anti-CD4 mAb Research Articles

Aire is not essential for regulating neuroinflammatory disease in mice transgenic for human autoimmune-diseases associated MHC class II genes HLA-DR2b and HLA-DR4

The human autoimmune disease-associated HLA alleles HLA-DR2b (DRB1*1501) and HLA-DR4 (DRB1*0401) are strongly linked to increased susceptibility for multiple sclerosis (MS) and rheumatoid arthritis (RA), respectively. The underlying mechanisms are not fully understood, but these MHC alleles may shape the repertoire of pathogenic T cells via central tolerance. The transcription factor autoimmune regulator (AIRE) promotes central T cell tolerance via ectopic expression of tissue-specific antigens (TSAs). Aire deficiency in humans causes autoimmune polyendocrinopathy syndrome type 1 (APS1), and Aire knockout mice (Aire−/−) develop spontaneous autoimmune pathology characterized by multi-organ lymphocytic infiltrates.Here, we asked whether impaired TSAs gene expression in the absence of Aire promoted spontaneous MS- or RA-like autoimmune pathology in the context of human HLA alleles in HLA-DR2b or HLA-DR4 transgenic (tg) mice.The results show that reduced TSAs gene expression in the thymus of Aire-deficient HLA-DR2b or HLA-DR4 tg mice corresponded to mild spontaneous inflammatory infiltrates in salivary glands, liver, and pancreas. Moreover, Aire-deficiency modestly enhanced experimental autoimmune encephalomyelitis (EAE) in HLA-DR tg mice, but the animals did not show signs of spontaneous neuroinflammation or arthritis. No significant changes were observed in CD4+ T cell numbers, T cell receptor (TCR) distribution, regulatory T cells (Treg), or antigen-induced cytokine production. Abrogating Treg function by treatment with anti-CTLA-4 or anti-CD25 mAb in Aire-deficient HLA-DR tg mice did not trigger EAE or other autoimmune pathology. Our results suggest a redundant role for Aire in maintaining immune tolerance in the context of autoimmune disease-associated human HLA alleles.

Highlights of This Issue

The efficacy of PD-1/L1 checkpoint blockade in cancer patients is limited by inadequate T-cell invigoration. Buchan and colleagues report synergism between PD-1/L1 blockade and anti-CD27 mAb for CD8+ T-cell activation and tumor therapy in multiple models, including in human CD27 transgenic mice. They find that combination therapy triggers a convergent program largely dependent on synergistic IL-2 signalling and Myc, but a division of labor such that PD-1/L1 blockade promotes a cytotoxic gene-expression profile, whereas anti-CD27 preferentially drives proliferation. These data provide a mechanistic basis for pursuing the combination of checkpoint blockade and anti-CD27 in the clinic.The central nervous system (CNS) is a sanctuary site from targeted drugs. While the multiple kinase inhibitor, entrectinib, is effective in the treatment of NTRK1-fusion positive cancers with CNS metastasis, resistance usually develops. Nishiyama and colleagues found that the NTRK1-G667C mutation, which induces moderate resistance to entrectinib, caused acquired entrectinib resistance of brain metastasis. Importantly, foretinib inhibited TRK-A with G667C mutation and overcame entrectinib resistance in the liver and brain metastasis models. These findings provide a rationale for clinical trials with foretinib in cancer patients with entrectinib-resistant tumors harboring the NTRK1-G667C mutation, including patients with brain metastases.Preoperative chemotherapy is used in certain groups of breast cancer patients, but many patients do not respond and/or suffer disease progression after treatment. Liu and colleagues show that cytotoxic chemotherapy induces monocytosis in the bone marrow, resulting in increased blood levels of monocyte chemoattractant proteins, which stimulate the cancer stem-like cell phenotype to promote tumor malignancy. The study suggests that pharmacologic inhibition of CCR2 may serve as a potential cotreatment during conventional chemotherapy to target therapy-induced cancer stemness.The prognostic value of the androgen receptor (AR) in breast cancer is controversial, due in part to differences in cohorts and methodology, including the cut-point used for AR positivity. To resolve this controversy, Ricciardelli and colleagues assessed AR positivity in two independent primary breast cancer cohorts and determined the optimal cut-point by ROC analysis. An AR cut-point of 78% positivity, approximating the median staining, provided optimal sensitivity and specificity to predict breast cancer survival. Testing this cut-point in prospective cohorts is now warranted for clinical implementation of AR as a prognostic factor in the management of breast cancer.

Role of the CD27/CD70 pathway in regulatory T cell function

Abstract There is evidence that the CD27/CD70 pathway plays a major role in the regulation of immunity versus tolerance in vivo. In particular, the transgenic expression of CD70 by dendritic cells breaks tolerance and induces a lethal autoimmune disorder. Although CD27 engagement on conventional T cells (Tconv) has been shown to induce Th1-type responses, the role of CD27, expressed at higher levels on regulatory T cells (Treg), remains elusive. Recent data from our laboratory have shown that CD27 expressed on Tregs was involved in the inhibition of CD70, suggesting that CD27 may display opposite functions, i.e. pro- or anti-inflammatory when expressed on Tconv and Treg, respectively. The objective of this work was therefore to decipher the role of CD27 on Treg function. Our data show that (i) injection of agonistic anti-CD27 mAb increased the development and function of Tregs, as assessed by increased proliferation and upregulation of hallmarks of Treg activation such as ICOS, GATA-3, Helios, and inhibitory molecules such as CTLA-4, PD-1 ; (ii) similarly, Tregs were activated in mice expressing CD70 in a constitutive manner (WT x CD70tg mice); (iii) CD27+ Tregs from WT mice were more activated and displayed higher suppressive capacity than CD27− Tregs. These observations suggest that the CD27/CD70 pathway potentiates the homeostasis and inhibitory function of Tregs. Experiments are under way to identify the transcriptomic profile induced by CD27 engagement in Tregs and to analyze their function in various settings in vivo, using a CD27fl/fl mouse to selectively delete CD27 in Tregs.

Long-term Nonhuman Primate Renal Allograft Survival Without Ongoing Immunosuppression in Recipients of Delayed Donor Bone Marrow Transplantation.

We have previously reported successful induction of renal allograft tolerance in nonhuman primates (NHP) after an initial posttransplant period of conventional immunosuppression (delayed tolerance) using a nonmyeloablative conditioning regimen consisting of anti-CD154 and anti-CD8 mAbs plus equine antithymocyte globulin (Atgam) and donor bone marrow transplantation (DBMT). Because these reagents are not currently clinically available, the protocol was revised to be applicable to human recipients of deceased donor allografts. Four cynomolgus monkeys received major histocompatibility complex-mismatched kidney allografts with conventional immunosuppression for 4 months. The recipients were then treated with a nonmyeloablative conditioning regimen consisting of thymoglobulin, belatacept, and DBMT. The results were compared with recipients treated with conditioning regimen consisting of Atgam and anti-CD154 mAb, with and without anti-CD8 mAb. In 4 consecutive NHP recipients treated with the modified conditioning regimen, homeostatic recovery of CD8 TEM was delayed until after day 20 and multilineage chimerism was successfully induced. Three of the 4 recipients achieved long-term allograft survival (>728, >540, >449 days) without ongoing maintenance immunosuppression. Posttransplant MLR showed loss of antidonor CD8 T cell and CD4 IFNγ responses with expansion of CD4FOXP3 regulatory T cells. However, the late development of donor-specific antibody in NHP recipients confirms the need for additional anti-B-cell depletion with agents, such as rituximab, as has been shown in our clinical trials. This study provides proof of principle that induction of mixed chimerism and long-term renal allograft survival without immunosuppression after delayed DBMT is possible with clinically available reagents.

Organ-specific Differences in a Thymoglobulin and Belatacept-based Protocol for Tolerance Induction in Non-human Primates

In non-human primate lung transplantation, allograft acceptance has been achieved using a delayed mixed-chimerism approach consisting of 4 months of triple-drug immunosuppression (FK, MMF, steroids) followed by donor bone marrow transplantation (DBMT) along with equine-ATG, anti-IL6R mAb, anti-CD154 mAb and anti-CD8 mAb. With this protocol, 3 out of 4 monkeys accepted their grafts long-term (>299, >813, >464 days). However, anti-CD154mAb and anti-CD8 mAb are not clinically available. In kidney transplantation, persistent allograft acceptance in Cynomolgus macaques was achieved using an alternative immunosuppression protocol relying on the clinically available reagents, rabbit-ATG and belatacept. Here, we have tested the same protocol in Cynomolgus macaques transplanted with fully allogeneic lung grafts.

Immune molecular profiling of whole blood drawn from a non‐human primate cardiac xenograft model treated with anti‐CD154 monoclonal antibodies

Most studies of xenografts have been carried out with complex immunosuppressive regimens to prevent immune rejection; however, such treatments may be fatal owing to unknown causes. Here, we performed immune molecular profiling following anti-CD154 monoclonal antibody (mAb) treatment in heterotopic abdominal cardiac xenografts from α-1,3-galactosyltransferase-knockout pigs into cynomolgus monkeys to elucidate the mechanisms mediating the undesirable fatal side effects of immunosuppressive agents. Blood samples were collected from healthy monkeys as control and then at 2days after xenograft transplantation and just before humane euthanasia; 94 genes related to the immune system were analyzed. The basic immunosuppressive regimen included cobra venom factor, anti-thymocyte globulin, and rituximab, with and without anti-CD154 mAbs. The maintenance therapy was followed with tacrolimus, MMF, and methylprednisolone. The number of upregulated genes was initially decreased on Day 2 (-/+ anti-CD154 mAb, 22/13) and then increased before euthanasia in recipients treated with anti-CD154 mAbs (-/+ anti-CD154 mAb, 30/37). The number of downregulated genes was not affected by anti-CD154 mAb treatment. Additionally, the number of upregulated genes increased over time for both groups. Interestingly, treatment with anti-CD154 mAbs upregulated coagulation inducers (CCL2/IL6) before euthanasia. In conclusion, immunosuppressive regimens used for cardiac xenografting affected upregulation of 6 inflammation genes (CXCL10, MPO, MYD88, NLRP3, TNFα, and TLR1) and downregulation of 8 genes (CCR4, CCR6, CD40, CXCR3, FOXP3, GATA3, STAT4, and TBX21).

S100A4 blockage alleviates agonistic anti-CD137 antibody-induced liver pathology without disruption of antitumor immunity

ABSTRACT Liver-related autoimmune toxicities triggered by agonistic anti-CD137 antibodies have greatly limited their use in clinical applications. Here, we found that anti-CD137 monoclonal antibody (mAb) treatment in mice induced the infiltration of a large number of S100A4+ macrophages into the liver. Depletion of these cells or deficiency of S100A4 decreased inflammatory cytokine profiles and drastically reduced the number of liver pathogenic CD8+ T cells. Mechanistically, soluble S100A4 directly activated the Akt pathway and specifically prolonged CD8+ T cell survival. Interestingly, one S100A4 neutralizing mAb selectively alleviated liver abnormalities but did not affect the antitumor immunity induced by anti-CD137 mAb therapy. Thus, our study presents a novel molecular link to the liver pathology induced by an immune stimulatory antibody and proposes that combinational immunotherapies targeting those pathways could potentially elicit optimal antitumor immunity with minimal side effects.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible “iTregs” because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy.

The effect of epitope-based ligation of ICAM-1 on survival and retransplantation of pig islets in nonhuman primates.

Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long-term survival of the graft. MD-3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM-1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long-term survival of adult wild-type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD-3 mAb and maintenance with anti-CD154 mAb and sirolimus. Eleven rhesus monkeys were assigned to three groups. Group 1 (n=4) involved treatment with MD-3 induction, short-term (<4months) administration of anti-CD154 mAb, and maintenance therapy with sirolimus. Group 2 (n=4) involved treatment with MD-3 induction and long-term maintenance therapy with anti-CD154 mAb and sirolimus. Group 3 (n=3) involved only maintenance therapy with anti-CD154 mAb and sirolimus. Diabetes was induced in monkeys by streptozotocin, and pig islets (61000-112000IEQ/kg for each transplant; up to 280000IEQ/kg per recipient) were infused through the portal vein. The in vivo functional potency of the isolated islets was tested by minimal model transplant in streptozotocin-induced diabetic NOD/SCID mice, and the mean AUC of blood glucose level divided by the number of follow-up days was calculated. The islet grafts survived more than 6months (between 225 and 727days) in nine of 12 transplants of MD-3-treated groups 1 and 2, whereas in the absence of MD-3 mAb, survival was <40days. In three transplants of the MD-3-treated Group 2, functional graft survival was only for 104, 125, and 154days. In these cases, a retrospective analysis suggested that the relatively short survival duration was associated with the relatively high AUC value in the NOD/SCID bioassay. Notably, when retransplantation was performed in Group 3, blood glucose control was extended up to 956days, which was supported by MD-3 mAb-based suppression of adaptive immunity. No replication of cytomegalovirus genes was observed. Long-term survival of pig islet xenografts and successful retransplantation were achieved with MD-3 mAb-based immunosuppression regimen in this pig-to-monkey transplantation model. It should be emphasized that these encouraging results were achieved following the transplantation of islets from pigs that had not been genetically modified. Considering that it is possible to further substantially reduce the destruction of grafted islet using genetically modified pig islet, the islet requirement could be reduced and much longer graft survival can be achieved.

Combined immunotherapy with anti-PDL-1/PD-1 and anti-CD4 antibodies cures syngeneic disseminated neuroblastoma

Anti-PD-1 or anti-PD-L1 blocking monoclonal antibodies (mAbs) have shown potent anti-tumor effects in adult cancer patients and clinical studies have recently been started in pediatric cancers, including high-risk/relapsing neuroblastoma (NB). Therefore, we studied the effects of anti-PD-1/PD-L1 mAbs in two syngeneic models of disseminated NB generated by the injection of either Neuro2a or NXS2 cells, which express PD-L1. In addition, we tested the combination of these agents with the immune-enhancing cytokine IL-21, the Ecto-NTPDase inhibitor POM-1, an anti-CD25 mAb targeting Treg cells, or an anti-CD4 mAb. We previously showed that CD4-transient depletion removes CD4+CD25+ Treg cells and other CD4+CD25− regulatory subsets. Here we show that mono-therapy with anti-PD-1/PD-L1 mAbs had no effect on systemic NB progression in vivo, and also their combination with IL-21, POM-1 or anti-CD25 mAb was ineffective. The combined use of anti-PD-1 with an anti-CD4 mAb mediated a very potent, CD8-dependent, synergistic effect leading to significant elongation of tumor-free survival of mice, complete tumor regression and durable anti-NB immunity. Similar results were obtained by combining the anti-PD-L1 and anti-CD4 mAbs. These findings indicate that both PD-1/PD-L1 and CD4+ T cell-related immune-regulatory mechanisms must be simultaneously blocked to mediate therapeutic effects in these models.

Pre-clinical results in pig-to-non-human primate islet xenotransplantation using anti-CD40 antibody (2C10R4)-based immunosuppression.

Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti-CD154 monoclonal antibody (mAb) in pig-to-non-human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti-CD40 (2C10R4) mAb-based studies have not been reported. Nine streptozotocin (STZ)-induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen-free (DPF) miniature pigs. They were treated with anti-CD40 mAb-based immunosuppressive regimen and were divided into 3 groups: anti-CD40 only group (n=2), belatacept group (anti-CD40 mAb+belatacept, n=2), and tacrolimus group (anti-CD40 mAb+tacrolimus, n=5). All monkeys received anti-thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C-peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted. All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow-up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C-peptide concentration of >0.15ng/mL, was 31, 27, and 60days for anti-CD40 only, belatacept, and tacrolimus groups, respectively. Anti-αGal IgG levels in serum and the number of interferon-γ secreting T cells in peripheral blood mononuclear cells did not increase in most animals. These results showed that anti-CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti-CD40 mAb was not as effective as anti-CD154 mAb in terms of preventing early islet loss.

CD80 Regulates Th17 Cell Differentiation in Coxsackie Virus B3-Induced Acute Myocarditis.

The cluster of differentiation protein complex, CD80/CD86, regulates Th1/Th2 differentiation in autoimmune disease. In order to establish the effects of CD80/CD86 on Th17 cell differentiation in acute viral myocarditis (VMC), we infected C57BL/6 mice with Coxsackie virus B3 (CVB3) and examined the effects of the treatment with anti-CD80/CD86 monoclonal antibodies (mAbs) on Th17 cell differentiation in vivo. The effects of anti-CD80/CD86 mAbs on Th17 cell differentiation were further evaluated in vitro. The treatment with anti-CD80 mAb induced marked suppression of Th17 cell differentiation and ROR-γt mRNA expression, whereas anti-CD86 mAb alone had no effect, both in vivo and in vitro. Our finding that CD80 regulates Th17 differentiation supports the potential utility of anti-CD80 mAb as an effective new immunotherapeutic target in acute VMC.

P003 Successful kidney transplantation across positive crossmatches with strong donor-specific HLA-DP antibodies

The role of anti-HLA-DP antibodies in renal transplantation is poorly defined. One of our kidney re-list candidates with 99% CPRA (+ many strong DP antibodies) received a kidney offer from a single DP1 mismatched deceased donor. The patient displayed strong DP1 antibodies (MFI = 10930) and thereby positive B cell pronase crossmatch (MCS = 125; cutoff is 120 MCS). Since the donor is highly matched and crossmatch was borderline positive, we move forward with this transplantation with anti-CD25 mAb induction and a single dose IVIG (2 g/kg) at day 1 post-tx. The graft was functioning and no rejection noticed in 6 month biopsy. The aim of the present study was to expand similar transplants for highly sensitized patients across DP donor-specific antibodies (DSA) with positive or negative B cell XM. 16 candidates (4 female listed for 1st tx, 6 females and 6 males listed for re-tx) with cPRA ranging from 75 to 100% (9 candidates had cPRA 100%) were transplanted with DP DSAs of greater than 5000 MFI (9 had MFI > 100,00). Retrospective FXM were done on 15 patients. Kidney biopsy data on 12 patients were evaluated for C4d deposition and AMR. One Lambda single antigen bead (SAB) assay was used to test HLA antibodies. 9 of 15 retrospective B FXM were positive, and the positive B FXM had DP DSAs of >10000 MFI (pronase MCS are generally ∼200). The levels of DP DSAs were plotted at pre- and post-tx (1 week, 1 month and 6 months) sera, which show significant decreases in DP DSAs in all 16 recipients (see figure). The 6 month protocol biopsy found no AMR in 9 recipients (8 were C4d- and 1 was C4d+), and C4d- AMR+ in 3 patients. One recipient developed de novo DSA to A32 (MFI = 14,000) but was undetectable at 6 months post-tx. The presence of HLA-DP antibodies particularly in broadly sensitized re-list candidates decrease the chance of finding a compatible donor. Results from this study show that patients can be transplanted across the HLA-DP DSAs without affecting graft survival, and therefore increasing access for broadly sensitized candidates.Download : Download high-res image (222KB)Download : Download full-size image

Brachytherapy attains abscopal effects when combined with immunostimulatory monoclonal antibodies

Purpose/ObjectivesPreclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lesions and on tumors at distant, nonirradiated sites. We have recently reported that external beam radiotherapy achieves abscopal effects when combined with antagonist anti-PD1 mAbs and agonist anti-CD137 (4-1BB) mAbs. The goal of this work is to study the abscopal effects of radiotherapy instigated by brachytherapy techniques. Methods and MaterialsMice bearing a subcutaneous colorectal carcinoma, MC38 (colorectal cancer), in both flanks were randomly assigned to receive brachytherapy or not (8 Gy × three fractions) to only one of the two grafted tumors, in combination with intraperitoneal immunostimulatory monoclonal antibodies (anti-PD1, anti-CD137, and/or their respective isotype controls). To study the abscopal effects of brachytherapy, we established an experimental set up that permits irradiation of mouse tumors sparing a distant site resembling metastasis. Such second nonirradiated tumor was used as indicator of abscopal effect. Tumor size was monitored every 2 days. ResultsAbscopal effects on distant nonirradiated subcutaneous tumor lesions of transplanted MC38-derived tumors only took place when brachytherapy was combined with immunostimulatory anti-PD1 and/or anti-CD137 mAbs. ConclusionsOur results demonstrate that immunotherapy-potentiated abscopal effects can be attained by brachytherapy. Accordingly, immunotherapy plus brachytherapy combinations are suitable for clinical translation.

Anti-CD25 monoclonal antibody enhances the protective efficacy of Schistosoma japonicum GST vaccine via inhibition of CD4+CD25+Foxp3+ regulatory T cells.

The effect of anti-CD25 monoclonal antibody (anti-CD25 mAb) on the protection efficacy of Schistosoma japonicum 26kDa GST (glutathione-S-transferase) vaccine was evaluated. Mice were immunized with GST before infection with S. japonicum cercariae and then injected with anti-CD25 mAb. The worm reduction rate was promoted from 24.18% in mice with GST immunization to 47.09% in mice with GST plus anti-CD25 mAb. Compared with the control group, the percentages of splenic CD4+CD25+Foxp3+ regulatory T cells (Tregs) were significantly lower after administration of anti-CD25 mAb; meanwhile, elevated levels of IFN-γ and IL-2 were secreted by splenocytes. These results indicate that the poor protective efficacy of the GST vaccine against S. japonicum results from the presence of CD4+CD25+Foxp3+ Tregs, while anti-CD25 mAb can partially block CD4+CD25+Foxp3+ Tregs and thus enhance the protective efficacy of the GST vaccine.

Abstract 3663: An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice

Abstract Administration of an anti-CD4 depleting antibody induces tumor-specific CD8+ T cell responses in tumor-bearing mice, although the mechanisms underlying this phenomenon remain unclear. CD4+ Foxp3+ regulatory T cells (Treg) impair antigen presentation by DCs through CTLA4-mediated down-regulation of CD80/CD86. In the present study, we investigated the effects of an anti-CD4 depleting monoclonal antibody (mAb) on DC responses in the B16F10 and LLC subcutaneous tumor models, and compared these effects with those of Treg targeting therapy. Administration of anti-CD4 mAb up-regulated CD80/CD86 in tumor-infiltrating DCs, and in migrating-type but not resident-type DC subsets in the tumor-draining lymph node. The upregulation of CD80/CD86 on these DC populations was correlated with increased proliferation of tumor-specific CD8+ T cells in the tumor-draining lymph node. Anti-CD4 mAb-induced CD80/CD86 up-regulation and tumor-specific CD8+ T cell responses were remarkably higher than that induced by an anti-CD25 depleting mAb or an anti-CTLA4 blocking mAb, and were equivalent to that induced by diphtheria-toxin-induced selective depletion of Tregs in Foxp3-DTR mice. Importantly, the activation of CD4+ conventional T cells and B cells in the peripheral lymph nodes that was observed following Treg depletion was not observed following anti-CD4 mAb administration. In conclusion, anti-CD4 depleting antibody therapy is an effective way to evoke anti-tumor CD8+ T cell responses by reversing regulatory T cell-induced suppression of dendritic cells, and to reduce the risk of severe autoimmunity in cancer patients. Citation Format: Satoshi Ueha, Haru Ogiwara, Shoji Yokochi, Yoshiro Ishiwata, Francis Shand, Shohei Hori, Kazuhiro Kakimi, Satoru Ito, Kouji Matsushima. An anti-CD4 depleting antibody reverses regulatory T-cell-induced suppression of dendritic cells while preventing nonspecific CD4+ T cell responses in tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3663. doi:10.1158/1538-7445.AM2017-3663

In Vivo Costimulation Blockade-Induced Regulatory T Cells Demonstrate Dominant and Specific Tolerance to Porcine Islet Xenografts.

Although islet xenotransplantation is a promising therapy for type 1 diabetes, its clinical application has been hampered by cellular rejection and the requirement for high levels of immunosuppression. The aim of this study was to determine the role of Foxp3 regulatory T (Treg) cells in costimulation blockade-induced dominant tolerance to porcine neonatal islet cell cluster (NICC) xenografts in mice. Porcine-NICC were transplanted under the renal capsule of BALB/c or C57BL/6 recipients and given a single dose of CTLA4-Fc at the time of transplant and 4doses of anti-CD154 mAb to day 6. Depletion of Foxp3Treg cell was performed in DEpletion of REGulatory T cells mice at day 80 posttransplantation. Foxp3Treg cell from spleens of treated BALB/c mice (tolerant Treg cell), and splenocytes were cotransferred into islet transplanted nonobese diabetic background with severe combined immunodeficiency mice to assess suppressive function. In treated mice, increased numbers of Foxp3Treg cell were identified in the porcine-NICC xenografts, draining lymph node, and spleen. Porcine-NICC xenografts from treated mice expressed elevated levels of TGF-β, IL-10 and IFN-γ. Porcine-NICC xenograft tolerance was abrogated after depletion of Foxp3Treg cell. Tolerant Treg cell produced high levels of IL-10 and had diverse T cell receptor Vβ repertoires with an oligoclonal expansion in CDR3 of T cell receptor Vβ14. These tolerant Treg cells had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to porcine-NICC xenografts that naive Treg cell. This study demonstrated that short-term costimulation blockade-induced dominant tolerance and that Foxp3Treg cell played an essential role in its maintenance. Foxp3Treg cells were activated and had more potent regulatory function in vivo than naive Treg cells.

Intratumoral injection of IFN-β induces chemokine production in melanoma and augments the therapeutic efficacy of anti-PD-L1 mAb

Despite recent advances in treatment for melanoma patients through using immune checkpoint inhibitors, these monotherapies have limitations and additional treatments have been explored. Type I IFNs have been used to treat melanoma and possess immunomodulatory effects including enhancement of T-cell infiltration. T-cell plays a critical role in immune checkpoint therapies via restoration of effector functions and tumor infiltration by T-cells predicts longer survival in a variety of cancer types. Moreover, tumor-infiltrating T-cells are associated with the expression of chemokines such as CCL5 and CXCR3 ligands in tumor tissues. We therefore investigated whether intratumoral injection of IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma cells and has additional antitumor effects when combined with anti-PD-L1 mAb treatment. IFN-β treatment enhanced CD8+ T-cell infiltration into tumors and CCL5 and CXCR3 ligand expression. In vivo studies using a mouse model showed that monotherapy with IFN-β, but not with anti-PD-L1 mAb, inhibited tumor growth in comparison to control. However, the therapeutic efficacy of IFN-β was significantly enhanced by the addition of anti-PD-L1 mAb. This antitumor response of combination therapy was abrogated by anti-CD8 mAb and IFN-β augmented the neoantigen-specific T-cell response of anti-PD-L1 mAb. Our findings suggest that IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma, which could play a role in T-cell recruitment, and enhances the efficacy of anti-PD-L1 mAb treatment in a CD8-dependent manner.

Development of a unique anti-CD137 therapeutic antibody: Efficacy and safety profiles in mono and combination therapy.

e23079 Background: Abstract: CD137 (4-1BB), a member of the TNFR super family, is expressed on several subsets of activated immune cells and acts as co-stimulatory receptor for T-cells to promote cellular proliferation, survival and cytokine production. Agonist antibodies against human CD137 in early clinical trials show strikingly different toxicology profiles, ranging from severe liver toxicity at low doses, to manageable adverse effects at modest doses. Whereas preclinical studies in mouse show intense liver T-cell infiltration and toxicity by surrogate agonist mAb. The preclinical efficacy and pharmacology of anti-CD137 were extensively tested and validated in various tumor models with intact immune system. Since none of the available anti-CD137 agonist antibodies display multiple species cross reactivity including rodent, the translational significance of these mAbs is limited concerning their efficacy and safety profile. Methods: Through our proprietary Dynamic Precision Library (DPL) technology, we have discovered multiple agonistic antibodies that exhibit high affinity and broad species cross-reactivity against a unique epitope shared among human, monkey and rodent CD137. Results: These mAbs, after crosslinking, strongly activate CD137 mediated cellular NFkB signaling, as well as stimulate T-cell proliferation and IFNg release in the presence of suboptimal concentrations of anti-CD3 antibodies. The broad species cross-reactivity by a single therapeutic agent enabled us to evaluate their potentiation of host immune responses that gives rise to the anti-tumor efficacy and potential toxicity in relevant rodent and monkey models. Our lead anti-CD137 agonist mAb has demonstrated robust anti-tumor activities in multiple mouse syngeneic tumor models, with durable antigen specific protective memory T-cell responses. Conclusions: In sharp contrast to surrogate anti-mouse CD137 mAbs, no toxicities including liver inflammation or other adverse effects were observed. These preclinical results warrant further clinical development of our lead as a novel immunotherapeutic anti-cancer agent.

IL-2 functionalized hydroxyethylstarch nanocapsules for targeting of human T cells with different IL-2 receptor affinities in vitro and in vivo

Abstract Cell type specific delivery of drugs by use of nanocapsules (NC) is a promising approach for induction of efficient anti-tumor immunotherapies. Here, we report the generation of IL-2 functionalized NC (HES-IL-2) to target CD25+ (IL-2 receptor α chain) T cells. In flow cytometry experiments HES-IL-2 exhibited an enhanced uptake by human CD25+ T cells compared to control capsules. The observed uptake was CD25 specific as CD25high T cells displayed a significantly enhanced NC incorporation compared to CD25− T cells with negligible internalization. In addition blocking of the IL-2 receptor dependent binding by an anti CD25 mAb significantly reduced the HES-IL-2 uptake by human CD25+ T cells. Furthermore, comparing and competitive studies of naïve CD25−, activated CD25+ and regulatory CD25high human T cells revealed a low incorporation of HES-IL-2 in naïve and a moderate and high uptake, respectively in activated or regulatory T cells. Additionally, in order to preferentially address different T cell populations by means of various IL-2 receptor affinities, we generated HES-IL-2 with distinct reduced amounts of IL-2 on their surface. Intriguingly, in contrast to naïve CD25− and activated CD25+ T cells, in which the NC uptake was clearly IL-2 dose-dependent we did not observe any differences when HES-IL-2 with a twofold and tenfold decreased amount of IL-2 were used for studies with regulatory CD25high T cell, indicating a high IL-2 sensitivity of regulatory T cells for a specific targeting. In addition, in vivo studies in human T cell reconstituted RAG2−/−γc−/− mice exhibited a significantly enhanced uptake of HES-D-IL-2 by CD25+ T cells. In summary, we generated IL-2 functionalized NC for targeting of human CD4+CD25+ T cells in vitro and in vivo.