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David M Kranz - One of the best experts on this subject based on the ideXlab platform.
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identification and engineering of human variable Regions that allow expression of stable single chain t cell receptors
Protein Engineering Design & Selection, 2011Co-Authors: David H Aggen, Adam S Chervin, Francis K Insaidoo, Kurt H Piepenbrink, Brian M Baker, David M KranzAbstract:Single-chain antibody fragments (scFv), consisting of two linked variable Regions (V(H) and V(L)), are a versatile format for engineering and as potential antigen-specific therapeutics. Although the analogous format for T cell receptors (TCRs), consisting of two linked V Regions (Vα and Vβ; referred to here as scTv), could provide similar opportunities, all wild-type scTv proteins examined to date are unstable. This obstacle has prevented scTv fragments from being widely used for engineering or therapeutics. To further explore whether some stable human scTv fragments could be expressed, we used a yeast system in which display of properly folded domains correlates with ability to express the folded scTv in soluble form. We discovered that, unexpectedly, scTv fragments that contained the human Vα2 Region (IMGT: TRAV12 family) were displayed and properly associated with different Vβ Regions. Furthermore, a single polymorphic residue (Ser(α49)) in the Framework Region conferred additional thermal stability. These stabilized Vα2-containing scTv fragments could be expressed at high levels in Escherichia coli, and used to stain target cells that expressed the specific pep-HLA-A2 complexes. Thus, the scTv fragments can serve as a platform for engineering TCRs with diverse specificities, and possibly for therapeutic or diagnostic applications.
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mapping the energy of superantigen staphylococcus enterotoxin c3 recognition of an α β t cell receptor using alanine scanning mutagenesis
Journal of Experimental Medicine, 2000Co-Authors: Hywyn R O Churchill, Peter S Andersen, Evan A Parke, Roy A Mariuzza, David M KranzAbstract:Binding of the T cell receptor (TCR) to a bacterial superantigen (SAG) results in stimulation of a large population of T cells and subsequent inflammatory reactions. To define the functional contribution of TCR residues to SAG recognition, binding by 24 single-site alanine substitutions in the TCR Vβ domain to Staphylococcus aureus enterotoxin (SE) C3 was measured, producing an energy map of the TCR–SAG interaction. The results showed that complementarity determining Region 2 (CDR2) of the Vβ contributed the majority of binding energy, whereas hypervariable Region 4 (HV4) and Framework Region 3 (FR3) contributed a minimal amount of energy. The crystal structure of the Vβ8.2–SEC3 complex suggests that the CDR2 mutations act by disrupting Vβ main chain interactions with SEC3, perhaps by affecting the conformation of CDR2. The finding that single Vβ side chain substitutions had significant effects on binding and that other SEC3-reactive Vβ are diverse at these same positions indicates that SEC3 binds to other TCRs through compensatory mechanisms. Thus, there appears to be strong selective pressure on SAGs to maintain binding to diverse T cells.
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selection of functional t cell receptor mutants from a yeast surface display library
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Michele C Kieke, David M Kranz, Eric V Shusta, Eric T Boder, Luc Teyton, Dane K WittrupAbstract:The heterodimeric αβ T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable Region residues. These residues are located in two Regions of the TCR, at the interface of the α- and β-chains and in the β-chain Framework Region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable Regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.
Mehdi Arbabighahroudi - One of the best experts on this subject based on the ideXlab platform.
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role of the non hypervariable fr3 d e loop in single domain antibody recognition of haptens and carbohydrates
Journal of Molecular Recognition, 2019Co-Authors: Kevin A Henry, Greg Hussack, Jyothi Kumaran, Michel Gilbert, Roger C Mackenzie, Traian Sulea, Mehdi ArbabighahroudiAbstract:Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between β-strands D and E of Framework Region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other Regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this Region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.
Ira Pastan - One of the best experts on this subject based on the ideXlab platform.
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lowering the isoelectric point of the fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity
Cancer Research, 2001Co-Authors: Masanori Onda, Robert J Kreitman, Byungkook Lee, Satoshi Nagata, Yasuo Tsutsumi, James J Vincent, Qingcheng Wang, Ira PastanAbstract:Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the Framework Region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.
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stabilization of the fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved Framework Regions
Biochemistry, 1994Co-Authors: Yoram Reiter, Robert J Kreitman, Ulrich Brinkmann, Sunhee Jung, Byungkook Lee, Ira PastanAbstract:Abstract Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved Framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved Framework Region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)
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design of interchain disulfide bonds in the Framework Region of the fv fragment of the monoclonal antibody b3
Proteins, 1994Co-Authors: Sunhee Jung, Ira Pastan, Byungkook LeeAbstract:The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the Framework Region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S. DHHS, NIH publication No. 91-3242, 1991). This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the Framework Region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.
Eli E Sercarz - One of the best experts on this subject based on the ideXlab platform.
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protection against experimental autoimmune encephalomyelitis generated by a recombinant adenovirus vector expressing the vβ8 2 tcr is disrupted by coadministration with vectors expressing either il 4 or 10
Journal of Immunology, 2003Co-Authors: Todd A Braciak, Brian Pedersen, Judy Chin, Clay Hsiao, Sally E Ward, Igor Maricic, Alex Jahng, Frank L Graham, Jack Gauldie, Eli E SercarzAbstract:Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR β-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vβ8.2 chain (Ad5E1 mVβ8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the Framework Region 3 determinant within the B5 peptide (aa 76–101) of the Vβ8.2 chain. This determinant is readily processed and displayed in an I-Au context, on ambient APC. Transient genetic delivery of the TCR Vβ8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVβ8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.
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induction or protection from experimental autoimmune encephalomyelitis depends on the cytokine secretion profile of tcr peptide specific regulatory cd4 t cells
Journal of Immunology, 1998Co-Authors: Vipin Kumar, Eli E SercarzAbstract:Autoimmune diseases can result from the breakdown of regulation and subsequent activation of self-antigenic determinant-reactive T cells. During the evolution of the autoimmune response to myelin basic protein (MBP) in B10.PL mice, several distinct T cell populations expand: the effectors mediating experimental autoimmune encephalomyelitis (EAE) are MBP-reactive, CD4 + , and predominantly TCR Vβ8.2 + ; in addition, at least two regulatory populations can be detected—one comprised of Vβ14 + CD4 T cells, reactive to a Framework Region 3 determinant on the Vβ8.2 chain, and a second that is CD8 + and reactive to another Vβ8.2 determinant. The combined action of these two regulatory cell types controls disease-causing effectors, resulting in spontaneous recovery from disease. In this report, we reveal that the cytokine secretion pattern of TCR peptide-specific regulatory CD4 T cells can profoundly influence whether a type 1 or type 2 population predominates among MBP-specific CD4 effectors. The priming of type 1 regulatory T cells results in deviation of the Ag-specific effector T cell population in a type 2 direction and protection from disease. In contrast, induction of type 2 regulatory T cells results in exacerbation of EAE, poor recovery, and an increased frequency of type 1 effectors. Thus, the encephalitogenic potential of the MBP-reactive effector population is crucially and dominantly influenced by the cytokine secretion phenotype of regulatory CD4 T cells. These findings have important implications in understanding peripheral tolerance to self-Ags as well as in the design of TCR-based therapeutic approaches.
Kevin A Henry - One of the best experts on this subject based on the ideXlab platform.
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role of the non hypervariable fr3 d e loop in single domain antibody recognition of haptens and carbohydrates
Journal of Molecular Recognition, 2019Co-Authors: Kevin A Henry, Greg Hussack, Jyothi Kumaran, Michel Gilbert, Roger C Mackenzie, Traian Sulea, Mehdi ArbabighahroudiAbstract:Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between β-strands D and E of Framework Region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other Regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this Region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.
