Lebanese UniversityFaculty of Sciences IDepartment of Biochemistry (2007-2008)

5T4 oncofetal antigen: Janus molecule in life and a novel target against tumors

Hilal Bakrihllbakri@hotmail.com

Table of Contents

Abstract:............................................................................................2

I. Introduction:................................................................................2

II. Generation & structure:.............................................................2

III. Function:.....................................................................................3

IV. A positive role in biological development:...............................3

V. Tumor associated expression of 5T4 glycoprotein:..................4

VI. A negative role in pathalogical events......................................5

VII. Passive immunotherapy:..........................................................5

VII.I. Monoclonal antibody.............................................................................5

VII.I.I. Scfv monoclonal antibody........................................................................6

VII.I.II. Fab monoclonal antibody.......................................................................6

VII.II. Antigen specific T cell infusion……………………………………..…...…7

VIII. Active immunotherapy:..........................................................8

VIII.I. Viral vector vaccines............................................................................8

VIII.II. Dentritic cell based vaccines..............................................................9

VIII.III. T reg cell and active DC therapy:.................................................10

IX. Conclusion:...............................................................................11

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Abstract:

Two decades ago, scientific studies and researches were done on the embryonic cells, where scientists first defined a highly glycosylated modified antigen on the surface of these cells and were given the name of 5T4 Oncotrophoblast Glycoprotein or simply Oncofetal Antigen. 5T4 is a transmembrane protein expressed on the surface of embryonic cells & many different malignant tumor cells. Studies showed that this antigen is involved in many biological and pathogenic processes, such as cell migration, invasion, and implantation (embryogenesis), and neoplastic metastasis in series of tumorigenesis. It is considered as tumor associated antigen due to its restricted expression profile, which makes it a contender for cancer immunotherapy. This helped explain the molecular function for discovering how tumor s diffuses and magnify, and understand the successful strategies of cancer therapy.

I. Introduction:

5T4 Oncofetal antigen is a type of heavily glycosylated transmembrane protein recognized by monoclonal antibodies secreted from human syncytiotrophoblast microvillus projections of the plasma membrane (StMPM), rose against wheat germ agglutinin-glycoprotein. It is isolated from the placenta, and chemically characterized as a 72 KDa product encoded by the trophoblast glycoprotein gene (TBGP gene).It is also expressed by tumor cells associated with metastasis and low cancer formation.

II. Generation & structure:

5T4 Oncofetal antigen is found on a variety of carcinomas and weakly on specialized epithelial cells. It is produced after the hatching of the blastocyst from the zona pellucida, due to the over expression of the TBGP gene. The gene-not like others-don’t have a TATA nor CAAT boxes, but a number of potential sp1 binding sites to help initiate the transcription of the gene to later produce the protein (or antigen). The 72 KDa product is encoded by the opening reading frame, having a 420 a.a, 46 KDa protein core-with 2 leucine rich repeat domains-separated by a hydrophilic stretch of amino acids with 8 potential N-glycosylation sites, and a 44 a.a cytoplasmic tail. The N-terminal act as a putative signal and the C-terminal acts as the anchorage sequence.

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Fig: 1 Generation of 5T4 on the trophoblast from the zona pellucida

III. Function:

5T4 Oncofetal antigen plays a role in many physiological & pathological cell events, and that is done through modulating the cells migration, adhesion, mobility, shape and morphology. We can be more perceive by stating that it increases cell mobility and migration (due to the increase in contact between actin & cadherin on the surface of the cell), enhances cell adhesion, and changes the cells shape and the morphology & integrity of its membrane. Changing in the morphology of the membrane includes the change of E-Cadherin on the cell surface of epithelial cell (important for embryo development after the blastocyst stage & formation of cell-cell adherence junctions) to N- Cadherin promotes migration and invasion of cells).

IV. A positive role in biological development:

Studies showed that the type I of the protein has the highest level of expression. It plays a positive role in the body due to its structural components. The extra cellular domain, having the domain of 7 leucine-rich repeat regions (LRRs), flanked with cysteine-rich region, helps in the intervention of certain protein-protein interactions. The cytoplasmic, having a PDZ domain-binding motif (containing: Ser/Asp/Val), is concerned in the course of embryogenesis.Concerning metastasis, during murine post-implantation, 5T4 Oncofetal antigen displays restricted expressions to diverse epithelial cell types formed from all 3 germ layers. This however is companied with the invasion of the trophoblast cells into the maternal host cells, in the uterus wall to aid in the adhesion and fixation of the embryo, in addition to the formation of the placenta.

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The antigen can be used in marking of the differentiation of premature embryonic stem cells. 5T4 positive epithelial cells can express formation of chimera more efficiently than 5T4 negative cells (7 times more).5T4 also plays an important role in the nervous system. It can help in the development of the epithelia of the nervous system. Many of the (LRRs) domains of the protein, present in the central nervous system, are involved in the formation of neural circuits.A novel function we can mention about the 5T4 Oncofetal antigen is that it is involved in the procedure of forming dendrodendritic synapse between granule cells (GCs) & mitral/tuftal cells. An example of this process can be located in the olfactory bulb, where a major population of interneurons-5T4 positive GCs-extend their peripheral dendrites preferentially toward the superficial stratum of external plexiform layer (EPL).As a summary we can say that the expression of the 5T4 antigen comes parallel to the formation of dendrodendritic synapse between GCs & mitral/tufted cells.

Fig: 2 5T4 right and control left

V. A negative role in pathological events:

The over expression of 5T4 antigen in both malignant and embryonic stem cells explains the acquirement of invasive and migratory phenotype. Placentation may share aspects of such mechanisms where foetal trophoblast cells, these aspect similarities was observed as avoiding host immunity and surveillancespindle-like (dendritic) morphology, increased motility, reduced cell-cell adhesion and proliferation rate, and loss of pluripotency of metastatic spread epithelia. Microvillus projections present in tumor cell (B16 F10 melanoma cells and A9 L fibroblasts) membranes which over expresses the cDNA encoding the human gene TBPG results the diversity of E-cadherin structure that influences the protection of the antigen against proteolytic attack and promotion of the interaction with other molecules responsible for invasion and migration and increased motility, that permits cancerous cells metastasis where the it interact with the N-linked glycosylated domain which contains PDZ binding domain that interacts with the PDZ domain protein and LRR motif which plays the role of cell signaling assemblies and remodulation the cytoskeleton thus alters the integrity of the cell membrane and cell adhesion. 5T4 antigen strongly it is over-expressed on many different types of human carcinoma such as gastric, colorectal and ovarian, cervical, renal, gastrial and breast cancer.

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VI. A novel promising target 5T4 to immunotherapy for cancers

Cancer immunotherapy is the use of the immune system to reject cancer the main premise is stimulating the patient's immune system to attack the malignant tumor cell that are responsible for the disease .Tumor antigen is a substance produced in tumor cells that triggers an immune response in the host. Tumor antigens are useful in identifying tumor cell and are potential candidates for use in cancer therapy. Mutation of other genes unrelated to the tumor formation may lead to synthesis of abnormal proteins which are called tumor associated antigens.A number of oncofoetal or tumor-associated antigens (TAAs) have been identified and characterized in human and animal tumors. Tumor cells have been observed to resume expression of TAAs, and the application of TAAs for tumor diagnosis, targeting and immunotherapy has therefore been suggested. 5T4 oncofetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed widely in carcinomas, but having a highly restricted expression pattern in normal adult tissues. It appears to be strongly correlated to metastasis in colorectal and gastric cancer. 5T4 satisfies the criteria for TAA and could prove to be effective antigens for immunotherapy against cancer that lead to cancer cell death depend on the relative immune mechanism included antibody-dependent cell-mediated cytoxicity (ADCC) and complement-dependent cytoxicity (CDC)

VII. Passive immunotherapy:

Passive immunotherapy is transfer of active humoral immunity in the form of ready made antibodies, from on individual to another. It can occur naturally, when maternal antibodies are transferred to the fetus through the placenta, and can also be induced artificially, when high levels of human antibodies specific for pathogen or toxins are transferred to non immune person, it may involve antigen specific T cell and cytokines .It is used when there is a high risk of infection and insufficient time for the body to develop its own immune response, or to reduce the effect of immunosuppressive diseases.

VII.I. Monoclonal antibody

Monoclonal antibody therapy is a form of passive immunotherapy because it uses antibodies made in large numbers outside the body (in the lab) rather than by a person's own immune system so it don't require the person's immune system to take an "active" role in fighting the cancer. When used as a treatment for cancer it may bind to the cancer cells having the tumor antigens on their surface, the immune system will see the cancer cells marked with bound antibodies as foreign and destroy them. A second strategy is to use the antibodies to block the binding of cytokines or other proteins that are needed by the cancerous cells to maintain their uncontrolled growth. Both strategies may help rid the body of the tumor cells.

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Fig: 3 Monoclonal antibodies for cancer

VII.I.I Scfv monoclonal antibody

The most well known example of monoclonal antibody is single chain antibody such as single chain antibody Fv (scfv) and the conjugated engineered antibody. Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effectors functions.

VII.I.II Fab monoclonal antibody

A phase 2 study of a tumor –targeted super antigen (TTS) (ABR-214936) recognizing the 5T4 oncofetal antigen in patients with advanced renal cell carcinoma shows that this tumor targeted super antigens activates and recruits T cells to tumors expressing the target antigen. This drug (ABR-214936) is a recombinant fusion of a fab of a monoclonal antibody fragment recognizing the oncofetal antigen 5T4 and the modified super Antigen SEA (D227A) .The cytotoxic effect is mediated by direct tumor cell killing (perfroin) and secretion of cytokines. This lead to tumor cell death, inflammation, vascular permeability and further recruitment of immune cells to the tumor site.

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Fig: 4 Action of ABR-214936 on tumor cell

VII.II. Antigen specific T cell infusion

Another effective strategy for passive immunotherapy is the infusion of tumor antigen-specific T cells since it plays a role in host response to a tumor. To prevent tumor cell from escaping T-cell killing genetical modification to T cell ex vivo to posses McAB specificity for protein epitope with a chimeric T-cell receptor is applied. Chimeric immune receptors (CIR) are engineered molecule encompass tumor- or virus-specific ligands or antibodies fused to the signaling domains of either the T cell receptor or Fc receptor.

Fig: 5 Antigen specific T cell infusion mechanisms

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VIII. Active immunotherapy:

A type of immunotherapy that attempts to stimulate the host's intrinsic immune response to the tumor, either nonspecifically or specifically. Active immunotherapy, also called therapeutic vaccines, aims to induce a therapeutic immune response through the patient own immune system. In particular, the use of active immunotherapy in cancer treatment is being evaluated. Most of the cancer vaccines currently under development aim at inducing a killer-cell response, whilst others attempt to generate antibody responses. It's characterized by long term specificity, long term efficacy and giving body protection against tumor genesis. There are two types of vaccines: viral vector vaccine and dentritic cell (DC) vaccines involved in active immunotherapy.

VIII.I. Viral vector vaccines

Many therapeutic viral vector are constructed based on TroVax virus which is a cancer immunotherapy product produced by oxford biomedical. It is a novel cancer vaccine in clinical development with potential application in most solid tumor types and delivers a novel proprietary tumor associated antigen, 5T4, using a pox virus vector, which is modified vaccinia virus Ankara (MVA) encoding the h5T4 protein .Vaccine viruses re engineered to express foreign genes are robust vectors for production of recombinant protein, the most common being a vaccine delivery system for antigens. Vaccination with recombinant modified vaccinia Ankara-5T4 vaccine may stimulate the host immune system to mount humoral and cytotoxic T lymphocyte (CTL) response against tumor cells expressing 5T4 fetal oncoprotein antigen, resulting in tumor cell lyses. Studies had shown that 5T4 immune response is boosted in the presence of MVA neutralizing antibodies. Vaccination with TroVax is safe and well tolerated, so 5T4 immune response can be induced without any evidence of autoimmune toxicity.

Fig: 6 (A) Production of the MVA-5T4 Constructs, (B) Mechanism of MVA strain encoding h5T4 protein.

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Other viral vaccine vectors are based on either replication- defective adenovirus like Adh5T4 or a lentivirial vectors, like the equine infectious anemia virus vector (EIVA), murine leukemia virus (MLV), human immunodeficiency virus (HIV)

Other therapeutic strategy targeting 5T4 antigen for destroying tumor cells take place through a viral vector encoding a fusion protein. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extra cellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4.The engineered retroviral constructs can produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effectors moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFvHIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers. Its propose that the combination of engineered T cell chimeric receptor with specific vaccination strategies can improve the active tumor therapy through mechanisms of enhancing Ag-specific cellular immune response.

VIII.II. Dentritic cell based vaccines

Lack of efficient tumor antigen presentation in DCs in cancer patients has led to the use of DC-based vaccines. As fig.5 shows the appropriate tumor antigen is bound antigen is bound to the DC cell surface. The tumor antigens are taken up by dendritic cells; they are processed and presented to the T cells along with the appropriate co stimulatory signal. Once activated by the DCs the cytotoxic T cells recognize and destroy the tumor cells and destroy the tumor cells expressing the tumor antigen.

Fig: 7 DC based vaccine

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Concerning the source of DC as fig.8 demonstrate they are collected from blood of the patients own tumor cells. This DC s is then reintroduced into the patient and stimulates the immune system.DC vaccines have been used in patients with metastatic melanoma, renal carcinoma and prostate cancer..

Fig: 8 Origin of DC used in DC based vaccine therapy.

Active immunotherapy strategy for tumor is more effective than passive one, since it increase the efficiency and improvement of the immune response against cancer cell also it involve the whole immune response activation and breaking the immune tolerance to tumor antigen involving different effecter cell interaction.

VIII.III. T reg cell and active DC therapy:

Regulatory T cell (sometimes known as suppressor T cells) is a specialized subpopulation of T cell that acts to suppress activation of the immune system most common is CD4+CD25+ regulatory T cells or "Tregs" and thereby maintain immune system homeostasis and tolerance to self antigens. In the tumor-bearing host, tumor-associated antigens (TAAs) could be taken up by tolerogenic dendritic cells present in the tumor microenvironment, plasmacytoid (pDC). PDCs have recently been shown to induce tolerance to an inhaled non-self-antigen. The allergen was taken up by pDCs, which presented the antigen to allergen-specific T cells that subsequently became T regulatory. These putative tumor-specific Treg cells may then circulate throughout the body and respond to self-antigens shed by tumors in the tumor-draining lymph nodes.

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Fig: 9 T reg cell and DC vaccine therapy

Subsequently, after activation, they may reside in the tumor-draining lymph node or infiltrate the tumor bed and suppress any precursor antitumor effectors CD8+ T lymphocytes from destroying their self-target, either through cell–cell contact by inhibiting T-cell help and activation (e.g., IL-2 deprivation or other mechanisms) or through release of immunosuppressive cytokines, such as IL-10 or TGF-β.TGF-β secreted by most melanomas could play a role in suppressive mechanisms by.Treg cells suppressed the expansion of co-transferred CD4+ Thelper cells, when suppressing T-cell help of tumor-reactive T cells; this will prevent CD8+ T cells from being properly activated. Knowing that CD8+ T cell recognizing 5T4 is important as anti-tumor effectors in the immune defense.

IX. Conclusion:

As we see 5T4 play a double role in biological and pathological progression .its a major candidate for immunotherapy for cancer. However information about the protein 5T4 activities in impanation, metastasis, interaction with immune effecter to decrease immunogenic is difficult to gather it by studying the human tissue but many research found that mouse and human 5T4 protein sequences show 81 % homology and with the possibility of cloning the coding region of the human and mouse 5T4 genes. This opens a wide view on study of 5T4 oncofetal antigen giving more opportunity to develop therapeutic strategy targeting this protein in cancer and other diseases.

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Glossary:

Antibody-dependent cell-mediated cytotoxicity: it is a mechanism of cell mediated immunity whereby an effecter cell of the immune system actively lyses a target cell that has been bound by specific antibodies. It is mediated by natural killer cell & monocytes

Carcinoma: is any malignant cancer that arises from epithelial cells.

MVA: (Modified Virus Ankara) It is a viral vector, derived from the replication-competent strain Ankara, is a highly attenuated, replicative-defective vaccinia strain incapable of virion assembly.

Lentiviral vectors: are types of retrovirus that can infect both dividing and non dividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell. .

ABR-214936: anatumomab mafenatox tumor targeted super antigen.

Adenovirus: any virus belonging to the family adenoviridae a family of DNA viruses with a double-stranded genome, generally with a narrow host ranges, and transferred by direct or indirect transmission.

Melanoma: is a malignant tumor of melanocytes which are found predominantly in skin but also in the bowel and the eye .It is one of the rarer types of skin cancer but causes the majority of skin cancer related deaths.

scFvHIgG1: single chain human immunoglobin (Ig) antibodies

TGF-β: Transforming growth factor beta controls proliferation, differentiation, and other functions in most cell types.

CD4: (cluster of differentiation 4) is a glycoprotein expressed on the surface of T helper cells, regulatory T cells. On T cells, CD4 is the co-receptor for the T cell receptor (TCR). It amplifies the signal generated by the TCR. It forms the T helper with ctyotoxic cell Tc.

CD8 :( cluster of differentiation 8) is a transmembrane glycoprotein which serves as a co-receptor for the T cell receptor (TCR). Cytotoxic T cells with CD8 surface protein are called CD8+ T cells.

LRR motif: leucine rich region motif, involved in cell signaling.

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