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Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi 1 – 5 . Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice 6 , the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions 7 – 9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported 10 , 11 , suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment 10 – 13 . Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-075 14 . Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy. ABBV-744, a selective inhibitor of the BD2 domains of BET family proteins, is effective against prostate cancer in mouse xenograft models, with lower toxicities than the dual-bromodomain BET inhibitor ABBV-075.

Melanotransferrin (MTf) is an iron-binding member of the transferrin superfamily that can be membrane-anchored or secreted in serum. On cells, it can mediate transferrin-independent iron uptake and promote proliferation. In serum, it is a transcytotic iron transporter across the blood–brain barrier. MTf has been exploited as a drug delivery carrier to the brain and as an antibody-drug conjugate (ADC) target due to its oncogenic role in melanoma and its elevated expression in triple-negative breast cancer (TNBC). For treatment of TNBC, an MTf-targeting ADC completed a phase I clinical trial (NCT03316794). The structure of its murine, unconjugated Fab fragment (SC57.32) is revealed here in complex with MTf. The MTf N-lobe is in an active and iron-bound, closed conformation while the C-lobe is in an open conformation incompatible with iron binding. This combination of active and inactive domains displays a novel inter-domain arrangement in which the C2 subdomain angles away from the N-lobe. The C2 subdomain also contains the SC57.32 glyco-epitope, which comprises ten protein residues and two N -acetylglucosamines. Our report reveals novel features of MTf and provides a point of reference for MTf-targeting, structure-guided drug design.

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4 CRBN , and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes. PTPN2 (protein tyrosine phosphatase non-receptor type 2) and PTPN1 are attractive immuno-oncology targets, however, targeting PTPN2/N1 poses significant challenges. Here, the authors report the development of potent PTPN2/N1 heterobifunctional degraders and reveal biochemical and structural insights into the formation of ternary structures with cereblon E3 ligase by X-ray diffraction, cryo-EM and MD simulations.

Glucocorticoid receptor modulators (GRMs) are an established and successful compound class for the treatment of multiple diseases. In addition, they are an area of high interest as payloads for antibody-drug conjugate s(ADCs) in both immunology and oncology. Solving the crystal structure of compound 2, the GRM payload from ABBV-3373 and ABBV-154, in the ligand binding domain of the glucocorticoid receptor (GR) revealed key information to facilitate optimal ADC payload design. All four critical H- bonds between the oxygen functional groups on the hexadecahydro-1H-cyclopenta[α]phenanthrene ring system of the small molecule and protein were shown to be made (carbonyl at C3 to Gln570 and Arg611 and Asn564, carbonyl atC20 to Thr739, hydroxyl at C21 to Asn 564 and Thr739). In addition, an extra H-bond between the linker attachment site on compound 2, the aniline in the biaryl region, was observed. Confirmation of the stereochemistry of the acetal in compound 2 as (R) was established. Finally, the importance of minimising the exposed hydrophobic surface area of a payload to reduce the negative impact on the properties of resulting ADCs, like aggregation, was rationalised by comparison of (R)-acetal compound 2 and (S)- acetal compound 3.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR + PD-1 + T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

BackgroundImmune checkpoint blockade is effective for a subset of patients across many cancers, but most patients are refractory to current immunotherapies and new approaches are needed to overcome resistance.1 2 The protein tyrosine phosphatase PTPN2 and the closely related PTPN1 are central regulators of inflammation, and their genetic deletion in either tumor cells or host immune cells promotes anti-tumor immunity.3–6 However, phosphatases are challenging drug targets and in particular, the active site has been considered undruggable. Here, we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2/N1 active site inhibitor.MethodsIn this study, we characterize AC484 and evaluate its effects in vitro and in vivo. We conduct in vitro experiments to investigate the interferon response and the activation and function of various immune cell subsets in response to AC484. We employ murine cancer models resistant to PD-1 blockade and assess the anti-tumor efficacy of AC484 monotherapy in these models. Additionally, through single-cell transcriptional profiling of tumor-infiltrating immune cells, we examine the transcriptional and functional effects of AC484 treatment, with a focus on CD8+ T cells.ResultsAC484 treatment demonstrates the ability to amplify the response to interferon and enhance the activation and function of multiple immune cell subsets in vitro. In murine cancer models resistant to PD-1 blockade, monotherapy AC484 treatment generates robust anti-tumor immunity. Transcriptomic and functional analyses of tumor-infiltrating immune cells reveal that AC484 treatment elicits broad effects on myeloid and lymphoid compartments, particularly influencing CD8+ T cells. Surprisingly, we find that AC484 treatment induces a unique transcriptional state in CD8+ T cells mediated by enhanced JAK-STAT signaling, whereby T cells display a highly cytotoxic effector profile, increased memory signatures, and reduced exhaustion and dysfunction.ConclusionsOur results demonstrate that oral administration of small molecule inhibitors of PTPN2/N1 can induce potent anti-tumor immunity. PTPN2/N1 inhibitors offer a promising new strategy for cancer immunotherapy and are currently being evaluated clinically in patients with advanced solid tumors (NCT04777994). More broadly, our study shows that small molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics targeting this important class of enzymes.ReferencesHugo W, et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell. 2017;168:542.Fares CM, Van Allen EM, Drake CG, Allison JP, Hu-Lieskovan S. Mechanisms of resistance to immune checkpoint blockade: why does checkpoint inhibitor immunotherapy not work for all patients? Am Soc Clin Oncol Educ Book. 2019;39:147–164.Manguso RT, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature. 2017;547:413–418.Wiede F, et al. PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours. EMBO J. 2020;39:e103637.LaFleur MW, et al. PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nat. Immunol. 2019;20:1335–1347.Flosbach M, et al. PTPN2 deficiency enhances programmed T cell expansion and survival capacity of activated T cells. Cell Rep. 2020;32:107957.Ethics ApprovalThe protocol, under which human blood samples were acquired, was approved by and is reviewed on an annual basis by WCG IRB (Puyallup, Washington). WCG IRB is in full compliance with the Good Clinical Practices as defined under the U.S. Food and Drug Administration (FDA) Regulations, U.S. Department of Health and Human Services (HHS) regulations and the International Conference on Harmonisation (ICH) Guidelines. All human research participants signed informed consent forms. All animal studies at AbbVie, were reviewed and approved by AbbVie’s Institutional Animal Care and Use Committee and in compliance with the NIH Guide for Care and Use of Laboratory Animals guidelines. Animal studies were conducted in an AAALAC accredited program where veterinary care and oversight was provided to ensure appropriate animal care. All in vivo studies conducted at the Broad Institute were approved by the Broad Institute IACUC committee and mice were housed in a specific-pathogen free facility. All in vivo studies at Calico were conducted according to protocols approved by the Calico Institutional Animal Care and Use Committee.

Casein kinase I (CKI) enzymes phosphorylate multiple proteins with diverse functions in eukaryotic organisms and may play an important regulatory role in a variety of biological processes. These protein kinases utilize ATP in a reaction forming a phosphate mono-ester that typically can be hydrolyzed by a protein phosphatase. Common features of certain substrates show that CKI activity has an unusual specificity for proteins that are already phosphorylated on an amino acid N-terminal to the site phosphorylated by CKI. Recently, cDNAs encoding several CKI enzymes have been cloned. Sequence analysis reveals that these enzymes constitute a unique family of protein kinases. While their catalytic domains have many similarities, regions outside of this domain vary in length and sequence. To understand the molecular basis for protein phosphorylation by CKI enzymes, experiments were designed to study the three-dimensional structure of a recombinant mammalian isoform of CKI expressed in Escherichia coli. Crystals of a truncation mutant of CKI$\\delta$ lacking a C-terminal autoinhibitory domain diffracted X-rays to 2.3 A and the structure of the catalytic domain was determined by X-ray crystallography. Like other protein kinases, the catalytic domain is composed of two lobes with a cleft between them for binding ATP. Comparison with the recent crystal structure of a CKI homolog from Schizosaccharomyces pombe suggests that a rotation of the N-terminal lobe occurs upon ATP binding. Furthermore, binding of a phosphate analog, tungstate, in a derivative structure reveals an anion binding site that likely contributes to the unique substrate specificity of CKI enzymes. Additional experiments resulted in crystallization of another truncation mutant of CKI$\\delta$, a mutant containing the autoinhibitory domain, and X-ray diffraction data were collected to 2.4 A. Structural analysis suggests that the inhibitory domain is disordered in these crystals, but a conserved intermolecular contact suggests formation of a dimer that would inhibit enzymatic activity. Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI$\\delta$ reveals a catalytic face that is especially conserved among a subset of CKI family members associated with the regulation of DNA repair.