Periodic Reporting for period 2 - OUTSOURCE (Outsourcing cancer immunity to healthy donors)
Okres sprawozdawczy: 2022-03-01 do 2023-08-31
Despite recent success of cancer immunotherapy, most patients with metastatic cancer are not cured. One of the most important reasons is lack of homogeneously and highly expressed molecules that can be safely targeted. CAR T cell therapies are now approved in acute B-cell leukemia and lymphoma, and have dramatically improved the outcome for patients with these cancers. These CAR T cells target B cell-specific surface antigens, such as CD19, expressed on normal as well as malignant B cells. The treatment is well tolerated, as we can live without normal B cells for years. We can, however, not live without normal T cells, myeloid cells and liver cells etc, which is why CAR T-cell therapies have not yet been approved for non-B-cell malignancies. CARs – Chimeric Antigen Receptors – are artificial immune receptors binding targets via an antibody domain. Thus CARs can only bind cell-surface molecules on cancer cells. In contrast, T-cell receptors (TCRs) can in principle recognize peptides derived from proteins with any subcellular location when presented in context of class I tissue type molecules – in humans called HLA class I. This dramatically increases the number of potential immunotherapy targets, as >80% of cellular proteins are inside the cell. A major challenge to identify TCRs that can strongly recognize self-antigens is, however, that our T-cells are educated during development to not recognize our own proteins, protecting us from autoimmunity. In OUTSOURCE, I am using technologies developed by my group whereby the mechanism of transplant rejection is exploited to identify TCRs from healthy donors that can strongly react to peptides derived from cell-type specific proteins when presented on foreign (allogeneic) HLA-molecules, in several cancer types. The immune response is “outsourced” to a healthy donor. The overall objective of the project is to demonstrate that T cells genetically equipped with such donor TCRs (dTCR T cell therapy) can efficiently “reject” primary cancer cells. Another important aim is to establish a preclinical pipeline for safety testing, and in vivo models for assessment of efficacy.
The work in OUTSOURCE has been divided into five subprojects (SP1-5), and significant progress has been made in each of these. In SP1 and 2, we established a bioinformatics and experimental proteomics pipeline to identify tissue-restricted, highly and homogeneously expressed proteins in hematopoietic lineages and prostate. The experimental platform involved establishment of mono-allelic cell lines expressing 11 of the most frequently expressed HLA-A and HLA-B alleles, as well as candidate proteins identified in the bioinformatics pipeline. Using immunopeptidomics, we have identified by mass spectrometry a number of peptides derived from these proteins presented on the HLA class I molecules. In SP3, we have used our patent-protected technology, exploiting healthy donor T cells primed with antigen-presenting cells expressing HLA-alleles that are foreign to the T cells in complex with peptides derived identified in SP1 and 2, to identify a large number of T-cell receptors. These TCRs have subsequently undergone /will undergo a careful screening for safety (off-target reactivity) and efficacy. To this end, we have in SP4 established a systematic and standardized pipeline for screening of off-target reactivity of the TCRs in vitro (manuscript positively reviewed and invited for re-submission). Such a pipeline has been missing in the field. In SP5, we have moreover established several disease-relevant mouse models for testing of TCR T cell efficacy in acute lymphoblastic leukemia and acute myeloid leukemia, using patient-derived xenograft models. In addition, we have established a humanized mouse model to assess potential toxicity of autologous TCR T cells on normal human hematopoiesis, developed from engrafted human CD34+ cord blood cells. These models also include a model to assess TCR T cell targeting of leukemia stem cells. During the project period, we have demonstrated that a TCR reactive to peptides derived from the nuclear enzyme Terminal deoxynucleotidyl transferase (TdT), which we have identified as a novel immunotherapy target, is safe and efficacious using our established safety pipeline and four different in vivo models. The data were published as a regular article in Nature Biotechnology in 2022. The TCR is restricted by HLA-A*02:01, expressed in 50% of the Caucasian population.
We have identified and validated a novel immunotherapy target, Terminal deoxynucleotidyl transferase (TdT). TdT is only transiently expressed during lymphoid differentiation, and it is not expressed in hematopoietic stem cells or in naïve and mature T and B cells. In contrast, it is overexpressed in 80-90% of acute lymphoblastic leukemias (ALL) of both T cell and B cell type. In our publication in Nature Biotechnology (Ali et al, 2022), we demonstrate that we by use of our patented technology (WO 2015/071763A2 and Kumari et al, PNAS 2014, Ali et al, Nature Protocols 2019) identify TCRs reactive to self-peptides derived from TdT, when presented on foreign HLA-A*0201. We next demonstrate that T cells expressing the TdT TCR (TdT TCR T cells) mediate efficacious killing of primary T-ALL and B-ALL patient cells in vitro, and in mouse models. Mapping of TCR fine-specificity followed by a bioinformatic search did not identify candidate cross-reactive peptides in the normal human proteome, and screening for reactivity against a large panel of TdT negative HLA-A*02:01 positive cell lines derived from a variety of tissues did not identify cross-reactivity. We found that human thymocytes do not express high levels of TdT and HLA class I molecules simultaneously, and consistent with this we could not detect toxicity of TdT TCR T cells on normal human thymocytes or normal hematopoiesis in a humanized mouse model engrafted with CD34+ cord blood cells. One of the TdT TCRs is currently in clinical development. Clinical grade virus for gene transfer has been produced, and funding for an investigator-initiated phase I clinical trial recruiting relapsed/refractory T-ALL and B-ALL patients has been acquired. A manuscript describing our stepwise and systematic safety pipeline for screening of off-target reactivity of TCRs in vitro has been completed. Such a platform has been missing in the field. To benchmark the technology we used a TCR that has been safely applied in the clinic – 1G4 targeting NY-ESO-1 - and a TCR developed by our group reactive to a peptide from the B-cell specific antigen CD20. The data described above contributed to a successful grant from the Research Council of Norway for a new Center of Excellence – the Precision Immunotherapy Alliance (PRIMA), for which I will be one of two co-Directors (one 5-yr term each). I was also awarded an ERC Proof-of-concept grant (April 2023) to pursue commercialization of this TCR.