Abstract 69P
Background
Cripto-1 (CR1) is an oncofetal protein required for implantation and expressed in the adult during wound repair, inflammation, and tumorigenesis. A second gene codes for a very homologous protein, Cripto-3 (CR3). Both proteins contain 188 amino acids and differ only in 6 residues. Heretofore, all available antibodies cannot discriminate between these two proteins, preventing any investigation on their differential contributions to cancer biology. Here, we present the generation, characterization, and clinical implications of highly specific monoclonal antibodies (MoAbs) for these two proteins.
Methods
Different peptide fragments of either protein were used as antigens to generate mouse MoAbs (233 clones for CR1, 319 clones for CR3) that were selected by their target protein specificity through SPR and ELISA assays. Selected MoAbs were used for immunohistochemistry on tissue arrays including normal tissues as well as cancers of the lung, breast, colon, ovary, and prostate. MoAbs were also used for Western blotting and ELISA characterization of human serum (breast cancer vs healthy controls).
Results
MoAbs NCI 5G1-1 and NCI 5G11-2 were highly specific for CR1 and CR3, respectively. No crossreactivity was observed with the other protein. Immunohistochemical analysis of cancer specimens showed differential staining patterns, where some tumor cells expressed both proteins, others expressed only CR3, and still others show vascular endothelial cells stained for CR1 while tumor cells express CR3. In prostate, colon, and breast cancer, CR1 and/or CR3 protein expression correlates with clinical parameters, such as TMN_N, TMN_M, tumor stage, tumor grade, or PR expression. Lastly, both CR1 and CR3 interact with established binding proteins Nodal, GRP78 and Alk4 and competitively interfere with one another for targeted binding.
Conclusions
We describe for the first time the development of MoAbs that discriminate human CR1 from CR3, confirming that CR3 is translated into a protein in human cells, thus removing its pseudogene status. We also confirm CR1, and propose CR3, as cancer prognosis and severity markers.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain.
Funding
Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain.
Disclosure
All authors have declared no conflicts of interest.
Resources from the same session
67P - The effect of non-viral gene-immune therapy via OX40L or 4-1BBL on murine subcutaneous CT26 colon cancer model
Presenter: Olga Rakitina
Session: Poster session 09
68P - Dendritic polylysine with paclitaxel and triptolide codelivery for enhanced NSCLC ferroptosis through the accumulation of ROS
Presenter: Huae Xu
Session: Poster session 09
70P - The human intratumor mycobiome is significantly influenced by an individual's race
Presenter: Dan Coster
Session: Poster session 09
71P - Preclinical characterization of ARX305: A next-generation anti-CD70 antibody drug conjugate for the treatment of CD70-expressing cancers
Presenter: Lillian Skidmore
Session: Poster session 09
72P - Impact of extended panel of genes for germline cancer testing
Presenter: Shaheenah Dawood
Session: Poster session 09
73P - Preclinical and clinical presentation of the nerve-driven tumor spread
Presenter: Dawid Sigorski
Session: Poster session 09
74P - Characterization of ERBB2 variation and their association with immune response in solid tumours
Presenter: Dong Wang
Session: Poster session 09
75P - Double-stranded RNA transfection induced anti-tumour effect mediated by dual RIG-I and TLR-3 immune pathways
Presenter: Jiayu Tai
Session: Poster session 09
76P - Improvement of whole-cell cancer vaccine anti-tumor effect by different injection methods
Presenter: Chin yang Chang
Session: Poster session 09
77P - Normative data on the sexual health questionnaires - QLQ-SHQ22, and the sexual domains of the QLQ-BR23/BR45 - for Norwegian general population with and without cancer
Presenter: Ragnhild Åsberg
Session: Poster session 09