Synonyms for scfv or Related words with scfv
Examples of "scfv"
The most common form of CARs are fusions of single-chain variable fragments (
) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. An example of such a construct is 14g2a-Zeta, which is a fusion of a
derived from hybridoma 14g2a (which recognizes disialoganglioside GD2).
The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form an
is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (cloven). A flexible spacer allows the
to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signalling endodomain that protrudes into the cell and transmits the desired signal.
/CD3-zeta hybrids result in the transmission of a zeta signal in response to recognition by the
of its target. When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g. neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.
The LD of the Cll1 toxin in mice is 85 μg/kg. A possible treatment for an intoxication by Cll1 toxin is the use of single chain variable fragments (
The variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (
), which is only half the size of the Fab fragment, yet retains the original specificity of the parent immunoglobulin.
Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., protein G) cannot be used to purify antibodies. These fragments can often be purified or immobilized using protein L, since protein L interacts with the variable region of kappa light chains. More commonly, scientists incorporate a six histidine tag on the c-terminus of the
molecule and purify them using immobilized metal affinity chromatography (IMAC). For unknown reasons, some
can also be captured by protein A.
Nine years later, Fukuda and colleagues chose mRNA display method for "in vitro" evolution of single-chain Fv (
) antibody fragments. They selected six different
mutants with five consensus mutations. However, kinetic analysis of these mutants showed that their antigen-specificity remained similar to that of the wild type. However, they have demonstrated that two of the five consensus mutations were within the complementarity determining regions (CDRs). And they concluded that mRNA display has the potential for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.
The disadvantage of using T7 is that the size of the protein that can be expressed on the surface is limited to shorter peptides because large changes to the T7 genome cannot be accommodated like it is in M13 where the phage just makes its coat longer to fit the larger genome within it. However, it can be useful for the production of a large protein library for
selection where the
is expressed on an M13 phage and the antigens are expressed on the surface of the T7 phage.
These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative,
can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors.
These employ the selective principles of specific antibody production but exploit micro-organisms (as in phage display) or even cell free extracts (as in ribosome display). These systems rely on the creation of antibody gene "libraries" which can be wholly derived from human RNA isolated from peripheral blood. The immediate products of these systems are antibody fragments, normally Fab or
A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternatives include the CHCH region of immunoglobulin and portions of CD3. For most
based constructs, the IgG1 hinge suffices. However the best spacer often is determined empirically.
A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to glycosylate and anchor in the cell membrane. Any eukaryotic signal peptide sequence usually works. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g. in a
with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).
The antigen recognition region is usually an
, although many alternatives exist. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). Almost anything that binds a given target with high affinity can be used as an antigen recognition.
SMIPs are single-chain proteins that comprise one binding region, one hinge region as a connector, and one effector domain. The binding region is a modified single-chain variable fragment (
), and the rest of the protein can be constructed from the fragment crystallizable region (Fc) and the hinge region of an immunoglobulin G (IgG). Genetically modified cells produce SMIPs as antibody-like dimers, which are about 30% smaller than real antibodies.
Chimeric antigen receptors (CARs) have been developed as a promising therapy for ALL. This technology uses a single chain variable fragment (
) designed to recognize the cell surface marker CD19 as a method of treating ALL. CD19 is a molecule found on all B-cells and can be used as a means of distinguishing the potentially malignant B-cell population in the patient. In this therapy, mice are immunized with the CD19 antigen and produce anti-CD19 antibodies. Hybridomas developed from the mouse spleen cells fused to a myeloma cell line can be developed as a source for the cDNA encoding the CD19 specific antibody. The cDNA is sequenced and the sequence encoding the variable heavy and variable light chains of these antibodies are cloned together using a small peptide linker. This resulting sequence encodes the
. This can be cloned into a transgene encoding what will become the endodomain of the CAR. There are varying arrangements of subunits used as the endodomain but they generally consist of the hinge region that attaches to the
, a transmembrane region, the intracellular region of a costimulatory molecule such as CD28, and the intracellular domain of CD3-zeta containing ITAM repeats. Other sequences frequently included are: 4-1bb and OX40. The final transgene sequence, containing the
and endodomain sequences is then inserted into immune effector cells that are obtained from the patient and expanded in vitro. In previous trials these have been a type of T-cell capable of cytotoxicity. Inserting the DNA into the effector cell can be accomplished by several methods. Most commonly, this is done using a lentivirus which encodes the transgene. Pseudotyped, self-inactivating lentiviruses have been shown to be an effective method for the stable insertion of a desired transgene into the target cell genomic DNA. Other methods include electroporation and transfection but these are limited in their efficacy as transgene expression will diminish over time. The gene-modified effector cells are then transplanted back into the patient. Typically this process is done in conjunction with a conditioning regimen such as cyclophosphamide which has been shown to potentiate the effects of infused T-cells. This effect has been attributed to making an immunologic space within which the cells populate. The process as a whole results in an effector cell, typically a T-cell, that can recognize a tumor cell antigen in a manner that is independent of the major histocompatibility complex, and which can initiate a cytotoxic response.
The first generation of CAR-modified T cells (CARTs) showed success in pre-clinical trials and have entered phase I clinical trials in ovarian cancer, neuroblastoma and various types of leukemia and lymphoma. To date, these clinical trials have shown little evidence of anti-tumor activity, with insufficient activation, persistence and homing to cancer tissue. Some anti-tumor responses have been reported in patients with B cell lymphoma (treated with alfaCD20-CD3zeta CAR-modified T cells) and some neuroblastoma patients (treated with
-CD3zeta CARTs) reported partial response, stable disease and remission.
A monoclonal antibody targeting the desired antigen can be developed the classical way, using hybridoma technology. The
is then constructed from the antibody's variable regions. A large number of different hinge regions and effector domains are taken from libraries of immunoglobulins, and the combined proteins are produced in genetically modified (transfected) cells and screened for clones with useful properties like high binding specificity. The selected protein is multiplied in transfected cells suitable for medium- or large-scale production, for example Chinese hamster ovary cells, and purified by chromatography.
In an experiment in 1995, display of Glutathione S-transferase was attempted on both pVII and pIX and failed. However, phage display of this protein was completed successfully after the addition of a periplasmic signal sequence (pelB or ompA) on the N-terminus. In a recent study, it has been shown that AviTag, FLAG and His could be displayed on pVII without the need of a signal sequence. Then the expression of single chain Fv's (
), and single chain T cell receptors (scTCR) were expressed both with and without the signal sequence.
A single-chain variable fragment (
) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (V) and light chains (V) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V with the C-terminus of the V, or "vice versa".
Heterodimeric antibodies, which are also asymmetrical and antibodies, allow for greater flexibility and new formats for attaching a variety of drugs to the antibody arms. One of the general formats for a heterodimeric antibody is the “knobs-into-holes” format. This format is specific to the heavy chain part of the constant region in antibodies. The “knobs” part is engineered by replacing a small amino acid with a larger one. It fits into the “hole”, which is engineered by replacing a large amino acid with a smaller one. What connects the “knobs” to the “holes” are the disulfide bonds between each chain. The “knobs-into-holes” shape facilitates antibody dependent cell mediated cytotoxicity. Single chain variable fragments (
) are connected to the variable domain of the heavy and light chain via a short linker peptide. The linker is rich in glycine, which gives it more flexibility, and serine/threonine, which gives it specificity. Two different
fragments can be connected together, via a hinge region, to the constant domain of the heavy chain or the constant domain of the light chain. This gives the antibody bispecificity, allowing for the binding specificities of two different antigens. The “knobs-into-holes” format enhances heterodimer formation but doesn’t suppress homodimer formation.
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