Preclinical and clinical development of neoantigen vaccines.

Cancer neoantigens are antigens that result from somatic mutations present in individual cancers. Neoantigens are considered important targets for cancer immunotherapy because of their immunogenicity and lack of expression in normal tissues. Next-generation sequencing technologies and computational analysis have recently made neoantigen discovery possible. Although neoantigens are important targets of checkpoint blockade therapy, neoantigen vaccines are currently being investigated in preclinical models and early-phase human clinical trials. Preliminary results from these clinical trials demonstrate that dendritic cell, synthetic long peptide, and RNA-based neoantigen vaccines are safe, and capable of inducing both CD8+ and CD4+ neoantigen-specific T-cell responses. We and others are testing neoantigen vaccines in melanoma, breast cancer, non-small-cell lung cancer and other cancer types. Since cancers have evolved mechanisms to escape immune control, it is particularly important to study the efficacy of neoantigen vaccines in combination with other immunotherapies including checkpoint blockade therapy, and immune therapies targeting the immunosuppressive tumor microenvironment.

A double-negative feedback loop between EpCAM and ERK contributes to the regulation of epithelial-mesenchymal transition in cancer.

Epithelial-mesenchymal transition (EMT) is an important biological process that has been implicated in cancer metastasis. Epithelial cell adhesion molecule (EpCAM) is expressed at the basolateral membrane of most normal epithelial cells but is overexpressed in many epithelial cancers. In our studies on the role of EpCAM in cancer biology, we observed that EpCAM expression is decreased in mesenchymal-like primary cancer specimens in vivo and following induction of EMT in cancer cell lines in vitro. Extracellular signal-related kinase (ERK) is a key regulator of EMT. We observed that EpCAM expression is decreased with activation of the ERK pathway in primary cancer specimens in vivo and in cancer cell lines in vitro. In experimental models, growth factor stimulation and/or oncogene-induced ERK2 activation suppressed EpCAM expression, whereas genetic or pharmacological inhibition of the ERK pathway restored EpCAM expression. In detailed studies of the EpCAM promoter region, we observed that ERK2 suppresses EpCAM transcription directly by binding to a consensus ERK2-binding site in the EpCAM promoter and indirectly through activation of EMT-associated transcription factors SNAI1, SNAI2, TWIST1 and ZEB1, which bind to E-box sites in the EpCAM promoter. Surprisingly, EpCAM appears to modulate ERK activity. Using multiple cell lines, we demonstrated that specific ablation of EpCAM resulted in increased ERK pathway activity and SNAI2 expression, migration and invasion, whereas forced expression of EpCAM resulted in decreased ERK pathway activity and SNAI2 expression, migration and invasion. These observations provide important insights into the regulation of EpCAM expression during EMT, demonstrate an unexpected role for EpCAM in the regulation of ERK and define a novel double-negative feedback loop between EpCAM and ERK that contributes to the regulation of EMT. These studies have important translational implications as both EpCAM and ERK are currently being targeted in human clinical trials.

Identification and translational validation of novel mammaglobin-A CD8 T cell epitopes.

Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80 % of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for a breast cancer prevention vaccine, and we recently initiated a phase 1 clinical trial of a MAM-A DNA vaccine. Previously, we have identified multiple MAM-A CD8 T cell epitopes using a reverse immunology candidate epitope approach based on predicted binding, but to date no attempt has been made to identify epitopes using an unbiased approach. In this study, we used human T cells primed in vitro with autologous dendritic cells expressing MAM-A to systematically identify MAM-A CD8 T cell epitopes. Using this unbiased approach, we identified three novel HLA-A2-restricted MAM-A epitopes. CD8 T cells specific for these epitopes are able to recognize and lyse human breast cancer cells in a MAM-A-specific, HLA-A2-dependent fashion. HLA-A2(+)/MAM-A(+) breast cancer patients have an increased prevalence of CD8 T cells specific for these novel MAM-A epitopes, and vaccination with a MAM-A DNA vaccine significantly increases the number of these CD8 T cells. The identification and translational validation of novel MAM-A epitopes has important implications for the ongoing clinical development of vaccine strategies targeting MAM-A. The novel MAM-A epitopes represent attractive targets for epitope-based vaccination strategies, and can also be used to monitor immune responses. Taken together these studies provide additional support for MAM-A as an important therapeutic target for the prevention and treatment of breast cancer.

Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer.

Recent studies in multiple epithelial cancers have shown that the inhibitory receptor programmed cell death 1 (PD-1) is expressed on tumor-infiltrating lymphocytes and/or programmed death ligand 1 (PD-L1) is expressed on tumor cells, suggesting that antitumor immunity may be modulated by the PD-1/PD-L1 signaling pathway. In addition, phase 1 clinical trials with monoclonal antibodies targeting PD-1 or PD-L1 have shown promising results in several human cancers. The purpose of this study was to investigate the impact of PD-L1 expression in human breast cancer specimens. We conducted an immunohistochemistry study using a tissue microarray encompassing 650 evaluable formalin-fixed breast cancer cases with detailed clinical annotation and outcomes data. PD-L1 was expressed in 152 (23.4 %) of the 650 breast cancer specimens. Expression was significantly associated with age, tumor size, AJCC primary tumor classification, tumor grade, lymph node status, absence of ER expression, and high Ki-67 expression. In univariate analysis, PD-L1 expression was associated with a significantly worse OS. In multivariate analysis, PD-L1 expression remained an independent negative prognostic factor for OS. In subset analyses, expression of PD-L1 was associated with significantly worse OS in the luminal B HER2(-) subtype, the luminal B HER2(+) subtype, the HER2 subtype, and the basal-like subtype. This is the first study to demonstrate that PD-L1 expression is an independent negative prognostic factor in human breast cancer. This finding has important implications for the application of antibody therapies targeting the PD-1/PD-L1 signaling pathway in this disease.

The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer.

Programmed death 1 (PD-1) is a co-inhibitory receptor in the CD28/CTL-4 family, and functions as a negative regulator of the immune system. Tumor-infiltrating lymphocytes (TIL) in many epithelial cancers express PD-1, suggesting that antitumor immunity may be modulated by the PD-1/PD-L1 signaling pathway, and promising results from two recent clinical trials with monoclonal antibodies targeting PD-1 or PD-L1 confirm the clinical relevance of this pathway in human cancer. To explore the role of PD-1(+) TIL in human breast cancer, we performed immunohistochemistry studies on a tissue microarray encompassing 660 breast cancer cases with detailed clinical annotation and outcomes data. PD-1(+) TIL were present in 104 (15.8 %) of the 660 breast cancer cases. Their presence was associated with tumor size, grade, and lymph node status, and was differentially associated with the intrinsic subtypes of breast cancer. In univariate survival analyses, the presence of PD-1(+) TIL was associated with a significantly worse overall survival (HR = 2.736, p < 0.001). In subset analyses, the presence of PD-1(+) TIL was associated with significantly worse overall survival in the luminal B HER2(-) subtype (HR = 2.678, p < 0.001), the luminal B HER2(+) subtype (HR = 3.689, p < 0.001), and the basal-like subtype (HR = 3.140, p < 0.001). This is the first study to demonstrate that the presence of PD-1(+) TIL is associated with poor prognosis in human breast cancer, with important implications for the potential application of antibody therapies targeting the PD-1/PD-L1 signaling pathway in this disease.

EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2(+), basal-like, and HER2 intrinsic subtypes of breast cancer.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is frequently expressed in breast cancer, and its expression has been associated with poor prognosis. Breast cancer can be subdivided into intrinsic subtypes, differing in prognosis and response to therapy. METHODS: To investigate the association between EpCAM expression and prognosis in the intrinsic subtypes of breast cancer, we performed immunohistochemical studies on a tissue microarray encompassing a total of 1365 breast cancers with detailed clinicopathological annotation and outcomes data. RESULTS: We observed EpCAM expression in 660 out of 1365 (48%) cases. EpCAM expression varied significantly in the different intrinsic subtypes. In univariate analyses of all cases, EpCAM expression was associated with a significantly worse overall survival. In the intrinsic subtypes, EpCAM expression was associated with an unfavourable prognosis in the basal-like and luminal B HER2(+) subtypes but associated with a favourable prognosis in the HER2 subtype. Consistently, specific ablation of EpCAM resulted in increased cell viability in the breast cancer cell line SKBR3 (ER(-), PR(-), and HER2(+)) but decreased viability in the breast cancer cell line MDA-MB-231 (ER(-), PR(-), and HER2(-) ). CONCLUSION: The differential association of EpCAM expression with prognosis in intrinsic subtypes has important implications for the development of EpCAM-targeted therapies in breast cancer.

PTP1B expression is an independent positive prognostic factor in human breast cancer.

Protein tyrosine phosphatase 1B (PTP1B) is a non-transmembrane protein tyrosine phosphatase that has come into focus as a critical regulator of multiple signaling pathways. The role of PTP1B in breast cancer remains unclear with evidence suggesting that PTP1B can exert both tumor-suppressing and tumor-promoting effects. To better define the role of PTP1B in human breast cancer, and its relationship with HER2, we performed immunohistochemical studies on a large cohort of functionally annotated primary breast cancer specimens. 683 of 1,402 (49 %) evaluable primary breast cancers are positive for PTP1B. There is no statistically significant association between PTP1B expression and age, tumor size, T stage, histologic grade, lymph node status, or histological subtype. Of note, there is no significant association between PTP1B expression and HER2 expression (PTP1B expression 53.1 % in HER2(+) cancers vs. 47.5 % in HER2(-) cancers, p = 0.0985). However, PTP1B expression is significantly associated with estrogen receptor expression (PTP1B expression 50.7 % in ER(+) cancers vs. 43.1 % in ER(-) cancers, p = 0.0137) and intrinsic molecular subtype (PTP1B expression 53.9 % in the luminal B HER2(+) subtype and 37.9 % in the basal-like subtype). Of note, multivariate analyses demonstrate that PTP1B is an independent predictor of improved survival in breast cancer (HR 0.779, p = 0.006). Taken together, we demonstrate in the largest study to date that (1) PTP1B is commonly expressed in breast cancer, (2) there is no association or functional impact of PTP1B expression in HER2(+) breast cancer, and (3) PTP1B expression in breast cancer is associated with significantly improved clinical outcome. Until additional studies are performed, caution should be exercised in using PTP1B inhibitors in human breast cancer.

Myeloid-derived suppressor cells: general characteristics and relevance to clinical management of pancreatic cancer.

Recent studies describe a heterogeneous population of cells of the myeloid lineage, termed myeloid derived suppressor cells (MDSC), which are observed with increased prevalence in the peripheral blood and tumor microenvironment of cancer patients, including pancreatic cancer. Accumulation of MDSC in the peripheral circulation has been related to extent of disease, and correlates with stage. MDSC have primarily been implicated in promoting tumor growth by suppressing antitumor immunity. There is also compelling evidence MDSC are also involved in angiogenesis and metastatic spread. Two main subsets of MDSC have been identified in cancer patients: a monocytic subset, characterized by expression of CD14, and a granulocytic subset characterized by expression of CD15. Both subsets of MDSC actively suppress host immunity through a variety of mechanisms including production of reactive oxygen species and arginase. Just as in humans, accumulation of monocytic and granulocytic MDSC has been noted in the bone marrow, spleen, peripheral circulation, and tumors of tumor bearing mice. Successful targeting of MDSC in mice is associated with improved immune responses, delayed tumor growth, improved survival, and increased efficacy of vaccine therapy. By further elucidating mechanisms of MDSC recruitment and maintenance in the tumor environment, strategies could be developed to reverse immune tolerance to tumor. We discuss here what is currently known about MDSC as well as some potential strategies targeting MDSC in the context of our work on pancreatic cancer and recent literature. Due to the number of new reports on MDSC, the most pertinent ones have been selected.

Loss of T cell-mediated antitumor immunity after construct-specific downregulation of retrovirally encoded T-cell receptor expression in vivo.

Adoptive T-cell therapy is clinically efficacious in the treatment of select cancers. However, it is often difficult to obtain adequate numbers of tumor-specific T cells for therapy. One method for overcoming this limitation is to generate tumor-specific T cells by retrovirally mediated T-cell-receptor (TCR) gene transfer. However, despite instances of therapeutic success, major obstacles remain, including attaining the survival of retrovirally modified T cells in vivo as well as inducing long-term and multi-gene retroviral expression. Using a murine model of adoptively transferred retrovirally modified CD8(+) T cells, where antitumor immunity was dependent on sustained, multigene expression, we found that in vitro assays are poor indicators of in vivo efficacy. Despite persisting for over 9 months in a nonlymphopenic environment, genetically modified T cells exhibited discordant retrovirally mediated gene expression in vivo not readily evident from initial in vitro assays. In particular, one of the two TCR subunit genes necessary for antigen specificity was selectively lost in vivo. As this discordant gene expression was associated with the loss of antitumor immunity, consideration of these findings may provide guidance in the design, evaluation and application of retroviral vectors for use in the treatment of cancer and other human disease.

Prostate-Specific Ets (PSE) factor: a novel marker for detection of metastatic breast cancer in axillary lymph nodes.

Prostate Specific Ets factor is a recently identified transcriptional activator that is overexpressed in prostate cancer. To determine whether this gene is overexpressed in breast cancer, we performed a virtual Northern blot using data available online at the Cancer Genome Anatomy Project website. Ninety-five SAGE libraries were probed with a unique sequence tag to the Prostate Specific Ets gene. The results indicate that Prostate Specific Ets is expressed in 14 out of 15 breast cancer libraries (93%), nine out of 10 prostate cancer libraries (90%), three out of 40 libraries from other cancers (7.5%), and four out of 30 normal tissue libraries (13%). To determine the possibility that the Prostate Specific Ets gene is a novel marker for detection of metastatic breast cancer in axillary lymph nodes, quantitative real-time RT-PCR analyses were performed. The mean level of Prostate Specific Ets expression in lymph nodes containing metastatic breast cancer (n=22) was 410-fold higher than in normal lymph node (n=51). A receiver operator characteristic curve analysis indicated that Prostate Specific Ets was overexpressed in 18 out of 22 lymph nodes containing metastatic breast cancer (82%). The receiver operator characteristic curve analysis also indicated that the diagnostic accuracy of the Prostate Specific Ets gene for detection of metastatic breast cancer in axillary lymph nodes was 0.949. These results provide evidence that Prostate Specific Ets is a potentially informative novel marker for detection of metastatic breast cancer in axillary lymph nodes, and should be included in any study that involves molecular profiling of breast cancer.

Sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific T-cell response.

The recent identification and molecular characterization of tumor antigens provides the opportunity to explore the rational development of peptide-based cancer vaccines. However, the response to these vaccines remains variable, and peptide-based cancer vaccines may even produce tolerance induction and enhanced tumor growth. The authors have developed a unique method for the isolation of a polysaccharide polymer of chemically pure poly- N -acetyl glucosamine (p-GlcNAc). This highly purified polysaccharide can be formulated into a stable gel matrix (designated F2 gel matrix) with unique properties of a sustained-release delivery system that has previously been shown to be an effective immune adjuvant. F2 gel matrix is capable of providing sustained release of antigenic peptide and cytokine in vitro. The purposes of this study were to characterize the ability of F2 gel matrix to provide sustained local release of cytokines in vivo and to test the hypothesis that such sustained release can enhance the microenvironment for antigen presentation, leading to a more effective antitumor response. Subcutaneous administration of F2 gel/cytokine matrix resulted in sustained release of cytokine at the vaccine site for up to 120 hours. Sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with an increased inflammatory infiltrate at the vaccine site and enhanced dendritic cell activation. Further, accination with F2 gel/SIINFEKL/GM-CSF matrix resulted in enhanced antigen-specific immunity. Addition of GM-CSF to the F2 gel matrix resulted in an increase in the percentage of antigen-specific T cells in the draining lymph nodes, enhanced cytotoxicity, a sustained presence of antigen-specific T cells in the peripheral blood, and protection from E.G7 tumor challenge. These results support the potential of an F2 gel matrix modular vaccine delivery system that can provide sustained local release of cytokine in vivo, and confirm the powerful effects of GM-CSF as an immune adjuvant.

Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel.

Real-time RT-PCR is a relatively new technology that uses an online fluorescence detection system to determine gene expression levels. It has the potential to significantly improve detection of breast cancer metastasis by virtue of its exquisite sensitivity, high throughput capacity and quantitative readout system. To assess the utility of this technology in breast cancer staging, we determined the relative expression levels of 12 cancer-associated genes (mam, PIP, mamB, CEA, CK19, VEGF, erbB2, muc1, c-myc, p97, vim and Ki67) in 51 negative-control normal lymph nodes and in 17 histopathology-positive ALNs. We then performed a receiver operating characteristic (ROC) curve analysis to determine the sensitivity and specificity levels of each gene. Areas under the ROC curve indicated that the most accurate diagnostic markers were mam (99.6%), PIP (93.3%), CK19 (91.0%), mamB (87.9%), muc1 (81.5%) and CEA (79.4.0%). mam was overexpressed in 16 of 17 lymph nodes known to contain metastatic breast cancer at levels ranging from 22- to 2.8 x 10(5)-fold above normal mean expression, whereas PIP was overexpressed from 30- to 2.2 x 10(6)-fold above normal in 13 lymph nodes. Real-time RT-PCR analysis of pathology-negative LN from breast cancer patients revealed evidence of overexpression of PIP (6 nodes), mam (3 nodes) and CEA (1 node) in 8 of 21 nodes (38%). Our results provide evidence that mam, PIP, CK19, mamB, muc1 and CEA can be applied as a panel for detection of metastatic and occult micrometastatic disease.

Melanoma thickness and histology predict sentinel lymph node status.

BACKGROUND: It remains unclear which patients with melanoma will benefit most from lymphatic mapping and sentinel lymphadenectomy. The purpose of this study is to determine whether primary melanoma histopathologic features could be applied to predict sentinel node status. METHODS: One hundred twelve patients underwent sentinel node biopsy between May 1995 and August 1999. Reported histologic features were assessed for predictive value by univariate and multivariate logistic regression. RESULTS: The sentinel node was located successfully in 105 of the 112 patients (94%). Twenty-one of these 105 patients (20%) had sentinel nodes that were positive for metastatic disease. Multivariate analyses revealed that tumor thickness greater than 1.5 mm (P = 0.01), ulceration (P <0.01), and lymphovascular invasion (P = 0.05) predicted the presence of micrometastases. CONCLUSIONS: The presence of unfavorable histopathology such as ulceration and lymphovascular invasion may identify a group of patients with thin melanomas who would benefit from sentinel lymphadenectomy.

Duodenopancreatic resections in patients with multiple endocrine neoplasia type 1.

OBJECTIVE: To review the authors' 7-year experience with a surgical approach for pancreatic and duodenal neuroendocrine tumors (NETs) in patients with multiple endocrine neoplasia type 1 (MEN 1) designed to remove all gross tumor with limited complications, preserving pancreatic function. SUMMARY BACKGROUND DATA: MEN 1 is an autosomal dominant familial neoplasia syndrome characterized by the development of NETs of the duodenum and pancreas. Some tumors are clinically insignificant or follow a benign course, although a subset pursues a malignant, lethal natural history; the risk of surgical management must be appropriate to the disease course. METHODS: The clinical, biochemical, genetic, and pathologic data were retrospectively reviewed for 21 consecutive MEN 1 patients undergoing pancreatic resection for NETs between 1993 and 1999 at one institution. Age at operation, presenting symptoms, results of preoperative and intraoperative localization studies, major and minor complications, and pathology, including metastases, were analyzed. RESULTS: The surgical approach was selected based on the location and size of the tumors. Five patients required pancreaticoduodenectomy, 11 patients underwent non-Whipple pancreatic resections, and 5 underwent simple enucleation of benign NETs. The incidence of regional lymph node metastases was 33%. CONCLUSIONS: Major pancreatic procedures can be performed safely in most patients with MEN 1 and NETs. Because NETs are the most common MEN 1-related cause of death in the authors' kindreds, an aggressive surgical approach, including early intervention before malignant spread and major pancreatic resection where indicated, appears justified.

Alloreactive and syngeneic CTL are comparably dependent on interaction with MHC class I alpha-helical residues.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.

Evidence for clonal deletion and clonal anergy after intrathymic antigen injection in a transplantation model.

Intrathymic (IT) antigen injection has been shown to induce antigen-specific systemic tolerance in the rodent. To delineate the mechanisms responsible for the induction of tolerance, we used the 2C line of T cell receptor transgenic mice. The majority of T cells in 2C mice express an antigen receptor specific for the major histocompatibility complex class I alloantigen Ld and can be identified with the clonotypic monoclonal antibody 1B2. IT injection of lymphoid cells expressing Ld was found to induce a significant prolongation in BALB/c skin allograft survival. The allograft prolongation was associated with a marked reduction in the number of developing 1B2+ thymocytes (clonal deletion), which occurred primarily at the CD4+ CD8+ stage of thymocyte development, as well as a reduction in the number of mature CD8+ 1B2+ 2C T cells in peripheral lymphoid tissue. In addition, CD8+ 1B2+ 2C T cells that survive deletion have decreased CD8 expression levels and a significantly reduced in vitro proliferative response to specific alloantigen (clonal anergy). Exogenous recombinant interleukin 2 restores the capacity of 2C T cells to respond in vitro to alloantigen. Experiments involving separation of cells by fluorescence-activated cell sorter indicate that there is a precise correlation between the reduction in CD8 expression and anergy induction. Collectively, these data indicate that IT antigen injection can induce antigen-specific systemic tolerance by both clonal deletion and clonal anergy.

Biochemical and functional characterization of soluble multivalent MHC L(d)/Fc gamma 1 and L(d)/Fc mu chimeric proteins loaded with specific peptides.

Central to the specificity of the immune system is the interaction between the T cell receptor and the major histocompatibility complex (MHC)-peptide ligand complex. To better understand the nature of this interaction, and to investigate possible avenues for specific therapeutic intervention, we have produced soluble recombinant molecules that can modulate antigen-specific T cells. Our approach involved the construction of recombinant murine genes composed of the MHC class I gene H-2L(d) and the Fc portion of immunoglobulin (Ig) heavy chain genes mu or gamma1. Stable transfectants of these L(d)/Fc gamma1 and L(d)/Fc mu genes generated correctly spliced transcripts and were capable of secreting chimeric protein. Immunoprecipitation analyses demonstrated the presence of chimeric L(d)/ Fc gamma1 and L(d)/Fc mu monomers of approximately 69 kDa and 90 kDa, respectively, as well as chimeric dimers under nonreducing conditions. The capacity of L(d)/Ig molecules to bind specific peptide ligands was demonstrated using radiolabeled peptides or with monoclonal reagents that specifically identify peptide-induced conformational changes in the L(d) ligand binding site. Soluble divalent L(d)/Fc gamma1 molecules were loaded with the murine cytomegalovirus-derived peptide and other L(d)-specific peptide ligands and subsequently isolated and purified. Peptide-loaded L(d)/Fc gamma1 molecules were capable of inhibiting the response of class I-restricted T cells in vitro in a peptide-specific fashion. The development of soluble multivalent chimeric proteins that possess unique properties of both the MHC class I and Ig molecules provides a valuable reagent for the study of potential mechanisms of in vitro and in vivo immune modulation.

Class I-restricted cytotoxic T cell recognition of split peptide ligands.

We developed a novel approach to probe the molecular basis of TCR recognition of the MHC class I-peptide complex and to determine how constraints placed on peptide binding by the class I molecule influence T cell recognition. We synthesized peptide pairs derived from the N- and C-terminal regions of class I peptide ligands in which the TCR contacts and dominant binding residues were placed together or were separated. Complementary peptide pairs derived from two well-characterized Ld peptide ligands, tum- (QNHRALDL) and p2Ca (LSPFPFDL), were tested for the ability to sensitize targets for recognition by peptide-specific cytotoxic T lymphocytes (CTL). The tum-derived tetramer QNHR, containing both primary TCR contact residues (H17 and R18), is recognized only when used in combination with ALDL which contains the primary binding residues (A19, D21 and L22). This suggests that both peptides of the pair contribute to positioning of the TCR contacts. Remarkably, CTL clone P24 recognized target cells sensitized with a trimer (QNH) combined with a pentamer (RALDL), demonstrating that TCR recognition can occur when the TCR contacts are separated (placed on separate peptide subunits). For the p2Ca peptide LSPFPFDL, the C-terminal tetramer PFDL, which contains both the primary TCR contact residue (P) and the dominant binding residue (L), is sufficient for recognition. In addition, PFDL was able to bind effectively to Ld and to activate naive antigen-specific T cells. These data suggest that peptide subunits and complementary peptide pairs composed of trimeric, tetrameric or pentameric peptides can bind independently to the Ld molecule in the same register and orientation as they do when contained within the parent peptide.

Definition of TCR recognition sites on Ld-tum- complexes.

The P911 variant of the P815 mastocytoma was shown by Lurquin et al. (Cell 58:293, 1989) to elicit rapid tumor rejection in a syngeneic host. This rejection was mediated by Ld-restricted cytotoxic T lymphocytes (CTL) for which targets could be sensitized by the synthetic peptide designated tum- (P91A-.12-24). In a previous study, T cell clones specific for Ld-tum- complexes displayed very restricted TCR usage and a characteristic TCR motif in the V alpha CDR3 region, predicted to interact with peptide. However, in contrast to the majority of Ld peptide ligands that are nonamers, the tum- peptide is a 13-mer and its sequence does not fit the Ld binding motif. Thus, to define shorter versions of the tum- 13-mer and residues involved in TCR recognition, nonamer derivatives were synthesized and compared in several different binding and functional assays. From these comparisons, the peptide TQNHRALDL was found to be the optimal nonamer. CTL recognition of Ala-substituted analogues of this peptide indicated that the His and Arg residues at positions 4 and 5 are important for TCR contact. We propose that these basic residues of the tum- peptide interact with the previously defined acidic residues in the CDR3 region of several TCR known to recognize Ld-tum- complexes.