Potent Control of Tumor Growth by CEA/CD3-bispecific Single-chain Antibody Constructs That Are Not Competitively Inhibited by Soluble CEA

Abstract

Carcinoembryonic antigen (CEA, CD66e) is a well-characterized tumor-associated antigen that is frequently overexpressed in tumors. Phospholipases release CEA from tumor cells resulting in high circulating serum levels of soluble CEA (sCEA) that has been validated as marker for progression of colorectal, breast, and lung cancers. sCEA also acts as a competitive inhibitor for anticancer strategies targeting membrane-bound CEA. As a novel therapeutic approach for treatment of tumors expressing CEA on their cell surface, we constructed a series of bispecific single-chain antibodies (bscAb) combining various single-chain variable fragments recognizing human CEA with a deimmunized single-chain variable fragments recognizing human CD3. CEA/CD3-bscAbs redirected human T cells to lyse CEA-expressing tumor cells in vitro and in vivo. Efficient tumor cell lysis was achieved in vitro at bscAb concentrations from 1 pg/mL (19 fM) to 8.9 pg/mL with preactivated CD8 T cells, and 200 to 500 pg/mL with unstimulated peripheral blood mononuclear cell. The cytotoxic activity of a subset of CEA/CD3-bscAbs was not competitively inhibited by sCEA at concentrations that exceeded levels found in the serum of most cancer patients. Treatment with CEA/CD3-bscAbs prevented the growth of human colorectal cancer lines in a severe combined immunodeficiency mouse model modified to show human T cell killing of tumors. A murine surrogate CEA/CD3-bscAb capable of recruiting murine T cells for redirected tumor lysis in immunocompetent mice prevented the growth of lung tumors expressing human CEA. Together, our results reveal a unique opportunity for targeting cytotoxic T cells toward CEA-expressing tumors without being competitively inhibited by sCEA and establish CEA/CD3-bscAb as a promising and potent therapeutic approach.

CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas

“…Characterization of the in vitro activity of MEDI-565 Similar to other described CEA-specific BiTE Ò antibody constructs, 63 the CEA-and CD3-binding arms of MEDI-565 were designed to redirect human T cells for the CEA-specific lysis of target cells. Parental CHO dihydrofolate reductase deficient (dhfr-) cells (CEA negative) and CHO cells stably expressing CEA were used in a flow cytometry-based assay to determine the cytotoxic activity of MEDI-565 against CEA-expressing cells with a non-mutated background.…”

“…We consider it unlikely that an increased amount of shed CEA in higher expressing cells is neutralizing MEDI-565 activity as this BiTE Ò antibody construct was selected based on its insensitivity toward soluble CEA. 63 Another explanation could be that higher surface density of CEA may hinder synapse formation by cytotoxic T cells, which could provide an explanation why certain cancer cells retain high level expression. The limitation of surface antigen expression has been overcome with other BiTE Ò antibody constructs when longer duration (96 hour) experiments were evaluated.…”

“…63 Briefly, a humanized CEACAM5-specific scFv fragment of A5B7 65 was fused via a short peptide linker to a de-immunized human CD3-specific scFv 66 derived from the murine monoclonal antibody L2K 67 in the orientation anti-human CEACAM5 VH-VL-glycine/serine linker-anti-human CD3 VH-VL. MEDI-565 was selected from a panel of these BiTE Ò antibody constructs, produced in CHO cells and affinity purified via a 6X histidine tag at the C-terminal end of the antibody.…”

“…MEDI-565 and control BiTE Ò antibody construct (bispecific monoclonal antibody with the same C-terminal CD3 binding scFv as MEDI-565 but with an irrelevant N-terminal scFv that binds the herbicide mecroprop) were produced as described. 63 Flow cytometry-based binding studies A total of 2.5 £ 105 cells, including CHO cell lines or human peripheral blood mononuclear cells (PBMCs) enriched for CD3C T cells, were suspended in phosphate-buffered saline, pH7.2 (PBS; Life Technologies, catalog #20012027) C 2% FBS and seeded into each well of 96-well non-tissue culture treated plates (VWR, catalog #29445-154). Each cell type was incubated with MEDI-565 or control BiTE Ò antibody construct in PBS C 2% FBS buffer in triplicate for 1 hour at 4 C in a total volume of 100 mL.…”

“…63 A series of studies with MEDI-565 demonstrated that the BiTE Ò antibody construct could induce patient T cells to kill metastatic CRC specimens derived from patients previously treated with conventional chemotherapy. 64 Herein we extend these initial findings by characterizing the in vitro and in vivo pharmacology of MEDI-565.…”