Value-Driven Analysis of New Paradigms in Space Architectures: An Ilities-Based Approach

Current commercial, civil, and military space architecture designs perform exquisitely and reliably. However, today’s architecture paradigms are also characterized by expensive launches, large and expensive high-performance spacecraft, long development cycles, and wide variations in ground architectures. While current assets provide high-quality services, and future assets are slated to improve performance within the same design frameworks, proposed future architectures may not be capitalizing on technology improvements, system innovations, or policy alternatives explored during the last two decades. This paper identifies five “trends” along which space architectures may develop, aimed at granting systems several “ilities,” such as resiliency, robustness, flexibility, scalability, and affordability. The trends examined include: commercialization of space, significant reductions in launch costs and the development of hybrid or reusable launch systems, development of on-orbit infrastructure and servicing, aggregation or disaggregation of orbital assets, and the automation and standardization of ground architectures. Further refinement of these key technological and system trends could result in major paradigm shifts in the development and fielding of space operations as well as lead to space architecture designs in the future that are radically different from those today. Within the framework of systems engineering ilities and risk management, this paper reviews current literature surrounding these new change trends and justifies their potential to cause significant paradigm shifts. By examining the work and research conducted so far through an ilities-based approach, systems engineers can more fully appreciate the value being offered by these trends

Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire

Phenotype of CAR⁺ T cell.

<p>(A) Expression of CD19RCD28 CAR on T cells day after electroporation (culture day 1) and after 28 days of co-culture on aAPC clone #4 along with lack of CD19⁺ aAPC. (B) CAR expression by western blot analysis using CD3-ζ specific antibody. Whole cell lysates were run on SDS-PAGE under reducing conditions. Molecular weight marker (M), Parental Jurkat cells (Lane 1), CD19RCD28⁺ Jurkat cells (Lane 2), CAR^neg control T cells (Lane 3) and CD19RCD28⁺ T cells (Lane 4). (C) Percent expression of CD3⁺, CD4⁺CAR⁺ and CD8⁺CAR⁺ T cells with in a lymphocyte gate in cultures over time. Each symbol represents a separate experiment; the solid lines are mean of the three validation experiments. (D) Immunophenotype of memory/naïve, adhesion, activation, cytolytic and exhaustion markers on CAR⁺ T cells at the end (d28) of co-culture.</p

Harvest and characterization of aAPC.

<p>(A, B) Sepax volume reduction. aAPC clone #4 grown in VueLife bags were harvested using CS-490.1 kit in Sepax II. The Sepax harvest (S, n = 4) was compared to manual (M, n = 1) procedure. The mean pre/post-processing cell-counts (4.9×10⁸ vs 5×10⁸) were similar using the Sepax system. (C) Phenotype of aAPC (clone #4). Flow cytometry analysis showing expression of CD19, CD64, CD86, CD137L and mIL-15 (expressed with EGFP) (mIL-15-EGFP) on K562 aAPC and K562 parental controls.</p

Safety profile associated with the SB system.

<p>(A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assay. Predominant T cell population at day 28 (V1 and V2, CD8⁺ T cells; V3, CD4⁺ T cells) was compared to respective miltenyi column purified subset of T cells from day 0. Mean ± SD of triplicates for each validation run is represented. (B) Genomic DNA from CAR⁺ T cells at day 28 was amplified using primers and probes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the CD19RCD28 target copy number was determined using normal donor PBMC as reference and endogenous RNaseP as a normalizer. Mean ± SD of triplicates for each validation run is shown. (C) TCR Vβ analysis of day 28 and day 35 CAR⁺ T cells. Data shows mean ± SD of three validation run CAR⁺ T cells as compared to day 0 unmanipulated controls. (D) A representative genomic PCR showing lack of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CAR^neg control T cells (lane 5) and CAR⁺ T cells (lane 7) amplified using SB11 primers; CAR^neg control T cells (lane 6), CAR⁺ T cells (lane 8) and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers were used as positive controls. (E) G-banded karyotypes of CAR⁺ T cells from the three validation runs reveal no structural or numeric alteration. A representative spread from validation 2 is shown.</p

Lack of autonomous cell growth by genetically modified T cells.

a<p>Days of culture when T cells were seeded.</p>b<p>Total number of T cells seeded in culture at the start of the experiment.</p>c<p>Total number of T cells counted in the absence of cytokines and aAPC.</p>d<p>Total (inferred) number of T cells counted in the presence of cytokines and aAPC (positive control).</p>e<p>Percent fold-change = [(c/b)÷(d/b)]*100.</p

Schematic of the process of generating clinical grade CD19-specific T cells.

<p>A MCB (PACT) and WCB (MDACC) were generated for K562-derived aAPC (clone #4). For the generation of CAR⁺ T cells, aAPC were numerically expanded in bags, harvested using the Sepax II system, irradiated (100 Gy), and cryopreserved for later use. CD19-specific T cells were manufactured as follows; PBMC were isolated from normal donor apheresis products using the Sepax II system and cryopreserved. The PBMC were later thawed, electroporated with the SB DNA plasmids (CD19RCD28 CAR transposon, SB11 transposase) using the Nucleofector System, co-cultured with thawed irradiated aAPC along with cytokines (IL-2 and IL-21) for a culture period of 28 days and cryopreserved.</p

Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using <i>Sleeping Beauty</i> System and Artificial Antigen Presenting Cells

<div><p>Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the <i>Sleeping Beauty</i> (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10¹⁰ T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.</p></div