Limited efficacy of APRIL CAR in patients with multiple myeloma indicate challenges in the use of natural ligands for CAR T-cell therapy

BACKGROUND: We used a proliferating ligand (APRIL) to construct a ligand-based third generation chimeric antigen receptor (CAR) able to target two myeloma antigens, B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor. METHODS: The APRIL CAR was evaluated in a Phase 1 clinical trial (NCT03287804, AUTO2) in patients with relapsed, refractory multiple myeloma. Eleven patients received 13 doses, the first 15×106 CARs, and subsequent patients received 75,225,600 and 900×106 CARs in a 3+3 escalation design. RESULTS: The APRIL CAR was well tolerated. Five (45.5%) patients developed Grade 1 cytokine release syndrome and there was no neurotoxicity. However, responses were only observed in 45.5% patients (1×very good partial response, 3×partial response, 1×minimal response). Exploring the mechanistic basis for poor responses, we then compared the APRIL CAR to two other BCMA CARs in a series of in vitro assays, observing reduced interleukin-2 secretion and lack of sustained tumor control by APRIL CAR regardless of transduction method or co-stimulatory domain. There was also impaired interferon signaling of APRIL CAR and no evidence of autoactivation. Thus focusing on APRIL itself, we confirmed similar affinity to BCMA and protein stability in comparison to BCMA CAR binders but reduced binding by cell-expressed APRIL to soluble BCMA and reduced avidity to tumor cells. This indicated either suboptimal folding or stability of membrane-bound APRIL attenuating CAR activation. CONCLUSIONS: The APRIL CAR was well tolerated, but the clinical responses observed in AUTO2 were disappointing. Subsequently, when comparing the APRIL CAR to other BCMA CARs, we observed in vitro functional deficiencies due to reduced target binding by cell-expressed ligand

Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity

Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor β-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies

IL13ra2-Targeted CAR T Cell Therapy for High-Grade Gliomas

IL13ra2 is a type I transmembrane protein. It is one of two receptors for IL13, alongside IL13r1. Unlike IL13r1, it is not ubiquitously expressed in the human body. However, it is expressed by many solid tumours, including high-grade gliomas. Being expressed on the cell surface of cancerous cells but not healthy tissues make IL13ra2 a suitable target for Chimeric Antigen Receptor (CAR) T cell therapy. This thesis describes the generation of an αIL13ra2 library and the panning process to select individual binders. The library was tested by Next Generation Sequencing (NGS) to elucidate the possibility of selecting a large and diverse pool of binders with out the necessity for manual picking. The isolated binders, both from manual picking and NGS-derived, were tested in-depth and characterised to test their specificity and binding kinetics. The specific binders were engineered into a second-generation CAR format and tested for their anti-tumoural activity, proliferation and cytokine secretion in a number of in vitro assays. Once the best performing binder was found, the CAR was further engineered by introducing immunosuppressive tumour microenvironment tackling modules. These were further tested in vitro and in vivo. An armoured CAR was developed that was shown to be efficacious both in vitro and in vivo. It was considered to be a great candidate for future use in clinical trial(s) targeting high-grade gliomas. To ensure the safety of using αIL13ra2 CAR T cells in a clinical trial, a tissue cross reactivity study was conducted to ensure the chosen antibody does not nonspecifically bind outside the tumour site. Finally, a novel strategy was proposed to disrupt the tumour microenvironment and prevent antigen-negative tumour escape which frequently hinders effectiveness of CAR T cell therapy. This was done by combining an expression of an enzyme capable of hydrolysing a non-cytotoxic, systemically administered prodrug into a cytotoxic drug at the tumour site by the CAR T cell

Anti-CCR9 Chimeric Antigen Receptor T cells for T Cell Acute Lymphoblastic Leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes, associated with higher rates of induction failure in comparison to B-ALL. The potent immunotherapeutic approaches applied in B-ALL, which have revolutionized the treatment paradigm, have proven more challenging in T-ALL, largely due to a lack of target antigens expressed on malignant but not healthy T cells. Unlike B cell depletion, T cell aplasia is highly toxic. Here, we demonstrate that the chemokine receptor CCR9 is expressed in >70% of cases of T-ALL, including >85% or relapsed/ refractory disease, and only on a small fraction (<5%) of normal T cells. Using cell line models and patient-derived xenografts, we show chimeric antigen receptor (CAR)-T cells targeting CCR9 are resistant to fratricide and have potent anti-leukemic activity both in vitro and in vivo, even at low target antigen density. We propose anti-CCR9 CAR-T cells could be a highly effective treatment strategy for T-ALL, avoiding T cell aplasia and the need for genome engineering that complicate other approaches

Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies

Mature T cell cancers are typically aggressive, treatment resistant and associated with poor prognosis. Clinical application of immunotherapeutic approaches has been limited by a lack of target antigens that discriminate malignant from healthy (normal) T cells. Unlike B cell depletion, pan–T cell aplasia is prohibitively toxic. We report a new targeting strategy based on the mutually exclusive expression of T cell receptor β-chain constant domains 1 and 2 (TRBC1 and TRBC2). We identify an antibody with unique TRBC1 specificity and use it to demonstrate that normal and virus-specific T cell populations contain both TRBC1+ and TRBC2+ compartments, whereas malignancies are restricted to only one. As proof of concept for anti-TRBC immunotherapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T cells, which recognized and killed normal and malignant TRBC1+, but not TRBC2+, T cells in vitro and in a disseminated mouse model of leukemia. Unlike nonselective approaches targeting the entire T cell population, TRBC-targeted immunotherapy could eradicate a T cell malignancy while preserving sufficient normal T cells to maintain cellular immunity