Cell-mediated Cytotoxic Response Research Articles

The Early Antibody-Dependent Cell-Mediated Cytotoxicity Response Is Associated With Lower Viral Set Point in Individuals With Primary HIV Infection.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune response largely mediated by natural killer (NK) cells that can lyse target cells and combat tumors and viral infections. However, the role of ADCC in response to primary HIV infection is poorly understood. In the present study, we explored the ADCC response and evaluated its characteristics in 85 HIV-infected individuals, including 42 with primary infections. Our results showed that ADCC occurs during acute infection, and the earliest ADCC response to a single peptide was detected at 52 days. Primary HIV-infected individuals exhibiting ADCC responses had lower viral set points than those with no ADCC response, and functional analyses demonstrated that the ADCC response could significantly inhibit viral infection during primary HIV infection. HIV epitopes that provoked the ADCC response were determined and three relatively conserved epitopes (HNVWATYACVPTDPNPQE, TSVIKQACPKISFDPIPI, and VVSTQLLLNGSLAEEEII) from the surface of the three-dimensional structure of the HIV Env protein were identified. Overall, our data indicate that ADCC responses may be significant for the control of HIV from an early stage during infection. These findings merit further investigation and will facilitate improvements in vaccines or therapeutic interventions against HIV infection.

Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane

Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.

Dissecting FcγR Regulation through a Multivalent Binding Model.

Many immune receptors transduce activation across the plasma membrane through their clustering. With Fcγ receptors (FcγRs), this clustering is driven by binding to antibodies of differing affinities that are in turn bound to multivalent antigen. As a consequence of this activation mechanism, accounting for and rationally manipulating immunoglobulin (Ig)G effector function is complicated by, among other factors, differing affinities between FcγR species and changes in the valency of antigen binding. In this study, we show that a model of multivalent receptor-ligand binding can effectively account for the contribution of IgG-FcγR affinity and immune complex valency. This model in turn enables us to make specific predictions about the effect of immune complexes of defined composition. In total, these results enable both rational immune complex design for a desired IgG effector function and the deconvolution of effector function by immune complexes.

Adverse outcome of AML with aberrant CD16 and CD56 NK cell marker expression.

Natural killer cells mediate antibody-dependent cell-mediated cytotoxicity, and CD16 exerts key functions to induce antibody-dependent cell-mediated cytotoxicity response. Because the prognostic relevance of aberrant CD16 expression in AML patients at diagnosis is unknown, we analyzed 325 AML patients undergoing intensive chemotherapy for aberrant CD16+ and CD56+ natural killer-cell marker expression. CD56+ AML patients had inferior median event-free (EFS; P=0.0699) and overall survival (OS; 10.9 versus 20.6months; P=0.0132). Patients expressing CD16 had worse median EFS (P=0.0622) and OS (13.0 versus 45.9months; P=0.0277). EFS for CD16+/CD56+ patients was 5.7months compared with 7.1months for CD16-/CD56- (P=0.3690), and OS was 10.6months for CD16+/CD56+ patients compared with 52.2months for CD16-/CD56- patients (P=0.0311). Patients with CD16+/CD56+ expression had a lower probability to achieve complete remission after 2 induction cycles (52% versus 72%). Our data suggest that AML patients with aberrant CD16 and CD56 expression have adverse survival outcomes.

Genes related to cell-mediated cytotoxicity and interferon response are induced in the retina of European sea bass upon intravitreal infection with nodavirus

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the central nervous system, is considered one of the most serious viral diseases in marine aquaculture. Although some studies have localized NNV in the retina cells, none has dealt with immunity in the retina. Thus, for the first time, we intravitreally infected healthy specimens of European sea bass (Dicentrarchus labrax) with NNV with the aim of characterizing the immune response in the retina. Ultrastructural analysis detected important retinal injuries and structure degradation, including pycnosis, hydropic degeneration and vacuolization in some cell layers as well as myelin sheaths in the optic nerve fibres. Immunohistochemistry demonstrated that NNV replicated in the eyes. Regarding retinal immunity, NNV infection elicited the transcription of genes encoding proteins involved in the interferon (IFN) and cell-mediated cytotoxicity (CMC) responses as well as B and T cell markers, demonstrating that viral replication influences innate and adaptive responses. Further studies are needed to understand the retina immunity and whether the main retinal function, vision, is affected by nodavirus.

Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function.

Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. End points included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell-mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids, and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures, and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol. We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

KIR Genes and Their Ligands Predict the Response to Anti-EGFR Monoclonal Antibodies in Solid Tumors.

Killer-cell immunoglobulin-like receptors (KIRs) regulate the killing function of natural killer cells, which play an important role in the antibody-dependent cell-mediated cytotoxicity response exerted by therapeutic monoclonal antibodies (mAbs). However, it is unknown whether the extensive genetic variability of KIR genes and/or their human leukocyte antigen (HLA) ligands might influence the response to these treatments. This study aimed to explore whether the variability in KIR/HLA genes may be associated with the variable response observed to mAbs based anti-epidermal growth factor receptor (EGFR) therapies. Thirty-nine patients treated with anti-EGFR mAbs (trastuzumab for advanced breast cancer, or cetuximab for advanced colorectal or advanced head and neck cancer) were included in the study. All the patients had progressed to mAbs therapy and were grouped into two categories taking into account time to treatment failure (TTF ≤6 and ≥10 months). KIR genotyping (16 genetic variability) was performed in genomic DNA from peripheral blood by PCR sequence-specific primer technique, and HLA ligand typing was performed for HLA-B and -C loci by reverse polymerase chain reaction sequence-specific oligonucleotide methodology. Subjects carrying the KIR/HLA ligand combinations KIR2DS1/HLAC2C2-C1C2 and KIR3DS1/HLABw4w4-w4w6 showed longer TTF than non-carriers counterparts (14.76 vs. 3.73 months, p < 0.001 and 14.93 vs. 4.6 months, p = 0.005, respectively). No other significant differences were observed. Two activating KIR/HLA ligand combinations predict better response of patients to anti-EGFR therapy. These findings increase the overall knowledge on the role of specific gene variants related to responsiveness to anti-EGFR treatment in solid tumors and highlight the importance of assessing gene polymorphisms related to cancer medications.

Abstract 4013: Transcriptionally mediated effects of radiation on the expression of immune susceptibility markers in murine and human melanoma

Abstract Radiation therapy (RT) may enhance tumor susceptibility to immune response. We and others have observed a cooperative interaction between RT and various immunotherapies, which appears dependent on the relative sequencing of each therapy. We reported a synergistic interaction between RT and the antibody-dependent cell-mediated cytotoxicity (ADCC) response to tumor-specific monoclonal antibodies (mAb) in murine melanoma. We reported enhanced cooperative effect when immunotherapy is administered 6-10 days after RT compared to administration on days 1-5 or 11-15. We hypothesized that this might reflect a delayed, transcriptionally-mediated effect of RT on the expression of tumor markers of immune susceptibility. In this study, we investigated the effect of RT on a variety of immune susceptibility markers at both transcriptional and post-translational levels to explore mechanisms behind the observed RT-induced changes. We used flow cytometry to examine the time course of phenotypic changes in immune susceptibility markers in B78 murine melanoma cells following in vitro RT. We observed a time- and RT dose-dependent increase in the expression of specific death receptors (Fas, DR5), as well as T cell co-stimulatory/co-repressor ligands (PD-L1, CD80). The timing of these changes correlated with tumor susceptibility to ADCC immune response in vivo. All protein expression changes observed by flow cytometry were found to occur over a similar time course by quantitative polymerase chain reaction (qPCR), suggesting that RT-induced protein expression changes were mediated by changes in transcriptional activity. Using high throughput qPCR, we observed a similar time course of transcriptional effects in additional markers of tumor cell immune susceptibility, including Fas, MHC I, CD40, and others. We compared the effect of RT on these markers in human and mouse melanoma cell lines and observed a comparable time course for transcript-level changes across species. This study sheds light on the mechanistic basis of time sensitivity in the interaction of RT with ADCC in vivo and suggests opportunities to enhance anti-tumor immune response by combining RT and immunotherapeutic agents. Such findings bear relevance for research investigating the potential role for RT in driving and optimizing the response to various cancer immunotherapies. Citation Format: Lauryn R. Werner, Monica Gressett, Maureen Riegert, Shyhmin Huang, Joseph G. Kern, Amy Erbe, Paul M. Harari, Paul M. Sondel, Zachary S. Morris. Transcriptionally mediated effects of radiation on the expression of immune susceptibility markers in murine and human melanoma. [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 4013.

Killer-cell Inmunoglobulin-like Receptor (KIR) genes as a predictor of response to the anti-HER therapy in solid tumors.

e13024Background: KIRs are an important receptor in natural killer (NK) cells; these cells play a role in antibody-dependent cell-mediated cytotoxicity (ADCC) response due to monoclonal antibodies therapy. It is unknown whether the extensive polymorphism KIRs and/or their human leukocyte antigen (HLA) ligands might influence the response to treatment with monoclonal antibodies (MABs). Methods: We select in this study 39 patients treated with MABs, anti-human epidermal growth factor receptor (anti-HER/EGFR: Trastuzumab for advanced breast cancer and Cetuximab for advanced colorectal and advanced head and neck cancer). All the patients had progressed to the MABs therapy and were grouped in two groups taking into account the time to treatment failure (TTF) duration ( or = 10m).KIR genotyping (16 polymorphisms) was performed by PCR sequence-specific primer technique and KIR-ligand typing was performed for HLA-B & -C loci by reverse PCRSSO methodology and for HLA-C alleles by direct sequencing....

Abstract A032: Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients

Abstract Background: Ganglioside GD2 is a glycolipid highly expressed on neuro-ectodermal tumors including melanoma and neuroblastoma (NB). It is ranked on position 12 of the list of tumor associated antigens by the NCI. Anti-GD2 antibody (Ab) ch14.18/CHO targets this antigen and effectively orchestrates innate immune effector mechanisms against GD2 expressing malignancies. However, one on target effect of anti-GD2 Abs is the induction of neuropathic pain when applied as short term infusion (STI) requiring co-medication with intravenous morphine. Here we investigated whether long-term infusion (LTI) of anti-GD2 Ab ch14.18/CHO may have an improved pain toxicity profile and at the same time mediate an effective immune response in patients (pts) with high risk relapsed/refractory NB which translates to objective clinical responses and an improved survival. Methods: 97 pts received 6x106 IU/m2 sc IL2 (d1-5; 8-12), LTI of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in an ongoing SIOPEN Phase II study (APN311-202) (NCT01701479) (44 pts) and a closed single center program (53 pts) (APN311-303). Response assessments followed INRG criteria. Serum ch14.18/CHO concentration-time curves were determined by validated ELISA methods and pharmacokinetics parameter were analyzed using standard non-compartmental methods. Fcγ;-receptor polymorphisms FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) and patient-specific antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and whole blood cytotoxicity (WBT) responses were determined. Results: LTI was associated with low pain scores and a decreasing morphine usage compared to STI. Clinical overall responses were 30% (APN311-303) and 31% (APN311-202). The survival update of the APN311-303 cohort revealed a 1-y &amp; 4-y OS of 94.2±3.2% &amp; 60.9±9.0% (median FU 2.9y [0.7-5.2y]) and a 1-y &amp; 4-y PFS of 54.4±6.9% &amp; 32.3%±6.9% (median FU 2.8y [0.7 - 4.9y]). Median TTP was 571d (95% CI: 232.7d). The comparator is the reported historical gold standard for relapsed refractory NB patients with 1-y &amp; 4-y PFS of 19±2% &amp; 8±3% and OS of 56±3% &amp; 14±4% and a median TTP of 63 d (95% CI: 56.8d). NB pts with high affinity FCGR alleles and an increase in ADCC (cut off 15%) are associated with longer PFS and OS rates (p&amp;lt;0.03; p&amp;lt;0.005), supporting NK-cell mediated ADCC as the mechanism of action. Parameters of immune modulation (CDC, and WBT) and PK of ch14.18/CHO were comparable between APN311-202 and -303 cohorts. PK of ch14.18/CHO was analyzed in cycle 1: Cmax=12.2±0.4μg/ml, t½=8.4±1.1 d, AUC=145.3± 5.8 μg*d/ml, Vdss=9.3±0.5L/m2. A pro-inflammatory cytokine response (IL-2, IL-6, IL-8, IFNγ) translated into the expansion of effector NK- (3x) and T-cells (2x). We observed HACA in 17/97 pts (17.5%) of which only 9/97 (9.3%) were neutralizing with respect to the inhibition of CDC and WBT activity. In HACA negative patients, levels of ch14.18/CHO and functional parameters (CDC and WBT) analyzed before subsequent treatment cycles indicate persistent anti-NB activity for the entire treatment period. Conclusion: LTI of ch14.18/CHO has an improved pain toxicity profile and at the same time is active and effective in high-risk relapsed/refractory NB. Citation Format: Holger N. Lode, Dominique Valteau-Couanet, Alberto Garaventa, Juliet Gray, Victoria Castel, Isaac Yaniv, Nikolai Siebert, Christian Jensen, Stefanie Endres, Lena Pill, Christin Eger, Diana Seidel, Madlen Jüttner, Silke Kietz, Karoline Ehlert, Evelyne Janzek, Carla Manzitti, Ina Müller, Hans Loibner, Ruth Ladenstein. Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A032.

Cancer Related Fatigue and Clinical Outcome of Dendritic Cell Vaccine in Combination with Cytokine-Induced Killer Cell Therapy in Cancer Patients

Cancer related fatigue significantly disrupts normal life for a portion of cancer survivors, and may persist for many years with cancer patients. Fatigue lasts longer than other treatment side-effects. There are increasing reports of using immunocyte therapy to treat cancer, and if it could be alternative relieve therapy in persons with cancer related fatigue is not so clear. Herein, we retrospectively evaluated the improvement of cancer related fatigue and safety of this therapy administered to cancer patients. 539 patients were treated with immunocyte therapy after conventional treatment. Methods: The delayed-type hyper-sensitivity (DTH) skin test was used to measure the immune response. Investigated into cancer related fatigue, physical strength, appetite and sleep as clinical efficacy. Results: The DTH skin tests showed that the positive cell-mediated cytotoxicity response rate was 82.7%. A large proportion of patients’ cancer related fatigue (80%); appetite (94.1%), sleeping status (96.6%) and metal state (92.9%) were improved. Adverse reactions were fever (35.1%), insomnia (21.4%), anorexia (15.7%), arthralgia (8%), rash (3.3%). DC (dendritic cell) therapy combined with CIK (cytokine-induced killer cell) was observed no toxicity in patients. Conclusion: This is the first time we reported combination of DC and CIK therapy to relieve the cancer related fatigue of patients after conventional therapy (surgery, radiotherapy, and chemotherapy). It may be an effective immunotherapy to reduce adverse reactions and a safety treatment. The data of CIK treatment time and CIK cell number provide strong support for further clinical treatment.

MEN1112/OBT357, an Anti Bst1/CD157 Humanized Antibody Inducing Acute Myelogenous Leukemia (AML) Blast Depletion in an Autologous Ex Vivo Assay: A Potential New Targeted Therapy for AML

Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. Bst1/CD157 is a protein belonging to the ADP-ribosyl-cyclase family expressed on monocytes and neutrophils. This antigen was shown to be also expressed in peripheral blood (PB) and bone marrow (BM) blasts of acute myeloid leukemia (AML) patients either at primary diagnosis or at relapse(1,2,3). MEN1112/OBT357 is a humanized, de-fucosylated antibody targeting Bst1/CD157 with high affinity and developed to generate antibody dependent cell-mediated cytotoxicity (ADCC) response against AML blasts. Peripheral blood (PB) and bone marrow (BM) samples of 38 AML patients (29 at diagnosis, 6 at relapse, 3 resistant), have been analyzed for the expression of Bst1/CD157 on AML blast cells by fluorescence-activated cell sorting (FACS) using a PE conjugated form of MEN1112/OBT357. Bst1/CD157 expression has been confirmed in 91% and 96% of PB and BM AML samples, respectively. Furthermore, statistical analysis demonstrated that monocyte-oriented blasts are characterized by a brighter expression of Bst1/CD157 compared to blasts of non-monocytic lineage. The efficacy of MEN1112/OBT357 in depleting AML blasts was evaluated through FACS analysis in an autologous ex vivo assay performed on whole blood. The assay was set up using blood from healthy donors exposed to 10 μg/ml Rituximab for 18 hours to induce B cell depletion. In the same conditions, the ability of 10 μg/ml MEN1112/OBT357 to induce blasts depletion was tested.In whole PB,MEN1112/OBT357 was able to deplete AML blasts in 15/32 evaluable cases (46%). In BM, MEN1112/OBT357 induced blast depletion in 9/24 evaluable cases (36%). Interestingly, higher depletion rate was observed in relapse/refractory patients. When CD16A-158Phe/Val polymorphisms were analyzed utilizing a sequence based typing (SBT) assay, it was demonstrated that AML blast depletion was independent by FcRg polymorphism. Furthermore, no significant shedding of Bst1/CD157 antigen was observed in sera from AML patients, compared to the sera from patients with other hematologic diseases or healthy donors.In summary, we confirmed the frequent expression of Bst1/CD157 on blasts from AML patients, with the brightest pattern of positivity observed in cases belonging to monocytic differentiation lineage. MEN1112/OBT357 also induced a promising ADCC against AML blasts in an autologous setting, which is independent from FcR g phenotype. Since in vivo the exposure of AML blasts to MEN1112/OBT357 largely exceeds the incubation time of the depletion assay, we expect a further improvement of its anti-leukemic effect in the clinical setting. Based on these results, a phase I study in patients with relapsed or refractory AML has been initiated in December 2014. DisclosuresBellarosa:Menarini Ricerche: Employment. Bressan:Menarini Ricerche: Employment. Wilson:Oxford Biotherapeutics: Employment. Manzini:Menarini Ricerche: Employment. Capriati:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche: Employment.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

Abstract CT129: Association of the FCGR2A and FCGR3A genotypes with trastuzumab benefit in NSABP B-31

Abstract Background: The FCGR3A-158V and FCGR2A-131H alleles are referred to as favorable alleles with regards to trastuzumab response because the FCGR3A-V/V and FCGR2A-H/H genotypes have been associated with higher trastuzumab induced antibody-dependent cell-mediated cytotoxicity (ADCC) response in vitro. The objective of this study was to assess the interaction between these genotypes and the degree of benefit from trastuzumab in a phase III trial which demonstrated the benefit of adding trastuzumab to the standard adjuvant chemotherapy in the treatment of HER2-positive breast cancer (NSABP B-31). Methods: Genotypes for FCGR3A-158V/F and FCGR2A-131H/R alleles were determined in a representative cohort of NSABP clinical trial B-31 which included all available pre-treatment blood samples (N = 1251). Genotypes were determined by Typeplex chemistry and mass spectrometry on the Seqeunom platform. A custom nested PCR design was required to enable specific amplification of the FCGR3A gene due to the extensive homology between the FCGR3A and FCGR3B genes. Cox regression analyses were used to test for genotype-treatment interactions. Endpoint was disease free survival (DFS). Results: The genotyped cohort resembled the entire B-31 cohort based on clinical variables and the degree of benefit from trastuzumab. Results are summarized in the table. While there were trends for interaction between polymorphisms and trastuzumab for both genes, only FCGR3A-158 polymorphism reached statistical significance for interaction (p = 0.005). Conclusion: In NSABP B-31 there was a trend for benefit in the favorable genotypes of the FCGR2A-131 SNP, but only the favorable FCGR3A-158 genotypes showed a significant association with trastuzumab benefit with genotype-treatment interaction. However, polymorphism alone could not identify a subgroup with no benefit from trastuzumab, since even the patients with less favorable genotypes received significant benefit. Support: NCI U10-CA-12027, -69651, -37377, -69974. PA DOH disclaims responsibility for analysis, interpretations, or conclusions. Citation Format: Patrick G. Gavin, Nan Song, Nicole L. Johnson, Corey Lipchik, Seong-Rim Kim, Melanie Finnigan, Hanna Bandos, Jong-Hyeon Jeong, Joseph P. Costantino, Priya Rastogi, Edward H. Romond, Louis Fehrenbacher, Eleftherios P. Mamounas, Sandra M. Swain, D. Lawrence Wickerham, Charles E. Geyer, Jr., Norman Wolmark, Soonmyung Paik, Katherine L. Pogue-Geile. Association of the FCGR2A and FCGR3A genotypes with trastuzumab benefit in NSABP B-31. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT129. doi:10.1158/1538-7445.AM2015-CT129

Dynamics of antigen delivery and the functional roles of L121-adjuvant

This study investigates the intracellular transport of protein antigens facilitated by L121-adjuvants and examines the associated cytotoxic T lymphocyte (CTL) effect. EL4 mouse thymoma cells were treated with L121-adjuvant and stained with AnnexinV-propidium iodide (PI) followed by flow cytometric analysis. The intracellular trafficking dynamics of bovine serum albumin (BSA)-FITC in the J774.A.1 macrophages, influenced by the L121-adjuvant, was visualized by confocal microscopy. The antigen-specific cytotoxic T lymphocyte (CTL) effect induced by the L121-adjuvant was determined by the cleavage-specific fluorogenic caspase substrate. The trafficking of BSA-FITC in the J774A.1 cells by confocal microscopy illustrated that the L121-adjuvant facilitated the intracellular transport of proteins to the subcellular compartments, including the lysosome, endoplasmic reticulum (ER), and the cis-Golgi apparatus. The L121-adjuvant also facilitated antigen delivery to the dendritic cells in the lymph nodes. Immunization of mice with the L121-adjuvant resulted in cell-mediated cytotoxic responses in the target cells, as detected by PhiPhiLux, a fluorogenic caspase substrate. Taken together, the L121-adjuvant improved the dynamics of protein delivery to antigen presenting cells, and also induced caspase activation, thereby illustrating the mechanism of antigen-specific CTL effects.

MEN1112, a Novel Humanized De-Fucosylated Monoclonal Antibody with High Affinity and Specificity for Bst1/CD157 Antigen and Enhanced CD16 Binding

Bst1/CD157 is a GPI-anchored transmembrane protein belonging to the ADP-ribosyl-cyclase family and it is expressed on some blood cells such as monocytes and neutrophils. Importantly, we demonstrated that this antigen is expressed in blast cells from almost 100% of Acute Myeloid Leukemia patients (either at primary diagnosis or at relapse).MEN1112 is a humanized, de-fucosylated antibody targeting the Bst1/CD157 antigen with high affinity and inducing potent in vitro and ex vivo Antibody Dependent Cell-mediated Cytotoxicity (ADCC) responses against AML cell lines and blasts, respectively. MEN1112 was discovered as a mouse Fab antibody from a phage display library raised against recombinant Bst1/CD157 protein. The Fab was selected base upon its ability to recognize recombinant Bst1/CD157 protein by ELISA and for its ability to recognize Bst1/CD157 positive endogenous cancer cell lines by FACS (fluorescence activated cell sorting). The Fab was also chosen based upon its ability to recognize cynomolgus Bst1/CD157 antigen on recombinant and endogenous cells. The selected Fab sequence was humanized and incorporated into the Biowa Potelligent cell line to produce a de-fucosylated full IgG1 version (MEN1112). The binding affinity of MEN1112 for the Bst1/CD157 antigen showed an EC50 of 1nM as assessed by flow cytometry (FACS) analysis using multiple cell lines. No binding was observed on antigen-negative cell lines demonstrating specificity of MEN1112. High affinity of MEN1112 for the recombinant antigen was confirmed in an ELISA assay (EC50 0.4 nM).Beyond the affinity for the antigen, it is of relevance for its ADCC activity that MEN1112 is characterized by an improved affinity for Fc receptors on effector cells over the parental antibody. In particular, MEN1112, lacking fucose residues in its Fc domain, showed an impressive high affinity binding to the Fc receptor CD16A both high affinity and low affinity alleles.Internalization studies demonstrate the slow internalization of the Bst1/CD157 antigen following binding to MEN1112 which favors the ADCC efficacy of the antibody. Overall our results indicate that MEN1112 has the potential to exert anti-leukemia actions through a powerful ADCC in view of: (i) superior affinity for blast cell membrane antigen Bst1/CD157, (ii) enhanced affinity for all variants of CD16A Fc receptor expressed on effector cells and (iii) minimal internalization following antigen binding. DisclosuresAud:Oxford BioTherapeutics: Employment. Dusek:Oxford BioTherapeutics: Employment. Bisht:Oxford BioTherapeutics: Employment. Swaminithan:Oxford BioTherapeutics: Employment. Awdew:Oxford BioTherapeutics: Employment. Trang:Oxford BioTherapeutics: Employment. Lin Lou:Oxford BioTherapeutics: Employment. Manzini:Menarini Ricerche SpA: Employment. Martinez Mogarra:Menarini Biotech srl: Employment. Bressan:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment.

Monoclonal antibody G250 targeting CA Ⅸ: Binding specificity, internalization and therapeutic effects in a non-renal cancer model.

G250 (Girentuximab) is a chimeric IgG1 monoclonal antibody (MAb) currently being evaluated as an immunotherapy for kidney cancer. It targets carbonic anhydrase protein (CA Ⅸ), a transmembrane carbonic anhydrase (CA) isoform, which is regulated by VHL/HIF pathway and hence expressed in the majority of renal cell carcinomas (RCCs) as well as in hypoxic non‑RCC tumours. CA Ⅸ functions in pH regulation and cell migration/invasion, and supports tumour cell survival in hypoxia and/or acidosis. It contains a highly active extracellular catalytic domain (CA) extended N-terminally with a proteoglycan-like region and C-terminally with short transmembrane and intracellular regions. Here we characterize the binding and internalization properties of G250, as well as its therapeutic effects in animal model, and discuss the impact of G250‑mediated immunotherapy in non‑RCC tumours. We demonstrated that G250 MAb recognizes a conformational epitope in the CA domain, detects the soluble CA Ⅸ ectodomain (ECD), but not the splicing variant, and does not cross-react with CA Ⅰ, Ⅱ, and Ⅻ isoforms. We showed that G250 internalizes via clathrin-coated vesicles, escapes degradation in lysosomes and enters the recycling pathway via the perinuclear compartment. This results in long intracellular persistence and enables consecutive internalization cycles. Moreover, the recycled antibody maintains an intact Fc portion potentially capable of continuous induction of antibody-dependent cell-mediated cytotoxicity (ADCC) response, thus explaining its therapeutic efficacy. Finally, we showed that G250 treatment is effective against HT-29 colorectal carcinoma xenografts that differ from RCC by more heterogeneous, hypoxia-related expression of CA Ⅸ. These results suggest potential therapeutic usefulness of the G250 MAb in non-RCC tumours.

Concurrent dendritic cell vaccine and strontium-89 radiation therapy in the management of multiple bone metastases.

In addition to its direct cytotoxic effects, radiation therapy renders tumor cells more susceptible to T cell-mediated cytotoxicity by modulating cell surface molecules involved in antigen presentation. The purpose of the present study was to determine the benefit of combined 89Sr radiation and dendritic cell (DC) vaccine therapy in bone metastasis patients. Patients were treated with intravenous 89Sr at a dose of 40 μCi/kg of body weight on the first day after the peripheral blood mononuclear cell collection. Seven days later, patients received DCs once a week for 6 weeks. The first three vaccines were administered by intravenous infusion, and the last three vaccines were administered by 24-point intradermal injection. Clinical response was evaluated by the number of bone metastatic foci demonstrated on bone scintigraphy; cell-mediated cytotoxicity response was evaluated by delayed-type hypersensitivity (DTH) reaction. All treatment-related toxicities including vaccine-induced fever and 89Sr-associated hematological toxicity were carefully monitored. Twenty-six patients with histologically diagnosed with primary cancers and multiple bone metastases demonstrated on bone scintigraphy were studied. The overall survival rate was 58.3%. The total positive DTH rate was 50%. The efficiency rate for pain relief was 60% (6/10), for quantity of life was 80%, and for clinic responses was 90%. Out of 10 cases, the Grade 1 or 2 of hematological depression in 4, erythema in 1, and fever in 7 were observed. The study has important implications for that combined 89Sr radiation, and DC vaccine therapy can benefit cancer patients with bone metastasis.

A critical role of non-classical MHC in tumor immune evasion in the amphibian Xenopus model.

Non-classical class Ib (class Ib) genes are found in all jawed vertebrates, including the amphibian Xenopus, which possesses at least 20 distinct Xenopus non-classical class Ib genes (XNCs). As an immune evasion strategy, tumors often downregulate surface expression of classical major histocompatibility complex class Ia molecules. In contrast, cancers commonly express class Ib molecules, presenting an alternative for tumor immune recognition. We characterized a novel XNC, XNC10, functionally similar to CD1d from a class Ia-deficient thymic lymphoid tumor (15/0), which grows aggressively in Xenopus LG-15 cloned animals. To investigate the roles of XNC10 in antitumor immunity, we generated stable 15/0-transfectants with silenced XNC10 mRNA and protein expression. Notably, XNC10 silencing resulted in acute tumor rejection by naturally class Ia-deficient syngeneic tadpoles, with greater potency of rejection in tumors with more efficient XNC10 knockdown. In vivo killing assays shows that the rejection of XNC10-deficient tumors is due to a cell-mediated cytotoxic immune response elicited by the tadpole host. Importantly, priming enhances XNC10-deficient tumor rejection. Flow cytometry reveals that XNC10-deficient tumor rejection is associated with an accumulation of XNC10-restricted invariant T cells and conventional CD8 T cells as well as other leukocytes. Similarly, semisolid tumor grafts in tadpoles also exhibit leukocytes infiltration. These findings suggest that XNC10 allows the 15/0-tumor to escape immune recognition and class Ia-independent cytotoxicity, thus emphasizing the critical roles of class Ibs in tumor immunity.