A single-chain fab derived drug conjugate for HER2 specific delivery.

Despite the development of antibody-drug conjugates, the fragment Fab-based drug conjugates offer some unique capabilities in terms of safety, clearance, penetration and others. Current methods for preparing Fab drug conjugates are limited by the availability and stability of Fab proteins, leaving reports on this rare. Here, we found that a single-chain scaffold of Fab enables stabilization of the paired structure and supports high-yield expression in bacteria cytoplasm. Furthermore, we conjugated anti-neoplastic agent SN38 to the C-terminus by sortase A ligation and generated a homogenous Fab conjugate with the drug-to-Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate demonstrated potent and antigen-dependent cell-killing ability with the aid of its special cathepsin-triggered cyclization-promoted release mechanism. In vivo, Fab-SN38 can prevent growths of HER2-positive tumors in athymic mice and be well tolerated to the treatment at 7 mg/kg per dose. Anti-tumor activity, high dose tolerance and penetration advantage observed in this study would merit Fab conjugate investigation in target chemotherapy.

Quantitative comparison of automated OCT and conventional FAF-based geographic atrophy measurements in the phase 3 OAKS/DERBY trials.

With the approval of the first two substances for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration (AMD), a standardized monitoring of patients treated with complement inhibitors in clinical practice is needed. Optical coherence tomography (OCT) provides high-resolution access to the retinal pigment epithelium (RPE) and neurosensory layers, such as the ellipsoid zone (EZ), which further enhances the understanding of disease progression and therapeutic effects in GA compared to conventional fundus autofluorescence (FAF). In addition, artificial intelligence-based methodology allows the identification and quantification of GA-related pathology on OCT in an objective and standardized manner. The purpose of this study was to comprehensively evaluate automated OCT monitoring for GA compared to reading center-based manual FAF measurements in the largest successful phase 3 clinical trial data of complement inhibitor therapy to date. Automated OCT analysis of RPE loss showed a high and consistent correlation to manual GA measurements on conventional FAF. EZ loss on OCT was generally larger than areas of RPE loss, supporting the hypothesis that EZ loss exceeds underlying RPE loss as a fundamental pathophysiology in GA progression. Automated OCT analysis is well suited to monitor disease progression in GA patients treated in clinical practice and clinical trials.

Rigid crosslinking of the CD3 complex leads to superior T cell stimulation.

Functionally bivalent non-covalent Fab dimers (Bi-Fabs) specific for the TCR/CD3 complex promote CD3 signaling on T cells. While comparing functional responses to stimulation with Bi-Fab, F(ab')2 or mAb specific for the same CD3 epitope, we observed fratricide requiring anti-CD3 bridging of adjacent T cells. Surprisingly, anti-CD3 Bi-Fab ranked first in fratricide potency, followed by anti-CD3 F(ab')2 and anti-CD3 mAb. Low resolution structural studies revealed anti-CD3 Bi-Fabs and F(ab')2 adopt similar global shapes with CD3-binding sites oriented outward. However, under molecular dynamic simulations, anti-CD3 Bi-Fabs crosslinked CD3 more rigidly than F(ab')2. Furthermore, molecular modelling of Bi-Fab and F(ab')2 binding to CD3 predicted crosslinking of T cell antigen receptors located in opposing plasma membrane domains, a feature fitting with T cell fratricide observed. Thus, increasing rigidity of Fab-CD3 crosslinking between opposing effector-target pairs may result in stronger T cell effector function. These findings could guide improving clinical performance of bi-specific anti-CD3 drugs.

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab'-DNA ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

A platform for the rapid screening of equine immunoglobins F (ab)2 derived from single equine memory B cells able to cross-neutralize to influenza virus.

Single B cells-based antibody platforms offer an effective approach for the discovery of useful antibodies for therapeutic or research purposes. Here we present a method for screening equine immunoglobins F(ab)2, which offers the potential advantage of reacting with multiple epitopes on the virus. Using equine influenza virus (EIV) as model, a hemagglutinin (HA) trimer was constructed to bait B cells in vaccinated horses. We screened 370 HA-specific B cells from 1 × 106 PBMCs and identified a diverse set of equine variable region gene sequences of heavy and light chains and then recombined with humanized Ig Fc. Recombinant equine Ig was then self-assembled in co-transfected 293 T cells, and subsequently optimized to obtain HA binding B-cell receptor (s). The recombinant antibodies exhibited a high binding affinity to the HA protein. Antibody H81 exhibited the highest cross neutralizing activities against EIV strains in vitro. Furthermore, it effectively protected EIV-challenged mice, resulting in significantly improved survival, reduced pulmonary inflammation and decreased viral titers. In silico predication identified a functional region of H81 comprising 27 key amino acids cross the main circulating EIV strains. The 12 amino acid residues in this region with the highest binding affinities were screened. Notably, the predicted epitopes of H81 encompassed the documented equine HA receptor binding site, validating its cross-neutralization. In summary, a rapid platform was successfully established to investigate the profiling of equine antigen-recognizing receptors (BCRs) following infection. This platform has the potential to optimize the screening of virus-neutralizing antibodies and aid in vaccine design.

TFAB002s, novel CD20-targeting T cell-dependent bispecific Fab-FabCH3 antibodies, exhibit potent antitumor efficacy against malignant B-cell lymphoma.

B-cell lymphoma, clinically, comprises a heterogeneous group of malignancies that encompass various subtypes. CD20 is an optimal target for therapeutic antibodies in B-cell lymphoma immunotherapy since approximately 90% of B-cell malignancies typically exhibit CD20 expression on their surface, while its presence is limited in normal tissues. In this study, we have developed a series of novel non-IgG-like T cell-dependent bispecific antibodies by constructing Fab-FabCH3, referred to as Tandem Antigen-binding Fragment 002 (TFAB002), which specifically target CD20 for the treatment of malignant B-cell lymphoma. TFAB002s display strong binding affinity with CD20 and moderate binding affinity with CD3, thereby triggering target-specific T-cell activation, cytokine release, and tumor cell lysis in vitro. Furthermore, TFAB002s exhibit potent cytotoxicity against B-cell malignancies that express varying levels of CD20. Besides, the TFAB002s show potent pharmacodynamic activity in vivo in the WIL2-S cells CDX mouse model. Collectively, these results underscore the potential of TFAB002s as a highly promising therapeutic approach for selectively depleting CD20-positive B cells, thereby warranting further clinical evaluation as a viable treatment option for CD20-expressing B-cell malignancies.

A next-generation Fab library platform directly yielding drug-like antibodies with high affinity, diversity, and developability.

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.

The Structural Basis for Recognition of Human Leukocyte Antigen Class I Molecules by the Pan-HLA Antibody W6/32.

The central immunological role of HLA class I (HLA-I) in presenting peptide Ags to cellular components of the immune system has been the focus of intense study for >60 y. A confounding factor in the study of HLA-I has been the extreme polymorphism of these molecules. The mAb W6/32 has been a fundamental reagent bypassing the issue of polymorphism by recognizing an epitope that is conserved across diverse HLA-I allotypes. However, despite the widespread use of W6/32, the epitope of this Ab has not been definitively mapped. In this study, we present the crystal structure of the Fab fragment of W6/32 in complex with peptide-HLA-B*27:05. W6/32 bound to HLA-B*27:05 beneath the Ag-binding groove, recognizing a discontinuous epitope comprised of the α1, α2, and α3 domains of HLA-I and ß2-microglobulin. The epitope comprises a region of low polymorphism reflecting the pan-HLA-I nature of the binding. Notably, the W6/32 epitope neither overlaps the HLA-I binding sites of either T cell Ag receptors or killer cell Ig-like receptors. However, it does coincide with the binding sites for leukocyte Ig-like receptors and CD8 coreceptors. Consistent with this, the use of W6/32 to block the interaction of NK cells with HLA-I only weakly impaired inhibition mediated by KIR3DL1, but impacted HLA-LILR recognition.

Synthesis and Activity of T-Cell Tumor-Directing MegaMolecules.

This paper describes the synthesis, characterization, and functional activity of 26 MegaMolecule-based bispecific antibody mimics for T-cell redirection toward HER2+ cancer cells. The work reports functional bispecific MegaMolecules that bind both receptor targets, and recruit and activate T-cells resulting in lysis of the target tumor cells. Changing the orientation of linkage between Fabs against either HER2 or CD3ε results in an approximately 150-fold range in potency. Increasing scaffold valency from Fab dimers up to tetramers improves the potency of the antibody mimics up to 5-fold, but with diminishing returns in effective dose beyond trimeric formats. Antibody mimics that present either one or two Fabs against either receptor target allows for initial engagement of one cell type over the other. Finally, the antibody mimics significantly reduce HER2+ tumor volumes in a humanized xenograft model of breast cancer.

An in situ bioadhesive foam as a large intestinal delivery platform for antibody fragment to treat inflammatory bowel disease.

Biologics have been widely used as injectables in the treatment of inflammatory bowel disease (IBD). Different local treatment attempts have been developed in recent years. However, maintaining systemic levels of biologics is still crucial for achieving colitis remission. An equilibrium between systemic and local concentrations of biologics is therefore essential for treatment of colitis. Current formulations struggle to create optimal balance between drug concentrations in plasma and the colonic wall. Addressing this challenge, we developed a rectally delivered in situ foam that generates CO2via a reaction between potassium bicarbonate (PB) and citric acid (CA) without the aid of an external device. An anti-TNF-α antibody fragment (Fab) was loaded into the foam formulation, which promoted prolonged colon retention and improved Fab distribution up to proximal colon following rectal administration to mice. In addition, we observed increased plasma Fab concentrations in mice receiving the rectal Fab foam compared to a Fab solution. In a non-everted rat gut ex vivo model, a single exposure to the CO2-containing foam improved macromolecule transepithelial flux across colonic tissue by over ten-fold. Foam efficacy for Fab was investigated in a range of colitis mouse models, from acute to chronic. This non-invasive formulation platform demonstrates potential to overcome existing limitations in delivering biologics to inflamed colonic tissue.

Structural Basis for the Recognition of GPRC5D by Talquetamab, a Bispecific Antibody for Multiple Myeloma.

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.

Rational design of environmentally responsive antibodies with pH-sensing synthetic amino acids.

Antibodies are widely used as therapeutic agents to tackle various diseases. In the present study, to enhance their clinical values, we rationally designed pH-responsivity by exploiting the idiosyncratic protonation/deprotonation profiles of non-natural amino acids. 3-Nitro-L-tyrosine, 3-cyano-L-tyrosine, and 3, 5-halogenated-L-tyrosine, each with near neutral pKa, were thus incorporated into Fab fragments in place of tyrosines and other residues in the variable regions. Cell-based assays showed that these modifications achieved up to 140-fold tighter binding to antigens and several-fold tighter cytotoxicity to antigen-expressing cell at pH 6.0 than pH 7.4. The pH-dependent binding effect was retained in full-length antibodies. In silico structural analyses revealed electrostatic repulsion at neutral pH between antigens and antibodies or inside the antibody as the underlying mechanisms of the acid preference, and this finding increases the designability of pH-dependent antigen binding. The development of antibodies responsive to the microenvironments of diseased tissues will allow more disease-related antigens to be targeted in treatments, because of the reduced cross-reactivity toward healthy tissues.

Multiantigen T-Cell Hybridizers: A Two-Component T-Cell-Activating Therapy.

Multispecific T-cell-engaging scaffolds have emerged as effective anticancer therapies for the treatment of hematological malignancies. Approaches that modulate cancer cell targeting and provide personalized, multispecific immunotherapeutics are needed. Here, we report on a modular, split antibody-like approach consisting of Fab' fragments modified with complementary morpholino oligonucleotides (MORFs). We synthesized a library of B-cell-targeting Fab'-MORF1 conjugates that self-assemble, via a Watson-Crick base pairing hybridization, with a complementary T-cell-engaging Fab'-MORF2 conjugate. We aptly titled our technology multiantigen T-cell hybridizers (MATCH). Using MATCH, cancer-specific T-cell recruitment was achieved utilizing four B-cell antigen targets: CD20, CD38, BCMA, and SLAMF7. The antigen expression profiles of various malignant B-cell lines were produced, and using these distinct profiles, cell-specific T-cell activation was attained on lymphoma, leukemia, and multiple myeloma cell lines in vitro. T-cell rechallenge experiments demonstrated the modular approach of MATCH by sequentially activating the same T-cell cohort against three different cancers using cancer antigen-specific Fab'-MORF1 conjugates. Furthermore, MATCH's efficacy was demonstrated in vivo by treating xenograft mouse models of human non-Hodgkin's lymphoma with CD20-directed MATCH therapy. In the pilot study, a single dose of MATCH allowed for long-term survival of all treated mice compared to saline control. In a second in vivo model, insights regarding optimal T-cell-to-target cell ratio were gleaned when a ratio of 5:1 T-cell-to-target cell MATCH-treated mice significantly delayed the onset of disease compared to higher and lower ratios.

Allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of IgE F(ab')2 epitopes.

Immunoglobulin E (IgE) plays pivotal roles in allergic diseases through interaction with a high-affinity receptor (FcεRI). We established that Fab fragments of anti-IgE antibodies (HMK-12 Fab) rapidly dissociate preformed IgE-FcεRI complexes in a temperature-dependent manner and inhibit IgE-mediated anaphylactic reactions, even after allergen challenge. X-ray crystallographic studies revealed that HMK-12 Fab interacts with each of two equivalent epitopes on the Cε2 homodimer domain involved in IgE F(ab')2. Consequently, HMK-12 Fab-mediated targeting of Cε2 reduced the binding affinity of Fc domains and resulted in rapid removal of IgE from the receptor complex. This unexpected finding of allosteric inhibition of IgE-FcεRI interactions by simultaneous targeting of two epitope sites on the Cε2 homodimer domain of IgE F(ab')2 may have implications for the development of novel therapies for allergic disease.

In silico evaluation of the role of Fab glycosylation in cetuximab antibody dynamics.

Introduction: N-glycosylation is a post-translational modification that is highly important for the development of monoclonal antibodies (mAbs), as it regulates their biological activity, particularly in terms of immune effector functions. While typically added at the Fc level, approximately 15-25% of circulating antibodies exhibit glycosylation in the Fab domains as well. To the best of our knowledge, cetuximab (Erbitux®) is the only therapeutic antibody presenting Fab glycosylation approved world-wide targeting the epidermal growth factor receptor for the treatment of metastatic-colorectal and head and neck cancers. Additionally, it can trigger antibody-dependent cell cytotoxicity (ADCC), a response that typically is influenced by N-glycosylation at Fc level. However, the role of Fab glycosylation in cetuximab remains poorly understood. Hence, this study aims to investigate the structural role of Fab glycosylation on the conformational behavior of cetuximab. Methods: The study was performed in silico via accelerated molecular dynamics simulations. The commercial cetuximab was compared to its form without Fab glycosylation and structural descriptors were evaluated to establish conformational differences. Results: The results clearly show a correlation between the Fab glycosylation and structural descriptors that may modulate the conformational freedom of the antibody, potentially affecting Fc effector functions, and suggesting a negative role of Fab glycosylation on the interaction with FcγRIIIa. Conclusion: Fab glycosylation of cetuximab is the most critical challenge for biosimilar development, but the differences highlighted in this work with respect to its aglycosylated form can improve the knowledge and represent also a great opportunity to develop novel strategies of biotherapeutics.

High titer expression of antibodies using linear expression cassettes for early-stage functional screening.

Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1 ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1 ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30 ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.

Compartment Syndrome Following Snake Envenomation in the United States: A Scoping Review of the Clinical Literature.

Introduction: Local tissue destruction following envenomation from North American snakes, particularly those within the Crotalinae subfamily, has the potential to progress to compartment syndrome. The pathophysiology of venom-induced compartment syndrome (VICS) is a debated topic and is distinct from trauma/reperfusion-induced compartment syndrome. Heterogeneity exists in the treatment practices of VICS, particularly regarding the decision to progress to fasciotomy. Associations with functional outcomes and evolution in clinical practice since the introduction of Crotalidae polyvalent immune Fab (FabAV) have not been well defined. Our goal was to identify the potential gaps in the literature regarding this phenomenon, as well as illuminate salient themes in the clinical characteristics and treatment practices of VICS. Methods: We conducted this systematic scoping-style review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Records were included if they contained data surrounding the envenomation and hospital course of one or more patients who were envenomated by a snake species native to North America and were diagnosed with compartment syndrome from 1980-2020. Results: We included 19 papers: 10 single- or two-patient case reports encompassing 12 patients, and nine chart reviews providing summary statistics of the included patients. In case reports, the median compartment pressure when reported was 60 millimeters of mercury (interquartile range 55-68), 66% underwent fasciotomy, and functional outcomes varied. Use of antivenom appeared to be more liberal with FabAV than the earlier antivenin Crotalidae polyvalent. Rapid progression of swelling was the most commonly reported symptom. Among the included retrospective chart reviews, important data such as compartment pressures, consistent laboratory values, and snake species was inconsistently reported. Conclusions: Venom-induced compartment syndrome is relatively rare. Existing papers generally describe good outcomes even in the absence of surgical management. Significant gaps in the literature regarding antivenom dosing practices, serial compartment pressure measurements, and functional outcomes highlight the need for prospective studies and consistent standardized reporting.

Venomous Snakebite Encounters in Liver Transplant Recipients: A Case Report and Review of Relevant Literature.

Envenomation of humans by snakes, a global health challenge, is poorly studied in liver transplant recipients. We report a case of rattlesnake envenomation in a 52-year-old female patient who had previously received a liver transplant to treat nonalcoholic steatohepatitis cirrhosis. Despite stable graft function since her transplant, she exhibited elevated liver enzymes on admission, with a mixed hepatocellular and cholestatic pattern. Treatment included CroFab Crotalidae polyvalent immune Fab (ovine) antivenom and close monitoring, with continuation of her standard immunosuppression regimen. Inpatient observation showed reduced swelling and pain but persistently elevated enzymes. Imaging indicated fatty infiltration with patent hepatic vasculature. Her liver enzymes improved spontaneously, and she was discharged after 5 days, with complete normalization of herliver enzyme levels as shown by repeated laboratory test results 1 month later. Our case emphasizes the risk of graftinjury in liver transplant recipients, as well as the need for vigilant monitoring and early antivenom administration. We urge furtherresearch to establish guidelines for optimal care in this unique population.

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies.

The third complementary-determining regions of the heavy-chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form ß-ribbon stalks that protrude from the surface of the antibody with a disulfide cross-linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B-cell sequencing, has been determined to 1.6 Šresolution. By swapping the D08 native light chain with that of an unrelated antigen-unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing.

Exploring How Antibody Format Drives Clearance from the Brain.

Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.