Cyril J. Cohen

Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC–peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC–peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC–peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC–peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen.

Critical Role for CD8 in Binding of MHC Tetramers to TCR: CD8 Antibodies Block Specific Binding of Human Tumor- Specific MHC-Peptide Tetramers to TCR

There are conflicting opinions about the role that the T cell coreceptors CD4 and CD8 play in TCR binding and activation. Recent evidence from transgenic mouse models suggests that CD8 plays a critical role in TCR binding and activation by peptide-MHC complex multimers (tetramers). Here we show with a human CTL clone specific for a tumor-associated MHC-peptide complex that the binding of tetramers to the TCR on these cells is completely blocked by anti-human CD8 Abs. Moreover, the staining of CTLs with specific MHC-peptide tetramers simultaneously with anti-CD8 Abs was completely blocked with three different anti-CD8 Abs. This blockage was mediated by anti-CD8 Abs but not anti-CD3 Abs and was dose dependent. The blocking effect of the anti-CD8 Abs was attributable to directly inhibiting tetramer binding and was not attributable to Ab-mediated TCR-CD8 internalization and down-regulation. Our results have important implications in TCR binding to MHC-peptide tetramers. MHC-peptide tetramers are widely used today in combination with anti-CD8 Abs for the phenotypic analysis of T cell populations and in the study of T cell responses under various pathological conditions such as infectious diseases and cancer. Our results indicate that also in the human system CD8 plays a critical role in the interaction of MHC-peptide multimers with TCR.

Increased Expression of Human T Lymphocyte Virus Type I (HTLV-I) Tax11-19 Peptide–Human Histocompatibility Leukocyte Antigen A*201 Complexes on CD4+ CD25+T Cells Detected by Peptide-specific, Major Histocompatibility Complex–restricted Antibodies in Patients with HTLV-I–associated Neurologic Disease

Human T lymphocyte virus type I (HTLV-I)–associated chronic inflammatory neurological disease (HTLV-I–associated myelopathy/tropical spastic paraparesis [HAM/TSP]) is suggested to be an immunopathologically mediated disorder characterized by large numbers of HTLV-I Tax–specific CD8+ T cells. The frequency of these cells in the peripheral blood and cerebrospinal fluid is proportional to the amount of HTLV-I proviral load and the levels of HTLV-I tax mRNA expression. As the stimulus for these virus-specific T cells are immunodominant peptide–human histocompatibility leukocyte antigen (HLA) complexes expressed on antigen-presenting cells, it was of interest to determine which cells express these complexes and at what frequency. However, until now, it has not been possible to identify and/or quantify these peptide–HLA complexes. Using a recently developed antibody that specifically recognizes Tax11-19 peptide–HLA-A*201 complexes, the level of Tax11-19–HLA-A*201 expression on T cells was demonstrated to be increased in HAM/TSP and correlated with HTLV-I proviral DNA load, HTLV-I tax mRNA load, and HTLV-I Tax–specific CD8+ T cell frequencies. Furthermore, CD4+ CD25+ T cells were demonstrated to be the major reservoir of HTLV-I provirus as well as Tax11-19 peptide–HLA-A*201 complexes. These results indicate that the increased detection and visualization of peptide–HLA complexes in HAM/TSP CD4+ CD25+ T cell subsets that are shown to stimulate and expand HTLV-I Tax–specific CD8+ T cells may play an important role in the pathogenesis of HTLV-I–associated neurological disease.

Resistance to Adjuvant Arthritis Is Due to Protective Antibodies Against Heat Shock Protein Surface Epitopes and the Induction of IL-10 Secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.

Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies

The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC‐peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single‐chain human MHC (scMHC)‐peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA‐A2 in the presence of tumor‐associated or viral peptides. The scMHC molecule was composed of β2‐microglobulin connected to the first three domains of the HLA‐A2 heavy chain through a 15‐amino acid flexible linker. Highly purified scMHC‐peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART‐1 or a viral peptide derived from HTLV‐1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA‐A2‐restricted peptides. We also generated scMHC‐peptide tetramers, which were biologically functional; they induced a peptide‐specific CTL clone to be activated and secrete IFN‐γ, and were able to stain specifically CTL lines. Such recombinant soluble scMHC‐peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen‐specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments

NY‐ESO‐1 is a germ cell antigen aberrantly expressed by different tumor types that elicits strong humoral and cellular immune responses, representing one of the most promising candidates for vaccination of cancer patients. A detailed analysis of CD8+ T cells generated in vaccine trials using NY‐ESO‐1‐derived peptides (157–165 and 157–167) revealed that the dominant immune response was directed against a cryptic epitope (159–167) diverting the immune response from tumor recognition. Only CTL reactivity to the NY‐ESO‐1157–165 peptide appeared to be capable of lysing NY‐ESO‐1/HLA‐A0201‐expressing tumor cells. To study the process of NY‐ESO‐1 peptide presentation by tumor cells in more detail we generated a high‐affinity (KD=60 nM) antibody fragment that specifically recognizes the NY‐ESO‐1157–165 peptide/HLA‐A0201 complex. Peptide variants such as the NY‐ESO‐1157–167 peptide or the cryptic NY‐ESO‐1159–167 peptide were not recognized. The antibody fragment blocked in a dose‐dependent fashion the recognition of NY‐ESO‐1/HLA‐A2‐positive tumor cells by NY‐ESO‐1157–165 peptide‐specific CD8+ T cells. This antibody fragment is a novel reagent that binds with TCR‐like specificity to the NY‐ESO‐1157–165/HLA‐A2 complex thus distinguishing between CTL responses against immunological meaningful or cryptic NY‐ESO‐1‐derived peptides. It may therefore become a useful monitoring tool for the development of NY‐ESO‐1‐based cancer vaccines.

Tax and M1 Peptide/HLA-A2-Specific Fabs and T Cell Receptors Recognize Nonidentical Structural Features on Peptide/HLA-A2 Complexes

Both TCRs and Ab molecules are capable of MHC-restricted recognition of peptide/MHC complexes. However, such MHC restriction is the predominant mode of recognition by T cells, but is extremely rare for B cells. The present study asks whether the dichotomy in Ag recognition modes of T and B cells could be due to fundamental differences in the methods by which TCRs and Abs recognize peptide/MHC complexes. We have compared MHC and peptide recognition by panels of CTL lines specific for the Tax and M1 peptides presented by HLA-A2 plus Tax and M1 peptide/HLA-A2-specific human Fabs that were selected from a naive phage display library. Collectively, the results indicate both striking similarities and important differences between Fab and TCR recognition of MHC and peptide components of the Tax and M1/HLA-A2 complexes. These findings suggest that these two classes of immunoreceptors have solved the problem of specific recognition of peptide/MHC complexes by nonidentical mechanisms. This conclusion is important in part because it indicates that Ab engineering approaches could produce second-generation Ab molecules that more closely mimic TCR fine specificity. Such efforts may produce more efficacious diagnostic and therapeutic agents.

Extended presentation of specific MHC-peptide complexes by mature dendritic cells compared to other types of antigen-presenting cells.

Dendritic cells are known as the most potent antigen‐presenting cells for the induction of T cell‐mediated immune responses. To discriminate between the presentation of antigens and the co‐stimulatory aspects of this high immunostimulatory capacity, we used recombinant antibodies with T cell receptor‐like specificity to detect defined MHC‐peptide complexes on living cells. Mature human dendritic cells (mDC) were compared with immature DC (iDC), monocytes, CD4+ T lymphocytes, melanoma cells, T2 cells and B lymphoblastoid cells for their capacity to present MHC class I‐restricted tumor‐associated T cell epitopes and were found to display the specific peptides two to six times longer than other cells. The most short‐lived peptide had an average half‐life of 8.7 h on mDCvs. 3.5 h on B lymphoblastoid cells, while the most long‐lived peptide had a half‐life of 118.5 h vs. 20.7 h on these two cell types. The decay kinetics of specific MHC‐peptide complexes on iDC were among the fastest observed. The high potency of dendritic cells to induce specific T cell responses is thus based, in addition to the expression of co‐stimulatory molecules, on an extended antigenic memory, which increases the likelihood and the extent of contacts between dendritic cells and antigen‐specific T cells.

Antitumor Activity of Immunotoxins with T-Cell Receptor–like Specificity against Human Melanoma Xenografts

In this study, we have explored the use of Fab-toxin proteins (immunotoxin) to target antigen-specific MHC-peptide complexes of in vitro and in vivo cancer cells. A human phage display library was used to screen for T-cell receptor (TCR)-like antibodies that are highly specific for the peptide melanoma-associated antigen MART-1(26-35) presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100(280-288) presented by HLA-A201. The recombinant immunotoxin constructs were generated by fusing the targeting Fab fragment to a truncated form of Pseudomonas exotoxin, PE38KDEL. These immunotoxins bound with high affinity to the EBV-transformed JY cell line pulsed with the aforementioned peptides and internalized within 30 min. A significant inhibition of protein synthesis, which resulted in cell death, was detected at 24 h. MART-1-specific and gp100-specific immunotoxins bound and killed HLA-A201 melanoma MART-1(+) and gp100(+) cell lines that were presented at natural levels but do not bind to HLA-A201(-) or to HLA-A201(+) MART-1(-) and gp100(-) cell lines. In severe combined immunodeficient mice, MART-1 and gp100 immunotoxins significantly and discriminately inhibited human melanoma growth. These results show that MHC class I/peptide complexes can serve as a specific target for passive immunotherapy of cancer.