Julie R. Ostberg

Antitransgene Rejection Responses Contribute to Attenuated Persistence of Adoptively Transferred CD20/CD19-Specific Chimeric Antigen Receptor Redirected T Cells in Humans

Immunotherapeutic ablation of lymphoma is a conceptually attractive treatment strategy that is the subject of intense translational research. Cytotoxic T lymphocytes (CTLs) that are genetically modified to express CD19- or CD20-specific, single-chain antibody-derived chimeric antigen receptors (CARs) display HLA-independent antigen-specific recognition/killing of lymphoma targets. Here, we describe our initial experience in applying CAR-redirected autologous CTL adoptive therapy to patients with recurrent lymphoma. Using plasmid vector electrotransfer/drug selection systems, cloned and polyclonal CAR(+) CTLs were generated from autologous peripheral blood mononuclear cells and expanded in vitro to cell numbers sufficient for clinical use. In 2 FDA-authorized trials, patients with recurrent diffuse large cell lymphoma were treated with cloned CD8(+) CTLs expressing a CD20-specific CAR (along with NeoR) after autologous hematopoietic stem cell transplantation, and patients with refractory follicular lymphoma were treated with polyclonal T cell preparations expressing a CD19-specific CAR (along with HyTK, a fusion of hygromycin resistance and HSV-1 thymidine kinase suicide genes) and low-dose s.c. recombinant human interleukin-2. A total of 15 infusions were administered (5 at 10(8)cells/m(2), 7 at 10(9)cells/m(2), and 3 at 2 x 10(9)cells/m(2)) to 4 patients. Overt toxicities attributable to CTL administration were not observed; however, detection of transferred CTLs in the circulation, as measured by quantitative polymerase chain reaction, was short (24 hours to 7 days), and cellular antitransgene immune rejection responses were noted in 2 patients. These studies reveal the primary barrier to therapeutic efficacy is limited persistence, and provide the rationale to prospectively define T cell populations intrinsically programmed for survival after adoptive transfer and to modulate the immune status of recipients to prevent/delay antitransgene rejection responses.

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells

An unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor(+) T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

The Roman god Janus: a paradigm for the function of CD43

Abstract Although the structural characterization of the major leukocyte cell surface mucin CD43 has proceeded rapidly, understanding its physiological function has been hindered by contradictory results. Here, Julie Ostberg and colleagues propose that the Roman god of two faces, Janus, provides a model that reconciles the roles attributed to CD43.

Regulatory effects of fever-range whole-body hyperthermia on the LPS-induced acute inflammatory response.

The thermal component of fever is one of the most poorly understood aspects of inflammation. To evaluate the role of fever‐range hyperthermia on acute inflammation, BALB/c and C57BL/6 mice were exposed to mild, long‐duration whole‐body hyperthermia (WBH), and serum concentrations of tumor necrosis factor α (TNF‐α), interleukin‐6 (IL‐6), IL‐1β, and the acute phase proteins (APPs) α1‐acid glycoprotein and haptoglobin were analyzed. WBH alone did not affect serum concentrations of these cytokines or APPs when compared with controls. In contrast, when WBH was applied just after intraperitoneal administration of lipopolysaccharide (LPS), serum concentrations of TNF‐α and IL‐6 were greater than or equal to threefold higher in BALB/c mice compared with LPS‐treated controls. LPS‐induced IL‐6 levels were also enhanced in WBH‐treated C57BL/6 mice. However, APP levels were prolonged only in WBH‐treated BALB/c mice. It is interesting thatin vitro hyperthermia treatment of LPS‐stimulated peritoneal cells resulted in decreased cytokine production compared with controls. These results suggest that fever‐range hyperthermia regulates acute inflammation in a mouse strain‐specific manner that is more complex than that observed in vitro.

Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale.

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8+ effector T cells derived from CD45RA− CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14+, CD45RA+, and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA− CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified TCM cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19+ B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.

A phase I study of fever-range whole body hyperthermia (FR-WBH) in patients with advanced solid tumours: correlation with mouse models.

Various studies in animal tumour models have revealed the potential of fever-range whole body hyperthermia (FR-WBH) to be used in cancer therapy. To determine the safety of FR-WBH treatment in the clinic, patients with advanced solid tumours were heated in the outpatient setting to 39-39.5°C for 3 or 6h, or 39.5-40°C for 6h using the Heckel-HT 2000 apparatus. These WBH treatments were well tolerated, with no significant adverse events related to cardiac, hepatic, renal or pulmonary systems. In the majority of patients, flow cytometric analysis of peripheral blood leukocyte populations indicated that there were transient decreases in the number of circulating T lymphocytes and a concomitant decrease in the number of L-selectin positive lymphocytes in the peripheral blood. These findings closely mimic the affects seen previously in pre-clinical murine studies in which this same fever-like treatment was shown to inhibit tumour growth. These studies have established the safety of this treatment and will allow for future clinical trials where application of FR-WBH treatment can be combined with other anti-cancer therapies, including immunotherapy and chemotherapy.

Enhancement of natural killer (NK) cell cytotoxicity by fever-range thermal stress is dependent on NKG2D function and is associated with plasma membrane NKG2D clustering and increased expression of MICA on target cells

Circulating NK cells normally experience temperature gradients as they move about the body, but the onset of inflammation can expose them and their targets to febrile temperatures for several hours. We found that exposure of human peripheral blood NK cells and target cells to fever‐range temperatures significantly enhances lysis of Colo205 target cells. A similar effect was not observed when NK cell lines or IL‐2‐activated peripheral blood NK cells were used as effectors, indicating that thermal sensitivity of effectors is maturation or activation state‐dependent. Use of blocking antibodies revealed that this effect is also dependent on the function of the activating receptor NKG2D and its ligand MHC class I‐related chain A (MICA). On NK cells, it was observed that thermal exposure does not affect the total level of NKG2D surface expression, but does result in its distinct clustering, identical to that which occurs following IL‐2‐induced activation. On tumor target cells, a similar, mild temperature elevation results in transcriptional up‐regulation of MICA in a manner that correlates with increased sensitivity to cytolysis. Overall, these data reveal that NK cells possess thermally responsive regulatory elements, which facilitate their ability to capitalize on reciprocal, stress‐induced changes simultaneously occurring on target cells during inflammation and fever.

Emerging evidence indicates that physiologically relevant thermal stress regulates dendritic cell function

SummaryElevations in temperature that are associated with inflammation or fever have been linked to improved survival from infections, enhanced immunological functions, and increased control of tumor growth. Over the past few years, several groups have begun to explore the possible linkage among these observations and have tested the hypothesis that various immune cells are especially sensitive to thermal stimulation. However, relatively little is known regarding the effects of thermal stimulation on antigen presenting cells (APCs), such as dendritic cells (DCs). Very recently, several groups have begun to examine the ability of thermal stimuli to regulate the function of these cells which are known to play a pivotal role in the efficacy of vaccines and other immunotherapies. In this review, we summarize what has been discovered about the role of mild thermal stress in regulating various Dendritic cell (DC) activities. Excitingly, it appears that mild elevations of temperature have the potential to enhance antigen uptake, activation associated migration, maturation, cytokine expression and T cell stimulatory activity of DCs. While these studies reveal that the timing, temperature and duration of heating is important, they also set the stage for essential questions that now need to be investigated regarding the molecular mechanisms by which elevated temperatures regulate DC function. With this information, we may soon be able to maximize the strategic use of thermal therapy as an adjuvant, i.e., combining its use with cancer immunotherapies such as vaccines, which depend upon the function of DCs. Several possible strategies and timepoints involving the clinical application of hyperthermia in combination with immunotherapy are presented.

Induction of stress proteins in a panel of mouse tissues by fever-range whole body hyperthermia

When exposed to environmental stress, cell survival is supported by the upregulation of stress proteins such as heat shock proteins (HSPs) or glucose regulated proteins (GRPs), which help prevent protein denaturation. To begin to characterize the ability of a physiologically relevant heat exposure to induce stress protein expression, the cerebellum, cerebrum, colon, heart, kidney, liver, lung, lymph nodes, muscle, serum and thymus were extracted from BALB/c mice at various times after fever-range whole body hyperthermia (FR-WBH, 39.5-40°C for 6h) treatment. The expression of three stress proteins, HSP70, HSP110 and GRP170, was determined in these tissues and serum and compared to constitutive levels in control tissues and serum using Western analysis. Constitutive expression of GRP170 was not affected by FR-WBH in any tissue. In contrast, FR-WBH did enhance HSP expression: HSP70 in heart, kidney, lung, lymph nodes and thymus; and HSP110 in lung, lymph nodes and thymus. The lymphoid tissues displayed the most consistent upregulation of both HSP70 and HSP110 upon FR-WBH treatment. The apparent sensitivity of immunologically relevant tissues to FR-WBH may relate to the enhanced immune responses that are observed during febrile temperatures.