Fc-mediated Effector Functions Research Articles

Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin

Fusion of therapeutic proteins with the antibody Fc domain is a strategy widely applied to increase protein half-life in plasma. In our previous study, we generated a recombinant human lactoferrin (hLF)-immunoglobulin G1 Fc fusion protein (hLF-hinge-CH2-CH3) with improved stability, biological activity, and pharmacokinetics ( Shiga , Y. et al. Eur J Pharm Sci. , 2015 , 67 , 136 - -143 ). However, the Fc domain in fusion proteins can potentially induce antibody-dependent and complement-dependent cytotoxicity and serious side effects. To overcome these drawbacks, we engineered an hLF-Fc fusion protein (hLF-CH2-CH3) without the Fc hinge region which is essential for engaging Fc receptors on immune cells and inducing complement-mediated cell lysis. The hLF-CH2-CH3 protein was stably expressed in Chinese hamster ovary (CHO) DG44 cells and compared for in vitro activities, thermal stability, pharmacokinetics, and attenuation of Fc-mediated immune effector functions with the conventional hinge-containing Fc fusion protein. Both hLF-hinge-CH2-CH3 and hLF-CH2-CH3 exhibited iron-binding activity, superior uptake by Caco-2 cells, similar thermal stability, and longer plasma half-life compared to recombinant hLF. However, in contrast to conventional hLF-hinge-CH2-CH3, hinge-deficient hLF-CH2-CH3 did not elicit Fc-mediated effector response potentially damaging for the target cells. Our findings demonstrate that conjugation of hinge-deficient Fc to therapeutic proteins is a promising strategy for improving their pharmacokinetic properties without enhancing effector functions. Cell-expressed hinge-deficient hLF-CH2-CH3 is a potential drug candidate with improved plasma half-life for parenteral administration.

Fc functional antibodies in humans with severe H7N9 and seasonal influenza.

Both seasonal and novel avian influenza viruses can result in severe infections requiring hospitalization. Anti-influenza antibodies (Abs) with Fc-mediated effector functions, such as Ab-dependent cellular cytotoxicity (ADCC), are of growing interest in control of influenza but have not previously been studied during severe human infections. As such, the objective of this study was to examine Fc-mediated Ab functions in humans hospitalized with influenza infection. Serum Ab response was studied in subjects hospitalized with either pandemic H7N9 avian influenza virus in China (n = 18) or circulating seasonal influenza viruses in Melbourne, Australia (n = 16). Recombinant soluble Fc receptor dimer ELISAs, natural killer (NK) cell activation assays, and Ab-dependent killing assays with influenza-infected target cells were used to assess the Fc functionality of anti-influenza hemagglutinin (HA) Abs during severe human influenza infection. We found that the peak generation of Fc functional HA Abs preceded that of neutralizing Abs for both severe H7N9 and seasonal influenza infections. Subjects who succumbed to complications of H7N9 infection demonstrated reduced HA-specific Fc receptor-binding Abs (in magnitude and breadth) immediately prior to death compared with those who survived. Subjects who recovered from H7N9 and severe seasonal influenza infections demonstrated increased Fc receptor-binding Abs not only against the homologous infecting strain but against HAs from different influenza A subtypes. Collectively, survivors of severe influenza infection rapidly generate a functional Ab response capable of mediating ADCC against divergent influenza viruses. Broadly binding HA Abs with Fc-mediated functions may be a useful component of protective immunity to severe influenza infection. The National Health and Medical Research Council ([NHMRC] grants 1023294, 1041832, and 1071916), the Australian Department of Health, and the joint University of Melbourne/Fudan University International Research and Research Training Fund provided funding for this study.

Abstract 4604: MEDI1873, a GITR ligand fusion protein (GITRL FP), induces effector T-cell proliferation, modulates T-regulatory cell function and has the potential to combine with checkpoint inhibitors

Abstract Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. MEDI1873 is a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerisation domain and the human GITRL extracellular domain (ECD) that is currently being assessed in a Phase 1 clinical study (NCT02583165) in patients with solid tumors. MEDI1873 exhibits in vitro superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Using in vitro assay systems, MEDI1873 recapitulates aspects of GITR targeting previously described in mice, including modulation of regulatory T-cell (Treg) suppression and the ability to increase the CD8:CD4 T-cell ratio via antibody-dependent T-cell cytotoxicity. Pharmacodynamic assessment of an agonistic mouse GITRL FP (mGITRL FP) in the CT26 model of colorectal cancer demonstrated activation and proliferation of peripheral CD4+ and CD8+ T cells coincident with an increased depletion of intratumoral Tregs, likely through Fc mediated effector functions. Furthermore, CT26 tumor growth studies indicated the mGITRL FP could result in significant antitumor activity. These data provide evidence that MEDI1873 is a novel, potent GITR agonist with the potential to modulate T-cell responses and enhance anti-tumor immunity. Combinations of immunotherapies are generating exciting results in the clinic, therefore, we sought to assess the potential for GITRL FPs to combine with antibodies targeting either anti-PD-L1 (durvalumab) or anti-CTLA-4 (tremelimumab) using both in vitro and in vivo systems. In vitro studies where MEDI1873 was combined with either durvalumab or tremelimumab showed that both combinations have the potential to enhance interleukin-2 release in a superantigen-stimulation of human peripheral blood mononuclear cells (PBMCs) compared to checkpoint blockade alone. Further evidence to support the potential for combinatorial antitumor activity was generated in the CT26 model where either 0.2mg/kg mGITRL combined with 10mg/kg anti-mouse PD-L1 or 0.1mg/kg mGITRL combined with 5mg/kg anti-mouse CTLA-4 antibodies resulted in enhanced antitumor activity versus monotherapies alone. Overall, our data suggest that therapeutically targeting GITR with a multimeric fusion protein, GITRL FP, may provide increased agonistic potential versus an antibody, and have the ability to both activate effector T-cells and modulate Tregs through suppression and/or depletion. Finally, combination studies provide preclinical evidence to support the rationale for combination of MEDI1873 with anti-PD-L1 or anti-CTLA-4 antibodies further reinforcing the potential of targeting the GITR pathway as a therapeutic approach to treating patients with cancer. Citation Format: Michelle Morrow, Rebecca Leyland, James Hair, Ross Stewart, Natalie Tigue, Lisa Bamber, Samantha Ireland, Nicholas Holoweckyi, Michael Oberst, Amanda Watkins, Emily Offer, David Perez-Martinez, Ching Ching Leow, Lesley Young, Tristan Vaughan, Philip Mallinder, Robert Wilkinson. MEDI1873, a GITR ligand fusion protein (GITRL FP), induces effector T-cell proliferation, modulates T-regulatory cell function and has the potential to combine with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4604. doi:10.1158/1538-7445.AM2017-4604

Abstract 3653: Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model

Abstract Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in Phase 1 clinical trial (NCT02748837). ERY974 consists of a common light chain and two different heavy chains that respectively recognize glypican-3 (GPC3) and CD3. The Fc portion of ERY974 is modified to lose FcγR binding to prevent GPC3-independent Fc-mediated effector function. However, binding activity to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface, and induces TRAB-dependent cellular cytotoxicity mediated by the potent effector function of T cells. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, and esophageal cancer-derived xenograft tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. Cancer immunotherapy, as represented by immune checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4 antibodies, has recently been demonstrating remarkable clinical benefit in various tumor types. However, the number of patients who have survival benefit is limited, and combining cancer immunotherapy with other agents is required to improve the efficacy. Although ERY974 monotherapy is expected to show clinical activity based on the preclinical data, we examined whether further improvement of ERY974-induced efficacy is attained by combination with chemotherapy. Method & Results: We evaluated the combination effect of ERY974 with chemotherapy against xenograft tumors of MKN45 (gastric cancer) or NCI-H446 (lung cancer) either in a NOD-SCID mouse model injected with human T cells or in a humanized non-obese diabetic/shi-scid/IL-2Rγnull model in which differentiated human T cells are constitutively supplied. Although ERY974 monotherapy shows only minor antitumor effect against MKN45 and NCI-H446, combination therapy remarkably enhanced efficacy. In particular, ERY974 in combination with paclitaxel or cisplatin in NCI-H446 tumors caused a tumor disappearance without regrowth for a long period. Conclusion: These preclinical data suggest the possibility that the strategy of combining ERY974 with chemotherapy may succeed in increasing the clinical benefit. Now the combination effect is being further investigated to clarify the mechanism. Citation Format: Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Kayukawa, Hironori Mutoh, Yoko Miyazaki, Takahiro Ishiguro, Shohei Kishishita, Yoshiki Kawabe, Mika Endo. Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2017-3653

Abstract 3633: Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cells in vitro and in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors

Abstract Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors (NETs) and small cell lung cancer (SCLC). Treatment options for NETs include somatostatin analogs and radionuclides; however, such therapies suffer from short half-life, modest efficacy, and toxicities due to inhibition of other SSTRs. Reasoning that a targeted immunotherapy against SSTR2 would provide a new therapeutic modality for NETs, we designed XmAb18087, a humanized and affinity-optimized bispecific antibody that engages T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of SSTR2+ tumor cells. XmAb18087 possesses an Fc domain that maintains long half-life, yet lacks binding to Fcγ receptors to reduce Fc-mediated effector functions. XmAb18087 stimulated robust RTCC of SSTR2+ cell lines including medullary thyroid carcinoma (TT), lung carcinoma (A549), and CHO cells overexpressing SSTR2, with EC50s of ~1 to 100 ng/ml. XmAb18087 also upregulated CD69 and CD25 activation markers on CD4 and CD8 T cells. T cell responses were target-specific, because SSTR2‾ cell lines were not depleted, and because a control bispecific (anti-RSV x CD3) was ineffective. In addition, XmAb18087 (3 mg/kg weekly) reduced tumor burden of an established A549 xenograft in NSG mice engrafted with 107 human PBMC. We next assessed XmAb18087 activity in cynomolgus monkeys. As SSTR2 is not expressed in peripheral blood, target cell depletion cannot be monitored in vivo. However, CD3 bispecifics induce effects such as lymphocyte extravasation, cytokine induction, and T cell activation, which can serve as pharmacologic markers for activity in target organs. XmAb18087 dosed once at 30 or 60 μg/kg rapidly activated peripheral T cells, as quantified by CD69 and CD25 induction (peaking at ~8-12 hr). T cells rapidly extravasated from blood, with a nadir by 8 hr. Cytokines IL6 and TNF were rapidly induced, peaking at ~1-8 hr and returning to baseline by 48 hr. To explore repeat dosing, XmAb18087 was dosed at 1 or 10 μg/kg on Days 0 and 7 in a second study. The first dose of both 1 and 10 μg/kg again stimulated peripheral T cell activation, extravasation, and cytokine induction. These responses decreased markedly after the second dose, suggesting that SSTR2+ target cells remained depleted for at least 7 days. In summary, these results on human cells, in mice, and in monkeys support clinical assessment of XmAb18087 in SSTR2+ cancers including NETs and SCLC. In monkeys, T cell activation, extravasation, and cytokine induction were readily measured in peripheral blood and are indicative of T cell-mediated depletion of SSTR2+ target cells. Importantly, these responses may also serve as useful surrogate markers of NET depletion in clinical trials of XmAb18087. Citation Format: Sung-Hyung Lee, Seung Y. Chu, Rumana Rashid, Sheryl Phung, Irene W. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, Suzanne Schubbert, Connie Ardila, Christine Bonzon, Paul Foster, David E. Szymkowski, John R. Desjarlais. Anti-SSTR2 × anti-CD3 bispecific antibody induces potent killing of human tumor cells in vitro and in mice, and stimulates target-dependent T cell activation in monkeys: A potential immunotherapy for neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3633. doi:10.1158/1538-7445.AM2017-3633

Abstract 4714: Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer

Abstract MK-4280 is a humanized IgG4 monoclonal antibody (mAb) that binds to the immune checkpoint receptor Lymphocyte Activation Gene-3 (LAG-3) to block the interaction with its ligand, Major Histocompatibility Complex (MHC) Class II. LAG-3 is frequently co-expressed with other immune checkpoint receptors, most notably programmed cell death protein-1 (PD-1), on T cells with an exhausted phenotype. LAG-3 and PD-1 cooperate to regulate peripheral immune tolerance in healthy individuals, and, conversely, play critical roles in several diseases, including autoimmunity, graft rejection, viral infections, and cancer. Co-blockade of LAG-3 and PD-1 in immunocompetent mouse tumor models have demonstrated augmented anti-tumor activity over single agents. However, the molecular mechanisms behind these combination effects have not been fully investigated. Here, preclinical proof-of-biology studies are presented for co-targeting LAG-3 and PD-1 in cancer. c28G10-mG1-[D265A] (abbreviated 28G10-mG1) is a rat:mouse chimera that mimics MK-4280 by its ability to directly block the mouse LAG-3:MHC Class II interaction without initiating Fc-mediated effector functions. As a single agent, 28G10-mG1 demonstrated modest anti-tumor activity across several syngeneic mouse tumor models, despite evidence of systemic drug exposure and target engagement (as assessed by sLAG-3-mAb complex accumulation). The combination of 28G10-mG1 and the anti-mouse PD-1 blocking antibody mDX400 resulted in greater tumor growth inhibition and increased numbers of complete responses (CR) over mDX400 alone in the MBT-2 tumor model. Furthermore, animals that had achieved CR to combination therapy were subsequently protected from MBT-2 re-challenge, suggesting the establishment of immune memory. RT-qPCR analyses revealed up-regulation of immune-related genes, primarily at Day 4 in the blood and Day 8 in the tumor with mDX400, but not 28G10-mG1, treatment. However, when combined with mDX400, 28G10-mG1 further altered the expression of many immune-related genes that were perturbed by mDX400 single agent therapy. Genes unique to combination treatment were also observed. Significantly, immune pathway signatures associated with clinical efficacy to Keytruda were upregulated with combination therapy. Tumor transcriptome and network analysis by RNAseq revealed enrichment in several immune- and cytokine-related pathways with combination therapy compared to mDX400 single agent therapy. Taken together, these preclinical oncology studies support the concept of co-targeting LAG-3 to increase the therapeutic efficacy of PD-1 blockade. Clinical investigation of MK-4280 in combination with anti-PD-1 therapy (pembrolizumab/Keytruda) is ongoing. Citation Format: Brian B. Haines, Sarah Javaid, Long Cui, Heather Hirsch, Saso Cemerski, Terri McClanahan, Manjiri Sathe, Shuli Zhang, Michael Rosenzweig, Brian Long, Rene de Waal Malefyt. Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4714. doi:10.1158/1538-7445.AM2017-4714

Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells

Interleukin-2 (IL-2) is an established therapeutic agent used for cancer immunotherapy. Since treatment efficacy is mediated by CD8+ and NK cell activity at the tumour site, considerable efforts have focused on generating variants that expand these subsets systemically, as exemplified by IL-2/antibody complexes and ‘superkines’. Here we describe a novel determinant of antitumour activity using fusion proteins consisting of IL-2 and the antibody fragment crystallizable (Fc) region. Generation of long-lived IL-2-Fc variants in which CD25 binding is abolished through mutation effectively prevents unwanted activation of CD25+ regulatory T-cells (Tregs) and results in strong expansion of CD25− cytotoxic subsets. Surprisingly, however, such variants are less effective than wild-type IL-2-Fc in mediating tumour rejection. Instead, we report that efficacy is crucially dependent on depletion of Tregs through Fc-mediated immune effector functions. Our results underpin an unexpected mechanism of action and provide important guidance for the development of next generation IL-2 therapeutics.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Beyond Viral Neutralization.

It has been known for more than 30 years that HIV-1 infection drives a very potent B cell response resulting in the production of anti-HIV-1 antibodies targeting several viral proteins, particularly its envelope glycoproteins (Env). Env epitopes are exposed on the surfaces of viral particles and infected cells where they are targets of potentially protective antibodies. These antibodies can interdict infection by neutralization and there is strong evidence suggesting that Fc-mediated effector function can also contribute to protection. Current evidence suggests that Fc-mediated effector function plays a role in protection against infection by broadly neutralizing antibodies and it might be important for protection by non-neutralizing antibodies. Fc-mediated effector function includes diverse mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation, antibody-dependent cellular phagocytosis, antibody-dependent cell-mediated virus inhibition, antibody-mediated trancytosis inhibition, and antibody-mediated virus opsonization. All these functions could be beneficial in fighting viral infections, including HIV-1. In this perspective, we discuss the latest developments in ADCC research discussed at the HIVR4P satellite session on non-neutralizing antibodies, with emphasis on the mechanisms of ADCC resistance used by HIV-1, the structural basis of epitopes recognized by antibodies that mediate ADCC, natural killer-cell education and ADCC, and murine models to study ADCC against HIV-1.

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

ABSTRACTWe developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Short Communication: Small-Molecule CD4 Mimetics Sensitize HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity by Antibodies Elicited by Multiple Envelope Glycoprotein Immunogens in Nonhuman Primates.

Recent studies have linked antibody Fc-mediated effector functions with control of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus infections. Interestingly, the presence of antibodies with potent antibody-dependent cellular cytotoxicity (ADCC) activity in RV144 vaccine trial participants correlated inversely with HIV-1 acquisition risk. These antibodies were recently found to recognize epitopes on the HIV-1 envelope (Env) glycoprotein exposed upon Env-CD4 binding. Accordingly, small-molecule CD4 mimetics (CD4mc) that induce Env to sample the CD4-bound conformation were shown to sensitize HIV-1-infected cells to ADCC mediated by sera from HIV-1-infected individuals. However, it remains unknown whether antibodies elicited through immunization can also mediate CD4mc-induced ADCC. In this study, we tested the capacity of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from Env-vaccinated nonhuman primates using a FACS-based ADCC assay. In parallel, we evaluated the ability of CD4mc to sensitize HIV-1 viral particles to neutralization by sera from these immunized animals. We found that the vaccine-induced antibodies were able to mediate ADCC and viral neutralization in the presence, but not the absence, of CD4mc. Thus, CD4mc are capable of sensitizing HIV-1-infected cells to ADCC and infectious viral particles to neutralization by easy-to-elicit antibodies that are otherwise unable to mediate these activities.

The first 24 h: targeting the window of opportunity for antibody-mediated protection against HIV-1 transmission.

I will review evidence that antibodies protect against HIV-1 transmission in a short window of opportunity, involving neutralization, Fc-mediated effector function, or both. The last decade witnessed a dramatic progress in the understanding of antibody-mediated protection against HIV-1, including active and passive immunization studies in nonhuman primates; association between reduced infection risk and the specificities and function of antibodies in the RV144 clinical trial; identification of potent, broadly neutralizing antibodies; high-resolution structural studies of the HIV-1 envelope trimer; and an increasing appreciation that Fc-mediated effector function is critical to protection against transmission for neutralizing and nonneutralizing antibodies. Less information is known about how antibodies protect in situ, except that they must do in the first 24 h after exposure. New evidence suggests that antibodies protect in an acute innate immune environment involving the NXLRX1 inflammasome and transforming growth factor beta (TGF-β) that favors infection and rapid dissemination of CCR6RORγ Th17 cells. These recent findings set the stage for understanding how antibodies can prevent the transmission of HIV-1. In this context, antibodies must prevent infection in an innate immune environment that strongly favors transmission. This information is key for the development of a vaccine against HIV-1.

Abstract IA25: Synergistic innate and adaptive integrin-targeted immunotherapy

Abstract We have determined that antitumor antibodies, when combined with extended systemic exposure to IL-2, strongly polarize an inflammatory tumor microenvironment and generate a vaccinal effect that significantly potentiates adoptive T cell therapies (1). We have broadened this approach by delivering activating Fc domains to tumors via a small peptide that targets RGD-binding integrins (i.e. alpha V beta 3 and alpha 5 beta1). Combining integrin-targeted Fc-mediated effector functions with extended IL-2 exposure leads to ablation of established syngeneic tumors, with formation of protective immunity against subsequent tumor rechallenge. The therapeutic index of this protocol is excellent in wild-type mice, with mild transient reversible toxicities attributable primarily to IL-2 exposure. These results provide a strong case for using tumor-opsonizing agents such as monoclonal antibodies or Fc fusions to synergize with T-cell directed immunotherapies.Reference1. Zhu EF, Gai SA, Opel CF, Kwan BH, Surana R, Mihm MC, Kauke MJ, Moynihan KD, Angelini A, Williams RT, Stephan MT, Kim JS, Yaffe MB, Irvine DJ, Weiner LM, Dranoff G, Wittrup KD. Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2. Cancer Cell 2015;27(4):489-501. Citation Format: Karl Dane Wittrup. Synergistic innate and adaptive integrin-targeted immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA25.

Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases.

Anti‐angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor‐A (VEGF‐A) have revolutionized treatment of retinal vascular diseases including age‐related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti‐VEGF‐A monotherapy efficacy by targeting both VEGF‐A and angiopoietin‐2 (ANG‐2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF‐A. Simultaneous VEGF‐A and ANG‐2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain‐exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF‐A and ANG‐2. RG7716 showed greater efficacy than anti‐VEGF‐A alone in a non‐human primate laser‐induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc‐mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next‐generation therapy for neovascular indications of the eye.

Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

ABSTRACTBroadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc–FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

A Functional Role for Antibodies in Tuberculosis

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remainsunclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.

Optimal activation of Fc-mediated effector functions by influenza virus hemagglutinin antibodies requires two points of contact

Influenza virus strain-specific monoclonal antibodies (mAbs) provide protection independent of Fc gamma receptor (FcγR) engagement. In contrast, optimal in vivo protection achieved by broadly reactive mAbs requires Fc-FcγR engagement. Most strain-specific mAbs target the head domain of the viral hemagglutinin (HA), whereas broadly reactive mAbs typically recognize epitopes within the HA stalk. This observation has led to questions regarding the mechanism regulating the activation of Fc-dependent effector functions by broadly reactive antibodies. To dissect the molecular mechanism responsible for this dichotomy, we inserted the FLAG epitope into discrete locations on HAs. By characterizing the interactions of several FLAG-tagged HAs with a FLAG-specific antibody, we show that in addition to Fc-FcγR engagement mediated by the FLAG-specific antibody, a second intermolecular bridge between the receptor-binding region of the HA and sialic acid on effector cells is required for optimal activation. Inhibition of this second molecular bridge, through the use of an F(ab')2 or the mutation of the sialic acid-binding site, renders the Fc-FcγR interaction unable to optimally activate effector cells. Our findings indicate that broadly reactive mAbs require two molecular contacts to possibly stabilize the immunologic synapse and potently induce antibody-dependent cell-mediated antiviral responses: (i) the interaction between the Fc of a mAb bound to HA with the FcγR of the effector cell and (ii) the interaction between the HA and its sialic acid receptor on the effector cell. This concept might be broadly applicable for protective antibody responses to viral pathogens that have suitable receptors on effector cells.