Human Flt3 Ligand Research Articles

A Minimal PBPK/PD Model with Expansion-Enhanced Target-Mediated Drug Disposition to Support a First-in-Human Clinical Study Design for a FLT3L-Fc Molecule.

FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule's mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.

Abstract 3217: Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA

Abstract Recent studies have highlighted the critical role of CD103+/CD141+ dendritic cells (DCs) in antitumor immunity. CDX-301 is a soluble, recombinant human FLT3 ligand (Flt3L). Flt3L is a hematopoietic cytokine which stimulates the proliferation and differentiation of various blood cell progenitors, including CD103+/CD141+ DCs. CDX-301 is in clinical development for multiple cancers and may hold significant opportunity for synergistic development in combination with other immunotherapies, in particular toll-like receptor 9 (TLR9) agonists that are known to promote maturation and activation of DCs. AST-008 is a TLR9 agonist oligonucleotide in a spherical nucleic acid (SNA) format and is in clinical development for multiple cancers in combination with pembrolizumab. SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. AST-008 induces potent TH1-type immune responses in vitro, in mice and non-human primates, and has shown potent antitumor activity as a monotherapy and enhanced checkpoint inhibitor activity in several murine tumor models. AST-008 increases tumor-infiltrating lymphocytes, interferon-inducible gene expression, and activation and expansion of CD8+ T cells with reduced T-regulatory cells in the tumor microenvironment. We examined the effects of the combination of CDX-301 and a murine version of AST-008, muAST-008, in mice on DCs and on antitumor efficacy in a tumor model. Mice were implanted with MC38 murine colon adenocarcinoma tumor cells (day 0) and animals were treated with either CDX-301 (5 μg, days 2-8 i.p.) only, muAST-008 only (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30 p.t.), or the combination of CDX-301 (5 μg, days 2-8) with muAST-008 (1 mg/kg or 3 mg/kg, days 9, 16, 23 and 30). Treatment with CDX-301 and muAST-008 showed an additive effect in retarding tumor growth and prolonging survival. To help understand the mechanisms involved in the antitumor effects, animals were treated with a similar course of CDX-301 and muAST-008 and spleens were harvested for flow cytometry evaluation 2 days after the last CDX-301 dose. For these studies, we also included a second DC activating agent, an agonist anti-CD40 mAb FGK45.5 (50 μg i.p. together with the last dose of CDX-301) among the combinations. We observed a significant increase in the percentage of CD103+CD8+ cDCs by the addition of muAST-008 to CDX-301 treatment. In addition, muAST-008 led to the up-regulation of activation markers on dendritic cells, which was markedly enhanced when combined with CD40 activation. These data demonstrate that muAST-008 leads to systemic activation of CDX-301 expanded DCs, leading to more potent anti-tumor immunity and support the potential of combining CDX-301 and AST-008 in augmenting the immunotherapy of cancers. Citation Format: Lawrence J. Thomas, Li-Zhen He, Lauren E. Gergel, Eric M. Forsberg, Elizabeth Q. Do, James M. Boyer, April R. Baronas, Mallary Rocheleau, Kathleen M. Borrelli, Anna Wasiuk, Jeffrey Weidlick, Henry C. Marsh, Bart R. Anderson, SubbaRao Nallagatla, Richard Kang, Ekambar R. Kandimalla, Tibor Keler. Preclinical evaluation of the recombinant dendritic cell growth factor CDX-301 (Flt3L), and AST-008, a TLR9 agonist SNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3217.

Overexpression of functional human FLT3 ligand in Pichia pastoris

Growth factors and cytokines including FLT3 Ligand (FLT3L) have many applications in cellular and molecular biology studies, and also as therapeutical agents. FLT3L was expressed in Pichia pastoris through cloning from human leukemic K562 cell line as well as using a codon-optimized synthetic construct. Codon adaptation index (CAI) was increased from 55 in the native sequence up to 95 after optimization. Significant changes occurred in the codons for Pro, Arg, Leu and Ser toward the favored codons in P. pastoris. Both forms of expressed FLT3L (from the native and optimized sequences) were capable of stimulating proliferation of the FDC-P1 cells expressing human wild-type FLT3 (FD–FLT3–WT). Sequence optimization resulted in 55-fold increase in the yield of active FLT3L (290 μg/mL of product in the crude supernatant). Amino acid residues 27–162 are sufficient for the biological function of human FLT3L. Pichia pastoris is a highly efficient and cost-effective system for expression of endotoxin-free FLT3L; however, codon optimization is necessary for its optimal expression.

The human CD34(−)CD117(+)CD56(−)NKp44(−) population in secondary lymphoid tissue serves as a common progenitor to NK cells and ILC2s

Abstract The developmental pathways of human innate lymphoid cells (ILCs) have not been fully elucidated. We reported that human natural killer (NK) cells can derive from progenitor cells naturally residing in tonsils. We hypothesized that Group 2 ILCs (ILC2s) also develop in tonsils through a shared pathway. In support of this, ex vivo flow cytometry analyses of enriched human tonsil ILCs revealed a putative pathway of human CD294(+) ILC2 development stemming from a lineage (Lin)(−)CD34(−)CD117(+) population from which non-overlapping CD94(+) NK cells also emerged. Furthermore, when purified tonsil Lin(−)CD34(−)CD117(+) cells were cultured for 28 days with murine OP9 stroma expressing the human Notch ligand, Delta-like 1, in the presence of human interleukin-7 and Flt3 ligand, they produced mutually distinct and functionally mature populations of CD94(+) NK cells and CD294(+) ILC2s. The tonsil Lin(−)CD34(−)CD117(+) population is heterogeneous for CD56 and NKp44. Evaluation of the in vitro lineage differentiation potentials of subsets within the Lin(−)CD34(−)CD117(+) population according to NKp44 and CD56 revealed that NKp44(−)CD56(−) cells gave rise to both NK cells and ILC2s, whereas fractions expressing NKp44 and/or CD56 gave rise to NK cells but only to negligible ILC2s. In fact, the NKp44(−)CD56(−) population gave rise to a significantly higher percentage of ILC2s (7.50 ± 0.6) compared to the NKp44(+)CD56(+/−) populations (1.22 ± 0.4) (n = 5, p < 0.001). These data support a model whereby human NK cells and ILC2s can develop from a common Lin(−)CD34(−)CD117(+)CD56(−)NKp44(−) progenitor in tonsils and that the acquisition of NKp44 and/or CD56 restricts lineage differentiation potential towards the NK cell lineage.

A Phase II Randomized Study of CDX-1401, a Dendritic Cell Targeting NY-ESO-1 Vaccine, in Patients with Malignant Melanoma Pre-Treated with Recombinant CDX-301, a Recombinant Human Flt3 Ligand.

9589Background: Patients (pts) with high-risk melanoma have a 20-60% recurrence rate with 5-year OS between 45% and 70%. The adjuvant setting is an opportunity to test prevention vaccines that may have efficacy against disease recurrence. We evaluated CDX301, a recombinant human Flt3 ligand (Flt3L), plus vaccination with CDX1401, a fusion protein consisting of human monoclonal IgG1 antibody targeting the dendritic cell (DC) receptor DEC-205 linked to the NY-ESO-1 tumor antigen, in a phase II, open-label, multicenter, randomized study of subjects with resected melanoma, to determine whether immune responses to NY-ESO-1 elicited by vaccination with CDX1401 + poly-ICLC are substantially increased by prior expansion of circulating DC with Flt3L therapy. Methods: 60 pts with resected melanoma were randomized to two cohorts: Cohort 1 received CDX301 pretreatment (25 ug/kg SC x 10 days) in 2 of 4 monthly cycles of vaccination with CDX1401 (1mg IC) + poly-ICLC (2mg SC, days 1 and 2). Cohort 2 received 4 monthly c...

An autologous in situ tumor vaccination approach for hepatocellular carcinoma. 2. Tumor-specific immunity and cure after radio-inducible suicide gene therapy and systemic CD40-ligand and Flt3-ligand gene therapy in an orthotopic tumor model.

Diffuse hepatocellular carcinoma (HCC) is a lethal disease that radiation therapy (RT) currently has a limited role in treating because of the potential for developing fatal radiation-induced liver disease. However, recently diffuse HCC, "radio-inducible suicide gene therapy" has been shown to enhance local tumor control and residual microscopic disease within the liver for diffuse HCC, by using a combination of chemoactivation and molecular radiosensitization. We have demonstrated that the addition of recombinant adenovirus-expressing human Flt3 ligand (Adeno-Flt3L) after radio-inducible suicide gene therapy induced a Th1-biased, immune response and enhanced tumor control in an ectopic model of HCC. We hypothesized that sequential administration of recombinant adenovirus-expressing CD40L (Adeno-CD40L) could further potentiate the efficacy of our trimodal therapy with RT + HSV-TK + Adeno-Flt3L. We examined our hypothesis in an orthotopic model of diffuse HCC using BNL1ME A.7R.1 (BNL) cells in Balb/c mice. BNL murine hepatoma cells (5 × 10(4)) transfected with an expression vector of HSV-TK under the control of a radiation-inducible promoter were injected intraportally into BALB/cJ mice. Fourteen days after the HCC injection, mice were treated with a 25 Gy dose of radiation to the whole liver, followed by ganciclovir (GCV) treatment and systemic adenoviral cytokine gene therapy (Flt3L or CD40L or both). Untreated mice died in 27 ± 4 days. Radiation therapy alone had a marginal effect on survival (median = 35 ± 7 days) and the addition of HSV-TK/GCV gene therapy improved the median survival to 47 ± 6 days. However, the addition of Adeno-Flt3L to radiation therapy and HSV-TK/GCV therapy significantly (P = 0.0005) increased survival to a median of 63 ± 20 days with 44% (7/16) of the animals still alive 116 days after tumor implantation. The curative effect of Flt3L was completely abolished when using immunodeficient nude mice or mice depleted for CD4, CD8 and natural killer cells. The addition of Adeno-CD40L further improved the median survival of animals to 80 ± 15 days and this effect was abolished only when using anti-CD8 antibodies. Chromium-51 (51Cr) release assay showed cytotoxic T lymphocyte (CTL) activation, suggesting efficient dendritic cell (DC) activation with CTL activation after the treatment. Furthermore, when surviving mice were rechallenged with BNL-ETK cells on the foot pad, RT + HSV-TK/GCV + Flt3L + CD40L-treated mice developed a small tumor on day 56 but the tumor eventually disappeared after 105 days. Mice treated with RT + HSV-TK/GCV + Flt3L showed a slowed tumor growth curve compared with untreated mice. Therefore, combination therapy using Flt3L to induce DC proliferation and CD40L to enhance DC maturation holds great promise for immunomodulation of radiation therapy to enhance HCC tumor control and prevent progression of disease in patients with diffuse HCC.

Establishment of hematological malignancy model in ex vivo cell culture

The aim of this study was to develop an ex vivo cell culture system for establishing the hematological malignancy model. Mouse bone marrow cells were transfected with GFP-expressed retroviral vectors encoding various leukemia/lymphoma-associated fusion proteins (TEL-PDGFR, Rabaptin5-PDGFR, p210BCR-ABL, AML1-ETO, NPM-ALK). After transfection, the cells were cultured in IMDM containing 10% FCS without growth factors, or with one of the following growth factor combinations: (1) murine c-kit ligand (KL) plus human flt3 ligand (FL); (2) IL-3, thrombopoietin, G-CSF, and hyper-IL-6 (3/T/G/H6); (3) KL/FL plus 3/T/G/H6. The flow cytometry was used to detect the ability of combinations of growth factors to complement the oncogene fusion protein to support self-renewal of the transfected cells. The results showed that the transfected cells could be amplified sustainably in the logarithmic growth way. The indicated combination of c-Kit ligand (KL) with flt-3 ligand (FL) supported the self-renewal of the marrow cells transfected with vectors encoding TEL-PDGFR, Rabaptin5-PDGFR, AML1-ETO and NPM-ALK, in addition to KL/FL, the self-renewal of p210 BCR-ABL transfected-marrow cells also required IL-3. The morphology of cells emerged from culture can be the predictor of the corresponding oncogene-associated malignancy. It is concluded that this study establishes a culture system ex vivo which provides a generalized method for studying hematological malignancies, and may facilitate the screening for therapeutic agents.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

In Vitro Expansion of Human Hematopoietic Cells with Delayed but Sustained Multi-Lineage Repopulating Activity

Abstract 1270In vivo expansion of hematopoietic stem cells (HSCs) involves local interactions with stimuli generated from non-hematopoietic niche environments, but the full spectrum of molecular mechanisms responsible have remained elusive. Initial experiments in mice showed that highly purified HSCs from adult mouse bone marrow are consistently expanded 3–5-fold with full maintenance of their long term (≥6 months), serially transplantable, multi-lineage repopulating ability when cultured for 7 days in serum-free UG26 stromal-cell conditioned medium (CM) supplemented with 100 ng/ml mouse Steel Factor (SF) and 20 ng/ml mouse IL-11. To explore the potential effects of this CM on HSCs in human cord blood, we conducted an initial experiment in which CD34+CD38− cells were cultured for 7 days in UG26 CM supplemented with 100 ng/ml human Flt3-ligand, 100 ng/ml human SF, and 20 ng/ml each of human IL-3, IL-6 and G-CSF. The results of limiting dilution transplants of the cultured cells in intravenously injected NSG mice showed retention of input numbers of cells with equivalent robust 6-month lympho-myeloid repopulating activity. To characterize the initial target cells and determine whether their proliferative responses might be predictive of their self-renewal behavior, we set up single cell cultures with the CD49f+ subset of CD34+CD38−CD45RA−CD90+Rho−cells with the 5 growth factors in the presence or absence of CM. Under both conditions, 7/13 and 4/13 input cells, respectively, died within the first 72 hours in culture. The subsequent rate of proliferation of the survivors was similar with all completing a first division after 96 hours and a second division 24–48 hours later. By day 8, clones of variable sizes were noted (6–1100 and 4–200 cells/clone, respectively). Clones generated under the same conditions were pooled and injected intravenously into 2 NSG mice each. We then looked for the presence of human cells in the mice by analysis of serial bone marrow aspirates starting 3 weeks post-transplant. Human cells were detected in only one of each of the 2 pairs of mice and, interestingly, in both cases, no evidence of human cells was detectable until 3 months post-transplant. In the positive mouse injected with cells generated in the absence of CM, this repopulation was transient, peaking at ∼0.1% of the mouse bone marrow compartment at 4 months post-transplant and undetectable a month later. In contrast, in the positive recipient of cells from the cultures that contained CM, both lymphoid and myeloid human cells reached much higher levels (together making up ∼20% of the mouse bone marrow compartment) which were maintained for another 3 months when the mouse was sacrificed. Transplants of cells obtained at this time from the marrow gave positive repopulation of secondary mice. In a subsequent experiment, in which similar cultures were initiated with CD34+ cord blood cells, evidence of a late continuing effect of the CM was obtained with a net absolute expansion of CD34+CD45RA−CD90+ cell numbers during the interval between 12 and 21 days in vitro. These findings highlight the important potential of as yet unidentified secreted stromal cell factors to stabilize the stem cell state in HSCs stimulated to proliferate in vitro by growth factors that favor their self-renewal. Disclosures:No relevant conflicts of interest to declare.

Functionalized Carbon Nanotubes Increase the Viability of Post-Thaw Cord Blood Cells and Enhance the Overall Hematopoietic Progenitor Cell Expansion in Ex Vivo Culture

Abstract 1327The clinical application of cord blood (CB) cells for transplantation is often hampered by the low cell number and viability from frozen cell sources. This problem could be partly resolved by ex vivo expansion and the improvement of CB viability through the use of a mesenchymal feeder cell layer or an appropriately designed material scaffold. Carbon-based nanomaterials have gained immense attention in biomedical applications in recent years, owing to their unique physicochemical characteristics. These novel materials have been applied as scaffolds in tissue engineering to increase the proliferation and differentiation of a wide range of cell types that include neurons and osteoblasts. In this study, we carried out a hitherto unreported use of functionalized carbon nanotubes to support the viability and ex vivo expansion of umbilical cord blood – mononucleated cells (UCB-MNCs). Specifically, the use of single-walled carbon nanotubes functionalized with carboxylic acid groups (fSWCNT-COOH) was selected, based on its improved aqueous dispersibility which constitutes better biocompatibility.UCB-MNCs cultured in a cocktail of human stem cell factor, Flt-3 ligand, thrombopoietin and insulin-like growth factor-binding protein 2 were exposed to fSWCNT-COOH at various concentrations (0 to 1 mg/mL). The effects of fSWCNT-COOH treatment were characterized based on UCB-MNC viability, CD45+CD34+CD38− phenotypic-based progenitor cell number, total nucleated cell count and functional progenitor cell number.In a concentration-dependent manner, fSWCNT-COOH was found to support the viability of UCB-MNCs, an effect that is similar to those observed with mesenchymal feeder co-culturing. After 3 days in culture, 0.5 and 1.0 mg/mL fSWCNT-COOH significantly (p<0.01) increased the percentage of viable CD45+ UCB-MNCs from 23.1±0.6% to 58.4±0.9% and 72.7±1.5%, respectively, as defined by Annexin-V/7AAD double negativity. Significant fold increases (p<0.01) in the phenotypically-defined progenitor cell numbers were observed after UCB-MNC exposure to 1 mg/mL fSWCNT-COOH for 11 days, with increases of 7.3±0.5-fold, 92.1±3.5 fold and 183.7±6.1-fold obtained respectively for viable CD45+, CD45+CD34+ and CD45+CD34+CD38− progenitor cell numbers. This finding was further supported by a significantly lower (p<0.01) side scatter value of 346.9±13.3 in the fSWCNT-COOH treated UCB-MNC as compared to the control treated with the cytokine cocktail alone (side scatter value of 405.6±4.9). Furthermore, a significant increase (p<0.01) in the expansion fold of functional CFU from 35.2±3.7 to 120.9±7.6 was obtained when UCB-MNC were treated with 1 mg/mL fSWCNT-COOH. Our results demonstrated that concentrations of fSWCNT-COOH as high as 1 mg/mL were non-toxic to UCB-MNCs. Similar to the mesenchymal feeder co-culture system, carbon-based nanomaterials such as fSWCNT-COOH could support UCB-MNC viability and progenitor expansion.In conclusion, we have demonstrated a novel method of ex vivo expansion of UCB-MNCs using carbon-based nanomaterials. Further investigation is required to elucidate the exact biological mechanism of the cytoprotective effect of carbon-based nanomaterials coupled with validation of the ex vivo expanded UCB-MNCs using in vivo immunodeficient mice models. Disclosures:No relevant conflicts of interest to declare.

Efficient Generation of Multipotent Mesenchymal Stem Cells from Umbilical Cord Blood in Stroma-Free Liquid Culture

BackgroundHaematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.Methods and FindingsUCB-derived mononuclear cells (MNC) or selected CD34+ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34+ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin+, CD133+ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.ConclusionsThis novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.

Renaturation with simultaneous purification of the recombinant human Flt3 ligand from inclusion bodies by high performance hydrophobic interaction chromatography

Flt3 ligand (FL) is a class of cytokines with the functions of promoting early hematopoiesis. It has important clinical value in promoting growth and development of hematopoietic cells and hematopoietic mobilization. In order to obtain large quantities of recombinant human FL (rhFL) by genetic engineering methods for clinic and research, in this work, rhFL was expressed in E. coli as inclusion bodies. The inclusion bodies were recovered, cleaned and solubilized in 8 mol/L urea, the solubilized rhFL was renatured by high performance hydrophobic interaction chromatography (HPHIC) with simultaneous purification, the retention feature and renaturation regularity were studied. The results showed that when the denatured protein concentration was 8.51 g/L, and the end group of stationary phase was PEG800, under the conditions of mobile phase of pH 7.0 and with the addition of 4 mol/L urea, 1.8 mmol/L glutathione (GSH) and 0.3 mmol/L oxidative glutathione (GSSG), a mass recovery of 36.9% and a purity of 94.5% were obtained after refolding with simultaneous purification. The obtained rhFL was successfully renatured with simultaneous purification in only one step of HPHIC, and it provided a foundation for the manufacturing of high quality rhFL.

Attenuation of vaccinia virus by the expression of human Flt3 ligand

BackgroundVaccinia virus, one of the best known members of poxvirus family, has a wide host range both in vivo and in vitro. The expression of Flt3 ligand (FL) by recombinant vaccinia virus (rVACV) highly influenced properties of the virus in dependence on the level of expression.ResultsHigh production of FL driven by the strong synthetic promoter decreased the growth of rVACV in macrophage cell line J774.G8 in vitro as well as its multiplication in vivo when inoculated in mice. The inhibition of replication in vivo was mirrored in low levels of antibodies against vaccinia virus (anti-VACV) which nearly approached to the negative serum level in non-infected mice. Strong FL expression changed not only the host range of the recombinant but also the basic protein contents of virions. The major proteins - H3L and D8L - which are responsible for the virus binding to the cells, and 28 K protein that serves as a virulence factor, were changed in the membrane portion of P13-E/L-FL viral particles. The core virion fraction contained multiple larger, uncleaved proteins and a higher amount of cellular proteins compared to the control virus. The overexpression of FL also resulted in its incorporation into the viral core of P13-E/L-FL IMV particles. In contrary to the equimolar ratio of glycosylated and nonglycosylated FL forms found in cells transfected with the expression plasmid, the recombinant virus incorporated mainly the smaller, nonglycosylated FL.ConclusionsIt has been shown that the overexpression of the Flt3L gene in VACV results in the attenuation of the virus in vivo.

Barley as a green factory for the production of functional Flt3 ligand

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Expression, purification and characterization of the extracellular domain of human Flt3 ligand in Escherichia coli

Fms-like tyrosine kinase 3 ligand (Flt3 ligand, FL) is a cytokine that affects the growth, survival and/or differentiation of hematopoietic cells through the activation of specific tyrosine kinase receptors, and is potentially useful for in vitro HSC amplification. To express the extracellular domain of human Flt3 ligand (hFL(ext)) in Escherichia coli, we cloned hFL(ext) and constructed the recombinant expression vector pET32a-hFL(ext). hFL(ext) was successfully expressed in E. coli as a Trx fusion protein (Trx-hFL(ext)) under IPTG (isopropyl-beta-D: -thiogalactopyranoside) induction for 12 h at 30 degrees C. The Trx-hFL(ext) protein, expressed in inclusion bodies even at a low induction temperature, was successfully refolded and purified using dialysis and affinity chromatography. The purified hFL(ext) was biologically active and could effectively stimulate the proliferation of mouse bone marrow nucleated cells revealed by cell proliferation assay and colony forming assay. In addition, in synergize with G-CSF and TPO, recombinant purified hFL(ext) could stimulate ex vivo expansion of murine Lin(-)Sca-1(+)c-Kit(+) cells. Therefore, using the E. coli expression system and an affinity chromatography system, we successfully expressed, refolded, and purified a biologically active Trx-hFL(ext) protein which might be potentially useful for in vitro HSC amplification.

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein.

We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34+ blood progenitors which express different levels of c-kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU-G) formation in the presence of granulocyte-colony stimulating factor (G-CSF) by peripheral blood (PB)-derived CD34+c-kit- cells which contained a large number of CFU-G. In addition, FL could synergistically increase the number of CFU-G supported by a combination of interleukin (IL)-3 and G-CSF, as did SCF. As we reported previously, SCF showed a significant burst-promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB-derived CD34+c-kithigh cells in which erythroid-burst (BFU-E) was highly enriched. However, FL could synergize with IL-3 or GM-CSF in support of erythrocyte-containing mixed (E-Mix) colony by PB-derived CD34+c-kithigh or low cells in the presence of Epo. Replating of E-Mix colonies derived from CD34+c-kithigh cells supported by IL-3+Epo+SCF yielded more secondary colonies than those supported by IL-3+Epo or IL-3+Epo+FL. When PB-derived CD34+c-kitlow cells which represent a more immature population than CD34+c-kithigh cells were used as the target, number of secondary colonies supported by IL-3+Epo, IL-3+Epo+SCF or IL-3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL-3+Epo+FL than in that containing IL-3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.

Fms-like tyrosine kinase 3 ligand increases a lung DC subset with regulatory properties in allergic airway inflammation

Dendritic cell (DC) subsets display different functional roles in regulating immune responses and lead to various outcomes, including T(H)1 versus T(H)2 or regulatory versus immunologic responses. Administration of Fms-like tyrosine kinase 3 (Flt3) ligand prevents and reverses allergic airway inflammation and airway hyperresponsiveness in a mouse model. However, the underlying mechanisms are unclear. We characterized and examined the role of lung DC subsets in the therapeutic effect of Flt3 ligand. DCs were isolated from the lungs of ovalbumin (OVA)-sensitized and OVA-challenged mice treated with recombinant human Flt3 ligand. Two populations of CD11c+ cells labeled with fluorochrome-conjugated antibodies were sorted. The ability of the purified cells to stimulate T-cell proliferation and cytokine secretion patterns by different DC subsets was examined. Also, DCs were adoptively transferred in mice to examine their effect on pulmonary function. Two DC populations, CD11c(high)CD11b(low) and CD11c(low)CD11b(high), were identified in the lungs of naive and OVA-sensitized and OVA-challenged mice with and without treatment with Flt3 ligand. The expression levels of CD8alpha, B220, CD19, F4/80, MHC II, CCR7, CD40, programmed death ligand 1, programmed death ligand 2, CD80, and CD86 were distinctly different between the 2 DC populations, which supports the notion that CD11c(high)CD11b(low) and CD11c(low)CD11b(high) DCs potentially have regulatory and immunogenic properties, respectively. Administration of Flt3 ligand increased the DCs with regulatory potential in the lungs of antigen-sensitized mice, and CD11c(high)CD11b(low) DCs acquired a maximum degree of regulatory capacity after Flt3 ligand treatment. These data suggest that Flt3 ligand reverses airway hyperresponsiveness by regulating the function of lung DCs in a mouse model of allergic airway inflammation.

Efficient Production of Bioactive Recombinant Human Flt3 Ligand in E. coli

Flt3 ligand (FL) is an early-acting hematopoietic cytokine that stimulates the proliferation and differentiation of hematopoietic progenitor cells by activating its cognate receptor, Flt3. Recently, FL was shown to potently contribute to the development and expansion of antigen-presenting dendritic cells and CD34(+) natural killer cell progenitors in vivo. Here, we report a comprehensive method for the production of bioactive recombinant human FL (rhFL) in E. coli, suitable for structural, biophysical and physiological studies. A soluble form of human FL capable of binding to the Ftl3 receptor could be overexpressed in the E. coli strain Rosetta-gami(DE3) as inclusion bodies. We have established protocols for the efficient in vitro refolding and ensuing purification of rhFL to homogeneity (>95%), with yields approaching 5 mg of pure rhFL per liter of culture. The ability of rhFL to adopt a bioactive conformation was confirmed via a cell-proliferation assay and the activation of the Flt3 receptor in the human leukemic cell line, OCI-AML3.

Flt-3 Ligand (FL) Treatment Upregulates PDL2 and Downregulates CCR7 in Distinct Myeloid Dendritic Cell (DC) Populations in the Lungs of Asthmatic Mice

RationaleWe observed that Flt-3 ligand reversed airway hyperresponsiveness (AHR) in a mouse model of asthma. FL preferentially affects the development and differentiation of myeloid DCs. In this study, we examined the phenotype of lung myeloid dendritic cells and their responses to ovalbumin (OVA) sensitization and FL treatment.MethodsA mouse model of asthma was established by administration of OVA and confirmed by AHR to methacholine. Sensitized and challenged mice were given either recombinant human FL or PBS. Non-sensitized mice received PBS as controls. CD11c+ lung DCs sorted by MACS were further labeled with CD11c, CD11b, CD8, PDL1, PDL2, and CCR7 antibodies conjugated with fluorochrome. FACS was used to identify phenotypic myeloid DC populations. The expression levels of cell surface markers were analyzed using flow cytometry.ResultsThree distinct lung myeloid DC populations, CD11c+CD11blow, CD11chiCD11bint, and CD11c+CD11bhi were identified. CD11chiCD11bint cells accounted for the majority of the lung myeloid DCs in non-sensitized condition. OVA sensitization significantly increased the proportion of CD11c+CD11bhi cells and decreased CD11chiCD11bint cells but FL treatment reversed the trend. CD11chiCD11bint cells had a higher expression of CD8, PDL1, and PDL2 as compared to CD11c+CD11bhi cells. CCR7 expression was higher in CD11c+CD11bhi cells. PDL2 was upregulated after sensitization and was further unregulated after FL treatment in CD11chiCD11bint cell population. CCR7 was upregulated after sensitization and downregulated after FL treatment in CD11c+CD11bhi cells.ConclusionsCD11chiCD11bint and CD11chiCD11bint DCs may be responsible for immune tolerance and immune response, respectively. FL reverses AHR in asthmatic mice possibly through upregulating PDL2 and downregulating CCR7 on DCs. RationaleWe observed that Flt-3 ligand reversed airway hyperresponsiveness (AHR) in a mouse model of asthma. FL preferentially affects the development and differentiation of myeloid DCs. In this study, we examined the phenotype of lung myeloid dendritic cells and their responses to ovalbumin (OVA) sensitization and FL treatment. We observed that Flt-3 ligand reversed airway hyperresponsiveness (AHR) in a mouse model of asthma. FL preferentially affects the development and differentiation of myeloid DCs. In this study, we examined the phenotype of lung myeloid dendritic cells and their responses to ovalbumin (OVA) sensitization and FL treatment. MethodsA mouse model of asthma was established by administration of OVA and confirmed by AHR to methacholine. Sensitized and challenged mice were given either recombinant human FL or PBS. Non-sensitized mice received PBS as controls. CD11c+ lung DCs sorted by MACS were further labeled with CD11c, CD11b, CD8, PDL1, PDL2, and CCR7 antibodies conjugated with fluorochrome. FACS was used to identify phenotypic myeloid DC populations. The expression levels of cell surface markers were analyzed using flow cytometry. A mouse model of asthma was established by administration of OVA and confirmed by AHR to methacholine. Sensitized and challenged mice were given either recombinant human FL or PBS. Non-sensitized mice received PBS as controls. CD11c+ lung DCs sorted by MACS were further labeled with CD11c, CD11b, CD8, PDL1, PDL2, and CCR7 antibodies conjugated with fluorochrome. FACS was used to identify phenotypic myeloid DC populations. The expression levels of cell surface markers were analyzed using flow cytometry. ResultsThree distinct lung myeloid DC populations, CD11c+CD11blow, CD11chiCD11bint, and CD11c+CD11bhi were identified. CD11chiCD11bint cells accounted for the majority of the lung myeloid DCs in non-sensitized condition. OVA sensitization significantly increased the proportion of CD11c+CD11bhi cells and decreased CD11chiCD11bint cells but FL treatment reversed the trend. CD11chiCD11bint cells had a higher expression of CD8, PDL1, and PDL2 as compared to CD11c+CD11bhi cells. CCR7 expression was higher in CD11c+CD11bhi cells. PDL2 was upregulated after sensitization and was further unregulated after FL treatment in CD11chiCD11bint cell population. CCR7 was upregulated after sensitization and downregulated after FL treatment in CD11c+CD11bhi cells. Three distinct lung myeloid DC populations, CD11c+CD11blow, CD11chiCD11bint, and CD11c+CD11bhi were identified. CD11chiCD11bint cells accounted for the majority of the lung myeloid DCs in non-sensitized condition. OVA sensitization significantly increased the proportion of CD11c+CD11bhi cells and decreased CD11chiCD11bint cells but FL treatment reversed the trend. CD11chiCD11bint cells had a higher expression of CD8, PDL1, and PDL2 as compared to CD11c+CD11bhi cells. CCR7 expression was higher in CD11c+CD11bhi cells. PDL2 was upregulated after sensitization and was further unregulated after FL treatment in CD11chiCD11bint cell population. CCR7 was upregulated after sensitization and downregulated after FL treatment in CD11c+CD11bhi cells. ConclusionsCD11chiCD11bint and CD11chiCD11bint DCs may be responsible for immune tolerance and immune response, respectively. FL reverses AHR in asthmatic mice possibly through upregulating PDL2 and downregulating CCR7 on DCs. CD11chiCD11bint and CD11chiCD11bint DCs may be responsible for immune tolerance and immune response, respectively. FL reverses AHR in asthmatic mice possibly through upregulating PDL2 and downregulating CCR7 on DCs.

Generation and characterization of an immunogenic dendritic cell population

Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c + cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-γ-producing CD4 + T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.