01; ***P < 0.001.
Mentions: We next asked if treatment with the inhibitory antibodies results in increased apoptosis in tumor cells. Apoptosis induction was followed by measuring Caspase 3/7 activity in H322M cells. As expected, only addition of antibody combinations elicited a significant increase of apoptosis after 2 days of incubation. Both tetraspecific antibodies significantly induced apoptosis at a concentration of 200 nM in this assay, to a similar degree as the combination treatment, which was almost equally potent as incubation with a 10 µM solution of the multi-kinase inhibitor staurosporine (Fig. 3).Fig. 3

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