Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells.

The success of immune system-based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti-CD3 scFv antibody) were previously shown to redirect CD8+ and CD4+ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma-specific protein, gp100, presented by HLA-A*0201) efficiently redirects and activates effector and memory cells from both CD8+ and CD4+ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8+ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4+ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8+ and CD4+ repertoires secrete key pro-inflammatory cytokines (tumour necrosis factor-α, interferon-γ, interleukin-6) and chemokines (macrophage inflammatory protein-1α-ß, interferon-γ-inducible protein-10, monocyte chemoattractant protein-1). At an individual cell level, IMCgp100-redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti-cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8+ T cell-mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.

Elimination of Latently HIV-infected Cells from Antiretroviral Therapy-suppressed Subjects by Engineered Immune-mobilizing T-cell Receptors.

Persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T-cells is a major barrier to eradication. Latency-reversing agents that induce direct or immune-mediated cell death upon reactivation of HIV are a possible solution. However, clearance of reactivated cells may require immunotherapeutic agents that are fine-tuned to detect viral antigens when expressed at low levels. We tested the antiviral efficacy of immune-mobilizing monoclonal T-cell receptors against viruses (ImmTAVs), bispecific molecules that redirect CD8+ T-cells to kill HIV-infected CD4+ T-cells. T-cell receptors specific for an immunodominant Gag epitope, SL9, and its escape variants were engineered to achieve supraphysiological affinity and fused to a humanised CD3-specific single chain antibody fragment. Ex vivo polyclonal CD8+ T-cells were efficiently redirected by immune-mobilising monoclonal T-cell receptors against viruses to eliminate CD4+ T-cells from human histocompatibility leukocyte antigen (HLA)-A*0201-positive antiretroviral therapy-treated patients after reactivation of inducible HIV in vitro. The efficiency of infected cell elimination correlated with HIV Gag expression. Immune-mobilising monoclonal T-cell receptors against viruses have potential as a therapy to facilitate clearance of reactivated HIV reservoir cells.

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 discussed.

Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

ImmTAC-redirected tumour cell killing induces and potentiates antigen cross-presentation by dendritic cells.

Antigen cross-presentation by dendritic cells (DCs) is thought to play a critical role in driving a polyclonal and durable T cell response against cancer. It follows, therefore, that the capacity of emerging immunotherapeutic agents to orchestrate tumour eradication may depend on their ability to induce antigen cross-presentation. ImmTACs [immune-mobilising monoclonal TCRs (T cell receptors) against cancer] are a new class of soluble bi-specific anti-cancer agents that combine pico-molar affinity TCR-based antigen recognition with T cell activation via a CD3-specific antibody fragment. ImmTACs specifically recognise human leucocyte antigen (HLA)-restricted tumour-associated antigens, presented by cancer cells, leading to T cell redirection and a potent anti-tumour response. Using an ImmTAC specific for a HLA-A*02-restricted peptide derived from the melanoma antigen gp100 (termed IMCgp100), we here observe that ImmTAC-driven melanoma-cell death leads to cross-presentation of melanoma antigens by DCs. These, in turn, can activate both melanoma-specific T cells and polyclonal T cells redirected by IMCgp100. Moreover, activation of melanoma-specific T cells by cross-presenting DCs is enhanced in the presence of IMCgp100; a feature that serves to increase the prospect of breaking tolerance in the tumour microenvironment. The mechanism of DC cross-presentation occurs via 'cross-dressing' which involves the rapid and direct capture by DCs of membrane fragments from dying tumour cells. DC cross-presentation of gp100-peptide-HLA complexes was visualised and quantified using a fluorescently labelled soluble TCR. These data demonstrate how ImmTACs engage with the innate and adaptive components of the immune system enhancing the prospect of mediating an effective and durable anti-tumour response in patients.

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-, NY-ESO-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-PCR was 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.

High-yield production and characterization of biologically active GST-tagged human topoisomerase IIα protein in insect cells for the development of a high-throughput assay.

DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase IIα proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase IIα using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase IIα was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase IIα inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ≥ 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.

Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.

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 muM at 37 degrees 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.

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.