mAbs最新文献

Monoclonal antibodies (mAbs) feature a conserved N-linked glycosylation site in the CH2 domain, which exhibits heterogeneities in both occupancy and glycan structures. Previous studies have suggested that the unoccupied (nonglycosylated) variant exhibits decreased thermal stability, potentially impacting the overall stability of mAb products. This hypothesis, however, has remained largely unconfirmed, due to the low abundance of nonglycosylated variants in typical mAb products and the lack of effective analytical tools for detailed characterization of large aggregates with glycoform-specific information. Here, we used a postcolumn denaturation-assisted size exclusion chromatography mass spectrometry technique (SEC-PCD-MS) to reevaluate the effects of the nonglycosylated mAb variant on the thermal stability of mAb drugs during forced degradation studies. Our findings confirmed the compromised thermal stability of the nonglycosylated variant and its increased propensity to form large aggregates at elevated temperatures relevant to mAb-forced degradation studies. We also showed that this thermal stress-induced, nonglycosylation-mediated aggregation pathway could be widely observed in a diverse group of mAb molecules with varying properties. This study offers valuable insights into the rationale of selecting the appropriate temperature for mAb-forced degradation studies and highlights key considerations for data interpretation.

Protein glycosylation at asparagine typically occurs at a consensus motif. However, recent studies have reported instances of N-glycosylation at non-consensus sites, though the mechanisms and implications of these atypical modifications remain unclear. In this study, we identified novel non-consensus N-glycosylation motifs with low glycosylation occupancy in the Fab region of human antibodies. We developed a computational workflow to predict the interaction between non-consensus peptides and the eukaryotic oligosaccharyltransferase (OST) complex. This model was validated through site-directed mutagenesis around the asparagine residue and glycosylation quantification via mass spectrometry. Our results show that glycan occupancy at non-consensus sites can be modulated by mutations that influence OST binding affinity. Pharmacological inhibition of OST activity reduced non-consensus and consensus glycosylation in both Fab and Fc regions. Additionally, we identified new non-consensus glycosylation sites in natural human antibodies, revealing the sequence preferences governing these modifications. These findings provide mechanistic insights into OST sequence specificity and establish a computational and analytical framework for assessing atypical N-glycosylation, aiding glycan profile control in therapeutic antibody development.

Single-chain antibodies (scAbs), derived from camelid antibodies, have gained attention as therapeutic candidates due to their small size and perceived low immunogenicity, but recent studies have reported immune responses to several scAbs. To better understand their immunogenicity, we investigated the T-cell responses induced by VHH76, a VHH-Fc engineered to target the SARS-CoV-2 RBD, along with its humanized and germlined variants. The humanized variant contains six human substitutions, while the germlined variant was obtained by screening of a combinatorial library of the VHH76 sequences, comprising human and wild-type substitutions at 12 different positions. The germlined variant finally contains 16 human substitutions. All VHH76 variants triggered CD4 T-cell responses from healthy donors, with the germlined VHH76 showing significantly reduced T-cell stimulation. Two epitope regions were identified: one overlapping CDR3 and another spreading from CDR1 to CDR2. Additional human substitutions at the VHH-conserved positions in FR2 compromised the biological properties of the germlined VHH76 and did not seem to reduce clearly the risk of T-cell response. In conclusion, using a sensitive T-cell assay, we showed that T cells specific for VHH76 variants were detected in the blood of healthy donors and that the frequency of responding T cells diminished with germlining. While epitopes in CDR3 are linked to VHH76 specificity, modifying the conserved FR2 region presents challenges for reducing VHH76 immunogenicity. This study contributes to the understanding of VHH76 immunogenicity and offers insights into strategies to mitigate immune responses.

Targeting checkpoint inhibitors is an effective therapy for treating cancer, with human programmed cell death protein 1 (hPD-1) being one of the most successful targets for developing antibody-based drugs. In this work, we isolated a panel of anti-PD-1 single-chain variable fragments with different binding and functional profiles from a fully synthetic human phage display library. Conversion of the best clone to hIgG1LALA and hIgG4PE formats, called UDIZ-007 and UDIZ-008, respectively, resulted in antibodies that effectively blocked the PD-1:PD-L1/L2 interaction and were highly selective as they did not cross-react with CD28 receptor family members. Doses of UDIZ-007 or UDIZ-008 at 10 mg/kg every 3 days for a total of six intraperitoneal administrations eradicated MC38-hPD-L1 colon tumors in B-hPD-1 transgenic mice for hPD-1 at day 17, with no relapse until the end of the study at day 56. Importantly, these antibodies bind hPD-1 in a unique region compared to the anti-PD-1 antibodies of known structure, which might have an impact on novel oncology indications when used as a standalone therapy or in combination with currently approved anti-PD-1 therapeutic antibodies. Therefore, UDIZ-007 and UDIZ-008 seem to be promising candidates for the development of antibody-based drugs targeting checkpoint inhibitors as a treatment for cancer.

Bispecific monoclonal antibodies (bsmAbs) are expected to provide targeted drug delivery that overcomes the dose-limiting toxicities often accompanying antibody-drug conjugates (ADC) in clinical practice. Much attention has been paid in the past to target selection, mAb affinities and the payload linker design, but challenges remain. Here, we demonstrate, by physiologically based pharmacokinetic (PBPK) in silico modeling and simulation, that the tissue-targeting accuracy of mono- and bispecific antibody therapeutics is substantially limited by normal physiological characteristics like organ volumes, blood flow rates, lymphatic circulation, and rates of extravasation. Only a small fraction of blood flows through solid tumor, where the diffusion-driven extravasation is relatively slow compared with many other organs. EGFR and HER2 are used as model antigens based on their experimentally measured tissue and tumor expression levels, but the approach is generic and can account for the cellular expression variation of targets. The model confirms experimental observations that only about 0.1-1% of the dosed mAb is likely to reach the tumor, while the rest ends up in healthy tissues due to target-mediated internalization and nonspecific uptake. The model suggests that the dual-positive tumor cell targeting specificity with bispecific antibodies is likely to be higher at lower drug concentrations and doses. However, this can be offset by elevated drug exposure in more accessible healthy tissues, primarily endothelium. The balance of exposure can be shifted toward tumor cells by using higher doses, albeit at the expense of more extensive target engagement elsewhere in the body, suggesting the need to adapt the toxicity of the payload if ADCs are considered. We suggest that PBPK modeling can guide and support biologics and bsmAb development, from target evaluation and drug optimization to therapeutic dose selection.

Therapeutic monoclonal antibodies (mAbs) are a successful class of biologic drugs that are frequently selected from phage display libraries and transgenic mice that produce fully human antibodies. However, binding affinity to the correct epitope is necessary, but not sufficient, for a mAb to have therapeutic potential. Sequence and structural features affect the developability of an antibody, which influences its ability to be produced at scale and enter trials, or can cause late-stage failures. Using data on paired human antibody sequences, we introduce a pipeline using a machine learning approach that exploits protein language models to identify antibodies which cluster with antibodies that have entered the clinic and are therefore expected to have developability features similar to clinically acceptable antibodies, and triage out those without these features. We propose this pipeline as a useful tool in candidate selection from large libraries, reducing the cost of exploration of the antibody space, and pursuing new therapeutics.

Cell lines that produce non-originator versions of the National Institute of Standards and Technology (NIST) monoclonal antibody reference material 8671 (NISTmAb) are invaluable to the biopharmaceutical industry because, unlike typical commercial cell lines, they can be used on a collaborative and noncompetitive basis for bioprocess development. NIST has generated NS0 clones, NISTCHO research-grade test material 10197 and reference material 8675 NISTCHO to fill this need. We set out to optimize seed train procedures, media and feeding strategies, and stirred tank and rocking bioreactor processes to facilitate our studies on the effects of cell substrate and bioreactor process parameters on non-originator NISTmAb quality attributes. For two NS0 clones and NISTCHO, we improved the baseline methods for seed train culture and demonstrated the critical roles of agitation and gassing strategies for stirred-tank bioreactor operations. For NISTCHO we also tested fed-batch and perfusion processes in rocking bioreactors, identifying several critical process parameters and in-process controls. In this work, for the NIST NS0-59 and NS0-66 clones, we demonstrated that shake flask geometry was critical for culturing a highly viable seed train with a high growth rate and exhibited impacts of feeds, agitation, and gassing during initial bioreactor process development. We identified agitation rates and gassing strategy as critical process parameters for NISTCHO stirred-tank bioreactor operations and established processes for fed-batch and perfusion rocking bioreactor operations. We anticipate this work to benefit the growing number of researchers employing non-originator NISTmAb-expressing cell lines to support precompetitive innovation in biomanufacturing.

The Observed Antibody Space provides the most abundant collection of annotated paired antibody variable domain sequences, thus offering a unique platform for the systematic investigation of the factors governing the pairing of antibody heavy and light chains. By examining a range of characteristics, including amino acid conservation, structural features, charge distribution, and interface residue identity, we challenge the prevailing assumption that pairing is random. Our findings indicate that specific physicochemical properties of single amino acid residues may influence the compatibility and affinity of heavy and light chain combinations. Further structural analyses based on antibody Fv fragments deposited in the Protein Data Bank (PDB) provide insights into the underlying structural features driving these pairing preferences, including a novel definition for the residues constituting the V_H-V_L interface, based on a collection of over 3500 structures. These results have significant implications for understanding antibody assembly and may guide the rational design of therapeutic antibodies with desired properties. Moreover, we provide a complete description and reference characterizing the various human germlines.

Co-formulated antibody cocktails are becoming an increasingly popular therapeutic class; however, they present analytical challenges over traditional single monoclonal antibody (mAb) formulations. One paramount concern is the formation of heteromeric species that have unknown impacts on safety and efficacy. Consequently, effective approaches for identifying and characterizing high-molecular weight (HMW) impurities are critical to the successful development of this therapeutic class. In this study, we used a multifaceted mass spectrometry approach to characterize a unique dimer species formed between two co-formulated mAbs under thermal stress, revealing an intriguing dimerization mechanism that is driven by complementarity-determining region clipping-induced domain swap. Size exclusion chromatography-mass spectrometry, complemented by post-column denaturation, was utilized at both intact and subunit levels to pinpoint the dimerization interface. Additionally, by probing the disulfide bond susceptibility changes via limited reduction and middle-down analysis, the structural changes of the involved domains were studied. These results highlight the critical role of sophisticated analytical methods in comprehending and addressing the complexities linked to co-formulated mAb cocktails.

Trial registration: ClinicalTrials.gov identifier, NCT05280717.