Background Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. Methods Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. Results Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1β in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. Conclusions MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.

AimsA common concern among pathologists scoring PD‐L1 immunohistochemical staining is interobserver and intraobserver variability. We assessed the interobserver and intraobserver reproducibility of PD‐L1 scoring among trained pathologists using a combined positive score (CPS; tumour cell and tumour‐associated immune cell staining).Methods and resultsData were collected for 2 years (2017–2019) from 456 pathologists worldwide. Digital training encompassed unique, tumour‐specific training, and test sets. Samples were stained using PD‐L1 IHC 22C3 pharmDx and evaluated at specific CPS cutoffs for gastric cancer (GC), cervical cancer (CC), urothelial cancer (UC), oesophageal cancer (OC), and head and neck squamous cell carcinoma (HNSCC). Pathologists underwent expert‐to‐peer training and scored 20 blinded samples on day 1 and 25 blinded samples on day 2 (including 15 of the day 1 samples). Interobserver and intraobserver reproducibility were assessed. For GC (120 observers) and CC (32 observers) samples assessed at CPS ≥1, average interobserver agreement was 91.5% and 91.0%, respectively, and average intraobserver agreement was 90.2% and 96.6%, respectively. For UC (139 observers) and OC (52 observers) samples measured at CPS ≥10, average interobserver agreement was 93.4% and 93.7%, respectively, and average intraobserver agreement was 92.0% and 92.5%, respectively. For HNSCC samples (113 observers), average interobserver agreement was 94.1% at CPS ≥1 and 86.5% at CPS ≥20; intraobserver agreement was 94.7% at CPS ≥1 and 90.5% at CPS ≥20.ConclusionThe consistently high interobserver and intraobserver concordance rates support the effectiveness of face‐to‐face training of many global pathologists for scoring PD‐L1 CPS across multiple indications at several specific cutoffs.

5024 Background: We assessed the association of baseline RNA-sequencing–based gene expression signatures and DNA alterations with response or resistance to pembrolizumab in patients with advanced renal cell carcinoma in cohorts A (clear cell; n = 110) and B (non-clear cell; n = 165) of the phase 2 KEYNOTE-427 study (NCT02853344). Methods: Using RNA-sequencing, we analyzed the association of gene expression signatures (18-gene T-cell–inflamed gene expression profile [GEP]; 10 non–T-cell–inflamed GEP canonical signatures [angiogenesis, gMDSC, glycolysis, hypoxia, mMDSC, MYC, proliferation, RAS, stromal/EMT/TGFβ, WNT]) quantifying tumor microenvironment elements (TME) with objective response rate (ORR) and progression-free survival (PFS). Canonical signatures were derived from 2 databases (TCGA, Moffit) using an algorithm that included genes based on their correlation to reference signatures in the literature. Signature definitions were finalized before linking to the clinical data, and significance was prespecified at 0.10 given the potential for limited power. Canonical signatures were analyzed through regression testing of response for association with consensus signatures after adjusting for T-cell–inflamed GEP and International Metastatic RCC Database Consortium scores in the model. P values were adjusted for multiplicity. Using whole exome sequencing, we also summarized the association of renal cell carcinoma driver gene mutations with ORR. Clinical data cutoff: Jan 30, 2019. Results: Patient characteristics for this analysis were comparable to the overall population. In cohort A, T-cell–inflamed GEP (n = 78) was statistically significantly associated with a better ORR ( P = 0.021; AUROC = 0.65) but not PFS ( P = 0.116). No other TME canonical signatures showed a correlation with ORR or PFS. ORR was estimated for mutations (Table). Conclusions: RNA-sequencing–based, T-cell–inflamed GEP was associated with ORR in patients with clear cell renal cell carcinoma receiving first-line pembrolizumab. Precision was limited by sample size for estimating ORR by specific gene mutation status. Evaluation of tissue-based biomarkers in larger studies are planned. Biomarker analyses from patients in cohort B will also be presented. Clinical trial information: NCT02853344 . [Table: see text]

The medical literature contains numerous examples of randomized clinical trials with time‐to‐event endpoints in which large numbers of events accrued over relatively short follow‐up periods, resulting in many tied event times. A generally common feature across such examples was that the logrank test was used for hypothesis testing and the Cox proportional hazards model was used for hazard ratio estimation. We caution that this common practice is particularly risky in the setting of many tied event times for two reasons. First, the estimator of the hazard ratio can be severely biased if the Breslow tie‐handling approximation for the Cox model (the default in SAS and Stata software) is used. Second, the 95% confidence interval for the hazard ratio can include one even when the corresponding logrank test p‐value is less than 0.05. To help establish a better practice, with applicability for both superiority and noninferiority trials, we use theory and simulations to contrast Wald and score tests based on well‐known tie‐handling approximations for the Cox model. Our recommendation is to report the Wald test p‐value and corresponding confidence interval based on the Efron approximation. The recommended test is essentially as powerful as the logrank test, the accompanying point and interval estimates of the hazard ratio have excellent statistical properties even in settings with many tied event times, inferential alignment between the p‐value and confidence interval is guaranteed, and implementation is straightforward using commonly used software.

3017Background: The relevance of RECIST v1.1 measures of progression in pts treated with immunotherapy is unclear. We conducted analyses of KEYNOTE-001 (KN; exploratory) and KN052 (confirmatory) to...

Therapies based on monoclonal antibodies (mAbs) have delivered an impressive success in the clinics due to their exquisite specificity, potential for agonistic or antagonistic responses, tunable effector function, and optimal pharmacokinetic properties. Building on these inherent antibody properties, the design and development of antibody-drug conjugates (ADCs) with improved or gained therapeutic activity and safety has been successfully demonstrated in oncological applications. There is enormous potential for this new type of hybrid biologics but there are also significant engineering, manufacturing and bioanalytical challenges. In this manuscript, we highlight the range and diversity of assays that are critical to characterize the individual components of ADCs-linker, carrier, and payload. We discuss a series of in vitro and in vivo preclinical experimental approaches we implemented to characterize two anti-inflammatory steroid bearing ADCs, and an ADC bearing a modified glucagon-like peptide 1 receptor/glucagon receptor co-agonist peptide.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.