Depletion of CD4 1 CD25 1 FoxP3 1 Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated $70% of CD4 1 FoxP3 1 cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcc receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcc receptor (FccR)III À/À mice were resistant to Treg depletion. FccRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependentcellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25 1 cells by WT or FccRIIB À/À , but not FccRIII À/À , macrophages. Altogether these data demonstrate the critical role of FccRIII 1 phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.Key words: CD25 . Fcc receptor . PC61 Ab . Phagocytes . Treg Supporting Information available online IntroductionNaturally occurring Treg play a critical role in the maintenance of peripheral tolerance. They can suppress not only CD4 1 and CD8 1 T cells, but also NK cells, macrophages and dendritic cells [1]. Treg are marked by expression of FoxP3 1 transcription factor (Foxp3), which confers the Treg's regulatory activity. Induction of Foxp3 expression in naïve T cells can convert them into Treg [2,3]. Before the discovery of Foxp3, Treg were identified by expression of CD25, an IL-2 receptor a subunit. Initially, CD25 was simply considered a Treg marker that was not associated with Treg function because of its expression on activated T cells without suppressive activity. More recently, however, it became clear that CD25, thus also IL-2, is essential for the generation, peripheral expansion and maintenance of Treg. CD25-deficient mice and mice lacking IL-2 or Foxp3 have a reduced Treg number and spontaneously develop a fatal lymphoproliferative autoimmune syndrome that can be rescued by adoptive transfer of Treg from WT mice [1]. Temporary neutralization of IL-2 using anti-IL-2 mAb also reduces Treg number, induces autoimmune gastritis in BALB/c mice and accelerates autoimmune diabetes in NOD mice [4]. Additionally, ectopic expression of IL-2 receptor b in the thymus increases Treg production [5]. A recent study using transgenic mice expressing GFP-linked Foxp3 shows that the peripheral expansion of CD25 À Treg is significantly reduced compared with that of CD25 Results and discussion Treg depletion by PC61 mAbWe first tested the extent of Treg depletion after CD25 mAb injection in C57BL/6 mice. In naïve mice (Fig. 1A left panel), around 70% of CD4 1 Fo...
Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.Sphingosine 1-phosphate (S1P) 2 receptor agonists are likely to be the next generation of pharmacologic agents used to modulate immune system function. The prototype drug of this class is FTY720, which is highly efficacious in prolonging allograft survival and in ameliorating autoimmune disease in a variety of animal models (1-4). FTY720 is being tested in human trials for the indications renal transplantation and multiple sclerosis (5). Further, there is mounting evidence that S1P agonists are efficacious in animal models of atherosclerosis (6), renal ischemia-reperfusion injury (7), and acute lung injury (8).FTY720 is a sphingosine analog that, after activation by phosphorylation (to FTY720-P), disrupts lymphocyte trafficking by decreasing lymphocyte egress from lymph nodes and the thymus (9, 10). Although the precise mechanisms that underlie this phenomenon are uncertain, the profound lymphopenia that is the index of FTY720 action is dependent on agonist action at lymphocyte S1P 1 receptors. Since FTY720-P is also a potent agonist at the S1P 3 , S1P 4 , and S1P 5 receptors (11, 12), it remains unknown whether the multiple therapeutic benefits of the drug correlate with agonist activity at the S1P 1 receptor. The propensity for S1P 1 receptor responses to desensitize (13) and the similar behaviors of S1P 1 receptor null thymocytes and FTY720-treated mouse lymphocytes have led to the suggestion that FTY720-P is a functional antagonist (14). In this scenario, the drug exaggerates S1P tone to the extent that the lymphocyte S1P 1 receptor signaling is chronically down-regulated.The kinase(s) responsible for FTY720 activation is the gateway whereby S1P signaling can be accessed readily with a therapeutic agent. Knowledge of this enzyme is important specifically to guide S1P prodrug design and generally to gain insight into the normal role of S1P in immune function. The identity of the kinase is not known currently; two candidates are sphingosine kinase 1 (SPHK1) and sphingosine kinase 2 (SPHK2). These enzymes, which are expressed widely, catalyz...
Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.
The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.
The CD4+ CD25+ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease-specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen-positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen-negative donors. Second, Tregs of antigen-negative adult mice respond to cognate antigen in vivo and rapidly gain disease-specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD-suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary-draining lymph node is the only location with evidence of profound and persistent (but reversible) host T-cell suppression. Fifth, from these nodes, highly potent AOD-suppressing Tregs are retrievable. We conclude that self-tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T-cell response is continuously negated by potent disease-specific Tregs that accumulate at the site of autoantigen presentation.
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