CD3epsilon Research Articles

Near missed aggressive natural killer cell leukaemia – importance of extensive immunophenotyping

Aggressive natural killer cell leukaemia (ANKL) is a rare neoplasm of NK cells with poor prognosis, varied morphology and positivity for CD2, CD3 epsilon and CD56, negative for surface CD3 and CD5. A 32-yearold man presented with high grade fever, generalized weakness, significant weight loss in the last 2 months and a lower limb mass, diagnosed elsewhere as Ewing sarcoma. He initially presented with TLC of 8000/uL which drastically increased to 52000/uL, with presence of atypical lymphoid cells. Bone marrow aspiration and biopsy was performed, bone marrow aspirate showed 44% atypical lymphoid cells that were 1.5 to 2 times the size of red blood cells with scant blue agranular cytoplasm, clumped chromatin and inconspicuous nucleoli, and flow cytometric immunophenotyping showed atypical lymphoid cells that were positive for CD45, CD38 and CD 56 and negative for CD34, CD3, CD4, CD5, CD7, CD8, CD20, CD10, CD19, CD138, kappa and lambda. Since a definitive diagnosis could not be reached on this, IHC on bone marrow biopsy was done, which showed the atypical lymphoid cells to be positive for CD45, CD3, CD2, CD56, CD38 and negative for CD117, CD5, CD7, CD30, CD20, CD57 and EBV. A diagnosis of aggressive natural killer lymphoma was reached. The diagnosis was difficult as the morphology and flow cytometry did not show any specific features of ANKL. The diagnosis was made after extensive immunophenotyping on bone marrow biopsy. This highlights the importance of doing NK cell markers when basic panel of antibodies are not supportive.

CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multi-institutional Phase 1 trials

Cibisatamab is a bispecific antibody-based construct targeting carcinoembryonic antigen (CEA) on tumour cells and CD3 epsilon chain as a T-cell engager. Here we evaluated cibisatamab for advanced CEA-positive solid tumours in two open-label Phase 1 dose-escalation and -expansion studies: as a single agent with or without obinutuzumab in S1 (NCT02324257) and with atezolizumab in S2 (NCT02650713). Primary endpoints were safety, dose finding, and pharmacokinetics in S1; safety and dose finding in S2. Secondary endpoints were anti-tumour activity (including overall response rate, ORR) and pharmacodynamics in S1; anti-tumour activity, pharmacodynamics and pharmacokinetics in S2. S1 and S2 enrolled a total of 149 and 228 patients, respectively. Grade ≥3 cibisatamab-related adverse events occurred in 36% of S1 and 49% of S2 patients. The ORR was 4% in S1 and 7% in S2. In S2, patients with microsatellite stable colorectal carcinoma (MSS-CRC) given flat doses of cibisatamab and atezolizumab demonstrated an ORR of 14%. In S1 and S2, 40% and 52% of patients, respectively, developed persistent anti-drug antibodies (ADAs). ADA appearance could be mitigated by obinutuzumab-pretreatment, with 8% of patients having persistent ADAs. Overall, cibisatamab warrants further exploration in immunotherapy combination strategies for MSS-CRC.

A novel TCGA-validated programmed cell-death-related signature of ovarian cancer

BackgroundOvarian cancer (OC) is a gynecological malignancy tumor with high recurrence and mortality rates. Programmed cell death (PCD) is an essential regulator in cancer metabolism, whose functions are still unknown in OC. Therefore, it is vital to determine the prognostic value and therapy response of PCD-related genes in OC.MethodsBy mining The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Genecards databases, we constructed a prognostic PCD-related genes model and performed Kaplan-Meier (K-M) analysis and Receiver Operating Characteristic (ROC) curve for its predictive ability. A nomogram was created via Cox regression. We validated our model in train and test sets. Quantitative real-time PCR (qRT-PCR) was applied to identify the expression of our model genes. Finally, we analyzed functional analysis, immune infiltration, genomic mutation, tumor mutational burden (TMB) and drug sensitivity of patients in low- and high-risk group based on median scores.ResultsA ten-PCD-related gene signature including protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), 8-oxoguanine-DNA glycosylase (OGG1), HECT and RLD domain containing E3 ubiquitin protein ligase family member 1 (HERC1), Caspase-2.(CASP2), Caspase activity and apoptosis inhibitor 1(CAAP1), RB transcriptional corepressor 1(RB1), Z-DNA binding protein 1 (ZBP1), CD3-epsilon (CD3E), Clathrin heavy chain like 1(CLTCL1), and CCAAT/enhancer-binding protein beta (CEBPB) was constructed. Risk score performed well with good area under curve (AUC) (AUC3 − year =0.728, AUC5 − year = 0.730). The nomogram based on risk score has good performance in predicting the prognosis of OC patients (AUC1 − year =0.781, AUC3 − year =0.759, AUC5 − year = 0.670). Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that the erythroblastic leukemia viral oncogene homolog (ERBB) signaling pathway and focal adhesion were enriched in the high-risk group. Meanwhile, patients with high-risk scores had worse OS. In addition, patients with low-risk scores had higher immune-infiltrating cells and enhanced expression of checkpoints, programmed cell death 1 ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO-1) and lymphocyte activation gene-3 (LAG3), and were more sensitive to A.443,654, GDC.0449, paclitaxel, gefitinib and cisplatin. Finally, qRT-PCR confirmed RB1, CAAP1, ZBP1, CEBPB and CLTCL1 over-expressed, while PPP1R15A, OGG1, CASP2, CD3E and HERC1 under-expressed in OC cell lines.ConclusionOur model could precisely predict the prognosis, immune status and drug sensitivity of OC patients.

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Modeling Flotetuzumab-Associated CRS in AML Using inVitro and inVivo Preclinical Models

Introduction: Flotetuzumab (FLZ) is a bispecific CD123 x CD3 DART® molecule that is being investigated for the treatment of relapsed and refractory acute myeloid leukemia (AML). FLZ induces artificial immunological synapses between CD3 on cytotoxic T-cells and CD123, which is expressed on AML blasts along with off-target hematopoietic and non-hematopoietic tissues. T-cell activation and expansion requires cytokine release from both innate and adaptive immune cells, which when excessive, can result in cytokine release syndrome (CRS), a potentially life-threatening toxicity that manifests with fever, hypotension, organ dysfunction, respiratory failure, and/or coagulopathy. FLZ-mediated anti-leukemia efficacy is often limited by CRS. Therefore, developing pre-clinical models by which to better understand the mechanisms of CRS induction, including collaborating cell types that produce IL-6 and other inflammatory cytokines, is essential to understanding the biology of CRS and developing mechanistically plausible preventative and treatment strategies. MacroGenics has developed a murine genetic knock-in model that expresses a chimeric murine CD3 epsilon (CD3e) chain bearing the human CD3e epitope recognized by FLZ (hCD3e). These mice are immune competent, maintain normal signaling through the T-cell receptor and expand and secrete cytokines upon binding to FLZ and human CD123. To validate this model, we explore whether T-cells harvested from the spleens of immune competent C57Bl/6 hCD3e mice will bind FLZ and kill human CD123-expressing target cells in vitro. Methods: A murine AML cell line (AML1-GFP FLT3-ITD, DNMT3AR878H) was transduced with human CD123 (hCD123) using a lentiviral vector construct and single cell clones expressing high levels of hCD123 were expanded (AMLGFP-hCD123). Spleens from hCD3eHOM C57Bl/6 mice were harvested for isolation of T-lymphocytes via negative immunomagnetic selection on an AutoMACS machine. In vitro killing assays were set up using purified C57Bl/6 hCD3eHOM T-cells and two clones of AMLGFP-hCD123 cells. The effector to target ratio was 1:1, with 2.5x105 cells per well in a 24-well plate. Flow cytometry/FACS was then performed at 24 hours to assess killing of AMLGFP-hCD123 clones 4 and 14 and T-cell subset number and phenotype. Antibodies to the following antigens were used for FACS: mCD4, hCD3 (epitope SP38), mCD25, mCD8, mCD44, mCD62L, hCD123, mCD3, mCD127, mCD69, and mCD45, in addition to endogenous GFP. Results: At 24 hours, target cell killing of clones 4 and 14 was noted (Figure 1). Compared to the controls treated with FLZ or T-cells alone, targets treated with T-cells + FLZ showed a 2.3- and 2.5-fold decrease in the number of AMLGFP-hCD123 targets in clones 4 and 14, respectively, at 24 hours (Figure 1). In terms of T-cell phenotypic changes, the percentage and MFI expression of activation markers, namely CD25 and CD69, increased for CD4+ and CD8+ subsets in the treatment group compared to controls (data not shown). T-cell subsets were characterized as naïve (CD62L+/CD44-), central memory (CD62L+/CD44+), and effector memory (CD62L-/CD44+), as shown in Figure 2. For CD4+ and CD8+ subsets co-cultured with targets, the addition of FLZ induced phenotypic changes away from naïve and towards central and effector memory subsets (Figure 2). In the two control groups, naïve T-cells predominated at the 24-hour time point (Figure 2). Conclusions: The in vitro killing assay shows that T-cells from the spleens of immune competent C57Bl/6 hCD123eHOM mice recognize FLZ and hCD123 on transduced murine leukemia cells. Co-culture with T-cells + target cells + FLZ showed killing at 24 hours compared to controls. T-cell activation and proliferation were noted based on cell surface markers. The shift in phenotype from naïve to central and effector memory subsets suggests T-cell expansion upon leukemia antigen recognition and portends killing of target cells. Pending in vivo studies will evaluate if FLZ can activate endogenous T cells, induce CRS, and kill AMLGFP-hCD123 targets in the huCD3eHOM mice. If successful, we will use the huCD3eHOM model to test the efficacy of various therapies to mitigate CRS and preserve FLZ-mediated T-cell cytotoxicity against AMLGFP-hCD123 targets in huCD3eHOM mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-07. HUMANIZED BI-SPECIFIC ANTIBODY SHOWS POTENT ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA

Abstract BACKGROUND Advancements in bioengineering have allowed bispecific Abs to develop with therapeutic applications in immuno-oncology. Here we describe a fully humanized bispecific t cell engager (BiTE) that binds the GBM-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and the T Cell receptor CD3 epsilon (CD3ε) to elicit an anti-tumor immune response in patient-derived models of GBM. METHODS In vitro, BiTE-mediated activation of T Cells was assessed via flow cytometry as measurements of T cell activation markers CD25 and CD69 and cytokines IFNγ and TNFα. BiTE-directed T cell-mediated cytotoxicity was assessed in GBM 6 and GBM12patient-derived xenograft lines. In vivo, GBM6 patient-derived xenograft cells were intracranially implanted in NSG-MHC I/II DKO mice, reconstituted with HLA-matched donor peripheral blood lymphocytes (PBMC), and subsequently treated with 2.5 mg/kg BiTE via i.p. injection x 4 days/week of therapeutic BiTE for three weeks. Animals were followed for survival, and the brains of long-term survivors were analyzed for residual disease and T cell infiltration. RESULTS In vitro, BiTE potently activated T cells as determined by a high expression of CD25 and CD69 and the production of TNFα and IFNγ. BiTE also directed T cell-mediated cytotoxicity against GBM cells in a dose-dependent manner, with significant activity still seen in the low nanogram range. In vivo, BiTE significantly extended the survival of tumor-bearing animals in GBM6patient-derived xenograft models. Histological analysis of long-term surviving mice shows complete absence of tumor and robust presence of CD8+ T cells localized to the original site of tumor implantation. CONCLUSIONS Our data demonstrate the efficacy of humanized BiTE in patient-derived GBM models, and our results support the investigation of this novel therapeutic in a clinical trial setting.FUNDING: R01 NS122395-01 and R01 NS095642-01

CD3e-immunotoxin spares CD62Llo Tregs and reshapes organ-specific T-cell composition by preferentially depleting CD3ehi T cells.

CD3-epsilon(CD3e) immunotoxins (IT), a promising precision reagent for various clinical conditions requiring effective depletion of T cells, often shows limited treatment efficacy for largely unknown reasons. Tissue-resident T cells that persist in peripheral tissues have been shown to play pivotal roles in local and systemic immunity, as well as transplant rejection, autoimmunity and cancers. The impact ofCD3e-ITtreatment on these local cells, however, remains poorly understood. Here, using a new murinetesting model, we demonstrate a substantial enrichment of tissue-resident Foxp3+ Tregs following CD3e-IT treatment. Differential surface expression of CD3e among T-cell subsets appears to be a main driver of Treg enrichment inCD3e-IT treatment. The surviving Tregs inCD3e-IT-treated micewere mostly the CD3edimCD62Lloeffector phenotype, but the levels of this phenotype markedly varied among different lymphoid and nonlymphoid organs. We also found notable variations in surfaceCD3e levels among tissue-resident T cells of different organs, and these variationsdrive CD3e-IT to uniquely reshape T-cell compositions in local organs. The functions of organs and anatomic locations (lymph nodes)also affected the efficacy of CD3e-IT. The multi-organ pharmacodynamics of CD3e-IT and potential treatment resistance mechanisms identified in this study may generate new opportunities to further improve this promising treatment.

Abstract 2078: CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells

Abstract Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with few options for curative treatment. In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable success. However, such immunotherapeutic strategies are not yet established for AML, likely due to a dearth of appropriate targets. The fms like tyrosine kinase 3 (FLT3)/CD135 surface receptor is expressed on AML cells in the large majority of AML patients and constitutes a highly selective target antigen for immunotherapy, as expression on healthy tissues is limited to low levels on dendritic cells, monocytes and hematopoietic progenitor cells. We report here on the development of CLN-049, a bispecific humanized antibody that binds to FLT3 on leukemic cells and the CD3 epsilon subunit of the T cell receptor complex on T cells. CLN-049 recognizes the membrane proximal domain of FLT3 and has a silenced Fc gamma 1 domain, with anti-CD3 single chain variable fragments (scFv) fused to the C-termini of the heavy chains. While FLT3 kinase inhibitors are limited to treatment of patients with specific intracellular FLT3 mutations, CLN-049 recognizes the extracellular domain of FLT3 and would therefore be more broadly applicable in AML therapy. CLN-049 showed low-nanomolar binding to both FLT3-expressing and CD3-expressing cells. The antibody induced target-dependent activation of CD4+ and CD8+ T cells as well as corresponding cytokine production by T cells only when co-cultured with FLT3+ AML cells. Human AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed upon treatment with sub-nanomolar concentrations of CLN-049 when co-cultured with heterologous PBMCs, regardless of the expression level or mutational status of FLT3. Intriguingly, FLT3-expressing hematopoietic progenitor cells and dendritic cells were not found to be sensitive to CLN-049 killing. The in vitro lysis of leukemic cells by CLN-049 was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft studies, CLN-049 was highly active against the MOLM-13 human AML model. In these studies, CLN-049 demonstrated significant dose-dependent activity as measured by survival as well as reduction of AML cells in the blood. In summary, CLN-049 is a promising FLT3-targeted T cell engaging antibody construct expected to have robust anti-tumor activity in the clinic against AML. CLN-049 is currently in a phase 1 clinical trial for the treatment of patients with relapsed/refractory AML. Citation Format: Kristan Meetze, Naveen K. Mehta, Martin Pfluegler, Bochong Li, Patrick A. Baeuerle, Jennifer S. Michaelson, Gundram Jung, Helmut Salih. CLN-049 is a bispecific T cell engaging IgG-like antibody targeting FLT3 on AML cells [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 2078.

Injection Effect of Anti-CD3 Monoclonal Antibody on Primo Vessel in Lymph Vessel of Rabbit with Lipopolysaccharide-Induced Inflammation.

The primo vascular system can be viewed as a circulatory system that plays a therapeutic function in regenerating the body tissue. The anti-CD3 monoclonal antibody was used as an immunotherapeutic agent to treat the novel coronavirus infection (COVID-19). Objectives: In this study, we observed the effect of injecting lymph nodes with Foralumab, an anti- human CD3 epsilon therapeutic monoclonal antibody, on primo vessels. The structure and atomic stoichiometry of the antibody were determined by transmission electron microscopy and energy dispersive spectroscopy. Alcian blue dying solution was injected into the lymph nodes of the abdominal vena cava of rabbits, and the solution further flowed into the lymph vessels. A primo vessel with primo nodes stained with Alcian blue was clearly visible in the lymph vessel. By injecting Foralumab into lymph nodes of rabbits with lipopolysaccharide-induced inflammation, the floating primo vessel in the lymph vessel appeared thicker and was distinctly visible. The observation of the primo vessel post-treated with Foralumab in the inflamed lymphatic system suggests the possibility of a functional role of the primo vascular circulatory system in pathophysiological conditions.

Surface Ligand Valency and Immunoliposome Binding: when More Is Not Always Better.

Nano-drug delivery systems are designed to contain surface ligands including antibodies for "active targeting". The number of ligands on each nanoparticle, known as the valency, is considered a critical determinant of the "targeting" property. We sought to understand the correlation between valency and binding properties using antibody conjugated liposomes, i.e. immunoliposomes (ILs), as the model. Anti-CD3 Fab containing a terminal cysteine residue were conjugated to DSPE-PEG-maleimide and incubated with preformed liposomes at 60°C. The un-incorporated antibodies were removed and the obtained ILs were characterized to contain in average 2-22 copies of anti-CD3 Fabs per liposome. The Biolayer Interferometry (BLI) probe surface was coated with various densities of CD3 epsilon&delta heterodimer (CD3D/E) to imitate different CD3 expression levels on target cells. The inference wavelength shifts upon anti-CD3 liposome binding were monitored and analyzed. The data indicated ILs may bind either monovalently or multivalently, determined mainly by the surface ligand density rather than the ILs antibody valency. The ILs valency indeed correlated with the dissociation rate constant (Koff), but not with the association rate constant (Kon). Their binding capabilities also did not necessarily increase with the surface anti-CD3 valency. We proposed a model for understanding the binding properties of ILs with different ligand valencies. The binding mode may change when the targeted surfaces had different antigen densities. The model should be important for the designing and optimization of active targeting drug delivery systems to fit different applications.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

APJS-2 Updates in research for CAR-T therapy in China

As we all know, more and more studies on CAR-T cell therapy were performed in China. And in 2020, although affected by the pandemic of COVID-19, great achievement has been made in both basic and clinical research in CAR-T cell therapy. As for basic research, a variety of new CAR-T related technologies was developed. For instance, CD3 epsilon cytoplasmic domain was incorporated into a second-generation chimeric antigen receptor (CAR), resulting increased antitumor activities and less cytokine release. Up to November 2nd, ten new INDs for CAR-T cell therapies were approved by National Medical Products Administration (NMPA). And, importantly, two New Drug Application of CAR-T cell programs were submitted to NMPA. Additionally, there are 397 clinical trials on CAR-T cell therapies in China. CAR-T cells redirected to new targets such as claudin18.2 and some next-generation of CAR-T cell programs are under clinical evaluation. We believe that all these researches and developments would advance CAR-T cell therapies to help more patients in the near future.

Molecular Imaging of Radiolabeled Bispecific T-Cell Engager 89Zr-AMG211 Targeting CEA-Positive Tumors.

Purpose: AMG 211, a bispecific T-cell engager (BiTE) antibody construct, targets carcinoembryonic antigen (CEA) and the CD3 epsilon subunit of the human T-cell receptor. AMG 211 was labeled with zirconium-89 (89Zr) or fluorescent dye to evaluate the tumor-targeting properties.Experimental Design: 89Zr-AMG211 was administered to mice bearing CEA-positive xenograft tumors of LS174T colorectal adenocarcinoma or BT474 breast cancer cells, as well as CEA-negative HL-60 promyelocytic leukemia xenografts. Biodistribution studies with 2- to 10-μg 89Zr-AMG211 supplemented with unlabeled AMG 211 up to 500-μg protein dose were performed. A BiTE that does not bind CEA, 89Zr-Mec14, served as a negative control. 89Zr-AMG211 integrity was determined in tumor lysates ex vivo Intratumoral distribution was studied with IRDye800CW-AMG211. Moreover, 89Zr-AMG211 was manufactured according to Good Manufacturing Practice (GMP) guidelines for clinical trial NCT02760199Results: 89Zr-AMG211 demonstrated dose-dependent tumor uptake at 6 hours. The highest tumor uptake was observed with a 2-μg dose, and the lowest tumor uptake was observed with a 500-μg dose. After 24 hours, higher uptake of 10-μg 89Zr-AMG211 occurred in CEA-positive xenografts, compared with CEA-negative xenografts. Although the blood half-life of 89Zr-AMG211 was approximately 1 hour, tumor retention persisted for at least 24 hours. 89Zr-Mec14 showed no tumor accumulation beyond background level. Ex vivo autoradiography revealed time-dependent disintegration of 89Zr-AMG211. 800CW-AMG211 was specifically localized in CEA-expressing viable tumor tissue. GMP-manufactured 89Zr-AMG211 fulfilled release specifications.Conclusions: 89Zr-AMG211 showed dose-dependent CEA-specific tumor targeting and localization in viable tumor tissue. Our data enabled its use to clinically evaluate AMG 211 in vivo behavior. Clin Cancer Res; 24(20); 4988-96. ©2018 AACR.

Abstract 962: Testing T cell co-potentiation as an antitumor therapeutic strategy in humanized mouse models

Abstract We recently described the concept of T cell co-potentiation as means to target antigen recognition by T cells to achieve antitumor immunotherapy in mouse models. Normally, T cell antigen receptor (TCR) engagement by a strong immunogenic antigen induces a CD3 conformational change that exposes a proline-rich sequence (PRS) in the cytosolic domain of CD3 epsilon (CD3e) to dock cytosolic proteins and contribute to the assembly of the signaling cascade that activates T cell immune function. While induction of CD3 conformational change is required by T cells to achieve productive TCR/CD3 signaling, this molecular event fails to occur when the TCR interacts with poorly immunogenic antigens, like most tumor-associated antigens (TAAs). We found that CD3 conformational change can be exogenously induced on T cells using monovalent Fab fragments (Mono-Fabs) specific for the CD3 complex. Anti-CD3 Mono-Fabs expose the CD3e PRS, but fail to crosslink the TCR/CD3, turning them incompetent to trigger T cell activation in the absence of additional stimulation. However, if TCR has bound a weak antigen, one that fails to induce CD3 conformational change intrinsically, then anti-CD3 Mono-Fabs enable TCR/CD3 signaling, and as a result co-potentiate T cell responses to weak antigenic stimulation. Using a mouse model for melanoma (B6 mice and B16-F10 melanoma cell line), we found that treatment of mice with anti-CD3e Mono-Fabs improved antitumor T cell responses against melanoma in an antigen-dependent manner, and dramatically improved the outcome of complementary T cell immunotherapies. Based on these observations, we hypothesize that T cell co-potentiation using anti-human CD3 Mono-Fabs can be exploited as a novel immunotherapy for cancer patients. We have recently identified one anti-human CD3 Mono-Fab that is capable to induce CD3 conformational change in human T cells and allows the co-potentiation of T cell response to weak antigenic stimulation. Here we present data from human T cells and humanized mouse models using this Mono-Fab that support the potential therapeutic value of exploiting T cell co-potentiation to treat cancer patients. Citation Format: Alfreda D. Nelson, Laura Elsbernd, Adam G. Schrum, Diana Gil Pages. Testing T cell co-potentiation as an antitumor therapeutic strategy in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 962.

Abstract 3814: TriTACs are novel T cell-engaging therapeutic proteins optimized for the treatment of solid tumors and for long serum half-life

Abstract T cell engagers are antibody-based therapeutics that transiently tether T cells via the T cell receptor complex (TCR) to surface antigens on tumor cells. This leads to activation of T cells and redirected lysis of the attached target cell. The therapeutic potential of this modality was demonstrated by blinatumomab, a CD19/CD3-bispecific T cell engager approved for the treatment of adult patients with relapsed/refractory acute lymphoblastic leukemia. Despite success of this T cell-engaging therapy in a hematologic malignancy, clinical studies in solid tumors with other T cell engagers have been less encouraging so far. The TriTAC (Tri-specific T cell Activating Construct) platform was developed to address shortcomings of existing T cell engagers, including short serum half-life, limited tissue penetration, and suboptimal activity. TriTAC constructs are made of a single polypeptide designed to bind to a cancer surface antigen, the CD3 epsilon subunit of the TCR, and to human serum albumin. CD3 is bound by a single-chain variable fragment (scFv) while both tumor targeting and albumin binding are achieved by single domain antibodies. The latter allow TriTACs to be very small, stable, and easily produced and purified. Noncovalent binding to serum albumin has been validated as an effective way to extend the serum half-life of other proteins up to several weeks. Even though TriTACs have three binding domains, their overall size is only ~50 kDa, one third of the size of a monoclonal antibody. This is expected to allow for faster diffusion into human tumor tissues than is possible with antibodies given the high interstitial pressure and dense extracellular matrix in solid tumors. TriTACs can induce T cell to kill tumor cells in vitro at single-digit picomolar to femtomolar concentrations with concomitant induction of inflammatory cytokine release and T cell proliferation. TriTACs can diffuse much faster across an extracellular matrix than antibodies, and eradicate tumors in mouse xenograft models supplemented with human T cells. In nonhuman primates, TriTAC molecules have serum half-lives of approximately 4 days, and appear well tolerated. Citation Format: Holger Wesche, Wade Aaron, Richard J. Austin, Patrick A. Baeuerle, Adrie Jones, Bryan Lemon, Kenneth Sexton, Timothy Yu. TriTACs are novel T cell-engaging therapeutic proteins optimized for the treatment of solid tumors and for long serum half-life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3814.

Mucosal administration of CD3-specific monoclonal antibody inhibits diabetes in NOD mice and in a preclinical mouse model transgenic for the CD3 epsilon chain

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

Application of a MABEL Approach for a T-Cell-Bispecific Monoclonal Antibody: CEA TCB.

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 μg was selected as a safe starting dose for clinical study.

Phase 1 Dose Escalation Study of MEDI-565, a Bispecific T-Cell Engager that Targets Human Carcinoembryonic Antigen, in Patients With Advanced Gastrointestinal Adenocarcinomas

IntroductionMEDI-565, a bispecific, single-chain antibody targeting human carcinoembryonic antigen on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex, showed antitumor activity in carcinoembryonic antigen-expressing tumors in murine models. Patients and MethodsThis phase I, multicenter, open-label dose escalation study enrolled adults with gastrointestinal adenocarcinomas. MEDI-565 was given intravenously over 3 hours on days 1 through 5 in 28-day cycles, with 4 single-patient (0.75-20 μg) and 5 standard 3 + 3 escalation (60 μg-3 mg; 1.5-7.5 mg with dexamethasone) cohorts. Primary objective was determining maximum tolerated dose; secondary objectives were evaluating pharmacokinetics, antidrug antibodies, and antitumor activity. ResultsThirty-nine patients were enrolled (mean age, 59 years; 56% male; 72% colorectal cancer). Four patients experienced dose-limiting toxicities (2 at 3 mg; 2 at 7.5 mg + dexamethasone): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Five patients reported grade 3 treatment-related adverse events: diarrhea, CRS, increased alanine aminotransferase, hypertension (all, n = 1), and hypoxia (n = 2); 6 experienced treatment-related serious adverse events: diarrhea, vomiting, pyrexia, CRS (all, n = 1), and hypoxia (n = 2). MEDI-565 pharmacokinetics was linear and dose-proportional, with fast clearance and short half-life. Nineteen patients (48.7%) had antidrug antibodies; 5 (12.8%) had high titers, 2 with decreased MEDI-565 concentrations. No objective responses occurred; 11 (28%) had stable disease as best response. ConclusionsThe maximum tolerated dose of MEDI-565 in this patient population was 5 mg administered over 3 hours on days 1 through 5 every 28 days, with dexamethasone. Pharmacokinetics were linear. No objective responses were observed.

Phase 1 dose escalation study of MEDI-565, a bispecific T-cell engager that targets human carcinoembryonic antigen (CEA), in patients with advanced gastrointestinal (GI) adenocarcinomas.

320 Background: MEDI-565, a bispecific single-chain antibody, targets human CEA on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex. In murine models, MEDI-565 showed antitumor activity in CEA-expressing tumors (J Immunother 2009;34:341-52). Methods: This phase 1, multicenter, open-label, dose-escalation study enrolled adults with GI adenocarcinomas (including esophageal, gastric, small intestine, colorectal, biliary tract, and pancreatic). MEDI-565 was given intravenously over 3 h on days 1–5 in 28-day cycles, with 4 single-patient (pt) (0.75–20 μg) and 5 standard 3+3 escalation (60 μg–3 mg; 1.5–7.5 mg with dexamethasone [dex]) cohorts. Primary objective was to determine the maximum tolerated dose (MTD); secondary objectives were to evaluate pharmacokinetics (PK), antidrug antibody (ADA), and antitumor activity. Results: Study enrolled39 pts: mean age 59 y; 56% male; 28 (72%) colorectal, 6 (15%) pancreatic, 5 (13%) other. Dose-limiting toxicities (grade ≥ 3 nonhematologic) were seen in 4 pts (2 at 3-mg; 2 at 7.5-mg + dex): hypoxia (n = 2), diarrhea, and cytokine release syndrome (CRS). Grade 3 treatment-related adverse events (AEs) seen in 5 pts: diarrhea, CRS, increased alanine aminotransferase, hypertension (all n = 1), and hypoxia (n = 2). Treatment-related serious AEs seen in 6 pts: diarrhea, vomiting, pyrexia, CRS (all n = 1), and hypoxia (n = 2). Five pts discontinued treatment due to AEs: diarrhea, CRS, central nervous system metastases, and hypoxia (n = 2). MEDI-565 exposures increased in approximately dose-proportional manner, with clearance (35–77 L/d) and half-life (2–7 h) typical of drug class. ADA had minor impact; 19 pts (48.7%) had ADAs, 5/39 (12.8%) with high titer, with decreased MEDI-565 concentrations in 2 pts. Plasma inflammatory cytokines were elevated posttreatment in several pts at 1.5- and 3-mg (no dex) dose levels. No objective responses were observed; 11 (28%) pts had stable disease as best response. Conclusions: The MTD of MEDI-565 in pts with GI adenocarcinomas was 5 mg with dex. PK was linear, with fast clearance and short half-life. No objective responses were observed. Clinical trial information: NCT01284231.

Contribution of CD3 <i>ε</i> Epitope and Oxidative Type of Copper-Zinc Superoxide Dismutase to the Degeneration Processes of Cerebellar Purkinje Cells in Patients with Multiple System Atrophy-Cerebellar Type (MSA-C: Olivopontocerebellar Atrophy, OPCA): An Immunohistochemical Study

Objective: This study aimed to investigate the contribution of CD3 epsilon (e) epitope and oxidative type of copper-zinc superoxide dismutase to the degeneration processes of cerebellar Purkinje cells in patients with Multiple System Atrophy-Cerebellar type (MSA-C). Methods: This retrospective study was carried out on autopsy specimens of 17 patients with sporadic MSA-C and 10 normal individuals. Paraffin sections of autopsied cerebella and pontes were immunostained with polyclonal antibodies against CD3 e epitope and oxidative modification to cysteine sulfonic acid of cys111 in human copper-zinc superoxide dismutase (Ox-SOD1). With respect to the areas of CD3-e-epitope expression, the immunohistochemical study and the quantitative statistical analysis between the areas of CD3-e-epitope expression in the surviving Purkinje cells of MSA-C patients and their disease duration were performed. Results: The cell bodies and dendritic arborization including primary, secondary, and tertiary dendrites of normal Purkinje cells were intensely immunostained by the antibody against CD3 e epitope. Both the immunohistochemical study and the quantitative statistical analysis revealed that the areas positive for CD3 e epitope disappeared in the order from tertiary dendrites, secondary dendrites, primary dendrites toward the cell bodies, along with the disease progression. In addition, Glial Cytoplasmic Inclusions (GCIs) and Neuronal Cytoplasmic Inclusions (NCIs) were strongly positive for CD3 e epitope. The surviving Purkinje cells in MSA-C showed immunostaining by the anti-Ox-SOD1 antibody, although normal Purkinje cells did not. Conclusion: Based on the oxidative stress that the surviving Purkinje cells in MSA-C express Ox-SOD1, the functions of morphogenesis and morphological maintenance related to CD3-e-epitope expression of the MSA-C Purkinje cells are impaired from the peripheral dendrites toward the cell bodies as the center of the Purkinje cell system. In addition, GCIs and NCIs that are pathological hallmarks of MSA also intensely express CD3 e epitope.