Sequential Infusion of CD146-Targeted and HER2-Targeted CAR T Cells in Patients With Advanced Sarcomas

A Phase 1/2 Open-Label, Multicenter Trial of Sequential Infusion of CD146-Targeted and HER2-Targeted CAR T Cells in Patients With Advanced Sarcomas

This is an open-label, non-randomized, multicenter Phase 1/2 trial evaluating a dual CAR-T cell therapy targeting CD146 and HER2 in patients with advanced sarcoma. Participants will receive lymphodepleting chemotherapy with cyclophosphamide and fludarabine, followed by sequential infusion of autologous CD146-specific and HER2-specific CAR-T cells. The Phase 1 portion will employ a dose-escalation design to assess safety and determine the recommended Phase 2 dose, while the Phase 2 expansion will evaluate preliminary efficacy (tumor response and survival outcomes). Approximately 40 patients (children and adults) with relapsed or refractory sarcomas will be enrolled across multiple centers. All participants will be followed for up to 36 months to monitor dose-limiting toxicities, objective response rates, progression-free survival, overall survival, and long-term safety.

Advanced sarcomas have poor prognoses with existing treatments, and novel immunotherapies are needed. HER2 is expressed in subsets of sarcomas (e.g. osteosarcoma), and a Phase 1 trial of HER2-targeted CAR T cells in sarcoma demonstrated that this approach is tolerable and can induce objective responses. However, in that study the median progression-free survival was only ~2.4 months and 1-year progression-free survival rate was ~21%, highlighting the need for enhanced efficacy. CD146 (MCAM) is a cell adhesion molecule highly expressed in many solid tumors (including sarcomas) and on tumor vasculature. Overexpression of CD146 is implicated in tumor progression and metastasis, so immunotherapy against CD146 is a promising strategy to inhibit metastatic spread. Dual targeting of HER2 and CD146 may therefore broaden anti-tumor activity by addressing tumor heterogeneity and the tumor microenvironment, potentially improving response durability.

Study Design: This trial consists of two phases. The Phase 1 portion is a dose-escalation (3+3 design) of sequential CD146 CAR-T and HER2 CAR-T cell infusions to evaluate safety, identify dose-limiting toxicities (DLTs), and determine the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D) for each CAR-T product when given sequentially. In Phase 1, small cohorts of patients will receive escalating doses of the CAR-T cells; safety data will be reviewed by a monitoring committee before dose progression. Once the RP2D is established, the trial will expand into Phase 2. The Phase 2 portion is a dose-expansion at the RP2D, treating a larger cohort of patients to evaluate preliminary efficacy and further characterize safety at that dose.

All enrolled participants will undergo lymphodepletion with cyclophosphamide (e.g. 500-1000 mg/m²/day for 2 days) and fludarabine (e.g. 30 mg/m²/day for 3-5 days) prior to CAR-T cell infusion, per standard CAR-T therapy protocols. Following lymphodepletion, patients will receive sequential infusions of two investigational CAR-T cell products: first an infusion of CD146-targeted CAR T cells, then an infusion of HER2-targeted CAR T cells. (The interval between the two CAR-T infusions may be on the same day or consecutive days, as defined in the protocol, to allow for safe administration of both products in sequence.) This sequential dual-CAR-T approach is intended to attack the tumor on two fronts: targeting CD146-expressing elements of the tumor and vasculature, as well as HER2-expressing tumor cells.

Endpoints and Follow-Up: Patients will be closely monitored for acute toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), which are known risks with CAR-T therapies. The primary endpoints focus on safety: incidence of DLTs within the first cycle (e.g. 28 days post-CAR T infusion) and the establishment of an MTD/RP2D. Secondary endpoints include measures of anti-tumor efficacy: objective response rate (ORR) per RECIST 1.1 or appropriate sarcoma response criteria, progression-free survival (PFS), overall survival (OS), and duration of response. Immune correlates (such as CAR T cell expansion and persistence, cytokine levels, and tumor biopsy analyses) will be explored to understand the mechanism of response or resistance.

Participants will be followed for up to 36 months after CAR-T administration. This long-term follow-up allows assessment of durable responses, late relapses, and delayed adverse events. Safety follow-up will include periodic physical exams, laboratory tests, and imaging. Responses will be assessed at regular intervals (e.g. every 8-12 weeks) using imaging (MRI/CT) to evaluate tumor size. Long-term monitoring will also watch for potential delayed toxicities such as late-onset cytopenias or secondary malignancies. The trial's multi-center design will facilitate the enrollment of a diverse patient population and ensure robust collection of safety and efficacy data. The ultimate goal is to determine whether the sequential infusion of CD146 and HER2 directed CAR T-cells can be safely administered and show sufficient anti-sarcoma activity to warrant further clinical development. Essen Biotech, as the sponsor, will oversee regulatory compliance, data collection, and analysis in alignment with GCP and ClinicalTrials.gov reporting requirements.

Inclusion Criteria:

• Expected survival time ≥3 months;
• Diagnosis: Histologically or cytologically confirmed sarcoma (soft tissue or bone sarcoma), that is metastatic, locally advanced, or refractory to standard therapy. This may include osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, or other high-grade sarcomas. Patients must have evidence of measurable disease as per RECIST or applicable criteria.
• Prior Treatment: Patients should have received and progressed on or not be candidates for standard first-line treatments. There is no limit on number of prior lines of therapy, but at least one prior systemic therapy for sarcoma is typically required (unless no standard therapy exists for the subtype). A minimum wash-out period (e.g. 2 weeks) from previous treatments (chemotherapy, radiation, or other immunotherapy) is required before lymphodepletion.
• Age and Performance Status: Participants age 12 years and older (both adolescent and adult patients are eligible; for minors, legal guardian consent is required). Upper age limit of ~75 years, or as determined by medical fitness. ECOG performance status 0-1 (or Karnofsky ≥70% for pediatric patients), indicating subjects are ambulatory and able to perform light work.
• Organ Function: Adequate organ function to undergo cytotoxic chemotherapy and cell transfer therapy, including: cardiac ejection fraction ≥50%; baseline oxygen saturation >92% on room air; adequate bone marrow reserves (absolute neutrophil count ≥1.0×10^9/L, platelets ≥75×10^9/L, hemoglobin ≥8 g/dL), hepatic function (e.g. bilirubin ≤1.5× ULN, AST/ALT ≤2.5× ULN), and renal function (e.g. creatinine clearance ≥50 mL/min or age-appropriate normal).
• Tumor Antigen Expression: Expression of CD146 and/or HER2 in the tumor is recommended (as assessed by immunohistochemistry or flow cytometry on a tumor sample). Note: At least one of the target antigens (CD146 or HER2) should be present on the tumor; if feasible, patients should have tumor tissue tested for these markers. (In cases where testing is unavailable, enrollment may proceed based on histology known to often express these targets, per investigator judgment.)
• Consent: Ability to understand and provide written informed consent (or assent for minors with consent of legal guardian). Patients (or guardians) must be willing to comply with trial procedures and follow-up.

Exclusion Criteria:

• Recent Therapies: Prior treatment with any CAR-T cell therapy or other gene-modified T-cell therapy is excluded. Also exclude patients who received any investigational drug, immunotherapy (e.g. checkpoint inhibitor), or major surgery within a certain interval (e.g. 4 weeks) prior to enrollment. Concurrent enrollment in another interventional clinical trial is not allowed.
• Recent Therapies: Prior treatment with any CAR-T cell therapy or other gene-modified T-cell Infections: Active uncontrolled infection, including active hepatitis B or C infection, or HIV infection with uncontrolled viral load. Patients must not have evidence of active tuberculosis or other severe infections. All patients will be screened for HBV, HCV, and HIV at baseline.
• Immunosuppression: Use of systemic immunosuppressive medications (such as chronic corticosteroids at >10 mg prednisone daily or equivalent) within 7 days prior to leukapheresis. (Physiologic replacement doses of steroids are permitted.) Patients with a history of allogeneic stem cell transplant or solid organ transplant are excluded (due to the need for immunosuppression and risk of graft-versus-host or graft rejection).
• Medical Comorbidities: Any significant uncontrolled medical condition that would, in the investigator's judgment, make the patient an unsuitable candidate for CAR-T therapy. For example: active autoimmune diseases requiring immunosuppression, clinically significant heart failure (NYHA class III-IV), unstable angina or myocardial infarction within 6 months, severe chronic respiratory disease requiring supplemental oxygen, or psychiatric conditions that would interfere with study participation and follow-up.
• Pregnancy/Breastfeeding: Women who are pregnant or breastfeeding are excluded due to unknown risks of the treatment to a fetus or infant. Female participants of childbearing potential and male participants with partners of childbearing potential must agree to use effective contraception during the study and for a suitable period after CAR-T infusion (e.g. 1 year), given the potential for sustained CAR T-cell activity. A negative serum pregnancy test is required for females of childbearing potential before starting lymphodepletion.