Research Activities Dr. Angel Cid-Arregui
Table of Contents
Projects
Our aim is to develop strategies that might help eradicate tumor cells. To this end, our work is focusing on (i) the construction of vaccines designed to stimulate the immune system against specific tumor antigens, and (ii) the investigation of new drug delivery systems, which could facilitate the administration of chemotherapy drugs specifically targeted to tumor cells thereby minimizing side effects.
1. Recombinant fusion proteins based on HBsAg virus-like particles (VLPs)
1a) Construction and characterization of recombinant fusion proteins designed to induce humoral and cellular immune responses against cells infected by human papillomavirus (HPV) types 16 and 18, which cause precancerous lesions in the uterine cervix leading to cervical cancer. In such lesions, the tumor cells express constitutively the transforming proteins of these virus types, namely E6 and E7, which represent specific tumor antigens.
We use the hepatitis B surface antigen (HBsAg) as carrier molecule of relevant HPV epitopes. We have shown previously that immunization with a recombinant adenovirus vector engineered to express a fusion of HBsAg with a truncated E7 mutant devoid of its transforming domains evokes strong B and T cell responses in mice.
Left picture: Coomassie blue staining of flow-through (FT), wash (W) and elution (E) fractions from an ion metal affinity chromatography (IMAC) of recombinant HBsAg-E7(D1-35) protein carrying a His-tag expressed in the yeast P. pastoris
Right picture: Confocal section of a transfected Vero cell expressing HBsAg-E7 (in green). The protein fills the ER and Golgi compartments. Counterstaining (in red) was performed with propidium iodide
1b) Generation of HBsAg fusion proteins carrying endogenous tumor antigens. In some tumors endogenous proteins are expressed at abnormally high levels. For instance, previous studies have revealed that melanoma cells express elevated levels of p53 with predominantly nuclear localization of the protein. Further, in many cancers, mutations inactivate the cytostatic function of certain proteins at the time that create non-self antigens. Stimulating the immune system against overexpressed proteins and/or neoantigens might help the organism to get rid of tumor cells. In collaboration with P. Beckhove and F. Momburg we are producing fusion HBsAg proteins carrying mutated antigens found in cancer patients.
2. Development of liposome formulations containing oncolytic drugs
Development and test in vitro and in vivo of formulations containing oncolytic drugs encapsulated into liposomes, which are additionally conjugated with peptides binding preferentially to cancer cells, such as alpha v beta 6 integrin binding peptides, peptides derived from the HPV L1 and L2 capsid proteins and others. These studies are aimed also at developing formulations in which liposomes are conjugated with antibodies binding preferentially to cervical cancer cells, including antibodies against v6 and ß3 integrins, HPV L1 and others.
Picture: Transmission electron micrograph of gemcitabine-containing liposomes. Bar = 100 nm
3. VLPs carrying cytotoxic drugs
Generation of engineered hollow nanoparticles targeted to specific tumor cells or tissues, which could be used as carriers of cytotoxic drugs. One part of this project is being carried out in the frame of a cooperative research agreement with the Cambridge-based company Aura Biosciences.
Picture: Electron micrograph of negatively stained hollow nanoparticles to be used as carriers for chemotherapeutic drugs
4. Innate immunity in cervical cancer
Study of soluble NKG2D ligands in cervical cancer patients. The innate immune response is considered to be the first line of defense at mucosal surfaces. NK cells represent an important arm of the innate immune system specialized in killing virus-infected and tumor cells through the NKG2D activating receptor, which can promote the lysis of target cells by binding to a recently identified family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes. The MIC family includes MICA, which is a highly polymorphic functional cell-surface protein. Similar to classical HLA class I molecules, MICA also contains three extracellular domains (a1-a3); however, MICA neither associates with a2-microglobulin nor binds antigen peptides. Under physiological conditions, MICA expression is almost restricted to the gastrointestinal epithelium. Nevertheless, MICA is overexpressed in several epithelial tumors. Available evidence suggests that NKG2D engagement by MICA induces proliferation, survival, and, cytotoxic activity in NK cells. Consequently, NKG2D/MICA interaction may represent an important activation pathway to trigger the immune attack against tumor cells. However, it has been demonstrated the shedding of MICA from tumor cell surface in a variety of malignant epithelial tumors, including advanced hepatocellular carcinoma, colon, prostate, renal, breast cancer, as well as hematopoietic tumors. Such phenomenon leads to accumulation of soluble MICA (sMICA) in serum leading to NKG2D down-modulation by facilitating its internalization and lysosomal degradation. This has been proposed to be a novel evasion mechanism of cancer cells to the NK-mediated tumor surveillance.
In collaboration with the group of Dr. del Toro we have recently shown that soluble MICA (sMICA) levels increase concomitantly during progression of HPV-induced premalignant lesions to cervical cancer. This study has provided evidence that sMICA may represent a novel immune evasion mechanism through the natural history of cervical cancer. Further studies are needed to better understand the mechanisms used by tumor cells to evade the innate immune responses, which should help to develop an effective immunotherapy for cervical cancer.
Infection with high-risk HPVs is more prevalent in Mexico, where cervical cancer is nearly two-fold more frequent and has a higher rate of mortality as compared to Germany. Therefore, we are investigating in detail the expression of NKG2D ligands (MICA, MICB, ULB-1 and ULBP-2) in patients of both countries to determine whether differences in expression of these ligands or their shedding from the surface of tumor cells might explain the differences in malignity of the tumors in both countries.
