Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn

Engineered antibody domains (eAds) are promising candidate therapeutics but their half-life is relatively short partly due to weak or absent binding to the neonatal Fc receptor (FcRn). We developed a novel approach to increase the eAd binding to FcRn based on a combination of structure-based design, computational modeling and phage display methodologies. By using this approach, we identified 2 IgG1 CH2-derived eAds fused to a short FcRn-binding motif derived from IgG1 CH3 that exhibited greatly enhanced FcRn binding with strict pH dependency. Importantly, the increased affinity resulted in significantly enhanced FcRn-mediated epithelial transcytosis and prolonged elimination half-life (mean 44.1 hours) in cynomolgus macaques. These results demonstrate for the first time that the half-life of isolated eAds can be prolonged (optimized) by increasing their binding to FcRn while maintaining their small size, which has important implications for development of therapeutics, including eAd-drug conjugates with enhanced penetration in solid tissues.     

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)₂, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.     

pH-dependent antigen-binding antibodies as a novel therapeutic modality

Monoclonal antibodies have become a general modality in therapeutic development. However, even with infinite binding affinity to an antigen, a conventional antibody is limited in that it can bind to the antigen only once, and this results in antigen-mediated antibody clearance when the a membrane-bound antigen is targeted, or in antibody-mediated antigen accumulation when a soluble antigen is targeted. Recently, a pH-dependent antigen-binding antibody that binds to an antigen in plasma at neutral pH and dissociates from the antigen in endosome at acidic pH has been reported to overcome this limitation and to reduce antigen-mediated antibody clearance and antibody-mediated antigen accumulation. A pH-dependent binding antibody against a soluble antigen can be further improved by Fc engineering to enhance the Fc receptor binding. Various approaches, including histidine-based engineering, direct cloning from immunized animals, and synthetic and combinatorial libraries, have been successfully applied to generate pH-dependent binding antibodies against various antigens. This review discusses the features, approaches, advantages, and challenges of developing a pH-dependent binding antibody as a novel therapeutic modality. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

IgG Fc engineering to modulate antibody effector functions

Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.     

Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.     

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')₂ (t_1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')₂, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t_1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t_1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.     

Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.     

A novel plant-made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS-CoV-2 Omicron variant

This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan-Hu-1 RBD of SARS-CoV-2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human-like, highly homogenous N-linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well-characterized mAbs, suggesting that it is a Class 4 RBD-binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS-CoV-2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety-friendly alternative platform for generating mAbs to address the evolving SARS-CoV-2 crisis.     

Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody

Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases.     

Pharmacokinetics and biodistribution of a light-chain-shuffled CC49 single-chain Fv antibody construct

Murine monoclonal antibodies to tumor-associated glycoprotein 72 (anti-TAG-72 mAb B72.3 and CC49) are among the most extensively studied mAb for immunotherapy of adenocarcinomas. They have been used clinically to localize primary and metastatic tumor sites; however, murine mAb generally induce potent human anti-(mouse antibody) responses. The immunogenicity of murine mAb can be minimized by genetic humanization of these antibodies, where non-human regions are replaced by the corresponding human sequences or complementary determining regions are grafted into the human framework regions. We have developed a humanized CC49 single-chain antibody construct (hu/muCC49 scFv) by replacing the murine CC49 variable light chain with the human subgroup IV germline variable light chain (Hum4 V_L). The major advantages of scFv molecules are their excellent penetration into the tumor tissue, rapid clearance rate, and much lower exposure to normal organs, especially bone marrow, than occur with intact antibody. The biochemical properties of hu/muCC49 scFv were compared to those of the murine CC49 scFv (muCC49 scFv). The association constants (K _a) for hu/muCC49 and muCC49 constructs were 1.1 × 10⁶ M⁻¹ and 1.4 × 10⁶ M⁻¹ respectively. Pharmacokinetic studies in mice showed similar rapid blood and whole-body clearance with a half-life of 6 min for both scFv. The biodistribution studies demonstrated equivalent tumor targeting to human colon carcinoma xenografts for muCC49 and hu/muCC49 scFv. These results indicate that the human variable light-chain subgroup IV can be used for the development of humanized or human immunoglobulin molecules potentially useful in both diagnostic and therapeutic applications with TAG-72-positive tumors. Received: 29 December 1999 / Accepted: 4 February 2000     

工程原核生物和酵母菌中生产单克隆抗体和抗体片段研究进展 *

抗体药物和抗体片段药物在药物市场占据了重要的地位,主要通过哺乳动物细胞系统进行生产,操作复杂并且成本高。为了能够克服哺乳动物细胞系统生产抗体药物的弊端,越来越多的抗体及抗体片段在原核细胞及酵母菌中生产,但是产率往往不高并且没有糖基化。从基因转录和翻译的优化、分子伴侣的共表达和抑制蛋白水解降解等方面概述了在原核生物表达系统及酵母菌中提高单克隆抗体和抗体片段产量的研究进展,为未来利用原核生物和酵母菌实现工业化生产单克隆抗体及抗体片段奠定基础。     

Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. D., Prabakaran, P., Veenstra, T. D., Tarasov, S. G., Ishima, R., and Dimitrov, D. S. (2009) J. Biol. Chem. 284, 14203-14210). We have hypothesized that the stability of this engineered antibody domain could be further increased by removing unstructured residues. To test our hypothesis, we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric, and remarkably stable, with a melting temperature (T(m)) of 82.6 °C, which is about 10 and 30 °C higher than those of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody that recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human neonatal Fc receptor at pH 6.0 more strongly than CH2. These data suggest that shortening the m01 N terminus significantly increases stability without disrupting its conformation and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins.     

Selection and characterisation of binders based on homodimerisation of immunoglobulin V(H) domains

The antigen-binding surface of antibodies is formed by the heterodimerisation of the two variable domains of the light (V(L)) and heavy (V(H)) chains. We have previously described the spontaneous formation of V(H) dimers (VHD) in both bacteria and mammalian cells. The self-association of a single domain produces a homo-VHD, in which the two identical V(H) domains generate a unique symmetric surface for antigen binding that is never found in the normal V(L)/V(H) antibody binding site. We developed a phagemid vector for the construction of phage display libraries in which a cysteine residue, introduced at the C-terminus of the only V(H) cloned, allowed display of homo-VHDs. Panning of the library on different proteins yielded antigen specific binders against lysozyme, glutathione S-transferase and streptavidin. A lysozyme specific homo-VHD was further characterised with an apparent affinity determined to be 216+/-6.6 nM. Importantly, the results showed that its binding activity was fully dependent on the dimerisation of both identical V(H) domains.     

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations. V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.     

Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.     

Bivalent Fv antibody fragments obtained by substituting the constant domains of a fab fragment with heterotetrameric molybdopterin synthase

The antibody Fv fragment is the smallest functional unit of an antibody but for practical use, the VH/VL interface requires stabilization, which is usually accomplished by a peptide linker that joins the two variable domains to form a single chain Fv fragment (scFv). An alternative format to scFv is proposed that (i) allows stabilization of the Fv fragment, and (ii) restores the bivalency of the antibody as a pseudo-F(ab')2 format. This new antibody fragment was constructed by replacing the CHI and CL domains of the Fab fragment with heterotetrameric molybdopterin synthase (MPTS). We found that this format, named MoaFv, improved significantly the cytoplasmic expression of the Fv as a soluble protein in BL21 or Origami Escherichia coli strains. This MoaFv format is expressed as a homogeneous heterotetrameric protein with a Mr value of 110 kDa containing two functional binding sites as revealed by active site titration. In its native condition at 37 degrees C or in the presence of urea, this format was nearly as stable as the corresponding scFv, indicating that non-covalent interactions between the MPTS subunits can replace the covalent peptide linker in scFv. Finally, this MoaFv construct could be a useful format when bivalency is desirable to improve the functional avidity.     

“Cleavable” hapten-biotin conjugates: Preparation and use for the generation of anti-steroid single-domain antibody fragments

Antibody engineering technology has the potential to provide artificial antibodies with higher performance than conventional antibodies. Filamentous phage particles are often used to express a vast diversity of mutated antibody fragments from which clones displaying improved fragments can be isolated. We recently showed that hapten-biotin conjugates, combined via a linker involving a reductively cleavable disulfide bond, are useful for isolating phage clones displaying high-affinity anti-hapten antibody fragments. Here we prepare cleavable hapten-biotin conjugates and use them to isolate anti-hapten antibody fragments with relatively low affinities. Three diagnostically important steroids (estradiol-17β [E₂], cortisol, and 17α-hydroxyprogesterone) were each coupled with a biotin derivative containing a disulfide bond. These conjugates could be bound simultaneously by their relevant anti-steroid antibody and NeutrAvidin, and their linkers were easily cleaved by dithiothreitol (DTT) treatment. The E₂-biotin conjugate was used to generate anti-E₂ single-domain antibody fragments (sdAbs). Random point mutations were introduced by error-prone PCR into the gene fragment encoding the V_H domain of a mouse anti-E₂ antibody, and these products were expressed as phagemid particles that were reacted with the E₂-biotin conjugates that had already been immobilized on a solid-phase via NeutrAvidin. Thorough washing off of nonspecific phages and subsequent DTT treatment provided a phagemid clone that displayed a mutated sdAb with improved binding properties.     

Development and characterization of a novel anti-IgE monoclonal antibody

IgE is the central macromolecular mediator responsible for the progression of allergic reactions. Omalizumab (Xolair) is a humanized monoclonal anti-IgE antibody directed at the FcεRI-binding domain of human IgE, which represents a novel therapeutic approach in the management of asthma. In this study, we developed a monoclonal antibody (7A5) against human IgE via hybridoma technique. Our data showed that 7A5 could inhibit free IgE molecules to bind to receptors without affecting IgE already bound to cellular receptors. Importantly, 7A5 was able to inhibit IgE-induced histamine release of basophilic leukemia cells. Next, the phage display peptide library technology was employed to select peptides binding to 7A5 and a striking peptide sequence motif was recovered, which is homologous to the sequence ³⁹¹KQR³⁹³ within the Cε3 domain of IgE-Fc, Our results further indicated that 7A5 specifically bound to the synthesized peptide “³⁸⁸KEEKQRN³⁹⁴” containing the ³⁹¹KQR³⁹³ motif in IgE-Fc. The epitope of 7A5 was found to be spatially close to the FcεRI-binding site, suggesting that 7A5 binding to IgE might block IgE binding to receptors via steric hindrance. The anti-IgE monoclonal antibody 7A5 may have the potential to be developed as a therapeutic agent for the treatment of allergic diseases.