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Research at Mass General
David Spriggs, MDProfessor of Medicine†Massachusetts General Hospital Cancer CenterHarvard Medical School
The Spriggs laboratory has been focused on the proteins on the ovarian cancer cell surface and how those proteins regulate function in health and cancer. The serum cancer marker, CA125, has been used to manage ovarian cancer since the 1980’s but its function is not known. CA125 has been identified as the product of the tethered mucin MUC16. Our studies over the past several years have provided insights into the function of MUC16. It is now apparent that the MUC16 regulates functions like growth, invasion and metastatic disease through the structure of sugars (glycosylation) on the surface of normal and cancer cells. This regulation requires interaction with specialized sugar binding proteins, Galectins which are key components of the tumor microenvironment. We are actively developing our antibodies against MUC16 and Galectin 3 for diagnosis, imaging and treatments. Our work has shown that antibodies which inhibit these cell – cell interactions can slow tumor growth and block the spread of cancer cells locally and inhibit the spread to new organs.
†Appointment process initiated
David Spriggs, MD Principal Investigator
Our research group is actively examining the role of glycosylation in tumor specific behaviors including uncontrolled growth, oncogene activation, invasion, immune system evasion angiogenesis, and metastatic spread. This work includes both inhibitory antibodies against MUC16 and Galectin-3 as sutiable clinical targets.
Our current MUC16 work concentrates on development of our human MUC16 antibodies for targeting ovarian cancer. Our antibodies uniquely target the most proximal, retained portion of the MUC16 following cleavage and release of the CA125 antigen into the circulation. This retained ectodomain is a 58 amino acid peptide, linked to the membrane via a short transmembrane domain and a 31 amino acid cytoplasmic tail which is linked to the cellular cytoskeleton for mobility. The introduction of elements from MUC16 We have shown that most of the adverse consequences related to MUC16 expression. In fact, as little as 114 amino acids from the carboxyl terminal of the intact MUC16 sequence is sufficient to transform immortalized 3T3 mouse fibroblasts. This transformation results in increased soft agar colony formation, Matrigel invasion with increased MMP2/MMP9 expression, activation of both AKT and ERK proto-oncogenes and enhanced growth in nude mice. Similar effects are observed in MUC16 negative ovarian cancer cells. Deletion experiments demonstrate that the MUC16 ectodomain is required for this effect but loss of the cytoplasmic domain is dispensable. If one examines the ectomain in greater detail, it is found to be highly homologous with MUC16 analogs in most mammals particularly the portion of the sequence containing 2 N-glycosylation sites.
Our work has been the first to shown that the oncogenic effects of MUC16 require MGAT5 dependent tetra-antennary glycosylation of the MUC16 ectodomain and interaction with Galactin 3 (LGALS3). This complex then binds to glycosylation sites on growth factors including EGFr, Integrins and immune receptors like CTLA4. This has provided us with opportunities for MUC16+ cancer cell targeting.
Based on this information, we have constructed a panel of anti-MUC16 antibodies to target portions of the ectodomain. Antibodies against the ectodomain confirm that it is co-located in with common growth factor receptors in lipid rafts, and undergoes internalization / cycling. Antibodies that block the N-Glycosylation sites on the ectodomain effectively block the oncogenic properties of MUC16. We are now adapting our murine anti-MUC16 antibodies for 1) direct ovarian cancer targeting; 2) ADC with toxins like MMAE linked to humanized anti-MUC16 antibodies; 3) Bispecific T-cell engaging constructs linking MUC16 expressing cells to CD3 + T cells; 4) T cells directed at MUC16 epitopes.
MUC16-directed Chimeric Antigen Receptor (CAR) T Cells
Chimeric Antigen Receptor (CAR) T cells have not been successful in the management of solid tumor malignancies. Reasons for this include; poor trafficking, the presence of an immunosuppressive tumor microenvironment, CAR T-cell dysfunction and immune escape via antigen-loss. In conjunction with Olapado Yeku, a newly recruited faculty member and former MSKCC collaborator, we are using our antibodies as MUC16 targeted CAR T cells. We are developing strategies to further modify CAR T cells to optimize their efficacy for ovarian cancer and gynecologic malignancies. Our approaches to further engineering these CAR T cells are informed by the ovarian cancer tumor microenvironment. Using syngeneic immune competent mouse models and subsequent validation in genetically engineered and xenograft models, we are able to effectively evaluate these rationally optimized CAR T cells as monotherapy or in combination with other immunomodulatory agents prior to initiation of clinical trials.
Galectin 3 Targeting
LGALS3 regulates the interaction of surface proteins with the extracellular membrane domain and mediates a signal cascade leading to invasion, oncogene activation and growth. While anti-MUC16 glycosylation site antibodies inhibit oncogenic properties, LGALS3 represents a more general strategy for targeting glycosylation dependent oncogenesis. We have developed high-affinity antigalectin-3 antibodies directed at the carbohydrate recognition domain (CRD) of the galectin-3 carboxyl-terminus (to block sugar binding). These antibodies are able to block the oncogenic effects of MUC16 expression including invasion, oncogene activation (AKT, ERK, SRC) and reduced growth in nude mice. In addition, these antibodies appear able to decrease metastatic behaviors in models lung metastasis.
Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci Rep. 2017 Sep 5;7(1):10541.
Rao TD, Fernández-Tejada A, Axelrod A, Rosales N, Yan X, Thapi S, Wang A, Park KJ, Nemieboka B, Xiang J, Lewis JS, Olvera N, Levine DA, Danishefsky SJ, Spriggs DR. Antibodies Against Specific MUC16 Glycosylation Sites Inhibit Ovarian Cancer Growth. ACS Chem Biol. 2017 Aug 18;12(8):2085-2096.
Rao TD, Tian H, Ma X, Yan X, Thapi S, Schultz N, Rosales N, Monette S, Wang A, Hyman DM, Levine DA, Solit D, Spriggs DR. Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion. PLoS One. 2015 May 12;10(5):e0126633.
Rao TD, Rosales N, Spriggs DR. Dual-fluorescence isogenic high-content screening for MUC16/CA125 selective agents. Mol Cancer Ther. 2011 Oct;10(10):1939-48.
Chekmasova AA, Rao TD, Nikhamin Y, Park KJ, Levine DA, Spriggs DR, Brentjens RJ. Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice following adoptive transfer of T cells genetically targeted to the MUC16 antigen. Clin Cancer Res. 2010 Jul 15;16(14):3594-606.
Dharma Rao T, Park KJ, Smith-Jones P, Iasonos A, Linkov I, Soslow RA, Spriggs DR. Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. ApplImmunohistochemMolMorphol. 2010 Oct;18(5):462-72.
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