Kita-Kyushu Lung Cancer Antigen-1 Research Articles

Abstract LB442: Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells

Abstract The prognostic benefit of immune checkpoint inhibitors (CPIs) is mainly restricted to specific cancers, such as melanoma and lung adenocarcinoma. For patients with cancers which are noninflamed (“cold”), or CPI-resistant, therapeutic options are currently limited. Recently, the US FDA approved the first bispecific molecule targeting a tumor peptide MHC. This highlights the potential of targeting MHC-presented tumor antigens to empower T cells to specifically recognize and eliminate solid tumors. While there is a growing number of agents targeting pMHCs, most of them are restricted to HLA-A*02. There is an urgent need to identify effective pMHC targeted therapies for solid tumors, especially on other alleles to increase the proportion of eligible patients. Kita-Kyushu lung cancer antigen-1 (KK-LC-1/CT83) peptide presented on HLA-A*01 is a promising tumor target as its expression is restricted to tumors and it is broadly expressed in many cancers including gastric, lung, breast, and cervical cancer. However, targeting the KK-LC-1/HLA-A*01 complex is challenging due to the high levels of similar off-target peptides presented in healthy tissues. Through in silico analysis we identified peptides within the human genome that share a high sequence identity to the target. These peptides were found in a database containing >1 million mass spectrometry (MS)-identified peptides presented by healthy tissues, cancer cell lines, and tumor tissue samples. A peptide derived from 6-pyruvoyltetrahydropterin synthase (PTS) was found to be abundantly presented in healthy tissues. MS confirmed that this peptide occurs at extremely high natural levels (620 - 3500 copies per cell) on HLA-A*01 cell lines and healthy tissue. Here, we describe a set of pMHC T-cell engager (TCE) antibodies with high specificity towards HLA-A*01 restricted KK-LC-1 epitope. ScFv phage display libraries were rigorously screened for binding to the pMHC target and counter-screened against predicted off-target peptides including PTS. Specific scFv hits were reformatted into TCEs using an anti-CD3 Fab effector arm. The TCEs showed efficient killing of KK-LC-1/HLA-A*01-positive human cell lines NCI-H1703 and EKVX in co-cultures with human PBMCs, while KK-LC-1-negative/HLA-A*01-positive cell lines SK-MEL-30 and PC-3 (known to present the risk peptides on the surface) were minimally affected. TCE-dependent T cell activation measured by IFNγ was significantly higher in presence of TAP-deficient, (empty) HLA-A*01-expressing T2 cells pulsed with the target peptide compared to the risk peptides. Alanine scans of isolated binders confirmed that at least 6 amino acids of the KK-LC-1 peptide are required for binding. Overall, the data show that these antibodies have promising anti-tumor activity and specificity for KK-LC-1/HLA-A*01 positive tumors. Future activities will focus on lead optimization for therapeutic development. Citation Format: Fabian Scheifele, Stephanie Jungmichel, Nagjie Alijaj, Athanasia Dasargyri, Romina Doerig, Philip Knobel, Hannes Merten, Philipp Richle, Thomas Schleier, Anna Sobieraj, Alessio Vantellini, Tim Fugmann, Rom Leidner, Swethajit Biswas, Leonardo Borras. Novel antibodies against a KK-LC-1-derived peptide presented on HLA-A*01 on tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB442.

An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Gastric cancer is a worldwide health problem. Chemotherapy and radiotherapy are of great importance in the management of advanced gastric cancer. However, their therapeutic efficacy is limited by off-target side effects. Peptide-drug conjugates (PDCs) are a novel strategy for tumor-targeted drug delivery to overcome the existing drug resistance mechanisms and improve antitumor effects. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is exclusively expressed in several types of cancer including gastric cancer, representing a promising target for drug delivery. Here, we suggested KK-LC-1 as a potential target for PDC design for the first time and reported the first KK-LC-1-targeting PDC product 1131-MMAE, which is composed of a KK-LC-1-targeting peptide and an antimitotic drug conjugated by an enzymatically cleavable linker. We observed that 1131-MMAE could be efficiently endocytosed by KK-LC-1 positive gastric cancer cells for subsequent drug release and arrest the cell cycle at the most radiosensitive G2/M phase. We demonstrated that 1131-MMAE could significantly delay tumor growth with reduced toxicity than free drugs as a monotherapy. We further confirmed that 1131-MMAE was also a potent radiosensitizer. 1131-MMAE could selectively enhance the radiation response of KK-LC-1 positive tumor cells and achieve improved tumor control when combined with low-dose radiation. Overall, our study proposed an optimized therapeutic regimen for precision chemoradiotherapy, which has translational potential in multiple types of cancer.

Abstract 2526: Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients

Abstract Introduction: KK-LC-1 is a cancer testis antigen expressed notably in lung, breast, and gastric cancer making it an attractive target for T-cell receptor therapy. Here, we evaluate cell densities of NSCLC tissue specimens with known KK-LC-1 gene expression to better characterize the tumor immune microenvironment in such patients. Methods: A seven-plex mIF panel (FoxP3, PDL1, PanCK, PD1, CD8, KK-LC-1, and CD68) was developed based on a previously established mIF Lung-cancer panel and addition of KK-LC-1 following rigorous optimization of KK-LC-1 antibody titer and panel order with qualitative assessment of monoplex immunofluorescent assays. Serial sections from 23 NSCLC cases (14 biopsy samples and 9 excision specimens) with known KK-LC-1 gene expression values were collected. Tissue sections were stained and imaged on an Akoya Biosciences Vectra Polaris multispectral imaging platform. The regions of interest were identified and stamped with the Phenochart mIF image management software. Spectral unmixing, tissue segmentation and cell phenotyping were performed with Akoya Biosciences InForm image analysis software using the multispectral fields workflow. The 4-tier positive staining thresholds for PD-L1 and KK-LC-1 were determined by a pathologist (WDW). Cell densities in the tumor and adjacent stroma as well as the KK-LC-1 H-score were assessed by linear regression analysis. Student’s t-test was used to compare between the top quartile of tumors based on KK-LC-1 gene expression (Q4) and the remaining 3 quartiles (Q1-Q3). Results: The study consisted of 17/23 adenocarcinoma (73.9%), 4/23 squamous cell carcinoma (SCC) (17.3%), and 2/23 (8.6%) unclear or mixed histology tumors. Adenocarcinoma trended to have higher KK-LC-1 gene expression compared to SCC tumors (mean 17.34 vs. 0.65, p-value = 0.204). 13/23 (56.5%) were pan-wild type, 6/23 (26.0%) were EGFR mutated, 2/23 (8.7%) were KRAS mutated, and 1/23 (4.4%) were MET Exon 14 skip and multiple alterations. We saw significantly higher cell density of FoxP3 in tumor cells in Q4 versus Q1-Q3 (mean 7.17 vs. 0.8 cells/mm2, p-value = 0.004), and there was a trend towards a higher FoxP3/CD8 cell density ratio (mean 3.28 vs. 0.05, p = .110) and higher PD-1 cell density (mean 186.7 vs. 32.8 cells/mm2, p=0.1548) in tumor cells in Q4 versus Q1-Q3. There was significant correlation towards increased CD8+ T cell density ratio between tumor and stroma cells in cases with higher KK-LC-1 gene expression (r2= 0.307, p = 0.011). Conclusion: KK-LC-1 expression in lung cancer is seen in multiple molecular subsets and is associated with increase in both regulatory T cells in tumor and in CD8+ T-cells in tumor relative to stroma. Strategies to deplete regulatory T cells and to further amplify CD8+ T cells should be pursued in the development of T-cell receptor therapy targeting KK-LC-1. Citation Format: Ainaz Dory Barkhordarzadeh, Robert C. Hsu, Rongfu Wang, Sue E. Martin, Jorge J. Nieva, William Dean Wallace. Immune infiltrates in Kita-Kyushu lung cancer antigen 1 (KK-LC-1) expressing non-small cell lung cancer (NSCLC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2526.

Abstract 5487: An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer

Abstract Gastric cancer is a worldwide health problem. Concurrent chemoradiotherapy (CRT) represents an important strategy to reduce recurrence and prolong survival for gastric cancer. However, non-targeted cytotoxic drugs capable of enhancing radiosensitization remain the main choice to be combined with radiotherapy, which has limited the anti-tumor efficacy of CRT by off-target side effects. Monomethyl auristatin E (MMAE) is a highly potent anti-tubulin agent and has been reported to act as an effective radiosensitizer recently. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer/testis antigen highly and exclusively expressed in multiple types of cancers including gastric cancer, representing a promising target for drug delivery. Previously, we identified a KKLC-1-targeting cyclic nonapeptide (1131 peptide) by screening a phage display peptide library. Here, we described a novel peptide-drug conjugate (PDC) 1131-MMAE by connecting the 1131 peptide with MMAE via a protease-sensitive linker for tumor-selective radiosensitization. We demonstrated that 1131-MMAE could be efficiently internalized by gastric cancer cells through KKLC1-mediating endocytosis and exhibited a comparable cytotoxicity profile with free MMAE. 1131-MMAE could arrest the cell cycle at the most radiosensitive G2/M phase in a dose-dependent manner and selectively enhance the radiation response of KK-LC-1 positive gastric cancer cells by increasing DNA double-strand break as indicated by γH2Ax immunofluorescence staining. Furthermore,1131-MMAE was able to inhibit tumor growth as a monotherapy at 2mg/kg with a good safety profile in vivo. Increased tumor control was achieved when 1131-MMAE was combined with radiotherapy at a relatively low dose of 0.3mg/kg. Overall, we proposed an MMAE-based PDC targeting a novel tumor marker KK-LC-1 and demonstrated its tumor-selective radiosensitization in gastric cancer models for the first time. This anti-KKLC1 PDC may help to promote the clinical progress in precision chemoradiotherapy for multiple types of cancer. Citation Format: Xiaotong Chen, Fangcen Liu, Xiaoxiao Yu, Lin Li, Jiayao Yan, Baorui Liu. An auristatin-based peptide-drug conjugate targeting Kita-Kyushu lung cancer antigen 1 for precision chemoradiotherapy in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5487.

A phase I trial of T-cell receptor gene therapy targeting KK-LC-1 for gastric, breast, cervical, lung and other KK-LC-1 positive epithelial cancers.

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

Detection of Kita-Kyushu Lung Cancer Antigen-1, a Cancer/Testis Antigen, in the Stomach Close to a Cancerous Condition.

Background: Kita-Kyushu lung cancer antigen-1 (KK-LC-1), encoded by CT83, is a cancer/testis antigen (CTA) and an attractive target for immunotherapy. Our previous study demonstrated frequent CT83 expression in gastric cancers (GCs) and non-tumor sites of the stomach with tumors. Additionally, there was a correlation with Helicobacter pylori (Hp) infection. Since it currently remains unclear whether KK-LC-1 is expressed in the stomach without GC, this study investigated KK-LC-1 expression in non-GC stomach. Methods: We investigated differences in CT83 gene expression at non-tumor sites of stomachs with or without tumors in 118 GC patients and 115 non-GC patients. Fisher's exact test was used for statistical analyses. Results: CT83 expression was detected in 77% of non-tumor sites in stomachs with tumors, which was significantly higher than in stomachs without tumors (7%, p < 0.0001). All patients with CT83 expression at non-tumor sites of their stomachs without tumors carried Hp. Conclusion: CT83 appears to be rarely expressed in the atrophic stomach, and furthermore, a part of patients positive for its expression will develop GC in the future, suggesting that CT83 expression is a useful marker for predicting GC.

Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1): A Promising Cancer Testis Antigen.

Cancer has always been a huge problem in the field of human health, and its early diagnosis and treatment are the key to solving this problem. Cancer testis antigens (CTAs) are a family of multifunctional proteins that are specifically expressed in male spermatozoa and tumor cells but not in healthy somatic cells. Studies have found that CTAs are involved in the occurrence and development of tumors, and some CTAs trigger immunogenicity, which suggests a possibility of tumor immunotherapy. The differential expression and function of CTAs in normal tissues and tumor cells can promote the screening of tumor markers and the development of new immunotherapies. This article introduces the expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1), a new member of the CTA family, in different types of tumors and its role in immunotherapy.

Relationship between Kita-Kyushu Lung Cancer antigen-1 expression and prognosis of cases with lung squamous cell carcinoma.

BackgroundPreviously, we identified the highly immunogenic cancer testicular antigen named Kita-Kyushu Lung Cancer antigen-1 (KK-LC-1). In this study, we analyzed the effect of KK-LC-1 expression on the prognosis of patients with resected squamous cell lung cancer.MethodsFifty squamous cell lung cancer patients, who received complete resection, were enrolled in this study. The expressions of KK-LC-1, CD8, human leukocyte antigen (HLA) class I, and programmed cell death protein ligand-1 (PD-L1) were assessed via immunohistochemistry staining using the specimens obtained from the participants. The association between the expression of the abovementioned molecules and patient prognosis was investigated.ResultsKK-LC-1 expression was observed in 21 of 50 recruited cases (42%). However, no significant correlation was found between KK-LC-1 expression and patient prognosis. The prognosis was significantly better in lung cancer cases with KK-LC-1 expression in which CD8+ T cells infiltrated the tumor. Regardless of the HLA class I expression or the PD-L1 expression, the KK-LC-1 expression in squamous cell lung cancer could not be detected as a significant prognostic factor. Furthermore, considering the polarity of the cancer tissue as epithelium, staining of KK-LC-1 tended to be strong in the area corresponding to the basal side of the tumor tissue. The Ki-67 expression was frequently observed in cancer cells on the basal side, which was consistent with the KK-LC-1 expression in representative four cases with KK-LC-1-positive squamous cell lung cancer.ConclusionsOur results indicated that lung squamous cell cancer patients with KK-LC-1 expression and the tumor infiltrating CD8+ T cells might exhibit better prognosis. KK-LC-1 might be highly expressed in cancer cells with high proliferative capacity. Larger cohort analysis is still required for further elucidation and validation of the results of this study.

Identification of a peptide binding to cancer antigen Kita-kyushu lung cancer antigen 1 from a phage-display library.

Kita‐kyushu lung cancer antigen 1 (KK‐LC‐1) is a kind of cancer‐testis antigen with anti‐tumor potential for clinical application. As a class of small‐molecule antigen conjugate, tumor‐targeting peptides have broad application prospects in gastric cancer diagnosis, imaging, and biological treatment. Here, we screened specific cyclic nonapeptides from a phage‐display library. The targeting peptide with the best affinity was selected and further verified in ex vivo tissue sections. Finally, enrichment of targeting peptides in tumor tissues was observed in vivo, and the dynamic biodistribution process was also observed with micro‐positron emission tomography (micro‐PET)/computed tomography (CT) imaging. Studies showed that the specific cyclic nonapeptide had a high binding capacity for KK‐LC‐1 protein. It has a strong affinity and specificity for KK‐LC‐1‐expressing positive tumor cells. Targeting peptides were significantly enriched at tumor sites in vivo, with very low normal tissue background. These findings demonstrated that the KK‐LC‐1 targeting peptide has high clinical potential.

Abstract 1496: KK-LC-1 targeting T cell receptor for adoptive T cell therapy

Abstract Introduction: As T cell receptor (TCR) therapy emerges as a powerful therapy in cancer treatment, the field is still limited by a relatively small number of targets that are expressed widely in tumor cells but not expressed healthy normal tissues. Kita-Kyushu lung cancer antigen 1 (KK-LC-1) is a cancer-testis antigen that is expressed only in the testis of normal adult tissues. KK-LC-1 is widely expressed across a large percentage of solid tumors. Transcriptional profiling reveals expression in triple-negative breast (TNBC, 53% of tumors), non-small cell lung (NSCLC, 40%), gastric (81%), and cervical cancers (40%). T-Cure is developing a TCR targeting KK-LC-1 (820TCR) that is restricted to HLA-A*01, which is expressed in approximately 25-30% of the U.S. population. The 820TCR was identified to be specific for KK-LC-152-60 peptide NTDNNLAVY from a responder cervical carcinoma patient. Methods: KK-LC-1 gene expression was measured by qPCR in donor total RNA. Jurkat cells and PBMCs from 5 healthy donors were transduced with a retroviral construct containing the 820TCR, and transduction efficiency was measured by flow staining of the mouse TCR-B constant region. Cell-free binding assays were performed using A*01 tetramers loaded with KK-LC-152-60 peptide labeled with fluorescent markers assayed by flow. TCR specificity was measured by co-culture of transduced Jurkat and T cells with peptide pulsed APCs and on/off-target tumor cell panels. Off-target reactivity was assayed by co-culture of transduced T cells with a panel of normal primary cells across multiple organ systems. All co-cultures measured cytokine release by ELISA and multi-cytokine Luminex panels. Results: Gene expression analysis of RNA from a panel of normal primary donor tissues shows that KK-LC-1 expression is restricted to testis. Cell lines derived from affected histologies also maintain high KK-LC-1 expression in vitro. 820TCR transduced Jurkat and donor T cells showed over 85% of transduced T cells exhibited A*01-KK-LC-152-60 tetramer binding. 820TCR-transduced donor T cells displayed high specificity in co-culture with a panel of cell lines of mixed A*01 and KK-CL-1 status. Reactivity was observed in all naturally expressing KK-LC-1 lines that are A*01+. Reactivity was observed across a wide range of expression levels, and high expression was not required for tumor reactivity. No reactivity was observed in any non-A*01 HLA type or in A*01+/KK-LC-1- tumors. Reactivity could be induced in these cells by exogenous expression of A*01 in non-A*01/KK-LC1+ cells and KK-LC-1 in A*01/KK-LC-1- cells. Off target analysis revealed no reactivity of 820TCR with any normal primary cells. Conclusion: KK-LC-1 is a promising target for adoptive TCR cell therapy, and the 820TCR targeting KK-LC-1 is a highly potent and specific TCR with no observed off-target effects. A phase I clinical trial with 820TCR is being planned for patients with cervical, NSCLC, TNBC, and gastric cancers. Citation Format: Nicholas S. Davis, Catherine Leites, Helicia Paz, Leslie Ryan, Nathaniel Magilnick, Christian Hinrichs, Gang Zeng, Erika von Euw. KK-LC-1 targeting T cell receptor for adoptive T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1496.

Reverse Vaccinology Approach in Constructing a Multi-Epitope Vaccine Against Cancer-Testis Antigens Expressed in Non-Small Cell Lung Cancer.

Background:The 5-year survival rate of non-small cell lung cancer (NSCLC) patients has not significantly improved despite advancements in the currently applied treatments. Thus, efforts are put forth in developing novel immunotherapeutic agents targeting cancer-testis antigens (CTA) in NSCLC. This work utilized reverse vaccinology approach in designing a novel multi-epitope vaccine targeting melanoma-associated antigen 3 (MAGEA3), MAGEA4, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), and Kita-Kyushu lung cancer antigen 1 (KK-LC1), being the most frequently expressed CTAs in NSCLC. Methods:Epitopes were mapped from the sequences of CTAs. The population coverage (PC) of identified CD4+ and CD8+ epitopes were estimated. Candidate linear B cell (BL), CD4+, and CD8+ epitopes were adjoined in a multi-epitope construct (Mvax) with flagellin domain as an adjuvant. Antigenicity, and cross-reactivity of Mvax were examined. The tertiary structure of Mvax was modelled, and validated. All epitopes included in the vaccine were docked with their human leukocyte antigen (HLA) binders. The immunogenicity of epitopes in Mvax was validated through molecular dynamics analysis. Results:Mvax contains 22 epitopes from MAGEA3, MAGEA4, NY-ESO-1, and KK-LC1. It is classified as antigenic, non-allergen, non-toxic, and possesses physicochemical stability. Epitopes have no significant hits with other human proteins, except for 2 other CTAs frequently expressed in NSCLC. The stretch of BL epitopes in Mvax confers flexibility, and accessibility emphasizing its antigenicity. The tertiary structure analysis showed that Mvax model has good structural quality. All epitopes included in the vaccine are highly immunogenic as indicated by favorable binding affinity, low binding energy, and acceptable root-mean-square deviation (RMSD). CD4+ and CD8+ epitopes have global PC of 81.81%, and 84.15%, respectively. Conclusion:Overall, in silico evaluations show that Mvax is a potential immunotherapeutic agent against NSCLC.

KK-LC-1 may be an effective prognostic biomarker for gastric cancer

BackgroundThe objective of the study was to detect the expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1) in gastric cancer (GC) specimens and analyse the associations between KK-LC-1 expression and clinicopathological parameters and clinical prognosis.MethodsAll of the 94 patients in this study were GC patients who underwent surgical resection. KK-LC-1 protein expression in GC tissue was detected by immunohistochemistry. This report applies the histological score (H-score) to evaluate KK-LC-1 expression. To calculate this indicator, the number of positive cells in each section and their staining intensity were converted to corresponding values. The expression of KK-LC-1 in the cytoplasm of cancer and normal tissues was scored to obtain their respective H values. The chi-square test, Kaplan-Meier method and Cox regression were used to analyse the linear association between KK-LC-1 expression and clinicopathological data and prognosis.ResultsIn the cytoplasm, KK-LC-1 expression in tumour tissues was significantly higher than that in normal tissues (P < 0.001). Using the median H-score as the cut-off value, we discovered that GC patients with high levels of KK-LC-1 expression in the cytoplasm had favourable overall survival (OS) (P = 0.016), and this result was statistically significant in the Cox regression analysis. Additionally, a negative correlation was found between KK-LC-1 protein expression and the pathological grade of the tumour (P = 0.036), with significantly more KK-LC-1 protein expression observed in the intestinal type of GC than in the diffuse type (P = 0.008).ConclusionsOur research data showed that KK-LC-1 expression was greater in GC tissues than in normal tissues, and higher KK-LC-1 expression was associated with longer OS of GC patients. KK-LC-1 can be used as a biomarker for a good prognosis in GC patients.

Development of adoptive immunotherapy with KK-LC-1-specific TCR-transduced γδT cells against lung cancer cells.

The present study analyzed the antitumor effect of γδT cells transduced with the TCR of cancer‐specific CTLs to establish forceful cancer‐specific adoptive immunotherapy. We cloned the TCRαβ genes from CTLs showing HLA‐B15 restricted recognition of Kita‐Kyushu lung cancer antigen‐1 (KK‐LC‐1), a cancer/germline gene antigen, identified in a lung adenocarcinoma case (F1121). The TCRαβ and CD8 genes were transduced into γδT cells induced from PBLs of healthy volunteers stimulated with zoledronate and IL‐2. The KK‐LC‐1‐specific TCRαβ‐CD8 γδT cells showed cytotoxic activity against the KK‐LC‐1 positive lung cancer cell line F1121L and produced IFN‐γ against F1121L and KK‐LC‐1 peptide‐pulsed F1121 EBV‐B cells. These responses were blocked by HLA class I and HLA‐B/C antibodies. An in vivo assay using NOD/SCID mice with xenotransplantation of human lung cancer cells was performed, and the TCRαβ‐CD8 transduced γδT cells (TCRαβ‐CD8 γδT cells) were intravenously injected. Growth inhibition of KK‐LC‐1+, HLA‐B15+ lung cancer cells was confirmed in mice with injection of the TCRαβ‐CD8 γδT cells from 1 wk after xenotransplantation of cancer cells but not in those treated 2 wk after xenotransplantation. The resected specimens of the tumor, 2 wk after xenotransplantation, highly expressed FasL but not programmed death ligand‐1 (PD‐L1) by immunohistochemical staining. FasL highly expressed cancer cells xenotransplanted 2 wk ago were resistant to TCRαβ‐CD8 γδT cells injection. These results suggested that apoptosis of Fas‐positive TCRαβ‐CD8 γδT cells may be induced by a Fas‐mediated signal after interacting with FasL‐positive cancer cells.

Expression of Kita-Kyushu Lung Cancer Antigen-1 as Detected by a Novel Monoclonal Antibody in Gastric Cancer.

Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a known cancer/testis antigen. Our group has previously shown KK-LC-1 gene expression in gastric cancer. However, could not be detected the KK-LC-1 protein due to the lack of an appropriate antibody. Here, we assessed our original monoclonal antibody (Kmab34B3) and, using it, assessed the expression of KK-LC-1 in gastric cancer. We evaluated an original monoclonal antibody against KK-LC-1 (Kmab34B3), and used this antibody to compare KK-LC-1 protein expression in tumour and non-tumour stomach cells from gastric cancer patients. Kmab34B3 stained testicular germ cells, and tumour cells in nine out of 11 (82%) specimens. In non-tumorous areas, Kmab34B3 stained 13 out of 29 (45%) pyloric gland specimens. Furthermore, Kmab34B3 also stained intestinal metaplasia positive and negative areas. Kmab34B3 was able to detect KK-LC-1 protein within tumour cells and the pyloric gland where the gene has been shown to be expressed. Therefore, it might be an attractive tool for detecting KK-LC-1 expression in precancerous and cancerous stomach cells.

Cancer targeting by TCR gene-engineered T cells directed against Kita-Kyushu Lung Cancer Antigen-1

T cell receptor (TCR) gene-engineered T cells have shown promise in the treatment of melanoma and synovial cell sarcoma, but their application to epithelial cancers has been limited. The identification of novel therapeutic TCRs for the targeting of these tumors is important for the development of new treatments. Here, we describe the preclinical characterization of a TCR directed against Kita-Kyushu Lung Cancer Antigen-1 (KK-LC-1, encoded by CT83), a cancer germline antigen with frequent expression in human epithelial malignancies including gastric cancer, breast cancer, and lung cancer. Gene-engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-Ts) demonstrated recognition of CT83+ tumor lines in vitro and mediated regression of established CT83+ xenograft tumors in immunodeficient mouse models. Cross-reactivity studies based on experimental determination of the recognition motifs for the target epitope did not demonstrate cross-reactivity against other human proteins. CT83 gene expression studies in 51 non-neural tissues and 24 neural tissues showed expression restricted exclusively to germ cells. CT83 was however expressed by a range of epithelial cancers, with the highest expression noted in gastric cancer. Collectively, these findings support the further investigation and clinical testing of KK-LC-1 TCR-Ts for gastric cancer and possibly other malignancies.

Abstract 1429: Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers

Abstract T cell receptor (TCR) gene therapy can mediate tumor regression in patients; however, successful treatment of epithelial cancers has been limited. We studied the targeting of Kita-Kyushu lung cancer antigen 1 (KK-LC-1), a cancer germline antigen expressed in multiple epithelial cancers, with TCR gene-engineered T cells. We thoroughly vetted KK-LC-1 expression in both cancerous and healthy tissue by pulling data from BioGPS and cBioPortal and analyzing a broad range of patient samples, primary cancer lines, and healthy tissue by both qRT-PCR and fluorescence in situ hybridization. Our findings show that KK-LC-1 is expressed in numerous epithelial cancers including breast, lung, cervical, ovarian, melanoma, and prostate whereas expression in healthy tissue is limited to immune-privileged sites including the epididymis and testis. We identified an HLA-A*01:01-restricted KK-LC-152-60-specific TCR isolated from the tumor infiltrating lymphocytes of a patient with metastatic cervical adenocarcinoma. T cells from two healthy donors were transduced with the KK-LC-1 TCR and tested for in vitro targeting of tumor cell lines. The KK-LC-1 TCR recognized the HLA-A*01:01+, KK-LC-1+ cell lines 4156, EKVX, and PC-3, as determined by IFN-γ release. The HLA-A*01:01- cell lines, HeLA, DU-145, and MDA-MD-468, were initially not recognized by the KK-LC-1 TCR; however, following stable transduction with HLA-A*01:01, these cell lines were targeted. The HLA-A*01:01+, KK-LC-1- cell lines, PC-3 and 3748, were not recognized by the KK-LC-1 TCR. To assess the TCR’s potential for off-target activity, we used amino acid scanning to determine the recognition motif, allowing for subsequent targeted in silico searches to identify peptides containing the same recognition motif. Using NCBI Blast and the ScanProsite database, 10 peptides with homologous essential but different non-essential residues were identified. A retroviral vector encoding the KK-LC-152-60 TCR was used to genetically engineer peripheral blood T cells. No peptide recognition by the TCR was observed when dendritic cells were pulsed with the individual peptides and co-cultured with KK-LC-1 TCR-transduced T cells. Finally, we assessed if human genetically engineered T cells expressing the KK-LC-1 TCR could mediate in vivo tumor regression in an NSG mouse model. Genetically engineered T cells expressing the KK-LC-1 TCR induced tumor regression in both a melanoma and cervical cancer line in a dose-dependent manner. Collectively, these findings indicate that KK-LC-1 is a suitable target for TCR gene therapy and they provide preclinical support for testing of the KK-LC-1 TCR in a phase I clinical trial. Citation Format: Bridget Marcinkowski, Carylinda Serna, Benjamin Jin, Scott Norberg, Christian Hinrichs. Preclinical characterization of a KK-LC-1-specific T cell receptor for the treatment of epithelial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1429.

Detection of KK-LC-1 Protein, a Cancer/Testis Antigen, in Patients with Breast Cancer.

Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen and predominant target for cancer immunotherapy. Its detection is only established based on gene expression. In this study, we established a monoclonal antibody against KK-LC-1 to detect its protein expression in formalin-fixed samples. The monoclonal antibody against KK-LC-1 was evaluated and the detection of KK-LC-1 between gene expression and protein expression was compared in patients with breast cancer. The monoclonal antibody clone 34B3, which we established, stained testicular germ cells positively. The rates of detection of KK-LC-1 gene and protein expression were 11.8% and 52.9%, respectively. Protein expression was detected in all triple-negative breast cancer cases studied (n=8). Furthermore, KK-LC-1 was detected in all tumours without oestrogen receptor expression. This study indicated that KK-LC-1 expression was detected in breast cancer, especially in oestrogen receptor-negative subtypes.

Expression of KK-LC-1, a cancer/testis antigen, at non-tumour sites of the stomach carrying a tumour

Kita-Kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen (CTA) and predominant target for cancer immunotherapy. Our previous study indicated that KK-LC-1 was expressed in 82% of gastric cancers, and also in 79% of early stage of gastric cancers, with a correlation to Helicobacter pylori (H. pylori) infection. In addition, we found that KK-LC-1 was occasionally expressed at non-tumour sites of stomachs carrying tumours. Here, we investigated the characteristics of KK-LC-1 expression at non-tumour sites and the clinical utility of these phenomena. The gene expression of KK-LC-1 was detected at the non-tumour sites including pyloric glands. The most detectable corpus/gland subset had a KK-LC-1 expression rate of 77% in the pyloric gland of the lower corpus where H. pylori preferentially exists. KK-LC-1 expression rates were 67% or 32% with or without intestinal metaplasia, which also induced by H. pylori, respectively. Consequently, KK-LC-1 would be detected at the pre-cancerous condition of the stomach, and may be a useful marker to predict gastric cancer.

Correlation Between Expression of the Cancer/Testis Antigen KK-LC-1 and Helicobacter pylori Infection in Gastric Cancer.

Our previous study indicated that Kita-kyushu lung cancer antigen-1 (KK-LC-1) is a cancer/testis antigen (CTA) expressed in 82% of gastric cancer cases. Here, we investigated the relationship between KK-LC-1 expression and Helicobacter pylori infection in Japanese patients with gastric cancer. We examined CTA expression in 25 surgical gastric cancer specimens and anti-H. pylori IgGs in the serum of each patient. KK-LC-1 was expressed in 80% of tumor samples, markedly higher than melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, synovial sarcoma, X breakpoint 4 (SSX4) and New York esophageal squamous cell carcinoma-1 (NY-ESO-1). Anti-H. pylori IgG titers from the KK-LC-1-positive patients were significantly higher (67.5±7.6) than those from KK-LC-1-negative patients (15.8±7.5, p<0.01) although there were no significant differences between patients positive and negative for MAGE-A1, -A3 and-A4, SSX4 and NY-ESO-1. As far as we are aware, this is the first report of a correlation between a carcinogen and CTA expression in clinical samples. KK-LC-1 was frequently expressed in gastric cancer caused by H. pylori infection. The risk diagnosis for gastric cancer might be more accurate if KK-LC-1 expression status were also considered.

Helicobacter Pylori Infection Induces Gastric Cancer and the Cancer/Testis Antigens Expression

Helicobacter pylori (HP) are known to induce gastritis, atrophy, intestinal metaplasia, dysplasia, and stomach malignancy. HP infection also induces cancer/testis antigens (CTAs) during or after gastric malignancy. This study aims to elucidate the relationship between HP and CTAs, especially melanoma antigen-A3 and Kitakyushu lung cancer antigen-1.