Melanoma Antigen Research Articles

Identification of Critical Biomarkers and Mechanisms of Fructus Ligustri Lucidi on Vitiligo Using Integrated Bioinformatics Analysis.

Vitiligo is an autoimmune disease of the skin that targets pigment-producing melanocytes and results in patches of depigmentation that are visible as white spots. Recent research studies have yielded a strong mechanistic understanding of this disease. Fructus Ligustri Lucidi (FLL) has been used for premature graying of hair since ancient China and is currently used to treat vitiligo. However, the key biomarkers and mechanisms underlying FLL in vitiligo remain unclear. This study aimed to identify the potential biomarkers and mechanisms of FLL in vitiligo using network pharmacology analysis. The expression profiles of GSE65127 and GSE75819 were downloaded from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between the vitiligo and healthy samples. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs were performed using R analyses. We performed R to further understand the functions of the critical targets. Cytoscape tools have facilitated network topology analysis. Molecular docking was performed using Auto Dock Vina software. The results showed that 13 DEGs were screened in vitiligo. Based on bioinformatics, network pharmacology and Western blot, we found that the critical targets of melanoma antigen recognized by 5,6-dihydroxyindole-2-carboxylic acid oxidase (TYRP1) may be related to the mechanism of action of FLL in the treatment of vitiligo. TYRP1, as a melanocyte molecular biomarker, may be closely related to the underlying mechanism of FLL in the treatment of vitiligo via the inhibition of melanocyte death.

PRAME immunohistochemistry in soft tissue tumors and mimics: a study of 350 cases highlighting its imperfect specificity but potentially useful diagnostic applications.

Preferentially expressed antigen in melanoma (PRAME) immunohistochemistry is currently used in pathology for the assessment of melanocytic neoplasms; however, knowledge of its expression patterns in soft tissue tumors is limited. PRAME immunohistochemistry (clone QR005) was assessed on whole tissue sections of 350 soft-tissue tumors and mimics (> 50 histotypes). PRAME immunoreactivity was evaluated as follows: 0 "negative" (0% positive cells); 1+ (1-25% positive cells); 2+ (26-50% positive cells); 3+ (51-75% positive cells), and 4+ "diffuse" (> 75% positive cells). PRAME was expressed in 111 lesions (0 benign, 6 intermediate malignancy, and 105 malignant), including fibrosarcomatous dermatofibrosarcoma protuberans (2/4, 0 diffuse), NTRK-rearranged spindle cell neoplasm (2/4, 0 diffuse), atypical fibroxanthoma (1/7, 0 diffuse), Kaposi sarcoma (1/5, 0 diffuse), myxoid liposarcoma (11/11, 9 diffuse), synovial sarcoma (11/11, 6 diffuse), intimal sarcoma (7/7, 5 diffuse), biphenotypic sinonasal sarcoma (3/3, 1 diffuse), angiosarcoma (10/15, 6 diffuse), malignant peripheral nerve sheath tumor (9/12, 4 diffuse), pleomorphic rhabdomyosarcoma (2/3, 2 diffuse), alveolar rhabdomyosarcoma (2/6, 0 diffuse), embryonal rhabdomyosarcoma (7/7, 4 diffuse), undifferentiated pleomorphic sarcoma (2/12, 1 diffuse), leiomyosarcoma (2/15, 1 diffuse), clear cell sarcoma of soft tissue (1/10, 0 diffuse), low-grade fibromyxoid sarcoma (1/5, 0 diffuse), Ewing sarcoma (2/10, 1 diffuse), CIC-rearranged sarcoma (8/8, 4 diffuse), BCOR-sarcoma (2/5, 1 diffuse), melanoma (20/20, 14 diffuse), and thoracic SMARCA4-deficient undifferentiated tumor (5/5, all diffuse). All tested cases of spindle cell lipoma, dedifferentiated/pleomorphic liposarcoma, dermatofibrosarcoma protuberans, solitary fibrous tumor, inflammatory myofibroblastic tumor, myxoinflammatory fibroblastic sarcoma, nodular fasciitis, myxofibrosarcoma, epithelioid hemangioendothelioma, atypical vascular lesion, hemangioma, lymphangioma, vascular malformation, papillary endothelial hyperplasia, GIST, gastrointestinal clear-cell sarcoma, malignant melanotic nerve sheath tumor, neurofibroma, schwannoma, granular cell tumor, alveolar soft part sarcoma, epithelioid sarcoma, extraskeletal myxoid chondrosarcoma, myoepithelioma, ossifying fibromyxoid tumor, angiomatoid fibrous histiocytoma, PEComa, dermatofibroma, pleomorphic dermal sarcoma, and chordoma were negative. PRAME shows imperfect specificity in soft-tissue pathology but may serve as a diagnostic adjunct in selected differential diagnoses that show contrasting expression patterns.

MAGE-B4, a binding partner of PRAMEF12, is dispensable for spermatogenesis and male fertility in mice

Melanoma antigen (MAGE)-B4 belongs to the MAGE-B family genes, which are located on the X chromosome. The MAGE-B family genes are classified as cancer-testis antigens, as they are primarily expressed in the testis and are aberrantly expressed in most cancers. Although a no-stop mutation in MAGE-B4 causes rare X-linked azoospermia and oligozoospermia phenotype in humans, the specific function of MAGE-B4 on spermatogenesis in mice remains unclear. In this study, we identified MAGE-B4 as a binding partner of PRAME family member 12, which plays an important role in the maintenance of mouse spermatogenic lineage in juvenile testes. Additionally, we found that Mage-b4 transcripts were restricted to the testis and that Mage-b4 was specifically expressed in spermatogonia. To explore the function of MAGE-B4 in spermatogenesis, we generated a Mage-b4 knockout (KO) mouse model using CRISPR/Cas9 technology. However, we found that Mage-b4 KO males displayed normal testicular morphology and fertility. Further histological analysis revealed that all stages of spermatogenic cells were present in the seminiferous tubules of the Mage-b4 KO mice. Altogether, our data suggest that Mage-b4 is dispensable for mouse spermatogenesis and male fertility.

Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen

The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ Tcell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur invivo, opening up the possibility that high-affinity CD8+ Tcells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance.

MAGEB2-Mediated Degradation of EGR1 Regulates the Proliferation and Apoptosis of Human Spermatogonial Stem Cell Lines.

Spermatogonial stem cells are committed to initiating and maintaining male spermatogenesis, which is the foundation of male fertility. Understanding the mechanisms underlying SSC fate decisions is critical for controlling spermatogenesis and male fertility. However, the key molecules and mechanisms responsible for regulating human SSC development are not clearly understood. Here, we analyzed normal human testis single-cell sequencing data from the GEO dataset (GSE149512 and GSE112013). Melanoma antigen gene B2 (MAGEB2) was found to be predominantly expressed in human SSCs and further validated by immunohistology. Overexpression of MAGEB2 in SSC lines severely weakened cell proliferation and promoted apoptosis. Further, using protein interaction prediction, molecular docking, and immunoprecipitation, we found that MAGEB2 interacted with early growth response protein 1 (EGR1) in SSC lines. Reexpression of EGR1 in MAGEB2 overexpression cells partially rescued decreased cell proliferation. Furthermore, MAGEB2 was shown to be downregulated in specific NOA patients, implying that abnormal expression of MAGEB2 may impair spermatogenesis and male fertility. Our results offer new insights into the functional and regulatory mechanisms in MAGEB2-mediated human SSC line proliferation and apoptosis.

Preclinical evaluation of targeting PRAME with TCR mimic CAR T cells in AML.

2553 Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, but has had limited success against AML in part due to overlap of cell surface antigens expressed in AML and normal hematopoietic cells. To identify AML-restricted targets, we interrogated the transcriptome from over 3000 AML patients and found PRAME (Preferentially Expressed Antigen in Melanoma), an intracellular protein, to be a highly expressed AML-restricted target. Using a novel approach to target intracellular antigens, we developed a PRAME CAR T cell using a TCR mimic (mTCR) antibody, which recognizes the PRAME peptide/HLA-A2 complex on the tumor cell surface. To conduct final IND-enabling studies, we generated an in vivo patient derived xenograft (PDX) model to treat with PRAME mTCRCAR T cells. Here we demonstrate the in vivo activity of PRAME mTCRCAR T cells against a PDX AML model. Methods: We used the VL and VH sequences from the PRAME TCR mimic antibody (Pr20) to construct the single chain fragment variable domain into the 41-BB/CD3ζ CAR vector. The PDX model was derived from a PRAME+/HLA-A2+ pediatric patient with AML. PDX cells were transduced with ffluciferase for noninvasive bioluminescent IVIS imaging to monitor leukemic progression. PDX leukemia-bearing mice were treated with unmodified T-cells or PRAME mTCRCAR T cells at 5x106 cells (1:1 CD4:CD8) per mouse 1 week after leukemia injection. Leukemia burden was measured by IVIS imaging and peripheral blood analysis. Results: Treatment with PRAME mTCRCAR T cells led to eradication of leukemia with all mice alive >100 days post-treatment, while control mice (unmodified T cells) had disease progression at Day 50 (p=0.001). Average leukemia burden in the control cohort was 3.5% at week 6 and 41.75% at week 11, while no leukemia was detected following CAR T cell treatment. In addition, the control arm had a marked expansion of leukemia with a 7.75 and 429.6 fold increase in radiance by IVIS at 6 and 11 weeks post-treatment. No significant increase in radiance was seen in the PRAME mTCRCAR T cell treated group. Human T cells (CD45+/CD3+) were detectable in the peripheral blood at Day 7 and 14 post-treatment in both the unmodified and CAR T cell treated groups. Conclusions: We demonstrate the therapeutic potential of targeting PRAME with mTCRCAR T cells in AML. We show potent efficacy with eradication of leukemia in PDX-bearing mice following treatment with PRAME mTCRCAR T cells resulting in prolonged survival. These results provide a novel approach to target PRAME with CAR T cells and provide compelling data to evaluate PRAME mTCRCAR T cells for use in clinical trials against AML. [Table: see text]

Chemical features of melanoma tumor resident TRG CDR3s associated with better survival probabilities.

We assessed the T-cell receptor gamma (TRG) recombination reads from the cancer genome atlas melanoma tumor exome files and the TRG recombination reads from an independent, melanoma exome file dataset, from the Moffitt Cancer Center. TRG complementarity determining region-3 (CDR3) amino acid (AA) sequences were assessed for chemical complementarity to cancer testis antigens, with such complementarity for FAM133A and CRISP2 associated with better survival probabilities for both datasets. These results, along with related TRG CDR3 AA chemical feature assessments provided in this report, have indicated opportunities for melanoma patient stratifications based on the recovery of TRG recombination reads from both tumor and blood samples, and the results may point towards novel, effective melanoma antigens.

Immunohistochemical expression of PRAME is a marker of poor prognosis in uveal melanoma: A clinico-pathologic and immunohistochemical study on a series of 85 cases

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, first isolated in tumor-reactive T-cell clones from a metastatic melanoma patient. It has been widely studied in skin pathology as an immunohistochemical marker capable of distinguishing between benign nevi and malignant melanomas. PRAME has been found to be also expressed in non-melanocytic tumors, including lung, breast, kidney and ovarian cancer. However, less is known about the diagnostic and/or prognostic role of this protein in uveal melanoma (UM); few studies have reported that PRAME expression seems to give to UM patients an additional metastatic risk beyond the other already-known prognostic parameters. In the present retrospective study, we aimed to correlate PRAME immunoreactivity to other clinico-pathologic features and follow-up data on a large series of 85 cases (45 non-metastasizing and 40 metastasizing tumors) of primary UM. A statistically significant correlation was found between PRAME expression and higher metastatic risk and lower metastasis-free survival. We propose to include PRAME in the immunohistochemical panel of UM as an easily usable marker capable of predicting higher metastatic risk and stratifying patients’ outcome.

Diagnostic utility of PRAME immunohistochemistry in PEComa family of tumors and morphologic mimics with emphasis on the gynecologic tract

Perivascular epithelioid cell tumors (PEComas), rare mesenchymal tumors with myomelanocytic differentiation, can be a diagnostic challenge, often requiring a panel of immunohistochemicalmarkers. Preferentially expressed antigen in melanoma (PRAME) is a relatively new antigen with utility in diagnosing melanomas. This study aimed to survey PRAME expression patterns in the PEComa family of tumors and morphologic mimics. Twenty cases of PEComas and 27 non-PEComas (10 leiomyosarcomas, 3 smooth muscle tumors of uncertain malignant potential [STUMPs], 11 leiomyomas, 1 uterine inflammatory myofibroblastic tumor [IMT], and 2 low-grade endometrial stromal sarcomas [LGESSs]) were stained with PRAME and compared to previously performed HMB45 and Melan-A stains, when available. Tumors showing no or barely perceptible PRAME staining at 10× were considered negative. Tumors were considered positive if there was full nuclear staining evident at 10× in at least one 10× field. Diffuse staining was defined as positivity in at least 80% of tumor nuclei. Overall, PRAME was expressed in 70% of PEComas, with diffuse positivity in 60%. However, PRAME was not specific for PEComas, with immunopositivity in the majority (70%) of uterine leiomyosarcoma cases, though negative in STUMP, leiomyoma, IMT, and LGESS cases. PRAME sensitivity was 70% and specificity was 74%, while HMB45 was more sensitive (90%) and specific (100%), but only 15% of PEComas showed diffuse staining. Melan-A staining was less common than HMB45 or PRAME, with only 18.8% sensitivity but 100% specificity. Among gynecologic PEComas, PRAME was expressed in 75% overall and enriched among malignant cases (85.7% positive). As part of an immunohistochemical panel, PRAME could be useful in the workup of PEComa cases. In the future, PRAME-specific immunotherapies may be beneficial in treating patients with malignant PEComas.

Melanoma Antigen Recognized by T Cells With Blue Chromogen Improves Identification of Melanocytes From Background Melanized Keratinocytes on Frozen Sections.

Fazio, Justin MD, FAAD*; Heras, Alfonso DVM, PhD†; Stein, Eric HT(ASCP)‡; Zitelli, John MD, FAAD, FACMS§,‖,¶ Author Information

Cancer testis antigen subfamilies: Attractive targets for therapeutic vaccine (Review).

Cancer‑testis antigen (CTA) is a well‑accepted optimal target library for cancer diagnosis and treatment. Most CTAs are located on the X chromosome and aggregate into large gene families, such as the melanoma antigen, synovial sarcoma X and G antigen families. Members of the CTA subfamily are usually co‑expressed in tumor tissues and share similar structural characteristics and biological functions. As cancer vaccines are recommended to induce specific antitumor responses, CTAs, particularly CTA subfamilies, are widely used in the design of cancer vaccines. To date, DNA, mRNA and peptide vaccines have been commonly used to generate tumor‑specific CTAs invivo and induce anticancer effects. Despite promising results in preclinical studies, the antitumor efficacy of CTA‑based vaccines is limited in clinical trials, which may be partially attributed to weak immunogenicity, low efficacy of antigen delivery and presentation processes, as well as a suppressive immune microenvironment. Recently, the development of nanomaterials has enhanced the cancer vaccination cascade, improved the antitumor performance and reduced off‑target effects. The present study provided an in‑depth review of the structural characteristics and biofunctions of the CTA subfamilies, summarised the design and utilisation of CTA‑based vaccine platforms and provided recommendations for developing nanomaterial‑derived CTA‑targeted vaccines.

MAGED4B is a Poor Prognostic Marker of Stomach Adenocarcinoma and a Potential Therapeutic Target for Stomach Adenocarcinoma Tumorigenesis.

Gastric cancer is the second most common cause of cancer death worldwide with poor overall prognosis. It is important to study the molecular mechanism of stomach adenocarcinoma (STAD). MAGED4B, a member of the melanoma antigen gene (MAGE) family, is highly expressed in many tumor cells and is associated with tumor progression. Its prognostic value in and the function of the encoded protein are still unclear. The data of 415 STAD tissues was retrieved from TCGA database, and the expression level of MAGED4B mRNA was evaluated. The correlation between the expression of MAGED4B mRNA and the progression free survival (PFS) time of STAD patients was evaluated by Kaplan Meier analysis. The STAD cell lines with overexpressed and silent MAGED4B were constructed, and the effects of MAGED4B on the viability, migration and proliferation were evaluated by the CCK-8, scratch test and EDU test. The flow cytometry was used to detect apoptosis with overexpressed and silent MAGED4B under the cisplatin treatment, and WB was used to detect the expressions of related proteins, such as TNF-α. The expression level of MAGED4B mRNA in the STAD tissues was higher than that in the normal tissues, and its high expression was related to poor PFS. The overexpression of MAGED4B in the STAD cell lines can promote the vitality, motility and proliferation of the STAD cells, while the silencing of MAGED4B can inhibit the above three cell functions of the STAD cells. The overexpression of MAGED4B can reduce the cisplatin induced apoptosis and increase the cisplatin IC50; the silencing of MAGED4B can promote the cisplatin induced apoptosis and reduce the cisplatin IC50. The overexpression of MAGED4B reduced the protein levels of TRIM27 and TNF- α. MAGED4B could be a valuable prognostic biomarker and a therapeutic target for gastric adenocarcinoma of great interest.

Heterologous prime-boost viral vector cancer vaccines have protective effects against a syngeneic mouse model of glioblastoma

Abstract Glioblastomas are immunologically “cold” tumors with intense immunosuppressive myeloid cell- and sparse cytotoxic T-cell infiltration. Therefore, viral vector vaccines may be a promising approach to boost the induction of glioblastoma-targeted T cells. It has been previously shown that heterologous prime-boost vaccination with chimpanzee-derived adenovirus ChAdOx1 and modified vaccinia Ankara (MVA) vectors can induce a high magnitude of CD8 +T cells specific for cancer-associated antigens and have therapeutic effects against mouse models of cancer. Therefore, we aimed to evaluate whether using ChAdOx1 and MVA vaccines targeting model antigen P1A, a mouse equivalent to human tumor-associated Melanoma Antigen GenE (MAGE)-type antigens, could have protective effects against a P1A-transfected BGL-1 glioblastoma model. Mice were vaccinated with ChAdOx1/MVA-P1A vaccines, then challenged subcutaneously with P1A-expressing BGL-1 cells. Prophylactic vaccination with ChAdOx1/MVA-P1A significantly prolonged the survival of syngeneic mice subcutaneously challenged with P1A-expressing BGL-1 tumors. Furthermore, different vaccination schedules significantly impacted the magnitude of antigen-specific CD8 +T-cell responses and protective efficacy. Future studies will investigate the protective effects of vaccination against intracranial P1A-expressing BGl-1 tumors. Supported by grants from the Ludwig Institute for Cancer Research, University of Oxford; the NIH Intramural Program (ZIA BC011877), the NIH Oxford-Cambridge Scholars Program, and the Marshall Scholarship Program.

Potential diagnostic utility of PRAME and p16 immunohistochemistry in melanocytic nevi and malignant melanoma.

PRAME (PReferentially expressed Antigen in MElanoma) is a tumor-associated antigen that has been studied in various cutaneous melanocytic lesions. p16, on the other hand, has been proposed to aid in distinguishing between benign and malignant melanocytic neoplasms. Studies on the diagnostic utility of PRAME and p16 in combination in differentiating nevi from melanoma are limited. We aimed to assess the diagnostic utility of PRAME and p16 in melanocytic tumors and their role in distinguishing between malignant melanomas and melanocytic nevi. This is a single-center retrospective cohort analysis over a 4-year period (2017-2020). We used the pathological database of malignant melanomas (77 cases) and melanocytic nevi (51 cases) specimens from patients who underwent shave/punch biopsies or surgical excisions and evaluated immunohistochemical staining percentage positivity and intensity for PRAME and p16. Most malignant melanomas showed positive/diffuse PRAME expression (89.6%); on the other hand, 96.1% of nevi did not express PRAME diffusely. p16 was expressed consistently in nevi (98.0%). However, p16 expression in malignant melanoma was infrequent in our study. PRAME had a sensitivity and specificity of 89.6% and 96.1%, respectively, for melanomas versus nevi; on the other hand, p16 had a sensitivity and specificity of 98.0% and 28.6%, respectively, for nevi versus melanoma. Also, a PRAME+/p16- melanocytic lesion is unlikely to be a nevus where most nevi were PRAME-/p16+. In conclusion, we confirm the potential utility of PRAME and p16 for distinguishing melanocytic nevi from malignant melanomas.

PRAME Is a Novel Target of Tumor-Intrinsic Gas6/Axl Activation and Promotes Cancer Cell Invasion in Hepatocellular Carcinoma.

(1) Background: Activation of the receptor tyrosine kinase Axl by Gas6 fosters oncogenic effects in hepatocellular carcinoma (HCC), associating with increased mortality of patients. The impact of Gas6/Axl signaling on the induction of individual target genes in HCC and its consequences is an open issue. (2) Methods: RNA-seq analysis of Gas6-stimulated Axl-proficient or Axl-deficient HCC cells was used to identify Gas6/Axl targets. Gain- and loss-of-function studies as well as proteomics were employed to characterize the role of PRAME (preferentially expressed antigen in melanoma). Expression of Axl/PRAME was assessed in publicly available HCC patient datasets and in 133 HCC cases. (3) Results: Exploitation of well-characterized HCC models expressing Axl or devoid of Axl allowed the identification of target genes including PRAME. Intervention with Axl signaling or MAPK/ERK1/2 resulted in reduced PRAME expression. PRAME levels were associated with a mesenchymal-like phenotype augmenting 2D cell migration and 3D cell invasion. Interactions with pro-oncogenic proteins such as CCAR1 suggested further tumor-promoting functions of PRAME in HCC. Moreover, PRAME showed elevated expression in Axl-stratified HCC patients, which correlates with vascular invasion and lowered patient survival. (4) Conclusions: PRAME is a bona fide target of Gas6/Axl/ERK signaling linked to EMT and cancer cell invasion in HCC.

Abstract LB027: In vivo delivery of novel CD89 fusion receptor to myeloid cells by mRNA activates anti-tumor immunity

Abstract Despite the revolution provided by immunotherapy, sustained clinical benefit has yet to be achieved for most of patients with advanced solid tumors. Myeloid cells readily accumulate in tumors, in some cases contributing up to 75% of the tumor mass. While supporting tumor growth and being associated with unfavorable prognosis, myeloid cells can mediate phagocytosis of cancer cells and cytotoxic tumor killing and promote adaptive immune responses through antigen presentation and co-stimulation, therefore representing promising targets in cancer therapy. The ability to engineer circulating and tumor associated myeloid cells to activate their ability to elicit anti-tumor adaptive immunity is an attractive approach to harness systemic anti-tumor immunity. However, it remains challenging to specifically target and activate myeloid cells in vivo. To overcome this hurdle, we have developed a novel in vivo myeloid cell engineering platform in which a chimeric antigen receptor (CAR) is generated by fusing a tumor recognition scFv with the alpha chain of human Fc receptors (CD89). The stable expression and function of these receptors requires the endogenously expressed common Fc receptor gamma chain (FcRγ), which expression is mostly restricted to myeloid cells. For in vivo engineering, the construct is encapsulated and delivered in lipid nanoparticles (LNP). Trophoblast cell surface antigen 2 (TROP2) is overexpressed in most human solid epithelial cancers, as compared to low expression in corresponding normal tissue. Increased TROP2 expression has been linked to increased tumor growth and has been implicated as a prognostic marker in these cancers, supporting the development of therapies targeting TROP2. Intravenous infusion of LNP encapsulating mRNA encoding the anti-TROP2-CD89 fusion protein results in the uptake of the LNPs and expression of the chimeric receptor fusion protein in myeloid cells. In immunodeficient xenograft models of hepatocellular carcinoma and triple negative breast cancer, delivery of LNP mRNA encoding GPC3-CD89 or TROP2-CD89 fusion proteins resulted in anti-tumor efficacy, confirming the ability of this approach to program myeloid cells. Repeat dosing studies showed significant anti-tumor efficacy following bi-weekly administration of TROP2-CD89. Furthermore, in the B16/10 syngeneic melanoma model, treatment with the melanoma antigen gp75-CD89 fusion protein was also associated with the initiation of broad systemic immune responses, characterized by tumor infiltration by activated CD8+ T cells, reduced tumor-associated Tregs and activation of antigen presenting cells in spleen. When infused in cynomolgus monkeys, TROP2-CD89 LNP led to cell surface expression of anti-TROP2 CAR in myeloid cells. Together these studies highlight the potential of in vivo delivery of CD89 fusion proteins to program myeloid cells to recognize and kill cancer. Citation Format: Thomas E. Prod'homme, Shannon Argueta, Hongyun Zhao, Michael Gorgievski, Neha Diwangi, Edward Cochran, Bruce McCreedy, Yuxiao Wang, Daniel Getts. In vivo delivery of novel CD89 fusion receptor to myeloid cells by mRNA activates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB027.

Abstract CT207: Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma

Abstract Oncologic viruses (OV) can induce tumor death through both direct oncolysis and immune-mediated destruction. Our study investigates the use of a novel OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-b) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNb-TYRP1). VSV is an attractive vector since humans are not naturally infected with VSV; thus, pre-existing immunity is minimal. IFN-b leads to increased T-cell responses and tumor cell apoptosis, while TYRP1, a melanocyte differentiation antigen, increases immunogenicity of the therapy. We conducted a Phase 1 clinical trial with a 3+3 design in patients with metastatic uveal melanoma. VSV-IFNb-TYRP1 was injected into a liver metastasis under image guidance, then administered on the same day as a single intravenous (IV) infusion. IV doses started at dose level (DL) 1 1 × 1010 TCID50, then escalated to DL2 3 × 10 10, DL3 1 × 1011, and DL4 3 × 1011. The dose delivered intratumorally (IT) varied based on the IV dose. The primary endpoints were safety and maximum tolerated dose (MTD). Efficacy was a secondary endpoint. Correlative studies focused on understanding viral pharmacokinetics (PK) and immunological responses induced by VSV-IFNb-TYRP1 therapy. Twelve patients with previously treated metastatic uveal melanoma were enrolled. Median follow up was 19.1 months. Four DLs were evaluated with one patient at DL4 experiencing dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). Non-DLTs at DL4 were fatigue, fever, CRS, hematological toxicities (decreased platelets and lymphocytes), and AST elevation. Four patients had stable disease (SD) and 8 had progressive disease (PD) as their best response. ELIspot data show that three of the four patients with response to TYRP1 also had a response to gp100, suggesting possible epitope spreading. These four patients received immune checkpoint inhibitor treatment as the next line of therapy, which led to clinical benefit for two patients. Our study evaluated VSV-IFNb-TYRP1 administered via IT and IV routes in a previously treated population of metastatic uveal melanoma patients and found no major safety events. Although there were no clear objective responses to VSV-IFNb-TYRP1, dose-dependent immunogenicity to melanoma antigens was seen. While OV response alone is not sufficient for clinical benefit, the evidence of epitope spreading is encouraging. Future studies of VSV-IFNb-TYRP1 will evaluate combinations with other therapies. Citation Format: Katherine E. Smith, Adam Weisbrod, Carrie Strand, Jacob Allerd, Jose S. Pulido, Alysha Newsom, Lianwen Zhang, Nandakumar Packiriswamy, Heather Montane, Lisa Kottschade, Anastasios Dimou, Yiyi Yan, Markovic Svetomir, Kah Whye Peng, Richard G. Vile, Matthew S. Block, Roxana S. Dronca. Phase I trial of vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 (VSV-IFNb-TYRP1) in metastatic ocular melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT207.

Hyaluronic acid-antigens conjugates trigger potent immune response in both prophylactic and therapeutic immunization in a melanoma model.

Immunotherapy of advanced melanoma has encountered significant hurdles in terms of clinical efficacy. Here, we designed a clinically translatable hyaluronic acid (HA)-based vaccine delivering a combination of major histocompatibility complex (MHC) class I- and class II-restricted melanoma antigens (TRP2 and Gp100, respectively) conjugated to HA. HA-nanovaccine (HA-TRP2-Gp100 conjugate) exhibited tropism in the lymph nodes and promoted stimulation of the immune response (2.3-fold higher than the HA+TRP2+Gp100). HA-nanovaccine significantly delayed the growth of B16F10 melanoma and extended survival in both the prophylactic and therapeutic settings (median survival of 22 and 27, respectively, vs 17days of the untreated group). Moreover, mice prophylactically treated with the HA-nanovaccine displayed significantly higher CD8+ and CD4+ T-cell/Treg ratios in both the spleen and tumor at day 16, suggesting that the HA-nanovaccine overcame the immunosuppressive tumor microenvironment. Superior infiltration of active CD4+ and CD8+ T cells was observed at the endpoint. This study supports the conclusion that HA potentiates the effect of a combination of MHC I and MHC II antigens via a potent immune response against melanoma.

Abstract 170: Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens

Abstract Introduction: Glioblastoma is resistant to standard treatment, leading to tumor recurrence and early death of patients. Using organoid models, we aim to explore the mechanisms of resistance to cancer therapies and identify novel therapeutic vulnerabilities, including stress protecting melanoma-associated antigens (MAGEs). MAGEs are normally restricted to expression in testis but are abnormally activated in cancer and are associated with therapy resistance. Methods: Organoids have been established from fresh tumor biopsies and are stored in Gliobank, a Slovenian collection of patient tumor samples with corresponding clinical data. Organoids were first characterized and compared with the corresponding parental tumor tissue by immunofluorescence and qPCR of selected markers. Organoids were then treated with a combination of irradiation, the chemotherapeutic agent temozolomide and/or a chemokine receptor antagonist, and response to therapy was assessed by immunofluorescence, qPCR, cell viability and invasion assays, multiplex ELISA and proximity ligation assay. Results: Organoids recapitulated the gene expression profile of parental glioblastoma tissues, including expression of genes related to cancer stem cells, DNA damage response, cell cycle progression, epithelial- to- mesenchymal transition, and cytokine signaling. In addition, the organoids preserved the cellular composition of the parental glioblastoma tissues, consisting of glioblastoma (stem) cells, macrophages, microglia, lymphocytes, and endothelial cells. Standard treatment decreased viability and invasion of organoids in only 2 of 6 patients, suggesting that organoids recapitulate tumor therapy resistance. The increased expression of MDM2 and CDKN1A in organoids after treatment with irradiation and temozolomide suggests that the p53 pathway and DNA damage response mechanisms may contribute to the therapy resistance. Next, we detected high abundance of secreted cytokines in the culture medium of the organoids, including CXCL12. However, treatment of the organoids with the CXCR4 antagonist Plerixafor, which blocked the interactions between CXCL12 and CXCR4 in the organoids, had no effect on organoid viability and invasion, suggesting resistance of glioblastoma to this immunotherapy. Since high expression of several type I MAGEs correlates with poor prognosis of glioma patients based on TCGA database analysis, we investigate the role of MAGEs in therapy resistance of glioblastoma organoids. Conclusions: Patient-derived tumor organoids provide a valuable tool to 1) identify novel therapeutic vulnerabilities in the context of the tumor microenvironment, 2) evaluate the effect of novel candidate treatments including immunotherapy, and 3) discover novel markers of therapy resistance. Citation Format: Barbara Breznik, Bernarda Majc, Anamarija Habič, Gloria Krapež, Andrej Porčnik, Jernej Mlakar, Tamara Lah Turnšek, Metka Novak, Klementina Fon Tacer. Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens [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 170.

Abstract 3469: Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immune tolerance

Abstract The majority of cancer-associated deaths result from distant organ metastasis, yet the mechanisms that enable this process remain poorly understood. For most solid tumors, colonization of regional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it remains unclear whether LN metastasis plays a functional role in disease progression. LNs are major sites of anti-tumor lymphocyte education, including in the context of immunotherapy, yet LN metastasis frequently correlates with further disease progression. Here, we find that LN metastasis represents a critical step in tumor progression through the capacity of such metastases to induce tumor-specific immune tolerance in a manner that promotes further dissemination of tumors to distant organs. Using an in vivo passaging approach of a non-metastatic syngeneic melanoma, we generated 300 unique cell lines exhibiting varying degrees of LN metastatic capacity. We show that the presence of these LN metastases enables distant organ seeding of metastases in a manner that the parental tumor cannot, and this effect is eliminated in mice lacking an adaptive immune response. Furthermore, this promotion of distant seeding by LN metastases is tumor specific. Using flow cytometry and single-cell sequencing to perform comprehensive immune profiling, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs). Furthermore, depletion of NK cells in vivo enables non-metastatic tumors to disseminate to LNs, and ablation of Tregs using FoxP3-DTR mice eliminates the occurrence of lymphatic metastases. Adoptive transfer of Tregs from the LNs of mice bearing LN metastasis to naïve mice facilitates metastasis in a manner that Tregs from mice without LN metastases cannot, and we find that these Tregs are induced in an antigen-specific manner. Whole exome sequencing revealed that neither the metastatic proclivity nor immunosuppression evolve through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I presentation, or decreases in melanoma antigen expression. Rather, by RNA-seq and ATAC-seq, we show that a conserved interferon signaling axis is upregulated in LN metastases and is rendered stable through epigenetic reprogramming of chromatin accessibility resulting from chronic exposure to interferons in vivo. Furthermore, using CRISPR/Cas9, we find that these pathways are required for LN metastatic seeding, and validate their conserved significance in additional mouse models of pancreatic ductal adenocarcinoma and head and neck squamous cell carcinoma and humans with LN metastatic disease. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immunosuppression. Citation Format: Nathan E. Reticker-Flynn, Weiruo Zhang, Julia A. Belk, Pamela A. Basto, Andrew J. Gentles, John B. Sunwoo, Ansuman T. Satpathy, Sylvia K. Plevritis, Edgar G. Engleman. Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immune tolerance [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 3469.