Allogeneic hematopoietic stem cell transplantation (allo-HSCT) aims to cure patients without inducing severe graft-versus-host disease (GVHD) or relapse. In prospective studies of mostly pediatric patients with haploidentical donors, ex vivo αβTCR/CD19 depletion has shown to have low incidences of GVHD, but data for adults with matched related (MRD) or unrelated donors (MUD) remain limited. We analyzed the outcomes of recipients who received a myeloablative regimen plus ATG, followed by an αβTCR/CD19-depleted allograft (cohort D+ATG (n = 122)), and compared outcomes to T cell-replete cohorts (cohort R (N = 60)); without ATG; R+ATG = with ATG (N = 129) in a single-center retrospective analysis. In D+ATG, the incidence of aGVHD grade III-IV was 7%, compared to 13% in R and 16% in R+ATG (p = 0.09). Extensive cGVHD was reduced from 23% in R and 10% in R+ATG to 2% in D+ATG (p < 0.001). The reduced incidence of cGVHD led to a superior GVHD-relapse-free survival (GRFS) of 56.7% in D+ATG versus 36.7% in R and 42.8% in R+ATG (p = 0.03) at 2 years. In conclusion, the combination of myeloablative conditioning, ATG, and ex vivo αβTCR/CD19 depletion appears to be a promising approach to enhance GRFS in adult patients up to 75 years of age undergoing allo-HSCT.

Background & Aim Despite recent successes in adoptive T cell therapies against hematological malignancies, response rates remain variable, and patients often experience relapse post-treatment. This phenomenon is potentially influenced by the immunosuppressive tumor microenvironment within the malignant bone marrow (BM), a complex niche where mesenchymal stem cells (MSCs) play a pivotal role. However, the specific contribution of MSC-mediated immunomodulation to the outcome of adoptive T cell therapies is not yet fully understood. Methodology Using a multicellular 3D biomimetic bone marrow niche consisting of cancer cells, MSCs and engineered T cells embedded in extracellular matrix we found distinct, MSC immunosuppressive (s-MSC) and permissive MSC (p-MSC) phenotypes, depending on the clinical sample. These MSC phenotypes respectively differ in their capacity to suppress or support engineered T cell migration and tumor cell killing in the 3D model and show distinct transcriptomic features. p-MSCs display dynamic transcriptomic profiles that adapt to the surrounding cellular environment, particularly in the presence of tumor and immune cells, while the transcriptomic flexibility of s-MSCs is more rigid. Results Importantly, we show that s-MSCs and p-MSCs have differential expression of various collagen genes. Indeed, by interfering with the binding of the inhibitory collagen receptor LAIR-1 on T cells to collagen, we can recover the migratory and cytolytic capacity of genetically engineered T cells in the immune suppressive bone marrow niche of sMSCs. This points to a direct immune suppressive effect of collagen on the engineered T cells. Conclusion The here-described MSCs signatures do not only reveal new fundamental insights into T cell modulation by stromal cells but also may guide designing future patient-tailored engineered immune cell therapy approaches.

Background: Preclinical data showed that Janus Kinase (JAK) signaling plays an important role in the pathogenesis of Graft versus Host Disease (GVHD) and several clinical studies showed remarkable clinical efficacy in both acute and/or chronic steroid-refractory (SR)-GVHD patients. However, these clinical results have not yet been repeated in real world cohorts with long term follow-up. Furthermore, infectious complications have not been highlighted in published clinical trials whilst these remain a significant problem in daily practice. Aims: To assess the response of acute and/or chronic SR-GVHD patients to ruxolitinib treatment and to assess side-effects of ruxolitinib treatment occurring in adult acute and/or chronic SR-GVHD patients. Methods: We performed a retrospective analysis of 121 consecutive patients treated with ruxolitinib for either acute or chronic GVHD. Patients were included from 3 academic centers in the Netherlands from 2014 to 2019. Patients provided written informed consent, with the exception of deceased patients in accordance with the local ethics committee. Patient data were extracted by personnel from an independent CRO to minimize reviewer bias. Patient data were anonymized. Results: 121 patients were included in the study of whom 54 were diagnosed with acute and 67 patients with chronic GVHD. Selected baseline characteristics are depicted in Table 1. The overall response rate at day 28 of acute GVHD was 70% (38/54) with a complete response in 50% of patients (27/54). At timepoint 6 months after initiation of ruxolitinib for chronic GVHD, the majority of patients showed stable disease. The overall response rate at 6 months in chronic GVHD was 28% (19/67) with a complete response in 13% of patients (9/67). Ruxolitinib use was mainly complicated by infections and hematological toxicity. Of the reported (S)AE’s possibly related to ruxolitinib, 38% were infectious and 13% were hematological. Severity of infections ranged from grade 1 to grade 5, median grade 2. Most infections were viral (43%), followed by bacterial (19%) and fungal or mould infections (14%). During follow up mortality was 14,9% (18 patients) of which 6 cases were due to uncontrolled GVHD, 5 cases due to infections and 3 patients died of relapse of their primary malignancy, 4 patients died of other causes. Image:Summary/Conclusion: In a real world cohort of GVHD patients treated with ruxolitinib we observed in patients with acute GVHD a complete response rate at day 28 of 50%. Overall response rate at 6 months in cGVHD patients was 28%. Infectious adverse events were common in both acute and chronic GVHD patients and were mainly caused by viral pathogens.

Background: The bispecific antibody blinatumomab (blina) is approved for patients(pts) with relapsed/refractory precursor B-cell ALL (ALL). It has also shown to be highly effective in MRD+ pts in first line treatment. TABLE - Pt characteristics Total, N(%) 71(100%) Age(y), median(range) 53(18-70) Age (y), N(%) ≤40 22(31%) 40-60 29(41%) >60 20(28%) BM blasts (%), N(%) ≤50% 4(6%) >50% 62(87%) Unknown 5(7%) Karyotype, N(%) Ph+ 26/71(37%) KMT2A 2/67(3%) Complex 23/67(34%) Hypodiploidy 5/67(7%) Aims: To evaluate whether blina added in upfront therapy to prephase and after consolidation (cons)-1 would increase MRD negativity measured by qPCR or flowcytometry at a cut-off of <10-4 (primary endpoint). Secondary endpoints included CR, EFS, OS, adverse events and treatment-related mortality. Methods: Pts, 18-70 years(y) old, with newly diagnosed CD19+ ALL (incl. Ph+), were included. Treatment was based on a pediatric inspired protocol (HOVON 70, Rijneveld et al., 2011) with reduced doses of anthracyclines, MTX, etoposide and PEG-ASP for pts ≥40y old. Prephase consisted of 10 days steroids, from day 5 combined with 14 days blina in the standard step-up dosing schedule. After cons-1 and after intensification 2, two 4-week blina courses were added irrespective of MRD. The protocol was amended twice due to toxicity. First, in 2018 the first PEG-ASP administration was omitted. Second, in 2021 doxorubicin, dexamethasone and PEG-ASP were reduced during intensification 1. Rituximab (if CD20+), prophylactic ITs and imatinib (if Ph+) were standard. AlloHSCT was offered to intermediate and high-risk pts. Trial was registered with ClinicalTrials.gov, identifier NCT03541083. Results: Seventy-one pts were enrolled. Pt characteristics are presented (Table). Fifteen pts discontinued treatment before blina cons-1 due to refractory disease (n=8), toxicity (n=7) or death (n=2). In the total study population, 55/71 pts (77%) achieved CR after (blina) cons-1. Among pts still on treatment after cons-1, 55/56 (98%) pts achieved CR, 50/55 (91%) reached MRD negativity. After prephase, CR was already reached in 63% and MRD negativity in 53%. Blina related AEs in prephase were as expected (83% of pts had ≥1 AE and 10% had ≥1 SAE (hepatotoxicity 3 pts, pain lymph node 1, CRS 1, pneumonia 1, renal insufficiency 1)). CRS was observed in 35% of pts, 32% of whom experienced grade 3 and no grade ≥4. During prephase 5 pts discontinued blina; during blina cons-1, 4 pts stopped blina. With a median follow-up of 17,6 months, the estimated 2-y EFS was 64% standard error (SE) ± 7% (≤60y 71% SE ± 9% and >60y 47% SE ± 12%). Overall, 14 (20%) pts died. OS after 2y was 73% SE ± 7% (≤60y 82% SE ± 8% and >60y 52% SE ± 14%) (Figure 1). For pts with Ph+ ALL, 2-y EFS was 88% SE ± 6%and OS also 88% SE ± 7%. For Ph- ALL, 2-y EFS and OS were 53% SE ± 9% and 68% SE ± 9%, resp. Among pts who reached CR on protocol (n=60), 5 (8%) had relapse, 6 (10%) died and 6 (10%) discontinued treatment due to toxicity. Until now, 22 pts proceeded to alloHSCT and 11 with maintenance. Image:Summary/Conclusion: Blina can safely be added to prephase of an intensified pediatric schedule for newly diagnosed ALL up to 70y of age, albeit with dose reductions for PEG-ASP, doxorubicin and dexamethasone. The combination increases CR and MRD negativity rate. The early addition of blina resulted in very early achievement of MRD negativity with 53% after prephase and 91% after blina cons-1. Further reductions of chemotherapy should be explored (especially for Ph+) if these results are maintained with longer follow-up.

Regulation of lymphocyte numbers is critical and under normal circumstances lymphocyte numbers remain relatively stable. It is generally assumed that when lymphocyte numbers decrease, the immune system responds homeostatically by increasing lymphocyte production and survival rates, thereby facilitating lymphocyte reconstitution. The widely-accepted concept of lymphopenia-induced proliferation is largely based on experiments in mice. In humans, it is well known that T-cell reconstitution tends to be very slow. This challenges the idea that increased lymphocyte production and survival aid recovery from lymphopenia in humans. Here, we investigated whether lymphocyte production and survival rates are increased in patients who underwent an autologous hematopoietic stem cell transplantation (autoHSCT), using in vivo deuterium labelling and mathematical modelling. We found that the production rate of most T- and B-cell subsets in these patients were 2 to 8-times higher than in healthy controls. Against our expectations, lymphocyte loss rates were also significantly increased, and increased lymphocyte production and loss rates were also observed for lymphocyte subsets of which cell numbers had already normalized. Thus, despite the slow reconstitution of lymphocytes in autoHSCT patients, T- and B-cell production rates are increased. This increased production, however, does not simply reflect a homeostatic mechanism, as it goes hand in hand with increased cell loss, and does not normalize when cell numbers do.

We performed a prospective phase II study to evaluate clinical safety and outcome in 48 patients with steroid-refractory grade II–IV acute graft-versus-host disease (aGVHD) treated with mesenchymal stromal cells (MSCs). Clinical outcomes were correlated to comprehensive analyses of soluble and cellular biomarkers. Complete resolution (CR) of aGVHD at day 28 (CR-28) occurred in 12 (25%) patients, CR lasting >1 month (CR-B) occurred in 24 (50%) patients. One-year overall survival was significantly improved in CR-28 (75 versus 33%, P=0.020) and CR-B (79 versus 8%, P<0.001) versus non-CR patients. A six soluble biomarker-panel was predictive for mortality (HR 2.924; CI 1.485–5.758) when measured before MSC-administration. Suppression of tumorigenicity 2 (ST2) was only predictive for mortality 2 weeks after but not before MSC-administration (HR 2.389; CI 1.144–4.989). In addition, an increase in immature myeloid dendritic cells associated with decreased mortality (HR 0.554, CI 0.389–0.790). Patients had persisting T-cell responses against defined virus- and leukemia-associated antigens. In conclusion, our data emphasize the need to carefully assess biomarkers in cohorts with homogeneous GVHD treatments. Biomarkers might become an additional valuable component of composite end points for the rapid and efficient testing of novel compounds to decrease lifecycle of clinical testing and improve the success rate of phase II/III trials.

Introduction: The outcome of allo-SCT in patients with poor risk leukemia is still hampered by GVHD and relapse. The innate immune system has been reported to contribute to tumor control, with lower incidence of GVHD. Specific depletion of αβ T- cells – key players in the development of GVHD – will render NK cells and γδ T cells within the allograft. Recently reported results have shown the great promise of this approach in haploindentical transplantations. Within this study, we aim to extend αβT- cell depleted allo-SCT to patients with a MRD or MUD. Methods: Patients with either ‘poor-risk’ or ‘very poor-risk’ leukemia were included in this phase I study. Either HLA matched siblings (MRD) or fully matched HLA matched (10/10) unrelated donors (MUD) were eligible. abT-cell reduction was performed by negative selection with anti-abTCR antibodies in combination with magnetic microbeads, using the automated CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The maximal contamination with αβT-cells for all dose levels was 5x105/kg. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. Within cohort II and III, no additional immune suppression was given after allo-SCT. Results: Products for 14 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ~4 log depletion of αβT-cells has been observed in the product with a recovery of ~75% of CD34+ cells. In cohort I and cohort II, 60% and 25% primary graft failures were observed, whereas in cohort III primary engraftment (chimerism > 95%) was observed in all patients. The combination of ATG/fludarabine/busilvex was well tolerated with a hematological recovery of within 3 weeks. In all 14 patients immune reconstitution primarily consisted of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. In addition, no increase in CMV or EBV reactivations has been observed so far under the profound “innate control”. Conclusion: ATG Busulfan Fludarabine is a low toxicity platform for abTCR-depleted transplantations, resulting in a swift reconstitution of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. This transplantation strategy can serve as a tool for future immunological interventions such as a low dose DLI or genetically modified T cells. Disclosures No relevant conflicts of interest to declare.