mAbs最新文献

Bispecific antibodies (BsAbs) capable of recognizing two distinct epitopes or antigens offer promising therapeutic options for various diseases by targeting multiple pathways. The favorable pharmacokinetic (PK) properties of monoclonal antibodies (mAbs) are crucial, as they directly influence patient safety and therapeutic efficacy. For numerous mAb therapeutics, optimization of neonatal Fc receptor (FcRn) interactions and elimination of unfavorable molecular properties have led to improved PK properties. However, many BsAbs exhibit unfavorable PK, which has precluded their development as drugs. In this report, we present studies on the molecular determinants underlying the distinct PK profiles of three IgG1-scFv BsAbs. Our study indicated that high levels of nonspecific interactions, elevated isoelectric point (pI), and increased number of positively charged patches contributed to the fast clearance of IgG1-scFv. FcRn chromatography results revealed specific scFv-FcRn interactions that are unique to the IgG1-scFv, which was further supported by molecular dynamics (MD) simulation. These interactions likely stabilize the BsAb FcRn interaction at physiological pH, which in turn could disrupt FcRn-mediated BsAb recycling. In addition to the empirical observations, we also evaluated the impact of in silico properties, including pI differential between the Fab and scFv and the ratio of dipole moment to hydrophobic moment (RM) and their correlation with the observed clearance. These findings highlight that the PK properties of BsAbs may be governed by novel determinants, owing to their increased structural complexity compared to immunoglobulin G (IgG) 1 antibodies.

Non-reduced peptide mapping provides essential data for characterizing therapeutic monoclonal antibodies by isolating disulfide connections between specific cysteines. However, conventional digestive strategies used throughout the biopharmaceutical industry have been shown to cause unintentional rearrangement of disulfide connections (disulfide scrambling), thus generating connectivity profiles that do not accurately represent the protein being analyzed. Common misconceptions (e.g. avoiding basic-pH digestion to prevent disulfide scrambling) have led to the development of alternative reagents and conditions that can alleviate this issue, but yield problematic digestion profiles. Herein, we systematically and comprehensively examine the primary considerations for accurate non-reduced peptide mapping, and provide effective, practical solutions to minimize undesired behavior while still yielding high-quality digests. Additionally, we present a method that exploits intentional disulfide scrambling as a reference tool to demonstrate the robustness of our proposed strategies. We also introduce maleimide as a cysteine-alkylating reagent and demonstrate its benefits over industry-leading analogs such as N-ethylmaleimide in terms of compatibility with regulatory reports.

Subcutaneous injection is the preferred route of administration for many antibody therapeutics for reasons that include its speed and convenience. However, the small volume limit (typically $\le$2 mL) for subcutaneous delivery often necessitates antibody formulations at high concentrations (commonly ≥100 mg/mL), which may lead to physicochemical problems. For example, antibodies with large hydrophobic or charged patches can be prone to self-interaction giving rise to high viscosity. Here, we combined X-ray crystallography with computational modeling to predict regions of an anti-glucagon receptor (GCGR) IgG₁ antibody prone to self-interaction. An extensive mutational analysis was undertaken of the complementarity-determining region residues residing in hydrophobic surface patches predicted by spatial aggregation propensity, in conjunction with residue-level solvent accessibility, averaged over conformational ensembles from molecular dynamics simulations. Dynamic light scattering (DLS) was used as a medium throughput screen for self-interaction of ~ 200 anti-GCGR IgG₁ variants. A negative correlation was found between the viscosity determined at high concentration (180 mg/mL) and the DLS interaction parameter measured at low concentration (2-10 mg/mL). Additionally, anti-GCGR variants were readily identified with reduced viscosity and antigen-binding affinity within a few fold of the parent antibody, with no identified impact on overall developability. The methods described here may be useful in the optimization of other antibodies to facilitate their therapeutic administration at high concentration.

Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.

T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.

Therapeutic efficacy with durable responses has been demonstrated with several antibody drugs that block key immune checkpoint receptors, including PD-1, PD-L1, and CTLA-4. Despite the success of these drugs, a substantial proportion of patients do not benefit. Targeting multiple inhibitory pathways simultaneously to augment anti-tumor immunity has proven to be a promising approach. The emergence of Repulsive Guidance Molecule b (RGMb), a ligand for PD-L2, as a novel co-inhibitory pathway in T cells, together with its regulation by the gut microbiome, encouraged the discovery and development of fully human anti-RGMb antibodies. Here, we describe phage display-derived monoclonal antibodies (mAbs) 2C11 and 5C10 that bind human RGMb with high affinities of 1.4 nM and 0.72 nM, respectively. Both mAbs 2C11 and 5C10 potently inhibited RGMb interaction with PD-L2. MAb 2C11 effectively inhibited RGMb interaction with bone morphogenetic proteins 2 and 4 (BMP2-4), while leaving RGMb interaction with Neogenin 1 (Neo1) unaffected. Conversely, mAb 5C10 disrupted RGMb interaction with Neo1 while maintaining RGMb binding to BMP2-4. These findings map the 2C11 epitope at the membrane-distal N-terminal region of RGMb, which coincides with both PD-L2- and BMP2-4-binding sites. The PD-L2 binding interface is likely positioned between RGMb's N-terminal BMP-binding and C-terminal Neo1-binding regions. The in vivo activity of mAb 2C11 in combination with anti-PD-1 or anti-PD-L1 was tested in MC38 and B16-OVA cancer models and demonstrated synergistic effects by significantly enhancing anti-tumor responses. These properties make mAb 2C11 a promising candidate for therapeutic use to overcome immune checkpoint inhibitor resistances, warranting further exploration in clinical settings.

Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63-69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Glycosylation affects the safety and efficacy of therapeutic proteins and is often considered a critical quality attribute (CQA). Therefore, it is important to identify and quantify glycans during drug development. Glycosylation is a highly complex post-translational modification (PTM) due to its structural heterogeneity, i.e. glycosylation site occupancy, glycan compositions, modifications, and isomers. Current analytical tools compromise either structural resolution or site specificity. Hydrophilic interaction liquid chromatography-fluorescence-mass spectrometry (HILIC-FLR-MS) is the gold standard for structural analysis of released glycans, but lacks information on site specificity and occupation. However, HILIC-FLR-MS often uses salt in the solvent, which impairs analysis robustness and sensitivity. Site-specific glycosylation analysis via glycopeptides, upon proteolytic digestion, is commonly performed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), but provides only compositional and limited structural glycan information. In this study, we introduce a salt-free, glycopeptide-based HILIC-tandem mass spectrometry (HILIC-MS/MS) method that provides glycan identification, glycan isomer separation and site-specific information simultaneously. Moreover, HILIC-MS/MS demonstrated comparable relative quantification results as released glycan HILIC-FLR-MS. Further, our new method improves the retention of hydrophilic peptides, allowing simultaneous analysis of important CQAs such as deamidation in antibodies. The developed method offers a valuable tool to streamline the site-specific glycosylation analysis of glycoproteins, which is particularly important for the expanding landscape of novel therapeutic formats in the biopharmaceutical industry.

Neurodegenerative diseases such as Alzheimer's disease (AD) pose substantial challenges to patients and health-care systems, particularly in countries with aging populations. Immunotherapies, including the marketed antibodies lecanemab (Leqembi®) and donanemab (Kisunla^TM), offer promise but face hurdles due to limited delivery across the blood-brain barrier (BBB). This limitation necessitates high doses, resulting in increased costs and a higher risk of side effects. This study explores transferrin receptor (TfR)-binding camelid single-domain antibodies (VHHs) for facilitated brain delivery. We developed and evaluated fusion proteins (FPs) combining VHHs with human IgG Fc domains or single-chain variable fragments (scFvs) of the anti-amyloid-beta (Aβ) antibody 3D6. In vitro assessments showed varying affinities of the FPs for TfR. In vivo evaluations indicated that specific VHH-Fc and VHH-scFv fusions reached significant brain concentrations, emphasizing the importance of optimal TfR binding affinities. The VHH-scFv fusions were further investigated in mouse models with Aβ pathology, showing higher retention compared to wild-type mice without Aβ pathology. Our findings suggest that these novel VHH-based FPs hold potential for therapeutic and diagnostic applications in AD, providing a strategy to overcome BBB limitations and enhance brain targeting of antibody-based treatments. Furthermore, our results suggest that a given bispecific TfR-binding fusion format has a window of "optimal" affinity where parenchymal delivery is adequate, while blood pharmacokinetics aligns with the desired application of the fusion protein.

Therapeutic mAbs show a specific "charge fingerprint" that may affect safety and efficacy, and, as such, it is often identified as a critical quality attribute (CQA). Capillary iso-electric focusing (cIEF), commonly used for the evaluation of such CQA, provides an analytical tool to investigate mAb purity and identity across the product lifecycle. Here, we discuss the results of an analysis of a panel of antibody products by conventional and whole-column imaging cIEF systems performed as part of European Pharmacopoeia activities related to development of "horizontal standards" for the quality control of monoclonal antibodies (mAbs). The study aimed at designing and verifying an independent and transversal cIEF procedure for the reliable analysis of mAbs charge variants. Despite the use of comparable experimental conditions, discrepancies in the charge profile and measured isoelectric points emerged between the two cIEF systems. These data suggest that the results are method-dependent rather than absolute, an aspect known to experts in the field and pharmaceutical industry, but not suitably documented in the literature. Critical implications from analytical and regulatory perspectives, are herein thoughtfully discussed, with a special focus on the context of market surveillance and identification of falsified medicines.