Search

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-126-35 presented by HLA-A201. We also used previously selected TCR-like antibodies specific for the peptide melanoma-associated antigen gp100280-288 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. [Cancer Res 2008;68(15):6360–7]

Dendritic cells (DCs) play a key role in the induction of cellular immune responses by harvesting antigens from peripheral tissue for cross-priming CD8(+) T cells. It has been demonstrated that apoptotic bodies, whole- or degraded-cell-associated or soluble antigens as well as heat shock protein-bound peptides can be taken up, processed and cross-presented by DCs. Since cells are continuously releasing peptides from their surface MHC molecules, DCs in the tissues are exposed to such peptides and might process and present them to T cells as an additional pathway for cross-priming. To investigate this possibility, we compared and characterized the presentation of exogenous peptides by DCs and other cell types employing novel recombinant antibodies with TCR-like specificities for specific peptide-MHC complexes (pMHCs). These analyses reveal that loading of immature and mature DCs with peptide is far less efficient than it is for monocytes, T and B lymphocytes, B-lymphoblastoid, melanoma and TAP-deficient T2 cells. This inefficiency of peptide transfer to the MHC molecules of DCs makes it unlikely that these cells recycle peptides released from the MHC molecules of other cells and may explain why cross-presentation of such peptides has not yet been observed.

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.

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.

The recent advent of peptide–MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide–MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide–MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide–MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF γ) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide–MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.

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.

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 beta2-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-gamma, 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.

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-1(157-165) 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 (K(D)=60 nM) antibody fragment that specifically recognizes the NY-ESO-1(157-165) peptide/HLA-A0201 complex. Peptide variants such as the NY-ESO-1(157-167) peptide or the cryptic NY-ESO-1(159-167) 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-1(157-165) peptide-specific CD8(+) T cells. This antibody fragment is a novel reagent that binds with TCR-like specificity to the NY-ESO-1(157-165)/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.

Short, tandemly repeated DNA motifs, termed SSRs (simple sequence repeats) are widely distributed throughout eukaryotic genomes and exhibit a high degree of polymorphism. The availability of size-based methods for genotyping SSRs has made them the markers of choice for genetic linkage studies in all higher eukaryotes. These genotyping methods are not efficiently applicable to mononucleotide repeats (MNRs). Consequently, MNRs, although highly frequent in the genome, have generally been ignored as genetic markers. In contrast to single nucleotide polymorphisms (SNPs), SSRs can be identified in silico once the genomic sequence or segment of interest is available, without requiring any additional information. This makes possible ad-hoc saturation of a target chromosomal region with informative markers. In this context, MNRs appear to have much to offer by increasing the degree of marker saturation that can be obtained. By using the human genome sequence as a model, computational analysis demonstrates that MNRs in the size of 9-15 bp are highly abundant, with an average appearance every 2.9 kb, exceeding di- and tri-nucleotide SSRs frequencies by two- and five-fold, respectively. In order to enable practical, high throughput MNR genotyping, a rapid method was developed, based on sizing of fluorescent-labeled primer extension products. Genotyping of 16 arbitrarily chosen non-coding MNR sites along human chromosome 22 revealed that almost two-thirds (63%) of them were polymorphic, having 2-5 alleles per locus, with 20% of the polymorphic MNRs having more than two alleles. Thus, MNRs have potential for in silico saturation of sequenced eukaryote genomes with informative genetic markers.

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.

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 antigen-specific CD8+ T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells or detect antigen-presenting cells (APCs) in tissues. Here we describe a new approach that enables study of human class I peptide-MHC ligand-presentation as well as TCR-peptide-MHC interactions. Such studies are facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant antibodies directed toward a large variety of tumor-associated as well as viral T-cell epitope peptides. Using a large human antibody phage display library, a large panel of recombinant antibodies that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner was isolated. These antibodies were used to directly visualize the specific MHC-peptide complex on tumor cells, antigen-presenting cells or virus-infected cells by flow cytometry. They enabled direct quantitation of the number of MHC-peptide complexes as well as in situ detection of the complex on the surface of APCs after naturally occurring active intracellular processing of the cognate antigen. These studies will enable also the development of a new class of targeting molecules to deliver drugs or toxins to tumor or virus-infected cells. Thus, we demonstrate our ability to transform the unique fine specificity but low intrinsic affinity of TCRs into high-affinity soluble antibody molecules endowed with a TCR-like specificity toward human tumor or viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

The recent characterization of MHC-displayed tumor-associated antigens that recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy, as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, specific T-cell epitopes derived from the Mucin-1 tumor-associated antigen (MUC1) that are widely expressed in many cancers were identified and shown to be recognized by CTLs. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying an antigenic T-cell epitope derived from MUC1. High frequency of anti-MHC-peptide binders was observed (84%), and surprisingly, a high percentage (80%) of antibodies was fully specific for the MUC1 epitope. We isolated a surprisingly large panel of 16 different high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and breast tumor cells. Therefore, these findings demonstrate the ability to transform the unique fine specificity but low intrinsic affinity of T-cell receptors on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells for structure-function studies of T-cell receptor-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.

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.

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.

The recent characterization of MHC-displayed tumor-associated antigensthat recognize effector cells of the immune system has created new perspectives for cancer therapy. Antibodies that recognize these tumor-associated MHC-peptide complexes with the same specificity as the T-cell antigen receptor will therefore be valuable tools for immunotherapy as well as for studying antigen presentation in human cancers. Most tumor-associated antigens are expressed in only one or a few tumor types; however, recently specific T-cell epitopes derived from the telomerase catalytic subunit (hTERT) that are widely expressed in many cancers were identified and shown to be recognized by CTLs derived from cancer patients. We selected a large nonimmune repertoire of phage Fab antibodies on recombinant human class I HLA-A2 complexes displaying two distinct antigenic T-cell epitopes derived from hTERT. We isolated a surprisingly large panel of high-affinity human recombinant Fab antibodies that exhibited peptide-specific, MHC-restricted binding characteristics of T cells. The analyzed Fabs not only recognize the cognate MHC-peptide complex in a recombinant soluble form but also the native complex as displayed on the surface of antigen-presenting cells and hTERT-expressing tumor cells. These findings demonstrate for the first time the ability to transform the unique fine specificity but low intrinsic affinity of TCRs on T cells into high-affinity soluble antibody molecules endowed with a T-cell antigen receptor-like specificity. These molecules may prove to be very important and widely applicable for monitoring the expression of specific MHC-peptide complexes on the surface of tumor and immune cells, for structure-function studies of TCR-peptide-MHC interactions, as well as for developing new targeting agents for immunotherapy.

Cpn60-2 is a member of a unique family of putative molecular chaperones homologous to GroEL (Cpn60) but of unknown function and found only in Mycobacterium tuberculosis and closely related species. Cpn60-2 has mainly been studied for its strong immunogenity. Here, the purification, crystallization and preliminary crystallographic analysis of M. tuberculosis Cpn60-2 are reported. The crystals belong to space group P2, with unit-cell parameters a = 57, b = 115.5, c = 81.5 A, beta = 95.5 degrees, and contain a dimer in the asymmetric unit. The crystals diffract to 4.0 A using a Cu rotating-anode X-ray generator.

Recombinant Fv-immunotoxins are a new class of biologic anticancer agents composed of a recombinant antibody fragment linked to a very potent bacterial toxin. These potent molecules are designed to specifically bind and kill cancer cells that express a specific target antigen on their cell surface. Recombinant Fv-immunotoxins are an excellent example for the concept of rational drug design. They combine the progress in understanding cancer biology, -the recent knowledge on the mechanisms of malignant transformation and the special properties of cancer cells, -with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant Fv immunotoxins were developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro and in vivo in animal models. The excellent in vitro and in vivo activities of recombinant Fv-immunotoxins have lead to their pre-clinical development and to the initiation of clinical trial protocols. Recent trials have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment. It is thus suggested that this strategy can be developed into a separate modality of cancer treatment with the basic rationale of specifically targeting cancer cells on the basis of their unique surface markers combined with potent effective biological toxic agents that directly kill the cancer cell. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. In this review, we will describe the rationale in designing Fv-immunotoxins and will review current progress made in using these agents for cancer treatment.