e15036 Background: Antibody–drug conjugates (ADCs) are an established therapeutic modality in solid tumors, although tumor heterogeneity and antigen loss can limit response durability. Bispecific ADCs targeting two tumor-associated antigens (TAAs) represent a promising strategy to improve tumor selectivity and potentially mitigate resistance mechanisms, and integrative in silico analyses may help inform the prioritization of biologically plausible TAA pairs. Methods: TAAs corresponding to ADCs approved by the FDA and/or EMA for solid tumors were selected as reference targets, including HER2, TROP2, NECTIN4, Tissue Factor, FOLR1 and c-MET. Gene expression correlations with an extended panel of clinically relevant TAAs were assessed using TIMER 3.0 across TCGA solid tumors. High-confidence associations were defined using a Spearman correlation coefficient ≥0.7, with tumor types annotated using standard TCGA nomenclature. Results: High-confidence co-expression patterns were identified between selected reference TAAs and an extended panel of TAAs across TCGA solid tumors; correlations observed in more than one tumor type were considered recurrent and are summarized in Table 1. HER2 showed recurrent high-confidence co-expression with ERBB3, together with additional tumor-specific associations. TROP2 and NECTIN4 displayed overlapping recurrent co-expression patterns with multiple partner antigens, alongside additional tumor-specific correlations. In contrast, Tissue Factor and FOLR1 exhibited exclusively tumor-specific high-confidence associations. c-MET demonstrated recurrent co-expression with several partner antigens, together with tumor-specific associations. Conclusions: This integrative in silico analysis identifies high-confidence co-expression patterns between clinically relevant TAAs across solid tumors, enabling the prioritization of biologically plausible TAA pairs. The identification of both recurrent and tumor-specific associations highlights distinct co-expression landscapes that may help inform pan-tumoral and indication-driven bispecific ADC design. TAA co-expression patterns supporting bispecific ADC design (r ≥ 0.7). Reference TAA Recurrent correlated TAAs Tumor-specific correlated TAAs TCGA tumor types HER2 ERBB3 B7-H3, TPBG, PTK7 PAAD, BLCA, THYM, KIRP, THCA TROP2 NECTIN4, ERBB3, FUT3, LYPD3, MUC1, NaPi-2b ITGB6, EGFR, FGFR2/3, Tissue Factor, CEACAM5, B7-H4 ESCA, SKCM, THCA, UCS, THYM, KIRP, HNSC, CESC NECTIN4 TROP2, LYPD3, ERBB3, FUT3 c-MET, EFNA4, EGFR, CDH3, B7-H4, SLC44A4, Tissue Factor ESCA, SKCM, THCA, UCS, PAAD, THYM Tissue Factor – AXL, ITGB6, LYPD3, NECTIN4, ROR1, TROP2 THYM, SKCM, TGCT FOLR1 – NaPi-2b, RORC, B7-H4, ROR2 LUSC, PRAD, UVM c-MET ADAM9, AXL, CD46, EGFR ERBB3, ITGB6, NaPi-2b, FGFR2, TPBG, CD71 LIHC, STAD, THCA, PRAD, UVM, KIRP, KICH, THYM

2559 Background: Adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) is a promising strategy in non-small cell lung cancer (NSCLC), although ex vivo expansion remains challenging due to variability in yield and product quality. Optimizing cytokine support during TIL expansion may improve manufacturing robustness while preserving a clinically relevant immunophenotype. Methods: Tumor tissue specimens were obtained from immediately resected NSCLC lesions and processed for TIL outgrowth and rapid expansion over 28 days using either standard IL-2 or an experimental IL-7/IL-15/IL-2 protocol. Manufacturing endpoints included expansion yield, viability and CD3⁺ proportion. Multiparametric flow cytometry assessed memory differentiation subsets and exhaustion-associated markers at baseline, pre-REP and post-REP. Clinical-pathological variables included disease stage, PD-L1 expression and prior systemic treatment. Results: 11 NSCLC TIL expansions were analyzed (experimental: n=7; IL-2: n=4). Median age was 73.1 years (range 48.5–92.3) and ECOG was 0–1 in 91% of patients; 64% had early-stage disease and 27% had advanced-stage (III–IV). PD-L1 was <1% in 73%, 9% showed PD-L1 1–49%, and none ≥50%. The cohort included pretreated patients, including prior anti–PD-1 exposure. Compared with IL-2, IL-7/IL-15/IL-2 resulted in a modest reduction in expansion yield (15–28%) and a lower CD4+/CD8+ ratio (6.62 vs 19.86), but showed improved manufacturing robustness, with all expansions meeting predefined quality criteria, whereas standard IL-2 expansions showed more variable product quality. Immunophenotypic profiling showed a more stable memory-oriented differentiation pattern with IL-7/IL-15/IL-2, including reduced naïve and TEMRA subsets and increased CD4+ TCM cells; CD8+ TCM remained predominant in both protocols. A polarisation towards the effector memory phenotype (CCR7⁻CD45RA⁻) was observed in the experimental protocol. CD28 remained highly expressed in both protocols, while CD134 tended to decrease with IL-7/IL-15. During pre-REP, PD-1 and TIM-3 were reduced in several subpopulations under the experimental protocol, suggesting a transiently less exhausted phenotype. After REP, these differences disappeared, with comparable levels between protocols. During REP, Th1 and Tfh increased in both protocols, while Th2 and Th17 remained low. Conclusions: Supplementation with IL-7 and IL-15 during NSCLC-derived TIL expansion improves consistency of product quality and supports a favorable memory-oriented and functional phenotype without increasing exhaustion markers at the end of manufacturing. This approach may enable more reliable generation of clinically applicable TIL products in heterogeneous and pretreated NSCLC populations, facilitating broader implementation of TIL-based therapies.

Loss of methylthioadenosine phosphorylase creates a therapeutically dependency on PRMT5, yet current strategies select patients based only on MTAP genomic deletion. We performed an integrated analysis of DNA methylation and gene expression using TCGA, TARGET and ENCODE data to identify alternative mechanisms of MTAP inactivation. A promoter-proximal CpG site (cg25162921) showed a frequent hypermethylation and strong inverse correlation with MTAP mRNA expression across multiple tumor types and cell lines, including lung squamous cell carcinoma and glioblastoma. These findings identify promoter hypermethylation as a copy-number–independent mechanism of MTAP silencing and support epigenetic profiling evaluation in tumor samples into MTAP-based precision oncology.

FGFR3-TACC3 fusion-positive IDH-wild-type (IDH-WT) glioblastoma (GB) is a rare GB subtype occurring in approximately 3% of cases. It is clinical behavior and molecular profile is different from those of fusion-negative IDH-WT GBs. Evidence on the role of FGFR inhibitors in FGFR-altered gliomas is limited. We present the case of a patient with a FGFR3-TACC3 fusion-positive IDH-WT GB that at its second recurrence was treated with the FGFR inhibitor erdafitinib through a compassionate use program. Although no objective response was achieved, an overt deceleration in tumor growth was evidenced and the patient remained on treatment for 5.5 months.

Antibody-drug conjugates (ADCs) represent a therapeutic modality that guides chemotherapies to tumoral cells by using antibodies against tumor-associated antigens (TAAs). The antibody and the chemotherapy or payload are attached by a chemical structure called the linker. The strategy for the development of this type of drug was based on several rational pillars, including the use of a very potent payload and the use of specific antibodies acting only on antigens expressed on tumoral cells. In this article, by using data from all approved ADCs that have received regulatory approval, we analyze the potential contribution of each ADC component to preclinical activity. We suggest that payload potency and the drug-to-antibody ratio (DAR) have a less relevant role in relation to efficacy than previously considered. Additionally, we have observed that some ADCs have been developed against antigens also present in non-transformed tissues, which could suggest that TAA specificity is not a mandatory requirement. Finally, we have identified that ADCs with payloads harboring more favorable physicochemical characteristics showed better potential activity. In this article, we also review other aspects that should be taken into consideration for ADC design, including linker structure, stability, conjugation type, pharmacokinetics, receptor internalization, and recycling. Based on currently available data, our study summaries different concepts that should be considered in the design of novel ADCs in the future.

The identification of surfaceome proteins is a main goal in cancer research to design antibody-based therapeutic strategies. T cell engagers based on KLK2, a kallikrein specifically expressed in prostate cancer (PRAD), are currently in early clinical development. Using genomic information from different sources, we evaluated the immune microenvironment and genomic profile of prostate tumors with high expression of KLK2. KLK2 was specifically expressed in PRAD but it was not significant associated with Gleason score. Additionally, KLK2 expression did not associate with the presence of any immune cell population and T cell activating markers. A mild correlation between the high expression of KLK2 and the deletion of TMPRSS2 was identified. KLK2 expression associated with high levels of surface proteins linked with a detrimental response to immune checkpoint inhibitors (ICIs) including CHRNA2, FAM174B, OR51E2, TSPAN1, PTPRN2, and the non-surface protein TRPM4. However, no association of these genes with an outcome in PRAD was observed. Finally, the expression of these genes in PRAD did not associate with an outcome in PRAD and any immune populations. We describe the immunologic microenvironment on PRAD tumors with a high expression of KLK2, including a gene signature linked with an inert immune microenvironment, that predicts the response to ICIs in other tumor types. Strategies targeting KLK2 with T cell engagers or antibody–drug conjugates will define whether T cell mobilization or antigen release and stimulation of immune cell death are sufficient effects to induce clinical activity.

Therapeutic strategies targeting non-adaptive immune cells are currently in clinical development. γδT cells are a small subtype of T cells (1-10% of total T cells) that mediate their effector function without the necessity of the antigen presenting machinery, and also share functional properties with innate cells. Among the different γδT subtypes, antibodies against Vγ9Vδ2T have reported signs of clinical efficacy in early clinical studies. In this review we describe the biology of this subtype of non-conventional T cells and provide insights into the mechanism of action of novel antibodies that activate these cells. We will focus on antibodies targeting the BTN3A ligand and bi-specific γδT cell engagers. We will review in detail the advantages of these strategies including the potential for overcoming mechanisms of resistance to check point inhibitors, or the much more adequate safety profile compared with agents activating classical T cells. Limitations identified during the first studies in humans and strategies to overcome them will be revised and discussed. Finally, clinical options for future clinical development will be suggested.

IntroductionIdentification of modulators of the immune response with inhibitory properties that could be susceptible for therapeutic intervention is a key goal in cancer research. An example is the human leukocyte antigen G (HLA-G), a nonclassical major histocompatibility complex (MHC) class I molecule, involved in cancer progression. MethodsIn this article we performed a systematic review and meta-analysis on the association between HLA-G expression and outcome in solid tumors. This study was performed in accordance with PRISMA guidelines and registered in PROSPERO.ResultsA total of 25 studies met the inclusion criteria. These studies comprised data from 4871 patients reporting overall survival (OS), and 961 patients, reporting disease free survival (DFS). HLA-G expression was associated with worse OS (HR 2.09, 95% CI = 1.67 to 2.63; P < .001), that was higher in gastric (HR = 3.40; 95% CI = 1.64 to 7.03), pancreatic (HR = 1.72; 95% CI = 0.79 to 3.74) and colorectal (HR = 1.55; 95% CI = 1.16 to 2.07) cancer. No significant differences were observed between the most commonly utilized antibody (4H84) and other methods of detection. HLA-G expression was associated with DFS which approached but did not meet statistical significance. DiscussionIn summary, we describe the first meta-analysis associating HLA-G expression and worse survival in a variety of solid tumors. Systematic Review Registrationhttps://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022311973.

Coronavirus disease 2019 (COVID‐19) pandemic is affecting a high percentage of the population at an unprecedented rate. Cancer patients comprise a subgroup especially vulnerable to this infection. Herein, we present a prospective analysis of epidemiological, clinical, radiological and laboratory data of consecutive adult cancer patients seen in the Clínico San Carlos University Hospital (Madrid, Spain), and admitted to hospital and tested for COVID‐19 between 21 February 2020 and 8 May 2020 due to clinical suspicion of infection. Data from 73 patients with confirmed COVID‐19 and active solid tumors or diagnosed within the previous 5 years were analyzed. The most frequent malignancy was lung cancer (19%) and 54 patients (74%) were on active cancer treatment. Most common findings on presentation included cough (55%), fever (52%) and dyspnea (45%), and 32 (44%) patients showed oxygen saturation levels below 95%. Radiologically, 54 (73%) patients presented an abnormal pattern, the most frequent being infiltrates (64%). 18 (24.7%) patients died in hospital and 55 (75.3%) were discharged with clinical resolution of the event. Multivariable logistic regression adjusted for age and tumor stage showed higher odds of in‐hospital death associated with a history of cardiovascular disease, hospitalization in the previous 30 days, and several features on admission including dyspnea, higher qSOFA score, higher C‐reactive protein levels and an abnormal neutrophil count. We present prospective, real‐world evidence that can help articulate cancer care protocols for patients infected with SARS‐CoV‐2, with special focus on features on admission that can stratify patients with a higher risk of death from COVID‐19.

During short-term periods of separation of rat pups from their mothers, the loss of certain sensory signals suppresses the increase in ornithine decarboxylase (ODC) gene expression induced by the growth-promoting hormones prolactin (PRL) and growth hormone (GH). Here, we identify a molecular mechanism through which maternal separation (MS) curtails ODC expression. Our results demonstrate that the absence of specific tactile stimuli provided by the mother limits PRL-evoked stimulation of ODC biosynthesis by interfering with sn-1,2-diacylglycerol's (DAG) ability to activate protein kinase Calpha (PKCalpha) and consequently c-myc mRNA and max mRNA expression. The proteins encoded by these proto-oncogenes function as direct transactivators of the ODC gene. As ODC activity is obligatory for normal cell replication and differentiation, PKCalpha activation by DAG represents an important control point at which 'nurturing touch' regulates growth and development of the neonate. Such a mechanism can explain the maladaptive consequences of disrupting mother-infant tactile interactions as occurs in isolated premature babies. Also, it could provide a basis for developing therapeutic interventions to maximize growth potential in children failing-to-thrive despite normal maternal care.

Previously, we have shown that subcutaneous administration of insulin stimulates ornithine decarboxylase (ODC) mRNA expression and enzymatic activity in the liver of infant control rats, but not in those pretreated intracerebroventricularly (i.c.v.) with beta-endorphin. This finding is consistent with the hypothesis that beta-endorphin synthesized in the brain plays a prime role in the control of postnatal development, in part, by modulating ODC gene transcription. We now report that insulin induced stimulation of hepatic ODC mRNA expression is accompanied by a concomitant increase in the expression of c-myc and max mRNAs, and that this effect is also inhibited by pretreatment with i.c.v. beta-endorphin. These results suggest that CNS beta-endorphin suppresses tissue ODC responsiveness to trophic hormones by downregulating the expression of c-myc and max mRNAs, the encoded proteins of which are known to act physiologically as transcriptional activators of the ODC gene.

Ornithine decarboxylase (ODC) is thought to play a critical role in pulmonary development. The purpose of this study was to characterize the effects of dexamethasone on ODC gene expression and enzyme activity in the lung of rat pups. Subcutaneous administration of dexamethasone (10 mgkg) was shown to suppress ODC activity in 2-, 6- and 10-day-old rats for as long as 24 h after injection. In contrast, dexamethasone treatment stimulated liver ODC activity indicating that the inhibition of lung ODC is tissue specific. Contrary to expectation, the glucocorticoid enhanced lung ODC expression as indicated by an increased accumulation of ODC mRNA transcripts. The latter effect was associated with an heightened expression of c-myc and max mRNAs, the encoded proteins of which act as transactivators of the ODC gene. Dexamethasone did not alter lung levels of “antizyme” (AZ), an inducible protein that specifically promotes the degradation of the ODC protein enzyme. However, the lack of AZ induction does not necessarily mean that ODC degradation is not the mechanism for the decrease in lung ODC activity of dexamethasone-treated animals. The results obtained indicate that glucocorticoids can downregulate lung ODC activity, and that the effect is mediated by post-transcriptional rather than transcriptional mechanisms. These findings are consistent with the idea that endogenous glucocorticoids play an important role in the modulation of ODC activity and early pulmonary development.

This study examined whether the developmental deficits usually observed in infants born to opiate addicted mothers could involve effects on ornithine decarboxylase, a growth-controlling enzyme. Intracerebroventricular (i.c.v.) injection of a single dose of morphine (2 μg) to 6-day-old rats markedly decreased basal brain and liver ornithine decarboxylase activity as well as the increases in hepatic ornithine decarboxylase activity produced by subcutaneously (s.c.) administered insulin, an important trophic hormone. Centrally applied morphine acts supraspinally to downregulate peripheral ornithine decarboxylase activity, since s.c. administration of the same dose as used i.c.v. decreased neither basal liver ornithine decarboxylase levels nor tissue responsiveness to insulin. This does not imply that the opiate is unable to affect ornithine decarboxylase when applied systemically. In fact, a robust inhibition of both basal and induced liver ornithine decarboxylase activity was obtained in rat pups given 20 μg of morphine s.c. This larger dose is able to trigger the hepatic ornithine decarboxylase effects presumably by stimulating opiate receptors located at central sites after crossing the blood–brain barrier and penetrating into the brain. Concomitant administration of naloxone plus morphine i.c.v. prevented morphine from downregulating ornithine decarboxylase activity, confirming the participation of supraspinal opioid receptors in morphine ornithine decarboxylase actions. Finally, as was the case for insulin induced stimulation of ornithine decarboxylase activity, i.c.v. injection of morphine markedly diminished insulin induced stimulation of hepatic ornithine decarboxylase mRNA accumulation. In turn, contrary to the inhibition of basal ornithine decarboxylase activity, morphine did not lower basal hepatic ornithine decarboxylase mRNA levels when given alone. Thus, CNS morphine can apparently suppress tissue ornithine decarboxylase expression through both transcriptional and posttranscriptional mechanisms. The evidence obtained suggest that postnatal exposure to opiate drugs might detrimentally affect development by altering normal tissue ornithine decarboxylase ontogeny.

We examined the effects of dietary cholesterol on cardiac and hepatic beta-adrenergic receptor functioning. Age-matched adult desert rodents (Psammomys obesus) were randomized to either a 5% cholesterol diet (CD, n = 20), or normal rabbit chow (RC, n = 18). After a 2-month exposure to the diets, animals were sacrificed and tissue from both heart and liver were retained for radioligand bindings studies. In heart tissue, cholesterol fed animals, relative to controls, showed an increased production of adenosine 3,5¢-cyclic monophosphate (cAMP) in response to isoproterenol. Cholesterol supplementation was not associated with an increase in heart beta-adrenergic receptor number. Animals fed the 5% cholesterol diet showed significant increases in the number of beta-adrenergic receptor sites in hepatic tissue (M = 13.2 vs. 10.4 pmol/mg protein, CD and RC, respectively). The increased number of receptor sites in the liver was accompanied by a significant increase in isoproterenol-stimulated cAMP production. Results are supportive of the hypothesis that dietary cholesterol contributes to an upregulation of beta-adrenergic receptor function in cardiac, as well as hepatic tissue. These findings may be relevant to the observations of excessive stress-induced cardiovascular reactivity in persons with high cholesterol levels.

Previously, we have shown that short-term (1 hr) separation of neonatal rats from their mother (MS) suppresses basal ornithine decarboxylase (ODC) synthesis and tissue ODC response to trophic factors. This effect in the pup is caused by absence of maternal tactile stimulation (touch) but not from lack of maternal nutrients (food). This study was performed to examine in 10-d-old rats whether maternal touch deprivation affects expression of certain hepatic proto-oncogenes, the protein products of which are known to interact with the regulatory region of the ODC gene. Prolactin (PRL) injected subcutaneously increased hepatic ODC activity as well as mRNA levels of ODC and the proto-oncogenes c-fos, c-jun, junB, junD, c-myc, and max. MS significantly suppressed PRL-induced increases in ODC enzyme activity and c-myc, max, and ODC mRNAs but had little effect on expression of the other proto-oncogenes. PRL-induced stimulation of ODC, c-myc, and max mRNAs also was depressed in neonates placed with an anesthetized lactating dam (touch-deprived) but not in pups placed with nipple-ligated dams (food-deprived). Furthermore, unlike its effect on preweanling-age pups (< 20 d old), MS did not alter expression of either ODC or c-myc mRNAs in 25-d-old pups acutely separated from their mother. These findings indicate that suppression of ODC gene transcription in the neonatal pup during MS may be mediated by downregulation of the ODC gene transactivator proto-oncogenes c-myc and max. They are also consistent with our previous observation that lack of maternal touch, but not maternal milk, initiates the physiological alterations induced by MS.