ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27.   ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 ({"type":"clinical-trial","attrs":{"text":"NCT01813539","term_id":"NCT01813539"}}NCT01813539).     

Effective Chemoimmunotherapy with Anti-TGFβ Antibody and Cyclophosphamide in a Mouse Model of Breast Cancer

TGFβ is reportedly responsible for accumulation of CD4⁺Foxp3⁺ regulatory T cells (Tregs) in tumor. Thus, we treated mouse 4T1 mammary carcinoma with 1D11, a neutralizing anti-TGFβ (1,2,3) antibody. The treatment delayed tumor growth, but unexpectedly increased the proportion of Tregs in tumor. In vitro, 1D11 enhanced while TGFβ potently inhibited the proliferation of Tregs. To enhance the anti-tumor effects, 1D11 was administered with cyclophosphamide which was reported to eliminate intratumoral Tregs. This combination resulted in long term tumor-free survival of up to 80% of mice, and the tumor-free mice were more resistant to re-challenge with tumor. To examine the phenotype of tumor infiltrating immune cells, 4T1-tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide. This treatment markedly inhibited tumor growth, and was accompanied by massive infiltration of IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic CD11b⁺Gr1⁺ cells, and increased their expression levels of MHC II and CD80. In a spontaneous 4T1 lung metastasis model with resection of primary tumor, this combination therapy markedly increased the survival of mice, indicating it was effective in reducing lethal metastasis burden. Taken together, our data show that anti-TGFβ antibody and cyclophosphamide represents an effective chemoimmunotherapeutic combination.     

Elimination of CD4+ T cells in mice bearing an advanced sarcoma augments the antitumor action of interleukin-2

Experiments were undertaken to determine whether the depletion of CD4⁺ T cells from mice bearing an advanced immunogenic SA-1 sarcoma would result in an enhanced ability of interleukin-2 (IL-2) to cause tumor regression. The results show that whereas IL-2 therapy given as a 5-day course starting on day 10 of tumor growth caused complete regression of the tumor, it failed to cause regression if started on day 15 of tumor growth. However, in mice depleted of CD4⁺ T cells by treatment with anti-CD4 monoclonal antibody (mAb), IL-2 therapy started on day 15 resulted in appreciable tumor regression in most animals, and the therapeutic effect was greatly increased if two consccutive courses of anti-CD4 mAb and IL-2 therapy were given. On the other hand, treatment with anti-CD4 mAb alone had no effect on tumor growth. It was shown that the therapeutic action of combination therapy with anti-CD4 mAb and IL-2 was mediated by CD8⁺ T cells, because the therapeutic effect was completely ablated in mice depleted of CD8⁺ T cells with anti-CD8 mAb. Taken together these results suggest that, at a late stage of growth of an immunogenic tumor, depletion of CD4⁺ T cells can enhance the antitumor effect of IL-2 therapy by releasing CD8⁺-T-cell-mediated immunity from T-cell-mediated suppression.     

Glucose metabolism characteristics and TLR8-mediated metabolic control of CD4+ Treg cells in ovarian cancer cells microenvironment

Immunotherapy is expected to become the most promising new treatment for ovarian cancer owing to its immunogenicity. However, immunosuppression in the tumor microenvironment is a major obstacle to the efficacy of tumor therapy. Studies have found different metabolism ways of regulatory T cells (Tregs) in the cancer environment may be related to the immunosuppression and Toll-like receptor 8 (TLR8) can reverse the suppression function of Tregs. But it is still unclear that if the TLR8-mediated function reversal is associated with the change of glucose metabolism of Tregs. It was found that the positive expression rates of Glut1, HIF-1α, and Ki67 in CD4⁺ Treg cells of OC were significantly higher than that in benign ovarian tumor and HC, and also significantly higher than that in CD4⁺ Teffs of OC. What’s more, compared with CD4⁺ Teff group, CD4⁺ Tregs highly expressed seven genes and three proteins related to glucose metabolism and had higher levels of glucose uptake and glycolysis. After activating TLR8 signal of CD4⁺ Tregs, the proliferation level of naive CD4⁺ T cells was higher than that of the control group. At the same time, the expression levels of eight genes and five proteins related to glucose metabolism in CD4⁺ Treg cells with TLR8 activated were decreased and levels of glucose uptake and glycolysis were also lower. Furthermore, TLR8 signaling also downregulated the mTOR pathway in CD4⁺ Tregs. CD4⁺ Tregs pretreated with 2-deoxy-d-Glucose (2-DG) and galloflavin also attenuated the inhibition of Teffs proliferation. Although CD4⁺ Tregs pretreated with 2-DG and galloflavin before activating TLR8 signal had no significant difference compared with the group only treated with inhibitors, which suggested TLR8-mediated reversal of CD4⁺ Treg cells inhibitory function in ovarian cancer cells co-cultured microenvironment had a causal relationship with glucose metabolism.Subject terms: Glycobiology, Tumour immunology     

CD27-mediated activation of murine NK cells

CD27, a member of the TNF receptor superfamily, has been implicated in T cell activation, T cell development, and T cell-dependent Ab production by B cells. In the present study we examined the expression and function of CD27 on murine NK cells. Murine NK cells constitutively expressed CD27 on their surface. Stimulation with immobilized anti-CD27 mAb or murine CD27 ligand (CD70) transfectans solely could induce proliferation and IFN-gamma production of freshly isolated NK cells and enhanced the proliferation and IFN-gamma production of anti-NK1.1-sutimulated NK cells. Although NK cell cytotoxicity was not triggered by anti-CD27 mAb or against CD70 transfectants, prestimulation via CD27 enhanced the cytotoxic activity of NK cells in an IFN-gamma-dependent manner. These results suggest that CD27-mediated activation may be involved in the NK cell-mediated innate immunity against virus-infected or transformed cells expressing CD70.     

Combination of rapamycin and IL-2 do not affect antigen presentation ability of rat B cell and could promote Tregs proliferation and inhibitory activity

Rapamycin (RPM), a powerful agent used clinically in transplant recipients, induces CD4⁺CD25⁺ regulatory T cells (Tregs) which play an important role in induction of immune tolerance. However, long-term use of RPM has negative side effects. In this report, we found that combination with the low dose RPM and high dose IL-2 did not affect antigen presentation of rat B cells to Tregs, and could efficiently promote Tregs proliferation and enhance their inhibitory activities in vitro. In addition, the combination of low dose RPM and high dose IL-2 enhanced mRNA expression of Foxp3, TGF-β1 and Pim-2 in Tregs but not in CD4⁺CD25⁻ T effector cells (Teffs). The Tregs inhibitory activity is positively associated with mRNA expressions of TGF-β1 and Pim-2 while unrelated to the Foxp3 mRNA expression. Our present study offers one approach to expand functional Tregs in vitro, which maybe used for clinical immune tolerance induction.     

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies

The conserved glycosylation site Asn²⁹⁷ of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn²⁹⁷ (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX? Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake.

Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose.

At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.

⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

Durable blockade of PD-1 signaling links preclinical efficacy of sintilimab to its clinical benefit

ABSTRACT

Blockade of immune checkpoint pathways by programmed cell death protein 1 (PD-1) antibodies has demonstrated broad clinical efficacy against a variety of malignancies. Sintilimab, a highly selective, fully human monoclonal antibody (mAb), blocks the interaction of PD-1 and its ligands and has demonstrated clinical benefit in various clinical studies. Here, we evaluated the affinity of sintilimab to human PD-1 by surface plasmon resonance and mesoscale discovery and evaluated PD-1 receptor occupancy and anti-tumor efficacy of sintilimab in a humanized NOD/Shi-scid-IL2rgamma (null) (NOG) mouse model. We also assessed the receptor occupancy and immunogenicity of sintilimab from clinical studies in humans (9 patients with advanced solid tumor and 381 patients from 4 clinical studies, respectively). Sintilimab bound to human PD-1 with greater affinity than nivolumab (Opdivo®, MDX-1106) and pembrolizumab (Keytruda®, MK-3475). The high affinity of sintilimab is explained by its distinct structural binding mode to PD-1. The pharmacokinetic behavior of sintilimab did not show any significant differences compared to the other two anti-PD-1 mAbs. In the humanized NOG mouse model, sintilimab showed superior PD-1 occupancy on circulating T cells and a stronger anti-tumor effect against NCI-H292 tumors. The strong anti-tumor response correlated with increased interferon-γ-secreting, tumor-specific CD8+ T cells, but not with CD4+ Tregs in tumor tissue. Pharmacodynamics testing indicated a sustained mean occupancy of ≥95% of PD-1 molecules on circulating T cells in patients following sintilimab infusion, regardless of infusion dose. Sintilimab infusion was associated with 0.52% (2/381 patients) of anti-drug antibodies and 0.26% (1/381 patients) neutralizing antibodies. These data validate sintilimab as a novel, safe, and efficacious anti-PD-1 mAb for cancer immunotherapy.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4+CD25+ regulatory T cells

Naturally occurring CD4⁺CD25⁺ regulatory T cells (Tregs) are required to limit immune‐induced pathology and to maintain homeostasis during the early‐phase of sepsis. This study aimed to investigate the role of interleukin (IL)‐38, a newly described member of the IL‐1 cytokine family, in mediated immune response of CD4⁺CD25⁺ Tregs in sepsis. Here, we provide evidence that expressions of IL‐38 and its receptor were detected in murine CD4⁺CD25⁺ Tregs. Stimulation of CD4⁺CD25⁺ Tregs with LPS markedly up‐regulated the expression of IL‐38. Treatment with rmIL‐38 dramatically enhanced the immunosuppressive activity of CD4⁺CD25⁺ Tregs after LPS stimulation and in septic mice induced by CLP, resulting in amplification of helper T cell (Th) 2 response and reduction in the proliferation of effector T cells. These effects were robustly abrogated when anti–IL‐38 antibody was administered. Administration of rmIL‐38 improved the survival rate of CLP mice. In addition, CD4⁺CD25⁺ Tregs depletion before the onset of sepsis obviously abolished IL‐38–mediated protective response. These findings suggest that IL‐38 enhances the immunosuppressive activity of CD4⁺CD25⁺ Tregs, which might contribute to the improvement of host immune function and prognosis in the setting of sepsis.     

Regulatory effect of vasoactive intestinal peptide on the balance of Treg and Th17 in collagen-induced arthritis

Vasoactive intestinal peptide (VIP) is a well-known anti-inflammatory neuropeptide. The capacity of VIP can be exhibited through inhibiting inflammatory responses, shifting the Th1/Th2 balance in favor of anti-inflammatory Th2 immunity and inducing regulatory T cells (Tregs) with suppressive activity. In addition to pro-inflammatory Th1 response, Th17 are also believed to play important roles in the pathogenesis of rheumatoid arthritis (RA). In this study, we used collagen-induced arthritis (CIA) model in Wistar rats to investigate the role of VIP in the balance of CD4⁺ CD25⁺ Tregs and Th17 on RA. Data presented here showed that administration of VIP decreased incidence and severity of CIA. Disease suppression was associated with the upregulation of CD4⁺ CD25⁺ Tregs, downregulation of Th17- and Th1-type response and influence on the RANK/RANKL/OPG system. The results provide novel evidence that the therapeutic effects of VIP on CIA rats were associated with the balance of CD4⁺ CD25⁺ Tregs and Th17.     

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/<Emphasis Type="Italic">neu</Emphasis>-tolerant mice

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT ⁺ (neuT ⁺) triple transgenic mice represent an improvement over neuT ⁺ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85–94) (p85) CTL and HER-2(776–790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT ⁺ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8⁺ CTLs and CD4⁺ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4⁺ and CD8⁺ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4⁺ and CD8⁺ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.     

Signaling through CD70 regulates B cell activation and IgG production

CD70, the cellular ligand of the TNF receptor family member CD27, is expressed transiently on activated T and B cells and constitutively on a subset of B cell chronic lymphocytic leukemia and large B cell lymphomas. In the present study, we used B cells constitutively expressing CD70 to study the functional consequences of signaling through CD70. In vitro, CD70 ligation with anti-CD70 mAbs strongly supported proliferation and cell cycle entry of B cells submitogenically stimulated with either anti-CD40 mAb, LPS, or IL-4. In this process, the cell surface receptors CD25, CD44, CD69, CD95, and GL7 were up-regulated, whereas the expression of CD21, CD62L, surface IgM (sIgM), and sIgD was decreased. Addition of CD70 mAb to low dose LPS-stimulated CD70-positive B cells strongly diminished IgG secretion and enhanced production of IgM. Signaling through CD70 on B cells was dependent on the initiation of both PI3K and MEK pathways. In vivo exposure to either CD70 mAb or the CD70 counterreceptor CD27 down-regulated CD62L and sIgM on CD70-positive B cells. CD70 signaling during T cell-dependent immune responses also decreased IgG-specific Ab titers. Together, the in vitro and in vivo data demonstrate that CD70 has potent reverse signaling properties in B cells, initiating a signaling cascade that regulates expansion and differentiation.     

Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-γ, IL-2 and IL-2R

Complex interactions between effector T cells and Foxp3⁺ regulatory T cells (Treg) contribute to clinical outcomes in cancer, and autoimmune and infectious diseases. Previous work showed that IL-12 reversed Treg-mediated suppression of CD4⁺Foxp3⁻ T cell (Tconv) proliferation. We and others have also shown that Tregs express T-bet and IFN-γ at sites of Th1 inflammation and that IL-12 induces IFN-γ production by Tregs in vitro. To investigate whether loss of immunosuppression occurs when IFN-γ is expressed by Tregs we treated mouse lymphocyte cultures with IL-12. IFN-γ expression did not decrease the ability of Tregs to suppress Tconv proliferation. Rather, IL-12 treatment decreased Treg frequency and Foxp3 levels in Tregs. We further showed that IL-12 increased IL-2R expression on Tconv and CD8 T cells, diminished its expression on Tregs and decreased IL-2 production by Tconv and CD8 T cells. Together, these IL-12 mediated changes favored the outgrowth of non-Tregs. Additionally, we showed that treatment with a second cytokine, IL-27, decreased IL-2 expression without augmenting Tconv and CD8 T cell proliferation. Notably, IL-27 only slightly modified levels of IL-2R on non-Treg T cells. Together, these results show that IL-12 has multiple effects that modify the balance between Tregs and non-Tregs and support an important role for relative levels of IL-2R but not for IFN-γ expression in IL-12-mediated reversal of Treg immunosuppression.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody

Background

The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents.

Methods

Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells.

Results

The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice.

Conclusions

A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.     

Tregs Modulate Lymphocyte Proliferation,Activation, and Resident-Memory T-Cell Accumulation within the Brain during MCMV Infection

Accumulation and retention of regulatory T-cells (Tregs) has been reported within post viral-encephalitic brains, however, the full extent to which these cells modulate neuroinflammation is yet to be elucidated. Here, we used Foxp3-DTR (diphtheria toxin receptor) knock-in transgenic mice, which upon administration of low dose diphtheria toxin (DTx) results in specific deletion of Tregs. We investigated the proliferation status of various immune cell subtypes within inflamed central nervous system (CNS) tissue. Depletion of Tregs resulted in increased proliferation of both CD8⁺ and CD4⁺ T-cell subsets within the brain at 14 d post infection (dpi) when compared to Treg-sufficient animals. At 30 dpi, while proliferation of CD8⁺ T-cells was controlled within brains of both Treg-depleted and undepleted mice, proliferation of CD4⁺ T-cells remained significantly enhanced with DTx-treatment. Previous studies have demonstrated that Treg numbers within the brain rebound following DTx treatment to even higher numbers than in untreated animals. Despite this rebound, CD8⁺ and CD4⁺ T-cells proliferated at a higher rate when compared to that of Treg-sufficient mice, thus maintaining sustained neuroinflammation. Furthermore, at 30 dpi we found the majority of CD8⁺ T-cells were CD127^hi KLRG1^- indicating that the cells were long lived memory precursor cells. These cells showed marked elevation of CD103 expression, a marker of tissue resident-memory T-cells (T_RM) in the CNS, in untreated animals when compared to DTx-treated animals suggesting that generation of T_RM is impaired upon Treg depletion. Moreover, the effector function of T_RM as indicated by granzyme B production in response to peptide re-stimulation was found to be more potent in Treg-sufficient animals. Taken together, our findings demonstrate that Tregs limit neuroinflammatory responses to viral infection by controlling cell proliferation and may direct a larger proportion of lymphocytes within the brain to be maintained as T_RM cells.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.