Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene.

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT remain major impediments. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells.1–5 CD19 is an attractive CAR target, expressed in most B cell malignancies as well as normal B cells.6,7 Clinical trails using autologous CD19-targeted T cells have shown remarkable outcomes in various B cell malignancies8–15. The use of allogeneic CAR T cells poses a concern of increased GVHD, which however has not been reported in selected patients infused with donor-derived CD19-CAR T cells after allo-HSCT.16,17 To understand the mechanism whereby allogeneic CD19-CAR T cells may mediate anti-lymphoma activity without significant GVHD, we studied donor-derived CD19-CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19-CARs experienced enhanced T cell stimulation, resulting in progressive loss of effector function and proliferative potential, clonal deletion, and significantly decreased GVHD. Concurrently, other CAR T cells present in bulk donor T cell populations retained their anti-lymphoma activity consistent with the requirement for engaging both the TCR and the CAR to accelerate T cell exhaustion. In contrast, first generation and 4-1BB-costimulated CARs increased GVHD. These findings could explain reduced risk of GVHD with cumulative TCR and CAR signaling.

Stable expression of human groups IIA and X secreted phospholipases A 2 (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum-and interleukin-1␤-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan-or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A 2 inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cellimpermeable, secreted phospholipase A 2 trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1␤-dependent release of arachidonate is promoted by secreted phospholipase A 2 expression and is completely dependent on cytosolic (group IVA) phospholipase A 2 . These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIAtransfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.Phospholipases A 2 (PLA 2 s) 1 are a class of enzymes that release fatty acids from the sn-2 position of glycero-phospholipids. Biomedical interest in these enzymes stems from the finding that the liberation of arachidonic acid for the biosynthesis of the eicosanoids (prostaglandins, leukotrienes, and others) is mediated in mammalian cells by one or more PLA 2 s. Current evidence favors a role for cytosolic phospholipase A 2 -␣ (cPLA 2 -␣, also known as group IVA PLA 2 ) as a major component of the arachidonate releasing signal transduction pathway (1-3). Mammals also contain a large number of secreted phospholipases A 2 (sPLA 2 s) (4, 5), and the possible participation of these enzymes in arachidonate release is under active investigation. For example, group V sPLA 2 is present in the macrophage-like cell line p388D1 and contributes a portion of the arachidonate released in response to lipopolysaccharide (6). Exogenous addition of groups V and X sPLA 2 s to a variety of mammalian cells leads to arachidonate release (7-10). There is some evidence to suggest that the action of cPLA 2 -␣ is a prerequisite for sPLA 2 function in cells (11,12) and even for the vice versa scenario (13-15), but such cross-talk between PLA 2 s remains poorly understood.To assess the arachidonate releasing capacity of PLA 2 s in mammalian cells, CHO and HEK293 cell lines that stably express the various enzymes have been established (16 -20). The behavior of human group IIA (hGIIA) and human group X (hGX) sPLA 2 s when transfected in HEK293 and CHO cells has been extensively studied. hGIIA is secreted from HEK293 cells, and most of the extracellular enzyme is a...

We previously demonstrated the feasibility of generating therapeutic numbers of cytotoxic T lymphocyte (CTL) clones expressing a CD20-specific scFvFc:CD3zeta chimeric T cell receptor (cTCR), making them specifically cytotoxic for CD20+ B lymphoma cells. However, the process of generating and expanding he CTL clones was laborious, the CTL clones expressed the cTCR at low surface density, and they exhibited suboptimal proliferation and cytotoxicity. To improve the performance of the CTLs in vitro and in vivo, we engineered "second-generation'' plasmid constructs containing a translational enhancer (SP163) and CD28 and CD137 costimulatory domains in cis with the CD3zeta intracellular signaling domain of the cTCR gene. Furthermore, we verified the superiority of generating genetically modified polyclonal T cells expressing the second-generation cTCR rather than T cell clones. Our results demonstrate that SP163 enhances the surface expression of the cTCR; that the second-generation cTCR improves CTL activation, proliferation, and cytotoxicity; and that polyclonal T cells proliferate rapidly in vitro and mediate potent CD20-specific cytotoxicity. This study provides the preclinical basis for a clinical trial of adoptive T cell immunotherapy for patients with relapsed CD20+ mantle cell lymphoma and indolent lymphomas.

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Therapeutic T-cell engineering is emerging as a powerful approach to treat refractory hematological malignancies. Its most successful embodiment to date is based on the use of second-generation chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule found in most B-cell leukemias and lymphomas. Remarkable complete remissions have been obtained with autologous T cells expressing CD19 CARs in patients with relapsed, chemo-refractory B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Allogeneic CAR T cells may also be harnessed to treat relapse after allogeneic hematopoietic stem cell transplantation. However, the use of donor T cells poses unique challenges owing to potential alloreactivity. We review different approaches to mitigate the risk of causing or aggravating graft-versus-host disease (GVHD), including CAR therapies based on donor leukocyte infusion, virus-specific T cells, T-cell receptor-deficient T cells, lymphoid progenitor cells, and regulatory T cells. Advances in CAR design, T-cell selection and gene editing are poised to enable the safe use of allogeneic CAR T cells without incurring GVHD.

We investigated relationships among chimeric T cell receptor (cTCR) expression density, target antigen density, and cTCR triggering to predict lysis of target cells by cTCR+ CD8+ T human cells as a function of antigen density. Triggering of chimeric and canonical TCR by antigen could be quantified by the same mathematical equation, but cTCR represented a special case in which serial triggering was abrogated. The magnitude of target lysis could be predicted as a function of cTCR-triggering and the predicted minimum cTCR density required for maximal target lysis by CD20-specific cTCR was experimentally tested. cTCR density below ~20,000 cTCR/cell impaired target lysis, but increasing cTCR expression above this density did not improve target lysis or antigen sensitivity. cTCR down-modulation to densities below this critical minimum by interaction with antigen-expressing targets limited the sequential lysis of targets in a manner that could be predicted based on the number of cTCR remaining. In contrast, acute inhibition of lysis of primary, intended targets (e.g. leukemic B cells) due to the presence of an excess of secondary targets (e.g. normal B cells) was dependent on the antigen density of the secondary target but occurred at antigen densities insufficient to promote significant cTCR down-modulation, suggesting a role for functional exhaustion rather than insufficient cTCR density. This suggests increasing cTCR density above a critical threshold may enhance sequential lysis of intended targets in isolation, but will not overcome the functional exhaustion of cTCR+ T cells encountered in the presence of secondary targets with high antigen density.