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Andrew M. Scott
Ludwig Institute for Cancer Research
Melbourne Tumour Biology Branch
Austin & Repatriation Medical Centre
Melbourne, Australia

Tumor Immunotherapy with Cytotoxic Humanized Antibodies

The premise that selective targeting of tumors will enable more potent cell killing mechanisms to be utilized with minimized normal tissue toxicity remains one of the most attractive therapeutic strategies in oncology. Our laboratory research has focussed on the utilization of potent immune effector functions (including complement-dependent cytotoxicity [CDC] and antibody-dependent cellular cytotoxicity [ADCC]), mediated by Fc regions of recombinant antibodies (mAb), and the mechanisms of upregulating these effector functions for more efficient tumor cell kill.

Recombinant mAbs directed against glycolipid (e.g. gangliosides GD3, GD2, GM2) and carbohydrate (eg Ley) antigens expressed on neurodermal and epithelial tumors have demonstrated the most potent immune cytotoxic activity in vitro and in vivo. In collaboration with scientists at Kyowa Hakko Kogyo Co. Ltd., we have explored GD3 targeting with mAb as a therapeutic strategy for melanoma. Anti-GD3 chimeric mAb KM871 has potent CDC (IC50 3.0mg/ml) and ADCC (IC50 1.0mg/ml) activity and produces tumor cures in xenograft models with GD3 expressing cell lines. We have shown that CD3-/CD16+56+ (NK cells) are primarily responsible for ADCC by KM871 with SK-Mel-28 cells (GD3+ve) compared with CD3+/CD4+, CD3+/CD8+, and CD14+ PBMC subsets. ADCC activity of PBMCs with KM871 could be observed up to 3 days when the effector cells to target cell ratio was 25:1. ADCC activity of KM871 is enhanced by IL-2 (10 ng/ml) and maintained by IL-12 (but not LAK cells) for up to 10 days in culture. Conjugation of KM87l with 111In via the bifunctional metal chelate CHX-A-DTPA has been achieved, with excellent retention of affinity. Biodistribution studies of 111In-CHX-A-DTPA KM871 in SK-Mel-28 xenografted BALB/c nude mice has shown maximal tumor uptake of 43% ID/gm at 72 hrs pi, with tumor:blood ratios to 5:1, which are the highest values reported for a mAb targeted to a glycolipid antigen. Analysis of KM871-GD3 antigen intracellular trafficking in this in vivo model has shown internalization and cytosolic retention of KM871 through a mechanism involving lysosomes. We have commenced clinical trials of KM871 in pts with metastatic melanoma, with the aim of determining the biodistribution, pharmacokinetics, immunogenicity, and targeting capabilities of this construct. Immunotherapy strategies involving immune effector function and cytokine upregulation of ADCC are planned.

Hu3S193 is a CDR grafted, humanized antibody against the Ley antigen expressed in over 70% of epithelial cancers (including breast, colon, ovary, and lung cancer). In an MCF-7 Leg +ve cell model, hu3S193 has potent CDC (IC50 1.0mg/ml) and ADCC (IC50 5.0mg/ml), superior to the murine form. Hu3S193 is internalized into MCF-7 cells in vitro and in vivo but retains cytotoxicity due to the high number of binding sites/cell (approx. lxl07) and the high mAb-Ag binding off rate. Due to the frequency of tyrosine residues in the CDR regions, hu3S193 has been conjugated with 111In-CHX-A-DTPA via lysine residues, with retention of immunoreactivity and excellent MCF-7 xenograft localization (mean 30%ID/gm) in a BALB/c nude mouse model. In view of the biologic activity of murine anti-Ley mAbs in clinical trials (limited by immunogenicity), hu3S193 is entering clinical trials for the therapy of epithelial tumors expressing this antigen. The development of tumor-targeting humanized antibodies that have potent immune cytotoxic activity is an emerging area of basic research and translational clinical trials in cancer immunology.