Low-affinity T-cell Receptors Research Articles

Abstract 3528: IMCmage1: A novel bi-specific biologic re-directing T cells to kill MAGE-A3/A6 presenting cancers

Abstract In a minority of cancer patients immunotherapy has shown the capacity to eradicate tumours leading to clinical remission and the promise of a cure. In the majority of patients however, a cure remains elusive due to active immune evasion by cancers; HLA-down-regulation and immunosuppression are two of the known mechanisms adopted by cancers to promote their survival and proliferation. To overcome these challenges we have developed bi-specific soluble biologics termed ImmTACs (Immune mobilising mTCR against cancer) to re-direct the immune system to recognise and kill cancers. Antigenic peptide fragments presented by HLA molecules on the surface of cancer cells constitute the largest class of cancer associated targets. T cells scan the HLA-peptide (pHLA) antigens being presented to them; sufficient recognition by the harboured T Cell Receptor (TCR) will result in T cell activation and killing of the antigen presenting cell. In cancer patients this process is inefficient partly due to the low affinity TCRs expressed by tumour specific T cells and the low abundance of pHLA on cancers. ImmTACs comprise a soluble TCR with an enhanced affinity for cancer associated pHLA (targeting end) fused to an anti-CD3 scfv, enabling potent T cell re-direction (effector end). Our pipeline constitutes a number of ImmTACs targeting various antigen pHLA complexes relevant to numerous cancer indications. IMCmage1 is a novel ImmTAC targeting MAGE-A3168-176 in the context of HLA-A1. MAGE-A3 is a well validated cancer testis antigen expressed in a variety of cancers including myeloma, NSCLC, prostate cancer, melanoma, bladder cancer, oesophageal cancer and others. IMCmage1 re-directs T cells from cancer patients or healthy donors to kill a range of MAGE positive cell-lines in vitro; this activity is observed against cells presenting as few as 40 epitopes per cell and is coupled with the release of pro-inflammatory cytokines including IFNα, TNFα, IL-2, MIP1α and others. We also demonstrate that IMCmage1 specifically targets and kills the myeloma associated population within CD138+ cells extracted from the marrow of a stage III myeloma patient. IMCmage1 specificity was confirmed by exposure to a panel of HLA-A1 MAGE negative primary cells derived from various organs such as the heart, skin, lung and others; no significant activity was detected. A phase I clinical trial in multiple myeloma to assess tolerability and establish a maximum tolerated dose is planned to commence in Q2 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3528. doi:1538-7445.AM2012-3528

Mechanisms of tolerance

Mechanisms of tolerance

Itk Derived Signals Regulate the Expression of Th-POK and Controls the Development of CD4+ T Cells

T cell development is critically dependent on both the environment and signals delivered by the T cell Receptor (TCR). The Tec family kinase Itk has been suggested to be an amplifier of signals emanating from the TCR and the loss of Itk partially affects most stages of thymopoiesis. Loss of Itk also differentially affects the development of conventional vs. non-conventional or innate memory phenotype T cells. Here, we examine whether these lineage choices are affected by a combination of TCR affinity and Itk by analyzing mice lacking Itk and carrying two TCR transgenes with differing affinities, OT-II and DO11.10. Our results show that developing thymocytes receive a gradient of signals, DO11.10>OT-II>DO11.10/Itk−/−>OT-II/Itk−/−. We also show that the development of CD4+ T cells is controlled by TCR signaling via Itk, which regulates the expression of the transcription factor, Th-POK, an enforcement factor for CD4 commitment. This results in a reduction in CD4+ T cell development, and an increase in the development of MHC class II restricted TCR transgenic CD8+ T cells that resemble non-conventional or innate memory phenotype CD8 T cells. This alteration accompanies increased expression of Runx3 and its target genes Eomesodermin, Granzyme B and Perforin in Itk null OT-II CD4+ thymocytes. All together, these data suggest that Itk plays an important role in CD4/CD8 commitment by regulating signal thresholds for the lineage commitment. Our data also suggest that the lower level of TCR signaling that occurs with a low affinity TCR in the absence of Itk can redirect some MHC class II restricted CD4+ T cell to class II-restricted CD8+ innate memory phenotype T cells.

Obstacles to and opportunities for more effective peptide-based therapeutic immunization in human melanoma

Melanoma cells can play a number of tricks to evade the host immune response. They can make themselves invisible to cells of the immune system poised to attack them, elaborate molecules that are frankly immunosuppressive, and can create a microenvironment that is hostile to cells of the immune system. Efforts are underway to institute measures that would make tumor cells more susceptible to immune attack, but these efforts have not been all that successful so far. This contribution reviews the history and the rationale of cancer vaccines, the major obstacles to peptide-based immunization, and a discussion on how to surmount them. Also included are the roles played by peripheral tolerance, low-affinity T-cell receptors, T-cell ignorance, activation-induced cell death, exhaustion of T cells and regulation of the immune response, helpless cytolytic T lymphocytes, and evasion by tumor cells.

Novel approaches for identifying target antigens of autoreactive human B and T cells

Antigen-specific immune responses in multiple sclerosis have been studied for decades, but the target antigens of the putatively autoaggressive B and T cells still remain elusive. Here, we summarize recent strategies which are based on the direct analysis of biopsy or autopsy specimens from patients. Since this material is extremely scarce, the experimental methods need to be exceptionally sensitive. We describe technologies to distinguish (auto) aggressive T cells from irrelevant bystander lymphocytes by analyzing clonal expansions in relation to the morphological location of the cells in the tissue lesions. We then discuss approaches to clone matching α- and β-chains of the antigen-specific T cell receptor (TCR) molecules from single T cells. This is necessary because usually, several clones are expanded and are diluted by many irrelevant cells. The matching TCR chains from individual T cells can be resurrected in hybridoma cells which may then be used for antigen searches. We discuss strategies to identify antigens of γδ- and αβ-TCR molecules, such as biochemical methods, candidate antigens, human leukocyte antigen requirements, synthetic peptide, and cDNA libraries. These strategies are tailored to characterize the antigens of the membrane-anchored, low-affinity TCR molecules. The strategies to identify (auto) reactive B cells or immunoglobulin (Ig) molecules are fundamentally different, because Ig molecules are water-soluble and have high affinities. We further discuss proteome-based approaches, techniques that analyze Ig-chains from single B cells, and a repertoire-based method that compares Ig-proteomes and Ig-transcriptomes. The first method detects Ig antigens directly, whereas the latter two methods allow reconstruction of Ig molecules, which can be used for antigen searches.

Detection of low avidity CD8 + T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8 + T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8 + T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A⁎0201 monomers engineered in the heavy chain α2 domain to enhance CD8 binding ( K D ≈ 85 μM) without impacting the TCR binding platform can detect cognate CD8 + T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A⁎0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8 + T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8 + T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8 + T cells.

Loss of CTL Function among High-Avidity Tumor-Specific CD8+ T Cells following Tumor Infiltration

A major problem in generating effective antitumor CTL responses is that most tumors express self-antigens to which the immune system is rendered unresponsive due to mechanisms of self-tolerance induction. CTL precursors (CTLp) expressing high-affinity T-cell receptors (TCR) are often functionally deleted from the repertoire, leaving a residual repertoire of CTLp having only low-affinity TCR. Furthermore, even when unique antigens are expressed, their presentation by dendritic cells (DC) may predispose to peripheral tolerance induction rather than the establishment of CTL responses that kill tumor cells. In this study, we examined both high-avidity (CL4) and low-avidity (CL1) CD8(+) T-cell responses to a murine renal carcinoma expressing, as a neoantigen, high and low levels of the hemagglutinin (HA) protein from influenza virus A/PR/8 H1N1 (PR8; RencaHA(high) and RencaHA(low)). Our data show that, following encounter with K(d)HA epitopes cross-presented by bone marrow-derived DC, low-avidity CL1 cells become tolerized within tumor-draining lymph nodes (TDLN), and in mice bearing either RencaHA(high) or RencaHA(low) tumors, very few form tumor-infiltrating lymphocytes (TIL). In marked contrast, high-avidity CL4 cells differentiate into effector CTL within the TDLN of mice bearing either RencaHA(high) or RencaHA(low) tumors, and although they form TIL in both tumors, they lose CTL effector function. Critically, these results show that anticancer therapies involving either adoptive transfer of high-avidity tumor-specific CTL populations or targeting of preexisting tumor antigen-specific memory CD8(+) T cells could fail due to the fact that CTL effector function is lost following tumor infiltration.

Role of TCR-restricting MHC density and thymic environment on selection and survival of cells expressing low-affinity T cell receptors.

H-Y-specific T cell receptor (TCR) transgenic mice express an MHC class I H-2D(b)-restricted TCR specific for a male antigen-derived peptide (H-Y). Selection and survival of cells expressing this low-affinity TCR were analyzed under optimal and sub-optimal conditions. Optimal conditions were provided by a thymic environment with high H-2D(b) density. Sub-optimal conditions were provided by a thymic environment with decreased H-2D(b) density or by an athymic environment with either high or low H-2D(b) density. Whereas negative selection was still guaranteed under sub-optimal conditions, selection and survival efficiency of cells expressing the transgenic TCR were strongly dependent on optimal conditions. These results indicated that both a thymic microenvironment and a high density of TCR-restricting MHC molecules were needed to ensure selection and maintenance of cells expressing TCR with low affinity and hence a more diverse T cell repertoire.

TCRs with high affinity for foreign pMHC show self-reactivity.

T cells with high-affinity T cell receptors (TCRs) for a foreign peptide-major histocompatibility complex (pMHC) appear to be negatively selected, even though they have never seen the foreign antigen. To examine how this process operates, we used in vitro yeast display to isolate high-affinity TCRs from the T cell clone 2C. The TCRs showed fast on-rates, which were consistent with reduced CDR (complementarity determining region) flexibility, and cross-reactivity with other cognate pMHCs. T cell hybridomas transfected with a high-affinity TCR were stimulated by endogenous self-pMHC, which suggested that T cells bearing the TCR would be negatively selected. The immune system appears to maintain a repertoire of flexible, low-affinity TCRs at the expense of more effective high-affinity TCRs.

Melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last 7 years significant progress has been made in the identification of melanoma-associated antigens recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: cancer/testis-specific antigens (MAGE, BAGE, GAGE, PRAME and NY-ESO-1), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2), and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). In this review we have summarized the available data concerning the characterization of melanoma-associated antigens, focusing on their immunogenic and protective properties. The development of a strong immune response to differentiation antigens is limited by the existence of tolerance to these "self"-antigens, permitting the involvement of only T cells with low affinity T-cell receptors. Among the melanoma differentiation antigens, only gp100 has been shown to be a tumor regression antigen. The cancer/testis-specific antigens such as MAGE and PRAME should potentially be highly immunogenic antigens. They contain several potential HLA class I binding epitopes and are present only in the testes, which are not accessible to the cells of the immune system owing to the lack of direct contact with the immune cells and the lack of HLA class I expression on the surface of germ cells. But only two patients have been found who responded to these antigens in vivo, indicating their genuinely low immunogenicity. A comparison of the predicted secondary structures of these two groups of antigens (cancer/testis-specific and differentiation antigens) revealed enrichment of long alpha-helical stretches in the cancer/testis-specific antigens. We hypothesize that such highly organized stable structures could, first, reduce denaturation of the protein and, thus, ubiquitinylation as a degradation signal, and, second, diminish the efficiency of the protein unfolding - a necessary step in the proteolytic cleavage by proteasomes. High structural stability could therefore be responsible for the low immunogenicity of these proteins. In this case, modifications decreasing the stability of these proteins might be a means of improving the immune response to these potentially therapeutically useful antigens.

CD4 dependence of activation threshold and TCR signalling in mouse T lymphocytes.

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.

Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy. Role of T cell actin cytoskeleton.

Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function. Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies. First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ([Ca2+]i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR. Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained [Ca2+]i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes. Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of [Ca2+]i rise, coincident with a block of the cyclic changes in T cell shape. Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion. These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.

Switch of autoreactive human T cell clones from a TH2 to a TGFβ1 secreting phenotype with low affinity T-cell receptor stimulating peptide/MHC complex

Switch of autoreactive human T cell clones from a TH2 to a TGFβ1 secreting phenotype with low affinity T-cell receptor stimulating peptide/MHC complex

The mechanism of anti-Lyt-2 inhibition of antibody-directed lysis by cytotoxic T lymphocytes.

Bifunctional antibodies specific for a determinant within the T cell receptor (TcR) complex of cytotoxic T lymphocytes (CTL) and a determinant expressed on the surface of the target cell will effectively mediate cytolysis. In such a lytic system anti-Lyt-2 antibody can block cytolysis. We have observed that the amount of inhibition varies considerably from clone to clone and surprisingly correlates well with inhibition of conjugate formation as mediated by bifunctional antibody. This implies that inhibition of antibody-mediated killing occurs as the result of reduction of the avidity of the effector cell for its target, the same mechanism responsible for inhibition of receptor-mediated lysis by anti-Lyt-2. In light of the similarity between the mechanism of inhibition by anti-Lyt-2 of receptor-mediated and antibody-mediated cytolysis, we compared the ability of anti-Lyt-2 to inhibit cytolysis in these two different assay systems by using a number of different CTL clones. Whereas the majority of secondary CTL clones (presumed to have high affinity TcR) are inhibited equally in both assay systems, most primary CTL (presumed to have low affinity TcR) are more susceptible to inhibition by anti-Lyt-2 in their receptor-specific than their antibody-directed cytolysis. These results, taken together with an apparent correlation between the amount of Lyt-2 expressed on the cell surface and susceptibility to inhibition, suggest anti-Lyt-2 may block CTL function by sterically inhibiting mobility of the TcR complex.