A Study to Evaluate the Safety, Tolerability, PK, PD, and Preliminary Efficacy of CS01 in Patients With Locally Advanced or Metastatic Solid Tumors

Overall Design

• Trial Overview This trial is an Investigator-Initiated Trial (IIT) clinical study evaluating CS01 injection as monotherapy for locally advanced or metastatic solid tumors. A Safety and Efficacy Data Assessment Committee (SMC), jointly formed by the Sponsor and the investigators, will evaluate data from all treated subjects in the dose escalation phase to screen and determine the clinically recommended dose. The trial consists of two phases: Dose Escalation (Phase I) and Dose Expansion (Phase II). The first phase involves monotherapy dose escalation of CS01, and the second phase involves monotherapy dose expansion of CS01.

Phase I: Dose Escalation The Dose Escalation Phase will first be conducted in subjects with locally advanced or metastatic solid tumors. The monotherapy dose-escalation study of CS01 is designed with four planned dose groups: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg. It aims to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary antitumor activity of CS01 monotherapy and to determine the maximum tolerated dose (MTD) (or minimum effective dose, MED) and the recommended Phase II dose (RP2D) for monotherapy.

Phase II: Dose Expansion The Dose Expansion Study will be conducted in potentially responsive populations identified during the Dose Escalation Phase (Phase I). Based on earlier findings, this phase aims to further validate the safety, efficacy, PK, PD, and biomarker profiles of CS01 injection as monotherapy at the RP2D in specific subjects with locally advanced or metastatic solid tumors.

• Detailed Design Phase I: Dose Escalation This phase will utilize an accelerated titration design (for the first dose group only) and a "3+3" design. It will enroll approximately 10 patients with locally advanced or metastatic solid tumors who have failed standard treatment, are intolerant, or lack effective treatment options. The four planned dose groups are: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg. All patients will receive CS01 injection via intravenous infusion once every two weeks (Q2W), with every 4 weeks constituting one treatment cycle. Dose-Limiting Toxicity (DLT) assessments will be conducted at the end of the 4th week for each dose group. Antitumor efficacy assessments (based on RECIST v1.1 criteria) will be performed every 8 weeks. Safety data will be collected throughout the treatment period, and blood samples for PK, PD, immunogenicity, and biomarker analyses will also be collected.

Symptoms and signs of Cytokine Release Syndrome (CRS) will be closely monitored. CRS is defined as a supraphysiological response resulting from the activation or engagement of endogenous or infused T cells and/or other immune effector cells following the administration of any immunotherapeutic agent. The American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading for CRS is as follows:

Grade Symptoms

• Feverª with or without constitutional symptoms (e.g., myalgia, arthralgia, malaise). ● No hypotension. ● No hypoxia.
• Feverª accompanied by at least one of the following: - Hypotension not requiring vasopressors. - Hypoxia requiring low-flow oxygen therapy via nasal cannula or simple facemaskb.
• Feverª accompanied by at least one of the following: - Hypotension requiring one vasopressor (with or without vasopressin). - Hypoxia requiring high-flow oxygen via nasal cannula, facemask, non-rebreather mask, or Venturi mask.
• Feverª accompanied by at least one of the following: - Hypotension requiring multiple vasopressors (excluding vasopressin). - Hypoxia requiring positive pressure ventilation (e.g., CPAP, BiPAP, intubation, and mechanical ventilation).

Death due to CRS, where other causes are not the primary factor leading to this outcome.

ASTCT=American Society for Transplantation and Cellular Therapy; BiPAP=Bilevel Positive Airway Pressure; CPAP=Continuous Positive Airway Pressure; CRS=Cytokine Release Syndrome.

• Fever is defined as temperature ≥38°C not attributable to any other cause. In subjects receiving antipyretics, anticytokine therapy, or corticosteroids after CRS onset, fever is no longer considered when determining the subsequent CRS severity grade. In such cases, the CRS grade is driven by the presence of hypotension and/or hypoxia.
• Low-flow is defined as oxygen delivered at ≤6 L/min; high-flow is defined as oxygen delivered at >6 L/min.

All subjects in this phase will undergo DLT evaluation during the DLT observation period (the first 28 days after the first dose of CS01). After completing the DLT evaluation, subjects with favorable safety profiles and clinical benefit may continue treatment at the original dose until endpoint events such as disease progression, death, intolerable toxicity, receipt of other antitumor therapy, withdrawal of informed consent, or study termination occur. Escalation to the next dose level may only commence after the current dose group meets the predefined DLT criteria for escalation.

Based on the acquired clinical data, combined with preclinical data and efficacy data observed in human studies of similar drugs domestically and internationally, the SMC will decide whether to extend or prematurely terminate dose escalation and adjust the dosing regimen until the MTD (or MED) or the clinically recommended dose is determined.

After the clinically recommended dose is defined, subjects currently receiving treatment may discuss with the investigator the option to adjust their dose to the clinically recommended dose.

Phase II: Dose Expansion Based on the safety, tolerability, preliminary efficacy, PK, and PD results from the Phase I dose escalation phase, the Sponsor and investigators will discuss and decide on the target tumor types for the Phase II dose expansion.

This phase will enroll tumor types in which one or more instances of partial response (PR), tumor-shrinking stable disease (SD), or biomarkers suggesting potential clinical benefit were observed during Phase I treatment. The CS01 dose will be the safety-assessed dose.

During treatment, subjects with favorable safety profiles and clinical benefit will continue treatment until endpoint events occur.

• Study Periods The study is divided into Screening, Treatment, and Follow-up periods. The Screening Period is defined as the time from the subject signing the ICF until before the first dose of the investigational product, with a maximum allowed duration of 28 days. One re-test is permitted per subject during screening. One re-screening is also allowed.

The Treatment Period is defined from the first dose of the study drug until the end of the last dose administration. The study drug is administered once every two weeks (Q2W), with each 4-week period constituting a treatment cycle.

• Phase I: Subjects undergoing DLT evaluation in the first cycle who, in the investigator's judgment, have a favorable safety profile and clinical benefit, may continue treatment at the original dose until endpoint events occur.
• Phase II: All subjects receive the validated dose until endpoint events occur. Safety examinations and tumor imaging assessments must be completed according to the trial schedule during the treatment period.

The Follow-up Period includes the End of Treatment (EOT) visit, safety follow-up, and survival follow-up.

• EOT Visit: If the investigator decides to discontinue the subject's study drug treatment, the treatment period is considered ended. All subjects (except those lost to follow-up, deceased, or who withdrew consent) should undergo an EOT visit within 7 days after the decision to stop treatment.
• Safety Follow-up: Occurs 28 ± 5 days after the last dose to collect concomitant medications/therapies and adverse events (AEs) after the last dose.
• Survival Follow-up: Follow-up for survival status occurs every 2 months after the last dose until death from any cause, loss to follow-up, or study end. If a subject discontinues treatment for reasons other than disease progression (excluding loss to follow-up, death, or withdrawal of consent) and has not started new antitumor therapy, efficacy assessments will continue during survival follow-up every 8 weeks (±7 days) until disease progression, death, withdrawal of consent, loss to follow-up, initiation of other new antitumor therapy, study end, or Sponsor termination, whichever occurs first.

• Discontinuation of Study Treatment

The investigator may decide to discontinue a subject's treatment based on emerging AEs. Subjects must discontinue study treatment if they meet ANY of the following criteria:

• Subject requests discontinuation of study drug/ or withdraws consent.
• Efficacy evaluation meets disease progression criteria (if the investigator believes continuation is beneficial, discussion with the Sponsor is required).
• Pregnancy occurs during the study.
• Any clinical AE, laboratory abnormality, or other medical condition arises where the risk of continuing treatment outweighs the benefit.
• Subject receives other (non-protocol) antitumor therapy.
• If study drug CS01 injection is suspended for more than 8 weeks, the subject should discontinue treatment.
• Subject is unable to tolerate the study drug.
• Other reasons deemed by the investigator to preclude continued study participation.

• Study Withdrawal Subjects may choose to withdraw from the clinical trial at any time. Reasons include: unwillingness/inability to continue (withdrawal of consent, not due to AE), loss to follow-up, death, Sponsor termination of the study.

• Study End The study end is defined as 12 months after the first dose of the last enrolled subject or the completion of the last visit for all subjects, whichever comes first. Post-study, if the investigator judges continued benefit, subjects may continue the study drug after providing fully informed consent until meeting treatment discontinuation criteria. SAEs, efficacy, and survival information should be collected per protocol during treatment and for 28 days (extendable to 28+5 days) after the last dose.

• Safety Assessment Safety and tolerability throughout the study will be assessed using CTCAE v5.0 for grading AE severity. Clinical lab tests, ECOG scores, physical exams, vital signs, ECG results, and AEs will be recorded for all subjects during the study, and the relationship of AEs to the study drug will be determined. Safety will be continuously evaluated.

• Efficacy Assessment Efficacy will be assessed per RECIST v1.1 every 2 cycles (8 weeks) until disease progression, receipt of other antitumor therapy, death from any cause, or study end (whichever occurs first). For initial disease progression, pseudo-progression must be ruled out. If clinically stable, the investigator may decide to continue CS01 treatment. Evaluation endpoints include Objective Response Rate (ORR), Clinical Benefit Rate (CBR), Disease Control Rate (DCR), Duration of Response (DoR), Time to Response (TTR), Progression-Free Survival (PFS), and Overall Survival (OS).

• Starting Dose Selection Non-GLP toxicology studies indicated a No Observed Adverse Effect Level (NOAEL) of 100 mg/kg. GLP toxicology studies indicated a Highest Non-Severely Toxic Dose (HNSTD) of 100 mg/kg. CS01 is a fully human monoclonal antibody. The intended route of administration for Phase I is intravenous injection. According to the "Guidance for Estimating the Maximum Recommended Starting Dose for Initial Clinical Trials of Drugs in Adult Healthy Volunteers," the formula for converting equivalent doses across species is: Human Equivalent Dose (HED in mg/kg) = Animal NOAEL (mg/kg). The Maximum Recommended Starting Dose (MRSD) is determined by dividing the HED by a safety factor. Considering this is a first-in-human study, a safety factor of 1000 is applied for full protection, i.e., MRSD = HED / 1000 = 0.1 mg/kg. As CS01 is intended for locally advanced or metastatic cancer patients, exposing many patients to doses unlikely to have clinical activity is undesirable. In vivo studies showed tumor growth inhibition (60% inhibition rate) in a colorectal adenocarcinoma model at 0.2 mg/kg. Therefore, 0.2 mg/kg is considered the minimal pharmacological active dose (PAD). The proposed human starting dose of 0.1 mg/kg is only half of the minimal PAD, offering both a sufficient safety window and the potential to observe clinical efficacy.

• Dose Escalation Criteria The dose escalation phase presets four escalating dose levels for CS01: 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, administered Q2W. This phase will use accelerated titration (for the first group only) and a "3+3" design.

To protect subject safety and avoid exposing many subjects to potentially low-benefit doses, accelerated titration will be used for the first dose group. One subject will be enrolled to receive CS01 0.03 mg/kg monotherapy. If this subject experiences no study drug-related Grade ≥2 AEs within 2 weeks after the first dose in the first cycle, escalation to the next group (0.1 mg/kg, using a "3+3" design) occurs. For subject benefit, this initial subject may continue treatment after the first cycle (continuation phase) until progression, death, intolerable toxicity, or consent withdrawal. If any study drug-related Grade ≥2 AE occurs (excluding specified exceptions), accelerated titration stops, three more subjects are enrolled at 0.03 mg/kg, switching to a "3+3" design for the next group (0.1 mg/kg).

Exceptions for triggering a stop to accelerated titration at the 0.03 mg/kg group upon occurrence of a related Grade ≥2 AE include:

• Isolated Grade ≥2 lab abnormalities unrelated to other clinically significant symptoms.

• Grade ≥2 amylase/lipase elevation without clinical symptoms of pancreatitis.

• Grade ≥2 nausea, vomiting, constipation, mucositis, fatigue, electrolyte abnormalities, or diarrhea resolving within 72 hours with supportive care.

Except for the accelerated titration group, the "3+3" design principle applies to all other dose groups.

• "3+3" Dose Escalation Design:

• Three subjects are initially enrolled per dose level.

• If 0 of 3 experience DLT, escalate to the next level. Current level subjects may continue treatment.

• If ≥2 of 3 experience DLT, stop escalation. SMC decides whether to test an intermediate dose or revert to the previous level for RP2D/MTD determination.

• If 1 of 3 experiences DLT, expand the cohort to 6 subjects. If ≥1 of the additional 3 experiences DLT, the MTD is exceeded; SMC decides on an intermediate dose or reversion. If 0 of the additional 3 experience DLT, escalate to the next level.

• When reverting, if the previous level has 6 evaluable subjects and only 0 or 1 DLT occurred, that level is the MTD. If it only has 3 subjects, expand to 6; if 0 or 1 DLT occurs, it is the MTD; if ≥2 DLTs occur, de-escalate further until MTD is determined.

• For safety, the first two subjects in a cohort should be enrolled at least 7 days apart (adjustable based on emerging data). Escalation occurs only after the last subject in a dose level completes the DLT observation period. Concurrent testing of multiple dose levels is prohibited.

• If the highest planned dose is reached without defining MTD, the SMC will decide on exploring higher doses or determining the RP2D based on acquired safety, tolerability, PK/PD data, other clinical data for CS01, and data from similar drugs.

• If a subject receives <90% of the planned dose during the DLT period for reasons unrelated to DLT, a replacement subject must be enrolled.

• The SMC may decide to add new dosing frequency groups based on acquired safety, tolerability, and PK/PD data.

• Dose-Limiting Toxicity (DLT)

According to CTCAE v5.0, DLT refers to toxicities occurring within the first treatment cycle (28 days) after the first dose, deemed related to CS01 by the Sponsor and investigator, and unrelated to the underlying cancer, disease progression, concomitant illness, or medications, including:

• Hematological Toxicity:

a. Grade 4 neutropenia lasting ≥7 days. b. Grade 3 neutropenia with fever (ANC <1.0 x 10⁹/L, with a single temperature >38.3°C or persistent temperature ≥38°C for >1 hour).

c. Grade 4 thrombocytopenia, or Grade 3 thrombocytopenia with bleeding or lasting >7 days.

d. Grade 4 anemia (life-threatening).

• Non-hematological Toxicity:

• Any Grade 4 immune-related adverse event (irAE).
• Grade 3 irAEs not improving to ≤ Grade 2 within 3 days with optimal supportive care (excluding Grade 3 thyroid/adrenal/pituitary insufficiency and Grade 3 flare reaction).
• Other Grade 3 or 4 non-hematological toxicities (excluding: Grade 3 electrolyte abnormalities; Grade 3 hypertension controllable within 7 days to baseline/≤G1; Grade 3 fatigue; Grade 3 vomiting/diarrhea; transient [≤6 hours] Grade 3 infusion reactions or fever), provided they:

• Require medical intervention.
• Involve clinically significant Grade 3+ lab abnormalities lasting >1 week or leading to hospitalization (excluding non-medical reasons).
• Grade 5 toxicity (e.g., death).
• Other toxicities of any grade necessitating premature discontinuation as agreed by the investigator and Sponsor.

Subjects who withdraw before completing the DLT observation period for reasons unrelated to DLT will be replaced and not included in the final DLT analysis for the dose group/overall; an additional subject must be enrolled at the same dose level as a replacement.

• Maximum Tolerated Dose (MTD) / Maximum Effect Dose (MED)

• MTD Definition: The MTD is defined as the highest dose at which DLTs occur in ≤33% of subjects during dose escalation. If the incidence is ≥33% at the starting dose level, escalation stops, and the SMC deliberates on adjusting the starting dose.

• MED Definition: The MED is defined as the dose level producing the maximum pharmacodynamic effect of CS01 during dose escalation.

Inclusion criteria

Subjects must meet ALL of the following criteria for enrollment:

• Age: 18 years or older (inclusive), male or female.
• ECOG Performance Status: 0 to 1.
• Disease Characteristics:

• Dose Escalation Phase: Subjects with histologically or cytologically confirmed unresectable or metastatic advanced solid tumors, who have failed standard treatment or are intolerant (disease progression, or intolerance to chemotherapy, targeted therapy, etc.), or who lack effective treatment options.
• Dose Expansion Phase: Tumor types for which ≥1 instance of partial response (PR), or tumor-shrinking stable disease (SD), or biomarkers suggesting potential clinical benefit are observed during the dose escalation phase, as decided by the Safety and Efficacy Data Assessment Committee.
• The subject must have at least one measurable lesion as defined by RECIST v1.1.
• Agreement to provide tumor tissue specimens (optional).

o Note: For the dose escalation phase, tumor tissue samples equivalent to 4 FFPE unstained slides, or an equivalent amount of FFPE archived (within 2 years) or newly obtained tissue blocks are required. For the dose expansion phase, at least 4 and up to 9 FFPE unstained slides, or an equivalent amount of FFPE archived (within 2 years) or newly obtained tissue blocks are required. The FFPE biopsy must be from a surgical resection or core needle biopsy.

• Life expectancy ≥ 3 months.
• Recovery from toxicities of previous anti-tumor therapy to ≤ Grade 1 per CTCAE v5.0 (except for toxicities judged by the investigator to pose no safety risk, alopecia, or Grade 2 peripheral neuropathy judged irreversible). Immune-related adverse reactions must have completely resolved to baseline or Grade 1.
• Normal function of major organs, meeting the following laboratory criteria:

• AST and ALT ≤ 2.5 × ULN; for subjects with liver metastases, AST and ALT ≤ 5 × ULN.
• Total Bilirubin (TBil) ≤ 1.5 × ULN; for subjects with liver metastases or confirmed/suspected Gilbert's syndrome, TBil ≤ 3 × ULN.
• Absolute Neutrophil Count (ANC) ≥ 1.5 × 10^9/L.
• Platelets ≥ 100 × 10^9/L, with no transfusion within 28 days prior to the start of the study.
• Hemoglobin ≥ 9.0 g/dL, with no transfusion within 14 days prior to the start of the study.
• Serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 50 mL/min (calculated by Cockcroft-Gault formula).
• International Normalized Ratio (INR) and Activated Partial Thromboplastin Time (APTT) ≤ 1.5 × ULN.
• For female subjects of childbearing potential, a negative serum pregnancy test is required prior to the first dose of the investigational product.
• Male or female subjects of childbearing potential (who have not undergone sterilization surgery or are not postmenopausal) must use highly effective contraception methods (such as oral contraceptives, intrauterine devices, sexual abstinence, or barrier methods combined with spermicide) during the study and continue for 6 months after the last dose.
• The subject voluntarily agrees to join the study, signs the informed consent form, is able to understand the study procedures and methods, and is willing to strictly comply with the clinical trial protocol to complete the study.

Exclusion criteria

Subjects who meet ANY of the following criteria will be excluded from the study:

• Previous treatment with anti-TNFR agonistic drugs.
• Active autoimmune disease requiring systemic treatment within 12 months prior to the initiation of study treatment. This includes, but is not limited to, myasthenia gravis, myositis, autoimmune hepatitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, antiphospholipid antibody syndrome, granulomatosis with polyangiitis (Wegener's granulomatosis), Guillain-Barré syndrome, multiple sclerosis, vasculitis, or glomerulonephritis. Exceptions are made for the following:

• Subjects with a history of autoimmune-related hypothyroidism who are on thyroid replacement hormone therapy are eligible.
• Subjects with type 1 diabetes mellitus controlled on an insulin regimen are eligible.
• Subjects with eczema, psoriasis, chronic lichen simplex, or vitiligo with dermatologic manifestations only (e.g., subjects with psoriatic arthritis are excluded) may be enrolled if they meet ALL of the following conditions:

i. The subject's rash covers <10% of body surface area. ii. The disease is well controlled at baseline, requiring only low-potency topical corticosteroids.

iii. There has been no acute exacerbation of the underlying condition within the past 12 months, or any acute exacerbation did not require treatment with psoralen plus ultraviolet A (PUVA) therapy, methotrexate, retinoids, biologics, oral calcineurin inhibitors, or high-potency/oral corticosteroids.

• Subjects with a history of two or more primary synchronous or metachronous malignancies are excluded, except for cured carcinoma in situ or basal cell carcinoma. Other malignancies that have been stably treated for over 5 years prior to enrollment are allowed.
• Within 4 weeks prior to the initiation of study treatment, subjects who have received the following treatments or medications:

• Participation in another clinical trial involving an investigational drug (including investigational vaccines) or invasive investigational medical device, or current enrollment in an interventional investigational study.
• Systemic anti-tumor therapy within 2 weeks prior to treatment initiation, or screening occurs within 5 half-lives of the previously administered drug (whichever is shorter).
• Major surgery or significant trauma, or being in the recovery period which, in the investigator's judgment, may affect the trial, or having a planned surgery during the study period.
• Chest radiotherapy or extensive field radiotherapy (defined as irradiation of >50% of pelvic bone marrow or equivalent) within 4 weeks, or palliative radiotherapy within 2 weeks prior to treatment initiation.
• Administration of a live attenuated vaccine within 4 weeks prior to the first dose of study treatment, or expectation of requiring such a vaccine during the study or within 5 months after the last dose of study treatment.
• Systemic treatment with corticosteroids (exceeding a dose equivalent to 10 mg prednisone per day) for more than 7 days, or other immunosuppressive agents, within 2 weeks prior to treatment initiation (use of inhaled or topical steroids, or adrenal replacement therapy at doses >10 mg prednisone equivalent is permitted in the absence of active autoimmune disease).
• Primary central nervous system (CNS) tumors or symptomatic CNS metastases (subjects with leptomeningeal metastases are excluded regardless of symptoms). Exceptions are made for subjects with asymptomatic CNS metastases or symptomatic CNS metastases deemed stable by the investigator, provided they meet ALL the following criteria:

• Clinical stability of neurological symptoms for ≥4 weeks prior to the first dose.
• No evidence of CNS disease progression on contrast-enhanced brain MRI within 4 weeks prior to the first dose.
• Discontinuation of anti-epileptic drugs for ≥2 weeks prior to the first dose.
• Presence of uncontrolled pleural effusion, ascites, or pericardial effusion.
• History of or current active interstitial lung disease.
• Uncontrolled hypertension, defined as systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥100 mmHg despite optimal medical therapy.
• Presence of clinically significant cardiovascular or cerebrovascular diseases, including:

• Heart failure classified as New York Heart Association (NYHA) Class II-IV, congestive heart failure, or second-degree or higher heart block.
• Myocardial infarction, unstable arrhythmia, or unstable angina occurring within the past 3 months.
• Baseline QT interval corrected for heart rate (QTcF) >480 ms, or history of long QT syndrome (Note: Based on the average of three 12-lead ECGs at rest, using Fridericia's formula for correction).
• Cerebral infarction occurring within the past 3 months.
• Percutaneous transluminal coronary angioplasty (PTCA) or coronary artery bypass grafting (CABG) within the past 6 months.
• Major vascular disease (e.g., aortic aneurysm requiring surgical intervention, any arterial thromboembolic event, or Grade 3 or higher venous thromboembolism per CTCAE v5.0) within 6 months prior to the first dose of study drug.
• Thrombolytic therapy within 10 days prior to treatment initiation (except for maintaining venous catheter patency).
• Any active infection requiring treatment, including but not limited to:

• Active hepatitis B (HBsAg positive and HBV-DNA level above the lower limit of normal) or hepatitis C (HCV antibody positive and HCV-RNA quantitative test result above the lower limit of normal).
• Known positive serology for human immunodeficiency virus (HIV).
• Active tuberculosis.
• History of allogeneic stem cell or solid organ transplantation.
• Women who are pregnant or breastfeeding. Pregnancy is defined as the state of a female from conception until termination of the pregnancy, confirmed by a positive laboratory human chorionic gonadotropin (hCG) test within 7 days prior to study initiation.
• Known or suspected inability to comply with the study protocol (e.g., due to alcoholism, drug dependency, or psychological disorders), or subjects who, in the opinion of the investigator, would be at risk by participating in this study; or the presence of any condition that, in the investigator's opinion, makes participation in this study not the best choice for the subject (e.g., compromises their health) or could affect, limit, or confound the protocol-specified assessments.