Trifunctional antibody
Encyclopedia
A trifunctional antibody is a monoclonal antibody with binding sites for two different antigens, typically CD3 and a tumor antigen
, making it a type of bispecific monoclonal antibody
. In addition, its intact Fc-part can bind to an Fc receptor
on accessory cells
like conventional monospecific antibodies. The net effect is that this type of drug links T cell
s (via CD3) and monocyte
s/macrophage
s, natural killer cell
s, dendritic cell
s or other Fc receptor expressing cells to the tumor cells, leading to their destruction.
At an equivalent dose a trifunctional antibody is more potent (more than 1,000-fold) in eliminating tumor cells than conventional antibodies. These drugs evoke the removal of tumor cells by means of (i) antibody-dependent cell-mediated cytoxicity, a process also described for conventional antibodies and more importantly by (ii) polyclonal cytotoxic T cell
responses with emphasis on CD8 T cells. These trifunctional antibodies also elicit individual anti-tumor immune responses in cancer patients treated with e.g. catumaxomab; i.e. autologous antibodies as well as CD4 and CD8 T cells directed against the tumor were detected. Furthermore, putative cancer stem cell
s from malignant ascites fluid were eliminated due to catumaxomab
treatment.
Examples include catumaxomab
(EpCAM / CD3), ertumaxomab
(HER2/neu
/ CD3), FBTA05 (CD20
/ CD3, proposed trade name Lymphomun) and TRBS07 (GD2
/ CD3, proposed trade name Ektomab), drugs against various types of cancer.
Furthermore, despite the development of anti-drug antibody responses after the first catumaxomab application cycle a repeated cycle of catumaxomab also leads to a treatment success in recurrent malignant ascites. Cross-linking leads to the release of cytokine
s, resulting in manageable adverse effects like fever, nausea
and vomiting, that were generally reversible and mainly related to the immunological mode of action (e.g. catumaxomab).
Catumaxomab, which was approved in 2009 for the treatment of malignant ascites in cancer patients, satisfies these conditions. It was the first, and as of May 2011 the only approved one of these antibodies in clinical use. Another way of immunotherapeutic intervention strategies is the exploration of bispecific antibodies with different structures, of which bi-specific T-cell engagers (BiTEs) have been produced since the mid-2000s.
cells whose monoclonal antibodies target one of the desired antigens are produced. Independently, rat hybridoma cells targeting the other antigen are produced. These two cell types are hybridised, yielding hybrid-hybridomas or quadromas, which produce hybrid (trifunctional) antibody as well as pure mouse and pure rat antibody. The trifunctional antibody is extracted chromatographically with protein A
.
Using two different species (mouse and rat) has the advantage that less mismatched antibodies are produced because rat light chains
preferably pair with rat heavy chains, and mouse light chains with mouse heavy chains. Single species (mouse/mouse or rat/rat) quadromas, by contrast, produce up to ten different kinds of antibody, most of which have mismatched heavy or light chains, or both.
Tumor antigen
Tumor antigen is an antigenic substance produced in tumor cells, i.e., it triggers an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for use in cancer therapy.- Mechanism of tumor antigenesis:...
, making it a type of bispecific monoclonal antibody
Bispecific monoclonal antibody
A bispecific monoclonal antibody is an artificial protein that is composed of fragments of two different monoclonal antibodies and consequently binds to two different types of antigen...
. In addition, its intact Fc-part can bind to an Fc receptor
Fc receptor
An Fc receptor is a protein found on the surface of certain cells - including natural killer cells, macrophages, neutrophils, and mast cells - that contribute to the protective functions of the immune system....
on accessory cells
Antigen-presenting cell
An antigen-presenting cell or accessory cell is a cell that displays foreign antigen complexes with major histocompatibility complex on their surfaces. T-cells may recognize these complexes using their T-cell receptors...
like conventional monospecific antibodies. The net effect is that this type of drug links T cell
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
s (via CD3) and monocyte
Monocyte
Monocytes are a type of white blood cell and are part of the innate immune system of vertebrates including all mammals , birds, reptiles, and fish. Monocytes play multiple roles in immune function...
s/macrophage
Macrophage
Macrophages are cells produced by the differentiation of monocytes in tissues. Human macrophages are about in diameter. Monocytes and macrophages are phagocytes. Macrophages function in both non-specific defense as well as help initiate specific defense mechanisms of vertebrate animals...
s, natural killer cell
Natural killer cell
Natural killer cells are a type of cytotoxic lymphocyte that constitute a major component of the innate immune system. NK cells play a major role in the rejection of tumors and cells infected by viruses...
s, dendritic cell
Dendritic cell
Dendritic cells are immune cells forming part of the mammalian immune system. Their main function is to process antigen material and present it on the surface to other cells of the immune system. That is, dendritic cells function as antigen-presenting cells...
s or other Fc receptor expressing cells to the tumor cells, leading to their destruction.
At an equivalent dose a trifunctional antibody is more potent (more than 1,000-fold) in eliminating tumor cells than conventional antibodies. These drugs evoke the removal of tumor cells by means of (i) antibody-dependent cell-mediated cytoxicity, a process also described for conventional antibodies and more importantly by (ii) polyclonal cytotoxic T cell
Cytotoxic T cell
A cytotoxic T cell belongs to a sub-group of T lymphocytes that are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses , or are otherwise damaged or...
responses with emphasis on CD8 T cells. These trifunctional antibodies also elicit individual anti-tumor immune responses in cancer patients treated with e.g. catumaxomab; i.e. autologous antibodies as well as CD4 and CD8 T cells directed against the tumor were detected. Furthermore, putative cancer stem cell
Cancer stem cell
Cancer stem cells are cancer cells that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs are therefore tumorigenic , perhaps in contrast to other non-tumorigenic cancer cells...
s from malignant ascites fluid were eliminated due to catumaxomab
Catumaxomab
Catumaxomab is a rat-mouse hybrid monoclonal antibody which is used to treat malignant ascites, a condition occurring in patients with metastasizing cancer. It binds to antigens CD3 and EpCAM. It is in clinical trials in the United States currently and is used in Europe...
treatment.
Examples include catumaxomab
Catumaxomab
Catumaxomab is a rat-mouse hybrid monoclonal antibody which is used to treat malignant ascites, a condition occurring in patients with metastasizing cancer. It binds to antigens CD3 and EpCAM. It is in clinical trials in the United States currently and is used in Europe...
(EpCAM / CD3), ertumaxomab
Ertumaxomab
Ertumaxomab is a rat-murine hybrid monoclonal antibody designed to treat some types of cancer. Phase II clinical trial evaluating the treatment of breast cancer was terminated due to change in Fresenius' development plans....
(HER2/neu
HER2/neu
HER-2 also known as proto-oncogene Neu, receptor tyrosine-protein kinase erbB-2, CD340 or p185 is an enzyme that in humans is encoded by the ERBB2 gene. Over expression of this gene is correlated with higher aggressiveness in breast cancers...
/ CD3), FBTA05 (CD20
CD20
B-lymphocyte antigen CD20 or CD20 is an activated-glycosylated phosphoprotein expressed on the surface of all B-cells beginning at the pro-B phase and progressively increasing in concentration until maturity....
/ CD3, proposed trade name Lymphomun) and TRBS07 (GD2
GD2
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans....
/ CD3, proposed trade name Ektomab), drugs against various types of cancer.
History
Trifunctional antibodies were the first type of bispecific monoclonal antibodies to be produced. The first concepts date back to the mid-1980s. For over twenty years, no such antibody was approved for clinical use, mainly because of manufacturing difficulties. Immunogenicity results from the fact that appropriate parental antibodies are obtained from rat and mice. After application, the patient's immune system usually produces anti-drug antibodies, which represent early indicators for a beneficial clinical outcome.Furthermore, despite the development of anti-drug antibody responses after the first catumaxomab application cycle a repeated cycle of catumaxomab also leads to a treatment success in recurrent malignant ascites. Cross-linking leads to the release of cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
s, resulting in manageable adverse effects like fever, nausea
Nausea
Nausea , is a sensation of unease and discomfort in the upper stomach with an involuntary urge to vomit. It often, but not always, precedes vomiting...
and vomiting, that were generally reversible and mainly related to the immunological mode of action (e.g. catumaxomab).
Catumaxomab, which was approved in 2009 for the treatment of malignant ascites in cancer patients, satisfies these conditions. It was the first, and as of May 2011 the only approved one of these antibodies in clinical use. Another way of immunotherapeutic intervention strategies is the exploration of bispecific antibodies with different structures, of which bi-specific T-cell engagers (BiTEs) have been produced since the mid-2000s.
Production
At first, mouse hybridomaHybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...
cells whose monoclonal antibodies target one of the desired antigens are produced. Independently, rat hybridoma cells targeting the other antigen are produced. These two cell types are hybridised, yielding hybrid-hybridomas or quadromas, which produce hybrid (trifunctional) antibody as well as pure mouse and pure rat antibody. The trifunctional antibody is extracted chromatographically with protein A
Protein A
Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in...
.
Using two different species (mouse and rat) has the advantage that less mismatched antibodies are produced because rat light chains
Immunoglobulin light chain
]The immunoglobulin light chain is the small polypeptide subunit of an antibody .A typical antibody is composed of two immunoglobulin heavy chains and two Ig light chains.-In humans:...
preferably pair with rat heavy chains, and mouse light chains with mouse heavy chains. Single species (mouse/mouse or rat/rat) quadromas, by contrast, produce up to ten different kinds of antibody, most of which have mismatched heavy or light chains, or both.