Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition.

Interleukin-6 (IL-6) is a cytokine that can bind to IL-6 receptor and induce pleiotropic effects. It serves as a critical biomarker, involved in inflammation amplification, tumor progression, and many other disease developments. Nanobodies, featuring small structure and high affinity, are a powerful and versatile tool in medical diagnostics and therapeutics. Here, based on a scaffold optimized for humanization and stability, we developed a synthetic phage display library that rapidly generated high-affinity and humanized nanobodies, negating the need for animal immunization. Using enhanced green fluorescent protein (eGFP) as a benchmark, we demonstrated that the library produced humanized nanobodies with high function and great intracellular stability. The library was then subjected to screening against IL-6. We identified a standout nanobody, NbL3, which exhibited high affinity (22.16 nM) and stability and significantly inhibited IL-6-enhanced migration on the human breast cancer cell MCF-7 at a relatively low concentration. NbL3's strong blocking activity provides a promising therapeutic alternative for the IL-6-targeted intervention strategy, underscoring the broader potential of our synthetic library as a versatile platform for the development of humanized nanobodies against multiple antigens.

68Ga Radiolabeling of NODASA-Functionalized Phage Display-Derived Peptides for Prospective Assessment as Tuberculosis-Specific PET Radiotracers.

This research presents the development of positron emission tomography (PET) radiotracers for detecting Mycobacterium tuberculosis (MTB) for the diagnosis and monitoring of tuberculosis. Two phage display-derived peptides with proven selective binding to MTB were identified for development into PET radiopharmaceuticals: H8 (linear peptide) and PH1 (cyclic peptide). We sought to functionalize H8/PH1 with NODASA, a bifunctional chelator that allows complexation of PET-compatible radiometals such as gallium-68. Herein, we report on the chelator functionalization, optimized radiosynthesis, and assessment of the radiopharmaceutical properties of [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1. Robust radiolabeling was achieved using the established routine method, indicating consistent production of a radiochemically pure product (RCP ≥ 99.6%). For respective [68Ga]Ga-NODASA-H8 and [68Ga]Ga-NODASA-PH1, relatively high levels of decay-corrected radiochemical yield (91.2% ± 2.3%, 86.7% ± 4.0%) and apparent molar activity (Am, 3.9 ± 0.8 and 34.0 ± 5.3 GBq/µmol) were reliably achieved within 42 min, suitable for imaging purposes. Notably, [68Ga]Ga-NODASA-PH1 remained stable in blood plasma for up to 2 h, while [68Ga]Ga-NODASA-H8 degraded within 30 min. For both 68Ga peptides, minimal whole-blood cell binding and plasma protein binding were observed, indicating a favorable pharmaceutical behavior. [68Ga]Ga-NODASA-PH1 is a promising candidate for further in vitro/in vivo evaluation as a tuberculosis-specific infection imaging agent.

Phage display for discovery of anticancer antibodies.

Antibodies and antibody-based immunotherapeutics are the mainstays of cancer immunotherapy. Expanding the repertoire of cancer-specific and cancer-associated epitopes targetable with antibodies represents an important area of research. Phage display is a powerful approach allowing the use of diverse antibody libraries to be screened for binding to a wide range of targets. In this review, we summarize the basics of phage display technology and highlight the advances in anticancer antibody identification and modification via phage display platform. Finally, we describe phage display-derived anticancer monoclonal antibodies that have been approved to date or are in clinical development.

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.

Rapid and sensitive detection of SARS-CoV-2 based on a phage-displayed scFv antibody fusion with alkaline phosphatase and NanoLuc luciferase.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent pandemic have led to devastating public health and economic losses. The development of highly sensitive, rapid and inexpensive methods to detect and monitor coronaviruses is essential for family diagnosis, preventing infections, choosing treatments and programs and laying the technical groundwork for viral diagnosis. This study established one-step immunoassays for rapid and sensitive detection of SARS-CoV-2 by using a single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) or NanoLuc (NLuc) luciferase. First, a high-affinity scFv antibody specific to the SARS-CoV-2 nucleocapsid (N) protein was screened from hybridoma cells-derived and phage-displayed library. Next, prokaryotic expression of the scFv-AP and scFv-NLuc fusion proteins were induced, leading to excellent antibody binding properties and enzyme catalytic activities. The scFv-AP fusion had a detection limit of 3 pmol per assay and was used to produce eye-readable biosensor readouts. Moreover, the scFv-NLuc protein was applied in a highly sensitive luminescence immunoassay, achieving a detection limit lower than 0.1 pmol per assay. Therefore, the scFv-AP and scFv-NLuc fusion proteins can be applied for the rapid and simple diagnosis of SARS-CoV-2 to safeguard human health and provide guidance for the detection of other pathogenic viruses.

Phage immunoprecipitation and sequencing-a versatile technique for mapping the antibody reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit-determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

An anti-sortilin affibody-peptide fusion inhibits sortilin-mediated progranulin degradation.

Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.

Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling.

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

Construction and validation of a synthetic phage-displayed nanobody library.

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phagedisplayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.

Selective Clearance of Circulating Histones Based on Dodecapeptide-Grafted Copolymer Material for Sepsis Blood Purification.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. Research indicates that circulating histones, as pathogenic factors, may represent a therapeutic target for sepsis. However, effectively clearing circulating histones poses a challenge due to their structural similarity to normal blood proteins, their low abundance in the bloodstream, and serious interference from other blood biomacromolecules. Here we design a dodecapeptide-based functional polymer that can selectively adsorb circulating histones from the blood. The peptide, named P1 (HNHHQLALVESY), was discovered through phage display screening and demonstrated a strong affinity for circulating histones while exhibiting negligible affinities for common proteins in the blood, such as human serum albumin (HSA), immunoglobulin G (IgG), and transferrin (TRF). Furthermore, the P1 peptide was incorporated into a functional polymer design, poly(PEGMA-co-P1), which was immobilized onto a silica gel surface through reversible addition-fragmentation chain transfer polymerization. The resulting material was characterized using solid nuclear magnetic resonance, thermogravimetric analysis, and X-ray photoelectron spectroscopy. This material demonstrated the ability to selectively and efficiently capture circulating histones from both model solutions and whole blood samples while also exhibiting satisfactory blood compatibility, good antifouling properties, and resistance to interference. Satisfactory binding affinity and efficient capture capacity toward histone were also observed for the other screened peptide P2 (QMSMDLFGSNFV)-grafted polymer, validating phage display as a reliable ligand screening strategy. These findings present an approach for the specific clearance of circulating histones and hold promise for future clinical applications in blood purification toward sepsis.

Evaluation of human antibodies from vaccinated volunteers for protection against Yersinia pestis infection.

Yersinia pestis has a broad host range and has caused lethal bubonic and pneumonic plague in humans. With the emergence of multiple resistant strains and the potential for biothreat use, there is an urgent need for new therapeutic strategies that can protect populations from natural or deliberate infection. Targeting F1 has been proven to be the main strategy for developing vaccines and therapeutic antibodies, but data on anti-F1 antibodies, especially in humans, are scarce. To date, three human anti-F1 monoclonal antibodies (m252, αF1Ig2, and αF1Ig8) from naive populations have been reported. Here, we constructed an antibody library from vaccinees immunized with the plague subunit vaccine IIa by phage display. The genetic basis, epitopes, and biological functions of the obtained mAbs were assessed and evaluated in plague-challenged mice. Three human mAbs, namely, F3, F19, and F23, were identified. Their biolayer responses were 0.4, 0.6, and 0.6 nm, respectively. The dissociation constants (KD) of the F1 antigen were 1 pM, 0.165 nM, and 1 pM, respectively. Although derived from distinct Ab lineages, that is, VH3-30-D3-10-JH4 (F3&F23) and VH3-43-D6-19-JH4 (F19), these mAbs share similar binding sites in F1 with some overlap with αF1Ig8 but are distinct from αF1Ig2. Each of them provided a significant protective effect for Balb/c mice against a 100 median lethal dose (MLD) challenge of a virulent Y. pestis strain when administered at a dose of 100 µg. No synergistic or antagonistic effects were observed among them. These mAbs are novel and excellent candidates for further drug development and use in clinical practice.IMPORTANCEIn this study, we identified three human monoclonal antibodies with a high affinity to F1 protein of Yersinia pestis. We discovered that they have relatively lower somatic hypermutations compared with antibodies, m252, αF1Ig2, and αF1Ig8, derived from the naive library reported previously. We also observed that these mAbs share similar binding sites in F1 with some overlapping with αF1Ig8 but distinct from that of αF1Ig2. Furthermore, each of them could provide complete protection for mice against a lethal dose of Yersinia pestis challenge. Our data provided new insights into the anti-F1 Ab repertories and their associated epitopes during vaccination in humans. The findings support the additional novel protective human anti-F1Abs for potential therapeutics against plaque.

Identification of Conserved Linear Epitopes on Viral Protein 2 of Foot-and-Mouth Disease Virus Serotype O by Monoclonal Antibodies 6F4.D11.B6 and 8D6.B9.C3.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67-78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115-126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

Synthetic antibodies for accelerated RNA crystallography.

RNA structure is crucial to a wide range of cellular processes. The intimate relationship between macromolecular structure and function necessitates the determination of high-resolution structures of functional RNA molecules. X-ray crystallography is the predominant technique used for macromolecular structure determination; however, solving RNA structures has been more challenging than their protein counterparts, as reflected in their poor representation in the Protein Data Bank (<1%). Antibody-assisted RNA crystallography is a relatively new technique that promises to accelerate RNA structure determination by employing synthetic antibodies (Fabs) as crystallization chaperones that are specifically raised against target RNAs. Antibody chaperones facilitate the formation of ordered crystal lattices by minimizing RNA flexibility and replacing unfavorable RNA-RNA contacts with contacts between chaperone molecules. Atomic coordinates of these antibody fragments can also be used as search models to obtain phase information during structure determination. Antibody-assisted RNA crystallography has enabled the structure determination of 15 unique RNA targets, including 11 in the last 6 years. In this review, I cover the historical development of antibody fragments as crystallization chaperones and their application to diverse RNA targets. I discuss how the first structures of antibody-RNA complexes informed the design of second-generation antibodies and led to the development of portable crystallization modules that have greatly reduced the uncertainties associated with RNA crystallography. Finally, I outline unexplored avenues that can increase the impact of this technology in structural biology research and discuss potential applications of antibodies as affinity reagents for interrogating RNA biology outside of their use in crystallography. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

A single-domain antibody library based on a stability-engineered human VH3 scaffold.

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues.

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Proteome-scale characterisation of motif-based interactome rewiring by disease mutations.

Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.

Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation.

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.

Phage Display Technology in Biomarker Identification with Emphasis on Non-Cancerous Diseases.

In recent years, phage display technology has become vital in clinical research. It helps create antibodies that can specifically bind to complex antigens, which is crucial for identifying biomarkers and improving diagnostics and treatments. However, existing reviews often overlook its importance in areas outside cancer research. This review aims to fill that gap by explaining the basics of phage display and its applications in detecting and treating various non-cancerous diseases. We focus especially on its role in degenerative diseases, inflammatory and autoimmune diseases, and chronic non-communicable diseases, showing how it is changing the way we diagnose and treat illnesses. By highlighting important discoveries and future possibilities, we hope to emphasize the significance of phage display in modern healthcare.

Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.

BACKGROUND: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. OBJECTIVES: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. METHODS: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen. RESULTS: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. CONCLUSIONS: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.