3.1 Production of CD19 CAR-T cells
CAR-T cells targeting CD19 utilized CAR lentivirus carrying a FMC63-derived CD19-specific single-chain variable fragment (scFv), a 4-1BB co-stimulatory domain that can improve the expansion, persistence, and antitumor effect of CAR-T cells[9, 10], and a CD3ζ signaling domain (Fig. 1A). The T cells were purified and transduced with CAR lentivirus and then cultured for 9–13 days to form CAR-T cells (Fig. 1B). Quality controls of CAR-T cells including viability, potency, copy number, replication-competent lentivirus (RCL), sterility, mycoplasma, and endotoxin were performed. The CAR-T cells used for all the pre-clinical and clinical studies met the defined specifications of Immunochina Pharmaceuticals.
3.2 CAR-T cells proliferated without target cells in NCG mice
Most people believed that without target cells in the mice, the CAR-T cells will disappear in a short time after infusion. To verify this point, we produced CAR-T cells targeting CD19 and transferred the cells to NCG mice through their tail vein. These mice were sacrificed at set times indicated in the Fig. 2. A and organs were collected for the CAR testing by qPCR. The results demonstrated that CAR-T cells still proliferated without target cells in mice. The number of CAR-T cells increased markedly in every tissue especially in spleen, peaking at 2 weeks (Fig. 2. B and Sup. Table 1). The AUC showed that the spleen had the most CAR copies which decreased significantly in the order of blood, lung, kidney, liver, heart, bone marrow. In the Brain, muscle and reproductive organs, low CAR copies were detected (Fig. 2. C and Sup.Table2).
Table 2
copies of CAR gene in blood at different time points(copies/µg DNA)
Time | Female | Male | Total |
Average | SD | Average | SD | 平均值 | SD |
5 min | 45135.5 | 29994.5 | 22675.7 | 6690.3 | 33905.6 | 23002.3 |
30 min | 5223.4 | 1193.7 | 4219.6 | 1909.1 | 4721.5 | 1526.5 |
1 h | 1615.1 | 889.4 | 1746.6 | 1045.9 | 1680.8 | 871.3 |
3 h | 2000.6 | 1853.7 | 1446.2 | 620.9 | 1778.8 | 1380.8 |
1d | 1664.2 | 410.8 | 2999.1 | 962.4 | 2331.7 | 986.2 |
2d | 1787.9 | 1328.3 | 3852.3 | 1586.7 | 2820.1 | 1729.5 |
7d | 1766.7 | 467.2 | 2023.3 | 466.4 | 1869.4 | 428.1 |
14d | 746.2 | 410.1 | 1037.4 | 69.5 | 891.8 | 293.2 |
28d | 711.6 | / | 641.3 | / | 676.5 | 49.7 |
42d | 2132.1 | 522.0 | 18925.6 | 10244.5 | 12208.2 | 11711.1 |
56d | 11348.7 | 15338.6 | 4543.3 | 4385.6 | 7946.0 | 10013.6 |
3.3 CAR-T distribution in the tumor-bearing mice
To evaluate the distribution of CD19 CAR-T cells with target cells, we took advantage of the xenograft mice model with NALM6 tumor infused with CD19 CAR-T cells derived from healthy human donors. At different time points after CAR-T cell infusion, the whole blood and tissues were gathered for CAR-T cell testing (Fig. 3A).
Copies of CAR gene were detected in the peripheral blood of all animals 5 minutes after CAR T cell infusion, which showed decreasing trend and dropped to the lowest level at day 28 after treatment, then increased at day 42, and decreased again at day 56 ( Fig. 3B and Table 2).
Three hours after administration, the CAR-T cells were mainly detected in heart, liver, lung, and the lung content was the highest. CAR copies in spleen were detected in all animals 2 days after administration, followed by a gradual overall increase to a peak at day 56. CAR-T cell detection in most of the other tissues, such as kidney, brain, stomach, duodenum, fat, muscle, colon, testis, epididymis, showed a decreasing trend after 2–14 days, and then gradually increased to the highest level at day 42. According to the results of CAR copy detection in various tissues, the number of CAR copies in most tissues increased at day 42 after administration, indicating that the activation and amplification of CAR-T cells appeared in most tissues (Fig. 3.C and Sup.Table 3 ).
Table 3
Summary of CAR-T parameters in tissues and whole blood(Average)
Type Tissue | Tmax | Cmax | AUC0-1344h |
h | copies/µg DNA | h*copies/µg DNA |
Heart | 1008 | 8290.2 | 3165207.4 |
Liver | 1008 | 30327.7 | 14164836.0 |
Spleen | 1344 | 39484.9 | 22287294.0 |
Lung | 1008 | 39389.1 | 20603898.0 |
Kidney | 1008 | 23099.1 | 9489641.0 |
Brain | 1008 | 3892.4 | 1581518.5 |
Uterus | 1344 | 9159.5 | 1538796.4 |
Testis | 1008 | 26255.8 | 9344230.5 |
Ovary | 1344 | 2157.9 | 362522.2 |
Epididymis | 1008 | 37075.9 | 14626642.0 |
Stomach | 1008 | 43912.5 | 15893940.0 |
Duodenum | 1008 | 15141.3 | 5315737.1 |
Fat | 1008 | 45655.4 | 17008538.0 |
muscle | 1008 | 34794.5 | 11934449.0 |
Colon | 1008 | 8374.4 | 2996989.2 |
Blood | 0.083 | 33905.6 | 6448893.4 |
Bone marrow | 1344 | 8535.2 | 4017144.8 |
The statistical data showed that CAR-T cells were most distributed in the spleen, followed by lung, fat, stomach, epididymis, liver, muscle, kidney, testis, blood, duodenum, bone marrow, heart and other tissues. The organ distribution of CAR-T cells in tumor-bearing mice was influenced by the animal model, drug administration and cell characteristics, and the results were consistent with the distribution of cell products in vivo (Fig. 3.D and Table 3).
3.4 CAR-T distribution and the safety in NHL patients
The clinical trial was launched for r/r NHL patients who showed primary resistance to prior chemotherapies. The study was approved by Beijing Cancer Hospital, which was carried out from May 2018 to Nov 2019. There were 13 patients with diffuse large B cell lymphoma ( DLBCL), follicular lymphoma ( FL ), marginal zone lymphoma (MZL) (Table 4) enrolled in this study.
Table 4
Patient characteristics and treatment
Patient No. | Diagnosis | Prior treatment | Conditioning regimen before T cell infusion | CAR-T dosage |
F0104 | FL/II | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0106 | DLBCL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0107 | FL/II | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0109 | DLBCL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0110 | MZL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0111 | DLBCL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0118 | DLBCL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0119 | DLBCL | ༞2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0121 | DLBCL | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0122 | DLBCL | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0123 | DLBCL | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0125 | DLBCL | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
F0126 | DLBCL | 2nd line | Flu25mg/m2 + CTX250mg/m2 | 1 × 106/kg |
Abbreviations: MZL, marginal zone lymphoma; DLBCL, diffuse large B cell lymphoma; FL, follicular lymphoma; Flu, fludarabine; Cy, cyclophosphamide; CR, complete response; PD, progressive disease |
All patients underwent preconditioning regimen to deplete endogenous lymphocytes before CAR-T cell infusion. After CAR-T cell infusion, patients were followed by monitoring disease response, peripheral CAR-T cell number, and adverse events including CRS, CRES, routine blood analysis, and blood biochemistry (Fig. 4. A). The first response was evaluated on day 28d, the CR rate was 46%(6/13), and 2 of the CR patients maintained remission over 15 months (Fig. 4. B). Expansion of CAR-T cells in peripheral blood was detected in all patients, which reached the peak on day 7–21. The persistence of CAR-T cells was detected up to 510 days (Fig. 4. C and Sup.Table 4). The average peak concentration of CAR-T cells was about 108/L (Fig. 4. D and Sup. Table 5).
About 44% (7/13) of the patients underwent grade 1 CRS and one of them developed grade 2. All the CRS patients recovered after receiving symptomatic treatment. None of the patients experienced CRES. Other side effects were summarized in Table 5. All the adverse events were effectively controlled within two months.
Table 5
List of other adverse events
Adverse events | Adverse events degrade |
I | II | III | IV |
Blood | | | | |
Decrease of leukocyte count | | 3 | 5 | 3 |
Decrease of lymphocyte count | | 3 | | 4 |
Decrease of neutrophil count | | 3 | 1 | 7 |
Decrease of platelet count | 1 | 3 | 1 | |
Increase of C-reactive protein | 2 | | | |
Degree III leukocyte bone marrow suppression | | | 1 | |
Anemia | 2 | 3 | 2 | |
Increase of serum ferritin | 1 | | | |
Liver | | | | |
Elevated Alanine aminotransferase | 1 | | | |
Elevated Aspartate aminotransferase | 1 | | | |
Pain | | | | |
Pain in the jaws | | | | 1 |
Muscular soreness | 1 | | | |
Pain in the left ilium | | 1 | | |
Immune globulin | | | | |
Decrease of immunoglobulin A | 3 | | | |
Decrease of immunoglobulin G | 3 | | | |
Decrease of immunoglobulin M | 3 | | | |
Digestive tract | | | | |
Diarrhea | | 2 | | |
Gastrointestinal reaction | 1 | | | |
Sick | | | 1 | |
Vomit | | | 1 | |
Dental ulcer | 1 | | | |
Respiratory tract | | | | |
Cough | 1 | | | |
Upper respiratory infection | 1 | 1 | | |
Pneumonia | | | 3 | |
Cardiovascular | | | | |
Nodal tachycardia | 2 | | | |
Others | | | | |
Insomnia | | 1 | | |
Elevated urinary white blood cells | | 1 | | |
Feeble | 1 | | | |
Headache | 1 | | | |
Elevated thyroid stimulating hormone | 1 | | | |