Despite the remarkable success in the treatment of some hematological cancers, the risk of serious adverse events and loss of potency remain major problems that threaten to curtail widespread application of chimeric antigen receptor (CAR) T-cell therapies to other cancer types including solid tumors. These problems, all of which are influenced by the binding affinity of the CAR targeting domain, result from the propensity for unpredictable hyperactivation that can lead to potentially fatal systemic toxicities such as cytokine release syndrome (CRS), tumor lysis syndrome (TLS) and neurotoxicity. Furthermore, on-target off-tumor toxicities can result from the indiscriminate binding of constitutively expressed CARs to tumor antigens on healthy tissues, whilst disease relapses can occur due to antigen escape and/or T cell exhaustion. Current and emerging CAR- T cell strategies and designs to address these problems are often too slow in terms of response times and onset of action. Furthermore, they prematurely and permanently eliminate the "high value" cells and often require the expression of additional proteins which may constrain the DNA payload capacity of current lenti- and retro- virus vectors. There is thus an urgent unmet need for in-vivo regulatable CAR-T cell platforms that are rapid, reversible and don't exert a substantial DNA burden on the CAR delivery vector. The objective of this proposal is to assess the feasibility to regulate the activation, effector functions and cytokine release of CAR T-cells using an exogenously administered ligand that modulates the affinity of the CAR T-cell targeting domain. To this end we will utilize our proprietary universal allosteric-linker and switch module for antibodies (UNASMA) technology to incorporate an ON/OFF affinity switch into a well characterized antibody single chain variable fragment (scFv) against a clinically validated hematological cancer target which will subsequently be formatted as a switchable affinity chimeric antigen receptor (SaefCAR). We postulate that the resulting SaefCAR following transduction into a T-cell, will enable the in-vivo regulation of the SaefCAR T-cells' activity via exogenous administration of a small molecule or peptide ligand, potentially solving several of the problems of current CAR-T cell therapies by: 1) Ameliorating intrinsic toxicities via enablement of a slow start to T-cell activation, 2) Preventing on-target off- tumor toxicities by switching off the CAR-T cell without eliminating it 3) Preventing exhaustion of tumor-specific CAR T cells caused by tonic signaling and other mechanisms via intermittent ON/OFF switching of the CAR. Specifically in Aim 1 we will develop switchable forms of the scFv which will subsequently be formatted into a CAR and transduced into a T-cell in Aim 2. In Aim 3 we will validate proof-of-concept of the regulated SaefCAR T-cells in in-vitro and in-vivo studies. We aim to demonstrate that a ligand-controllable affinity switch in the binding domain of SaefCAR T-cells can regulate it's activation and effector functions against a tumor. Importantly due to the universal character of our approach, it provides a blueprint to improve any scFv-based CAR T-cell therapy in the future by adding a capability to regulate the CAR affinity for the tumor antigen.
Public Health Relevance Statement: Project narrative: The widespread application of chimeric antigen receptor (CAR) T-cell therapies to other cancer types including solid tumors is curtailed by the risk of serious adverse events and loss of potency all of which are influenced by the binding affinity of the CAR targeting domain. In this project, Mabswitch will develop switchable affinity chimeric antigen receptor (SaefCAR) T-cells based on a commercially validated CAR-T cell therapy with the aim to demonstrate the in-vivo regulatability of the CAR-T cells activation, effector function and cytokine release upon exogenous administration of a small molecule or peptide ligand that modulates the affinity of the CAR T-cell targeting domain. Importantly due to the universal character of the approach, it provides a blueprint to improve any scFv-based CAR-T cell therapy by adding a capability to regulate the CAR affinity for the tumor antigen.
Project Terms: Antibodies; immunogen; Antigens; Architecture; Engineering / Architecture; Biological Products; Biologic Products; Biological Agent; biologics; biopharmaceutical; biotherapeutic agent; Calmodulin; Calcium-Dependent Activator Protein; Calcium-Dependent Regulator; Phosphodiesterase Activating Factor; Phosphodiesterase Protein Activator; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Discrimination; Cognitive Discrimination; Disease; Disorder; DNA; Deoxyribonucleic Acid; Engineering; Equilibrium; balance; balance function; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Future; Human; Modern Man; Immunoglobulin Fragments; Antibody Fragments; Immunotherapy; Immune mediated therapy; Immunologically Directed Therapy; immune therapeutic approach; immune therapeutic interventions; immune therapeutic regimens; immune therapeutic strategy; immune therapy; immune-based therapies; immune-based treatments; immuno therapy; In Vitro; Libraries; Ligands; Patients; Peptides; Proteins; Reaction Time; Response RT; Response Time; psychomotor reaction time; Risk; Safety; Signal Transduction; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Specificity; T-Lymphocyte; T-Cells; thymus derived lymphocyte; Technology; Tissues; Body Tissues; Tumor Antigens; Tumor-Associated Antigen; cancer antigens; tumor-specific antigen; Tumor Lysis Syndrome; Work; Writing; cytokine; improved; Clinical; prematurity; premature; Phase; Variation; Variant; Physiologic; Physiological; anti-cancer immunotherapy; anticancer immunotherapy; immune-based cancer therapies; immunotherapy for cancer; immunotherapy of cancer; cancer immunotherapy; Solid Tumor; Solid Neoplasm; Letters; Reporter; Hematologic Neoplasms; Hematologic Cancer; Hematologic Malignancies; Hematological Malignancies; Hematological Neoplasms; Hematological Tumor; Hematopoietic Cancer; Malignant Hematologic Neoplasm; exhaustion; experience; success; Animal Model; Animal Models and Related Studies; model of animal; Toxic effect; Toxicities; Modality; Regulation; Adverse event; Adverse Experience; antigen bound; antigen binding; activate T cells; T-Cell Activation; Molecular Interaction; Binding; preventing; prevent; small molecule; CD19 gene; CD19; Address; Dose; Affinity; Retroviral Vector; Retrovirus Vector; in vivo; Antigen Targeting; Serious Adverse Event; Severe Adverse Event; serious adverse experience; serious adverse reaction; Signal Transduction Induction; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; designing; design; cancer type; neuron toxicity; neuronal toxicity; neurotoxicity; tumor; FDA approved; screenings; screening; chimeric antigen T cell receptor; chimeric antigen receptor; systemic toxicity; clinically translatable; clinical translation; pre-clinical development; preclinical development; cytokine storm; cytokine release syndrome; engineered T cells; CAR T cells; CAR modified T cells; CAR-T; CAR-Ts; T cells for CAR; chimeric antigen receptor (CAR) T cells; chimeric antigen receptor fusion protein T-cells; chimeric antigen receptor modified T cells; chimeric antigen receptor T cells; CAR T therapy; chimeric antigen receptor (CAR) T cell therapy; chimeric antigen receptor T cell therapy; CAR T cell therapy; delivery vehicle; delivery vector
