Anti-Claudin-2 Antibody-Drug Conjugates for the Treatment of Colorectal Cancer Liver Metastasis.

We have demonstrated that Claudin-2 is required for colorectal cancer (CRC) liver metastasis. Expression of Claudin-2 in primary CRC is associated with poor survival and is highly expressed in liver metastases. Claudin-2 also promotes breast cancer liver metastasis by enabling seeding and cancer cell survival. These observations support Claudin-2 as a potential therapeutic target for managing patients with liver metastases. Antibody-drug conjugates (ADCs) are promising anti-tumor therapeutics that combine the specific targeting ability of monoclonal antibodies with the potent cell killing activity of cytotoxic drugs. Here we report the generation of twenty-eight anti-Claudin-2 antibodies for which the binding specificities, the cross-reactivity with Claudin family members and the cross-species reactivity were assessed by flow cytometry analysis. Multiple drug conjugates were tested and PNU was selected for conjugation with anti-Claudin-2 antibodies binding either extracellular loop 1 or extracellular loop 2. Anti-Claudin-2 ADCs were efficiently internalized and effective at killing Claudin-2-expressing CRC cancer cells in vitro. Importantly, PNU-conjugated-anti-Claudin-2 ADCs impaired the development of replacement type CRC liver metastases in vivo, using established CRC cell lines and patient-derived xenograft (PDX) models of CRC liver metastases. Our results suggest that the development of ADCs targeting Claudin-2 is a promising therapeutic strategy for managing CRC liver-metastatic patients that present with replacement type liver metastases.

An Automated Single-Cell Droplet-Digital Microfluidic Platform for Monoclonal Antibody Discovery.

Monoclonal antibody (mAb) discovery plays a prominent role in diagnostic and therapeutic applications. Droplet microfluidics has become a standard technology for high-throughput screening of antibody-producing cells due to high droplet single-cell confinement frequency and rapid analysis and sorting of the cells of interest with their secreted mAbs. In this work, a new method is described for on-demand co-encapsulation of cells that eliminates the difficulties associated with washing in between consecutive steps inside the droplets and enables the washing and addition of fresh media. The new platform identifies hybridoma cells that are expressing antibodies of interest using antibody-characterization assays to find the best-performing or rare-cell antibody candidates.

TNX-1500, a crystallizable fragment-modified anti-CD154 antibody, prolongs nonhuman primate renal allograft survival.

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa-dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

Mechanism and function of monoclonal antibodies targeting siglec-15 for therapeutic inhibition of osteoclastic bone resorption.

The use of monoclonal antibodies to target functionally important cell-surface proteins on bone-resorbing osteoclasts represents a promising approach for treatment of cancer-associated bone loss and other skeletal pathologies. Previously, we identified Siglec-15, a little studied sialic acid-binding receptor, as a candidate target that is highly up-regulated during osteoclast differentiation induced by the cytokine receptor activator of NF-κB ligand (RANKL). In this report, we confirm that Siglec-15 is localized to the plasma membrane where it can be targeted by monoclonal antibodies to inhibit differentiation of functional osteoclasts in vitro. Furthermore, we found that treatment of mice with these antibodies led to a marked increase in bone mineral density, consistent with inhibition of osteoclast activity. Interestingly, osteoblast numbers were maintained despite the anti-resorptive activity. At the molecular level, Siglec-15 interacts with the adapter protein DAP12 and can induce Akt activation when clustered on the osteoclast cell surface, which likely represents its normal signaling function. Importantly, we discovered that monoclonal antibodies induce rapid internalization, lysosomal targeting, and degradation of Siglec-15 by inducing receptor dimerization. This study defines a key regulatory node that controls osteoclast differentiation and activity downstream of RANKL and supports further development of Siglec-15 antibodies as a novel class of bone loss therapeutics.

The co-repressor hairless protects RORalpha orphan nuclear receptor from proteasome-mediated degradation.

RORalpha is a constitutively active orphan nuclear receptor essential for cerebellar development and is previously shown to regulate genes involved in both myogenesis and adipogenesis. The transcriptional activity of RORalpha is dependent on the presence of a ubiquitous ligand and can be abolished by interaction with Hairless (Hr), a ligand-oblivious nuclear receptor co-repressor. In this study, we first demonstrate that RORalpha is a short-lived protein and that treatment with the MG-132 proteasome inhibitor results in the accumulation of ubiquitin-conjugated receptor and inhibition of transcription. These data show that RORalpha transcriptional activity and degradation are intrinsically linked. In addition, the introduction of inactivation mutations in the ligand-binding pocket and co-regulator-binding surface of RORalpha significantly increases protein stability, indicating that ligand and/or co-regulator binding perpetuates RORalpha degradation. Strikingly, expression of the co-repressor Hr results in the stabilization of RORalpha because of an inhibition of proteasome-mediated degradation of the receptor. Stabilization of RORalpha by Hr requires intact nuclear receptor recognition LXXLL motifs within Hr. Interestingly, the co-repressor nuclear receptor co-repressor (NCoR) has no effect on RORalpha protein turnover. This study shows that stabilization of RORalpha is an essential component of Hr-mediated repression and suggests a molecular mechanism to achieve transcriptional repression by a liganded receptor-co-repressor complex.

Novel mechanism of nuclear receptor corepressor interaction dictated by activation function 2 helix determinants.

Transcriptional regulation by nuclear receptors is controlled by the concerted action of coactivator and corepressor proteins. The product of the thyroid hormone-regulated mammalian gene hairless (Hr) was recently shown to function as a thyroid hormone receptor corepressor. Here we report that Hr acts as a potent repressor of transcriptional activation by RORalpha, an orphan nuclear receptor essential for cerebellar development. In contrast to other corepressor-nuclear receptor interactions, Hr binding to RORalpha is mediated by two LXXLL-containing motifs, a mechanism associated with coactivator interaction. Mutagenesis of conserved amino acids in the ligand binding domain indicates that RORalpha activity is ligand-dependent, suggesting that corepressor activity is maintained in the presence of ligand. Despite similar recognition helices shared with coactivators, Hr does not compete for the same molecular determinants at the surface of the RORalpha ligand binding domain, indicating that Hr-mediated repression is not simply through displacement of coactivators. Remarkably, the specificity of Hr corepressor action can be transferred to a retinoic acid receptor by exchanging the activation function 2 (AF-2) helix. Repression of the chimeric receptor is observed in the presence of retinoic acid, demonstrating that in this context, Hr is indeed a ligand-oblivious nuclear receptor corepressor. These results suggest a novel molecular mechanism for corepressor action and demonstrate that the AF-2 helix can play a dynamic role in controlling corepressor as well as coactivator interactions. The interaction of Hr with RORalpha provides direct evidence for the convergence of thyroid hormone and RORalpha-mediated pathways in cerebellar development.