Overcoming Resistance to Natural Killer Cell Based Immunotherapies for Solid Tumors

Despite advances in the diagnostic and therapeutic modalities, the prognosis of several solid tumor malignancies remains poor. Different factors associated with solid tumors including a varied genetic signature, complex molecular signaling pathways, defective cross talk between the tumor cells and immune cells, hypoxic and immunosuppressive effects of tumor microenvironment result in a treatment resistant and metastatic phenotype. Over the past several years, immunotherapy has emerged as an attractive therapeutic option against multiple malignancies. The unique ability of natural killer (NK) cells to target cancer cells without antigen specificity makes them an ideal candidate for use against solid tumors. However, the outcomes of adoptive NK cell infusions into patients with solid tumors have been disappointing. Extensive studies have been done to investigate different strategies to improve the NK cell function, trafficking and tumor targeting. Use of cytokines and cytokine analogs has been well described and utilized to enhance the proliferation, stimulation and persistence of NK cells. Other techniques like blocking the human leukocyte antigen-killer cell receptors (KIR) interactions with anti-KIR monoclonal antibodies, preventing CD16 receptor shedding, increasing the expression of activating NK cell receptors like NKG2D, and use of immunocytokines and immune checkpoint inhibitors can enhance NK cell mediated cytotoxicity. Using genetically modified NK cells with chimeric antigen receptors and bispecific and trispecific NK cell engagers, NK cells can be effectively redirected to the tumor cells improving their cytotoxic potential. In this review, we have described these strategies and highlighted the need to further optimize these strategies to improve the clinical outcome of NK cell based immunotherapy against solid tumors

A humanized orthotopic mouse model for preclinical evaluation of immunotherapy in Ewing sarcoma

The advent of novel cancer immunotherapy approaches is revolutionizing the treatment for cancer. Current small animal models for most cancers are syngeneic or genetically engineered mouse models or xenograft models based on immunodeficient mouse strains. These models have been limited in evaluating immunotherapy regimens due to the lack of functional human immune system. Development of animal models for bone cancer faces another challenge in the accessibility of tumor engraftment sites. Here, we describe a protocol to develop an orthotopic humanized mouse model for a bone and soft tissue sarcoma, Ewing sarcoma, by transplanting fresh human cord blood CD34+ hematopoietic stem cells into young NSG-SGM3 mice combined with subsequent Ewing sarcoma patient derived cell engraftment in the tibia of the humanized mice. We demonstrated early and robust reconstitution of human CD45+ leukocytes including T cells, B cells, natural killer cells and monocytes. Ewing sarcoma xenograft tumors successfully orthotopically engrafted in the humanized mice with minimal invasive procedures. We validated the translational utility of this orthotopic humanized model by evaluating the safety and efficacy of an immunotherapy antibody, magrolimab. Treatment with magrolimab induces CD47 blockade resulting in significantly decreased primary tumor growth, decreased lung metastasis and prolonged animal survival in the established humanized model. Furthermore, the humanized model recapitulated the dose dependent toxicity associated with the CD47 blockade as observed in patients in clinical trials. In conclusion, this orthotopic humanized mouse model of Ewing sarcoma represents an improved platform for evaluating immunotherapy in bone and soft tissue sarcoma, such as Ewing sarcoma. With careful design and optimization, this model is generalizable for other bone malignancies

Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine

Stem cells are unspecialized cells capable of renewing themselves and giving rise to differentiated and specialized cell subtypes. There are two general categories of stem cells, i.e., pluripotent stem cells capable of differentiation into any cell type in the human body and multipotent adult stem cells maintaining tissue homeostasis in postnatal life. Investigations in both these categories of stem cells have expanded our knowledge on human organogenesis and tissue regeneration and have suggested potential therapeutic functions of stem cells in regenerative medicine. The advent of induced pluripotent stem cell (iPSC) technology a decade ago further revolutionized stem cell biology and has given rise to the translation of stem cell-based therapies. This chapter will summarize some of the exciting progress and challenges in the applications of iPSC-derived stem cells and adult stem cells and the potential of translational and clinical research of these stem cells in regenerative medicine

Improved Survival After Acute Graft-Versus-Host Disease Diagnosis in the Modern Era

Acute graft-versus-host disease remains a major threat to a successful outcome after allogeneic hematopoietic cell transplantation. While improvements in treatment and supportive care have occurred, it is unknown whether these advances have resulted in improved outcome specifically among those diagnosed with acute graft-versus-host disease. We examined outcome following diagnosis of grade II-IV acute graft-versus-host disease according to time period, and explored effects according to original graft-versus-host disease prophylaxis regimen and maximum overall grade of acute graft-versus-host disease. Between 1999 and 2012, 2,905 patients with acute myeloid leukemia (56%), acute lymphoblastic leukemia (30%) or myelodysplastic syndromes (14%) received a sibling (24%) or unrelated donor (76%) blood (66%) or marrow (34%) transplant and developed grade II-IV acute graft-versus-host disease (n=497 for 1999–2001, n=962 for 2002–2005, n=1,446 for 2006–2010). The median (range) follow-up was 144 (4–174), 97 (4–147) and 60 (8–99) months for 1999–2001, 2002–2005, and 2006–2010, respectively. Among the cohort with grade II-IV acute graft-versus-host disease, there was a decrease in the proportion of grade III-IV disease over time with 56%, 47%, and 37% for 1999–2001, 2002–2005, and 2006–2012, respectively (P\u3c0.001). Considering the total study population, univariate analysis demonstrated significant improvements in overall survival and treatment-related mortality over time, and deaths from organ failure and infection declined. On multivariate analysis, significant improvements in overall survival (P=0.003) and treatment-related mortality (P=0.008) were only noted among those originally treated with tacrolimus-based graft-versus-host disease prophylaxis, and these effects were most apparent among those with overall grade II acute graft-versus-host disease. In conclusion, survival has improved over time for tacrolimus-treated transplant recipients with acute graft-versus-host disease

GvHD After Umbilical Cord Blood Transplantation for Acute Leukemia: an Analysis of Risk Factors and Effect on Outcomes

Using the Center for International Blood and Marrow Transplant Research (CIBMTR) registry, we analyzed 1404 umbilical cord bloodtransplantation (UCBT) patients (single (\u3c18 years)=810, double (⩾18 years)=594) with acute leukemia to define the incidence of acuteGvHD (aGvHD) and chronic GvHD (cGvHD), analyze clinical risk factors and investigate outcomes. After single UCBT, 100-day incidence of grade II-IV aGvHD was 39% (95% confidence interval (CI), 36-43%), grade III-IV aGvHD was 18% (95% CI, 15-20%) and 1-year cGvHD was 27% (95% CI, 24-30%). After double UCBT, 100-day incidence of grade II-IV aGvHD was 45% (95% CI, 41-49%), grade III-IV aGvHD was 22% (95% CI, 19-26%) and 1-year cGvHD was 26% (95% CI, 22-29%). For single UCBT, multivariate analysis showed that absence of antithymocyte globulin (ATG) was associated with aGvHD, whereas prior aGvHD was associated with cGvHD. For double UCBT, absence of ATG and myeloablative conditioning were associated with aGvHD, whereas prior aGvHD predicted for cGvHD. Grade III-IV aGvHD led to worse survival, whereas cGvHD had no significant effect on disease-free or overall survival. GvHD is prevalent after UCBT with severe aGvHD leading to higher mortality. Future research in UCBT should prioritize prevention of GvHD

Efficacy of Human Placental-Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal-epidermal junction (DEJ). We recently demonstrated that human cord blood-derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1(-/-) ) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental-derived stem cells (HPDSCs), currently being used as human leukocyte antigen-independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full-term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1(-/-) mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short-term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1(-/-) mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti-C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018

Inflammation-mediated fibroblast activation and immune dysregulation in collagen VII-deficient skin

Inflammation is known to play a critical role in all stages of tumorigenesis; however, less is known about how it predisposes the tissue microenvironment preceding tumor formation. Recessive dystrophic epidermolysis bullosa (RDEB), a skin-blistering disease secondary to COL7A1 mutations and associated with chronic wounding, inflammation, fibrosis, and cutaneous squamous cell carcinoma (cSCC), models this dynamic. Here, we used single-cell RNA sequencing (scRNAseq) to analyze gene expression patterns in skin cells from a mouse model of RDEB. We uncovered a complex landscape within the RDEB dermal microenvironment that exhibited altered metabolism, enhanced angiogenesis, hyperproliferative keratinocytes, infiltration and activation of immune cell populations, and inflammatory fibroblast priming. We demonstrated the presence of activated neutrophil and Langerhans cell subpopulations and elevated expression of PD-1 and PD-L1 in T cells and antigen-presenting cells, respectively. Unsupervised clustering within the fibroblast population further revealed two differentiation pathways in RDEB fibroblasts, one toward myofibroblasts and the other toward a phenotype that shares the characteristics of inflammatory fibroblast subsets in other inflammatory diseases as well as the IL-1-induced inflammatory cancer-associated fibroblasts (iCAFs) reported in various cancer types. Quantitation of inflammatory cytokines indicated dynamic waves of IL-1α, TGF-β1, TNF, IL-6, and IFN-γ concentrations, along with dermal NF-κB activation preceding JAK/STAT signaling. We further demonstrated the divergent and overlapping roles of these cytokines in inducing inflammatory phenotypes in RDEB patients as well as RDEB mouse-derived fibroblasts together with their healthy controls. In summary, our data have suggested a potential role of inflammation, driven by the chronic release of inflammatory cytokines such as IL-1, in creating an immune-suppressed dermal microenvironment that underlies RDEB disease progression

Ruxolitinib Significantly Enhances in Vitro Apoptosis in Hodgkin Lymphoma and Primary Mediastinal B-Cell Lymphoma and Survival in a Lymphoma Xenograft Murine Model

Hodgkin lymphoma (HL) and primary mediastinal B-cell lymphoma (PMBL) share similar molecular features by gene expression profiling. Frequent gains of chromosome 9p exhibit higher Janus Kinase 2 (JAK2) transcript levels with increased JAK2 activity, suggesting aberrant activity of JAK2 and STAT pathways. This signaling pathway alteration may in part play an important role in the pathogenesis and/or chemoradiotherapy resistance in HL and PMBL. Ruxolitinib is a potent and selective JAK1/JAK2 inhibitor, with activity against myeloproliferative neoplasms (MPNs) including those harboring the JAK2V617F mutation. We investigated the in vitro and in vivo efficacy of ruxolitinib and changes in downstream signaling pathways in HL and PMBL. We demonstrated that ruxolitinib significantly inhibited STAT signaling in both HL and PMBL with constitutively active JAK2 signaling. We also observed that ruxolitinib significantly induced in vitro anti-proliferative effects (p \u3c 0.05) and increased programmed cell death (p \u3c 0.05) against both HL and PMBL cells. Importantly, ruxolitinib significantly inhibited tumor progression by bioluminescence (p \u3c 0.05) and significantly improved survival in HL (p = 0.0001) and PMBL (p \u3c 0.0001) xenograft NSG mice. Taken altogether, these studies suggest that ruxolitinib may be a potential adjuvant targeted agent in the therapeutic approach in patients with high risk HL and PMBL

Graft-Versus-Host Disease in Recipients of Male Unrelated Donor Compared with Parous Female Sibling Donor Transplants

Optimal donor selection is critical for successful allogeneic hematopoietic cell transplantation (HCT). Donor sex and parity are well-established risk factors for graft-versus-host disease (GVHD), with male donors typically associated with lower rates of GVHD. Well-matched unrelated donors (URDs) have also been associated with increased risks of GVHD as compared with matched sibling donors. These observations raise the question of whether male URDs would lead to more (or less) favorable transplant outcomes as compared with parous female sibling donors. We used the Center for International Blood and Marrow Transplant Research registry to complete a retrospective cohort study in adults with acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome, who underwent T-cell replete HCT from these 2 donor types (parous female sibling or male URD) between 2000 and 2012. Primary outcomes included grade 2 to 4 acute GVHD (aGVHD), chronic GVHD (cGVHD), and overall survival. Secondary outcomes included disease-free survival, transplant-related mortality, and relapse. In 2813 recipients, patients receiving male URD transplants (n = 1921) had 1.6 times higher risk of grade 2 to 4 aGVHD (P \u3c .0001). For cGVHD, recipient sex was a significant factor, so donor/recipient pairs were evaluated. Female recipients of male URD grafts had a higher risk of cGVHD than those receiving parous female sibling grafts (relative risk [RR] = 1.43, P \u3c .0001), whereas male recipients had similar rates of cGVHD regardless of donor type (RR = 1.09, P = .23). Donor type did not significantly affect any other end point. We conclude that when available, parous female siblings are preferred over male URDs