Pyrrolobenzodiazepine dimers are an emerging class of warhead in the field of antibody-drug conjugates (ADCs). Tesirine (SG3249) was designed to combine potent antitumor activity with desirable physicochemical properties such as favorable hydrophobicity and improved conjugation characteristics. One of the reactive imines was capped with a cathepsin B-cleavable valine-alanine linker. A robust synthetic route was developed to allow the production of tesirine on clinical scale, employing a flexible, convergent strategy. Tesirine was evaluated both in stochastic and engineered ADC constructs and was confirmed as a potent and versatile payload. The conjugation of tesirine to anti-DLL3 rovalpituzumab has resulted in rovalpituzumab-tesirine (Rova-T), currently under evaluation for the treatment of small cell lung cancer.

Human CD19 antigen is a 95-kDa type I membrane glycoprotein in the immunoglobulin superfamily whose expression is limited to the various stages of B-cell development and differentiation and is maintained in the majority of B-cell malignancies, including leukemias and non-Hodgkin lymphomas of B-cell origin. Coupled with its differential and favorable expression profile, CD19 has rapid internalization kinetics and is not shed into the circulation, making it an ideal target for the development of antibody-drug conjugates (ADCs) to treat B-cell malignancies. ADCT-402 (loncastuximab tesirine) is a novel CD19-targeted ADC delivering SG3199, a highly cytotoxic DNA minor groove interstrand crosslinking pyrrolobenzodiazepine (PDB) dimer warhead. It showed potent and highly targeted in vitro cytotoxicity in CD19-expressing human cell lines. ADCT-402 was specifically bound, internalized, and trafficked to lysosomes in CD19-expressing cells and, following release of the PBD warhead, resulted in formation of DNA crosslinks that persisted for 36 hours. Bystander killing of CD19 cells by ADCT-402 was also observed. In vivo, single doses of ADCT-402 resulted in highly potent, dose-dependent antitumor activity in several subcutaneous and disseminated human tumor models with marked superiority to comparator ADCs delivering tubulin inhibitors. Dose-dependent DNA crosslinks and γ-H2AX DNA damage response were measured in tumors by 24 hours after single dose administration, whereas matched peripheral blood mononuclear cells showed no evidence of DNA damage. Pharmacokinetic analysis in rat and cynomolgus monkey showed excellent stability and tolerability of ADCT-402 in vivo. Together, these impressive data were used to support the clinical testing of this novel ADC in patients with CD19-expressing B-cell malignancies.

Sphingosine-1-phospate (S1P) is a bioactive lysophospholipid signaling molecule that serves important roles in normal development and physiological processes, including modulating the immune, cardiovascular, and central nervous systems ( 1-4 ). S1P is a key player in the sphingolipid signaling cascade and is produced from ceramide (CER) and sphingosine (SPH) through the action of sphingosine kinase (SPHK). While CER and SPH are intracellular promoters of apoptosis, S1P has opposite action and, in general, protects cells from apoptotic stimuli. Several experimental fi ndings from independent research groups implicate S1P as a key mediator of multiple survival and growth-promoting pathways ( 5 ). The extracellular functions of S1P are initiated by the binding of the bioactive lipid to a set of fi ve G protein-coupled receptors (GPCRs) belonging to the S1P receptor family ( 6 ). The balance between CER/SPH levels versus S1P provides a rheostat that determines whether a cell is sent into the death Abstract Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and infl ammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affi nity and specifi city for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can outcompete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte traffi cking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, fi rst-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be benefi cial.

A challenge in obstetrics is to distinguish pathological symptoms from those associated with normal changes of pregnancy, typified by the need to differentiate whether gestational pruritus of the skin is an early symptom of intrahepatic cholestasis of pregnancy (ICP) or due to benign pruritus gravidarum. ICP is characterized by raised serum bile acids and complicated by spontaneous preterm labor and stillbirth. A biomarker for ICP would be invaluable for early diagnosis and treatment and to enable its differentiation from other maternal diseases. Three progesterone sulfate compounds, whose concentrations have not previously been studied, were newly synthesized and assayed in the serum of three groups of ICP patients and found to be significantly higher in ICP at 9‐15 weeks of gestation and prior to symptom onset (group 1 cases/samples: ICP n = 35/80, uncomplicated pregnancy = 29/100), demonstrating that all three progesterone sulfates are prognostic for ICP. Concentrations of progesterone sulfates were associated with itch severity and, in combination with autotaxin, distinguished pregnant women with itch that would subsequently develop ICP from pruritus gravidarum (group 2: ICP n = 41, pruritus gravidarum n = 14). In a third group of first‐trimester samples all progesterone sulfates were significantly elevated in serum from low‐risk asymptomatic women who subsequently developed ICP (ICP/uncomplicated pregnancy n = 54/51). Finally, we show mechanistically that progesterone sulfates mediate itch by evoking a Tgr5‐dependent scratch response in mice. Conclusion: Our discovery that sulfated progesterone metabolites are a prognostic indicator for ICP will help predict onset of ICP and distinguish it from benign pruritus gravidarum, enabling targeted obstetric care to a high‐risk population. Delineation of a progesterone sulfate‐TGR5 pruritus axis identifies a therapeutic target for itch management in ICP. (Hepatology 2016;63:1287–1298)

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

Despite the many advances in the treatment of hematologic malignancies over the past decade, outcomes in refractory lymphomas remain poor. One potential strategy in this patient population is the specific targeting of IL2R-α (CD25), which is overexpressed on many lymphoma and leukemic cells, using antibody-drug conjugates (ADC). ADCT-301 is an ADC composed of human IgG1 HuMax-TAC against CD25, stochastically conjugated through a dipeptide cleavable linker to a pyrrolobenzodiazepine (PBD) dimer warhead with a drug-antibody ratio (DAR) of 2.3. ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and selective cytotoxicity against a panel of CD25-expressing human lymphoma cell lines. Once internalized, the released warhead binds in the DNA minor groove and exerts its potent cytotoxic action via the formation of DNA interstrand cross-links. A strong correlation between loss of viability and DNA cross-link formation is demonstrated. DNA damage persists, resulting in phosphorylation of histone H2AX, cell-cycle arrest in G-M, and apoptosis. Bystander killing of CD25-negative cells by ADCT-301 is also observed. In vivo, a single dose of ADCT-301 results in dose-dependent and targeted antitumor activity against both subcutaneous and disseminated CD25-positive lymphoma models. In xenografts of Karpas 299, which expressed both CD25 and CD30, marked superiority over brentuximab vedotin (Adcetris) is observed. Dose-dependent increases in DNA cross-linking, γ-H2AX, and PBD payload staining were observed in tumors in vivo indicating a role as relevant pharmacodynamic assays. Together, these data support the clinical testing of this novel ADC in patients with CD25-expressing tumors. Mol Cancer Ther; 15(11); 2709-21. ©2016 AACR.

Since their emergence as independent drugs, antibodies and chemotherapy are being brought together in effective synergy. The conjunction is timely: many of the technical challenges in preparing antibodies have been addressed; potent new drugs are available and linker technology is advancing apace. ADCs however are not just a sum of their individual parts. The current challenge is in understanding the holistic nature of this exciting class of drugs that promise a new avenue for cancer treatment. Target selection, the interaction of ADC with tumour and off-tumour targets and the internalisation of ADCs, are critical to the effective maturation of ADC technology. Ongoing recent developments in attachment sites and linker chemistry can provide fine-tuning of drug loading, elements of ADC PK and off-target ADC toxicity.

The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type‐1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN‐β treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL‐6, CCL5, IFN‐β) in the presence of immune stimulatory DNA and RNA. The production of IL‐6 is partially dependent on the interferon receptor as is IFN‐β itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis. GLIA 2015;63:812–825