Easy assessment of the avidity of polyclonal allergen-specific serum antibodies.

INTRODUCTION: Allergen-specific IgE-blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody-allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT-induced blocking and non-blocking Mald1-specific antibodies in sera from individuals with or without reduced apple allergy were compared. METHODS: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4-3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (KD) of mAbs determined by surface plasmon resonance. RESULTS: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with KD values and were unaffected by epitope specificity or the presence of serum proteins. The inter-assay variability was <4% CV. Protective Mald1-specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non-protective antibodies of individuals without reduced apple allergy. CONCLUSION: Acidic disruption of allergen-antibody complexes may be used to estimate the net-binding force of polyclonal serum antibodies and eases the investigation of affinity-related research questions in AIT.

Development of a Cytotoxic Antibody-Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells.

High numbers of membrane immunoglobulin E (IgE)-positive cells are characteristic of allergic conditions, atopic dermatitis, or IgE myeloma. Antibodies targeting the extracellular membrane-proximal domain of the membranous IgE-B-cell receptor (BCR) fragment can be used for specific depletion of IgE-BCR-positive cells. In this study, we derivatized such an antibody with a toxin and developed an antibody-drug conjugate (ADC) that showed strong cytotoxicity for an IgE-positive target cell line. Site-specific conjugation with maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl-auristatin E via a newly introduced single cysteine residue was used to prepare a compound with a drug-antibody ratio of 2 and favorable biophysical properties. The antibody was rapidly taken up by the target cells, showing almost complete internalization after 4 h of treatment. Its cytotoxic effect was potentiated upon cross-linking mediated by an anti-human IgG F(ab')2 fragment. Because of its fast internalization and strict target specificity, this antibody-drug conjugate presents a valuable starting point for the further development of an anti-IgE cell-depleting agent, operating by the combined action of receptor cross-linking and toxin-mediated cytotoxicity.

Trispecific antibodies produced from mAb2 pairs by controlled Fab-arm exchange.

Bispecific antibodies and antibody fragments are therapeutics of growing importance. They are clinically applied for effector cell engagement, enhanced targeting selectivity, addressing of multiple cellular pathways and active transfer of certain activities into difficult-to-reach compartments. These functionalities could profit from a third antigen specificity. In this work we have employed symmetrical bispecific parental antibodies of mAb2 format, which feature a novel antigen binding site in the CH3 domains, and engineered them with a minimal number of point mutations to guide the formation of a controlled Fab-arm exchanged trispecific antibody at a high yield after reduction and re-oxidation. Two model antibodies, one reactive with EGFR, Her2 and VEGF, and one with Fab-arms binding to Ang2 and VEGF and an Fc fragment binding to VEGF, were prepared and examined for heterodimeric status, stability, antigen binding properties and biological activity. Resulting molecules were of good biophysical characteristics and retained antigen reactivity and biological activity of the parental mAb2 constructs.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.

Structural studies of several clinically important oncology drugs in complex with human serum albumin.

BACKGROUND: Serum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin. METHODS: Protein crystal growth and low temperature X-ray crystallography RESULTS: These large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IBd) and the other between L1 and α-helix h9 (proximal site: IBp). Camptothecin (2.4Å) and 9 amino camptothecin (2.0Å) are clearly bound as the open lactone form (IBp). Idarubicin (2.8Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IBd. Bicalutamide (2.4Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IBd similar to idarubicin. Teniposide (2.7Å) and etoposide (2.7Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IBp-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network. CONCLUSIONS: Subdomain IB is a major binding site for complex heterocyclic molecules. GENERAL SIGNIFICANCE: The structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.

Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments.

Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and ß1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast.

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.

Selection of High-Affinity Heterodimeric Antigen-Binding Fc Fragments from a Large Yeast Display Library.

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the CH3 domain, function as parts of bispecific IgG-like symmetrical antibodies when they replace their wild-type Fc. Their homodimeric structure typically leads to bivalent antigen binding. In particular, biological situations monovalent engagement, however, would be preferred, either for avoiding agonistic effects leading to safety issues, or the attractive option of combining a single chain (i.e., one half) of an Fcab fragment reactive with different antigens in one antibody. We present the strategies for construction and selection of yeast libraries displaying heterodimeric Fcab fragments and discuss the effects of altered thermostability of the basic Fc scaffold and novel library designs that lead to isolation of highly affine antigen binding clones.

A C-terminal interdomain disulfide bond significantly stabilizes the Fc fragment of IgG.

We describe the stabilization of human IgG1 Fc by an engineered interdomain disulfide bond at the C-terminal end of the molecule. Covalently interconnecting the C-termini of the CH(3) domains led to an increase of the melting temperatures by 5.6 and 9.1 °C respectively as compared to CH(3) domains in the context of the wild-type Fc. Combined with a recently described additional intradomain disulfide bond, both novel disulfide bonds led to an increase of the Tm by about 18.1 °C to 100.7 °C. The interdomain disulfide bond had no impact on the thermal stability of the CH(2) domain. Far- and near-UV CD spectroscopy showed very similar overall CD profiles, indicating that secondary and tertiary structure of the Fc was not negatively affected. When introduced into an Fc fragment that had been engineered to bind to Her2/neu via a novel antigen binding site located at the C-terminus of the CH(3) domain, the novel inter- and intra-domain bonds also brought about a significant increase in thermostability. Using them in combination, the Tm of the CH(3) domain was raised by 18 °C and thus restored to the Tm of the wild-type CH(3) domain. Importantly, antigen binding of the modified Fc was not affected by the engineered disulfide bonds.

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions.

Construction of Yeast Display Libraries for Selection of Antigen-Binding Variants of Large Extracellular Loop of CD81, a Major Surface Marker Protein of Extracellular Vesicles.

Over the last two decades, yeast display methodology has served as a popular tool for discovery, humanization, stability improvement, and affinity maturation of antibodies and antibody fragments, but also for development of diverse non-antibody protein scaffolds towards the ability of antigen recognition. Yeast display is particularly well suited for multiparametric analysis of properties of derivatized proteins, allowing the evolution of most diverse protein structures into antigen binding entities with favorable expression, stability, and folding properties. Here we present the methodological basics of a novel yeast display-based approach for the functionalization of the large extracellular loop of CD81 into a de novo antigen binding unit. CD81 is intrinsically overrepresented on the surface of extracellular vesicles (EVs), naturally occurring nanoparticle units that act as cell-to-cell messengers by delivering their intracellular cargo from the source cell into a recipient cell. This amazing feature makes them of highest biotechnological interest, yet methods for their targeted delivery are still in their infancy. As a novel approach for introducing EV surface modifications enabling specific target cell recognition and internalization, we have prepared yeast display libraries of CD81 large extracellular loop mutants, which are selected towards specific antigen binding and resulting mutants conveniently clicked into the full-length EV surface protein. Resulting EVs display wild-type-like characteristics regarding the expression level and distribution of recombinant proteins and are hence promising therapeutic tools.

Efficient spontaneous site-selective cysteine-mediated toxin attachment within a structural loop of antibodies.

BACKGROUND: Site-specific coupling of toxin entities to antibodies has become a popular method of synthesis of antibody-drug conjugates (ADCs), as it leads to a homogenous product and allows a free choice of a convenient site for conjugation. METHODS: We introduced a short motif, containing a single cysteine surrounded by aromatic residues, into the N-terminal FG-loop of the CH2 domain of two model antibodies, cetuximab and trastuzumab. The extent of conjugation with toxic payload was examined with hydrophobic interaction chromatography and mass spectrometry and the activity of resulting conjugates was tested on antigen-overexpressing cell lines. RESULTS: Antibody mutants were amenable for rapid coupling with maleimide-based linker endowed toxin payload and the modifications did not impair their reactivity with target cell lines or negatively impact their biophysical properties. Without any previous reduction, up to 50% of the antibody preparation was found to be coupled with two toxins per molecule. After the isolation of this fraction with preparative hydrophobic interaction chromatography, the ADC could elicit a potent cytotoxic effect on the target cell lines. CONCLUSION: By fine-tuning the microenvironment of the reactive cysteine residue, this strategy offers a simplified protocol for production of site-selectively coupled ADCs. GENERAL SIGNIFICANCE: Our unique approach allows the generation of therapeutic ADCs with controlled chemical composition, which facilitates the optimization of their pharmacological activity. This strategy for directional coupling could in the future simplify the construction of ADCs with double payloads ("dual warheads") introduced with orthogonal techniques.

A Tetravalent Biparatopic Antibody Causes Strong HER2 Internalization and Inhibits Cellular Proliferation.

The overexpression of tyrosine kinase HER2 in numerous cancers, connected with fierce signaling and uncontrolled proliferation, makes it a suitable target for immunotherapy. The acquisition of resistance to currently used compounds and the multiplicity of signaling pathways involved prompted research into the discovery of novel binders as well as treatment options with multiple targeting and multispecific agents. Here we constructed an anti-HER2 tetravalent and biparatopic symmetrical IgG-like molecule by combining the Fab of pertuzumab with a HER2-specific Fcab (Fc fragment with antigen binding), which recognizes an epitope overlapping with trastuzumab. In the strongly HER2-positive cell line SK-BR-3, the molecule induced a rapid and efficient reduction in surface HER2 levels. A potent anti-proliferative effect, specific for the HER2-positive cell line, was observed in vitro, following the induction of apoptosis, and this could not be achieved with treatment with the mixture of pertuzumab and the parental Fcab. The inhibitory cytotoxic effect of our antibody as a single agent makes it a promising contribution to the armory of anti-cancer molecules directed against HER2-addicted cells.

Binding Characteristic of Various Antibody Formats Against Aflatoxins.

The application of recombinant antibodies for the analysis of foods and food contaminants is now a major focus, given their capacity to be engineered to tailor their specificity, enhance their stability, and modify their structural formats to fit the desired analytical platform. In this study, human scFv antibody fragments generated against aflatoxin B1 (AFB1) were selected as the model antibody to explore the effect of antibody formats on their binding activity and to evaluate their potential use as immunoreagents for food contaminant analysis. Four human scFv antibody fragments against aflatoxin B1 (AFB1), previously isolated and engineered by chain shuffling, were converted into various formats, that is, scFv-AP fusions, scFv-Fc, and whole IgG molecules. The result indicated that the effects of the antibody format on the binding property varied, depending on the sequence of scFv. For all of the scFv clones, the scFv-AP fusion format showed the highest sensitivity by competitive ELISA, while the effects on the binding activity after conversion to scFv-Fc or IgG format varied, depending on the amino acid sequence of the antibodies. The sAFH-3e3 antibodies that showed the best performance by competitive ELISA were selected for further investigation. The sAFH-3e3 was converted to the scFv-GFP format and tested by fluorescence-linked immunosorbent assay (FLISA), which showed that its binding property was equivalent to those of scFv-Fc and IgG formats. The potential applications of the sAFH-3e3 in a rapid test kit format based on ELISA (scFv-AP) and in a lateral flow immunochromatography assay (LFIA) (IgG) were demonstrated. A comparison of methods for the extraction of AFB1 from matrices for use with these assay formats indicated that PBS and TBST are better than 70% methanol.

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains.

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a "Fab-like" format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites.

Bispecific T-Cell Engagers Targeting Membrane-Bound IgE.

The increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells.

An engineered CD81-based combinatorial library for selecting recombinant binders to cell surface proteins: Laminin binding CD81 enhances cellular uptake of extracellular vesicles.

The research of extracellular vesicles (EVs) has boomed in the last decade, with the promise of them functioning as target-directed drug delivery vehicles, able to modulate proliferation, migration, differentiation, and other properties of the recipient cell that are vital for health of the host organism. To enhance the ability of their targeted delivery, we employed an intrinsically overrepresented protein, CD81, to serve for recognition of the desired target antigen. Yeast libraries displaying mutant variants of the large extracellular loop of CD81 have been selected for binders to human placental laminin as an example target. Their specific interaction with laminin was confirmed in a mammalian display system. Derived sequences were reformatted to full-length CD81 and expressed in EVs produced by HeLa cells. These EVs were examined for the presence of the recombinant protein and were shown to exhibit an enhanced uptake into laminin-secreting mammalian cell lines. For the best candidate, the specificity of antigen interaction was demonstrated with a competition experiment. To our knowledge, this is the first example of harnessing an EV membrane protein as mediator of de novo target antigen recognition via in vitro molecular evolution, opening horizons to a broad range of applications in various therapeutic settings.

Methods for Construction of Yeast Display Libraries of Four-Domain T-Cell Receptors.

Since two decades, yeast display methodology is a popular tool for discovery, stability improvement, and affinity maturation of diverse protein scaffolds, intended for antigen recognition. Yeast display is particularly well suited for the selection of heterodimeric proteins, such as antibodies and T-cell receptors (TCRs), as it allows rapid library creation via gap-repair-driven homologous recombination and subsequent construction of a combinatorial library after mating of yeast of opposite mating types. Certain properties of the TCR scaffold, such as its stability, inferior to most antibody fragments, require modifications of traditional antigen selection strategies. Their selection can be monitored and guided upon staining with the soluble versions of their original antigen, peptide-major histocompatibility complex (MHC), or clonotypic antibodies, whose binding is critically dependent on the TCR structural integrity. Overall, this chapter underlines the importance of the versatile yeast display technique for the diversification of the TCR scaffold for antigen recognition and optimization of its stability.

Stabilization of soluble high-affinity T-cell receptor with de novo disulfide bonds.

Soluble T-cell receptors (TCRs) have recently gained visibility as target-recognition units of anticancer immunotherapeutic agents. Here, we improved the thermal stability of the well-expressed high-affinity A6 TCR by introducing pairs of cysteines in the invariable parts of the α- and ß-chain. A mutant with a novel intradomain disulfide bond in each chain also tested superior to the wild-type in the accelerated stability assay. Binding of the mutant to the soluble cognate peptide (cp)-MHC and to the peptide-loaded T2 cell line was equal to the wild-type A6 TCR. The same stabilization motif worked efficiently in TCRs with different specificities, such as DMF5 and 1G4. Altogether, the biophysical properties of the soluble TCR molecule could be improved, without affecting its expression level and antigen-binding specificity.