An Exploratory Clinical Trial of Autologous Humanized Anti-cluster of Differentiation Antigen 19/20(CD19/CD20) Dual Specific CAR-T Cells Injection

This is a single-arm, open-label, dose escalation, phase I study, aiming to evaluate the safety and efficacy of Autologous Humanized Anti-CD19 and Anti-CD20 Dual Specific Chimeric Antigen Receptor (CAR) T-cells in patient with relapsed or refractory diffuse B cell lymphoma.

CD19 CAR-T cell therapy has made breakthroughs in the treatment of B cell lymphoma and leukemia, but 30% of patients still have antigen escape, which may be related to variants in tumor cells and the expansion of CD19-negative tumor cells after treatment with CD19 CAR-T cells. CD19/CD20 bispecific CAR-T cell targeting multiple antigens can attack tumor cells while overcoming tumor antigen escape caused by a single target, maximizing efficacy and duration of treatment, and can also solve the problem of uneven distribution or low expression of single target on the tumor surface.

Eligibility Criteria:
Inclusion Criteria:

1. The subject or her/his legally guardian(s) must sign the informed consent form approved by the Institutional Ethics Committee (IEC) prior to any screening procedures;
2. Subjects aged 18 years or older with relapsed or refractory DLBCL (including primary mediastinal large B-cell lymphoma and transformed follicular lymphoma), of which refractory is defined as:

   * Have no response to the recent treatment including:

     * The best response to the treatment regimen is progressive disease (PD) ,or;
     * stable disease(SD) which maintained less than 6 months after the last treatment, or;
   * not suitable for autologous hematopoietic stem cell transplantation (ASCT), or ASCT refractory, including:

     * progressive disease after ASCT or relapse within 12 months (relapse must be confirmed by biopsy), or;
     * If remedial treatment is given after ASCT, the subject must have no response or relapse after the last treatment.
3. Subjects who have previously received ≥2 lines treatment, and at least including:

   * Anti-CD20 monoclonal antibody(rituximab), unless the CD20 negative;
   * A chemotherapy regimen containing anthracyclines;
   * The DLBCL patients who transformed from follicular lymphoma must have previously received chemotherapy for follicular lymphoma and have developed chemotherapy-refractory diseases after transform to DLBCL.
4. Confirmation for either CD19 or CD20 positivity using immunohistochemistry or flow cytometry(accepting the previous results from the a third-level grade A hospitals before the collection of peripheral blood mononuclear cells or peripheral blood. For CD20 positive only, the investigator needs to determine whether the treatment benefit);
5. According to the preliminary assessment of Hodgkin's lymphoma and non-Hodgkin's lymphoma, staging and response assessment recommendations (2014 version), there is at least one measurable lesion at baseline;
6. If the subject has received a single target in the past, such as CD19-CAR cell therapy, it must be confirmed that the disease has progressed or relapsed after treatment and is at least 1 month from the planned single collection period
7. Life expectancy ≥12 weeks;
8. Eastern Cooperative Oncology Group (ECOG) performance status that is either 0 or 1 at screening;
9. Adequate organ function:

   * Renal function defined as:

     * A serum creatinine of ≤1.5 × Upper Limit of Normal(ULN), or;
     * Estimated Glomerular Filtration Rate (eGFR) ≥60 ml/min/1.73m2；\[eGFR=186×（age）-0.203×SCr-1.154（mg/dl）， female ×0.742 on the basis of the calculation results\]；
   * Liver function defined as:

     * Alanine aminotransferase (ALT)≤ 5 × Upper Limit of Normal(ULN) for age, and;
     * Total bilirubin ≤ 2.0 mg/dl with the exception of patients with Gilbert-Meulengracht syndrome; patients with Gilbert-Meulengracht syndrome may be included if their total bilirubin is ≤ 3.0 × ULN and direct bilirubin ≤ 1.5 × ULN.
   * Must have a minimum level of pulmonary reserve defined as ≤ Grade 1 dyspnea and blood oxygen saturation \> 91% on room air;
10. Hemodynamically stable and Left Ventricle Ejection Fraction (LVEF) ≥ 45% confirmed by echocardiogram or Multigated Radionuclide Angiography (MUGA);
11. Adequate bone marrow reserve without transfusions defined as:

    * Absolute neutrophil count (ANC) \>1×10\^9 /L；
    * Absolute lymphocyte count (ALC) ≥0.3×10\^9 /L；
    * Platelets ≥50×10\^9 /L;
    * Hemoglobin \> 8.0 g/dl;
12. Subjects who use the following drugs should meet the following criteria:

    * Steroids: Therapeutic doses of steroids must be stopped 2 weeks prior to A-02 infusion. However, the following physiological replacement doses of steroids are allowed: \< 6 - 12 mg/m2/day hydrocortisone or equivalent;
    * Immunosuppression: Any immunosuppressive medication must be stopped ≥ 4 weeks prior to sign the informed consent form;
    * Anti-proliferative therapy other than pretreatment chemotherapy within 2 weeks of A-02 infusion;
    * CD20 antibody-related treatment must be discontinued within 4 weeks of A-02 infusion or 5 half-lives (whichever is longer);
    * CNS disease prophylaxis must be stopped \> 1 week prior to A-02 infusion (e.g. intrathecal methotrexate);
13. The investigator judged that the subject recovered from the toxicity of the previous anti-tumor treatment to grade 1 or below (except for special grade 2 or below toxicity that cannot be recovered in a short period of time, such as hair loss), suitable for pretreatment. Chemotherapy and treatment of CAR-T cells;
14. Women of child-bearing potential and all male subjects must agree to use highly effective methods of contraception for at least 12 months following A-02 infusion and until CAR-T cells are no longer present by Polymerase chain reaction(PCR) on two consecutive tests.

Exclusion Criteria:

Subjects who meet any of the following criteria will not be enrolled:

1. Subjects who have received any CD19/CD20 dual-target cell therapy products before signing the informed consent form;
2. Subjects with detectable cerebrospinal fluid malignant cells or brain metastases, or with a history of central nervous system (CNS) lymphoma or primary CNS lymphoma;
3. Subjects with current or previous history of central nervous system disease, such as seizures, cerebral ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune disease involving the central nervous system;
4. Subjects who have previously received allogeneic hematopoietic stem cell transplantation (HSCT); or suitable and consenting to Autologous hematopoietic stem cell transplantation (ASCT);
5. Chemotherapy other than lymphodepleting chemotherapy within 2 weeks of A-02 infusion;
6. Investigational medicinal product within the last 30 days prior to sign the informed consent form;
7. Subjects with active hepatitis B（defined as hepatitis B surface antigen positive, or hepatitis B core antibody positive with hepatitis B virus DNA detection value \> 1000 copies/ml）or hepatitis C(HCV RNA positive);
8. Subjects positive for HIV antibody or treponema pallidum antibody;
9. Uncontrolled acute life threatening bacterial, viral or fungal infection (e.g. blood culture positive ≤ 72 hours prior to A-02 infusion);
10. Unstable angina and/or myocardial infarction within 6 months prior to sign the informed consent form;
11. Previous or concurrent malignancy with the following exceptions:

    * Adequately treated basal cell or squamous cell carcinoma (adequate wound healing is required prior to sign the informed consent form);
    * In situ carcinoma of the cervix or breast, treated curatively and without evidence of recurrence for at least 3 years prior to sign the informed consent form;
    * A primary malignancy which has been completely resected and in complete remission for ≥ 5 years;
12. Pregnant or nursing women (women of childbearing age were tested positive for pregnancy during screening period);
13. Subjects with active neurological auto immune or inflammatory disorders (e.g. Guillain Barre Syndrome, Amyotrophic Lateral Sclerosis);
14. Other conditions that the investigator thinks he/she should not be included in this clinical trial, such as poor compliance.