anti-CD3 Monoclonal Antibody Research Articles

Digital Microscopy Versus Conventional Microscopy In Assessment Of Jejune Intraepithelial Lymphocytes

Introduction/ Background Leukocytes infiltrates assessment in the lining epithelium of the digestive tract is a common request in current pathology practice both in terms of diagnosis and quantification of the therapeutic response. Frequently analyzed are lymphocytes positive for CD4 and CD8 in the context of gluten sensitive enteropathy. Microscopic quantification remains a time consuming and a laborious work. The possibility of human error puts at question the accuracy of data provided. Implementation of digital image acquisition techniques brings a relative comfort in delimitation the areas of interest, quantifying those areas and introduces digital algorithms that can determine the number of positive signals on the area of interest. Aims Establishing quantitative differences between the data acquired using optical microscopy versus digital evaluation of intraepithelial leukocytes (CD3+, CD4+, CD8+). Methods We analyzed paraffin embedded jejune segments fixed in buffered formalin (10%), taken from healthy patients (n= 15), monitoring the positive lymphocyte count and CD4 + CD8 + ratio. From each block, 4 sections of 3 µm thickness were taken. One section was stained with hematoxylin and eosin, and the other 3 sections were stained immunohistochemically using three steps-indirect streptavidin method for anti CD3 monoclonal antibodies, anti CD4 and CD8. The sections were referred to five independent pathologists in order to quantify positive leukocyte number in 100 enterocytes using optical microscopy and subsequent transmission microscopy images projected on high-definition monitors. Readings were performed on optical microscopes Leica DM750 objective 10X 40X eyepieces FN20. Image acquisition was made with 5-megapixel Leica microscope camera, ICC50 model, in direct transmission (stream) USB 2.0, monitors Dell 27 “HDMI IPS. For normalization and comparison of data, reporting was done in an area of 1 mm2. The variation between observers, for the two methods, were calculated (k). Results Interobserver variability for CD3 in optical was estimated as slight agreement (k0.36) in traditional microscopy compared to substantial agreement for digital assessment (0.68). For CD4 and CD8 markers the differences were less significant, substantial agreement in both categories (CD4 0.72 for optical evaluation vs 0.78 in digital evaluation data and for CD8 0.65 vs 0.72 respectively). In our opinion the use of digital image acquisition techniques in quantification of intraepithelial leukocytes increases concordance degree among observers through diminishing the subjective factors and by choosing identical ROIs.

Modulation of human T cell cytokines by the Mycobacterium tuberculosis-secreted protein Wag31

Mycobacterium tuberculosis secretes a number of proteins into the extracellular milieu during growth. Several of these proteins have been associated with modulation of the host immune response. Antigen 84, or Wag31, is one such protein that is conserved among all mycobacterial species and is recognized by the sera from tuberculosis and leprosy patients. Here, we examined the effect of Wag31 on the ability of activated human T cells to produce cytokines such as IL-10, IL-17 and IFN-γ in response to combined anti-CD3 and anti-CD28 stimulation. Purified recombinant Wag31 inhibited the secretion of IL-10 and IL-17, but not IFN-γ, by human T cells stimulated with plate-bound anti-CD3 and anti-CD28 monoclonal antibodies. Furthermore, the C-terminal domain, but not the N-terminal domain, inhibited the production of IL-10 and IL-17 without a significant effect on the production of IFN-γ. These data suggest that Wag31 may modulate human T cell immune responses during tuberculosis infection through its C-terminal domain.

Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4(+) CD25(+) regulatory T cells population.

Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3−/−) as skin graft donors or recipients. Compared with C3+/+ B6 allografts, C3−/− B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2bm12 B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3+/+ allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3−/− allografts. Moreover, C3−/− allografts caused attenuated Th1/Th17 responses, but increased CD4+CD25+Foxp3+ regulatory T (Treg) cell expression markedly in local intragraft and H-2bm12 recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4+ CD25+ Treg cell population expansion and attenuated Th1/Th17 response.

CD95-mediated apoptosis in Burkitt's lymphoma B-cells is associated with Pim-1 down-regulation

B-cells of the high-grade non-Hodgkin lymphoma Burkitt's lymphoma (BL) overexpress survival oncoproteins, including the proviral integration site for Moloney murine leukaemia virus kinase (Pim)-1, and become apoptosis resistant. Activated death receptor CD95 after ligation with anti-CD95 monoclonal antibody (mAb) resulted in the regression of BL via induction of apoptosis, suggesting a decrease of survival protein expression. Here, CD95-mediated apoptotic pathways in BL B-cell lines (Raji and Daudi) following treatment with anti-CD95 mAb was investigated with the cause-and-effects on pim-1 gene expression, in comparison with leukemic cell line (K562) used as CD95-negative cells. Immunohistochemical staining for CD95 and Pim-1 was performed, and the effects of anti-CD95 mAb on apoptotic signalling using western blotting, on caspase activity and cell survival of BL B-cell and leukemic cell lines were determined. We showed that Raji cells expressed more CD95 receptors than Daudi cells. Half of each population underwent apoptosis accompanied by decreased cell viability after anti-CD95 mAb treatment. Distinct extrinsic and intrinsic CD95-mediated apoptotic pathways in Raji and Daudi cells were revealed by high caspase activity and mitochondrial outer membrane permeabilization, respectively. We observed decreased Pim-1 transcript and protein expression levels with increased heat-shock protein (Hsp)70 and decreased Hsp90 expression in anti-CD95 mAb-treated cells. Throughout the study, K562 cells did not undergo apoptosis upon anti-CD95 mAb treatment. Pim-1 knockdown following to stable transfection with plasmid vectors induced apoptosis and decreased viability of BL and K562 cells. Therefore, CD95-mediated apoptosis induces Pim-1 down-regulation in BL B-cells, but Pim-1 down-regulation cannot fully eradicate BL and leukaemia.

Rituximab induces phenotypical and functional changes of NK cells in a non-malignant experimental setting.

BackgroundRituximab has broad and increasing application in rheumatic diseases. It is known from lymphoma studies that natural killer (NK) cells can lyse rituximab-coated transformed B cells. However, the role of NK cells in mediating rituximab-induced depletion of non-malignant B cells is unknown. The purpose of this study was to provide fundamental data on rituximab-mediated effects on NK cells in PBMCs without tumor cells, in order to simulate effects that could be relevant in patients with rheumatic disease.MethodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured overnight with therapeutic antibodies. NK cells were isolated using a commercial kit or depleted from PBMCs using anti-CD56 and anti-CD16 monoclonal antibodies and magnetic beads. Cells were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using 51Cr-labeled K562 target cells.ResultsAddition of rituximab to PBMCs resulted in depletion of B cells, which was dependent on NK cells and serum factors. The extent of B cell depletion correlated with the percentage of NK cells. Following incubation with rituximab, NK cells within PBMCs were activated, degranulated and downregulated the low affinitiy Fc-γ-receptor CD16 (FcγRIIIA). The co-activating receptor CD137 (41BB) was upregulated on a fraction of NK cells. NK cell function was altered in some donors in whom we observed rituximab-dependent reduction in NK cell cytotoxicity towards K562 tumor cells.ConclusionsNK cells mediate rituximab-induced B cell depletion. Rituximab induces altered NK cell phenotype and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1101-3) contains supplementary material, which is available to authorized users.

Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

Molecular immunology profiles of monkeys following xenografting with the islets and heart of α-1,3-galactosyltransferase knockout pigs.

Effective immunosuppression strategies and genetically modified animals have been used to prevent hyperacute and acute xenograft rejection; however, the underlying mechanisms remain unknown. In this study, we evaluated the expression of a comprehensive set of immune system-related genes (89 genes, including five housekeeping genes) in the blood of cynomolgus monkeys (~5 yr old) used as graft recipients, before and after the xenografting of the islets and heart from single and double α-1,3-galactosyltransferase (GalT) knockout (KO) pigs (<6 weeks old). The immunosuppressive regimen included administration of cobra venom factor, anti-thymocyte globulin, rituximab, and anti-CD154 monoclonal antibodies to recipients before and after grafting. Islets were xenografted into the portal vein in type 1 diabetic monkeys, and the heart was xenografted by heterotopic abdominal heart transplantation. Genes from recipient blood were analyzed using RT(2) profiler PCR arrays and the web-based RT(2) profiler PCR array software v.3.5. Recipients treated with immunosuppressive agents without grafting showed significant downregulation of CCL5, CCR4, CCR6, CD4, CD40LG, CXCR3, FASLG, CXCR3, FOXP3, GATA3, IGNG, L10, IL23A, TRAF6, MAPK8, MIF, STAT4, TBX21, TLR3, TLR7, and TYK2 and upregulation of IFNGR1; thus, genes involved in protection against viral and bacterial infection were downregulated, confirming the risk of infection. Notably, C3-level control resulted in xenograft failure within 2 days because of a 7- to 11-fold increase in all xenotransplanted models. Islet grafting using single GalT-KO pigs resulted in upregulation of CXCL10 and MX1, early inflammation, and acute rejection-associated signals at 2 days after xenografting. We observed at least 5-fold upregulation in recipients transplanted with islets grafts from single (MX1) or double (C3, CCR8, IL6, IL13, IRF6, CXCL10, and MX1) GalT-KO pigs after 77 days; single GalT-KO incurred early losses owing to immune attacks. Our results suggest that this novel, simple, non-invasive, and time-efficient procedure (requiring only 1.5 ml blood) for evaluating graft success, minimizing immune rejection, and blocking infection.

IL-7 Mediated Homeostatic Expansion of Human CD4+CD25+FOXP3+ Regulatory T Cells After Depletion With Anti-CD25 Monoclonal Antibody.

The maintenance or expansion of regulatory T (Treg) cells has a fundamental role in the achievement of immunological tolerance after transplantation. Here we aimed to determine mechanisms of human Treg cell depletion and reconstitution after anti-CD25 monoclonal antibody (mAb) treatment. Seventeen patients with type 1 diabetes who received pancreatic islet transplantation and anti-CD25 mAb as induction therapy were studied. We observed an almost complete depletion of Treg cells after injection of anti-CD25 mAb. The kinetic of Treg cell depletion did not parallel the disappearance of CD25+ T cells as CD25 is also rapidly downregulated and internalized. Regulatory T cell reconstitution is completed within 6 months posttransplantation and appeared to be driven by IL-7-mediated homeostatic T cell proliferation. Anti-CD25 mAb treatment sensitizes Treg cell to the biological effect of IL-7, possibly rendering more common γc-chain available to interact with CD127. Homeostatic Treg cell proliferation is resistant to the inhibitory effect of rapamycin and FK506 but can be blocked by the presence of mycophenolate mofetil. Our data suggest that a compensatory mechanism of IL-7-mediated homeostatic proliferation can restore the inhibitory network of Treg cell after anti-CD25 induction therapy in islet allotransplantation.

Abstract 4866: Development and characterization of novel CD40 antibody agonists for cancer immunotherapy

Abstract CD40 is a key molecule in the regulation of immune responses and its activity can be modulated using antibodies. In particular, agonist CD40 antibodies are highly effective in preclinical tumor models either through direct interaction with CD40-expressing lymphomas, or indirectly through the activation of an adaptive anti-tumor immune response. To date, limited clinical data have been reported with strong CD40 agonist antibodies; nonetheless it seems likely that targeting this pathway will require a balance between the benefits of immune stimulation to drive anti-tumor responses, and the damage that can result from non-specific immune cell activation. We set out to develop novel human anti-CD40 antibodies with different levels of agonist activity to identify a lead candidate for systemic application. Anti-CD40 monoclonal antibodies (mAbs) were generated by immunization of human Ig transgenic mice with recombinant and cell surface expressed human CD40. Hybridomas developed from these mice were screened using CD40 binding assays and activity on a reporter cell line engineered to express CD40 and NFêB-responsive luciferase. The variable regions of lead antibodies that displayed differential activity were cloned into vectors containing human IgG1 or IgG2 constant domains and expressed in CHO cells. These human CD40 mAbs were further characterized by analysis of binding affinity, CD40L blocking activity, B cell and dendritic cell activation, and anti-tumor activity in xenograft tumor models. We found a wide range of activities among the CD40 mAbs that is linked to epitope specificity as well as the isotype. In general, the IgG2 isotype mAbs had greater signaling activity than their IgG1 counterparts. The lead candidate mAbs are undergoing additional testing related to functional and toxicity parameters before a final candidate is nominated for clinical development. Citation Format: Laura A. Vitale, Thomas O’Neill, Jenifer Widger, Andrea Crocker, Li-Zhen He, Jeffrey Weidlick, Karuna Sundarapandiyan, James Storey, Lawrence Thomas, Joel Goldstein, Henry C. Marsh, Tibor Keler. Development and characterization of novel CD40 antibody agonists for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4866.

Failure of transplantation tolerance induction by autologous regulatory T cells in the pig-to-non-human primate islet xenotransplantation model.

Islet allotransplantation is a promising way to treat some type 1 diabetic (T1D) patients with frequent hypoglycemic unawareness, and islet xenotransplantation is emerging to overcome the problem of donor organ shortage. Our recent study showing reproducible long-term survival of porcine islets in non-human primates (NHPs) allows us to examine whether autologous regulatory T-cell (Treg) infusion at peri-transplantation period would induce transplantation tolerance in xenotransplantation setting. Two diabetic rhesus monkeys were transplanted with porcine islets from wild-type adult Seoul National University (SNU) miniature pigs with immunosuppression by anti-thymoglobulin (ATG), cobra venom factor, anti-CD154 monoclonal antibody (mAb), and sirolimus. CD4(+) CD25(high) CD127(low) autologous regulatory T cells from the recipients were isolated, ex vivo expanded, and infused at the peri-transplantation period. Blood glucose and porcine C-peptide from the recipients were measured up to 1000 days. Maintenance immunosuppressants including a CD40-CD154 blockade were deliberately discontinued to confirm whether transplantation tolerance was induced by adoptively transferred Tregs. After pig islet transplantation via portal vein, blood glucose levels of diabetic recipients became normalized and maintained over 6 months while in immunosuppressive maintenance with a CD40-CD154 blockade and sirolimus. However, the engrafted pig islets in the long-term period were fully rejected by activated immune cells, particularly T cells, when immunosuppressants were stopped, showing a failure of transplantation tolerance induction by autologous Tregs. Taken together, autologous Tregs infused at the peri-transplantation period failed to induce transplantation tolerance in pig-to-NHP islet xenotransplantation setting.

Your nose knows how to target brain inflammation

Your nose knows how to target brain inflammation

Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement

While engagement of the inhibitory Fcγ-receptor (FcγR) IIB is an absolute requirement for invivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its FcγRs and CD40, we revealed that FcγRIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating FcγRIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to FcγRIIB, but not to FcγRIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human FcγR.

Anti–CD3 Antibody Treatment: a Promising Immunotherapeutic for Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by spontaneous pancreatic beta cells destruction. The central cause of T1D is mainly the weakening of central and peripheral tolerance. So, the induction of tolerance is a major goal for most of the immunotherapies. Investigations over the last couple of decades have shown that anti–CD3 monoclonal antibodies (mAbs) and their therapeutic variants effectively treat autoimmune disease in animal models, and have also shown promise in clinical trials. Tolerance induction by anti–CD3 mAbs is mainly related to the induction of Th2 type immune response and regulatory T cells (Tregs) development that control pathogenic autoimmune responses. Here, we review main findings of last couple of decades research, and current scenario of anti–CD3 mediated immune tolerance, and preclinical and clinical studies in which anti–CD3 mAbs have been used.

A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells.

Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.

Specific deletion of the progesterone receptor on CD11c dendritic cells reveals a progesterone-DC-crosstalk necessary to sustain fetal development

T cells are involved in the maintenance of immunocompetence and in the development of alloimmune responses in solid organ transplant recipients. The kinetics of functionally distinct T-cell subsets in peripheral blood has received little attention in the field of heart transplantation. We performed a simultaneous analysis of the maturation, activation, and regulatory profiles of T cells using 4-color flow cytometry in a study of 77 heart recipients. Induction therapy included 2 doses of anti-CD25 monoclonal antibodies (daclizumab). Lymphocyte subsets were prospectively evaluated at different times before and up to 1 year after transplantation in 46 heart recipients. A separate cross-sectional study was performed in 33 heart recipients who had received a transplant more than 1 year previously to evaluate abnormalities persisting in the long term. As compared with baseline values, a decrease in regulatory CD4 + T-cell percentages (CD4+CD127lowCD25highFoxP3 +) was observed from day 7 to 12 months after transplantation. Interestingly, T cells expressing the beta chain of IL-2 (CD122 +) remained stable during the first 3 months. A significant decrease in the activation status of CD4 T cells was documented from day 7 to 1 year after transplantation, while the activation status of CD8 + T cells remained stable during follow-up. Compared with values for healthy controls (n = 36), higher CD8 + terminally differentiated effector memory percentages (CD8+CD45RA+CCR7 −) were observed from baseline up to more than 1 year after transplantation. Rejection was associated with higher levels of these cells during the first 6 months after transplant. We characterized the abnormalities in distinct functional T-cell subsets at different times before and after heart transplantation. Some of these abnormalities should be further investigated as biomarkers of clinical complications.

Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

Therapeutic anti-CD3 monoclonal antibodies: from bench to bedside.

The induction of tolerance is a major goal of immunotherapy. Investigations over the last 20 years have shown that anti-CD3 monoclonal antibodies (mAbs) effectively treat autoimmune disease in animal models and have also shown promise in clinical trials. Tolerance induction by anti-CD3 mAbs is related to the induction of Tregs that control pathogenic autoimmune responses. Here, we review preclinical and clinical studies in which intravenous or mucosal administration of anti-CD3 mAbs has been employed and provide an outlook on future developments to enhance the efficacy of this promising therapeutic approach.

Efficacy and safety of otelixizumab use in new-onset type 1 diabetes mellitus

ABSTRACTIntroduction: Type 1 diabetes (T1DM) is an immune-mediated disease induced by antigen-specific T cells infiltrating pancreatic beta cells leading to the progressive loss of endogenous insulin secretion.Areas covered: The identification of specific components of the autoimmune response favoured the implementation of several immunomodulatory therapies including antiCD3 monoclonal antibody (mAb) called otelixizumab. Otelixizumab is a chimeric monoclonal antibody that targets the ε-chain of the CD3T-lymphocyte surface receptor that has been developed with the aim of short therapeutic courses capable of inducing a remission of T1DM. Clinical trials have been carried out with otelixizumab to evaluate its safety and efficacy, but despite positive results of Phase I and II studies, the results of Phase III studies have been contradictory.Expert opinion: High doses of otelixizumab have shown beneficial effects on beta cell function whereas a lower dose, which was tested to avoid the adverse effects associated with higher doses, was not effective on beta cells preservation. We believe that otelixizumab is a drug of potential interest for treating new onset T1DM patients and its use in combination with other immunomodulatory agents should be considered as a solution to circumvent adverse effects while maintaining efficacy.

A Depleting Anti-CD45 Monoclonal Antibody as Isolated Conditioning for Bone Marrow Transplantation in the Rat.

ObjectiveA monoclonal antibody (mAb) against the leukocyte common antigen CD45 (RT7 in rats) could facilitate bone marrow transplantation (BMT). This study in rats evaluates a depletive rat anti-RT7a mAb as isolated tool for BMT conditioning without using irradiation or any chemotherapeutic / immunosuppressive agent.MethodsThe model used a CD45 di-allelic polymorphism (RT7a/RT7b). The anti-RT7a mAb was intravenously administered to LEW.1W rats (RT1uRT7a) at 5, 10 and 15 mg/kg. 1x108 BM cells of MHC syngeneic (RT1u), MHC disparate (RT1l) or MHC haploidentical (RT1u/l) donors were transplanted. All BM donor strains carried the RT7b allele so that their CD45+ cells were not affected by the anti-RT7a mAb. Recipients were monitored for reconstitution and donor-chimerism in blood leukocytes.ResultsmAb dosages of 5 or 10 mg/kg were myelosuppressive, whereas 15 mg/kg was myeloablative. Multi-lineage donor-chimerism at day 100 indicated engraftment of MHC syngeneic BM after any used mAb dosage (5 mg/kg: 46+/-7%; 10 mg/kg: 62+/-5%; 15 mg/kg: 80+/-4%). MHC disparate BM resulted in autologous reconstitution after conditioning by 10 mg/kg of the mAb and caused transient chimerism ending up in death associated with aplasia after conditioning by 15 mg/kg of the mAb. MHC haploidentical BM (F1 to parental) engrafted only after conditioning by 15 mg/kg (chimerism at day 100: 78+/-7%). Abandonment of α/β TCR+ cell depletion from BM grafts impaired the engraftment process after conditioning using 15 mg/kg of the mAb in the MHC syngeneic setting (2 of 6 recipients failed to engraft) and the MHC haploidentical setting (3 of 6 recipients failed).ConclusionThis depletive anti-RT7a mAb is myelosuppressive and conditions for engraftment of MHC syngeneic BM. The mAb also facilitates engraftment of MHC haploidentical BM, if a myeloablative dose is used. RT7b expressing, BM-seeded α/β TCR+ cells seem to impair the engraftment process after myeloablative mAb conditioning.

287. Production of KTE-C19 (Anti-CD19 CAR T Cells) for ZUMA-1: A Phase 1/2 Multi-Center Study Evaluating Safety and Efficacy in Subjects with Refractory Aggressive Non-Hodgkin Lymphoma (NHL)

Introduction: An ongoing anti-CD19 CAR T cell study with KTE-C19 in subjects with refractory, aggressive NHL achieved objective responses in 5/7 subjects, including 4 complete remissions (Locke, ASH 2015). To support this trial (ZUMA-1, NCT02348216) and other multicenter trials in lymphoma, we developed a robust and efficient strategy to generate autologous anti-CD19 CAR-engineered T cell products. The newly developed process aims to minimize the time between subject leukapheresis and product administration, and generate a KTE-C19 lot for every enrolled subject. Methods: Upon confirmation of eligibility, leukapheresis was performed to process 12-15 L of blood targeting collection of 5-10 × 109 peripheral blood mononuclear cells (PBMC). After collection at the investigational site, subject apheresis material was shipped to the central manufacturing site, where it was processed to enrich for the T cell-containing PBMC fraction on a closed system Ficoll™ gradient. In a closed bag system, T cells in the PBMC fraction were then activated using an anti-CD3 monoclonal antibody and cultured in serum-free medium containing 300 IU/ml of IL-2. Magnetic beads were not used for either cell selection or activation. Activated T cells were transduced with a gamma retroviral vector that contains the anti-CD19 CAR gene and further expanded for 4 to 6 days to achieve the target cell dose of 2 × 106 CAR-positive T cells/kg (minimum of 1 × 106). Final KTE-C19 product was washed, cryopreserved and tested for identity, potency, and adventitious agents. In-process samples were collected for analysis by flow cytometry for CAR expression and other characteristics. After meeting acceptance criteria, the KTE-C19 product was shipped back to the clinical sites using a validated cryo-shipper. Results: 7 subjects were dosed in the phase 1 portion of ZUMA-1. KTE-C19 was successfully manufactured in all cases despite a broad range of baseline leukocyte counts (median 5.4×103 cells/µl, 2.1-11.1) and low numbers of baseline lymphocytes (median 0.9×103 lymphocytes/µl, 0.1-1.4) prior to apheresis. Apheresed cell populations used to generate clinical lots had a broad range of phenotypic characteristics including lymphocyte / monocyte ratio (median 1.1, 0.04-3.5), CD8/CD4 ratio (median 4.2, 0.3-7.7), and naïve plus central memory (Tcm) / more differentiated T cells (median 0.3, 0.04-1.3). Fold-expansion of T cells was consistent among the 7 product lots (average 6-fold) from transduction to harvest. All KTE-C19 lots contained predominantly CD3+ T cells (median 96%; 90-99%), with CD8+ T cells (median 57%, 27-82%) and CD4+ T cells (median 43%, 18-73%). Both CD4+ and CD8+ T cells expressed CAR at similar levels. The product lots contained predominantly effector memory (median 42%, range 30-56%) and Tcm cells (median 34%, 15-58%), with the remainder being naïve (median 14%, 6-19%) and effector T cells (median 5%, 1-22%). All KTE-C19 lots met release specifications and were available for clinical administration within ~2 weeks of apheresis. Conclusions: We developed a robust KTE-C19 manufacturing process that successfully generated biologically and clinically active product irrespective of the characteristics of the starting cell populations processed to date. Product lots met release specifications and were available for subject treatment within the target timeframe. This centralized manufacturing process is well suited to support multicenter clinical trials.