Effect of framework and complementarity determining region H1 charge on the human VH-B1a domain expression, folding, stability, and solubility
Bibliographic record
Abstract
Many difficulties related to using antibodies in diagnostic and therapeutic applications can sometimes be circumvented by using smaller, less complex single domain antibodies based on variable heavy chain (VH) domain constructs such as camelid VHH domains. However, VH domains have their own limitations, including an increased tendency to aggregate. VH domains often contain hydrophobic residues within their complementarity-determining regions (CDRs) that facilitate binding to target antigens but can also mediate VH domain aggregation, which is a concern for therapeutic applications since this can trigger immune responses. In this study, we engineered the human VH-1Ba domain to improve its stability and solubility by introducing charged amino acids in the VH domain framework region and CDRH1. We followed two strategies to improve the stability and solubility of VH domains. First, we introduced positive and negatively charged amino acids in the framework region of an autonomous human VH domain (VH-B1a) and observed the effect of framework net charge on VH domain refolding, stability, and solubility. Introducing positive charge into the VH-B1a framework increased its thermostability but slightly lowered its refolding ability and solubility. We were not able to obtain correctly folded negatively charged (-VH) VH domains. Second, we introduced a series of positive and negatively charged amino acids in the CDRH1 loop of near-neutral (VH-B1a) and positively charged (+VH) VH domains, and observed their effect on expression, refolding, stability, and solubility. For both the VH-B1a and +VH domains, we observed a decrease in melting temperature (Tm) and room temperature solubility as more negative or positive charged amino acids were added to the CDRH1. The VH-B1a domain had higher room temperature solubility for negative and slightly positive CDRH1 net charges. The + VH domain had higher Tms for all CDRH1 net charges and was better able to tolerate the adverse effects of adding positive charge to CDRH1. We observed a similar response in refolding and solubility of VH-B1a and +VH in response to changes in CDRH1 net charge after temperature-induced denaturation for negative and neutral CDRH1s. We observed a positional effect with a single Lys (31K) and double Lys (31, 32 KK) substitutions in CDRH1, which promoted VH-B1a aggregation and was partially overcome by the +VH domain. In summary, this study provides information for designing VH domains with improved biophysical properties and a +VH domain that will be useful for applications where positive surface charge and CDRH1 are desirable.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".