Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer, for which effective therapies are urgently needed. Chimeric antigen receptor (CAR)-based immunotherapy represents a promising therapeutic approach, but it is often impeded by highly immunosuppressive tumor microenvironments (TME). Here, in an immunocompetent, orthotopic GBM mouse model, we show that CAR-T cells targeting tumor-specific epidermal growth factor receptor variant III (EGFRvIII) alone fail to control fully established tumors but, when combined with a single, locally delivered dose of IL-12, achieve durable anti-tumor responses. IL-12 not only boosts cytotoxicity of CAR-T cells, but also reshapes the TME, driving increased infiltration of proinflammatory CD4+ T cells, decreased numbers of regulatory T cells (Treg), and activation of the myeloid compartment. Importantly, the immunotherapy-enabling benefits of IL-12 are achieved with minimal systemic effects. Our findings thus show that local delivery of IL-12 may be an effective adjuvant for CAR-T cell therapy for GBM.
Invasive fungal disease (IFD) is a major cause of infectious mortality in hemato-oncology patients due to their underlying disease and its treatment, which lead to periods of prolonged immunosuppression (1). Invasive pulmonary aspergillosis (IPA) is the most common cause of mortality due to mold disease (2, 3), and early diagnosis and treatment are vital for improving outcomes (4). However, early diagnosis is hampered by the limitations of current biomarker tests and a lack of consensus on the best samples to be tested (i.e., blood, bronchoalveolar [BAL] fluid, or tissue biopsy specimens) in terms of both test sensitivity and practicability. Direct examination or culture of pulmonary tissue remains the "gold standard" for the diagnosis of IPA (5, 6). However, lung biopsy in this acute care setting is rarely performed due to the associated risks. Consequently, in view of the diagnostic challenges and worse outcomes with late treatment, an empirical strategy has been-and remains-the standard of care in many hematology units (7). This approach leads to overtreatment with antifungal drugs, which have significant side effects and drugdrug interactions. Furthermore, health care systems are exposed to spiraling drug costs (8). Improving the means of diagnosis of IPA is an urgent clinical need.Attempts have been made to optimize and standardize existing tests (5,6,(9)(10)(11)
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.
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