Digital Receptor Occupancy Assay in Quantifying On- and Off-Target Binding Affinities of Therapeutic Antibodies.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant (Kd) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

Interferon-inducible protein IFIXalpha1 functions as a negative regulator of HDM2.

The 200-amino-acid repeat (HIN-200) gene family with the hematopoietic interferon (IFN)-inducible nuclear protein encodes highly homologous proteins involved in cell growth, differentiation, autoimmunity, and tumor suppression. IFIX is the newest member of the human HIN-200 family and is often downregulated in breast tumors and breast cancer cell lines. The expression of the longest isoform of IFIX gene products, IFIXalpha1, is associated with growth inhibition, suppression of transformation, and tumorigenesis. However, the mechanism underlying the tumor suppression activity of IFIXalpha1 is not well understood. Here, we show that IFIXalpha1 downregulates HDM2, a principal negative regulator of p53, at the posttranslational level. IFIXalpha1 destabilizes HDM2 protein and promotes its ubiquitination. The E3 ligase activity of HDM2 appears to be required for this IFIXalpha1 effect. Importantly, HDM2 downregulation is required for the IFIXalpha1-mediated increase of p53 protein levels, transcriptional activity, and nuclear localization, suggesting that IFIXalpha1 positively regulates p53 by acting as a negative regulator of HDM2. We found that IFIXalpha1 interacts with HDM2. Interestingly, the signature motif of the HIN-200 gene family, i.e., the 200-amino-acid HIN domain of IFIXalpha1, is sufficient not only for binding HDM2 but also for downregulating it, leading to p53 activation. Finally, we show that IFIX mediates HDM2 downregulation in an IFN-inducible system. Together, these results suggest that IFIXalpha1 functions as a tumor suppressor by repressing HDM2 function.

Interferon-inducible protein IFIXalpha inhibits cell invasion by upregulating the metastasis suppressor maspin.

IFIXalpha, a member of the interferon-inducible HIN-200 family, has been identified as a putative tumor suppressor. However, the molecular mechanisms underlying IFIXalpha-mediated tumor suppression are poorly understood. In the present study, we demonstrated that the metastasis suppressor maspin acts as the downstream target of IFIXalpha. IFIXalpha suppressed the invasion activity of MDA-MB-468 breast cancer cells, and its inhibitory effect was reversed by the knockdown of maspin. Both Maspin mRNA and protein were upregulated by IFIXalpha. Histone deacetylase (HDAC) inhibitors, but not DNA methyltransferase inhibitor upregulated maspin, and HDAC1 inhibited the transactivation of maspin promoter. Although the HDAC1 protein was downregulated in IFIXalpha-expressing cells, IFIXalpha did not affect HDAC1 mRNA levels. Conversely, a proteasome inhibitor restored the level of HDAC1 protein in IFIXalpha-expressing cells, and the polyubiqutination of HDAC1 was promoted by IFIXalpha, suggesting that HDAC1 is regulated by IFIXalpha through a ubiquitin-proteasome pathway. Together, these data provide novel insights into the tumor-suppressive function of IFIXalpha.

mMAPS: a flow-proteometric technique to analyze protein-protein interactions in individual signaling complexes.

Signal transduction is a dynamic process that regulates cellular functions through multiple types of biomolecular interactions, such as the interactions between proteins and between proteins and nucleic acids. However, the techniques currently available for identifying protein-protein or protein-nucleic acid complexes typically provide information about the overall population of signaling complexes in a sample instead of information about the individual signaling complexes therein. We developed a technique called "microchannel for multiparameter analysis of proteins in a single complex" (mMAPS) that simultaneously detected individual target proteins either singly or in a multicomponent complex in cell or tissue lysates. We detected the target proteins labeled with fluorophores by flow proteometry, which provided quantified data in the form of multidimensional fluorescence plots. Using mMAPS, we quantified individual complexes of epidermal growth factor (EGF) with its receptor EGFR, EGFR with signal transducer and activator of transcription 3 (STAT3), and STAT3 with the acetylase p300 and DNA in lysates from cultured cells with and without treatment with EGF, as well as in lysates from tumor xenograft tissue. Consistent with the ability of this method to reveal the dynamics of signaling protein interactions, we observed that cells treated with EGF induced the interaction of EGF with EGFR and the autophosphorylation of EGFR, but this interaction decreased with longer treatment time. Thus, we expect that this technique may reveal new aspects of molecular interaction dynamics.