Abstract
A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.
Highlights
Signals transmitted through cell surface growth factor receptors upon binding to cognate ligands are essential for regulating normal cell growth and differentiation, and contribute to the development, proliferation, survival, motility, and metastasis of diverse types of malignant cells, as exemplified by the well-studied insulin-like growth factors (IGFs), and their main signaling receptor, insulin-like growth factor type 1 receptor (IGF-1R) [1,2,3,4]
CR1 failed to block the binding of IGF-1 or IGF-2 to immobilized rhIGF-1R in the bead assay (Figure S5), contrary to the earlier observation that its murine counterpart could partially inhibit the binding of 125I-IGF-1 to MCF-7L cells
MAB391 had no effect on the binding of PE-labeled R1 to immobilized rhIGF-1R (Figure S8A), R1 substantially reduced the binding of PE-labeled MAB391 (Figure S8B), suggesting that R1 may inhibit the binding of MAB391 to immobilized rhIGF-1R allosterically
Summary
Signals transmitted through cell surface growth factor receptors upon binding to cognate ligands are essential for regulating normal cell growth and differentiation, and contribute to the development, proliferation, survival, motility, and metastasis of diverse types of malignant cells, as exemplified by the well-studied insulin-like growth factors (IGFs), and their main signaling receptor, IGF-1R [1,2,3,4]. The cytoplasmic region of the bsubunit harbors a tyrosine kinase domain, as well as a docking site for members of the insulin receptor substrate (IRS) family, and the SH2-containing adaptor protein, Shc [5]. The engagement of IGF-1R by IGFs induces autophosphorylation of the three tyrosine residues in the kinase domain of b-subunit [7], which further phosphorylates other tyrosine residues in the cytoplasmic domain, thereby leading to recruitment of IRS and Shc, with subsequent activation of both phosphoinositide 3-kinase (PI3K)-Akt and the mitogen-activated protein kinase (MAPK) pathways [8]. IRB recognizes only insulin, its splice variant, IRA, which is most commonly expressed by tumors, binds to IGF-2 [11] with high affinity, resulting in mitogenic effects and increased survival, motility, and invasiveness of cancer cells [12]. The complexity of the IGF-signaling system is further compounded by the ability of IGF-2 to stimulate IRA and IRA/IRB, the ability of both IGF-1 and IGF-2 to stimulate IGF-1R, IGF-1R/IRA, and IGF-1R/IRB, and the crosstalk between IGF-1R and EGFR [13,14,15], all of which appear to constitute pathways for certain cancer cells to escape IGF-1R-targeted therapies, and provide the rational for cotargeting IGF-1R with IR [16,17] or EGFR/HER2 [18,19] to enhance treatment efficacy
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