No abstract

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...

Two neutralizing epitopes were defined for MAbs to SARS-CoV S glycoprotein. Antibodies to both epitopes protected mice against SARS-CoV challenge. Clinical trials are planned to test MAb 201, a fully human MAb specific for the epitope within the receptor-binding region.

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.

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.