Safety and Efficacy of an autologous HER2-Targeting CAR-T therapy in Patients with HER2-positive recurrent or refractory Breast Cancer: An analysis of a Phase I clinical study

Safety and Efficacy of an autologous HER2-Targeting CAR-T therapy in Patients with HER2-positive recurrent or refractory Breast Cancer: An analysis of a Phase I clinical study | 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 Research Article Safety and Efficacy of an autologous HER2-Targeting CAR-T therapy in Patients with HER2-positive recurrent or refractory Breast Cancer: An analysis of a Phase I clinical study Ning Liu, Peng Yan, Chunsheng Yang, Chuanyong Liu, Xinyu Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8829992/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 Background Chimeric Antigen Receptor (CAR)-engineered T cells constitute a novel immunotherapeutic approach. CAR-T therapy has achieved remarkable breakthroughs in the treatment of hematological malignancies, yet its application to solid tumors confronts enormous challenges. HER2-CAR-T therapy may serve as a promising treatment option for breast cancer patients who have failed anti-HER2 therapy. Here we reported the Phase I clinical trials results of C406 in HER2-positive recurrent or refractory breast cancer. Methods The Phase I was a dose-escalation study to evaluate the safety, efficacy and pharmacokinetics of C406 in breast cancer patients. Patients received a single dose of 3×107cells/kg (Cohort 1) or 1×108cells/kg (Cohort 2) following bridging and lymphodepleting chemotherapy. The primary endpoint was to evaluate the incidence of treatment emergent adverse events. The secondary endpoint was to evaluate the efficacy and pharmacokinetics of C406. The study also explored diversified bridging therapy and conditioning chemotherapy for lymphodepletion. Results By cut-off date of 2025 July 1st, totally 8 patients were enrolled, 6 patients received C406 at a dose of 3×10⁷ cells/kg, and the remaining 2 patients were administered 1×10⁸ cells/kg. Results showed that C406 was well tolerated and demonstrated a favorable toxicity profile. No case of grade 3 or higher cytokine release syndrome. No case of grade 3 or higher CAR-T related adverse events. Grade 3 or higher adverse events were hematological disorders by lymphodepletion therapy. DCR (disease control rate) was 75% and mPFS (median progression-free survival) was 9 weeks. Conclusion In patients with HER2-positive relapsed or refractory breast cancer, C406 has demonstrated a tolerable safety profile, providing a rationale for subsequent research; however, its efficacy requires further confirmation. Trial registration Trial No.MR-37-23-019847. Registered 17 January 2025, https://www.medicalresearch.org.cn . HER2 Breast cancer CAR-T therapy Clinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Immunotherapy represents a highly promising strategy for cancer treatment. Chimeric Antigen Receptor (CAR)-engineered T cells constitute a novel immunotherapeutic approach. Synthetic CARs can redirect T cells to recognize and eliminate tumor cells expressing specific antigens, and advances in CAR-T cell therapy have transformed the therapeutic landscape of targeted immunotherapy[ 1 ]. CAR-T cells have achieved remarkable clinical efficacy in the treatment of hematological malignancies, and more than ten CAR-T cell products targeting CD19 or B-cell Maturation Antigen (BCMA) have been approved to date[ 2 – 4 ]. While CAR-T therapy has achieved major breakthroughs in the treatment of hematological malignancies, translating this success to solid tumors faces formidable challenges. The main obstacles of which include tumor antigen heterogeneity, poor trafficking, T-cell dysfunction induced by the suppressive tumor microenvironment, and so on[ 1 , 5 – 7 ]. At present, relevant clinical studies of CAR-T have been initiated for various solid tumors, such as EGFR-CAR-T for lung cancer, GPC3-CAR-T for hepatocellular carcinoma, PSMA-CAR-T for prostate cancer, MSLN-CAR-T for ovarian cancer, IL3Rα2-CAR-T, EGFR/IL3Rα2 dual-target CAR-T and GD2-specific 4SCAR-T for glioma, as well as Claudin18.2-CAR-T for gastrointestinal cancers which has shown relatively rapid development[ 7 – 17 ]. Since the discovery of the HER2 gene [ 18 ], research on this proto-oncogene has been ongoing. The incidence of HER2 positive breast cancer in China accounts for approximately 25%[ 19 – 21 ]. At present, three categories of drugs have been approved for clinical treatment, including monoclonal antibodies (trastuzumab, pertuzumab, etc.), small-molecule tyrosine kinase inhibitors (lapatinib, pyrotinib, neratinib), and antibody-drug conjugates (T-DM1 and DS-8201)[ 22 – 28 ]. Based on the DESTINY-Breast02 study, patients previously treated with HER2-ADC can still derive benefits from anti-HER2 therapy[ 29 ]. Therefore, HER2-CAR-T therapy may serve as a promising treatment option for breast cancer patients who have failed anti-HER2 therapy. In this study, breast cancer patients with HER2 positivity who experienced failure of standard therapy were enrolled, aiming to investigate the safety and tolerability, as well as the anti-tumor efficacy and pharmacokinetic characteristics of C406 in patients with HER2 positive advanced breast cancer. Meanwhile, this study explored the value of different bridging therapy regimens, different lymphodepletion doses and multiple infusion regimens in enhancing CAR-T expansion and efficacy. Considering that HER2 expression levels play a critical role in CAR-T activation, the study also collected and detected HER2 levels in patients prior to enrollment to clarify the correlation between antigen expression levels and therapeutic efficacy. Study Design and Study Methods Study Design This is a single-arm, open-label Phase I trial designed to evaluate the safety and tolerability of C406 in patients with HER2-positive recurrent or refractory breast cancer. This Phase I clinical study has been approved by the Medical Ethics Committee of the Central Hospital Affiliated to Shandong First Medical University and registered on ClinicalTrials.gov with the registration number ChiCTR2500096093. Main Inclusion Criteria. 1. Aged 18 to 70 years old. 2. Patients with advanced solid tumors confirmed by histology or cytology who have failed standard treatment. 3. Presence of at least one measurable lesion. 4. HER2-positive status, defined as HER2 IHC 2+ with positive FISH testing or HER2 IHC 3+. For patients with recurrence after anti-HER2 targeted therapy, HER2 expression must be confirmed based on pathological results post-recurrence or via repeat biopsy and IHC testing. 5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. 6. Adequate organ and hematopoietic function, meeting the following requirements: Hemoglobin≥90 g/L, White blood cell count ≥ 2.5 × 10⁹/L, Absolute neutrophil count ≥ 1.5 × 10⁹/L, Platelet count ≥ 80 × 10⁹/L, Total bilirubin (TBIL) ≤3.0 mg/dL or≤1.5×upper limit of normal (ULN), Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)≤2.5×ULN; for abnormal liver function caused by hepatocellular carcinoma or hepatic tumor metastasis, AST and ALT≤5×ULN, Serum creatinine≤1.5×ULN or creatinine clearance (CrCl)≥50 mL/min. 7. Left ventricular ejection fraction (LVEF)≥50% as assessed by echocardiography. Main Exclusion Criteria. 1. Pregnancy, lactation, or concurrent participation in other clinical trials. 2. Active infection; hepatitis or human immunodeficiency virus (HIV) infection; other severe autoimmune diseases; uncontrolled cardiovascular and cerebrovascular diseases; severe hepatic or renal dysfunction, etc. 3. Meningeal metastasis or central nervous system (CNS) metastasis. After meeting the screening criteria, subjects will undergo blood collection and cell processing, and receive a bridging therapy regimen. Subjects receive lymphodepletion therapy 5 days prior to CAR-T cell infusion, followed by a 28-day hospitalization for safety monitoring and pharmacokinetics (PK) assays after CAR-T cell infusion. Adverse events (AEs) were coded using International Conference on Harmonization Medical Dictionary for Regulatory Activities codes version 23.1 and were graded according to CTCAE version 5.0. The first tumor imaging examination during the screening period will be conducted within 28 days (inclusive) prior to the first CAR-T cell infusion. Tumor imaging assessment will be repeated on the day of cell infusion and serve as the baseline. During the study period, tumor assessments will be performed on the 28th day after infusion and then every 4 weeks subsequently, and the patients' therapeutic efficacy will be evaluated in accordance with the irRECIST 1.1 criteria. Patient Baseline Characteristics All 8 enrolled patients were female with HER2-positive metastatic breast cancer, with a median age of 58 years, and all had received prior anti-HER2 therapy (Table 1). Seven patients received bridging therapy while awaiting CART cell manufacturing. The bridging therapy regimens were promptly adjusted according to the patients' differential treatment responses (Table 2). Table 1 Baseline Patient Characteristics Table 2 Bridging regimens, lymphodepletion regimens, and details of C406 reinfusion Preparation of CAR-T Cells All 8 patients underwent apheresis of autologous peripheral blood mononuclear cells (PBMCs) at the study center. CAR-T cells were manufactured via lentiviral vector transduction, followed by in vitro expansion for 7 to 10 days, and cryopreserved once the release criteria were met. Functional CAR-T cell products were successfully generated for all patients. Treatment Procedure The lymphodepletion regimen uniformly consisted of cyclophosphamide combined with fludarabine. Specifically, fludarabine (30 mg/m², once daily for 3 consecutive days) and cyclophosphamide (300~500 mg/m², once daily for 3 consecutive days) were co-administered on Days -5, -4 and -3 prior to intravenous CAR-T cell infusion. The doses of fludarabine and cyclophosphamide could be adjusted according to the subjects' estimated glomerular filtration rate (eGFR). All patients received and completed C406 CAR-T cell therapy between 2023 2025 July 1 st and 2025 July 1 st . In accordance with the study protocol, all patients were intravenously administered C406 CAR-T cells on Day 0. Safety assessments were conducted on Day 28 post-infusion, and serum biochemistry, PK and cytokine levels were evaluated on Days 2, 4, 6, 8, 10, 12, 14, 17, 21, and 28, respectively. After Day 28, follow-up visits were scheduled every 4 weeks to assess the patients' safety and therapeutic efficacy. Two of the patients received a second CAR-T cell infusion, with intervals of 70 days and 62 days from the first infusion, respectively. Study Endpoints Safety The primary objective of this study was to evaluate the safety and tolerability of C406 following infusion. No predefined dose-limiting toxicities (DLTs) were observed in any patient within 28 days after the first infusion. AEs occurred in all patients, and AEs of all grades were monitored and documented (Table 3). The most common grade ≥3 AEs were preconditioning-related hematological events, including lymphopenia in 8 patients (100%), leukopenia in 8 patients (100%), neutropenia in 8 patients (100%), anemia in 2 patients (25%), and thrombocytopenia in 5 patients (62.5%). These hematological AEs occurred within 28 days after the first infusion, with recovery typically occurring at approximately 7 days to 3 months. According to the criteria of the American Society for Transplantation and Cellular Therapy (ASTCT), 2 patients developed grade 1-2 cytokine release syndrome (CRS) with symptoms of fever and hypoxemia; no grade 3 or higher CRS was observed. One of these patients had an inadequate response to nonsteroidal anti-inflammatory drugs (NSAIDs) after the onset of fever, with a maximum body temperature of 39.4 °C. Both patients showed resolution of symptoms on the second day after treatment with tocilizumab, and no glucocorticoid therapy was administered. One patient experienced an infusion reaction with chills after infusion, which resolved following antihistamine administration. Additionally, one patient developed hypothyroidism during subsequent follow-up (more than 6 months after CAR-T infusion). No CRS was observed in the two patients who received the second CAR-T infusion. Immune effector cell-associated neurotoxicity syndrome (ICANS) was not observed in any enrolled patient, and no treatment-related deaths occurred. Table3-1 Summary of AEs TEAE: Treatment emergent adverse event; TRAE: Treatment related adverse event; SAE: serious adverse event Table3-2 AEs that occurred after T cell administration Efficacy of C406 The secondary objectives of this study included objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). Radiological examinations were performed and compared for all 8 patients both before and after CAR-T cell infusion. According to the investigator assessment based on the RECIST 1.1 criteria, 6 patients achieved stable disease (SD), with a DCR of 75%. The median progression-free survival (mPFS) for all patients was 9 weeks, and the median overall survival (mOS) was 9.5 months. The 6-month OS rate was 62.5%, and the 12-month OS rate was 37.5%. Among them, the patient with the longest disease control had a PFS of 8 months. As of the last follow-up in December 2025, 2 patients remained alive (Table 4). Considering that HER2 expression levels play a critical role in CAR-T activation, the study also collected and detected HER2 levels in patients prior to enrollment to clarify the correlation between antigen expression levels and therapeutic efficacy. Patient 002, enrolled with extensive ulcerated cutaneous metastases from breast cancer, experienced a rapid improvement in the ulcerated skin within the first few days after CAR-T cell infusion (Figure 1). For Patient 003, radiological examinations at 2 months post CAR-T cell infusion revealed a certain degree of reduction in metastatic lesions (Figure 2). However, the duration of disease response was short in both subjects, which may be attributed to the subsequent restricted in vivo expansion of CAR-T cells. Table 4 Efficacy of enrolled patients Fig. 1 Comparison of Patient 002 before treatment (left) and 1 month after C406 infusion (right) Fig. 2 Patient 003 before treatment (left) vs. 2 months after C406 infusion (right) Levels and Persistence of C406 CAR-T Cells in Peripheral Blood This was a secondary objective of this clinical study. PK and cytokine assays were performed immediately after C406 CAR-T cell infusion and on Days 2, 4, 6, 8, 10, 12, 14, 17, 21 and 28 post-infusions. Based on the detection of vector copy number (VCN), the study evaluated the expansion of CAR-T cells in subjects following C406 infusion. The assessments revealed that CAR-T cells were detectable in all patients after infusion: CAR-T cell levels dropped to an extremely low level on Day 2 post-infusion, subsequently rose on Days 6–10 post-infusion, and then declined thereafter (Figure 3). Fig. 3 CAR-T cell proliferation kinetics after infusion Cytokine changes: Cytokine profiles (IL‑2, IL‑4, IL‑6, IL‑10, TNF‑α, and IFN‑γ) were analyzed in 8 patients following C406 CAR‑T infusion. Plasma levels of IL‑2, IL‑4, IL‑10, and TNF‑α remained largely unchanged and lacked obvious clinical significance. IFN‑γ has been well documented as a critical antitumor effector molecule[30]. In the present study, IFN‑γ increased approximately 35‑fold on day 2 in patient 005 after C406 infusion, then rapidly declined and normalized by day 6. In all other patients, only minor fluctuations in IFN‑γ were observed. IL‑6 is widely recognized as the primary cytokine associated with immune reactions in cellular immunotherapy[31, 32]. In this study, IL‑6 rose dramatically to 272‑fold on day 4 in patient 001 and 67‑fold on day 10 in patient 002, with mild elevations in the remainder (Figure 4). No clear correlation was identified between the magnitude of IL‑6 increase and clinical efficacy. Discussion CAR-T therapy is a highly promising immunotherapeutic approach for the management of malignant tumors. At present, non-Hodgkin's lymphoma, leukemia and multiple myeloma can be treated with CAR-T therapy to prolong patients' survival time[ 3 , 33 , 34 ]. CAR-T therapy has achieved great success in hematologic malignancies, paving the way for its application in solid tumors. To date, no CAR‑T products for solid tumors have been approved for marketing worldwide. Among the most advanced programs is CT041, a Claudin18.2‑targeting CAR‑T therapy developed by CARsgen Therapeutics. Based on the results of the pivotal phase II trial (CT041‑ST‑01), a New Drug Application (NDA) has been submitted to China’s National Medical Products Administration (NMPA) for the treatment of patients with Claudin18.2‑positive advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma who have failed at least two lines of prior therapy. However, the development of CAR-T therapy in solid tumors remains challenging at present. First, the antigen issue: solid tumors lack targets such as CD19 and BCMA that are relatively specifically expressed in hematologic malignancies and critical for cell survival. Many solid tumor-associated antigens are also expressed in normal tissues, leading to the occurrence of side effects[ 35 , 36 ]. In addition, the expression of tumor cell antigens is heterogeneous in solid tumors. CAR-T cells only act on tumor cells that express the antigens, which subsequently leads to tumor recurrence[ 37 ]. Even if the antigens are tumor-specific, a large tumor burden or a critical anatomical location will result in massive tumor lysis by CAR-T cells, which itself can trigger severe inflammatory responses such as CRS and ICANS, both of which can be life-threatening[ 38 ]. Second is the issue of the tumor microenvironment (TME). A large number of immunosuppressive cells (regulatory T cells, myeloid-derived suppressor cells [MDSCs]) and a high level of immunosuppressive factors are present in the tumor microenvironment. In addition, the upregulation of immune checkpoint molecules on tumor cells and other cells within the TME accelerates the exhaustion of CAR-T cells, shortens their in vivo survival time, and impairs the persistence of their anti-tumor efficacy[ 39 ]. Third is the issue with CAR-T cells themselves. CAR-T cells are derived from T cells collected from patients, whose T cells may exhibit an exhausted and dysfunctional state. The proliferative and survival capacities of T lymphocytes, which are mainly the result of their differentiation status, are closely associated with the anti-tumor activity of adoptively transferred T cells[ 40 ]. In addition, after CAR-T cells are infused into the body, the dense structure of solid tumor tissues hinders their effective infiltration. Furthermore, the absence of precise mechanisms to recruit CAR-T cells to tumor tissues prevents their complete accumulation and infiltration within the tumor mass[ 39 ]. C406 is designed with a conditional activation mechanism, remaining inactive in normal tissues and exerting its effects only upon activation within tumors. Preclinical studies have demonstrated that the cytotoxicity and cytokine release of C406 are significantly inhibited under a normal pH environment, while C406 exerts a marked cytotoxic effect on cell lines with high or low HER2 expression (including NCI-N87, AGS and SNU-16) under a low pH environment. In this study, we evaluated the safety and anti-tumor activity of C406 in patients with advanced HER2-positive breast cancer who had failed prior anti-HER2 therapy. No DLTs, treatment-related deaths, or AEs leading to study withdrawal occurred in any subject following infusion. Most AEs were grade 1–2; all grade 3 or higher AEs were hematological adverse events caused by lymphodepletion therapy, which were expected adverse events. These data preliminarily demonstrate that the C406 regimen has a favorable safety profile. The DCR was 75% among subjects receiving C406 treatment, with both subjects in the high-dose group achieving disease control, suggesting a potential dose-related therapeutic response. Given the limited number of treated patients to date, further confirmation is required with additional data. This study also investigated the use or non-use of bridging therapy, the application of different bridging therapy regimens, as well as bridging therapy regimens combined with radiotherapy, and no definitive impact of different regimens on the efficacy of C406 was observed in the study. However, given the small number of enrolled patients, the efficacy of CAR‑T combination therapy warrants further investigation. Some patients achieved tumor reduction after C406 treatment, but the duration of response was short in all cases. In addition, C406 exhibited limited in vivo expansion and short persistence, resulting in an inability to exert sustained antitumor activity. To clarify the correlation between antigen expression level and therapeutic efficacy, we assessed HER2 expression in the enrolled patients. All patients tested FISH-positive, but showed variable IHC expression levels of HER2. We observed no significant correlation between the intensity of HER2 expression and clinical response. This study also investigated the impact of repeated infusions on therapeutic efficacy. The PFS of the two patients after repeated infusions was 12 weeks and 22 weeks, respectively, which was higher than the mPFS of 9 weeks. This indicates that repeated infusions may help improve the durability of therapeutic efficacy. No significant effect of repeated infusions on tumor regression was observed. All patients received subsequent new anti-tumor treatment after disease progression, with a mOS of 9.5 months, confirming that all patients maintained a good general condition following CAR-T cell infusion. Therefore, further development is still needed in the future to address the challenges of CAR-T cells in solid tumors. Firstly, regarding the antigen issue, efforts should be made to identify more tumor-specific antigens. Alternatively, CAR-T cells can be engineered into dual-target or multi-target constructs that are only activated when two or more targets are simultaneously present, which would reduce damage to normal tissues[ 41 ]. Another approach is to make CAR-T cell activation dependent on the administration of a small-molecule drug, allowing CAR-T activity to be pharmacologically controlled. In the event of intolerable toxicity, CAR-T cells can be inactivated accordingly[ 42 ]. To address the immunosuppressive nature of the tumor microenvironment, CAR-T cells can be engineered to secrete cytokines endogenously, thereby modifying and ameliorating the surrounding microenvironment.[ 43 , 44 ]. Genetic engineering of CAR-T cells to overexpress FLT3L and XCL1 can promote the recruitment of dendritic cells (DCs) and antigen spreading, thereby enhancing the therapeutic efficacy of CAR-T cells against solid tumors[ 45 ]. Knocking out the PD-1 gene, combining with PD-1/PD-L1 inhibitors, or engineering CAR-T cells to express non-functional TGF-β receptors to counteract the suppressive effects of TGF-β may also be viable strategies. A recent study has genetically engineered CAR-T cells to express and secrete a bifunctional fusion protein, αPD-L1–IL-12, in situ. This simultaneously relieves immunosuppression and provides activating signals locally in the tumor, thereby significantly enhancing the therapeutic efficacy and safety of CAR-T cells in solid tumor models[ 46 ]. To improve infiltration capacity, CAR-T cells can be engineered to express specific chemokine receptors that recognize chemokines secreted by tumors, guiding CAR-T cells to accurately target tumor sites[ 39 ].Alternatively, combination with drugs that disrupt the tumor stroma can enhance the infiltration of CAR-T cells into tumor tissues. In addition, consideration can be given to combining CAR-T therapy with other treatment modalities. Besides PD-1/PD-L1 inhibitors, radiotherapy can also be used in combination. In the present study, we also added local radiotherapy to the bridging therapy for subsequent patients. Theoretically, radiotherapy can alter the tumor microenvironment, disrupt physical barriers, promote the release of tumor-associated antigens, alleviate immunosuppression, and enhance the infiltration and cytotoxicity of CAR-T cells in tumors[ 47 ].Although no definitive impact of different combination regimens of bridging therapy on the efficacy of C406 was observed in this study, further investigation is warranted given the small sample size. Concerning the exhaustion or functional impairment of patients' own T lymphocytes, an early apheresis strategy can be adopted. Early isolation not only ensures the availability of robust T cells for CAR‑T cell manufacturing but also allows timely administration of CAR‑T therapy in the event of disease progression[ 48 ]. In summary, this study is a phase I, single-center trial with a small sample size and a short follow-up period. Further validation of the safety and anti-tumor activity of C406 in a larger patient population is therefore required. As a form of immunotherapy, CAR-T therapy may drastically reshape the current landscape of anti-tumor treatment and drive more extensive innovative research compared with conventional anti-tumor approaches. In addition, different combination strategies warrant further exploration in future research. Abbreviations CAR-T Chimeric Antigen Receptor T-cell BCMA B-cell Maturation Antigen ECOG Eastern Cooperative Oncology Group TBIL Total bilirubin ULN upper limit of normal AST Aspartate aminotransferase ALT alanine aminotransferase CrCl creatinine clearance LVEF Left ventricular ejection fraction CNS central nervous system PK pharmacokinetics AEs adverse effects PBMCs peripheral blood mononuclear cells eGFR estimated glomerular filtration rate DLTs dose-limiting toxicities ASTCT American Society for Transplantation and Cellular Therapy CRS cytokine release syndrome NSAIDs nonsteroidal anti-inflammatory drugs ICANS Immune effector cell-associated neurotoxicity syndrome ORR objective response rate DCR disease control rate, PFS progression-free survival SD stable disease DCR disease control rate mPFS median progression-free survival mOS median overall survival VCN vector copy number Declarations Acknowledgements I would like to thank the Central Hospital Affiliated to Shandong First Medical University and Shanghai Perhum Therapeutics for their support and assistance during this research. Author contributions MLS and RG designed this study. NL, PY, CSY, CYL, XYW, ML and HC performed the experiments and collected and analyzed the data. NL and RG wrote the manuscript. MLS and RG provided technical support for the analysis and critical revision of the manuscript. All authors contributed to this manuscript. All authors have read and agreed to the published version of the manuscript. Fundings This study was funded by Science and Technology Development Program of Jinan Municipal Health Commission (2024201001); Wu Jieping Medical Foundation (320.6750.2025-02-31/ 320.6750.2025-06-157/320.6750.2025-13-116); Shandong Medical Association (YXH2025YS099); Beijing Bethune Charitable Foundation(2024-YJ-237-J-027); Scientific Research Foundation for the Introduced Talents of Jinan Central Hospital (YJRC2022005). Data availability All the data and materials were presented in the main paper. Ethics approval and consent to participate All participants provided informed consent. Research protocol using human samples was approved by the Medical Ethics Committee of the Central Hospital Affiliated to Shandong First Medical University and complied with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. Consent for publication All authors have agreed to publish this manuscript. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest Author details 1. Department of Oncology, Central Hospital Affiliated to Shandong First Medical University, Jinan 250013, China 2. Shanghai Perhum Therapeutics, LTD, Shanghai, 201314, China References Patel KK, et al. From concept to cure: The evolution of CAR-T cell therapy. Mol Ther. 2025;33(5):2123–40. https://doi.org/10.1016/j.ymthe.2025.02.015 . Ahmad A, Uddin S, Steinhoff M. 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Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med. 2018;24(6):739–48. https://doi.org/10.1038/s41591-018-00102-3 . Liu D, Zhao J. Cytokine release syndrome: Grading, modeling, and new therapy. J Hematol Oncol. 2018;11(1):121. https://doi.org/10.1186/s13045-018-0645-5 . van de Donk N, Usmani SZ, Yong K. CAR T-cell therapy for multiple myeloma: State of the art and prospects. Lancet Haematol. 2021;8(6):e446–61. https://doi.org/10.1016/S2352-3026(21)00129-9.=-8195091 . Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531–44. https://doi.org/10.1056/NEJMoa1705937 . Lamers CH, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: First clinical experience. J Clin Oncol. 2006;24(13):e20–2. https://doi.org/10.1200/JCO.2005.04.7818 . Morgan RA, et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther. 2010;18(4):843–51. https://doi.org/10.1038/mt.2010.32 . O'Rourke DM, et al. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med. 2017;9(399):eaah5868. https://doi.org/10.1126/scitranslmed.aah5868 . Lee DW, et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014;124(2):188–95. https://doi.org/10.1182/blood-2014-03-556491 . Lamplugh ZL, et al. Microenvironmental regulation of solid tumour resistance to CAR T cell therapy. Nat Reviews Immunol Adv online publication. 2025. https://doi.org/10.1038/s41577-025-00896-7 . Kaczanowska S, et al. Immune determinants of CAR-T cell expansion in solid tumor patients receiving GD2 CAR-T cell therapy. Cancer Cell. 2024;42(1):35–e518. https://doi.org/10.1016/j.ccell.2023.11.005 . Kloss CC, et al. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol. 2013;31(1):71–5. https://doi.org/10.1038/nbt.2464 . Wu CY, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science. 2015;350(6258):aab4077. https://doi.org/10.1126/science.aab4077 . Yeku OO, Brentjens RJ. Armored CAR T-cells: Utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy. Biochem Soc Trans. 2016;44(2):412–8. https://doi.org/10.1042/BST20150211 . Kagoya Y. Cytokine signaling in chimeric antigen receptor T-cell therapy. Int Immunol. 2024;36(2):49–56. Xiao Z, et al. Engineered T cells stimulate dendritic cell recruitment and antigen spreading for potent anti-tumor immunity. Cell Rep Med. 2025;6(9):102307. https://doi.org/10.1016/j.xcrm.2025.102307 . Murad JP, et al. Solid tumour CAR-T cells engineered with fusion proteins targeting PD-L1 for localized IL-12 delivery. Nat Biomedical Eng Adv online publication. 2025. https://doi.org/10.1038/s41551-025-01123-9 . Wallington DG, et al. The role of radiotherapy in lymphoma patients undergoing CAR T therapy: Past, present, and future. Semin Radiat Oncol. 2025;35(1):99–109. https://doi.org/10.1016/j.semradonc.2024.09.005 . Li J, et al. Optimizing CAR T cell therapy for solid tumours: A clinical perspective. Nat Reviews Clin Oncol. 2025;22(12):953–68. https://doi.org/10.1038/s41571-025-00812-3 . 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. 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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-8829992","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":589820826,"identity":"e76f9fb9-8a13-472c-b1e9-85966b51e711","order_by":0,"name":"Ning Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvElEQVRIiWNgGAWjYBACA2bmhgMMDBIMDOyNjQ8/EKeFEaqF53CzsQRRWhgYGyAsifQ2AR5itJizMzYe5t1hkScf+bANaJmdnG4DAS2WzYwNh3nPSBQb3k5se1DAkGxsdoCQww6DtLRJJG6cndhuIMFwIHEb8VpmHmyT4CFJy3wJRhK0HJwL1LKBJxEYyAbE+OX84cMf3rbVJc5vP/7w4YcKOzmCWhB6wSoNiFUOAvINpKgeBaNgFIyCEQUAl7xFDWrSlKsAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0007-6775-6186","institution":"Central Hospital Affiliated to Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ning","middleName":"","lastName":"Liu","suffix":""},{"id":589820827,"identity":"7439f258-ba90-4148-8c65-c409b02649ae","order_by":1,"name":"Peng Yan","email":"","orcid":"","institution":"Central Hospital Affiliated to Shandong First Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Wang","suffix":""},{"id":589820831,"identity":"cb4aebf3-02dd-4f75-bacf-4416a58407f3","order_by":5,"name":"Meng Ai","email":"","orcid":"","institution":"Central Hospital Affiliated to Shandong First Medical Uniersity","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Ai","suffix":""},{"id":589820832,"identity":"705c6015-b582-484a-8788-4d3f8457c922","order_by":6,"name":"Hua Chen","email":"","orcid":"","institution":"Central Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Chen","suffix":""},{"id":589820833,"identity":"4b277eaa-2aec-46d1-a64d-d48d65bea552","order_by":7,"name":"Rang Gao","email":"","orcid":"","institution":"Shanghai Perhum Therapeutics, LTD","correspondingAuthor":false,"prefix":"","firstName":"Rang","middleName":"","lastName":"Gao","suffix":""},{"id":589820834,"identity":"8d996efd-faf3-4624-9d89-0e1d6fd1a451","order_by":8,"name":"Meili Sun","email":"","orcid":"https://orcid.org/0000-0002-4914-6508","institution":"Central Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meili","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-02-09 11:43:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8829992/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8829992/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102854181,"identity":"58bb9d25-b8f3-4f93-9de0-8802f763d671","added_by":"auto","created_at":"2026-02-17 14:47:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146188,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Patient 002 before treatment (left) and 1 month after C406 infusion (right)\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8829992/v1/9754bce99ffd863bce2c0c10.jpg"},{"id":102854183,"identity":"62c70bcf-755c-4b1c-a352-96801bcd7f76","added_by":"auto","created_at":"2026-02-17 14:47:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71936,"visible":true,"origin":"","legend":"\u003cp\u003ePatient 003 before treatment (left) vs. 2 months after C406 infusion (right)\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8829992/v1/ce4c3de867a47e9a7bbd9624.jpg"},{"id":102854180,"identity":"64c98f4e-aa50-4533-94e3-e6b51c2a62f4","added_by":"auto","created_at":"2026-02-17 14:47:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39211,"visible":true,"origin":"","legend":"\u003cp\u003eCAR-T cell proliferation kinetics after infusion\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8829992/v1/c8fedbbe47bd05ab2b1ec72f.jpg"},{"id":102854182,"identity":"232ffa32-2fbd-47ba-a7b9-7f9791af7d10","added_by":"auto","created_at":"2026-02-17 14:47:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24247,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of changes in IL‑6 levels after C406 infusion\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8829992/v1/38473a8b2977136e01655e48.jpg"},{"id":105728192,"identity":"f84ada1f-d1ec-4d3b-a16a-a7fd77972420","added_by":"auto","created_at":"2026-03-30 11:10:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1554914,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8829992/v1/5022046e-11a8-445d-b698-e0aa9a5a228f.pdf"}],"financialInterests":"","formattedTitle":"Safety and Efficacy of an autologous HER2-Targeting CAR-T therapy in Patients with HER2-positive recurrent or refractory Breast Cancer: An analysis of a Phase I clinical study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmunotherapy represents a highly promising strategy for cancer treatment. Chimeric Antigen Receptor (CAR)-engineered T cells constitute a novel immunotherapeutic approach. Synthetic CARs can redirect T cells to recognize and eliminate tumor cells expressing specific antigens, and advances in CAR-T cell therapy have transformed the therapeutic landscape of targeted immunotherapy[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. CAR-T cells have achieved remarkable clinical efficacy in the treatment of hematological malignancies, and more than ten CAR-T cell products targeting CD19 or B-cell Maturation Antigen (BCMA) have been approved to date[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile CAR-T therapy has achieved major breakthroughs in the treatment of hematological malignancies, translating this success to solid tumors faces formidable challenges. The main obstacles of which include tumor antigen heterogeneity, poor trafficking, T-cell dysfunction induced by the suppressive tumor microenvironment, and so on[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At present, relevant clinical studies of CAR-T have been initiated for various solid tumors, such as EGFR-CAR-T for lung cancer, GPC3-CAR-T for hepatocellular carcinoma, PSMA-CAR-T for prostate cancer, MSLN-CAR-T for ovarian cancer, IL3Rα2-CAR-T, EGFR/IL3Rα2 dual-target CAR-T and GD2-specific 4SCAR-T for glioma, as well as Claudin18.2-CAR-T for gastrointestinal cancers which has shown relatively rapid development[\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the discovery of the HER2 gene [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], research on this proto-oncogene has been ongoing. The incidence of HER2 positive breast cancer in China accounts for approximately 25%[\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. At present, three categories of drugs have been approved for clinical treatment, including monoclonal antibodies (trastuzumab, pertuzumab, etc.), small-molecule tyrosine kinase inhibitors (lapatinib, pyrotinib, neratinib), and antibody-drug conjugates (T-DM1 and DS-8201)[\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Based on the DESTINY-Breast02 study, patients previously treated with HER2-ADC can still derive benefits from anti-HER2 therapy[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, HER2-CAR-T therapy may serve as a promising treatment option for breast cancer patients who have failed anti-HER2 therapy. In this study, breast cancer patients with HER2 positivity who experienced failure of standard therapy were enrolled, aiming to investigate the safety and tolerability, as well as the anti-tumor efficacy and pharmacokinetic characteristics of C406 in patients with HER2 positive advanced breast cancer.\u003c/p\u003e \u003cp\u003eMeanwhile, this study explored the value of different bridging therapy regimens, different lymphodepletion doses and multiple infusion regimens in enhancing CAR-T expansion and efficacy. Considering that HER2 expression levels play a critical role in CAR-T activation, the study also collected and detected HER2 levels in patients prior to enrollment to clarify the correlation between antigen expression levels and therapeutic efficacy.\u003c/p\u003e"},{"header":"Study Design and Study Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is a single-arm, open-label Phase I trial designed to evaluate the safety and tolerability of C406 in patients with HER2-positive recurrent or refractory breast cancer. This Phase I clinical study has been approved by the Medical Ethics Committee of the Central Hospital Affiliated to Shandong First Medical University and registered on ClinicalTrials.gov with the registration number ChiCTR2500096093.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMain Inclusion Criteria.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Aged 18 to 70 years old.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Patients with advanced solid tumors confirmed by histology or cytology who have failed standard treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Presence of at least one measurable lesion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. HER2-positive status, defined as HER2 IHC 2+ with positive FISH testing or HER2 IHC 3+. For patients with recurrence after anti-HER2 targeted therapy, HER2 expression must be confirmed based on pathological results post-recurrence or via repeat biopsy and IHC testing.\u003c/p\u003e\n\u003cp\u003e5. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e6. Adequate organ and hematopoietic function, meeting the following requirements: Hemoglobin\u0026ge;90 g/L, White blood cell count\u0026nbsp;\u0026ge;\u0026nbsp;2.5 \u0026times; 10⁹/L, Absolute neutrophil count\u0026nbsp;\u0026ge;\u0026nbsp;1.5 \u0026times; 10⁹/L, Platelet count\u0026nbsp;\u0026ge;\u0026nbsp;80 \u0026times; 10⁹/L, Total bilirubin (TBIL)\u0026nbsp;\u0026le;3.0 mg/dL or\u0026le;1.5\u0026times;upper limit of normal (ULN), Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)\u0026le;2.5\u0026times;ULN; for abnormal liver function caused by hepatocellular carcinoma or hepatic tumor metastasis, AST and ALT\u0026le;5\u0026times;ULN, Serum creatinine\u0026le;1.5\u0026times;ULN or creatinine clearance (CrCl)\u0026ge;50 mL/min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e7. Left ventricular ejection fraction (LVEF)\u0026ge;50% as assessed by echocardiography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMain Exclusion Criteria.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Pregnancy, lactation, or concurrent participation in other clinical trials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Active infection; hepatitis or human immunodeficiency virus (HIV) infection; other severe autoimmune diseases; uncontrolled cardiovascular and cerebrovascular diseases; severe hepatic or renal dysfunction, etc.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Meningeal metastasis or central nervous system (CNS) metastasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter meeting the screening criteria, subjects will undergo blood collection and cell processing, and receive a bridging therapy regimen.\u0026nbsp;Subjects receive lymphodepletion therapy 5 days prior to CAR-T cell infusion, followed by a 28-day hospitalization for safety monitoring and pharmacokinetics (PK) assays after CAR-T cell infusion. Adverse events (AEs) were coded using International Conference on Harmonization Medical Dictionary for Regulatory Activities codes version 23.1 and were graded according to CTCAE version 5.0.\u003c/p\u003e\n\u003cp\u003eThe first tumor imaging examination during the screening period will be conducted within 28 days (inclusive) prior to the first CAR-T cell infusion. Tumor imaging assessment will be repeated on the day of cell infusion and serve as the baseline. During the study period, tumor assessments will be performed on the 28th day after infusion and then every 4 weeks subsequently, and the patients\u0026apos; therapeutic efficacy will be evaluated in accordance with the irRECIST 1.1 criteria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient Baseline Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 8 enrolled patients were female with HER2-positive metastatic breast cancer, with a median age of 58 years, and all had received prior anti-HER2 therapy (Table 1). Seven patients received bridging therapy while awaiting CART cell manufacturing. The bridging therapy regimens were promptly adjusted according to the patients\u0026apos; differential treatment responses (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Baseline Patient Characteristics\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1771332885.png\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eBridging regimens, lymphodepletion regimens, and details of C406 reinfusion\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1771332891.png\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of CAR-T Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 8 patients underwent apheresis of autologous peripheral blood mononuclear cells (PBMCs) at the study center. CAR-T cells were manufactured via lentiviral vector transduction, followed by in vitro expansion for 7 to 10 days, and cryopreserved once the release criteria were met. Functional CAR-T cell products were successfully generated for all patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment Procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lymphodepletion regimen uniformly consisted of cyclophosphamide combined with fludarabine. Specifically, fludarabine (30 mg/m\u0026sup2;, once daily for 3 consecutive days) and cyclophosphamide (300~500 mg/m\u0026sup2;, once daily for 3 consecutive days) were co-administered on Days -5, -4 and -3 prior to intravenous CAR-T cell infusion. The doses of fludarabine and cyclophosphamide could be adjusted according to the subjects\u0026apos; estimated glomerular filtration rate (eGFR).\u003c/p\u003e\n\u003cp\u003eAll patients received and completed C406 CAR-T cell therapy between 2023 2025 July 1\u003csup\u003est\u003c/sup\u003e and 2025 July 1\u003csup\u003est\u003c/sup\u003e. In accordance with the study protocol, all patients were intravenously administered C406 CAR-T cells on Day 0. Safety assessments were conducted on Day 28 post-infusion, and serum biochemistry, PK and cytokine levels were evaluated on Days 2, 4, 6, 8, 10, 12, 14, 17, 21, and 28, respectively. After Day 28, follow-up visits were scheduled every 4 weeks to assess the patients\u0026apos; safety and therapeutic efficacy. Two of the patients received a second CAR-T cell infusion, with intervals of 70 days and 62 days from the first infusion, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Endpoints\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSafety\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary objective of this study was to evaluate the safety and tolerability of C406 following infusion. No predefined dose-limiting toxicities (DLTs) were observed in any patient within 28 days after the first infusion. AEs occurred in all patients, and AEs of all grades were monitored and documented (Table 3). The most common grade\u0026nbsp;\u0026ge;3 AEs were preconditioning-related hematological events, including lymphopenia in 8 patients (100%), leukopenia in 8 patients (100%), neutropenia in 8 patients (100%), anemia in 2 patients (25%), and thrombocytopenia in 5 patients (62.5%). These hematological AEs occurred within 28 days after the first infusion, with recovery typically occurring at approximately 7 days to 3 months. According to the criteria of the American Society for Transplantation and Cellular Therapy (ASTCT), 2 patients developed grade 1-2 cytokine release syndrome (CRS) with symptoms of fever and hypoxemia; no grade 3 or higher CRS was observed. One of these patients had an inadequate response to nonsteroidal anti-inflammatory drugs (NSAIDs) after the onset of fever, with a maximum body temperature of 39.4\u0026nbsp;\u0026deg;C. Both patients showed resolution of symptoms on the second day after treatment with tocilizumab, and no glucocorticoid therapy was administered. One patient experienced an infusion reaction with chills after infusion, which resolved following antihistamine administration. Additionally, one patient developed hypothyroidism during subsequent follow-up (more than 6 months after CAR-T infusion). No CRS was observed in the two patients who received the second CAR-T infusion. Immune effector cell-associated neurotoxicity syndrome (ICANS) was not observed in any enrolled patient, and no treatment-related deaths occurred.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable3-1\u003c/strong\u003e Summary of AEs\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1771332912.png\"\u003e\u003c/p\u003e\n\u003cp\u003eTEAE: Treatment emergent adverse event; TRAE: Treatment related adverse event; SAE: serious adverse event\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable3-2\u003c/strong\u003e AEs that occurred after T cell administration\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1771332921.png\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficacy of C406\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe secondary objectives of this study included objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). Radiological examinations were performed and compared for all 8 patients both before and after CAR-T cell infusion. According to the investigator assessment based on the RECIST 1.1 criteria, 6 patients achieved stable disease (SD), with a DCR of 75%. The median progression-free survival (mPFS) for all patients was 9 weeks, and the median overall survival (mOS) was 9.5 months. The 6-month OS rate was 62.5%, and the 12-month OS rate was 37.5%. Among them, the patient with the longest disease control had a PFS of 8 months. As of the last follow-up in December 2025, 2 patients remained alive (Table 4). Considering that HER2 expression levels play a critical role in CAR-T activation, the study also collected and detected HER2 levels in patients prior to enrollment to clarify the correlation between antigen expression levels and therapeutic efficacy. Patient 002, enrolled with extensive ulcerated cutaneous metastases from breast cancer, experienced a rapid improvement in the ulcerated skin within the first few days after CAR-T cell infusion (Figure 1). For Patient 003, radiological examinations at 2 months post CAR-T cell infusion revealed a certain degree of reduction in metastatic lesions (Figure 2). However, the duration of disease response was short in both subjects, which may be attributed to the subsequent restricted in vivo expansion of CAR-T cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eEfficacy of enrolled patients\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1771332934.png\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1\u0026nbsp;\u003c/strong\u003eComparison of Patient 002 before treatment (left) and 1 month after C406 infusion (right)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u0026nbsp;\u003c/strong\u003ePatient 003 before treatment (left) vs. 2 months after C406 infusion (right)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLevels and Persistence of C406 CAR-T Cells in Peripheral Blood\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis was a secondary objective of this clinical study. PK and cytokine assays were performed immediately after C406 CAR-T cell infusion and on Days 2, 4, 6, 8, 10, 12, 14, 17, 21 and 28 post-infusions. Based on the detection of vector copy number (VCN), the study evaluated the expansion of CAR-T cells in subjects following C406 infusion. The assessments revealed that CAR-T cells were detectable in all patients after infusion: CAR-T cell levels dropped to an extremely low level on Day 2 post-infusion, subsequently rose on Days 6\u0026ndash;10 post-infusion, and then declined thereafter (Figure 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3\u0026nbsp;\u003c/strong\u003eCAR-T cell proliferation kinetics after infusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine changes:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokine profiles (IL‑2, IL‑4, IL‑6, IL‑10, TNF‑\u0026alpha;, and IFN‑\u0026gamma;) were analyzed in 8 patients following C406 CAR‑T infusion. Plasma levels of IL‑2, IL‑4, IL‑10, and TNF‑\u0026alpha;\u0026nbsp;remained largely unchanged and lacked obvious clinical significance.\u0026nbsp;IFN‑\u0026gamma; has been well documented as a critical antitumor effector molecule[30]. In the present study, IFN‑\u0026gamma;\u0026nbsp;increased approximately 35‑fold on day 2 in patient 005 after C406 infusion, then rapidly declined and normalized by day 6. In all other patients, only minor fluctuations in IFN‑\u0026gamma; were observed. IL‑6 is widely recognized as the primary cytokine associated with immune reactions in cellular immunotherapy[31, 32]. In this study, IL‑6 rose dramatically to 272‑fold on day 4 in patient 001 and 67‑fold on day 10 in patient 002, with mild elevations in the remainder (Figure 4). No clear correlation was identified between the magnitude of IL‑6 increase and clinical efficacy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCAR-T therapy is a highly promising immunotherapeutic approach for the management of malignant tumors. At present, non-Hodgkin's lymphoma, leukemia and multiple myeloma can be treated with CAR-T therapy to prolong patients' survival time[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CAR-T therapy has achieved great success in hematologic malignancies, paving the way for its application in solid tumors. To date, no CAR‑T products for solid tumors have been approved for marketing worldwide. Among the most advanced programs is CT041, a Claudin18.2‑targeting CAR‑T therapy developed by CARsgen Therapeutics. Based on the results of the pivotal phase II trial (CT041‑ST‑01), a New Drug Application (NDA) has been submitted to China\u0026rsquo;s National Medical Products Administration (NMPA) for the treatment of patients with Claudin18.2‑positive advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma who have failed at least two lines of prior therapy.\u003c/p\u003e \u003cp\u003eHowever, the development of CAR-T therapy in solid tumors remains challenging at present. First, the antigen issue: solid tumors lack targets such as CD19 and BCMA that are relatively specifically expressed in hematologic malignancies and critical for cell survival. Many solid tumor-associated antigens are also expressed in normal tissues, leading to the occurrence of side effects[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, the expression of tumor cell antigens is heterogeneous in solid tumors. CAR-T cells only act on tumor cells that express the antigens, which subsequently leads to tumor recurrence[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Even if the antigens are tumor-specific, a large tumor burden or a critical anatomical location will result in massive tumor lysis by CAR-T cells, which itself can trigger severe inflammatory responses such as CRS and ICANS, both of which can be life-threatening[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Second is the issue of the tumor microenvironment (TME). A large number of immunosuppressive cells (regulatory T cells, myeloid-derived suppressor cells [MDSCs]) and a high level of immunosuppressive factors are present in the tumor microenvironment. In addition, the upregulation of immune checkpoint molecules on tumor cells and other cells within the TME accelerates the exhaustion of CAR-T cells, shortens their in vivo survival time, and impairs the persistence of their anti-tumor efficacy[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Third is the issue with CAR-T cells themselves. CAR-T cells are derived from T cells collected from patients, whose T cells may exhibit an exhausted and dysfunctional state. The proliferative and survival capacities of T lymphocytes, which are mainly the result of their differentiation status, are closely associated with the anti-tumor activity of adoptively transferred T cells[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, after CAR-T cells are infused into the body, the dense structure of solid tumor tissues hinders their effective infiltration. Furthermore, the absence of precise mechanisms to recruit CAR-T cells to tumor tissues prevents their complete accumulation and infiltration within the tumor mass[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eC406 is designed with a conditional activation mechanism, remaining inactive in normal tissues and exerting its effects only upon activation within tumors. Preclinical studies have demonstrated that the cytotoxicity and cytokine release of C406 are significantly inhibited under a normal pH environment, while C406 exerts a marked cytotoxic effect on cell lines with high or low HER2 expression (including NCI-N87, AGS and SNU-16) under a low pH environment.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the safety and anti-tumor activity of C406 in patients with advanced HER2-positive breast cancer who had failed prior anti-HER2 therapy. No DLTs, treatment-related deaths, or AEs leading to study withdrawal occurred in any subject following infusion. Most AEs were grade 1\u0026ndash;2; all grade 3 or higher AEs were hematological adverse events caused by lymphodepletion therapy, which were expected adverse events. These data preliminarily demonstrate that the C406 regimen has a favorable safety profile. The DCR was 75% among subjects receiving C406 treatment, with both subjects in the high-dose group achieving disease control, suggesting a potential dose-related therapeutic response. Given the limited number of treated patients to date, further confirmation is required with additional data.\u003c/p\u003e \u003cp\u003eThis study also investigated the use or non-use of bridging therapy, the application of different bridging therapy regimens, as well as bridging therapy regimens combined with radiotherapy, and no definitive impact of different regimens on the efficacy of C406 was observed in the study. However, given the small number of enrolled patients, the efficacy of CAR‑T combination therapy warrants further investigation. Some patients achieved tumor reduction after C406 treatment, but the duration of response was short in all cases. In addition, C406 exhibited limited in vivo expansion and short persistence, resulting in an inability to exert sustained antitumor activity. To clarify the correlation between antigen expression level and therapeutic efficacy, we assessed HER2 expression in the enrolled patients. All patients tested FISH-positive, but showed variable IHC expression levels of HER2. We observed no significant correlation between the intensity of HER2 expression and clinical response.\u003c/p\u003e \u003cp\u003eThis study also investigated the impact of repeated infusions on therapeutic efficacy. The PFS of the two patients after repeated infusions was 12 weeks and 22 weeks, respectively, which was higher than the mPFS of 9 weeks. This indicates that repeated infusions may help improve the durability of therapeutic efficacy. No significant effect of repeated infusions on tumor regression was observed.\u003c/p\u003e \u003cp\u003eAll patients received subsequent new anti-tumor treatment after disease progression, with a mOS of 9.5 months, confirming that all patients maintained a good general condition following CAR-T cell infusion.\u003c/p\u003e \u003cp\u003eTherefore, further development is still needed in the future to address the challenges of CAR-T cells in solid tumors. Firstly, regarding the antigen issue, efforts should be made to identify more tumor-specific antigens. Alternatively, CAR-T cells can be engineered into dual-target or multi-target constructs that are only activated when two or more targets are simultaneously present, which would reduce damage to normal tissues[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Another approach is to make CAR-T cell activation dependent on the administration of a small-molecule drug, allowing CAR-T activity to be pharmacologically controlled. In the event of intolerable toxicity, CAR-T cells can be inactivated accordingly[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To address the immunosuppressive nature of the tumor microenvironment, CAR-T cells can be engineered to secrete cytokines endogenously, thereby modifying and ameliorating the surrounding microenvironment.[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Genetic engineering of CAR-T cells to overexpress FLT3L and XCL1 can promote the recruitment of dendritic cells (DCs) and antigen spreading, thereby enhancing the therapeutic efficacy of CAR-T cells against solid tumors[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Knocking out the PD-1 gene, combining with PD-1/PD-L1 inhibitors, or engineering CAR-T cells to express non-functional TGF-β receptors to counteract the suppressive effects of TGF-β may also be viable strategies. A recent study has genetically engineered CAR-T cells to express and secrete a bifunctional fusion protein, αPD-L1\u0026ndash;IL-12, in situ. This simultaneously relieves immunosuppression and provides activating signals locally in the tumor, thereby significantly enhancing the therapeutic efficacy and safety of CAR-T cells in solid tumor models[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. To improve infiltration capacity, CAR-T cells can be engineered to express specific chemokine receptors that recognize chemokines secreted by tumors, guiding CAR-T cells to accurately target tumor sites[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].Alternatively, combination with drugs that disrupt the tumor stroma can enhance the infiltration of CAR-T cells into tumor tissues.\u003c/p\u003e \u003cp\u003eIn addition, consideration can be given to combining CAR-T therapy with other treatment modalities. Besides PD-1/PD-L1 inhibitors, radiotherapy can also be used in combination. In the present study, we also added local radiotherapy to the bridging therapy for subsequent patients. Theoretically, radiotherapy can alter the tumor microenvironment, disrupt physical barriers, promote the release of tumor-associated antigens, alleviate immunosuppression, and enhance the infiltration and cytotoxicity of CAR-T cells in tumors[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].Although no definitive impact of different combination regimens of bridging therapy on the efficacy of C406 was observed in this study, further investigation is warranted given the small sample size. Concerning the exhaustion or functional impairment of patients' own T lymphocytes, an early apheresis strategy can be adopted. Early isolation not only ensures the availability of robust T cells for CAR‑T cell manufacturing but also allows timely administration of CAR‑T therapy in the event of disease progression[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, this study is a phase I, single-center trial with a small sample size and a short follow-up period. Further validation of the safety and anti-tumor activity of C406 in a larger patient population is therefore required. As a form of immunotherapy, CAR-T therapy may drastically reshape the current landscape of anti-tumor treatment and drive more extensive innovative research compared with conventional anti-tumor approaches. In addition, different combination strategies warrant further exploration in future research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCAR-T Chimeric Antigen Receptor T-cell\u003c/p\u003e\n\u003cp\u003eBCMA B-cell Maturation Antigen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eECOG Eastern Cooperative Oncology Group\u003c/p\u003e\n\u003cp\u003eTBIL Total bilirubin\u003c/p\u003e\n\u003cp\u003eULN upper limit of normal\u003c/p\u003e\n\u003cp\u003eAST Aspartate aminotransferase\u003c/p\u003e\n\u003cp\u003eALT alanine aminotransferase\u003c/p\u003e\n\u003cp\u003eCrCl creatinine clearance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLVEF Left ventricular ejection fraction\u003c/p\u003e\n\u003cp\u003eCNS central nervous system\u003c/p\u003e\n\u003cp\u003ePK pharmacokinetics\u003c/p\u003e\n\u003cp\u003eAEs adverse effects\u003c/p\u003e\n\u003cp\u003ePBMCs peripheral blood mononuclear cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eeGFR estimated glomerular filtration rate\u003c/p\u003e\n\u003cp\u003eDLTs dose-limiting toxicities\u003c/p\u003e\n\u003cp\u003eASTCT American Society for Transplantation and Cellular Therapy\u003c/p\u003e\n\u003cp\u003eCRS cytokine release syndrome\u003c/p\u003e\n\u003cp\u003eNSAIDs nonsteroidal anti-inflammatory drugs\u003c/p\u003e\n\u003cp\u003eICANS Immune effector cell-associated neurotoxicity syndrome\u003c/p\u003e\n\u003cp\u003eORR objective response rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDCR disease control rate,\u003c/p\u003e\n\u003cp\u003ePFS progression-free survival\u003c/p\u003e\n\u003cp\u003eSD stable disease\u003c/p\u003e\n\u003cp\u003eDCR disease control rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emPFS median progression-free survival\u003c/p\u003e\n\u003cp\u003emOS median overall survival\u003c/p\u003e\n\u003cp\u003eVCN vector copy number\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to thank the Central Hospital Affiliated to Shandong First Medical University and Shanghai Perhum Therapeutics for their support and assistance during this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMLS and RG designed this study. NL, PY, CSY, CYL, XYW, ML and HC performed the experiments and collected and analyzed the data. NL and RG wrote the manuscript. MLS and RG provided technical support for the analysis and critical revision of the manuscript. All authors contributed to this manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by\u0026nbsp;Science and Technology Development Program of Jinan Municipal Health Commission (2024201001); Wu Jieping Medical Foundation (320.6750.2025-02-31/\u0026nbsp;320.6750.2025-06-157/320.6750.2025-13-116);\u0026nbsp;Shandong Medical Association (YXH2025YS099); Beijing Bethune Charitable Foundation(2024-YJ-237-J-027); Scientific Research Foundation for the Introduced Talents of Jinan Central Hospital (YJRC2022005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data and materials were presented in the main paper.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided informed consent. Research protocol using human samples was approved by the Medical Ethics Committee of the Central Hospital Affiliated to Shandong First Medical University and complied with the ethical standards of the 1964 Declaration of Helsinki and its later amendments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have agreed to publish this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare 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\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Department of Oncology, Central Hospital Affiliated to Shandong First Medical University,\u003c/p\u003e\n\u003cp\u003eJinan 250013, China\u003c/p\u003e\n\u003cp\u003e2. Shanghai Perhum Therapeutics, LTD, Shanghai, 201314, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePatel KK, et al. From concept to cure: The evolution of CAR-T cell therapy. Mol Ther. 2025;33(5):2123\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ymthe.2025.02.015\u003c/span\u003e\u003cspan address=\"10.1016/j.ymthe.2025.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad A, Uddin S, Steinhoff M. CAR-T cell therapies: An overview of clinical studies supporting their approved use against acute lymphoblastic leukemia and large B-cell lymphomas. 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Nat Reviews Clin Oncol. 2025;22(12):953\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41571-025-00812-3\u003c/span\u003e\u003cspan address=\"10.1038/s41571-025-00812-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"HER2, Breast cancer, CAR-T therapy, Clinical research","lastPublishedDoi":"10.21203/rs.3.rs-8829992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8829992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChimeric Antigen Receptor (CAR)-engineered T cells constitute a novel immunotherapeutic approach. CAR-T therapy has achieved remarkable breakthroughs in the treatment of hematological malignancies, yet its application to solid tumors confronts enormous challenges. HER2-CAR-T therapy may serve as a promising treatment option for breast cancer patients who have failed anti-HER2 therapy. Here we reported the Phase I clinical trials results of C406 in HER2-positive recurrent or refractory breast cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe Phase I was a dose-escalation study to evaluate the safety, efficacy and pharmacokinetics of C406 in breast cancer patients. Patients received a single dose of 3\u0026times;107cells/kg (Cohort 1) or 1\u0026times;108cells/kg (Cohort 2) following bridging and lymphodepleting chemotherapy. The primary endpoint was to evaluate the incidence of treatment emergent adverse events. The secondary endpoint was to evaluate the efficacy and pharmacokinetics of C406. The study also explored diversified bridging therapy and conditioning chemotherapy for lymphodepletion.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBy cut-off date of 2025 July 1st, totally 8 patients were enrolled, 6 patients received C406 at a dose of 3\u0026times;10⁷ cells/kg, and the remaining 2 patients were administered 1\u0026times;10⁸ cells/kg. Results showed that C406 was well tolerated and demonstrated a favorable toxicity profile. No case of grade 3 or higher cytokine release syndrome. No case of grade 3 or higher CAR-T related adverse events. Grade 3 or higher adverse events were hematological disorders by lymphodepletion therapy. DCR (disease control rate) was 75% and mPFS (median progression-free survival) was 9 weeks.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn patients with HER2-positive relapsed or refractory breast cancer, C406 has demonstrated a tolerable safety profile, providing a rationale for subsequent research; however, its efficacy requires further confirmation.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003eTrial No.MR-37-23-019847. Registered 17 January 2025, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.medicalresearch.org.cn\u003c/span\u003e\u003cspan address=\"https://www.medicalresearch.org.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e","manuscriptTitle":"Safety and Efficacy of an autologous HER2-Targeting CAR-T therapy in Patients with HER2-positive recurrent or refractory Breast Cancer: An analysis of a Phase I clinical study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 14:47:32","doi":"10.21203/rs.3.rs-8829992/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":"7bc59736-0feb-4a27-8dc4-62323a27a8ba","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T15:01:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 14:47:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8829992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8829992","identity":"rs-8829992","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}