Center for Cancer Research

Bacteriophage-based display has been utilized for a variety of purposes, such as to assemble protein libraries and conduct biopanning. We have created a modified lambda (λ) bacteriophage platform, ideal for the display and delivery of proteins. Our system utilizes counter-selection recombineering for versatile modification, temperature-sensitive induction for timely lysate production, and an arabinose-inducible mechanism for high-titer, stable yield. Here, we investigated the ability of this specialized λ phage display platform to stimulate highly specific antibodies in mice against the displayed cancer-variant cell-surface receptor EGFRvIII, demonstrating its potential in cancer immunotherapy and broader vaccine development. λ display immunogenicity was explored by generating fusion proteins between the λ head protein D and a 13-mer peptide from the N terminus of glioblastoma variant cell-surface receptor, EGFRvIII. The 13-mer peptide was fused to either the N or C terminus of the λD protein while λ remained a dormant lysogen in the bacterial host chromosome. Recombinant phage lysates were then generated with ~420 displayed fusion proteins per phage particle. Mice were injected with purified recombinant λ phage without an adjuvant via both intraperitoneal and intramuscular routes, and sera harvested at various timepoints were profiled for immunogenicity. Analysis of serum samples by ELISA and Western blotting demonstrated the ability of the λD~EGFRvIII phage display, especially in the C-terminal fusion construction, to elicit a robust anti-EGFRvIII humoral response by either injection route. Notably, the antibody response was highly specific to EGFRvIII without exhibiting cross-reactivity to wild-type EGFR. The data generated in this study demonstrate the λ system's immunotherapeutic potential as a high-titer, stable, self-adjuvanting vector for the stimulation of robust antibody titers with defined specificity.

Cytotoxic CD8+ T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8+ T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8+ T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8+ T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8+ T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8+ T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8+ T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8+ T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.

The drug development process in cancer faces significant challenges due to high failure rates in translational studies despite promisingin vitroresults. Additionally, conventional animal models exhibit inherent limitations and ethical concerns, constraining their relevance to cancer studies. Recognizing the pivotal role of the tumor microenvironment (TME) on cancer development and treatment outcomes, recent advancements in 3D microfluidic devices and tumor-on-a-chip models enabled researchers to explore the TME with enhanced accuracy and reliability, yielding novel insights. Notably, the emergence of physiological tumor models, particularly 3D models such as organoids derived from human tissues, provides a more accurate representation ofin vivotumor features. Moreover, 3D tumor models hold promise for diverse applications, including high-throughput drug testing, disease modeling, and regenerative medicine. Meanwhile, combining artificial intelligence (AI) with patient-derived tumor organoids has become a key strategy in predictive oncology and personalized cancer treatment. Furthermore, incorporating quantitative systems pharmacology and physiologically based pharmacokinetic modeling, and pharmacokinetics/pharmacodynamics analysis with generative AI (Gen-AI) has revolutionized predictive oncology by enabling precise simulations of drug interactions and patient-specific responses, thereby enhancing the predictive accuracy of personalized cancer treatments. These advanced methodologies harness the power of AI algorithms to analyze intricate datasets derived from patient-specific tumor organoids. Moreover, the predictive modeling capabilities of Gen-AI facilitate the development of personalized treatment strategies customized for each patient, thereby revolutionizing oncology practice. This review explores the synergistic impact of tumor-on-a-chip models, organoids derived from patient tumors, and Gen-AI. Together, these technologies mark a significant advancement in precision medicine, offering promising opportunities to improve therapeutic effectiveness and treatment outcomes in cancer care.

Unlike genomic instability, the implications of proteomic instability in cancer remain ambiguous. By governing the proteotoxic stress response, heat shock factor 1 (HSF1) sustains proteomic stability upon environmental insults. Apart from its importance to stress resistance and survival, HSF1 is emerging as a powerful oncogenic enabler. In the Neurofibromatosis type I (NF1)-deficient malignant peripheral nerve sheath tumor (MPNST) cells, HSF1 depletion triggered protein polyubiquitination, aggregation, and even tumor-suppressive amyloidogenesis. In contrast, HSF1 is dispensable for the proteome of non-transformed human Schwann cells. Mechanistically, HSF1 defends the essential mitochondrial chaperone HSP60 against the direct assault from soluble amyloid oligomers. To survive and adapt to compromised protein quality, owing to HSF1 deficiency, MPNST cells mobilized JNK to repress mTORC1 and protein translation, thereby attenuating protein quantity to alleviate proteomic imbalance. mTORC1 stimulation, via either pharmacological JNK blockade, genetic TSC2 depletion, or leucine supplementation, markedly aggravated the proteomic imbalance elicited by HSF1 deficiency. This catastrophic imbalance instigated pronounced cell death partly through unchecked amyloidogenesis, thereby exerting tumor suppression in both MPNST and melanoma models in vivo. Thus, HSF1 safeguards the cancer proteome to enable the oncogenic potential of mTORC1. This proof-of-principle study highlights provoking proteomic catastrophe as a next-generation therapeutic concept for combating malignancy.

33 Background: Historically, PSA DT can be prognostic for metastasis free survival in BCR (as defined on computed tomography (CT) and bone scan). PSA DT is also an important tool in risk stratification in BCR to identify which patients (pts) require therapy (e.g. pts with a PSA DT>6 months). The emerging use of PSMA imaging creates another tool to assess BCR pts, but there is no data on the association of PSA DT and PSMA findings. Methods: NCT05588128 enrolls BCR pts after definitive +/- salvage therapies. Pts must have a PSA>0.5 ng/ml, testosterone >100 ng/dL, and negative CT/bone scans. Lymph nodes (LNs) up to 1.5 cm and prior therapies are permitted. All patients undergo a baseline PSMA PET scan, along with measurement of PSA and PSA DT. Here we describe the relationship between baseline PSA metrics and baseline PSMA imaging findings in patients with BCR. Results: 130 patients are currently evaluable with a median age= 71 years, baseline PSA of 1.95 ng/dL (range: 0.5 to 71) and PSA DT of 10.6 months (range 1.2 to 132) off therapy. Of all participants, 16.9% (n=22) had findings limited to the prostate bed and 34.6% (n=45) had PSMA+ avidity in the prostate bed with other findings. 42.3% (n=55) had PSMA + LNs, and 13.8% (n=18) had PSMA+ bone lesions. Four patients had serosal deposits with PSMA avidity, and one patient had a PSMA+ lung nodule. Conclusions: These data are the first to compare PSMA imaging results with corresponding PSA levels and PSA DT in a large cohort of patients with BCR. Results show that pts with historically favorable/long PSA DT may have high volume/bone+ findings on PSMA. There is no data to suggest treatment escalation is required in BCR pts with high volume PSMA findings, but long/favorable PSA DT. These results highlight the caveats of using PSMA imaging alone to drive treatment decisions in BCR without further data for how baseline PSMA imaging corresponds with long-term outcomes. Clinical trial information: NCT05588128 . PSMA PET+ lesion locations by PSA doubling time in patients with BCR prostate cancer (total n=130). PSA DT # Pts Median PSA PSMA Neg Prostate+ only Lymph Nodes+ ≥ 4 Lymph Nodes+ Bone+ ≥12 mo 52 3.55 9 (17.3%) 16 (30.8%) 24 (46.2%) 12 (23%) 6 (11.5%) 9 to <12 mo 20 1.56 2 (10%) 4 (20%) 11 (55%) 3 (15%) 3 (15%) 6 to <9 mo 17 0.9 5 (29.4%) 0 (0%) 12 (70%) 8 (47.1%) 2 (11.8*) <6 mo 41 2.1 5 (12.2%) 2 (4.9%) 28 (68.3) 15 (36.6) 7 (17.1)

508 Background: C+N is approved as first-line treatment in aRCC based on the CheckMate 9ER trial, which demonstrated superior progression-free survival (PFS) and overall survival (OS) versus sunitinib in this pt population. In this exploratory, post hoc analysis, we characterized the depth and durability of response with long-term follow-up (based on the final analysis of CheckMate 9ER) in pts receiving C+N. Methods: Pts received C (40 mg) once daily plus N (240 mg) every 2 weeks. Depth of response (DepOR) subgroups were based on best overall response (blinded independent central review [BICR] per RECIST v1.1) and best tumor reduction threshold, as follows: complete response (CR); partial response subdivided by a tumor reduction of ≥80% (PR1), ≥60%–<80% (PR2), or ≥30%–<60% (PR3); stable disease (SD); and progressive disease (PD). PFS (per BICR) and OS were analyzed by DepOR subgroups after a 6-month post-randomization landmark. Results: Of 323 pts randomized to C+N, 293 pts were alive, and 234 pts were alive and progression-free at the 6-month landmark and were categorized by DepOR subgroup. With a median follow-up of 67.6 months (range: 60.2–80.2), pts with a CR exhibited a durable response and prolonged benefit in DOR, PFS, and OS (Table). Pts who experienced a response (CR or any PR) included those with liver metastases (16%– 29%), sarcomatoid features (6%–15%), ≥2 metastatic sites (60%–78%), and poor IMDC risk (7%–17%). Median duration of therapy was 30.4 (CR), 29.9 (PR1), 35.6 (PR2), 23.0 (PR3), 15.3 (SD), and 9.0 (PD) months. Any-grade treatment-related adverse events were generally consistent across DepOR subgroups. Conclusions: This exploratory analysis of CheckMate 9ER revealed durable responses in pts who experienced a CR. Pts who responded to treatment (CR or any PR) included those with poor prognostic characteristics. Clinical trial information: NCT03141177 . Clinical outcomes by DepOR subgroup in pts treated with C+N. DepOR Median DOR a (95% CI), months PFS DepOR pop n=234n (%) 48-month b PFS (95% CI), % Median PFS b (95% CI), months OS DepOR popn=293n (%) 48-month b OS (95% CI), % Median OS b (95% CI), months CR NR(30.5, NE) 45 (19) 54(38, 68) NR(30.4, NE) 45 (15) 84(70, 92) NR(NE, NE) PR1 22.1(15.1, 26.0) 25 (11) 12(3, 28) 18.8(13.3, 32.7) 27 (9) 56(35, 72) 51.6(32.2, NE) PR2 21.7(14.1, 30.4) 36 (15) 24(11, 39) 18.9(13.3, 27.4) 38 (13) 67(50, 80) 63.2(47.0, NE) PR3 10.8(7.0, 17.3) 63 (27) 3(0, 13) 9.4(5.5, 17.0) 70 (24) 42(30, 53) 41.9(33.8, 49.5) SD NA 65 (28) 4(1, 11) 5.9(3.9, 10.0) 98 (33) 29(20, 39) 28.7(17.5, 35.0) PD NA NA NA NA 15 (5) 20(5, 42) 11.0(4.8, 25.1) a Calculated in the OS DepOR response (CR or any PR) pop. b From the 6-month landmark.DOR, duration of response;

Abstract Background Immune checkpoint blockade (ICB) treatment, alone or in combination with cisplatin and fluorouracil (FU) chemotherapy, is used as first-line treatment for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Yet, a significant proportion of patients with carcinogen-associated HNSCC develop disease relapse or progression within 2 years. Effective treatments for patients who have failed standard of care (SOC) treatment are lacking. STAR0602, a selective, bifunctional T cell agonist consisting of an antibody targeting Vβ6 and Vβ10 T cell receptors fused to human interleukin-2, has demonstrated clinical activity in anti-PD(L)-1 resistant tumors. The murine surrogate, mSTAR1302, has been shown to induce tumor regression in multiple syngeneic tumor models and provided preclinical evidence for enhanced antitumor activity in ICB-refractory settings. This study investigates the therapeutic benefit of mSTAR1302 combined with SOC in the mouse oral carcinoma MOC1 and MOC2 HNSCC tumor models. Methods C57BL/6 mice bearing MOC1 or MOC2 tumors were treated once a week with mSTAR1302, cisplatin and α-programmed cell death protein 1 (PD-1) to determine antitumor efficacy and survival benefit. Immune populations and their effector functions were characterized by flow cytometry and cytokine assays. The tumor microenvironment’s immune architecture was analyzed by multiplex immunofluorescence. Gene expression analysis derived a comprehensive understanding of the multimodal therapy’s mechanism of action. Results Combination therapy with mSTAR1302, cisplatin and α-PD-1 induced a robust antitumor activity, significantly prolonged survival and yielded the highest percentage of tumor resolution in comparison with mSTAR1302 monotherapy or SOC treatment in MOC1 and MOC2 tumors. Tumor-free animals from cohorts treated with the combination therapy demonstrated immune protection against tumor rechallenge and an overall increase in antigen-specific T cells. Tumor growth inhibition was associated with Vβ13 CD4+ and CD8+ T cells expansion and heightened cytotoxic activity. The improved therapeutic effect was strongly dependent on interferon-γ expression. The combination of mSTAR1302 and α-PD-1 enabled control of tumors that progressed following platinum-containing treatment. Conclusion These findings provide a rationale for the combination of STAR0602 and SOC therapy in the clinical setting for patients with recurrent or metastatic carcinogen-associated HNSCC. Citation Format: Francesca Rosato, Ginette S. Santiago-Sanchez, Kellsye P. Fabian, Michelle R. Padget, Jonelle K. Lee, Clint T. Allen, Zhen Su, Jacques Moisan, Madan Katragadda, Andrew Bayliffe, Jeffrey Schlom, James W. Hodge. Combination therapy with a TCR Vβ-directed bifunctional molecule, cisplatin and anti-PD-1 promotes antitumor activity in immune checkpoint blockade-refractory head and neck murine tumor models [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A028.

Abstract Background: RIB demonstrated clinically and statistically significant PFS and overall survival (OS) benefit in all of its phase 3 trials in HR+/HER2− advanced breast cancer (ABC; ML-2, -3, -7). We studied clinical characteristics and biomarkers of pts who derived long-term treatment benefit. We present an exploratory analysis of the ML trials of all 1L pts with long-term response (LTR; PFS >4 y) to RIB and 1L postmenopausal pts with very long-term response (VLTR; PFS >5 y). Methods: In the ML trials, pre- (ML-7) and postmenopausal (ML-2, -3) pts with HR+/HER2− ABC received RIB + ET or placebo + ET. Only pts who received 1L RIB were included in this pooled analysis. Pts with early relapse (treatment-free interval [TFI] ≤12 months on [neo]adjuvant ET) and those in ML-7 treated with tamoxifen were excluded. Overall median follow-up (mfu) was 6.1 y (ML-2, 6.7 y; ML-3, 5.9 y; ML-7, 4.5 y). Given the median PFS of ≈2 y reported with CDK4/6i in 1L ABC, more than doubling that time (PFS >4 y) was considered LTR. VLTR was defined as PFS >5 y, calculated only for postmenopausal pts (ML-2, -3) as mfu in ML-7 was <5 y. Biomarker analyses were performed using baseline ctDNA and tumor samples. Nominal P values (unadjusted) were calculated to compare characteristics of pts with LTR and non-LTR. Results: Of 666 pts treated with 1L RIB + ET included in the analysis, 109 were on treatment at data cutoff. A total of 153 pts (23.0%) had LTR; 164 pts (24.6%) were censored before LTR cutoff. Median PFS (mPFS) in pts with LTR was 6.8 y (95% CI: 6.4 y-NE [not estimable]); median OS (mOS) was NE (95% CI: 7 y-NE). Age, body mass index, and menopausal status were balanced between pts with LTR and non-LTR ( Table ). Similar proportions of premenopausal (34/150 [22.7%]) and postmenopausal pts (119/516 [23.1%]) had LTR. Pts with LTR were less likely than those with non-LTR to have liver metastasis (16.3% vs 25.5%) and less likely to have ≥3 metastatic sites (30.1% vs 43.0%). Similar proportions of pts with LTR vs non-LTR had bone-only disease (24.2% vs 19.5%). ctDNA analysis used a panel of 558 genes; gene expression analysis used 800 genes. Pts with LTR had a lower mean ctDNA fraction and were less likely to have CCND1 or TP53 alteration detected by ctDNA. Additionally, they had lower gene expression of CCNE1 and a higher proportion of luminal A disease (PAM50 based). Of 516 postmenopausal pts, 88 (17.1%) had VLTR; further analysis will be provided in the presentation. Conclusions: In the ML studies, 1 of 4 pts treated with 1L RIB were able to derive LTR (PFS >4 y). Pts with LTR had mPFS of 6.8 y, and mOS was NE. Although LTR was more evident in pts with better prognostic factors, noticeably, some pts with unfavorable prognostic factors were still able to achieve LTR. This exploratory analysis suggests long-term benefit with 1L RIB in keeping a good number of pts with HR+/HER2− ABC free of disease progression for more than 4 y. Citation Format: F. Andre, P. A. Fasching, A. Prat, P. Neven, Y. Lu, A. Bardia, G. Curigliano, V. Kaklamani, P. Razavi, Y. Yap, F. Su, E. Roux, K. Pantoja, J. Wu, A. Lteif, D. Juric. Pooled analysis of patients (pts) treated with 1st-line (1L) ribociclib (RIB) + endocrine therapy (ET) in the MONALEESA (ML) studies: long-term progression-free survival (PFS) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD5-10.