Use Of Altered Peptide Ligands Research Articles

T-Cell Responses to Immunodominant Listeria Epitopes Limit Vaccine-Directed Responses to the Colorectal Cancer Antigen, Guanylyl Cyclase C.

The Gram-positive bacterium Listeria monocytogenes (Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8+ T-cell responses towards the dominant H-2Kd-restricted epitope, GUCY2C254-262. However, Lm-GUCY2C produced robust CD8+ T-cell responses towards Lm-derived peptides suggesting that GUCY2C254-262 peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C254-262 immunity completely suppressed GUCY2C254-262 responses. Comparison of immunogenic Lm-derived peptides to GUCY2C254-262 revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2Kd compared to GUCY2C254-262 peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2Kd binding, producing GUCY2CF255Y, significantly improved stability with H-2Kd and rescued GUCY2C254-262 immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines.

Peptide Super-Agonist Enhances T-Cell Responses to Melanoma.

Recent immunotherapeutic approaches using adoptive cell therapy, or checkpoint blockade, have demonstrated the powerful anti-cancer potential of CD8 cytotoxic T-lymphocytes (CTL). While these approaches have shown great promise, they are only effective in some patients with some cancers. The potential power, and relative ease, of therapeutic vaccination against tumour associated antigens (TAA) present in different cancers has been a long sought-after approach for harnessing the discriminating sensitivity of CTL to treat cancer and has seen recent renewed interest following cancer vaccination successes using unique tumour neoantigens. Unfortunately, results with TAA-targeted “universal” cancer vaccines (UCV) have been largely disappointing. Infectious disease models have demonstrated that T-cell clonotypes that recognise the same antigen should not be viewed as being equally effective. Extrapolation of this notion to UCV would suggest that the quality of response in terms of the T-cell receptor (TCR) clonotypes induced might be more important than the quantity of the response. Unfortunately, there is little opportunity to assess the effectiveness of individual T-cell clonotypes in vivo. Here, we identified effective, persistent T-cell clonotypes in an HLA A2+ patient following successful tumour infiltrating lymphocyte (TIL) therapy. One such T-cell clone was used to generate super-agonist altered peptide ligands (APLs). Further refinement produced an APL that was capable of inducing T-cells in greater magnitude, and with improved effectiveness, from the blood of all 14 healthy donors tested. Importantly, this APL also induced T-cells from melanoma patient blood that exhibited superior recognition of the patient's own tumour compared to those induced by the natural antigen sequence. These results suggest that use of APL to skew the clonotypic quality of T-cells induced by cancer vaccination could provide a promising avenue in the hunt for the UCV “magic bullet.”

Altered peptide ligands can modify the Th2 T cell response to the immunodominant 161-175 peptide of LACK ( Leishmania homolog for the receptor of activated C kinase)

Following Leishmania major infection, the early LACK ( Leishmania homolog of receptors for activated C kinase)-induced IL-4 response appears to determine disease susceptibility in BALB/c mice. Therefore, we sought to manipulate the pathogenic T cell responses to the immunodominant epitope with the use of altered peptide ligands (APLs). Conservative and non-conservative substitutions for each amino acid of the LACK 161-175 peptide determinant were tested for their stimulatory capacity in four different LACK-reactive T cell systems. From these results, we propose a likely LACK 163-171/I-A d core peptide register and show that APLs with changes at putative T cell receptor (TCR) contacts provide the greatest potential for immune deviation. In particular, the TCR-contact H164V APL expanded Th1 cells upon in vitro recall of naïve splenocytes from LACK-specific BV4 T cell receptor transgenic mice and stimulated IFN-γ secretion from a Th2-committed LACK-reactive T cell line. We also observed that non-conservative substitutions flanking the core determinant had strong agonistic effects for proliferation and Th1/Th2 modulation. However, upon immunization, the H164V APL considerably downregulated proliferation and cytokine responses to the wild type LACK 161-175 peptide, while immunization with the weak agonist, MHC contact APL S171K, increased the IFN-γ/IL-4 ratio to the wild type peptide. In these instances, a hyporesponsive T cell response to the wild-type peptide was achieved by immunizing with an APL possessing non-conservative substitutions at TCR contact sites, while immune deviation was accomplished using a weak-agonist APL that retained the core determinant. Thus, certain LACK-APLs are able to induce T cell responses with a protective phenotype in an infectious disease such as leishmaniasis.

A high‐throughput genetic system for identifying enhanced altered peptide ligands of tumor associated antigens: implications for enhancing adoptive immunotherapy

Cancer immunotherapy offers a promising approach for eradicating metastatic tumors that fail to fully respond to existing therapies. Unfortunately, efforts are frequently hampered by tolerance mechanisms that act to prevent autoimmunity. A potential means for circumventing these mechanisms is the use of altered peptide ligands (APL). APLs are mutant T cell epitopes that can either enhance or dampen a T cell response. Existing methods for identifying effective APLs are either limited in scope or are expensive and technically restrictive. The objective of this study is to develop a comprehensive genetic approach for identifying enhanced APLs of CTL epitopes. With this procedure every point mutant of a CTL epitope can be generated and screened, and the effective APL easily identified. In this study we utilize saturation mutagenesis to independently mutagenize every codon of Mart‐126–35 A27L. Recombinant mini‐gene products of the APLs are cross‐presented to Mart‐126–35 ‐specific CTL clones via dendritic cells in a 96‐well based assay format. Here we identify three novel superagonist peptides of Mart‐126–35, which can generate higher frequencies of Mart‐126–35 ‐specific CTL from peripheral blood leukocytes of melanoma patients. This work has implications for the identification of APLs that may prove clinically beneficial in generating tumor‐specific CTLs for use in adoptive T cell immunotherapy.

Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

Truncation of the neuritogenic peptide bP2(60–70) results in the generation of altered peptide ligands with the potential to interfere with T cell activation

Due to the central role of T cells in the pathogenesis of inflammatory diseases of the peripheral nervous system like the Guillain–Barré syndrome, specific immunotherapies aim at modifying T cell responses. Use of truncated mutants of the neuritogenic peptide of myelin basic protein (MBP) has been shown to anergize autoreactive T cells and to reverse experimental autoimmune encephalitis (EAE). To establish a rationale basis for the use of altered peptide ligands (APLs) in the treatment of autoimmune diseases we designed a set of N- and C-terminally truncated mutants of the minimal experimental autoimmune neuritis (EAN) inducing bovine P2 (bP2) (60–70) peptide and compared them for the ability to induce immune responses and T cell receptor (TCR) cell signaling. Truncated peptides bound to MHC class II molecules and induced TCR internalization and expression of interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) with decreasing potency. None of the shortened mutants elicited a proliferative response in P2-specific T cells. Stimulation of these antigen-specific T cells with peptide bP2(62–69) using antigen presenting cells (APCs) prepulsed with bP2(60–70) resulted in a significant decrease of the proliferative response. In agreement with the observed effects on T cell activation, analysis of TCR signaling demonstrated a lack of CD3ε phosphorylation and MAPK activation. Moreover, repeated injection of bP2(62–69) significantly slowed progression of adoptive transfer EAN (AT-EAN). Taken together, these findings strongly suggest that peptide bP2(62–69) can favorably modulate the antigen-induced response of neuritogenic T cells.

Antigen-specific immunotherapy for autoimmune disease: fighting fire with fire?

The defining characteristic of an autoimmune disease is the existence of T- and B-cell autoreactivity directed against self proteins (autoantigens). The early events that precipitate autoreactivity in human autoimmune disease remain obscure. Once established, the autoreactive process itself may or may not be directly relevant to the pathological events involved in the disease. In patients with established rheumatoid arthritis, for example, on-going joint damage is most likely the result of a self-perpetuating inflammatory process that has few features of tissue-specific autoreactivity. In type 1 diabetes mellitus (DM), on the other hand, autoreactivity directed against the target cell in the pancreas reaches its peak of intensity around the time of clinical diagnosis and typically wanes only after the target cell has been destroyed. These are examples at opposite ends of a continuum but most theoretical models used to explain autoimmune disease invoke a period of autoreactivity against a specified protein autoantigen(s) as a critical phase in disease development. Work in animal models indicates that this phase may be susceptible to regulation by administration of the inciting autoantigen. In this review several key questions will be addressed in relation to antigen-specific immunotherapy (ASI) for autoimmune disease. First, does it work in animal models, and by what mechanism(s) of action? How should we best harness these effects for human disease? How will we know when it has worked? And finally, will it work in established disease or will prophylaxis be required? In terms of autoantigen administration, we will focus mainly upon systemic rather than mucosal routes, which have been discussed extensively in other recent reviews.1,2 A particular emphasis will be placed upon the issues to be faced in transposing ASI as a therapeutic option from animal into clinical studies.

Use of altered peptide ligands to modulate immune responses as a possible immunotherapy for allergies

Allergies are dramatically increasing in prevalence, and the management of these diseases is a heavy burden on the health‐care systems of developed countries. In recent years, many efforts have been made to improve the therapy of allergies and to develop new approaches for immunotherapy. Here we briefly review the use of peptides to modulate T‐cell responses to allergens. We focus mainly on the possibility of using altered peptide ligands (APLs), i.e., peptides tailored on immunodominant T epitopes and bearing a single amino‐acid substitution, as a tool to modulate immune responses to allergens. These peptides may be recognized by the specific T cells triggered by the agonist peptides, but they are unable to elicit T‐cell responses; thus, they could be ideal candidates to modulate immune responses to allergens. The availability of these peptides could allow new approaches for immunotherapies.

Absence of co-stimulation and not the intensity of TCR signaling is critical for the induction of T cell unresponsiveness in vivo.

Understanding the mechanisms of T cell activation versus induction of unresponsiveness is of critical importance for the rational modulation of immune responses. Efficient T cell activation is critical for vaccination purposes, while the inhibition of T cell responses is potentially important for the ablation of autoimmune diseases. Modulation of co-stimulation and changing TCR-mediated signaling using altered peptide ligands (APL) have been shown to result in clonal T cell unresponsiveness. This study compares for the first time the efficiency of the two approaches for the induction of CD8+ T cell unresponsiveness in vivo for naive and memory T cells using TCR-transgenic mice. The results demonstrate that inhibition of CD28-mediated co-stimulation in the presence of a strong TCR-mediated signal most efficiently induces T cell unresponsiveness. In contrast, APL that are capable of weak TCR triggering fail to interfere with T cell responsiveness in vivo and are ignored by T cells. Thus, short-term blockage of CD28 during antigenic stimulation rather than the use of APL is the most promising way to actively down-modulate responsiveness of naive CD8+ T cells at least in the particular TCR-transgenic mouse model analyzed in this study.

Mechanisms of central and peripheral T-cell tolerance: lessons from experimental models of multiple sclerosis.

Studies of experimental autoimmune encephalomyelitis in conventional and transgenic mouse models have clarified mechanisms of central and peripheral tolerance to myelin antigens. It is now clear that myelin antigens are expressed in the thymus and their expression can influence generation of the potential autoimmune T-cell repertoire. How autoreactive T cells escape tolerance in the thymus is largely unclear. One mechanism has been revealed through the use of a transgenic mouse expressing a T-cell receptor specific for a myelin antigen. T cells specific for the N-terminal epitope of myelin basic protein escape tolerance through low avidity interaction. The affinity of antigen binding to MHC also proves to be important for induction of peripheral tolerance. Peptides may be administered in solution to adults in order to reinstate peripheral tolerance and suppress disease. Induction of antigen-specific suppression with synthetic peptides can result in either linked or bystander suppression and this appears to involve the generation of T cells secreting suppressive cytokines. The use of altered peptide ligands for induction of peripheral tolerance has been investigated. This can be achieved but the complexity of the approach argues against its use for treatment of human autoimmune diseases.

Therapeutic potential of TCR antagonists is determined by their ability to modulate a diverse repertoire of autoreactive T cells.

The use of altered peptide ligands (APL) with TCR antagonist properties holds promise as an antigen-specific therapy for autoimmune disorders. We are investigating the therapeutic potential of APL in experimental autoimmune encephalomyelitis (EAE) using the Ac1-9 peptide of myelin basic protein in H-2u mice. Encephalitogenic T cells recognize Ac1-9 using residues 3Gln and 6Pro as the major TCR contact sites. Use of position 6 APL is compromised by the heterogeneous nature of the Ac1-9-specific repertoire. Here we identify two position 3 APL that act as TCR antagonists on transgenic T cells expressing Ac1-9-specific TCR and that inhibit EAE in H-2u mice. However, the therapeutic capacity of these two APL correlated directly with the ability to maximally inhibit activation of a heterogeneous T cell pool. The implications of these findings for the requirements for EAE induction, the relative contribution of a given T cell subpopulation to pathology and the mechanism underlying EAE inhibition in this model are discussed.

Fine Specificity of the Myelin-Reactive T Cell Repertoire: Implications for TCR Antagonism in Autoimmunity

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1-9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1-9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1-9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.

Strongly increased efficiency of altered peptide ligands by mannosylation.

Altered peptide ligands (APL) have the potential to modulate pathogenic T cell reactivity. High concentrations of APL, however, are required to achieve efficient blocking of the T cell response. We have therefore investigated whether improved delivery of APL to professional antigen-presenting cells (APC) can lead to more efficient application of such peptides. For this purpose APL were bis-mannosylated in order to facilitate their uptake by mannose receptor-positive dendritic cells (DC) in vitro. We present evidence that a 100- to 1000-fold lower concentration of bis-mannosylated APL was sufficient for complete blocking of the proliferative T cell response against the agonist peptide compared to the non-mannosylated APL. Moreover, bis-mannosylated APL were similarly effective in the inhibition of the T cell response against whole protein antigens. In contrast, unrelated, bis-mannosylated class II binding peptides were ineffective, indicating that the increased efficiency of the mannosylated APL was not due to competition for binding to class II molecules. Furthermore, a strong increase in the efficiency of presentation of APL was also observed when macrophages and peripheral blood mononuclear cells were used as APC. Thus, bis-mannosylation of APL greatly increases their potency to inhibit proliferative T cell responses. Moreover, it is likely that the use of bis-mannosylated APL will result in preferential presentation by mannose receptor-positive, professional APC. These results may be of relevance for more effective use of APL for immunoregulation in vivo.

A gene therapy approach to treatment of autoimmune disease.

New insights into the underlying mechanisms for the development of autoimmune diseases in humans and various animal models continue to increase with our understanding of factors that drive polarization of T helper (Th) responses and tolerance. This information has led to the development of new treatment strategies, including oral tolerance clinical trails and the use of altered peptide ligands in animal models. These approaches have shown some promise and provided additional insight into the disease processes. The use of gene therapy in many disease states continues to increase. We are starting to see the application of gene therapy in chronic diseases in humans. Gene therapy has been used in several animal models of autoimmune disease with promising preliminary results. In this article, an overview will be provided for the use of gene therapy in autoimmune disease.