The Experts below are selected from a list of 219 Experts worldwide ranked by ideXlab platform
Inhee Chung - One of the best experts on this subject based on the ideXlab platform.
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transferrin receptor tfr trafficking determines brain uptake of tfr Antibody Affinity variants
Journal of Experimental Medicine, 2014Co-Authors: Nga Bienly, Joy Y Yu, Daniela Bumbaca, Justin Elstrott, Andrew C Boswell, Yin Zhang, Yanmei Lu, Mark S Dennis, Robby M Weimer, Inhee ChungAbstract:Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood–brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR Affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR Antibody Affinity alters the intracellular trafficking of TfR. Comparing high- and low-Affinity TfR bispecific antibodies in vivo, we found that high-Affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-Affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-Affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-Affinity anti-TfR bispecific. Thus, high-Affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.
Ira Pastan - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Antibody Affinity Maturation Targeting Germline Hotspots
2009Co-Authors: Mitchell Ho, Ira PastanAbstract:Affinity-matured antibodies can exhibit increased biological efficacy. Regardless of whether an Antibody is isolated from a hybridoma or a human Fv phage library, the Antibody Affinity for its target may need improvement for therapeutic applications. An increased Affinity may allow for a reduced dosage of a therapeutic Antibody; toxic side effects may also be reduced. In the immune system, Affinity maturation is a process involving somatic hypermutations in B cells. Therefore, germline hotspot residues are most likely to have a major impact on Antibody Affinity. Here, we describe procedures for germline hotspot mutagenesis with an emphasis on strategies for randomizing hotspots with PCR and phage display, using as an example the anti-CD22 monoclonal Antibody.
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Improving Antibody Affinity by mimicking somatic hypermutation in vitro
Nature Biotechnology, 1999Co-Authors: Partha S. Chowdhury, Ira PastanAbstract:In vivo Affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve Antibody Affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in Affinity compared with a single clone with a fourfold increased Affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-Affinity mutants of Fvs, Fabs, and other recombinant antibodies from Antibody phage libraries that are small in size.
Nga Bienly - One of the best experts on this subject based on the ideXlab platform.
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transferrin receptor tfr trafficking determines brain uptake of tfr Antibody Affinity variants
Journal of Experimental Medicine, 2014Co-Authors: Nga Bienly, Joy Y Yu, Daniela Bumbaca, Justin Elstrott, Andrew C Boswell, Yin Zhang, Yanmei Lu, Mark S Dennis, Robby M Weimer, Inhee ChungAbstract:Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood–brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR Affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR Antibody Affinity alters the intracellular trafficking of TfR. Comparing high- and low-Affinity TfR bispecific antibodies in vivo, we found that high-Affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-Affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-Affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-Affinity anti-TfR bispecific. Thus, high-Affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.
Partha S. Chowdhury - One of the best experts on this subject based on the ideXlab platform.
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Improving Antibody Affinity by mimicking somatic hypermutation in vitro
Nature Biotechnology, 1999Co-Authors: Partha S. Chowdhury, Ira PastanAbstract:In vivo Affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve Antibody Affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in Affinity compared with a single clone with a fourfold increased Affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-Affinity mutants of Fvs, Fabs, and other recombinant antibodies from Antibody phage libraries that are small in size.
Robby M Weimer - One of the best experts on this subject based on the ideXlab platform.
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transferrin receptor tfr trafficking determines brain uptake of tfr Antibody Affinity variants
Journal of Experimental Medicine, 2014Co-Authors: Nga Bienly, Joy Y Yu, Daniela Bumbaca, Justin Elstrott, Andrew C Boswell, Yin Zhang, Yanmei Lu, Mark S Dennis, Robby M Weimer, Inhee ChungAbstract:Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood–brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR Affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR Antibody Affinity alters the intracellular trafficking of TfR. Comparing high- and low-Affinity TfR bispecific antibodies in vivo, we found that high-Affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-Affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-Affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-Affinity anti-TfR bispecific. Thus, high-Affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.
