CC-96673 (BMS-986358), an affinity-tuned anti-CD47 and CD20 bispecific antibody with fully functional fc, selectively targets and depletes non-Hodgkin's lymphoma.

Cluster of differentiation 47 (CD47) is a transmembrane protein highly expressed in tumor cells that interacts with signal regulatory protein alpha (SIRPα) and triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis and enabling tumor escape from immunosurveillance. The CD47-SIRPα axis has become an important target for cancer immunotherapy. To date, the advancement of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hematologic toxicity including anemia. To overcome those challenges a bispecific approach was taken. CC-96673, a humanized IgG1 bispecific antibody co-targeting CD47 and CD20, is designed to bind CD20 with high affinity and CD47 with optimally lowered affinity. As a result of the detuned CD47 affinity, CC-96673 selectively binds to CD20-expressing cells, blocking the interaction of CD47 with SIRPα. This increased selectivity of CC-96673 over monospecific anti-CD47 approaches allows for the use of wild-type IgG1 Fc, which engages activating crystallizable fragment gamma receptors (FcγRs) to fully potentiate macrophages to engulf and destroy CD20+ cells, while sparing CD47+CD20- normal cells. The combined targeting of anti-CD20 and anti-CD47 results in enhanced anti- tumor activity compared to anti-CD20 targeting antibodies alone. Furthermore, preclinical studies have demonstrated that CC-96673 exhibits acceptable pharmacokinetic properties with a favorable toxicity profile in non-human primates. Collectively, these findings define CC-96673 as a promising CD47 × CD20 bispecific antibody that selectively destroys CD20+ cancer cells via enhanced phagocytosis and other effector functions.

Discovery and Preclinical Activity of BMS-986351, an Antibody to SIRPα That Enhances Macrophage-mediated Tumor Phagocytosis When Combined with Opsonizing Antibodies.

In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). SIGNIFICANCE: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.

Identification of tyrosine sulfation in the variable region of a bispecific antibody and its effect on stability and biological activity.

Despite tyrosine sulfation being a relatively common post-translational modification (PTM) on the secreted proteins of higher eukaryotic organisms, there have been surprisingly few reports of this modification occurring in recombinant monoclonal antibodies (mAbs) expressed by mammalian cell lines and even less information regarding its potential impact on mAb efficacy and stability. This discrepancy is likely due to the extreme lability of this modification using many of the mass spectrometry methods typically used within the biopharmaceutical industry for PTM identification, as well as the possible misidentification as phosphorylation. Here, we identified sulfation on a single tyrosine residue located within the identical variable region sequence of a 2 + 1 bispecific mAbs heavy and heavy-heavy chains using a multi-enzymatic approach in combination with mass spectrometry analysis and examined its impact on binding, efficacy, and physical stability. Unlike previous reports, we found that tyrosine sulfation modestly decreased the mAb cell binding and T cell-mediated killing, primarily by increasing the rate of antigen disassociation as determined from surface plasmon resonance-binding experiments. We also found that, while this acidic modification had no significant impact on the mAb thermal stability, sulfation did modestly increase its rate of aggregation, presumably by lowering the mAb's colloidal stability as indicated by polyethylene glycol induced liquid-liquid phase separation experiments.

Structure of the human marker of self 5-transmembrane receptor CD47.

CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1-S Checkpoint.

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1-S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1-S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1-S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727-38. ©2018 AACR.

Caspase-8 association with the focal adhesion complex promotes tumor cell migration and metastasis.

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

FERM control of FAK function: implications for cancer therapy.

Integrins are transmembrane receptors that bind to extracellular matrix proteins and convey anchorage-dependent signals regulating normal cell proliferation. Integrin signals within the tumor micro-environment also impact cancer cell survival and invasion during tumor progression. These integrin-associated signaling events are transduced in part through the activation of non-receptor protein-tyrosine kinases. Focal adhesion kinase (FAK) is activated by beta-subunit integrins in both normal and transformed cells. As genetic inactivation of beta1 integrin or FAK yield early embryonic lethal phenotypes associated with decreased cell proliferation, and dominant-negative inhibition of FAK can cause increased cell apoptosis, there is a concern that FAK inhibition may have cytotoxic effects on cell growth or survival. However, FAK-specific small molecule inhibitors do not directly impact cell growth in culture, but yet show potent anti-tumor growth effects in vivo. Additionally, recent studies have shed new insight into the FAK kinase-independent regulation of cell proliferation and survival mediated by the FAK N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain. Herein, we review the role of the FAK FERM domain in both the intrinsic regulation of FAK kinase activity and how FERM-mediated nuclear localization of FAK promotes enhanced cell survival through the inhibition of tumor suppressor p53 activation during development and under conditions of cellular stress. As we find that FAK FERM-mediated regulation of p53 occurs in human carcinoma cells, elevated FAK expression in tumors may promote both kinase-dependent and -independent survival mechanisms. We discuss how the pharmacological inhibition of FAK kinase activity may impact tumor progression through combined effects of blocking both tumor- and stromal-associated signaling regulating neo-vascularization.

Hepatitis C virus stabilizes hypoxia-inducible factor 1alpha and stimulates the synthesis of vascular endothelial growth factor.

Hepatitis C virus (HCV) infection is one of the major causes of chronic hepatitis, liver cirrhosis, which subsequently leads to hepatocellular carcinoma (HCC). The overexpression of the angiogenic factors has been demonstrated in HCC. In this study, we investigated the potential of HCV gene expression in inducing angiogenesis. Our results show that HCV infection leads to the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha). We further show that this stabilization was mediated via oxidative stress induced by HCV gene expression. The activation of NF-kappaB, STAT-3, PI3-K/AkT, and p42/44 mitogen-activated protein kinase was necessary for HIF-1alpha stabilization. HIF-1alpha induction in turn led to the stimulation of vascular endothelial growth factor. By using the chick chorioallantoic membrane assay, we show that HCV-infected cells released angiogenic cytokines, leading to neovascularization in vivo. These results indicate the potential of HCV gene expression in angiogenesis.

Potentiation of neuroblastoma metastasis by loss of caspase-8.

Neuroblastoma, the most common paediatric solid tumour, arises from defective neural crest cells. Genetic alterations occur frequently in the most aggressive neuroblastomas. In particular, deletion or suppression of the proapoptotic enzyme caspase-8 is common in malignant, disseminated disease, although the effect of this loss on disease progression is unclear. Here we show that suppression of caspase-8 expression occurs during the establishment of neuroblastoma metastases in vivo, and that reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed their metastases. Caspase-8 status was not a predictor of primary tumour growth; rather, caspase-8 selectively potentiated apoptosis in neuroblastoma cells invading the collagenous stroma at the tumour margin. Apoptosis was initiated by unligated integrins by means of a process known as integrin-mediated death. Loss of caspase-8 or integrin rendered these cells refractory to integrin-mediated death, allowed cellular survival in the stromal microenvironment, and promoted metastases. These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.

Activation of lipid metabolism contributes to interleukin-8 production during Chlamydia trachomatis infection of cervical epithelial cells.

Chlamydia trachomatis infection is the most common cause of bacterial sexually transmitted diseases. Infection of the urogenital tract by C. trachomatis causes chronic inflammation and related clinical complications. Unlike other invasive bacteria that induce a rapid cytokine/chemokine production, chlamydial infection induces delayed inflammatory response and proinflammatory chemokine production that is dependent on bacterial growth. We present data here to show that the lipid metabolism required for chlamydial growth contributes to Chlamydia-induced proinflammatory chemokine production. By gene microarray profiling, validated with biochemical studies, we found that C. trachomatis LGV2 selectively upregulated PTGS2 (COX2) and PTGER4 (EP4) in cervical epithelial HeLa 229 cells. COX2 is an enzyme that catalyzes the rate-limiting step of arachidonic acid conversion to prostaglandins, including prostaglandin E2 (PGE2) and other eicosanoids, whereas EP4 is a subtype of cell surface receptors for PGE2. We show that Chlamydia infection induced COX2 protein expression in both epithelial cells and peripheral blood mononuclear cells and promoted PGE2 release. Exogenous PGE2 was able to induce interleukin-8 release in HeLa 229 epithelial cells. Finally, we demonstrated that interleukin-8 induction by Chlamydia infection or PGE2 treatment was dependent on extracellular signal-regulated kinase/mitogen-activated protein activity. Together, these data demonstrate that the host lipid remodeling process required for chlamydial growth contributes to proinflammatory chemokine production. This study also highlights the importance of maintaining a balanced habitat for parasitic pathogens as obligate intracellular organisms.

Angiogenesis assays in the chick CAM.

The growth of new blood vessels from pre-existing vascular elements, or angiogenesis, involves coordinated signals to the adhesion, migration, and survival machinery within the target endothelial cell. Agents that interfere with any of these processes may therefore influence angiogenesis. Here, we describe the angiogenesis assay in the chick chorioallantoic membrane (CAM). The CAM is a useful tool to studying angiogenesis because 1) it is amenable to both intravascular and topical administration of study agents, 2) it is a relatively rapid assay, and 3) it can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Importantly, the CAM provides a physiological setting that permits investigation of pro- and anti-angiogenic agent interactions in vivo.