Circulating senescent CD8+ T (T8sen) cells are characterized by a lack of proliferative capacities but retain cytotoxic activity and have been associated to resistance to immunotherapy in patients with advanced non-small cell lung cancer (aNSCLC). We aimed to better characterize T8sen and to determine which factors were associated with their accumulation in patients with aNSCLC. Circulating T8sen cells were characterized by a higher expression of SA-βgal and the transcription factor T-bet, confirming their senescent status. Using whole virome profiling, cytomegalovirus (CMV) was the only virus associated with T8sen. CMV was necessary but not sufficient to explain high accumulation of T8sen (T8senhigh status). In CMV+ patients, the proportion of T8sen cells increased with cancer progression. Last, CMV-induced T8senhigh phenotype but not CMV seropositivity itself was associated with worse progression-free and overall survival in patients treated with anti-PD-(L)1 therapy but not with chemotherapy. Overall, CMV is the unique viral driver of T8sen-driven resistance to anti-PD-(L)1 antibodies in patients with aNSCLC.

BackgroundOur group developed a strategy for generating an ‘off-the-shelf’ multivalent proteasome-blocked autophagosome vaccine that contains proteins for many genes commonly overexpressed in adenocarcinoma and squamous cell cancers. This strategy exploits in vitro manipulation of the antigen presentation pathway to concentrate the dominant epitopes presented by MHC, including short-lived proteins (SLiPs), defective ribosomal products (DRiPs), and Dark Matter, the short-lived non-canonical peptides that are not expressed in the thymus and represent potential shared alternative cancer neoantigens.1 In preclinical models this vaccine strategy provides significant protection as a single agent,2 and significantly increased therapeutic efficacy when combined with anti-GITR and anti-PD-1.3 Based on these studies we hypothesize that addition of anti-GITR to the human vaccine, DPV-001, and anti-PD-1, will augment expansion and limit contraction of the anti-cancer immune response. A phase I clinical trial was initiated, and preliminary immunological monitoring data will be presented.MethodsPatients received DPV-001, with sequenced checkpoint inhibition (aPD-1 mAb; retifanlimab), with or without aGITR agonist mAb (INCAGN1876), in recurrent or metastatic HNSCC (NCT04470024). Tumor biopsies were taken pre-treatment, week 2 and 8. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Tumor biopsies and blood were assayed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluorescence (mIF) was performed on biopsies.ResultsIn the first 4 patients evaluated, tumor-infiltrating T cells at week 8 increased from pre-treatment levels by an average of 4.3 fold (range 2.9 – 6.7, p=0.032). The density of CD39/CD103 double positive cells, previously shown to identify tumor-reactive T cells,4 also increased in all week 8 biopsies (mean 14.7 fold, range 5–40). All patients showed increased numbers of cells expressing IFN-γ and GZMB and increased numbers of T cells expressing LAG3+ in week 8 biopsies. Preliminary TCR evaluation of the tumor identified proliferation of clones previously undetected in PBL, including αβ T cells, iNKT and MAIT cells. Expansion of clones that predated treatment was also identified.ConclusionsAn increase in intra-tumoral T cells expressing activation and effector molecules is encouraging and studies are underway to expand the number of patients analyzed. Increased expression of LAG3 by T cells that infiltrate and have expanded in the tumor, provide a rationale for including anti-LAG-3 in this treatment strategy. Future plans include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001 and whether antibody responses identify targets of cellular immunity.AcknowledgementsSupport: Incyte, UbiVac, Providence Medical Foundation, The Chiles Foundation, The Harder Family, Nancy Lematta, Robert W. Franz and Elsie Franz Finley, The Murdoch Memorial TrustTrial RegistrationNCT04470024ReferencesFox BA, Urba WJ, Jensen SM, Page DB, Curti BD, Sanborn RE, Leidner RS. Cancer’s Dark Matter: Lighting the Abyss Unveils Universe of New Therapies. Clinical Cancer Research 2023;29:2173–2175. 10.1158/1078–0432.CCR-23–0422.Twitty CG, Jensen SM, Hu H-M, Fox BA. Tumor-Derived Autophagosome Vaccine: Induction of Cross-Protective Immune Responses against Short-lived Proteins through a p62-Dependent Mechanism. Clinical Cancer Research 2011;17:6467–6481. 10.1158/1078–0432.CCR-11–0812.Patel JM, Cui Z, Wen Z-F, Dinh CT, Hu H-M. Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism. J Immunother Cancer 2019;7:311. 10.1186/s40425–019-0786–7.Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724. 10.1038/s41467–018-05072–0.Ethics ApprovalPSJH IRB# 2020000480

We have developed a pipeline to express, purify, and characterize HIV envelope protein (Env) gp145 from Chinese hamster ovary cells, to accelerate the production of a promising vaccine candidate. First in shake flasks, then in bioreactors, we optimized the growth conditions. By adjusting the pH to 6.8, we increased expression levels to 101 mg/L in a 50 L bioreactor, nearly twice the previously reported titer value. A battery of analytical methods was developed in accordance with current good manufacturing practices to ensure a quality biopharmaceutical. Imaged capillary isoelectric focusing verified proper glycosylation of gp145; dynamic light scattering confirmed the trimeric arrangement; and bio-layer interferometry and circular dichroism analysis demonstrated native-like properties (i.e., antibody binding and secondary structure). MALDI-TOF mass spectrometry was used as a multi-attribute platform for accurate mass determination, glycans analysis, and protein identification. Our robust analysis demonstrates that our gp145 product is very similar to a reference standard and emphasizes the importance of accurate characterization of a highly heterogeneous immunogen for the development of an effective vaccine. Finally, we present a novel guanosine microparticle with gp145 encapsulated and displayed on its surface. The unique properties of our gp145 microparticle make it amenable to use in future preclinical and clinical trials.

To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.

Due to poor compliance and uptake of LDCT screening among high-risk populations, lung cancer is often diagnosed in advanced stages where treatment is rarely curative. Based upon the American College of Radiology's Lung Imaging and Reporting Data System (Lung-RADS) 80-90% of patients screened will have clinically "non-actionable" nodules (Lung-RADS 1 or 2), and those harboring larger, clinically "actionable" nodules (Lung-RADS 3 or 4) have a significantly greater risk of lung cancer. The development of a companion diagnostic method capable of identifying patients likely to have a clinically actionable nodule identified during LDCT is anticipated to improve accessibility and uptake of the paradigm and improve early detection rates. Using protein microarrays, we identified 501 circulating targets with differential immunoreactivities against cohorts characterized as possessing either actionable (n = 42) or non-actionable (n = 20) solid pulmonary nodules, per Lung-RADS guidelines. Quantitative assays were assembled on the Luminex platform for the 26 most promising targets. These assays were used to measure serum autoantibody levels in 841 patients, consisting of benign (BN; n = 101), early-stage non-small cell lung cancer (NSCLC; n = 245), other early-stage malignancies within the lung (n = 29), and individuals meeting United States Preventative Screening Task Force (USPSTF) screening inclusion criteria with both actionable (n = 87) and non-actionable radiologic findings (n = 379). These 841 patients were randomly split into three cohorts: Training, Validation 1, and Validation 2. Of the 26 candidate biomarkers tested, 17 differentiated patients with actionable nodules from those with non-actionable nodules. A random forest model consisting of six autoantibody (Annexin 2, DCD, MID1IP1, PNMA1, TAF10, ZNF696) biomarkers was developed to optimize our classification performance; it possessed a positive predictive value (PPV) of 61.4%/61.0% and negative predictive value (NPV) of 95.7%/83.9% against Validation cohorts 1 and 2, respectively. This panel may improve patient selection methods for lung cancer screening, serving to greatly reduce the futile screening rate while also improving accessibility to the paradigm for underserved populations.

B7 homolog 3 (B7-H3; CD276), a tumor-associated antigen and possible immune checkpoint, is highly expressed in prostate cancer (PCa) and is associated with early recurrence and metastasis. Enoblituzumab is a humanized, Fc-engineered, B7-H3-targeting antibody that mediates antibody-dependent cellular cytotoxicity. In this phase 2, biomarker-rich neoadjuvant trial, 32 biological males with operable intermediate to high-risk localized PCa were enrolled to evaluate the safety, anti-tumor activity and immunogenicity of enoblituzumab when given before prostatectomy. The coprimary outcomes were safety and undetectable prostate-specific antigen (PSA) level (PSA0) 1 year postprostatectomy, and the aim was to obtain an estimate of PSA0 with reasonable precision. The primary safety endpoint was met with no notable unexpected surgical or medical complications, or surgical delay. Overall, 12% of patients experienced grade 3 adverse events and no grade 4 events occurred. The coprimary endpoint of the PSA0 rate 1 year postprostatectomy was 66% (95% confidence interval 47-81%). The use of B7-H3-targeted immunotherapy in PCa is feasible and generally safe and preliminary data suggest potential clinical activity. The present study validates B7-H3 as a rational target for therapy development in PCa with larger studies planned. The ClinicalTrials.gov identifier is NCT02923180.

Abstract The Uropathogenic Specific Protein (USP) is an E. coli genotoxin nuclease primarily associated with urinary tract infections. Previous efforts in our laboratory found the USP gene in stool samples; more frequently found in stool samples from adenoma and CRC patients than in samples from healthy individuals. New efforts in our laboratory aim to find the USP protein in the same population of patient samples and this may require anti-USP antibodies, which at present are not commercially available. In this work, we report the first anti-USP monoclonal antibodies (mAbs) for the detection of USP in human samples. A total of 38 hybridoma supernatants containing mAbs were assayed by immunoblotting and biolayer light interferometry (BLI) for their ability to recognize purified USP(∆HNH). Results from BLI showed that the pair AB-A10 and AB-D5 presented the highest affinity with slow dissociation, moving forward for the development of a sandwich ELISA. Using AB-A10 as the capture antibody, and AB-D5 as the top antibody, results from the ELISA showed signals that were concentration-dependent above 6 ug/mL of USP. We also used the AB-D5 and AB-D10 in immunoblots for the detection of USP in proteins extracted from stool samples of patients with adenoma and CRC. The immunoblots reveal the detection of a 25 kDa protein that remains unidentified but is present only in some of the samples. This could be the first detection of USP protein directly in a biological sample. Efforts are underway to identify this protein band and confirm its identity as USP. Additional efforts are aimed at increasing the number of stool samples analyzed to establish whether USP protein is a risk factor for CRC or adenoma. Citation Format: Rachell Martínez-Ramírez, Valeria Toro-Díaz Toro-Díaz, Pearl Akamine, Kimberly Ruiz-Rosado, Ignacio Pino, Abel Baerga-Ortiz. Monoclonal antibodies for the detection of the uropathogenic specific protein (USP): A bacterial genotoxin linked to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 713.

5015 Background: B7-H3/CD276, a member of the B7 superfamily, is highly expressed in prostate cancer (PCa) and is associated with rapid biochemical recurrence and early metastases. B7-H3 is the only checkpoint candidate to have a presumptive androgen receptor binding site, suggesting interaction with the androgen axis. Enoblituzumab (MacroGenics) is an investigational humanized Fc-optimized B7-H3–targeting antibody that induces antibody dependent cellular cytotoxicity (ADCC). Methods: In this phase 2 single-arm biomarker-rich neoadjuvant trial, men with operable intermediate- and high-risk localized prostate cancer (Grade Groups 3-5) were enrolled to evaluate the safety, anti-tumor efficacy, and immunogenicity of enoblituzumab when given prior to prostatectomy. Patients received enoblituzumab (15 mg/kg IV weekly x 6) prior to surgery. Prostate glands were harvested 2 weeks after the last dose, and were examined for pathologic and immunologic endpoints. The co-primary outcomes were safety and PSA0 at 1 year post-op. Pre-planned secondary outcomes were PSA and Gleason grade group change from biopsy to prostatectomy. Results: 32 men were enrolled. Grade 3/4 adverse events occurred in 12% of patients. One patient developed a grade-3 infusion reaction, and one had immune myocarditis that improved with steroids. Pre-prostatectomy PSA declines of >10% were observed in 31% of patients (95% CI: 16-50%). PSA0 at 1 year post-op was seen in 66% of men (95% CI: 47-81%). Median time to PSA recurrence was not reached, with a median follow-up of 30 months. Gleason group upgrade, no change, and downgrade was observed in 13%, 37%, and 50% of patients. Gleason grade group changes were significantly associated with enoblituzumab treatment compared to 1:1 matched historical controls (p=0.023). Tumor microenvironment profiling by NanoString GeoMx spatial proteomics and PanCancer IO 360 mRNA expression analysis revealed post-treatment upregulation of CD8+ T cells, PD-1/PD-L1 expression, and immune activation (granzyme B, IFN signaling, myeloid inflammation). There was a significant association between CD8+ T-cell increases and Gleason grade group declines. First-in-human antigen spread profiling revealed no safety concerns. TCR sequencing showed focused peripheral expansion of tumor associated T-cell clones that correlated with PSA0 at 1 year. Whole exome and RNAseq data, and clinical correlations, will be presented. Conclusions: In this neoadjuvant trial, inhibition of B7-H3 with enoblituzumab demonstrated favorable safety and encouraging activity in localized PCa patients. Data suggest robust intratumoral induction (adaptive upregulation) of immune checkpoints, T-cell activation, and myeloid inflammation. Enoblituzumab-induced peripheral expansion of tumor associated T-cell clones may be associated with tumor control. Clinical trial information: NCT02923180.

Autoantibodies are present in healthy individuals and altered in chronic diseases. We used repeated samples collected from participants in the NYU Women’s Health Study to assess autoantibody reproducibility and repertoire stability over a one-year period using the HuProt array. We included two samples collected one year apart from each of 46 healthy women (92 samples). We also included eight blinded replicate samples to assess laboratory reproducibility. A total of 21,211 IgG and IgM autoantibodies were interrogated. Of those, 86% of IgG (n = 18,303) and 34% of IgM (n = 7,242) autoantibodies showed adequate lab reproducibility (coefficient of variation [CV] < 20%). Intraclass correlation coefficients (ICCs) were estimated to assess temporal reproducibility. A high proportion of both IgG and IgM autoantibodies with CV < 20% (76% and 98%, respectively) showed excellent temporal reproducibility (ICC > 0.8). Temporal reproducibility was lower after using quantile normalization suggesting that batch variability was not an important source of error, and that normalization removed some informative biological information. To our knowledge this study is the largest in terms of sample size and autoantibody numbers to assess autoantibody reproducibility in healthy women. The results suggest that for many autoantibodies a single measurement may be used to rank individuals in studies of autoantibodies as etiologic markers of disease.