An antidote approach to reduce risk and broaden utility of antibody-based therapeutics.

Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs.

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.

Quantifying the importance of pMHC valency, total pMHC dose and frequency on nanoparticle therapeutic efficacy.

Nanoparticles (NPs) coated with ß-cell-specific peptide major histocompatibility complex (pMHC) class I molecules can effectively restore normoglycemia in spontaneously diabetic nonobese diabetic mice. They do so by expanding pools of cognate memory autoreactive regulatory CD8+ T cells that arise from naive low-avidity T-cell precursors to therapeutic levels. Here we develop our previously constructed mathematical model to explore the effects of compound design parameters (NP dose and pMHC valency) on therapeutic efficacy with the underlying hypothesis that the functional correlates of the therapeutic response (expansion of autoregulatory T cells and deletion of autoantigen-loaded antigen-presenting cells by these T cells) are biphasic. We show, using bifurcation analysis, that the model exhibits a 'resonance'-like behavior for a given range of NP dose in which bistability between the healthy state (possessing zero level of effector T-cell population) and autoimmune state (possessing elevated level of the same population) disappears. A heterogeneous population of model mice subjected to several treatment protocols under these new conditions is conducted to quantify both the average percentage of autoregulatory T cells in responsive and nonresponsive model mice, and the average valency-dependent minimal optimal dose needed for effective therapy. Our results reveal that a moderate increase (≥1.6-fold) in the NP-dependent expansion rate of autoregulatory T-cell population leads to a significant increase in the efficacy and the area corresponding to the effective treatment regimen, provided that NP dose ≥8 µg. We expect the model developed here to generalize to other autoimmune diseases and serve as a computational tool to understand and optimize pMHC-NP-based therapies.

Neuroprotective Effects of IVIG against Alzheimer' s Disease via Regulation of Antigen Processing and Presentation by MHC Class I Molecules in 3xTg-AD Mice.

BACKGROUND: The results of clinical trials for Alzheimer's disease (AD) patients treated with Intravenous immunoglobulin (IVIG) revealed inconsistency in efficacy. OBJECTIVE: To explore the neuroprotective effects and possible mechanisms of different IVIG in 3xTg-AD mice. METHODS: 3-month-old 3xTg-AD mice were administered intraperitoneally with different IVIG (A/B/C) for 3 months and then the therapeutic effects were observed and tested at 9 months of age. The bioavailability of IVIG and Aß40/42 concentrations in parietotemporal cortex was measured by ELISA. Behavioral tests were performed to examine cognitive functions. Immunohistochemistry was utilized to examine the deposition of Aß, the phosphorylation of tau, the levels of GFAP and Iba-1 in the hippocampus. Proteomics, Luminex assay and parallel reaction monitoring were performed to identify and verify the proteins that showed a marked change in the hippocampus. RESULTS: IVIG-C was more effective than IVIG-A and IVIG-B in counteracting cognitive deficits, ameliorating Aß deposits and tau phosphorylation, attenuating the activation of microglia and astrocytes in the hippocampus and inhibiting the secretion of pro-inflammatory factors. IVIG-C affected innate immunity and suppressed the activation of antigen processing and presentation by MHC class I molecule (APP-MHC-I). CONCLUSION: The efficacy of different IVIG on AD was significantly different, and only IVIG-C has been confirmed to possess significant neuroprotective effects, which are related to the inhibition of APP-MHC-I. IVIG may be a potential therapeutic for AD but further research is needed to evaluate the functional of IVIG before clinical trials of AD treatment.

HLA-G in organ transplantation: towards clinical applications.

HLA-G plays a particular role during pregnancy in which its expression at the feto-maternal barrier participates into the tolerance of the allogenic foetus. HLA-G has also been demonstrated to be expressed in some transplanted patients, suggesting that it regulates the allogenic response. In vitro data indicate that HLA-G modulates NK cells, T cells, and DC maturation through its interactions with various inhibitory receptors. In this paper, we will review the data reporting the HLA-G involvement of HLA-G in human organ transplantation, then factors that can modulate HLA-G, and finally the use of HLA-G as a therapeutic tool in organ transplantation.

Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the main pathogens associated with lower respiratory tract infections in infants and young children worldwide. Exosomes secreted by antigen presenting cells (APCs) can elicit immune responses by carrying major histocompatibility complex (MHC) class I molecules complexed with antigenic peptides and other co-stimulating factors. Therefore, we developed novel immunomagnetic nanographene particles to sequentially isolate, surface engineer, and release intact dendritic cell (DC) exosomes for use as a potential vaccine platform against RSV. The H-2Db-restricted, immunodominant peptides from RSV (M187-195 and NS161-75) were introduced to MHC-I on DC-derived exosomes to express peptide/MHC-I (pMHC-I) complexes. A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses. Ex vivo assays demonstrated that engineered exosomes carrying RSV-specific peptides can elicit interferon-gamma (IFN-γ) production by virus-specific CD8+ T cells isolated from RSV-infected C57BL/6 mice. In vivo assays demonstrated that subcutaneous administration of both M187-195 and NS161-75 engineered exosomes to mice, with or without additional adjuvant, appeared safe and well tolerated, however, did not prime antigen-specific CD8+ T cell responses. Surface engineered exosomes are immunogenic and promising for further development as a vaccine platform.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.

Beyond the increasing complexity of the immunomodulatory HLA-G molecule.

Human leukocyte antigen G (HLA-G) is a nonclassic major histocompatibility complex (MHC) class I molecule that functions as an immunomodulatory molecule capable of protecting fetal tissues from the maternal immune system. The relevance of HLA-G in other contexts was investigated soon afterward. Numerous studies have sought (and some have shown) the relevance of HLA-G in pathologic conditions, such as transplantation, autoimmunity, and cancer and hematologic malignancies. One of the main goals of the current research on HLA-G is now to use it in the clinic, either for diagnosis or as a therapeutic tool/target. For this, precise knowledge on the nature and functions of HLA-G is critical. We highlight here what we consider are recent key basic findings on the immunomodulatory function of HLA-G. These strengthen the case for considering HLA-G as clinically relevant.

Global immunoglobulin supply: steaming towards the iceberg?

PURPOSE OF REVIEW: This review describes how plasma is sourced for fractionation into plasma-derived medicinal products (PDMPs), such as immunoglobulin (Ig) together with differences between plasma from whole blood (recovered plasma) and from plasmapheresis (source plasma) in terms of global plasma supply. Specific areas of growth in immunoglobulin use are identified alongside novel therapies, which may reduce demand for some immunoglobulin indications. RECENT FINDINGS: There has been a 6--8% annual growth in immunoglobulin use. Secondary immunodeficiency alongside improved recognition and diagnosis primary immunodeficiency disorders are drivers whereas the novel neonatal Fc receptor inhibitors (FcRni) may reduce demand for some immunomodulatory indications. SUMMARY: There is a significant geographical imbalance in global supply of plasma with 65% collected in the United States. This results in a dependency of other countries on United States supply and argues for both more plasma supply and greater regionally balanced plasma collection. In addition, progress towards a transparent, regulated and well tolerated framework for the coexistence of unpaid and compensated plasma donations is needed as unpaid donation will not be sufficient. These discussions should be informed by the needs of patients for this life-saving therapy, the care of donors and the safety of plasma and PDMPs.

Mechanisms of survival prolongation of murine cardiac allografts using the treatment of CTLA4-Ig and MR1.

BACKGROUND: [corrected] The present study was undertaken to determine the role of costimulatory blockade in a murine cardiac transplant model. MATERIALS AND METHODS: We blocked the CD28/B7 and CD154/CD40 costimulatory pathways by transient administration of CTLA4-Ig and MR1 antibody to study the effects on allograft survival time, deviation of Th1 and Th2 cytokine secretion, and other mechanisms related to prolonged survival. RESULTS: Costimulatory blockade prolonged the mean survival time (MST) of cardiac allografts to 43 days for the treated group vs 8 days for the untreated group (P < .01). The costimulatory blockade down-regulated the expression of 2 Th1 cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and 2 Th2 cytokines (IL-4 and IL-10), reduced the numbers of graft-infiltrating CD4+ and CD8+ lymphocytes, and inhibited the expression of both perforin/GrB and FasL in allografts. CONCLUSIONS: Combined administration of CTLA4-Ig/MR1 inhibited acute rejection reactions in murine cardiac allografts, prolonging the survival of cardiac grafts through several mechanisms, including inhibition of Th1 and Th2 cytokine expression, graft infiltration by CD4+ and CD8+ T lymphocytes, and reduced both perforin/GrB and Fas-FasL.

KIR-like activating natural killer cell receptors and their association with complicated malaria in north India.

Killer immunoglobulin-like receptors (KIRs) genomic regions have been suggested to influence malaria pathogenesis and infection susceptibility. KIRs are known as activating natural killer (NK) cell receptors, which upon binding to their corresponding human leukocyte antigen (HLA) ligands cause lysis of any infected cell. We have examined the potential association of KIR genes with complicated malaria (CM) among north Indians in this study and further evaluated the KIR receptor-HLA ligand association on the severity of the disease considering the uncomplicated malaria (UCM) subjects as control. Molecular profiling of KIR and HLA was carried out using the PCR-SSP method. Susceptible association was found for individuals possessing KIR2DS2 (OR=1.76, p-value=0.0390), KIR2DL1 (OR=2.87, p-value=0.0005) and KIR2DL3 (OR=2.74, p-value=0.0011) genes with CM. This was supported by the strong linkage disequilibrium observed for 2DS2-2DL2 (D́=0.87, r2=0.54) with CM. Whereas the receptor-ligand association has revealed risk association against KIR2DS2-HLAC1 (OR=2.08, p-value=0.0229), KIR2DL3-HLAC1 (OR=1.79, p-value=0.0301), and KIR2DL1-HLAC2 (OR=2.10, p-value=0.0175) combinations for complicated malaria. The frequency of different KIR genes are more or less similar to that observed in African population showing not much genetic diversity at KIR level in context to malarial infection. In conclusion, our data indicates KIR gene loci differentially influenced the malarial outcome in north Indians and in particular the KIR2DS2 gene appeared to be associated with disease severity.

Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells.

Manipulation of human natural killer (NK) cell repertoires promises more effective strategies for NK cell-based cancer immunotherapy. A subset of highly differentiated NK cells, termed adaptive NK cells, expands naturally in vivo in response to human cytomegalovirus (HCMV) infection, carries unique repertoires of inhibitory killer cell immunoglobulin-like receptors (KIR), and displays strong cytotoxicity against tumor cells. Here, we established a robust and scalable protocol for ex vivo generation and expansion of adaptive NK cells for cell therapy against pediatric acute lymphoblastic leukemia (ALL). Culture of polyclonal NK cells together with feeder cells expressing HLA-E, the ligand for the activating NKG2C receptor, led to selective expansion of adaptive NK cells with enhanced alloreactivity against HLA-mismatched targets. The ex vivo expanded adaptive NK cells gradually obtained a more differentiated phenotype and were specific and highly efficient killers of allogeneic pediatric T- and precursor B-cell acute lymphoblastic leukemia (ALL) blasts, previously shown to be refractory to killing by autologous NK cells and the NK-cell line NK92 currently in clinical testing. Selective expansion of NK cells that express one single inhibitory KIR for self-HLA class I would allow exploitation of the full potential of NK-cell alloreactivity in cancer immunotherapy. In summary, our data suggest that adaptive NK cells may hold utility for therapy of refractory ALL, either as a bridge to transplant or for patients that lack stem cell donors. Cancer Immunol Res; 5(8); 654-65. ©2017 AACR.

Engineered clearing agents for the selective depletion of antigen-specific antibodies.

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor.

The role of blood-brain barrier (BBB) transport in clearance of amyloid beta-peptide (Abeta) by Abeta immunotherapy is not fully understood. To address this issue, we studied the effects of peripherally and centrally administered Abeta-specific IgG on BBB influx of circulating Abeta and efflux of brain-derived Abeta in APPsw(+/-) mice, a model that develops Alzheimer's disease-like amyloid pathology, and wild-type mice. Our data show that anti-Abeta IgG blocks the BBB influx of circulating Abeta in APPsw(+/-) mice and penetrates into the brain to sequester brain Abeta. In young mice, Abeta-anti-Abeta complexes were cleared from brain to blood by transcytosis across the BBB via the neonatal Fc receptor (FcRn) and the low-density lipoprotein receptor-related protein (LRP), whereas in older mice, there was an age-dependent increase in FcRn-mediated IgG-assisted Abeta BBB efflux and a decrease in LRP-mediated clearance of Abeta-anti-Abeta complexes. Inhibition of the FcRn pathway in older APPsw(+/-) mice blocked clearance of endogenous Abeta40/42 by centrally administered Abeta immunotherapy. Moreover, deletion of the FcRn gene in wild-type mice inhibited clearance of endogenous mouse Abeta40/42 by systemically administered anti-Abeta. Our data suggest that the FcRn pathway at the BBB plays a crucial role in IgG-assisted Abeta removal from the aging brain.

RDP58 does not prevent graft-versus-host disease after dog leukocyte antigen-nonidentical canine hematopoietic cell transplantation.

Graft-versus-host disease (GVHD) remains a cause of substantial morbidity for patients undergoing allogeneic hematopoietic cell transplantation (HCT). The present study was undertaken to investigate the effectiveness of RDP58, a peptide derived from the human leukocyte antigen class I heavy chain, in preventing GVHD in the established dog leukocyte antigen (DLA)-nonidentical canine model. Dogs underwent HCT from unrelated DLA-nonidentical donors after conditioning with 920 cGy total body irradiation. Engraftment and achievement of full donor chimerism was seen in five of six dogs, whereas one dog showed rejection and died of marrow aplasia. All five dogs with engraftment developed acute GVHD and were euthanized at an average of 20.6 days after HCT. Compared with historical controls, the Suse of RDP58 neither prevented acute GVHD nor significantly prolonged survival of DLA-nonidentical HCT recipients.

A comprehensive review of the neonatal Fc receptor and its application in drug delivery.

Advances in the understanding of neonatal Fc receptor (FcRn) biology and function have demonstrated that this receptor, primarily identified for the transfer of passive immunity from mother infant, is involved in several biological and immunological processes. In fact, FcRn is responsible for the long half-life of IgG and albumin in the serum, by creating an intracellular protein reservoir, which is protected from lysosomal degradation and, importantly, trafficked across the cell. Such discovery has led researchers to hypothesize the role for this unique receptor in the controlled delivery of therapeutic agents. A great amount of FcRn-based strategies are already under extensive investigation, in which FcRn reveals to have profound impact on the biodistribution and half-life extension of therapeutic agents. This review summarizes the main findings on FcRn biology, function and distribution throughout different tissues, together with the main advances on the FcRn-based therapeutic opportunities and model systems, which indicate that this receptor is a potential target for therapeutic regimen modification.

The effectiveness of pretreatment with soluble or membrane-bound donor class I major histocompatibility complex antigens in the induction of unresponsiveness to a subsequent rat renal allograft.

We have examined the ability of two physical forms of RT1.A class I molecules to induce immunologic unresponsiveness to renal allografts in the rat. Both preparations of class I MHC antigen were derived from rat liver. Class I MHC antigen was presented either as purified membrane-bound molecules incorporated into protein micelles or as a water-soluble preparation containing soluble RT1.A class I molecules. The amount of RT1.A class I contained in each preparation was compared with the amount of class I antigen expressed by whole viable liver cells by quantitative absorption analysis using F16.4.4.11 mAb. The results demonstrated that DA recipients pretreated with a single dose of 1.75 x 10(10) cellular equivalents or multiple doses of 5 x 10(9) cellular equivalents of purified LEW membrane-bound class I molecules, delivered in aggregated micelle form, accepted their LEW renal allografts indefinitely (MST greater than 100 days). In contrast, no prolongation of graft survival was observed using the liver cell cytosol preparation containing soluble RT1.A class I molecules (MST 10 days) at the concentrations tested (10(8) -3 x 10(8) cell equivalents). However, when preoperative treatment with single (greater than or equal to 5 x 10(7) cellular equivalents of soluble class I MHC antigen) or multiple doses (greater than or equal to 10(7) cellular equivalents per dose) of the liver cell cytosol preparation was combined with a subtherapeutic dose of CsA given postoperatively (day +2, 10 mg/kg), suppression of renal allograft rejection was achieved with long-term survival (MST greater than 100 days). The immunologic unresponsiveness observed in both cases was donor specific.

The combined effect of perioperative donor spleen cells or KCl-extracted antigen and cyclosporine on renal allograft survival in the rat.

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).

Use of allochimeric proteins to mitigate graft-versus-host and host-versus-graft immune responses to rat small bowel allografts.

BACKGROUND: We aimed to identify the polymorphic epitopes that mitigate graft-versus-host disease (GvHD) and host-versus-graft response (HvGR) toward rat small bowel allografts in rats. METHODS: We tailored class I major histocompatibility complex (MHC) allochimeric antigens encoding 10 al-helical (alpha(1h)l58-80-RT1.Aa) or 4 (alpha(1h)l/u62-69-RT1.Aa) polymorphic amino acids. In the GvHD model, ACI (RT1a) donors were pretreated (day -14) with an intrathymic injection of alpha(1h)l58-80-RT1.Aa, alpha(1h)l/u62-69-RT1.Aa, or RT1.Al protein, with or without simultaneous intravenous injection of anti-T-cell receptor R73 monoclonal antibodies. Wistar-Furth (WF; RT1u) donors were tested with a similar protocol. In the HvGR model, ACI recipients were treated with a protocol designed to induce transplantation tolerance toward WF heart allografts: a portal vein injection of alpha(1h)l/u62-69-RT1.Aa protein and cyclosporine (4 mg/kg, intramuscular; days 0-6). RESULTS: GvHD was prevented in all (ACI x LEW) F1 recipients (RT1a/l) by pretreating ACI donors with R73 monoclonal antibody and recipient RT1.Al or alpha(1h)l58-80-RT1.Aa protein. Similarly, pretreatment of WF donors with RT1.Aa protein also prevented GvHD in (ACI x WF) F1 recipients. However, in a combined GvHD/HvGR model, ACI recipient perioperative treatment designed to prevent HvGR only modestly prolonged WF small bowel allograft survival (27.7+/-5.3 days compared to 17.4+/-4.6 days in the cyclosporine-alone group). In contrast, application of the two protocols significantly prolonged WF allograft survival (55.6+/-34.6 days), with two of seven recipients surviving more than 100 days. CONCLUSION: Simultaneous inhibition of GvHD and HvGR significantly prolongs small bowel allograft survival.