Expression on cultured human tumour cells of placental trophoblast membrane antigens and placental alkaline phosphatase defined by monoclonal antibodies

The cellular reactivity of six monoclonal antibodies (McAbs) produced to isolated human placental syncytiotrophoblast microvillous plasma membranes has been examined using a variety of normal and malignant cell types. Two McAbs reacted with antigenic determinants common to most normal human cells. Two other McAbs (H310 and H316) reacted predominantly with normal placental trophoblast and with lymphocytic cells, as well as with most transformed or neoplastic cultured cell lines. Two further McAbs (H315 and H317) identified foetal differentiation antigens expressed only on the membranes of normal placental trophoblast and of certain tumor cell lines. H317 has been shown to be specific for the heatstable L-phenylalanine-inhibitable placental-type alkaline phosphatase isoenzyme. These latter McAbs (H315 and H317) may prove useful in monitoring of some human cancers.     

Humoral and cellular immunity to paternal antigens in trophoblastic neoplasia.

Antibody-dependent cellular cytotoxicity (ADCC) was employed to detect and characterize an unusually high-titered antibody to a paternal HLA antigen in a patient with gestational choriocarcinoma. Choriocarcinoma is a trophoblastic neoplasm of embryonic origin, and therefore genetically competent to express paternal histocompatibility antigens. However, normal human trophoblast is reported to lack HLA antigens. The demonstration of a high-titre antibody to a paternal antigen is of interest for two reasons: it suggests that HLA antigens develop in trophoblast as a consequence of malignancy, and might therefore constitute a marker for neoplasia. Secondly, it indicates that a highly immunogenic antigen of tumor origin is provoking a humoral response, but that the disease is progressing in the face of such immunity. The antibody is distinguishable from that provoked by normal pregnancy in three ways. It is of exceedingly high titre (> 1:32,000 against paternal lymphoblast targets); it is monospecific for the paternal HLA B locus antigen 12; and it demonstrates a pronounced prozone indicative of the presence of immune complexes (ICs). Post-partum sera from multiparous women frequently contain HLA antibodies demonstrable in ADCC. However, titres are comparatively low, the antibodies are not monospecific, but recognize HLA and other (Ia) antigens, and they do not show a marked prozone, or the presence of immune complexes. ICs were indeed demonstrable at a concentration of >25 μg/ml (aggregated human gamma globulin equivalents) by a modified Raji cell technique. Histological examination of the uterine tumor following hysterectomy revealed a high degree of mononuclear cell infiltration, suggestive of cell-mediated immunity. The patient was quite capable of generating cytotoxic T-cells to paternal lymphocytes on challenge in mixed lymphocyte culture, indicating that no general anergy or suppressor function was present. The possibility that suppression might be limited to the single B locus specificity sensitizing the patient's humoral response was not investigated. Since immune complexes interfere with both ADCC cytotoxicity and T-cell cytotoxicity it is suggested that they might contribute to the lack of resistance to a demonstrably immunogenic allograft.     

Induction of interleukin-2, natural killer cell activity and anti-melanoma antibodies resulting from immunization of mice with formalinized extracellular antigens (FECA) of murine melanoma.

Extracellular products from melanoma cells may play an important role in the pathogenesis of metastatic melanoma. Studies were designed to evaluate the effect of vaccination with formalinized extracellular antigens (FECA) of murine melanoma cells (MMM B16-F10) on survival and immune response of C57BL/6 mice. The cellular immune response was evaluated by assessing interleukin-2 (IL-2) production and natural killer cell activity, whereas the humoral immune response was examined by measuring the production of specific antibodies to extracellular antigens (ECA). IL-2 production by the splenocytes from immunized animals was significantly higher (4.7 U/ml and 3.7 U/ml) than that of controls (1.38 U/ml). The splenocytes from immunized mice revealed significantly higher natural killer cell activity. Similarly, immunized animals responded by producing specific antibodies against the extracellular melanoma antigens as detected by ELISA. The peak production of antibodies against ECA was observed on the 21st day post-immunization. These results suggest that FECA are immunogenic and may enhance active cellular and humoral anti-melanoma immunity.     

T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy

Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex-peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide-major histocompatibility complex ligand presentation, as well as T-cell receptor peptide-major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.     

Direct identification of human tumor-associated peptide antigens and a preclinical model to evaluate their use

Although the arsenal of a healthy immune system includes both circulating antibodies and cellular components such as T cells, the latter seem to be particularly important in tumor immunology. Under normal conditions, the immune system does not react to the body's cells, which may be described as expressing "self" antigens on the cell surface. When a cell becomes cancerous, however, novel antigens are expressed on the cell surface. These novel "tumor" antigens are recognized as foreign by the body's immune system, and the cells that express them are destroyed or incapacitated. Whereas antibodies may react directly with protein antigens, T cells instead recognize peptide antigens presented by class I and class II molecules of the major histocompatibility complex (MHC). All cells normally break down proteins that they have made. The class I antigen-processing pathway has evolved to display peptides produced by this breakdown process as a way to provide information to cytotoxic T cells about what the cell is making. The display of new peptides as a result of infection or transformation can stimulate cytotoxic T cells to kill the cell. In addition, antigen-processing cells such as dendritic cells engulf dead or dying cells and degradeproteins into peptide fragments. These peptides are then displayed by the MHC class II molecules and presented to T helper cells, which augment the activity of the cytotoxic T cells. Cytotoxic T lymphocytes have recently been isolated from human tumors (especially melanoma) and are critical to the development of promising immunotherapeutic agents. As we shall discuss, these cells can recognize antigens that are common to tumors from different patients. We shall also explore how advances in instrumentation and the use of transgenic mice have increased our understanding of tumor-associated peptides to the point where we can begin to strive for a peptide-based therapeutic vaccine. The caveats for such therapy will also be addressed.     

T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy

Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex–peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex–peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide–major histocompatibility complex ligand presentation, as well as T-cell receptor peptide–major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.     

Prediction of the immunogenic potential of frameshift-mutated antigens in microsatellite instable cancer

Microsatellite instable (MSI) cancers express frameshift-mutated antigens, the C-terminal polypeptides of which are foreign to the immune system. Consequently, these antigens constitute a unique pool of tumor-specific antigens that can be exploited for patient diagnosis and selective, immune-mediated targeting of cancers. However, other than their sequence, very little is known about the characteristics of the majority of these proteins. We therefore developed a methodology for predicting their immunogenic behavior that is based on a gene-expression system, in which each of the proteins was fused to a short C-terminal polypeptide comprising two epitopes that can be readily detected by T-cells and antibodies, respectively. In this manner, accumulation of the antigens and processing of peptides derived thereof into MHC can be monitored systematically. The antigens, which accumulate in the cells in which they are synthesized, are of primary interest for cancer immunotherapy, because peptide epitopes derived thereof can be presented by dendritic cells in addition to the tumor cells themselves. As a result, these antigens constitute the best targets for a coordinated immune response by both CD8+ and CD4+ T-cells, which increases the likelihood that tumor-induced immunity would be detectable against these antigens in cancer patients, as well as the potential value of these antigens as components of anticancer vaccines. Our data indicate that, of 15 frameshift-mutated antigens examined in our study, 4 (TGFbetaR2-1, MARCKS-1, MARCKS-2 and CDX2-2) are of primary interest, and 4 additional antigens (TAF1B-1, PCNXL2-2, TCF7L2-2 and Baxalpha+1) are of moderate interest for further tumor immunological research.     

Placental metastasis (author's transl)]

Rarity of placental metastasis is only apparent, for only few placentas of cancerous mothers have been examined histologically. However, it may show biological and immunological conditions which are characteristics of foeto-placental unit. During metastatic spread of solid tumors or hematologic malignancies in the mother, tumor emboli may be localized in intervillous spaces, without being real placental metastasis. Rarely tumor emboli are able to invade the struma of chorionic villi and produce true placental metastases: twelve such observations have been published, seven of which were malignant melanomas. It is even more exceptional that metastatic spread reaches the foetus. In most of the cases, it is thus protected against maternal cancer. This historical observation holds true. The fear of transplacental graft to the foetus is not an argument favorable of terminating a cancer associated pregnancy and foetal metastasis of maternal origin are not among the causes of congenital cancers in children.     

Breast cancer stroma frequently recruits fetal derived cells during pregnancy

Introduction

Breast carcinomas associated with pregnancy display a high frequency of inflammatory types, multifocal lesions and lymph node metastasis. Because pregnancy results in transfer to mothers of foetal stem cells that can migrate and differentiate into various tissues, we addressed the issue of whether such cells are present in breast carcinoma associated with pregnancy.

Methods

We analyzed women presenting with such tumours who were pregnant with male foetuses using fluorescence in situ hybridization (FISH), targeting X and Y chromosomes. The foetal cell phenotype was then determined by combining FISH and immunohistochemistry with various antibodies. Statistical analysis was performed using t-test or nonparametric Wilcoxon's test.

Results

We found that foetal cells were present in nine out of 10 carcinomas, in contrast with none of four benign mammary lesions (P < 0.05). Counting foetal and maternal cells showed that the mean number of foetal cells per million maternal cells was 36 in breast cancers and 0 in control samples (P < 0.01). By combining FISH and immunolabelling, we found that foetal cells expressed mainly mesenchymal or, to a lesser degree, epithelial or endothelial markers, but never leucocytes.

Conclusion

These findings demonstrate the frequent presence of foetal derived cells essentially in tumour stroma. Given the role played by stroma in tumour proliferation, these findings raise the issue of whether foetal cell can be targeted to influence tumour behaviour.     

Sera from patients with malignant mesothelioma can contain autoantibodies

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.     

The serologic response of patients with stage II melanoma to allogeneic melanoma cell vaccines

Seventeen patients with Stage II malignant melanoma were treated with vaccines prepared from three allogeneic melanoma cell lines in an attempt to induce a humoral immune response against melanoma cell surface antigens. The patients were free of detectable melanoma at the time of vaccination. Vaccines were prepared from three melanoma cell lines that expressed highly restricted melanocyte differentiation antigens. One of these cell lines also expressed an antigen found only on this particular line. The antigens were initially identified by antibodies in autologous serum; they were thus known to be recognized by the human immune system. In addition, two of the cell lines expressed HLA-A, -B, -C, and -DR antigens; no HLA antigens were detectable on the third line. The vaccines were administered sequentially by subcutaneous injection, mixed with bacillus Calmette-Guerin (BCG) or Corynebacterium parvum. The patients' sera were tested for antibodies against cell surface antigens of the vaccine cells in protein A assays and immune adherence assays, and the specificity of observed reactions was defined by absorption tests. Antibodies against alloantigens of the vaccine cells developed in 16 patients and in 15 patients, against antigens related to fetal calf serum in the culture medium. The magnitude of the antibody response to alloantigens varied considerably, with no difference between patients who received BCG or C. parvum with their vaccines. Antibodies against the restricted melanocyte differentiation antigens or the unique melanoma antigen expressed by the vaccine cells were not detected.     

肿瘤免疫治疗的研究进展和发展趋势

随着免疫学技术的进步,大量肿瘤抗原不断被发现。DC摄取肿瘤抗原诱导免疫激活还是抑制,取决于肿瘤细胞释放危险信号（GMCSF、单核趋化蛋白1、MCP1及热休克蛋白等）还是抑制性信号（TGFβ、IDO和iNOS等）。在危险信号的调节下,激活Th1细胞免疫应答清除肿瘤;而在抑制性信号的作用下,激活Th2应答,不能有效清除肿瘤。肿瘤免疫治疗方面的进展主要表现在抗体疗法、T细胞疗法及肿瘤疫苗。目前至少有7种抗体与化疗药物合用的临床效果已经被证实;尽管抗不同种癌症的抗体种类在逐渐增加,但还需进一步探讨用于抗体治疗的新靶点、开发新抗体及扩大抗体应用的抗肿瘤范围。而T细胞疗法治疗效果不十分理想。大多数肿瘤疫苗处于Ⅰ期和Ⅱ临床试验,但为数不多的Ⅲ期临床试验结果不理想,尚需进一步完善。抗体在免疫监视中的重要作用被逐渐认识,肿瘤免疫预防最终可能成为现实。     

Surface antigens on human melanoma cells studied with heterologous antisera

Antisera were raised in rabbits to 6 human melanoma cell lines. The cell-surface antigens recognized by these antisera were examined using cell-surface labelling with ¹²⁵I, followed by immunoprecipitation of soluble extracts of the cells and polyacrylamide-gel electrophoresis of the immunoprecipitates in the presence of sodium dodecyl sulphate (SDS). Up to 16 cell-surface antigens were recognized by these antisera, and 5 of the melanoma cell lines had a similar profile of cell-surface antigens. Digestion of labelled melanoma cells with neuraminidase before immunoprecipitation revealed that 8 of the larger antigens were sialoglycoproteins. The melanoma antisera produced haemagglutination of human erythrocytes at high dilutions, but the antigens involved could not be detected by iodination. In contrast, absorption of the melanoma sera with lymphocytes and fibroblasts revealed that these cells did contain some cell-surface glycoproteins in common with melanoma cells. The melanoma antisera contained antibodies to foetal calf serum proteins, but the amounts of these proteins on the surface of melanoma-cells were very low. Immunoprecipitation of labelled melanoma cell extracts with monospecific antiserum to β₂-microglobulin produced 2 bands with mol. wts corresponding to β₂-microglobulin and the HLA-determinant polypeptide chain. After absorption of melanoma antisera with cross-linked foetal calf serum, erythrocytes, lymphocytes and fibroblasts, antibodies against 10 labelled antigens remained in the absorbed antisera. However, antibodies against 8 of these antigens were still detected after absorption of the melanoma antisera with melanoma cells.     

Pregnancy in women who had cancer in childhood

The majority of female cancer survivors will have normal reproductive function and would be expected to have a successful pregnancy. For the minority of young women who have received significant cytotoxic insult to the reproductive organs and yet still manage to conceive, pregnancy must be considered a high risk condition and these patients should be managed by a multidisciplinary specialist team. Female survivors of childhood cancer who are able to become pregnant carry an excess risk of preterm delivery and low birth weight baby. This restricted foetal growth and inability of the uterus to carry the foetus to term is associated with radiation-induced damage to the uterus. Chemotherapy does not appear to be associated with adverse pregnancy outcomes. However, prospective follow-up of cohorts of patients treated with contemporary therapies, frequently involving more intensive therapies are required to determine the risk. A number of large multi-centre studies, are underway and will provide new insights into pregnancy outcomes in survivors of childhood cancer.     

Immunity to tumor antigens: potential implications in human neuroblastoma.

Immune reactions to tumor-specific and tumor-associated antigens have been demonstrated in animals with neoplasms with in vitro and in vovo techniques. Some of the antigens detected in vitro induce transplantation resistance in vivo, while others do not. Human neuroblastoma cells cultivated in vitro have been shown to possess common antigens to which lymphocytes from neuroblastoma patients react. Whether it is possible to augment the immune reactivity of patients with neuroblastoma to these common antigens and, if so, whether this heightened immune reactivity would have clinically beneficial effects are as yet unknown. These reactions are complex, involving both cellular and humoral mechanisms. The fact that one type of immune response can be detected to one type of antigen present in a tumor in vitro does not necessarily mean that the immune response is effective in vivo. Responses to other tumor antigens may be deficient, or the immune response may be depressed. This may be due to active suppression of and/or selective deficiencies in critical cell populations required for an augmented immune response; this possibility may be evaluated with techniques allowing for in vitro sensitization to tumor antigens.     

Antigens in human glioblastomas and meningiomas: Search for tumour and onco-foetal antigens. Estimation of S-100 and GFA protein.

Extracts of glioblastomas and meningiomas were analysed by quantitative immunoelectrophoresis for the presence of foetal brain antigens and tumour-associated antigens, and levels of 2 normal brain-specific proteins were also determined. The following antibodies were used: monospecific anti-S-100 (glia specific); monospecific anti-GFA (glial fibrillary acidic protein), (astroglia specific); polyspecific anti-foetal brain (12-16th week of gestation); a polyspecific anti-glioblastoma antiserum, absorbed with insolubilized serum, haemolysate and normal brain extract; polyspecific anti-alpha-foetoprotein; and monospecific anti-ferritin. Using the antibodies raised against the tumours, several antigens not present in foetal or adult normal brain were found in the glioblastomas and the meningiomas. These antigens cross-reacted with antigens present in normal liver and were therefore not tumour-associated. S-100 was found in glioblastomas in approximately one tenth the amount in whole brain homogenate, whereas GFA was found 2-4 times enriched. The 2 proteins were absent in meningiomas. The possible use of the GFA protein as a marker for astroglial neoplasia is discussed. Five foetal antigens were found in foetal brain, but none in the tumours. alpha-Foetoprotein could only be demonstrated in foetal tissue extracts, including foetal brain, but not in tumours. Ferritin was detected in all tumour extracts, although the amounts determined were unrelated to histological tumour type.     

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)-gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient-derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell-surface antigens. However, selections using the non-immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial-cell specific target antigens.     

Immune response of athymic nude mice to papovavirus SV40 tumor-associated antigens.

The requirement of T cell functions in the induction of immune response to SV40-specific transplantation rejection antigen and intranuclear tumor antigen was studied using athymic nude mice. The results obtained indicate that virus-immunized athymic nude mice were unable to reject SV40 tumor cell challenge, and sensitized lymphocytes capable of inhibiting tumor growth in vivo could not be demonstrated in the spleens of virus-immunized mice. Athymic nude mice bearing tumor induced by virus-free SV40-transformed BALB/c cells failed to develop antibodies to intranuclear T antigen. Athymic nude mice also failed to respond to viral antigens. Thus it can be concluded that T cell functions are required in the induction of cellular immune response to SV40-specific transplantation rejection antigen and in humoral immune response to SV40-specific T antigen and virion antigen.     

An immunohistological study of HLA antigen expression by gestational choriocarcinoma

Two cases of gestational choriocarcinoma have been examined for the expression of HLA and trophoblast antigens using the indirect immunoperoxidase technique on frozen tissue sections. Approximately 40-70% of tumour cells express MHC Class I antigen as detected by a panel of antibodies to monomorphic, or framework, MHC Class I antigenic determinants. Evidence in one case suggests that most of these cells may not express the paternal, polymorphic HLA antigenic determinants but that a small subpopulation do carry the fully antigenically active molecule. These latter may give rise to patient anti-paternal HLA antibodies. Class II (HLA DR or DC) antigens are expressed by none, or very few, tumour cells.     

Immunostimulatory CpG oligodeoxynucleotides and antibody therapy of cancer

Synthetic oligodeoxynucleotides containing CG motifs (CpG ODN) have potent immunostimulatory properties, and have potential as immunotherapeutic agents in cancer. Animal models suggest CpG ODN can activate a variety of immune effector cells such as natural killer (NK) cells, and also enhance the efficacy of tumor immunization when used as immune adjuvants or to directly activate antigen-presenting cells. CpG ODN are also capable of altering the expression of a number of antigens by malignant B-cells, including those targeted by monoclonal antibodies (moAbs) and those involved in communication with T cells. The ability of CpG ODN to activate the immune effector cells that participate in antibody-dependent cellular cytotoxicity (ADCC), upregulate target antigen, and perhaps induce development of an active immune response, suggest these agents may be capable of enhancing the efficacy of antitumor moAb therapy. Such enhanced efficacy has been demonstrated in animal models and is now undergoing evaluation in clinical trials.