Fc Portion Research Articles

Economic Impact of Immune Tolerance Induction (ITI) with Recombinant Factor VIII Fc Fusion Protein (rFVIIIFc) Compared to Conventional Recombinant Factor VIII (rFVIII)

Introduction:Hemophilia A results from a clotting protein factor VIII (FVIII) deficiency, which leads to the need for the use of exogenous FVIII. Such therapy is effective unless alloantibodies (inhibitors) develop and render FVIII replacement ineffective. Patients with high-titer inhibitors may try to eradicate the presence of these inhibitors by undergoing ITI, an often costly process that requires the prolonged use of frequent doses of FVIII to induce FVIII antigen-specific tolerance. There is no consensus on the best approach to achieve tolerance and no product has been approved by any regulatory agency for ITI treatment in hemophilia A patients with inhibitors. The Fc portion of rFVIIIFc has shown immunomodulatory properties in mice, and chart reviews and case reports suggest that tolerization with rFVIIIFc in first-attempt ITI patients can potentially be achieved rapidly (i.e., ≤18 months) and therefore may possibly offer cost savings compared to conventional rFVIII. This analysis assessed the economic consequences and budget impact of using rFVIIIFc vs. conventional rFVIII for first-attempt immune tolerance induction (ITI) in hemophilia A patients with inhibitors.Methods:A literature-based model was developed to estimate the effect of rFVIIIFc vs conventional rFVIII on drug cost of first-attempt ITI, based on modelled ITI duration and outcome (rates and time to ITI success or failure) for US patients with varying clinical profiles (e.g., historical peak titer, FVIII dose) over a 3-year period. In the model, at any given time after ITI initiation (on either rFVIIIFc or rFVIII), a patient was categorized as ongoing ITI, post-ITI as success, or post-ITI as failure. The duration and outcomes for patients treated with rFVIIIFc ITI were based on observed data for first-attempt ITI patients with varied clinical profiles and ITI regimens from in chart reviews and case reports (n = 9). In the absence of direct head-to-head observations, the duration and outcome of ITI for the exact same patients in a scenario in which they received rFVIII (instead of rFVIIIFc) at the same doses were estimated indirectly using a previously published regression model (Bojke et al. 2009, Value in Health, 12(7), A378-A379) based on data for 113 patients from international and national registries that adjusts for historical and pre-ITI titer levels, time from inhibitor diagnosis to ITI start, and ITI factor dose. To place the cost comparison results in the perspective of a third-party US payer, a budget impact analysis was undertaken on a population of typical hemophilia A patients (after adjusting for patient characteristics) who are embarking on first-attempt ITI in a plan of 10 million insured members. In this analysis, the number of patients starting ITI each year was assumed constant, and patients who successfully eradicated inhibitors with ITI would transition back to FVIII prophylaxis.Results:The model predicted that, compared to rFVIII, rFVIIIFc would result in a reduction in estimated time to ITI success (rFVIIIFc: 9.43 months, rFVIII: 16.80 months), increased success probability at 20 months (rFVIIIFc: 67%, rFVIII: 25%), and lower 3-year per-patient costs (rFVIIIFc: $1,709,000, rFVIII: $3,630,000), respectively. In the budget impact analysis for a US health plan of 10 million insured members, 11.2 patients were expected to be newly diagnosed hemophilia A patients, among whom 1.17 patients were expected to develop inhibitors to FVIII and initiate ITI each year. By year 3, the predicted number of successful ITI patients increased by 23% and the budget savings for the plan were $103,738 per patient per year (for the ~1 patient who started ITI each year) after the inclusion of rFVIIIFc for ITI and subsequent prophylaxis when patients successfully eradicated inhibitors.Conclusions:Based on this mathematical economic model of first-attempt ITI patients, the modeled faster time to tolerization with rFVIIIFc vs. rFVIII, resulted in estimated per-patient and overall budget savings in ITI from a US payer perspective. Current prospective studies (e.g. verITI-8) will provide additional evidence on the efficacy of rFVIIIFc for ITI in Hemophilia A patients with inhibitors. DisclosuresLi:Bioverativ: Employment. Wilson:SOBI: Employment. Botteman:Daiichi Sankyo Incorporated: Research Funding; BMS: Research Funding; Pharmerit International: Employment, Equity Ownership, Research Funding; Celgene: Research Funding; Bioverativ: Consultancy, Other: Provided consulting to Bioverativ, Research Funding. Nicoloso:Bioverativ: Other: an employee of Pharmerit, which provided consulting to Bioverativ. Krishnan:Bioverativ: Employment. Su:Bioverativ: Employment.

Induced Pluripotent Stem Cell-Derived NK Cells Genetically Modified to Express NKG2C/DAP12 Mediate Potent Function When Targeted through an NKG2C/IL-15/CD33 Tri-Specific Killer Engager (TriKE)

Adoptive transfer of haplo-donor NK cells can achieve remission in patients with relapsed or refractory acute myeloid leukemia (AML). This strategy is limited by NK cell heterogeneity, lack of specificity, and single dosed strategies. To overcome these barriers, we genetically modified induced pluripotent stem cells (iPSCs) to stably over-express NKG2C alone or along with DAP12. CD94/NKG2C is a heterodimeric receptor that binds to HLA-E and associates with DAP12, a protein containing an immunoreceptor tyrosine-based activating motif. Efficient expression of CD94/NKG2C on the cell surface requires the presence of DAP12, and charged amino acids in the transmembrane domains of DAP12 and NKG2C mediate this interaction. Physiologic NKG2C expression is limited to subsets of NK and CD8+ T cells that expand in response to cytomegalovirus (CMV) infection.Engineered iPSCs were differentiated into CD34+ hematopoietic progenitor cells and subsequently differentiated into CD56+ NK cells (iNK cells) using a stepwise differentiation and expansion strategy. Surface NKG2C expression was low on unmodified iNK cells (10.7%) with higher levels of NKG2C on the surface of iNK cells derived from the NKG2C only line (65.1%). These results demonstrate that transgenic NKG2C can pair with endogenous CD94 and DAP12 to form stable structures on the cell surface. Importantly, surface NKG2C expression was highest on iNK cells derived from the NKG2C/DAP12 line (84.1%) with higher MFI, demonstrating the importance of engineering a line with both components (Figure 1A). To validate function via engagement of NKG2C on iNK cells, we used a redirected antibody dependent cellular cytotoxicity (ADCC) assay where P815 cells (which bind the Fc portion of antibodies) were coated with a monoclonal anti-NKG2C agonist antibody at a range of concentrations (0.01-10 mg/ml) and co-cultured with unmodified iNK cells, NKG2C iNK cells or NKG2C/DAP12 iNK cells. We found that at all concentrations tested, NKG2C/DAP12 iNK cells demonstrated the strongest functional response as measured by the frequencies of surface CD107a expression, interferon (IFN)-g production and tumor necrosis factor (TNF) production. Intermediate functional responses were observed for NKG2C iNK cells (Figure 1B).Having demonstrated stable expression and intact signaling of NKG2C/DAP12 in iNK cells, we next created a Tri-specific Killer Engager (TriKE) molecule with single chain Fv fragments specific for NKG2C (on iNK cells) and CD33 (on AML cells), as well as IL-15 (to support iNK cell survival and proliferation). This NKG2C/CD33/IL-15 TriKE was tested in functional assays where NKG2C iNK cells or NKG2C/DAP12 iNK cells were used as effectors against CD33+ THP1 cells (an AML cell line shown to be refractory to NK cell cytotoxicity). Without the addition of NKG2C/CD33/IL-15 TriKE, the frequencies of both NKG2C iNK cells and NKG2C/DAP12 iNK cells producing IFN-g was relatively low (< 10%). Addition of NKG2C/IL-15/CD33 TriKE in the assay markedly increased the frequency of IFN-g production by both NKG2C iNK cells (19.1%) and NKG2C/DAP12 iNK cells (25.8%) (Figure 1C). Finally, we directly tested the ability of the NKG2C/IL-15/CD33 TriKE molecule to trigger target killing by engineered iNK cells monitored over a 24-hour period. THP1 target cells were dye-labeled and plated in wells with either NKG2C iNK cells or NKG2C/DAP12 iNK cells at a 5:1 effector-to-target ratio with or without NKG2C/CD33/IL-15 TriKE. We found that in the absence of TriKE, there was no killing of THP1 targets. In contrast, both NKG2C iNK cells and NKG2C/DAP12 iNK cells mediated robust THP1 killing with the addition of NKG2C/CD33/IL-15 TriKE, with NKG2C/DAP12 iNK cells exhibiting the strongest response (Figure 1D). Our results provide a proof-of-concept that iNK cells engineered to express NKG2C/DAP12 can be used in combination with a novel NKG2C/IL-15/CD33 TriKE molecule to effectively target cancer cells. Engaging NK cells through NKG2C, restricted to adaptive NK cells or genetically modified iPSC will be more specific than CD16, which will bind to CD16A on NK cells but also have off-target binding to CD16B on neutrophils. Because of the enormous expansion and engineering capacity of the iNK cell platform, we are in a unique position to create an “off-the-shelf” NK cellular therapy that is targeted, specific and efficacious. DisclosuresCichocki:Fate Therapeutics Inc.: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding. Chu:Fate Therapeutics Inc.: Employment. Ge:Fate Therapeutics Inc.: Employment. Bjordahl:Fate Therapeutics Inc.: Employment. Kaufman:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Valamehr:Fate Therapeutics Inc.: Employment.

Enhanced anti-metastatic bioactivity of an IGF-TRAP re-engineered to improve physicochemical properties

The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG1 (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin. We showed that the IGF-TRAP had potent anti-cancer activity in several pre-clinical models of aggressive carcinomas. Here we report on the re-engineering of the IGF-TRAP with the aim of improving physicochemical properties and suitability for clinical applications. We show that cysteine-serine substitutions in the Fc hinge region of IGF-TRAP eliminated high-molecular-weight oligomerized species, while a further addition of a flexible linker, not only improved the pharmacokinetic profile, but also enhanced the therapeutic profile of the IGF-TRAP, as evaluated in an experimental colon carcinoma metastasis model. Dose-response profiles of the modified IGF-TRAPs correlated with their bio-availability profiles, as measured by the IGF kinase-receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. This study provides a compelling example of structure-based re-engineering of Fc-fusion-based biologics for better manufacturability that also significantly improved pharmacological parameters. It identifies the re-engineered IGF-TRAP as a potent anti-cancer therapeutic.

Nonclinical safety assessment of a human interleukin-22FC IGfusion protein demonstrates invitro to invivo and cross-species translatability.

Although Interleukin‐22 (IL‐22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL‐22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL‐22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL‐22 recombinant fusion protein that links the human cytokine IL‐22 with the Fc portion of a human immunoglobulin. To assess the pharmacological activity of UTTR1147A, STAT3 activation was evaluated in primary hepatocytes isolated from human, cynomolgus monkey, minipig, rat, and mouse after incubation with UTTR1147A. UTTR1147A activated STAT3 in all species evaluated, demonstrating that all were appropriate nonclinical species for toxicology studies.The nonclinical safety profile of UTTR1147A was evaluated in rats, minipigs, and cynomolgus monkeys to establish a safe clinical starting dose for humans in Phase I trials and to support clinical intravenous, subcutaneous and/or topical administration treatment regimen. Results demonstrate the cross‐species translatability of the biological response in activating the IL‐22 pathway as well as the translatability of findings from in vitro to in vivo systems. UTTR1147A was well tolerated in all species tested and induced the expected pharmacologic effects of epidermal hyperplasia and a transient increase in on‐target acute phase proteins. These effects were all considered to be clinically predictable, manageable, monitorable, and reversible.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

Stimulation of natural killer cells with rhCD137 ligand enhances tumor-targeting antibody efficacy in gastric cancer.

Although many anticancer agents for gastric cancer have been developed, the prognosis for many patients remains poor. Recently, costimulatory immune molecules that reactivate antitumor immune responses by utilizing the host immune system have attracted attention as new therapeutic strategies. CD137 is a costimulatory molecule that reportedly potentiates the antitumor activity of tumor-targeting monoclonal antibodies (mAbs) by enhancing antibody-dependent cellular cytotoxicity. However, it remains unclear whether CD137 stimulates tumor-regulatory activity in gastric cancer. In this study, we investigated the antitumor effects of CD137 stimulation on gastric cancer cells administered tumor-targeting mAbs. Our results showed that human natural killer (NK) cells were activated by expressing CD137 after encountering trastuzumab-coated gastric cancer cells, and that stimulation of activated NK cells in the presence of trastuzumab and recombinant human CD137 ligand (rhCD137L) enhanced cytotoxicity and release of cytokines (IFN-γ, TNF, granzyme A, or granzyme B) as compared with activated NK cells with trastuzumab alone (p < 0.05). By combination treatment with rhCD137L, similar effects were obtained regarding cancer cell cytotoxicity in the presence of cetuximab (p < 0.01). Moreover, we revealed that CD137 expression was dependent upon the affinity between the Fc portion of the antibodies and FcγRIIIa of NK cells based on results indicating that human IgG1 and IgG3 subclasses enhanced CD137 expression (p < 0.001). These results confirmed that FcγRIIIA polymorphisms (158 V/V) enhanced CD137 expression to a greater degree than 158 F polymorphisms (p = 0.014). Our results suggested that CD137 stimulation could promote the effects of tumor-targeting mAbs in gastric cancer, and that further investigation of antibody binding affinity and in vivo activities might improve therapeutic strategies related to the treatment of gastric cancer patients.

A Novel Role for the Interleukin-1 Receptor Axis in Resistance to Anti-EGFR Therapy.

Cetuximab (CTX) is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), commonly used to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, objective remissions occur only in a minority of patients and are of short duration, with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous study on CRC xenopatients associated poor response to CTX with increased abundance of a set of pro-inflammatory cytokines, including the interleukins IL-1A, IL-1B and IL-8. Stemming from these observations, our current work aimed to assess the role of IL-1 pathway activity in CTX resistance. We employed a recombinant decoy TRAP IL-1, a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of the IL-1-receptor (IL-1R1), able to sequester IL-1 directly from the medium. We generated stable clones expressing and secreting a functional TRAP IL-1 into the culture medium. Our results show that IL-1R1 inhibition leads to a decreased cell proliferation and a dampened MAPK and AKT axes. Moreover, CRC patients not responding to CTX blockage displayed higher levels of IL-1R1 than responsive subjects, and abundant IL-1R1 is predictive of survival in patient datasets specifically for the consensus molecular subtype 1 (CMS1). We conclude that IL-1R1 abundance may represent a therapeutic marker for patients who become refractory to monoclonal antibody therapy, while inhibition of IL-1R1 by TRAP IL-1 may offer a novel therapeutic strategy.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation

BackgroundTEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown.ResultsHere we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections.ConclusionsTaken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.

Comparative Glycopeptide Analysis for Protein Glycosylation by Liquid Chromatography and Tandem Mass Spectrometry: Variation in Glycosylation Patterns of Site-Directed Mutagenized Glycoprotein

Glycosylation is one of the most important posttranslational modifications for proteins, including therapeutic antibodies, and greatly influences protein physiochemical properties. In this study, glycopeptide mapping of a reference and biosimilar recombinant antibodies (rAbs) was performed using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and an automated Glycoproteome Analyzer (GPA) algorithm. The tandem mass analyses for the reference and biosimilar samples indicate that this approach proves to be highly efficient in reproducing consistent analytical results and discovering the implications of different rAb production methods on glycosylation patterns. Furthermore, the comparative analysis of a mutagenized rAb glycoprotein proved that a single amino acid mutation in the Fc portion of the antibody molecule caused increased variations in glycosylation patterns. These variations were also detected by the mass spectrometry method efficiently. This mapping method, focusing on precise glycopeptide identification and comparison for the identified glycoforms, can be useful in differentiating aberrant glycosylation in biosimilar rAb products.

Abstract 5611: Inhibition of IL-6 trans-signaling in HNSCC

Abstract Various types of cancers including head and neck squamous cell carcinomas (HNSCCs) are highly inflammatory, and the cytokine interleukin-6 (IL-6) is believed to be associated with this inflammatory phenotype. Unfortunately, global inhibition of IL-6 signaling has demonstrated little to no clinical efficacy in various types of cancers. Previous studies in the field have reported major differences between IL-6 classical signaling through the membrane bound IL-6R (mIL-6R) and IL-6 trans-signaling through the soluble IL-6R (sIL-6R), and the idea that these 2 pathways may have vastly different functions. Recent studies in models of inflammation have proposed that IL-6 classical signaling serves an anti-inflammatory/homeostatic role while IL-6 trans-signaling stimulates proinflammatory events. Therefore, IL-6 trans-signaling (and not classical signaling) may be associated with the proinflammatory phenotype observed in HNSCCs and may also be responsible for the increased tumor progression and treatment resistance frequently encountered with this disease. The goal of this work is to investigate if selective inhibition of IL-6 trans-signaling will demonstrate superior antitumor efficacy compared to global inhibition of IL-6 signaling in HNSCC cells and if selective inhibition of IL-6 trans-signaling will enhance the antitumor efficacy of chemo/radiotherapy in HNSCC cells. We observed that the HNSCC cell lines SQ20B, SCC-25, FaDu and Cal-27 all express mIL-6R and secrete IL-6, sIL-6R, and the natural inhibitor of sIL-6R signaling–soluble glycoprotein 130 (sgp130)–at varying levels. Simultaneous inhibition of IL-6 classical and trans-signaling using the IL-6R antagonist tocilizumab, and selective inhibition of IL-6 trans-signaling using the extracellular portion of gp130 fused to the Fc portion of human IgG1 (sgp130-Fc), showed no significant effects on cell survival in vitro. However, sgp130-Fc significantly suppressed SQ20B tumor growth in athymic nude mice while tocilizumab had no effect. Significant antitumor effects of sgp130-Fc were also observed in a murine SCCVII/C3H syngeneic mouse model. Cell lines treated with cisplatin and/or X-ray radiation (0-4 Gy) increased IL-6, sIL-6R and sgp130 in a dose-dependent manner and sgp130-Fc significantly increased SQ20B tumor response to chemo/radiotherapy in vivo. Altogether, this work suggests that selective inhibition of IL-6 trans-signaling using sgp130-Fc may be more effective than global IL-6 inhibition with respect to suppressing HNSCC tumor growth and warrants further study as an adjuvant to standard HNSCC therapy. Citation Format: Andrean L. Simons, Rachel A. Dahl, Madelyn Espinosa-Cotton, Samuel N. Rodman, Stefan Rose-John. Inhibition of IL-6 trans-signaling in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5611.

Abstract 3833: SEA-BCMA: A highly active enhanced antibody for multiple myeloma

Abstract Despite a number of new therapies for multiple myeloma (MM) most patients relapse, require multiple lines of therapy, and ultimately succumb to disease. Therapies that are well tolerated and active remain an unmet need. Currently, successful MM therapies combine agents with different mechanisms of action and safety profiles. Therapeutic antibodies have recently altered the MM treatment paradigm, allowing patients to achieve deeper responses with minimal added toxicity. SEA-BCMA is a humanized afucosylated IgG1 antibody targeting BCMA, which shows preclinical evidence of encouraging activity and tolerability. BCMA is expressed at the surface of plasma cells, and induces proliferative signals through the binding of its ligands APRIL and BAFF. SEA-BCMA acts through three mechanisms of action. One, it engages in increased binding to FcγRIII through SEA technology leading to enhanced antibody dependent cellular cytotoxicity. Two, it mediates antibody dependent cellular phagocytosis. Three, it blocks the proliferative signals from BCMA ligand binding. This antibody shows activity in all seven tumor xenograft models tested, inducing tumor delays at doses as low as 0.1mg/kg, and generating prolonged survival and durable regressions with repeat dosing. SEA-BCMA is active on tumor xenografts expressing as few as 2000 surface copies of BCMA antigen. Both effector function and ligand blocking contribute to overall in vivo activity. In the absence of effector cell recruitment, ligand blocking alone can induce prolonged durable regressions. In addition, SEA-BCMA can target MM cells in the presence of soluble BCMA. SEA-BCMA is tolerated up to 100mg/kg in cynomolgus monkey, reflecting lack of toxicity from cellular interactions with the Fc portion of the Ab, since SEA-BCMA does not bind to the cynomolgus monkey antigen. SEA-BCMA displays a 12-day half-life in these animals. In vitro testing with human PBMCs or bone marrow mononuclear cells induced minimal cytokine production in the presence of BCMA target. In summary, SEA-BCMA is highly active and well tolerated in preclinical models and is a strong candidate for treatment of MM patients. Citation Format: Heather Van Epps, Martha Anderson, Changpu Yu, Kerry Klussman, Lori Westendorf, Chris Carosino, Luke Manlove, Julia Cochran, Jason Neale, Dennis Benjamin, Maureen Ryan. SEA-BCMA: A highly active enhanced antibody for multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3833.

PO-263 Assessment of the accumulation of hyaluronan in the tumour microenvironment (TME) of solid tumours

IntroductionHyaluronan (HA), a primary component of the extracellular matrix, can accumulate in a variety of solid malignancies, where histological analyses of patient tumour tissues demonstrated that HA levels may correlate with patient outcomes. HA accumulation in the TME may be associated with poor prognosis. Preclinical studies demonstrated elevated TME HA levels increased tumour interstitial pressure, reduced tumour blood flow, and resulted in poor penetration of anticancer agents and immune cells. A PEGylated recombinant human hyaluronidase, pegvorhyaluronidase alfa (PEGPH20), depolymerizes HA, increases tumour vascular perfusion, and may increase access and anti-tumour efficacy of cytotoxic and immunotherapies.Material and methodsPegvorhyaluronidase alfa is being evaluated in clinical studies for pancreatic, breast, lung, cholangiocarcinoma, and gastric cancers. To broaden the spectrum of possible tumour types that may benefit from pegvyorhyaluronidase alfa, we assessed the prevalence and accumulation of HA in 3633 human histological samples in 22 solid tumour types. HA levels were measured using tissue microarrays (TMAs) stained with a proprietary HA probe, HTI–601, a recombinant immunoadhesin containing a modified HA–specific link domain from TNF-Stimulated Gene 6 protein (TSG–6) and the Fc portion of human IgG1. The resulting HA probe was biotinylated for use in an affinity-based histochemistry method. HA staining was evaluated with research-grade histological image analysis software. HA levels associated with distinct tumour compartments (stroma or tumour cells) were assessed. HA accumulation associated with immune infiltrates present in the TMA cores was also evaluated.Results and discussionsDistribution of HA accumulation across compartments fell into 4 main arrangements:- For desmoplastic tumour indications (pancreatic ductal adenocarcinoma, cholangiocarcinoma), the stroma was associated with high percentage of surface area stained with HTI-601 relative to the tumour cells;- Comparable distribution of HA staining in both tumour cells and stroma (gall bladder and glioblastoma);- Sub-clusters of HA staining within a specific tumour type (NSCLC non-squamous);- Distribution bias towards increased or decreased HA intensity staining (head and neck excluding nasopharyngeal cavities and renal cell carcinoma, respectively).ConclusionUnderstanding HA prevalence and accumulation in solid tumours may potentially identify malignancies that may benefit from pegvorhyaluronidase alfa therapy targeting HA.

Abstract 5609: ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression

Abstract Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in a Phase 1 clinical trial (NCT02748837) in patients with solid tumors that are glypican-3 (GPC3)-positive. ERY974 consists of a common light chain but has two different heavy chains that each recognize a different protein, GPC3 or CD3. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface to induce cellular cytotoxicity mediated by the potent effector function of T cells. The Fc portion of ERY974 is modified to eliminate FcγR binding and prevent GPC3-independent Fc-mediated effector function. However, binding to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head &amp; neck, hepatic, and esophageal cancer-derived tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. In a cohort expansion of the Phase 1 trial, the activity of ERY974 will be examined in gastric cancer; however, individual clinical samples do not show uniform expression levels of GPC3 in tumor lesions, reflecting the heterogenous and complex structure of patients' tumors. To predict the potential efficacy of ERY974 in gastric cancer patients, we examined whether ERY974 alone or in combination with chemotherapy, could show substantial activity against heterogenous tumors with different GPC3 expression profiles using patient-derived xenografts (PDX) of gastric cancer. Method &amp; results: We evaluated the antitumor effect of ERY974 in PDX gastric cancer tumors that have high, moderate, or low expression levels of GPC3 in a NOD-SCID mouse model injected with human T cells that were expanded in vitro using CD3/CD28 beads. The expression of GPC3 was evaluated by immunohistochemistry and quantitative RT-PCR. The efficacy of ERY974 monotherapy seemed to correlate with the GPC3 expression levels in each PDX tumor. The combination of ERY974 and chemotherapy, such as paclitaxcel, cisplatin or capecitabine, showed increased antitumor activity compared with ERY974 or chemotherapy alone. Conclusion: These preclinical data support the possibility that ERY974 alone or in combination with chemotherapy will demonstrate activity in patients with gastric cancer, and the GPC3 expression profile might be a useful predictive biomarker of ERY974 efficacy for patient selection. Citation Format: Azuma Yumiko, Yuji Sano, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Miyazaki, Junko Shinozuka, Etsuko Fujii, Atsuhiko Kato, Takahiro Ishiguro, Shohei Kishishita, Junichi Nezu, Yoshiki Kawabe, Mika Endo. ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5609.

Differences in IgG Fc Glycosylation Are Associated with Outcome of Pediatric Meningococcal Sepsis.

ABSTRACTPediatric meningococcal sepsis often results in morbidity and/or death, especially in young children. Our understanding of the reasons why young children are more susceptible to both the meningococcal infection itself and a more fulminant course of the disease is limited. Immunoglobulin G (IgG) is involved in the adaptive immune response against meningococcal infections, and its effector functions are highly influenced by the glycan structure attached to the fragment crystallizable (Fc) region. It was hypothesized that IgG Fc glycosylation might be related to the susceptibility and severity of meningococcal sepsis. Because of this, the differences in IgG Fc glycosylation between 60 pediatric meningococcal sepsis patients admitted to the pediatric intensive care unit and 46 age-matched healthy controls were investigated, employing liquid chromatography with mass spectrometric detection of tryptic IgG glycopeptides. In addition, Fc glycosylation profiles were compared between patients with a severe outcome (death or the need for amputation) and a nonsevere outcome. Meningococcal sepsis patients under the age of 4 years showed lower IgG1 fucosylation and higher IgG1 bisection than age-matched healthy controls. This might be a direct effect of the disease; however, it can also be a reflection of previous immunologic challenges and/or a higher susceptibility of these children to develop meningococcal sepsis. Within the young patient group, levels of IgG1 hybrid-type glycans and IgG2/3 sialylation per galactose were associated with illness severity and severe outcome. Future studies in larger groups should explore whether IgG Fc glycosylation could be a reliable predictor for meningococcal sepsis outcome.

Past, Present, and Future of Rituximab-The World's First Oncology Monoclonal Antibody Therapy.

Rituximab is a chimeric mouse/human monoclonal antibody (mAb) therapy with binding specificity to CD20. It was the first therapeutic antibody approved for oncology patients and was the top-selling oncology drug for nearly a decade with sales reaching $8.58 billion in 2016. Since its initial approval in 1997, it has improved outcomes in all B-cell malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia. Despite widespread use, most mechanistic data have been gathered from in vitro studies while the roles of the various response mechanisms in humans are still largely undetermined. Polymorphisms in Fc gamma receptor and complement protein genes have been implicated as potential predictors of differential response to rituximab, but have not yet shown sufficient influence to impact clinical decisions. Unlike most targeted therapies developed today, no known biomarkers to indicate target engagement/tumor response have been identified, aside from reduced tumor burden. The lack of companion biomarkers beyond CD20 itself has made it difficult to predict which patients will respond to any given anti-CD20 antibody. In the past decade, two new anti-CD20 antibodies have been approved: ofatumumab, which binds a distinct epitope of CD20, and obinutuzumab, a mAb derived from rituximab with modifications to the Fc portion and to its glycosylation. Both are fully humanized and have biological activity that is distinct from that of rituximab. In addition to these new anti-CD20 antibodies, another imminent change in targeted lymphoma treatment is the multitude of biosimilars that are becoming available as rituximab’s patent expires. While the widespread use of rituximab itself will likely continue, its biosimilars will increase global access to the therapy. This review discusses current research into mechanisms and potential biomarkers of rituximab response, as well as its biosimilars and the newer CD20 binding mAb therapies. Increased ability to assess the effectiveness of rituximab in an individual patient, along with the availability of alternative anti-CD20 antibodies will likely lead to dramatic changes in how we use CD20 antibodies going forward.

A carbon ink screen-printed immunoelectrode for Dengue virus NS1protein detection based on photosynthesized amine gold nanoparticles

Dengue virus (DENV) is a reemerging mosquito-borne disease that is endemic in more than 125 countries, affecting 200 million people per year. Screening testing has been a good attempt to minimize the impact caused by high morbity and mortality rates of DENV. In this study, a simple and disposable label-free electrochemical immunosensor based on a carbon ink graphite screen-printed electrode (SPE) one-step fabricated was developed for detection of non-structural 1 protein (NS1). The SPE surface was modified by drop casting, depositing a colloidal suspension containing amine-functionalized gold nanoparticles (AuNP-NH2). AuNPs were synthetized by a photoinduced physical method, illuminating preformed gold seeds with a light-emitting diode (LED,) at blue region, by using the polyethyleneimine (NH2) as reductor and stabilizing agent. UV-VIS spectroscopy and Transmission Electron Microscopy (TEM) were used to characterize the amine AuNPs. Electrocatalytic activity of AuNPs allowed more sensitivity for a label-free detection of NS1 by square wave voltammetry (SWV), with linear response from 0.1 to 2 µg mL-1. It was found a good linearity (coefficient of correlation of 0.995 (p&lt;0.01) and a limit of detection of 0.03 µg mL-1 NS1 for analytical responses. AuNP-NH2 synthesis provided an easy oriented immobilization of anti-NS1 antibodies by Fc portion, resulting in a simple fabrication immunosensor with relative high performance and feasibility for early diagnostic of DENV.

Evolution of Therapeutic Antibodies, Influenza Virus Biology, Influenza, and Influenza Immunotherapy

This narrative review article summarizes past and current technologies for generating antibodies for passive immunization/immunotherapy. Contemporary DNA and protein technologies have facilitated the development of engineered therapeutic monoclonal antibodies in a variety of formats according to the required effector functions. Chimeric, humanized, and human monoclonal antibodies to antigenic/epitopic myriads with less immunogenicity than animal-derived antibodies in human recipients can be produced in vitro. Immunotherapy with ready-to-use antibodies has gained wide acceptance as a powerful treatment against both infectious and noninfectious diseases. Influenza, a highly contagious disease, precipitates annual epidemics and occasional pandemics, resulting in high health and economic burden worldwide. Currently available drugs are becoming less and less effective against this rapidly mutating virus. Alternative treatment strategies are needed, particularly for individuals at high risk for severe morbidity. In a setting where vaccines are not yet protective or available, human antibodies that are broadly effective against various influenza subtypes could be highly efficacious in lowering morbidity and mortality and controlling unprecedented epidemic/pandemic. Prototypes of human single-chain antibodies to several conserved proteins of influenza virus with no Fc portion (hence, no ADE effect in recipients) are available. These antibodies have high potential as a novel, safe, and effective anti-influenza agent.

Human IgG Increases Virulence of Streptococcus pyogenes through Complement Evasion

Streptococcus pyogenes is an exclusively human pathogen that can provoke mild skin and throat infections but can also cause fatal septicemia. This gram-positive bacterium has developed several strategies to evade the human immune system, enabling S. pyogenes to survive in the host. These strategies include recruiting several human plasma proteins, such as the complement inhibitor, C4b-binding protein (C4BP), and human (hu)-IgG through its Fc region to the bacterial surface to evade immune recognition. We identified a novel virulence mechanism whereby IgG-enhanced binding of C4BP to five of 12 tested S. pyogenes strains expressed diverse M proteins that are important surface-expressed virulence factors. Importantly, all strains that bound C4BP in the absence of IgG bound more C4BP when IgG was present. Further studies with an M1 strain that additionally expressed protein H, also a member of the M protein family, revealed that binding of hu-IgG Fc to protein H increased the affinity of protein H for C4BP. Increased C4BP binding accentuated complement downregulation, resulting in diminished bacterial killing. Accordingly, mortality from S. pyogenes infection in hu-C4BP transgenic mice was increased when hu-IgG or its Fc portion alone was administered concomitantly. Electron microscopy analysis of human tissue samples with necrotizing fasciitis confirmed increased C4BP binding to S. pyogenes when IgG was present. Our findings provide evidence of a paradoxical function of hu-IgG bound through Fc to diverse S. pyogenes isolates that increases their virulence and may counteract the beneficial effects of IgG opsonization.

Augmenting vaccine immunogenicity through the use of natural human anti-Rha antibodies and monoclonal Fc domains

Abstract In order to obtain an effective cellular and humoral immune response, a vaccine construct has to be captured, processed and presented by antigen presenting cells (APCs) through their major histocompatibility complex (MHC) molecules. One effective way to enhance antigen presentation is through an antibody-dependent antigen uptake mechanism by targeting FcγR receptors of APCs that can bind with the Fc portion of an immune complexed antibody. Anti-Rhamnose (Anti-Rha) antibody is among the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. Binding rhamnose of a rhamnose-containing vaccine with murine anti-Rha antibody has previously been proven effective for capturing and processing of antigen by APCs via stimulatory Fc receptors. We show that human anti-Rha antibodies enhance the uptake of rhamnose-containing antigen by murine APCs in vitro. In-vivo, passive transfer of human anti-Rha antibodies into mice improves the priming of T cells to Rha-antigen. We synthesized and tested an anti-cancer vaccine containing a lipoglycopeptide cancer antigen in a Rha-bearing liposome and demonstrated improved immunogenicity. Also, we showed that direct conjugation of the Fc domain of an antibody to a liposomal vaccine instead of rhamnose can be a good approach for efficient uptake and presentation, as we can bypass the need for anti-Rha antibodies. A liposomal vaccine bearing IgG3Fc has been found to be equally effective as the Rha-vaccine at priming CD4+ T cells. Priming of CD8+ T cells for IFN-γ release and tumor cytotoxicity was promising in comparison to anti-Rha antibody targeting.

How does abatacept really work in rheumatoid arthritis?

The purpose of this review is to summarize the current knowledge concerning the mechanisms of action of Abatacept in patients with rheumatoid arthritis. Abatacept (CTLA-4Ig) represents a soluble, recombinant, fully humanized fusion protein, comprising the extracellular domain of CTLA-4 and the Fc portion of IgG1. Abatacept binds to the costimulatory molecules CD80 and CD86 on antigen-presenting cells (APC), thereby blocking interaction with CD28 on T cells. In humans, Abatacept treatment was shown to be effective in patients with various autoinflammatory diseases including rheumatoid arthritis. Although the prevention of T-cell activation by interfering with signaling via CD28 still represents the main mechanism of action Abatacept acts on additional cell populations including regulatory T cells (Treg), monocytes/macrophages, osteoclasts, and B cells. Effects of Abatacept on other cell populations besides T cells have to be taken into account and might represent a valuable contribution to the therapeutic success.