Receptor Cross-linking Research Articles

Abstract 64: Meditope SnAP-body technology facilitates enhanced internalization and in vivo efficacy of antibody therapeutics

Abstract Meditope Biosciences has developed a way to use SnAP technology to promote internalization of antibody receptor complexes through a novel construct called a SnAP-body. Meditope’s SnAP platform functionalizes monoclonal antibodies to bind to specific meditope peptides; this property can be used to directly facilitate receptor crosslinking when meditope-enabled antibodies are bound to cell surface receptors. SnAP-bodies are self-crosslinking meditope-enabled antibodies which permit a meditope peptide to specifically interact with a meditope-enabled antibody at the cell surface upon antigen engagement. Antigen-antibody complex formation through the specific contact of the SnAP-bodies and their cognate epitopes on the target antigen can increase clustering or co-localization of a cell surface antigen on a cell, which can promote enhanced internalization of the SnAP-body. The increased internalization can be used to clear or reduce the concentration of a cell surface antigen from the surface of a target cell or tumor. Enhancing internalization can increase the efficacy of antibody-based therapeutics, and in the case of antibody-drug conjugates, which require receptor internalization to deliver cytotoxic payloads, can reduce the amount of drug necessary to achieve a therapeutic effect. In vitro studies of SnAP-body binding to cell surface antigens demonstrate accelerated internalization of the antibody. In vivo, this translates into enhanced efficacy. The ability to see equivalent efficacy in the absence of toxin suggests a unique role for SnAP-body constructs in cancer therapeutics, especially in cases where lack of efficacy for a specific target is correlated with poor receptor internalization. Citation Format: Karin Forster, Elisabeth Gardiner, Stephanie Hsieh. Meditope SnAP-body technology facilitates enhanced internalization and in vivo efficacy of antibody therapeutics [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 64. doi:10.1158/1538-7445.AM2017-64

Quantitative in vitro and in vivo models to assess human IgE B cell receptor crosslinking by IgE and EMPD IgE targeting antibodies

Targeting plasma IgE by therapeutic mABs like Omalizumab (Xolair®) is current clinical practice for severe allergic conditions or other IgE related diseases like chronic urticaria. As an alternative to soluble IgE targeting, IgE supply can be lowered by targeting the Extracellular Membrane Proximal Domain (EMPD) of the IgE B cell receptor (BCR) present on IgE switched B cells. This ultimately leads to apoptosis of these cells upon IgE BCR crosslinking. Since tools to selectively assess the efficacy of IgE BCR crosslinking by IgE targeting antibodies are limited, a readily quantifiable cell model was developed that allows to specifically address IgE BCR crosslinking activity in vitro. The new cell model allowed for a direct quantitative comparison of anti-EMPD IgE therapeutic prototype antibody 47H4 with anti-IgE(Ce3) directed therapeutic antibody Omalizumab and with a newly selected anti-human EMPD IgE monoclonal antibody, designated mAB 15cl12. Furthermore, a complementing mouse model was developed that allows for in vivo validation of antibodies addressing human EMPD IgE. It carries a targetable humanized EMPD IgE sequence that has been introduced by seamless genomic replacement of the endogenous EMPD encoding sequence. The model allowed to directly compare IgE lowering activity of two anti-human EMPD IgE therapeutic antibodies in vivo. Our tools provide the means for quantitative assessment of IgE BCR crosslinking activity which is increasingly gaining attention with respect to forthcoming second generation anti-IgE clinical candidates such as Ligelizumab or other clinical candidates featuring additional effector functions such as IgE BCR crosslinking activity.

Relative efficacy of T cell stimuli as inducers of productive HIV-1 replication in latently infected CD4 lymphocytes from patients on suppressive cART

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

Antibody Functional Assays as Measures of Fc Receptor-Mediated Immunity to HIV - New Technologies and their Impact on the HIV Vaccine Field.

Background:There is now intense interest in the role of HIV-specific antibodies and the engagement of FcγR functions in the control and prevention of HIV infection. The analyses of the RV144 vaccine trial, natural progression cohorts, and macaque models all point to a role for Fc-dependent effector functions, such as cytotoxicity (ADCC) or phagocytosis (ADCP), in the control of HIV. However, reliable assays that can be reproducibly used across different laboratories to measure Fc-dependent functions, such as antibody dependent cellular cytotoxicity (ADCC) are limited.Method:This brief review highlights the importance of Fc properties for immunity to HIV, particular-ly via FcγR diversity and function. We discuss assays used to study FcR mediated functions of HIV-specific Ab, including our recently developed novel cell-free ELISA using homo-dimeric FcγR ecto-domains to detect functionally relevant viral antigen-specific antibodies.Results:The binding of these dimeric FcγR ectodomains, to closely spaced pairs of IgG Fc, mimics the engagement and cross-linking of Fc receptors by IgG opsonized virions or infected cells as the es-sential prerequisite to the induction of Ab-dependent effector functions. The dimeric FcγR ELISA reli-ably correlates with ADCC in patient responses to influenza. The assay is amenable to high throughput and could be standardized across laboratories.Conclusion:We propose the assay has broader implications for the evaluation of the quality of anti-body responses in viral infections and for the rapid evaluation of responses in vaccine development campaigns for HIV and other viral infections.

Vaccinia virus preferentially infects primary human memory B cells

Abstract Vaccinia virus (VACV) has shown an extreme preference for binding to and infection of primary human antigen-presenting cells including monocytes, macrophages, and dendritic cells in the peripheral blood. Although VACV infection in monocytes, macrophages, and dendritic cells has been examined, few studies have evaluated VACV infection in B cells. In this study, we characterized VACV binding to and infection of primary human B cells. Flow cytometric analysis revealed that B cells isolated from human peripheral blood mononuclear cells exhibited two distinct populations of B cells, referred to as Bsmall and Blarge. Phenotypic analysis demonstrated that the Bsmall compartment was more quiescent compared to the Blarge population and enriched with CD138-positive plasma cells. VACV preferentially entered into and infected memory B cells. Especially, VACV preferentially infected ex vivo CXCR5+ B cells, particularly CXCR5+HLA-DRhigh B cells. Assessment of VACV gene expression by RT-PCR in total B cells to evaluate permissiveness of VACV infection in B cells revealed early and intermediate viral gene expression, but little to no late viral gene expression, suggesting that VACV infection was aborted at the late viral gene expression stage in unstimulated B cells. Additionally, preliminary data demonstrated enhanced VACV binding to and infection of B cells stimulated by B cell receptor crosslinking and TLR-9 agonist treatment. With the risk of smallpox or monkeypox being used as bioterrorism agents and the multitude of adverse reactions to the current vaccine, understanding VACV infection in B cells is important for developing a better smallpox vaccine.

TGFβ Receptor mutations alter mast cell development and function

Abstract Mutations in TGFBRI, encoding the receptor for TGFβ, cause Loeys-Dietz syndrome (LDS), a Mendelian disorder associated with an increased risk of developing nearly all forms of allergic disease, including food allergy. To better understand the mechanisms responsible for this association, knock-in mice harboring LDS mutations have been developed, which recapitulate nearly all aspects of the human phenotype. Mast cells are the main effector cells of allergic disease and several studies have identified TGFβ as a critical mediator of mast cell development and function. We hypothesized that LDS mast cells are altered in a manner that would promote greater susceptibility to allergic outcomes. To address this possibility, in vitro assays were performed with peritoneal mast cells (pMCs) cultured in IL-3 and stem cell factor and stimulated with IL-33, TGFβ, or IgE crosslinking agents. LDS pMCs expressed higher levels of ST2, the receptor for IL-33, and greater secretion of IL-9 following IL-33 stimulation. Furthermore, LDS pMCs were more resistant to apoptosis, exhibited greater degranulation following IgE receptor crosslinking, and showed less downregulation of FcɛRI and c-kit following exposure to recombinant TGFβ1. This was associated with reduced pSmad2/3 levels, a key signaling molecule in the TGFβ pathway. Furthermore, we demonstrated that following food allergy induction, LDS mice exhibited greater MC hyperplasia in the small intestinal lamina propria as well as in the peritoneum where LDS peritoneal mast cells showed enhanced secretion of IL-9. In vivo studies are ongoing to further examine the role of altered mast cell frequency and/or function resulting from altered TGFβ signaling in promoting food-induced anaphylaxis.

The Multimerization State oftheAmyloid-β42 Amyloid Peptide Governs its Interaction Network withtheExtracellular Matrix.

The goals of this work were i) to identify the interactions of amyloid-β (Aβ)42 under monomeric, oligomeric, and fibrillar forms with the extracellular matrix (ECM) and receptors, ii) to determine the influence of Aβ42 supramolecular organization on these interactions, and iii) to identify the molecular functions, biological processes, and pathways targeted by Aβ42 in the ECM. The ECM and cell surface partners of Aβ42 and its supramolecular forms were identified with protein and glycosaminoglycan (GAG) arrays (81 molecules in triplicate) probed by surface plasmon resonance imaging. The number of partners of Aβ42 increased upon its multimerization, ranging from 4 for the peptide up to 53 for the fibrillar aggregates. The peptide interacted only with ECM proteins but their percentage among Aβ42 partners decreased upon multimerization. Aβ42 and its supramolecular forms recognized different molecular features on their partners, and the partners of Aβ42 fibrillar forms were enriched in laminin IV-A, N-terminal, and EGF-like domains. Aβ42 oligomerization triggered interactions with receptors, whereas Aβ42 fibrillogenesis promoted binding to GAGs, proteoglycans, enzymes, and growth factors and the ability to interact with perineuronal nets. Fibril aggregation bind to further membrane proteins including tumor endothelial marker-8, syndecan-4, and discoidin-domain receptor-2. The partners of the Aβ42 supramolecular forms are enriched in proteins contributing to cell growth and/or maintenance, involved in integrin cell surface interactions and expressed in kidney cancer, preadipocytes, and dentin. In conclusion, the supramolecular assembly of Aβ42 governs its ability to interact in vitro with ECM proteins, remodeling and crosslinking ECM enzymes, proteoglycans, and receptors.

Small Fluorescent Probes Show iGluRs are in the Synapses of Transfected Neurons under Basal Conditions

We imaged transiently transfected live glutamate receptors (iGluRs), namely AMPAR and NMDAR, with super-resolution in three-dimension, labeled with differently sized fluorophores. We used small organic fluorescent dyes (∼ 4 nm), small quantum dots (sQD, ∼10 nm in diameter), or big (commercial) quantum dots (bQD, ∼ 20 nm in diameter). The iGluRs were imaged along with a synaptic protein, Homer1c, using fiducial markers that eliminate stage drift, and under conditions where the receptor cross-linking is monitored or eliminated. With small probes under basal conditions, we find that both AMPAR and NMDAR are predominantly within the synapse (∼84-95%), in contrast to bQDs, which show only 5-10% within the synapse. The results can likely be explained by cross-linking and inhibited mobility of the iGluRs labeled with bQDs, but not with sQDs or organic fluorophores. Hence, this data indicates that there is not a highly diffusible pool of extrasynaptic iGluRs in the plasma membrane, as has been widely reported. In addition, within a synapse, the distribution is non-homogeneous, perhaps due to the non-homogeneous distribution of glutamate.

The roles of mast cells in allergic inflammation and mast cell-related disorders

Mast cells, which are major effector cells in allergic reactions, are found in the perivascular spaces of most tissues and contain pro-in-flammatory and vasoactive mediators. These mediators are released after IgE receptor cross-linking induced by allergens or other stimuli, including anaphylatoxins (C3a and C5a), aggregated IgG, certain drugs, venoms, and physical stimuli (pressure and tempera-ture changes), as well as cytokines and neuropeptides. The excess release of these mediators can cause variable allergic symptoms and signs, such as bronchospasm, itching, flushing, nausea, vomiting, diarrhea, abdominal pain, vascular instability, and anaphylaxis. Furthermore, mast cell disorders may involve either excessive proliferation of mast cells or abnormal mast cell reactivity. Mast cell disorders can be broadly divided into 3 types: primary, secondary, and idiopathic. All of these disorders present with signs and symp-toms of mast cell activation and differ in severity and involvement of various organ systems. The best characterized primary disorder is mastocytosis. Systemic and cutaneous forms of the disease are well described. Secondary disorders include typical allergic dis-eases and some types of urticarial diseases. In this article, the biochemical characteristics of mast cells and the role of mast cells in al-lergic inflammation, as well as the classification, diagnosis, and management of mast cell-related disorders, will be reviewed. (Allergy Asthma Respir Dis 2017;5:248-255)

Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells

Recently, we developed a new paradigm in macromolecular therapeutics that avoids the use of low molecular weight drugs. The activity of the “drug-free macromolecular therapeutics” is based on the biorecognition of complementary motifs at cell surface resulting in receptor crosslinking and apoptosis induction. The system is composed of two nanoconjugates: (1) a single-stranded morpholino oligonucleotide (MORF1) attached to an anti-CD20 Fab′ fragment (Fab′-MORF1); (2) multiple copies of complementary oligonucleotide MORF2 grafted to a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) – P-(MORF2)x. The two conjugates crosslink CD20 antigens via MORF1-MORF2 hybridization at the surface of CD20+ malignant B-cells and induce apoptosis. Preclinical studies in a murine model of human non-Hodgkin's lymphoma showed cancer cells eradication and long-term survivors. The aim of this study was to determine the relationship between the detailed structure of the nanoconjugates and apoptosis induction in Raji cells to allow system optimization. The factors studied include the length of the MORF sequence, the valence of P-(MORF2)x (varying x), molecular weight of P-(MORF2)x, incorporation of a miniPEG spacer between Fab′ and MORF1 and between polymer backbone and pendant MORF2, and comparison of two Fab′ fragments, one from 1F5 antibody (Fab′1F5), the other from Rituximab (Fab′RTX). The results of apoptosis induction in human Burkitt's B-cell non-Hodgkin's lymphoma (NHL) Raji cells as determined using three apoptotic assays (Annexin V, Caspase 3, and TUNEL) indicated that: a) An improvement of apoptotic activity was observed for a 28 base pair MORF sequence when compared to MORFs composed of 20 and 25 base pairs. The differences depended on type of assay, concentration and exposure schedule (consecutive vs. premixed). b) The higher the valence of P-(MORF2)x the higher the levels of apoptosis. c) Higher molecular weight of P-(MORF2)x induced higher levels of apoptosis. d) A miniPEG8 spacer was effective in enhancing apoptotic levels in contrast to a miniPEG2 spacer. e) There was not a statistically significant difference when comparing Fab′1F5-MORF1 with Fab′RTX-MORF1.

A flavanone derivative from the Asian medicinal herb (Perilla frutescens) potently suppresses IgE-mediated immediate hypersensitivity reactions

Perilla frutescens is a dietary leafy herb consumed as a traditional Japanese condiment as well as used for Chinese medicine with anti-inflammatory activity. Here we report a hitherto-unrecognized P. frutescens phytochemical that potently suppresses IgE-mediated type I hypersensitivity reactions. Structural analysis reveals that the purified anti-allergic compound (Perilla-derived methoxyflavanone, PDMF) is identified as 8-hydroxy-5,7-dimethoxyflavanone. PDMF significantly inhibits IgE-mediated histamine release from RBL-2H3 rat basophilic leukemia cells as compared with those seen in known P. frutescens-derived anti-inflammatory polyphenols. We also show that oral administration of PDMF not only suppresses passive cutaneous anaphylaxis, but also prevents allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis. Mechanistically, PDMF negatively regulates Akt phosphorylation and intracellular Ca2+ influx, both of which are essential for mast cell secretory granule translocation and its exocytosis upon high-affinity IgE receptor (FcεRI) cross-linking. These results represent PDMF as a new potent anti-allergic phytochemical useful for prevention of IgE-driven hypersensitivity reactions.

CDX-1140, a Novel Agonist CD40 Antibody with Potent Anti-Lymphoma Activity

CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is essential for activating both innate and adaptive immune systems. CD40 is expressed by antigen-presenting cells (APC) including dendritic cells and B cells. The engagement by its natural ligand (CD40L) on T cells activates APC thus enhancing immune responses. CD40 is also expressed on many cancer cells, in particular B cell lymphomas, and agonist CD40 antibodies can lead to apoptosis and impaired growth of cancer cells due to direct signaling effects. There are several CD40 agonist antibodies in clinical development, which are differentiated by their level of agonist activity, route of administration, and associated side effect profile. We have generated a panel of CD40-specific fully human antibodies derived from human immunoglobulin transgenic mice with the aim to select a clinical candidate with strong agonist activity and a safety profile that allows for systemic dosing. CDX-1140 is a human IgG2 antibody that stimulates CD40 signaling without the requirement for cross-linking or Fc receptor interactions. CDX-1140 is a potent stimulator of human B cells and dendritic cells in vitro, and has potent direct anti-tumor activity against B cell lymphoma cell lines transplanted into immunocompromised mice. To demonstrate the contribution of immune cell activation to the anti-lymphoma activity in vivo, we included human PBMCs in subsequent xenograft studies. Using human B cell lymphoma cell lines Raji and Ramos as tumor models and severe combined immunodeficiency mice (SCID-beige) as hosts, we found that addition of human PBMC alone had a minor impact on tumor growth (median survival 32 days, compared to 26 days with no treatment, p<0.01). Administration of CDX-1140 alone showed partial anti-tumor activity (median survival 39 days, p< 0.001), while the combination of human PBMCs and CDX-1140 provided a significant increase in survival with all mice alive at day 45. These studies provide evidence that CDX-1140 is able to induce in vivo activation and anti-tumor activity of effector cells derived from injected human PBMC, in addition to the direct signaling effects on lymphoma cells. We are investigating anti-lymphoma efficacy of combining CDX-1140 with other immunotherapies including varlilumab, our CD27 agonist antibody, which is currently in clinical development. Together with our prior preclinical studies characterizing this antibody, these results suggest that CDX-1140 is a promising candidate for clinical development in patients with lymphomas. DisclosuresHe:Celldex Therapeutics, Inc.: Employment. Testa:Celldex Therapeutics, Inc.: Employment. Anna:Celldex Therapeutics, Inc.: Employment. Jeffery:Celldex Therapeutics, Inc.: Employment. Sisson:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Crocker:Celldex Therapeutics, Inc.: Employment. Widger:Celldex Therapeutics, Inc.: Employment. Goldstein:Celldex Therapeutics, Inc.: Employment. Marsh Jr.:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership.

Endotoxemia contributes to CD27+ memory B-cell apoptosis via enhanced sensitivity to Fas ligation in patients with Cirrhosis.

Peripheral CD27+ memory B-cells become quantitatively reduced and dysfunctional in patients with cirrhosis through poorly characterized mechanisms. We hypothesized that the disappearance of CD27+ memory B-cells results from enhanced sensitivity to apoptosis caused by exposure to gut microbial translocation products. Using isolated naïve and memory B-cells from patients with cirrhosis and age-matched controls, ex vivo and activation-induced sensitivity to Fas-mediated apoptosis was assessed under relevant experimental conditions. We observed differential expression of CD95(Fas) in CD27+ B-cells from cirrhotic patients that was inversely correlated with peripheral CD27+ B-cell frequency. While memory B-cells from cirrhotic patients were resistant to Fas-mediated apoptosis ex vivo, Toll-like receptor 4(TLR4)-ligation restored Fas-sensitivity. Sensitivity to Fas-mediated apoptosis could be transferred to healthy donor memory B-cells by co-culturing these cells with plasma from cirrhotic patients, a sensitivity partially mediated by Fas and TLR4 signaling, and partially rescued via B-cell receptor crosslinking. We conclude that peripheral CD27+ memory B-cells in cirrhosis exhibit increased sensitivity to Fas-induced apoptosis in an activation-dependent manner to which endotoxin contributes, associated with reduced frequency of circulating memory B-cells. Destruction of this critical cell subset may contribute to the cirrhotic immunodeficiency state and heightened risk of systemic infections in advanced liver disease.

Abstract B046: Therapeutic mechanisms of anti-4-1BB antibodies in cancer: agonism versus regulatory T cell depletion

Abstract 4-1BB (CD137) is a T cell activating receptor belonging to the TNF receptor superfamily that can be targeted by mAb to eradicate established tumors. 4-1BB is expressed on a multitude of hematopoietic cells, including activated conventional CD4 and CD8 T cells, regulatory T (Treg) cells, dendritic cells and NK cells. 4-1BB signaling promotes the proliferation, survival and effector function of conventional activated T cells and therapeutic 4-1BB antibodies are thought to mediate their effects through receptor agonism. However, whilst investigating the expression pattern of a panel of activated T cell markers on tumor infiltrating T cells, we unexpectedly found that intra-tumoral Treg cells express high levels of 4-1BB when compared with conventional T cells. This finding raised the possibility that anti-4-1BB antibodies mediate their antitumor effects through depletion of intra-tumoral Treg cells. Moreover, precedence for a Treg cell dependent mechanism exists for mAb targeting the related receptors OX40 and GITR. The choice of antibody isotype is critical as the therapeutic mechanism of action is dependent on the engagement of activatory and inhibitory FcγRs. Murine IgG1 mAb confer good agonistic potential through co-engagement of inhibitory FcγRs leading to receptor cross-linking and activation to promote CD8+ T cell mediated antitumor immunity. In contrast, murine IgG2a mAb have the potential to deplete target cells through activation of FcγR-expressing macrophages within the tumor microenvironment. To address the question of whether tumor protection with anti-4-1BB antibodies is mediated by agonism or selective Treg cell depletion, we generated a novel mouse strain with conditional deletion of 4-1BB on Foxp3+ Treg cells (4-1BBFoxp3-/−). The phenotype was validated by showing complete loss of 4-1BB expression on intra-tumoral CD4+ Foxp3+ cells whilst being conserved on conventional T cell subsets. To examine whether 4-1BB expression on intra-tumoral Treg cells was required for the therapeutic activity of anti-4-1BB mAb, 4-1BBFoxp3-/- or control mice were inoculated with EG7 thymoma cells and subsequently treated with either mIgG1 or mIgG2a anti-4-1BB mAb. The EG7 tumors grew equally well in 4-1BBFoxp3-/- and control mice given IgG isotype control Ab, indicating that the loss of 4-1BB expression on Treg cells does not alter the tumor immune environment. Treatment with mIgG1 or mIgG2a anti-4-1BB mAb resulted in tumor regression then complete clearance in the majority of control mice. Equivalent response to therapy was observed in the 4-1BBFoxp3-/- mice, suggesting that 4-1BB expression on Treg cells is not essential for the therapeutic activity of mIgG1 or mIgG2a anti-4-1BB mAb. Specifically, the mIgG2a anti-4-1BB mAb maintained its antitumor activity in the absence of 4-1BB-expressing Treg cells, suggesting that the therapeutic activity is driven by effector T cell costimulation and not differential intra-tumoral Treg cell deletion. These results establish T cell costimulation as a key mechanism for the therapeutic activity of anti-4-1BB mAb in cancer and have clinical relevance in optimizing human anti-4-1BB mAb for delivering costimulation. Citation Format: Marcus Remer, Anne Rogel, Sarah Buchan, Peter W.M. Johnson, Aymen Al-Shamkhani. Therapeutic mechanisms of anti-4-1BB antibodies in cancer: agonism versus regulatory T cell depletion [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 B046.

Tumor-targeted delivery of a C-terminally truncated FADD (N-FADD) significantly suppresses the B16F10 melanoma via enhancing apoptosis

Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182–205 aa) named N-FADD (m-FADD, 1–181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma.

Revisiting the Immunoglobulin Intramolecular Signaling Hypothesis

A generation ago, the immunoglobulin intramolecular signaling, or allosteric, hypothesis was abandoned in favor of the associative hypothesis, which posited that Fc receptor crosslinking produced the increased affinity of antigen-antibody complexes. This essay argues that there is sufficient evidence to resuscitate the allosteric hypothesis, at least for some antibodies.

Virus Like Nanoparticles: Display Matters

An efficacious way to avoid atleast a bunch of infections by microbes is to neutralize the microbial infection right at the very beginning. This is well achieved by vaccination against many infectious agents. While vaccine is available for many pathogenic micro-organisms, we are still struggling to achieve success to develop prophylactic strategies for many others. Most commonly vaccines are developed by attenuation/ inactivation of infectious agents or by using killed pathogens. Yet another variety of vaccines include the subunit vaccine. In this review a subcategory of subunit vaccine has been discussed where the viral surface antigens when displayed in a rigid multivalent fashion at high densities, induce very potent and robust neutralizing antibody response as a result of very strong and extensive cross-linking of B-cell receptors.

IL-4 rescues surface IgM expression in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) cells express poor levels of surface immunoglobulin (sIg), and many are minimally activated or anergic in response to B-cell receptor (BCR) crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express a low level of total CD79b protein but normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that interleukin-4 (IL-4) markedly rescues CD79b and sIgM protein in CLL samples. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in immunoglobulin heavy chain variable (IGHV)-unmutated CLL cells than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of total CD79b protein in CLL cells. IL-4 substantially restores CD79b protein expression, sIgM expression, and BCR signaling.

Abstract 598: Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization

Abstract Therapeutic antibodies can bind to cell-surface receptors, prompting receptor internalization and effectively blocking further stimulation by the cognate receptor ligands. Receptor internalization is also exploited by antibody-drug conjugates (ADC) to promote effective delivery of cytotoxic payload to the inside of the cell. Meditope Biosciences has developed a way to use its SnAP (Site-specfi novel Antibody Platform) technology to promote enhanced internalization of antibody receptor complexes. The Meditope's SnAP technology functionally enables antibodies to bind to specific peptides, termed “meditopes’, and this property can be used to directly facilitate receptor crosslinking when meditope enabled antibodies are bound to cell surface receptors. Here we show that the affinity of the meditope enabled antibodies (trastuzumab anti-HER2 and gemtuzumab anti-CD33) for cell surface receptors results in the formation of an antibody receptor complex that can be detected by native gel electrophoresis and gel filtration. Binding of the meditope peptide to the enabled antibody does not interfere with normal antigen-antibody interaction and the formation of the peptide bound antibody:receptor complexes results in accelerated internalization of the complex, as shown by decreased surface fluorescence of Alexa 488-labeled SnAP peptides as well as by increased pHrodo-Red increased fluorescence of internalized antibodies when they reach the endosomal compartment. Finally, as a result this increased internalization, the downregulation of specific antibody mediated signaling events are much more effectively downregulated in the presence of the meditope peptide complex as compared to the antibody alone. Citation Format: Calin D. Dumitru, Elisabeth Gardiner, John Williams, Cindy Zer. Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 598.

Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection

A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4(+) T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses.