Clinical Immunology (2014) 155, 160–175

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma treated by CD20-directed chimeric antigen receptor-modified T cells Yao Wang a,1 , Wen-ying Zhang b,1 , Qing-wang Han a,1 , Yang Liu c , Han-ren Dai a , Ye-lei Guo a , Jian Bo d , Hui Fan b , Yan Zhang b , Ya-jing Zhang b , Mei-xia Chen b , Kai-chao Feng b , Quan-shun Wang d , Xiao-bing Fu a , Wei-dong Han a,⁎ a

Department of Immunology, Institute of Basic Medicine, School of Life Sciences, Chinese PLA General Hospital, China Department of Bio-therapeutic, Chinese PLA General Hospital, China c Department of Geriatric Hematology, Chinese PLA General Hospital, China d Department of Hematology, Chinese PLA General Hospital, China b

Received 29 June 2014; accepted with revision 3 October 2014 KEYWORDS Anti-CD20 chimeric antigen receptor (CAR) T cells; Refractory advanced; Diffuse large B-cell lymphoma (DLBCL); Delayed toxicities

Abstract 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. © 2014 Elsevier Inc. All rights reserved.

⁎ Corresponding author at: Biotherapeutic Department/Medical Clinical Immunology Department, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China. Fax: + 86 10 6823 7516. E-mail address: [email protected] (W. Han). 1 Contributed equally to this work.

http://dx.doi.org/10.1016/j.clim.2014.10.002 1521-6616 © 2014 Elsevier Inc. All rights reserved.

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma

1. Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) histology, representing 25% to 35% of new cases annually [1]. Unlike indolent lymphomas, DLBCL is an aggressive type characterized by a rapidly enlarging mass if left untreated. Furthermore, extranodal involvement or associated constitutional symptoms indicate a more aggressive subtype [2]. CD20 antigen, a tetra-transmembrane protein present in N 90% of B-cell lymphomas, is a well-established target for NHL treatment. Anti-CD20 treatment using antibody therapy combined with chemotherapy will produce superior clinical effects in most NHL patients compared to chemotherapy alone [3]. Even though most NHL DLBCL patients have an objective response after initial treatment, a portion of them will inevitably relapse and subsequently develop resistance to chemotherapy and anti-CD20 therapy and ultimately deteriorate with a poor prognosis [4]. Adoptive cell transfer (ACT) typically represented by tumor-specific chimeric antigen receptor modified T cells (CART) holds great promise for tumor immunotherapy [5,6]. Most of the dramatic results of CART-based ACT have been achieved in hematological malignancies, in particular by targeting the CD19 antigen. The use of the second generation CART cells targeting CD19 (CART-19) has shown unprecedented clinical efficacy even in some of relapsed and/or refractory patients with acute lymphoblastic leukemia (ALL) [7,8], chronic lymphocytic leukemia (CLL) [9] and NHL [10], providing a potentially curable strategy for B-cell malignancies [9,11]. Although most cases of conspicuous clinical efficacy of CART-19 were achieved in patients with B-cell diseases, no more than 10 DLBCL patients have been treated by this protocol thus far according to our review of the public literature. One trial showed that almost none of the studied advanced DLBCL patients had an objective clinical response after CART-19 treatment in the absence of conditioning regimens [12]. In contrast, another preliminary trial reported at the 2013 American Society of Hematology (ASH) meeting demonstrated effective clinical outcomes in some of the refractory DLBCL patients treated by CART-19 who had received prior cytoreductive chemotherapies [13]. Under the premise that CD20 is also an ideal target for NHL treatment, the second generation CART-20 was previously used by Till's group in a pilot trial. Clinical observations derived from three non-DLBCL patients with stable and minimal lesions revealed the in vivo anti-tumor activity of CART-20 cells [14]. In this study, to test whether the second generation CART-20 could have clinical benefits for DLBCL patients, especially for those in advanced stage, seven refractory patients were initially recruited into our trial. Treatment with CART-20 in combination with conditioning chemotherapies resulted in 3- to 6-month tumor regressions in four out of five evaluable patients. One patient, characterized as high-risk and with a poor prognosis, experienced a 14-month durable and ongoing complete remission resulting from CART-20 infusion alone. Except for tumor lysis syndrome and cytokine release syndrome, which have been previously linked to bulky tumor burden, the unusually delayed toxicities such

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as normal tissue damage at special tumor-localized sites associated with CART cell infusion are reported first in this study.

2. Methods 2.1. Clinical design and protocol eligibility requirements All enrolled patients gave informed consent upon enrollment in accordance with the Declaration of Helsinki. The protocol (ClinicalTrials.gov identifier NCT 01735604) was approved by the Institutional Review Board at the Chinese PLA General Hospital. No commercial sponsor was involved in the study. Patients were eligible if they had a pathologically confirmed diagnosis of CD20+ DLBCL, had active relapsed or refractory disease after at least four prior chemotherapies, and were deemed not to be candidates for stem cell transplantation (SCT). We defined refractory as progression or no response 1 month after the end of the most recent chemotherapy or anti-CD20 Abs therapy. Patients enrolled in this protocol are not precluded from undergoing additional chemotherapy after the modified T cell infusion in the case of aggressive disease. Patients were excluded if they received anti-CD20 Abs within 4 weeks of cell infusion and had human anti-mouse Ab seropositivity. The patients' autologous peripheral blood mononuclear cells (PBMCs) were collected for the production of CART-20 cells. During this interval, patients underwent cytoreductive chemotherapy if necessary for tumor debulking or to maintain disease control. Patients subsequently received CART-20 cells split into 3 to 5 consecutive daily intravenous infusions in an escalating dose. No post-infusion cytokines were administered. An excisional biopsy of a palpable lymph node and a tissue biopsy were performed on patients at least 3 weeks after the final CART-20 cell infusion. We assessed toxicity according to the National Institutes of Health Common Terminology Criteria for Adverse Events Version 3.0 (http://ctep.cancer.gov/). Clinical responses were assessed according to International Working Group criteria [15].

2.2. Modification and expansion of CD20-specific T cells 2.2.1. Constructs and lentiviral packaging The DNA sequences of the scFv domain targeting the CD20 and CD138 antigens were derived from AY160760.1 and NM_001006946.1, respectively. The CAR.20-CD137ζ vector harboring anti-CD20 scFv, and human CD137 and CD3ζ signaling domains were generated (Fig. 1A). The cassette was cloned into a lentiviral backbone. A pseudotyped, clinical-grade lentiviral vector was produced according to current good manufacturing practices with a three-plasmid production approach. The green fluorescence protein (GFP)-harboring vector CAR.20-CD137ζ-GFP was also constructed to verify transduction efficiency.

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2.2.2. Generation and expansion of CAR T cells CAR T cell expansion was carried out mainly according to the procedure of cytokine-induced killer (CIK) cells as described previously [16]. Lentivirus-mediated CAR transduction was performed twice on days 2 and 3 of cell culture, respectively. Composition and purity were assessed by fluorescentactivated cell sorting (FACS) and were harvested beginning on days 10–12.

Standard 6-hour carboxyfluorescein succinimidyl ester (CSFE) cytotoxicity assays were performed as previously described [16], using the following targets: Molt-4 (negative control), Molt-4/CD20+ (Molt-4 cells with CD20 transduction), Raji and Ramos (naturally expressed CD20 molecules).

2.3. Flow cytometry

2.5. Quantitative PCR (Q-PCR)

The following anti-human antibodies were used in this study: CD4 (fluorescein isothiocyanate, FITC), CD8 (phycoerythrin, PE), CD3 (chlorophyll protein complex, PerCP, CD56 (allophycocyanin, APC), CD19-APC, CD45-PerCP, and CD20-PE. All of these antibodies and isotype-matched monoclonal antibodies were purchased from BD Biosciences (CA, USA). Data acquisition was performed using a FACSCalibur flow cytometer (BD Biosciences).

We used real-time PCR to quantify the level of the CAR gene according to the protocol described previously [14]. A 153-bp (base pair) fragment containing portions of the CD8a chain and adjacent CD137 chain were amplified using the forward primer 5′-GGTCCTTCTCCTGTCACTGGTT-3′ and reverse primer 5′-TCTTCTTCTTCTGGAAATCGGCAG-3′. Amplification of β-actin was used as an internal control and for the normalization of DNA quantities.

A

2.4. Cytotoxicity assays

Anti-CD20 ScFv VL

VH

CD8a hinge and TM

CD137

CD3

Linker

B

C

D

E

Figure 1 Expansion and cytotoxicity of CART-20 cells. (A) Schematic representation of the CAR.20-CD137ζ chimeric T-cell receptor cDNA plasmid, not to scale. (B) Median expansion (− fold) of control (NT; not transfected) and CART-20 cells generated from all patients that were cultured for approximately 13 days. Mean and SDs are shown for seven different T-cell lines. (C) Phenotypic analyses of PBMCs, NT and CART-20 cells. Phenotypic comparisons were performed in samples from seven patients, and the results are expressed as the means ± SD. (D) The total cell count of CD3+ cells and verified CART-20 cell infusion (107/kg) in all seven patients (E) Cytotoxic activity of control NT, CART-20 and CART138 cells obtained from all 7 patients, using the following target cells: Molt-4 cell line (acute lymphoblastic leukemia, CD20−), Molt-4 (CD20+) cell line (transfected to express the human CD20 molecule), Raji cell line (Burkitt's lymphoma) and Ramos cell line (Burkitt's lymphoma). Cytotoxic activity was evaluated in a 6-hour CFSE-staining assay, and the results are shown at effector:target (E:T) ratios of 5:1, 10:1, 20:1 and 40:1. The data are represented as the mean of triplicate values from each patient, and error bars represent SEM. * represents p b 0.05; ** represents p b 0.01.

Baseline demographic and clinical patient characteristics.

UPN

Age, y

Gender

Diagnosis/stage

The no. of prior therapies

Disease status at study entry

Conditioning regimen before T-cell infusions

1

85

Male

DLBCL/III-B

10

PD

COED

2

47

Male

DLBCL/IV-B

6

PD

COD

3 4

70 37

Male Male

DLBCL/IIIX-B DLBCL/IV-A

8 and Radiotherapy 14

PR PD

None CHODE

5

76

Male

DLBCL/II-A

5

PD

COED

6

57

Male

DLBCL/IV-B

12

PD

ESHAP

7

65

Female

DLBCL/IV-S

12

PD

CHODE

day1, Cyclophosphamide, 500 mg/m2 day1, Vincristine, 2 mg day1–3, Etoposide, 40 mg/m2/d day1–3, Dexamethasone, 10 mg/m2/d day1, Cyclophosphamide, 500 mg/m2 day1, Vincristine, 2 mg, day1–3, Dexamethasone, 10 mg/m2/d day1, Cyclophosphamide, 500 mg/m2 day1, Doxorubicin 35 mg/m2 day1, Vincristine, 2 mg day1–3, Dexamethasone, 10 mg/m2/d day1–3, Etoposide 40 mg/m2/d day1, Cyclophosphamide, 500 mg/m2 day1, Vincristine, 2 mg day1–3, Etoposide, 40 mg/m2/d day1–3,Dexamethasone, 10 mg/m2/d day1–3, Methylprednisolone100mg/m2/d day1–4, Etoposide 40 mg/m2/d day1–4, Carboplatin 80 mg/m2/d day5, High-dose cytosine arabinoside 1 g/m2 day1, Cyclophosphamide, 500 mg/m2 day1, Doxorubicin 35 mg/m2 day1, Vincristine, 2 mg day1–3, Dexamethasone, 10 mg/m2/d day1–3, Etoposide 40 mg/m2/d

Response and time since treatment (mon) SD (2), PR (4), died of MOF

NE, died of massive hemorrhage of alimentary tract CR (14 +) PR (3), PD

PR (6)

PD

PR (3), PD

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma

Table 1

DLBCL: diffuse large B-cell lymphoma; X: lesions diameter N 10 cm; S: spleen involvement. CHODE: cyclophosphamide, doxorubicin, vincristine, dexamethasone and etoposide. ESHAP: etoposide, carboplatin, high-dose cytosine arabinoside and methylprednisolone. COED: cyclophosphamide, vincristine, etoposide and dexamethasone. COD: cyclophosphamide, vincristine and dexamethasone. MOF: Multiple organ failure. “+” indicates an ongoing response as of the time of manuscript submission. NE: non evaluable. PD: progress disease. NE: non evaluable. PR: partial response. SD: stable disease. CR: complete response.

163

164

Days after infusion

ESHAP

CD3+ T cells (102/µl)

CD3+ T cells (102/µl)

GDP

Days after infusion

UPN6

UPN5

Days after infusion

Days after infusion

ICE

Days after infusion

Days after infusion

UPN4

UPN7

UPN6

ICE

UPN7 GDP

The ratio CD8+ /CD4+ cell

UPN3

Days after infusion

CD3+ T cells (102/µl)

Days after infusion

The ratio CD8+ /CD4+ cell

UPN1

TLS

The ratio CD8+ /CD4+ cell

The ratio CD8+ /CD4+ cell

B

Days after infusion

ESHAP

UPN5

The ratio CD8+ /CD4+ cell

Days after infusion

UPN4 CD3+ T cells (102/µl)

TLS

UPN3

The ratio CD8+ /CD4+ cell

UPN1

CD3+ T cells (102/µl)

CD3+ T cells (102/µl)

A

Days after infusion

Days after infusion

Days after infusion

Days after infusion

Days after infusion

Days after infusion

UPN6

Days after infusion

CD20+ cells in PB (/µl)

UPN5

CD20+ cells in PB (/µl)

UPN4

CD20+ cells in PB (/µl)

UPN3

CD20+ cells in PB (/µl)

UPN1

CD20+ cells in PB (/µl)

CD20+ cells in PB (/µl)

C UPN7

Leukemia

Days after infusion

Figure 2 Altered T and B cell counts in the PB of patients after CART-20 cell infusion. (A) CD3+ cell count changes in the PB of UPN1, 3, 4, 5, 6 and 7 after the infusion of CART-20 cells detected by FACS. (B) The ratio of CD8+/CD4+ cells in the PB after CART-20 cell infusion. The red line on the plots is the baseline ratio of each patient. (C) The change of CD20+ B cell in the PB after the infusion of CART-20 cells. The dot line on the plots represents the minimum reference value of B cells in PB of healthy people. TLS indicates Tumor Lysis Syndrome; ICE indicates the chemotherapy regimen: ifosfamide, etoposide and carboplatin; ESHAP indicates the chemotherapy regimen: etoposide, carboplatin, high-dose cytosine arabinoside and methylprednisolone; GDP indicates the chemotherapy regimen: gemcitabine, cisplatin and dexamethasone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Medrol TLS

UPN2

Days after infusion

ESHAP Medrol

Medrol

Days after infusion

Transgene copies (no./g gDNA)

Transgene copies (no./g gDNA)

UPN4

Transgene copies (no./g gDNA)

Leukemia

UPN6

UPN5

ICE

Medrol GDP

Days after infusion

Days after infusion

B UPN7

Days after infusion Transgene copies (no./g gDNA)

Days after infusion Medrol

UPN3 Death

Transgene copies (no./g gDNA)

Medrol

Transgene copies (no./g gDNA)

Medrol

Transgene copies (no./g gDNA)

Transgene copies (no./g gDNA)

UPN1

Biopsy Tissue

Days after infusion

165

Figure 3 CART-20 persists in the peripheral blood and localize to tumor sites. (A) Quantitative real-time PCR was performed on genomic DNA harvested from each patient's PBMCs collected before and at serial time points after CART-20 cell infusion using primers specific for the transgene. (B) The same quantitative PCR assay was used to detect modified cells in the genomic DNA harvested from ultrasound-guided needle biopsies of lymph nodes or tumor tissues. For patient UPN1, lymph nodes were collected at 17, 38 and 73 days after the CART-20 cell infusion. For patient UPN4, lung and liver tissues were collected at 53 and 61 days after the CART-20 cell infusion. For patient UPN5, mucosa-like necrotic material was collected at 246 days after the CART-20 cell infusion. The data are represented as the mean values (± SD) of at least two assays per time point, with each sample assessed in triplicate. Ly indicates lymph nodes; Li indicates liver tissue; Lu indicates lung tissue; MLE indicates mucosa-like necrotic material. TLS indicates Tumor Lysis Syndrome; ICE indicates the chemotherapy regimen: ifosfamide, etoposide and carboplatin; ESHAP indicates the chemotherapy regimen: etoposide, carboplatin, high-dose cytosine arabinoside and methylprednisolone; GDP indicates the chemotherapy regimen: gemcitabine, cisplatin and dexamethasone. (C) Immunohistochemical examination (diaminobenzidine with hematoxylin counterstaining) of a punch biopsy of lymph nodes from UPN1 before and 17, 38 and 73 days after CART-20 cell infusion showed that tumor cells were CD20+ (shown), CD10+, BCL2+, and BCL6+, which is consistent with involvement by follicular lymphoma with large cell transformation. Notably, scattered CD3+ and CD8+ cells infiltrated the tumor after infusion, whereas CD3+ cells did not infiltrate the tumor at the time of initial diagnosis, and approximately 50% CD3+ cells had dramatically infiltrated the tumor at 73 days after infusion. Scale bar: 50 μm.

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma

A

CD20

166

C

CD3

Initial diagnosis

CD20

CD3 6.8%

CD4

CD8

CD20

CD3 8.9%

CD4

CD8

CD20

CD3 42%

CD4

CD8

17days after infusion

38days after infusion

73days after infusion

Figure 3 (continued).

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Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma

2.6. Cytokine measurements Serum IL-2, IL-6, IL-10, IL-12p70, IL-12/IL23p40, IFN-γ, TNF-α, VEGF, and Granzyme A levels were batch analyzed using a BD Biosciences microbead sandwich immunoassay according to the manufacturer's instructions. Briefly, the analyte concentrations were determined using a standard curve prepared with each assay.

2.7. Statistics The results are shown as the mean ± standard error of the mean (SEM) of triplicate determinants (wells). Data were plotted using GraphPad Prism version 5.0. Two-way analysis of variance (ANOVA) was used to determine the significance of the differences between the means in all experiments. A P value b 0.05 was considered to be statistically significant.

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cytotoxicity on the CD20+ cells of the CART-20 cells prepared from patients.

3.3. In vivo increase of CD8+ T cells and decrease of CD20+ B cells post CART20 infusion The T cell subpopulation and CD20+ cells in patients' PB were serially measured post CART-20 infusion. The total T cells (CD3+) nearly in all patients post CART-20 cell infusion were markedly increased within 1 to 2 months relative to their baseline levels (Fig. 2A), and further phenotypic analysis revealed the main contribution of the CD8+ subpopulation to this elevation (Fig. 2B). Meanwhile, during this period, CART-20 cells resulted in an at least a 2-fold decrease of CD20+ B cells (Fig. 2C), although this result was achieved in an absolutely reduced background caused by their previous anti-CD20 therapy, demonstrating the in vivo CD20-directed cytotoxicity effects of CART-20 cells.

3. Results 3.1. Characteristics of patients Seven patients with refractory advanced CD20+ DLBCL from stage IIIB to IVB were enrolled in this study. Patients UPN 1, 2, 4, 6 and 7 were defined as having bulky tumor burden according to our definition that includes those with lesions with a longest diameter greater than 5 cm or patients with more than three lesions. The patients had all received extensive prior treatments including anti-CD20 Abs, and none of them had ever obtained complete remission. Before their enrollment into our trial, six patients were defined as either refractory or progressive according to their responses to recent chemotherapeutic regimens. Except for UPN3, the other six patients received preconditioning chemotherapy for disease control or debulking before CART-20 cell infusion. The general clinopathological characteristics, preconditioning regimens and the corresponding clinical responses of patients prior to CART-20 infusions are summarized in Tables 1 and A.1.

3.2. Generation, characterization and specific cytotoxicity of patient-derived CART-20 cells in vitro CART-20 cells were initially generated from the PBMCs of 50 ml PB of each patient. After 10 to 12 days of culture, the total number of cells was expanded 26- to 84-fold, and the cells were then ready for infusions (Fig. 1B). A mean of 95 ± 12.7% of the infused cells was CD3+ cells principally composed of the CD8+ subset (average 76%) (Fig. 1C), and a mean of 33% (range 15–50%) of the cells expressed CAR. The detailed data of the total infused CD3+ cells and CART-20 cells for each patient are summarized in Fig. 1D, Tables A.2 and A.3 and Fig. A.1. As illustrated in Fig. 1E, CART-20 cells had an approximately identical cytotoxic activity as CART-138 and nontransduced T (NT) cells against Molt-4 (CD20−) cells. The prominent cytolytic activities of CART-20 cells were observed in Ramos, Raji, and Molt-4 cells transduced with CD20 as compared to the control cells, indicating a specific ex vivo

3.4. The CAR molecule was persistently detected in PB and target tissues at a high copy number We measured the copy numbers of the CAR molecule to determine the persistence of CART-20 cells in PB at serial time points. A high copy number of the CAR gene (N 1000/μg gDNA) was found to be persistently maintained for at least 4 weeks in nearly all patients (Fig. 3A), which was reversely correlated with the decreased CD20+ B cell count in PB. Similarly, as shown in Fig. 2C, CD20+ B cell count increased 30 days after CART-20 transfusion in most patients along with the decrease of CAR gene copy number in PB. Among these patients, UPN1 had the highest number of copies of the CAR gene, which was continuously sustained for more than 5 months. The CAR gene level was drastically increased in UPN7 after she progressed into lymphoma leukemia. Intriguingly, the reduced level of CAR rapidly recovered after the cessation of the use of methylprednisolone and corticosteroid-containing chemotherapeutic regimens in UNP 1, 4 and 6. Based on Q-PCR assays, high copy numbers of the CAR gene were also detected in biopsy tissues derived from UPN 1, 4 and 5, even after 10 weeks of cell infusion (Fig. 3B), indicating the effective trafficking and persistence of the infused cells into the target tissues. In addition, a serial immunohistochemical staining on biopsies from a submandibular lesion of UPN1 was performed (Fig. 3C), showing an increasing augmentation of T cells scatted within the tumor parenchyma, strongly suggesting the increasingly accumulation or in situ expansion of CART-20 cells in target sites. This finding is in agreement with the synchronous augmentation of CAR copy numbers, as illustrated in Fig. 3B.

3.5. Altered serum cytokine levels associated with CART-20 treatment We evaluated patient serum samples from serial posttreatment time points and then analyzed the changes in the serum levels of cytokines correlated with the CART-20 treatment (Fig. 4). For patients without bulky tumor burden,

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fold change

UPN4

UPN6

fold change

UPN5

fold change

fold change

Days after infusion

Days after infusion

Days after infusion

Days after infusion

UPN3

UPN2 fold change

fold change

UPN1

Days after infusion

Days after infusion

fold change

UPN7

Days after infusion

Figure 4 Changes in the patients' serum cytokine levels before and after CART-20 cell infusion. Serum was harvested from each patient PB collected before and at serial time points after CART-20 cell infusion. Serum cytokines were determined by FACS. The x-axis represents the treatment time course with day 0 being the day of CART-20 cell infusion.

such as UPN3, all cytokines fluctuated within normal ranges during and after cell therapy. Robust increases in IFN-γ, IL-6, VEGF, IL-10 and Granzyme A were only detected during the cell infusion period in UPN5, who developed infusionassociated Grade 4 chills and fever 0.5 to 1 h after CART-20 treatment. All patients with bulky tumor burden (UPN 1, 4, 6, 7) developed delayed elevations of at least three cytokines, which were highly associated with the adverse effects observed in the clinic (Table 2).

3.6. Toxicities associated with CART-20 treatment 3.6.1. Infusion-associated acute toxicities Infusion-associated adverse effects were not observed in UPN1. UPN5 developed Grade 4 chills and fever accompanied by temporary and well-tolerated respiratory distress, which improved overnight. Five other patients experienced transient and well-tolerated Grade 1 to 2 chills and fever occurring within 1 to 2 h post infusion. 3.6.2. Delayed toxicities related to CART-20 The delayed adverse events related to CART-20 therapy are summarized in Table 2. Consistent with the previous reports, most of toxicities were significantly associated with bulky tumor burdens; however, normal tissue damage in sites

around the lesions and organ dysfunction were observed in UPN 1, 2, 4 and 5. Cytokine elevation and the related toxicities including febrile syndrome, serous cavity effusion, capillary leak syndrome, and other systemic syndromes were confirmed in four out of five patients with bulky tumor burden, three of whom developed a continuous elevation of cytokines that had to be resolved by the discontinuous administration of corticosteroid and/or etanercept (anti-TNFα). UPN1 and UPN2 exhibited submucosal involvement of stomach lymphoma for the first experienced massive alimentary tract hemorrhage, which was the direct cause of death for UPN2 (Fig. A.2A). UPN5 experienced once expectoration containing mucus-like necrotic material from a pharyngeal tumor after 8 months of CART-20 infusion. Local mucosal necrosis and scattered petechia were observed in the glossopharyngeal region by laryngopharyngeoscopy on the following day. Further local biopsy and laboratory analysis revealed a high level of the CAR molecule in his damaged mucosal tissue as indicated in Fig. 3B. UPN4, who had primary extranodal lymphoma involving multiple organs including the right intrapulmonary tissue, gradually developed dyspnea and respiratory distress, which was temporarily mitigated by the intravenous use of glucocorticoid. The patient's respiratory distress ultimately progressed

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma to the point of requiring bed rest and the use of an oxygen mask. X-ray examination showed the gradually exacerbated “consolidation” of his right lung middle lobe (Fig. 5A). Histopathologic and immunohistochemical analyses on biopsy tissue extracted from right middle lobus pulmonis demonstrated a substantial infiltration of CD3+ lymphocytes in the pulmonary interstitial region and inflammatory exudate filling the alveolus. However, only weaker CD20-expressing cells were detected, which is in contrast to what was observed in the biopsy taken from a liver lesion (Fig. 5B). In addition, high-copy CAR molecules were detected in both lung and liver biopsy tissues, as illustrated in Fig. 3B, fundamentally indicating a predominant trafficking of CART0-20 cells into tumor sites and providing a reasonable interpretation for the subsequent damage of normal lung tissue around the tumors. UPN4 was then subjected to continuous moderate elevation of multiple cytokines from the 9th week post infusion as shown in Fig. 4, which may promote the exacerbation of lung consolidation.

3.7. Overall clinical responses to CART-20 therapy Six of seven patients were available to evaluate the objective clinical responses after CART-20 therapy, five of whom experienced tumor regression during this trial (Table 1). Of the two patients without bulky tumor burden, UPN3, who had only residual disease at the time of enrollment, attained a 14-month ongoing complete remission at the time of the publishing of this manuscript (Fig. 6A). UPN5 obtained a 10-month durable and ongoing tumor disappearance for a

Table 2

169

lesion in the hilus of the lung, but only a 6-month disease regression in the glossopharyngeal lesion (Fig. 6B). Upon the obvious enlargement of his glossopharyngeal lesion, UPN5 was transferred to a local radiotherapy treatment regimen. For four evaluable patients with defined bulky burdens, UPN1 remained a more than 4-month drastic tumor diminution after a sudden occurring of tumor lysis at the 8th week post cell infusion (Figs. 6C and A.2B). This patient then died of multiple organ failure evoked by recurrent attacks of pulmonary infection within the previous 2 years. UPN4, characterized by multiple bulky extranodal lesions involving the chest and abdominal walls, the right middle lobus pulmonis, the left lobe of liver, the left kidney and others, experienced only a 3-month tumor regression after CART-20 infusion. UPN7, the only lymphoma patient with detectable bone marrow involvement when she was enrolled into our trial, achieved a 6-month dramatic tumor regression (Fig. 6D). Before she progressed into lymphoma leukemia, this patient received an unknown traditional Chinese herb therapy for 2 months outside our institution and then entered an alternative clinical trial of ours. UPN6, the only patient with a weak response to a debulking regimen and refractory to subsequent cell treatment as well, was transferred to last salvage therapy and then palliative therapy.

4. Discussion CD20 antigen has been identified as an ideal therapeutic target for most DLBCL patients for decades. This clinical trial

Delayed adverse events after T cell infusion.

UPN Adverse events

Grade a Time of occurrence Description

Duration

1

Cytokine release syndrome

3

Continuous

Alimentary tract hemorrhage

3

Sudden tumor lysis syndrome

3

Capillary leak syndrome

2

Acute alimentary tract hemorrhage None Cytokine release syndrome

4

2

Lung dysfunction

3

glossopharyngeal mucus damage Cytokine release syndrome

1 2

Serous cavity effusion

3

Cytokine release syndrome

1

2 3 4

5 6

7 a

8 weeks after infusion 8 weeks after infusion 8 weeks after infusion 10 weeks after infusion 3 weeks after infusion

Anorexia, fatigue, diaphoresis and low fever, the peak temperature to 37.7 °C. Amount of bleeding to 800 ml and hypotension

2 days

Electrolyte abnormalities, LDH elevation

1 week

Edema, pleural effusion

5 weeks

Acute refractory massive hemorrhage of alimentary tract

1 day

9 weeks after infusion 7 weeks after infusion

Fatigue, diaphoresis and low fever, the peak temperature to 37.5 °C. Pleural effusion Dyspnea, hyoxemia, aggressive right lung tissue inflammation and “consolidation” and respiratory distress Spitting black mucus-like material and 2–5 ml blood without pain and cough. Fever, the peak temperature to 38.3 °C.

Continuous

8 months after infusion 4 weeks after infusion 4 weeks after infusion 3 weeks after infusion

Continuous

1 day 1 week

Dyspnea, abdominal distension

Continuous

Fever, the peak temperature to 38.0 °C.

1 week

The maximum grade experienced for the corresponding toxicity for a given patient.

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A

UPN4

Before CART-20 infusion

4 weeks after CART-20 infusion

8 weeks after CART-20 infusion

12 weeks after CART-20 infusion

B UPN4

HE

CD20

CD3

HE

CD20

CD3

53 days (Lung)

CD8

61 days (Liver)

Figure 5 Delayed advance effects occurred in UPN4 after CART-20 therapy. (A) UPN4 experienced cytokine release syndrome. X-ray scans were performed at 4, 8 and 12 weeks post infusions. The films show the right lung surrounding the extronodal lesion as a high-density shadow that enlarged gradually during the 3 months after the infusions. (B) Pathological and immunohistochemical examinations of an extranodal lesion biopsy from UPN4 collected post infusion. HE: numerous lymphocytes can be seen in lung consolidation tissue on the 53rd day (top) and can be seen in a liver lesion on the 61rt day post infusion (bottom). The tumor cells were CD20+, which was consistent with involvement by DLBCL. However, the lung and liver lesions had different distribution densities of tumor cells. Scattered CD3+ lymphocytes were seen in both lesions; the CD8+ lymphocytes were found in the liver lesion.

was designed to test the clinical efficacy of autologous CD20-directed CAR T cell infusion in chemotherapy and anti-CD20 Ab resistant/refractory DLBCL patients and the related toxicities. The fact that five out of six evaluable advanced patients experienced objective responses in this trial not only revealed the potent in vivo anti-tumor activity of CART-20 cells, but suggested the promising applicability of CART-20 in CD20+ B-cell malignant diseases. Importantly, the unusual side effects involved cell therapy-associated normal tissue damage and the corresponding dysfunction in patients were initially reported in this trial as well. Accumulated evidence suggests an enhanced in vivo anti-tumor activity of transferred cells and an improved

clinical response to the conditioning chemotherapy prior to ACT in both hematological malignancies and solid tumors [17,18]. It has been previously confirmed that most patients did not show any objective response to CART-19 therapy in the absence of conditioning chemotherapy [12,19]. In this study, five out of six evaluable patients received COP-based conditioning regimens, which were previously used before enrollment, for transient disease control or debulking. As expected, four of the patients (UPN 1, 4, 5, 7) obtained a N 30% estimated reduction of tumor burden when CART-20 cells were infused, and they subsequently attained a 3- to 6-month PR. Nevertheless, one (UPN6) patient had nearly no response to the prior conditioning chemotherapy and further

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma

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A UPN3

At diagnosis

1 month after 8 courses of chemotherapy

2 month post radiotherapy and before CART-20 infusion

1 month after CART-20 infusion

14+ months after CART-20 infusion

B UPN5

Tougue root lesion

Before CART-20 infusion

6 months after CART-20 infusion

8 months after CART-20 infusion

Before CART-20 infusion

3 months after CART-20 infusion

10+ months after CART-20 infusion

Lung lesion

Figure 6 The regression of adenopathy occurred after the infusion of CART-20 cells into UPN 3 and 5 patients (without bulky tumor burden) and in UPN 1 and 7 (with bulky tumor burden). (A) The positron emission tomography-computerized tomography (PET-CT) scans of UPN3 demonstrated abnormal fluorodeoxyglucose (FDG) uptake in abdominal lesions before enrollment in our trial. The follow-up scans at 1 month and 14 months after infusion were normal. (B) Magnetic resonance imaging (MRI) images of UPN5 before treatment and those taken 6 months and 8 months after treatment showed a marked reduction in glossopharyngeal lesions at 6th month, until the 8th month after CART-20 therapy. The CT scan of UPN5 showed extensive adenopathy before treatment in the left hilum of lung (arrow). The 3-month follow-up scan showed that the lesion had disappeared and remained in remission for 11 months after CART-20 cell infusion. (C) The response in submandibular, axilla and retroperitoneal lymph nodes (arrow) as imaged by a CT scan of UPN1 at baseline, 7 and 9 weeks after CART-20 infusion. The B-ultrasonic examination showed that the submandibular lymph node continued to substantially regress between 9 and 21 weeks post cell therapy. (D) CT scans of UPN7 were performed at baseline and 3 months post T-cell infusion. A decrease in adenopathy of the submandibular and tonsil lesions occurred at the locations indicated by the arrows.

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C UPN1

Submandibular lymph node

Axillary lymph nodes

Retroperitoneal lymph node

Before CART-20 infusion

7 weeks after CART-20 infusion

9 weeks after CART-20 infusion

+ Submandibular lymph node

+

LD:3.3cm 9 weeks after CART-20 infusion

D

LD:1.2cm 21 weeks after CART-20 infusion

UPN7 Submandibular lymph nodes

Tonsil lesion

Before CART-20 infusion

Figure 6 (continued).

3 months after CART-20 infusion

Effective response and delayed toxicities of refractory advanced diffuse large B-cell lymphoma progressed even after CART-20 cell treatment. A series of pilot studies such as those conducted in metastatic melanoma patients demonstrated that increased conditioning chemotherapy prior to cell infusion is well correlated to clinical benefit [20]. Our clinical observations also supported the notion that an increased intensity of the conditioning regimens may be required before CAR-engineered T cell infusion, if possible, to improve clinical response for refractory advanced DLBCL patients, especially for those with bulky tumor burden. Many mechanisms have been proposed to underline the enhanced in vivo anti-tumor activity of transferred T cells through the prior conditioning chemotherapy. One of the widely accepted mechanisms is the lymphocyte homeostatic mechanism, i.e., the in vivo engraftment of infused T cells during the baseline restoration term disrupted by conditioning agents [17,18,21]. The alterative mechanism is that the prior chemotherapy promotes the trafficking of transferred cytotoxic T cells to tumor lesion sites and also potentiates the ability of cells to kill stressed tumor cells that would otherwise survive the chemotherapy [21–23]. Herein, we observed a more durable functional persistence of CART-20 cells in the PB of most patients at a modest to high level (Fig. 3), as compared with those reported elsewhere, in which advanced DLBCL patients were treated with CART-19CD28ζ alone [12]. Meanwhile, we also observed that the first peak value of CAR gene copy number after CART-20 infusion in the PB of UPN3, the only patient who did not receive conditioning treatment prior to cell infusion, was relatively lower than that of the others who received conditioning chemotherapy, with the exception of UPN7. In addition, in comparison with those patients treated by CART-19 in the absence of a prior regimen [12], prior conditioning treatment resulted in a seemingly higher rate of CD3/ CD8-positive cells and a higher level of the CAR molecule in the tumor biopsy tissues collected from UPN 1, 4, and 5 (Figs. 3B and C). Certainly, the durable persistence and robust trafficking of CART cells were not only affected by the prior chemotherapy, but might be affected by different costimulatory signaling mechanisms (CD137 versus CD28) in engineered T cells [24,25]. UPN3, who was high-risk with a poor prognosis but had only residual disease before enrollment, was the only patient who did not receive conditioning chemotherapy before CART-20 infusion in this trial. His durable complete remission suggested the superiority of CAR-redirected T cell treatment strategy for patients with lower tumor burden, as was endorsed by previous reports [14,26]. Meanwhile, the question was raised whether a conditioning or lymphodepletion regimen is indispensable for patients with residual disease. In fact, the durable and high level persistence of the cells and a high rate of transferred cells trafficking to the tumor site are also affected by other factors, such as the quality and quantity [27] of transferred cells which rely on different expansion assays [28–30], immunosuppressive factors such as regulatory T cells [31,32] preexisting in patient PB and tumor sites, and tumor localization. Phenotype analysis revealed a considerable percentage of CD20-directed T cells and CD8+ cytotoxic T cells in our output cells (Fig. 1C), which are comparable to the cell phenotypes detected in other promising trials [7,9,33]. Because a higher effector/target ratio is correlated with potent

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cytotoxicy in vitro, the absolute number of effector T cells is almost always correlated with better responses in clinical settings [27]. Due to the continuous elevation of serum cytokines or disease progression, three patients (UPN 1, 4, 6) in our study were later discontinuously administered corticosteroid or corticosteroid-containing regimens post CART-20 infusion. The levels of the CAR gene in PB were transiently reduced, but they recovered after the cessation of corticosteroid administration (Fig. 3A). These observations recapitulated the previous animal model-based findings demonstrating that although corticosteroid treatment radically decreased the number of native CD8+, it did not affect the numbers of CD8+ cells derived from ACT [34]. Considering the phenotypic traits of CART-20 enriched with CD8+ cells, we conceived that these cells were largely derived from the central memory cells of the patients' PBMCs under the premise that central memory cell-derived CD8+ cells can persist much longer in vivo than those derived from effector memory cells [35,36]. Given that CAR-redirected T cells may survive corticosteroid-containing chemotherapy, we raised the possibility of a combinatorial application of a single treatment with CART and a multiple-cycle induction regimen in early diagnosed NHL patients in future clinical trials, especially for patients with more aggressive DLBCL subtypes. Cytokine release syndrome and tumor lysis syndrome associated with tumor burden have been frequently reported in several trials [9,37]. Normal tissue damage resulting from on-target off-tumor effects triggered by CARredirected transferred T lymphocytes was occasionally observed [38,39]. In this study, cytokine release syndrome (four patients) and tumor lysis syndrome (one patient) were observed in all five of the patients with bulky DLBCL burden after CART-20 treatment. Unexpectedly, three patients (UPN 1, 2 and 5) with tumors localized within the submucosal tissue of the alimentary tract experienced bleeding of the involved sites. Another patient (UPN4) with an extranodal tumor deposited within intrapulmonary tissue gradually developed an inflammatory reaction extending to normal tissue (Fig. 6B), which was associated with the clinical presentation of aggressive respiratory distress. These CART20 infusion-related toxicities led us to postulate that the transferred T cells that traffic into the tumor parenchyma were increasingly stimulated, even by weaker CD20+ tumor cells, to proliferate and activate. We believe that the cells then extravasated outside the tumor parenchyma and into the surrounding normal tissue due to the floppy structural features in areas such as the lung and mucosa. The cells subsequently induced a local inflammatory reaction by producing cytokines, which finally led to the damage of normal tissue. Important lessons can be learned from these four patients with the unusual off-target normal tissue damage toxicities correlated to CART-20 therapy. Some antigens that are safely targeted with antibodies may not be suitable for targeting with CAR-redirected T cell transfer, which is a theory that is also supported by the finding that the activation of CART-20 requires a lower threshold of CD20 antigen expression [40]. Targeting lesions located within some special sites with floppy anatomic structural features, including the intrapulmonary and alimentary tract mucosal tissues, must be approached with great caution or with sufficient prophylactic treatment.

174 IL-6 and IL-12/IL23p40 are believed to be principally produced from activated macrophages [41,42]. A parallel elevation of serum IL-6 and IL-12/IL23p40 levels in UPN 1, 4, 6, and 7 was observed in this trial. Despite the fact that anti-IL-6 therapy was frequently used to block severe cytokine cascade-related cytotoxicity induced by ACT [42,43], the elevation of IL-12/IL23p40 may enhance the in vivo anti-tumor activity possibly by effectively promoting the proliferation and activation of memory/effector T cells [44].

5. Conclusion Adoptive immunotherapy with anti-CD20 CAR-modified T cells is a feasible and possibly effective treatment modality for patients with relapsed or refractory aggressive DLBCL. Prior treatment with an effective debulking conditioning regimen is a prerequisite for inducing a prolonged tumor regression by CART-20. Patients with residual disease may have a favorable clinical response even with CART-20 treatment alone. Finally, but importantly, it should be re-emphasized that the targeting of lesions localized in special sites, such as sites within intrapulmonary tissue and the submucosa of the alimentary tract, by CAR T cells must be undertaken with extreme caution.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (Nos. 31270820, 81230061, 81121004 and 81402566) and was partially supported by a grant from the National Basic Science and Development Programme of China (Nos. 2012CB518103, 2012AA020502 and 2013BAI01B00).

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clim.2014.10.002.

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