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Immunization With the CSF-470 Vaccine Plus BCG and rhGM-CSF Induced in a Cutaneous Melanoma Patient a TCR? Repertoire Found at Vaccination Site and Tumor Infiltrating Lymphocytes That Persisted in Blood.

ABSTRACT: The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-?2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCR? repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-? ELISPOT assay, were performed to analyze the T-cell immune response dynamics throughout the immunization protocol. Histopathological analysis of the VAC-SITE revealed a highly-inflamed granulomatous structure encircled by CD11c+ nested-clusters, brisk CD8+ and scarce FOXP3+, lymphocytes with numerous Langhans multinucleated-giant-cells and macrophages. A large tumor-regression area fulfilled the C-MTS with brisk lymphocyte infiltration, mainly composed of CD8+PD1+ T-cells, CD20+ B-cells, and scarce FOXP3+ cells. Increasing DTH score and IFN-? ELISPOT assay signal against the CSF-470 vaccine-lysate was evidenced throughout immunization. TCR? repertoire analysis revealed for the first time the presence of common clonotypes between a VAC-SITE and a C-MTS; most of them persisted in blood by the end of the immunization protocol. In vitro boost with vaccine-lysate revealed the expansion of persistent clones that infiltrated the VAC-SITE and/or the C-MTS; other persistent clones expanded in the patient's blood as well. We propose that expansion of such persistent clonotypes might derive from two different although complementary mechanisms: the proliferation of specific clones as well as the expansion of redundant clones, which increased the number of nucleotide rearrangements per clonotype, suggesting a functional antigenic selection. In this patient, immunization with the CSF-470 vaccine plus BCG and rhGM-CSF induced a T-cell repertoire at the VAC-SITE that was able to infiltrate an emerging C-MTS, which resulted in the expansion of a T-cell repertoire that persisted in blood by the end of the 2-year treatment.

SUBMITTER: Aris M

PROVIDER: S-EPMC6759869 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Immunization With the CSF-470 Vaccine Plus BCG and rhGM-CSF Induced in a Cutaneous Melanoma Patient a TCRβ Repertoire Found at Vaccination Site and Tumor Infiltrating Lymphocytes That Persisted in Blood.

Aris Mariana M Bravo Alicia Inés AI Garcia Alvarez Heli Magalí HM Carri Ibel I Podaza Enrique E Blanco Paula Alejandra PA Rotondaro Cecilia C Bentivegna Sofia S Nielsen Morten M Barrio María Marcela MM Mordoh José J

Frontiers in immunology 20190918

The CSF-470 cellular vaccine plus BCG and rhGM-CSF increased distant metastases-free survival in cutaneous melanoma patients stages IIB-IIC-III relative to medium dose IFN-α2b (CASVAC-0401 study). Patient-045 developed a mature vaccination site (VAC-SITE) and a regional cutaneous metastasis (C-MTS), which were excised during the protocol, remaining disease-free 36 months from vaccination start. CDR3-TCRβ repertoire sequencing in PBMC and tissue samples, along with skin-DTH score and IFN-γ ELISPO ...[more]

PMID: 31620131

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Project description:The allogeneic therapeutic vaccine CSF-470 has demonstrated a significant benefit over medium-dose IFN?2b in the distant metastasis-free survival for stages IIB-IIC-III cutaneous melanoma patients in a randomized phase II/III clinical trial (CASVAC-0401, NCT01729663). At the end of the 2-year CSF-470 immunization protocol, patient #006 developed several lung and one subcutaneous melanoma metastases; this later was excised. In this report, we analyzed the changes throughout vaccination of immune populations in blood and in the tumor tissue, with special focus on the T-cell repertoire. Immunohistochemistry revealed a marked increase in CD8+, CD4+, and CD20+ lymphocytes infiltrating the metastasis relative to the primary tumor. Lymphocytes were firmly attached to dying-tumor cells containing Granzyme-B granules. Whole-exon sequencing assessment indicated a moderate-to-high tumor mutational burden, with BRAFV600E as the main oncogenic driver. Mutational signature presented large numbers of mutations at dipyrimidines, typical of melanoma. Relevant tumor and immune-related genes from the subcutaneous metastasis were addressed by RNA-Seq analysis, revealing expression of typical melanoma antigens and proliferative tumor-related genes. Stimulatory and inhibitory immune transcripts were detected as well as evidence of active T-cell effector function. Peripheral blood monitoring revealed an increase in CD4+ and CD8+ cells by the end of the immunization protocol. By CDR3-T-cell receptor ? (TCR?) sequencing, generation of new clones and an increase in oligoclonality was observed in the peripheral T-cells immune repertoire throughout immunization. A shift, with the expansion of selected preexisting and newly arising clones with reduction of others, was detected in blood. In tumor-infiltrating lymphocytes, prevalent clones (50%) were both new and preexisting that were expanded in blood following CSF-470 immunization. These clones persisted in time, since 2?years after completing the immunization, 51% of the clones present in the metastasis were still detected in blood. This is the first report of the modulation of the TCR? repertoire from a melanoma patient immunized with the CSF-470 vaccine. After immunization, the changes observed in peripheral immune populations as well as in the tumor compartment suggest that the vaccine can induce an antitumor adaptive immune repertoire that can reach tumor lesions and persists in blood for at least 2?years.

Project description:The CSF-470 vaccine consists of lethally-irradiated allogeneic cells derived from four cutaneous melanoma cell lines administered plus BCG and GM-CSF as adjuvants. In an adjuvant phase II study vs. IFN-α2b, the vaccine significantly prolonged the distant metastasis-free survival (DMFS) of stages IIB-IIC-III melanoma patients with evidence of the induction of immune responses against vaccine cells. Purpose: The aim of this study was to analyze the antigens against which the immune response was induced, as well as the T-helper profile and lytic ability of immune cells after CSF-470 treatment. Methods: HLA-restricted peptides from tumor-associated antigens (TAAs) were selected from TANTIGEN database for 13 evaluable vaccinated patients. In addition, for patient #006 (pt#006), tumor somatic variants were identified by NGS and candidate neoAgs were selected by predicted HLA binding affinity and similarity between wild type (wt) and mutant peptides. The patient's PBMC reactivity against selected peptides was detected by IFNγ-ELISPOT. T-helper transcriptional profile was determined by quantifying GATA-3, T-bet, and FOXP3 mRNA by RT-PCR, and intracellular cytokines were analyzed by flow cytometry. Autologous tumor cell lysis by PBMC was assessed in an in vitro calcein release assay. Results: Vaccinated patient's PBMC reactivity against selected TAAs derived peptides showed a progressive increase in the number of IFNγ-producing cells throughout the 2-yr vaccination protocol. ELISPOT response correlated with delayed type hypersensitivity (DTH) reaction to CSF-470 vaccine cells. Early upregulation of GATA-3 and Foxp3 mRNA, as well as an increase in CD4+IL4+cells, was associated with a low DMFS. Also, IFNγ response against 9/73 predicted neoAgs was evidenced in the case of pt#006; 7/9 emerged after vaccination. We verified in pt# 006 that post-vaccination PBMC boosted in vitro with the vaccine lysate were able to lyse autologous tumor cells. Conclusions: A progressive increase in the immune response against TAAs expressed in the vaccine and in the patient's tumor was induced by CSF-470 vaccination. In pt#006, we demonstrated immune recognition of patient's specific neoAgs, which emerged after vaccination. These results suggest that an initial response against shared TAAs could further stimulate an immune response against autologous tumor neoAgs.

Project description:High levels of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) are associated with a survival benefit in various cancer types and the targeted (re)activation of TILs is an attractive therapeutic anti-cancer approach that yields curative responses. However, current T cell targeting strategies directed at known immune checkpoints have not increased objective response rates for all cancer types, including for epithelial ovarian cancer (EOC). For this reason, the identification of new immune checkpoints that regulate T cell immunity remains of great interest. One yet largely uninvestigated checkpoint of potential interest is the G protein-coupled receptor 56 (GPR56), which belongs to the adhesion GPCR family. GPR56 was originally reported to function in cerebral cortical development and in anti-depressant response, but also in cancer. Recently, GPR56 was identified as an inhibitory receptor expressed on human NK cells that by cis-interaction with the tetraspanin CD81 attenuated the cytotoxic activity of NK cells. This NK cell checkpoint could be blocked by an GPR56 antibody, leading to increased cytotoxicity. Interestingly, GPR56 expression has also been reported on cytokine producing memory CD8 T lymphocytes and may thus represent a T cell checkpoint as well. Here, GPR56 mRNA expression was characterized in the context of TILs, with GPR56 expression being detected predominantly in tumor infiltrating CD8 T cells with a cytotoxic and (pre-)exhausted phenotype. In accordance with this mRNA profile, TILs from ovarian cancer patients expressed GPR56 primarily within the effector memory and central memory T cell subsets. On T cells from healthy donors the expression was limited to effector memory and terminally differentiated T cells. Notably, GPR56 expression further increased on TILs upon T cell receptor (TCR)-mediated stimulation in co-cultures with cancer cells, whereas GPR56 expression on healthy primary human T cells did not. Further, the ectopic expression of GPR56 significantly reduced the migration of GPR56-positive T cells. Taken together, GPR56 is a potential immune-checkpoint in EOC found on (pre-)exhausted CD8 TILs that may regulate migratory behavior.

Dataset Information

Immunization With the CSF-470 Vaccine Plus BCG and rhGM-CSF Induced in a Cutaneous Melanoma Patient a TCR? Repertoire Found at Vaccination Site and Tumor Infiltrating Lymphocytes That Persisted in Blood.

Publications

Immunization With the CSF-470 Vaccine Plus BCG and rhGM-CSF Induced in a Cutaneous Melanoma Patient a TCRβ Repertoire Found at Vaccination Site and Tumor Infiltrating Lymphocytes That Persisted in Blood.

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