Post-translational modifications such as citrullination are excellent targets for cancer therapy.

Under conditions of cellular stress, proteins can be post-translationally modified causing them to be recognized by the immune system. One such stress-induced post-translational modification (siPTM) is citrullination, the conversion of arginine residues to citrulline by peptidylarginine deiminase (PAD) enzymes. PAD enzymes are activated by millimolar concentrations of calcium which can occur during apoptosis, leading to precipitation of proteins, their subsequent uptake by B cells and stimulation of antibody responses. Detection of anti-citrullinated protein antibodies (ACPAs) is a diagnostic of rheumatoid arthritis (RA), where immune complexes stimulate inflammation around the joints. More recently, autophagy has been shown to play a role in the presentation of citrullinated peptides on MHC class II molecules to CD4+ helper T cells, suggesting that citrullination may be a way of alerting immune cells to cellular stress. Additionally, inflammation-induced IFNγ and concomitant MHC class II expression on target cells contributes to immune activation. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a pro-survival mechanism. Cancer cells also over express PAD enzymes and in light of this the hypothesis that citrullinated peptides stimulate CD4+ T cell responses that would recognize these siPTM's produced during autophagy has been investigated. The induction of potent citrullinated peptide-specific CD4 responses has been shown in both humans and HLA transgenic mouse models. Responses in mouse models resulted in potent anti-tumour responses against tumours expressing either constitutive or IFNγ-inducible MHC class II. The anti-tumour effect relied upon direct recognition of tumours by specific CD4 T cells suggesting that citrullinated peptides are attractive targets for cancer vaccines.

The chemokine, CXCL12, is an independent predictor of poor survival in ovarian cancer.

BACKGROUND: The chemokine CXCL12 and its cognate receptor, CXCR4, have been implicated in numerous tumour types where expression promotes tumour growth, angiogenesis, metastasis and suppresses tumour immunity. METHODS: Using a tissue microarray of 289 primary ovarian cancers coupled to a comprehensive database of clinicopathological variables, the expression of CXCL12 and CXCR4 was assessed by immunohistochemistry and its impact in terms of survival and clinicopathological variables was determined. RESULTS: Patients whose tumours expressed high levels of CXCL12 had significantly poorer survival (P=0.026) than patients whose tumours failed to produce this chemokine. Lack of CXCL12 expression within tumours was associated with a 51-month survival advantage for patients when compared with patients whose tumours expressed high levels of CXCL12. FIGO stage, adjuvant chemotherapy and the absence of macroscopic disease after surgery were all shown to predict prognosis independently of each other in this cohort of patients. CXCL12 was independently predictive of prognosis on multivariate analysis (P=0.016). There was no correlation between CXCL12 and any clinicopathological variable. CONCLUSION: The chemokine CXCL12 is an independent predictor of poor survival in ovarian cancer. High expression of CXCL12 was seen in only 20% of the tumours, suggesting a role for anti-CXCL12/CXCR4 therapy in the management of these patients.

Immunology in the clinic review series; focus on cancer: glycolipids as targets for tumour immunotherapy.

Research into aberrant glycosylation and over-expression of glycolipids on the surface of the majority of cancers, coupled with a knowledge of glycolipids as functional molecules involved in a number of cellular physiological pathways, has provided a novel area of targets for cancer immunotherapy. This has resulted in the development of a number of vaccines and monoclonal antibodies that are showing promising results in recent clinical trials.

Native/citrullinated LL37-specific T-cells help autoantibody production in Systemic Lupus Erythematosus.

LL37 exerts a dual pathogenic role in psoriasis. Bound to self-DNA/RNA, LL37 licenses autoreactivity by stimulating plasmacytoid dendritic cells-(pDCs)-Type I interferon (IFN-I) and acts as autoantigen for pathogenic Th17-cells. In systemic lupus erythematosus (SLE), LL37 also triggers IFN-I in pDCs and is target of pathogenic autoantibodies. However, whether LL37 activates T-cells in SLE and how the latter differ from psoriasis LL37-specific T-cells is unknown. Here we found that 45% SLE patients had circulating T-cells strongly responding to LL37, which correlate with anti-LL37 antibodies/disease activity. In contrast to psoriatic Th17-cells, these LL37-specific SLE T-cells displayed a T-follicular helper-(TFH)-like phenotype, with CXCR5/Bcl-6 and IL-21 expression, implicating a role in stimulation of pathogenic autoantibodies. Accordingly, SLE LL37-specific T-cells promoted B-cell secretion of pathogenic anti-LL37 antibodies in vitro. Importantly, we identified abundant citrullinated LL37 (cit-LL37) in SLE tissues (skin and kidney) and observed very pronounced reactivity of LL37-specific SLE T-cells to cit-LL37, compared to native-LL37, which was much more occasional in psoriasis. Thus, in SLE, we identified LL37-specific T-cells with a distinct functional specialization and antigenic specificity. This suggests that autoantigenic specificity is independent from the nature of the autoantigen, but rather relies on the disease-specific milieu driving T-cell subset polarization and autoantigen modifications.

Human leukocyte antigen class I expression is an independent prognostic factor in advanced ovarian cancer resistant to first-line platinum chemotherapy.

BACKGROUND: Loss of HLA class I is important in ovarian cancer prognosis but its role as a prognostic indicator in relation to therapy remains unproven. We studied the prognostic potential of this antigen and its significance in relation to platinum therapy. METHODS: A total of 157 primary ovarian cancers were assessed for HLA class I immunohistochemically and linked to a comprehensive database of clinicopathological variables, treatment details, and platinum sensitivity. RESULTS: Tumours expressing high levels of HLA class I had significantly improved survival (P=0.044). There was a 19-month difference in the median overall survival between tumours with high and low antigen expression. HLA class I antigen expression, stage, and platinum sensitivity were independently predictive of prognosis on multivariate analysis. HLA class I antigen was shown to be expressed at higher levels in patients with good overall survival in platinum-resistant patients (P=0.042). HLA class I significantly correlated with overall survival on multivariate analyses (P=0.034). CONCLUSION: Low-level HLA class I expression is an independent prognostic indicator of poor clinical outcome in ovarian cancer. The survival advantage of patients with platinum-resistant tumours expressing high levels of HLA class I suggests that immunotherapy may be of use in these ovarian cancers resistant to standard chemotherapy.

T-cell responses in osteosarcoma patients vaccinated with an anti-idiotypic antibody, 105AD7, mimicking CD55.

We assessed T-cell responses in young osteosarcoma patients vaccinated with 105AD7, 1-6 months after having received chemotherapy. 105AD7 is a human anti-idiotypic antibody mimicking CD55, a glycoprotein that protects from attack by complement and which is overexpressed on osteosarcoma cells. Seven out of 21 investigated patients made a IFN-gamma T-cell response against the vaccine, 105AD7 as assessed by ELISPOT. Cytokine secretion was analysed using Luminex assays and revealed TNF-alpha and GM-CSF responses not only to the vaccine but also towards the native antigen, CD55, in 5 / 14 (36%) of investigated patients. Importantly, the Luminex assay was found to be more sensitive than the more established T-cell assays (ELISPOT and proliferation assay), since responses towards the native antigen were recorded in this assay. Clinical responses and induction of immune responses to both the anti-idiotype and the native CD55 antigen support the use of CD55 as a target in cancer treatment.

Flow cytometric screening of monoclonal antibodies for drug or toxin targeting to human cancer.

Monoclonal antibodies (MAbs) that are candidates for antibody-directed therapy were evaluated by a flow cytometric method. This method accurately quantitates the intensity of staining and the percentage of cells from freshly derived primary tumors expressing the relevant cell surface antigens. This method was applied to human colorectal, gastric, and ovarian carcinomas. It allowed calculations of the number of drug molecules that potentially could be delivered by each MAb as well as selection of the optimal combinations of antibodies for treatment of each type of cancer. The binding of all the MAbs varied among the tumors, although combinations of antibodies reduced this problem. A combination of MAbs C14 and NCRC-23 recognized 97% of colorectal tumors. A combination of C14, NCRC-23, and 791T/36 recognized 95% of gastric tumors. Combinations of either 791T/36 and C14 or 791T/36 and NCRC-11 recognized 80% of ovarian tumors. The number of cells binding with a single MAb varied within the tumor. The optimal anti-colorectal tumor antibody was NCRC-23 (anti-carcinoembryonic antigen), which recognized a mean of 65% of the large cells within a tumor at a mean antigen density of 4.9 X 10(5) sites/cell. The optimal anti-gastric tumor antibody was C14 (anti-Y hapten), which recognized a mean of 66% of the large cells within a tumor at a mean antigen density of 4.4 X 10(5) sites/cell. The optimal anti-ovarian antibody was 115/D8, which recognized 54% of the large cells at a mean antigen density of 4.2 X 10(5) sites/cell. These antigen densities were similar to those calculated for HLA/ABC antigens in colorectal and ovarian cancers. However, the gastric tumors expressed elevated levels of major histocompatibility complex class I antigens, with a mean density of 7.3 X 10(5) sites/cell. Combinations of antibodies that recognize a high proportion of tumor cells are likely to be necessary for MAb-drug targeting to prevent tumor recurrence and/or metastases.

Induction of delayed hypersensitivity to human tumor cells with a human monoclonal anti-idiotypic antibody.

There is considerable interest in the development of anti-idiotypic antibodies as vaccines in a number of diseases, including cancer. We have developed a human anti-idiotypic monoclonal antibody (105AD7) which binds at or very near to the binding site of mouse antitumor monoclonal antibody 791T/36. The 791T/36 antibody binds to a tumor-associated antigen (gp72) expressed on a number of human tumors, including colorectal and ovarian carcinomas and osteogenic sarcoma. This study shows that, in rats and mice, 105AD7 induces delayed-type hypersensitivity to human tumor cells bearing the gp72 antigen. Local transfer of delayed hypersensitivity was also demonstrated using lymphocytes from mice primed with 105AD7. These findings show that the human monoclonal anti-idiotypic antibody 105AD7 is likely to induce cellular immune responses to tumors in cancer patients.

Intracellular gastrin in human gastrointestinal tumor cells.

Flow cytometry and immunohistochemical analyses of the human gastric adenocarcinoma cell line MKN45G identified an intracellular peptide recognized by an anti-gastrin-17 (G17) antiserum but not by an anti-cholecystokinin-specific antiserum. Staining was not associated with the parental line MKN45, of which MKN45G is a clonal variant. The MKN45G cell line had elevated in vitro growth in serum-free medium in which the proliferation of MKN45G cells but not MKN45 cells was reduced to 58% of the control value by treatment with a rabbit anti-G17 antiserum. This inhibition of proliferation was reversed by preabsorbing the antiserum with excess G17. Disaggregated primary human gastric and colorectal tumors were screened for gastrin immunoreactivity by flow cytometry, and 6 of 28 colorectal and 8 of 22 gastric tumors had greater than 20% positively staining cells.

Decay accelerating factor (CD55): a target for cancer vaccines?

The 791Tgp72 antigen has been used successfully as a target for tumor imaging and T-cell immunotherapy. We have characterized this antigen using the monoclonal antibody 791T/36 as a 72/66 kDa doublet. NH2-terminal protein sequencing of the two bands revealed identity with the complement regulatory protein CD55. Antibodies recognizing different domains of CD55 were also shown to bind to the purified 791Tgp72, although sequence analysis of the cDNA cloned from 791T tumor cells showed 100% homology with CD55 and transfection of the cDNA into antigen-negative CHO cells resulted in binding of 791T/36. This identifies the tumor antigen 791Tgp72 as CD55. This protein protects cells from complement attack; however, it can also transduce signals in lymphocytes and is a ligand for CD97, expressed by activated T cells. These results suggest that CD55 plays a role in signaling between the innate and adaptive immune responses. It is therefore a very intriguing target, because absence of the molecule makes the tumor cells susceptible to complement, whereas protective overexpression results in the antigen being a target for T-cell immunotherapy.

Association of antigen expression and DNA ploidy in human colorectal tumors.

Fifty colorectal tumors were screened by indirect immunofluorescence and flow cytometry for antigen expression using a panel of monoclonal antibodies that recognize determinants preferentially expressed on tumor cells (carcinoembryonic antigen, Y haptenic blood group, 791T/36 defined antigen 791T-P72). Fifty % of the tumors expressed all three antigens, 41%, two, and 9%, one. Over a third reacted strongly with at least one monoclonal antibody, although the majority of tumors stained with a moderate intensity. Extranuclear membranes from tumors showed similar antigen expression to disaggregated tumor cells and were particularly useful for providing the relative tumor:normal tissue binding ratios. The carcinoembryonic antigen specific monoclonal antibody showed the strongest tumor selectivity with a tumor:normal tissue ratio of 24 +/- 7:1. Lack of correlation between expression of the three antigens suggested that the monoclonal antibodies recognizing them may have potential as a "cocktail." One-third of the tumors contained cells with an aneuploid DNA content and expressed elevated levels of carcinoembryonic antigen and Y haptenic blood group antigen when compared to tumors with diploid DNA content. Aneuploid cells within a tumor were also preferentially stained with all of the monoclonal antibodies.

Vaccine-induced apoptosis: a novel clinical trial end point?

The functional end point of immunotherapy is to induce tumor regression. Because immune effector mechanisms usually result in apoptosis, the aim of this study was to determine whether measurement of tumor apoptosis ex vivo is a good end point to evaluate the efficacy of cancer vaccines. A prototype vaccine, 105AD7, was administered to colorectal cancer patients before resection of their primary tumors. There was a significant increase in apoptosis of tumor cells within immunized patients compared with control patients as assessed by immunohistochemistry (P = 0.005; n = 16) or by flow cytometry (P = 0.003; n = 34). Preoperative immunization and measurement of tumor cell apoptosis may be a valuable clinical end point for evaluation of new vaccine and other biological approaches.

Enhanced cell-mediated tumor killing in patients immunized with human monoclonal antiidiotypic antibody 105AD7.

A human antiidiotypic monoclonal antibody (105AD7) has been shown to induce antitumor cellular responses in animals and appears to prolong survival in patients with metastatic colorectal cancer without associated toxicity. Proliferative leukocyte responses to the targeted tumor antigen gp72 were observed in these patients and plasma interleukin 2 levels were increased following immunization. Autologous tumor tissue was not available in these patients, so antitumor cytotoxicity could not be measured. This issue has now been addressed in an adjuvant clinical study in primary rectal cancer patients. Six patients with rectal cancer were immunized preoperatively with 105AD7. Peripheral blood lymphocytes taken prior to immunization were tested against tumor cells extracted from biopsies also obtained prior to immunization or from natural killer (NK)-sensitive target cells. Cryopreserved lymphocytes taken before and after tumor immunization, fresh peripheral blood lymphocytes taken immediately prior to surgery, and lymphocytes from tumor-draining lymph nodes were tested against autologous cells from the resected specimen or NK-sensitive target cells. Significant killing of autologous tumor cells, which was not due to NK activity, was seen with cryopreserved lymphocytes or lymph node cells of three patients at 1-2 weeks postimmunization with 105AD7 but not on pretreatment biopsies. Enhanced NK activity was seen 2-3 weeks postimmunization in 3 of 6 patients. These results indicate that 105AD7 human monoclonal antibody immunization enhances cytotoxicity in rectal cancer patients by specific and nonspecific effector mechanisms.

Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody.

The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.

Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer.

Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.

A neoadjuvant clinical trial in colorectal cancer patients of the human anti-idiotypic antibody 105AD7, which mimics CD55.

Thirty-five patients received 105AD7 human anti-idiotype vaccination prior to surgery for colorectal carcinoma. Patients were immunized before and also received one to two immunizations after surgical resection of their colorectal cancer. The vaccine was well tolerated with no associated toxicity. Lymphocytic infiltration within the resected tumors was quantified by immunohistochemistry and image analysis. Enhanced infiltration of helper T cells (CD4) and natural killer (NK) cells (CD56) were observed in the tumors from immunized patients when compared with tumors from stage, grade, site, age, and sex matched unimmunized patients. NK activity was increased in the blood, peaking 7-10 days post immunization and then dropping rapidly and correlating with NK extravasation within the tumor. Comparison of the amino acid sequences of 105AD7 anti-idiotype and the antigen it mimics, CD55, has predicted that patients with HLA-DR1, HLA-DR3, and HLA-DR7 haplotypes should show helper T cell responses following 105AD7 vaccination. Eighty-three percent of patients expressing these haplotypes responded to 105AD7, whereas 88% of patients who failed to express these haplotypes were nonresponders. With a median follow-up of 4 years (range, 2.5-6 years) 65% of patients remained disease free. This trial shows that 105AD7 stimulates antitumor inflammatory responses allowing extravasation within tumor deposits of both helper T cells and NK cells. This represents a way of evaluating immune responses in patients both within the blood and at the tumor site. The study confirms that immunization with a human anti-idiotypic antibody results in immune responses in 83% of patients with a permissive haplotype.

Production of monoclonal antibodies recognising the peptide core of MUC2 intestinal mucin.

A peptide based on the tandem repeat sequence of MUC2 mucin was used to produce a series of monoclonal antibodies (MAb). The fine specificity of these antibodies and their implications for MUC2 expression are presented. Three of the MAbs, 996/1, 996/7 and 995/25, were specific to the MUC2p and failed to bind to peptides based on the MUC1,3,4 tandem repeat sequences whereas three others, 994/152, 994/91 and 996/36, cross reacted with the MUC2p and the MUC3 tandem repeat peptide but not the MUC1 and MUC4 peptides. An antigen, affinity purified from a colorectal tumour on one of the MUC2p-specific MAbs, 996/1, was shown to be a high molecular weight polydisperse, mucin-like antigen. Two of the MAbs, 996/1 and 994/152, recognised MUC2 in tissue sections, although the fine specificity varied between the two MAbs, with 994/152 strongly staining gastric, ileum and kidney epithelia, and MAb 996/1 intensely staining colon, liver and prostate tissues. These antibodies also stained a colorectal cell line, and MAb 994/152 also stained a gastric and an ovarian cell line. Six of the MAbs were used to stain colorectal tumour and adjacent 'normal' colonic mucosa sections. All six stained normal mucosa, but only two of the MAbs, 996/1 and 994/91, stained tumour tissue. The staining probably reflects exposure of cryptic epitopes due to varying levels of glycosylation in different tissues. These anti-MUC2p MAbs may help in determining the normal role of MUC2 mucin and how it is subverted in malignancy.

Inhibition of gastrin-stimulated growth of gastrointestinal tumour cells by octreotide and the gastrin/cholecystokinin receptor antagonists, proglumide and lorglumide.

The rat pancreatic cell line, AR42J possessed high-affinity gastrin and somatostatin receptors and its growth was stimulated by physiological gastrin-17 concentrations between 5 x 10(-11) mol/l and 10(-9) mol/l as measured by [75Se]selenomethionine uptake. The somatostatin analogue, octreotide (2 x 10(-7) to 2 x 10(-11) mol/l), reduced this stimulated growth. Gastrin-stimulated AR42J growth was also inhibited by proglumide (3 x 10(-4) mol/l) and lorglumide (3 x 10(-5) mol/l) at maximal G17 concentrations of 5 x 10(-11) and 10(-10) mol/l, respectively, and the analogues competed with [125I] gastrin-17 (5 x 10(-10) mol/l) for binding to gastrin receptors on AR42J (50% inhibitory concentrations, less than or equal to 10(-3) mol/l and 4 x 10(-6) mol/l, respectively. Octreotide reduced the basal growth of the human gastric cell line, MKN45G, (which is associated with intracellular gastrin immunoreactivity) in serum-free medium to 73% of control at a concentration of 2 x 10(-8) mol/l, which was reversed by gastrin-17 (10(-10) mol/l). Lorglumide (3 x 10(-5) mol/l) also reduced the basal growth to 30% of control, which was reversed to 78% by 10(-5) mol/l gastrin. Proglumide had no effect on the basal growth of MKN45G.

A rapid method for separating tumour infiltrating cells and tumour cells from colorectal tumours.

As solid tumours are composed of a heterogeneous mixture of cells the role of any particular cell type is difficult to analyse. A rapid method of sorting freshly disaggregated highly viable single cell suspensions of colorectal tumours has therefore been developed. Infiltrating cells were initially sorted from tumour cell suspensions using magnetic beads coated with a cocktail of monoclonal antibodies recognising leucocyte common antigen, CD45 (lymphocytes), CDw14 (macrophages and granulocytes) and Thy-1 antigen (stromal cells). Between 4-10 x 10(6) cells, 70-87% pure, were rapidly sorted. The tumour cells were then sorted from the remaining cell suspension using a cocktail of monoclonal antibodies which recognise 100% of colorectal tumours. Between 2-7 x 10(6) tumour cells, 70-87% pure, were rapidly sorted. Both populations of cells were 95% viable and able to grow in vitro.

Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates.

Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.