CD6 Regulates T-Cell Responses through Activation-Dependent Recruitment of the Positive Regulator SLP-76

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 μM at 37°C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5

Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor

Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discusse

An approved in vitro approach to preclinical safety and efficacy evaluation of engineered T cell receptor anti-CD3 bispecific (ImmTAC) molecules.

Robust preclinical testing is essential to predict clinical safety and efficacy and provide data to determine safe dose for first-in-man studies. There are a growing number of examples where the preclinical development of drugs failed to adequately predict clinical adverse events in part due to their assessment with inappropriate preclinical models. Preclinical investigations of T cell receptor (TCR)-based immunotherapies prove particularly challenging as these biologics are human-specific and thus the conventional testing in animal models is inadequate. As these molecules harness the full force of the immune system, and demonstrate tremendous potency, we set out to design a preclinical package that would ensure adequate evaluation of these therapeutics. Immune Mobilising Monoclonal TCR Against Cancer (ImmTAC) molecules are bi-specific biologics formed of an affinity-enhanced TCR fused to an anti-CD3 effector function. ImmTAC molecules are designed to activate human T lymphocytes and target peptides within the context of a human leukocyte antigen (HLA), thus require an intact human immune system and peptidome for suitable preclinical screening. Here we draw upon the preclinical testing of four ImmTAC molecules, including IMCgp100, the first ImmTAC molecule to reach the clinic, to present our comprehensive, informative and robust approach to in vitro preclinical efficacy and safety screening. This package comprises a broad range of cellular and molecular assays using human tissues and cultured cells to test efficacy, safety and specificity, and hence predict human responses in clinical trials. We propose that this entirely in vitro package offers a potential model to be applied to screening other TCR-based biologics

Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors

NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NYESO- 1157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCRwas used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers

Casting a wider net: Immunosurveillance by nonclassical MHC molecules.

Most studies of T lymphocytes focus on recognition of classical major histocompatibility complex (MHC) class I or II molecules presenting oligopeptides, yet there are numerous variations and exceptions of biological significance based on recognition of a wide variety of nonclassical MHC molecules. These include αβ and γδ T cells that recognize different class Ib molecules (CD1, MR-1, HLA-E, G, F, et al.) that are nearly monomorphic within a given species. Collectively, these T cells can be considered "unconventional," in part because they recognize lipids, metabolites, and modified peptides. Unlike classical MHC-specific cells, unconventional T cells generally exhibit limited T-cell antigen receptor (TCR) repertoires and often produce innate immune cell-like rapid effector responses. Exploiting this system in new generation vaccines for human immunodeficiency virus (HIV), tuberculosis (TB), other infectious agents, and cancer was the focus of a recent workshop, "Immune Surveillance by Non-classical MHC Molecules: Improving Diversity for Antigens," sponsored by the National Institute of Allergy and Infectious Diseases. Here, we summarize salient points presented regarding the basic immunobiology of unconventional T cells, recent advances in methodologies to measure unconventional T-cell activity in diseases, and approaches to harness their considerable clinical potential