CD3epsilon Research Articles

BI 836909, a Novel Bispecific T Cell Engager for the Treatment of Multiple Myeloma Induces Highly Specific and Efficacious Lysis of Multiple Myeloma Cells in Vitro and Shows Anti-Tumor Activity in Vivo

BI 836909 is a Bispecific T cell Engager (BiTE®), designed to redirect the body's endogenous T cells towards cells expressing B cell maturation antigen (BCMA) on the cell surface.BCMA is a highly plasma cell specific antigen and shows homogeneous expression on the cell surface of multiple myeloma, plasma cell leukemia and plasmacytoma cells. In normal tissues, BCMA expression is restricted to plasma cells, while other normal tissues do not express BCMA. This highly selective expression pattern makes BCMA an ideal target for T cell redirecting therapy.The pharmacological effect of BI 836909 depends on its simultaneous binding to both the CD3 epsilon subunit of the T cell receptor complex on T cells as well as to BCMA on multiple myeloma cells, resulting in the lysis of the BCMA-expressing cells.In vitro, unstimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with several multiple myeloma cell lines and increasing concentrations of BI 836909, and tumor cell lysis, T cell activation, and induction of cytokine release were assessed. BI 836909 induced dose-dependent redirected lysis of human multiple myeloma cell lines with EC90 values ranging from 16 to 810 pg/mL. Viability of BCMA-negative cells was not affected, demonstrating the specificity of BI 836909 for BCMA. The expression of the activation markers CD69 and CD25 on T cells and the release of cytokines by T cells were target-dependent and occurred only in the presence of BCMA-positive cells.In vivo anti-tumor activity of BI 836909 was assessed in NOD/SCID mice reconstituted with human T cells and bearing subcutaneous or orthotopic xenografts derived from human multiple myeloma cell lines.In the subcutaneous NCI-H929 xenograft model, animals were treated with BI 836909 by daily intravenous or subcutaneous bolus injections. Statistically significant dose-dependent anti-tumor activity was observed at doses of 50 µg/kg/day and higher. The efficacy of BI 836909 was comparable after intravenous and subcutaneous administration, when the difference in bioavailability of the different routes was considered.In an orthotopic L-363 xenograft model, treatment with BI 836909 resulted in a statistically significant prolonged survival at doses of 5 µg/kg/day and higher.BI 836909 shows comparable cross-reactive binding to both BCMA and CD3 epsilon of human and macaque origin at picomolar and low nanomolar affinities respectively, thus allowing the assessment of pharmacodynamics, pharmacokinetics, and safety in non-human primates. In toxicity studies, cynomolgus monkeys were administered doses of up to 135 µg/kg/day of BI 836909 via continuous intravenous infusion, and up to 405 µg/kg/day via daily subcutaneous injection for up to 28 days. A dose- dependent decrease in plasma cells was observed in the bone marrow of treated animals compared to the vehicle control group, consistent with BCMA expression on cynomolgus monkey plasma cells, this demonstrated the pharmacological activity of BI 836909.These pre-clinical data demonstrate that BI 836909 is a highly potent, efficacious and BCMA-selective T cell redirecting agent and support clinical testing of BI 836909 in multiple myeloma patients. DisclosuresHipp:Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Deegen:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Wahl:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Blanset:Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA: Employment. Thomas:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Rattel:Amgen Research (Munich) GmbH, Staffelseestrasse 2, 81477 Munich, Germany: Employment. Adam:Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria: Employment. Friedrich:Amgen Research (Munich) GmbH: Employment.

Abstract A85: Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors

Abstract Background: AMG 211 is a novel, bispecific single-chain antibody of the bispecific T-cell engager (BiTE® antibody construct) class. AMG 211 targets carcinoembryonic antigen (CEA) on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex on T-cells. CEA is a glycosylated human oncofetal antigen and is abundantly expressed by a variety of tumors; especially those in the gastrointestinal tract. We radiolabeled AMG 211 with zirconium-89 (89Zr) to study the tumor targeting and tissue distribution of AMG 211 in preclinical mouse tumor xenograft models. Material and Methods: AMG 211 was conjugated with desferal for 89Zr labeling and quality control was performed. A tracer amount of 10 μg (5 MBq) 89Zr-AMG 211 was administered to mice bearing subcutaneously-implanted CEA-expressing LS174T human colorectal adenocarcinoma. MicroPET imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after tracer injection. A dose-escalation biodistribution study was performed 6 h after injection of 2, 10, 50, 100 or 500 μg 89Zr-AMG 211. 89Zr-AMG 211 was also evaluated in a CEA-negative HL60 promyelocytic leukemia xenograft. Furthermore, the non-CEA-binding BiTE® antibody construct, MEC 14, served as a negative control in LS174T tumors. Ex vivo gel electrophoresis and autoradiography was used to determine 89Zr-AMG 211 integrity in plasma and tumor lysate. Results: In vitro analysis of desferal-conjugated AMG 211 showed similar CEA binding as unlabeled AMG 211. Radiochemical purity of 89Zr-AMG 211 exceeded 95%, with a maximum specific activity of 500 MBq/mg. Size-exclusion high-performance liquid chromatography showed no degradation products and less than 5% of aggregates. MicroPET imaging revealed time-dependent tumor uptake of 89Zr-AMG 211 in LS174T bearing mice with the highest uptake 3 h after injection and a prolonged imageable tumor retention up to 48 h. 89Zr-AMG 211 rapidly decreased in blood (T1/2 = 1 h). Dose-escalation showed a dose-dependent tumor uptake 6 hours after injection with the highest tumor uptake observed with 2 μg (7.5 ± 1.5%ID/g) and the lowest tumor uptake with 500 μg (3.9 ± 0.1%ID/g). Mice bearing CEA-positive LS174T versus CEA-negative HL60 tumors showed higher tumor uptake (6.0 ± 1.3 vs 0.5 ± 0.1%ID/g) and tumor-to-blood ratios (21.0 ± 4.0 vs 1.6 ± 0.2) after injection of 10 μg 89Zr-AMG 211. In addition, the non-specific 89Zr-MEC 14 showed low tumor (0.5 ± 0.2%ID/g) uptake and tumor-to-blood ratio (5.5 ± 2.1) that is similar to tissue background in the LS174T model. Ex vivo autoradiography showed intact 89Zr-AMG 211 in all plasma samples. Moreover, in line with biodistribution data, intact 89Zr-AMG 211 was present only in the LS174T xenograft lysates and not detected in HL60 lysate. 89Zr-MEC14 did not bind CEA and was not detected in LS174T lysates. Conclusions: This study showed dose-dependent CEA-specific targeting of 89Zr-AMG 211. In addition, intact labeled AMG 211 was present in plasma and LS174T tumor lysates indicating in vivo tracer integrity. Our data support the use of 89Zr-ImmunoPET in the clinical setting to assess the distribution and tumor-targeting properties of AMG 211 and BiTE® antibody constructs. Citation Format: S.J.H. Waaijer, F.J. Warnders, M.N. Lub-de Hooge, S. Stienen, M. Friedrich, A. Sternjak, P.C. Pieslor, K. Cheung, A.G.T. Terwisscha van Scheltinga, C.P. Schröder, E.G.E. de Vries. Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A85.

Abstract 2481: CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors

Abstract T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumor cells and T cells trigger strong T cell activation resulting in the killing of tumor cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumors (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumor antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumor and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro, CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumor cells with high expression of CEA. In vivo, CEA TCB induces dose- and time-dependent regression of CEA-expressing tumors with variable amounts of immune cell infiltrate. In fully humanized NOG mice, CEA TCB is efficacious in poorly-infiltrated tumors and converts non-inflamed into highly-inflamed tumors. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumors and/or expands pre-existing ones and is able to induce T cell re-localization from the tumor periphery into the tumor bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoral T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumor-targeted T cell bispecific antibody with promising anti-tumor activity and the novel ability to modify the tumor microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Citation Format: Marina Bacac, Tanja Fauti, Sara Colombetti, Johannes Sam, Valeria Nicolini, Nathalie Steinhoff, Oliver Ast, Peter Bruenker, Ralf Hosse, Thomas Hofer, Ekkehard Moessner, Christiane Jaeger, Jose Saro, Vaios Karanikas, Christian Klein, Pablo Umana. CEA TCB, a novel T-cell bispecific antibody with potent in vitro and in vivo antitumor activity against solid tumors. [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 2481. doi:10.1158/1538-7445.AM2015-2481

CD20‐Positive nodal natural killer/T‐cell lymphoma with cutaneous involvement

CD20-positive natural killer (NK)/T-cell lymphoma is extremely rare. We describe a case of a CD20-positive nodal NK/T-cell lymphoma with cutaneous involvement in a 32-year-old man. The patient presented with fever, night sweats, right inguinal lymphadenopathy and multiple violaceous to erythematous nodules and plaques on the back and bilateral legs. Immunohistochemical analysis showed diffusely and strongly positive staining for CD3, CD3 epsilon, CD43, CD56, TIA-1 and CD20 but negative staining for other B-cell markers, including CD79a and PAX-5 and T-cell markers CD5 and CD7. The tumor cell nuclei were diffusely positive for Epstein-Barr virus-encoded RNA in situ hybridization. A partial clinical response was observed after chemotherapy, indicated by the decreased size of the lymph nodes and skin lesions. It is a diagnostic challenge to deal with lymphoma cells that present with the surface proteins of both T- and B-cells.

ITOC2 – 037. CEA TCB, A novel T-cell bispecific antibody with potent in vitro and in vivo antitumour activity against solid tumours

T cell bispecific antibodies (TCBs) are potent molecules that upon simultaneous binding to tumour cells and T cells trigger strong T cell activation resulting in the killing of tumour cells. CEA TCB (RG7813) is a novel bispecific antibody targeting carcinoembryonic antigen (CEA), often overexpressed on solid tumours (e.g. colorectal, gastric, pancreatic, lung carcinoma etc.), and the CD3 epsilon chain present on T cells. CEA TCB bears several innovative technological features that distinguish it from other bispecific antibodies currently in (pre-)clinical development: (a) bivalency for tumour antigen translating into higher avidity, superior potency and better differentiation between high and low antigen-expressing cells; (b) head-to-tail fusion geometry for anti-tumour and CD3-binding domains, resulting in higher potency compared to conventional IgG-based TCBs; (c) extended half-life compared to non-Fc-based TCBs; (d) fully silent Fc ensuring lower risk of FcgR-mediated infusion reactions; and (e) robust production using standard manufacturing processes (enabled by “CrossMAb” and knob-into-hole bispecific antibody technologies). In vitro , CEA TCB mediates potent target-dependent T cell cytotoxicity, T cell activation, proliferation, and cytokine release in killing assays, exclusively in the presence of CEA-expressing target-cells. CEA TCB activity correlates with CEA expression level, showing higher potency against tumour cells with high expression of CEA. In vivo , CEA TCB induces dose- and time-dependent regression of CEA-expressing tumours with variable amounts of immune cell infiltrate. In fully humanised NOG mice, CEA TCB is efficacious in poorly-infiltrated tumours and converts non-inflamed into highly-inflamed tumours. Histological and FACS analyses revealed that CEA TCB recruits new T cells into tumours and/or expands pre-existing ones and is able to induce T cell re-localisation from the tumour periphery into the tumour bed. Surprisingly, CEA TCB treatment also qualitatively alters the composition of intratumoural T cells resulting in an increased frequency of activated (CD69, CD25), proliferating (Ki67) and differentiated T cells (having effector memory phenotype) that are ready to kill (express high levels of Granzyme B). Taken together, these preclinical data show that CEA TCB is a novel tumour-targeted T cell bispecific antibody with promising anti-tumour activity and the novel ability to modify the tumour microenvironment. Phase 1 clinical trials with CEA TCB are currently ongoing. Future studies will focus on identification of combination partners that inhibit T cell suppression and unleash the full potential of T cell activity. Presented in Plenary Session 4 : Immunomodulatory agents.

A mouse strain defective in both T cells and NK cells has enhanced sensitivity to tumor induction by plasmid DNA expressing both activated H-Ras and c-Myc.

As part of safety studies to evaluate the risk of residual cellular DNA in vaccines manufactured in tumorigenic cells, we have been developing in vivo assays to detect and quantify the oncogenic activity of DNA. We generated a plasmid expressing both an activated human H-ras gene and murine c-myc gene and showed that 1 µg of this plasmid, pMSV-T24-H-ras/MSV-c-myc, was capable of inducing tumors in newborn NIH Swiss mice. However, to be able to detect the oncogenicity of dominant activated oncogenes in cellular DNA, a more sensitive system was needed. In this paper, we demonstrate that the newborn CD3 epsilon transgenic mouse, which is defective in both T-cell and NK-cell functions, can detect the oncogenic activity of 25 ng of the circular form of pMSV-T24-H-ras/MSV-c-myc. When this plasmid was inoculated as linear DNA, amounts of DNA as low as 800 pg were capable of inducing tumors. Animals were found that had multiple tumors, and these tumors were independent and likely clonal. These results demonstrate that the newborn CD3 epsilon mouse is highly sensitive for the detection of oncogenic activity of DNA. To determine whether it can detect the oncogenic activity of cellular DNA derived from four human tumor-cell lines (HeLa, A549, HT-1080, and CEM), DNA (100 µg) was inoculated into newborn CD3 epsilon mice both in the presence of 1 µg of linear pMSV-T24-H-ras/MSV-c-myc as positive control and in its absence. While tumors were induced in 100% of mice with the positive-control plasmid, no tumors were induced in mice receiving any of the tumor DNAs alone. These results demonstrate that detection of oncogenes in cellular DNA derived from four human tumor-derived cell lines in this mouse system was not possible; the results also show the importance of including a positive-control plasmid to detect inhibitory effects of the cellular DNA.

Feasibility Study on Measuring Selected Proteins in Malignant Melanoma Tissue by SRM Quantification

Currently there are no clinically recognized molecular biomarkers for malignant melanoma (MM) for either diagnosing disease stage or measuring response to therapy. The aim of this feasibility study was to develop targeted selected reaction monitoring (SRM) assays for identifying candidate protein biomarkers in metastatic melanoma tissue lysate. In a pilot study applying the SRM assay, the tissue expression of nine selected proteins [complement 3 (C3), T-cell surface glycoprotein CD3 epsilon chain E (CD3E), dermatopontin, minichromosome maintenance complex component (MCM4), premelanosome protein (PMEL), S100 calcium binding protein A8 (S100A8), S100 calcium binding protein A13 (S100A13), transgelin-2 and S100B] was quantified in a small cohort of metastatic malignant melanoma patients. The SRM assay was developed using a TSQ Vantage triple quadrupole mass spectrometer that generated highly accurate peptide quantification. Repeated injection of internal standards spiked into matrix showed relative standard deviation (RSD) from 6% to 15%. All nine target proteins were identified in tumor lysate digests spiked with heavy peptide standards. The multiplex SRM peptide assay panel was then measured and quantified on a set of frozen MM tissue samples obtained from the Malignant Melanoma Biobank collected in Lund, Sweden. All nine proteins could be accurately quantified using the new SRM assay format. This study provides preliminary data on the heterogeneity of biomarker expression within MM patients. The S100B protein, which is clinically used as the pathology identifier of MM, was identified in 9 out of 10 MM tissue lysates. The use of the targeted SRM assay provides potential advancements in the diagnosis of MM that can aid in future assessments of disease in melanoma patients.

A role for CD3 conformation in the preTCR/CD3 complex (P4428)

Abstract Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.

Variable presentation of primary immune deficiency: Two cases with CD3 gamma deficiency presenting with only autoimmunity

CD3 chain expression defects including CD3 gamma, epsilon, delta, and zeta chain subunits, are autosomal recessive inherited severe combined immunodeficiencies (SCID). The phenotype is usually T-B+NK+ SCID with lymphopenia where the clinical findings may be mild (CD3γ) or severe (CD3δ, ε, ζ) owing to the underlying molecular defect. There is limited information about the disease in literature. Here, we present two siblings from non-consanguineous family with autoimmunity including Evans syndrome, autoimmune hepatitis, nephrotic syndrome, and Hashimoto's thyroiditis and with no previous history of infections. To define the molecular basis of the disease, we performed linkage analysis around the CD3 receptor cluster and found consistent linkage to this region. The patient one displayed low TCRαβ expression, low IgG, low IgA, low IgM, low CD3, low CD4, low CD8. The patient two also displayed low TCRαβ expression and low anti-HBs titer. We went onto identify a homozygous splicing mutation (IVS2-1G>C) in the two affected individuals in the CD3γ gene. To date, only four cases have been reported with CD3γ deficiency. Occasionally, the patients present with only autoimmunity including autoimmune hemolytic anemia, vitiligo, Hashimoto's thyroiditis, and autoimmune enteropathy. However, Evans syndrome, autoimmune hepatitis, and nephrotic syndrome have not been reported in previous cases. We believe that our cases will contribute to the literature.

Regression of Human Prostate Cancer Xenografts in Mice by AMG 212/BAY2010112, a Novel PSMA/CD3-Bispecific BiTE Antibody Cross-Reactive with Non-Human Primate Antigens

For treatment of patients with prostate cancer (PCa), we developed a novel T cell-engaging (BiTE) antibody designated AMG 212 or BAY2010112 that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. AMG 212/BAY2010112 induced target cell-dependent activation and cytokine release of T cells, and efficiently redirected T cells for lysis of target cells. In addition to Chinese hamster ovary cells stably expressing human or cynomolgus monkey PSMA, T cells redirected by AMG 212/BAY2010112 also lysed human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC-3-huPSMA, and LnCaP at half maximal BiTE concentrations between 0.1 and 4 ng/mL (1.8-72 pmol/L). No lysis of PSMA-negative human PCa cell lines PC-3 and DU145 was observed. The subcutaneous (s.c.) formation of tumors from PC-3-huPSMA cells in NOD/SCID mice was significantly prevented by once daily intravenous (i.v.) injection of AMG 212/BAY2010112 at a dose level as low as 0.005 mg/kg/d. Rapid tumor shrinkage with complete remissions were observed in NOD/SCID mice bearing established s.c. 22Rv1 xenografts after repeated daily treatment with AMG 212/BAY2010112 by either the i.v. or s.c. route. Of note, 22Rv1 tumors were grown in the absence of human T cells followed by intraperitoneal injection of T cells 3 days before BiTE treatment. No effects on tumor growth were observed in the absence of human T cells or AMG 212/BAY2010112. On the basis of these preclinical results, AMG 212/BAY2010112 appears as a promising new BiTE antibody for the treatment of patients with PSMA-expressing PCa.

Abstract 3523: Preclinical characterization of MT114, a novel CD33/CD3-bispecific BiTE antibody for the treatment of acute myeloid leukemia (AML)

Abstract There has been little improvement of standard of care for AML patients over the past 20 years, creating a large demand for therapies with improved benefit/risk profile and potential to cure. The CD19/CD3-bispecific, T cell-engaging (BiTE) antibody blinatumomab has shown high response rates and durable remissions in patients with acute lymphocytic leukemia and non-Hodgkin lymphoma. In order to employ this novel therapeutic modality for the treatment of patients with CD19-negative AML, we have constructed a panel of CD33/CD3-bispecific BiTE antibodies and selected MT114 as lead candidate for pre-clinical development for its superior pharmacological and pharmaceutical properties. Its target antigen CD33 (SIGLEC-3) is frequently expressed on AML blasts and leukemic stem cells, and extensive clinical experience has been gained by the CD33-targeting antibodies Mylotarg®, lintuzumab and AVE9633. Here, we report on the biological activities of MT114. MT114 recognized epitopes in the V domain of CD33 and of CD3 epsilon that are conserved between human and macaque antigen, allowing pharmacological and nonclinical safety investigations of the BiTE antibody in macaques as a relevant primate species. MT114-induced redirected lysis of 10 human AML cell lines by unstimulated peripheral blood mononuclear cell (PBMC) at half-maximal concentrations of MT114 between 23 and 328 pg/ml (0.4-7 pM). The 10 AML lines expressed CD33 at 20,000-50,000 molecules/cell. Redirected lysis was specific for CD33-expressing cells, reached completion after 28-h of co-culture, and was effective even at PBMC-to-target ratios of α1:2. MT114 induced the expression of activation markers CD69 and CD25 on T cells and the transient release of IFN-γ, TNF-α and IL-2, -6 and -10 by T cells, but only in the presence of CD33+ target cells. Redirected lysis and T cell activation by MT114 was not affected by up to 100 ng/ml of shed CD33, serum levels of which were found to be associated with AML progression. Neoexpression of CD33 on BiTE-activated T cells did not exceed 8% of total activated T cells when tested with 10 human T cell donors. The BiTE antibody significantly delayed growth of the subcutaneously injected human AML cell line HL60, and induced a robust infiltration of T cells into tumor tissue. MT114 is a highly active novel BiTE antibody with promise for the treatment of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3523. doi:1538-7445.AM2012-3523

Abstract 3526: Subcutaneous administration of PSMA/CD3-bispecific BiTE antibody MT112/BAY 2010112 leads to complete remission of human prostate cancer xenografts in mice

Abstract Blinatumomab, a CD19/CD3-bispecific BiTE antibody has shown high response rates and durable remissions in patients with relapsed or refractory acute lymphocytic leukemia and non-Hodgkin's lymphoma. For treatment of patients with prostate cancer (PCa), we have developed a novel BiTE antibody, MT112/BAY2010112, that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. MT112/BAY 2010112 binds PSMA and CD3 of human and macaque origin allowing for assessment of safety, pharmacodynamics and pharmacokinetics in a relevant animal species. PSMA expressing human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC3-PSMA and LNCaP were lysed by freshly isolated, unstimulated human T cells redirected by MT112/BAY2010112 at EC50 values ranging between 0.1 and 4 ng/ml (1.8-72 pM). Efficacy of lysis correlated with levels of surface expressed PSMA. No lysis was observed in PSMA-negative PCa cell lines PC3 and DU145, showing the target antigen-specific activity of MT112/BAY2010112. The relevance of cynomolgus monkeys for further nonclinical safety evaluation of MT112/BAY 2010112 was demonstrated by in vitro side-by-side comparison of the BiTE antibody's pharmacological characteristics in human and cynomolgus monkey assay systems. For studying the activity of MT112/BAY 2010112 after subcutaneous (s.c.) administration, human 22Rv1 PCa xenografts were grown s.c. in mice to sizes of >200 mm3 in the absence of human T cells. Three days prior to treatment with BiTE, ex-vivo expanded human T cells were intraperitoneally injected. Animals were treated s.c. at 2.5 mg/kg/d or intravenously (i.v.) at 0.5 mg/kg/d for 28 consecutive days. S.c. administration of MT112/BAY 2010112 resulted in a similar drug exposure, rapid shrinkage and complete remission of established PCa xenografts as seen upon i.v. administration of the BiTE antibody. Treatment with human T cells or vehicle alone had no effect on tumor growth. Compared to i.v. administration, the relative bioavailability of the PSMA BiTE in serum after s.c. injection in mice was approximately 18%. Initial results will be presented showing that 14C-labeled MT112/BAY 2010112 accumulates in s.c. implanted LNCaP PCa tumors of mice after tail vein injection. Based on these preclinical in vivo pharmacology results, treatment of PCa patients with s.c. administered MT112/BAY2010112 appears to be justified. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3526. doi:1538-7445.AM2012-3526

Role of Disorder in T-Cell Signaling

The T cell receptor plays a central role in the initiation of adaptive immune responses by recognizing microbial peptides bound to MHC molecules. Signaling through the receptor occurs through three associated signaling dimers (the CD3 dimers). We have shown that the cytoplasmic domain of the CD3 epsilon chain is membrane bound in live cells. Furthermore, we determined the NMR structure of the CD3 epsilon cytoplasmic domain in its lipid-bound state. The structure showed that the two critical tyrosines are inserted into the hydrophobic part of the lipid bilayer. We therefore propose that membrane binding prevents premature receptor activation.

Clinicopathological analysis of pulmonary marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue

背景与目的肺粘膜相关淋巴组织边缘区B细胞淋巴瘤（pulmonary marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, PMZL-MALT）极少见，易误诊。本文旨在总结该病特征，以提高临床医生的认识水平。方法回顾性分析四川大学华西医院2008年11月-2010年11月确诊的7例PMZL-MALT患者的临床病理资料。结果女性5例，男性2例；中位年龄62岁（37岁-74岁）。以咳嗽、咳痰为主要症状（6例）；CT多表现为肺部实变影（6例），误诊为肺炎。纤维支气管镜是主要的侵入性检查方法（6例）；7例患者肿瘤细胞免疫表型均为CD20和CD79a细胞膜弥漫强阳性表达，CD3ε、CD5、CyclinD1、CD10、Bcl-2和CD30不表达，Ki-67指数 < 10%；6例IgH基因重排分析可见克隆性条带。3例患者予以COP方案（环磷酰胺、长春新碱、泼尼松）化疗，其中1例加利妥昔单抗（RCOP）；1例予以CHOP方案（环磷酰胺、阿霉素、长春新碱、泼尼松）化疗；1例因糖尿病血糖控制不佳予以CTX（环磷酰胺）化疗；2例拒绝化疗。随访至2010年12月2日，仅1例既往有慢性阻塞性肺疾病史且未化疗者在确诊12个月后因呼吸衰竭死亡，其余患者的病情控制可。结论PMZL-MALT患者的临床表现无特异性，组织病理学检查是确诊的唯一手段，患者的预后较好。

Paraneoplastic pemphigus due to natural‐killer/T‐cell lymphoma

A 70-year-old woman presented with a 4-month history of right leg ulcers (upper left panel). Biopsy showed infiltration by abnormal medium to large size lymphoid cells (upper left middle panel, haematoxylin eosin). There was no obvious angiocentricity or angioinvasion. The abnormal lymphoid cells were positive for cytoplasmic CD3 epsilon (upper right middle panel, immunoperoxidase), CD56 (upper right panel), and T-cell intracellular antigen-1. In-situ hybridization for EpsteinBarr virus encoded RNA was positive. Overall features were consistent with extranodal natural killer (NK)/T-cell lymphoma, nasal type. She was treated with chemotherapy. After the second course, blistering skin eruptions occurred over the trunk, buttocks and legs (lower left panel). A skin biopsy revealed erythema multiforme-like areas with scattered cytoid bodies (white arrow) throughout all levels of the epidermis, and areas with pemphigus vulgaris-like changes showing early suprabasal acantholysis (black arrow) (lower right panel). Direct immunofluorescence studies showed weak intercellular and basement membrane staining with both C3 and IgG, confirming the diagnosis of paraneoplastic pemphigus (PNP). She deteriorated progressively and died 3 months after presentation despite immunosuppression and further chemotherapy. Paraneoplastic pemphigus characteristically presents as painful oral and mucosal ulcerations, with concomitant skin involvement simulating pemphigus vulgaris or erythema multiforme. Bronchiolitis obliterans may occur and can be fatal. PNP is caused by serum autoantibodies against epithelial proteins, particularly the plakin family, leading to epithelial injury in the skin, and mucosal and bronchial linings. Therefore, the underlying neoplasms are almost exclusively of B-cell lineage. However, T-cells and CD56+ NK cells have also been postulated to be effectors of PNP. This case illustrated the rare association between NK cell lymphoma and PNP, suggesting therefore that NK cells might be involved in the pathogenesis of PNP. Clinicians should also be alert to the possibility of PNP in lymphomas not of B-cell lineage.

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)-associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR-induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate-treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in-gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3 epsilon, WASP, and WIPF1) and identified several novel putative Nck-binding proteins. We subsequently verified the SH2 domain binding to the actin-binding protein HIP55 and to FYB/ADAP, and the SH3-mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR-to-cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.

Extranodal Natural-Killer/T-Cell Lymphoma, Nasal Type

The World Health Organization (WHO) classification recognizes 2 main categories of natural killer (NK) cell-derived neoplasms, namely, extranodal NK/T-cell lymphoma, nasal type, and aggressive NK-cell leukaemia. Extranodal nasal NK/T-cell lymphoma is more frequent in the Far East and Latin America. Histopathological and immunophenotypical hallmarks include angiocentricity, angiodestruction, expression of cytoplasmic CD3 epsilon (ε), CD56, and cytotoxic molecules and evidence of Epstein-Barr virus (EBV) infection. Early stage disease, in particular for localized lesion in the nasal region, is treated with chemotherapy and involved-field radiotherapy. On the other hand, multiagent chemotherapy is the mainstay of treatment for advanced or disseminated disease. L-asparaginase-containing regimens have shown promise in treating this condition. The role of autologous hematopoietic stem cell transplantation is yet to be clearly defined. Allogeneic hematopoietic stem cell transplantation, with the putative graft-versus-lymphoma effect, offers a potentially curative option in patients with advanced disease.

Pyoderma gangrenosum treated successfully with visilizumab in patients with ulcerative colitis

Pyoderma gangrenosum is a misleading designation for an idiopathic ulcerating cutaneous disease. The activity of pyoderma gangrenosum may or may not follow the activity of the inflammatory bowel disease. Visilizumab (Nuvion, PDL Bio Pharma, USA), a humanized IgG2 monoclonal antibody that binds to the human CD3 epsilon chain expressed on human T cells, induces the rapid production of selective chemokines, and reduces the CXCR3-mediated chemotaxis of the resting peripheral blood lymphocytes. In activated, but not resting T cells, visilizumab leads to rapid apoptosis. During a period of 20 years, a 40-year-old male took part in more than 30 courses of treatment for a dermatological condition misdiagnosed as crural eczema, mycosis and infected wounds. This ulcerative process was very severe in both crural regions. Ulcerative colitis was diagnosed a decade ago. He has been steroid-dependent since 1997. In 2005, pyoderma gangrenosum was diagnosed. In 2006, the patient participated in the visilizumab study and received 2x375 mcg study drug (5 mcg/kg/dose) intravenously. Six months after visilizumab administration, his leg ulcers healed. After the administration of visilizumab, pyoderma gangrenosum had not relapsed. In this severe pyoderma case, visilizumab also treated the skin disease. Although the colitis later worsened, the pyoderma gangrenosum has not recurred to date in this steroid-dependent patient. Pyoderma gangrenosum may be a T cell-mediated disease. The fact that biological therapy proved dramatically effective in this patient may suggest the use of these agents as first line of therapy in such cases.

Diagnosis and Treatment of Chronic T-Lymphocytic Leukemia in a Green Tree Monitor (Varanus prasinus)

A 13-year-old male green tree monitor (Varanus prasinus) presented with lethargy and chronic weight loss. Complete blood count (CBC) revealed marked leukocytosis (total leukocyte count = 278.1 × 103/μl) and peripheral blood films were characterized by marked lymphocytosis comprised of small, well-differentiated lymphocytes. Bone marrow aspiration revealed increased cellularity with a predominance of well-differentiated lymphocytes. Using immunocytochemistry, neoplastic lymphocytes in peripheral blood films were positive for CD3 epsilon and negative for CD79a and BLA.36, consistent with T-cell chronic lymphocytic leukemia (CLL). Distal tail necrosis was noted 17 days post-presentation and treated with partial caudectomy. Treatment was initiated for CLL with 0.8 mg/kg prednisone q 48 h 22 days post-presentation. CBC on days 94 to 151 post-presentation showed marked reduction in leukocytosis (counts remaining below 40.0 × 103/μL). The animal displayed improvement in activity, appetite, and gained weight during the initial treatment period. Marked leukocytosis (253.4 × 103/μL) returned on day 203 post-presentation, and on day 206 post-presentation the monitor received 0.08 mg/kg chlorambucil PO once in addition to ongoing prednisone administration. The animal died 210 days post-presentation. Histopathologic examination revealed neoplastic lymphocyte infiltration in the bone marrow at multiple sites as well as in liver, spleen, gall bladder, pancreas, kidneys, esophagus, and the oral cavity. Lymphocyte infiltration in multiple organs was primarily associated with areas of dense lymphocytic infiltrates surrounding blood vessels. This report describes the first published attempt of CLL treatment in a saurian species.

The αβ T Cell Receptor Is an Anisotropic Mechanosensor

Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric alphabeta, CD3 epsilon gamma, CD3 epsilon delta, and CD3 zeta zeta subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3 epsilon lobe that they approach diagonally, adjacent to the lever-like C beta FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3 epsilon and CD3 gamma in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force ( approximately 50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.