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Introduction: Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1)., Results: In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo ., Discussion: Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors., Competing Interests: The study was sponsored by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and that the authors are Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA employees., (Copyright © 2022 Tan, Zybina, McKenna, Olow, Rukmini, Wong, Sadekova, Chackerian and Bauché.)

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity., Competing Interests: Authors SR, NT-S, AS, MS, EP and AC were employed by Merck & Co., Inc., Kenilworth, NJ, USA. CK and RW were employed by Merck & Co., Inc., Kenilworth, NJ, USA at the time of the study and are currently employed by Synthekine. WB, EM, and TR are currently employed by Merck & Co., Inc., Kenilworth, NJ, USA., (Copyright © 2021 Raghavan, Tovbis-Shifrin, Kochel, Sawant, Mello, Sathe, Blumenschein, Muise, Chackerian, Pinheiro, Rosahl, Luche and de Waal Malefyt.)

Background: Programmed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events., Methods: We have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody., Results: Sustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit., Conclusion: These data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade., Competing Interests: Competing interests: The study was sponsored by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and that the authors are Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA employees. A patent related to this work was published in September 2020 (WO2020185722A2)., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Undesired immune responses against protein therapeutics may adversely affect the pharmacokinetics, efficacy, and safety of the product. The presence of anti-drug-antibodies (ADA) has been the key determinant of immunogenic responses. Here we describe the use of a capillary electrophoresis platform for the identification of ADAs against several experimental camelid VHH biologics (Nanobodies®). Hereafter, we refer to this assay as WESADA. We modified the Wes platform by ProteinSimple to screen serum samples for ADA against covalently linked multi-modular Nanobodies and compared it to standard ADA methodologies. We were able to identify ADA positive samples and determine which individual VHH module in a multivalent Nanobody construct stimulated the predominant ADA response. WESADA requires denaturation of the experimental immobilized drug, which could affect recognition of the immunogenic epitope and alter ADA signal. To address this issue, we demonstrated that signal can be immunodepleted by pre-incubation of serum samples with native Nanobody. This capillary electrophoresis based approach allows for rapid analysis without the need for individually tailored assay optimization or reagent labeling, while consuming small amounts of sample and drug. It also allows for the simultaneous ADA analysis of multiple targets of different molecular size in the same experimental sample. WESADA is not intended to replace traditional ADA assay formats, but it facilitates the expedient immunogenic assessment of a large number of experimental drug candidates in the early developmental space., Competing Interests: Declaration of Competing Interest All authors are current or former employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. All authors declare that they have no professional and financial affiliation with ProteinSimple., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

The T-cell immune response to Mycobacterium tuberculosis is critical in preventing clinical disease. While it is generally accepted that both major histocompatibility complex class I (MHC-I)-restricted CD8(+) and MHC-II-restricted CD4(+) T cells are important for the immune response to M. tuberculosis, the role of non-MHC-restricted T cells is still not clearly delineated. We have previously reported that CD1d(-/-) mice do not differ from CD1d(+/+) mice in their survival following infection with M. tuberculosis. We now show that, although CD1d-restricted NKT cells are not required for optimum immunity to M. tuberculosis, specific activation of NKT cells by the CD1d ligand alpha-galactosylceramide protects susceptible mice from tuberculosis. Treatment with alpha-galactosylceramide reduced the bacterial burden in the lungs, diminished tissue injury, and prolonged survival of mice following inoculation with virulent M. tuberculosis. The capacity of activated NKT cells to stimulate innate immunity and modulate the adaptive immune response to promote a potent antimicrobial immune response suggests that alpha-galactosylceramide administration could have a role in new strategies for the therapy of infectious diseases.