anti-ICOS Monoclonal Antibodies Research Articles

Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy.Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®).Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry.Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS.Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered.ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1).Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031).Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53–27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001).Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab.Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. DisclosuresLopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Molecular Imaging of Chimeric Antigen Receptor T Cells By ICOS-Immunopet

Introduction: Immunomonitoring of chimeric antigen receptor (CAR) T cells relies primarily on their quantification in the peripheral blood, which inadequately quantifies their biodistribution and activation status in the tissues. Non-invasive molecular imaging of CAR T cell therapy by positron emission tomography (PET) is a promising approach providing spatial, temporal and functional information. Reported strategies for PET-based monitoring of CAR T cells rely on additional manipulation of the cell product such as the incorporation of reporter transgenes or ex vivo biolabeling, which significantly limits the wider application of CAR T cell molecular imaging. In the present study, we assessed the ability of antibody-based PET (immunoPET) to non-invasively visualize CAR T cells in vivo. Methods: For analysis of human CAR T cell activation, we analyzed publicly available RNA sequencing data (GSE136891) obtained at serial time points during in vitro culture of CD19.CD28z CAR T cells. We analyzed by mass cytometry (CyTOF) the ex vivo ICOS expression on human CD19-28z CAR T cells obtained from 31 patients receiving axicabtagene ciloleucel (Axi-cel) for relapsed/refractory diffuse large B-cell lymphoma (DLBCL). For in vivo murine experiments, CD19-expressing B-cell lymphoma A20 cells (2.5×10e5 cells) were injected by tail vein intravenously (i.v.) into sub-lethally (4.4 Gy) irradiated Thy1.2+ BALB/c mice. Seven days later, murine CD19.CD28z Luc+ Thy1.1+ CAR T cells (1×10e6) were i.v. injected. ICOS expression was analyzed by flow cytometry on CAR T cells recovered from spleen and bone marrow 5 days after injection. For imaging studies, anti-ICOS monoclonal antibody (mAb) specific for murine ICOS (clone:7E.17G9, BioXcell) was modified with the bifunctional chelator deferoxamine (DFO/p-SCN-Bn-Deferoxamine). The DFO-ICOS mAb conjugate was radiolabeled with 37 MBq (~1 mCi) of 89Zr-oxalate (final specific activity 6 µCi/µg/ml and radiochemical purity of 99%). 89Zr-DFO-ICOSmAb (45 μCi ± 3.6, 7.5 μg± 0.6) was injected i.v. 5 days post-CAR T cell administration and PET-CT imaging performed 48 hours later. Following PET-CT, mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter. Results: Analysis of RNA-sequencing data from human CAR T cells identified ICOS as an activation marker whose transcription was up-regulated and sustained during in vitro culture. ICOS was preferentially expressed on CAR+ T cells recovered at day 7 from axi-cel treated patients compared with CAR- cells (p&amp;lt;0.001; Figure 1A). Phenotypic analysis in a murine model of B cell lymphoma infiltrating the spleen and the bone marrow confirmed preferential ICOS expression on murine CAR T cells compared to resident cells in both spleen (p=0.003) and bone marrow (p=0.008). Figure 1B shows representative volume-rendered technique (VRT) PET/CT images of 89Zr-DFO-ICOS mAb-injected tumor-bearing mice either untreated (left panels) or that received mCD19.28z CAR T cells (right panels). 89Zr-DFO-ICOS mAb similarly accumulated in highly vascularized organs (heart, liver and spleen) of both untreated and CAR T cell treated mice, consistent with the biodistribution and clearance of intact antibodies. We detected pronounced 89Zr-DFO-ICOS mAb-PET signals in the bones of CAR T cell treated mice, particularly prominent in the lumbar spine, iliac bones, femur, tibia and humeral heads (Figure 1B). Region of interest analysis confirmed markedly increased radiotracer uptake in bones rich in bone marrow from CAR T treated mice compared with those of untreated mice (lumbar spine vertebrae p&amp;lt;0.001; iliac bones p=0.001; femur p=0.002; tibia p=0.002). Moreover we observed a slight, but statistically significant increase in radiotracer accumulation in the heart of CAR T cell-treated mice (p=0.004) while no significant differences were detected in spleen and liver. As expected, there was no significant signal difference in the muscle, considered background. Biodistribution analysis using gamma counting of tissues confirmed the PET results. Conclusions: We describe for the first time an immunoPET approach to monitor the in vivo dynamics of CAR T cell migration, expansion, and persistence that does not require the addition of reporter genes or ex vivo labeling, being therefore applicable to the clinical setting for the study of any commercially available and investigational CAR T cell products. Disclosures Miklos: Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents &amp; Royalties, Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Lyell Immunopharma: Consultancy, Current equity holder in private company; NeoImmune Tech: Consultancy; Nektar Therapeutics: Consultancy; Apricity Health: Consultancy, Current equity holder in private company. Gambhir:CellSight Inc: Current equity holder in private company. Negrin:Amgen: Consultancy; BioEclipse Therapeutics: Current equity holder in private company; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; Biosource: Current equity holder in private company; KUUR Therapeutics: Consultancy; UpToDate: Honoraria.

ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells

AbstractThe treatment of advanced-stage cutaneous T-cell lymphoma (CTCL) remains an unmet medical need. Mogamulizumab, anti-KIR3DL2, and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled with monomethyl-auristatin-E (MMAE), provided encouraging results, but new targeted therapies are needed. Inducible T-cell costimulator (ICOS), a T-cell costimulatory receptor, is a promising therapeutic target, not only because it is expressed by malignant T cells in CTCL but also because of its connection with the suppressive activity of regulatory T (Treg) cells. Immunohistochemical analysis revealed that ICOS was widely expressed by malignant cells in skin biopsy specimens from 52 patients with mycosis fungoides and Sézary syndrome (SS), as well as in involved node biopsy specimens from patients with SS. Furthermore, flow cytometry demonstrated its strong expression by circulating tumor cells in all our patients with SS. Percentages of ICOS+ Treg cells were significantly higher in patients with SS than in healthy donors. We then investigated the preclinical efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE and pyrrolobenzodiazepine. In 3 CTCL cell lines (Myla, MJ, and HUT78), we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In addition, anti-ICOS-MMAE ADCs had an in vitro and in vivo efficacy superior to BV in a mouse xenograft model (MyLa). Finally, we assessed the efficacy of anti-ICOS ADCs in ICOS+ patient-derived xenografts from patients with SS and angioimmunoblastic T-cell lymphoma. Collectively, our findings provide the preliminary basis for a therapeutic trial.

Effect of expression of ICOS in cutaneous T-cell lymphoma and its targeting on killing of malignant cells.

e20040 Background: Advanced stage cutaneous T-cell lymphomas (CTCLs) remain an unmet medical need. Immunomodulatory agents such as mogamulizumab, anti-KIR3DL2 and brentuximab vedotin (BV), an anti-CD30 antibody–drug conjugate (ADC) coupled to monomethyl-auristatin-E (MMAE), provided encouraging results but new targeted therapies are needed. Inducible Co-Stimulator (ICOS), a T-cell costimulatory receptor involved in the development of CTCLs, arouses interest. Methods: We used immunohistochemistry to study ICOS expression in skin biopsies of 23 patients with early-stage mycosis fungoides (MF), 12 with transformed MF (TMF) and 17 with Sézary Syndrome (SS), at diagnosis or in relapse. ICOS expression by circulating Sézary cells and regulatory T cells (Tregs) in patients with SS was evaluated using flow cytometry, and compared to healthy donors (HD) lymphocytes. In 5 patients with SS, we also analyzed concomitant biopsies from involved nodes. Then, we investigated the efficacy of anti-ICOS ADCs generated by coupling murine anti-ICOS monoclonal antibodies with MMAE, in comparison to BV. We used ICOS+ CTCL cell lines (MyLa and MJ), murine xenograft models with MyLa and ICOS+ Patient Derived Xenografts (PDXs) from patients with SS and angioimmunoblastic T-cell lymphoma (AITL). Results: ICOS was highly expressed by the cutaneous atypical lymphocytic infiltrates in respectively 61%, 75% and 88% of patients with early-stage MF, TMF and SS, such as in the 5 patients with node involvement. ICOS expression by circulating Sézary cells was strong: 69±7.3% versus 38.8±7.1% of non-tumoral CD4+ cells ( p&lt; 0.009; CI95%: 8.7-51.6); and 31±3.2% of CD4+ cells in HD ( p&lt; 0.0001; CI95%:20.3-46.3). Percentages of ICOS+ Tregs were significantly higher in patients with SS than in HD. In CTCL cell lines, we observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS ADCs. In a mouse xenograft model (MyLa), anti-ICOS ADCs provided a longer overall survival (OS) than BV (HR = 15.2;CI95%:3.2-71.1; p&lt; 0.0006). Finally, in ICOS+ PDXs anti-ICOS ADCs significantly improved OS, and reduced the number of tumor cells in the blood, bone marrow and spleen. No evidence of ADC toxicity was observed in treated mice. Conclusions: All together our results show that ICOS is a therapeutic target of interest in CTCLs and provide the preliminary basis for a therapeutic trial

Abstract 573: Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody

Abstract ICOS (inducible co-stimulator molecule) is a co-stimulatory molecule and a member of the CD28 superfamily expressed primarily on T lymphocytes. Analysis of cancer patient samples as well as rodent preclinical data have implicated a role for the ICOS pathway in cancer immunotherapy. We have generated a panel of anti-ICOS monoclonal antibodies with in vitro agonistic properties. The anti-ICOS antibodies are efficacious as monotherapies and in combination with anti-PD1 in multiple syngeneic tumor models. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8:T regulatory (Treg) cells as well as preferential reduction in ICOS-high Tregs in the tumor microenvironment. JTX-2011, a species cross-reactive high affinity humanized agonist monoclonal antibody, has been selected for development. Evaluation of JTX-2011 in nonhuman primate models will be presented, including data informing safety and PK parameters. Our preclinical data provides rational for clinical development of JTX-2011 as a cancer immunotherapeutic to be tested as both a monotherapy as well as in combination with immunotherapies in solid tumor indications. Citation Format: Jennifer S. Michaelson, Christopher J. Harvey, Kutlu G. Elpek, Ellen Duong, Matthew Wallace, Chengyi J. Shu, Sriram Sathyanarayanan, Robert Mabry, Lindsey Shallberg, Tong Zi, Amit Deshpande, Stephen L. Sazinsky, Joshua Apgar, Deborah Law. Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody. [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 573.

Abstract A059: Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics

Abstract ICOS (inducible co-stimulator molecule) is a T cell encoded member of the extended B7/CD28 superfamily that is up-regulated upon early T cell activation. Previous studies have shown that higher ICOS expression on circulating T cells, post treatment with ipilimumab, is associated with better clinical outcome. In complementary pre-clinical studies, efficacy observed with a whole cell vaccine consisting of ICOS-L expressing B16 melanoma (IVAX) suggests that agonism of this pathway could provide therapeutic benefit in the cancer setting. In accordance with these observations, we generated a panel of anti-ICOS monoclonal antibodies with in vitro agonist properties. A lead series of mAbs were shown to be efficacious as monotherapies in syngeneic tumors models with enhanced tumor inhibition observed in combination with anti-PD1. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8/regulatory T (Tregs) cells and that this change is related to preferential reduction in ICOS high Tregs in the tumor microenvironment. These data provided the rationale for development of a high affinity humanized agonist monoclonal antibody to be tested in both mono therapy and in combination with other T cell checkpoints. Our lead anti-ICOS antibody is currently in IND-enabling studies and will be tested for activity in solid tumor indications. Citation Format: Kutlu Elpek, Christopher Harvey, Ellen Duong, Tyler Simpson, Jenny Shu, Lindsey Shallberg, Matt Wallace, Sriram Sathy, Robert Mabry, Jennifer Michaelson, Michael Briskin. Efficacy of anti-ICOS agonist monoclonal antibodies in preclinical tumor models provides a rationale for clinical development as cancer immunotherapeutics. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A059.

Increased production of interleukin-17 over interleukin-10 by treg cells implicates inducible costimulator molecule in experimental spondyloarthritis.

HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic (B27-transgenic) rats develop an inflammatory disorder resembling spondyloarthritis, with accumulation of proinflammatory Th17 cells. Because Treg cells and Th17 cells have opposing effects in inflammatory disorders, we sought to determine whether biased expansion of Th17 cells could result from altered Treg cell frequency and/or function in B27-transgenic rats. We characterized the phenotype and function of Treg cells from B27-transgenic rats in comparison with those from control rats, by examining their expression of cell surface markers, suppressive activity, cytokine production, and differentiation pattern. In B27-transgenic rats, the preferential accumulation of CD4+ Teff cells over Treg cells was not associated with a defect in Treg cell differentiation or suppressive activity. The expression of Treg cell markers was similar between B27-transgenic and control rats, with the exception of the inducible costimulator (ICOS) molecule, which was overexpressed in B27-transgenic rats. High levels of ICOS are considered to be a hallmark of Treg cells with heightened suppressive activity and interleukin-10 (IL-10) expression. Paradoxically, the production of IL-10 by Treg cells was reduced in B27-transgenic rats, whereas the production of IL-17 was enhanced. Moreover, the addition of anti-ICOS monoclonal antibodies during Treg cell differentiation in the presence of dendritic cells from B27-transgenic rats reversed this cytokine profile, restoring the balance between IL-10 and IL-17 in Treg cells from B27-transgenic rats. We observed dysregulated production of IL-10 and IL-17 by Treg cells from B27-transgenic rats, which may contribute to disease development. Moreover, our data highlight a key role for ICOS signaling in the generation of imbalanced production of IL-10 and IL-17 by Treg cells in this experimental model of spondyloarthritis.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice

AbstractFormation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

ICOS/B7RP-1 Interference in Mouse Kidney Transplantation

Activated T cells play a key role in allograft rejection. T cell activation requires signaling via the T cell receptor as well as costimulatory signals. Inducible costimulatory molecule (ICOS), with its ligand B7RP-1, is a recently discovered costimulatory molecule of the CD28 family. The role of this signaling pathway during the early phases of kidney allograft rejection is not clear so far. Kidneys were orthotopically transplanted from BALB/c to C57BL/6 mice. Animals were assigned to five experimental groups: blocking anti-ICOS monoclonal antibody, ICOS fusion protein, anti-B7RP1 monoclonal antibody, B7RP-1 fusion protein, and control immunoglobulin G. Survival was significantly reduced in animals treated with ICOS monoclonal antibody (mAb) and B7RP-1 Fc as compared with controls. These animals had also a lower number of apoptotic graft infiltrating T cells, whereas the expression of intracellular interferon-gamma in CD3CD4 T cells was increased. Animals treated with ICOS Fc and B7RP-1 mAb had similar survival and numbers of apoptotic T cells as compared with controls. In summary, the blockade of ICOS with ICOS mAb or B7RP-1 Fc reduced the amount of apoptosis of infiltrating lymphocytes and resulted in continuous inflammatory processes with progressive tissue damage and graft failure.

Inhibition of Chronic Rejection and Development of Tolerogenic T Cells after ICOS-ICOSL and CD40-CD40L Co-stimulation Blockade

: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells down-regulates graft-versus-host disease (GVHD) and facilitates engraftment of allogeneic bone marrow (BM)

AbstractInducible costimulator (ICOS), a CD28/cytotoxic T lymphocyte antigen 4 (CTLA-4) family member, is expressed on activated T cells. ICOS ligand, a B7 family member, is constitutively expressed on B cells, macrophages, and dendritic cells and is up-regulated on antigen-presenting cells (APCs) and some nonlymphoid tissues by tumor necrosis factor α (TNFα) or lipopolysaccharide (LPS). Thus, ICOS: ICOS ligand (ICOSL) blockade could reduce alloreactive T cell-APC interactions responsible for graft-versus-host disease (GVHD) and bone marrow (BM) graft rejection. ICOS blockade, achieved with ICOS-/- mice or anti-ICOS monoclonal antibody (mAb) administration, resulted in significant inhibition of GVHD in multiple strain combinations whether mediated by CD4+ and/or CD8+ T cells, alloantigen-specific T-cell receptor (TCR) transgenic (Tg) T cells, or CD28-, T helper 1 (Th1)-, or Th2-deficient T cells. Anti-ICOS significantly delayed GVHD mortality even when mAb infusions were delayed until day 5 after transplantation. ICOS blockade reduced the number of alloantigen-specific effector cells but did not prevent their activation. Imaging of green fluorescent protein-positive (GFP+) effectors indicated that ICOS blockade inhibited expansion of GVHD-causing effector T cells in secondary lymphoid and GVHD target organs. Engraftment rates were significantly higher in ICOS-/- versus wild-type (WT) mice receiving allogeneic BM, and ICOS blockade significantly inhibited expansion of host antidonor alloantigen-specific BM graft-rejecting T cells. These data suggest that the ICOS pathway may be a beneficial therapeutic target for GVHD inhibition, GVHD therapy, and BM graft promotion. (Blood. 2005; 105:3372-3380)

Hyperexpression of inducible costimulator on lamina propria mononuclear cells in rat dextran sulfate sodium colitis

The authors have previously shown that a third member of the CD28 family, inducible costimulator (ICOS), was increased in the inflamed intestinal mucosa of murine experimental colitis, and that the blockade of ICOS ameliorated the development of colitis. However, the role of ICOS in rat intestinal inflammation and its expression profile remains unclear. In the present study, the authors investigated the involvement of ICOS in the development of rat dextran sulfate sodium (DSS)-induced colitis, and the therapeutic potential of anti-ICOS monoclonal antibody (mAb) in colitis. The authors first examined expression of ICOS protein in normal rat by immunohistochemistry and flow cytometry. Sprague-Dawley rats were fed 3.0% DSS. The expression of ICOS on infiltrating lamina propria mononuclear cells and splenocytes were examined. The DSS-fed rats were then administered anti-ICOS mAb to test its effect on the development of colitis. Unlike mice and human, ICOS was expressed on a part of CD4+ T-cells from the thymus, spleen, mesenteric lymph nodes and lamina propria. Levels of ICOS on CD4+ T-cells from the spleen and colonic lamina propria were significantly upregulated after Concanavalin A (Con A) stimulation. In addition, ICOS was also upregulated on CD4+ T-cells from DSS-fed rats compared with those from non DSS-fed rats. However, anti-ICOS mAb did not ameliorate the development of both acute and chronic DSS colitis. These results suggest that the different expression of ICOS in rats plays a distinct role in rat intestinal inflammation.

Ameliorating effect of anti-inducible costimulator monoclonal antibody in a murine model of chronic colitis

Background & Aims: Inducible costimulator (ICOS)/B7RP-1 represents a newly described receptor/ligand pair involved in costimulation of T cells by antigen-presenting cells. We investigated the involvement of the ICOS/B7RP-1 interaction in the pathogenesis of colitis and the therapeutic potential of anti-ICOS monoclonal antibody (mAb) in experimental colitis Methods: We administered anti-ICOS or anti–B7RP-1 mAb to mice with experimental colitis induced by transfer of CD4 +CD45RB high T cells from normal mice into SCID mice. The ability of CD4 +CD45RB high cells derived from ICOS−/− mice to induce colitis was assessed. Th2 cytokine production and apoptosis in infiltrating T cells was examined after administration of anti-ICOS mAb. Results: ICOS was strongly induced on CD4 + T cells, and B7RP-1 was expressed by macrophages in the inflamed mucosa of colitic mice. Anti-ICOS mAb, but not anti–B7RP-1, ameliorated chronic colitis when administered in prevention or therapeutic protocols. Transfer of CD4 +CD45RB high T cells from ICOS−/− mice induced colitis. Treatment with anti-ICOS mAb did not enhance the production of Th2 cytokines, but a single dose of anti-ICOS mAb induced massive apoptosis of infiltrating ICOS-expressing T cells. Conclusions: ICOS/B7RP-1 interactions are not required for the development of colitis. However, treatment with anti-ICOS mAb can prevent and reverse intestinal inflammation by inducing apoptosis of ICOS-expressing T lymphocytes.