Figure S1. Mutation signatures and altered canonical pathways in GBC.

Table S2. Oncogenic/Likely-oncogenic structural variants in GBC

Table S1. Sample list (cBioPortal identifiers, ‘DMP_SAMPLE_ID’). Only the primary tumor (if available) or the earliest collected metastasis are included (n=233)

Metastatic castration-resistant prostate cancer (mCRPC) continues to pose a significant clinical challenge and is associated with poor survival rates in patients who failed previous lines of androgen receptor axis-targeted therapies and taxanes. Antibody drug conjugates (ADCs) provide a novel approach to traditional chemotherapy by targeting tumor-specific antigens to deliver cytotoxic payloads while sparing normal tissue and enhancing the therapeutic index. Herein, we describe the preclinical characterization of AZD0516, a first-in-class ADC directed against six-transmembrane epithelial antigen of the prostate-2 (STEAP2), a novel tumor associated antigen that is highly and homogenously expressed across all stages of prostate cancer. The anti-STEAP2 monoclonal antibody (mAb) is conjugated via interchain cysteines to a maleimide-reactive, β-glucuronidase-cleavable linker bearing the topoisomerase 1 inhibitor (TOP1i) payload, exatecan. The STEAP2 antibody binds specifically to the extracellular domains of human, cynomolgus monkey, mouse, and rat STEAP2, but exhibits no detectable binding to other STEAP family members. The fragment crystallizable (Fc) domain of the antibody binder portion carries three amino acid point mutations designed to reduce Fc-mediated immune effector functions. In vitro characterization of the STEAP2 mAb in prostate cancer cells demonstrated specific binding, rapid internalization, and efficient lysosomal trafficking. In vitro cytotoxicity assays with AZD0516 revealed IC50 values in the low nM range and the ADC induced both single- and double-strand DNA breaks, characteristic of the proposed primary mechanism of action (MoA) of the TOP1i payload, namely, DNA damage and apoptotic cell death. Exatecan-driven bystander cell killing from intracellular release of payload was demonstrated in co-culture systems involving STEAP2-positive and negative cell lines. Murine plasma pharmacokinetic data suggest minimal impact of linker-payload conjugation on antibody clearance, while maintaining high plasma stability. The mechanistic and pharmacodynamic effects of AZD0516 were observed in a prostate cancer cell line-derived xenograft (CDX) model via a dose-dependent increase in positive staining for γH2AX foci, indicative of DNA damage. AZD0516 monotherapy administration in prostate cancer CDX and patient-derived xenograft models led to sustained tumor responses. AZD0516 was well-tolerated in both rats and cynomolgus monkeys with no unexpected toxicities observed. Safety findings included hematological and gastrointestinal effects, consistent with the MoA and known effects of TOP1i payloads. Together, these data support exploring AZD0516 clinically for patients with mCRPC. Darlene Monlish, Vanessa Muniz-Medina, Mel Ehudin, Dewald van Dyk, Claire Myers, Rachel Lawrence, Liang Zhang, Linda Irons, Chara Stavraka, Wardha Qureshi, Ruoyan Chen, Asurayya Worrede, Nicolas Giraldo, Miljenka Vuko, Melody Handali, Ali Saleh, Balakumar Vijayakrishnan, Maximillian Lee, Jeong Min Han, Crystal Cheung, Ariel Endlich-Frazier, Amber Lee, Mark Hutchinson, Andrew Dippel, Gilad Kaplan, Keith Rickert, Shraddha Kale, Ryan Fleming, Clare Hoover, Benedicte Recolin, Jan Zaucha, Sreedevi Kesavan, John` Meekin, Aida Mariani, Christian Eisen, John Bullen, Eric Gangl, Jay Harper, Andreas Maderna, Edward Rosfjord, Frank Comer, Elaine Hurt, Neil Gibson, Puja Sapra. Preclinical characterization of AZD0516, a novel STEAP2 antibody-drug conjugate (ADC) for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1158.

Figure S2. Microbiome analysis in GBC.

AZD2284 is a novel, targeted alpha therapeutic directed against six-transmembrane epithelial antigen of prostate-2 (STEAP2), a receptor highly overexpressed in prostate cancer. AZD2284 is comprised of a humanized, STEAP2-targeting rodent cross-reactive IgG1 monoclonal antibody conjugated with a DOTA chelate and radiolabeled with the alpha-particle emitting radionuclide, actinium-225 [225Ac]. Leveraging high STEAP2 expression in prostate cancer and the high linear energy transfer of the alpha emitter, AZD2284 is presumed to deliver the radioisotope to targeted tumor cells, generating clustered DNA double strand breaks and other cellular damage that ultimately leads to cell death. Preclinical pharmacological assessments were conducted with the lutetium-177 [177Lu] labeled analog of AZD2284, FPI-2281. In vitro binding and internalization assays with FPI-2281 were performed in STEAP2-positive prostate cancer cell lines with varying levels of target expression. In vivo biodistribution and uptake were evaluated ex vivo across multiple timepoints following FPI-2281 administration to tumor-bearing cell-line derived xenograft (CDX) models. To assess the radiotherapeutic efficacy of AZD2284, a single dose was administered intravenously to prostate cancer CDX and patient-derived xenograft (PDX) models. Binding and internalization studies demonstrated FPI-2281 effectively binds to the STEAP2-positive prostate cancer cell line models (LNCaP, C4-2, and 22Rv1) with over 30% cellular retention at 24 hours. Corresponding in vivo biodistribution profiles showed sustained tumor uptake in xenograft models, with peak tumor uptakes of 60% ID/g in the 22Rv1 model and 83% ID/g in the C4-2 model at 14 days, respectively, with limited normal organ uptake observed at all timepoints. AZD2284 elicited target- and dose-dependent anti-tumor efficacy and sustained tumor regressions in CDX and PDX models with varying levels of STEAP2 expression. In preclinical models, AZD2284 effectively binds to and internalizes in prostate cancer cells, thereby delivering the radioisotope, [225Ac]. Biodistribution studies confirm sustained tumor uptake of AZD2284 with low normal organ uptake. Single-dose administration of AZD2284 leads to durable anti-tumor efficacy in xenograft models. These data suggest AZD2284 is a promising therapeutic candidate in prostate cancer and support the initiation of clinical trials. Darlene Monlish, Brigitte Thériault, Mel Ehudin, Vanessa Muniz-Medina, Dewald van Dyk, Lisa Godfrey, Dipal Patel, William Turnbull, Liang Zhang, Moditha Nawinne, Teresa Collins, Kenneth Thress, Douglas Ferguson, Rajiv Bhalla, Nicolas Giraldo, Melody Handali, Jeong Min Han, Crystal Cheung, Asurayya Worrede, Clare Hoover, Sujiet Puthenveetil, Edward Rosfjord, Frank Comer, Elaine Hurt, Darshan Dalal, Christopher Leamon, John Valliant, Puja Sapra. AZD2284: A novel, alpha-particle emitting radioconjugate targeting STEAP2 in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4303.

Evaluation of CLDN18.2-targeting CAR-T cells in vivo. A, NSG mice bearing PaTu8988s HS xenografts (CLDN18.2 H-score = 268) were dosed by tail vein with 9e6 CAR+ CLDN18.2 Bz CAR-T cells; total T-cell infusion number was matched across groups. Tumor volume and body weight were measured biweekly (n = 9). Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). B, Schematic representation of a second-generation CAR-T design modified to replace 4-1BB with a CD28 costimulatory domain (28z). The average transduction efficiency (CAR+, day 9) of multiple healthy donors for clone 9 28z is shown. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. C, NSG mice bearing PaTu8988s HS xenografts were dosed as described in A with clone 9 CD28z or Bz CAR-T (n = 6) at indicated doses. Serum levels of IFNγ were measured at 4, 7, and 14 days after infusion (n = 3). D, Representative images of CLDN18.2 (top row) and CD3 (bottom row) staining in the stomachs of mice dosed with clone 9 CAR-T cells from C at indicated time points. All data represent mean ± SEM of replicate experiments or animals.

Development and evaluation of CLDN18.2-targeting CAR-T cells in vitro. A, Schematic representation of second-generation CAR-T lentivirus design, which includes a 4-1BB costimulatory domain (Bz). The table shows for each CLDN18.2-reactive clone the relative binding affinity (human and mouse), reactivity to mutant CLDN18.2 (M149L), and average transduction efficiency (CAR+, day 9) of multiple healthy donors. Representative FC plots of CAR surface expression at day 9 after lentivirus transduction were compared with UT control for a single donor. B, CLDN18.2 cell surface expression of various cell lines as determined by FC with 5 μg/mL CLDN18.2-reactive clones compared with nonspecific isotype antibody (R347). C, Epitope characterization of CLDN18.2-reactive clones. The AlphaFold structure on the far left (red) represents all sites of point mutation in HEK293 cells that vary between CLDN18.1 and CLDN18.2 in the first extracellular loop; the color-coded diagrams represent sites that influence respective clone binding. D, Percent cytolysis of HEK293 + huCLDN18.1, HEK293 + huCLDN18.2, HEK293 + muCLDN18.1, HEK293 + muCLDN18.2, and PaTu8988s HS cells determined by xCELLigence RTCA assay after 48 hours of co-culture with CLDN18.2 CAR-T cells at a 1:1 E:T ratio. The supernatants from the xCELLigence assay were collected at 24 hours for cytokine assessment (Meso Scale Discovery) assay. All data represent mean ± SEM of replicate experiments.

Optimized manufacturing protocol, STAR-T, for the generation of the CAR-T product. A, Baseline characteristics of donor-matched dnTGFβRII CAR-T cells with traditional manufacture (day 10) vs. AZD6422 (day 4), including CAR+ expression, percent CD4 and CD8 expression, and T-cell phenotypic status as determined by cell surface expression of CCR7 and CD45RO. Results are shown for naïve (CCR7+/CD45RO−, Tn), central memory (CCR7+/CD45RO+, Tcm), effector memory (CCR7−/CD45RO+, Tem), and effector (CCR7−/CD45RO−, Teff) cells. Data are shown as mean ± SEM of representative donors. B, Comparison of bioenergetic profiles of traditionally manufactured dnTGFβRII CAR-T cells vs. AZD6422. Spare respiratory capacity was determined as the differential between basal and maximum respiration. 2-DG, 2-deoxy-D-glucose; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone; OCR, oxygen consumption rate; Oligo, oligomycin; Rot/AA, rotenone and antimycin A. C, Serial restimulation assay to examine cytotoxicity and persistence of dnTGFβRII CAR-T cells and AZD6422. CAR-T cells were co-cultured at a ratio of 1:2 with BXPC3 + CLDN18.2, tumor lysis was measured every 3 to 4 days, and IFNγ was profiled at 24 hours after each new co-culture. Representative of multiple donors. D, Results of quantitative FC to determine cell surface expression of CLDN18.2 across multiple cancer cell lines. Percent cytolysis was determined by xCELLigence RTCA assay after 48 hours of co-culture with AZD6422 at an E:T ratio of 1:1. Data represent mean ± SEM of replicate experiments.

4089 Background: Despite recent advances in treatment, gastric cancer (GC) remains the fourth leading cause of cancer-related mortality worldwide. Claudin 18.2 (CLDN18.2) has emerged as a promising therapeutic target as its expression is mostly restricted to the gastric epithelium and persists in a significant fraction of GC and other upper gastrointestinal tumors (e.g. pancreatic ductal adenocarcinoma). The goal of this study was to compare the analytical performance of multiple CLDN18/CLDN18.2 IHC assays and understand the prevalence of CLDN18.2 with other GC-relevant biomarkers (PD-L1, HER2) and the tumor immune microenvironment. Methods: Cell lines (n=11) and primary resection specimens from patients with GC (n=91) were studied. To compare the analytical performance of three CLDN18/CLDN18.2 IHC assays, FFPE samples were stained with pan-CLDN18 (43-14A) and CLDN18.2-specific (SP455 and EPR19202) clones. Staining results were validated using orthogonal methods i.e., flow cytometry and mass spectrometry. The relationship of CLDN18.2 expression with PD-L1 (SP263) and HER2 (HercepTest) as well as T cell contexture (CD3/CD8/PanCK; CD3/PD-L1/Ki67) were explored by IHC and chromogenic multiplex IHC. Association of these markers with clinicopathological features was also investigated. Results: Comparison of the three CLDN18/CLDN18.2 IHC assays showed similar analytical performance across cell lines and GC samples. Using orthogonal methods, we determined that the limit of detection and linear range was comparable amongst assays. In GC, samples classified as CLDN18/CLDN18.2 positive at a cutoff of 75% with 2-3+ intensity included 22% (SP455), 22% (43-14A), and 15% (EPR19202) of samples. Classification at other CLDN18/CLDN18.2 cutoffs were also explored and will be presented. Only one case (1%) showed significantly discrepant staining patterns between pan-CLDN18 and CLDN18.2-specific assays (H score 235 vs. 105, respectively). CLDN18.2 positivity did not correlate with HER2 expression, PD-L1 expression, T cell densities (cells/mm2), or any of the tested clinicopathological characteristics i.e., tumor grade, histologic subtype, or TNM stage. Conclusions: We demonstrated a concordant analytical performance between the tested CLDN18/CLDN18.2 IHC assays in GC. Their implementation in the clinical setting might help identify patient candidates that could benefit from treatment with CLDN18.2 targeted therapies. Additional research is needed to confirm similar analytical performance between the three IHC assays in other indications.

AbstractPurpose: Claudin 18.2 (CLDN18.2) is a surface membrane protein that is crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers. Thus, CLDN18.2 is suited for exploration as a clinical target for chimeric antigen receptor T-cell (CAR-T) therapy in these indications. Although CAR-T therapies show promise, a challenge faced in their development for solid tumors is the immunosuppressive tumor microenvironment, which is often characterized by the presence of immune and stromal cells secreting high levels of TGFβ. The addition of TGFβ armoring can potentially expand CAR-T activity in solid tumors. We report on the preclinical development of a CLDN18.2-targeting CAR-T therapy showing effectiveness in patient models with CLDN18.2-positive gastric, esophageal, and pancreatic tumors. Experimental Design: The lead lentivirus product contains a unique single-chain variable fragment; CD28 and CD3z costimulatory and signaling domains; and dominant-negative TGF-β receptor armoring, enhancing targeting and safety and counteracting suppression. We developed a shortened cell manufacturing process to enhance the potency of the final product AZD6422. Results: AZD6422 exhibited significant antitumor activity and tolerability in multiple patient-derived tumor xenograft models with various CLDN18.2 and TGF-β levels, as determined by IHC. The efficacy of armored CAR-T cells in tumor models with elevated TGFβ was increased in vitro and in vivo. In vitro restimulation assays established greater persistence and cytolytic function of AZD6422 compared with a traditionally manufactured CAR-T. Conclusions: AZD6422 was safe and efficacious in patient-derived, CLDN18.2-positive murine models of gastrointestinal cancers. Our data support further clinical development of AZD6422 for patients with these cancers.

In vivo antitumor activity of AZD6422 in PDX models of gastric cancer, PDAC, and esophageal adenocarcinoma. Activity and tolerability of AZD6422 are shown in various PDX models of esophageal adenocarcinoma (A, ES11085; D, ES_9500), gastric cancer (E, GA_9275), and PDAC (B, PANC_22; C, PANC_12; F, PANC_24). Each model was selected to represent a range of CLDN18.2 (shown at 10× scan) and TGFβ expression. NSG MHC-DKO mice received a single tail-vein infusion of 1e6 AZD6422, donor-matched UT, or vehicle when the average tumor volume reached 150 mm3. Tumor volumes and body weights were measured biweekly until study completion on day 35, and blood was collected for cytokine analysis on days 7 and 14. Data are shown as mean ± SEM (n = 5).

Table S3. Survival analysis according to various cut-offs. Log-rank test p-values for CD8+, PD-1+ and LAG-3+ cell densities at the optimal, median and third quartile cut-offs

Table S4. Univariate Cox regression analysis for OS and DFS in primary and metastatic ccRCC. P-values and HR of significant variables are highlighted in bold font.

Supplemental Figure 1-3. Figure S1: Expression of immune modulators among the 4 molecular subgroups and normal Samples; Figure S2: Expression of cell-type specific metagenes among the different subgroups and normal samples; Figure S3: CD8+ cell infiltration according to the molecular ccRCC subtype classification.

Table S2. Antibodies used for the IHC and IF studies. Antibodies and conditions used for the IHC and IF studies

Figure S1. Test of the specificity of anti-immune checkpoints : Immunohistochemical staining of immune checkpoints on sections from paraffin embedded cell pellets of untransfected and transfected cell lines used as negative and positive controls respectively and from paraffin embedded tonsils and placenta.

Levels of expression of genes related to inflammation, angiogenesis and immunomodulation in CRC cohorts