Excessive activated T-cell proliferation after anti-CD19 CAR T-cell therapy.

Excessive activated T-cell proliferation was observed in vivo in one patient after an anti-CD19-chimeric antigen receptor (CAR) T-cell infusion. The patient, who had chemotherapy refractory and CD19+ diffuse large B-cell lymphoma (DLBCL), received an anti-CD19 CAR T-cell infusion following conditioning chemotherapy (fludarabine/cyclophosphamide). The lymphocyte count in the peripheral blood (PB) increased to 77 × 109/L on day 13 post infusion, and the proportion of CD8+ actived T cells was 93.06% of the lymphocytes. Then, the patient suffered from fever and hypoxaemia. Significant increases in serum cytokine, lactate dehydrogenase, aspartate aminotransferase (AST), alanine transaminase (ALT), and glutamic-oxalacetic transaminase (γ-GT) levels were observed. A high-throughput sequencing analysis for T-cell receptors (TCRs) and whole-genome sequencing were used to explore the mechanisms underlying this excessive T-cell proliferation. TCR diversity was demonstrated, but no special gene mutation was found. The patient was found to be infected with the John Cunningham polyomavirus (JCV). It cannot be ruled out the bystander activation pathway induced by JCV infections related the excessive activated T-cell proliferation. Although the clinical and laboratory data do not fully explain the reason for excessive T-cell proliferation after the anti-CD19 CAR T-cell infusion, the risk of this type of toxicity should be emphasized. This study was registered at www.clinicaltrials.gov as NCT01864889.

Cocktail treatment with EGFR-specific and CD133-specific chimeric antigen receptor-modified T cells in a patient with advanced cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is one of the most fatal malignant tumors with increasing incidence, mortality, and insensitivity to traditional chemo-radiotherapy and targeted therapy. Chimeric antigen receptor-modified T cell (CART) immunotherapy represents a novel strategy for the management of many malignancies. However, the potential of CART therapy in treating advanced unresectable/metastatic CCA is uncharted so far. CASE PRESENTATION: In this case, a 52-year-old female who was diagnosed as advanced unresectable/metastatic CCA and resistant to the following chemotherapy and radiotherapy was treated with CART cocktail immunotherapy, which was composed of successive infusions of CART cells targeting epidermal growth factor receptor (EGFR) and CD133, respectively. The patient finally achieved an 8.5-month partial response (PR) from the CART-EGFR therapy and a 4.5-month-lasting PR from the CART133 treatment. The CART-EGFR cells induced acute infusion-related toxicities such as mild chills, fever, fatigue, vomiting and muscle soreness, and a 9-day duration of delayed lower fever, accompanied by escalation of IL-6 and C reactive protein (CRP), acute increase of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase, and grade 2 lichen striatus-like skin pathological changes. The CART133 cells induced an intermittent upper abdominal dull pain, chills, fever, and rapidly deteriorative grade 3 systemic subcutaneous hemorrhages and congestive rashes together with serum cytokine release, which needed emergent medical intervention including intravenous methylprednisolone. CONCLUSIONS: This case suggests that CART cocktail immunotherapy may be feasible for the treatment of CCA as well as other solid malignancies; however, the toxicities, especially the epidermal/endothelial damages, require a further investigation. TRIAL REGISTRATION: ClinicalTrials.gov NCT01869166 and NCT02541370 .

Autologous T Cells Expressing CD30 Chimeric Antigen Receptors for Relapsed or Refractory Hodgkin Lymphoma: An Open-Label Phase I Trial.

Purpose: Relapsed or refractory Hodgkin lymphoma is a challenge for medical oncologists because of poor overall survival. We aimed to assess the feasibility, safety, and efficacy of CD30-targeting CAR T cells in patients with progressive relapsed or refractory Hodgkin lymphoma.Experimental Design: Patients with relapsed or refractory Hodgkin lymphoma received a conditioning chemotherapy followed by the CART-30 cell infusion. The level of CAR transgenes in peripheral blood and biopsied tumor tissues was measured periodically according to an assigned protocol by quantitative PCR (qPCR).Results: Eighteen patients were enrolled; most of whom had a heavy treatment history or multiple tumor lesions and received a mean of 1.56 × 107 CAR-positive T cell per kg (SD, 0.25; range, 1.1-2.1) in total during infusion. CART-30 cell infusion was tolerated, with grade ≥3 toxicities occurring only in two of 18 patients. Of 18 patients, seven achieved partial remission and six achieved stable disease. An inconsistent response of lymphoma was observed: lymph nodes presented a better response than extranodal lesions and the response of lung lesions seemed to be relatively poor. Lymphocyte recovery accompanied by an increase of circulating CAR T cells (peaking between 3 and 9 days after infusion) is a probable indictor of clinical response. Analysis of biopsied tissues by qPCR and immunohistochemistry revealed the trafficking of CAR T cells into the targeted sites and reduction of the expression of CD30 in tumors.Conclusions: CART-30 cell therapy was safe, feasible, and efficient in relapsed or refractory lymphoma and guarantees a large-scale patient recruitment. Clin Cancer Res; 23(5); 1156-66. ©2016 AACR.

Treatment of CD20-directed Chimeric Antigen Receptor-modified T cells in patients with relapsed or refractory B-cell non-Hodgkin lymphoma: an early phase IIa trial report.

Patients with relapsed or refractory non-Hodgkin lymphoma have a dismal prognosis. Chimeric Antigen Receptor (CAR)-modified T cells (CART cells) that targeted CD20 were effective in a phase I clinical trial for patients with advanced B-cell lymphomas. We performed a phase IIa trial to further assess the safety and efficacy of administering autologous anti-CD20 CART (CART-20) cells to patients with refractory or relapsed CD20+ B-cell lymphoma. Eleven patients were enrolled, and seven patients underwent cytoreductive chemotherapy to debulk the tumors and deplete the lymphocytes before receiving T-cell infusions. The overall objective response rate was 9 of 11 (81.8%), with 6 complete remissions (CRs) and 3 partial remissions; no severe toxicity was observed. The median progression-free survival lasted for >6 months, and 1 patient had a 27-month continuous CR. A significant inverse correlation between the levels of the CAR gene and disease recurrence or progression was observed. Clinically, the lesions in special sites, specifically the spleen and testicle, were refractory to CART-20 treatment. Collectively, these results together with our data from phase I strongly demonstrated the feasibility and efficacy of CART-20 treatment in lymphomas and suggest large-scale patient recruitment in a future study. This study was registered at www.clinicaltrials.org as NCT01735604.

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma treated by CD20-directed chimeric antigen receptor-modified T cells.

We conducted a trial testing a CD20-specific CAR coupled with CD137 and the CD3ζ moiety in patients with chemotherapy refractory advanced diffuse large B cell lymphomas (DLBCL). Seven patients were enrolled. One of the two patients with no bulky tumor obtained a 14-month durable and ongoing complete remission by cell infusion only, and another attained a 6-month tumor regression. Four of five patients with bulky tumor burden were evaluable for clinical efficacy, three of which attained 3- to 6-month tumor regression. Delayed toxicities related to cell infusion are directly correlated to tumor burden and tumor-harboring sites, and mainly included cytokine release symptoms, tumor lysis symptoms, massive hemorrhage of the alimentary tract and aggressive intrapulmonary inflammation surrounding extranodal lesions. These results show firstly that anti-CD20 CART cells can cause prolonged tumor regression in combination with debulking conditioning regimens for advanced DLBCL. This study is registered at www.clinicaltrials.gov as NCT01735604.

[Study on the stability of the anergic CD8(+);NKT cells in vitro].

AIM: To study the CD8(+);NKT cell stability function. METHODS: Splenic lymphocytes from C57BL/J mice were treated with Staphylococcal enterotoxin B (SEB) and then cultured in vitro. Day 10, 20, 30 and freezen effector cells were harvested and used. The cells were cultured in medium containing ConA and LPS for 72 hours and measured their response to mitogens. The inhibitory action of the effector cells were examined. The effector cells were co-cultured with normal lymphocytes and above mitogens for 72 hours. The cells proliferation was assessed with MTT method. The effector cells were cultured with heterogenic lymphocytes and were assessed with MTT method. The NKT cell subsets among these effector cells were analyzed by flow cytometry. RESULTS: The response of these effector cells to ConA and LPS was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.67 and 0.61 to 0.30 and 0.31, 0.28 and 0.20, 0.26 and 0.24, 0.22 and 0.23 (P<0.05, n=3). The inhibitory ability of effector cells aginst the response of normal lymphocytes to ConA and LPS were clearly observed. They inhibited the response of normal lymphocytes to above mitogens. And the A values of cell proliferation were decreased from 0.67 and 0.61 to 0.33 and 0.39, 0.30 and 0.43, 0.36 and 0.43, 0.26 and 0.29(P<0.05, n=3). The response of effector cells to heterogenic lymphocytes was significantly decreased compared with normal lymphocytes. The A values of cell proliferation were decreased from 0.70 to 0.42, 0.42 and 0.54 on day 20, 30 and freezen effector cells respectively(P<0.05, n=3). The CD8(+);NKT cell subsets among these effector cells with tolerance function were significantly increased(P<0.05, n=3). CONCLUSION: The anergic CD8(+);NKT cells could culture in vitro. And the anergic function of these cells were still existing.

[Identification of cell division of CD8+ NKT cells in vitro].

AIM: To measure the in vitro cell division ability of CD8(+);NKT cells by CFSE staining and flow cytometry(FCM). METHODS: Fresh spleen lymphocytes of C57BL/J mice were separated and stained with CFSE, and then stimulated by ConA and LPS for 3 d, and by SEB for 5 d and 10 d respectively. The stimulated cells were harvested and analyzed for CD69 expression on the cell surface and the ability of cell division using FCM. The SEB-activated effector cells for 10 d further stimulated with IL-2 for the consecutive 10 days, and were analyzed for their cell division ability, CD69 expression and NKT cell subsets by FCM. RESULTS: ConA, LPS and SEB stimulated the proliferation of spleen cells. ConA and LPS made the cells divide 3 times within 3 d, and increased CD69 expression up to 74.19% and 41.56% respectively. SEB made the cells divide 5 times within 5 d and 7 times within 10 d respectively, with increased CD69 expression of 32.09% and 48.66% respectively. Ten-day IL-2 stimulation of SEB-activated cells caused population expansion for 7 times with the CD8(+);NKT cell subsets significantly increased from 0.36% to 38.58% and CD69 expression significantly increased from 0.11% to 83.74%. CONCLUSION: The SEB-activated CD8(+);NKT cells proliferated in vitro and their cell division capability could be determined by CFSE staining and FCM.

[Morphology and differentiation pathway of superantigen SEB-activated NKT subsets].

AIM: This study is to explore the biological characteristics of NKT subsets through investigating their morphology and differentiation pathways. METHODS: Splenic lymphocytes from C57BL/J mice were treated with either Staphylococcal entertoxin B (SEB) or ConA and then cultured in vitro. On day 5 and day 10, the lymphocytes were harvested. Normal adherent macrophages and normal lymphocytes were harvested on day 0. Expression of CD69 molecule on the cell surfaces of the lymphocytes and large lymphocytes by SEB-activated was measured using staining of fluorescent antibody and flow cytometry (FACS). And the percent (%) of NKT subsets was determined and analyzed the differentiation pathways of the NKT subsets. Appearance of subsets was observed and compared under a light microscope at x400 magnification. RESULTS: Expression levels of CD69 molecule on the SEB-activated lymphocytes and the giant lymphocytes were significantly increased to 55% and 68.95% respectively as compared to the level (0.11%) detected on the normal lymphocytes (both P<0.01; n=3). The percent (%) of CD8(+) NK1.1(+) and TcRVbeta8(+) NK1.1(+) NKT subsets in the giant lymphocytes was significantly enhanced to 30.29% and 31.48% (84.0 or 38.9 folds) originally from 0.36% and 0.81%, respectively. Based on the cell distribution shown in the upper part of the FACS picture, they should be large-scale selection. The SEB-activated lymphocytes in size were larger than not only the ConA-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. They are no-adherent cells. There were a few granule seen in cytoplasm . The value of cytoplasm vs nuclei was less than 1.0. The SEB-activating CD8(+) and TcRVbeta8(+) NKT subsets were not relative to a NK source. They were produced directly from T cells differentiation. CONCLUSION: The SEB-activating NKT cells are giant lymphocytes in size, consisting predominantly of CD8(+) NK1.1(+) NKT cells. They are considered as a subsets of T lymphocytes.

[Study of the NKT cell subsets with superantigen SEB-activated tolerance].

AIM: To study the NKT cell subsets and their differentiation. METHODS: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry. RESULTS: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population. CONCLUSION: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.