scholarly journals Improved Patient-Derived Xenograft Model for Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3491-3491 ◽  
Author(s):  
Xiaochuan Shan ◽  
Cedric Dos Santos ◽  
Chenghui Zhou ◽  
Georges Habineza Ndikuyeze ◽  
Anthony Secreto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Xenograft Model ◽  
Mouse Strains ◽  
Mouse Bone Marrow ◽  
Tissue Samples ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Patient Derived Xenograft

Abstract We previously demonstrated that the NSG (NOD-Scid-IL2Rgcnull) xenotransplantation is an improved model for human AML samples, allowing us to better understand and characterize AML biology, especially in the context of drug therapy studies (Sanchez et al., Leukemia 2009). However, we observed that approximately half of AML patients’ samples either did not engraft in NSG mice (based on <0.1% human blasts in mouse bone marrow) or showed low (0.1 to 1% blasts) and highly variable engraftment. Recently, NSG mice expressing human SCF, GM-CSF and IL-3 transgenes (NSG-S) have been reported to enhance the engraftment of normal hematopoietic stem cells and primary AML cells, although only a few AML patients were evaluated (Wunderlich M et al, Leukemia 2010). This report describes a comprehensive paired analysis of engraftment of AML samples in NSG and NSG-S mice. T-cell depleted AML cells (5 -10 x 106 per mouse) were injected intravenously in sub-lethally irradiated mice (n=5/AML sample). Leukemia engraftment was assessed up to 16 weeks after injection in peripheral blood (PB), spleen (SPL) and bone marrow (BM) based on the percentage and absolute number of human leukemic blasts (huCD45+CD33+/-CD3-) in each tissue. Samples from 71 AML patients, representing all FAB and prognosis groups, were injected in NSG mice and only 35 samples (49%) engrafted based on human blasts >0.5% in mouse BM. From these 35 NSG-engrafting samples, 14 were also injected in NSG-S mice and all of them engrafted. Leukemic burden was significantly (p ≤ 0.05) increased in NSG-S versus NSG mice: 39±21% vs 22±23% BM blasts, 21±15% vs 7±10% SPL blasts, 2,732±6,488 vs 141±221 blasts/ml PB. Interestingly, the dramatic increase in peripheral blast count observed in NSG-S mice provides new opportunities to use PB to monitor drug response for the many patient samples that show no or very low peripheral engraftment in NSG mice. However, for 7 of these 14 NSG-engrafting AML samples, the use of NSG-S mice as recipients was associated with rapid engraftment, excessive leukemic burden, anemia, weight loss and lethargy requiring early sacrifice and leading to shorter overall survival (54±26 days in NSG-S vs >90 days in NSG). Out of the 36 patients’ samples that failed to engraft in NSG mice, 19 were tested for engraftment in NSG-S mice. Remarkably, 14 out 19 (74%) samples engrafted (17±16% BM blasts, 8±12% SPL, and 1,418±4,609/ml PB blasts at Day 77 post-transplant) and the kinetics of engraftment were slower compared to AML samples that can engraft in both mouse strains. These results suggest that the presence of human SCF, GM-CSF and IL-3 in NSG-S is sufficient to rescue leukemia-initiating cells for most AML samples that fail to engraft in NSG mice. Only 5 out of 33 samples (15%) failed to engraft in NSG and NSG-S mice, indicating that the NSG-S BM microenvironment remains suboptimal for a small minority of AML samples. We are investigating if NSG-S engraftment is correlated to CD116, CD117, CD123 expression, cytogenetics, mutations, and prognosis. Overall, our results show that NSG-S mice represent a significant improvement over previous patient-derived xenograft models since they can (1) accelerate and enhance leukemic engraftment compared to NSG mice, and (2) support engraftment for 85% of our AML patients, making this model particularly useful for pre-clinical studies. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 143-143 ◽  
Author(s):  
Saar Gill ◽  
Sarah K Tasian ◽  
Marco Ruella ◽  
Olga Shestova ◽  
Yong Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Research Funding ◽  
Conditioning Regimen ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1653-1653
Author(s):  
Nobuyuki Onai ◽  
Aya Onai ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Type I Interferon ◽  
Receptor Expression ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions. To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity. In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.

Human CD8+/TCR−/CD56dim/- Facilitating Cells Facilitate Homing and Engraftment of Human HSC in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4093-4093
Author(s):  
Yiming Huang ◽  
Mary J Elliott ◽  
Thomas Miller ◽  
Deborah R Corbin ◽  
Larry D. Bozulic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Mouse Bone Marrow ◽  
Common Procedure ◽  
Peripheral Blood Mononuclear ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Nsg Mice ◽  
Hsc Transplantation

Abstract Abstract 4093 Hematopoietic stem cell (HSC) transplantation has become a common procedure for treatment of hematopoietic malignancies and autoimmune disease. Despite significant advances in HSC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain limitations to application in the clinic. However, these risks can be overcome through less toxic nonmyeloablative conditioning and cell depletion strategies to remove GVHD causing-cells while retaining engraftment enhancing-tolerogeneic cells. We were the first to discover CD8+/TCR− graft facilitating cells (FC) in mouse bone marrow. The addition of as few as 30,000 FC to 10,000 HSC significantly enhances engraftment of HSC in allogeneic recipients without causing GVHD. FC also potently enhance engraftment of limiting numbers of syngeneic HSC. Human CD8+/TCR- FC comprised 1.1% ± 0.27% of total G-CSF-mobilized peripheral blood mononuclear cells (mPBMC). In the CD8+/TCR- FC, 48% of cells expressed CD3ε+, 43% were FoxP3+, 43% were CD11c+, 19% were CD19+, and 30% were HLA-DR+. Approximately 55% of FC are also CD56dim/-, and the remaining population is CD56bright. The morphology of human CD8+/TCR− FC with Wright-Giemsa staining under light microscopy suggested that the human FC population is heterogeneous. Here we evaluated if human FC enhance human HSC or progenitor homing to bone marrow of NOD/SCID/IL-2rγnull (NSG) mouse recipients. CD45+CD34+ HSC and CD8+/TCR−/CD56dim/-FC were sorted from mPBMC. NSG recipients were conditioned with 1100 cGy of total body irradiation (TBI). 24 hours after TBI, 100,000 HSC with or without 300,000 CD8+/TCR−/CD56dim/- FC were transplanted into conditioned NSG recipients. Recipients were euthanized 16 hours after transplantation. Bone marrow was harvested from femurs and tibias of recipients and plated in Colony Forming Culture (CFC) Assays. Recipients of HSC plus FC generated significantly more colony formation (colonies = 110) compared with HSC alone (colonies = 65) (P = 0.011), suggesting that CD8+/TCR−/CD56dim/- FC enhanced homing of HSC or progenitors to bone marrow. To test if human CD8+/TCR−/CD56dim/- FC facilitate engraftment of human HSC in NSG mice, 300,000 CD8+/TCR−/CD56dim/- FC were mixed with 100,000 HSC and transplanted into NSG recipient mice conditioned with 325 cGy TBI. Mice that received HSC alone served as controls. At 30 days after transplantation, PBL typing showed that 34% (10 of 29) recipients of HSC alone engrafted. In contrast, 78% of recipients (n = 23) of HSC plus CD8+/TCR−/CD56dim/- FC engrafted, and donor chimerism in PB was 1.1% ± 0.8% and 4.1% ± 1.3% (P <0.05), respectively. At 6 months after transplantation, NSG recipients of HSC + CD8+/TCR−CD56dim/- FC exhibited persistent donor chimerism in PB (9.1% ± 6% vs. 3.8% ± 3.5%) (P <0.05) and significantly higher levels of donor chimerism in spleen (26.3% ± 11.8% vs. 12.3% ± 9.8%) (P <0.05) and BM (11.6% ± 4.8% vs. 2.9% ± 1.3%) (P <0.05) compared to recipients of HSC alone. Our data indicate that CD8+/TCR−/CD56dim/- FC facilitate homing of human HSC or progenitors and enhance engraftment of human HSC in NSG recipient mice. Disclosures: Bozulic: Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

ΜΙSΤRG Mice Support Good-Risk AML Engraftment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3808-3808 ◽  
Author(s):  
Jana M. Ellegast ◽  
Yasuyuki Saito ◽  
Richard A. Flavell ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Genetic Alterations ◽  
Therapeutic Strategies ◽  
Live Cells ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Nsg Mice ◽  
Cell Counts ◽  
Good Risk

Abstract Introduction Acute myeloid leukemia (AML) is a hematopoietic stem cell derived disease with still unsatisfactory treatment options. Its broad biological heterogeneity needs to be dissected by deeper mechanistic understanding to translate functional insights into novel therapeutic strategies. AML engraftment in murine models and subsequent in vivo studies have been limited to clinically aggressive AML and cell lines. In fact there is no published xenograft model for good risk AML. The hypothesis of a humanized environment being favorable for AML engraftment motivates the development of humanized mouse models for faithful in vivo analysis including biologically less aggressive, so called good risk AML. Material & methods We transplanted sublethally irradiated newborn MISTRG mice (Rongvaux et al. Nat. Biotech. 2014) that express as knock-ins human M-CSF, IL-3, GM-CSF, TPO and human SIRPa as transgene as well as NSG mice as controls i.h. with human AML samples. AML blasts were purified prior to transplantation by immunomagnetical CD3 and CD19 depletion to avoid xenogeneic graft versus host disease (GvHD) and EBV-driven B-cell proliferation/lymphoma, respectively. We selected AML samples with known genetic alterations representing good risk subgroups (AML with isolated NPM1 mutation or t(8;21)). Engraftment was assessed at different time points by peripheral blood analysis (cell counts and immunophenotyping) and bone marrow aspiration. Final analysis was completed by quantitative and histologic bone marrow analysis such as molecular diagnostics. Results Robust engraftment of good risk AML could be observed upon transplantation of patient samples into MISTRG mice: 87% of animals (n=15) engrafted (>1% hCD45+/ live cells) with a mean engraftment level of 61%. Time to and extent of engraftment depends on transplanted cell numbers, aggressiveness of disease and reaches its maximum around 16 to 20 weeks post transplantation in good risk AML when disease burden tends to limit survival. Gender of transplanted mice did not appear to influence engraftment. Engraftment was associated with a distinct phenotype including peripheral pancytopenia with leading anemia and splenomegaly with a significant positive correlation between engraftment level and spleen weight (Spearman r=0.6, p=0.03). We were able to show engraftment by immunophenotyping and molecular prove of preexisting genetic alterations. Engraftment levels in NSG mice were similar or inferior at any time (67% engraftment (n=6) with a mean engraftment level of 37% hCD45+/ live cells) and side to side comparison suggests an AML immunophenotype closer to human AML presentation in MISTRG mice. Engraftment did not seem to depend on SIRPa expression while preliminary data points towards a critical role of IL-3/GM-CSF and M-CSF. Conclusions Humanised mouse strains with differential human knock-ins provide suitable models to further dissect the disease supporting environment, which can then be translated into the development of direct therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

scholarly journals XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer

Oncogene ◽  
2015 ◽  
Vol 35 (3) ◽  
pp. 290-300 ◽  
Author(s):  
J J Morton ◽  
G Bird ◽  
S B Keysar ◽  
D P Astling ◽  
T R Lyons ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Head And Neck Cancer ◽  
Head And Neck ◽  
Neck Cancer ◽  
Xenograft Model ◽  
Mouse Bone Marrow ◽  
Patient Derived Xenograft

Repairing of Homing Defect in Cord Blood Hematopoietic Stem Cell Transplantation–Comparison of Fucosyltransferase VII with Fucosyltransferase VI.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2988-2988
Author(s):  
Wan Xiang ◽  
Hiromasa Miyaji ◽  
Hidetaka Sato ◽  
Len Miller ◽  
Sun Zimin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Flow Conditions ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Abstract 2988 Objective: Relatively slower speed of engraftment in bone marrow of recipients makes cord blood (CB) transplantation (CBT) more difficult in a clinical setting. Selectins and their ligands are known to play a role in the homing of hematopoietic stem/progenitor cell (HSPC) to the bone marrow. Fucosyltransferase VI (FTVI) mediated ex vivo fucosylation improves human CB engraftment in NOD-SCID IL-2Rγnull (NSG) mice. FTVI is expressed in many cell types, while fucosyltransferase VII (FTVII) is primarily expressed in hematopoietic cells. FTVII deficient leukocytes do not bind the P- and E-selectins and are not recruited to sites of inflammation. We hypothesize that FTVII is more efficient than FTVI in surface fucosylation of CB-HSPC and thus enhances homing and engraftment of CB-HSPC (CD34+) in NSG mice. Method: Purified CB CD34+ cells were obtained from fresh CB in healthy full-term newborns by immunomagnetic microbeads sorting. CD34+ cells were treated with FTVI or FTVII respectively, the expression level of sLex (HECA-452) and binding to P- or E-selectin were detected by flow cytometry. Interactions of FTVI-, FTVII- or combined FTVI/FTVII-fucosylated CB CD34+cells with P- or E-selectin were measured under physiological flow conditions using a parallel-plate flow chamber assay. CBT was performed in NSG mice of 5 groups (negative control, sham-control, FTVI-treated, FTVII-treated, and FTVI/FTVII-treated). From 2 weeks post transplantation, mice were bled every 2 weeks for anti-human CD45 screening by flow cytometry. We used this method to evaluate the engraftment progress of CB-HSPC with different treatments. Ten weeks after transplantation, all mice were sacrificed and peripheral blood and bone marrow cells were isolated. Hematopoiesis reconstitution of CB cells was analyzed by flow cytometry after staining the cells with antibodies to various human hematopoietic subset markers, which included CD45 for total leukocytes, CD19 for B-cells, CD33 for myeloid cells, CD41 for megakaryocytes, CD3 for T-cells, and CD34 for HSPCs. Result: Under in vitro conditions, surface fucosylation with FTVI or FTVII results in an enhanced expression of HECA-452 epitopes on CD34+ cells from fresh CB. Increased P- and E-selectin binding to fresh CB CD34+ cells after either FTVI or FTVII treatment compared to sham-treated. However, FTVII-treated fresh CB CD34+ cells had a much higher binding to P-selectin than that of FTVI-treated fresh CB CD34+ cells. FTVI/FTVII-fucosylated CD34+ cells isolated from fresh CB accumulated more on P- or E-selectin coated plates than that of single enzyme fucosylated CD34+ cells under flow conditions. Although it is not statistically significant, more FTVII-treated fresh CB CD34+ cells rolled on both P- and E-selectin surface than FTVI-treated fresh CB CD34+ cells did. FTVII- or FTVI/FTVII-treated CD34+cells from fresh CB engrafted significantly better in NSG mice than the FTVI-treated cells at 4 and 6 weeks. Ten weeks after transplantation, the bone marrows of mice that received FTVII-treated cells had significantly improved engraftment of human hematopoietic progenitors of some lineages (CD3, CD33, and CD41) compared to that of mice that received FTVI-treated cells. Conclusion: FTVII-treated CB-HSPCs have greater engraftment progress during early time after transplantation compared to FTVI. FTVII mediated fucosylation did not affect self-renewal of CB-HSPC. Hematopoiesis reconstitution of T-lymphocytes, myeloid cells, and megakaryocytes of FTVII is better than FTVI. Finally, FTVI and FTVII may have a cooperative effect on CB-HSPC homing to bone marrow. Disclosures: Miyaji: Kyowa Hakko Kirin Co., Ltd.: Research Funding. Sato:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Miller:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Xia:Kyowa Hakko Kirin Co., Ltd.: Research Funding.

The Antibody-Drug Conjugate (ADC) IMGN779 Is Highly Active in Vitro and in Vivo Against Acute Myeloid Leukemia (AML) with FLT3-ITD Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2321-2321 ◽  
Author(s):  
Kathleen R Whiteman ◽  
Paul Noordhuis ◽  
Russell Walker ◽  
Krystal Watkins ◽  
Yelena Kovtun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Research Funding ◽  
Xenograft Model ◽  
Hematopoietic Stem ◽  
Highly Active ◽  
Ic50 Values

Abstract IMGN779 is a CD33-targeted ADC utilizing DGN462, a novel DNA-alkylating agent consisting of an indolino-benzodiazepine dimer containing a mono-imine moiety. CD33 is expressed on the surface of about 90% of AML cases, with elevated levels of CD33 found in cases having molecular markers associated with poor prognosis, including mutations in FMS-like tyrosine kinase 3 (FLT3). The internal tandem duplication mutation (FLT3-ITD) is the most common FLT3 mutation, present in about 20-25% of AML cases. Patients with FLT3-ITD AML have a worse prognosis than those with wild-type (WT) FLT3, with an increased rate of relapse and a shorter duration of response to induction chemotherapy. IMGN779 was found to demonstrate targeted activity against AML cell lines in vitro, with IC50 values ranging from 2-3,000 pM. The MV4-11 cell line, which has a FLT3-ITD mutation, was the most sensitive to IMGN779 of the cell lines tested, with an IC50 of 2 pM. We evaluated the in vivo activity of IMGN779 against MV4-11 xenografts in SCID mice; IMGN779 was highly active (T/C = 1 %) at a single dose of 0.6 mg/kg (conjugate dose, 10 µg/kg DGN462 dose), resulting in complete tumor regressions (CR) in 3/6 animals and partial regressions (PR) in 6/6 animals. A DGN462-ADC to a non-relevant target was inactive (T/C = 95%) at the same dose, demonstrating that the activity of IMGN779 was due to its CD33 targeting. IMGN779 has previously been shown to be highly active against AML xenograft models without FLT3-ITD mutations, at minimally efficacious doses of 0.6 mg/kg (10 µg/kg DGN462), demonstrating that the presence of FLT3-ITD does not confer resistance to IMGN779 treatment. IMGN779 was also highly active in vitro against primary patient AML cells isolated from peripheral blood or bone marrow samples. Patient AML cells with FLT3-ITD were more sensitive to IMGN779 compared with FLT3 WT AML samples. IC50 values in FLT3-ITD samples ranged from 10 to 300 pM. CD33 expression was generally greater on FLT3-ITD leukemic blast cells than on FLT3 WT blasts, which likely contributed to their increased sensitivity to IMGN779. In long term cultures, IMGN779 showed a dose dependent decrease in leukemic stem cell (LSC) colony formation using an AML patient sample with both FLT3-ITD and NPM1 mutations, which are an even worse prognostic marker than FLT3-ITD alone. In contrast, colony formation increased in normal bone marrow, indicating that normal hematopoietic stem cells (HSCs) were spared. The differential expression of CD33 on LSC compared to HSCs makes CD33 an attractive target for treatment of AML, with the potential to eliminate LSCs and, thus, minimal residual disease in FLT3-ITD AML. The potent in vitro activity of IMGN779 against FLT3-ITD AML cell lines and primary patient FLT3-ITD AML progenitor cells and LSCs and its high level of CD33-targeted in vivo activity in a FLT3-ITD AML xenograft model support the advancement of IMGN779 as a potential treatment for AML, including FLT3-ITD AML. Disclosures Whiteman: ImmunoGen, Inc.: Employment. Noordhuis:ImmunoGen, Inc.: Research Funding. Walker:ImmunoGen, Inc.: Employment. Watkins:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Johnson:ImmunoGen, Inc.: Employment. Schuurhuis:ImmunoGen, Inc.: Research Funding. Ossenkoppele:ImmunoGen, Inc.: Research Funding. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

scholarly journals The Effects ofParacoccidioides brasiliensisInfection on GM-CSF- and M-CSF-Induced Mouse Bone Marrow-Derived Macrophage from Resistant and Susceptible Mice Strains

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Calliandra de Souza Silva ◽  
Aldo Henrique Tavares ◽  
Marcio Sousa Jeronimo ◽  
Yasmin Soares de Lima ◽  
Lorena da Silveira Derengowski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Expression Profiles ◽  
Paracoccidioides Brasiliensis ◽  
Gene Expression Profiles ◽  
Mouse Strains ◽  
Mouse Bone Marrow ◽  
Strongly Correlated ◽  
Gm Csf ◽  
Susceptibility And Resistance ◽  
Innate Tendency

Considering the importance of macrophages as the first line of defense against fungal infection and the different roles played by the two M1- and M2-like polarized macrophages, we decided to evaluate the effects ofParacoccidioides brasiliensisinfection on GM-CSF- and M-CSF-induced bone marrow-derived macrophages (BMM) from the A/J and B10.A mouse strains, an established model of resistance/susceptibility to PCM, respectively. Upon differentiation, the generated GM- or M-BMMs were characterized by morphological analyses, gene expression profiles, and cytokines production. Our main results demonstrate that GM-BMMs derived from A/J and B.10 produced high levels of pro- and anti-inflammatory cytokines that may contribute to generate an unbalanced early immune response. In accordance with the literature, the B10.A susceptible mice lineage has an innate tendency to polarize into M1-like phenotype, whereas the opposite phenotype occurs in A/J resistance mice. In this context, our data support that susceptibility and resistance are strongly correlated with M1 and M2 polarization, respectively.

Recent approaches for isolating and culturing mouse bone marrow-derived mesenchymal stromal stem cells

2020 ◽  
Vol 15 ◽  
Author(s):  
Basem M. Abdallah ◽  
Hany M. Khattab
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Molecular Mechanisms ◽  
Replicative Senescence ◽  
Cellular Heterogeneity ◽  
High Yield ◽  
Mouse Strains ◽  
Mouse Bone Marrow ◽  
Differentiation Potential ◽  
Stromal Stem Cells

: The isolation and culture of murine bone marrow-derived mesenchymal stromal stem cells (mBMSCs) have attracted great interest in terms of the pre-clinical applications of stem cells in tissue engineering and regenerative medicine. In addition, culturing mBMSCs is important for studying the molecular mechanisms of bone remodelling using relevant transgenic mice. Several factors have created challenges in the isolation and high-yield expansion of homogenous mBMSCs; these factors include low frequencies of bone marrow-derived mesenchymal stromal stem cells (BMSCs) in bone marrow, variation among inbred mouse strains, contamination with haematopoietic progenitor cells (HPCs), the replicative senescence phenotype and cellular heterogeneity. In this review, we provide an overview of nearly all protocols used for isolating and culturing mBMSCs with the aim of clarifying the most important guidelines for culturing highly purified mBMSC populations retaining in vitro and in vivo differentiation potential.

Sign in / Sign up

Export Citation Format

Share Document