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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 proliferation inhibition of NSCLC co-cultured cell lines. (A) RNA expression of cMet, HER1, HER3 and IGF1R in A549, H596, H322M and H441 cells measured by affymetrix analysis. (B) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells grown individually and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. (C) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells cultivated in various combinations (shown as ± symbols below the graph) and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. Statistical significance was evaluated versus the BsAb showing the strongest growth inhibitory effect; *P < 0.05; **P < 0.01; ***P < 0.001
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gzw037F5: TsAb3v1 and TsAb2v2 proliferation inhibition of NSCLC co-cultured cell lines. (A) RNA expression of cMet, HER1, HER3 and IGF1R in A549, H596, H322M and H441 cells measured by affymetrix analysis. (B) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells grown individually and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. (C) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells cultivated in various combinations (shown as ± symbols below the graph) and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. Statistical significance was evaluated versus the BsAb showing the strongest growth inhibitory effect; *P < 0.05; **P < 0.01; ***P < 0.001

Mentions: Additional selection criteria for the cell lines were the relative expression levels of the targets. Presence of target mRNA was confirmed by affymetrix analysis (Fig. 5A). In addition, receptor presence on the cell surface was confirmed by flow cytometry (data not shown). Growth inhibition in dependence of antibody treatment was initially evaluated on each cell line individually and compared to the BsAbs. TsAb2v2 and TsAb3v1 showed higher efficacy in comparison to the BsAbs in A549, H596 and H322M cells (Fig. 5B). A potential synergistic activity of the tetraspecific antibodies was observed in H322M where treatment with the two simultaneously given BsAbs resulted in a significantly (P < 0.01) lower effect compared to what was observed with each of the two tetraspecific antibodies (data not shown). Inhibitory activity in H441 was overall limited and tetraspecific antibodies do not add to the effect observed with a HER1/IGF1R BsAb. The extent of inhibition is similar to what has been observed with the TKI afatinib and implies a dependency on HER1-mediated signaling (Chao et al., 2015). For H596, we and others had previously shown that a cMet/HER1 BsAb inhibits cellular growth and receptor-mediated signaling (Castoldi et al., 2013; Spiess et al., 2013). A cMet/HER3 BsAb is in this setting similarly active (Fig. 5B).Fig. 5


TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations
TsAb3v1 and TsAb2v2 proliferation inhibition of NSCLC co-cultured cell lines. (A) RNA expression of cMet, HER1, HER3 and IGF1R in A549, H596, H322M and H441 cells measured by affymetrix analysis. (B) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells grown individually and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. (C) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells cultivated in various combinations (shown as ± symbols below the graph) and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. Statistical significance was evaluated versus the BsAb showing the strongest growth inhibitory effect; *P < 0.05; **P < 0.01; ***P < 0.001
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gzw037F5: TsAb3v1 and TsAb2v2 proliferation inhibition of NSCLC co-cultured cell lines. (A) RNA expression of cMet, HER1, HER3 and IGF1R in A549, H596, H322M and H441 cells measured by affymetrix analysis. (B) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells grown individually and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. (C) Percentage growth inhibition versus control of A549, H596, H322M, H441 cells cultivated in various combinations (shown as ± symbols below the graph) and treated for 5 days with cMetHER3 BsAb, HER1/IGF1R BsAb, TsAb3v1 and TsAb2v2. Cell viability was evaluated in a Cell Titer Glo assay. Statistical significance was evaluated versus the BsAb showing the strongest growth inhibitory effect; *P < 0.05; **P < 0.01; ***P < 0.001
Mentions: Additional selection criteria for the cell lines were the relative expression levels of the targets. Presence of target mRNA was confirmed by affymetrix analysis (Fig. 5A). In addition, receptor presence on the cell surface was confirmed by flow cytometry (data not shown). Growth inhibition in dependence of antibody treatment was initially evaluated on each cell line individually and compared to the BsAbs. TsAb2v2 and TsAb3v1 showed higher efficacy in comparison to the BsAbs in A549, H596 and H322M cells (Fig. 5B). A potential synergistic activity of the tetraspecific antibodies was observed in H322M where treatment with the two simultaneously given BsAbs resulted in a significantly (P < 0.01) lower effect compared to what was observed with each of the two tetraspecific antibodies (data not shown). Inhibitory activity in H441 was overall limited and tetraspecific antibodies do not add to the effect observed with a HER1/IGF1R BsAb. The extent of inhibition is similar to what has been observed with the TKI afatinib and implies a dependency on HER1-mediated signaling (Chao et al., 2015). For H596, we and others had previously shown that a cMet/HER1 BsAb inhibits cellular growth and receptor-mediated signaling (Castoldi et al., 2013; Spiess et al., 2013). A cMet/HER3 BsAb is in this setting similarly active (Fig. 5B).Fig. 5

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