Antibody OKT3 Research Articles

A multi-functional drug delivery nanosystem release of TLR-7 immunostimulant and OKT3 induced efficient cancer immunotherapy

Immunotherapy has some shortcomings such as off-target toxicity, treatment time and poor immunogenicity, which limit its therapeutic effect. Nanomaterials are particularly attractive in immunotherapy due to their drug delivery capabilities. Nano drug delivery system loaded with Toll-like receptor (TLR) agonist imiquimod (IMQ) and CD3 immune antibody OKT3 is constructed by using polydopamine (PDA) and CaCO3. While PDA-IMQ@CaCO3-OKT3 (PICO NPs) drug delivery system has the advantages of high biocompatibility, low toxicity, degradability. Antitumor studies in vitro and in vivo have shown that the system can effectively inhibit the proliferation of mouse breast cancer cells and the activity of Regulatory T Cells (Tregs), activate immunogenic cell death (ICD), and enhance the activity of antigen-presenting cells (APCs). Effectively eliminate tumor immunosuppression and fully activate immune function.

Human Antibody VH Domains Targeting GPNMB and VCAM-1 as Candidate Therapeutics for Cancers

The elevated expression of GPNMB and VCAM-1 has been observed in many cancers including breast cancer, melanoma, and prostate cancers. Such overexpression of GPNMB and VCAM-1 has been associated with poor prognosis and increased cancer metastasis. Thus, GPNMB and VCAM-1 are potential targets for immunotherapies across multiple cancers. In this study, two high-affinity specific human VH domain antibody candidates, 87 (GPNMB) and 1B2 (VCAM-1), were isolated from our in-house proprietary phage-displayed human VH antibody domain libraries. The avidity was increased after conversion to VH-Fc. Domain-based bispecific T-cell engagers (DbTE) based on these two antibodies combined with the anti-CD3ε OKT3 antibody exhibited potent killing against GPNMB and VCAM-1-positive cancer cells, respectively. Hence, these two domain antibodies are promising therapeutic candidates for cancers expressing GPNMB or VCAM-1.

Abstract 4240: In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™

Abstract Introduction: CD3 bispecific antibodies have transformed the therapeutic landscape of immunotherapy for some hematological malignancies, however, the clinical therapeutic effects of CD3 bispecific therapies is still under investigation for solid tumors. Moreover, CD3 bispecific antibody-mediated tumor targeting as well as T cell engagement have resulted in cytokine release syndrome (CRS), increased on-target/off-tumor toxicities and poor T cell infiltration, which may affect safety and limit therapeutic efficacy. To better understand the relationship between T cell activation and systemic cytokine release in the context of CD3 bispecific antibody treatment, a homozygous human CD3EDG HuGEMM࣪ preclinical model was developed. Methods: The homozygous CD3EDG HuGEMM mice, which express human CD3ε/CD3δ/CD3γ on the surface of T cells, were subcutaneously inoculated with MC38 or MC38-hEpCAM HuCELL™ syngeneic tumors and treated with vehicle, anti-mPD-1 (RMP1-14), OKT3, and anti-hCD3×hEpCAM. For efficacy studies, mouse body weight and tumor volume were measured twice weekly and tumor growth inhibition (TGI%) was determined. To investigate OKT3 or CD3 bispecifics-induced cytokine release, mouse body weight was monitored daily and several inflammatory and proinflammatory cytokines in the serum were detected by MSD. Results: Firstly, anti-mPD-1 treatment (5 mg/kg and 15 mg/kg i.p.) of homozygous CD3EDG mice engrafted with MC38 syngeneic tumors led to 42% and 62% TGI, respectively. OKT3 treatment of MC38-hEpCAM bearing homozygous CD3EDG HuGEMM promoted tumor growth (TGI= -80%), likely due to activation-induced cell death (AICD) of T cells. In addition, CRS was induced within 24 hours of OKT3 dosing, with IL-2, IL-6, and TNF-α peaking 2 hr post the 1st dose, and IFN-γ, IL-10 and IL-5 peaking 6 hr post the 1st dose. In contrast, no significant cytokine release was observed upon anti-hCD3×hEpCAM treatment despite remarkable TGI, indicating that the delicate balance of in vivo T cell activation and T cell cytotoxicity is crucial for effective tumor killing by CD3 bispecific antibodies or engagers. Conclusions: Our CD3EDG HuGEMM and HuCELL models provide a predictive platform for in vivo efficacy and safety assessment of CD3 bispecific or trispecific antibodies. The uncoupling of systemic cytokine release and T cell cytotoxicity via CD3 bispecific or trispecific antibodies provides a biological rationale to clinically explore preventative treatment approaches to avoid or reduce CRS-related toxicity, as well as combination approaches to increase selectivity and efficacy. Citation Format: Daniel XF He. In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4240.

Establishment of a cytokine release syndrome associated with chimeric antigen receptor T cell treatment in SCID/Beige mice model

Objective: To establish a cytokine release syndrome (CRS) mouse model related to CAR-T cell therapy and provide a research model for the clinical phenomena. Methods: CAR-T cells targeting human CD19 molecule were constructed by molecular cloning and lentiviral transfection. Flow cytometry (FACS) was used to detect the transfection efficiency of CAR-T cells. The tumor-killing efficiency of CAR-T cells was detected by ELISA and flow cytometry. The CAR-T cells were injected into the tumor-bearing SCID/Beige mice through tail vein, and divided into phosphate buffered solution (PBS) group, low-burden group (1×10(5) Raji-Luc2 cells) and high-burden group (5×10(5) Raji-Luc2 cells). The tumor treatment effect was detected by animal in vivo imaging. Serum levels of cytokines including human IFN-γ, human IL-2, mouse IL-6, and mouse GM-CSF were measured by ELISA. The health status of the mice was evaluated by pathological examination. Results: The health scores of T cell group and T cell+ OKT-3 group were (1.15±0.08) and (2.90±0.15), respectively, after the injection of human T cell and T cell + OKT-3 antibody through tail vein, and the difference was statistically significant (P<0.001). The serum levels of human IL-2, human IFN-γ, human IL-15, mouse IL-6 and mouse GM-CSF in T cell+ OKT-3 group were (1 064.00±50.14), (1 285.00±193.90), (202.4±18.76), (1 478.00±289.20) and (350.70±42.27) pg/ml, respectively, higher than (22.67±6.36), (23.67±3.71), (44.33±14.45), (147.30±36.20), (138.00±22.74) pg/ml in T cell group (P<0.05). OKT-3 combined with human T cells caused a rapid increase in serum levels of human IL-2, human IFN-γ, mouse IL-6 and mouse GM-CSF, accompanied by an increase in body temperature and weight loss. CD19-targeting CAR-T cells were successfully constructed, and the positive rate of CAR-T cells was >30% detected by flow cytometry. ELISA results showed that in the presence of CD19 antigen, IL-2 and IFN-γ secreted by CAR-T19 cells co-incubated with Raji and Nalm were (561.00±37.07), (680.30±71.27), (369±25.71) and (523.00±26.31) pg/ml, respectively, higher than (55.00±20.53) and (64.00±7.55) pg/ml in the co-incubated with K562 group (P<0.001). Activated CAR-T19 cells were reinjected through the tail vein on the seventh day after tumor formation. Imaging experiments in mice showed that on the thirteenth day after tumor formation, the fluorescence intensities of tumors in the low-burden and high-burden groups were lower than on the seventh day of tumor inoculation, and the fluorescence intensity of tumors in the high-burden group decreased from 144.00±24.69 to 5.02±2.35 (P=0.005). The fluorescence intensity of low burden group decreased from 58.47±9.36 to 3.48±1.67 (P=0.004). The serum levels of T cell activation related cytokines IL-2, IL-15 and IFN-γ increased rapidly, and the secretion of monocyte related cytokines IL-16 and GM-CSF increased, accompanied by the typical characteristics of CRS such as increased body temperature and weight loss at 72 hours after injection of CAR-T19 cells. Conclusions: CAR-T cells targeting CD19 molecule are successfully constructed, and CRS phenomenon is verified in tumor-bearing mice by CAR-T cell re-infusion, providing an animal model for the mechanism of CAR-T treatment-related CRS and CRS prevention strategies.

T-cell surface generation of dual bivalent, bispecific T-cell engaging, RNA duplex cross-linked antibodies (dbBiTERs) for re-directed tumor cell lysis.

Genetic engineered Bispecific T-cell engagers (BiTEs) generate potent cytotoxic effects. Alternately, click chemistry engineered, dual specific bivalent Bispecific T-cell engaging antibodies (dbBiTEs) on T-cell surfaces can be generated from parent monoclonal antibodies. We show the formation of dbBiTEs on the surface of T-cells along with the introduction of complementary 2'-OMe RNA 32-mer oligonucleotides allowing duplex formation between antibodies, designated as dbBiTERs. dbBiTERs generated in solution from anti-CEA and anti-CD3 OKT3 antibodies retained specific binding to CEA positive versus CEA negative cancer cells and to CD3 positive T-cells comparable to dbBiTEs. When T-cells were precoated with dbBiTEs or dbBiTERs and mixed with CEA positive versus CEA negative cancer cells, similar dose dependent and specific cytotoxicity were observed in redirected cell lysis assays. On-cell generated dbBiTERs exerted potent cytotoxic responses against CEA positive targets and were localized at the cell surface by immuno-gold EM. In addition, we demonstrate that target and T-cells, each coated separately with complementary 2'OMe-RNA-linked antibodies can be cross-linked by RNA duplex formation in vitro to generate redirected cell lysis. The facile generation of dbBiTERs with specific cytolytic activity from intact antibodies and their generation on-cell offers a new avenue for antigen specific T-cell therapy.

Abstract 3354: Preclinical evaluation of potential cytokine release syndrome and efficacy of a BCMA-CD3 bispecific antibody for the treatment of multiple myeloma in humanized mice

Abstract Bispecific antibodies (BsAbs) are now emerging as the next promising class of immunotherapies. BsAbs can bind simultaneously two different antigens or epitopes, which leads to a wide range of applications including redirecting T cells or NK cells to tumor cells and eradicate them more precisely and effectively. BCMA is a cell surface protein that is expressed on the malignant plasma cells, a substantial number of lymphoma and at low levels on normal B cells. This cell surface protein has emerged as a very selective antigen of targeted therapy for the treatment of multiple myeloma. As BsAbs can have significant adverse effects such as cytokine release syndrome (CRS), we decided to evaluate the CRS related safety profile, in addition to efficacy, of this BCMA-CD3 BsAb in pre-clinical studies. We first evaluated the potential CRS after this BCMA-CD3 bispecific antibody treatment. As in vitro human PBMC assays presently in use do not reliably predict the occurrence of CRS in patients, we have developed a rapid, sensitive and reproducible PBMC humanized mouse model that can measure cytokine release induced by bispecifics in vivo. NSG™ and NSG-MHC Class I/II KO mice, which is a new NSG™ strain useful for studying human immunity in the absence of acute GVHD, are used in this mouse model. The mice were engrafted with human PBMCs for 6 days before being treated with BCMA-CD3 BsAb, OKT3 and anti-CD28 antibodies. Human cytokines IFN-γ, IL-2, IL-4, IL-6, IL-10 and TNFα in serum were measured 6 hours after antibody treatments. Clinical observations of mice were recorded with OKT3, BCMA-CD3 BsAb and other treatments. Dose-dependent cytokine release was observed following BCMA-CD3 BsAb treatment with the highest concentration of 10 mg/kg showing a significant increased cytokine levels over that of 1 or 0.1 mg/kg. BCMA-CD3 BsAb was tested in a total of 8 PBMC donors, demonstrating donor dependent cytokine release. Donor variability for the cytokine release was observed with some donors more sensitive than others. 0.5-1 mg/kg BCMA-CD3 treatment was chosen for evaluating both efficacy and CRS using BCMA expressing NCI-H929 multiple myeloma cells in humanized NSG™ and the results will be discussed. Together, the data suggests that the BCMA-CD3 BsAb may have cytokine release activity at high doses and supports our humanized mouse platform to evaluate potential CRS and efficacy studies of novel bispecific antibodies. Citation Format: Jing Jiao, Danying Cai, Li-Chin Yao, Kyle Draheim, Hongyuan Yang, Mingshan Cheng, James Keck. Preclinical evaluation of potential cytokine release syndrome and efficacy of a BCMA-CD3 bispecific antibody for the treatment of multiple myeloma in humanized mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3354.

Abstract B36: TGFβ blockade and epigenetic modulation for cancer treatment: Efficient breast cancer targeted therapy with TCR-T cell transfer

Abstract Introduction: The cancer testis (CT) antigens, such as NY-ESO1, are considered ideal targets for immunotherapy as they are highly immunogenic and their restricted expression malignancies and not normal tissue except testis. However, the expression of CT antigen is downregulated in many tumor types, including breast cancer due to DNA methylation. DNA methylation is a common epigenetic mechanism to regulate gene expression and phenotype and maintain DNA integrity and stability. DNA methylation inhibition can induce NY-ESO1 expression on tumor cells and sensitize them to NY-ESO1 specific T cells. NY-ESO1 targeted T cell receptors (TCR) have been successfully used to engineer T cells for adoptive T cell therapy (ACT). We hypothesize that incorporating a dominant-negative TGFβ receptor II (dnTGFβRII) into the NY-ESO-1 TCR construct to transduce T cells combined with epigenetic modulator treatment may improve T cell effector function in immunosuppressive tumor microenvironment. Methods: Seven different human breast cancer cell lines were treated with epigenetic modulators at different doses and analyzed by flow cytometry to check the increase in expression of NY-ESO1 antigen and HLA-A2.1. Eight-weeks or older NSG mice were inoculated with HLA-A2.1+, NY-ESO negative at baseline breast cancer cells subcutaneously. PBMCs from healthy donors were transduced with a HLA-A2.1 NY-ESO1 TCR with/without dnTGFβRII two day after activation with OKT3 antibody plus IL-2. The transduction efficiency was confirmed by flow cytometry prior to ACT. Equal numbers of TCR+ cells were injected 10 days after tumor inoculation. Mice were then monitored for tumor growth till endpoint. The spleens and tumors were collected for further analysis of immunophenotype and function. Results: The expression of NY-ESO1 was inducible with epigenetic modulation in the majority of breast cancer cell-lines tested (5/7). More than 60% of Vβ+ (marker for surface expression of specific TCR) cells were observed after retroviral transduction to PBMCs. The transduced NY-ESO1 TCR/dnTGFβRII cells with epigenetic modulator were successfully able to infiltrate into tumor microenvironment in higher numbers and control cancer growth of inoculated tumor significantly (p&amp;lt;0.01) in comparison to control tumor and control NY-ESO1 TCR only groups. The tumor infiltrated lymphocytes (TILs) from NY-ESO1 TCR/dnTGFβRII with treatment showed that regulatory T cells and cytotoxic T cells were significantly lower and higher, respectively. Conclusions: Epigenetic modification enhances the cytotoxicity of TCR engineered T cells with dnTGFβRII. Our results demonstrate the feasibility of combined epigenetic modulation and TCR-T treatment for breast cancer patients. Citation Format: Satoko Matsueda, Kunle Odunsi, Richard C. Koya. TGFβ blockade and epigenetic modulation for cancer treatment: Efficient breast cancer targeted therapy with TCR-T cell transfer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B36.

Research Highlights

Research Highlights

Stimulation of Mononuclear Cells Through Toll-Like Receptor 9 Induces Release of Microvesicles Expressing Double-Stranded DNA and Galectin 3-Binding Protein in an Interferon-α-Dependent Manner.

Background: Microvesicles (MVs) expressing the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP) may play a pathogenic role in systemic lupus erythematosus (SLE). Co-expression of double-stranded DNA (dsDNA) on such MVs may render them immunogenic and targets for anti-dsDNA antibodies. Little is known about the mechanisms underlying generation of this MV population. In this study, we investigated how Toll-like receptors (TLRs), IFN-α, and T cells are involved in this process in healthy subjects.Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy donors were stimulated in-vitro for 24 h with a series of TLR-agonists or the T cell activating antibody OKT3 or were subjected to apoptosis by incubation with staurosporine. MVs in the supernatants were subsequently isolated by differential centrifugation and were quantified and characterized with respect to expression of G3BP and dsDNA by flow cytometry.Results: Stimulation of PBMCs with the TLR9-agonist and strong IFN-α inducer ODN2395 significantly increased the release of MVs expressing G3BP. The production of MVs with this phenotype was markedly enhanced by co-stimulation of T cells. Furthermore, dependency on IFN-α in the generation of G3BP-expressing MVs was indicated by a marked reduction following addition of the IFN-α inhibitor IFN alpha-IFNAR-IN-1 hydrochloride.Conclusion: Release of G3BP-expressing MVs from healthy donor PBMCs is induced by stimulation of TLR9 in an IFN-α-dependent manner and is enhanced by co-stimulation of T cells.

Abstract 1496: Novel hCD3e mouse models for preclinical pharmacology studies of therapeutic bi-specific antibodies

Abstract Recently it has been demonstrated that T cell-mediated immunotherapy is an attractive treatment strategy for various diseases. For this therapeutic approach, the CD3 complex is one of the key targets to modulate T cell activation; however, in many instances we cannot directly evaluate antibody candidates in mice because the monoclonal antibodies specific to human CD3 cannot cross-react to mouse endogenous CD3. In order to overcome the current model limitation, we have generated CD3e-extracellular domain KI mice for anti-human CD3e bispecific antibody in vivo efficacy evaluation. In this model, the mouse CD3e extracellular domain was replaced by human CD3e. We used conventional mouse ES/HR technology to knock-in human CD3e genomic DNA from the 3’ end of exon 2 to the 5’ end of exon7 to replace murine exon 2-exon 6. CD3e is one of four subunits of the CD3 co-receptor, which plays an important role in the immune response, associating with the T cell receptor (TCR) to couple antigen recognition to intracellular signal transduction, leading to T cell activation. We have characterized our B-hCD3e mice with the following features: 1.Mouse CD3e+ cells were detected in the wildtype C57BL/6 mice, while human CD3e+ cells were only detected in the homozygous B-hCD3e mice. 2.T cell development was normal in B-hCD3e homozygous mice. 3.T cell subtypes in the spleen, thymus, lymph nodes, and PBMC in B-hCD3e mice were similar and comparable to that in wildtype C57BL/6 mice. 4.Normal in vitro T cell proliferation and cytokine production were found in anti-hCD3e antibody treated humanized B-hCD3e mice 5.Mouse anti-PD-1 antibody can significantly repress the growth of MC38 tumor cells engrafted on B-hCD3e mice, suggesting that T cells in B-hCD3e mice are functionally normal 6.Treatment of B-hCD3e mice with anti-human CD3e antibody OKT leads to T cell depletion, resulting in more aggressive tumor growth. Furthermore, efficacy and toxicity evaluation of CD3e based bispecific antibodies in B-hCD3e mice are ongoing. Based on our data from extensive phenotypical analyses of B-hCD3e mice, we believe this novel humanized CD3e mouse model will be very useful for in vivo efficacy studies of CD3- mediated therapy, especially anti-CD3e bi-specific antibodies. Citation Format: Jie Xiang, Yanan Li, Yuelei Shen, Yanan Guo. Novel hCD3e mouse models for preclinical pharmacology studies of therapeutic bi-specific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1496.

Immunosuppressive Total Nodal Irradiation–Based Reconditioning Regimens After Graft Rejection or Graft Failure in Pediatric Patients Treated With Myeloablative Allogeneic Hematopoietic Cell Transplantation

This retrospective analysis aimed to address the efficacy of total nodal irradiation (TNI)-based reconditioning regimens in pediatric patients with graft failure/rejection after allogeneic hematopoietic cell transplantation. Thirty-three pediatric patients with malignant (n=25) and nonmalignant diseases (n=8) were treated with a TNI-based reconditioning regimen. All patients received a 7-Gy single dose combined with anti-T lymphocyte antibody OKT3 (n=16), anti-thymocyte globulin (n=24), fludarabine (n=31), and/or thiotepa (n=28), followed by an infusion of peripheral blood stem cells (n=31) or bone marrow transplant (n=2). Twenty-eight of 33 patients had haploidentical family donors. After a median of 11days, engraftment was seen in 32 of 33 children. Two children died 34days after retransplantation because of either disease relapse or treatment-related multiple organ failure. Severe acute toxicity was reported in only 1 child (systemic inflammatory response syndrome-like reaction; recovery after cortisone treatment). The average follow-up was 60.2months (range, 1.1-162.5months). Event-free and overall survival rates at 2/5years follow-up were 62.0%/58.6% and 65.1%/61.7%, respectively. Despite sustained engraftment, 12 patients died from disease relapse (n=3), Moschkowitz syndrome (n=1), or multiple organ failure (n=8). Follow-up data were available for 18 of 21 survivors, with a median follow-up of 92.8months (range, 3.6-162.5months). Hypothyroidism was present in 78.6% of patients, and sex/growth hormonal insufficiencies were reported for 37.5%. Mean forced expiratory volume in 1second after TNI was 84%; mean vital capacity was 79%. Severe growth failure (<3rd percentile) occurred in 28.6% (height) and 35.7% (weight) of patients. No secondary malignancies were reported. In the high-risk group of patients with graft failure/rejection after allogeneic hematopoietic cell transplantation, the TNI-based reconditioning regimen seems to allow sustained engraftment combined with a favorable toxicity profile, leading to long-term event-free and overall survival. Late toxicity after a median follow-up of over 7.5years includes growth failure, manageable hormonal deficiencies, and a low risk of decrease of lung function.

Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer.

Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX. Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells. TFIL demonstrated high antibody-liposome conjugation ratios (100-130μg protein/μmol phospholipid) and cargo capacity (0.21mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150nm), and good stability (up to 3months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX. Novel anti-HER2xCD3 + DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.

Resident T Cells in Resolved Psoriasis Steer Tissue Responses that Stratify Clinical Outcome

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.

Abstract 5098: NG-348: a novel oncolytic virus designed to mediate anti-tumour activity via the potent and selective polyclonal activation of tumor-infiltrating T-cells

Abstract NG-348 is a transgene-modified variant of enadenotucirev, a chimeric oncolytic group B adenovirus with potent and selective anti-tumor activity against a range of epithelial cancer cells. Enadenotucirev has a blood stability profile that enables systemic dosing and has been administered intravenously to over 90 cancer patients. These studies have demonstrated that IV dosed enadenotucirev is delivered to tumors and subsequent virus activity is associated with CD8+ T-cell infiltration in tumor cell nests, consistent with immune stimulation within the tumor. NG-348 encodes two immunomodulatory proteins in its genome: full-length human CD80, and a membrane anchored single chain variable fragment of the mouse anti-human CD3ϵ monoclonal antibody OKT3. Together these membrane proteins provide both T-cell receptor (signal 1) and costimulatory (signal 2) activation signals required to polyclonally activate tumor-infiltrating T-cells. When expressed on the surface of NG-348 infected tumor cells the transgenes therefore enhance the potency of the virus by driving local T-cell immune responses selectively in the tumor microenvironment. The expression of both transgenes encoded in the NG-348 virus is controlled by the endogenous virus major late promoter. This restricts expression of the proteins to the surface of cells permissive to virus infection (i.e. tumor cells) and prevents off-target expression in the cells from healthy tissues. Using co-cultures of human T-cells with human tumor cell lines, we have shown that NG-348 infected tumor cells potently activate both CD4 and CD8 T-cells. This was demonstrated by analysis of activation marker expression (CD25, CD69), intracellular and secreted cytokines (IL-2, TNF, IFNγ) and induction of T-cell mediated tumor cell death by apoptosis (prior to oncolytic death by the virus). Treatment of different human non-tumor cells (e.g. fibroblasts, T-cells, PBMCs) with NG-348 did not lead to transgene protein expression or activation of T-cells in co-cultures. NG-348 has also been shown to activate human T-cells in vivo, using a human tumor xenograft model system in immunodeficient mice reconstituted with human PBMCs. Collectively these data indicate that following delivery to tumor tissues of patients, NG-348 oncolytic virus can selectively replicate and express it’s payload of T-cell activating ligands. NG-348 should therefore stimulate potent antigen-independent, polyclonal activation of T-cells already present in the tumor, as well as those recruited into the tumor in response to the virus infection, to drive effective anti-tumor immunity. NG-348 is currently in preclinical development with a first phase I study planned to initiate in Q4 2017. Citation Format: Brian R. Champion, Matthieu Besneux, Nalini Marino, Darren Plumb, Prithvi Kodialbail, Sam Illingworth, Rochelle Lear, Alice C. Brown. NG-348: a novel oncolytic virus designed to mediate anti-tumour activity via the potent and selective polyclonal activation of tumor-infiltrating T-cells [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 5098. doi:10.1158/1538-7445.AM2017-5098

MicroRNA expression profiles in human CD3+ T cells following stimulation with anti-human CD3 antibodies

BackgroundAnti-CD3 therapy can induce immunosuppression by several non mutually exclusive mechanisms that have been proposed to explain the therapeutic effect the administration anti-CD3 mAb, but its immunoregulatory mechanism is still not completely clear. In T cells, microRNAs (miRNAs) regulate several pathways, including those associated with immune tolerance. Here, we report changes in miRNA expression in T cells following treatment with anti-human CD3 antibodies. Peripheral blood mononuclear cells were cultured in the presence of the monoclonal antibody OKT3 or a recombinant fragment of humanized anti-CD3. Following these treatments, the expression profiles of 31 miRNA species were assessed in T cells using TaqMan arrays.ResultsEight of the tested miRNAs (miR-155, miR-21, miR-146a, miR-210, miR-17, miR-590-5p, miR-106b and miR-301a) were statistically significantly up- or down-regulated relative to untreated cells.ConclusionsStimulation of T cells with anti-human CD3 antibodies alters miRNA expression patterns, including of miRNA species associated with immune regulatory pathways.

Abstract No. 129 - Development of a novel bispecific antibody for targeted immunotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an ideal target for novel immunotherapies due to its otherwise immunosuppressive intratumoral environment. While the efficacy of bispecific antibodies for immunotherapy is well established for hematological malignancies, their limited circulatory half-lives make them challenging to use for solid tumors. Intratumoral delivery can bypass this issue, potentially making transcatheter-administered bispecific antibody therapy an exciting new paradigm for HCC treatment. We have developed a bispecific antibody that can simultaneously bind to both HCC cells and cytotoxic T cells in order to enhance T-cell mediated tumor lysis. The bispecific antibody targeting the HCC surface antigen glypican-3 (GPC-3) and the T-cell antigen CD-3 was designed as a fusion protein (IDT DNA) and expressed recombinantly in E. coli expression system (Origami Cells) according to previous reports. [1] Specific binding of the antibody against both HCC cells (HepG2) and T cells (Jurkat) was confirmed using cell-surface ELISA assays. [2] Bispecific-triggered activation of T cells (donor CD4 cells) was assessed in vitro using IFN-gamma release ELISA. The bispecific antibody is designed as a 33kD fusion protein consisting of a GPC-3 binding peptide (RLNVGGTYFLTTRQ), previously shown to bind specifically to HCC cells, connected to a CD3-binding single variable fragment domain (ScFV), derived from an OKT3 antibody. [3] The antibody was successfully expressed and purified. It binds to both HCC cells (31.6nM, SD 0.008nM) and T cells (13.2nM, SD 0.006nM) with affinities that are comparable to values reported for other bispecific constructs. [4] The binding to both cell lines were specific and can be inhibited by using excess of either pure GPC-3 binding peptide or full-length OKT3 antibody. The activity of the bispecific antibody is demonstrated by its ability to activate T cells in vitro, resulting in robust IFN-gamma release as measured using ELISA (0.196 Bispecific vs 0.008 Cell Only Control, p = 0.0003). A novel bispecific antibody targeting GPC-3 and CD-3 was successfully synthesized and validated in vitro demonstrating the potential for targeted immunotherapy of HCC.

English

Heart transplantation has emerged as the definitive therapy for patients with end-stage cardiomyopathy. The two most common forms of cardiac disease that lead to transplantation are ischemic cardiomyopathy and dilated cardiomyopathy, which together comprise approximately 90% of cases. The other less common forms of heart disease include viral cardiomyopathy, infiltrative cardiomyopathy, postpartum cardiomyopathy, valvular heart disease and congenital heart disease. KEYWORDS: Anesthetic management; Cardiomyopathy; Cardiopulmonary bypass; Heart failure; Heart transplantation; Pulmonary artery hypertension; INTRODUCTION: Heart transplantation has emerged as the definitive therapy for patients with end- stage cardiomyopathy. The two most common forms of cardiac disease that lead to transplantation are ischemic cardiomyopathy and dilated cardiomyopathy, which together comprise approximately 90% of cases. The other less common forms of heart disease include viral cardiomyopathy, infiltrative cardiomyopathy, postpartum cardiomyopathy, valvular heart disease and congenital heart disease. The introduction of cyclosporine in 1981 and monoclonal antibody okT3 in 1983 for heart transplant patients greatly accelerated the progress of organ transplantation.1 The peak period of heart transplantation was mid-1980s.The pace slowed down due to the limitations in the availability of donor organs. We have performed five heart transplant surgeries at Rajiv Gandhi Government General Hospital, Chennai, India during 2009 to 2011. The corresponding author was a member of the team during three cardiac transplant surgeries while the co-author was a member during all the five transplant surgeries. This article is intended to share our experiences and anesthetic challenges during cardiac transplantations.

Insights into the interaction of CD4 with anti-CD4 antibodies

Knowledge about the mechanism by which some antibodies can block HIV-1 entry is critical to our understanding of their function and may offer new avenues for controlling the adhesion of HIV-1 to the host cells. While much progress has been made, this mechanism remains unclear. Here, atomic force microscopy, isothermal titration calorimetry (ITC), and circular dichroism spectroscopy were used to measure some biophysical characteristics of the interaction of four-domains (D1–D4) membrane protein CD4 with anti-D3 antibody OKT4 and with HIV-1 entry blocking anti-D1 antibody Leu3a. The results showed that at 37°C they bind with similar binding strength, thermodynamics, and kinetics but with different assembly states. Further analyzing the interactions at different temperatures by ITC showed that binding of CD4 with Leu3a is characteristic for specific hydrophobic binding as well as for protein folding while with OKT4 comes from an extensive additional hydration upon binding and charge-related interactions within the binding site. Comparing these characteristics with those of HIV-1 gp120-CD4 interaction revealed that Leu3a binds to CD4 faster than HIV-1 followed by changing local structure of D1 to which HIV-1 binds leading to a prevention of viral entry.

Liver Allograft Provides Immunoprotection for the Cardiac Allograft in Combined Heart-Liver Transplantation.

When transplanted simultaneously, the liver allograft has been thought to have an immunoprotective role on other organs; however, detailed analyses in simultaneous heart-liver transplantation (SHLT) have not been done to date. We analyzed patient outcomes and incidence of immune-mediated injury in 22 consecutive SHLT versus 223 isolated heart transplantation (IHT) recipients between January 2004 and December 2013, by reviewing 3912 protocol- and indication-specific cardiac allograft biopsy specimens. Overall survival was similar (86.4%, 86.4%, and 69.1% for SHLT and 93.3%, 84.7%, and 70.0% for IHT at 1, 5, and 10 years; p = 0.83). Despite similar immunosuppression, the incidence of T cell-mediated rejection (TCMR) was lower in SHLT (31.8%) than in IHT (84.8%) (p < 0.0001). Although more SHLT patients had preexisting donor-specific HLA antibody (22.7% versus 8.1%; p = 0.04), the incidence of antibody-mediated rejection was not different in SHLT compared with IHT (4.5% versus 14.8%, p = 0.33). While the left ventricular ejection fraction was comparable in both groups at 5 years, the incidence and severity of cardiac allograft vasculopathy were reduced in the SHLT recipients (42.9% versus 66.8%, p = 0.03). Simultaneously transplanted liver allograft was associated with reduced risk of TCMR (odds ratio [OR] 0.003, 95% confidence interval [CI] 0-0.02; p < 0.0001), antibody-mediated rejection (OR 0.04, 95% CI 0-0.46; p = 0.004), and cardiac allograft vasculopathy (OR 0.26, 95% CI 0.07-0.84; p = 0.02), after adjusting for other risk factors. These data suggest that the incidence of alloimmune injury in the heart allograft is reduced in SHLT recipients.