scholarly journals Effect of Humanizing Mutations on the Stability of the Llama Single-Domain Variable Region

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 163
Author(s):  
Miguel A. Soler ◽  
Barbara Medagli ◽  
Jiewen Wang ◽  
Sandra Oloketuyi ◽  
Gregor Bajc ◽  
...  
Keyword(s):  
Molecular Level ◽  
Variable Region ◽  
Clinical Applications ◽  
Antigen Binding ◽  
Structural Elements ◽  
Sequence Variability ◽  
Primary Sequence ◽  
Vh Domain ◽  
The Stability

In vivo clinical applications of nanobodies (VHHs) require molecules that induce minimal immunoresponse and therefore possess sequences as similar as possible to the human VH domain. Although the relative sequence variability in llama nanobodies has been used to identify scaffolds with partially humanized signature, the transformation of the Camelidae hallmarks in the framework2 still represents a major problem. We assessed a set of mutants in silico and experimentally to elucidate what is the contribution of single residues to the VHH stability and how their combinations affect the mutant nanobody stability. We described at molecular level how the interaction among residues belonging to different structural elements enabled a model llama nanobody (C8WT, isolated from a naïve library) to be functional and maintain its stability, despite the analysis of its primary sequence would classify it as aggregation-prone. Five chimeras formed by grafting CDRs isolated from different nanobodies into C8WT scaffold were successfully expressed as soluble proteins and both tested clones preserved their antigen binding specificity. We identified a nanobody with human hallmarks that seems suitable for humanizing selected camelid VHHs by grafting heterologous CDRs in its scaffold and could serve for the preparation of a synthetic library of human-like single domains.

Site-Specific in vivo Targeting of Magnetoliposomes Using Externally Applied Magnetic Field

2000 ◽  
Vol 55 (3-4) ◽  
pp. 278-281 ◽  
Author(s):  
Melánia Babincová ◽  
Veronika Altanerová ◽  
Miloš Lampert ◽  
Čestmír Altaner ◽  
Eva Machová ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Drug Targeting ◽  
Left Kidney ◽  
Clinical Applications ◽  
Magnetic Drug Targeting ◽  
Site Specific ◽  
Magnetite Particles ◽  
The Stability ◽  
The Right

Abstract Human serum albumin labeled with technetium-99m was encapsulated together with magnetite particles into phosphatidylcholine/cholesterol liposomes. In order to investigate the stability of this complex and its ability to be used for magnetic drug targeting, the in-vivo distribution after intravenous administration in rats was estimated. For in-vivo targeting an SmCo permanent magnet with intensity ~ 0.3 5 T was attached near the right kidney. Difference between the relative radioactivity in the magnetically targeted right kidney (25.92±5.84%) and non-targeted left kidney (0.93±0.05%) is sufficiently high for relevant clinical applications.

scholarly journals An in vivo method for diversifying the functions of therapeutic antibodies

2021 ◽  
Vol 118 (10) ◽  
pp. e2025596118
Author(s):  
Ming Tian ◽  
Hwei-Ling Cheng ◽  
Michael T. Kimble ◽  
Kelly McGovern ◽  
Peyton Waddicor ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Mouse Models ◽  
De Novo ◽  
Variable Region ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Therapeutic Antibodies ◽  
Ligand Interaction

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin (Ig) heavy chain (IgH) and light chain (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modified the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting antitumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies.

scholarly journals An In Vivo Method for Diversifying the Functions of Therapeutic Antibodies

2020 ◽  
Author(s):  
Ming Tian ◽  
Hwei-Ling Cheng ◽  
Michael T Kimble ◽  
Kelly McGovern ◽  
Peyton Waddicor ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Mouse Models ◽  
De Novo ◽  
Variable Region ◽  
Affinity Maturation ◽  
Antigen Binding ◽  
Therapeutic Antibodies ◽  
Ligand Interaction

V(D)J recombination generates mature B cells that express huge repertoires of primary antibodies as diverse immunoglobulin heavy (IgH) and light chains (IgL) of their B cell antigen receptors (BCRs). Cognate antigen binding to BCR variable region domains activates B cells into the germinal center (GC) reaction in which somatic hypermutation (SHM) modifies primary variable region-encoding sequences, with subsequent selection for mutations that improve antigen-binding affinity, ultimately leading to antibody affinity maturation. Based on these principles, we developed a humanized mouse model approach to diversify an anti-PD1 therapeutic antibody and allow isolation of variants with novel properties. In this approach, component Ig gene segments of the anti-PD1 antibody underwent de novo V(D)J recombination to diversify the anti-PD1 antibody in the primary antibody repertoire in the mouse models. Immunization of these mouse models further modifies the anti-PD1 antibodies through SHM. Known anti-PD1 antibodies block interaction of PD1 with its ligands to alleviate PD1-mediated T cell suppression, thereby boosting anti-tumor T cell responses. By diversifying one such anti-PD1 antibody, we derived many new anti-PD1 antibodies, including anti-PD1 antibodies with the opposite activity of enhancing PD1/ligand interaction. Such antibodies theoretically might suppress deleterious T cell activities in autoimmune diseases. The approach we describe should be generally applicable for diversifying other therapeutic antibodies.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

Aggregation prone regions in antibody sequences raised against Vibrio cholerae: A bioinformatic approach

2020 ◽  
Vol 15 ◽  
Author(s):  
Zakia Akter ◽  
Anamul Haque ◽  
Md. Sabir Hossain ◽  
Firoz Ahmed ◽  
Md Asiful Islam
Keyword(s):  
Vibrio Cholerae ◽  
Binding Sites ◽  
Bioinformatic Analysis ◽  
Antigen Binding ◽  
Short Term ◽  
Protein Database ◽  
The Stability ◽  
Bioinformatic Approach ◽  
Self Aggregation ◽  
Complementarity Determining Region

Background: Cholera, a diarrheal illness causes millions of deaths worldwide due to large outbreaks. Monoclonal antibody used as therapeutic purposes of cholera are prone to be unstable due to various factors including self-aggregation. Objectives: In this bioinformatic analysis, we identified the aggregation prone regions (APRs) of different immunogens of antibody sequences (i.e., CTB, ZnM-CTB, ZnP-CTB, TcpA-CT-CTB, ZnM-TcpA-CT-CTB, ZnP-TcpA-CT-CTB, ZnM-TcpA, ZnP-TcpA, TcpA-CT-TcpA, ZnM-TcpA-CT-TcpA, ZnP-TcpA-CT-TcpA, Ogawa, Inaba and ZnM-Inaba) raised against Vibrio cholerae. Methods: To determine APRs in antibody sequences that were generated after immunizing Vibrio cholerae immunogens on Mus musculus, a total of 94 sequences were downloaded as FASTA format from a protein database and the algorithms such as Tango, Waltz, PASTA 2.0, and AGGRESCAN were followed to analyze probable APRs in all of the sequences. Results: A remarkably high number of regions in the monoclonal antibodies were identified to be APRs which could explain a cause of instability/short term protection of anticholera vaccine. Conclusion: To increase the stability, it would be interesting to eliminate the APR residues from the therapeutic antibodies in a such way that the antigen binding sites or the complementarity determining region loops involved in antigen recognition are not disrupted.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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