A Calreticulin Neoepitope-Directed Monoclonal Antibody Can Overcome JAK Inhibitor Resistance and Block TPO-Independent Megakaryocyte Differentation

Introduction: Mutations within the gene encoding calreticulin (CALR) are the second most common genetic aberration associated with primary myelofibrosis (PMF), observed in 70% of non-JAK2 V617F cases. Importantly, patients with CALR mutations do not effectively respond to JAK inhibitor therapy and no mutation specific therapy is currently in use. Virtually all CALR mutations identified in PMF are small insertions or deletions clustered within exon 9 leading to a neo-epitope peptide sequence which is thought to directly or indirectly activate the thrombopoietin receptor (TpoR) by a poorly defined mechanism. Here we engineered a neo-epitope specific monoclonal antobody that has striking biological activity against ruxolitinib persistent cells. Methods TF-1 TpoR cells expressing TpoR were supplemented with 20 ng/mL of TPO. Rats were immunised with a CALR mutant peptide coupled to KLH. Serum from the immunised rats was screened by enzyme linked immunoassay, to verify a strong titre to the peptide immunogen. Primary PMF CD34+ cells were cultured in StemCell Pro with human SCF, IL-6 and IL-9. NSG mice were used to for engraftment studies after 150 cGy irradiation. Results: We engineered a panel of rat monoclonal antibodies after immunization with a 30 amino acid peptide corresponding to the C-terminal mutant CALR neoepitope sequence with an extra cysteine residue. Clone 4D7 showed superior activity of detecting mutant but not wild type CALR protein with a binding affinity of 13.5 pM and dissociation constant of 1.53 nM as measured by I 125-Scatchard. Treatment with 4D7 resulted in a significant (5-7-fold) increase in the amount of full-length mutant CALR protein in conditioned media. 4D7 inhibited Tpo-independent cell growth over 6 days in TF-1 cells expressing MPL and mutant CALR at 2, 10 and 20 µg. 4D7 blocked constitutive factor-independent phospho-STAT5 and phospho-ERK after incubation exclusively in mutant CALR cells but not in TF-1 cells expressing TpoR alone and increased the sub-G 0 fraction was observed compared to IgG control (P = 0.001, n = 3 independent experiments) consistent with induction of an apoptotic response. We tested activity in purified primary CD34+ cells obtained from patients with CALR mutant myelofibrosis using two orthogonal assays: - (i) Tpo-independent megakaryocyte differentiation in liquid culture and (ii) Tpo-independent megakaryocyte colony formation on a collagen-based medium. 4 out of 4 patient samples that displayed robust Tpo-independent growth of CD41+CD61+ megakaryocyte progenitors showed inhibition by 4D7 of at least 50%. Similarly, we saw dramatic reduction in the absolute numbers of primary Tpo-independent megakaryocyte colonies cultured on collagen (colony-forming unit-mega) treated with 4D7 in multiple patient samples (decrease of 46%, P = 0.0001, Student's t-test, n = 4 independent patient samples) Importantly, secretion of mutant CALR protein was neither upregulated nor downregulated by ruxolitinib, indicating ruxolitinib is unlikely to alter mutant CALR trafficking in patients. 4D7 had strong inhibitory activity on cells that were resistant to ruxolitinib, in both liquid culture at 96 hours or colony formation. To test whether 4D7 could block mutant CALR-dependent proliferation in vivo, we developed two distinct xenograft models, a bone marrow engraftment model, which measures mutant CALR dependent proliferation in the bone marrow microenvironment, and a chloroma model, which mimics extravascular infiltration of mutant CALR leukaemia, by injection of TPO-independent TF-1 cells in NSG mice. In the bone marrow engraftment model 4D7 treatment (12 mg/kg twice weekly via intraperitoneal injection) lowered peripheral blood engraftment of human CD33 myeloid cells at 3 weeks, bone marrow engraftment and significantly prolonged survival compared to IgG control (P=0.004, HR=0.2). In the chloroma model, 4D7 treatment resulted in significant decrease in tumour growth measured at 3 weeks (P<0.01) and improved overall survival (P=0.02, HR=0.07) compared to IgG control Conclusion: Together, these results suggest an immunotherapeutic approach may have clinical utility CALR-driven myeloproliferative neoplasms and CALR mutant acute myeloid leukaemia, as well as activity in CALR mutant patients that develop resistance/persistence to ruxolitinib. Disclosures Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Antifungal prophylaxis with micafungin three times a week in children after allogeneic bone marrow transplantation

BACKGROUND The use of azoles for antifungal prophylaxis after familial allogeneic stem cell transplantation in children (SCT) is hindered by adverse events and drug interactions especially in children affected by sickle cell disease. Intermittent, higher dose of micafungin could be an alternative. METHODS A prospective, observational, longitudinal, single-center study was conducted between May 2015 and June 2018. The study included 30 patients between 2 and 18 years old who underwent allogeneic SCT and received prophylaxis with micafungin on alternating days after the bone marrow engraftment phase. FINDINGS Fifty transplants performed, 30 included prophylaxis against IFIs, with micafungin in an alternating pattern according to the previously described protocol. The indication for HSCT was hemoglobinopathies in 76.7%, acute leukemia in 20.0% and Fanconi anemia in 3.3%. The prophylaxis duration was 2.33 months (1.53 to 3.98). In our study, 40.0% (12/30) of the patients had acute GVHD, and 6.7% (2/30) had chronic GVHD, which prolonged the duration of alternating prophylaxis. No serious adverse effects of the use of micafungin were observed in any of the patients. There was also no breakthrough Invasive fungal infection (IFI) during alternating prophylaxis. CONCLUSION: In selected patients, micafungin was well tolerated without breakthrough IFI in our study.

Conditioned Medium from Human Tonsil-Derived Mesenchymal Stem Cells Enhances Bone Marrow Engraftment via Endothelial Cell Restoration by Pleiotrophin

Cotransplantation of mesenchymal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been widely reported to promote HSC engraftment and enhance marrow stromal regeneration. The present study aimed to define whether MSC conditioned medium could recapitulate the effects of MSC cotransplantation. Mouse bone marrow (BM) was partially ablated by the administration of a busulfan and cyclophosphamide (Bu–Cy)-conditioning regimen in BALB/c recipient mice. BM cells (BMCs) isolated from C57BL/6 mice were transplanted via tail vein with or without tonsil-derived MSC conditioned medium (T-MSC CM). Histological analysis of femurs showed increased BM cellularity when T-MSC CM or recombinant human pleiotrophin (rhPTN), a cytokine readily secreted from T-MSCs with a function in hematopoiesis, was injected with BMCs. Microstructural impairment in mesenteric and BM arteriole endothelial cells (ECs) were observed after treatment with Bu–Cy-conditioning regimen; however, T-MSC CM or rhPTN treatment restored the defects. These effects by T-MSC CM were disrupted in the presence of an anti-PTN antibody, indicating that PTN is a key mediator of EC restoration and enhanced BM engraftment. In conclusion, T-MSC CM administration enhances BM engraftment, in part by restoring vasculature via PTN production. These findings highlight the potential therapeutic relevance of T-MSC CM for increasing HSC transplantation efficacy.

A Comparison of the Effect of Granulocyte-Colony Stimulating Factor (G-CSF) in Chemotherapy Versus Plerixafor for Hematopoietic Stem Sell Transplantation (HSCT) and Mobilization

INTRODUCTION: Peripheral Hematopoietic Stem Cells have been widely used as a source for Autologous Hematopoietic Stem Cell Transplantation (HSCT). The use of G-CSF associated with chemotherapy increases the number of circulating Hematopoietic Progenitor Cells; however, there is no harmony about the best medical strategy for the use of G-CSF associated with the mobilization conduct. The use of plerixafor for poorly mobilized patients has provided a safe and effective option for optimizing apheresis procedures despite the high cost. Thus, the evaluation of efficacy becomes essential for the incorporation of plerixafor into the HSCT routine. OBJECTIVE: To evaluate the mobilization of autologous HSCT in chemotherapy associated with G-CSF and cyclophosphamide (QT) versus plerixafor. METHODS: Retrospective evaluation of 66 patients mobilized for autologous HSCT with G-CSF associated with chemotherapy versus plerixafor (plerixafor was used in the first mobilization), also to evaluate the number of apheresis procedures, CD34-positive cells, cell viability and for the bone marrow engraftment date. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA), using the Mann-Whitney test for nonparametric data (p <0.05). RESULTS: A mobilization protocol was performed in 83 patients (60% male), with a median age of 55 years (17 to 68 years). Multiple myeloma (55.7%), Hodgkin's lymphoma (14.7%), Non-Hodgkin's lymphoma (16.39%), primary amyloidosis (4.9%) were the most common hematologic malignancies. There was no statistical difference between G-CSF and QT group versus plerixafor groups regarding age, sex, type of disease and previous QT. The plerixafor group had better CD34 positive cell counts compared to G-CSF and QT (median 6.2 versus 3.8 x106, p<0.004), shorter sessions of apheresis procedure (median 2 versus 4, p < 0.0002) and a better cell viability rate (98 versus 95%, p <0.0034). The bone marrow engraftment rate was 10 days for plerixafor group versus 15.05 days for G-CSF and QT group, p <0.0055 (Figure 1).CONCLUSION: Our results showed the mobilization group using plerixafor had superior data and statistical significance regarding the number of apheresis procedures, cell viability, number of CD34 positive stem cells and earlier bone marrow engraftment rates when compared to the G-CSF + QT. These findings suggest and encourage the use of plerixafor in the first mobilization, as well as it should be incorporated into the standard routine. In addition, the number of apheresis procedures will be reduced, cell viability and CD34-positive cells rate will be increased. As a result, there will be a better medullary recovery and the reducing of both hospital costs and infectious complication rates. Disclosures No relevant conflicts of interest to declare.

Pre-Clinical Efficacy of CD123-Targeting Antibody-Drug Conjugate IMGN632 in Blastic Plasmacytoid Dentritic Cell Neoplasm (BPDCN) Models

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy of plasmacytoid dendritic precursor cells. Lacking standard therapy due to limited understanding of biology and historical rarity of the disease, clinical outcomes for BPDCN patients remain poor. Surface overexpression of CD123/IL3Rα is seen 70-80% of patients with AML (Han et.al, Clin Cancer Res, 2017) and in nearly 100% of BPDCN cases, with a markedly high intensity compared to normal hematopoietic stem cells, thus making CD123 an attractive target for BPDCN treatment. IMGN632 is a CD123-targeting antibody-drug conjugate (ADC) comprised of a novel humanized anti-CD123 antibody G4723A linked to a DNA mono-alkylating payload of the indolindobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. IMGN632 demonstrates potent activity in AML samples at low concentrations with minimal impact on normal bone marrow progenitors, and anti-leukemia effects in AML xenograft models (Kovtun et.al., Blood Advances, 2018). A phase I clinical trial of IMGN632 in relapse/ refractory AML (NCT033865) is now being performed and achieved encouraging results. We thus explored the anti-tumor effect of IMGN632 in BPDCN. We first tested the in vitro activity of IMGN632 in a BPDCN cell line CAL-1. Cells were exposed to control antibody or IMGN632 at various concentration for 96 hours, after which viable cell numbers were measured using AnnexinV /DAPI assay with counting beads by flow cytometry. IMGN632 demonstrated cytotoxic activity at 0.2μg/ml, with stronger effects at concentrations of 2μg/ml and above (Figure 1A). Next, the in vivo efficacy of IMGN632 was evaluated in BPDCN patient-derived-xenograft (PDX) models. NSG mice (6-8 weeks old) were injected with 1e6/mouse BPDCN cells by tail vein 24 hours after 250cGY sublethal irradiation. Upon confirmation of the bone marrow engraftment of hCD45/hCD123 cells by bone marrow aspiration, mice were randomized to 5 or 8 mice/group and received weekly intravenous vehicle, control antibody or IMGN632 by tail vein injection 24 hours following FcR blockade (400mg/kg naked antibody by intraperitoneal injection) for 3 weeks. Bone marrow engraftment and survival were monitored. For model A, the mice received either 24μg/kg or 240μg/kg of IMGN632. For model B, the mice received 240μg/kg IMGN632. Both samples responded to the IMGN632 treatment, with the engraftment lower than 1% in the bone marrow after 3 doses, compared with over 90% in vehicle and control antibody group (Figure 1B). All mice from vehicle and control antibody groups in model A died by 52 days after cell injection; the 24μg/kg IMGN632 treated mice had prolonged survival with median survival of 111 days (p<0.0001), and the 240μg/kg IMGN632 treated mice are still alive after more than 140 days (p<0.0001) (Figure 1C). In the aggressive model B, a fraction of mice in each group died before efficacy could be established; the rest of the mice survived a median of 39 days in the control arm and 60 days in IMGN632-treated animals (p=0.0343) (Figure 1D). No evidence of treatment-related toxicity was observed using murine weight as a read-out. In summary, our preliminary data demonstrate pre-clinical proof of targeting CD123 in BPDCN using a novel antibody-drug conjugate. IMGN632 exhibited promising anti-tumor effects in BPDCN in vitro and in vivo, even at a dose 10-fold lower than the anticipated therapeutically active dose. This large therapeutic window would be expected to be advantageous when used in combination with other treatments in BPDCN. Disclosures Adams: ImmunoGen Inc.: Employment. Callum:ImmunoGen Inc.: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy. Kovtun:ImmunoGen Inc.: Employment. Daver:Daiichi-Sankyo: Research Funding; Novartis: Consultancy; Novartis: Research Funding; Incyte: Consultancy; BMS: Research Funding; Kiromic: Research Funding; Otsuka: Consultancy; Karyopharm: Consultancy; Alexion: Consultancy; Sunesis: Consultancy; Pfizer: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; ARIAD: Research Funding; Pfizer: Consultancy; ImmunoGen: Consultancy; Karyopharm: Research Funding. Pemmaraju:Affymetrix: Research Funding; SagerStrong Foundation: Research Funding; plexxikon: Research Funding; daiichi sankyo: Research Funding; samus: Research Funding; celgene: Consultancy, Honoraria; abbvie: Research Funding; cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; novartis: Research Funding. McKay:ImmunoGen Inc.: Employment. Konopleva:Stemline Therapeutics: Research Funding.

Self-repopulating recipient bone marrow resident macrophages promote long-term hematopoietic stem cell engraftment

Key Points Recipient macrophages persist in hematopoietic tissues and self-repopulate via in situ proliferation after syngeneic transplantation. Targeted depletion of recipient CD169+ macrophages after transplant impaired long-term bone marrow engraftment of hematopoietic stem cells.