Potential causes of mortality for horseshoe crabs (Limulus polyphemus) during the biomedical bleeding process

Biomedical companies catch and bleed horseshoe crabs for the production of Limulus amebocyte lysate (LAL), a product used for protecting public health (Berkson and Shuster, 1999). LAL is a clotting agent, derived solely from horseshoe crab blood cells, which is used to detect the presence of pathogenic gramnegative bacteria in injectable drugs and implantable medical and dental devices (Mikkelsen, 1988; Novitsky, 1991). In addition, LAL is used in many diagnostic tests for such illnesses as gram-negative bacterial meningitis and typhoid fever (Ding and Ho, 2001). Because the LAL test allows one to detect femtogram levels of endotoxin (Ding and Ho, 2001), it is the most effective test for detecting endotoxin contamination, and its increasing use in medical and pharmaceutical laboratories makes it a highly valued product

TETHERED IL-15 TO AUGMENT THE THERAPEUTIC POTENTIAL OF T CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTOR: MAINTAINING MEMORY POTENTIAL, PERSISTENCE, AND ANTITUMOR ACTIVITY

Tethered IL-15 to augment the therapeutic potential of T cells expressing chimeric antigen receptor: Maintaining memory potential, persistence, and antitumor activity Adoptive immunotherapy can retarget T cells to CD19, a tumor-associated antigen (TAA) expressed on B-cell malignancies, by the expression of a chimeric antigen receptor (CAR). Infusion of CAR-modified T cells for the treatment B-cell malignancies has demonstrated promise in preclinical and clinical trials. These data highlight the ability of infused CD19-specific T cells to be synchronously activated by large burdens of CD19+ leukemia and lymphoma. This can lead to dramatic antitumor effects, but also exposes the recipient to toxicity associated with tumor-cell lysis and cytokine storm. Clinical trials will now be addressing the targeting of minimal burdens of CD19+ malignancy as patients are enrolled earlier in their disease course and receive concomitant chemotherapy. It is likely that the existing populations of CAR T cells generated ex vivo to address relapsed disease may not be able to address minimal residual disease (MRD). Therefore, we have developed a clinically appealing approach to sustaining the persistence of CAR T cells independent of TAA by providing signaling through the common gamma chain receptor (gc). Administration of exogenous soluble recombinant cytokines that signal through the gc, such as interleukin (IL)-2, have been used clinically to sustain the persistence of adoptively transferred T cells. However, systemic high-dose administration has resulted in dose-limiting toxicities. Unlike IL-2, IL-15 possesses numerous attributes desirable for adoptive therapy and has been ranked among the most valuable immunotherapeutic agent for cancer treatment. It is a pro-survival cytokine that promotes the survival of long-lived T-cell memory subsets and in vivo antitumor activity. Unlike other gc family cytokines, IL-15 is transpresented to responding T cells in the context of IL-15 receptor alpha (IL-15Ra). Therefore, we hypothesized that a membrane-bound IL-15 fusion protein (mIL15) tethered to the cell surface would enhance T-cell costimulation to support persistence independent of CAR activation by preserving T-cell memory potential and maintain antitumor activity in the presence of low TAA. Using clinically compliant methods, the generated mIL15-CAR T cells mimicked the physiologic mechanism of transpresentation to sustain costimulation via phosphorylation of signal transducer and activator of transcription (pSTAT5). In contrast to conventional CD19-specific CAR T cells, mIL15-CAR T cells persisted in mice independent of the presence of TAA and mediated potent rejection of a systemically distributed CD19+ leukemia. The potential for sustained immunity against B-cell malignancies was shown as, in the absence of antigen, mIL15-CAR T cells were long-lived and adopted a desirable CD45ROnegCCR7+ “low-differentiation” state with a memory-like molecular profile and phenotype. These results have direct implications for the design of an adoptive immunotherapy clinical trial evaluating mIL15-CAR T cells in the setting of MRD and warrants further investigation of mIL15 to engineer T cells targeting other tumor cells that have sequestered or low levels of TAA

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

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

Schema for NK cell manufacturing with aAPCs.

<p>Artificial antigen-presenting cells (aAPCs) were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells <i>ex</i> vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the aAPCs as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use.</p