In this section, I have copied the Workpackage (WP) description from the original application and I have indicates some of the main results achieved. As you can see, for all eight Workpackages we have made considerable progress, which is documented in articles which have been published or in manuscripts which have been submitted for publication.
WP1: Design, implementation and testing (in vitro and in vivo) of modular antibody-cytokine fusion proteins, capable of re-assembly at the tumor site. Initially, the focus will be on immunocytokines, based on the p40 and p35 subunits of murine interleukin-12, but the work will expand to include cytokine-masking strategies and allosteric regulation.
We have generated and tested in vivo novel classes of antibody-cytokine fusion proteins with activity on demand (e.g. split cytokine fusion proteins)
WP2: Investigation of the synergistic therapeutic activity of immunocytokine pairs, first by the co-administration of immunocytokine products and, if successful, by the simultaneous engineering of two cytokine payloads into the same antibody-based fusion protein.
We have investigated the combination of various antibody-cytokine fusions (e.g. those based on IL2 and TNF, or IL4 and IL12) in various models of tumor-bearing mice) and published the resulting findings in numerous articles
WP3: Investigation of the interplay between administration modality (i.e. rate of infusion), tolerability and therapeutic activity of two IL2-based and TNF-based immunocytokines in mouse models of cancer.
We have performed several studies on the impact of administration modalities, both from a theoretical and from a practical viewpoint. Also this WP has led to numerous publications
WP4: Study of ADCC potentiation by the co-administration of therapeutic antibodies in IgG format and IL2- based immunocytokines. Some of these studies may be performed in nude mice bearing subcutaneously-grafted tumors, as these immunodeficient mice still contain functional NK cells.
We have developed novel products that work through ADCC. We have shown that they can be particularly useful for the treatment of hematological malignancies and/or minimal residual disease. We have studied the combination of intact immunoglobulins with immunocytokines and published the findings in many articles.
WP5: Design and experimental implementation of “release on demand” strategies for non-internalizing antibody-drug conjugates, based on the use of reducing agents, tetrazines or other chemical methodologies for the cleavage of antibody-drug linkers.
We have focused on the generation and characterization of antibody-drug conjugates (ADCs) and of small molecule-drug conjugates (SMDCs). We have published the first ever pairwise comparison of ADCs and SMDCs directed against the same tumor associated antigen
WP6: Design and experimental implementation of “release on demand” strategies for non-internalizing small molecule-drug conjugates, based on the use of reducing agents, tetrazines or other chemical methodologies for the cleavage of antibody-drug linkers.
We have mainly focused on non-internalizing small molecule-drug conjugates (SMDCs), specific to carbonic anhydrase IX (CAIX). This membrane protein is expressed in the majority of renal cell carcinomas, but also in other malignancies (e.g. colorectal tumors). In normal tissues, expression is confined to stomach, gallbladder and duodenum.
We have generated and characterized in vivo (both using quantitative biodistribution studies with radiolabeled material and near-infrared fluorescence imaging) various CAIX ligands and identified two molecules, which were more promising for subsequent SMDC studies.
As for the ADC experience described in WP5, the results obtained with exogenous drug release triggering agents were disappointing, whereas enzymatic activities present in the tumor environment (e.g. certain proteases) allowed an efficient and selective mechanism of SMDC activation.
WP7: Experimental determination of the MHC-I peptidome analysis of murine tumor cell lines, which are used in preclinical therapy studies in the lab, in fully immunocompetent syngeneic settings.
We have extablished methodologies for the characterization of the MHC class I and class II peptidome, both on murine and human material.
This work has already led to various publications
In addition, we have used the methodology in order to identify and validate tumor-rejection antigens in mouse models of cancer. This work has led to numerous publications.
WP8: Generation of multiplex tetramers based on the MHC-I peptidomes, identified in WP7, for the FACS- based analysis of T-cell specificities before and after therapeutic intervention.
We have capitalized on our knowledge of tumor rejection antigens in order to characterize the number and activity of tumor-specific T cells, using tetramer technology and a panel of other experimental techniques. Also this WP has led to numerous publications.
