Preclinical testing of bispecific T cells attracting monoclonal antibodies for immunotherapy of colorectal cancer

Thesis

Immunotherapy using bispecific monoclonal antibodies (BiMAb) is a promising cancer therapy. Such BiMAbs bind simultaneously to immune effector cells and to a cancer-specific antigen on tumour cells, resulting in killing of the latter.

The heterogeneity of tumour associated antigen (TAA) expression in a tumour as well as poor infiltration of the T cells into the tissue can pose challenges to the use of BiMAb in solid tumours, raising the need for mechanistic insights into the mode of BiMAb-dependent cell killing. It has been shown that IFNγ secreted by antigen specific T cells can act on bystander tumour cells resulting in contact independent cell killing; for efficient eradication of tumours a bystander effect is essential.

Placental alkaline phosphatase (PLAP) is an enzyme expressed on the cell surface only in the placenta not in other normal tissues. PLAP is ectopically expressed in some tumours such as ovarian tumours, seminomas, and about 20% of colon carcinoma, making it a good target for BiMAb immunotherapy.

Colorectal cancer (CRC) causes one of the highest numbers of cancer-related deaths worldwide. That emphasises the urgency to find an effective treatment to treat colon cancer. We have a collection of 116 different CRC cell lines in our lab, well characterised in terms of gene expression and mutations, providing a reliable tool to test potential therapeutic agents.

The specificity of an anti-PLAP monoclonal antibody (H17E2) produced by our lab was used to develop a BiMAb that binds to T cell co-receptor CD3ε chain and to PLAP (CD3 x PLAP BiMAb (RPLP3)).

Microarray analysis of our CRC cell lines showed that 21.5% of them express PLAP. Interestingly, immunofluorescence and FACS analysis showed that some of the lines are heterogeneous in their expression, with levels ranging from PLAP-negative to PLAP-high.

We found that the CD3-PLAP BiMAb induced specific killing of PLAP-positive colorectal cancer cell lines cultured with peripheral blood mononuclear cells (PBMCs) as source of T cells, and that the killing depends on PLAP expression.

Moreover, we found that the effect of CD3-PLAP BiMAb (RPLP3) treatment was extended to PLAP-negative cells, when co-cultured with PLAP-positive ones, indicated a bystander effect. The bystander effect on PLAP-negative cells is only visible after 48 hours of treatment, suggesting a different mechanism for the indirect killing. Understanding the mechanism behind the bystander killing would be key to improving antibody therapy for colorectal cancer. As well as understanding the cause of PLAP expression on CRC cell lines.

Authors: Alrishedan, NS

• DOI: 10.5287/ora-nge8zmzze
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: IHC; placental alkaline phosphatase; ELISA M30; bispecific monoclonal antibodies; caspase 3; EpCAM; colorectal cancer cell lines; monoclonal antibody; T cells; PLAP; PBMCs; H17E2; epithelial cell adhesion molecule or CD326; T cells attracting monoclonal antibodies; apoptosis; CRC cell lines; immunofluorescence; TCRm; lipid nanoparticles (LNPs); cancer heterogeneity; immunotherapy; bystander killing; cancer antigen; tissue staining; colorectal cancer; carT cells
• Subjects: Rectum--Cancer; Bispecific T Cells Attracting Monoclonal Antibodies for Immunotherapy of Colorectal cancer; Bystander tumour cells killing; Cancer; Immunotherapy; Bystander killing