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IgGA: A "Cross-Isotype" Engineered Human Fc Antibody Domain that Displays Both IgG-like and IgA-like Effector Functions
Abstract
All clinically approved antibodies are of the IgG isotype and mediate the clearance of target cells via binding to Fcγ receptors and complement (C1q). Even though IgA can elicit powerful cytotoxic action via FcαRI receptor binding, IgA antibodies have not been amenable to therapeutic development. Here, we report the engineering of a "cross-isotype" antibody, IgGA, which combines the effector functions of both IgG and IgA. IgGA binds to FcαRI with an affinity comparable to that of IgA, and to the activating Fcγ receptors, FcγRI and FcγRIIa, with high affinity, and displays increased binding to C1q compared to IgG. Unlike trastuzumab-IgG, trastuzumab-IgGA potently activates both neutrophils and macrophages to kill Her2(+) cancer cells. Furthermore, IgGA mediates greater complement-dependent cytotoxicity than IgG1 or IgA antibodies. The multitude of IgGA effector functions could be important for therapeutic purposes and highlights the concept of engineering antibodies that combine effector functions from multiple antibody isotypes.
Copyright © 2014 Elsevier Ltd. All rights reserved.
... IgA can activate neutrophils and macrophages through the engagement of FcαRI (CD89) on Ivyspring International Publisher neutrophils and macrophages. The activation of neutrophils by IgA results in more effective tumor cell killing than the activation of neutrophils by IgG antibodies [3][4][5][6][7]. The GD2 targeting IgA antibody improved neutrophil-mediated lysis of neuroblastoma and circumvented CDC-associated pain in a preclinical mouse model [8]. ...
... IgGA chimeric Fc displayed high affinity for FcαRI and retained binding to the activating Fcγ receptors CD64 and CD32a, leading to potent activation of both neutrophils and macrophages for cancer cell killing [5]. However, IgGA did not bind to CD16a or FcRn. ...
... Prior to this study, several efforts were made to combine IgG Fc and IgA Fc with other groups, including IgGA [5], tandem IgG/IgA [4,30], and TrisomAb [9]. TrisomAb has achieved significant anti-tumor effects in vitro and in vivo. ...
Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood, but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore, we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA, which can effectively recruit and activate NK cells, macrophages, and neutrophils to kill tumor cells.
Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body, tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil, macrophage, and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets.
Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart, and no obvious adverse effect is observed upon X-body treatment. Moreover, the X-body has a serum half-life and drug stability comparable to IgG.
Conclusions: The X-body, as a myeloid-cell-centered therapeutic strategy, holds promise for the development of more effective cancer-targeting therapies than the current state of the art.
... Despite its high abundancy, knowledge concerning the functional role of serum IgA is not adequate. The reasons are: (1) limitations in animal models (e.g., for rodents: missing FcαRI, polymeric IgA molecule instead of monomeric IgA in human); (2) recombinant production of IgA is difficult by the high and heterogeneous glycosylation; and (3) the low stability as well as the short serum half-life compared to IgG [5,[10][11][12][13]]. ...
... IgA mediates stronger activation of ADCC on neutrophils and ADCP on macrophages than IgG. In particular, the IgA-mediated activation of neutrophils could be a powerful tool in cancer therapy [11,44,[56][57][58][59]. On the other hand, overwhelming inflammation by neutrophils is a harmful factor for tissue damage in severe infectious diseases [60][61][62]. ...
... Despite the lower concentration of IgA on the trimodulin IC (compared to IgG and IgM), the results of blocking experiments highlight the importance of IgA for phagocytosis. An explanation for the observed stronger ADCP might be the interaction of IgA with two FcαRI molecules, thereby mediating stronger ADCP compared to IgG [11,44,45]. These data show for the first time the anti-pathogenic activity of the trimodulin IgA component. ...
In comparison to human immunoglobulin (Ig) G, antibodies of IgA class are not well investigated. In line with this, the functional role of the IgA component in IgM/IgA-enriched immunoglobulin preparations is also largely unknown. In recent years, powerful anti-pathogenic and immunomodulatory properties of human serum IgA especially on neutrophil function were unraveled. Therefore, the aim of our work is to investigate functional aspects of the trimodulin IgA component, a new plasma-derived polyvalent immunoglobulin preparation containing ~56% IgG, ~23% IgM and ~21% IgA. The functional role of IgA was investigated by analyzing the interaction of IgA with FcαRI, comparing trimodulin with standard intravenous IgG (IVIG) preparation and investigating Fc receptor (FcR)-dependent functions by excluding IgM-mediated effects. Trimodulin demonstrated potent immunomodulatory, as well as anti-pathogenic effects in our neutrophil model (neutrophil-like HL-60 cells). The IgA component of trimodulin was shown to induce a strong FcαRI-dependent inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) signaling, counteract lipopolysaccharide-induced inflammation and mediate phagocytosis of Staphylococcus aureus. The fine-tuned balance between immunomodulatory and anti-pathogenic effects of trimodulin were shown to be dose-dependent. Summarized, our data demonstrate the functional role of IgA in trimodulin, highlighting the importance of this immunoglobulin class in immunoglobulin therapy.
... To improve antibody effector function the Fc of a single antibody can be engineered to bind to Fc receptors for multiple antibody isotypes. This concept has led to the design of cross-isotype IgGA antibodies (center) where the IgG1 CH2 a1 loop residues 245-258 and the IgG1 CH3 domain (cyan) were exchanged with the structurally analogous regions of IgA (54). The regions inside the dashed box were combined to create a chimeric cross-isotype Fc. ...
... The IgG1 segments are colored light and dark cyan (right, PDB: 3DO3), and the IgA segments are colored light and dark red (left, PDB: 1OW0). The cross-isotype Fc is capable of binding to FcγRI and FcαRI, hence either of these Fc receptors can be used to recruit diverse effector cells to target cells (54). ...
... Lastly, in BALB/c mice, tandem IgG/IgA circulated with a halflife similar to that of IgG1. In a third design, Kelton et al. created a cross-isotype antibody by exchanging the CH3 domain and CH2 α1 loop residues 245-258 (PKPKDTLMISRTPE) of the gamma 1 constant region with that of the alpha constant region [ Figure 2C; (54)]. The chimeric Fc possessed the ability to bind to FcγRI, FcγRIIa, and FcαRI (54). ...
Antibodies and Fc-fusion antibody-like proteins have become successful biologics developed for cancer treatment, passive immunity against infection, addiction, and autoimmune diseases. In general these biopharmaceuticals can be used for blocking protein:protein interactions, crosslinking host receptors to induce signaling, recruiting effector cells to targets, and fixing complement. With the vast capability of antibodies to affect infectious and genetic diseases much effort has been placed on improving and tailoring antibodies for specific functions. While antibody:antigen engagement is critical for an efficacious antibody biologic, equally as important are the hinge and constant domains of the heavy chain. It is the hinge and constant domains of the antibody that engage host receptors or complement protein to mediate a myriad of effector functions and regulate antibody circulation. Molecular and structural studies have provided insight into how the hinge and constant domains from antibodies across different species, isotypes, subclasses, and alleles are recognized by host cell receptors and complement protein C1q. The molecular details of these interactions have led to manipulation of the sequences and glycosylation of hinge and constant domains to enhance or reduce antibody effector functions and circulating half-life. This review will describe the concepts being applied to optimize the hinge and crystallizable fragment of antibodies, and it will detail how these interactions can be tuned up or down to mediate a biological function that confers a desired disease outcome.
... In an attempt to combine the advantages of targeting multiple sites, Kelton et al. reported the development of an "IgGA", a hybrid antibody with the ability to bind both FcγR (such as IgG) and FcαR (such as IgA) [82]. These "cross-isotype" constructions, which were initially developed in combination with the anti-HER2 antibody trastuzumab but also described with anti-CD20, enhance the effector functions of myeloid cells (ADCC, ADCP and CDC) both in vitro and in vivo and could therefore represent a new area of research for improved myeloid cell immunotherapy [82][83][84]. ...
... In an attempt to combine the advantages of targeting multiple sites, Kelton et al. reported the development of an "IgGA", a hybrid antibody with the ability to bind both FcγR (such as IgG) and FcαR (such as IgA) [82]. These "cross-isotype" constructions, which were initially developed in combination with the anti-HER2 antibody trastuzumab but also described with anti-CD20, enhance the effector functions of myeloid cells (ADCC, ADCP and CDC) both in vitro and in vivo and could therefore represent a new area of research for improved myeloid cell immunotherapy [82][83][84]. ...
By binding to multiple antigens simultaneously, multispecific antibodies are expected to substantially improve both the activity and long-term efficacy of antibody-based immunotherapy. Immune cell engagers, a subclass of antibody-based constructs, consist of engineered structures designed to bridge immune effector cells to their target, thereby redirecting the immune response toward the tumor cells or infected cells. The increasing number of recent clinical trials evaluating immune cell engagers reflects the important role of these molecules in new therapeutic approaches for cancer and infections. In this review, we discuss how different immune cell types (T and natural killer lymphocytes, as well as myeloid cells) can be bound by immune cell engagers in immunotherapy for cancer and infectious diseases. Furthermore, we explore the preclinical and clinical advancements of these constructs, and we discuss the challenges in translating the current knowledge from cancer to the virology field. Finally, we speculate on the promising future directions that immune cell engagers may take in cancer treatment and antiviral therapy.
... Similar approaches may now be generalizable to improve the functional potency of future blood-stage mAbs. Using a similar cross-isotype approach, antibody trastuzumab that targets the Her2 antigen expressed by Her2+ cancer cells was engineered and showed higher Fc-mediated function (46). The mechanism by which the IgG-IgA cross isotype may induce higher levels of ADRB is not completely understood. ...
... These strengthen the possible use case for a potent bi-isotype antibody. The clinical utility of the IgG-IgA cross isotype may be hindered due to the poor serum half-life suggested by low FcgRIIIa/CD16a and FcRn interaction observed with the IgG-IgA-trastuzumab cross isotype (46). However, other chimeric antibody strategies have been developed that may overcome this and potentially allow FcRn interaction. ...
Background
Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay.
Methods
We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG–IgA Fc region chimera would enhance the level of ADRB activity.
Results
We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG–IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31.
Conclusions
We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites.
... IgG-IgA bi-isotype Fc chimerism was initially explored with the socalled 'IgGA cross-isotype' format, in which the Fc domain of the anti-HER2 IgG1 therapeutic, Trastuzumab, was genetically engineered to acquire the ability to bind to both the FcgR and FcaR ( Figure 4C) (124). The authors elegantly determined the residues controlling the engagement of Fca and g with their cognate FcRs by first using alanine scanning approaches. ...
... Due to its ability to bind Fca-and FcgR, the IgGA-Trastuzumab chimera mediated higher ADCC and ADCP than the parental IgG, but did not reach the level mediated by Trastuzumab-IgA. Furthermore, the cross-isotype format showed a higher CDC compared to IgG and IgA isotypes (124). Unexpectedly, Fc engineering in the IgGA chimera abolished commitment to FcgRIIIa/ CD16a and FcRn, suggesting low bioavailability in vivo (24). ...
Recent advances in the development of therapeutic antibodies (Abs) have greatly improved the treatment of otherwise drug-resistant cancers and autoimmune diseases. Antibody activities are mediated by both their Fab and the Fc. However, therapeutic Abs base their protective mechanisms on Fc-mediated effector functions resulting in the activation of innate immune cells by FcRs. Therefore, Fc-bioengineering has been widely used to maximise the efficacy and convenience of therapeutic antibodies. Today, IgG remains the only commercially available therapeutic Abs, at the expense of other isotypes. Indeed, production, sampling, analysis and related in vivo studies are easier to perform with IgG than with IgA due to well-developed tools. However, interest in IgA is growing, despite a shorter serum half-life and a more difficult sampling and purification methods than IgG. Indeed, the paradigm that the effector functions of IgG surpass those of IgA has been experimentally challenged. Firstly, IgA has been shown to bind to its Fc receptor (FcR) on effector cells of innate immunity with greater efficiency than IgG, resulting in more robust IgA-mediated effector functions in vitro and better survival of treated animals. In addition, the two isotypes have been shown to act synergistically. From these results, new therapeutic formats of Abs are currently emerging, in particular chimeric Abs containing two tandemly expressed Fc, one from IgG (Fcγ) and one from IgA (Fcα). By binding both FcγR and FcαR on effector cells, these new chimeras showed improved effector functions in vitro that were translated in vivo. Furthermore, these chimeras retain an IgG-like half-life in the blood, which could improve Ab-based therapies, including in AIDS. This review provides the rationale, based on the biology of IgA and IgG, for the development of Fcγ and Fcα chimeras as therapeutic Abs, offering promising opportunities for HIV-1 infected patients. We will first describe the main features of the IgA- and IgG-specific Fc-mediated signalling pathways and their respective functional differences. We will then summarise the very promising results on Fcγ and Fcα containing chimeras in cancer treatment. Finally, we will discuss the impact of Fcα-Fcγ chimerism in prevention/treatment strategies against infectious diseases such as HIV-1.
... These range from designing a bispecific IgG antibody, with one binding arm targeting FcαRI, 29 to engineering IgG/A hybrid and chimeric molecules aimed at combining long half-life and proven developability of IgG with the effector functionality of IgA. 25,30 Taken together, IgA shows potential for a wide array of therapeutic applications ranging from immuno-oncology and infectious diseases to autoimmunity. Similar to how a heterodimeric Fc expanded the accessible formats for IgG, a solution for a heterodimeric IgA Fc could open a new chapter in antibody-based therapeutics. ...
... The previously reported binding of FcαRI to IgA found apparent K D values ranging from 122 to 455 nM. 21,25,30,32,33 We further investigated the effect that different immobilized FcαRI ligand densities had on the measured apparent K D for the IgA OAA variants with SPR. Consistent with the model for bivalent IgA Fc binding to FcαRI, increasing the density of FcαRI resulted in higher apparent affinities for wild-type and heterodimeric IgA Fc variants containing two FcαRI binding sites due to avidity (Figure 4b, Table S4). ...
Immunoglobulin G (IgG) has served as a traditional framework for antibody-based biology, and engineering approaches for creating multispecific therapeutics have greatly expanded its applicability. Despite these developments, there are limits to the functionality of IgG with respect to effector cells that can be activated and paratope valency that can be obtained. Other Ig isotypes have distinct functions that can engage and activate different effector cells, and some can be found naturally in higher-order assemblies. In an effort to expand the repertoire of multispecific designs for other antibody isotypes, we present here engineering of the first heterodimeric IgA Fc that can be produced at high purity with native IgA-like thermal stability. The crystal structure confirmed the accuracy of the in silico model used for engineering and that the mutations introduced at the CH3 interface do not perturb the overall IgA Fc structure. Affinity measurements and on-cell neutrophil binding demonstrated that the heterodimeric IgA Fc retains the ability to bind FcαRI, an important prerequisite for IgA-based therapeutics designed to interact with effector cells, such as neutrophils. Given the ability of IgA antibodies to multimerize via interaction with the J-chain, the designs presented here could also be used to generate multispecific, multimeric scaffolds that leverage valency to increase clustering and specificity via avidity. Taken together, the newly developed heterodimeric IgA Fc platform allows for the development of novel, multifunctional, and multimeric molecules that have the potential to transform the next generation of antibody therapeutics.
Abbreviations
CE-SDS: capillary electrophoresis sodium dodecyl sulfate; DSC: differential scanning calorimetry; FACS: fluorescence-activated cell sorting; FSA: full-sized antibody; Her2: human epidermal growth factor receptor 2; MFI: mean fluorescent intensity; OAA: one-armed antibody; PBS: phosphate-buffered saline; PDB: Protein Data Bank; SEC: size-exclusion chromatography; prepSEC (preparative SEC); RMSD: root-mean-square deviation; RU: resonance units; SPR: surface plasmon resonance; TAA: tumor-associated antigen; WT: wild-type.
... To combine functions of IgA and IgG, antibodies were developed with a cross-isotype Fc domain in which residues from IgG1 and IgA were merged (IgGA) [83]. The fab domains of the anti-HER-2 mAb trastuzumab were fused to the IgGA Fc region (trastuzumab-IgGA). ...
... The fab domains of the anti-HER-2 mAb trastuzumab were fused to the IgGA Fc region (trastuzumab-IgGA). Human macrophages showed more effective phagocytosis of MDA-MB-453 breast cancer cells (expressing HER-2) in the presence of trastuzumab-IgGA (0.05-5 µg/ml) compared to trastuzumab-IgG [83]. Similarly, enhanced phagocytosis of SK-BR-3 HER-2 + and MDA-MB-453 HER-2 + cells was observed in the presence of anti-HER-2-IgG1/ IgA2 chimeric antibodies compared to IgG1 anti-HER-2 mAbs in vitro [84]. ...
Introduction:
Most bispecific antibody (BsAb) therapies focus on stimulating the adaptive immune system, in particular T cells, to promote tumor cell killing. Another method to promote tumor eradication is through the engagement of myeloid cells, including macrophages and neutrophils, which are abundantly present and possess intrinsic cytotoxic mechanisms for tumor cell killing, making them interesting effector cells to recruit for BsAb therapy.
Areas covered:
In this review, we describe the evolving knowledge of the role of macrophages and neutrophils in cancer in scientific literature. Moreover, we address the BsAbs that have been developed over the years to recruit these cell types as effector cells in immunotherapy of cancer. This includes the discussion of BsAbs that target Fc receptors (i.e. FcγR and FcαRI) to induce antibody-dependent cellular phagocytosis (ADCP) by macrophages or trogoptosis via neutrophils, as well as BsAbs that interfere with checkpoint inhibition, including the SIRPα-CD47 pathway.
Expert opinion:
Elucidating the complexity of macrophage and neutrophil heterogeneity in cancer may help to specifically enlist the cytotoxic ability of these cells through targeting Fc receptors and checkpoint pathways, which may further enhance anti-cancer immunity.
... 16 However, it has been shown that the addition of both IgG and IgA antibodies further enhances tumor cytotoxicity. 17 Here, we have developed a Fc-fusion peptide against PD-L1 consisting of a cross-hybrid Fc region containing constant regions of an IgG1 and an IgA1, termed IgGA, 18 connected to a PD-1 ectodomain, carrying mutations that increase its affinity towards PD-L1 via a glycine linker. However, carrying a competent/functional Fc region can be a double-edged sword since immune checkpoints are expressed ubiquitously and such antibodies are systemically administered, frequently causing irAEs. ...
... Polymorphonuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats as previously described. 18 The PBMC and PMN layers were subsequently removed between serum and Ficoll or in the Histopaque layer, respectively, and cultured in 1xRPMI (Roswell Park Memorial Institute, Gibco, Cat# 21875034)- ...
Background
Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms.
Methods
The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids.
Results
Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations.
Conclusion
Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.
... However, like FcαRI Fab-based BsAbs, IgA antibodies have a short half-life, which may limit clinical use (47). To improve the half-life of IgA, antibody engineering has resulted in various fusion antibodies where (parts of) the heavy chain of IgA2 was linked to IgG1 (59)(60)(61). For instance, an IgGA α-HER-2/neu "cross-type" antibody induced both neutrophil-and macrophage-mediated killing of tumor cells in vitro (60). ...
... To improve the half-life of IgA, antibody engineering has resulted in various fusion antibodies where (parts of) the heavy chain of IgA2 was linked to IgG1 (59)(60)(61). For instance, an IgGA α-HER-2/neu "cross-type" antibody induced both neutrophil-and macrophage-mediated killing of tumor cells in vitro (60). Similarly, a tandem IgG/IgA2 α-HER-2/ neu antibody had improved half-life and induced tumor cell killing in vitro (59). ...
Most clinically used anti-cancer monoclonal antibodies (mAbs) are of the IgG isotype, which can eliminate tumor cells through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI), but are unable to activate NK cells and have poorer half-life. Here, we combine the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-epidermal growth factor receptor (EGFR) TrisomAb, but were less efficient in mediating killing in the presence of IgG α-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide high potential alternatives for cancer patients that do not benefit from current IgG mAb therapy.
... (E) Albumin-binding domain (ABD) in yellow fused to IgA2m (1) [27]. Illustration of hybrid IgG-IgA molecules: (F) Tandem IgG1-IgA [52], (G) strand-exchange engineered domain (SEED)-bodies [53], and (H) Cross-isotype IgGA [54]. (I) FcαRI-engaging (red/orange) × CD20 (pink/purple) bispecific antibody [55]. ...
... Another IgG-IgA hybrid molecule combining critical domains from IgA engrafted into IgG has been described. An in-depth Alanine-scanning analysis determined which residues were essential for FcαRI binding [54]. Out of several variants, a single IgG-IgA hybrid antibody displayed proper assembly and demonstrated both binding to FcαRI and certain FcγRs. ...
In the past three decades, a great interest has arisen in the use of immunoglobulins as therapeutic agents. In particular, since the approval of the first monoclonal antibody Rituximab for B cell malignancies, the progress in the antibody-related therapeutic agents has been incremental. Therapeutic antibodies can be applied in a variety of diseases, ranging from cancer to autoimmunity and allergy. All current therapeutic monoclonal antibodies used in the clinic are of the IgG isotype. IgG antibodies can induce the killing of cancer cells by growth inhibition, apoptosis induction, complement activation (CDC) or antibody-dependent cellular cytotoxicity (ADCC) by NK cells, antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages, or trogoptosis by granulocytes. To enhance these effector mechanisms of IgG, protein and glyco-engineering has been successfully applied. As an alternative to IgG, antibodies of the IgA isotype have been shown to be very effective in tumor eradication. Using the IgA-specific receptor FcαRI expressed on myeloid cells, IgA antibodies show superior tumor-killing compared to IgG when granulocytes are employed. However, reasons why IgA has not been introduced in the clinic yet can be found in the intrinsic properties of IgA posing several technical limitations: (1) IgA is challenging to produce and purify, (2) IgA shows a very heterogeneous glycosylation profile, and (3) IgA has a relatively short serum half-life. Next to the technical challenges, pre-clinical evaluation of IgA efficacy in vivo is not straightforward as mice do not naturally express the FcαR. Here, we provide a concise overview of the latest insights in these engineering strategies overcoming technical limitations of IgA as a therapeutic antibody: developability, heterogeneity, and short half-life. In addition, alternative approaches using IgA/IgG hybrid and FcαR-engagers and the impact of engineering on the clinical application of IgA will be discussed.
... Strikingly, these formulations are able to neutralize bacterial toxins in vitro or prevent gastrointestinal infections in animals [148][149][150]. Since cost hurdles might impede mAb IgA drug commercialization, future work should focus on simple and low-cost manufacturing [126] (dark blue round arrow), albuminbinding domain [127] or IgG Fc domains [128,129] have been generated. Removal of N-linked glycosylation sites (N166G, N337T, green arrows) decreases IgA clearance by the asialoglycoprotein receptor and thereby increasing serum half-life [130]. ...
... processes. Finally, in order to capitalize on the advantages of IgA and IgG isotypes, the engineering of either crossisotype molecules [129] or bispecific antibodies [151,152] might be considered for therapeutic applications. ...
Background:
Extensive efforts have been made in optimizing monoclonal immunoglobulin (Ig)G antibodies for use in clinical practice. Accumulating evidence suggests that IgA or anti-FcαRI could also represent an exciting avenue toward novel therapeutic strategies.
Summary:
Here, we underline that IgA is more effective in recruiting neutrophils for tumor cell killing and is potently active against several pathogens, including rotavirus, poliovirus, influenza virus, and SARS-CoV-2. IgA could also be used to modulate excessive immune responses in inflammatory diseases. Furthermore, secretory IgA is emerging as a major regulator of gut microbiota, which impacts intestinal homeostasis and global health as well. As such, IgA could be used to promote a healthy microbiota in a therapeutic setting. Key messages: IgA combines multifaceted functions that can be desirable for immunotherapy.
... 110 For instance, the breadth of antibody effector mechanisms has been increased using an IgG1 framework containing IgA1 sequences in the lower Fc domain. 111 The IgG1/A1 hybrid elicited strong ADCC, ADCP, and CDC by binding to FcaRI in addition to FcgRs and C1q. Conversely, the clinically approved eculizumab combined the IgG2 C H 1 and hinge with the IgG4 C H 2 and C H 3 to abrogate both FcgR and C1q binding and serve as an immunologically silent cross-subtype. ...
... Although both of these ARCs target CD20 for treatment of non-Hodgkin's lymphoma, it is thought that b emitters may be suitable for elimination of high-volume solid tumors because of the large crossfire effect that damages cells distant to the radionuclide. 212,213 Conversely, a emitters (e.g., 211 At, 213 Bi) and Auger emitters (e.g., 111 In, 125 I), which have a higher linear energy transfer but a lower range, may be more apt for treatment of small tumors or metastasized clusters of cells. For all types of emitters, another important consideration is the physical half-life of the radionuclide, which should ideally be similar to the biological half-life of the ARC. ...
Antibody-based proteins have become an important class of biologic therapeutics, due in large part to the stability, specificity, and adaptability of the antibody framework. Indeed, antibodies not only have the inherent ability to bind both antigens and endogenous immune receptors; they have also proven extremely amenable to protein engineering. Thus, several derivatives of the monoclonal antibody format, including bispecific antibodies, antibody-drug conjugates, and antibody fragments, have demonstrated efficacy for treating human disease, particularly in the fields of immunology and oncology. Reviewed here are considerations for the design of antibody-based therapeutics, including immunological context, therapeutic mechanisms, and engineering strategies. First, characteristics of antibodies are introduced, with emphasis on structural domains, functionally important receptors, isotypic and allotypic differences, and modifications such as glycosylation. Next, aspects of therapeutic antibody design are discussed, including identification of antigen-specific variable regions, choice of expression system, utility of multispecific formats, and design of antibody derivatives based on fragmentation, oligomerization, or conjugation to other functional moieties. Finally, strategies to enhance antibody function through protein engineering are reviewed while highlighting the impact of fundamental biophysical properties on protein developability.
... Calcein-loaded cancer cells were washed twice, resuspended in RPMI medium, and seeded into a 96-well plate at 10,000 cells/well with various concentrations of IgG variants. As previously described (40,48), PBMCs and PMNs were isolated from healthy anonymous donors using Histopaque (Sigma-Aldrich). CD14 + monocytes were isolated from PBMCs by magnetic bead separation (EasySep by STEMCELL Inc.) (40). ...
... gmMφ express hFcγRI at moderate, hFcγRIIa/b at high and hFcγRIIIa V158 at low levels, as determined by flow cytometry (Figure 3A) (30)(31)(32)35). Raji cells were labeled with fluorescent PKH67 and then opsonized with Fc-engineered Rituximab variants, followed by incubation with gmMφ, as previously reported (40,48). The extent of phagocytosis was evaluated using flow cytometry by determining the percentage of cells staining double positive for PKH67 and anti-CD14-APC or anti-CD11b-APC over the total number of PKH67 + cancer cells. ...
IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIaV158 and hFcγRIIIaF158, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIaV158. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIaV158 allotype with a somewhat lower KD than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIaV158 was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIaF158 variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFcγRIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFcγRs as tools for the detailed molecular understanding of hFcγR biology.
... For these reasons, there have been efforts to engineer novel mAbs containing the Fc regions of both IgG and IgA, with a view that the resulting molecule will harness the beneficial properties of both Ig classes. Kelton et al. grafted relevant regions of IgA into the Fc region of an anti-HER2 mAb to form a so-called "cross-isotype" IgGA mAb (167). The resulting IgGA mAbs were capable of binding to both FcɑRI and FcγR, and induced neutrophil ADCC and macrophage ADCP of HER2 + targets similar to IgA molecules, and to a greater extent than parental IgG mAb. ...
... This is likely related to the "accessory CDC" mechanism (166) mentioned above. Notably, the IgGA construct did not bind to FcγRIIIA or FcRn (167). As this would be predicted to negatively impact ADCC/ADCP and IgG recycling, respectively, the functionality of IgGA molecules in vivo would be interesting to assess. ...
Therapeutic monoclonal antibodies (mAbs) have become one of the fastest growing classes of drugs in recent years and are approved for the treatment of a wide range of indications, from cancer to autoimmune disease. Perhaps the best studied target is the pan B-cell marker CD20. Indeed, the first mAb to receive approval by the Food and Drug Administration for use in cancer treatment was the CD20-targeting mAb rituximab (Rituxan®). Since its approval for relapsed/refractory non-Hodgkin’s lymphoma in 1997, rituximab has been licensed for use in the treatment of numerous other B-cell malignancies, as well as autoimmune conditions, including rheumatoid arthritis. Despite having a significant impact on the treatment of these patients, the exact mechanisms of action of rituximab remain incompletely understood. Nevertheless, numerous second- and third-generation anti-CD20 mAbs have since been developed using various strategies to enhance specific effector functions thought to be key for efficacy. A plethora of knowledge has been gained during the development and testing of these mAbs, and this knowledge can now be applied to the design of novel mAbs directed to targets beyond CD20. As we enter the “post-rituximab” era, this review will focus on the lessons learned thus far through investigation of anti-CD20 mAb. Also discussed are current and future developments relating to enhanced effector function, such as the ability to form multimers on the target cell surface. These strategies have potential applications not only in oncology but also in the improved treatment of autoimmune disorders and infectious diseases. Finally, potential approaches to overcoming mechanisms of resistance to anti-CD20 therapy are discussed, chiefly involving the combination of anti-CD20 mAbs with various other agents to resensitize patients to treatment.
... Genes encoding 801-Fc and 802-Fc were cloned in-frame into the mammalian expression vector pcDNA3.4 with (or without) Fab of Rituximab or ofatumumab using Gibson Assembly cloning (NEB) 52 . Expression plasmids encoding histidine-tagged mouse FcγRI-IV (His-mFcγRI-IV) were constructed by cloning gene blocks of the respective genes synthesized by IDT into pcDNA3.4 ...
... Complement-dependent cytotoxicity (CDC) assays. CDC assays were performed as described previously 52 . In brief, serially diluted antibodies were incubated with 25% pooled human serum and cancer cells loaded with calcein AM (Life Technologies) at 37 °C for 1 h in 96-well plates. ...
Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.
... We designed a series of His-tagged antigens that linked ovine IgG2a Fc domains or ovine GM-CSF cytokine sequences to the C-terminus of SARS-CoV-2 RBD or full-length spike protein (Fig. 1a, b; Supplementary Table S1, Additional File 1). Due to incomplete information about functional ovine IgG2a hinge region sequences, we performed an alignment to the human IgG1 sequence and selected ovine residues that align to the "DKTH" motif (residues 221-224 by the Eu numbering system), which we have previously used to generate functional Fc domains that retain Fc receptor binding [32,33]. The unstructured hinge region of IgG2a Fc was hypothesized to act as a flexible linker sequence between the antigen and the Fc domain. ...
Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.
... For example, secretory VHH-IgA fusions were protective against ETEC infection unlike related VHH-IgG fusions [24]. Another engineered fusion antibody which has been explored is an IgG backbone with IgA Fc sequences inserted-this demonstrated binding to both the IgA receptor (FcαRI) and IgG receptors (FcγRI/FcγRIIa/ FcγRIIb), possessing both classes of effector function [25]. ...
The dawn of antibody therapy was heralded by the rise of IgG therapeutics. However, other antibody classes are at our disposal—one of the most exciting is IgA and is the most abundant antibody class within humans. Unlike IgG, it is uniquely specialized for mucosal applications due to its ability to form complex Secretory IgA (SIgA) molecules. Since the mucosa is constantly exposed to potential infectious agents, SIgA is pivotal to disease prevention as an important component of the mucosal barrier. Compared to IgG, SIgA has proven superior effectiveness in mucosal surfaces, such as the airway epithelium or the harsh gut environment. Despite this, hurdles associated with low yield and challenging purification have blocked SIgA therapeutic advancement. However, as a result of new antibody engineering strategies, we are approaching the next generation of (IgA-based) antibody therapies. Strategies include fine-tuning SIgA assembly, exploring different production platforms, genetic engineering to improve purification, and glycoengineering of different components. Due to its stability in mucosal environments, SIgA therapeutics would revolutionize passive mucosal immunotherapy—an avenue still underexploited by current therapeutics. This chapter will focus on the current perspectives of SIgA engineering and explore different approaches to unlocking the full therapeutic potential of SIgAs.
... This BsAb was able to recruit neutrophils (via the FcαRI arm) and NK cells and macrophages (via the IgG1 Fc tail) as effector cells in a B16F10 melanoma mouse model [115]. Similar effects can be achieved by genetically fusing IgG and IgA Fc regions or by creating a chimeric variant of the two regions (IgGA) [116][117][118]. ...
Monoclonal antibody (mAb) therapy has successfully been introduced as treatment of several lymphomas and leukemias. However, solid tumors reduce the efficacy of mAb therapy because of an immune-suppressive tumor micro-environment (TME), which hampers activation of effector immune cells. Pro-inflammatory cytokine therapy may counteract immune suppression in the TME and increase mAb efficacy, but untargeted pro-inflammatory cytokine therapy is limited by severe off-target toxicity and a short half-life of cytokines. Antibody-cytokine fusion proteins, also referred to as immunocytokines, provide a solution to either issue, as the antibody both acts as local delivery platform and increases half-life. The antibody can furthermore bridge local cytotoxic immune cells, like macrophages and natural killer cells with tumor cells, which can be eliminated after effector cells are activated via the cytokine. Currently, a variety of different antibody formats as well as a handful of cytokine payloads are used to generate immunocytokines. However, many potential formats and payloads are still left unexplored. In this review, we describe current antibody formats and cytokine moieties that are used for the development of immunocytokines, and highlight several immunocytokines in (pre-)clinical studies. Furthermore, potential future routes of development are proposed.
... Increasing the binding affinity against an antigen by 'affinity maturation' in vivo or in vitro has been the most common approach to improve the antibody efficacy [67,68]. However, in recent years, various methods have been used to improve the efficacy of antibodies [69][70][71][72]. ...
Monoclonal antibodies (mAb) have been used as therapeutic agents for various diseases, and immunoglobulin G (IgG) is mainly used among antibody isotypes due to its structural and functional properties. So far, regardless of the purpose of the therapeutic antibody, wildtype IgG has been mainly used, but recently, the engineered antibodies with various strategies according to the role of the therapeutic antibody have been used to maximize the therapeutic efficacy. In this review paper, first, the overall structural features and functional characteristics of antibody IgG, second, the old and new techniques for antibody discovery, and finally, several antibody engineering strategies for maximizing therapeutic efficacy according to the role of a therapeutic antibody will be introduced.
... 250,251 Engineering of a cross-isotype antibody that contains merged Fc domain residues from IgG1 and IgA, combining the effector functions of both isotypes, mediated increased complementdependent cytotoxicity and Her2 + tumor cell killing by both neutrophils and macrophages. 252 Furthermore, it was shown that anti-epidermal growth factor receptor-2 IgG1/IgA2 cross-isotype antibodies induced increased recruitment of neutrophils, resulting in enhanced ADCC of human breast cancer cells by neutrophils. 253 Pharmacokinetics of IgG and IgA cross-isotype antibodies were similar to the parental IgG antibodies and may therefore augment IgG-based antibody therapies. ...
Mucosal surfaces constitute the frontiers of the body and are the biggest barriers of our body for the outside world. Immunoglobulin A (IgA) is the most abundant antibody class present at these sites. It passively contributes to mucosal homeostasis via immune exclusion maintaining a tight balance between tolerating commensals and providing protection against pathogens. Once pathogens have succeeded in invading the epithelial barriers, IgA has an active role in host-pathogen defense by activating myeloid cells through divers receptors, including its Fc receptor, FcαRI (CD89). To evade elimination, several pathogens secrete proteins that interfere with either IgA neutralization or FcαRI-mediated immune responses, emphasizing the importance of IgA-FcαRI interactions in preventing infection. Depending on the IgA form, either anti- or pro-inflammatory responses can be induced. Moreover, the presence of excessive IgA immune complexes can result in continuous FcαRI-mediated activation of myeloid cells, potentially leading to severe tissue damage. On the one hand, enhancing pathogen-specific mucosal and systemic IgA by vaccination may increase protective immunity against infectious diseases. On the other hand, interfering with the IgA-FcαRI axis by monovalent targeting or blocking FcαRI may resolve IgA-induced inflammation and tissue damage. This review describes the multifaceted role of FcαRI as immune regulator between anti- and pro-inflammatory responses of IgA, and addresses potential novel therapeutic strategies that target FcαRI in disease.
... Similar to IgG1/IgG3 chimeras, attempts to construct IgG1/IgA chimeras have been made with the intention of combining the advantages of the two different isotypes [69,70]. Binding to FcRn, FcγRs and C1q with the IgG1 part as well as to FcαR with the IgA part, has been successfully achieved [70]. ...
The clinical application of monoclonal antibodies (mAbs) has revolutionized the field of cancer therapy, as it has enabled the successful treatment of previously untreatable types of cancer. Different mechanisms play a role in the anti‐tumour effect of mAbs. These include blocking of tumour‐specific growth factor receptors or of immune modulatory molecules as well as complement and cell‐mediated tumour cell lysis. Thus, for many mAbs, Fc‐mediated effector functions critically contribute to the efficacy of treatment. As immunoglobulin (Ig) isotypes differ in their ability to bind to Fc receptors on immune cells as well as in their ability to activate complement, they differ in the immune responses they activate. Therefore, the choice of antibody isotype for therapeutic mAbs is dictated by its intended mechanism of action. Considering that clinical efficacy of many mAbs is currently achieved only in subsets of patients, optimal isotype selection and Fc optimization during antibody development may represent an important step towards improved patient outcome. Here, we discuss the current knowledge of the therapeutic effector functions of different isotypes and Fc‐engineering strategies to improve mAbs application.
... 49,50 Combinations of IgG and IgA mAbs can enhance tumor killing and work on "cross-type antibodies" such as IgGA and tandem antibodies combines the best of both IgG (complement binding) and IgA (cytotoxicity/phagocytosis) antitumor effects. 51 BACTERIA Invasive bacterial infections can cause severe disease such as sepsis and meningitis. Early research from the 1970s through to the early 2000s highlights the role of serum IgA in the second (serum) and potentially third line (liver) of defense from bacteria that enter the blood and tissues. ...
Immunoglobulin A (IgA) is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralisation) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated, in contrast serum IgA is comparatively understudied. IgA binding to Fc alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy.
... Similarly, isolated Fc candidates were cloned in-frame into the mammalian expression vector pcDNA3.4-IgH-Trastuzumumab using Gibson Assembly cloning (NEB) 66 and were produced in Expi293 cells (Invitrogen) grown for 5 days at 37°C with 8% CO 2. All IgG variants proteins purified by Protein A high capacity agarose resin (Thermo Scientific) according to the manufacturer's instructions. Purity of antibody variants was confirmed by SDS-PAGE and size exclusion chromatography and was over 95%. ...
The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability. Lee et al. report an engineered IgG1 Fc domain that behaves like an hFcRn binding pH toggle switch. The authors show that this new half-life extension Fc domain confers improved pharmacokinetics in new humanized knock-in mouse strains that recapitulate the key processes for antibody persistence in circulation.
... [4][5][6] The cytotoxic activity of IgA could be further increased via dual engagement of both FcγR and FcαRI by IgG/A fusion or hybrid molecules. 7,8 For infectious disease, IgA multivalent target engagement enabled superior antigen binding and neutralization in influenza infection models. 9 Additionally, human IgA dimer (dIgA) could be effectively delivered to the kidney lumen in a polycystic kidney disease mouse model via binding to the polymeric immunoglobulin receptor (pIgR), whereas IgG molecules could not. ...
IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance that is thought to be mediated by glycan receptor scavenging of recombinantly produced IgA monomer bearing incompletely sialylated N-linked glycans. Here, we present a comprehensive biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA: removal of all N-linked glycosylation sites creating an aglycosylated polymeric IgA and engineering in FcRn binding with the generation of a polymeric IgG-IgA Fc fusion. While previous reports and the results presented in this study indicate that glycan-mediated clearance plays a major role for monomeric IgA, systemic clearance of polymeric IgA in mice is predominantly controlled by mechanisms other than glycan receptor clearance, such as pIgR-mediated transcytosis. The developed IgA platform now provides the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure.
... Importantly, IgA can facilitate antibodydependent cellular phagocytosis and antibody-dependent cellular cytotoxicity. 32 Due to its size, mIgA should have greater diffusion than pIgA, sIgA and IgM. Together with the FcαRI/CD89-mediated anti-inflammatory effects, 28 mIgA could protect host tissues from an excessive injury. ...
Recurrent and persistent airway infections remain prevalent in patients with primary immunodeficiency (PID), despite restoration of serum immunoglobulin levels by intravenous or subcutaneous plasma-derived IgG. We investigated the effectiveness of different human Ig isotype preparations to protect mice against influenza when delivered directly to the respiratory mucosa. Four polyvalent Ig preparations from pooled plasma were compared: IgG, monomeric IgA (mIgA), polymeric IgA-containing IgM (IgAM) and IgAM associated with the secretory component (SIgAM). To evaluate these preparations, a transgenic mouse expressing human FcαRI/CD89 within the myeloid lineage was created. CD89 was expressed on all myeloid cells in the lung and blood except eosinophils, reflecting human CD89 expression. Intranasal administration of IgA-containing preparations was less effective than IgG in reducing pulmonary viral titres after infection of mice with A/California/7/09 (Cal7) or the antigenically distant A/Puerto Rico/8/34 (PR8) viruses. However, IgA reduced weight loss and inflammatory mediator expression. Both IgG and IgA protected mice from a lethal dose of PR8 virus and for mIgA, this effect was partially CD89 dependent. Our data support the beneficial effect of topically applied Ig purified from pooled human plasma for controlling circulating and non-circulating influenza virus infections. This may be important for reducing morbidity in PID patients.
... This modified IgA showed improved half-life, and was equally effective as IgG1 anti-EGFR antibodies in reducing tumor outgrowth. Thus, IgA anti-tumor mAbs have prominent therapeutic efficacy in vivo, especially after modifications have been made to improve half-life.Additionally, it was demonstrated that a "cross-isotype" antibody (referred to as IgGA), in which properties of IgG and IgA1 had been combined, bound to FcγRI, FcγRIIa and FcαRI with similar affinities compared to IgG and IgA1, respectively54 . IgG anti-HER-2 antibodies were unable to activate neutrophils, but IgGA anti-HER-2 antibodies potently induced killing of SKBR3 breast cancer cells by neutrophils. ...
Neutrophils represent the most abundant population of circulating cytotoxic effector cells. Moreover, their number can be easily increased by treatment with granulocyte‐colony stimulating factor or granulocyte macrophage‐colony stimulating factor, without the need for ex vivo expansion. Because neutrophils express Fc receptors they have the potential to act as effector cells during monoclonal antibody therapy of cancer. Additionally, since neutrophils play a role in the regulation of adaptive immune responses, exploiting neutrophils in mAb therapy may result in long term anti‐tumor immunity. There is limited evidence that neutrophils play a prominent role in current immunoglobulin G‐based immunotherapy. However, as IgA induces neutrophil recruitment, novel therapeutic strategies that aim to target the IgA Fc receptor FcαRI may fully unleash the potential of enlisting neutrophils as cytotoxic effector cells in antibody therapy of cancer.
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... IgA (IgA1 and IgA2) is the major immunoglobulin isotype in adaptive mucosal immunity [12][13][14][15] and is responsible for several disease pathologies such as IgA nephropathy when they polymerize or self-aggregate [16,17]. Recently, a chimeric IgG-A antibody (with engineered CHγ2-CHα3 Fc region [18]) showed greater killing of Her2+ cancer cells by higher levels of complement-dependent cytotoxicity and activations of both neutrophils and macrophages. Although the chimeric IgG-A utilized only the CHα3 domain, this example clearly showed that the Fc of antibodies could be engineered towards various effector effects. ...
Therapeutic antibodies have shifted the paradigm of disease treatments from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. Recent findings showed that the antibody constant region can govern localization effects that are useful in reducing side effects due to systemic circulation by the commonly used IgG isotypes. Given their localized mucosal effects, IgA antibodies are increasingly promising therapeutic biologics. While the antibody Fc effector cell activity has been a focus point, recent research showed that the Fc could also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To investigate this, we analysed the IgA antibody constant region and its distal effects on the antigen binding regions using recombinant Pertuzumab IgA1 and IgA2 variants. We found that mutations in the C-region reduced Her2 binding experimentally, and computational structural analysis showed that allosteric communications were highly dependent on the antibody hinge, providing strong evidence that we should consider antibodies as whole proteins rather than a sum of functional regions.
... These so-called cross-isotypes demonstrated interesting profiles of effector functions. For example, Kelton and colleagues [65] reported the engineering of an IgGA 'cross-isotype' antibody which combines selected effector functions of both IgG and IgA ( fig. 3B). ...
In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.
... To combine the best of both worlds, 'cross-isotype' IgGA antibodies were developed. These engineered antibodies elicited effector functions of both IgA and IgG, inducing complement-dependent cytotoxicity and tumor killing by both neutrophils and macrophages [58]. In a similar fashion, using tandem IgG1/IgA2 Fc format antibodies increased neutrophil activation and recruitment compared to IgA2, while prolonging serum perseverance [59]. ...
Background:
Neutrophils participate in the first line of defense by executing several killing mechanisms, including phagocytosis, degranulation and the release of neutrophil extracellular traps. Additionally, they can orchestrate the adaptive immune system by secreting cytokines and chemokines. Opsonization with antibodies aids in the recognition of pathogens, via binding to Fc receptors on the neutrophil surface. Immunoglobulin A (IgA) is the most abundant antibody at mucosal sites and has multiple functions in homeostasis and immunity. Neutrophils and IgA can interact via the IgA Fc receptor Fc?RI (CD89), leading to pro- or anti-inflammatory responses.
Aims:
The aim of this review is to give a concise overview of the interplay between IgA, Fc?RI and neutrophils and to explore potential therapies for autoimmune diseases and cancer.
Results:
Crosslinking of FcαRI by IgA-immune complexes yields potent neutrophil activation and pro-inflammatory effector functions, including the recruitment of neutrophils. This can lead to neutrophil accumulation and tissue destruction during IgA-autoantibody mediated diseases. Conversely, for cancer treatment, the myriad of powerful neutrophil effector functions after targeting FcαRI may contribute to effective immunotherapy.
Conclusion:
By interfering with or actively promoting the interaction between IgA and FcαRI, therapies for multiple maladies could be developed.
... The "IgGA" hybrid trastuzumab mediated an enhanced ADCC/ ADCP activity against Her2 overexpressing cells and destroyed up to 50% SkBr3 breast cancer cells (via ADCC) and MDA-MB-453 cells (via ADCP) (60). Similarly, "IgGA" hybrid rituximab lysed ~70% of the CD20 + calcein-AM-loaded Raji tumor cells when compared to the WT counterparts (60). ...
Today, monoclonal immunoglobulin gamma (IgG) antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs) is achieved through both its antigen-binding fragment (Fab) and crystallizable fragment (Fc). Fab can specifically recognize tumor-associated antigen (TAA) and thus modulate TAA-linked downstream signaling pathways that may lead to the inhibition of tumor growth, induction of tumor apoptosis, and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis. Moreover, Fc is the region interacting with the neonatal Fc receptor in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anticancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce the cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering-based mAbs under clinical trials.
... Although the engineering strategy described above resulted in altered glycosylation and enhanced pharmacokinetic properties of the IgA2.0 antibody, additional improvements appear reasonable. For example, C-terminal fusion of human albumin or FcRn-binding motifs to IgA antibodies, or the construction of IgG/A hybrid antibodies (46). Also, further glyco-engineering strategies or the combination of these novel approaches may further enhance the serum half-life and therapeutic efficacy of IgA antibodies (47). ...
... Binding to albumin extended the half-life of the IgA antibodies and also slightly improved their anti-tumor activity (226). Finally, an IgGA antibody has been developed that has the capacity to bind to both FccRs and FcaRI (227). This so-called tandem IgG1/IgA2 antibody with a trastuzumab variable domain had a comparable half-life in vivo in mice as regular IgG antibodies and showed enhanced ADCC by human neutrophils in vitro (228). ...
Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients.
There have been major advances in the immunotherapy of cancer in recent years, including the development of T cell engagers - antibodies engineered to redirect T cells to recognize and kill cancer cells - for the treatment of haematological malignancies. However, the field still faces several challenges to develop agents that are consistently effective in a majority of patients and cancer types, such as optimizing drug dose, overcoming treatment resistance and improving efficacy in solid tumours. A new generation of T cell-targeted molecules was developed to tackle these issues that are potentially more effective and safer. In addition, agents designed to engage the antitumour activities of other immune cells, including natural killer cells and myeloid cells, are showing promise and have the potential to treat a broader range of cancers.
Inflammatory lung diseases represent a persistent burden for patients and the global healthcare system. The combination of high morbidity, (partially) high mortality and limited innovations in the last decades, have resulted in a great demand for new therapeutics. Are therapeutic IgA antibodies possibly a new hope in the treatment of inflammatory lung diseases? Current research increasingly unravels the elementary functions of IgA as protector against infections and as modulator of overwhelming inflammation. With a focus on IgA, this review describes the pathological alterations in mucosal immunity and how they contribute to chronic inflammation in the most common inflammatory lung diseases. The current knowledge of IgA functions in the circulation, and particularly in the respiratory mucosa, are summarized. The interplay between neutrophils and IgA seems to be key in control of inflammation. In addition, the hurdles and benefits of therapeutic IgA antibodies, as well as the currently known clinically used IgA preparations are described. The data highlighted here, together with upcoming research strategies aiming at circumventing the current pitfalls in IgA research may pave the way for this promising antibody class in the application of inflammatory lung diseases.
Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation.
Interactions between the crystallizable fragment (Fc) domain of antibodies and a plethora of cellular Fc receptors (FcRs) or soluble proteins form a critical link between humoral and innate immunity. In particular, the immunoglobulin G Fc domain is critical for the clearance of target cells by processes that include ( a) cytotoxicity, phagocytosis, or complement lysis; ( b) modulation of inflammation; ( c) antigen presentation; ( d) antibody-mediated receptor clustering; and ( e) cytokine release. More than 30 Fc-engineered antibodies aimed primarily at tailoring these effects for optimal therapeutic outcomes are in clinical evaluation or have already been approved. Nonetheless, our understanding of how FcR engagement impacts various immune cell phenotypes is still largely incomplete. Recent insights into FcR biology coupled with advances in Fc:FcR structural analysis, Fc engineering, and mouse models that recapitulate human biology are helping to fill in existing knowledge gaps. These advances will provide a blueprint on how to fine-tune the Fc domain to achieve optimal therapeutic efficacy.
Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 24 is June 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
According to "The Proteome Code" concept introduced by J. Biro and our early development of affinity peptide calculation method it was studied the possibility of high affinity peptide chromatographic gels development for IgG1-4 separation from the donor plasma. Given the next step of virus inactivation of IgG directly in the chromatographic column, the affinity gel had bind IgG at several spatially spaced points in order to limit the degree of freedom of the protein for retention IgG at high buffer flow rate or elevated buffer temperatures without denaturation. In addition, the possibility of creating highly specific affinity sense-antisense peptides against Rubella virus in order to increase the titer of aRIgG in plasma or even its isolation in highly purified form was studied. Based on previous experiments, an affinity multi-peptide chromatographic gel with the following properties was developed: the DBC with enough residence time 10 min was around 50-54 mg × mL-1 of total 98.0% purity of IgG with natural proportion of the 1-4 subclasses, any other immunoglobulins were not found. The virus inactivation/elimination on this gel directly in chromatographic column shown a highly effective virus elimination (log10>9) for both nonenveloped and lipid enveloped viruses. Using RV sequence from UniProt_KB and dates from more than 20 literature sources on the virus proteins interaction, affinity peptides were calculated against virus proteins C and E1,2. Then these peptides were modified to reach more affinity enhancement and affinity-peptide chromatographic gel was synthetized. By this gel from total mass IgG1-4 contained 6644 IU anti-Rubella IgG with specificity 6.64 IU × mg-1 were isolated 5382 IU aRIgG (> 80%) with a specificity of 791 IU × mg-1.
Early humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are dominated by IgM and IgA antibodies, which greatly contribute to virus neutralization at mucosal sites. Given the essential roles of IgM and IgA in the control and elimination of SARS-CoV-2 infection, the mucosal immunity could be exploited for therapeutic and prophylactic purposes. However, almost all neutralizing antibodies that are authorized for emergency use and under clinical development are IgG antibodies, and no vaccine has been developed to boost mucosal immunity for SARS-CoV-2 infection. In addition to IgM and IgA, bispecific antibodies (bsAbs) combine specificities of two antibodies in one molecule, representing an important alternative to monoclonal antibody cocktails. Here, we summarize the latest advances in studies on IgM, IgA and bsAbs against SARS-CoV-2. The current challenges and future directions in vaccine design and antibody-based therapeutics are also discussed.
Antibody-based immunotherapy is a promising strategy in cancer treatment. Antibodies can directly inhibit tumor growth, induce complement-dependent cytotoxicity and induce Fc receptor-mediated elimination of tumor cells by macrophages and natural killer cells. Until now, however, neutrophils have been largely overlooked as potential effector cells, even though they are the most abundant type of immune cells in the circulation. Neutrophils display heterogeneity, especially in the context of cancer. Therefore, their role in cancer is debated. Nevertheless, neutrophils possess natural anti-tumor properties and appropriate stimulation, i.e. specific targeting via antibody therapy, induces potent tumor cell killing, especially via targeting of the immunoglobulin A Fc receptor (FcαRI, CD89). In this review we address the mechanisms of tumor cell killing by neutrophils and the role of neutrophils in induction of anti-tumor immunity. Moreover, possibilities for therapeutic targeting are discussed.
Antibodies have unique specificity, tolerability, and long half‐life, which make them ideal therapeutic agents. Over the last 35 years, ever‐increasing understanding of the multiple immunological roles that antibodies play has led to creative and diverse targeting modalities for the treatment of human diseases. Approved antibody therapies are now available for cancers, autoimmune disorders, and infectious diseases in formats ranging from small antibody subunits to patient‐derived T cells armed with antibody‐based targeting molecules. Physiological understanding of specific disease states is required to generate effective antibody‐based therapies, and this must be combined with biophysical considerations for the development and manufacture of these complex proteins. Here, we review the latest progress in the field, focusing on antibody‐based therapy formats, discovery methods, functional optimization, and production considerations.
The humanization of antibodies for therapeutics is a critical process that can determine the success of antibody drug development. However, the science underpinning this process remains elusive with different laboratories having very different methods. Well-funded laboratories can afford automated high throughput screening methods to derive their best binder regardless of many other parameters, yet this often involves a very expensive initial set of equipment affordable only to a few. Often within these high-throughput processes, only standard key parameters such as production, binding, and aggregation can and are analysed. Given the lack of suitable animal models, it is only at clinical trials can immunogenicity and allergy adverse effects be detected through anti-human antibodies as per FDA guidelines. While some occurrences that slip through can be mitigated by additional desensitization protocols, such adverse reactions to grafted humanized antibodies can be prevented at the humanization step. Considerations such as better antibody localization, avoidance of unspecific interactions to superantigens, and the tailoring of antibody dependent triggering of immune responses, the antibody persistence on cells, can all be considered through a holistic sagacious approach, allowing for better outcomes for therapy and even for research and diagnostic purposes.
The humanization of antibodies for therapeutics is a critical process that can determine the success of antibody drug development. However, the science underpinning this process remains elusive with different laboratories having very different methods. Well-funded laboratories can afford automated high throughput screening methods to derive their best binder regardless of many other parameters, yet this often involves a very expensive initial set of equipment affordable only to a few. Often within these high-throughput processes, only standard key parameters such as production, binding, and aggregation can and are analysed. Given the lack of suitable animal models, it is only at clinical trials can immunogenicity and allergy adverse effects be detected through anti-human antibodies as per FDA guidelines,. While some occurrences that slip through can be mitigated by additional desensitization protocols, such adverse reactions to grafted humanized antibodies can be prevented at the humanization step. Considerations such as better antibody localization, avoidance of unspecific interactions to superantigens, and the tailoring of antibody dependent triggering of immune responses, the antibody persistence on cells, can all be considered through a holistic sagacious approach, allowing for better outcomes for therapy and even for research and diagnostic purposes.
Antibody‐dependent complement activity is associated with autoimmune morbidity, but also anti‐tumor efficacy. In infectious disease, both recombinant monoclonal antibodies and polyclonal antibodies generated in natural adaptive responses can mediate complement activity to protective, therapeutic, or disease‐enhancing effect. Recent advances have contributed to the structural resolution of molecular complexes involved in antibody‐mediated complement activation, defining the avid nature of participating interactions, and pointing to how antibody isotype, subclass, hinge flexibility, glycosylation state, amino acid sequence, and the contextual nature of the cognate antigen/epitope are all factors that can determine complement activity through impact on antibody multimerization and subsequent recruitment of C1q. Beyond the efficiency of activation, complement activation products interact with various cell types that mediate immune adherence, trafficking, immune education, and innate functions. Similarly, depending on the anatomical location and extent of activation, complement can support homeostatic restoration or be leveraged by pathogens or neoplasms to enhance infection or promote tumorigenic microenvironments, respectively. Advances in means to suppress complement activation by intravenous immunoglobulin (IVIG), IVIG mimetics, and complement‐intervening antibodies represent proven and promising exploratory therapeutic strategies, while antibody engineering has likewise offered frameworks to enhance, eliminate, or isolate complement activation to interrogate in vivo mechanisms of action. Such strategies promise to support the optimization of antibody‐based drugs that are able to tackle emerging and difficult‐to‐treat diseases by improving our understanding of the synergistic and antagonistic relationships between antibody mechanisms mediated by Fc receptors, direct binding, and the products of complement activation.
Therapeutic monoclonal antibodies (mAbs), directed towards either tumor antigens or inhibitory checkpoints on immune cells, are effective in cancer therapy. Increasing evidence suggests that the therapeutic efficacy of these tumor antigen-targeting mAbs is mediated - at least partially - by myeloid effector cells, which are controlled by the innate immune checkpoint interaction between CD47 and SIRPα. We and others have previously demonstrated that inhibiting CD47-SIRPα interactions can substantially potentiate antibody-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC) of tumor cells by IgG antibodies both in vivo and in vitro. IgA antibodies are superior in killing cancer cells by neutrophils compared to IgG antibodies with the same variable regions, but the impact of CD47-SIRPα on IgA-mediated killing has not been investigated. Here, we show that checkpoint inhibition of CD47-SIRPα interactions further enhances destruction of IgA antibody-opsonized cancer cells by human neutrophils. This was shown for multiple tumor types and IgA antibodies against different antigens, i.e. HER2/neu and EGFR. Consequently, combining IgA antibodies against HER2/neu or EGFR with SIRPα inhibition proved to be effective in eradicating cancer cells in vivo. In a syngeneic in vivo model, the eradication of cancer cells was predominantly mediated by granulocytes, which were actively recruited to the tumor site by SIRPα blockade. We conclude that IgA-mediated tumor cell destruction can be further enhanced by CD47-SIRPα checkpoint inhibition. These findings provide a basis for targeting CD47-SIRPα interactions in combination with IgA therapeutic antibodies to improve their potential clinical efficacy in tumor patients.
Therapeutic antibodies are effective for tumor immunotherapy and exhibit prominent clinical effects. All approved antibody therapeutics utilize IgG as the molecular format. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a key mechanism for tumor cell killing by antibodies. For IgG antibodies, ADCC depends on FcγR-expressing cells, such as natural killer (NK) cells. However, in patients with a high tumor burden, antibody therapeutics may lose efficacy owing to exhaustion of FcγR-expressing effector cells as well as the inhibitory effects of certain FcγRs on effector cells. To achieve more potent effector functions, we engineered an anti-CD20 antibody to contain both IgG Fc and IgA Fc domains. These engineered antibodies interacted with both IgG and IgA Fc receptors (FcγR and FcαR) and recruited a broader range of effector cells, including monocytes, macrophages, neutrophils, and NK cells, thereby enhancing antibody-dependent cellular phagocytosis. Using transgenic mice expressing the FcαRI (CD89) in macrophages, we demonstrated that recombinant antibodies bearing the chimeric IgG and IgA Fc exhibited potent in vivo antitumor activity. Additionally, in a short-term peritoneal model using CD20-transfected LLC target cells, the in vivo cytotoxic activity of hybrid recombinant antibodies was mediated by macrophages with significant reduction in the absence of FcαRI. Our findings supported targeting of FcαRI on monocytes and macrophages for improved tumor immunotherapy.
Non-human primate (NHP) studies are often an essential component of antibody development efforts prior to human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity towards human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared to human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models prior to human clinical studies when either efficacy or toxicity may be associated with FcγR engagement.
The Fc region of the IgG antibody recruits immune leukocytes or serum complement molecules, which in turn triggers the clearance of defective cells such as tumor cells or infected cells. In addition, the Fc region is crucial for the prolonged serum persistence of circulating IgG antibodies through an intracellular trafficking and recycling mechanism. Recently, the utility of antibody Fc has been further expanded to include a new class of antigen-binding scaffolds. This review presents the recent progress in the field of antibody Fc engineering and highlights new biomolecular, cellular, and evolutionary approaches to overcome the limitations of conventional monoclonal therapeutic antibodies by engineering the antibody Fc domain.
Variation in assay sensitivity was studied in more than 90 laboratories that assayed 4 formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/neu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories. Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB11 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods. While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays,for HER-2/neu, regardless of the antibody used is studied in more than 90 laboratories that assayed 4 formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/heu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories. Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB1 1 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods. While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays,for HER-2/neu, regardless of the antibody used.
Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.
Currently all approved anti-cancer therapeutic monoclonal antibodies (mAbs) are of the IgG isotype, which rely on Fcgamma receptors (FcγRs) to recruit cellular effector functions. In vitro studies showed that targeting of FcαRI (CD89) by bispecific antibodies (bsAbs) or recombinant IgA resulted in more effective elimination of tumour cells by myeloid effector cells than targeting of FcγR. Here we studied the in vivo anti-tumour activity of IgA EGFR antibodies generated using the variable sequences of the chimeric EGFR antibody cetuximab. Using FcαRI transgenic mice, we demonstrated significant in vivo anti-tumour activity of IgA2 EGFR against A431 cells in peritoneal and lung xenograft models, as well as against B16F10-EGFR cells in a lung metastasis model in immunocompetent mice. IgA2 EGFR was more effective than cetuximab in a short-term syngeneic peritoneal model using EGFR-transfected Ba/F3 target cells. The in vivo cytotoxic activity of IgA2 EGFR was mediated by macrophages and was significantly decreased in the absence of FcαRI. These results support the potential of targeting FcαRI for effective antibody therapy of cancer.
The study reveals that IgA antibodies directed against EGFR and engaging Fcalpha receptor (FcαRI) on effector cells, have in vivo anti-cancer activity. These data support the development of novel immunotherapeutic strategies based on targeting FcαRI.
Treatment with monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma. Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression. We demonstrate, however, that the activity of anti-CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions. Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.
Monoclonal antibodies of the immunoglobulin G (IgG) isotype have become a well-established therapeutic tool for the targeting of malignant cells in tumor patients. Despite tremendous success in the treatment of lymphoma and breast cancer, it has also become clear that we may not be able to further improve antibody therapy of cancer by simply generating more tumor-specific antibodies with a higher affinity. Instead, the work of many groups in the past years suggests that optimizing the recruitment of effector functions provided by the adaptive and innate immune systems via engineering of the IgG constant domain may hold great promise to achieve enhanced therapeutic activities. A major goal in cancer therapy would be to initiate adaptive immune responses to the patient's tumor that would result in long-term protection against recurrence. The use of immunostimulatory antibodies shows great promise in stimulating adaptive immune responses. Surprisingly, recent studies also implicate an important role for the antibody constant domain in the activity of these molecules in vivo, opening up new possibilities to further improve the activity of immunomodulatory antibodies by Fc engineering.
Therapeutic properties of antibodies strongly depend on the composition of their glycans. Most of the currently approved antibodies are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans, but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs. The recent approval in Japan of mogamulizumab (POTELIGEO (®) ), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies. Mogamulizumab is a humanized mAb derived from Kyowa Hakko Kirin's POTELLIGENT (®) technology, which produces antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The approval was granted April 30, 2012 by the Japanese Ministry of Health, Labour and Welfare for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.
Background:
While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1.
Design and methods:
In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells.
Results:
We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils.
Conclusions:
We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.
Background: IgA constitutes a promising antibody isotype, which requires optimization before immunotherapeutic application.
Results: P221R-mutated and wild type IgA2m(1) antibodies were similarly effective in killing tumor cells and in recruiting myeloid effector cells.
Conclusion: Improved IgA antibodies constitute promising next generation antibodies for tumor therapy.
Significance: These studies support the clinical development of therapeutic IgA antibodies.
The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.
Neutrophils have long been viewed as the final effector cells of an acute inflammatory response, with a primary role in the clearance of extracellular pathogens. However, more recent evidence has extended the functions of these cells. The newly discovered repertoire of effector molecules in the neutrophil armamentarium includes a broad array of cytokines, extracellular traps and effector molecules of the humoral arm of the innate immune system. In addition, neutrophils are involved in the activation, regulation and effector functions of innate and adaptive immune cells. Accordingly, neutrophils have a crucial role in the pathogenesis of a broad range of diseases, including infections caused by intracellular pathogens, autoimmunity, chronic inflammation and cancer.
Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.
Polymorphonuclear neutrophils (PMN) are the most abundant circulating immune cells and represent the first line of immune defense against infection. This review of the biomedical literature of the last 40 years shows that they also have a powerful antitumoral effect under certain circumstances. Typically, the microenvironment surrounding a solid tumor possesses many of the characteristics of chronic inflammation, a condition considered very favorable for tumor growth and spread. However, there are many circumstances that shift the chronic inflammatory state toward an acute inflammatory response around a tumor. This shift seems to convert PMN into very efficient anticancer effector cells. Clinical reports of unexpected antitumoral effects linked to the prolonged use of granulocyte colony-stimulating factor, which stimulates an intense and sustained neutrophilia, suggest that an easy way to fight solid tumors would be to encourage the development of intense peritumoral PMN infiltrates. Specifically designed clinical trials are urgently needed to evaluate the safety and efficacy of such drug-induced neutrophilia in patients with solid tumors. This antitumoral role of neutrophils may provide new avenues for the clinical treatment of cancer.
Although trastuzumab (Herceptin) is an important advance in the treatment of breast cancer, a significant proportion of patients do not respond to trastuzumab either alone or in combination with chemotherapy. In this study, we observe that epidermal growth factor receptor (EGFR) and HER3 expression is substantially increased after long-term trastuzumab exposure of HER2-positive breast carcinoma-derived cell lines that show primary resistance to trastuzumab. Furthermore, long-term trastuzumab exposure of trastuzumab-resistant cell lines induces de novo sensitivity to the EGFR-targeted agents gefitinib or cetuximab in two of three cell lines accompanied by increased EGFR expression. Together, these results indicate that primary trastuzumab resistance is not synonymous with lack of responsiveness to trastuzumab and, importantly, suggest that trastuzumab priming may sensitize trastuzumab-resistant tumors to other HER family-directed therapeutics.
Distinct genes encode 6 human receptors for IgG (hFcgammaRs), 3 of which have 2 or 3 polymorphic variants. The specificity and affinity of individual hFcgammaRs for the 4 human IgG subclasses is unknown. This information is critical for antibody-based immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3, and IgG4 to FcgammaRI; FcgammaRIIA, IIB, and IIC; FcgammaRIIIA and IIIB; and all known polymorphic variants. Wild-type and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found that (1) IgG1 and IgG3 bind to all hFcgammaRs; (2) IgG2 bind not only to FcgammaRIIA(H131), but also, with a lower affinity, to FcgammaRIIA(R131) and FcgammaRIIIA(V158); (3) IgG4 bind to FcgammaRI, FcgammaRIIA, IIB and IIC and FcgammaRIIIA(V158); and (4) the inhibitory receptor FcgammaRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other hFcgammaRs. We also identified parameters that determine the specificity and affinity of hFcgammaRs for IgG subclasses. These results document how hFcgammaR specificity and affinity may account for the biological activities of antibodies. They therefore highlight the role of specific hFcgammaRs in the therapeutic and pathogenic effects of antibodies in disease.
Rituximab, a genetically engineered chimeric monoclonal antibody specifically binding to CD20, was the first antibody approved by the U.S. Food and Drug Administration for the treatment of cancer. Rituximab significantly improves treatment outcome in relapsed or refractory, low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL). However, there are also some challenges for us to overcome: why approximately 50% of patients are unresponsive to rituximab in spite of the expression of CD20, and why some responsive patients develop resistance to further treatment. Although the antitumor mechanisms of rituximab are not completely understood, several distinct antitumor activities of rituximab have been suspected, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), apoptosis, and direct growth arrest. To counteract resistance to rituximab therapy, several strategies have been developed to: (a) augment the CDC effect by increasing CD20 expression, heteroconjugating rituximab to cobra venom factor and C3b, and inhibiting membrane complement regulatory protein, especially CD59, function; (b) enhance the ADCC effect through some immunomodulatory cytokines and CR3-binding beta-glucan; and (c) reduce the apoptotic threshold or induce apoptotic signaling on the tumor. Extensive studies indicate that rituximab combined with these approaches is more effective than a single rituximab approach. Herein, the mechanism of action of and resistance to rituximab therapy in B-cell NHL, in particular, the involvement of the complement system, are extensively reviewed.
Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.
The human serum immunoglobulins IgG and IgA1 are produced in bone marrow and both interact with specific cellular receptors
that mediate biological events. In contrast to IgA1, the glycosylation of IgG has been well characterized, and its interaction
with various Fc receptors (FcγRs) has been well studied. In this paper, we have analyzed the glycosylation of IgA1 and IgA1
Fab and Fc as well as three recombinant IgA1 molecules, including twoN-glycosylation mutants. Amino acid sequencing data of the IgA1 Fc O-glycosylated hinge region indicated thatO-glycans are located at Thr228, Ser230, and Ser232, while O-glycan sites at Thr225 and Thr236 are partially occupied. Over 90% of the N-glycans in IgA1 were sialylated, in contrast to IgG, where <10% contain sialic acid. This paper contains the first report
of Fab glycosylation in IgA1, and (in contrast to IgG Fab, which contains only N-linked glycans) both N- and O-linked oligosaccharides were identified. Analysis of the N-glycans attached to recombinant IgA1 indicated that the Cα2 N-glycosylation site contained mostly biantennary glycans, while the tailpiece site, absent in IgG, contained mostly triantennary
structures. Further analysis of these data suggested that processing at one FcN-glycosylation site affects the other. Neutrophil FcαR binding studies, using recombinant IgA1, indicated that neither the
tailpiece region nor the N-glycans in the Cα2 domain contribute to IgA1-neutrophil FcαR binding. This contrasts with IgG, where removal of the Fc N-glycans reduces binding to the FcγR. The primary sequence and disulfide bond pattern of IgA1, together with the crystal structures
of IgG1 Fc and mouse IgA Fab and the glycan sequencing data, were used to generate a molecular model of IgA1. As a consequence
of both the primary sequence and S–S bond pattern, the N-glycans in IgA1 Fc are not confined within the inter-α-chain space. The accessibility of the Cα2N-glycans provides an explanation for the increased sialylation and galactosylation of IgA1 Fc over that of IgG FcN-glycans, which are confined in the space between the two Cγ2 domains. This also suggests why in contrast to IgG Fc, the IgA1N-glycans are not undergalactosylated in rheumatoid arthritis.
Immunoglobulin A (IgA) is by far the most abundant immunoglobulin in humans.1 It is also the most heterogeneous. Serum IgA is mainly monomeric. It is produced by B lymphocytes in the bone marrow and in some lymphoid organs. Humans produce as much serum IgA as they do IgG. Most of the IgA in mammals is, however, found in mucosal secretions. Secretory IgA is dimeric or polymeric IgA associated with J chain and with secretory component, a part of the receptor involved in the secretion process.
Recent research2 has suggested that secretory IgA in the gut comes from two sources. Approximately 75% is from B2 lymphocytes in organised germinal centres of mucosal lymphoid tissues such as Peyer's patches. This IgA production is T lymphocyte dependent. A second source, possibly contributing around 25% of the secretory IgA, is produced by B1 lymphocytes that develop in the peritoneal cavity and are distributed diffusely in the intestinal lamina propria. This IgA may represent a primitive T lymphocyte independent source of IgA recognising commensal bacteria. Given the abundance and complexity of IgA, it is difficult to envisage that it does not perform important functions in health and disease.
Identification and characterisation of a leucocyte Fc receptor for IgA (FcαR, CD89) present on neutrophils, eosinophils, and monocytes demonstrated an active role for IgA in …
Variation in assay sensitivity was studied in more than 90 laboratories that assayed 4formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/neu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories. Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB11 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods. While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays for HER-2/neu, regardless of the antibody used.
Antitumor Abs are promising therapeutics for cancer. Currently, most Ab-based therapies focus on IgG Ab, which interact with IgG FcR (FcgammaR) on effector cells. In this study, we examined human and mouse neutrophil-mediated tumor cell lysis via targeting the IgA FcR, FcalphaRI (CD89), in more detail. FcalphaRI was the most effective FcR in triggering tumor cell killing, and initiated enhanced migration of neutrophils into tumor colonies. Importantly, immature neutrophils that are mobilized from the bone marrow upon G-CSF treatment efficiently triggered tumor cell lysis via FcalphaRI, but proved incapable of initiating tumor cell killing via FcgammaR. This may provide a rationale for the disappointing results observed in some earlier clinical trials in which patients were treated with G-CSF and antitumor Ab-targeting FcgammaR.
The vast surfaces of the gastrointestinal, respiratory, and genitourinary tracts represent major sites of potential attack by invading micro-organisms. Immunoglobulin A (IgA), as the principal antibody class in the secretions that bathe these mucosal surfaces, acts as an important first line of defence. IgA, also an important serum immunoglobulin, mediates a variety of protective functions through interaction with specific receptors and immune mediators. The importance of such protection is underlined by the fact that certain pathogens have evolved mechanisms to compromise IgA-mediated defence, providing an opportunity for more effective invasion. IgA function may also be perturbed in certain disease states, some of which are characterized by deposition of IgA in specific tissues. This review details current understanding of the roles played by IgA in both health and disease.
IgA is the most abundantly produced Ab isotype in humans, but its potential as immunotherapeutic reagent has hardly been explored. In this study, we describe anti-tumor mechanisms of mouse/human chimeric IgA Abs against the epidermal growth factor receptor (EGF-R). EGF-R Abs of IgG isotype are currently approved for the treatment of colon or head and neck cancers. As expected, the human IgG1, IgA(1), and IgA(2) variants of the 225 Ab demonstrated similar binding to EGF-R. Furthermore, IgA Abs were as effective as IgG in mediating direct effector mechanisms such as blockade of EGF binding, inhibition of EGF-R phosphorylation, and induction of growth inhibition. None of the three variants induced complement-mediated lysis. Human IgG1 effectively recruited MNC for ADCC, but activated PMN only weakly, whereas both IgA isoforms proved to be effective in triggering neutrophils. Interestingly, the IgA(2) isoform was significantly superior to its IgA(1) counterpart in recruiting PMN as effector cells. Because neutrophils constitute the most abundant effector cell population in human blood, this enhanced neutrophil recruitment lead to increased killing of EGF-R expressing tumor cells in whole blood assays. This killing was further enhanced when blood from G-CSF-primed donors was compared with healthy donor blood. Together, these data suggest EGF-R Abs of human IgA isotype to bear promise for therapeutic use in cancer.
Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA. Indeed, since the C alpha 2 N-linked glycosylation site lies near the Fab-distal pole of C alpha 2, the inability of a mutant IgA1 lacking C alpha 2 N-glycosylation to bind its cognate receptor suggested that the monocyte Fc alpha receptor (mFcalphaR) recognizes IgA at a hinge-distal site encompassing the boundary between the C alpha 2 and C alpha 3 domains. In this report, we utilize both domain-swapped IgA/IgG and point-mutated IgA chimeras to verify the above hypothesis. Using an antigen-specific rosetting assay and a mFc alpha R-expressing cell line, we show that (a) C alpha 2 and C alpha 3 together are necessary and sufficient for binding; (b) neither the IgA hinge nor the tailpiece is necessary for binding; (c) mutations away from the interdomain boundary do not affect binding; and (d) mutations located near the three-dimensional boundary between C alpha 2 and C alpha 3 completely disrupt binding. Taken together, these results localize the mFc alpha R recognition site on IgA to the boundary region between the second and third constant domains--a site analogous to that recognized by Staphylococcus aureus protein A on IgG. The use of this hinge-distal site is, to date, unique among Fc receptors of the Ig superfamily.
Immunoglobulin-α (IgA)-bound antigens induce immune effector
responses by activating the IgA-specific receptor FcαRI (CD89) on
immune cells. Here we present crystal structures of human FcαRI
alone and in a complex with the Fc region of IgA1 (Fcα).
FcαRI has two immunoglobulin-like domains that are oriented at
approximately right angles to each other. Fcα resembles the Fcs of
immunoglobulins IgG and IgE, but has differently located interchain
disulphide bonds and external rather than interdomain N-linked
carbohydrates. Unlike 1:1 FcγRIII:IgG and FcɛRI:IgE
complexes, two FcαRI molecules bind each Fcα dimer, one at
each Cα2-Cα3 junction. The FcαRI-binding site on IgA1
overlaps the reported polymeric immunoglobulin receptor (pIgR)-binding
site, which might explain why secretory IgA cannot initiate phagocytosis
or bind to FcαRI-expressing cells in the absence of an integrin
co-receptor.
Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (FcγRI) and the IgA FcR (FcαRI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-γ, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to FcαRI or FcγRI on MDM. Although FcαRI and FcγRI share a common signaling pathway contingent on association with the γ-chain (FcRγ subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by FcαRI and FcγRI; however, IFN-γ-treated MDM phagocytosed tumor cells only with the FcγRI-directed bispecific Abs. Similarly, IFN-γ-cultured MDM lysed tumor cells more efficiently via FcγRI then by FcαRI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via FcαRI than FcγRI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-γ-mediated enhancement of FcγRI expression and FcγRI γ-chain complexes, the regulation of FcγRI- or FcαRI-mediated activity occurred without significant change in either receptor expression or total complexes with γ subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.
The use of monoclonal antibodies (mAbs) has become a mainstream strategy for the treatment of cancer. After binding to the target antigen, they mediate a plethora of effector functions for elimination of tumour cells. These range from direct effects like induction of apoptosis or growth inhibition of tumour cells to activation of immune system components, including the initiation of complement-dependent lysis or recruitment of immune effector cells. Neutrophils are generally not considered as potential effector cells. However, the most abundant population of circulating white blood cells consists of neutrophils, which express Fc receptors for both immunoglobulin (Ig) G and IgA. In the presence of mAbs that are directed against tumour cells, they execute potent cytotoxic functions. Moreover, as they play a role in regulating adaptive immunity, neutrophil activation may lead to the generation of anti-tumour immune responses as well. This article addresses the possibility of exploiting neutrophils for mAb-based immunotherapy of cancer.
Glycans anchored to residue N297 of the antibody IgG Fc domain are critical in mediating binding toward FcγRs to direct both adaptive and innate immune responses. However, using a full length bacterial IgG display system, we have isolated aglycosylated Fc domains with mutations that confer up to a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous human inhibitory receptor, FcγRIIb. The mutant Fc domain (AglycoT-Fc1004) contained a total of 5 amino acid substitutions that conferred an activating to inhibitory ratio of 25 (A/I ratio; FcyRIIa-R131:FcγRIIb). Incorporation of this engineered Fc into trastuzumab, an anti-Her2 antibody, resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with both medium and low Her2-expressing cancer cells. A mathematical model has been developed to help explain how receptor affinity and the A/I ratio relate to improved antibody dependent cell-mediated phagocytosis. Our model provides guidelines for the future engineering of Fc domains with enhanced effector function.
The direct or indirect targeting of antibody Fc receptors (FcRs) presents unique opportunities and interesting challenges for the treatment of inflammatory diseases, cancer and infection. Biological responses induced via the Fc portions of antibodies are powerful, complex and unusual, and comprise both activating and inhibitory effects. These properties can be exploited in the engineering of therapeutic monoclonal antibodies to improve their activity in vivo. FcRs have also emerged as key participants in the pathogenesis of several important autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. Therapeutic approaches based on antagonizing FcR function with small molecules or biological drugs such as monoclonal antibodies and recombinant soluble FcR ectodomains have gained momentum. This Review addresses various strategies to manipulate FcR function to overcome immune complex-mediated inflammatory diseases, and considers approaches to improve antibody-based anticancer therapies.
Several novel technologies have evolved over the last decade for the modification of antibodies to enhance their inherent effector functions. All focus on the constant Fc domain and utilize either amino acid substitutions or glycoform perturbations to modulate their interaction with Fc receptors and the effector cells that bear them. We review these technologies with an emphasis on their validation with animal models and human clinical data.
Studies have begun to emerge showing critical roles for neutrophils in tumorigenesis. Neutrophils can have a significant impact on the tumor microenvironment via their production of cytokines and chemokines, which influence inflammatory cell recruitment and activation. Additionally, products secreted from neutrophils, such as reactive oxygen species and proteinases, have defined and specific roles in regulating tumor cell proliferation, angiogenesis, and metastasis. Although evidence suggests that neutrophils act in a decidedly protumor capacity in vivo, recent studies indicate that neutrophils may be manipulated to exhibit cytotoxicity against tumors. Herein, we explore the idea of targeting tumor-associated neutrophils as a means of antitumor therapy and the important ramifications such manipulation could pose to host tissues.
The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.
Monoclonal antibodies directed at tumor-associated antigens have been used clinically since 1981. In most of the completed clinical trials, the McAbs were of murine origin. Twenty-six of 184 patients (14%) receiving McAbs demonstrated a major clinical response, including 3 complete responders. Toxicities are primarily related to immune responses. The immune response triggering the toxic manifestations may be mediated by interactions between the administered McAb and the target tumor-associated antigens, or between the McAb and host antibodies made in response to its administration. Analysis of the mechanism of action, pharmacokinetics and toxicity of McAb administration should lead to improved design of clinical protocols, and provide a basis for the use of human McAbs and McAbs conjugated to radioisotopes, toxins or chemotherapeutic agents.
The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly.
Even though IgA is considered to play an important role in immunity, surprisingly little is known about the presence of IgA Fc receptor (Fc alpha R)-expressing effector cells in tissues. Difficulties in obtaining human tissue macrophages, led us to study peritoneal macrophages in a human Fc alpha RI transgenic (Tg) mouse model. Naive peritoneal macrophages did not express hFc alpha RI. Expression, however, could be induced by overnight culture, and was upregulated by GM-CSF. In addition, the receptor proved functional since macrophage-mediated phagocytosis and tumor cell kill were effectively triggered via hFc alpha RI. To assess necessity of the FcR gamma-chain signaling molecule for hFc alpha RI function in macrophages, Tg mice were crossed with mice deficient in FcR gamma-chain (gamma-/-). Tg, gamma-/- macrophages were unable to kill tumor cells. This, because Tg macrophages failed to express hFc alpha RI in the absence of FcR gamma-chain, and overnight culture did not overcome this lack of expression. Further studies with the transgenic mouse model presented in this study will help to define the precise conditions under which Fc alpha RI is expressed on macrophages. It will, furthermore, represent a useful tool to study Fc alpha RI function in immune defense.
IgA has traditionally been regarded a non-inflammatory antibody. This might indeed be true for secretory IgA (SIgA), which exerts its function at mucosal surfaces where commensal microorganisms and dietary antigens prevail. Serum IgA, however, potently triggers (pro)-inflammatory activity upon binding to the myeloid IgA receptor, FcalphaRI. Here, new insights in the roles of IgA and FcalphaRI are addressed and a model integrating the various functions of IgA in immunity is discussed.
Antibody-based therapy is a new treatment option for selected tumor patients. Today, human IgG(1) is the most widely used isotype, because it effectively activates human complement, recruits NK cells for ADCC, and has an extended plasma half life. In recent work, however, neutrophils--the most populous cytotoxic cells in humans--were more effectively recruited by human IgA than by IgG antibodies. IgA antibodies may have the additional advantages of forming natural dimers with improved signaling capacity on tumor cells, and being actively transported into mucosal secretions with the potential for improved targeting of certain carcinomas from the luminal surface.
Immunoglobulin (Ig) A and IgG are the principal immune effector molecules at mucosal surfaces and in blood, respectively. Mucosal IgA is polymeric and bound to secretory component, whereas serum IgG is monomeric. We have now produced IgA2/IgG1 hybrid antibodies that combine the properties of IgA and IgG. Antibodies with Calpha3 at the end of the IgG H chain resemble IgA and form polymers with J chain that bind the polymeric Ig receptor. Like IgG, the hybrid proteins activated complement and bound FcgammaRI and protein A. Though the hybrid proteins contained both Cgamma2 and Cgamma3, they have a short in vivo half-life. Surprisingly, this decreased half-life correlated with a higher avidity than that of IgG for murine FcRn. Interestingly, antibodies with Calpha1 replacing Cgamma1 were resistant to extremes of pH, suggesting that Calpha1 increases antibody stability. These results provide insights into engineering antibodies with novel combinations of effector functions.
FcalphaRI, the receptor specific for the Fc region of immunoglobulin A (IgA), is responsible for IgA-mediated phagocytosis, oxidative burst, and antibody-dependent cellular cytotoxicity. Using the techniques of analytical ultracentrifugation and equilibrium gel-filtration, we show that two FcalphaRI molecules bind to a single Fcalpha homodimer. Surface plasmon resonance studies confirm the 2:1 stoichiometry of binding, with equilibrium dissociation constants of 176 nM and 431 nM for the first and second binding events, respectively. The binding affinity decreases at acidic pH in a manner consistent with protonation of a single histidine residue in the binding site. A thermodynamic analysis indicates that the histidine residue does not participate in a salt-bridge in the complex; in fact, less than 10% of the free energy of binding was contributed by electrostatic interactions. The bivalent, pH-dependent interaction between FcalphaRI and IgA has important implications for cytokine-dependent phagocytosis of IgA and the FcalphaRI-mediated degradation or recycling of IgA.
Immunoglobulin-alpha (IgA)-bound antigens induce immune effector responses by activating the IgA-specific receptor FcalphaRI (CD89) on immune cells. Here we present crystal structures of human FcalphaRI alone and in a complex with the Fc region of IgA1 (Fcalpha). FcalphaRI has two immunoglobulin-like domains that are oriented at approximately right angles to each other. Fcalpha resembles the Fcs of immunoglobulins IgG and IgE, but has differently located interchain disulphide bonds and external rather than interdomain N-linked carbohydrates. Unlike 1:1 FcgammaRIII:IgG and Fc epsilon RI:IgE complexes, two FcalphaRI molecules bind each Fcalpha dimer, one at each Calpha2-Calpha3 junction. The FcalphaRI-binding site on IgA1 overlaps the reported polymeric immunoglobulin receptor (pIgR)-binding site, which might explain why secretory IgA cannot initiate phagocytosis or bind to FcalphaRI-expressing cells in the absence of an integrin co-receptor.
Neutrophils in the activation and regulation of innate and adaptive immunity
- A Mantovani
- M A Cassatella
- C Costantini
Mantovani, A., Cassatella, M.A., Costantini, C., and Jaillon, S. (2011).
Neutrophils in the activation and regulation of innate and adaptive immunity.
Nat. Rev. Immunol. 11, 519-531.
Neutrophils in the activation and regulation of innate and adaptive immunity
- Mantovani
The function of immunoglobulin A in immunity
- Woof