Lee W. Thompson

Terminal modifications inhibit proteolytic degradation of an immunogenic mart-127–35 peptide: Implications for peptide vaccines

Peptide epitopes for tumor‐reactive cytotoxic T‐lymphocytes (CTL) have been identified on human cancers and are being used in tumor vaccine trials. However, the pharmacokinetics and pharmacodynamics of such peptides have been inadequately studied. It is predicted that immunogenic tumor peptides would have short half‐lives in vivo. The goal of the present work was to evaluate the stability of the immunogenic peptide MART‐127–35 in fresh normal human plasma (NHP) and to identify modifications that convey protection against enzymatic destruction without loss of immunogenicity. We evaluated the stability of the MART‐127–35 peptide (AAGIGILTV) and modified forms of that peptide for stability and immune recognition in an in vitro model. The peptides were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART‐127–35‐reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 μM MART‐127–35 peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half‐life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART‐127–35 was markedly prolonged by C‐terminal amidation and/or N‐terminal acetylation (peptide capping), or by polyethylene‐glycol modification (PEGylation) of the C‐terminus. These modified peptides were recognized by CTL. The MART‐127–35 peptide is very unstable in plasma. It is probable that it and other immunogenic peptides will be similarly unstable in vivo. Immunogenicity of these peptides might be enhanced by creating modifications that enhance stability. Int. J. Cancer 83: 326–334, 1999.

Melanomas with concordant loss of multiple melanocytic differentiation proteins : immune escape that may be overcome by targeting unique or undefined antigens

Abstract Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Sequential Immune Escape and Shifting of T Cell Responses in a Long-Term Survivor of Melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse “edited” tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.

Identification of Novel and Widely Expressed Cancer/Testis Gene Isoforms That Elicit Spontaneous Cytotoxic T-Lymphocyte Reactivity to Melanoma

Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84–88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.

Competition among peptides in melanoma vaccines for binding to MHC molecules.

The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2+ cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100154-162 (KTWGQYWQV), gp100280-288 (YLEPGPVTA), and tyrosinase369-377D (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.

Preventing the spontaneous modification of an HLA-A2-restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity

Abstract: The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

OBJECTIVES:There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery.METHODS:WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity.RESULTS:We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion.CONCLUSIONS:WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer.

Abstract B4: Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions.

Pancreatic ductal secretions are an abundant source of proteins that may serve as biomarkers for the early detection of pancreatic adenocarcinoma. Most pancreatic ductal secretion proteomes published in the literature are dominated by serum proteins. Due to the invasive nature of procedures used to sample pancreatic secretions, it remains uncertain whether the serum proteins present in the samples are artifacts of blood introduced during sampling or true indicators of disease. In addition, serum proteins are not generally useful as specific disease markers and mask other less abundant pancreas-specific proteins of interest in the sample. The enrichment of pancreas-specific proteins in pancreatic ductal secretions increases the opportunity for uncovering specific biomarkers of pancreatic cancer. Pancreatic ductal secretions were collected from ten patients with pancreatic adenocarcinoma undergoing pancreatectomy during surgery according to IRB-approved protocols. Protease inhibitors were added and samples were stored at -80°C. Samples were extracted 3x with chloroform to remove hydrophobic contaminants followed by IgA depletion using SSL-7 agarose (Invivogen) and depletion with a Vivapure Anti-HSA human albumin depletion (Sartorius) or Seppro IgY 14 Spin Column (Sigma) serum protein depletion column. Depleted samples were precipitated with methanol/chloroform/water, dissolved into 2M urea with 50mM ammonium bicarbonate/ 10mM TCEP and digested with trypsin. The digest was processed on a C-18 column on an HPLC, dried, resuspended in 0.1% TFA and characterized by ESI-LC-MS/MS on an LTQ-Orbitrap. Data were searched using Mascot against the NCBI RefSeq database. The top 20 proteins in pancreatic secretions taken from pancreatic adenocarcinoma patients in order of abundance without depletion were albumin, transferrin, beta globin, alpha 2 globin, IgA-1 chain constant region, alpha-1 antitrypsin, complement component 3, carboxyl ester lipase, keratin 1, apolipoprotein A-1, haptoglobin, delta globin, Predicted protein similar to complement component C3, alpha-2 macroglobulin, pancreatic carboxypeptidase B1, IgA-2 chain constant region, apolipoprotein E, polymeric immunoglobulin receptor, IgG-1 constant region, and Ig lambda chains. Mascot scores for albumin were about 20 times higher than scores for the pancreas-specific proteins and about three times higher than scores for the other serum proteins. Twelve of the fourteen proteins which are depleted by the Seppro IgY 14 depletion columns are present as dominant proteins in most pancreatic secretion samples. Of the remaining top proteins, alpha-2, beta and delta globin are blood proteins which are not depleted. These are most likely the result of hemolyzed red blood cells during freezing. Carboxyl ester lipase, pancreatic carboxypeptidases A1, B1, elastase 3A and 3B, trypsin, regenerating islet-derived 1 alpha and beta, pancreatic lipase, chymotrypsin B1 and B2, pancreatitis-associated protein, pancreatic amylase alpha 2A and B, mesotrypsin, and phospholipase A2 are pancreas specific proteins which are detected with increased sensitivity once the interfering serum proteins are depleted. Data will be shown comparing the performance of the two columns in depletion efficiency as well as the effect of the addition of an additional depletion step of secretory IgA from the samples, which depleted IgA-1, IgA-2, polymeric immunoglobulin receptor, Ig-lambda and Ig-kappa chain proteins. In conclusion, IgA, albumin, and IgY 14 serum protein depletion improve the LC-MS analysis of pancreas-derived proteins in pancreatic secretions by reducing interference from abundant serum proteins. Citation Format: Jana D. Rocker, Carlo Contreras, Lee W. Thompson, Russell E. Brown, Jack A. DiPalma, Lewis K. Pannell. Depletion of antibodies and serum-derived proteins to facilitate LC-MS detection of pancreas-specific proteins in pancreatic ductal secretions. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B4.