The BTLA-HVEM axis restricts CAR T cell efficacy in cancer.

The efficacy of T cell-based immunotherapies is limited by immunosuppressive pressures in the tumor microenvironment. Here we show a predominant role for the interaction between BTLA on effector T cells and HVEM (TNFRSF14) on immunosuppressive tumor microenvironment cells, namely regulatory T cells. High BTLA expression in chimeric antigen receptor (CAR) T cells correlated with poor clinical response to treatment. Therefore, we deleted BTLA in CAR T cells and show improved tumor control and persistence in models of lymphoma and solid malignancies. Mechanistically, BTLA inhibits CAR T cells via recruitment of tyrosine phosphatases SHP-1 and SHP-2, upon trans engagement with HVEM. BTLA knockout thus promotes CAR signaling and subsequently enhances effector function. Overall, these data indicate that the BTLA-HVEM axis is a crucial immune checkpoint in CAR T cell immunotherapy and warrants the use of strategies to overcome this barrier.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Aryl hydrocarbon receptor inhibition promotes hematolymphoid development from human pluripotent stem cells.

The aryl hydrocarbon receptor (AHR) plays an important physiological role in hematopoiesis. AHR is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and inhibition of AHR results in a marked expansion of human umbilical cord blood-derived HSPCs following cytokine stimulation. It is unknown whether AHR also contributes earlier in human hematopoietic development. To model hematopoiesis, human embryonic stem cells (hESCs) were allowed to differentiate in defined conditions in the presence of the AHR antagonist StemReginin-1 (SR-1) or the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We demonstrate a significant increase in CD34+CD31+ hematoendothelial cells in SR-1-treated hESCs, as well as a twofold expansion of CD34+CD45+ hematopoietic progenitor cells. Hematopoietic progenitor cells were also significantly increased by SR-1 as quantified by standard hematopoietic colony-forming assays. Using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-engineered hESC-RUNX1c-tdTomato reporter cell line with AHR deletion, we further demonstrate a marked enhancement of hematopoietic differentiation relative to wild-type hESCs. We also evaluated whether AHR antagonism could promote innate lymphoid cell differentiation from hESCs. SR-1 increased conventional natural killer (cNK) cell differentiation, whereas TCDD treatment blocked cNK development and supported group 3 innate lymphoid cell (ILC3) differentiation. Collectively, these results demonstrate that AHR regulates early human hematolymphoid cell development and may be targeted to enhance production of specific cell populations derived from human pluripotent stem cells.

Single Cell Resolution of Human Hematoendothelial Cells Defines Transcriptional Signatures of Hemogenic Endothelium.

Endothelial-to-hematopoietic transition (EHT) is an important stage in definitive hematopoietic development. However, the genetic mechanisms underlying human EHT remain poorly characterized. We performed single cell RNA-seq using 55 hemogenic endothelial cells (HECs: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c+ ), 47 vascular endothelial cells without hematopoietic potential (non-HE: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c- ), and 35 hematopoietic progenitor cells (HPCs: CD34+ CD43+ RUNX1c+ ) derived from human embryonic stem cells (hESCs). HE and HP were enriched in genes implicated in hemogenic endothelial transcriptional networks, such as ERG, GATA2, and FLI. We found transcriptional overlap between individual HECs and HPCs; however, these populations were distinct from non-HE. Further analysis revealed novel biomarkers for human HEC/HPCs, including TIMP3, ESAM, RHOJ, and DLL4. Collectively, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE are more heterogeneous and transcriptionally distinct. Our findings provide a novel strategy to test new genetic targets and optimize the production of definitive hematopoietic cells from human pluripotent stem cells. Stem Cells 2018;36:206-217.

Advances in the role of the aryl hydrocarbon receptor to regulate early hematopoietic development.

PURPOSE OF REVIEW: We summarize current advances to define the role the aryl hydrocarbon receptor (AHR) plays in mammalian hematopoiesis. We emphasize approaches to modulate AHR throughout human hematopoietic development in vitro to support the production of clinically relevant blood products suitable for patient care. RECENT FINDINGS: Initial data demonstrate that both pharmacologic AHR inhibition and genetic deletion from human pluripotent stem cells provide useful strategies to enhance the yield of hematopoietic stem and progenitor cells. AHR hyperactivation following the induction of CD34 megakaryocyte-erythroid progenitors skews developed toward erythroid lineages, whereas AHR inhibition supports platelet production. At the level of lymphoid specification, AHR inhibition enhances the proliferation and differentiation of functional human natural killer cells, whereas hyperactivation leads to production of Group 3 innate lymphoid cells and provides a novel platform for studying human innate lymphoid cell development. SUMMARY: Modulation of AHR in human hematopoietic cells in vitro is a promising tool to mediate development of terminal hematopoietic cell populations with significant clinical implications to generate cells suitable for antitumor immunotherapy and bone marrow transplantation.

Pluripotent stem cell applications for regenerative medicine.

PURPOSE OF REVIEW: In this review, we summarize the current status of clinical trials using therapeutic cells produced from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We also discuss combined cell and gene therapy via correction of defined mutations in human pluripotent stem cells and provide commentary on key obstacles facing widescale clinical adoption of pluripotent stem cell-based therapy. RECENT FINDINGS: Initial data suggest that hESC/hiPSC-derived cell products used for retinal repair and spinal cord injury are safe for human use. Early-stage studies for treatment of cardiac injury and diabetes are also in progress. However, there remain key concerns regarding the safety and efficacy of these cells that need to be addressed in additional well designed clinical trials. Advances using the clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system offer an improved tool for more rapid and on-target gene correction of genetic diseases. Combined gene and cell therapy using human pluripotent stem cells may provide an additional curative approach for disabling or lethal genetic and degenerative diseases wherein there are currently limited therapeutic opportunities. SUMMARY: Human pluripotent stem cells are emerging as a promising tool to produce cells and tissues suitable for regenerative therapy for a variety of genetic and degenerative diseases.

Progress and Pitfalls of Chimeric Antigen Receptor T Cell Immunotherapy against T Cell Malignancies.

Chimeric antigen receptor T cell (CAR-T) immunotherapy has revolutionized the treatment of relapsed and refractory B cell-derived hematologic malignancies. Currently, there are 6 Food and Drug Administration-approved commercial CAR-T products that target antigens exclusively expressed on malignant B cells or plasma cells. However, concurrent advancement for patients with rarer and more aggressive T cell-derived hematologic malignancies have not yet been achieved. CAR-T immunotherapies are uniquely limited by challenges related to CAR-T product manufacturing and intrinsic tumor biology. In this review tailored for practicing clinician-scientists, we discuss the major barriers of CAR-T implementation against T cell-derived neoplasms and highlight specific scientific advancements poised to circumvent these obstacles. We summarize salient early-stage clinical trials implementing novel CAR-T immunotherapies specifically for patients with relapsed and/or refractory T cell neoplasms. Finally, we highlight novel manufacturing and treatment strategies that are poised to have a meaningful future clinical impact.

Hemangiosarcoma Cells Promote Conserved Host-derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. SIGNIFICANCE: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Advances and Personalized Approaches in the Frontline Treatment of T-Cell Lymphomas.

Peripheral T-cell lymphomas (PTCLs) are a rare and heterogenous subset of non-Hodgkin lymphoma characterized by an aggressive clinical course. Historically, the treatment of PTCLs have been analogous to that of aggressive B-cell lymphomas; however, it has been well-established that overall responses and complete remission rates are far inferior using near-identical chemotherapy strategies. Recently, there has been a plethora of newer agents designed to target distinguishing cellular and molecular features of specific PTCL subtypes. These agents have been proven to yield superior anti-lymphoma responses and, in some cases, overall survival in the relapsed, refractory, and frontline treatment setting. In this review, we will summarize and highlight the most influential clinical trials leading to the Food and Drug Administration (FDA) approval of several novel therapeutic agents against PTCL, with an emphasis on emerging studies and strategies to expand their potential use in the frontline treatment setting.

Dynamic adhesion of umbilical cord blood endothelial progenitor cells under laminar shear stress.

Late outgrowth endothelial progenitor cells (EPCs) represent a promising cell source for rapid reendothelialization of damaged vasculature after expansion ex vivo and injection into the bloodstream. We characterized the dynamic adhesion of umbilical-cord-blood-derived EPCs (CB-EPCs) to surfaces coated with fibronectin. CB-EPC solution density affected the number of adherent cells and larger cells preferentially adhered at lower cell densities. The number of adherent cells varied with shear stress, with the maximum number of adherent cells and the shear stress at maximum adhesion depending upon fluid viscosity. CB-EPCs underwent limited rolling, transiently tethering for short distances before firm arrest. Immediately before arrest, the instantaneous velocity decreased independent of shear stress. A dimensional analysis indicated that adhesion was a function of the net force on the cells, the ratio of cell diffusion to sliding speed, and molecular diffusivity. Adhesion was not limited by the settling rate and was highly specific to α(5)ß(1) integrin. Total internal reflection fluorescence microscopy showed that CB-EPCs produced multiple contacts of α(5)ß(1) with the surface and the contact area grew during the first 20 min of attachment. These results demonstrate that CB-EPC adhesion from blood can occur under physiological levels of shear stress.

Intra-articular treatment of knee osteoarthritis: from anti-inflammatories to products of regenerative medicine.

OBJECTIVES: Knee osteoarthritis (OA) is a debilitating condition that may ultimately require total knee arthroplasty (TKA). Non-operative treatments are bracing, oral analgesics, physical therapy, and intra-articular knee injection (IAKI). The objective of this paper is to provide a systematic literature review regarding intra-articular treatment of knee OA and insight into promising new products of regenerative medicine that may eventually have a substantial effect on treatment. METHODS: A literature search was executed using Medline, Cochrane, and Embase with keywords "knee osteoarthritis" and "injection." Specifically, 45 articles that discussed intra-articular knee injection using corticosteroids, hyaluronic acid, analgesics, local anesthetics, and newer products of regenerative medicine, such as platelet-rich plasma (PRP) and mesenchymal stem cells (MSC), were analyzed. Of these, eleven were level 1, three were level 2, twelve were level 3, two were level 4, and seventeen were level 5 evidence. Papers included animal models. RESULTS: Local anesthetics have potential side effects and may only be effective for a few hours. Morphine and ketorolac may provide significant pain relief for 24 hours. Corticosteroids may give patients weeks to months of effective analgesia, but complications may occur, such as systemic hyperglycemia, septic arthritis, and joint degradation . Hyaluronic acid is a natural component of synovial fluid, but efficacy with respect to analgesia is controversial. Platelet-rich plasma formulations, autologous conditioned serum, autologous protein solution, and mesenchymal stem cell injections contain anti-inflammatory molecules and have been proposed to attenuate joint destruction or potentially remodel the joint. CONCLUSIONS: Currently, knee OA treatment does not address the progressively inflammatory environment of the joint. More investigation is needed regarding products of regenerative medicine, but they may ultimately have profound implications in the way knee OA is managed.

Characterization of umbilical cord blood-derived late outgrowth endothelial progenitor cells exposed to laminar shear stress.

Endothelial progenitor cells isolated from umbilical cord blood (CB-EPCs) represent a promising source of endothelial cells for synthetic vascular grafts and tissue-engineered blood vessels since they are readily attainable, can be easily isolated, and possess a high proliferation potential. The objective of this study was to compare the functional behavior of late outgrowth CB-EPCs with human aortic endothelial cells (HAECs). CB-EPCs and HAECs were cultured on either smooth muscle cells in a coculture model of a tissue-engineered blood vessels or fibronectin adsorbed to Teflon-AF-coated glass slides. Late outgrowth CB-EPCs expressed endothelial cell-specific markers and were negative for the monocytic marker CD14. CB-EPCs have higher proliferation rates than HAECs, but are slightly smaller in size. CB-EPCs remained adherent under supraphysiological shear stresses, oriented and elongated in the direction of flow, and expressed similar numbers of alpha(5)beta(1) and alpha(v)beta(3) integrins and antithrombotic genes compared to HAECs. There were some differences in mRNA levels of E-selectin and vascular cell adhesion molecule 1 between CB-EPCs and HAECs; however, protein levels were similar on the two cell types, and CB-EPCs did not support adhesion of monocytes in the absence of tumor necrosis factor-alpha stimulation. Although CB-EPCs expressed significantly less endothelial nitric oxide synthase protein after exposure to flow than HAECs, nitric oxide levels induced by flow were not significantly different. These results suggest that late outgrowth CB-EPCs are functionally similar to HAECs under flow conditions and are a promising cell source for cardiovascular therapies.