CD3 Monoclonal Antibody Research Articles

Abstract 2884: Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment

Abstract Tumor-targeted CD28 bispecific antibodies (bsAbs) are designed to co-stimulate T cells specifically within the tumor microenvironment. By bridging T cells to malignant cells expressing a selected tumor-associated antigen (TAA), CD28 bsAbs deliver the so-called signal 2 to T cells unleashing their full cytotoxic potential. Contrary to bivalent CD28 monoclonal antibody (mAb) superagonists, the activation of CD28 with monovalent CD28 bispecifics requires the binding of a TAA expressed by the cancer cells to induce CD28 clustering. Preclinical studies have shown the benefit of adding costimulatory CD28 bsAbs for the treatment of solid tumors, boosting the efficacy of bispecific T cell engagers (CD3 bsAbs) or PD-(L)1 checkpoint inhibitors. Using our proprietary fully human κλ body antibody platform, which relies on a common heavy chain and on two distinct light chains driving specificity and affinity (i.e., one kappa and one lambda), we have identified a panel of agonist anti-CD28 kappa arms. Using our existing lambda anti-TAA arms (e.g., targeting CD19, MSLN, HER2, EGFR), an array of TAA-CD28 κλ bodies targeting different tumors were generated. The capacity of these CD28 κλ bodies to provide signal 2 to T cells was assessed both in vitro and in vivo in combination with CD3 bsAbs. In the presence of cancer cells and corresponding TAA-CD3 bsAbs, the CD28 κλ bodies enabled the activation of primary human CD4 and CD8 T cells, leading to T cell proliferation and secretion of cytokines such as IL-2 and IFNγ, and granzyme B. Importantly, when used alone or in combination with an untargeted CD3 bsAb (that is, in the absence of signal 1), CD28 κλ bodies showed no sign of T cell activation, potentially limiting peripheral toxicity. The resulting change in the T cell activation status led to a potent in vitro T cell-dependent cellular cytotoxicity (TDCC) against TAA-expressing cancer cell lines. CD28 κλ bodies targeting gastrointestinal tumors were tested in combination with CD3 bsAbs in xenogeneic tumor models and synergistically improved the antitumoral activity of T cell engagers, leading to an enhanced tumor control. Thus, via the identification of optimal agonist anti CD28 arms, CD28 bsAbs targeting multiple TAAs were designed for tumor-specific activation of the immune system. The resulting TAA-CD28 κλ bodies can enhance the antitumor response induced by CD3-retargeting bsAbs via the induction of T cell proliferation and activation, increased cytokine secretion and boosted anti-tumoral cytotoxicity. Other anti-TAA arms are being explored, to expand the panel of TAAs that could be easily paired with our anti-CD28 platform arms. Citation Format: Sara Majocchi, Pauline Lloveras, Coline Burnet-Merlin, Lise Nouveau, Nicolas Pleche, Pauline Malinge, Claudia Batista, Christelle Daviet, Maud Charreton, Guillemette Pontini, Franck Gueneau, Valery Moine, Giovanni Magistrelli, Limin Shang, Walter Ferlin, Krzysztof Masternak. Optimized CD28 bispecific antibodies for targeted activation of T cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2884.

Anticipation of Relapse and Acute Graft-Versus-Host Disease after Allogeneic Peripheral Blood Stem Cell Transplantation: The Fundamental Role of Antigen-Presenting (Dendritic) Cells.

BackgroundDendritic cells (DCs) are antigen-presenting cells. In humans two distinct lineages of DCs exist: DC1 and DC2. Efforts to explore the role of DCs in acute graft-versus-host disease (aGVHD) after allogeneic peripheral blood stem–cell transplantation (PBSCT) are gaining traction. However, further research is needed to identify particular lineages and their values in terms of developing an evidence-based aGVHD- or relapse-prevention strategy. We monitored DC counts and subsets in PBSC grafts while harvesting stem cells in recipients to elucidate their value in anticipating disease relapse or aGVHD.MethodsWe enrolled 29 participants. Using fluorescence-activated cell sorting, total counts/kg of CD34+, DCs, and DC subsets were analyzed in 29 PBSC-graft components using CMRF44, CD11c, and CD4 monoclonal antibodies (MoAbs).ResultsIn the 29 grafts, we detected a significant positive correlation (P<0.01) between DCs and both DC1 and DC2. Significantly higher counts (P<0.01) of DCs and DC1 in those who had developed aGVHD (nine cases) were also observed. Relapsed cases (two) were also associated with higher counts of DCs and DC2. A significant positive correlation (P<0.05), was recorded between DCs and DC1 counts and the day of myeloid engraftment, while this was not detected on the day of platelet engraftment. Myeloid engraftment transpired earlier in patients without aGVHD. Increased DC-graft numbers, particularly DC1 measured by CD11c Moabs, were associated with aGVHD. Recipients of higher numbers of CD4bright DCs had an increased risk of relapse after allogeneic PBSCT.ConclusionThis study analyzed DCs in PBSC grafts, using novel specific MoAbs and flow cytometry. Our data showed that higher donor DC1 counts were linked to the incidence of aGVHD and DC2 with relapse. We propose a fundamental role for DC-graft monitoring in anticipating aGVHD and disease relapse.

Deficiency of Leukocyte Surface Glycoproteins Mol, LFA-1, and Leu M5 in a Dog With Recurrent Bacterial Infections: An Animal Model

A dog with severe recurrent bacterial infections, impaired pus formation, delayed wound healing, and severe persistent leukocytosis was the result of a mother-son mating. Assessment of leukocyte function revealed profound abnormalities in adherence-dependent activities including impaired granulocyte adhesion to glass/plastic surfaces or nylon wool, decreased granulocyte aggregation and chemotaxis, and diminished lymphocyte blastogenesis, but normal neutrophil oxidative activity, serum immunoglobulin, and complement levels. By immunofluorescence analysis, CD11b and CD18 monoclonal antibodies specific for the 155-kd alpha polypeptide of Mo1 (gp 155, 94) and the 94 kd beta peptide common to Mo1, LFA-1 (gp 170, 94), and Leu M5 (p 150, 94) (surface molecules that promote leukocyte adhesion) failed to bind to unstimulated and A23187 calcium ionophore-stimulated granulocytes or mononuclear cells of the affected dog as compared with strong specific binding to canine control cells. The Mo1 glycoproteins were only barely detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of immunoprecipitates from lysates of 125I surface-labeled neutrophils from the affected dog as compared with intense bands seen with canine control cell precipitates. We conclude that this dog has a severe leukocyte surface glycoprotein deficiency syndrome that is similar, if not identical, to that recently recognized in humans. Dogs with deficiency of leukocyte Mo1, LFA-1, and Leu M5 expression may represent a useful animal model to characterize further the molecular basis for an inherited disorder in leukocyte effector function.

Bio-conjugation of anti-human CD3 monoclonal antibodies to magnetic nanoparticles by using cyanogen bromide: A potential for cell sorting and noninvasive diagnosis

The conjugation of monoclonal antibodies with superparamagnetic iron oxide nanoparticles (SPIONs) has appeared as a potential multifunctional clinical tool, which can effectively diagnose cancers and monitor their treatment, specifically. Despite the presence of different methods for conjugating antibodies to iron oxide nanoparticles, novel cost-effective and simpler conjugation techniques should be performed in this regard. In current study, an anti-CD3 monoclonal antibody was conjugated to the Fe3O4 coated by carboxymethyl dextran (CMD) using cyanogen bromide (CNBr). Moreover, EDC/NHS techniques were applied as a positive control. The experimental results showed that the Conjugation was performed and the presence of the antibody conjugated to the MNPs in human xenograft tumors was confirmed using Prussian blue (PB) staining, following magnetic resonance imaging (MRI), 30 min after injection. This conjugation method was shown to be able to separate CD3+ T lymphocytes efficiently from whole blood with high purity. Accordingly, this type of bio-conjugation method can be utilized in the future for cell sorting, and can be applied for adopted cell therapies such as CAR-T cell (Chimeric antigen receptor T cell) therapy, as well as targeted MRI imaging.

Combination of oncolytic adenovirus ZD55 harboring TRAIL-IETD-MnSOD and cytokine-induced killer cells against lung cancer.

BackgroundOur study aimed to investigate the effect of cancer-targeting gene-virotherapy and cytokine-induced killer (CIK) cell immunotherapy on lung cancer.MethodsCIK cells were obtained from peripheral blood mononuclear cells using interferon (IFN)-γ, interleukin (IL)-2, and CD3 monoclonal antibody. The CIK cells were infected with oncolytic adenovirus ZD55 harboring tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), manganese-containing superoxide dismutase (MnSOD), and TRAIL-isoleucine-aspartate-threonine-glutamate (IETD)-MnSOD. The cells were then cocultured with lung cancer cell lines A549 and NCI-H1650, normal cell line BEAS-2B, or injected into an A549 xenograft mouse model.ResultsProliferation, colony formation, and invasion of A549 and NCI-H1650 cells were significantly inhibited by co-cultivation with CIK cells carrying oncolytic adenoviruses (in order) ZD55-TRAIL-IETD-MnSOD > ZD55-TRAIL + ZD55-MnSOD > ZD55-MnSOD > ZD55-TRAIL. Compared to BEAS-2B cells, the production of IFN-γ, TNF-α, and lactate dehydrogenase (LDH) in tumor cells was increased. Tumor volume in the xenograft model and Ki-67 expression in tumor samples were reduced after injection of CIK cells carrying oncolytic adenoviruses, in the same order as the in vivo experiments. Levels of IFN-γ, TNF-α, and LDH contents were also increased in the same order.ConclusionsOur studies confirmed the high efficacy of combined oncolytic adenovirus ZD55 harboring TRAIL-IETD-MnSOD and CIK cells against lung cancer.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

Anti-tumour necrosis factor α antibodies and circulating lymphocyte phenotypes in inflammatory bowel disease

ObjectiveCirculating lymphocyte subtypes are not fully explored parameters for monitoring chronic T cell activation during inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα), one of the main mediators of IBD related inflammation induces expression of CD70 on T cells. CD70 limits T cell expansion and controls CD27 receptor on activated B lymphocytes. Aim of this study was to assess the number and the frequency of CD70+ T cells and CD27+ B cells in IBD patients during inactive phase of the disease under or without anti-TNFα treatment. DesignWe studied 91 patients with inactive IBD, 31 untreated, 29 treated with infliximab (IFX), and 31 treated with adalimumab (ADA). Lymphocyte phenotypes were assessed by flow cytometry using anti-CD45, CD19, CD27, CD3, and CD70 monoclonal antibodies. IFX and ADA actual capacity of TNFα neutralization in serum was estimated by the recoveryELISA technique. ResultsWhereas CD3+ T cells were increased in treated compared to untreated patients, the percentage of the CD70+ T cells was significantly lower in treated patients indicating a ‘cooling’ effect of the biological therapy. This effect differs between samples according to the therapeutic range of the circulating drug. Although the CD19+ B-cell percentage tended to be lower in treated patients, CD19+27+ memory B cells did not show significant differences between groups. ConclusionsFrequency of peripheral blood CD70+ T cells was significantly reduced by treatment with anti-TNFα antibodies. Monitoring of this parameter of T cells can give better insight to the disease progression and therapy application in IBD patients.

Abstract CT005: T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial

Abstract Deploying CD40 activation to stimulate T cell responses upstream of immune checkpoint molecules is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological treatment effects especially treatment-related modulation of the tumor microenvironment (TME), remain poorly understood. Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy (gemcitabine and nab-paclitaxel) to 16 resectable patients with pancreatic ductal adenocarcinoma (PDAC) prior to surgery followed by adjuvant chemotherapy and CD40 mAb. The toxicity profile was acceptable, including only grade 1 or 2 cytokine release syndrome and expected toxicities from chemotherapy. Disease-free survival was 13.8 months (95% CI 2.9 - 24.8 months) and median overall survival was 23.4 months (95% CI 18.0 - 28.8), with 8 patients alive at a median of 20.0 months after surgery (follow-up range 12.2 to 34.8 months). Neoadjuvant selicrelumab induced major pharmacodynamic differences in the TME, as revealed by a multiplex imaging platform auditing the immune ecosystem, compared to resection specimens from PDAC patient previously untreated or given neoadjuvant chemotherapy/chemoradiotherapy only. For tumors resected after selicrelumab, 82% (9/11) were T-cell enriched, compared to 37% (38/104) (p=0.004) of untreated tumors and 23% (93/13) of chemotherapy/chemoradiation-treated tumors (p=0.012). Moreover, for selicrelumab tumors, tumor-associated fibrosis was less, “M2” macrophages were fewer, dendritic cells were more mature, and T cells were activated and proliferative, compared to the non-selicrelumab groups. In the periphery, CD8+ and CD4+ T cells were more activated and proliferative, and serum inflammatory cytokines CXCL10 and CCL22 increased after treatment. This study provides proof-of-concept in patients that agonistic CD40 mAb alters the TME, enhances T-cell infiltration, and modulates systemic inflammatory responses. These findings inform design of next-generation CD40 clinical trials. Citation Format: Katelyn T. Byrne, Courtney B. Betts, Rosemarie Mick, Shamilene Sivagnanam, David L. Bajor, Daniel A. Laheru, E. Gabriela Chiorean, Mark H. O'Hara, Shannon M. Liudahl, Craig Newcomb, Cécile Alanio, Ana P. Ferreira, Byung S. Park, Takuya Ohtani, Austin P. Huffman, Sara A. Väyrynen, Andressa Dias Costa, Judith C. Kaiser, Andreanne M. Lacroix, Colleen Redlinger, Martin Stern, Jonathan A. Nowak, E. John Wherry, Martin A. Cheever, Brian M. Wolpin, Emma E. Furth, Elizabeth M. Jaffee, Lisa M. Coussens, Robert H. Vonderheide. T cell inflammation in the tumor microenvironment after agonist CD40 antibody: Clinical and translational results of a neoadjuvant clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT005.

Donor-Derived Marrow Mesenchymal Stem Cells Co-Transplantation Following Haploidentical Hematopoietic Stem Cell transplantation: a Multicenter Clinical Study in 35 Severe Aplastic Anemia Children

BackgroundHaploidentical hematopoietic stem-cell transplantation (haplo-HSCT) is associated with an increased risk of graft failure and severe graft-versus-host disease (GVHD). Marrow mesenchymal stromal cells (MSCs) have been shown to support in vivo normal hematopoiesis and to display potent immunosuppressive effects. We launched a multi-center clinical study to examine the safety and feasibility of co-transplantation of MSCs (from third party donors) and haploidentical HSCs into 35 children with severe aplastic anemia (SAA).MethodsA total of 35 children with SAA were enrolled in this multi-center study between January 2014 and December 2016. All patients met the criteria of HLA-mismatched with ⩾5/10 HLA-matched loci. The conditioning regimen for haploidentical hematopoietic stem cell transplantation consisted of busulfan (Bu), cyclophosphamide and ATG. BM and peripheral blood CD34+ cells were infused intravenously ⩾5×108 cells/kg and ⩾2×106 cells/kg of recipient weight on day 01 and day 02, respectively. MSCs (1×106cells/kg) were administered by venous infusion within 6 h proceeding stem cell infusion on day 01 then on day +14 (figure 1).ResultsOf the 35 patients,16 (45.71%) were considered to have very severe SAA. The patients' median age was 11.5 years (range 3-18). The median mononuclear cell and CD34+ counts were 15.4 × 108/kg and 3.7 × 106/kg, respectively.All 35 patients (100%) achieved hematopoietic reconstitution and sustained full donor chimerism within 30 days after HSCT. The median time for myeloid engraftment was 14 days (range 10-22 days) and for platelets engraftment 18 days (range 9-36 days).Almost half of patients (17, 48.57%) experienced aGVHD after transplantation, including 8 (22.86%) with grade I, 5 (14.29%) with grade II, 2 (5.71%) with grade III and 2(5.71%)with grade IV. At 100 days after transplantation, the cumulative incidence of grades II-IV aGvHD was 25.71% and that of grades III-IV was 11.42% (Figure 2). The four patients with grade III or IV aGVHD received additional treatment with methylprednisolone (2 mg/kg/day for 5 days), CD25 monoclonal antibody and the immunosuppressant CsA was changed to FK-506. Four patients were cured, but one patient died.All 35 patients survived for more than 100 days after transplantation, the majority of them (25, 71.43%) remained disease-free and 8 patients (22.86%) developed cGVHD. Among these cGVHD patients, 5 patients (14.29%) showed limited cGVHD and 3 patients (8.57%) showed extensive cGVHD (Figure 3).All patients received the conditioning regimen on schedule without encountering any significant transplantation-related, bowel, renal or liver toxicity. No patients experienced infusional toxicity during the infusion of MSCs. Of the 35 patients, 22 developed varying degrees of fever associated with hematopoietic dysfunction which was resolved after treatment with anti-inflammatory medication. In addition, 11 patients developed a CMV infection and 17 developed an EB virus infection and 2 developed an infection with both CMV and EBV. One patient progressed to EBV-associated PTLD and died of viral infection 130 days after transplantation, and another patient progressed to EBV-associated veno-occlusive disease (VOD) and died of viral infection 115 days after transplantation. Two patients developed reversible posterior leukoencephalopathy syndrome (RPLS) died of 120 days and 105 days respectively. Viral infections in the remaining patients were treated with ganciclovir or foscarnet and gamma globulin and, all patients fully recovered.Patients were followed-up for a median of 22 months (range 3.5-37 months). None of the patients were lost to follow-up. Five patients died within the follow-up period. The causes of death included GVHD (1 patient) and infection (4 patients). The mean survival time was 21.4 months (Figure 4). All 30 of the surviving patients (beyond 8 months) achieved a hematologic complete remission(CR).ConclusionsOur results indicate that co-transplantation of MSCs with haplo-HSCT is a relatively safe therapy regime for children with SAA. Despite the lack of a comparative control group and limited cases enrolled in the study, our findings support previous studies showing that alternative donor HSCT combined with MSCs infusion could be an effective approach to improve survival rates for patients with SAA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Co-Stimulatory versus Cell Death Aspects of Agonistic CD40 Monoclonal Antibody Selicrelumab in Chronic Lymphocytic Leukemia.

Simple SummaryPrevious observations have shown that CD40 activation of CLL cells via coculture with CD40L-expressing fibroblasts increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. We studied the activity of the fully human-agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile and sensitization for cell death by aCD20 mAbs. We found that the pro-survival effect of selicrelumab is minimal, while cell death by combined selicrelumab plus anti-CD20 antibody treatment is maintained. Thus, further investigation of applying selicrelumab combined with anti-CD20 mAbs in a therapeutic setting might be considered.Objectives: Chronic lymphocytic leukemia (CLL) is a common form of leukemia with a heterogeneous clinical course that remains incurable due to the development of therapy resistance. In lymph node proliferation centers, signals from the microenvironment such as CD40 ligation through interaction with follicular T helper cells shield CLL cells from apoptosis. Previous observations have shown that, despite CD40-induced changes in apoptotic mediators resulting in cell survival, CD40 activation also increases sensitivity to cell death by CD20 mAbs rituximab and obinutuzumab. To further investigate these observations, we here studied the activity of the fully human agonistic CD40 mAb selicrelumab in primary CLL cells in relation to cell activation, induced pro-survival profile, and sensitization for cell death by aCD20 mAbs, in vitro. Methods: CLL cells from peripheral blood were isolated by the Ficoll density method. The expression of activation markers and cytokine production following CD40 stimulation was quantified by flow cytometry and ELISA. The anti-apoptotic profile of CLL induced by stimulation was evaluated by the expression of BCL-2 proteins with Western blot, and resistance to venetoclax with flow cytometry. Cell death induced by the combination of selicrelumab and aCD20 mAbs was quantified by flow cytometry. Results: CLL cells treated with selicrelumab upregulated co-stimulatory molecules such as CD86, TNF-α and death receptor CD95/Fas. In contrast to the CD40 ligand-transfected NIH3T3 cells, induction of resistance to venetoclax by selicrelumab was very moderate. Importantly, selicrelumab stimulation positively sensitized CLL cells to CD20-induced cell death, comparable to CD40 ligand-transfected NIH3T3 cells. Conclusions: Taken together, these novel insights into selicrelumab-stimulatory effects in CLL may be considered for developing new therapeutic strategies, particularly in combination with obinutuzumab.

Histopathological and immunohistochemical features of facial papules in frontal fibrosing alopecia.

Facial papules (FPs) are considered to be created by the inflammatory process, which involves facial vellus hairs, in frontal fibrosing alopecia. To demonstrate the histopathological features of FPs and the composition of the inflammatory infiltrate. In total, 18 patients with FPs were enrolled in the study after histopathological confirmation of lichen planopilaris. Histopathological evaluation of the specimens was performed by two dermatopathologists. The samples were immunostained with CD4, CD8 and CD123 monoclonal antibodies, and the percentage and proportion of cells stained with these markers were investigated. A follicular lichenoid reaction and perifollicular fibrosis were present in all cases. Vellus hairs were detected in 83.3% of biopsy specimens (15 cases), all of which were involved by the inflammation. The majority of the follicles (72%) revealed follicular plugs. Reduction and destruction of elastic fibres were visible in the perifollicular (adventitial) and the papillary dermis (100% and 78% of specimens, respectively). Prominent sebaceous glands and dilated ducts were detected in 78% and 72% of samples, respectively. CD4-positive T cells formed 67.72% and CD8-positive T cells 32.28% of the infiltrate, and the mean CD4/CD8 ratio was 2.48. In 13 (72.2%) biopsy specimens <10% of the infiltrate was positive for CD123 marker. Perifollicular inflammation, fibrosis and elastic-fibre destruction were constant histopathological features of FPs; furthermore, prominent sebaceous glands were present in the majority of samples. We also observed a CD4-positive predominance in the infiltrate.

Pre-clinical evaluation of immunoPET imaging using agonist CD40 monoclonal antibody in pancreatic tumor-bearing mice

BackgroundA novel [64Cu]Cu-NOTA-aCD40 immunoPET tracer was developed to image a CD40+ pancreatic tumor model in C57BL/6 mice and to study the biodistribution profile of the agonist CD40 (aCD40) monoclonal antibody (mAb) alone or combined with other mAbs. ProceduresCopper-64 ([64Cu]Cu) labeled NOTA-aCD40 and NOTA-IgG (10 μg; 7 MBq) were injected intravenously into C57BL/6 mice with subcutaneous mT4 tumors to assess specificity 48 h post injection (p.i.) through positron emission tomography/computed tomography (PET/CT) imaging and biodistribution studies (n = 5). [64Cu]Cu-NOTA-aCD40 was injected alone or simultaneously in combination with a therapeutic mass of cold aCD40 (100 μg), aPD-1 (200 μg) and aCTLA-4 (200 μg) mAbs. A group of mice with or without tumor received the second round of injections 1 or 3 weeks apart, respectively. PET/CT imaging and biodistribution studies were performed at 48 h p.i. The organ dose for [64Cu]Cu was estimated based on biodistribution studies with 2 μg [64Cu]Cu-NOTA-aCD40 (corresponds to 5 mg patient dose) in non-tumor bearing mice. Results[64Cu]Cu-NOTA-aCD40 accumulation was 2.3- and 7.8-fold higher than [64Cu]Cu-NOTA-IgG in tumors and spleen, respectively, indicating the specificity of aCD40 mAb in a mouse pancreatic tumor model. Tumor accumulation of [64Cu]Cu-NOTA-aCD40 was 21.2 ± 7.3%ID/g at 48 h after injection. Co-injection of [64Cu]Cu-NOTA-aCD40 with cold aCD40 mAb alone or with PD-1 and CTLA-4 mAbs reduced both spleen and tumor uptake, whereas liver uptake was increased. With the second round of injections, the liver was the only organ with substantial uptake. With a 2 μg administered dose of [64Cu]Cu-NOTA-aCD40 in a dosimetry study, the liver to spleen ratio was greater compared to the 10 μg dose (2.8 vs 0.37; respectively). The human equivalent for the highest dose organ (liver) was 198 ± 28.7 μSv/MBq. ConclusionsA CD40-immunoreactive [64Cu]Cu-NOTA-aCD40 probe was developed. The ratio of spleen to liver accumulation exceeded that of the IgG isotype and was greatest with a single small, injected mass. The safety of human patient imaging with [64Cu]Cu was established based on extrapolation of the organ specificity to human imaging.

Prior transient exposure to interleukin-21 delivered by recombinant adeno-associated virus vector protects mice from hepatitis B virus persistence

Chronic infection of hepatitis B virus (HBV) is a high risk factor for hepatic diseases, such as liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-responders and hyporesponders to HBV vaccine are not protected from HBV infection. Patients that achieve autonomous or treatment-induced recovery are at risk of reactivation due to persistence of HBV covalently closed circular DNA (cccDNA) in hepatocytes. Interleukin 21 (IL-21) is a key regulator of HBV clearance in mouse models of HBV persistence: IL-21-based therapies effectively induces HBV clearance and protects mice from subsequent re-challenge. In this study, we explore the possibility of using IL-21 as prophylaxis against HBV by using mouse models of HBV persistence. HBV-naïve mice were transiently exposed to exogenous IL-21 through injection with recombinant adeno-associated virus expressing mouse IL-21 (AAV-IL-21). After extraneous IL-21 protein and DNA had become undetectable, mice were challenged with persistence-inducing HBV replicon plasmid through hydrodynamic injection. Viral persistence was analyzed by measuring viral antigens and DNA markers in serum and intrahepatic HBV DNA. For mechanistic studies, CD8+ T cell functions were blocked by repeated intraperitoneal injections of CD8 monoclonal antibodies in HBV-challenged mice. AAV-IL-21-injected mice quickly cleared HBV after HBV replicon challenge. In contrast, untreated mice and mice injected with control virus (AAV-Ctrl) allowed establishment of HBV persistence. Mechanistically, mice with prior IL-21 exposure displayed marked intrahepatic CD8+ T cell infiltrations, and CD8 blocking experiments demonstrated that CD8+ T cell responses functionally contributed toward clearance.

Immunosuppresive Sesterterpenoids and Norsesterterpenoids from Colquhounia coccinea var. mollis.

A pair of new C-14 epimeric sesterterpenoids, colquhounoid D (1) and 14-epi-colquhounoid D (2), and five degradation products featuring new C20 and C21 frameworks, norcolquhounoids A-E (3-7), were isolated from Colquhounia coccinea var. mollis. Their structures were elucidated by comprehensive spectroscopic analysis and single-crystal X-ray diffraction. Degradation of the C25 skeleton to the C21 skeleton was also achieved using aqueous NaIO4 and RuCl3. Compounds 1 and 2 showed significant immunosuppressive activity on the cytokine IFN-γ secretion of mouse splenocytes induced by anti-CD3/CD4 monoclonal antibodies, with IC50 of 8.38 and 5.79 μM, respectively, and compounds 5 and 6 were moderately active.

Ex Vivo High Salt Activated Tumor-Primed CD4+T Lymphocytes Exert a Potent Anti-Cancer Response.

Simple SummaryCell based immunotherapy is rapidly emerging as a promising cancer treatment. Salt (sodium chloride) treatment to immune cell cultures is known to induce inflammatory activation. In our current study, we analyzed the anti-cancer ability of salt treatment on immune cells outside the host followed by reinfusion into the host. Using a pre-clinical breast cancer model, we demonstrated that external salt treatment on T-cell subset of immune cells produced a viable anti-cancer response, which may have future human clinical application.Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.

Immune signature of CCR7+ central memory T cells associates with disease severity and Immunoglobulin E in bronchial asthma.

Objective. CD4+T cell subtypes are the central orchestrators of airway inflammation in bronchialasthma (BA); however, the mechanisms that regulate their accumulation in asthmatic airways are still a challenging subject. In addition, neutrophils play a significant role in the development of airway remodeling and their presence may influence clinical presentation of BA being linked to the development of severe BA. Neutrophils have also been found to acquireantigen presenting functions, enabling them to directly activate T cells. The study aimed to evaluate the possible association of chemokine receptor 7 (CCR7)+ memory CD4+T cells andCCR4+ effector T cells with disease severity and immunoglobulin E (IgE) production as well as to explore the relationship between these cells and neutrophil function in both allergic andnon-allergic asthmatic patients. Methods. Flow cytometry was used to determine the expression of different T cell subset phenotypes (CCR7 memory CD4+ and CCR4+T cells using anti-human CD3, CD4, CD45RO, CCR4 and CCR7 monoclonal antibodies) utilizing peripheral blood mononuclear cells (PBMCs) isolated from 78 allergic asthmatic patients, 41 non-allergic asthmatic patients, and 40 healthy individuals. Moreover, neutrophils' phagocytic activity was assessed by ingestion of candida particles. Results. We demonstrated increased percentages of CCR7+ memory CD4+T cells and CCR4+CD4+T cells in patients compared to control, where this up regulation was significantly higher in allergic than non-allergic asthmatic patients. Additionally, these cells were negatively correlated with improved pulmonary tests and significantly associated with disease severity scores and IgE levels. The neutrophil phagocytic activity was markedly increased in patients compared to control, showing a significant positive correlation with disease severity. Conclusions. These findings suggest that increased CCR4+ CD4+ T cells and CCR7+ memory CD4+ T cells (Tcm) may be associated with BA severity, especially in allergic BA patients and can potentially contribute to the rational design of new therapeutic approaches for asthma in the future.

APX005M, a CD40 monoclonal antibody, for patients with pancreatic adenocarcinoma.

Despite progress, chemotherapy yields disappointing results in metastatic pancreatic ductal adenocarcinoma and median overall survival is less than 12 months. Mark H O’Hara and colleagues1 investigated how to improve this figure by building on a nab-paclitaxel plus gemcitabine chemotherapy backbone in a phase 1b trial assessing the recommended phase 2 dose of the CD40 agonistic monoclonal antibody APX005M, with and without nivolumab, in an attempt to sensitise an immunologically cold disease. The authors provide an example of a smart strategic project of drug development and multicentre cooperation aimed at boosting and fostering the acquisition of sound and reliable data by rapid execution of a well designed and well managed trial.

PRESENCE OF GRAFT-INFILTRATING REGULATORY T CELLS ARE ASSOCIATED WITH LONG TERM CARDIAC XENOGRAFT SURVIVAL IN NON-HUMAN PRIMATE

Introduction: Regulatory T (CD4+CD25HiFoxP3+) cells (Treg) are a very small subset of CD4+ T cells that have been shown to play an important role in immune regulation and induce tolerance in transplantation. Previously we have reported the increased number of Treg cells in peripheral blood of long term cardiac xenograft survival recipients. However, in both kidney and liver transplantation in humans, FoxP3+ Tregs have also been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In this study, we have examined the FoxP3+ Tregs in graft-infiltrating lymphocytes (GILS) from cardiac xenograft. Materials and Methods: Genetically engineered (GE) donor pig heart was heterotopically transplanted in 3-4-year-old specific pathogen-free baboon (n=6). These GE pigs were alpha 1-3 galactosidase knockout and had multiple combinations of transgenic expression of human complement inhibitory protein (CD46), thrombomodulin (TBM), endothelial protein C receptor (EPCR), tissue factor pathway inhibitor (TFPI), and decay-accelerating factor (DAF) and protective gene against phagocytosis (CD47). Immunosuppression consisted of targeted T and B cell depletion and conventional anti-rejection agents. Immuno-phenotyping on GILS from explanted xenograft was performed to analyze the percentage of Treg cells with anti-human CD3, CD4, CD25, CD127, FoxP3, and Helios monoclonal antibodies. Results and Discussion: Cardiac xenotransplantation were performed without complication. Baboons were extubated immediately following surgery and were active, eating, and generally well soon after. WBC and lymphocytes counts were low in recipients after the xenotransplantation due to immunosuppression but recovered in a few days to a normal level. Three of six cardiac xenografts were explanted after 115 days of cardiac xenotransplantation and GILS were examined. A small population of lymphocytes was seen in all the explanted xenograft and CD4+CD25HiFoxP3+ Tregs were examined. Conclusion: We found that long term survivors had an increased percentage of FoxP3+ Treg cells compared to the GILS in cardiac xenografts with earlier graft failure. Tregs were not identified in GILS from recipients with early graft failure. The presence of Tregs was associated with improved graft function. This suggests a TReg-dependent mechanism of survival for xenografts and supports future experiments investigating this mechanism to bring xenotransplantation to clinical reality.

High Expression of Cannabinoid Receptor 2 on Cytokine-Induced Killer Cells and Multiple Myeloma Cells.

Multiple myeloma (MM) is characterized by aberrant bone marrow plasma cell (PC) proliferation and is one of the most common hematological malignancies. The potential effect of cannabinoids on the immune system and hematological malignancies has been poorly characterized. Cannabidiol (CBD) may be used to treat various diseases. CBD is known to exert immunomodulatory effects through the activation of cannabinoid receptor 2 (CB2), which is expressed in high levels in the hematopoietic system. Cytokine-induced killer (CIK) cells are a heterogeneous population of polyclonal T lymphocytes obtained via ex vivo sequential incubation of peripheral blood mononuclear cells (PBMCs) with interferon-γ (IFN-γ), anti CD3 monoclonal antibody, and IL-2. They are characterized by the expression of CD3+ and CD56+, which are surface markers common to T lymphocytes and natural killer (NK) cells. CIK cells are mainly used in hematological patients who suffer relapse after allogeneic transplantation. Here, we investigated their antitumor effect in combination with pure cannabidiol in KMS-12 MM cells by lactate dehydrogenase LDH cytotoxicity assay, CCK-8 assay, and flow cytometry analysis. The surface and intracellular CB2 expressions on CIK cells and on KMS-12 and U-266 MM cell lines were also detected by flow cytometry. Our findings confirm that the CB2 receptor is highly expressed on CIK cells as well as on MM cells. CBD was able to decrease the viability of tumor cells and can have a protective role for CIK cells. It also inhibits the cytotoxic activity of CIKs against MM at high concentrations, so in view of a clinical perspective, it has to be considered that the lower concentration of 1 µM can be used in combination with CIK cells. Further studies will be required to address the mechanism of CBD modulation of CIK cells in more detail.

Regulatory T Cells License Macrophage Pro-Resolving Functions During Atherosclerosis Regression.

Regression of atherosclerosis is an important clinical goal; however, the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, yet the numbers of these immunosuppressive cells decrease with disease progression, and whether they contribute to atherosclerosis regression is not known. We investigated the roles of Tregs in the resolution of atherosclerotic inflammation, tissue remodeling, and plaque contraction during atherosclerosis regression. Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. Single-cell RNA-sequencing of plaque immune cells revealed that unlike Tregs from progressing plaques that expressed markers of natural Tregs derived from the thymus, Tregs in regressing plaques lacked Nrp1 expression, suggesting that they are induced in the periphery during lipid-lowering therapy. To test whether Tregs are required for resolution of atherosclerotic inflammation and plaque regression, Tregs were depleted using CD25 monoclonal antibody in atherosclerotic mice during apolipoprotein B antisense oligonucleotide-mediated lipid lowering. Morphometric analyses revealed that Treg depletion blocked plaque remodeling and contraction, and impaired hallmarks of inflammation resolution, including dampening of the T helper 1 response, alternative activation of macrophages, efferocytosis, and upregulation of specialized proresolving lipid mediators. Our data establish essential roles for Tregs in resolving atherosclerotic cardiovascular disease and provide mechanistic insight into the pathways governing plaque remodeling and regression of disease.