CAR T-cell-mediated delivery of bispecific innate immune cell engagers for neuroblastoma.

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a.

Targeting GPC2 on Intraocular and CNS Metastatic Retinoblastomas with Local and Systemic Delivery of CAR T Cells.

Purpose: Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy.

Dietary vitamin D is a novel modulator of tumor engraftment through regulation of GC protein abundance.

The vitamin D binding protein, the GC protein, is a multifunctional protein that binds circulating vitamin D and also increases macrophage killing of tumor cells. Injecting exogenous GC protein concurrent with experimental tumor implant decreases tumor engraftment rate. Until now serum abundance of this protein was thought to be controlled by estrogen, glucocorticoids and inflammatory cytokines, but, not by vitamin D itself(1, 2).

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination.

Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants.

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta.

Background: Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.

Monocyte lineage-derived IL-6 does not affect chimeric antigen receptor T-cell function.

Background Aims: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated remarkable success in targeting B-cell malignancies but is often complicated by serious systemic toxicity in the form of cytokine release syndrome (CRS). CRS symptoms are primarily mediated by interleukin 6 (IL-6), and clinical management has focused on inhibition of IL-6 signaling. The cellular source and function of IL-6 in CRS remain unknown.

Hematopoietic derived cells do not contribute to osteogenesis as osteoblasts.

Despite years of extensive investigation, the cellular origin of heterotopic ossification (HO) has not been fully elucidated. We have previously shown that circulating bone marrow-derived osteoblast progenitor cells, characterized by the immunophenotype CD45-/CD44+/CXCR4+, contributed to the formation of heterotopic bone induced by bone morphogenetic protein (BMP)-2. In contrast, other reports have demonstrated the contribution of CD45+ hematopoietic derived cells to HO.

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies.

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

Unlabelled: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2.

Genomic and functional comparison of mesenchymal stromal cells prepared using two isolation methods.

Background Aims: Mesenchymal stromal cells (MSCs) have been applied to patients in cell therapy for various diseases. Recently, we introduced a novel MSC separation filter device which could yield approximately 2.5-fold more MSCs from bone marrow in a closed system compared with the conventional open density gradient centrifugation method.

Tendon progenitor cells in injured tendons have strong chondrogenic potential: the CD105-negative subpopulation induces chondrogenic degeneration.

To study the cellular mechanism of the tendon repair process, we used a mouse Achilles tendon injury model to focus on the cells recruited to the injured site. The cells isolated from injured tendon 1 week after the surgery and uninjured tendons contained the connective tissue progenitor populations as determined by colony-forming capacity, cell surface markers, and multipotency. When the injured tendon-derived progenitor cells (inTPCs) were transplanted into injured Achilles tendons, they were not only integrated in the regenerating area expressing tenogenic phenotype but also trans-differentiated into chondrogenic cells in the degenerative lesion that underwent ectopic endochondral ossification.

Delayed marrow infusion in mice enhances hematopoietic and osteopoietic engraftment by facilitating transient expansion of the osteoblastic niche.

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours.

IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation.

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation.

Megakaryocytes promote murine osteoblastic HSC niche expansion and stem cell engraftment after radioablative conditioning.

Successful hematopoietic stem cell (HSC) transplantation requires donor HSC engraftment within specialized bone marrow microenvironments known as HSC niches. We have previously reported a profound remodeling of the endosteal osteoblastic HSC niche after total body irradiation (TBI), defined as relocalization of surviving megakaryocytes to the niche site and marked expansion of endosteal osteoblasts. We now demonstrate that host megakaryocytes function critically in expansion of the endosteal niche after preparative radioablation and in the engraftment of donor HSC.

Transplanted murine long-term repopulating hematopoietic cells can differentiate to osteoblasts in the marrow stem cell niche.

Bone marrow transplantation (BMT) can give rise to donor-derived osteopoiesis in mice and humans; however, the source of this activity, whether a primitive osteoprogenitor or a transplantable marrow cell with dual hematopoietic and osteogenic potential, has eluded detection. To address this issue, we fractionated whole BM from mice according to cell surface immunophenotype and assayed the hematopoietic and osteopoietic potentials of the transplanted cells. Here, we show that a donor marrow cell capable of robust osteopoiesis possesses a surface phenotype of c-Kit(+) Lin(-) Sca-1(+) CD34(-/lo), identical to that of the long-term repopulating hematopoietic stem cell (LTR-HSC).

Improved isolation and expansion of bone marrow mesenchymal stromal cells using a novel marrow filter device.

Background Aims: Mesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and malignant transformation.

Transplanted bone marrow mononuclear cells and MSCs impart clinical benefit to children with osteogenesis imperfecta through different mechanisms.

Transplantation of whole bone marrow (BMT) as well as ex vivo-expanded mesenchymal stromal cells (MSCs) leads to striking clinical benefits in children with osteogenesis imperfecta (OI); however, the underlying mechanism of these cell therapies has not been elucidated. Here, we show that non-(plastic)-adherent bone marrow cells (NABMCs) are more potent osteoprogenitors than MSCs in mice. Translating these findings to the clinic, a T cell-depleted marrow mononuclear cell boost (> 99.

Cytokine-induced osteopoietic differentiation of transplanted marrow cells.

Transplantation of whole bone marrow (BMT) leads to engraftment of both osteoprogenitor cells and hematopoietic cells; however, the robust osteopoietic chimerism seen early after BMT decreases with time. Using our established murine model, we demonstrate that a post-BMT regimen of either granulocyte-colony stimulating factor, growth hormone, parathyroid hormone, or stem cell factor each stimulates greater donor osteoblast chimerism at 4 months posttransplantation than saline-treated controls and approximates the robust osteopoietic chimerism seen early after BMT; however, only growth hormone led to significantly more donor-derived osteocytes than controls. Importantly, there were no adverse hematologic consequences of the different treatments.

Osteopoietic engraftment after bone marrow transplantation: effect of inbred strain of mice.

Objective: Transplantable osteoprogenitors, as well as hematopoietic progenitors, reside in bone marrow. We previously reported the first clinical trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. Although the patients demonstrated striking clinical benefits after transplantation, measured osteopoietic engraftment was low and did not seem to be durable.

A strategy for single nucleotide polymorphism analysis of chimerism for somatic cell therapy.

Background Aims: Chimerism is an important outcome measure in hematopoietic cell transplantation as well as somatic cell therapy. Commonly used methods to estimate chimerism are restricted by either gender or inefficient sensitivity. In principle, real-time polymerase chain reaction (PCR)-based assays can be used to assess single nucleotide polymorphisms (SNP), which are a vast resource of molecular markers, and such assays demonstrate a substantially higher sensitivity (0.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation.

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche.

Donor cell-derived osteopoiesis originates from a self-renewing stem cell with a limited regenerative contribution after transplantation.

In principle, bone marrow transplantation should offer effective treatment for disorders originating from defects in mesenchymal stem cells. Results with the bone disease osteogenesis imperfecta support this hypothesis, although the rate of clinical improvement seen early after transplantation does not persist long term, raising questions as to the regenerative capacity of the donor-derived mesenchymal progenitors. We therefore studied the kinetics and histologic/anatomic pattern of osteopoietic engraftment after transplantation of GFP-expressing nonadherent marrow cells in mice.

Transplantable marrow osteoprogenitors engraft in discrete saturable sites in the marrow microenvironment.

Objective: Based on the recognition that marrow contains progenitors for bone as well as blood, we undertook the first trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. While we documented striking clinical benefit soon after transplantation, the measured level of osteopoietic engraftment was low. To improve the efficacy of BMT for bone disorders, we sought to gain insight into the cellular mechanism of engraftment of transplantable marrow osteoprogenitors.

Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel surface marker for the identification of MSCs.

Mesenchymal stromal cells (MSCs) have enormous potential for the regeneration of bone, cartilage, and other tissues derived from primitive mesoderm. Despite extensive research, there is still no single marker that reliably identifies MSCs within the bone marrow. Using immunocytochemistry and flow cytometry, we demonstrate here that the neural ganglioside GD2 is expressed by MSCs either newly isolated from bone marrow or expanded in tissue culture; this finding was supported by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showing expression of the mRNA for GD2 synthase, an essential enzyme for GD2 biosynthesis.