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The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis–Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein–protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1–A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

Chimeric antigen receptor (CAR) T-cell therapy has been widely successful in the treatment of B-cell malignancies, including B-cell lymphoma, mantle cell lymphoma, and multiple myeloma; and three generations of CAR designs have led to effective FDA approved therapeutics. Traditionally, CAR antigen specificity is derived from a monoclonal antibody where the variable heavy (VH) and variable light (VL) chains are connected by a peptide linker to form a single-chain variable fragment (scFv). While this provides a level of antigen specificity parallel to that of an antibody and has shown great success in the clinic, this design is not universally successful. For instance, issues of stability, immunogenicity, and antigen escape hinder the translational application of some CARs. As an alternative, natural receptor- or ligand-based designs may prove advantageous in some circumstances compared to scFv-based designs. Herein, the advantages and disadvantages of scFv-based and natural receptor- or ligand-based CAR designs are discussed. In addition, several translational aspects of natural receptor- and ligand-based CAR approaches that are being investigated in preclinical and clinical studies will be examined.

The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI) and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

BACKGROUND: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. OBJECTIVES: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. METHODS: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. RESULTS: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. CONCLUSIONS: These improved structures yield a more confident model for next generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels and lower immunogenicity.

Adoptive therapy with ex vivo–expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27 + CD28 + T cells vs 27.4% in control cultures; P

A difficulty in the field of gene therapy is the need to increase the susceptibility of hematopoietic stem cells (HSCs) to ex vivo genetic manipulation. To overcome this obstacle a high-throughput screen was performed to identify compounds that could enhance the transduction of target cells by lentiviral vectors. Of the 1280 compounds initially screened using the myeloid-erythroid-leukemic K562 cell line, 30 were identified as possible enhancers of viral transduction. Among the positive hits were known enhancers of transduction (camptothecin, etoposide and taxol), as well as the previously unidentified phorbol 12-myristate 13-acetate (PMA). The percentage of green fluorescent protein (GFP)-positive-expressing K562 cells was increased more than fourfold in the presence of PMA. In addition, the transduction of K562 cells with a lentiviral vector encoding fVIII was four times greater in the presence of PMA as determined by an increase in the levels of provirus in genetically modified cells. PMA did not enhance viral transduction of all cell types (for example, sca-1(+) mouse hematopoietic cells) but did enhance viral transduction of human bone marrow-derived CD34(+) cells. Notably, the percentage of GFP-positive CD34(+) cells was increased from 7% in the absence of PMA to greater than 22% in the presence of 1 nM PMA. PMA did not affect colony formation of CD34(+) cells or the expression of the hematopoietic markers CD34 and CD45. These data demonstrate that high-throughput screening can be used to identify compounds that increase the transduction efficiency of lentiviral vectors, identifying PMA as a potential enhancer of lentiviral HSC transduction.

We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha (EF1α), cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

Gene therapy has promised clinical benefit to those suffering with hemophilia A, but this benefit has not yet been realized. However, during the past two decades, basic and applied gene therapy research has progressed and the goal of gene therapy for hemophilia A is once again in our sights. The hemophilia A patient population suffers from a disease that requires invasive, lifelong management, is exorbitantly expensive to treat, has geographically limited treatment access and can become untreatable due to immune reactions to the treatment product. Subsequent to the cloning of the factor VIII gene and cDNA in the early 1980s, academic and commercial research laboratories began to pursue gene transfer-based therapies to supplement or supplant the available protein replacement therapy. However, to date, clinical trials for gene therapy of hemophilia A have been unsuccessful. Three trials have been conducted with each having tested a different gene-transfer strategy and each demonstrating that there is a considerable barrier to achieving sustained expression of therapeutic amounts of factor VIII. Recent progress has been made in gene-transfer technology and, relevant to hemophilia A, towards increasing the biosynthetic efficiency of factor VIII. These advances are now being combined to develop novel strategies to treat and possibly cure hemophilia A.

INTRODUCTION: Conventional treatment strategies for high-grade gliomas have been uniformly dismal. We have previously shown that both primary and Temozolomide (TMZ)-resistant glioblastoma (GBM) cell lines upregulate stress-associated NKG2D ligands (NKG2DL) during the first several hours following exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapy, particularly γδ T cells that directly recognize these stress-associated antigens. Using a human/mouse patient-derived tumor xenograft (PDTX) model, we combined TMZ chemotherapy and TMZ-resistant ex vivo expanded/activated γδ T cells as Drug Resistant Immunotherapy (DRI). Drug resistance in this example is conferred by O-6-methylguanine-DNA-methyltransferase (MGMT) gene transfer, thereby enabling cytotoxic lymphocyte function in a chemotherapy-rich environment when the tumor is maximally stressed. METHODS: A total of five GBM PDTX were examined, three parent (X12P, X22P, X59P) and two TMZ-resistant (X12T, X22T). Tumor NKG2DL expression and cytotoxicity of DRI were assessed using flow cytometry with cultured human astrocytes as controls. Intracranial (IC) glioma xenografts were established using either an unmodified (P) or a TMZ resistant clone (T) of human GBM explants passaged exclusively in immunodeficient mice. Tumor-bearing mice received intraperitoneal 60mg/kg TMZ on days 6, 8,13, and 15 and received IC injection of 1.5 x 106 DRI 4 hours following TMZ injection. Control mice received DRI T cells alone, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis. RESULTS: All xenografts were found to constitutionally express NKG2DL which were upregulated upon exposure to TMZ. DRI T cells were cytotoxic to all tumors in vitro, showing 65%-80% specific lysis at an E:T ratio of 20:1 with no evidence of toxicity against cultured human astrocytes. Median survival (MS) for all groups of untreated mice was approximately 25 days, and γδ T cell therapy alone did not improve survival in the absence of TMZ. For the parent tumors, TMZ therapy significantly improved MS over untreated controls for both X12P and X59P (29 vs. 59 days and 20 vs 51 days respectively, p =0.0001) and eliminated tumors in X22P. DRI γδ T cells + TMZ significantly increased median survival additionally over TMZ alone with 80% of animals in both X12P and X59P surviving long-term (p=0.0001 and 0.05 respectively). DRI had no clear effect over TMZ for X22T (p=0.46), however, for X12T, DRI + TMZ significantly increased median survival from 22 to 38 days (72.7%) over TMZ alone (p = 0.04). CONCLUSIONS: Combined TMZ chemotherapy and drug-resistance modified γδ T cell therapy can produce a significant increase in time to progression and improvement in median and overall survival for both primary and TMZ-resistant GBM using a strategy is readily adaptable to the clinical setting. Citation Format: Samantha B. Langford, Harold T. Spencer, Anindya Dasgupta, George Y. Gillespie, Kathryn Sutton, Larisa Pereboeva, Lawrence S. Lamb. Improved outcomes with drug-resistant immunotherapy in a human xenograft model of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2017-1638

INTRODUCTION: Conventional treatment strategies for Temozolomide (TMZ)-resistant high-grade gliomas have been uniformly dismal. We have shown that TMZ-resistant tumors up-regulate stress-associated NKG2D ligands (NKG2DL) during the first hours after exposure to TMZ, thereby creating an opportunity for NKG2DL-directed cell therapies. We report improved survival using a combination of TMZ chemotherapy and gene modified TMZ-resistant γΔ T cells which we term Drug Resistant Immunotherapy (DRI). TMZ resistance is conferred by lentivector transfer of methylguanine methyltransferase (MGMT) enabling cytotoxic lymphocyte function during high-dose chemotherapy.METHODS: Tumor NKG2DL expression and DRI cytotoxicity were assessed by flow cytometry. Intracranial (IC) glioma xenografts were established on Day 0 with either unmodified (P) or a TMZ resistant clone (T) of human primary GBM-X12 passaged exclusively in immunodeficient mice. Tumor-bearing mice were treated with intraperitoneal TMZ 60mg/kg on days 6, 8,13, and 15 followed by IC injection of 1.5 × 106 DRI 4h after each TMZ dose × 2 weeks. Control mice received non-modified cells, TMZ, or no therapy. Survival was assessed using Kaplan-Meier analysis.RESULTS: Both GBM-X12P and X12T express NKG2DL MIC-A, MIC-B, and ULBP-4. DRI cells killed both tumors in vitro at approximately 80% at an effector: target ratio of 20:1 with no toxicity against cultured human astrocytes. Cell therapy alone did not improve survival beyond that of untreated mice. For the parent tumor X12P (Figure 1), TMZ therapy significantly improved median survival over untreated controls (29 vs. 59 days, p < 0.0001), and addition of DRI increased median survival to 120 days with 80% long term survivors. For the TMZ-resistant X12T (Figure 2), TMZ alone did not improve survival over untreated controls (22 vs. 27 days p = 0.4827). Addition of DRI significantly increased median survival from 22 days to 38 days (p = 0.0004) with 10% of animals surviving to day +119. View Large Image | Download PowerPoint Slide View Large Image | Download PowerPoint SlideCONCLUSIONS: This is the first report of combined TMZ chemotherapy and drug-resistance modified γΔ T cell therapy resulting in a significant increase in time to progression and improvement in median and overall survival in immunodeficient mice bearing otherwise impervious human primary xenograft tumors using a strategy is readily adaptable to the clinical setting

Conventional treatment strategies for high-grade primary brain tumors have failed to consistently extend median survival beyond two years. Using a human/mouse xenograft model, we report improved survival using a combination of Temozolomide (TMZ) chemotherapy with a Drug Resistant Cellular Immunotherapy (DRI) consisting of activated MGMT-transduced γδ T cells and NK cells. MGMT genetic engineering enables cytotoxic lymphocyte function in a chemotherapy-rich environment at a time when the tumor is maximally stressed. Intracranial (IC) glioma xenografts were established using either unmodified (P) or a TMZ-resistant clone (T) of human GBM-X12 primary explants propagated in mice. Tumor-bearing mice were treated i.p. with 60mg/kg TMZ on days 6, 8,13, and 15 post-tumor placement and received IC injection of 1 x 106 MGMT-modified DRI on post-injection days 7 and 14. Control mice received non-modified cells, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis. Both GBM-X12P and X12T express stress antigens MIC-A, MIC-B, and ULBP-4 with only ULBP-4 slightly upregulated on X12T upon exposure to TMZ. Both tumors were killed by expanded/activated γδ T cells in vitro at approximately 80% at an effector : target ratio of 20:1. Cell therapy alone did not improve survival beyond that of untreated mice for either tumor. For the unmodified tumor X12P, both TMZ therapy and TMZ + DRI significantly improved survival over untreated controls (p < 0.001), and the combined therapy increased median survival from 57 to 75 days over TMZ alone. Combined therapy with TMZ + DRI also improved survival for X12T (p = 0.0147) over untreated controls, and showed a marginal improvement over TMZ alone (p = 0.0966) and improvement in median survival from 22 to 25 days. In summary, the combination of chemotherapy-induced stress antigen expression and targeted DRI significantly increases time to progression and improves survival in tumor-bearing immunodeficient mice.