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A safe and effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be required to end the coronavirus disease 2019 (COVID-19) pandemic1-8. For global deployment and pandemic control, a vaccine that requires only a single immunization would be optimal. Here we show the immunogenicity and protective efficacy of a single dose of adenovirus serotype 26 (Ad26) vector-based vaccines expressing the SARS-CoV-2 spike (S) protein in non-human primates. Fifty-two rhesus macaques (Macaca mulatta) were immunized with Ad26 vectors that encoded S variants or sham control, and then challenged with SARS-CoV-2 by the intranasal and intratracheal routes9,10. The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete protection in bronchoalveolar lavage and nasal swabs after SARS-CoV-2 challenge. Titres of vaccine-elicited neutralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate robust single-shot vaccine protection against SARS-CoV-2 in non-human primates. The optimal Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in clinical trials.

Introduction: Prostate cancer therapy involves the use of androgen deprivation therapy, chemotherapy, targeted therapy and immunotherapy. Chemotherapy involves the use of anti-tubulins (docetaxel, cabazitaxel), platinum salts, and topoisomerase inhibitors (TOPO1, TOPO2A). There is no biomarker of chemotherapy that is routinely used. We examined 87 prostate cancer samples using targeted proteomics for biomarkers of response or resistance to chemotherapy agents. Biomarkers of resistance include ERCC1 (Platinum), TUBB3 (taxanes), ALDH1A1 (cyclophosphamide), while response biomarkers include TOPO1 (irinotecan, topotecan), TOPO2A (doxorubicin, epirubicin), and hENT1 (Gemcitabine). We also measured markers for several antibody-drug conjugates targets (Her2, Her3, Trop2) in our clinical proteomics (CLIA) platform and several ADC/CAR-T proteins (PSMA, STEAP1, Nectin4, Claudin 18.2. in our research platform Methods: Tumor areas from Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from clinical samples of prostate cancer received at our CLIA certified laboratory were microdissected and a quantitative proteomic analysis of 72 biomarkers were conducted using selected reaction monitoring mass spectrometry (SRM-MS). Discussion:[WL1] Androgen receptor was detected in majority of the samples (83%) with a 19x range of distribution (329 amol/ug - 24063 amol/µg). Majority of prostate cancer samples expressed a range of resistance markers for anti-tubulin inhibitors (TUBB3: 69% detection with a 14x range) indicating that the high expressors is likely to be resistant to docetaxel/cabazitaxel based regimens. ERCC1, a marker for resistance to platinum-based agents, was not detected in 26% of cases, potentially enabling a cisplatin/carboplatin-based regimen in 1/4th of prostate cancer patients. Cyclophosphamide could be effective in 10% of cases where ALDH1A1 was not detected. TOPO1, a marker for irinotecan-based therapy was observed in 94% of cases with a range of ~6x (488 - 2760 amol/µg). Other chemotherapy agents that are not routinely used in prostate cancer was observed in select groups. These include doxorubicin biomarker TOPO2A in 34% of cases with 10x range, gemcitabine biomarker hENT1 in 36% of cases (4x range), temozolomide biomarker MGMT (ND in 12% of cases). ADC biomarker analysis revealed HER2 which was observed in 59% of the cases with range of expression from 301 - 1255 amol/µg including a significant population of low HER2 (84%, Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Amanda Strasbaugh, Marya Melkie, Negin Ghafourian, Xuefeng B. Ling. Quantitative proteomics of antibody-drug conjugates and chemotherapy targets in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2160.

671 Background: Pancreatic cancer (PaC) is a highly fatal disease with a 5-year survival rate of 5-10%. Effective screening is not available, and most patients (50-55%) present with metastatic (50%-55%) disease at diagnosis. For patients with advanced PaC, the standard chemotherapy combinations include FOLFIRINOX and/or gemcitabine/nab-paclitaxel which has results in a overall survival of 7-11 months. The lack of effective therapies underscores the importance of exploring other agents. We propose that quantitating therapy-associated protein biomarkers including markers of antibody-drug conjugates (ADC) can improve treatment personalization for PaC. Methods: FFPE tumor tissues from 185 clinical PaC patients were microdissected and solubilized for mass spectrometry-based targeted proteomic analysis. We quantified biomarkers for ADCs simultaneously from 2-3 section of FFPE. These biomarkers include antibody targets such as EGFR, HER2, HER3, FRalpha, and Trop2 and payload biomarkers of sensitivity (TOPO1) and resistance (TUBB3). The multiplexed assay also quantified additional 65 clinically relevant protein biomarkers for chemotherapy, immunotherapy and targeted therapy. Results: Expression of EGFR was observed in majority of samples (88%) while only overexpressed ( > 1000 amol/µg) in 3% of samples. HER2 was expressed in half of patients (52%) and overexpressed ( > 750 amol/µg) in 5% of cases while the rest of HER2 protein expression ranged from 300 -750 amol/µg which corresponds to low Her2 expression. Trop2 was expressed in majority of patients (91%) with a 25x distribution between lowest and highest expressor. Other ADC biomarkers include HER3(55%, 5x), Axl (24%, 12x), Mesothelin (65%, 58x), Folate receptor alpha (10%, 17x). Expression of TUBB3 (77%, 8x) and TOPO1 (92%, 8x) in antibody target-positive subset may suggest resistance or response for several known payloads, such as taxanes and irinotecan/deruxtecan/govitecan, respectively. Conclusions: There is currently no approved ADC for pancreatic cancer, but several ADC clinical trials are underway. Quantitative proteomics identified antibody targets as well as markers of resistance or response to payloads for a variety of approved and investigational ADC therapies, which could aid in patient stratification in ADC clinical trials.

Introduction: Trop2 is overexpressed in many cancers and currently Trop2 ADC is approved in TNBC. In ADC drug design, it is imperative to assess not only the levels of the receptor but also the payload biomarkers. We have developed a multiplex mass spectrometry method to quantitate 72 actionable proteins from FFPE samples that requires minimal tissue (2-3 sections). This panel includes chemotherapy, targeted therapy and immunotherapy agents. The test is run in our CAP, CLIA, and NYSDOH approved laboratory. For this study, we examined a subset of samples run in the clinical lab for the levels of Trop2 (target biomarker) and payload biomarkers (Topo1, TUBB3). Topo1 is a chemosensitive marker for irinotecan, while TUBB3 is a chemoresistance marker for tubulin inhibitors. Methods: FFPE tissue sections from 1140 clinical samples from a variety of cancers were examined. These include breast (n=318), colorectal (n=228), ovarian (n=199), GBM (n=69), NSCLC (n=169), HNSCC (n=91), and Gastric cancer (n=66). Two sections (10 µ) of FFPE tissue were cut on DIRECTOR slides and only the tumor areas were laser microdissected for downstream analysis which resulted in tryptic peptides. 1µg of peptides (~4000 cells) along with heavy peptides was injected into a triple quad mass spec and 72 biomarkers were quantitated concurrently. Results: Trop2 showed a wide range of expression in various cancers. Almost all (95%)breast cancer samples expressed Trop2 which exhibited a wide range (93x; 157 - 14650 amol/µg). Topo1 and TUBB3 were expressed in 93% and 60% of samples respectively. 1/4th of NSCLC does not express Trop2 and there is a 106x difference in expression of Trop2 in NSCLC. Topo1 was expressed in almost all samples while TUBB3 was expressed in 80% of NSCLC. Majority of ovarian cancer samples (85%), HNSCC (89%), Gastric cancer (88%) samples expressed Trop2 with a 113x, 134x, 47x difference in expression. Chemosensitive biomarker Topo1 was expressed in almost all ovarian (96%), HNSCC (91%) and Gastric (99%) cancer samples. Chemoresistant marker TUBB3 was expressed in 66% of ovarian cancer, 44% of HNSCC and 45% of gastric cancer samples. In contrast to above cancers, only 10% of Glioblastoma samples express Trop2 and only 3/4th of GBMs express Topo1. Discussion: In a randomly selected group of cancers, we have found Trop2 is expressed in majority of Breast, Ovarian, Lung, HNSCC and gastric cancers and minimal expression in GBM. Given the range of expression of anti-tubulin resistance marker in many solid tumors, a payload biomarker guided clinical trial is highly recommended in ADCs that employ anti-tubulin inhibitors. In contrast, wide expression of chemosensitive biomarker for Topo1 payload makes it a promising candidate for many solid tumors. Further studies are warranted to determine the level of target and payload biomarkers that will be required for a clinical response. Citation Format: Sheeno P. Thyparambil, Wei-Li Liao, Robert Heaton, Guolin Zhang, Amanda Strasbaugh, Marya Melkie, Xuefeng B. Ling. Clinical survey of Trop2 antibody drug conjugate target and payload biomarkers in multiple cancer indications using multiplex mass spectrometry [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 4099.

Summary SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19., Highlights • mRNA vaccines induce robust type 1 CD4+ and CD8+ T cells in the spleen and lung • Vaccine-induced T cells readily exit the vasculature and enter the lung parenchyma • mRNA vaccines elicit strong long-lived plasma cell and memory B cell responses • mRNA vaccines induce antibodies with potent anti-SARS-CoV-2 neutralization activity, SARS-CoV-2 mRNA-based vaccines are among the most promising vaccine candidates against COVID-19. Laczkó et al. evaluate two nucleoside-modified mRNA vaccine candidates in mice and demonstrate that they induce potent T and B cell immune responses and high levels of neutralizing antibodies after administration of a single vaccine dose.

Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates. Methods: Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens. Results: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID50) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. Conclusions: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.).