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.

CAR-T cell therapy against CD19 has changed the treatment landscape in relapsed/refractory (r/r) B-ALL. R/r T-ALL has a dismal prognosis, with an unmet need for effective targeted therapies. Several unique challenges mean that CAR-T cell therapy has yet to be successfully translated to T-ALL. Most strategies have targeted pan-T cell antigens (CD7, CD5) but these are limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen surface expression during manufacture. An ideal target would be exclusively or largely confined to the malignant T cell component but published examples of these (CD1a and TRBC1) are expressed in only minor T-ALL subsets. We previously showed that CD21 is expressed in a NOTCH-dependent manner in T-ALL (Leukemia. 2013, 27:650) and have developed it as a potential immunotherapy target, being primarily expressed on normal B cells, with minimal expression on mature T cells. 70% of human T-ALL cell lines (9/16) expressed surface CD21 by flow cytometry (FACS), with a median antigen density in positive lines of 2545/cell. In primary T-ALL, 57% of presentation samples (n=58) expressed CD21 (median antigen density 1168/cell). 45% of relapse (n=11) and 20% of primary refractory cases (n=30) expressed CD21, with a similar antigen density to presentation samples. CD21 positivity varied by maturational stage, with highest expression in cortical T-ALL (80% of cases) followed by pre-T (72%), mature (67%), ETP (25%) and pro-T (17%). Healthy donor blood (n=14) showed CD21 expression limited to B cells and a low proportion (11%) of T cells (10-fold lower intensity v B cells, 316 antigens/cell). T cell CD21 expression was not up-regulated upon activation with CD3/CD28 antibodies (n=6) and was not associated with markers of differentiation/exhaustion. To target CD21, DNA gene-gun vaccination of rats with a plasmid encoding full-length CD21, followed by phage display was performed and multiple anti-CD21 scFvs isolated. These were cloned into 4-1BBζ CARs and expressed in primary T cells but failed to kill or secrete cytokines in response to CD21+ SupT1 cells. CD21 is a bulky molecule, with 15/16 sushi repeats in the extracellular domain. All isolated scFvs were found to bind membrane-distal domains. We hypothesized that ineffective signalling due to inadequate synapse formation was responsible for poor performance of anti-CD21 CAR-T, and that binders to membrane-proximal epitopes would signal more efficiently. We re-vaccinated rats with the first 5 sushi repeats of CD21 and generated a library of binders which bound CD21 at this membrane-proximal region. Multiple candidate binders expressed as CARs were functional, with cytotoxicity and interferon-γ secretion in response to CD21+ target cells. However, non-specific background cytokine secretion was seen against CD21 negative cells, and no IL-2 secretion was seen. Re-cloning binders into a fragment antigen binding (Fab)-CAR architecture yielded constructs capable of specific cytotoxicity, IFN-γ and IL2 secretion against a CD21+ cell line but not its CD21 negative counterpart (n=6). Our lead anti-CD21 candidate CAR specifically proliferated in vitro, without fratricide or premature exhaustion/ differentiation, and was active against low-density CD21-positive cell lines (n=3) and primary cells from 2 T-ALL patients. Improved functionality of Fab v scFv-based CAR was not driven by higher affinity binding or CAR surface expression. We tested anti-CD21 CAR in murine models of T-ALL. NSG mice were injected with SupT1-luciferase cells and treated with aCD19 or aCD21 CAR-T on day +5. At 2 weeks post treatment, markedly lower disease burden was seen in CD21 CAR-T v CD19 recipients by bioluminescence imaging (median radiance 71700 v 790000 p=0.0079). Further, we injected primary T-ALL blasts in another cohort, treating with aCD19 or aCD21 CAR-T on D+20. Serial bleeds from day 27 post CAR-T showed tumour control in aCD21 CAR treated mice (p=0.024) with an overall survival advantage (median OS 44 days vs undefined, HR = 19.8, p = 0.0069, n=4/group). In summary, we propose CD21 as a novel target for CAR-T cell therapy in T-ALL. Its expression is largely restricted to the malignant T cell compartment, overcoming issues with fratricide and on-target off-tumour effects seen in many T-ALL CAR-T strategies to date. Despite the complexity of the target, we have successfully generated an aCD21 CAR that is functional both in vitro and in vivo. Disclosures Maciocia: Autolus: Current equity holder in publicly-traded company. Onuoha: Autolus: Ended employment in the past 24 months. Khwaja: Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company.

The prognosis of relapsed/refractory T acute lymphoblastic leukemia (T-ALL) is poor with a dire lack of new treatment options which impart meaningful survival benefits. This is in stark contrast with B-ALL, where CD19-directed CAR-T cell therapy has revolutionized the treatment of relapsed/ refractory (r/r) disease. However, CAR-T for T-ALL is challenging since target antigens described to date are expressed on normal T cells. This leads to two main problems: loss of essential normal T cells and self-kill 'fratricide' of CAR-T. To avoid these issues, we sought to identify potential immunotherapy targets for T-ALL which are not expressed on normal T cells or other essential cell types. We analysed the collated gene expression profiles of 35 normal tissues (n=172 samples) as compared to MOLT-4, a T-ALL cell line. Using subtractive transcriptomics, we identified 12 transcripts uniquely expressed in MOLT-4 but not in normal mature tissues. Of these, CCR9 (C-C Motif Chemokine Receptor 9) was the most attractive, being cell-surface resident and thus potentially amenable to immunotherapy. Further, publicly available RNA-seq data confirmed CCR9 was expressed in >70% of cases of paediatric T-ALL. CCR9 is a G-protein coupled receptor for the natural ligand CCL25, and is expressed in gut intraepithelial γδ T cells, some plasmacytoid dendritic cells and double-positive thymocytes, but in less than 5% of normal circulating T and B cells. CCR9 is not expressed in hematopoietic stem cells (HSCs) or myeloid cells. Using flow cytometry, 74/102 cases (73%) of primary T-ALL expressed CCR9, with expression enriched in cases of relapsed/ refractory disease - 64% diagnostic v 85% relapsed v 86% primary refractory (Figure 2a). The median antigens/ cell was 1732 and expression was preserved or increased upon relapse (1320 diagnostic v 1889 relapsed v 2175 refractory). On normal blood cells, expression was limited to ~9% of B cells and <3% of T cells, at low density (<500 antigens/cell). We developed a novel rat-derived anti-CCR9 scFv, and generated a second-generation CAR with 4-1BB-CD3ζ endodomain, expressed in a gamma-retroviral vector. T cells transduced with anti-CCR9 CAR (CARCCR9) expanded similarly to control anti-CD19 CAR (CAR19), with no evidence of fratricide. No CCR9+ cells were detected following transduction, suggesting 'purging' of CCR9+ T cells. We co-cultured CARCCR9 or control CAR19 for 48hrs with multiple T-ALL cell lines, including CCR9-negative variants generated using CRISPR-Cas9. We showed specific cytotoxicity, cytokine secretion (interferon-gamma and IL-2) and proliferation of CARCCR9 against CCR9+ cell lines, including at low target density of ~400 copies/ cell. In addition, in 72hr co-cultures at a 1:1 ratio, CARCCR9 but not NT or CAR19 T cells secreted interferon-gamma and lysed primary blasts from 3 patients with T-ALL. We tested CARCCR9 in vivo. We intravenously (IV) injected NSG mice with 3 x 10^6 MOLT-4 cells, engineered to express luciferase. Nine days later (D+9), mice received 8 x 10^5 non-transduced (NT), CAR19 or CARCCR9 cells IV. While untreated mice and NT or CAR19 recipients experienced disease progression, weight loss and death, mice receiving CARCCR9 had disease regression, continued weight gain and prolonged survival beyond day 80 (median OS NT 16 days, CAR19 16 days, CARCCR9 NR, p = 0.003). Further, these mice were re-injected with 1 x 10^6 MOLT4-Fluc on D+49, and in 3/4 (75%), no increasing BLI signal was detected, suggesting continued anti-leukemic immunosurveillance. We also tested CARCCR9 in 2 patient-derived xenograft (PDX) models of T-ALL. NSG mice were injected with 1 x 10^6 primary blasts, then received 8 x 10^5 NT, CAR19 or CARCCR9 cells IV on D+20. All recipients of NT or CAR19 displayed increasing ALL burden in peripheral blood over time, with weight loss, splenomegaly and eventual leukaemic death. By contrast, all CARCCR9 recipients had undetectable leukemia and long-term disease-free survival (median OS NT 42 days, CAR19 42 days, CARCCR9 NR, p = 0.003), with no detectable blasts in marrow or spleen at necropsy. Thus, we have demonstrated potent anti-leukemic function of anti-CCR9 CAR-T cells both i n vitro and in vivo. Further, efficacy was not associated with loss of essential normal T cells or with CAR-T fratricide. We propose that anti-CCR9 CAR-T cells could be a safe and effective treatment strategy for T-ALL, and potentially a major advance in a neglected clinical area. Disclosures Maciocia: Autolus: Current equity holder in publicly-traded company, Research Funding. Maciocia: Autolus: Current equity holder in publicly-traded company. Leon: BenevolentAI: Current Employment. Pule: Autolus: Current Employment, Current equity holder in publicly-traded company. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy.

Relapsed/refractory (R/r) T cell acute lymphoblastic leukemia (T-ALL) has a dismal prognosis, with an unmet need for effective novel therapies. The successes seen in chimeric antigen receptor (CAR)-T cell therapy for B-ALL have yet to be fully translated to T-ALL. Most strategies have targeted pan-T antigens (CD7, CD5) but these may be limited by T cell aplasia and fratricide, requiring elimination of CAR-T antigen expression during manufacture and salvage hematopoietic stem cell transplantation (HSCT). Here, we describe CD21 as a novel immunotherapeutic strategy for the treatment of T-ALL. CD21 is largely confined to malignant T cells with expression in 57% of diagnostic T-ALL but only on a minor fraction of mature T cells (10%). While anti-CD21 CAR-T targeting membrane distal epitopes were ineffective, CAR-T cells utilising a novel Fab-CAR architecture and binding to membrane proximal epitopes showed no fratricide and were potent against low antigen density cell line and patient-derived xenograft models of T-ALL in vitro and in vivo. Further, we showed that CD21 expression in T-ALL can be upregulated by inhibition of the PI3K/ axis. CD21 is a novel target for CAR-T cell therapy in T-ALL, avoiding the fratricide and T cell aplasia seen with many T-ALL CAR-T strategies.

Cancer-associated fibroblasts (CAFs) play a fundamental role in the development of cancers and their response to therapy. In recent years, CAFs have returned to the spotlight as researchers work to unpick the mechanisms by which they impact tumour evolution and therapy responses. However, study of CAFs has largely been restricted to a select number of common cancers, whereas research into CAF biology in bladder cancer has been relatively neglected. In this review, we explore the basics of CAF biology including the numerous potential cellular origins of CAFs, alongside mechanisms of CAF activation and their diverse functionality. We find CAFs play an important role in the progression of bladder cancer with significant implications on tumour cell signaling, epithelial to mesenchymal transition and the capacity to modify components of the immune system. In addition, we highlight some of the landmark papers describing CAF heterogeneity and find trends in the literature to suggest that the iCAF and myCAF subtypes defined in bladder cancer share common characteristics with CAF subtypes described in other settings such as breast and pancreatic cancer. Moreover, based on findings in other common cancers we identify key therapeutic challenges associated with CAFs, such as the lack of specific CAF markers, the paucity of research into bladder-specific CAFs and their relationship with therapies such as radiotherapy. Of relevance, we describe a variety of strategies used to target CAFs in several common cancers, paying particular attention to TGFβ signaling as a prominent regulator of CAF activation. In doing so, we find parallels with bladder cancer that suggest CAF targeting may advance therapeutic options in this setting and improve the current poor survival outcomes in bladder cancer which sadly remain largely unchanged over recent decades.

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