mAbs最新文献

Antibodies recognize antigens via complementary and structurally dependent mechanisms. Therefore, inclusion of antibody inputs is crucial for accurate epitope prediction. Given the limited availability of antibody-antigen complex structures, any epitope prediction model will require minimal yet sufficient antibody inputs to ensure precise epitope identification. To address this need, we introduce Epi4Ab, an antibody-specific epitope prediction model that focuses on identifying unique in-contact antigen residues for a given antibody. Epi4Ab requires minimal antibody inputs, specifically VH/VL families and complementarity-determining region sequences.

Here, we describe a new VHH library for therapeutic discovery which optimizes humanness, stability, affinity, diversity, developability, and facile purification using protein A in the absence of an Fc domain. Four therapeutic humanized VHHs were used as scaffolds, into which we inserted human HCDR1s, HCDR2s and HCDR3s. The HCDR1 and HCDR2 sequences were derived from human VH3 family next-generation sequencing datasets informatically purged of sequence liabilities, synthesized as array-based oligonucleotides, cloned as single CDR libraries into each of the parental scaffolds and filtered for protein A binding by yeast display to ensure correct folding and display. After filtering, the CDR1 and CDR2 libraries were combined with amplified human HCDR3 from human CD19⁺ IgM⁺ B cells. This library was further improved by eliminating long consecutive stretches of tyrosines in CDR3 and enriching for CDR1-2 diversity with elevated tolerance to high temperatures. A broad diversity of high affinity (100 pM-10 nM), developable binders was directly isolated, with developability evaluated for most assays using the isolated VHHs, rather than fused to Fc, which is customary. This represents the first systematic developability assessment of isolated VHH molecules.

Artificial intelligence and machine learning models have been developed to engineer antibodies for specific recognition of antigens. These approaches, however, often focus on the antibody complementarity-determining region (CDR) whilst ignoring the immunoglobulin framework (FW), which provides structural rigidity and support for the flexible CDR loops. Here we present an integrated computational-experimental workflow, combining static structure analyses, molecular dynamics simulations and in vitro physicochemical and functional assays to generate rational designs of FW mutations for modulating antibody stability and activity. We first showed that recent antibody-specific language models lacked insights in FW mutagenesis, in comparison to approaches that use antibody structure information. Using the widely used breast cancer therapeutic trastuzumab as a use case, we designed stabilizing mutants which were distal to the CDR and preserved the antibody's functionality to engage its cognate antigen (HER2) and induce antibody-dependent cellular cytotoxicity. Interestingly, guided by local backbone motions predicted using molecular dynamics simulations, we designed a FW mutation on the trastuzumab light chain that retained antigen-binding effects, but lost Fab-mediated and Fc-mediated effector functions. This highlighted the effects of FW on immunological functions engendered in distal areas of the antibody, and the importance of considering attributes other than binding affinity when assessing antibody function. Our approach incorporates interdomain dynamics and distal effects between FW and the Fc domains, expands the scope of antibody engineering beyond the CDR, and underscores the importance of a holistic perspective that considers the entire antibody structure in optimizing antibody stability, developability and function.

Nanobodies (Nbs) are antigen-binding fragments derived from unique heavy-chain-only antibodies. In recent years, the development of Nbs has progressed rapidly due to their therapeutic potential. Here we present a comprehensive patent landscape of Nb technologies, focusing on uncovering innovation trends, identifying novel drug candidates, and analyzing opportunities and challenges for research, development, and commercialization. Using B-cell maturation antigen (BCMA) as an example drug target, we summarize the features, physicochemical properties, modification sites, and epitope-binding tendencies of patented sequences of Nb drugs, highlighting the importance of structural-level patent protection, and offering a theoretical foundation for Nb design and experimental validation. Through patent landscape and patent sequence analysis, our study provides valuable insights for Nb drug development and supports decision-making in patent strategy.

Proteins are an important class of therapeutics for combatting a wide variety of diseases. The increasing demand for convenient, patient-centric treatment options has propelled the development of subcutaneously delivered protein therapies and increased the interest in novel formulations and delivery methods. However, subcutaneous delivery of protein therapeutics remains a challenge due to the high protein concentrations ( >100 mg/mL) required to circumvent lower bioavailability and the smaller injection volumes required to enable the use of mature and cost-effective devices, such as standard prefilled syringes and autoinjectors. At high concentrations, protein solutions exhibit elevated viscosity, which poses injectability and manufacturing challenges. Here, we review the state of the art in experimental and computationally predictive formulation development approaches for viscosity mitigation of high-concentration protein solution therapeutics, and we suggest new directions for expanding the utility of these approaches beyond traditional monoclonal antibodies. Innovative approaches should leverage and combine advances in both experimental and computational methods, including machine learning and artificial intelligence, to rapidly identify formulation compositions for viscosity reduction, and subsequently facilitate the development of patient-centric biotherapeutics.

Snakebite envenoming is a neglected tropical disease that afflicts millions of people globally, leading to substantial morbidity and mortality. Developing novel antivenoms, particularly recombinant antivenoms based on broadly neutralizing monoclonal antibodies, offers a promising strategy to address the challenge posed by venom variability. However, the extensive diversity of snake venom toxins across species and geographical regions makes this goal inherently complex. Consequently, there is a pressing need for robust discovery methodologies capable of identifying broadly neutralizing antibodies with high affinity and functional potency against a wide range of toxin families. In this study, we engineered a short-chain consensus (SCC) α-neurotoxin to serve as an antigen for a phage display - based antibody discovery campaign. The SCC was expressed using a yeast system, enabling the identification of seven variable domains of heavy-chain-only antibodies (V_HHs) from immune libraries. These V_HHs exhibited nanomolar-binding affinities and low dissociation rates across a panel of short-chain α-neurotoxins, which translated into in vitro neutralization, protecting the target receptor. The best two V_HHs also conferred protection against lethality in a rodent model. These results highlight the unexpected value of consensus toxins in antibody discovery and offer a viable route for developing recombinant antivenoms with broad-spectrum efficacy.

Immunoglobulin (Ig) A has attracted interest as a proposed therapeutic agent due to its ability to engage cell groups differently compared to an IgG scaffold and elicit tumor eradication. Further, its multimeric forms enable increased flexibility in the design of available paratopes. The latter is particularly advantageous for bi- and multispecific antibody formats, which are unparalleled in their enhanced selectivity and unique biological functions. We engineered bispecific heterodimeric IgA-based antibodies using the strand-exchanged engineered domain (SEED) technology, which relies on intertwined segments of IgA and IgG in the C_H3 domain, and applied mutagenesis to introduce two additional binding sites to enable the interaction of IgA-Fc with the myeloid cell-activating receptor CD89 (FcαR). These antibodies exhibited good biophysical properties and thermostability similar to the parental SEED molecule. Binding capacity to both antigens recognized by variable domains, epidermal growth factor receptor (EGFR) and receptor tyrosine kinase like orphan receptor 1 (ROR1), was not impaired, and in contrast to the original SEED-IgA, trispecific mutants could bind to CD89-expressing cells, mediate tumor cell-effector cell clustering, and induce neutrophil-mediated specific lysis of tumor cells. Trispecific design was applicable to both SEED-IgA1 and -IgA2 scaffolds. Interestingly, HEK-expressed mutants featured a CH2-linked N-glycan pattern more similar to wild-type IgA, with reduced core fucosylation in comparison with IgA-SEED. Collectively, the presented format combines the mobilization of CD89-positive effector cells with the flexibility of incorporating antigen specificities of choice into the variable domains, and thus is a promising basis for biochemically stable multispecific IgA with high therapeutic potential.

Acute myeloid leukemia (AML) is a heterogeneous malignancy with poor clinical outcome. Aberrant expression of CD7 in AML patients is linked to shorter overall survival and lack of response to standard of care therapy. CD33/CD7 co-expression on leukemic blasts occurs in approximately one-third of AML patients and is known to be absent in normal myeloid cells. We propose that CD33⁺CD7⁺ AML constitutes an aggressive subgroup characterized by poorer prognosis and enrichment in stem-cell associated gene signatures. To address the substantial unmet need in this patient cohort, we developed the antibody-drug conjugate BVX001, a CD33xCD7-targeted bispecific antibody-binding fragment linked to an auristatin payload. Importantly, BVX001 relies on simultaneous binding to CD33 and CD7 in cis through an 'AND-gated' design, for optimal delivery of its cytotoxic payload. Consequently, BVX001 did not affect healthy myeloid progenitors or T cells at concentrations at which its monospecific counterparts showed toxicity. BVX001 induced significant tumor regression in AML cell line and patient-derived xenografts and increased overall survival. Finally, BVX001 showed significant blast ablation and reduced leukemic stem cell frequency in AML patient samples with both high and low target co-expression. Together, our findings support BVX001 as a new and promising approach for the treatment of this aggressive CD33⁺CD7⁺ AML subtype, currently lacking targeted therapeutic options.

Ricin, a ribosome-inactivating lectin from Ricinus communis seeds, has been used as a bioterrorism agent in multiple cases. While passive immunotherapy with anti-ricin antibodies shows promise in preclinical studies, no approved countermeasure exists. Developing effective monoclonal antibodies (mAbs) is challenging, requiring epitope targeting that ensures neutralization of the two most dominant natural ricin isoforms (D and E). Moreover, high-affinity binding does not always correlate with toxin neutralization, highlighting the importance of epitope specificity in driving protection. Here, we characterized a panel of 17 anti-ricin antibodies, including VHH and IgG mAbs, to determine their affinities, selectivity, and epitopes. Using surface plasmon resonance (SPR) and biolayer interferometry (BLI), we evaluated antibody affinities for the two ricin isoforms (D and E), as well as for ricin agglutinin, a related lectin with markedly lower toxicity. Epitope determination was performed using (1) SPR-based epitope binning, enhanced by network analysis for streamlined bin visualization, and (2) deep mutational scanning with yeast surface display to identify key epitope residues. BLI effectively distinguished low- and high-affinity interactions, while SPR provided superior resolution for determining the highest affinities and lowest dissociation rates. Both epitope-mapping strategies yielded highly consistent results, allowing the identification of critical epitopes associated with potent neutralization and cross-reactivity between ricin isoforms. This study advances our understanding of ricin neutralization by this panel of antibodies, providing key insights into their affinity, epitope specificity, and cross-reactivity. These findings contribute to the rational design of antibody-based therapeutics for ricin intoxication.