EU project: Profiling Responders In Antibody Therapies (PRIAT)
What are general and long term goals of the project?
The vast majority of pharmaceutical agents can be divided into two categories: small organic drugs (typically < 500 Da) and biotech drugs, mainly consisting of therapeutic proteins. About 50% of product sales in this latter category is represented by antibody-based products. Seven out of ten of top-selling therapeutic proteins are antibodies or antibody derivatives. Furthermore, Humira™ (a monoclonal antibody directed against TNF and approved for the treatment of various inflammatory conditions) is the best-selling pharmaceutical agents in all categories [Walsh (2014) Nat. Biot., 32, 992]. In spite of their commercial success, antibody products are not always as active as one would like them to be. For example, Avastin™ and Herceptin™, used in combination with chemotherapy, have been approved for the treatment of metastatic colorectal cancer and of metastatic breast cancer, respectively, on the basis of a prolongation of median overall survival for only few months. However, patients treated with these antibody drugs are typically not cured and the few extra months of life come at a very high cost for the healthcare system. TNF blockers (such as Humira™, Enbrel™ and Remicade™) provide a substantial benefit to about 50% of patients with rheumatoid arthritis. However, these drugs do not cure the disease. Thus, there is a need to develop better antibody-based drugs and to understand which patients are likely to benefit from antibody therapy most.
In the PRIAT Project, we focused on three main indications for antibody-based therapeutics (cancer, rheumatoid arthritis and graft rejection), with the aim to experimentally address three issues, which are crucial for the understanding of antibody activity and for the design of the drugs of the future: The main goal of the PRIAT Project consists in the development and use of HLA peptidome analysis for the study of interpatient and intrapatient variations in peptides associated with HLA class I and, potentially, HLA class II in response to antibody treatment. The main therapeutic focus will be cancer, rheumatoid arthritis and heart transplant rejection.
A second goal of the PRIAT Project will focus on the understanding of the interplay between antibody accumulation at the site of disease and therapeutic efficacy, with a special emphasis on armed antibodies. Philogen and ETH Zürich are also developing an anti-inflammatory immunocytokine (“F8-IL10”, currently in Phase I clinical trials) for the treatment of patients with rheumatoid arthritis (RA). Immuno-PET imaging methodologies, based on the radiolabeling of armed antibody products with iodine-124 developed at VUMC Amsterdam, promise to facilitate patient selection, based on the detection of antibody uptake at site of disease using PET methodologies [Tijink et al. (2009) Eur. J. Nucl. Med. Mol. Imaging, 36, 1235].
A third goal of the PRIAT Project will be the multi-colour immunofluorescence study of biopsies taken from cancer patients undergoing therapy with armed antibodies (or animal specimens), in order to monitor the accumulation of therapeutic antibodies at site of disease and patterns of leukocyte infiltration.
Please state a few more specific objectives of the project.
In more detail, we will work on the following experimental Objectives, which correspond to the main experimental Work Packages of the Project:
I. Implementation of the methodology to study HLA class I peptide complexes
II. Monitoring of patients with metastatic melanoma using HLA-peptidome analysis and correlation with response to treatment with L19-IL2
III. Monitoring of rats in a heart transplant rejection model, using HLA-peptidome analysis, and evaluating markers in this model under treatment with antibody-cytokine conjugates
IV. Development of HLA-peptidome methodologies for the study of circulating HLA class II – peptide complexes and validation in CIA and RA
V. Monitoring of mice with collagen-induced arthritis (CIA) and patients with RA using HLA-peptidome analysis and correlation with response to treatment with TNF-blocking antibodies
VI. Development of non-invasive immuno-PET methodologies for the radiolabeling of armed antibody products and their imaging in patients with rheumatoid arthritis and cancer
VII. Integration of HLA-peptidome analysis data, imaging data and response to treatment, for the implementation of patients’ stratification strategies
Describe the methodology, approach and technologies used.
The PRIAT project relies on three innovative methodologies for the profiling of patients’ responses to antibody treatment:
HLA-peptidome analysis HLA-peptidome analysis, as recently described [Bassani-Sternberg et al. (2010) Proc. Natl. Acad. Sci. U.S.A., 107, 18769], represents the main technological component for the PRIAT Project. Thanks to recent discoveries and improved MS methodologies, several hundred HLA-associated peptides can be detected from only 3-5 ml of cancer patients’ plasma. Signatures from HLA-bound peptides will be studied, in order to assess whether the presentation of certain peptides distinguishes responders from non-responders, and to study whether the HLA-peptidome evolves during antibody treatment in an intra-patient analysis. HLA-peptidome analysis will be applied not only to the study of plasma samples from patients and rodents with cancer and rheumatoid arthritis, but also for the study of plasma samples of rats which reject a heart transplant [Franz et al. (2010) J. Heart Lung Transplant., 30, 86]. Indeed, antibody products targeting the IL2 receptor are used to prevent rejection in transplantation and there is a considerable research interest to profile patients who are likely to reject a transplanted heart, in a process which is dominated by a CD8+ T cell response to peptides presented on HLA class I.
Immuno-PET Analysis Armed antibodies (e.g., immunocytokines, radiolabelled antibodies and antibody-drug conjugates) exhibit a clear dose-response profile for the treatment of cancer, arthritis and other diseases. The labelling of armed antibodies with iodine-124 and the implementation of immuno-PET methodologies may allow convenient patient’s stratification, as those patients with a higher uptake of antibody product are more likely to respond to treatment. In the PRIAT Project, we will demonstrate the feasibility of immuno-PET analysis in the clinic, by studying arthritis patients with the clinical-stage anti-inflammatory immunocytokine F8-IL10 (“Dekavil”) labelled with iodine-124, a radionuclide which has only recently become available in clinical-grade quality thanks to the work of the Amsterdam group [Tijink et al. (2009) Eur. J. Nucl. Med. Mol. Imaging, 36, 1235].
Analysis of biopsies Some patients with metastatic melanomas have cutaneous or sub-cutaneous metastases which can be biopsied, thus offering a unique opportunity to correlate HLA-peptidome data and immuno-PET images with the nature of leukocyte infiltrate within the metastatic tumour mass. Based on our previous experience in the analysis of animal models of cancer and of biopsy specimens from patients, we expect that patients with a stronger response in the systemic or intralesional trial will exhibit a rich infiltrate of leukocytes into the tumour mass. At the same time, we will stain for Tregs, CD4 and CD8, NK cells and other leukocytes with multi-colour immunofluorescence methodologies, in order to gain insight about dynamic changes in the cell infiltrate in relation to response to therapy.
Systems biology approach finally, for Cancer, Rheumatoid Arthritis and Transplant Rejection, respectively, the PRIAT Project will have the unique opportunity to study data from all three methodologies and correlate it with clinical parameters in a systems biology approach to identify molecular mechanisms underlying targeting efficiency and treatment response.
How is the project progressing, any results you wish to highlight?
Characterization of the disease-targeting performance of antibody products.
In animal models of disease, we have performed extensive quantitative biodistribution studies, using radiolabeled preparations of therapeutic antibody products. This work has led to numerous publications and, collectively, has provided us with a quantitative understanding of the selectivity which antibody products can reach in vivo in rodent models of cancer, rheumatoid arthritis and graft rejection.
A priori, there is no guarantee that biodistribution and imaging studies in animal models may faithfully reflect the performance of the same products in patients. In order to bridge this gap of knowledge, we have made two contributions to the field, which we believe to be important:
(i) we have published a dosimetric estimate of radiation doses in organs and neoplastic lesions for a pan-tumoral antibody (L19, specific to the alternatively-spliced EDB domain of fibronectin), which has been studied in patients with solid tumors or with lymphomas. These studies have shown that the L19 antibody preferentially localizes on neosplastic lesions, but also that the tumor-homing selectivity varies greatly (i.e., up to 40-fold) from lesion to lesion and from patient to patient
(ii) we have developed and clinically implemented a methodology for the radiolabeling of antibody products in GMP conditions with iodine-124, a PET radionuclide which is particularly suitable for the study of antibody products in PET procedures. The methodology has been used for the characterization of Dekavil (F8-IL10), an armed antibody developed by Philogen, currently being investigated in randomized double-blind Phase IIb clinical trials in patients with rheumatoid arthritis. In addition to the specific information gained in this study about the arthritis homing properties of the product, the Project has demonstrated the feasibility of mechanistic immuno-PET studies, which should facilitate product development in the future Multiplex analysis of biomarkers and of leukocyte infiltration.
We have implemented the routine analysis of leukocyte infiltration at the site of disease and the multiplex analysis of cytokine levels (in serum and at the site of disease) for many preclinical experiments, aimed at the characterization of the activity and mechanism of action of various antibody-based therapeutics. These studies have been published in several articles and have facilitated the identification of product candidates for clinical and industrial development programs (see below).
Furthermore, we have studied in substantial detail the infiltration of lymphocytes into melanoma lesions in patients receiving antibody drugs and the specificities of cytotoxic T-lymphocytes which account for long-term survival, following pharmacological treatments. These investigations have led to the identification of certain tumor rejection antigens (e.g., NY-ESO-1), which are efficiently recognized by T cells in patients that respond well to treatment. Furthermore, the demonstration that certain antibody-based therapeutic interventions (e.g., treatment with IL2-based immunocytokines) led to a dramatic increase of certain leukocytes (e.g., T cells and NK cells) in the neoplastic mass has prompted the design of novel combination therapies, which are about to be studied in clinical trials (see Sections below).
HLA peptidome analysis. We have developed, implemented and perfected methodologies for the determination of “atlases” of peptides bound to MHC-I or MHC-II (HLA-I and HLA-II in man). Thanks to the use of state-of-the-art mass spectrometers and to the implementation of efficient antibody-based pulldown procedures, these methodologies have allowed the determination of complex peptidomes, comprising hundreds of MHC-bound peptides, in complex biological samples, such as cell lines or patients’ sera.
For the first time, it has been possible to identify hundreds of HLA-I-bound peptides from sera of melanoma patients. The identified peptides included previously reported melanoma rejection antigens, thus providing confidence about the robustness of the procedure. In mice with arthritis, collagen-derived peptides were identified as MHC-II ligands, thus providing a direct analytical access to antigens which may trigger autoimmune conditions.
The applications opened by the successful implementation of methods for the determination of HLA peptidomes are numerous and, in our opinion, important. For example, on the basis of the knowledge of HLA-I peptidome in melanoma patients, it now becomes possible to synthesize the corresponding peptides and probe T-cell specificities in the same patients, using either peptide-based stimulation or multiplex tetramer technology, pioneered by Ton Schumacher in Amsterdam. Furthermore, we now have the opportunity to go back to experiments, in which we could cure tumor-bearing mice with innovative antibody-based therapeutics (e.g., immunocytokines based on IL2 or TNF payloads) and determine which are the tumor rejection antigens recognized by cytotoxic T-cells, providing a protective immunity against the tumor.
The importance of HLA peptidome analysis and of multiplex tetramer technology analysis for the study of the response of melanoma patients to antibody drug treatment [Kvistborg et al. (2014) Sci. Transl. Med., 6, 254ra128] has become a particularly important and timely topic, with the approval of anti-CTLA4 and anti-PD1 immunostimulatory antibody products.
Funding source and funding duration:
European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 305309 (PRIAT), 2 years.
Prof. Dario Neri