Accumulating clinical evidences over recent years support the notion that the immune system can cure cancers. The potential of T cell therapy against cancer cells is documented by the persistent clinical responses observed after transfer of tumor specific T cells in some cancer patients. However, tumor-associated antigens are often self-antigens, and thus high-avidity tumor-specific lymphocytes are often deleted or anergized to prevent detrimental autoimmunity. This hurdle can be overcome by the transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells. Initial clinical studies have demonstrated the feasibility of TCR gene transfer, but the therapeutic results have been thus far suboptimal. This likely reflects limitations of current gene transfer approaches, which may fail to establish stable high-level TCR expression because the exogenous tumor-specific TCR α and β chains compete with endogenous TCR chains for surface expression. This problem is further aggravated by the potential for inappropriate pairing of exogenous and endogenous TCR chains, which leads to the assembly of novel TCRs with unpredictable, and possibly auto-reactive, specificities. Finally, the presence of endogenous TCR limits the use of TCR gene transfer in the allogeneic setting due to the risk of graft-versus-host disease (GvHD).
To overcome these limitations, in collaboration with the group of Chiara Bonini, San Raffaele Scientific Institute, we developed the first strategy based on engineered nucleases to edit T cell specificity at the DNA level. Our approach combines somatic knockout of the endogenous TCR genes by transient exposure to α and β chain specific Nucleases, with the introduction of the desired tumor-specific TCR by lentiviral vector gene transfer. The resulting ‘TCR-edited’ lymphocytes showed enhanced tumor killing activity with sharply reduced non-specific alloreactivity, as compared to matched cells undergoing conventional TCR gene transfer (Provasi*, Genovese* et al., Nature Medicine 2012). This work was the first proof that gene editing can be used to genetically re-write the endogenous antigen specificity of cytotoxic T cells and enable the feasibility of a safe allogeneic T cell transplantation, thus providing the basis for several other studies in the rapidly expanding cancer immunotherapy field, some of which already entered clinical testing.
To simplify the TCR gene editing procedure in view of clinical development, we developed the TCR single editing approach which enables a rapid generation of highly performing tumor specific T cells (Mastaglio, Genovese et al., Blood 2017). This innovative approach is now widely used in the immunotherapy field for generating allo-compatible T cells or to express CAR genes under the control of endogenous TCR promoter.
To simplify the TCR gene editing procedure in view of clinical development, we developed the TCR single editing approach which enables a rapid generation of highly performing tumor specific T cells (Mastaglio, Genovese et al., Blood 2017). This innovative approach is now widely used in the immunotherapy field for generating allo-compatible T cells or to express CAR genes under the control of endogenous TCR promoter.
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Chimeric antigen receptor T cells (CAR-T), bispecific, and toxin-conjugated antibodies represent emerging approaches to overcome cancer immune evasion and dampen alloreactive immune responses. Ideally, the target antigen should be strictly tumor-specific to avoid toxicity to healthy tissues and essential for tumor survival to reduce the occurrence of antigen-loss variants. Yet, despite significant efforts in advancing immunophenotype profiling and high-throughput genetic screening of cancer cells, the identification of targets remains a major hurdle to the full realization of these approaches' potential. To address this unmet medical need, we recently developed a compelling novel "Epitope Editing" strategy that generates multiple tumor-specific immunotherapy targets by removing them from the healthy tissue.
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See highlights on ACIR here.
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We found that specific epitopes on essential genes can be engineered in healthy hematopoietic stem/progenitor cells (HSPCs) to avoid binding by therapeutic antibodies (Ab) while preserving physiologic protein expression, regulation, and function. This strategy will create “stealth” blood lineages resistant to Ab-based drugs or CAR-T cells, thus enabling prolonged and more aggressive immunotherapy for high-risk patients, such as those affected by aggressive forms of acute myeloid leukemia (AML). Differently from other proposed strategies aimed at abrogating dispensable lineage markers, such as CD33, or targeting surface markers that are downregulated on AML blasts, such as the CD45, our epitope editing strategy will enable targeting genes directly involved in malignant transformation, upregulated in leukemic stem cells, and essential for leukemia survival, thus minimizing the risks of tumor immune escape by antigen loss or downregulation. To this goal, we selected the cytokine receptors FLT3, KIT, and CD123, whose expression, either in wild-type or mutated forms, is found in more than 85% of AML cases and is associated with poor prognosis. By transposon-based library screenings, we identified a set of orthologous/not-conserved single amino acid (aa) substitutions on the extracellular domain of each target gene that can be introduced by adenine base-editors (ABE) and confers our desired features (Casirati et al., Nature 2023). Our strategy will dramatically increase the therapeutic potential of these approaches by enabling prolonged/more intensive treatments while sparing the risks of on-target/off-tumor toxicity, thus obtaining high chances to significantly improve patient survival rates and quality of life. Moreover, the use of base editing, which does not introduce DNA breaks into the genome, will allow co-targeting of two or multiple genes, thus enabling combination therapies that will further reduce the risks of relapse. We envision a clear path to clinical translation, given that good manufacturing processes for HSPC gene editing are currently under development in several facilities, including our Gene Therapy Program.
Although CAR T cells have demonstrated highly compelling efficacy in patients with CD19+ hematological malignancies, comparable therapeutic activity of CAR T cells has not yet been achieved in solid tumors, mainly because of the difficulties of engineered T cells to circumvent the immunosuppressive tumor microenvironment. Thus, innovative approaches and suitable models have to be exploited to broaden the applicability of these revolutionary technologies for the treatment of highly aggressive malignancies.