Functional humanization of immunoglobulin heavy constant gamma 1 Fc domain human FCGRT transgenic mice.

A major asset of many monoclonal antibody (mAb)-based biologics is their persistence in circulation. The MHC class I family Fc receptor, FCGRT, is primarily responsible for this extended pharmacokinetic behavior. Engagement of FCGRT with the crystallizable fragment (Fc) domain protects IgG from catabolic elimination, thereby extending the persistence and bioavailability of IgG and related Fc-based biologics. There is a need for reliable in vivo models to facilitate the preclinical development of novel IgG-based biologics. FcRn-humanized mice have been widely accepted as translationally relevant surrogates for IgG-based biologics evaluations. Although such FCGRT-humanized mice, especially the mouse strain, B6.Cg-Fcgrttm1Dcr Tg(FCGRT)32Dcr (abbreviated Tg32), have been substantially validated for modeling humanized IgG-based biologics, there is a recognized caveat - they lack an endogenous source of human IgG that typifies the human competitive condition. Here, we used CRISPR/Cas9-mediated homology-directed repair to equip the hFCGRT Tg32 strain with a human IGHG1 Fc domain. This replacement now results in mice that produce human IgG1 Fc-mouse IgG Fab2 chimeric antibodies at physiologically relevant levels, which can be further heightened by immunization. This endogenous chimeric IgG1 significantly dampens the serum half-life of administered humanized mAbs in an hFCGRT-dependent manner. Thus, such IgG1-Fc humanized mice may provide a more physiologically relevant competitive hFCGRT-humanized mouse model for the preclinical development of human IgG-based biologics.

In Vivo Analysis of Human Immune Responses in Immunodeficient Rats.

BACKGROUND: Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described. METHODS: We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line. RESULTS: RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection. CONCLUSIONS: hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.

Genetic Removal of the CH1 Exon Enables the Production of Heavy Chain-Only IgG in Mice.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Protective efficacy of Fc targeting conserved influenza virus M2e antigen expressed by Lactobacillus plantarum.

The influenza A (H1N1) virus is a highly contagious acute respiratory disease affecting pigs and humans. This disease causes severe economic loss in many countries, and developing mucosal vaccines is an efficient strategy to control the influenza virus. The neonatal Fc receptor (FcRn) plays an important role in transferring IgG across polarized epithelial cells. In the present study, an oral vaccine was developed using Lactobacillus plantarum to deliver the internal influenza viral protein M2e fused to an IgG Fc fragment. Oral vaccination with recombinant L. plantarum expressing 3M2e-Fc elicited Peyer's patch (PP) DC activation, improved the number of gamma interferon (IFN-γ)-producing T cells and increased the frequency of CD8+IFN-γ+ cells in the mesenteric lymph nodes (MLNs). In addition, the recombinant L. plantarum can induce PP B220+IgA+ expression and enhance specific sIgA secretion and the shaping of growth centers (GCs) in PPs. Furthermore, the data demonstrated that immunization with recombinant L. plantarum expressing 3M2e-Fc markedly reduced the viral load in the lung and protected against H1N1 influenza virus and mouse-adapted H9N2 avian influenza virus (AIV) challenge in BALB/c mice. Collectively, the data also showed that this vaccine strategy provided effective protective immunity against infection with homologous and heterologous influenza viruses in a mouse model and may be useful for future influenza vaccine development.

Antibodies from donor B cells perpetuate cutaneous chronic graft-versus-host disease in mice.

Cutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4(+) T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)H(µÎ³1) DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgH(µÎ³1) grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgH(µÎ³1) grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgH(µÎ³1) grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells.

Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

T lymphocyte antigen 4-modified dendritic cell therapy for asthmatic mice guided by the CCR7 chemokine receptor.

The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.

An uncommon tail about the common γ-chain.

The common γ-chain (γc) is a key component of multiple cytokine receptors. In this issue of Immunity, Hong et al. (2014) demonstrate a unique function of γc as a secreted protein capable of inhibiting cytokine responses and promoting autoimmunity.

Activated T cells secrete an alternatively spliced form of common γ-chain that inhibits cytokine signaling and exacerbates inflammation.

The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rß and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.

TRIF signaling is essential for TLR4-driven IgE class switching.

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.

Cloning and expression of the functional human anti-vascular endothelial growth factor (VEGF) using the pcDNA3.1 vector and the human chronic myelogenous leukemia cell line K562.

In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.

Genetic variants of immunoglobulin γ and κ chains influence humoral immunity to the cancer-testis antigen XAGE-1b (GAGED2a) in patients with non-small cell lung cancer.

GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour-associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE-1b, a highly immunogenic lung tumour-associated cancer-testis antigen. Sera from 89 patients with non-small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE-1b protein. The distribution of various GM phenotypes was significantly different between XAGE-1b antibody-positive and -negative patients (P = 0·023), as well as in the subgroup of XAGE-1b antigen-positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE-1b antibody. In patients with antigen-positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody-positive group than in those who lacked the XAGE-1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE-1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer-testis antigen, which has important implications for XAGE-1b-based immunotherapeutic interventions in lung adenocarcinoma.

Production of a neutralizing mouse-human chimeric antibody against botulinum neurotoxin serotype E.

A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

Proliferative glomerulonephritis with discrete deposition of monoclonal immunoglobulin γ1 CH 2 heavy chain and κ light chain: a new variant of monoclonal immunoglobulin deposition disease.

A 45-year-old man presented with moderate proteinuria and hematuria. A renal biopsy showed mesangial/endocapillary proliferative glomerulonephritis, linear deposition of monoclonal immunoglobulin γ1 C(H) 2 heavy chain along glomerular and tubular basement membranes (GBMs and TBMs), granular deposition of κ light chain within the mesangial area, and continuous linear deposits of finely granular electron-dense materials along GBMs and TBMs. Dual immunostaining showed essentially discrete glomerular localization of γ1 C(H) 2 heavy chain and κ light chain. Monoclonal protein was not detected in urine and serum. A bone marrow aspiration showed no abnormalities. Steroid therapy led to the improvement of proteinuria and hematuria. We would classify this case as a new variant of monoclonal immunoglobulin deposition disease, light chain/heavy chain deposition disease. In contrast with light and heavy chain deposition disease, the remarkable characteristics of this variant are separate deposition of monoclonal heavy chain and light chain, deposition of largely deleted γ heavy chain lacking the C(H) 1 domain, and good response to steroid therapy.

Brain protection from stroke with intravenous TNFα decoy receptor-Trojan horse fusion protein.

Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood-brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke.

Therapeutic use of an anti-ErbB3 monoclonal antibody.

Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers and some mental disorders. Here we report the generation of a specific anti-ErbB3 antibody intended for use in diagnosing disease or therapeutic application. By using the hybridoma technique, one cell line (2E(12)C(3)) stably producing anti-ErbB3 antibody was obtained. Its molecular weight was about 185 kDa and its isotype was IgG 2a and κ, respectively. The affinity constant (Kaff) of the anti-ErbB3 MAb was 5.83×10(10) M(-1). This antibody may become a useful tool for diagnostic and therapeutic targeting of ErbB3-expressing cancers or helpful in highlighting the etiology of schizophrenia.

Gamma heavy chain disease in a patient with underlying lymphoplasmacytic lymphoma of the thyroid. Report of a case and comparison with other reported cases with thyroid involvement.

BACKGROUND: Gamma heavy chain disease with underlying thyroid pathology is rare. There are 5 reported cases in the English literature, including the present case of an elderly female with γ heavy chain disease with underlying lymphoplasmacytic lymphoma of the thyroid who initially presented with long-standing goiter and chronic thyroiditis. METHODS: The protein studies and histopathologic findings in her thyroid are described. Her case is compared with reported cases of γ heavy chain disease with thyroid involvement. RESULTS: Initial impression on most cases was chronic thyroiditis; however pathology showed 3 cases with plasmacytoma and 2 with lymphoplasmacytic infiltrate. All were diagnosed and followed up using serum and urine electrophoresis. CONCLUSION: Gamma heavy chain disease has a protean manifestation; however there appears to be a more uniform pattern of the disease when it is associated with the thyroid. The inclusion of protein studies in cases diagnosed with chronic thyroiditis by FNA may aid in establishing γ heavy chain disease with underlying thyroid involvement. In this case serum and urine electrophoresis, and immunofixation studies which are simple and affordable tests facilitated the hematologic workup and follow up.