Adoptive T-Cell Therapy in Solid Tumor with KRASG12V/G12D Mutation

Adoptive T-Cell Therapy in Solid Tumor with KRASG12V/G12D Mutation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adoptive T-Cell Therapy in Solid Tumor with KRAS G12V/G12D Mutation Hong-Ming Hu, Zhifen Zeng, Tao Qin, Guangjie Yu, Shih-Ting Tsao, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7496490/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract KRAS, the most commonly mutated oncogene, plays a central role in the pathogenesis of many cancers. Identifying T cell receptors (TCRs) reactive to mutant KRAS enables the exploration of TCR gene-modified T cell (TCR-T) therapy for solid tumors with KRAS mutations. An investigator-initiated trial (IIT) evaluated adoptive TCR-T therapy using autologous T cells engineered to target KRAS G12V or KRAS G12D mutations in HLA-A*11:01-positive patients. Eligible individuals had advanced pancreatic or other solid tumors with these mutations and received a single infusion of 1 × 10⁹ to 1 × 10¹⁰ TCR-T cells. The primary endpoint was progression-free survival (PFS); secondary endpoints included objective response rate (ORR), overall survival (OS), duration of response (DOR), disease control rate (DCR), and safety. So far, we observed that ORR (100%), PFS (10, 8, and 27 months), DOR (10, 8, and 13 months), and OS (17, 10, and 30 months) in two patients with PDAC and NSCLC. No grade 3–4 toxicities occurred. All patients exhibited cytopenia, characterized by anemia, transient thrombocytopenia, and a reduction in white blood cells due to the lymphodepletion required for TCR-T therapy. These results demonstrate that TCR-T therapy targeting KRAS G12V/D is feasible and beneficial in patients with advanced solid tumors. The IIT trial is ongoing to recruit more patients with KRAS G12V/D mutations (NCT05438667). Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Immunology/Tumour immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 5 Figure 6 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pancreatic cancer and non-small cell lung cancer (NSCLC) rank among the ten most common cancers globally. It is well-known that pancreatic cancer is associated with poor prognosis, exhibiting a 5-year survival rate of less than 10% 1 . In addition, lung cancer is the leading cancer globally, accounting for 20% of cancer-related mortality worldwide and 22% in China 1 , 2 . NSCLC accounts for 80–85% of all lung cancer cases. Despite advances in immune checkpoint inhibitors and targeted therapies using small-molecule inhibitors, treating advanced metastatic NSCLC beyond second-line therapy remains challenging. The Chinese Society of Clinical Oncology (CSCO) recommends anlotinib as a third-line treatment for advanced NSCLC. However, the prognosis remains poor, and there is a significant unmet need in treating NSCLC with KRAS mutations. KRAS mutation, an important biomarker in solid tumors, was found in NSCLC in 1984, and chemotherapy and immunotherapy were still the standard of care. Both in PAC and NSCLC, the KRAS gene mutation is commonly found at rates of 90% and 30%, respectively 3 , which is regarded as a gene associated with poor survival in these tumors. The most common mutation sites in the KRAS gene are found in codon 12, which represent over 80% of all variants, including G12A, G12C, G12D, G12R, G12S, and G12V mutations. Efforts to target these KRAS mutations had been largely unsuccessful for decades until the recent development of a small-molecule inhibitor targeting the KRAS G12C mutation. In patients with KRAS G12C-mutated pancreatic cancer treated with sotorasib, the median progression-free survival was 4.0 months, and the median overall survival was 6.9 months 4 . However, the KRAS G12C mutation is relatively rare in pancreatic cancer, occurring in only 1–2% of cases, which is significantly lower than its prevalence in lung or colon cancer 5 , compared to the more dominant G12D or G12V mutations 6 . Currently, there is no targeted therapy available for patients with KRAS G12D or G12V mutations. Notably, patients with NSCLC and actionable gene mutations who received a combination of chemotherapy and immunotherapy demonstrated a median survival of 12.6 months, as reported in the Keynote 189 study. Clinical outcomes have improved significantly for NSCLC patients with EGFR mutations and ALK fusions, with median survivals of 18.9 months 7 and 36.7 months 8 , respectively. However, there remains an unfavorable prognosis and significant unmet need in the treatment of NSCLC with KRAS mutations. Adoptive cell therapy utilizing T-cells that recognize mutant KRAS represents another potential strategy for targeting KRAS mutations 9 . This approach is bolstered by findings from Tran et al., who documented substantial tumor regression in a patient with widely metastatic colorectal cancer following the infusion of tumor-infiltrating lymphocytes (TILs) that recognized the KRAS G12D mutation in association with the HLA-C08:02 allele 10 . More recently, Tran and colleagues achieved regression of metastatic pancreatic cancer in a patient after administering a single infusion of autologous T cells. These T cells had been genetically modified to express HLA-C08:02-restricted TCRs specifically targeting the KRAS G12D mutation, which were cloned from TILs initially obtained from the aforementioned colorectal cancer patient 11 . However, the prevalence of the HLA-C08:02 allele is exceedingly low, particularly among Chinese patients (1%). Conversely, the HLA-A11:01 allele is the most common in the Chinese population, occurring in 30% of individuals. Therefore, targeting patients with prevalent HLA alleles and either G12D or G12V mutations could extend the benefits of this therapy to a broader demographic. Here, we present three cases of advanced solid tumors that were effectively treated with autologous T cells engineered to express exogenous T cell receptors (TCRs) targeting the KRAS G12V or KRAS G12D mutations within the context of the HLA-A*11:01 allele. The cohort included two cases of PDAC and one case of metastatic NSCLC. Tumor regression was observed in all three patients, and the treatment was well-tolerated by each individual. Results Patients and clinical assessment Three HLA-A*11:01 positive patients with either KRAS G12V or KRAS G12D mutations were enrolled in the TCR-T cell therapy study. Two of these patients had advanced metastatic PDAC, and one had advanced NSCLC. Before TCR-T therapy, they had received a median of two lines of systemic therapy. Each patient was infused with an average dose of 8.2 × 10^ 9 autologous T cells engineered to express TCRs targeting the specific KRAS mutations. Tumor response was evaluated post-infusion of TCR-T cells. Both PDAC patients achieved objective partial responses within one month of infusion, with PFS durations of 10 and 8 months, respectively. The NSCLC patient, who had undergone extensive prior treatments, was assessed as having achieved partial remission. Safety assessments were conducted to monitor the occurrence of adverse events. All patients experienced cytopenia, including anemia (≤ grade 2), transient reductions in platelet count (≤ grade 2), and transient decreases in white blood cell counts (grade 2–3), likely resulting from the lymphodepletion preconditioning regimen. Additionally, two patients experienced moderate fevers lasting less than 48 hours after the infusion, and one patient reported swelling and redness at the IL-2 injection sites. No infections related to the therapy were observed, and no fatal adverse events occurred. This profile suggests that TCR-T therapy, while potent, is manageable in terms of toxicity. Case report of each patient Patient 001 (Fig. 2 , Table 1 ) Table 1 Response in Individual Patient with Various Tumor Types Patient ID Age(y) Sex Tumor Type KRAS Mutation Sites of Metastases Lymphodepletion regimen IL-2 usage Cell Dose mTCR+% Response PFS (months) OS (months) 001 59 M PDAC G12V Abdominal lymph nodes Flu 30mg/m 2 ×3d CTX 300mg/m 2 ×2d 500,000IU/m 2 q12h*14d 5.1×10 9 84.8% TCR 051 PR 10 17 68.3% TCR PCV 002 63 F PDAC G12D Liver Flu 30mg/m 2 ×3d CTX 300mg/m 2 ×2d 500,000IU/m 2 q12h*14d 8×10 9 90.9% TCR-2 PR 8 10 003 71 F NSCLC G12D Supraclavicular lymph nodes Flu 30mg/m 2 ×3d CTX 300mg/m 2 ×2d 500,000IU/m 2 q12h*14d 1×10 10 46.7% TCR-2 PR 27 30 PDAC, pancreatic ductal adenocarcinoma; NSCLC, non-small cell lung cancer; PR, partial response; PD, progression disease; SD, stable disease; OS, overall survival; PFS, progression free survival; mTCR+%, percentage of CD3 + T cells express engineered TCR A 57-year-old male was diagnosed with adenocarcinoma of the pancreatic head via EUS-guided fine-needle biopsy after presenting with jaundice and an elevated CA19-9 level (454.0 U/ml, reference ≤ 34 U/ml) in June 2019. He underwent pylorus-preserving open Whipple surgery in the same month. Post-operative pathology indicated poorly differentiated PDAC with a disease stage of IIB (pT1N1M0). The patient subsequently received 12 cycles of adjuvant mFOLFIRINOX chemotherapy, resulting in normalization of his CA19-9 levels and a disease-free status for 16 months post-surgery. In October 2020, new mesenteric root lymphadenopathy was identified via MRI and PET-CT, and his CA19-9 level increased to 222.3 U/ml as of November 3, 2020, suggesting metastatic recurrence. He underwent stereotactic body radiation therapy (SBRT) targeting the mesenteric root lymphadenopathy. This was followed by a second-line AG chemotherapy regimen, which was discontinued after the first day due to severe bone marrow suppression and skin rashes. Subsequently, his CA19-9 level temporarily decreased to 81.7 U/ml but began to rise again. By March 2021, the irradiated mesenteric root lymphadenopathy remained stable; however, new metastases were discovered in the hepatic portal and retroperitoneal lymph nodes, with CA19-9 increasing to 403 U/ml. A second course of SBRT was administered targeting these newly identified metastatic lymph nodes. In May 2021, another enlarged lymph node metastasis was detected in the abdominal mesenteric region, accompanied by a sharp increase in CA19-9 to 927.9 U/ml. Following this, the patient was enrolled in a TCR-T therapy program. This patient underwent NGS testing of his tumor, which revealed missense mutations of KRAS Exon 2 p.G12V (c.35G > T, abundance: 29.93%) and TP53 exon 6 p.Y220C (c.659A > C). HLA typing of his peripheral blood samples confirmed the presence of the HLA-A*11:01 allele. In June 2021, following preparatory lymphodepletion with fludarabine (30 mg/m 2 per day for three days) and cyclophosphamide (300 mg/m 2 per day for two days), he received an infusion of 5.1×10 9 TCR-gene positive T cells via intravenous administration over 30 minutes. This was supplemented with low-dose subcutaneous IL-2 injections (500,000 IU/m 2 every 12 hours) for 14 days to enhance the in vivo persistence of the transferred cells. Rapid regression of metastatic lymph nodes was observed on MRI within the first month post-infusion, with the target measurable metastatic lymph node shrinking from 16 mm to 4.2 mm, a 75% reduction by the sixth month. However, by the ninth month, the short axis of the target lesion had increased to 9 mm, though no other metastases were yet detected. CA19-9 levels peaked at 1763 U/ml one week post-infusion, halved within a month, and reached a nadir of 40 U/ml by the fourth month, maintaining minimal fluctuations for the subsequent six months (Fig. 2 ). The patient achieved a 75% tumor reduction and remained progression-free for ten months post-TCR-T therapy. However, ten months post-infusion, he was hospitalized due to mild back pain and abdominal distension, and MRI scans indicated slight lymph node enlargement in the hepatic portal, retroperitoneal, and mesenteric regions, suggesting disease progression. Twelve months after the infusion, MRI and subsequent needle biopsy confirmed newly developed multiple liver metastases, displaying a higher abundance (60.47%) of the KRAS p.G12V mutation compared to the initial tumor (Fig. 3 ). Despite initial responsiveness to TCR-T therapy, the patient's condition deteriorated due to progressive liver metastases, leading to his death in November 2022, with an overall survival of 17 months post-therapy. Patient 002 A 63-year-old female presented with abdominal pain and jaundice and was diagnosed with pancreatic cancer. She underwent tumor resection in July 2021. Postoperative pathology identified the tumor as moderately differentiated PDAC with a pathological staging of pT4N1M0. IHC results were positive for CK7, CK19, focal CK20, Muc-1, and some Muc-5AC, with a high proliferation index shown by Ki67 at approximately 60%. P53 and Muc-2 were negative. A NGS test of the tumor revealed a KRAS Exon 2 p.G12D missense mutation with an abundance of 8.14%. Subsequently, the patient underwent two cycles of AG chemotherapy, after which MRI imaging revealed new liver metastases, the largest located in the S7 segment with a diameter of 16 mm, indicating disease progression. As a result, AG chemotherapy was discontinued. In November 2021, after receiving pretreatment with the FC regimen, the patient received a single infusion of 8 × 10^ 9 transgenic TCR-positive cells. Post-infusion, IL-2 injections were administered as per the protocol. Within one month, the diameter of the largest liver metastasis decreased to 11 mm, became almost undetectable at three months post-infusion, and the condition remained stable for an additional three months (Fig. 2 ). However, eight months after the TCR-T infusion, in July 2022, there was a progression of liver metastases, and the patient subsequently passed away in September 2022. The patient achieved an objective partial response with a PFS of 8 months and an OS of 10 months following the single infusion of TCR-T cells. This case underscores the transient efficacy of TCR-T therapy in treating aggressive metastatic PDAC with specific KRAS mutations, highlighting the need for ongoing assessment and management of disease progression post-therapy. Patient 003 A 71-year-old female with a history of cough and chest pain was diagnosed with stage IV NSCLC, cT3N3M1, in January 2021. A biopsy from a neck lymph node confirmed adenocarcinoma, showing positive results for TTF-1, Napsin A, E-cadherin, and an immune cell (10%+), with a Ki67 proliferation index of 70%. The tumor was negative for EGFR, ALK, ROS1, estrogen and progesterone receptors, and PD-L1 (22C3) TPS, while HER-2 was equivocally expressed at 2 + and strongly at 3 + in 80% of cells. The patient also had a KRAS p.G12D mutation, a TP53 p.E198X mutation, a tumor mutational burden (TMB) of 4.2 mutations per megabase (Muts/Mb), and stable microsatellites (MSS). Her medical history included hypertension, diabetes, coronary heart disease, and invasive breast cancer treated with mastectomy and axillary lymph node resection in 2016, followed by anastrozole without adjuvant chemotherapy. We then initiated treatment with docetaxel, carboplatin, and the anti-PD1 antibody Camrelizumab on March 8, 2021, achieving stable disease after two cycles. However, she experienced severe fatigue, musculoskeletal pain, and reactive cutaneous capillary endothelial proliferation (RCCEP), leading her to refuse further chemotherapy. She then received another anti-PD1 antibody, Tislelizumab. Despite this, the primary lung tumor progressed by August 26, 2021. She underwent two cycles of second-line chemotherapy with pemetrexed and carboplatin starting September 30, 2021, during which her supraclavicular and mediastinal lymph nodes enlarged, causing superior vena cava syndrome. This was managed with endovascular therapy and radiation, leading to significant symptomatic relief. However, her condition was complicated by severe thrombocytopenia, and she could not tolerate further chemotherapy. Disease progression was noted on November 12, 2021, with increased lymph node metastases. Subsequently, she was enrolled in a clinical trial and received TCR-T cell therapy after lymphodepletion. On November 17, 2021, she was infused with 1 x 10^ 10 TCR-T cells, followed by a two-week course of IL-2. She experienced only mild fatigue and a fever after the infusion. One month later, her CEA levels dropped from 12.9 ng/mL to 9.7 ng/mL, and the targeted metastatic mediastinal lymph nodes decreased in size, classified as partial remission (Fig. 2 ). Despite initial success, an MRI on February 14, 2024, revealed multiple brain metastases, indicating disease progression. Her progression-free survival was 27 months post-TCR-T infusion. She underwent surgical intervention for brain metastases but did not receive further systemic therapy and ultimately succumbed to lung cancer on May 11, 2024. Her OS from the first TCR-T infusion was approximately 30 months. In vivo persistence of transduced KRAS G12V or G12D-reactive T Cells Post-Infusion The in vivo persistence of infused T cells is a crucial determinant of the antitumor efficacy of adoptive T cell therapies, including CAR-T for hematopoietic malignancies and TILs for solid tumors. For the three patients treated, the persistence of engineered TCR-T cells post-infusion was closely monitored. The first patient received an infusion containing two TCR constructs, PCV and 051, both targeting G12V mutations. Flow cytometry analysis, using an antibody against the mouse TCR constant region to identify exogenous TCR-expressing cells, showed that 68.3% and 84.8% of CD3 + cells were positive for PCV TCR and 051 TCR, respectively (Fig. 4 A). The percentage of CD3 + , mouse TCR-positive T cells peaked at 56.89% week post-infusion and then decreased to 9.88% after two weeks. A minor resurgence to 12.11% was observed at three weeks, with a gradual decline to 1.42% and 0.32% by weeks 9 and 16, respectively. By the six-month mark, the transgenic T cells had become nearly undetectable (Fig. 4 B, 4 C). Consistent with these findings, the persistence of infused T cells detectable by primers specific for the retroviral vector backbone (RVV) showed a significant drop by the 12th week post-infusion (Fig. 4 D). Moreover, the in vivo persistence of the two individual TCRs was assessed using digital droplet PCR (ddPCR) with TCR-specific primers, revealing that the 051 TCR exhibited superior persistence compared to the PCV TCR. At weeks 1 and 2 post-infusion, both TCRs were found in equal numbers, but by weeks 7 and 8, the persistence of the 051 TCR was approximately three times higher than that of the PCV TCR. Given these observations, the differential persistence of these TCRs could significantly impact their antitumor activities. Therefore, subsequent patients will be treated with autologous T cells modified solely with the 051 TCR gene, anticipating that it may offer enhanced persistence and more effective in vivo antitumor activity. For Patient 002 , who received TCR-modified T-cell therapy, a notable expansion of mainly CD8 + T cells was observed during the first month following infusion. The peak expansion level reached 32% of total T cells, as indicated in Fig. 5 A. Despite this significant early expansion, the modified T cells were undetectable by flow cytometry analysis 120 days after the cell infusion, suggesting a decline in the persistence of these cells over time. Contrary to the significant expansion observed in the first two patients, both of whom had PDAC, the expansion level of T cells in the third patient with NSCLC was considerably lower. This observation is illustrated in Fig. 5 B. Despite this lower level of initial T-cell expansion, the infused T cells in the NSCLC patient exhibited an interesting persistence pattern, maintaining a stable presence at a level comparable to the first two weeks for over 90 days post-infusion. Several factors, including differences in the tumor microenvironment, the specific characteristics of the infused T cells, or the underlying immune status of each patient, could influence this variation in T-cell expansion and persistence between patients. The sustained persistence of T cells in the NSCLC patient, despite low expansion, suggests that even a modest number of infused T cells may maintain a therapeutic effect over a prolonged period. These observations underscore the complexity of predicting responses in adoptive T-cell therapies and highlight the need for further studies to optimize these treatments based on individual patient and tumor characteristics. Transient Cytokine Release in Peripheral Blood after Cell Infusion For Patient 001 , following a lymphodepletion regimen with adjusted doses of fludarabine and cyclophosphamide, only a transient episode of lymphopenia was observed, with stable neutrophil and platelet counts, as depicted in Fig. 6 . This modified regimen likely contributed to the patient's well-being post-infusion, as no prophylactic antibiotics or antiviral drugs were administered, and the patient experienced fewer complications. Significantly, no major adverse events typically associated with T-cell infusion therapies, such as CRS, neurotoxic effects, or prolonged cytopenia, were observed in this patient. These conditions are often reported in treatments involving CAR-T cells, where they occur with high frequency and severity. The lymphocyte count for patient 001 showed a rapid increase by day seven post-infusion, suggesting a robust proliferation of the infused T cells, followed by a decline to pre-therapy levels. In terms of cytokine levels, patient 001 exhibited elevated levels of IL-6, TNF-α, and IL-1β one week after cell infusion (Fig. 7 A). Although the patient experienced a brief fever within 48 hours of the therapy, which resolved quickly, no other symptoms indicative of CRS were noted. This contrasts with Patient 002 , who did not exhibit a significant rise in cytokine levels, possibly correlating with a lower percentage of CD3-positive, mouse TCR-positive T cells compared to Patient 001. This difference suggests that higher cytokine levels in Patient 001 could be associated with better proliferation and persistence of the transduced modified T cells. Unfortunately, for patient 003, cytokine release data could not be comprehensively measured due to logistical challenges in blood sample collection resulting from the COVID-19 pandemic, limiting a complete assessment of cytokine dynamics for this patient. This gap highlights the influence of external factors on clinical monitoring and underscores the importance of consistent sample collection in assessing immunological responses in T-cell therapy trials. Continuous monitoring of circulating tumor DNA (ctDNA) in peripheral blood after TCR-T cell infusion. Continuous monitoring of ctDNA is a critical component in assessing the early molecular response and potential relapse in patients undergoing TCR-T cell therapy. In the case of Patient 001 , ctDNA levels were routinely measured in the peripheral blood post-infusion. Notably, seven months after receiving the TCR-T cell therapy, an increase in the abundance of the KRAS G12V mutation was observed, as illustrated in Table 2 . This rise in ctDNA occurred before any radiological evidence of disease progression was visible on MRI scans. Table 2. ctDNA was detected in the peripheral blood of Patient 001 . The abundance of the KRAS G12V mutation began to increase seven months after the infusion of TCR-T cells in this patient. Date Abundance of KRAS G12V mutation 2022.1.14 0.12% 2022.1.20 0.17% 2022.2.13 0.20% 2022.4.20 0.22% 2022.4.30 1.00% Discussion Our study demonstrated that cancer-antigen-specific TCR-T cell therapy is an effective treatment modality for solid tumors, including pancreatic cancer and NSCLC. We observed comparable efficacy of TCR-T cells in treating pancreatic cancer cases. Notably, this research also marks the first report of TCR-T therapy being applied to treat NSCLC harboring the KRAS G12V mutation, showing promising anti-tumor efficacy and contributing to prolonged survival. These findings highlight the potential of TCR-T cells to target specific cancer antigens, providing a promising therapeutic approach for tumors that are traditionally resistant to other forms of treatment. This approach also underscores the significance of genetic profiling in identifying suitable targets for TCR-T therapy, enabling more personalized and potentially more effective treatments for cancer patients. Pancreatic cancer remains one of the most lethal malignancies, with a 5-year survival rate under 10% 1 . Traditional second-line treatments offer limited benefits for patients who experience recurrence or progression after first-line chemotherapy. For instance, in the Sotorasib clinical trial targeting KRAS p.G12C–mutated advanced pancreatic cancer, the mOS was reported as 6.9 months, and the mPFS was 4.0 months 4 . Patients with other KRAS mutations, such as KRAS G12D or KRAS G12V, typically have no targeted therapy options available, aside from chemotherapy. Regardless of the chosen second-line regimen—whether S1 as monotherapy, or double or triple regimens involving 5-Fu and Oxaplatin with or without irinotecan—the mOS ranged from 4.9 to 9.2 months, while the mPFS ranged from 2.1 to 5.2 months 12 , 13 . In our study, two patients with metastatic PDAC who had undergone multiple lines of prior treatments achieved an OS of 17 and 10 months and a PFS of 10 and 8 months, respectively. When compared to conventional therapies, our approach, which utilizes autologous T cells engineered to target KRAS G12V or KRAS G12D mutations, demonstrates superior efficacy and minimal toxicity. Given the typically poor prognosis of late-stage pancreatic cancer, these outcomes are particularly promising. These results support the potential for further research into the use of autologous T cell therapies targeting specific KRAS mutations in pancreatic cancer. This could pave the way for more personalized and effective treatment strategies, offering hope for improved outcomes in a patient population in dire need of better therapeutic options. In the treatment of NSCLC, targeted therapy is applied specifically to patient subtypes bearing actionable gene mutations. For those lacking such mutations, chemotherapy and immunotherapy are the most commonly employed treatments. A Phase 3, double-blinded, randomized clinical trial assessed the efficacy and safety of anlotinib as a third-line or subsequent therapy in advanced NSCLC, where the median overall survival (mOS) and median progression-free survival (mPFS) were recorded at 9.6 months and 5.4 months, respectively. Recent advancements have highlighted antibody-drug conjugates (ADCs) as promising treatments for NSCLC. For instance, the TROPION-Lung01 study reported a 31.2% response rate and a 5.6-month PFS for NSCLC patients treated with a TROP2-ADC (NCT04656652). Furthermore, differential responses have been noted in patients based on HER2 status; those with HER2 overexpression treated with T-DxT showed no response, unlike those with HER2 mutations, who exhibited high response rates 15 . In our study, we explored the efficacy of TCR-T cell therapy targeting the KRAS G12D mutation as a third-line treatment for advanced NSCLC. This approach demonstrated promising anti-tumor efficacy and contributed to long-term survival following the first TCR-T therapy. These findings suggest that adoptive TCR-T cell therapy targeting specific KRAS mutations could represent a valuable treatment alternative for advanced NSCLC, in addition to its application in PDAC. This expands the therapeutic landscape and provides a potentially effective option for patients with NSCLC who have exhausted other treatments. To date, targeted therapies for KRAS mutations in cancer have largely been limited to the KRAS G12C mutation. Our study represents the first report on the efficacy and safety of engineered TCR-T cells targeting the KRAS G12D mutation specifically in the treatment of patients with NSCLC. This advancement is notable considering the historical challenge of effectively targeting KRAS mutations due to their high variability and the general resilience of the KRAS protein to small-molecule inhibitors. Previously, there have been limited reports of TCR-T cell therapies targeting other antigens in NSCLC. For instance, a case report indicated a short-term partial response in advanced NSCLC using TCR-T cells targeting the cancer-testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1) 16 . In another clinical trial, 11 patients with advanced NSCLC treated with TCR-T cells targeting the melanoma-associated antigen A10 (MAGE-A10) achieved a median progression-free survival (mPFS) of 58 days and a median overall survival (mOS) of 132 days, with only one patient reporting an objective clinical response (partial response, PR). The role of T-cell responses against neoantigens is well-established in enhancing the antitumor efficacy of immune checkpoint inhibitors (ICIs 18 . TCRs that recognize these neoantigens could potentially be used for personalized adoptive cell transfer therapies 19 . However, a significant challenge remains, as most neoantigens are patient-specific, and only a minority (less than 5%) are immunogenic 20 . Given these challenges, the success of TCR-T cells targeting KRASG12D in our study suggests a promising new direction for personalized cancer therapy in NSCLC, especially for patients whose tumors harbor specific KRAS mutations that have previously been difficult to target with conventional therapies. This opens up potential for broader applications of TCR-T cell therapies, emphasizing the importance of continued research and development in this area. The deployment of cancer-antigen-specific TCR-T cells in the fight against malignant tumors has become a prominent focus in recent years. High-profile targets, such as NY-ESO-1, MART-1, and HPV E6 or E7, have been extensively studied and implemented in clinical settings. However, the exploration of TCR-T therapy specifically targeting KRAS mutations is relatively nascent, with primarily anecdotal evidence and a few case reports substantiating its use 21 . Our study makes a significant contribution to this emerging field by providing long-term follow-up data for each patient, offering invaluable insights into the practical application of KRAS-specific TCR-T cells. This is particularly crucial for treating pancreatic cancer, a malignancy characterized by a dismal prognosis and a high incidence of KRAS mutations, where traditional treatment options frequently fall short. Previous research on TCR-T therapy in other types of cancer, such as melanoma , , myeloma, hepatocellular carcinoma, HPV-associated epithelial cancers, and synovial sarcoma, underscores that the proliferation and persistence of gene-modified TCR-T cells are critical to achieving a substantial anti-cancer effect. In our study, for example, Patient 001 demonstrated a notably successful outcome; the transduced TCR-T cells not only expanded to a high level but also maintained their presence for over three months, correlating with a superior therapeutic result and the most prolonged survival among the cohort. The varying peak percentages of mTCR + CD3 + cells among the patients (ranging from 1.42–56.89%) highlight an area for further investigation. The challenge of maintaining robust expansion and persistent viability of infused TCR-T cells is critical to their long-term efficacy in cancer treatment. After infusion, the gene-modified TCR-T cells often exhibit a rapid decline in number, becoming barely detectable within a few months, which may potentially lead to tumor recurrence or progression. To counteract this, repeated infusions of TCR-T cells could theoretically sustain or enhance the therapeutic effects. The feasibility of this approach has been demonstrated in various clinical trials. For instance, in a phase I trial of immunotherapy using HBV-specific TCR-redirected T cells for advanced hepatocellular carcinoma 25 , patients received between one and four treatment cycles, spaced over 30-day intervals. The patient who underwent a maximum of four cycles showed the longest time to progression (TTP), reaching 27.7 months. Conversely, in a trial targeting synovial cell sarcoma with NY-ESO-1-reactive TCR-engineered T cells 27 , while 61% of patients responded to the first infusion, only one out of the six who received a second infusion achieved a sustained response, lasting an additional nine months following the second treatment. Interestingly, this patient did not respond to a third infusion, highlighting the variable outcomes associated with repeated TCR-T cell treatments. The utility and timing of subsequent TCR-T infusions need further investigation to optimize benefits. In this context, ctDNA emerges as a highly promising biomarker for guiding such decisions. ctDNA, which reflects tumor-specific genetic and epigenetic features with high tissue and cell-type specificity, serves as a sensitive and specific indicator for detecting recurrence. Monitoring ctDNA levels could provide critical insights into tumor dynamics and help clinicians determine the most opportune moments for additional TCR-T infusions, thereby enhancing treatment strategies and potentially improving patient outcomes. This approach not only could extend the benefits of TCR-T therapy but also help in customizing surveillance and treatment modifications in response to the evolving tumor landscape. Conclusion Our study highlights the significant anticancer effects of autologous T cells modified with TCRs that specifically target KRAS G12V or KRAS G12D mutations in patients with PDAC and NSCLC. The implications of these findings extend beyond these two cancer types, offering potential benefits to a broader patient cohort. Recent advancements have expanded the repertoire of available TCRs, with a dozen new TCRs identified that are specific for various KRAS mutations, including G12D, G12V, G12R, G12C, and G13D. These TCRs are compatible with a range of class I and II HLA molecules, significantly broadening the potential applicability of this therapy. The development of a comprehensive TCR library targeting these mutations opens up new avenues for treating a variety of advanced, lethal cancers. The broader availability of targeted TCRs marks a substantial leap forward in precision medicine, offering hope for improved treatment outcomes in cancers traditionally associated with poor prognoses. With these tools, we may soon realize the promise of engineered T cells as a powerful therapeutic option against a wide array of malignancies, making a significant impact on the field of oncology and patient care. Participants and Methods Study design and participants Patients with advanced pancreatic cancer or other solid tumors harboring KRAS G12V or KRAS G12D mutations, who have undergone at least one standard treatment regimen and declined further chemotherapy, may be eligible for recruitment. These patients must also express the compatible HLA-A*11:01 allele and meet additional inclusion criteria, which include being at least 18 years old, having an expected survival of more than three months, an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, and at least one measurable lesion according to RECIST version 1.1 criteria. This study received approval from the Institutional Review Board at Sun Yat-sen Memorial Hospital of Sun Yat-sen University. The study was conducted in accordance with the Declaration of Helsinki and the guidelines of Good Clinical Practice. All participants provided written informed consent before enrollment. Procedures A flow chart of the trial procedures is shown in Fig. 1 . Peripheral blood mononuclear cells (PBMCs) are collected from eligible patients through leukapheresis to manufacture TCR-T cells. Five days before the readiness of the TCR-T infusion product, patients undergo preparatory lymphodepletion (LD) treatment, administered with fludarabine (30 mg/m ² per day for three days) and cyclophosphamide (300 mg/m ² per day for two days). Following this, patients receive an infusion of 1 × 10^ 9 to 1 × 10^10 total TCR-gene-positive T cells via intravenous ( i.v. ) administration over 30 minutes, followed by low-dose subcutaneous ( s.c. ) IL-2 injections (500,000 IU/m 2 every 12 hours) for 14 days to enhance the in vivo persistence of the transferred cells. Imaging assessments, including computed tomography (CT) or magnetic resonance imaging (MRI) of the chest, abdomen, and pelvis, are conducted at baseline, the 4th week, the 3rd month, and every three months thereafter to monitor tumor response according to RECIST version 1.1. Safety evaluations are performed at each follow-up visit. All adverse events (AEs) are monitored continuously and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 5.0. Outcomes The primary endpoint of this clinical trial is progression-free survival (PFS), defined as the time from the infusion of TCR-T cells to the first occurrence of disease progression or death. Secondary endpoints include the objective response rate (ORR), calculated as the proportion of patients achieving either a complete response (CR) or a partial response (PR); overall survival (OS), measured as the duration from TCR-T cell infusion to death or loss to follow-up; duration of response (DOR), defined as the period from the initial documentation of an objective response to the first occurrence of disease progression or death; and the disease control rate (DCR), which is the proportion of patients exhibiting CR, PR, or stable disease (SD). Toxicity assessments focus on the frequency and severity of adverse events (AEs). The pharmacokinetic (PK) and pharmacodynamic (PD) evaluations will measure the peak concentration (C max ), the time to reach peak concentration (T max ), and the area under the curve from 0 to 28 days (AUC 0 − 28 ) of the infused TCR-T cells and cytokines in the peripheral blood. These metrics will help elucidate the bioactivity and exposure profile of the therapeutic cells and their effects. Generation of TCR-T Infusion Products We utilized the MSGV1 retroviral vector to transduce autologous peripheral blood mononuclear cells (PBMCs) before in vitro expansion of the transduced T cells, employing anti-CD3 and media supplemented with recombinant interleukin-2 (IL-2 10 . The resulting TCR, named PCV, recognizes a 9-mer RASG12V peptide (VVGAVGAGK) 28 . At the same time, TCR 051 is specific to a 10-mer RASG12V peptide (VVVGAVGAGK) in the context of HLA-A11:01, as detailed in the Supplementary Methods section of the Supplementary Appendix. Additionally, TCR-2, isolated from colorectal cancer tumor-infiltrating lymphocytes (TILs), was reconstructed to recognize a 10-mer RASG12D peptide (VVVGADGVGK) in the context of HLA-A11:01 29 . TCR gene modification was conducted by the transduction of autologous PBMCs using recombinant replication-incompetent retroviruses post-stimulation with OKT-3 antibodies within a retronectin-coated cell culture bag (Origen) and subsequent expansion in GREX flasks (Wilson Wolf) as previously described 30 . In vivo Tracking of Transgenic TCR-positive Cells To monitor the in vivo persistence of infused T cells, we employed both flow cytometry analysis and digital droplet PCR (ddPCR). The murine TCRα and β chains were substituted for their human equivalents in the engineered T cells, serving both to ensure the correct pairing of the exogenous TCRs and to act as a marker for tracking the transgenic T cells. The frequency of these transgenic T cells in vivo was determined by flow cytometry analysis, utilizing anti-mouse TCR β chain antibodies to identify the cells in isolated PBMC. Concurrently, digital droplet PCR was utilized to assess the DNA copy number of the transgenic T cells. By using specific primer pairs, we were able to accurately evaluate the relative persistence of two distinct populations of T cells, each engineered to express a different TCR recognizing either the 9-mer (PCV) or 10-mer (051) epitopes of the KRAS G12V mutation. This approach provided a comprehensive assessment of the longevity and distribution of the therapeutic T cells post-infusion. Cytokine Release in PBMC after Cell Infusion To assess cytokine release in PBMC following cell infusion, we utilized a cytokine bead array assay. This method was employed to quantify the levels of several cytokines known to contribute to cytokine release syndrome (CRS). Blood samples were collected at various time intervals post-infusion. The measurement process involved using flow cytometry to analyze beads that were coated with antibodies specific to different cytokines. After incubation with diluted patient sera, these coated beads were then labeled with fluorescent secondary antibodies to enable the detection and quantification of cytokine levels. This assay provides a detailed profile of cytokine dynamics, which is critical for understanding and managing potential CRS in patients undergoing cell therapy. Declarations Data Availability Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. Acknowledgements We remember Yandan Yao, a great clinician, researcher, colleague, friend, husband, and father. This study was supported by Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (Grant Number: SYS-5010Z-202303). Contribution Z Zeng, T Qin, and G Yu write the original manuscript, collect and analysis data, and writing, review, and edit the manuscript. X Wu, S Huang, T Chen, and X Chen collect clinical data. W Wu, X Zhang, C Gan, and X Jiang collect and analyze clinical data. M Zhang, H Yao, and H Hu conceptualization, supervision, writing–review and editing, and funding and approving the final manuscript. Conflict of Interests The authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Zhang Meng was funded by the Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (SYS-5010Z-202303). Zhifen Zeng, Wenxia Wu, Tonghua Chen, and Xueru Chen are her subordinates. Dr. Herui Yo is the director of the Oncology Department, and QinTao is his subordinate. Songyin Huang is the director of the Biological Therapy Center, and XiaohuaWu is her subordinate. Dr. Hong-Ming Hu was funded by the National Key Research and Development Program of China (2023YFC3403800) and is the founder and CSO of ImmuXell Biotech. Guangjie Yu, Shih-Ting Tsao, Xiaohui Zhang, Chi Gan, and Xiaochun Jiang are employees of ImmuXell Biotech. References Siegel, R. 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Front Immunol 13, 835762, doi: 10.3389/fimmu.2022.835762 (2022). Morgan, R. A. et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126–129, doi: 10.1126/science . 1129003 (2006). Johnson, L. A. et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114, 535–546, doi: 10.1182/blood-2009-03-211714 (2009). Rapoport, A. P. et al. NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat Med 21, 914–921, doi: 10.1038/nm.3910 (2015). Meng, F. et al. Immunotherapy of HBV-related advanced hepatocellular carcinoma with short-term HBV-specific TCR-expressing T cells: results of dose escalation, phase I trial. Hepatology international 15, 1402–1412, doi: 10.1007/s12072-021-10250-2 (2021). Nagarsheth, N. B. et al. TCR-engineered T cells targeting E7 for patients with metastatic HPV-associated epithelial cancers. Nat Med 27, 419–425, doi: 10.1038/s41591-020-01225-1 (2021). Ramachandran, I. et al. Systemic and local immunity following adoptive transfer of NY-ESO-1 SPEAR T cells in synovial sarcoma. Journal for immunotherapy of cancer 7, 276, doi: 10.1186/s40425-019-0762-2 (2019). Additional Declarations There is a conflict of interest The authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Zhang Meng was funded by the Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (SYS-5010Z-202303). Zhifen Zeng, Wenxia Wu, Tonghua Chen, and Xueru Chen are her subordinates. Dr. Herui Yo is the director of the Oncology Department, and QinTao is his subordinate. Songyin Huang is the director of the Biological Therapy Center, and XiaohuaWu is her subordinate. Dr. Hong-Ming Hu was funded by the National Key Research and Development Program of China (2023YFC3403800) and is the founder and CSO of ImmuXell Biotech. Guangjie Yu, Shih-Ting Tsao, Xiaohui Zhang, Chi Gan, and Xiaochun Jiang are employees of ImmuXell Biotech. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7496490","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":508177179,"identity":"57a7c1d6-4be7-4e42-b4fa-737d2a28b131","order_by":0,"name":"Hong-Ming 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University","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-08-30 16:25:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7496490/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7496490/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90485265,"identity":"fdfdebd4-7ad9-4e17-b6fa-cd00f424a5a9","added_by":"auto","created_at":"2025-09-03 08:45:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30500,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic Representation of the Study Design. The major steps include patient screening for KRAS mutation and HLA compatibility, leukapheresis, lymphodepletion, cell infusion, and administration of IL-2.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/a7a6efce9c8dc6d3807ca5e3.png"},{"id":90485269,"identity":"1023375a-8c60-4ba5-a22f-e9603c8e63dd","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1788803,"visible":true,"origin":"","legend":"\u003cp\u003eA comparison of the primary targeted lesions on MR/CT images before and after adoptive TCR-T therapy in three patients. The short axis of the unirradiated recurrent mesenteric lymph node metastasis in \u003cstrong\u003ePatient 001\u003c/strong\u003edecreased from 16 mm to 4.2 mm at six months post-TCR-T cell infusion, reflecting a 75% reduction. The diameter of the liver metastasis in \u003cstrong\u003ePatient 002\u003c/strong\u003e reduced from 16 mm to 11 mm at one month and became nearly undetectable at three and six months post-infusion. The large lung tumor in \u003cstrong\u003ePatient 003\u003c/strong\u003eremained stable for the first three months, with slow progression observed thereafter.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/7ed5db414f493e37ad27d24a.png"},{"id":90485266,"identity":"723b6594-ca08-4418-a372-29fd3370ffc0","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31209,"visible":true,"origin":"","legend":"\u003cp\u003eCA19-9 Levels in \u003cstrong\u003ePatient 001\u003c/strong\u003e Before and After Adoptive TCR-T Therapy. The CA19-9 level peaked at 1763 U/ml one week after infusion, declined to half of the original level within one month, dropped to a minimum of 40 U/ml by the fourth month, and then fluctuated within a narrow range for the following six months.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/cdb5d0757502e8efaf1762bc.png"},{"id":90485267,"identity":"10eea3a2-5cdb-4f95-af1c-edc62edfa065","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":265588,"visible":true,"origin":"","legend":"\u003cp\u003eT-cell Pharmacokinetics in \u003cstrong\u003ePatient 001\u003c/strong\u003e. Panel 4A shows the percentage of CD3\u003csup\u003e+\u003c/sup\u003e T cells in the final cell infusion products that express the mouse TCR β chain (mTCR) after transduction with PCV TCR (left) or 051 TCR (right). Panel 4B illustrates \u003cem\u003ein vivo\u003c/em\u003e persistence in the blood following the infusion of 5.1 × 10\u003csup\u003e9\u003c/sup\u003e autologous T cells modified with TCR genes targeting the G12V mutant KRAS. Panel 4C displays the DNA copy number, determined by measuring the total integrated retroviral DNA (RVV) isolated from PBMCs at various time points post-infusion. Finally, Panel 4D shows the DNA copy number of two different TCR-T products found in PBMCs at various time points after infusion.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/1f50fda43b5dd7dbab2c1471.png"},{"id":90486649,"identity":"8b2b5551-0dc4-4ebd-915a-2426d3e35869","added_by":"auto","created_at":"2025-09-03 08:53:59","extension":"pdf","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141246,"visible":true,"origin":"","legend":"T-cell Pharmacokinetics in . Panel 4A shows the percentage of CD3 T cells in the final cell infusion products that express the mouse TCR β chain (mTCR) after transduction with PCV TCR (left) or 051 TCR (right). Panel 4B illustrates persistence in the blood following the infusion of 5.1 \u0026times; 10 autologous T cells modified with TCR genes targeting the G12V mutant KRAS. Panel 4C displays the DNA copy number, determined by measuring the total integrated retroviral DNA (RVV) isolated from PBMCs at various time points post-infusion. Finally, Panel 4D shows the DNA copy number of two different TCR-T products found in PBMCs at various time points after infusion.","description":"","filename":"FIGURE4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/dc82d41645b6fdefe3912d7d.pdf"},{"id":90485272,"identity":"e49651d5-7f71-4164-9f47-d3ea5c5cbcaf","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190851,"visible":true,"origin":"","legend":"\u003cp\u003eT-cell pharmacokinetics of \u003cstrong\u003ePatients 002\u003c/strong\u003e (panel A) and \u003cstrong\u003ePatient 003\u003c/strong\u003e (panel B). At various time points post-cell infusion, the mTCR-positive T cells among CD3\u003csup\u003e+\u003c/sup\u003e, CD4\u003csup\u003e+\u003c/sup\u003e, or CD8\u003csup\u003e+\u003c/sup\u003e T cells in PBMC were measured by flow cytometry.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/5eeb47ed64daeecd4140b7c7.png"},{"id":90487280,"identity":"aa2f6f94-c4e7-42a2-9a05-3e05667eaaca","added_by":"auto","created_at":"2025-09-03 09:01:59","extension":"pdf","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103979,"visible":true,"origin":"","legend":"T-cell pharmacokinetics of (panel A) and (panel B). At various time points post-cell infusion, the mTCR-positive T cells among CD3, CD4, or CD8 T cells in PBMC were measured by flow cytometry.","description":"","filename":"FIGURE5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/1e9193dcbb33837acc197096.pdf"},{"id":90486647,"identity":"f6b09e65-a83b-4e73-9278-56ec60d6ea85","added_by":"auto","created_at":"2025-09-03 08:53:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":159298,"visible":true,"origin":"","legend":"\u003cp\u003eLymphodepletion with Minimal Cytopenia and Thrombocytopenia. No prolonged cytopenia was observed following lymphodepletion and T cell infusion. Routine blood tests were performed to measure the counts of white blood cells (WBC), neutrophils (NEU), and lymphocytes (LYM) (Panel A). Additionally, platelet (PLT) counts were also assessed (Panel B).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/aa936a2d45c1445d9e008c05.png"},{"id":90485274,"identity":"3abefb4a-6421-4e56-af95-d3cb32f7c99b","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":222326,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine Levels in Peripheral Blood Samples at Various Time Points After Cell Infusion. Cytokine release into the blood at different time points after cell infusion was measured using a CBA assay. (A) Cytokine release data for \u003cstrong\u003ePatient 001\u003c/strong\u003e. Elevated levels of IL-6, TNF-α, and IL-1β were detected one week after cell infusion, with a rapid decline thereafter. No IFN-γ was detected in the blood, and low levels of IL-10 and granzyme B were observed. (B) Cytokine release data for \u003cstrong\u003ePatient 002\u003c/strong\u003e. No significant rise in cytokine levels was detected.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/518c1e4d24e3b4a521d0a231.png"},{"id":90485277,"identity":"d712da4b-5ef9-4a73-8961-380ffb98a3b0","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"pdf","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":161236,"visible":true,"origin":"","legend":"Lymphodepletion with Minimal Cytopenia and Thrombocytopenia. No prolonged cytopenia was observed following lymphodepletion and T cell infusion. Routine blood tests were performed to measure the counts of white blood cells (WBC), neutrophils (NEU), and lymphocytes (LYM) (Panel A). Additionally, platelet (PLT) counts were also assessed (Panel B).","description":"","filename":"FIGURE6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/64b9fcbe88170d00b3e9b1da.pdf"},{"id":90485278,"identity":"4d8e0ba5-b465-4fa4-9055-546bc2d0e74a","added_by":"auto","created_at":"2025-09-03 08:45:59","extension":"pdf","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":596729,"visible":true,"origin":"","legend":"Cytokine Levels in Peripheral Blood Samples at Various Time Points After Cell Infusion. Cytokine release into the blood at different time points after cell infusion was measured using a CBA assay. (A) Cytokine release data for . Elevated levels of IL-6, TNF-α, and IL-1β were detected one week after cell infusion, with a rapid decline thereafter. No IFN-γ was detected in the blood, and low levels of IL-10 and granzyme B were observed. (B) Cytokine release data for . No significant rise in cytokine levels was detected.","description":"","filename":"FIGURE7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/2ceb1c75af75ff0ff476a2a0.pdf"},{"id":90607718,"identity":"8cdb152b-ef22-419b-8807-7bf675f98479","added_by":"auto","created_at":"2025-09-04 16:04:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3657611,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7496490/v1/33100ce8-39e1-496a-869f-d1bdfd7d80cc.pdf"}],"financialInterests":"There is a conflict of interest\nThe authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\r\nDr. Zhang Meng was funded by the Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (SYS-5010Z-202303). Zhifen Zeng, Wenxia Wu, Tonghua Chen, and Xueru Chen are her subordinates. Dr. Herui Yo is the director of the Oncology Department, and QinTao is his subordinate. Songyin Huang is the director of the Biological Therapy Center, and XiaohuaWu is her subordinate.\r\nDr. Hong-Ming Hu was funded by the National Key Research and Development Program of China (2023YFC3403800) and is the founder and CSO of ImmuXell Biotech. Guangjie Yu, Shih-Ting Tsao, Xiaohui Zhang, Chi Gan, and Xiaochun Jiang are employees of ImmuXell Biotech.","formattedTitle":"Adoptive T-Cell Therapy in Solid Tumor with KRAS\u003csup\u003eG12V/G12D\u003c/sup\u003e Mutation","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer and non-small cell lung cancer (NSCLC) rank among the ten most common cancers globally. It is well-known that pancreatic cancer is associated with poor prognosis, exhibiting a 5-year survival rate of less than 10%\u003csup\u003e1\u003c/sup\u003e. In addition, lung cancer is the leading cancer globally, accounting for 20% of cancer-related mortality worldwide and 22% in China\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. NSCLC accounts for 80\u0026ndash;85% of all lung cancer cases. Despite advances in immune checkpoint inhibitors and targeted therapies using small-molecule inhibitors, treating advanced metastatic NSCLC beyond second-line therapy remains challenging. The Chinese Society of Clinical Oncology (CSCO) recommends anlotinib as a third-line treatment for advanced NSCLC. However, the prognosis remains poor, and there is a significant unmet need in treating NSCLC with KRAS mutations. KRAS mutation, an important biomarker in solid tumors, was found in NSCLC in 1984, and chemotherapy and immunotherapy were still the standard of care. Both in PAC and NSCLC, the KRAS gene mutation is commonly found at rates of 90% and 30%, respectively\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, which is regarded as a gene associated with poor survival in these tumors. The most common mutation sites in the KRAS gene are found in codon 12, which represent over 80% of all variants, including G12A, G12C, G12D, G12R, G12S, and G12V mutations. Efforts to target these KRAS mutations had been largely unsuccessful for decades until the recent development of a small-molecule inhibitor targeting the KRAS G12C mutation. In patients with KRAS G12C-mutated pancreatic cancer treated with sotorasib, the median progression-free survival was 4.0 months, and the median overall survival was 6.9 months\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the KRAS G12C mutation is relatively rare in pancreatic cancer, occurring in only 1\u0026ndash;2% of cases, which is significantly lower than its prevalence in lung or colon cancer\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, compared to the more dominant G12D or G12V mutations\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Currently, there is no targeted therapy available for patients with KRAS G12D or G12V mutations. Notably, patients with NSCLC and actionable gene mutations who received a combination of chemotherapy and immunotherapy demonstrated a median survival of 12.6 months, as reported in the Keynote 189 study. Clinical outcomes have improved significantly for NSCLC patients with EGFR mutations and ALK fusions, with median survivals of 18.9 months\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and 36.7 months\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, respectively. However, there remains an unfavorable prognosis and significant unmet need in the treatment of NSCLC with KRAS mutations.\u003c/p\u003e\u003cp\u003eAdoptive cell therapy utilizing T-cells that recognize mutant KRAS represents another potential strategy for targeting KRAS mutations\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This approach is bolstered by findings from Tran et al., who documented substantial tumor regression in a patient with widely metastatic colorectal cancer following the infusion of tumor-infiltrating lymphocytes (TILs) that recognized the KRAS \u003csup\u003eG12D\u003c/sup\u003e mutation in association with the HLA-C08:02 allele\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. More recently, Tran and colleagues achieved regression of metastatic pancreatic cancer in a patient after administering a single infusion of autologous T cells. These T cells had been genetically modified to express HLA-C08:02-restricted TCRs specifically targeting the KRAS G12D mutation, which were cloned from TILs initially obtained from the aforementioned colorectal cancer patient\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the prevalence of the HLA-C08:02 allele is exceedingly low, particularly among Chinese patients (1%). Conversely, the HLA-A11:01 allele is the most common in the Chinese population, occurring in 30% of individuals. Therefore, targeting patients with prevalent HLA alleles and either G12D or G12V mutations could extend the benefits of this therapy to a broader demographic.\u003c/p\u003e\u003cp\u003eHere, we present three cases of advanced solid tumors that were effectively treated with autologous T cells engineered to express exogenous T cell receptors (TCRs) targeting the KRAS \u003csup\u003eG12V\u003c/sup\u003e or KRAS \u003csup\u003eG12D\u003c/sup\u003e mutations within the context of the HLA-A*11:01 allele. The cohort included two cases of PDAC and one case of metastatic NSCLC. Tumor regression was observed in all three patients, and the treatment was well-tolerated by each individual.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePatients and clinical assessment\u003c/h2\u003e\n \u003cp\u003eThree HLA-A*11:01 positive patients with either KRAS\u003csup\u003eG12V\u003c/sup\u003e or KRAS\u003csup\u003eG12D\u003c/sup\u003e mutations were enrolled in the TCR-T cell therapy study. Two of these patients had advanced metastatic PDAC, and one had advanced NSCLC. Before TCR-T therapy, they had received a median of two lines of systemic therapy. Each patient was infused with an average dose of 8.2 \u0026times; 10^\u003csup\u003e9\u003c/sup\u003e autologous T cells engineered to express TCRs targeting the specific KRAS mutations. Tumor response was evaluated post-infusion of TCR-T cells. Both PDAC patients achieved objective partial responses within one month of infusion, with PFS durations of 10 and 8 months, respectively. The NSCLC patient, who had undergone extensive prior treatments, was assessed as having achieved partial remission.\u003c/p\u003e\n \u003cp\u003eSafety assessments were conducted to monitor the occurrence of adverse events. All patients experienced cytopenia, including anemia (\u0026le;\u0026thinsp;grade 2), transient reductions in platelet count (\u0026le;\u0026thinsp;grade 2), and transient decreases in white blood cell counts (grade 2\u0026ndash;3), likely resulting from the lymphodepletion preconditioning regimen. Additionally, two patients experienced moderate fevers lasting less than 48 hours after the infusion, and one patient reported swelling and redness at the IL-2 injection sites. No infections related to the therapy were observed, and no fatal adverse events occurred. This profile suggests that TCR-T therapy, while potent, is manageable in terms of toxicity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eCase report of each patient\u003c/p\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003cp\u003ePatient 001 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eResponse in Individual Patient with Various Tumor Types\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatient\u003c/p\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAge(y)\u003c/p\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTumor Type\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKRAS\u003c/p\u003e\n \u003cp\u003eMutation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSites of Metastases\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLymphodepletion regimen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIL-2 usage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCell Dose\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emTCR+%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eResponse\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePFS\u003c/p\u003e\n \u003cp\u003e(months)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOS\u003c/p\u003e\n \u003cp\u003e(months)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e59 M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePDAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eG12V\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAbdominal lymph nodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eFlu 30mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;3d\u003c/p\u003e\n \u003cp\u003eCTX 300mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e500,000IU/m\u003csup\u003e2\u003c/sup\u003e q12h*14d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e5.1\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.8% TCR 051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.3% TCR PCV\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63 F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePDAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlu 30mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;3d\u003c/p\u003e\n \u003cp\u003eCTX 300mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500,000IU/m\u003csup\u003e2\u003c/sup\u003e q12h*14d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026times;10\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90.9% TCR-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71 F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNSCLC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eG12D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSupraclavicular lymph nodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlu 30mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;3d\u003c/p\u003e\n \u003cp\u003eCTX 300mg/m\u003csup\u003e2\u003c/sup\u003e\u0026times;2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500,000IU/m\u003csup\u003e2\u003c/sup\u003e q12h*14d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026times;10\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.7% TCR-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"12\"\u003e\n \u003cp\u003ePDAC, pancreatic ductal adenocarcinoma; NSCLC, non-small cell lung cancer; PR, partial response; PD, progression disease; SD, stable disease; OS, overall survival; PFS, progression free survival; mTCR+%, percentage of CD3\u0026thinsp;+\u0026thinsp;T cells express engineered TCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eA 57-year-old male was diagnosed with adenocarcinoma of the pancreatic head via EUS-guided fine-needle biopsy after presenting with jaundice and an elevated CA19-9 level (454.0 U/ml, reference\u0026thinsp;\u0026le;\u0026thinsp;34 U/ml) in June 2019. He underwent pylorus-preserving open Whipple surgery in the same month. Post-operative pathology indicated poorly differentiated PDAC with a disease stage of IIB (pT1N1M0). The patient subsequently received 12 cycles of adjuvant mFOLFIRINOX chemotherapy, resulting in normalization of his CA19-9 levels and a disease-free status for 16 months post-surgery.\u003c/p\u003e\n \u003cp\u003eIn October 2020, new mesenteric root lymphadenopathy was identified via MRI and PET-CT, and his CA19-9 level increased to 222.3 U/ml as of November 3, 2020, suggesting metastatic recurrence. He underwent stereotactic body radiation therapy (SBRT) targeting the mesenteric root lymphadenopathy. This was followed by a second-line AG chemotherapy regimen, which was discontinued after the first day due to severe bone marrow suppression and skin rashes. Subsequently, his CA19-9 level temporarily decreased to 81.7 U/ml but began to rise again. By March 2021, the irradiated mesenteric root lymphadenopathy remained stable; however, new metastases were discovered in the hepatic portal and retroperitoneal lymph nodes, with CA19-9 increasing to 403 U/ml. A second course of SBRT was administered targeting these newly identified metastatic lymph nodes.\u003c/p\u003e\n \u003cp\u003eIn May 2021, another enlarged lymph node metastasis was detected in the abdominal mesenteric region, accompanied by a sharp increase in CA19-9 to 927.9 U/ml. Following this, the patient was enrolled in a TCR-T therapy program. This patient underwent NGS testing of his tumor, which revealed missense mutations of KRAS Exon 2 p.G12V (c.35G\u0026thinsp;\u0026gt;\u0026thinsp;T, abundance: 29.93%) and TP53 exon 6 p.Y220C (c.659A\u0026thinsp;\u0026gt;\u0026thinsp;C). HLA typing of his peripheral blood samples confirmed the presence of the HLA-A*11:01 allele. In June 2021, following preparatory lymphodepletion with fludarabine (30 mg/m\u003csup\u003e2\u003c/sup\u003e per day for three days) and cyclophosphamide (300 mg/m\u003csup\u003e2\u003c/sup\u003e per day for two days), he received an infusion of 5.1\u0026times;10\u003csup\u003e9\u003c/sup\u003e TCR-gene positive T cells via intravenous administration over 30 minutes. This was supplemented with low-dose subcutaneous IL-2 injections (500,000 IU/m\u003csup\u003e2\u003c/sup\u003e every 12 hours) for 14 days to enhance the \u003cem\u003ein vivo\u003c/em\u003e persistence of the transferred cells. Rapid regression of metastatic lymph nodes was observed on MRI within the first month post-infusion, with the target measurable metastatic lymph node shrinking from 16 mm to 4.2 mm, a 75% reduction by the sixth month. However, by the ninth month, the short axis of the target lesion had increased to 9 mm, though no other metastases were yet detected. CA19-9 levels peaked at 1763 U/ml one week post-infusion, halved within a month, and reached a nadir of 40 U/ml by the fourth month, maintaining minimal fluctuations for the subsequent six months (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe patient achieved a 75% tumor reduction and remained progression-free for ten months post-TCR-T therapy. However, ten months post-infusion, he was hospitalized due to mild back pain and abdominal distension, and MRI scans indicated slight lymph node enlargement in the hepatic portal, retroperitoneal, and mesenteric regions, suggesting disease progression. Twelve months after the infusion, MRI and subsequent needle biopsy confirmed newly developed multiple liver metastases, displaying a higher abundance (60.47%) of the KRAS p.G12V mutation compared to the initial tumor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Despite initial responsiveness to TCR-T therapy, the patient\u0026apos;s condition deteriorated due to progressive liver metastases, leading to his death in November 2022, with an overall survival of 17 months post-therapy.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePatient 002\u003c/h3\u003e\n\u003cp\u003eA 63-year-old female presented with abdominal pain and jaundice and was diagnosed with pancreatic cancer. She underwent tumor resection in July 2021. Postoperative pathology identified the tumor as moderately differentiated PDAC with a pathological staging of pT4N1M0. IHC results were positive for CK7, CK19, focal CK20, Muc-1, and some Muc-5AC, with a high proliferation index shown by Ki67 at approximately 60%. P53 and Muc-2 were negative. A NGS test of the tumor revealed a KRAS Exon 2 p.G12D missense mutation with an abundance of 8.14%. Subsequently, the patient underwent two cycles of AG chemotherapy, after which MRI imaging revealed new liver metastases, the largest located in the S7 segment with a diameter of 16 mm, indicating disease progression. As a result, AG chemotherapy was discontinued.\u003c/p\u003e\n\u003cp\u003eIn November 2021, after receiving pretreatment with the FC regimen, the patient received a single infusion of 8 \u0026times; 10^\u003csup\u003e9\u003c/sup\u003e transgenic TCR-positive cells. Post-infusion, IL-2 injections were administered as per the protocol. Within one month, the diameter of the largest liver metastasis decreased to 11 mm, became almost undetectable at three months post-infusion, and the condition remained stable for an additional three months (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, eight months after the TCR-T infusion, in July 2022, there was a progression of liver metastases, and the patient subsequently passed away in September 2022. The patient achieved an objective partial response with a PFS of 8 months and an OS of 10 months following the single infusion of TCR-T cells. This case underscores the transient efficacy of TCR-T therapy in treating aggressive metastatic PDAC with specific KRAS mutations, highlighting the need for ongoing assessment and management of disease progression post-therapy.\u003c/p\u003e\n\u003ch3\u003ePatient 003\u003c/h3\u003e\n\u003cp\u003eA 71-year-old female with a history of cough and chest pain was diagnosed with stage IV NSCLC, cT3N3M1, in January 2021. A biopsy from a neck lymph node confirmed adenocarcinoma, showing positive results for TTF-1, Napsin A, E-cadherin, and an immune cell (10%+), with a Ki67 proliferation index of 70%. The tumor was negative for EGFR, ALK, ROS1, estrogen and progesterone receptors, and PD-L1 (22C3) TPS, while HER-2 was equivocally expressed at 2\u0026thinsp;+\u0026thinsp;and strongly at 3\u0026thinsp;+\u0026thinsp;in 80% of cells. The patient also had a KRAS p.G12D mutation, a TP53 p.E198X mutation, a tumor mutational burden (TMB) of 4.2 mutations per megabase (Muts/Mb), and stable microsatellites (MSS). Her medical history included hypertension, diabetes, coronary heart disease, and invasive breast cancer treated with mastectomy and axillary lymph node resection in 2016, followed by anastrozole without adjuvant chemotherapy.\u003c/p\u003e\n\u003cp\u003eWe then initiated treatment with docetaxel, carboplatin, and the anti-PD1 antibody Camrelizumab on March 8, 2021, achieving stable disease after two cycles. However, she experienced severe fatigue, musculoskeletal pain, and reactive cutaneous capillary endothelial proliferation (RCCEP), leading her to refuse further chemotherapy. She then received another anti-PD1 antibody, Tislelizumab. Despite this, the primary lung tumor progressed by August 26, 2021. She underwent two cycles of second-line chemotherapy with pemetrexed and carboplatin starting September 30, 2021, during which her supraclavicular and mediastinal lymph nodes enlarged, causing superior vena cava syndrome. This was managed with endovascular therapy and radiation, leading to significant symptomatic relief. However, her condition was complicated by severe thrombocytopenia, and she could not tolerate further chemotherapy. Disease progression was noted on November 12, 2021, with increased lymph node metastases.\u003c/p\u003e\n\u003cp\u003eSubsequently, she was enrolled in a clinical trial and received TCR-T cell therapy after lymphodepletion. On November 17, 2021, she was infused with 1 x 10^\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e TCR-T cells, followed by a two-week course of IL-2. She experienced only mild fatigue and a fever after the infusion. One month later, her CEA levels dropped from 12.9 ng/mL to 9.7 ng/mL, and the targeted metastatic mediastinal lymph nodes decreased in size, classified as partial remission (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Despite initial success, an MRI on February 14, 2024, revealed multiple brain metastases, indicating disease progression. Her progression-free survival was 27 months post-TCR-T infusion. She underwent surgical intervention for brain metastases but did not receive further systemic therapy and ultimately succumbed to lung cancer on May 11, 2024. Her OS from the first TCR-T infusion was approximately 30 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003epersistence of transduced KRAS G12V or G12D-reactive T Cells Post-Infusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e persistence of infused T cells is a crucial determinant of the antitumor efficacy of adoptive T cell therapies, including CAR-T for hematopoietic malignancies and TILs for solid tumors. For the three patients treated, the persistence of engineered TCR-T cells post-infusion was closely monitored. The first patient received an infusion containing two TCR constructs, PCV and 051, both targeting G12V mutations. Flow cytometry analysis, using an antibody against the mouse TCR constant region to identify exogenous TCR-expressing cells, showed that 68.3% and 84.8% of CD3\u003csup\u003e+\u003c/sup\u003e cells were positive for PCV TCR and 051 TCR, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The percentage of CD3\u003csup\u003e+\u003c/sup\u003e, mouse TCR-positive T cells peaked at 56.89% week post-infusion and then decreased to 9.88% after two weeks. A minor resurgence to 12.11% was observed at three weeks, with a gradual decline to 1.42% and 0.32% by weeks 9 and 16, respectively. By the six-month mark, the transgenic T cells had become nearly undetectable (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Consistent with these findings, the persistence of infused T cells detectable by primers specific for the retroviral vector backbone (RVV) showed a significant drop by the 12th week post-infusion (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Moreover, the in vivo persistence of the two individual TCRs was assessed using digital droplet PCR (ddPCR) with TCR-specific primers, revealing that the 051 TCR exhibited superior persistence compared to the PCV TCR. At weeks 1 and 2 post-infusion, both TCRs were found in equal numbers, but by weeks 7 and 8, the persistence of the 051 TCR was approximately three times higher than that of the PCV TCR.\u003c/p\u003e\n\u003cp\u003eGiven these observations, the differential persistence of these TCRs could significantly impact their antitumor activities. Therefore, subsequent patients will be treated with autologous T cells modified solely with the 051 TCR gene, anticipating that it may offer enhanced persistence and more effective in vivo antitumor activity. For \u003cstrong\u003ePatient 002\u003c/strong\u003e, who received TCR-modified T-cell therapy, a notable expansion of mainly CD8\u003csup\u003e+\u003c/sup\u003e T cells was observed during the first month following infusion. The peak expansion level reached 32% of total T cells, as indicated in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA. Despite this significant early expansion, the modified T cells were undetectable by flow cytometry analysis 120 days after the cell infusion, suggesting a decline in the persistence of these cells over time.\u003c/p\u003e\n\u003cp\u003eContrary to the significant expansion observed in the first two patients, both of whom had PDAC, the expansion level of T cells in the third patient with NSCLC was considerably lower. This observation is illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB. Despite this lower level of initial T-cell expansion, the infused T cells in the NSCLC patient exhibited an interesting persistence pattern, maintaining a stable presence at a level comparable to the first two weeks for over 90 days post-infusion. Several factors, including differences in the tumor microenvironment, the specific characteristics of the infused T cells, or the underlying immune status of each patient, could influence this variation in T-cell expansion and persistence between patients. The sustained persistence of T cells in the NSCLC patient, despite low expansion, suggests that even a modest number of infused T cells may maintain a therapeutic effect over a prolonged period. These observations underscore the complexity of predicting responses in adoptive T-cell therapies and highlight the need for further studies to optimize these treatments based on individual patient and tumor characteristics.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eTransient Cytokine Release in Peripheral Blood after Cell Infusion\u003c/h2\u003e\n \u003cp\u003eFor \u003cstrong\u003ePatient 001\u003c/strong\u003e, following a lymphodepletion regimen with adjusted doses of fludarabine and cyclophosphamide, only a transient episode of lymphopenia was observed, with stable neutrophil and platelet counts, as depicted in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e. This modified regimen likely contributed to the patient\u0026apos;s well-being post-infusion, as no prophylactic antibiotics or antiviral drugs were administered, and the patient experienced fewer complications. Significantly, no major adverse events typically associated with T-cell infusion therapies, such as CRS, neurotoxic effects, or prolonged cytopenia, were observed in this patient. These conditions are often reported in treatments involving CAR-T cells, where they occur with high frequency and severity. The lymphocyte count for patient 001 showed a rapid increase by day seven post-infusion, suggesting a robust proliferation of the infused T cells, followed by a decline to pre-therapy levels.\u003c/p\u003e\n \u003cp\u003eIn terms of cytokine levels, patient 001 exhibited elevated levels of IL-6, TNF-\u0026alpha;, and IL-1\u0026beta; one week after cell infusion (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Although the patient experienced a brief fever within 48 hours of the therapy, which resolved quickly, no other symptoms indicative of CRS were noted. This contrasts with \u003cstrong\u003ePatient 002\u003c/strong\u003e, who did not exhibit a significant rise in cytokine levels, possibly correlating with a lower percentage of CD3-positive, mouse TCR-positive T cells compared to Patient 001. This difference suggests that higher cytokine levels in \u003cstrong\u003ePatient 001\u003c/strong\u003e could be associated with better proliferation and persistence of the transduced modified T cells. Unfortunately, for patient 003, cytokine release data could not be comprehensively measured due to logistical challenges in blood sample collection resulting from the COVID-19 pandemic, limiting a complete assessment of cytokine dynamics for this patient. This gap highlights the influence of external factors on clinical monitoring and underscores the importance of consistent sample collection in assessing immunological responses in T-cell therapy trials.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eContinuous monitoring of circulating tumor DNA (ctDNA) in peripheral blood after TCR-T cell infusion.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eContinuous monitoring of ctDNA is a critical component in assessing the early molecular response and potential relapse in patients undergoing TCR-T cell therapy. In the case of \u003cstrong\u003ePatient 001\u003c/strong\u003e, ctDNA levels were routinely measured in the peripheral blood post-infusion. Notably, seven months after receiving the TCR-T cell therapy, an increase in the abundance of the KRAS\u003csup\u003eG12V\u003c/sup\u003e mutation was observed, as illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. This rise in ctDNA occurred before any radiological evidence of disease progression was visible on MRI scans.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e ctDNA was detected in the peripheral blood of \u003cstrong\u003ePatient 001\u003c/strong\u003e. The abundance of the KRAS\u003csup\u003eG12V\u003c/sup\u003e mutation began to increase seven months after the infusion of TCR-T cells in this patient.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbundance of KRAS\u003csup\u003eG12V\u003c/sup\u003e mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e2022.1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e0.12%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e2022.1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e0.17%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e2022.2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e0.20%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e2022.4.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e0.22%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e2022.4.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 284px;\"\u003e\n \u003cp\u003e1.00%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study demonstrated that cancer-antigen-specific TCR-T cell therapy is an effective treatment modality for solid tumors, including pancreatic cancer and NSCLC. We observed comparable efficacy of TCR-T cells in treating pancreatic cancer cases. Notably, this research also marks the first report of TCR-T therapy being applied to treat NSCLC harboring the KRAS G12V mutation, showing promising anti-tumor efficacy and contributing to prolonged survival. These findings highlight the potential of TCR-T cells to target specific cancer antigens, providing a promising therapeutic approach for tumors that are traditionally resistant to other forms of treatment. This approach also underscores the significance of genetic profiling in identifying suitable targets for TCR-T therapy, enabling more personalized and potentially more effective treatments for cancer patients. Pancreatic cancer remains one of the most lethal malignancies, with a 5-year survival rate under 10%\u003csup\u003e1\u003c/sup\u003e. Traditional second-line treatments offer limited benefits for patients who experience recurrence or progression after first-line chemotherapy. For instance, in the Sotorasib clinical trial targeting KRAS p.G12C\u0026ndash;mutated advanced pancreatic cancer, the mOS was reported as 6.9 months, and the mPFS was 4.0 months\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Patients with other KRAS mutations, such as KRAS\u003csup\u003eG12D\u003c/sup\u003e or KRAS\u003csup\u003eG12V,\u003c/sup\u003e typically have no targeted therapy options available, aside from chemotherapy. Regardless of the chosen second-line regimen\u0026mdash;whether S1 as monotherapy, or double or triple regimens involving 5-Fu and Oxaplatin with or without irinotecan\u0026mdash;the mOS ranged from 4.9 to 9.2 months, while the mPFS ranged from 2.1 to 5.2 months\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our study, two patients with metastatic PDAC who had undergone multiple lines of prior treatments achieved an OS of 17 and 10 months and a PFS of 10 and 8 months, respectively. When compared to conventional therapies, our approach, which utilizes autologous T cells engineered to target KRAS\u003csup\u003eG12V\u003c/sup\u003e or KRAS\u003csup\u003eG12D\u003c/sup\u003e mutations, demonstrates superior efficacy and minimal toxicity. Given the typically poor prognosis of late-stage pancreatic cancer, these outcomes are particularly promising. These results support the potential for further research into the use of autologous T cell therapies targeting specific KRAS mutations in pancreatic cancer. This could pave the way for more personalized and effective treatment strategies, offering hope for improved outcomes in a patient population in dire need of better therapeutic options. In the treatment of NSCLC, targeted therapy is applied specifically to patient subtypes bearing actionable gene mutations. For those lacking such mutations, chemotherapy and immunotherapy are the most commonly employed treatments. A Phase 3, double-blinded, randomized clinical trial assessed the efficacy and safety of anlotinib as a third-line or subsequent therapy in advanced NSCLC, where the median overall survival (mOS) and median progression-free survival (mPFS) were recorded at 9.6 months and 5.4 months, respectively.\u003c/p\u003e\u003cp\u003eRecent advancements have highlighted antibody-drug conjugates (ADCs) as promising treatments for NSCLC. For instance, the TROPION-Lung01 study reported a 31.2% response rate and a 5.6-month PFS for NSCLC patients treated with a TROP2-ADC (NCT04656652). Furthermore, differential responses have been noted in patients based on HER2 status; those with HER2 overexpression treated with T-DxT showed no response, unlike those with HER2 mutations, who exhibited high response rates\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In our study, we explored the efficacy of TCR-T cell therapy targeting the KRAS\u003csup\u003eG12D\u003c/sup\u003e mutation as a third-line treatment for advanced NSCLC. This approach demonstrated promising anti-tumor efficacy and contributed to long-term survival following the first TCR-T therapy. These findings suggest that adoptive TCR-T cell therapy targeting specific KRAS mutations could represent a valuable treatment alternative for advanced NSCLC, in addition to its application in PDAC. This expands the therapeutic landscape and provides a potentially effective option for patients with NSCLC who have exhausted other treatments. To date, targeted therapies for KRAS mutations in cancer have largely been limited to the KRAS G12C mutation. Our study represents the first report on the efficacy and safety of engineered TCR-T cells targeting the KRAS\u003csup\u003eG12D\u003c/sup\u003e mutation specifically in the treatment of patients with NSCLC. This advancement is notable considering the historical challenge of effectively targeting KRAS mutations due to their high variability and the general resilience of the KRAS protein to small-molecule inhibitors.\u003c/p\u003e\u003cp\u003ePreviously, there have been limited reports of TCR-T cell therapies targeting other antigens in NSCLC. For instance, a case report indicated a short-term partial response in advanced NSCLC using TCR-T cells targeting the cancer-testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In another clinical trial, 11 patients with advanced NSCLC treated with TCR-T cells targeting the melanoma-associated antigen A10 (MAGE-A10) achieved a median progression-free survival (mPFS) of 58 days and a median overall survival (mOS) of 132 days, with only one patient reporting an objective clinical response (partial response, PR).\u003c/p\u003e\u003cp\u003eThe role of T-cell responses against neoantigens is well-established in enhancing the antitumor efficacy of immune checkpoint inhibitors (ICIs\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. TCRs that recognize these neoantigens could potentially be used for personalized adoptive cell transfer therapies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, a significant challenge remains, as most neoantigens are patient-specific, and only a minority (less than 5%) are immunogenic\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Given these challenges, the success of TCR-T cells targeting KRASG12D in our study suggests a promising new direction for personalized cancer therapy in NSCLC, especially for patients whose tumors harbor specific KRAS mutations that have previously been difficult to target with conventional therapies. This opens up potential for broader applications of TCR-T cell therapies, emphasizing the importance of continued research and development in this area. The deployment of cancer-antigen-specific TCR-T cells in the fight against malignant tumors has become a prominent focus in recent years. High-profile targets, such as NY-ESO-1, MART-1, and HPV E6 or E7, have been extensively studied and implemented in clinical settings. However, the exploration of TCR-T therapy specifically targeting KRAS mutations is relatively nascent, with primarily anecdotal evidence and a few case reports substantiating its use\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our study makes a significant contribution to this emerging field by providing long-term follow-up data for each patient, offering invaluable insights into the practical application of KRAS-specific TCR-T cells. This is particularly crucial for treating pancreatic cancer, a malignancy characterized by a dismal prognosis and a high incidence of KRAS mutations, where traditional treatment options frequently fall short.\u003c/p\u003e\u003cp\u003ePrevious research on TCR-T therapy in other types of cancer, such as melanoma\u003csup\u003e,\u003c/sup\u003e, myeloma, hepatocellular carcinoma, HPV-associated epithelial cancers, and synovial sarcoma, underscores that the proliferation and persistence of gene-modified TCR-T cells are critical to achieving a substantial anti-cancer effect. In our study, for example, \u003cb\u003ePatient 001\u003c/b\u003e demonstrated a notably successful outcome; the transduced TCR-T cells not only expanded to a high level but also maintained their presence for over three months, correlating with a superior therapeutic result and the most prolonged survival among the cohort. The varying peak percentages of mTCR\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e cells among the patients (ranging from 1.42\u0026ndash;56.89%) highlight an area for further investigation. The challenge of maintaining robust expansion and persistent viability of infused TCR-T cells is critical to their long-term efficacy in cancer treatment. After infusion, the gene-modified TCR-T cells often exhibit a rapid decline in number, becoming barely detectable within a few months, which may potentially lead to tumor recurrence or progression. To counteract this, repeated infusions of TCR-T cells could theoretically sustain or enhance the therapeutic effects. The feasibility of this approach has been demonstrated in various clinical trials. For instance, in a phase I trial of immunotherapy using HBV-specific TCR-redirected T cells for advanced hepatocellular carcinoma\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, patients received between one and four treatment cycles, spaced over 30-day intervals. The patient who underwent a maximum of four cycles showed the longest time to progression (TTP), reaching 27.7 months. Conversely, in a trial targeting synovial cell sarcoma with NY-ESO-1-reactive TCR-engineered T cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, while 61% of patients responded to the first infusion, only one out of the six who received a second infusion achieved a sustained response, lasting an additional nine months following the second treatment. Interestingly, this patient did not respond to a third infusion, highlighting the variable outcomes associated with repeated TCR-T cell treatments.\u003c/p\u003e\u003cp\u003eThe utility and timing of subsequent TCR-T infusions need further investigation to optimize benefits. In this context, ctDNA emerges as a highly promising biomarker for guiding such decisions. ctDNA, which reflects tumor-specific genetic and epigenetic features with high tissue and cell-type specificity, serves as a sensitive and specific indicator for detecting recurrence. Monitoring ctDNA levels could provide critical insights into tumor dynamics and help clinicians determine the most opportune moments for additional TCR-T infusions, thereby enhancing treatment strategies and potentially improving patient outcomes. This approach not only could extend the benefits of TCR-T therapy but also help in customizing surveillance and treatment modifications in response to the evolving tumor landscape.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study highlights the significant anticancer effects of autologous T cells modified with TCRs that specifically target KRAS\u003csup\u003eG12V\u003c/sup\u003e or KRAS\u003csup\u003eG12D\u003c/sup\u003e mutations in patients with PDAC and NSCLC. The implications of these findings extend beyond these two cancer types, offering potential benefits to a broader patient cohort. Recent advancements have expanded the repertoire of available TCRs, with a dozen new TCRs identified that are specific for various KRAS mutations, including G12D, G12V, G12R, G12C, and G13D. These TCRs are compatible with a range of class I and II HLA molecules, significantly broadening the potential applicability of this therapy. The development of a comprehensive TCR library targeting these mutations opens up new avenues for treating a variety of advanced, lethal cancers. The broader availability of targeted TCRs marks a substantial leap forward in precision medicine, offering hope for improved treatment outcomes in cancers traditionally associated with poor prognoses. With these tools, we may soon realize the promise of engineered T cells as a powerful therapeutic option against a wide array of malignancies, making a significant impact on the field of oncology and patient care.\u003c/p\u003e"},{"header":"Participants and Methods","content":"\u003ch2\u003eStudy design and participants\u003c/h2\u003e\n\u003cp\u003ePatients with advanced pancreatic cancer or other solid tumors harboring KRAS\u003csup\u003eG12V\u003c/sup\u003e or KRAS\u003csup\u003eG12D\u003c/sup\u003e mutations, who have undergone at least one standard treatment regimen and declined further chemotherapy, may be eligible for recruitment. These patients must also express the compatible HLA-A*11:01 allele and meet additional inclusion criteria, which include being at least 18 years old, having an expected survival of more than three months, an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, and at least one measurable lesion according to RECIST version 1.1 criteria. This study received approval from the Institutional Review Board at Sun Yat-sen Memorial Hospital of Sun Yat-sen University. The study was conducted in accordance with the Declaration of Helsinki and the guidelines of Good Clinical Practice. All participants provided written informed consent before enrollment.\u003c/p\u003e\n\u003ch2\u003eProcedures\u003c/h2\u003e\n\u003cp\u003eA flow chart of the trial procedures is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Peripheral blood mononuclear cells (PBMCs) are collected from eligible patients through leukapheresis to manufacture TCR-T cells. Five days before the readiness of the TCR-T infusion product, patients undergo preparatory lymphodepletion (LD) treatment, administered with fludarabine (30 mg/m\u003csup\u003e\u0026sup2;\u003c/sup\u003e per day for three days) and cyclophosphamide (300 mg/m\u003csup\u003e\u0026sup2;\u003c/sup\u003e per day for two days). Following this, patients receive an infusion of 1 \u0026times; 10^\u003csup\u003e9\u003c/sup\u003e to 1 \u0026times; 10^10 total TCR-gene-positive T cells via intravenous (\u003cem\u003ei.v.\u003c/em\u003e) administration over 30 minutes, followed by low-dose subcutaneous (\u003cem\u003es.c.\u003c/em\u003e) IL-2 injections (500,000 IU/m\u003csup\u003e2\u003c/sup\u003e every 12 hours) for 14 days to enhance the \u003cem\u003ein vivo\u003c/em\u003e persistence of the transferred cells. Imaging assessments, including computed tomography (CT) or magnetic resonance imaging (MRI) of the chest, abdomen, and pelvis, are conducted at baseline, the 4th week, the 3rd month, and every three months thereafter to monitor tumor response according to RECIST version 1.1. Safety evaluations are performed at each follow-up visit. All adverse events (AEs) are monitored continuously and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), version 5.0.\u003c/p\u003e\n\u003ch2\u003eOutcomes\u003c/h2\u003e\n\u003cp\u003eThe primary endpoint of this clinical trial is progression-free survival (PFS), defined as the time from the infusion of TCR-T cells to the first occurrence of disease progression or death. Secondary endpoints include the objective response rate (ORR), calculated as the proportion of patients achieving either a complete response (CR) or a partial response (PR); overall survival (OS), measured as the duration from TCR-T cell infusion to death or loss to follow-up; duration of response (DOR), defined as the period from the initial documentation of an objective response to the first occurrence of disease progression or death; and the disease control rate (DCR), which is the proportion of patients exhibiting CR, PR, or stable disease (SD). Toxicity assessments focus on the frequency and severity of adverse events (AEs). The pharmacokinetic (PK) and pharmacodynamic (PD) evaluations will measure the peak concentration (C\u003csub\u003emax\u003c/sub\u003e), the time to reach peak concentration (T\u003csub\u003emax\u003c/sub\u003e), and the area under the curve from 0 to 28 days (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;28\u003c/sub\u003e) of the infused TCR-T cells and cytokines in the peripheral blood. These metrics will help elucidate the bioactivity and exposure profile of the therapeutic cells and their effects.\u003c/p\u003e\n\u003ch2\u003eGeneration of TCR-T Infusion Products\u003c/h2\u003e\n\u003cp\u003eWe utilized the MSGV1 retroviral vector to transduce autologous peripheral blood mononuclear cells (PBMCs) before \u003cem\u003ein vitro\u003c/em\u003e expansion of the transduced T cells, employing anti-CD3 and media supplemented with recombinant interleukin-2 (IL-2\u003csup\u003e10\u003c/sup\u003e. The resulting TCR, named PCV, recognizes a 9-mer RASG12V peptide (VVGAVGAGK)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. At the same time, TCR 051 is specific to a 10-mer RASG12V peptide (VVVGAVGAGK) in the context of HLA-A11:01, as detailed in the Supplementary Methods section of the Supplementary Appendix. Additionally, TCR-2, isolated from colorectal cancer tumor-infiltrating lymphocytes (TILs), was reconstructed to recognize a 10-mer RASG12D peptide (VVVGADGVGK) in the context of HLA-A11:01\u003csup\u003e29\u003c/sup\u003e. TCR gene modification was conducted by the transduction of autologous PBMCs using recombinant replication-incompetent retroviruses post-stimulation with OKT-3 antibodies within a retronectin-coated cell culture bag (Origen) and subsequent expansion in GREX flasks (Wilson Wolf) as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eTracking of Transgenic TCR-positive Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo monitor the \u003cem\u003ein vivo\u003c/em\u003e persistence of infused T cells, we employed both flow cytometry analysis and digital droplet PCR (ddPCR). The murine TCR\u0026alpha; and \u0026beta; chains were substituted for their human equivalents in the engineered T cells, serving both to ensure the correct pairing of the exogenous TCRs and to act as a marker for tracking the transgenic T cells. The frequency of these transgenic T cells \u003cem\u003ein vivo\u003c/em\u003e was determined by flow cytometry analysis, utilizing anti-mouse TCR \u0026beta; chain antibodies to identify the cells in isolated PBMC. Concurrently, digital droplet PCR was utilized to assess the DNA copy number of the transgenic T cells. By using specific primer pairs, we were able to accurately evaluate the relative persistence of two distinct populations of T cells, each engineered to express a different TCR recognizing either the 9-mer (PCV) or 10-mer (051) epitopes of the KRAS G12V mutation. This approach provided a comprehensive assessment of the longevity and distribution of the therapeutic T cells post-infusion.\u003c/p\u003e\n\u003ch2\u003eCytokine Release in PBMC after Cell Infusion\u003c/h2\u003e\n\u003cp\u003eTo assess cytokine release in PBMC following cell infusion, we utilized a cytokine bead array assay. This method was employed to quantify the levels of several cytokines known to contribute to cytokine release syndrome (CRS). Blood samples were collected at various time intervals post-infusion. The measurement process involved using flow cytometry to analyze beads that were coated with antibodies specific to different cytokines. After incubation with diluted patient sera, these coated beads were then labeled with fluorescent secondary antibodies to enable the detection and quantification of cytokine levels. This assay provides a detailed profile of cytokine dynamics, which is critical for understanding and managing potential CRS in patients undergoing cell therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analysed during the current study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe remember Yandan Yao, a great clinician, researcher, colleague, friend, husband, and father. This study was supported by Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (Grant Number: SYS-5010Z-202303).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ Zeng, T Qin, and G Yu write the original manuscript, collect and analysis data, and writing, review, and edit the manuscript. X Wu, S Huang, T Chen, and X Chen collect clinical data. W Wu, X Zhang, C Gan, and X Jiang collect and analyze clinical data. M Zhang, H Yao, and H Hu conceptualization, supervision, writing–review and editing, and funding and approving the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eDr. Zhang Meng was funded by the Sun Yat-Sen Memorial Hospital Clinical Research 5010 Program (SYS-5010Z-202303). Zhifen Zeng, Wenxia Wu, Tonghua Chen, and Xueru Chen are her subordinates. Dr. Herui Yo is the director of the Oncology Department, and QinTao is his subordinate. Songyin Huang is the director of the Biological Therapy Center, and XiaohuaWu is her subordinate.\u003c/p\u003e\n\u003cp\u003eDr. Hong-Ming Hu was funded by the National Key Research and Development Program of China (2023YFC3403800) and is the founder and CSO of ImmuXell Biotech. Guangjie Yu, Shih-Ting Tsao, Xiaohui Zhang, Chi Gan, and Xiaochun Jiang are employees of ImmuXell Biotech.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel, R. L., Miller, K. D. \u0026amp; Jemal, A. Cancer statistics, 2020. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e 70, 7\u0026ndash;30, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21590\u003c/span\u003e\u003cspan address=\"10.3322/caac.21590\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eViale, P. H. 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Identifying T cell receptors (TCRs) reactive to mutant KRAS enables the exploration of TCR gene-modified T cell (TCR-T) therapy for solid tumors with KRAS mutations.\u003c/p\u003e\n\u003cp\u003eAn investigator-initiated trial (IIT) evaluated adoptive TCR-T therapy using autologous T cells engineered to target KRAS G12V or KRAS G12D mutations in HLA-A*11:01-positive patients. Eligible individuals had advanced pancreatic or other solid tumors with these mutations and received a single infusion of 1 × 10⁹ to 1 × 10¹⁰ TCR-T cells. The primary endpoint was progression-free survival (PFS); secondary endpoints included objective response rate (ORR), overall survival (OS), duration of response (DOR), disease control rate (DCR), and safety. So far, we observed that ORR (100%), PFS (10, 8, and 27 months), DOR (10, 8, and 13 months), and OS (17, 10, and 30 months) in two patients with PDAC and NSCLC. No grade 3–4 toxicities occurred. All patients exhibited cytopenia, characterized by anemia, transient thrombocytopenia, and a reduction in white blood cells due to the lymphodepletion required for TCR-T therapy.\u003c/p\u003e\n\u003cp\u003eThese results demonstrate that TCR-T therapy targeting KRAS\u003csup\u003eG12V/D\u003c/sup\u003e is feasible and beneficial in patients with advanced solid tumors. The IIT trial is ongoing to recruit more patients with KRAS\u003csup\u003eG12V/D\u003c/sup\u003e mutations (NCT05438667).\u003c/p\u003e","manuscriptTitle":"Adoptive T-Cell Therapy in Solid Tumor with KRASG12V/G12D Mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 08:45:54","doi":"10.21203/rs.3.rs-7496490/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ccefef42-7a09-4612-919d-07edfe2803a6","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53966082,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":53966083,"name":"Biological sciences/Immunology/Tumour immunology"}],"tags":[],"updatedAt":"2025-09-04T15:56:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 08:45:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7496490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7496490","identity":"rs-7496490","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}