Myeloma patients who become refractory to immunomodulatory agents (IMiDs) and bortezomib have poor survival, with limited therapeutic options. Pomalidomide has shown improved survival and good tolerability in this patient cohort in clinical trials, but real world data are scarce. We retrospectively analysed all patients treated with pomalidomide at five UK centres between 2013 and 2016. Of 85 patients identified, 70 had sufficient information for response assessments. Median age was 66 years [40-89], 96·5% were refractory to IMiDs, 72·9% were refractory to both an IMiD and bortezomib and 92·9% were refractory to their last treatment. Of 45 patients with fluorescence in situ hybridization results 64% had adverse risk, 19 patients (22·4%) had an estimated glomerular filtration rate <45 ml/min. Grade ≥3 non-haematological toxicities occurred in 42·4%, and grade ≥3 neutropenia and thrombocytopenia in 38% and 24% respectively, but only 18·8% had dose reductions. The overall response rate was 52·9%. At a median follow-up of 13·2 months, median progression-free survival was 5·2 months [95% confidence interval (CI) 4·150-6·238], and median overall survival was 13·7 months (95% CI 11·775-15·707). No significant difference was seen in response, survival or tolerability by renal function, age or cytogenetic risk. This real-world data support the results seen in published clinical trials.
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.
Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma are precursor conditions of symptomatic multiple myeloma (MM). Diagnostic principles are aimed at excluding MM requiring therapy, other conditions associated with paraproteins that may require different management, and risk stratifying patients for the purposes of tailored follow-up and investigation. The International Myeloma Working Group have recently published a revised definition of MM, which singles out a small group of patients with smoldering multiple myeloma who are at very high risk of progression and organ damage; such patients are now included under the definition of MM and recommended to start anti-myeloma treatment. Furthermore, the recently published National Institute of Health and Care Excellence guideline recommends cross-sectional imaging techniques in place of skeletal survey. These recent recommendations are discussed, and practical guidance for investigation and management are presented.
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.
Introduction. The outlook for myeloma patients who relapse after or become refractory to bortezomib and IMiDs is poor, with limited therapeutic options and a median survival (OS) of 9 months. In the phase 3 MM-003 study, pomalidomide plus low-dose dexamethasone resulted in a significant PFS (median 4 vs 1.9 months) and OS (median 13.1 vs 8.1 months) benefit, compared to high-dose dexamethasone. Information on real-world outcomes of pomalidomide therapy is limited. We carried out a retrospective analysis of patients receiving pomalidomide in the UK, to compare outcomes and tolerability with published clinical trial data, and focus on high risk subgroups. Methods. All patients treated with pomalidomide at 5 major UK centres between August 2013 and March 2016 were identified from chemotherapy records, and clinical data including toxicity and survival from patient records. Disease response and adverse FISH were defined as per IMWG. Survival was estimated using Kaplan-Meier, and correlations made using log-rank methods. Key subgroups: eGFR <45ml/min, adverse genetics, and older age were assessed. Results. A total of 85 patients were identified. Of these, 70 (82%) had measurable disease (IMWG criteria) and received ≥1 cycle so were included in response analyses. Baseline patient characteristics are reported in Table 1. 96.5% of patients were refractory to one or more IMiDs, and 72.9% were refractory to both IMiDs and bortezomib. 92.9% were refractory to their last treatment. The median dose of pomalidomide was 4mg (2-4). Grade 3-4 non-haematological toxicities occurred in 42.4%: pneumonia (16.5%), neutropenic sepsis (8.2%), and acute kidney injury (7.1%), were most common. Grade 3-4 neutropenia occurred in 38% and thrombocytopenia in 24%. Seven patients died on treatment, 6 during the first cycle (2 PD and sepsis, 2 neutropenic sepsis, 1 PD and AKI, 2 pneumonia). In the 70 patients assessable for response, ORR was 52.9% (5.7% VGPR, 47.1% PR, 38.6% SD). Median duration of response (DoR) was 4 months. With median follow-up of 13 months, median PFS was 5 months (95%CI 3.6-6.4), and median OS 13 months (95%CI 10.8-15.2). Patients with renal failure (eGFR <45ml/min) had PFS and OS that were similar to those with eGFR ≥45ml/min ((HR=0.95, 95%CI 0.49-1.82, and HR=1.22, 0.59-2.53). Patients with adverse FISH (44%) had similar outcomes to those with standard FISH (ORR 45.8 vs 50%, median PFS 6 vs 5 months (HR=0.86, 95%CI 0.44-1.67, and median OS 10 vs 8 months, HR=1.223, 0.55-2.68). Patients aged >65yrs had similar outcomes to younger patients,(ORR 54.1 vs 51.5%, median PFS and OS comparable between groups). Rates of toxicity were also not influenced by renal impairment, adverse genetics, or older age. The most important predictors of PFS and OS were depth and durability of response. PFS was 6 months for patients achieving PR, 4 months for SD and 1 month for PD, while OS was 18 months in patients achieving PR, 13 months for SD and 3 months for PD. For patients with DoR >4 months, PFS was 11 months and OS 23 months. In contrast, in patients whose DoR was < 4 months or who did not respond, OS was 9 months. Conclusions. Our real-world data on the characteristics and outcomes of patients receiving pomalidomide for relapsed/refractory myeloma in the UK reflect results of published clinical trials. The ORR of 52.9% in our cohort is higher than in MM-003 and MM-010, but PFS (5 months) and OS (13 months) were remarkably similar. Rates of haematological toxicity and infections are low, confirming the good tolerability of pomalidomide in this patient group. Depth and sustainability of response were important predictors of survival: achievement of PR was associated with improved PFS and OS, while patients who achieved SD still derived a survival benefit. Patients who maintained a response for at least 4 months had an estimated survival of nearly 2 years. No difference in response, survival or tolerability was seen in key subgroups, including those with moderate renal impairment, adverse cytogenetics and older age. Our findings confirm the efficacy of pomalidomide in these heavily pre-treated patients and add to the evidence for the benefit of pomalidomide in high risk patient groups. Table Patient characteristics and comparison with MM003 trial Table. Patient characteristics and comparison with MM003 trial Figure 1 PFS and OS for the edited group of 70 patients Figure 1. PFS and OS for the edited group of 70 patients Disclosures Maciocia: Autolus: Equity Ownership, Patents & Royalties: TRBC1 and 2 Targeting for the Diagnosis and Treatment of T-cell Malignancies. Ramasamy:Celgene: Honoraria, Research Funding. Jenner:Novartis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other: Travel support; Amgen: Consultancy, Honoraria, Other: Travel support; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding. Schey:Celgene, Takeda: Honoraria; Celgene, Johnson & Johnson: Speakers Bureau; Celgene: Consultancy. Yong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding.
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