LOXHD1 is an oncofusion-regulated antigen of ewing sarcoma

Blanchard, Tatiana; Faridi, Pouya; Xu, Chong; Bear, Adham S.; Rasool, Reyaz ur; Huang, Grace; Lim, Terry C. C.; Ayala, Rochelle; Gabunia, Khatuna; Ji, Mei; Posey, Avery D.; Scholler, John; Asangani, Irfan A.; Purcell, Anthony W.; Linette, Gerald P.; June, Carl H.; Carreno, Beatriz M.

doi:10.1038/s41598-025-96877-9

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Published: 15 April 2025

LOXHD1 is an oncofusion-regulated antigen of ewing sarcoma

Tatiana Blanchard¹,
Pouya Faridi^2,3,4,5,
Chong Xu⁶,
Adham S. Bear^7,12,
Reyaz ur Rasool⁸,
Grace Huang^2,3,
Terry C. C. Lim^2,3,4,
Rochelle Ayala⁵,
Khatuna Gabunia⁶,
Mei Ji⁶,
Avery D. Posey Jr^1,6,9,
John Scholler⁶,
Irfan A. Asangani^8,10,11,
Anthony W. Purcell⁵,
Gerald P. Linette^6,7,12,14,
Carl H. June^{6,10,12,13,14} &
…
Beatriz M. Carreno^6,13,14

Scientific Reports volume 15, Article number: 13007 (2025) Cite this article

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Abstract

Ewing Sarcoma (EwS) is a rare pediatric malignancy characterized by a unique t(11:22) (q24;q12) translocation resulting in the pathognomonic EWSR1::FLI1 fusion. Recent reports indicate that the EWSR1::FLI1 oncofusion drives aberrant expression of numerous transcripts, including Lipoxygenase Homology Domains 1 (LOXHD1). Given its highly restricted protein expression pattern and role in EwS tumorigenesis and metastasis, LOXHD1 may serve as a novel immunotherapeutic target in this malignancy. LOXHD1 immunogenic epitopes restricted to HLA-A*02:01 allowed for the isolation of a high avidity αβTCR. LOXHD1-specific TCR engineered CD8⁺ T cells conferred cytotoxic activity against a panel of HLA-A*02:01⁺ EwS tumor cell lines and adoptive transfer led to tumor eradication in a mouse xenograft model of EwS. This study nominates LOXHD1 as an oncofusion regulated, non-mutated tumor associated antigen (TAA) with expression limited to inner hair cells of the cochlea, adult testis, and EwS.

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SIX1 and EWS/FLI1 co-regulate an anti-metastatic gene network in Ewing Sarcoma

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Introduction

EwS is the second most common malignant bone tumor predominantly affecting children, adolescents, and young adults¹. Intensive multimodal treatment regimens, including high-dose chemotherapy² have yielded poor survival rates for patients with metastatic disease which contrasts with the more favorable 5-year overall survival rate of 70% for patients with localized disease^3,4. EwS is most commonly driven by the t(11:22) (q24;q12) chromosomal translocation resulting in the EWSR1::FLI1 oncofusion (85% of cases)⁵, with the remaining cases driven by EWSR1::ERG⁶, and EWSR1::FEV fusions⁷. These chimeric fusion proteins act as aberrant transcription factors deregulating the expression of hundreds of genes in the setting of a malignancy with few somatic mutations or additional genomic alterations⁸.

Recent success in the field of cellular immunotherapy for the treatment of hematological malignancies have revitalized efforts to investigate adoptive cell therapies (ACT) for solid tumors⁹. In synovial sarcoma, the recent approval of a TCR therapy against the cancer testis antigen melanoma-associated antigen A4 (MAGE-A4) in patients with unresectable or metastatic disease as well as the promising results with a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) TCR validate the potential of ACT in sarcoma^10,11,12. In contrast, the paucity of antigens in EwS has hampered development of ACT against this disease¹³. Novel epitopes resulting from the EWSR1::FLI1 breakpoint have been examined as immunotherapeutic targets^14,15,16; however, these oncofusion-encoded peptides display weak MHC class I binding affinities and have failed to induce durable T cell immunity^17,18. Chimeric antigen receptor (CAR) T cells targeting TAAs have been explored in the pre-clinical setting, but clinical results have not been reported^{19,20,21,22,23,24}.

Unique transcripts driven by the EWSR1::FLI1 oncofusion could serve as TAAs in EwS. Among these unique transcripts is Lipoxygenase Homology Domains 1 (LOXHD1), a protein with expression restricted to cochlea inner hair cells, the testis and EwS^25,26. LOXHD1 is a highly conserved 2211 amino acid protein characterized by 15 PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domains which have been postulated to target proteins to the plasma membrane. Inherited mutations in LOXHD1 have been identified in individuals with familial deafness reaffirming a role in the mechanosensory function of the inner ear^27,28. Here, we sought to test the hypothesis that LOXHD1 represents an actionable immunological target of EwS. Utilizing computational prediction algorithms coupled with immunopeptidomic analysis, several high affinity HLA-A*02:01 restricted LOXHD1 peptides were identified. Select LOXHD1 epitopes were found to be immunogenic enabling the molecular cloning and isolation of a LOXHD1-specific TCR, TCR353. CD8⁺ T cells engineered to express TCR353 demonstrated cytolytic function against a panel of HLA-A*02:01⁺ EwS cell lines, and the adoptive transfer of these TCR engineered T cells into mice bearing cutaneous/subcutaneous or lung EwS tumors led to complete tumor clearance or transient partial response, respectively. Altogether, these findings support LOXHD1 as a EwS immunological target and TCR directed at this protein for the development of I-O in EwS.

Results

Identification of LOXHD1 candidate epitopes

LOXHD1 exhibits a restricted expression pattern in the healthy adult (cocheal hair cells and testis) and is transcriptionally regulated in EwS by the EWSR1::FLI1 oncofusion protein²⁵. Transcriptome data from EwS tumors (ICGC database) derived from 56 patients confirms comparable expression of LOXHD1 mRNA in primary tumor and metastatic specimens (Supplementary Fig. 1A). Thus, LOXHD1 may represent a conserved, non-mutated TAA in EwS. A multiomics strategy that combined computational epitope prediction algorithms coupled with mass spectrometry-based immunopeptidomics was developed to identify candidate LOXHD1 epitopes under basal and inflammatory (+ IFN-γ) conditions in the EwS cell line SK-N-MC engineered to expressed HLA-A*02:01 (SK-N-MC^A*02:01). Computational algorithms (NetMHC4.0; NetMHCstabpan1.0, DTU Health Tech)²⁹predicted twenty-seven nonamer and thirty decamer high-affinity HLA-A*02:01 -restricted LOXHD1 peptides³⁰. These putative candidates (n = 57) displayed predicted HLA-A*02:01 affinities ranging from 9.51nM to 197.8nM and predicted p-MHC-I complex stabilities (half-life) from 8.1 h to 0.4 h (Supplemental Tables 1, 2).

To identify processed and presented LOXHD1 candidate epitopes, immunopeptidomic analysis of SK-N-MC^A*02:01 cells under basal and inflammatory conditions was performed³¹. Three HLA-A*02:01 restricted LOXHD1-derived epitopes of the 57 predicted candidates were identified: VLLSPLSRV (aa353-361), FLGSVQIRV (aa766-774), KMADVDISTV (aa1604-1613). Notably, peptides VLLSPLSRV and KMADVDISTV were detected under both basal and inflammatory (IFN-γ treatment) conditions, while peptide FLGSVQIRV was detected exclusively under inflammatory conditions. MS/MS fragmentation pattern comparison of eluted and synthetic peptides ensured accurate assignment of AA sequence identity for all three LOXHD1 epitopes (Fig. 1A and B). The binding affinity of these epitopes was experimentally determined using fluorescence polarization competitive peptide binding assays which validated these peptides as high-affinity HLA-A*02:01 binders (Log IC₅₀ < 3.7)³²(Fig. 1C and D).

Treatment of SK-N-MC^A*02:01 and other EwS cell lines with IFN-γ (100U/ml, 24 h) induced de novo expression of immunoproteasome subunits as assessed by Western blots using mAbs specific for β1i/LMP2, β5i/LMP7, and β2i/PSMB10 (Fig. 1E, Supplementary Fig. 2)³³. Additionally, IFN-γ treatment induced MHC class I upregulation as detected by flow cytometry with the pan-MHC W6/32 mAb as well as the HLA-A2 specific BB7.2 mAb (Fig. 1F, Supplementary Fig. 1B). Changes in proteosome composition as well as class I upregulation may contribute to the distinct LOXHD-1 peptide profiles observed by immunopeptidomics and will be a matter of further investigation in the future.

Immunogenicity of LOXHD1 peptides and isolation of LOXHD1-specific TCR353

Next, a workflow was developed for evaluating the immunogenicity of LOXHD1 peptides VLLSPLSRV, FLGSVQIRV, and KMADVDISTV and isolation and validation of LOXHD1-specific TCRs (Fig. 2A). Purified CD8⁺ T cells were stimulated with peptide-loaded autologous monocyte-derived mature dendritic cells, followed by stimulation with peptide-loaded HLA-A*02:01 + artificial antigen presenting cells (APC) and responses to peptides evaluated in IFN-γ ELISPOT assays. T cell responses to peptides VLLSPLSRV (n = 2/6) were observed, while no responses were observed to peptides FLGSVQIRV and KMADVDISTV in the 6 healthy donors studied (Fig. 2B). These findings emphasize the challenges of identifying T cell responses to non-mutated self HLA class I epitopes. Weak immunogenicity of non-mutated high affinity self MHC-I epitopes is generally thought to be due to central (thymic) tolerance. Antigen specificity of T cell responses to VLLSPLSRV was confirmed by flow cytometry via staining with a custom HLA-A*02:01/ aa353-361 peptide (p-MHC) multimers (Fig. 2C).

The antigenicity of LOXHD1 peptide VLLSPLSRV allowed for the isolation of a TCR directed against this epitope. CD8⁺/p-MHC multimer⁺ T cells specific for VLLSPLSRV were flow cytometrically sorted to > 99% purity and the TCRαβ repertoire was examined by next-generation DNA and RNA sequencing. A dominant TCRα/β pair was identified in the VLLSPLSRV- specific T cell population and the TCR defined by this αβ pair designated as TCR353 (Fig. 2D, Supplemental Table 3).

For the initial characterization of TCR353, lentivirus encoding the TCRαβ heterodimer construct was transduced into J^ASP90, to generate J^ASP90-^TCR353. J^ASP90 reporter cells are derived from CD8⁺/TCRαβ^null Jurkat E6.1 cells engineered to express a NFAT-inducible eGFP reporter as a readout for TCR signaling³⁴. The expression of this LOXHD1-specific TCRs was confirmed by flow cytometry with p-MHC multimer staining (Fig. 2E). To functionally characterize TCR353, J^ASP90-^TCR353 were cocultured with SK-N-MC^A*02:01 or SK-N-MC/HLA class I null (SK-N-MC\(^{{\upbeta}_2}\)^M_KO) cells under basal, inflammatory, or peptide-pulsed conditions. J^ASP90-^TCR353 activation is observed under basal conditions and further enhanced under inflammatory conditions relative to the control cell line SK-N-MC\(^{{\upbeta}_2}\)^M_KO (Fig. 2F). This data supports TCR353 recognition of processed and presented VLLSPLSRV epitope by SK-N-MC^A*02:01 under basal and inflammatory conditions.

LOXHD1 expression in EwS cell lines

A panel of 10 EwS cancer cell lines collectively representing the three EwS oncofusion variants (EWSR1::FLI1, EWSR1::FEV or EWSR1::ERG) was characterized for LOXHD1 RNA transcript and/or protein expression^{35,36,37,38,39}. All EwS cell lines expressed LOXHD1 transcript as determined by qRT-PCR (Supplemental Fig. 3A). Similarly, all cell lines expressed LOXHD1 protein as assessed by Western blot with a detected band between 160 and 260 kDa, consistent with the molecular weight of LOXHD1 AA1-1891 isoform (Supplemental Fig. 3B).

TCR353-T cells are cytolytic against EwS cell lines

To further characterize TCR353 functionality, CD8⁺ T cells from healthy donors were lentivirus transduced with TCR353 and gene edited using CRISPR/Cas9 to delete endogenous TCRα and β loci to minimize TCR mispairing^40,41,42. After expansion, TCR353-T cells were 91.1% TCR positive as determined by p-MHC staining (Fig. 3A). In a 4 h⁵¹Cr-release assay, TCR353-T cells were co-cultured with HLA-A*02:01 monoallelic K562 cells pulsed with titrated amounts of cognate peptide and a functional EC₅₀ (50% of maximal lysis) avidity of 24nM determined (Fig. 3B)⁴³.

To investigate TCR353-T cell recognition of EwS, endogenous HLA-A*02:01 cell surface expression was confirmed in A673, CHLA-9, TC-32, TC-71, TC-205, and COG-E-352 EwS cancer cell lines. In the CHLA-10, CHLA-258, RD-ES, and SK-N-MC EwS cell lines, a GFP-tagged HLA-A*02:01/β2M single-chain dimer (A*02:01/SCD) was inserted using a lentivirus to provide the appropriate HLA restricting molecule (Supplemental Fig. 4A-4 C). In short-term 4 h⁵¹Cr-release assays, TCR353-T cells kill EwS cell lines under basal and inflammatory (IFN-γ ) conditions. Under basal conditions, TCR353-T cells exhibited modest to minimal recognition of most EwS cell lines. IFN-γ exposure improved antigen recognition by TCR353-T cells independent o whether HLA-A*02:01 is endogenous or engineered (Fig. 3C-E). TNF-α, another inflammatory cytokine, also promotes increased antigen recognition by TCR353-T cells as demonstrated by enhanced SK-N-MC^A*02:01 and RD-ES^A*02:01 lysis in the presence of this cytokine (Supplemental Fig. 5A-5B). TCR353-T cells did not display lytic activity against MHC-I deficient (β2M^null) EwS cell lines (Supplemental Fig. 6A-6B), or non-EwS (LOXHD1 negative) HLA-A*02:01 cell lines (Supplemental Fig. 6C). The requirement of LOXHD1 as a secondary driver in EWS²⁵, did not allow for the generation of EWS cell lines deficient in this protein for antigen specificity examination.

The lytic activity of TCR353-T cells against EwS was further evaluated in live cell imaging and cellular impedance assays. These orthogonal assays allow for complementary measurement of cell death for visualization of TCR353 mediated lysis over a 48 h period (Fig. 3F). At an E:T ratio of 3:1, TCR353-T cells demonstrated enhanced lysis of IFN-γ treated cancer cell lines (Fig. 3G and I) manifested as a decreased time to achieved 50% of tumor cell killing (KT₅₀) (Fig. 3J and L). These findings agree with results in short-term assays and highlight the contribution of inflammatory conditions to improved presentation of antigen in EwS. Indeed, quantitative immunopeptidomics to assess the VLLSPLSRV/HLA-A*02:01 complexes on SK-N-MC^A*02:01 cells demonstrated that while 3 p-MHC complexes/cell were detected under basal conditions, IFN-γ treatment of cell line increased the number to 43 p-MHC complexes /cell (Supplemental Table 4).

TCR353 T cell therapy promotes durable regression of EwS

The in vivo activity of TCR353-T cells was tested in a NSG xenograft model of cutaneous/subcutaneous EwS using SK-N-MC^A*02:01 cells^44,45. Subcutaneous (SQ) tumors were implanted in the right flank of mice on day − 5 and tumor growth monitored using standard caliper measurements and bioluminescence (BLI). On day − 1, mice were randomized and received either PBS (basal group) or a single dose of IFN-γ (500IU) by intratumoral injection (i.t.). Twenty-four hr (day 0) and every 48 h (day 2,4,6) thereafter for a total of 4 doses, mice were treated i.t with either 5 × 10⁶ TCR353-T cells, TCRαβ^null (TCR KO)-T cells or PBS (Mock) (Fig. 4A). Representative BLI imaging of 3 mice per group are shown (Fig. 4B and C). Mice treated with TCR353-T cells demonstrated complete SK-N-MC^A*02:01 tumor rejection under both basal (n = 8/8) (Fig. 4D) and inflammatory conditions (n = 6/8) (Fig. 4E). The response to TCR353-T cell therapy was durable with no evidence of tumor recurrence 100 days after treatment. The control animals that received either TCRαβ^null -T or Mock had a median survival of ~ 40d with a total BLI flux > 10¹⁰ (Fig. 4F and G) and tumor volumes of ≥ 1000mm³ (Supplemental Fig. 7A-7B). Moreover, TCR353-T cell therapy confers prolonged overall survival with no weight loss or apparent toxicity under either condition (Supplemental Fig. 7C-7D). As an additional test of TCR353-T cell therapy, a lung metastasis model of EwS was employed (Supplemental Fig. 8A). In our pilot studies, various EwS cancer cell lines were tested for lung engraftment in NSG animals over a 3-week period and TC-71 was selected for further study due to its consistent engraftment and rate of serial growth (data not shown). Under basal conditions, TCR353-T cells showed low lytic activity in vitro against TC-71 in a 4 h⁵¹Cr release assay (Fig. 3C); while expression of the LOXHD1 epitope (VLLSPLSRV)/HLA-A*02:01 single chain trimer (SCT) by TC-71 (TC-71^SCT) cells as expected led to rapid lysis by TCR353-T cells (Supplemental Fig. 8B). In long-term (~ 25 h) impedance assays, TCR353-T cells lyse parental TC-71 cells and TC-71^SCT cells with similar kinetics (Supplemental Fig. 8C). In the NSG lung metastasis model, 5 × 10⁵ tumor cells (TC-71 or TC-71^SCT) were administered by tail vein injection and 10 days later (day 0), 1 × 10⁷ TCR353 CD8 + T cells were adoptively transferred by iv route. TC-71 grew exponentially despite the transfer of TCR353-T cells and no effect on tumor growth was evident. In contrast, a single dose of TCR353-T cells led to tumor regression in all 5 mice bearing TC-71^SCT tumors for the initial 2 weeks (Supplemental Fig. 8D). However, by day 20, slow tumor growth was evident in all animals. Disease control of TC-71^SCT metastases suggested that a higher antigen density was necessary for TCR353-T cell recognition which achieves some degree of equilibrium over the 35-day observation period. Tumor bearing animals left untreated (control group) expired by day 20 due to rapid disease progression.

Failure of TCR353-T cells to lyse hair cells of the inner ear

LOXHD1 is a stereociliary protein with a restricted pattern of expression to the inner ear hair cells and testis^25,26. The UB-OC2 (organ of Corti) cell line which is often employed as a model system for investigating inner ear development and ototoxicity studies was selected to test for TCR353-T cell mediated destruction of auditory hair cells^46,47. UB-OC2 is a committed hair cell precursor line derived from primary murine (E13, H2-K1-tsA58 strain) embryonic cultures of auditory sensory epithelia^46,47. To assess recognition by TCR353-T cells, UB-OC2 cells were transduced with lentivirus encoding HLA-A*02:01-SCD to generate UB-OC^A*02:01 (Supplemental Fig. 9A). RT-PCR analysis showed no expression of LOXHD1 in hair precursor cells maintained at 33 C. Upon temperature shift to 39 C and addition of IFN-g, LOXHD1 mRNA was now detectable but at significantly lower expression levels compared to SK-N-MC (Supplemental Fig. 9B). TCR353-T cells failed to recognize UB-OC^A*02:01 target cells under basal conditions or in the presence of interferon-g suggesting the density of LOXHD1 353–361 (VLLSPLSRV) epitope, if presented, is below the limit of detection by this TCR (Supplemental Fig. 9C). As a positive control, UB-OC^A*02:01 cells pulsed with LOXHD1 353–361 peptide are lysed by TCR353-T cells in a dose dependent manner.

Discussion

Pediatric malignancies present a major challenge for the field of cancer immunotherapy. The paucity of genomic alterations creates a significant bottleneck for cancer antigen discovery and impedes the development of T cell-based therapies. EwS typifies most pediatric solid tumors with a low mutational burden (0.15/Mb) and is notable for a recurrent chromosomal translocation involving the EWSR1 breakpoint region (22q12) and ETS family partner^13,48. In this report, LOXHD1 is identified as a novel TAA unique to EwS and TCR-T cell (TCR353) therapy directed at this TAA is described as an approach to target EwS. All EwS cell lines examined regardless of their ETS partner were sensitive to lysis by TCR353-T cells and lysis was augmented, in most cases, upon IFN-γ treatment of cancer lines. This enhancement may result from increase in tumor cell surface MHC-I and/or switch from constitutive to immunoproteosome. Adoptive transfer of TCR353-T CD8 + T cells to NSG mice bearing the SK-N-MC^A*02:01 EwS xenograft promotes tumor clearance of subcutaneous tumors and long-term survival with no signs of toxicity. Thus, LOXHD1 is an oncofusion driven lineage-restricted TAA that may be amenable to targeting by immunotherapy approaches.

Identified in 2009 by Grillet and co-workers, LOXHD1 was described as a stereociliary protein required for auditory hair cell mechanotransduction and, mutations in LOXHD1 are associated with autosomal recessive non-syndromic hearing loss²⁸. In a recent study, Deng et al. reported on a 32 gene signature driven by the EWSR1::FLI1 oncofusion protein that stratifies EWS from pan-cancer²⁵. Among genes in this signature, LOXHD1 is the most highly expressed and ablating its expression in EwS cell lines impairs hypoxia response and cytoskeletal reorganization as well as retards EWS growth and metastases in xenograft models²⁵. Additionally, a survey of 18 EwS cell lines confirms LOXHD1 as a member of the EWSR1::ETS core induced gene set²⁶. These observations suggest that LOXHD1 plays a critical role as a secondary non-canonical driver in EwS metastasis and hence might be more than a typical bystander TAA worthy of examination as a cancer-specific, high prevalence antigen amenable to therapeutic targeting.

In our study, we identified three high affinity/stability epitopes by immunopeptidomics that were derived from a candidate list of 57 nonamer and decamer peptides predicted to bind a high prevalence MHC class I allele, HLA-A*02:01. Antigenicity of VLLSPLSRV (aa353-361) epitope allow for the isolation of the LOXHD1-specific TCRαβ heterodimer, TCR353. TCR353-T cells permitted in-depth functional testing and validation of LOXHD1 as a TAA in a panel of cancer cell lines. Inflammatory cytokines promote changes in proteosome composition and upregulation of MHC class I, processes that enhanced sensitivity of EWS to immune targeting. However, further studies are needed to address how this information can be harnessed to improved immune targeting this malignancy. Similarly, it is well known EWSR1::FLI1 oncofusion transcript levels dictate EWS heterogeneity and control the proliferation and metastatic potential of individual tumor cell clones⁴⁹. Although, LOXHD1 transcript levels appear similar in primary tumors and metastatic lesions (Supplemental Fig. 1), an open question remains as to intratumoral LOXHD1 heterogeneity and the potential emergence of LOXHD1 negative subclones. Analysis of single cell tumor clones by scRNA-seq that survive TCR-T cell treatment in a larger panel of EwS tumors should be informative to reveal potential mechanisms of resistance. Failure to eradicate diffuse lung metastases with a single bolus dose of TCR353-T cells (Supplemental Fig. 7) underscores the need to target multiple tumor epitopes as well as improve the persistence of engineered T cells. Even though HLA-A*02:01 is present in ~ 45% of the general population, most EwS patients would not benefit from TCR353-T cell therapy which necessitates the identification of additional HLA-I restricted LOXHD1 epitopes to broaden the population coverage. Finally, since LOXHD1 expression is restricted to the inner hair cells of the cochlea and testis, ototoxicity induced by TCR-T cell therapy is a potential concern that should be considered in future translational studies. As an initial in vitro test for ototoxicity, TCR353-T cells failed to recognize UB-OC2/HLA-A*02:01 + hair cells which were shown to express low levels of LOXHD1 mRNA. Immune-related adverse events in the testis are mitigated by the lack of MHC-I expression in the male germ cells.

Finally, it has been estimated that ~ 16.5% of all malignancies⁵⁰ are driven by oncofusion proteins which are typically associated with leukemias, lymphomas, and sarcomas. In certain diseases defined by recurrent chromosomal translocations, small molecule inhibitors provide durable therapeutic benefit and long-term remission; for example, imatinib as treatment for CML defined by the BCR-ABL1 oncofusion protein^51,52. Previous attempts to target the recurrent t(11;22)(q24;q12) translocation in EwS with small molecule inhibitors have had limited success to date, so new approaches are urgently needed^53,54. Our study supports the continued pursuit of new immunological approaches for solid tumor malignancies that have been deemed undruggable, or in certain instances, resistant to targeted agents. In summary, we describe LOXHD1 as an oncofusion driven TAA specific to EwS and suggest that identification of oncofusion driven TAAs in other malignancies may serve as immunological targets for the development of new cancer therapeutics.

Materials and methods

Primary cells and cell lines

Peripheral blood mononuclear cells (PBMCs) and purified CD8⁺ T cells were obtained from healthy volunteers. Informed consent was obtained from all subjects in this study and apheresis was collected under University of Pennsylvania IRB-approved protocol 705906 and processed by the Human Immunology Core (HIC). All experiments using materials from humans were in accordance with the approved operating procedures and guidelines of University of Pennsylvania’s Office of Environmental Health and Radiation Safety (EHRS). The cell lines CHLA-9, CHLA-10, CHLA-258, COG-E-352, TC-32, TC-71, TC-205 ( all obtained from Childhood Cancer Repository/Alex’s Lemonade Stand, Texas Tech University) and CFPAC-1 (ATCC CRL-1918) were cultured in IMDM media, 20% fetal bovine serum (FBS), 2mM L- glutamine, 1% ITS (Insulin-Transferrin-Selenium), and 1% Penicillin/streptomycin (P/S). The cell lines SK-N-MC (ATCC-HTB-10) and PANC-1 (ATCC, CRL-1469) were cultured in EMEM media, 10% FBS, 2nM L-glutamine and 1% P/S. The cell lines A-673 (ATCC-CRL-1598) and MDA-MB-231 (ATCC HTB-26) were cultured in DMEM media, 10% FBS, 2nM L-glutamine and 1% P/S. The cell lines RD-ES (ATCC-HTB-166), K562 (ATCC CCL-243) and Jurkat E6.1 (ATCC TIB-152) were cultured in RPMI media, 15% FBS, 2nM L-glutamine and 1% P/S. The cell line UB-OC2 (CancerTools, cat # 153623) was cultured at 33°C in DMEM, 10% FCS, 10% glutamine to express the small tumour antigen (Tag) gene. To induce differentiation cell cultures are switched to 39°C in media with 50 U/mL IFNγ for 14 days⁴⁶. HLA-A*02:01 negative tumor cell lines were transduced with lentiviral particles expressing HLA-A*02:01 single-chain dimer (SCD, eGFP⁺).Generation of β₂MKO EwS cell lines was carried out using single guide RNA (5 ug) targeting β2M (5’-TCACGTCATCCAGCAGAGAA-3’, Integrated DNA Technologies) and TrueCut Cas9 Protein V2 (10 ug, ThermoFisher Scientific) as previously described³⁴. Ablation of β2M was confirmed by flow cytometry. All cell lines were determined to be Mycoplasma free by the University of Pennsylvania Department of Genetics Core Services using the MycoAlert (Cambrex) kit.

Antibodies and flow cytometry

The following antibodies were utilized: anti-human pan HLA-A, B, C (W6/32) APC (BioLegend, 311410), anti-human HLA-A*02:01 PE (BD, 558570), anti-human β2-Microglobulin (BD, 551337) CD99 (Biolegend, 371305), anti-human CD3 FITC (Invitrogen, MHCD0301), anti-human CD8 APC (ThermoFisher Scientific, MHCD0805), anti-human CD8 (SK1) APC (BioLegend, 344722), anti-human TCRαβ (IP26) APC (BioLegend, 306717), Purified anti-PE Ab (Biolegend, PE001). Custom VLLSPLSRV/HLA-A*02:0 p-HLA dextramers were obtained from Immudex. Samples were acquired on a BD LSRFortessa flow cytometer using BD FACSDiva software (version 8.0.2) and analyzed using FlowJo software (version 10.0.7).

Western blot analysis

Cell lines were lysed in RIPA buffer (Cell Signaling Technology 9806 S), incubated on ice for 30 min and centrifugated at 14,000xg for 10 min. Protein was quantitated using BIO-RAD DC Protein Assay Kit (reagent package,500 − 0116). 50 ug of protein lysate was loaded onto 4–12% SDS-PAGE gel (Invitrogen NP0335) and transferred onto nitrocellulose membranes (BioRad, 1620177). Membrane blots were blocked in blocking buffer (Licor, 927-60001) followed by O/N incubation with diluted primary antibodies at 4 °C. The following antibodies were used: anti-proteasome 20 S LMP2 [EPR22042] ab242061⁵⁵, anti-proteasome 20 S LMP7 [EPR14482(B)] ab180606⁵⁵, and anti-PSMB10/MECL1 [EPR14902] ab183506⁵⁵, β-actin Mouse mAb (Cell Signaling 8H10D10)⁵⁶and GAPDH (Cell Signaling 3683) ab1642205²⁵. The following secondary antibodies were used IRDye 680RD goat anti-mouse (LI-COR, 926-68070), IRDye 800CW goat anti-rabbit (LI-COR, 926-32211). Blots were scanned by Odyssey Clx Imaging System (LI-COR Bioscience). For LOXHD1 protein expression²⁵, an anti-LOXHD1 primary antibody (gift from Dr. Nicolas Grillet, Stanford University)²⁵ was employed followed by HRP-conjugated secondary antibody. Blots were visualized using a chemiluminescent substrate (GE Healthcare).

In silico prediction of LOXHD1 epitopes

NetMHC4.0 was employed to identify high affinity (< 200nM) nonamer and decamer HLA-A*02:01 restricted -LOXHD1 peptides (short isoform 1, 1891 AA). Synthetic peptides (> 95% purity, LifeTein) were reconstituted in 10% DMSO (Mylan Cryoserv 67457-178-50) and peptide binding to HLA-A*02:01 evaluated using a fluorescence polarization-based competition assay (Pure Protein, L.L.C.) as previously described³⁴.

Immunopeptidome analysis

HLA-I peptides were extracted from 5 × 10⁸ SK-N-MC^A*02:01 cells before and after exposure to IFN-γ (100IU/mL, 24 h) (n = 6) as previously described³¹. For peptide identification, a Data-Dependent Acquisition (DDA) strategy was employed³¹. Peptides were chromatographically separated by a gradient of 80% acetonitrile (ACN) and 0.1% formic acid (FA) at a flow rate of 250 nl/min over a 65-minute period and subsequently, analyzed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific). MS/MS spectra were subjected to analysis using PEAKS Studio X software. An initial de novo search was conducted on all MS/MS spectra against peptide sequences, followed by a search against the human proteome database (Uniprot Human Reviewed Proteins downloaded as of October 2021). To validate the authenticity of peptide identifications, candidate LOXHD1 peptides were synthesized (Mimotopes, Melbourne, Australia) and analyzed using the same methodology described above. To assess the number of p-HLA complexes in the SK-N-MC^A*02:01 cell line, LC-MS/MS analysis was conducted on known concentrations (1, 2.5, 5, 7.5, 10, and 20 fmol) of the synthesized labelled peptide. The resulting data, characterized by the area under the curve (AUC) for each peptide concentration, was employed to generate a standard curve. This standard curve was subsequently utilized to calculate the estimated numbers of VLLSPLSRV/HLA-A*02:01 complexes in SK-N-MC^A*02:01 cell line.

Generation of LOXHD1-reactive T cell lines and LOXHD1-specific TCRs isolation

The generation of monocyte-derived dendritic cells mDC and antigen-specific T cells was performed as previously described³⁴. Briefly, purified CD8⁺ T cells were cultured with (2500 Rads) irradiated/peptide-pulsed autologous mDC (20:1 ratio) in T cell media [Optimizer CST media (Gibco), 5% AB human, glutamine] with IL-2 (50IU/well) added every 48 h. On day 14, T cell cultures were re-stimulated with (10,000 Rads) irradiated/peptide-pulsed HLA-SCD/4-1BBL expressing K562 cells (1:1 ratio) with IL-2 (500IU/mL) added every 48 h until day 10–14. IFN-γ ELISPOT assay and p-HLA multimer staining were performed by evaluate T cell responses. A positive IFN-γ response was defined as > 100 IFN-γ spot forming cells (SFCs) / 5 × 10⁵ CD8 + T cells. Sorted (97–99% p-HLA/CD8+) LOXHD1-specific CD8⁺ T cells underwent a rapid expansion method³⁴. Expanded T cells were sorted, RNA and DNA isolated and TCRα/TCRβ sequencing was performed³⁴ Raw sequences were quality filtered as previously described³⁴.

Lentiviral TCR constructs and production

All lentiviral constructs were generated using the third-generation lentiviral transfer vector pTRPE-eGFP-T2A-mCherry (provided by Michael C. Milone, University of Pennsylvania, Philadelphia, PA). Synthetic TCR DNA vector constructs were designed (TWIST Bioscience, San Francisco, CA) to include the TCRα chain followed by the TCRβ chain separated by a T2A sequence.

TCR353-engineered T cells

TCR353 functionally was assessed using the J^ASP90 Jurkat (NFAT/eGFP) reporter system or primary CD8 + T cells³¹. J^ASP90_TCR353 reporter cell line (2.5 × 10⁵ cells/well) was cocultured with SK-N-MC^A*02:01 cell line (1:1 ratio). After 16–20 h, cells were analyzed by flow cytometry to determine the percentage of eGFP⁺ cells as a surrogate of T cell activation. For TCR353 CD8⁺ T cells, primary CD8 + T cells were activated with anti-CD3/CD28 microbeads (Life Technologies) and transduced with TCR353 lentiviral vector particles at an MOI of 5. Cell cultures were grown in IL-7 (5 ng/ml; R&D Systems) / IL-15 (5 ng/ml; R&D Systems) supplemented T cell media. On day 5, microbeads were removed and T cells electroporation with single guide RNAs targeting TRAC (5’-TGTGCTAGACATGAGGTCTA-3’) and TRBC1/TRBC2 (5’-GGAGAATGACGAGTGGACCC-3’, Integrated DNA Technologies) complexed with TrueCut Cas9 Protein V2 (10 ug, ThermoFisher Scientific). Edited T cells were cultured in IL-7/IL-15 supplemented T cell media for a total of 14 days followed by rapid expansion method. LOXHD1-specific T cell frequencies were determined by p-HLA multimer flow cytometric assays.

Cytotoxicity assays-⁵¹Cr-release assay

Tumor cell lines were labeled with 25 µCi⁵¹Cr for 1 h at 37 °C and tested as targets in a standard 4 h⁵¹Cr-release assay³⁴. Assays were performed, in triplicate, at various effector: target ratios. Data were collected using a MicroBeta2 LumiJET Microplate Counter (PerkinElmer). Data are represented as percent-specific lysis reported as mean ± standard deviation (SD). Real-time apoptotic cell death analysis: Real-time apoptotic cell death analysis (live cell imaging) was performed using the xCELLigence Real Time Cell Analysis eSight system (ACEA Biosciences). Target tumor cells were plated (5 × 10⁴ cells/well) and allowed to adhere for 48 h. Effector T cells were added at E: T ratios 3:1. Concurrent time lapse video monitoring was performed with acquisition of brightfield, green (GFP) windows every hour over a ~ 48 h duration.

Animal model

All experiments were reviewed and performed accordaning to University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) protocol 804,226 and all animal procedures were performed in the animal facility at the University of Pennsylvania in accordance with Federal and Institutional IACUC requirements. Mice were subject to routine veterinary assessment for signs of overt illness and were euthanized at experimental termination or when predetermined IACUC rodent health endpoints were reached. The maximal tumor size of 2 cm in diameter permitted by the institutional review board was not exceeded. Additionally, in vivo experiments were also performed in accordance with ARRIVE guidelines. The SK-N-MC^A*02:01 xenograft model was established by subcutaneous injection of 2.5 × 10⁵ CBR/luciferase + SK-N-MC^A*02:01 cells (1:1 PBS/Matrigel, Corning 356234) in NSG (NOD/scid/γcnull) female mice (6–8 weeks age). Four days after tumor implantation, mice were either injected with PBS or 500IU of IFN-γ intratumorally followed by treatment with 5 × 10⁶ TCR353-T cells at 24 (day 0), 72 (day 2), 96 (day 4) and 144 (day 6) hr. Control mice either received no treatment (Mock) or 5 × 10⁶ TCRαβ^null (TCRKO) CD8⁺ T cells. Tumor growth was monitored weekly by caliper measurements (mm³) and BLI (total flux). The TC-71^SCT lung metastasis model was established by of tail vein injection of 5 × 10⁵ CBR/luciferase + TC-71^SCT or TC-71 cells in NSG (NOD/scid/γcnull) female mice (6–8 weeks age). Ten days after tumor implantation, 1 × 10⁷ TCR353-T cells were given iv and tumor growth monitored weekly by BLI. Data was analyzed using Living Image Version 4.5.2 software (PerkinElmer). Animals were also monitored for signs of general health and toxicity, including weight loss, loss of fur, diarrhea, and hind limb paralysis.

Statistical analysis

Statistical analysis (Graphpad, Prism 7.0) for multiple comparisons was performed using one-way ANOVA with Tukey’s HST post-test. Two-group comparisons were analyzed using Student’s unpaired t-test. Significance of overall survival was determined via Kaplan–Meier analysis with log-rank (Mantel-Cox) analysis. Tumor growth and survival curves were analyzed using linear mixed-effects model with Tukey’s HSD post-test using the Ime4 and the survival package in R. All data presented are representative of two or more independent experiments.

Data availability

LOXHD1 gene expression values reported in Supplementary Figure 1 A are supported by data obtained from the International Cancer Genome Consortium (ICGC) data portal (data release 28).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD046803 and 10.6019/PXD046803. Username: reviewer_pxd046803@ebi.ac.uk Password: OnhDJSIM.

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Acknowledgements

We gratefully acknowledge the Human Immunology Core at the University of Pennsylvania for providing leukocytes for research, The Cell and Animal Radiation Core (SCR_022377) at the University of Pennsylvania, Alex’s Lemonade Stand Foundation for Childhood Cancer (Cell Line and Xenograft Repository), Drs Nina Luning Prak and Wenzhao Meng for assistance in TCR sequencing, Drs Rico Buchli for assistance in biochemical characterization of peptide epitopes, Monash Proteomics and Metabolomics Platform (MPMP) for expertise in sample preparation and mass spectrometry method development, and Dr. Grillet (Stanford University) for LOXHD1 antibody. Computational resources were supported by the R@CMon/Monash Node of the NeCTAR Research Cloud, an initiative of the Australian Government’s Super Science Scheme and the Education Investment Fund. This work was supported by the Parker Institute for Cancer Immunotherapy (to B.M.C., G.P.L., C.H.J.), U54CA244711 (to G.P.L., A.P. and C.H.J.), Predoctoral Training Grants in Pharmacology and in Immmunology of Normal and Neoplastic Lymphocytes T32GM007086 and NIH T32 CA099140 (to T.B.), The Abramson Cancer Center Paul Calabresi Career Development Award for Clinical Oncology NIH K12 CA076931 (to A.S.B.), NHMRC Investigator Award (#2016596, to A.P.), Victorian Cancer Agency (grant 2001870, to P.F), Cancer Australia and the Australian Lions Childhood Cancer Research Foundation and My Room Children’s Cancer Charity Ltd. ( to P.F.) Sarcoma Foundation of America Research Grants and American Cancer Society Research Scholar Grants (to I.A.A.)

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Authors and Affiliations

Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Tatiana Blanchard & Avery D. Posey Jr
Centre for Cancer Research, Hudson Institute of Medical Research, Monash University, Clayton, VIC, 3168, Australia
Pouya Faridi, Grace Huang & Terry C. C. Lim
Department of Molecular and Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, 3168, Australia
Pouya Faridi, Grace Huang & Terry C. C. Lim
Monash Proteomics and Metabolomics Platform, Department of Medicine, School of Clinical Sciences, Monash University, Clayton, VIC, Australia
Pouya Faridi & Terry C. C. Lim
Department of Biochemistry and Molecular Biology and Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
Pouya Faridi, Rochelle Ayala & Anthony W. Purcell
Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Chong Xu, Khatuna Gabunia, Mei Ji, Avery D. Posey Jr, John Scholler, Gerald P. Linette, Carl H. June & Beatriz M. Carreno
Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Adham S. Bear & Gerald P. Linette
Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, BRBII/III, 421 Curie Boulevard, Philadelphia, PA, 19104, USA
Reyaz ur Rasool & Irfan A. Asangani
Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, USA
Avery D. Posey Jr
Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Irfan A. Asangani & Carl H. June
Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Irfan A. Asangani
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA
Adham S. Bear, Gerald P. Linette & Carl H. June
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Carl H. June & Beatriz M. Carreno
Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Gerald P. Linette, Carl H. June & Beatriz M. Carreno

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Tatiana Blanchard
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Conceptualization: B.Carreno, C.June, G.Linette. Methodology: T.Blanchard, P. Faridi, A.Bear, R.Rasool, G.Huang, T.Lim, R.Ayala, A.Purcell, J.Scholler, K.Gabunia, I.A.Asangani, B.Carreno, G.Linette. Validation: T.Blanchard, C.Xu, P.Faridi. Investigation: T, Blanchard, P. Faridi, A.Bear, C.Xu, A.Purcell, A.Posey, Formal Analysis: P. Faridi, A.Purcell, I.A.Asangani, B.Carreno. Resources: A.Posey, P.Faridi, A.Purcell, I.A.Asangani Data Curation: T.Blanchard, P.Faridi, G.Linette, B.Carreno. Writing Original Draft: T.Blanchard, B.Carreno, G.Linette. Writing – review and editing: T.Blanchard, P. Faridi, A. Purcell, B.Carreno, C.June, G.Linette. Visualization: T.Blanchard, P. Faridi, B.Carreno, G.Linette. Supervision: B.Carreno, C.June, G.Linette. Funding: P. Faridi, I.A.Asangani, A.Purcell, B.Carreno, C.June, G.Linette. Corresponding author: B.Carreno.

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Correspondence to Beatriz M. Carreno.

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Competing interests

T.B, I.A.A., G.P.L., and B.M.C. are inventors on a patent (63/516,178) related to the targeting of LOXHD1 for immunotherapy entitled “Compositions and Methods for Targeting LOXHD1-Associated Disease”. All other authors have no competing interest to declare.

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Blanchard, T., Faridi, P., Xu, C. et al. LOXHD1 is an oncofusion-regulated antigen of ewing sarcoma. Sci Rep 15, 13007 (2025). https://doi.org/10.1038/s41598-025-96877-9

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Received: 22 November 2024
Accepted: 01 April 2025
Published: 15 April 2025
DOI: https://doi.org/10.1038/s41598-025-96877-9