Therapeutic vaccine generated by electrofusion of dendritic cells and tumour cells.

Immunotherapy with fusion of dendritic cells (DCs) and tumour cells potentially confers the advantages of DC antigen-presenting functionality and a continuous source of unaltered tumour antigens. However, fusion using chemical or viral fusogens has been inefficient. We have recently developed a high throughput electrofusion technique with which very efficient fusion rates (15-54%) were observed in over 300 experiments, using a variety of murine and human tumour cell lines. The fused cells display a mature DC phenotype and express tumour-associated antigens. In two pre-clinical animal models (B16 melanoma transduced with the LacZ gene and the MCA 205 fibrosarcoma), a single vaccination of mice bearing tumours established in the lung, brain and skin resulted in tumour regression and prolongation of life. However, therapeutic efficacy required the administration of adjuvants such as IL-12 and OX-40R mAbs. Effective immunotherapy also required the delivery of fusion cells directly into lymphoid organs (spleen or lymph nodes). Using five defined human T cell lines derived from melanoma patients, allogeneic DCs of HLA-A2, HLA-DR4 and HLA-DR7 haplotypes fused with MART-1, gp100, tyrosinase and TRP-2 expressing 888 mel melanoma cells were analysed for their ability to stimulate specific cytokine (IFN-gamma and GM-CSF) secretion. DC-888 mel hybrids presented all tumour-associated epitopes to both CD4 and CD8 T cell lines in the context of MHC class II and I molecules, respectively. The therapeutic efficacy of a DC-tumour fusion vaccine is now being evaluated for the treatment of metastatic melanoma.

Identification of a new shared HLA-A2.1 restricted epitope from the melanoma antigen tyrosinase.

Tyrosinase has many advantages as a target antigen for the immunotherapy of patients with melanoma because it is expressed in nearly all melanoma specimens with a high degree of cellular homogeneity, and its distribution in normal tissues is limited to melanocytes. To broaden our ability to direct cellular immune responses against this protein, we pursued an investigation to identify new shared human leukocyte antigen (HLA)-A2.1 restricted epitopes from tyrosinase. Peptides were synthesized that fit a permissive HLA-A2.1 binding motif and did not span common sites of polymorphism. The binding affinity of each peptide to HLA-A2.1 relative to a standard peptide with intermediate binding affinity was evaluated in a competitive inhibition assay. Twelve peptides were selected that had binding affinities within 80% of that of the standard peptide, and these were used to stimulate peripheral blood mononuclear cells (PBMC) in vitro from three HLA-A2.1+ patients with metastatic melanoma. Cytotoxic T lymphocytes that specifically recognized peptide-pulsed target cells as well as HLA-A2.1+ tyrosinase+ melanoma cells were raised from one patient with tyrosinase:8-17 (CLLWSFQTSA). To evaluate further the immunogenicity of this peptide, PBMC from 23 HLA-A2.1+ patients were stimulated in vitro with tyrosinase:8-17. Eleven bulk T-cell cultures demonstrated specific peptide recognition, and six of these also recognized HLA-A2.1+ tyrosinase+ melanoma cells. These data suggest that tyrosinase:8-17 may be clinically useful for the treatment of patients with melanoma.

Recombinant virus vaccination against "self" antigens using anchor-fixed immunogens.

To study the induction of anti-"self" CD8+ T-cell reactivity against the tumor antigen gp100, we used a mouse transgenic for a chimeric HLA-A*0201/H-2 Kb molecule (A2/Kb). We immunized the mice with a recombinant vaccinia virus encoding a form of gp100 that had been modified at position 210 (from a threonine to a methionine) to increase epitope binding to the restricting class I molecule. Immunogens containing the "anchor-fixed" modification elicited anti-self CD8+ T cells specific for the wild-type gp100(209-217) peptide pulsed onto target cells. More important, these cells specifically recognized the naturally presented epitope on the surface of an A2/Kb-expressing murine melanoma, B16. These data indicate that anchor-fixing epitopes could enhance the function of recombinant virus-based immunogens.

Antibodies to CD18 influence neutrophil migration through extracellular matrix.

Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another.

MHC class I-restricted recognition of a melanoma antigen by a human CD4+ tumor infiltrating lymphocyte.

It is generally considered that MHC class I-restricted antigens are recognized by CD8+ T cells, whereas MHC class II-restricted antigens are recognized by CD4+ T cells. In the present study, we report an MHC class I-restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. TIL 1383 I recognized HLA-A2+ melanoma cell lines but not autologous transformed B cells or fibroblasts. The antigen recognized by TIL 1383 I was tyrosinase, and the epitope was the 368-376 peptide. Antibody blocking assays confirmed that TIL 1383 I was MHC class I restricted, and the CD4 and CD8 coreceptors did not contribute significantly to antigen recognition. TIL 1383 I was weakly cytolytic and secreted cytokines in a pattern consistent with it being a Th1 cell. The avidity of TIL 1383 I for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. These CD4+ T cells may represent a relatively rare population of T cells that express a T-cell receptor capable of cross-reacting with an MHC class I/peptide complex with sufficient affinity to allow triggering in the absence of the CD4 coreceptor.

Identification of new melanoma epitopes on melanosomal proteins recognized by tumor infiltrating T lymphocytes restricted by HLA-A1, -A2, and -A3 alleles.

To isolate melanoma Ags recognized by T cells, cDNA libraries made from melanoma cell lines were screened with four CTLs derived from tumor infiltrating lymphocytes (TIL) that were able to recognize melanoma cells in a HLA-A1, -A2, or -A3 restricted manner. Although cDNAs encoding the previously identified melanoma Ags, tyrosinase and gp100, were isolated, these TIL were found to recognize previously unidentified peptides. An HLA-A1-restricted CTL, TIL1388, was found to recognize a tyrosinase peptide (SSDYVIPIGTY), and an HLA-A3-restricted CTL, TIL1351, recognized a gp100 peptide (LIYRRRLMK). CTL clones isolated from the HLA-A2-restricted TIL1383 recognized a gp100 peptide (RLMKQDFSV). HLA-A2-restricted CTL, TIL1200, recognized a gp100 peptide (RLPRIFCSC). Replacement of either cysteine residue with alpha-amino butyric acid in the gp100 peptide, RLPRIFCSC, enhanced CTL recognition, suggesting that the peptide epitope naturally presented on the tumor cell surface may contain reduced cysteine residues. Oxidation of these cysteines might have occurred during the course of the synthesis or pulsing of the peptide in culture. These modifications may have important implications for the development of efficient peptide-based vaccines. These newly identified peptide epitopes can extend the ability to perform immunotherapy using synthetic peptides to a broader population of patients, especially those expressing HLA-A1 or HLA-A3 for whom only a few melanoma epitopes have previously been identified.

Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2).

Tyrosinase-related protein 2 (TRP2) is a melanosomal enzyme expressed in most mammalian melanocytes and melanomas. This protein has been identified as a melanoma antigen recognized by tumor reactive CTLs derived from tumor infiltrating lymphocytes in the context of HLA-A31 and HLA-A33. The frequencies of these HLA-A alleles among melanoma patients in the United States is low (approximately 6% for HLA-A31 and approximately 2% for HLA-A33) compared with that of HLA-A*0201 (approximately 46%). Therefore, to extend significantly the use of TRP2-based immunotherapies for the treatment of patients with melanoma, we searched for new HLA-A*0201-restricted epitopes from this protein by screening TRP2-derived peptides for the induction of melanoma-reactive CTL. Fifty-one peptides were selected from TRP2 based on a permissive HLA-A*0201 binding motif, and the 21 peptides with the highest experimentally determined binding affinities were used to stimulate peripheral blood lymphocytes from HLA-A*0201+ melanoma patients in vitro. One peptide, TRP2(180-188) (SVYDFFVWL), induced CTLs from three of four patients that specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and TRP2, and HLA-A2+ TRP2+ melanomas. TRP2(180-188) is identical to a previously identified TRP2 epitope recognized by murine melanoma-reactive CTLs in the context of H-2Kb. These results suggest that TRP2 may be useful for the development of murine tumor immunotherapy models and for the treatment of melanoma patients who are diverse in HLA expression.

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells. Although a recombinant vaccinia virus (rVV) encoding the mouse homologue of gp100 was nonimmunogenic, immunization of normal C57BL/6 mice with the rVV encoding the human gp100 elicited a specific CD8(+) T cell response. These lymphocytes were cross-reactive with mgp100 in vitro and treated established B16 melanoma upon adoptive transfer. To understand the mechanism of the greater immunogenicity of the human version of gp100, we characterized a 9-amino acid (AA) epitope, restricted by H-2Db, that was recognized by the T cells. The ability to induce specific T cells with human but not mouse gp100 resulted from differences within the major histocompatibility complex (MHC) class I-restricted epitope and not from differences elsewhere in the molecule, as was evidenced by experiments in which mice were immunized with rVV containing minigenes encoding these epitopes. Although the human (hgp10025-33) and mouse (mgp10025-33) epitopes were homologous, differences in the three NH2-terminal AAs resulted in a 2-log increase in the ability of the human peptide to stabilize "empty" Db on RMA-S cells and a 3-log increase in its ability to trigger interferon gamma release by T cells. Thus, the fortuitous existence of a peptide homologue with significantly greater avidity for MHC class I resulted in the generation of self-reactive T cells. High-affinity, altered peptide ligands might be useful in the rational design of recombinant and synthetic vaccines that target tissue differentiation antigens expressed by tumors.

Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.

The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9154 (KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2+ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9154, G9209, and G9280 at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2+ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9209 or G9280 peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.

Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen.

Tumor infiltrating lymphocytes (TILs) derived from tumor-bearing patients recognize tumor-associated antigens presented by major histocompatibility complex (MHC) class I molecules. The infusion of TIL586 along with interleukin (IL) 2 into an autologous patient with metastatic melanoma resulted in the objective regression of tumor. A gene encoding a tumor antigen recognized by TIL586 was recently isolated and shown to encode gp75. Here we report that an antigenic peptide, MSLQRQFLR, recognized by TIL586 was not derived from the normal gp75 protein. Instead, this nonamer peptide resulted from translation of an alternative open reading frame of the same gene. Thus, the gp75 gene encodes two completely different polypeptides, gp75 as an antigen recognized by immunoglobulin G antibodies in sera from a patient with cancer, and a 24-amino acid product as a tumor rejection antigen recognized by T cells. This represents the first demonstration that a human tumor rejection antigen can be generated from a normal cellular gene using an open reading frame other than that used to encode the normal protein. These findings revealed a novel mechanism for generating tumor antigens, which may be useful as vaccines to induce tumor-specific cell-mediated immunity against cancer.

Leukocytes migrate through three-dimensional gels of midcycle cervical mucus.

The migration of leukocytes through mucus must be an essential factor in the mucosal immune response, particularly during mucosal invasion by pathogens. We used two methods to evaluate the ability of neutrophils to migrate through human midcycle cervical mucus: (i) direct visualization of cell migration within freshly collected mucus samples and (ii) quantification of cell penetration through a layer of mucus overlying a monolayer of cultured intestinal epithelial cells from the T84 cell line. Neutrophils migrated rapidly through human midcycle cervical mucus: the random motility coefficient (mu) ranged from 1.2 to 5.6 x 10(-8) cm2/sec in different mucus samples. The rate and pattern of neutrophil migration in cervical mucus were identical to those in a structurally similar collagen gel (0.4 mg/ml). Therefore, we also quantitatively examined the migratory behavior of human neutrophils, monocytes, and lymphocytes from peripheral blood, as well as leukocytes isolated from semen, in collagen gels as a model of cell migration in cervical mucus. In the absence of stimulation, mu was approximately 10(-8) cm2/s for neutrophils and lymphocytes; monocytes and seminal leukocytes were immotile. In all cases, mu increased with the addition of FMLP. Furthermore, neutrophils were able to penetrate layers of mucus overlying monolayers of T84 cells when the gel thickness was less than approximately 500 microns. These results suggest that leukocytes migrate effectively through mucus, a process which may be related to their ability to function in the mucosal immune system or to serve as vectors for the translocation of infectious pathogens like HIV.

Three-dimensional cell cultures mimic tissues.

Three-dimensional cell culture using gels of type I collagen is a flexible method for studying cell behavior in a tissuelike environment. With only small changes in the basic protocol, we were able to encapsulate neutrophils, hepatocytes, and PC12 cells. As demonstrated by cell-specific assays for migration, protein secretion, and growth factor induction, the encapsulated cells were viable and functional. In future studies, we will focus on using these cell cultures to study cell movement, cell growth, and cell function in carefully controlled tissuelike environments.

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.