Development of a latex agglutination test based on VH antibody fragment for detection of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (SS2) is an important porcine pathogen that causes diseases in both swine and human. For rapid SS2 identification, a novel latex agglutination test (LAT) based on heavy-chain variable domain antibody (VH) was developed. Firstly, the soluble 47B3 VH antibody fragment from a phage display library, in which cysteine residues were engineered at the C-terminus, was expressed in Escherichia coli. The purified protein was then gently reduced to form monomeric soluble 47B3 VH subsequently used to coat with latex beads by means of site-specific conjugation. The resulting VH-coated beads gave a good agglutination reaction with SS2. The LAT was able to distinguish S. suis serotype 2 from serotype 1/2, which shares some common sugar residues, and showed no cross-reaction with other serotypes of S. suis or other related bacteria. The detection sensitivity was found to be as high as 1.85x106 cells. The LAT was stable at 4°C for at least six months without loss of activity. To the best of our knowledge, this is the first LAT based on a VH antibody fragment that can be considered as an alternative for conventional antibody-based LAT where VHs are the most favored recombinant antibody.

Mixed-mode size-exclusion silica resin for polishing human antibodies in flow-through mode.

The polishing step in the downstream processing of therapeutic antibodies removes residual impurities from Protein A eluates. Among the various classes of impurities, antibody fragments are especially challenging to remove due to the broad biomolecular diversity generated by a multitude of fragmentation patterns. The current approach to fragment removal relies on ion exchange or mixed-mode adsorbents operated in bind-and-gradient-elution mode. However, fragments that bear strong similarity to the intact product or whose biophysical features deviate from the ensemble average can elude these adsorbents, and the lack of a chromatographic technology enabling robust antibody polishing is recognized as a major gap in downstream bioprocessing. Responding to this challenge, this study introduces size-exclusion mixed-mode (SEMM) silica resins as a novel chromatographic adsorbent for the capture of antibody fragments irrespective of their biomolecular features. The pore diameter of the silica beads features a narrow distribution and is selected to exclude monomeric antibodies, while allowing their fragments to access the pores where they are captured by the mixed-mode ligands. The static and dynamic binding capacity of the adsorbent ranged respectively between 30-45 and 25-33 gs of antibody fragments per liter of resin. Selected SEMM-silica resins also demonstrated the ability to capture antibody aggregates, which adsorb on the outer layer of the beads. Optimization of the SEMM-silica design and operation conditions - namely, pore size (10 nm) and ligand composition (quaternary amine and alkyl chain) as well as the linear velocity (100 cm/h), ionic strength (5.7 mS/cm), and pH (7) of the mobile phase - afforded a significant reduction of both fragments and aggregates, resulting into a final antibody yield up to 80% and monomeric purity above 97%.

The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments.

BACKGROUND: Chimeric antigen receptor-T (CAR-T) cells therapy is one of the novel immunotherapeutic approaches with significant clinical success. However, their applications are limited because of long preparation time, high cost, and interpersonal variations. Although the manufacture of universal CAR-T (U-CAR-T) cells have significantly improved, they are still not a stable and unified cell bank. METHODS: Here, we tried to further improve the convenience and flexibility of U-CAR-T cells by constructing novel modular universal CAR-T (MU-CAR-T) cells. For this purpose, we initially screened healthy donors and cultured their T cells to obtain a higher proportion of stem cell-like memory T (TSCM) cells, which exhibit robust self-renewal capacity, sustainability and cytotoxicity. To reduce the alloreactivity, the T cells were further edited by double knockout of the T cell receptor (TCR) and class I human leukocyte antigen (HLA-I) genes utilizing the CRISPR/Cas9 system. The well-growing and genetically stable universal cells carrying the CAR-moiety were then stored as a stable and unified cell bank. Subsequently, the SDcatcher/GVoptiTag system, which generate an isopeptide bond, was used to covalently connect the purified scFvs of antibody targeting different antigens to the recovered CAR-T cells. RESULTS: The resulting CAR-T cells can perform different functions by specifically targeting various cells, such as the eradication of human immunodeficiency virus type 1 (HIV-1)-latenly-infected cells or elimination of T lymphoma cells, with similar efficiency as the traditional CAR-T cells did. CONCLUSION: Taken together, our strategy allows the production of CAR-T cells more modularization, and makes the quality control and pharmaceutic manufacture of CAR-T cells more feasible.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (ß-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide ß-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions.

The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

Mechanistic model-based characterization of size-exclusion-mixed-mode resins for removal of monoclonal antibody fragments.

Although antibody fragments are a critical impurity to remove from process streams, few platformable purification techniques have been developed to this end. In this work, a novel size-exclusion-mixed-mode (SEMM) resin was characterized with respect to its efficacy in mAb fragment removal. Inverse size-exclusion chromatography showed that the silica-based resin had a narrow pore size distribution and a median pore radius of roughly 6.2 nm. Model-based characterization was carried out with Chromatography Analysis and Design Toolkit (CADET), using the general rate model and the multicomponent Langmuir isotherm. Model parameters were obtained from fitting breakthrough curves, performed at multiple residence times, for a mixture of mAb, aggregates, and an array of fragments (varying in size). Accurate fits were obtained to the frontal chromatographic data across a range of residence times. Model validation was then performed with a scaled-up column, altering residence time and feed composition from the calibration run. Accurate predictions were obtained, thereby illustrating the model's interpolative and extrapolative capabilities. Additionally, the SEMM resin achieved 90% mAb yield, 37% aggregate removal, 29% [Formula: see text] removal, 54% Fab/Fc removal, 100% Fc fragments removal, and a productivity of 72.3 g mAbL×h. Model predictions for these statistics were all within 5%. Simulated batch uptake experiments showed that resin penetration depth was directly related to protein size, with the exception of the aggregate species, and that separation was governed by differential pore diffusion rates. Additional simulations were performed to characterize the dependence of fragment removal on column dimension, load density, and feed composition. Fragment removal was found to be highly dependent on column load density, where optimal purification was achieved below 100 mg protein/mL column. Furthermore, fragment removal was dependent on column volume (constant load mass), but agnostic to whether column length or diameter was changed. Lastly, the dependence on feed composition was shown to be complex. While fragment removal was inversely related to fragment mass fraction in the feed, the extent depended on fragment size. Overall, the results from this study illustrated the efficacy of the SEMM resin in fragment and aggregate removal and elucidated relationships with key operational parameters through model-based characterization.

Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Electrostatic properties of human germlines and biodistribution of small biologics.

Off-target biodistribution of biologics bears important toxicological consequences. Antibody fragments intended for use as vectors of cytotoxic payloads (e.g. antibody-drug conjugates, radiotherapy) can accumulate at clearance organs like kidneys and liver, where they can cause dose-limiting toxicities. Renal and hepatic uptakes are known to be affected by protein electrostatics, which promote protein internalization through pinocytosis. Using minibodies as a model of an antibody fragment lacking FcRn recycling, we compared the biodistributions of leads with different degrees of accumulation at the kidney and liver. We identified a positive electrostatic patch highly conserved in a germline family very commonly used in the humanization of approved biologics. Neutralization of this patch led to a drastic reduction in the kidney uptake, leading to a biodistribution more favorable to the delivery of highly cytotoxic payloads. Next, we conducted a high throughput study of the electrostatic properties for all combinations of VH and VL germlines. This analysis shows how different VH/VL combinations exhibit varying tendencies to create electrostatic patches, resulting in Fv variants with different isoelectric points. Our work emphasizes the importance of carefully selecting germlines for humanization with optimal electrostatic properties in order to control the unspecific tissue uptake of low molecular weight biologics.

Recombinant Antibody Fragments for Immunotherapy of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. Multiple genetic and environmental factors leading to progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) and consequent depletion of dopamine were described. Current clinical approaches, such as dopamine replacement or deep brain stimulation using surgically implanted probes, provide symptomatic relief but cannot modify disease progression. Therefore, disease-modifying therapeutic tools are urgently needed. Immunotherapy approaches, including passive transfer of protective antibodies and their fragments, have shown therapeutic efficacy in several animal models of neurodegenerative diseases, including PD. Recombinant antibody fragments are promising alternatives to conventional full-length antibodies. Modern computational approaches and molecular biology tools, directed evolution methodology, and the design of tissue-penetrating fusion peptides allowed for the development of recombinant antibody fragments with superior specificity and affinity, reduced immunogenicity, the capacity to target hidden epitopes and cross the blood-brain barrier (BBB), higher solubility and stability, the ability to refold after heat denaturation, and inexpensive large-scale production. In addition, antibody fragments do not induce microglia Fcγ receptor (FcγR)-mediated proinflammatory response and tissue damage in the central nervous system (CNS), because they lack the Fc portion of the immunoglobulin molecule. In the present review, we summarized data on recombinant antibody fragments evaluated as immunotherapeutics in preclinical models of PD and discussed their potential for developing therapeutic and preventive protocols for patients with PD.

Rapid discovery of high-affinity antibodies via massively parallel sequencing, ribosome display and affinity screening.

Developing therapeutic antibodies is laborious and costly. Here we report a method for antibody discovery that leverages the Illumina HiSeq platform to, within 3 days, screen in the order of 108 antibody-antigen interactions. The method, which we named 'deep screening', involves the clustering and sequencing of antibody libraries, the conversion of the DNA clusters into complementary RNA clusters covalently linked to the instrument's flow-cell surface on the same location, the in situ translation of the clusters into antibodies tethered via ribosome display, and their screening via fluorescently labelled antigens. By using deep screening, we discovered low-nanomolar nanobodies to a model antigen using 4 × 106 unique variants from yeast-display-enriched libraries, and high-picomolar single-chain antibody fragment leads for human interleukin-7 directly from unselected synthetic repertoires. We also leveraged deep screening of a library of 2.4 × 105 sequences of the third complementarity-determining region of the heavy chain of an anti-human epidermal growth factor receptor 2 (HER2) antibody as input for a large language model that generated new single-chain antibody fragment sequences with higher affinity for HER2 than those in the original library.

An antibody fragment-decorated liposomal conjugate targets Philadelphia-like acute lymphoblastic leukemia.

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive B-ALL malignancy associated with high rates of relapse and inferior survival rate. While targeted treatments against the cell surface proteins CD22 or CD19 have been transformative in the treatment of refractory B-ALL, patients may relapse due to antigen loss, necessitating targeting alternative antigens. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in half of Ph-like ALL cases conferring chemoresistance and enhancement of leukemia cell survival. Therefore, targeting CRLF2 may reduce the likelihood of relapse associated with antigen loss. We developed a CRLF2-targeting single-chain variable fragment modified by the fragment crystallizable region (CRLF2 scFv-Fc) conjugated to a drug maytansinoid 1 (DM1)-DOPC liposomal conjugate, creating homogeneous CRLF2-targeted liposomes (CRLF2-DM1 LIP). Cellular association and internalization studies in a Ph-like ALL cell line, MHH-CALL-4, compared to its lentivirally transduced CRLF2-knockdown counterpart (KD-CALL-4) revealed excellent CRLF2-targeting efficiency of CRLF2-DM1 LIP. Moreover, CRLF2-DM1 LIP showed selective association and internalization ex vivo using Ph-like ALL patient-derived xenograft (PDX) cells with minimal reactivity with non-target cells. Cell apoptosis assays demonstrated the CRLF2-dependent potency of CRLF2-DM1 LIP in Ph-like ALL cell lines. This study is the first to highlight the therapeutic potential of a CRLF2-directed scFv-Fc-liposomal conjugate for targeting Ph-like ALL.

A Generic Antibody-Blocking Protein That Enables pH-Switchable Activation of Antibody Activity.

Molecular strategies that allow for reversible control of antibody activity have drawn considerable interest for both therapeutic and diagnostic applications. Protein M is a generic antibody-binding protein that binds to the Fv domain of IgGs and, in doing so, blocks antigen binding. However, the dissociation of protein M is essentially irreversible, which has precluded its use as an antibody affinity reagent and molecular mask to control antibody activity. Here, we show that introduction of 8 histidine residues on the Fv binding interface of protein M results in a variant that shows pH-switchable IgG binding. This protein M-8his variant provides an attractive and universal affinity resin for the purification of IgGs, antibody fragments (Fab and single-chain variable fragments (scFv)), and antibody conjugates. Moreover, protein M-8his enables the pH-dependent blocking of therapeutic antibodies, allowing the selective targeting of cells at pH 6.0.

Framework-Directed Amino-Acid Insertions Generated over 55-Fold Affinity-Matured Antibody Fragments That Enabled Sensitive Luminescent Immunoassays of Cortisol.

We generated three single-chain Fv fragments (scFvs) specific to cortisol according to our original affinity-maturation strategy and verified their utility in developing immunoassays. These scFv mutants (m-scFvs) had insertion of one, four, or six amino acid(s) in the framework region 1 of the VH-domain and showed >55-fold higher affinity (Ka, 2.0 - 2.2 × 1010 M-1) than the unmodified scFv (wt-scFv). Each m-scFv was fused with NanoLuc luciferase (NLuc) for the use in enzyme-linked immunosorbent assays (ELISAs). In these ELISA, the m-scFv-NLuc fusions were competitively reacted with immobilized cortisol residues and cortisol standards, and then the bound NLuc activity was monitored luminometrically. The luminescent ELISAs generated dose-response curves with extremely low midpoints (approx. 3 pg/assay) and were >150-fold more sensitive than the colorimetric ELISAs using wt-scFv and >8000-fold more sensitive than the ELISA using the parental native antibody. The luminescent ELISAs showed acceptable cross-reactivity patterns with related steroids, and the determination of control sera afforded cortisol levels in the reference range with satisfactory parallelism.

[Single chain antibody fragment display systems: a review].

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Specific IgG glycosylation differences precede relapse in PR3-ANCA associated vasculitis patients with and without ANCA rise.

Introduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares. Methods: Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV. Results: Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02). Discussion: Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre.

The role of airway mucus and diseased pulmonary epithelium on the absorption of inhaled antibodies.

Inhaled antibody therapy for the treatment of respiratory diseases is a promising strategy to maximize pulmonary exposure and reduce side effects associated with parenteral administration. However, the development of inhaled antibodies is often challenging due to a poor understanding of key mechanisms governing antibody absorption and clearance in healthy and diseased pulmonary epithelium. Here, we utilize well established Human Bronchial Epithelial Cell (HBEC) models grown at air-liquid interface to study the absorption process of antibodies and antibody fragments. With these cellular models, we recapitulate the morphology and function of healthy and diseased pulmonary epithelium, and incorporate the mucosal barrier to enable the investigation of both cellular permeability as well as mucodiffusion. We studied the saturation of antibody transport across the HBEC barriers and estimated the impact of disease-like epithelial barriers on antibody paracellular transport. Additionally, we identified a potential role of neonatal Fc receptor (FcRn)-independent and target-mediated transcytosis in the transport of Fragment antigen-binding (Fab) and F(ab)2 antibody fragments. Lastly, our models were able to pinpoint an impaired antibody diffusion across mucus gels. These mechanistic cellular models are promising in vitro tools to inform Physiologically-based Pharmacokinetic (PBPK) computational models for dose prediction toward de-risking the development of inhaled biologics.

EGFR-targeted humanized single chain antibody fragment functionalized silica nanoparticles for precision therapy of cancer.

The combination of highly specific targeting ability and potent killing effect has made antibody-drug conjugates (ADCs) a popular area of focus in the development of anti-cancer drugs. However, the large molecular weight of IgG antibodies (∼ 150 kDa) often faces challenges in penetrating capillaries and stroma in tumor tissue. Moreover, when the drug-antibody ratio (DAR) is too low (DAR < 2) or too high (DAR > 6) it decreases the effectiveness of the ADC and further increases the potential for aggregation, overall clearance of the early system payload, and release rate. In this study, an EGFR-based single-chain antibody fragment (husA)-human serum albumin (HSA)-coupled FITC-labeled mesoporous silica nanoparticle (FMSN-DOX-H-husA) was developed. Chinese hamster ovarian cells express the husA, which is a single chain antibody fragment of the EGFR that has been humanized. The small molecular weight of the single chain antibody allows for shorter penetration into solid tumors and the absence of adverse effects of the Fc fragment. The modification of HSA improves the safety of the antibody nanoparticle couples by both improving the biocompatibility of the nanoparticles, prolonging the circulation time of the nanoparticles, and avoiding early release of the payload. Also, the humanization substantially reduces the immunogenicity. More importantly, the ratio of drug antibodies on nanoparticles was experimentally and computationally derived to be 11.8, providing a more accurate guide for clinical trials. The results of both in vivo and in vitro experiments indicated promising antitumor activity and safety of FMSN-DOX-H-husA. Thus, this antibody-drug conjugate provided a hopeful option for cancer treatment.

Construction of Synthetic Antibody Phage Display Libraries.

Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

Antibody Batch Cloning.

The antigen-binding ability of each antibody clone selected by phage display is usually initially ranked by a screening ELISA using monovalent scFv antibody fragments. Further characterization often requires bivalent antibody molecules such as IgG or scFv-Fc fusions. To produce these, the V region encoding genes of selected hits have to be cloned into a mammalian expression vector and analyzed as a bivalent molecule, requiring a laborious cloning procedure. We established a high-throughput procedure allowing rapid screening of candidates in bivalent formats. This protocol allows for the parallelized cloning of all selected antibody fragments into a mammalian expression vector in the 96-well plate format. The bivalent antibody molecules can then be produced and purified in 96-well plates for further analysis in microtiter plate assays.

Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy.

Multiplexed high-density label super-resolution microscopy image reconstruction by integrating exchangeable single-molecule localization (IRIS) enables elucidating fine structures and molecular distribution in cells and tissues. However, fast-dissociating binders are required for individual targets. Here, we present a protocol for generating antibody-based IRIS probes from existing antibody sequences. We describe steps for retrieving antibody sequences from databases. We then detail the construction, purification, and evaluation of recombinant probes after site-directed mutagenesis at the base of complementarity-determining region loops. The protocol accelerates dissociation rates without compromising the binding specificity. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.