CD38 x ICAM-1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma.

The cluster of differentiation 38 (CD38) is a well-validated target for treating multiple myeloma. Although anti-CD38 mAbs have demonstrated outstanding initial responses in patients with multiple myeloma, nearly all patients eventually develop resistance and relapse. In addition, currently approved CD38 targeting therapies have failed to show monotherapy efficacy in lymphomas, where CD38 expression is present but at lower levels. To effectively target CD38 on tumor cells, we generated an antibody-dependent cellular cytotoxicity (ADCC) enhanced bispecific CD38 x intercellular cell adhesion molecule 1 (ICAM-1) antibody, VP301. This bispecific antibody targets unique epitopes on CD38 and ICAM-1 on tumor cells with reduced red blood cell binding compared with the benchmark CD38 antibody daratumumab. VP301 demonstrated potent ADCC and antibody-dependent cellular phagocytosis activities on a selected set of myeloma and lymphoma cell lines even those with low CD38 expression. In an ex vivo drug sensitivity assay, we observed responses to VP301 in multiple myeloma primary samples from relapsed/refractory patients. Moreover, VP301 demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma models. Interestingly, combination of VP301 with the immunomodulatory drug, lenalidomide, led to synergistic antitumor growth activity in an in vivo efficacy study. In conclusion, the CD38 x ICAM-1 bispecific antibody VP301 demonstrated promising efficacy and specificity toward CD38+ and ICAM-1+ tumor cells and represents a novel approach for treating multiple myeloma and lymphoma.

RN765C, a low affinity EGFR antibody drug conjugate with potent anti-tumor activity in preclinical solid tumor models.

Epidermal growth factor receptor (EGFR) is a clinically validated target and often overexpressed in some solid tumors. Both EGFR tyrosine kinase inhibitors and ligand-blocking antibodies have been approved for treatment of NSCLC, head and neck cancers and colorectal cancers. However, clinical response is limited and often accompanied by significant toxicities due to normal tissue expression. To improve the effectiveness of targeting EGFR while minimizing the toxicities on normal tissues, we developed a low-affinity anti-EGFR antibody drug conjugate (ADC), RN765C. Potent in vitro cytotoxicity of RN765C, with nanomolar to subnanomolar EC50, was observed on a panel of cancer cell lines expressing moderate to high level of EGFR. In contrast, RN765C was less effective in killing normal human keratinocytes, presumably due to its lower receptor expression. Mechanistically, RN765C has multiple modes of action: inducing payload mediated mitotic arrest and cell death, blocking EGFR pathway signal and mediating antibody dependent cell cytotoxicity. In preclinical studies, a single dose of RN765C at 1.5-3 mg/kg was generally sufficient to induce tumor regression in multiple cell line and patient-derived xenograft models, including those that are resistant to EGFR-directed tyrosine kinase inhibitors. Our data support further investigation of RN765C in the clinic to treat EGFR expressing solid tumors.

RN927C, a Site-Specific Trop-2 Antibody-Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models.

Trop-2, also known as TACSTD2, EGP-1, GA733-1, and M1S1, is frequently expressed on a variety of human carcinomas, and its expression is often associated with poor prognosis of the diseases. However, it is also present on the epithelium of several normal tissues. A comprehensively designed Trop-2-targeting antibody-drug conjugate (ADC), balancing both efficacy and toxicity, is therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2 ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a proprietary microtubule inhibitor (MTI) linker-payload, PF-06380101. Robust in vitro cytotoxicity of RN927C was observed on a panel of Trop-2-expressing tumor cell lines, with IC50 generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death. The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft tumor models, including pancreatic, lung, ovarian, and triple-negative breast tumor types. Single-dose administration of RN927C at 0.75 to 3 mg/kg was generally sufficient to induce sustained regression of Trop-2-expressing tumors and showed superior efficacy over standard treatment with paclitaxel or gemcitabine. Administration of RN927C in nonhuman primate toxicity studies resulted in target-mediated effects in skin and oral mucosa, consistent with Trop-2 expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. On the basis of the combined efficacy and safety results, RN927C is postulated to have a favorable therapeutic index for treatment of solid tumors. Mol Cancer Ther; 15(11); 2698-708. ©2016 AACR.

Generating bispecific human IgG1 and IgG2 antibodies from any antibody pair.

Bispecific antibodies and antibody fragments are a new class of therapeutics increasingly utilized in the clinic for T cell recruitment (catumaxomab anti-EpCAM/CD3 and blinatumomab anti-CD19/CD3), increase in the selectivity of targeting, or simultaneous modulation of multiple cellular pathways. While the clinical potential for certain bispecific antibody formats is clear, progress has been hindered because they are often difficult to manufacture, may suffer from suboptimal pharmacokinetic properties, and may be limited due to potential immunogenicity issues. Current state-of-the-art human IgG-like bispecific technologies require co-expression of two heavy chains with a single light chain, use crossover domains to segregate light chains, or utilize scFv (single-chain fragment variable)-Fc fusion. We have engineered both human IgG1 and IgG2 subtypes, with minimal point mutations, to form full-length bispecific human antibodies with high efficiency and in high purity. In our system, the two antibodies of interest can be expressed and purified separately, mixed together under appropriate redox conditions, resulting in a formation of a stable bispecific antibody with high yields. With this approach, it is not necessary to generate new antibodies that share a common light chain, therefore allowing the immediate use of an existing antibody regardless of whether it has been generated via standard hybridoma or display methods. We demonstrate the generality of the approach and show that these bispecific antibodies have properties similar to those of wild-type IgGs, and we further demonstrate the utility of the technology with an example of a CD3/CD20 bispecific antibody that effectively depletes B cells in vitro and in vivo.

Homologous recombination repair is essential for repair of vosaroxin-induced DNA double-strand breaks.

Vosaroxin (formerly voreloxin) is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective double-strand breaks (DSB), G2 arrest and apoptosis. Objective responses and complete remissions were observed in phase 2 studies of vosaroxin in patients with solid and hematologic malignancies, and responses were seen in patients whose cancers were resistant to anthracyclines. The quinolone-based scaffold differentiates vosaroxin from the anthracyclines and anthracenediones, broadly used DNA intercalating topoisomerase II poisons. Here we report that vosaroxin induces a cell cycle specific pattern of DNA damage and repair that is distinct from the anthracycline, doxorubicin. Both drugs stall replication and preferentially induce DNA damage in replicating cells, with damage in G2 / M > S >> G1. However, detectable replication fork collapse, as evidenced by DNA fragmentation and long tract recombination during S phase, is induced only by doxorubicin. Furthermore, vosaroxin induces less overall DNA fragmentation. Homologous recombination repair (HRR) is critical for recovery from DNA damage induced by both agents, identifying the potential to clinically exploit synthetic lethality.

Interconvertible lac repressor-DNA loops revealed by single-molecule experiments.

At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.

Cdk1 phosphorylation of BubR1 controls spindle checkpoint arrest and Plk1-mediated formation of the 3F3/2 epitope.

Accurate chromosome segregation is controlled by the spindle checkpoint, which senses kinetochore- microtubule attachments and tension across sister kinetochores. An important step in the tension-signaling pathway involves the phosphorylation of an unknown protein by polo-like kinase 1/Xenopus laevis polo-like kinase 1 (Plx1) on kinetochores lacking tension to generate the 3F3/2 phosphoepitope. We report here that the checkpoint protein BubR1 interacts with Plx1 and that phosphorylation of BubR1 by Plx1 generates the 3F3/2 epitope. Formation of the BubR1 3F3/2 epitope by Plx1 requires a prior phosphorylation of BubR1 on Thr 605 by cyclin-dependant kinase 1 (Cdk1). This priming phosphorylation of BubR1 by Cdk1 is required for checkpoint-mediated mitotic arrest and for recruitment of Plx1 and the checkpoint protein Mad2 to unattached kinetochores. Biochemically, formation of the 3F3/2 phosphoepitope by Cdk1 and Plx1 greatly enhances the kinase activity of BubR1. Thus, Cdk1-mediated phosphorylation of BubR1 controls checkpoint arrest and promotes the formation of the kinetochore 3F3/2 epitope.

Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins.

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.

Plx1 is the 3F3/2 kinase responsible for targeting spindle checkpoint proteins to kinetochores.

Dynamic attachment of microtubules to kinetochores during mitosis generates pulling force, or tension, required for the high fidelity of chromosome separation. A lack of tension activates the spindle checkpoint and delays the anaphase onset. A key step in the tension-response pathway involves the phosphorylation of the 3F3/2 epitope by an unknown kinase on untensed kinetochores. Using a rephosphorylation assay in Xenopus laevis extracts, we identified the kinetochore-associated Polo-like kinase Plx1 as the kinase both necessary and sufficient for this phosphorylation. Indeed, Plx1 is the physiological 3F3/2 kinase involved in checkpoint response, as immunodepletion of Plx1 from checkpoint extracts abolished the 3F3/2 signal and blocked association of xMad2, xBubR1, xNdc80, and xNuf2 with kinetochores. Interestingly, the kinetochore localization of Plx1 is under the control of the checkpoint protein xMps1, as immunodepletion of xMps1 prevents binding of Plx1 to kinetochores. Thus, Plx1 couples the tension signal to cellular responses through phosphorylating the 3F3/2 epitope and targeting structural and checkpoint proteins to kinetochores.

Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1.

The abnormal expression of breast cancer-specific gene 1 (BCSG1) in malignant mammary epithelial cells is highly associated with the development and progression of breast cancer. A series of in vitro and in vivo studies performed in our laboratory and others have demonstrated that BCSG1 expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. However, currently little is known about how BCSG1 exerts its oncogenic functions. To elucidate the cellular mechanisms underlying the effects of BCSG1 in breast cancer cells, we used a yeast two-hybrid system to screen for proteins that could associate with BCSG1. Through this screening, we identified the mitotic checkpoint protein BubR1 as a novel binding partner of BCSG1. The specific association of BCSG1 with BubR1 in breast cancer cells was demonstrated by immunoprecipitation and GST pull-down assays. Intriguingly, experiments conducted in four different cell lines all showed that exogenous expressions of BCSG1 consistently reduce the cellular levels of the BubR1 protein without affecting BubR1 mRNA expression. The tendency of endogenous BCSG1 expression coinciding with lower BubR1 protein levels was also observed in seven out of eight breast cancer cell lines. We further showed that the reducing effect of BCSG1 on BubR1 protein expression could be prevented by treating BCSG1-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the BCSG1-induced reduction of the BubR1 protein. Accompanied with a reduction of BubR1 protein level, BCSG1 expression resulted in multinucleation of breast cancer cells upon treatment with spindle inhibitor nocodazole, indicating an impaired mitotic checkpoint. Taken together, our novel findings suggest that BCSG1 may accelerate the progression of breast cancer at least in part by compromising the mitotic checkpoint control through inactivation of BubR1.