MIF, as a cytokine, is implicated in several disease classes, including cancer, autoimmunity, and viral infection, making it an attractive target for therapeutic development. Many biophysical techniques can provide estim...MIF, as a cytokine, is implicated in several disease classes, including cancer, autoimmunity, and viral infection, making it an attractive target for therapeutic development. Many biophysical techniques can provide estimates of binding affinity, which are helpful as compound screening techniques and invaluable in validating biological assay results. Among these techniques, Heteronuclear Single Quantum Coherence spectroscopy (HSQC) NMR offers significant advantages that justify its inherent technical challenges, including its high sensitivity, robustness to false positives, and the ability to determine binding affinities at micromolar to millimolar concentrations. Furthermore, by mapping the amino acids whose NMR chemical shift resonances are perturbed by compound binding onto the 3D structure of MIF, researchers can infer the binding site of the compound. Here, we present an isotopic N labeling technique for MIF. We also present conditions for obtaining high-quality N-HSQC NMR spectra and explain how to use chemical shift mapping to determine binding sites and binding affinities.
Molecular dynamics (MD) simulations are a powerful computational tool grounded in classical mechanics and quantum principles that enables the detailed study of atomic and molecular interactions. MD simulations have evolv...Molecular dynamics (MD) simulations are a powerful computational tool grounded in classical mechanics and quantum principles that enables the detailed study of atomic and molecular interactions. MD simulations have evolved throughout the years to become indispensable for investigating complex macromolecular systems, such as proteins. In addition to insights into protein structure, dynamics, and function, MD simulations very often complement experimental data, providing in-depth analysis for findings that are otherwise challenging to explain. Herein, we describe an MD simulation protocol focusing on D-Dopachrome tautomerase (D-DT or MIF-2), a protein with diverse biological activities. Our protocol provides a step-by-step procedure for structure preparation, minimization, equilibration, and production run. Additionally, we describe how the conformational flexibility and correlated movements of D-DT are examined through the use of root mean square fluctuation (RMSF) and correlation analyses, respectively. With minor modifications, this protocol is applicable to proteins with diverse sizes and biological assemblies.
Mass spectrometry (MS) is the analytical technique of choice when information about molecular mass and structural characteristics of minute sample quantities are desired. Here, we report a generally applicable, robust, a...Mass spectrometry (MS) is the analytical technique of choice when information about molecular mass and structural characteristics of minute sample quantities are desired. Here, we report a generally applicable, robust, and fast method for sample preparation and matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) analysis of D-dopachrome tautomerase (D-DT or MIF-2) in the presence of a small molecule ligand. We show that noncovalent protein-ligand complexes can be detected. Our method will allow researchers to quickly screen large libraries of ligands against a given protein target and should be considered a supplement to other detection techniques for noncovalent protein-ligand complexes.
The cytokine MIF is implicated in several autoimmune and inflammatory conditions and is elevated in several types of cancer, making it a promising therapeutic target. Small-molecule MIF inhibitors provide tools to elucid...The cytokine MIF is implicated in several autoimmune and inflammatory conditions and is elevated in several types of cancer, making it a promising therapeutic target. Small-molecule MIF inhibitors provide tools to elucidate MIFs' immune regulatory roles, and their further optimization could lead to potential therapeutics. Surface plasmon resonance (SPR) is a relatively high-throughput and sensitive biophysical technique used in drug discovery for screening small organic compounds (termed fragments) for their interactions with macromolecules. SPR analysis can provide the association and dissociation rate constants and the equilibrium binding constant for protein interactions. As such, SPR also provides a valuable orthogonal technique to determine compound binding (K and K) and correlate it with inhibitory activity (IC) observed in functional assays or cell culture experiments. This helps researchers discriminate false positive results from off-target effects or compound misbehavior. Establishing SPR conditions required to immobilize active and stable proteins for analysis can be challenging. Hence, we present a robust SPR assay for MIF, outlining basic approaches and tips for screening and validating fragments.
Protein crystallography is a key technique of structural biology that allows researchers to determine the three-dimensional structure of proteins, protein-protein, protein-ligand, and protein-nucleic acid complexes at th...Protein crystallography is a key technique of structural biology that allows researchers to determine the three-dimensional structure of proteins, protein-protein, protein-ligand, and protein-nucleic acid complexes at the atomic level. Whereas obtaining high-quality crystals is the first milestone for the structural characterization of a protein, the establishment of a detailed crystallization protocol promotes reproducibility of the crystallization process. Here, we describe a versatile crystallization method for human D-dopachrome tautomerase (D-DT or MIF-2), a small pleotropic protein of increasing interest due to its key role in human pathophysiology. The protocol described here provides a step-by-step procedure for generating high-quality protein crystals for a wide range of D-DT variants, including aggressive truncations and mutants with mechanistic value.
The determination of experimental X-ray crystal structures of macrophage migration inhibitory factor (MIF) with bound ligands is a crucial step in understanding their binding modes and steering medicinal chemistry effort...The determination of experimental X-ray crystal structures of macrophage migration inhibitory factor (MIF) with bound ligands is a crucial step in understanding their binding modes and steering medicinal chemistry efforts. Here, a generic method for the crystallization of MIF and subsequent structural analysis with ligands is described.
D-dopachrome tautomerase (D-DT) is a multifunctional protein with various physiological roles, including immune system regulation and protection from oxidative stress. However, D-DT is also implicated in the progression...D-dopachrome tautomerase (D-DT) is a multifunctional protein with various physiological roles, including immune system regulation and protection from oxidative stress. However, D-DT is also implicated in the progression of inflammatory conditions, cancers, and neurodegenerative diseases, where oxidative stress contributes to neuronal damage. Despite its importance, D-DT's role in cellular processes and disease remains to be fully elucidated. For both basic research and exploring D-DT as a potential therapeutic target, access to recombinant, purified D-DT is invaluable. We present a detailed methodology for the bacterial expression of D-DT and its purification using ion exchange and size exclusion chromatography. Our systematic approach yields 8 mg of purified protein per liter of bacterial culture.
Solid phase peptide synthesis is a method used to generate peptides through the sequential addition of amino acids attached to an immobilized resin. Due to the procedure followed, excess reagents and byproducts are remov...Solid phase peptide synthesis is a method used to generate peptides through the sequential addition of amino acids attached to an immobilized resin. Due to the procedure followed, excess reagents and byproducts are removed while synthesis is ongoing, resulting in a final product that is reproducibly obtained at high purity. Herein, we describe the synthetic procedure of peptides derived from the pleotropic protein macrophage migration inhibitory factor (MIF). Notably, this protocol is valid for the generation of peptides regardless of their sequence and length. Whereas MIF has diverse activities and protein partners, the generation of thoughtfully designed MIF peptides may have applications in drug discovery and structural biology.
Recombinant proteins are crucial in biomedical research, highlighting the importance of efficient expression and purification methods. Here, we present a protocol for the expression of recombinant macrophage migration in...Recombinant proteins are crucial in biomedical research, highlighting the importance of efficient expression and purification methods. Here, we present a protocol for the expression of recombinant macrophage migration inhibitory factor (MIF), followed by its purification. Our optimized approach incorporates three chromatographic purification steps: anion and then cation exchange chromatography followed by size exclusion chromatography (SEC). Protein validation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting confirms the production of pure MIF protein.
Bioconjugation of linker-payloads on monoclonal antibodies is achieved to target the action of a small molecule towards a cell of interest. Herein, the bioconjugation of a vedotin linker-payload to an immunoglobulin G (I...Bioconjugation of linker-payloads on monoclonal antibodies is achieved to target the action of a small molecule towards a cell of interest. Herein, the bioconjugation of a vedotin linker-payload to an immunoglobulin G (IgG) is described along with the subsequent analysis of the obtained antibody-drug conjugate (ADC).
Interleukin-18 (IL-18) is a promising cytokine for immunotherapy. Besides exploiting the cytokine itself or mutant versions thereof for biomedical applications, bi- and multispecific antibody scaffolds can be harnessed t...Interleukin-18 (IL-18) is a promising cytokine for immunotherapy. Besides exploiting the cytokine itself or mutant versions thereof for biomedical applications, bi- and multispecific antibody scaffolds can be harnessed that mimic the function of IL-18 by triggering receptor signaling. In addition to potential advantages with respect of manufacturability, a major benefit in using bispecific antibody derivatives relies in the different structure and sequence of the antibody compared to the cytokine. The activity of IL-18 is normally regulated in a negative feedback loop via IL-18 binding protein (IL-18BP) that binds with high affinities to IL-18 and prevents IL-18 from binding to its receptor. This physiological process is hijacked in certain types of cancer. We have generated VHH-derived bispecific surrogate agonists that are naturally resistant to inhibition by the decoy receptor IL-18BP. In this chapter, we detail protocols about the generation and characterization of IL-18 mimicking bispecifics.
CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds...CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.
Bispecific antibodies show therapeutic potential; thus, their screening campaign has attracted attention for the development of biotherapeutics. However, many bispecific formats require engineering or fine-tuned purifica...Bispecific antibodies show therapeutic potential; thus, their screening campaign has attracted attention for the development of biotherapeutics. However, many bispecific formats require engineering or fine-tuned purification processes optimized for each molecule, hindering simple screening, especially when impurities are not allowed. In this chapter, we describe a method for producing IgG-like bispecific antibodies via intein-mediated protein trans-splicing, fusing two polypeptide units post-translationally. This method allows the separate expression of two antigen-binding fragments and eliminates the light chain problem. Homobivalent species are not contaminated. In addition, the common purification process finalized by size-exclusion chromatography enables the systematic production of bispecific antibodies suitable for in vitro screening.
Monoclonal antibodies have revolutionized cancer therapy and are integral part of standard therapies. Despite their clinical efficacy, not all cancer patients benefit from established antibody therapies. Preclinical and...Monoclonal antibodies have revolutionized cancer therapy and are integral part of standard therapies. Despite their clinical efficacy, not all cancer patients benefit from established antibody therapies. Preclinical and clinical findings revealed that Fc-mediated effector functions play an essential role in antibody therapy. Therefore, current antibody-based immunotherapies can be optimized by antibody Fc engineering to generate next-generation monoclonal antibodies with tailor-made effector functions. The enhancement of Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) may increase efficiency of prospective antibody-based immunotherapies.
Immunoreagents, such antibodies, must often be functionalized before being used for a specific application, and this modification can strongly affect their biophysical characteristics. In contrast, recombinant antibody f...Immunoreagents, such antibodies, must often be functionalized before being used for a specific application, and this modification can strongly affect their biophysical characteristics. In contrast, recombinant antibody fragments can be engineered with elevated precision at selected residues to obtain customized reagents. Several alternative strategies can be used that exploit different principles and require different effort to reach this objective. Direct labeling can be obtained by fusing the antibody fragment to a fluorescent protein; otherwise, tag sequences can be appended to the binders and then these are modified by chemical or enzymatic reactions. In this contribution, a model system will be illustrated and compared with other options to illustrate what factors should be considered to choose the functionalization strategy.
Antibody humanization is the process by which animal species' antibodies (usually murine) are engineered to more closely resemble human antibodies so that they are less likely to elicit anti-drug antibody responses when...Antibody humanization is the process by which animal species' antibodies (usually murine) are engineered to more closely resemble human antibodies so that they are less likely to elicit anti-drug antibody responses when used clinically. The process involves selection of human antibody acceptor templates, followed by the identification of amino acids from the murine sequence that should be retained in the humanized sequence in order to preserve antigen binding; these amino acids comprise CDR residues plus additional framework resides important for maintaining the overall structure of the CDRs. Further consideration is also given to the physicochemical characteristics of the antibody and how these can be optimized during the humanization process.
Antibodies with affinities in the lower nanomolar or sub-nanomolar range or specific off-rate binding kinetic (K) are favored for many research, diagnostic, and therapy applications. Many protocols for affinity maturatio...Antibodies with affinities in the lower nanomolar or sub-nanomolar range or specific off-rate binding kinetic (K) are favored for many research, diagnostic, and therapy applications. Many protocols for affinity maturation exist but often fail to produce desired results due to inefficient exploration of the sequence space or because the developability of the parental molecule is compromised. Here we describe a two-phase protocol to explore the sequence space for a given antibody efficiently, enabling simultaneous affinity and developability maturation.
The internalization of antibody via pinocytosis and its pH-dependent interaction with the neonatal Fc receptor (FcRn) play crucial roles in antibody recycling within circulation. To achieve an optimal half-life, the bind...The internalization of antibody via pinocytosis and its pH-dependent interaction with the neonatal Fc receptor (FcRn) play crucial roles in antibody recycling within circulation. To achieve an optimal half-life, the binding of antibodies to FcRn at acidic pH and their release at physiological pH must be carefully characterized. Here, we describe the use of surface plasmon resonance (SPR) to measure the affinity of human IgG1 antibodies to human FcRn in both acidic (pH 6.0) and physiological (pH 7.4) conditions to evaluate the recycling behavior of therapeutic candidates.
Early-stage screening of antibody developability properties is important for identifying monoclonal antibody (mAb) candidates with favorable biophysical properties in addition to potent biological activities. Nevertheles...Early-stage screening of antibody developability properties is important for identifying monoclonal antibody (mAb) candidates with favorable biophysical properties in addition to potent biological activities. Nevertheless, this process is challenging given the large numbers of mAb candidates, as well as their low quantities, concentrations, and purities. Here, we report methods for assessing the developability of mAbs in terms of their levels of self-association and non-specific binding at ultra-dilute concentrations (~0.01-0.05 mg/ml). The self-association measurements using the AC-SINS and CS-SINS assays are performed by capturing mAbs on immunogold conjugates and evaluating their plasmon shifts in either formulation (CS-SINS) or physiological (AC-SINS) solution conditions. The non-specific binding measurements using the PSP assay are performed by capturing mAbs on Protein A-functionalized magnetic beads and evaluating their interactions with protein reagents in physiological solution conditions using flow cytometry. Using these methods in concert enables the identification of mAbs with reduced risk for development challenges associated with high self-association (e.g., high viscosity or low solubility) and high non-specific binding (e.g., fast antibody clearance).
Antibodies play crucial roles in both basic research and clinical practice. The binding interface between an antibody and its antigen, i.e., the epitope, is a defining feature of the antibody. Accurate epitope mapping is...Antibodies play crucial roles in both basic research and clinical practice. The binding interface between an antibody and its antigen, i.e., the epitope, is a defining feature of the antibody. Accurate epitope mapping is essential for developing antibody-based reagents and therapies and safeguarding intellectual property. Here, we present the detailed procedure of a novel high-throughput technology, namely, Antibody binding epitope Mapping (AbMap) version 1.0. Leveraging versatile phage-displayed peptide library and next-generation sequencing (NGS), AbMap enables the comprehensive profiling of hundreds to thousands of antibody-binding epitopes in a single run.