Use of CAR-T cell therapy, PD-1 blockade, and their combination for the treatment of hematological malignancies

Clinical Immunology 214 (2020) 108382

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Review Article

Use of CAR-T cell therapy, PD-1 blockade, and their combination for the treatment of hematological malignancies Wenting Songa,b, Mingzhi Zhanga, a b

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Department of Oncology, The First Affiliated Hospital of Zhengzhou University, 450052 Zhengzhou City, Henan Province, China Academy of Medical Sciences of Zhengzhou University, 450052 Zhengzhou City, Henan Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: CAR-T PD-1 blockade Combination Hematological malignancies

With the successful treatment of B-cell lymphomas using rituximab, a monoclonal antibody targeting CD20, novel immunotherapies have developed rapidly in recent years. Immune checkpoint blockade and chimeric antigen receptor-T (CAR-T) cell therapy, which are antibody-based therapy and cell-based therapy, respectively, show promising efficacy and have been approved by the Food and Drug Administration for treating hematological malignancies. However, considering severe side effects and short-term clinical remission, the combination of CAR-T cell therapy and programmed cell-death protein-1 (PD-1) blockade has been applied to enhance therapeutic efficacy in preclinical models and clinical trials. Herein, we review the mechanism of the two therapies, show their toxicities and clinical use respectively, address their combined application, and discuss the scope of further investigations of this mechanism-based combination therapy.

1. Introduction Hematological malignancies consist of all types of leukemia, multiple myelomas, and malignant lymphomas, including Hodgkin's lymphomas (HL) and non-Hodgkin's lymphomas (NHL), wherein remission can be achieved with classic chemotherapy and hematopoietic stem cell transplantation (HSCT). However, most remission can be temporary. A high risk of relapse which is often observed in refractory disease is still a challenge faced in the treatment of such diseases [1]. After the success of rituximab, a monoclonal antibody targeting CD20, in the treatment of CD20-positive B-cell lymphomas, several studies to find novel therapies have been conducted, and corresponding clinical trials are ongoing. Among these, immune checkpoint blockade, represented by programmed cell-death protein-1 (PD-1) blockade, and chimeric antigen receptor-T (CAR-T) cell therapy have attracted great attention [2]. Nevertheless, on account of the poor expansion and shortterm persistence of T cells and other reasons [3,4], the application of the two novel immunotherapies has been limited. The pathways of glycolysis, exhaustion, and apoptosis are upregulated in non-responders treated with CAR-T cell therapy [5]; moreover, CAR-T demonstrates target-stimulated enhancement of PD-1 expression [6]. Therefore, adding PD-1 blockade to CAR-T therapy may escalate CAR-T function and to some extent, improve prognosis and efficacy. In this review, we will retrospect each mechanism and clinical use, elucidate the possible

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combination and ongoing clinical trials, and discuss further exploration. 2. CAR-T cell therapy 2.1. Establishment of CAR-T cells CAR consists of an extracellular antigen binding domain of a singlechain variable fragment of an antibody, a transmembrane domain, and an intracellular signaling or co-stimulating domain, typically the CD3ξ [2,7]. CAR-T is genetically engineered to specifically recognize and eliminate tumor cells. After the design was initially proposed in 1989 [8], its efficacy and persistence has been advanced by modifying its construction. Single CD3ξ structure from the intracellular signaling domain is characteristic for first-generation CAR-T, which could provide T cells and other effector lymphocytes with antibody-specific recognition and directly render their activation [9]. However, as relevant clinical trials have reported, this design could not produce adequate interleukin-2 (IL2) [8,9] and lacks significant antitumor activity on account of poor CAR-T persistence [10,11]. Hence, second-generation CAR-T, containing one costimulatory domain, such as CD28, 4-1BB, or OX-40, is equipped to deliver not only an activation signal but also a costimulatory signal. Furthermore, third-generation CAR-T, containing two co-

Corresponding author at: No.1, Jianshedong Road, The First Affiliated Hospital of Zhengzhou University, Zhengzhou City, Henan Province, China. E-mail address: [email protected] (M. Zhang).

https://doi.org/10.1016/j.clim.2020.108382 Received 26 December 2019; Received in revised form 5 February 2020; Accepted 9 March 2020 Available online 10 March 2020 1521-6616/ © 2020 Elsevier Inc. All rights reserved.

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W. Song and M. Zhang

generated, producing lower levels of cytokines, expressing higher levels of antiapoptotic molecules, and notably resulting in no CRS or NT in any treated patients, with more than 50% treated patients acquired complete remission (CR) [26]. There are also a number of strategies of overcoming toxicities, such as suicide gene switch, combinatorial target-antigen recognition, inhibitory CAR-T cell therapy, and bispecific T cell engager therapy [47].

stimulatory domains, CD28, in addition to 4-1BB or OX-40, shows strikingly enhanced proliferation and longer persistence than the second-generation CAR-T [12–14]. Nevertheless, due to off-target effects, inadequate T-cell proliferation and persistence, and heightened suppressive tumor microenvironment [15,16], there still remain patients who are uncured, with severe side effects, and insufficient clinical data. Fourth-generation CAR-T is based on the second-generation construction, but additionally expresses cytokines, including IL-2, IL-15, and IL-12 [17]. Furthermore, the fourth-generation CAR-T, also called T cell redirected for universal cytokine-mediated killing (TRUCK), aids the treatment of cancer by enhancing expansion and persistence of the CAR-T and is also effective against virus infections, auto-immune diseases, and metabolic disorders [18]. These advanced generations of CAR-T extend the traditional application of CAR-T cell therapy and can help cure cancer in the future.

3. PD-1 blockade therapy 3.1. Mechanism of blockading PD-1 signaling pathways The immune activation of T cells originated from antigen recognition between T-cell receptor (TCR) and major histocompatibility complex (MHC), is regulated by the balance between co-stimulatory and inhibitory signals, which is also called immune checkpoints [48,49]. Normally, the expression of inhibitory signals is under control. However, when tumor initiates, tumor and nontumor cells from their microenvironment commonly overexpress the inhibitory signals, resulting in immune escape and tumor development [48]. This suppression has been partly reversed by relevant blockade therapy, which has been a therapeutic strategy in treating hematological malignancies [50–54], most typically PD-1 blockade. The inner mechanism of PD-1 blockade is distinct from other blockade inhibitory signals [2]. The role of PD-1 in the immune response to tumors is well known; activated PD-1 recognizes its ligands, PD-L1/PD-L2, and subsequently antagonizes phosphatidylinositol 3kinase (PI3K)/protein kinase B (Akt) and RAS/MEK/ERK pathways, hindering the function of protein kinase C-theta (PKC-θ), glycolysis and ZAP70 phosphatidylinositol, leading to decreased cell cycle progression, T cell activation, effector T-cell development and increased apoptosis of T cells [55,56]. Additionally, the theoretical foundation of PD-1 blockade is that PD-L1/PD-L2 is expressed in a variety of lymphomas [56]. A few studies show that PD-L1 is greatly expressed in Reed-Sternberg (RS) cells of classical HL (cHL) [54], PMBCL [57], primary central nervous system lymphoma (PCNSL), primary testicular lymphoma (PTL) [58], non-germinal center B cell (GCB)-like phenotype of DLBCL [59], and some Epstein-Barr virus (EBV)-associated types [60]. Similarly, PD-L2 expression is also observed in RS cells of cHL, PMBL, PCNSL, and PTL, irrespective of EBV status [56,58,61,62]. (See Fig. 1) Thus, PD-1 has been an ideal target in the treatment of some hematological neoplasms, single or combined with other therapies.

2.2. Clinical application of CAR-T cell therapy in recent years Since CAR-T cell therapy proven to be efficient in many preclinical models, several clinical trials have been conducted for treating hematological malignancies, especially HL, B cell lymphomas and leukemia. We listed partial relevant clinical trials in recent years (Table 1) and extracted diseases and their remission rate of each study. 2.3. Toxicities and limitations of CAR-T cell therapy Although there are promising results observed in clinical trials of CAR-T cell therapy in the context of hematological malignancies, resolving relevant severe adverse effects, such as cytokine release syndrome (CRS) and neurological toxicity (NT) remains a challenge. CRS, the most common acute toxicity of CAR-T cell therapy, occurs due to cytokine release by infused CAR-T or other immune cells, such as macrophages, that may produce cytokines in response to infused CAR-T [33]. The primary cytokines include IL-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-β, IL-2, IL-8, and IL-10, which can be precisely detected in the serum of patients. Generally, patients suffer severe symptoms, affecting a wide variety of organs, including but not limited to fever, rash, tachycardia, hypotension, pulmonary edema, hypoxia, reduced renal perfusion, hepatic damage, anemia, leukopenia and thrombocytopenia [33–36]. Because of the diversity of clinical manifestations during CAR-T cell therapy, it is crucial to accurately grade CRS and select corresponding treatment algorithm. We recommend the grading, which has been modified and revised by Lee et al. [37,38] (Table 2). Based on reported cases [35,39–41], the incidence rate of NT ranges from 0% to 5%, including a range of symptoms, such as cerebral edema, encephalopathy, confusion, agitated delirium, somnolence, abulia, headache, asterixis, autonomic instability, aphasia, focal weakness, vision changes, seizure, allodynia, apraxia, stroke, intracranial hemorrhage, dizziness, dyskinesia, hallucination and restlessness. However, the exact etiology of this syndrome still remains unclear. Similar to CRS, NT has been associated with high CAR-T cell doses, as well as higher peak blood CAR-T cell numbers and serum levels of cytokines, including IL-2, IL-6, IL-15, IL-18, TNF-β, IFN-γ, CRP, ferritin, TIM-3, and granzyme B [42–44]. A study provides evidence that cytokine-induced central nervous system (CNS) endothelial cell activation leading to disruption of the blood-brain barrier plays an early and critical role in this syndrome [45]. Additionally, there is a correlation between severe NT and abnormal laboratory markers, including prolonged thrombocytopenia and elevated prothrombin time, activated partial thromboplastin time and d-dimer, a reflection of disseminated intravascular coagulation [46]. Thus, the above-mentioned mechanism likely contributes to NT with current research progress. Severe side effects seriously impede the therapeutic application of CAR-T cell therapy, and great effort is spent in alleviating or even eliminating them. In a study, a new anti-CD19 CAR molecule was

3.2. Clinical application and limitations of PD-1 blockade Currently, there are two PD-1 blockades approved for clinical use by the Food and Drug Administration (FDA) in the treatment of hematological malignancies: nivolumab and pembrolizumab. Because of their remarkable and long-term responses according to many clinical trials, a range of cancers have been cured. However, any kind of therapy can be a double-edged sword. While we recommend its effectiveness, we also acknowledge its limitations [63]. 3.2.1. Nivolumab Nivolumab has been conducted for the treatment of hematological malignancies since its first application to clinical practice in 2012. While in treating HL, a study reported in 2015 enrolling 23 patients with relapsed/refractory (RR) HL, achieved 87% ORR, consisting of 17% CR and 80% PR. Drug-related adverse events (AEs) of any grade and of grade 3 were 78% and 22% [54]. In 2017, 3 researches were reported concerning treatment of RR HL. They were designed as respectively nivolumab combined with allogeneic hematopoietic cell transplantation (allo-HCT) [64], nivolumab with brentuximab vedotin [65], and nivolumab as a monotherapy for Japanese patients [66], and the corresponding ORR were 95%, 82%, 81.3%. Moreover, the 2

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Table 1 Clinical application of CAR-T cell therapy for treating hematological malignancies in recent years. NCT number

Targeted disease

Interventions

Patients distribution

Phase

Response

Date of publication

NCT02348216 [19]

· · · · · ·

· DLBCL [77] · PMBCL/ transformed FL [24]

I, II

2019

· DLBCL [93] · Pediatric B-cell ALL [14]

II I

· All patients: ORR (82%), CRR (54%) · DLBCL: ORR (81%), CRR (49%) · PMBCL/ transformed FL: ORR (83%), CRR (71%) · All: OS (52%), CR (40%), PR (12%) · 12 months: EFS (46%), OS (63%)

· B-cell lymphoma [11] · B-cell leukemia [4]

I, II

NCT02132624 [22]

· B-cell lymphoma · B-cell leukemia

· Axicabtagene Ciloleucel (CAR-T cell) · Fludarabine · Cyclophosphamide · CTL019 · CD19 CAR T -cells · Fludarabine · Cyclophosphamide · Autologous 3rd generation CD19targeting CAR-T cells

NCT02435849 [23]

· B-cell ALL

· Single dose of CTL019

· B-cell ALL [75]

II

NCT01044069 [24]

· ALL

· Gene-modified T cells targeted

· ALL [53]

I

NCT01416974 [25]

· CLL

· CLL [8]

I

NCT02842138 [26]

· B-cell lymphoma

· Cyclophosphamide · Modified T cells · Autologous anti-CD19 CAR-T cells

I

· DLBCL: CR (31%), PRR (25%), SD (6%), PD (38%) · FL/ transformed FL: CR (25%), PR (37.5%), SD (12.5), PD (25%)

2017

NCT01316146 [27]

· Hodgkin lymphoma · ALCL

· CAR.CD30 T cells

I

· CLL

· Autologous anti-CD19CAR-4-1BBCD3zeta-EGFRt-expressing T lymphocytes

NCT01865617 [29]

· De novo aggressive B cell lymphoma · Transformed large B cell lymphoma · FL · MCL

· Autologous anti-CD19CAR-4-1BBCD3zeta-EGFRt-expressing T lymphocytes

· De novo aggressive B cell lymphoma [11] · Transformed large B cell lymphoma [11] · FL [6] · MCL [4]

I. II

NCT02259556 [30] NCT01815749 [31]

· HL · DLBCL · MCL

· CART30 · Autologous CD19CAR-CD28CD3zeta-EGFRt-expressing Tcmenriched T cells

I I

NCT00924326 [32]

· · · · ·

· Fludarabine · Cyclophosphamide · Anti-cluster of differentiation 19 (CD19)-CAR PBL · Aldesleukin

· HL [18] · NHL1: DLBCL [7], MCL [1] · NHL2: DLBCL [4], MCL [4] · PMBCL [3] · DLBCL [5] · CLL [4] · SMZL [1] · Low-grade NHL [1]

· Hodgkin lymphoma: CR (29%), SD (43%) · ALCL: CR (50%) · All: OS (71%) · Fludarabine, cyclophosphamide and CAR-T [20]: OR (74%), CR (21%), PR (53%) · All: ORR (72%), CR (50%) · De novo aggressive B cell lymphoma: ORR (86%), CR (29%) · Transformed large B cell lymphoma: ORR (83%), CR (83%) · FL: ORR (67%), CR (67%) · MCL: ORR (0%), CR (0%) · ORR (39%), PR (39%), SD (33%) · NHL1: continuing CR/CR (63%), continuing PR/PR (25%) · NHL2: continuing CR/CR (100%)

2017

NCT01865617 [28]

· DLBCL [16] · FL/ transformed FL [8] · High grade B-cell lymphoma [1] · Hodgkin lymphoma [7] · ALCL [2] · CLL [24]

NCT02445248 [20] NCT02443831 [21]

Refractory DLBCL Relapsed DLBCL Transformed FL PMBCL DLBCL Pediatric B-cell ALL

PMBCL DLBCL CLL SMZL Low-grade NHL

I, II

I. II

· · · · · · · ·

· · · · · ·

All: CR (40%) B-cell lymphoma: CR (36%) B-cell leukemia: CR (50%) 6 months: EFS (73%), OS (90%) 12 months: EFS (50%), OS (76%) CR (83%) Median OS (12.9 months) All: ORR (38%), CR (25%)

All: CR (53%), PR (27%), SD (7%) PMBCL: CR (67%), SD (33%) DLBCL: CR (40%), PR (40%) CLL: CR (75%), PR (25%) SMZL: PR (100%) Low-grade NHL: CR (100%)

2019 2019

2018

2018 2018 2018

2017

2016

2016 2016

2015

Abbreviations: DLBCL: diffuse large B cell lymphoma, FL: follicular lymphoma, PMBCL: primary mediastinal B cell lymphoma, ORR: objective response rate, CRR: complete response rate, PR: partial remission, SD: stable disease, PD: progressive disease, MCL: mantle cell lymphoma, CLL: chronic lymphocytic leukemia, SMZL: splenic marginal zone lymphoma, ALL: acute lymphoblastic lymphoma, EFS: event-free survival, OS: overall survival, OR: overall response, ALCL: anaplastic large cell lymphoma.

When referring to multiple myeloma, a multicenter phase I clinical trial was conducted. The ORR was 47% of data available patients, and in the first 30 days, 28.6% of all patients had grade 3 drug-related AEs [71]. To assess the safety and efficacy while treating B cell malignant diseases with the combined regimen of ibrutinib and nivolumab, 141 patients were enrolled in a two-part, phase I/IIa study since 2015. The ORR was 61%, 33%, 36% and 65% in chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), FL, DLBCL and Richter transformation (RT), respectively. The most common grade 3–4 AEs were neutropenia, occurring from 18% in DLBCL to 53% in CLL/SLL, and anemia, ranged from 13% in FL to 35% in RT [72].

nivolumab-induced graft versus host disease (GVHD) after allo-HCT was manageable and most AEs were grade 1–2. For two clinical trials designed for RR HL after autologous stem cell transplantation (ASCT) [67,68], patients were both treated with nivolumab of 3 mg/kg (FDAapproved dose) every 2 weeks and achieved objective response 66.3% and 69% respectively. Similarly, the most common drug-related AEs of grade 3–4 were lipase increases and neutropenia. For the treatment of relapsed acute myeloid leukemia (AML) after allo-HCT, a letter reported the experience regrading efficacy and tolerability with nivolumab in 3 patients in 2016. One patient achieved ongoing CR, one was in a stage of SD, and the rest failed to respond to nivolumab. The only side effects were immune-related pancytopenia and GVHD, which was well-tolerated with lower doses of nivolumab [69]. Another study for newly diagnosed AML or high-risk myelodysplastic syndrome, while treated with combination of idarubicin, cytarabine and nivolumab, the median OS was 18.54 months and 13.6% of patients suffered grade 3–4 immune-related AEs (irAEs) [70].

3.2.2. Pembrolizumab Compared with another PD-1 blockade pembrolizumab, the condition did not greatly improve. For the treatment of HL, especially classic HL, several studies have been conducted since 2013. In a study reported 3

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Table 2 Grading of CRS. Symptoms or signs of CRS

CRS grade 1

CRS grade 2

CRS grade 3

CRS grade 4

Vital symptoms Fever (temperature ≥ 38 °C) Hypotension (systolic blood pressure < 90 mmHg)

Yes No No

Any Needs high-dose or multiple vasopressors FiO2 ≥ 40%

Any Life-threatening

Hypoxia (needing oxygen for SaO2 > 90%)

Any Responds to IV fluids or lowdose vasopressors FiO2 < 40%

Needs ventilator support

Grade 1

Grade 2

Grade 3 or grade 4 transaminitis

Grade 4 except grade 4 transaminitis

Organ toxicities · Cardiac: tachycardia, arrhythmia, heart block, low ejection fraction · Respiratory: tachypnea, pleural effusion, pulmonary edema · GI: nausea, vomiting, diarrhea · Hepatic: increased serum ALT, AST, or bilirubin levels · Renal: acute kidney injury (increased serum creatinine levels), decreased urine output · Dermatological: rash (less common) · Coagulopathy: disseminated intravascular coagulation (less common)

Notes: Grade 5 indicates death. The grading of organ toxicities refers to that of Lee et al. [38]. The CRS grade should be determined at least twice a day, and whenever a change in the patient's status is observed. Abbreviations: SaO2, arterial oxygen saturation; IV, intravenous; FiO2, fraction of inspired oxygen; GI, gastrointestinal; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

achieving 69% of ORR and 22.4% of CR, irAEs and infusion-related reactions were reported in 60 patients (28.6%), and 9 patients (4.3%) discontinued the treatment [73]. In a phase II trial reported recently, with 200 mg of pembrolizumab every 3 weeks in treating the patients of RR cHL after ASCT, 18-month OS was 100% and the toxicity was

in 2014, 31 patients with cHL with brentuximab vedotin failure, with dose of 10 mg/kg every 2 weeks, the CR rate was 16%, for an ORR was 65%. 5 patients (16%) had grade 3 drug-related AEs with no grade 4 AEs or death related to treatment [53]. Another study reported in 2017, 210 patients with cHL treated with dose of 200 mg every 3 weeks,

Fig. 1. Mechanism of activated PD-1 in malignancies. Connected with overexpressed PD-L1/2, PD-1, after recruiting SHP-1 and SHP-1/2 by activated ITIM and ITSM respectively, eventually leads to blocking PI3K/Akt and RAS/MEK/ERK pathways, and dysfunction of PKC-θ, glycolysis and ZAP70 phosphatidylinositol. Abbreviations: PD-1: Programmed cell-death protein-1; PD-L1/2: Programmed cell-death ligand protein-1/2; PD-L1/2: The encoding gene of PD-L1/L2; ITIM: Immunoreceptor tyrosine-based inhibitory motif; ITSM: Immunoreceptor tyrosine-based switch motif; SHP-1/2: Scr homology 2 domain-containing phosphatases; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; PKC-θ: Protein kinase C-theta; MHC: Major histocompatibility complex; TCR: T-cell receptor 4

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Fig. 2. Mechanism of the combination in treating malignancies. T cells can be designed to express CARs to recognize TAA existing on cell surface and then attack malignant cells specifically. Antigen recognition through PD-1-PDL1 axis can activate the inhibitory signal, resulting anergic T cells, immune escape and tumor development. PD-1 blockade can stop this recognition and then refresh the immune system. Moreover, combination with PD-1 blockade will benefit CAR-T cells with PD-1 expression. In recent years studies also designed the CAR-T cells with anti-PD-1 self-secreting or PD-1 silencing. Abbreviations: CAR: Chimeric antigen receptor; TAA: Tumor-associated antigen; PD-1: Programmed cell-death protein-1; PD-L1/2: Programmed cell-death ligand proein-1/2

disorder, liver damage, pyrexia, and fatigue [77].

manageable, 30% of patients experiencing at least 1 grade 3 or higher AEs, while 40% of patients having at least 1 grade 2 or higher irAEs [74]. While in treating PMBCL, in a phase Ib clinical trial started from 2013, enrolling 18 RR PMBCL patients receiving 10 mg/kg every 2 weeks, the ORR was 41% and 35% of patients revealed SD. 11 patients (61%) had drug-related AEs, mostly grade 1–2, and none discontinued treatment due to AEs [50]. Another study reported in 2019 consisting of two clinical trials of RR PMBCL, summarized that the ORR was 48% in 21 patients and 45% in 53 patients, while the treatmentrelated AEs was 24% and 23% respectively [75]. In a study for the treatment of ipilimumab-refractory advanced melanoma, patients were divided into 2 groups: patients who received 2 mg/kg (FDA-approved dose) and those who received 10 mg/kg every 3 weeks. The ORR was 26% at both doses and safety profiles were similar between the two groups. The most common drug-related AEs were fatigue (33% vs 37%), pruritus (23% vs 16%), and rash (18% vs 18%) [76]. Moreover, for the treatment of CLL and RT or with relapsed CLL, the ORR was 44% in patients and 0% in CLL patients. Treatmentrelated grade 3 AEs was observed in 15% patients and were manageable [52]. Generally, the application of PD-1 blockade for the treatment of hematological malignancies is worthy to be confirmed. However, there still remains uncured patients and severe AEs, characterized by T-cell infiltration in various organs, including skin lesion, endocrine organ

4. Combined therapy 4.1. Rationale for combining CAR-T cell therapy and PD-1 blockade Although the two above mentioned novel immune therapies have been met with variable success, relevant limitations still hamper their application, such as short-term clinical remission, severe associated toxicities, and high cost. A study designed CAR-T cells secreting checkpoint inhibitors targeting PD-1 (CAR.αPD1-T) and evaluated their efficacy in a lung carcinoma xenograft mouse model. They found with anti-PD-1, CAR.αPD1T were more expandable, and more effective in tumor regression than the parental CAR-T cells [78]. For the treatment of hepatocellular carcinoma, the CAR-T cells with deficient PD-1 showed greater CARdependent anti-tumor efficacy compared with the wild-type CAR-T cells [79]. Moreover, while in treating hematological malignancies like follicular lymphoma, a case reported in 2019 demonstrated that the unsuccessful CAR-T 19 cell therapy could be saved with a low-dose PD-1 blockade, achieving remission of more than 10 months and reduced adverse effects [80]. Several studies demonstrate that compared with CAR-T-based monotherapy, the combined regimen shows greater strength. Furthermore, there does exist synergy between CAR-T cell therapy and PD-1 blockade for the treatment of malignancies. A study 5

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functional capacity of tumor-infiltrated lymphocyte, and condition of the mouse model to test its efficacy. As shown in the results, the modified CAR-T cells reversed the inhibitory effect of PD-1/PD-L1 interaction on T cell function. PD-1 blockade by continuously secreting anti-PD-1 attenuated the inhibitory T cell signaling and enhanced T cell expansion and effector function both in vitro and in vivo [78]. Furthermore, Rafiq et al. employed CAR-T cells to secrete PD-1-blockading single-chain variable fragments (scFv), which acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematological and solid tumors. The efficacy was similar or even better than that achieved by simple combination of CAR-T cell therapy and PD-1 blockade, with benefits of secreted scFv remaining localized to tumor and protecting CAR-T cells from PD-1 blockade [89]. Moreover, silencing the expression of PD-1 is also an effective strategy. In 2017, Rupp et al. combined Cas9 ribonucleoprotein-mediated gene editing and lentiviral transduction, which have generated anti-CD19 CAR-T cells with PD-1 deficiency, leading to augmented killing of tumor cells in vitro and enhanced elimination of PD-L1+ tumor xenografts in vivo [86]. Similarly, in 2018, Wanghong et al. created a reduced PD-1 CAR-T population, and found that CAR-T cells with PD-1 disruption showed enhanced tumor control and relapsed prevention [88].

was designed for Her-2+ tumor cell lines, and T cell transduced with anti-Her-2 CAR or control vector alone were predominantly CD8+. Interestingly, an increased PD-1 expression was only observed on CD8+ T cells when cocultured with Her-2+ target cells, contrast to the stimulation with parental tumor cells. Moreover, the addition of PD-1 blockade could not only enhance the proliferative and functional capacity of CAR-T cells in vitro, but also promote regression of established tumor in vivo, which would not cause autoimmunity in mouse models [81]. Thus, it is proposed that the stimulation of CAR may escalate the expression of PD-1 inhibitory signaling, and interference of PD-1 pathway may restore the effector function of CAR-T cells, indicating that PD-1 blockade would be an effective strategy in improving the potency of CAR-T cell therapy [78,81–88], which may also exhibit the mechanism of short lives of CAR-T cells after infusion. Also, enhanced therapeutic effects correlated with the combination therapy, were indicated by not only increased function of anti-Her-2 T cells but also decreased percentage of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment [81,84]. While treating metastatic melanoma, the study designed a third-generation CAR-T, GD-2 specific CART, which had highly potent immediate effector functions. Significant activation-induced cell death of the CAR-T cells was observed after repeated antigen stimulation, and enhanced CAR-T survival and increased death of PD-L1+ tumor cell lines were followed by addition of PD-1 blockade [85]. Considering the safety and efficacy of this theoretical combination, there is another attempt of combining in recent years, designing anti-PD-1 self-secreting CAR-T cells in mediating tumor eradication effectively and making CAR-T cells more functional and expandable [78,89]. (See Fig. 2)

4.3. Ongoing clinical trials of combination therapy for hematological malignancies Referring to CliniclTrial.gov, the ongoing clinical trials concerning combination therapy of CAR-T cells and PD-1 blockade are presented in Table 3. Although most relevant trials are designed for solid tumor treatment, which are not shown in the table, two different combined types are applied to treat a variety of lymphomas, especially B-cell lymphomas.

4.2. Different combined types of CAR-T cell therapy and PD-1 blockade Since John et al. first found an increase of PD-1 expression in transduced T-cell population following specific antigen stimulation and increased efficacy of anti-Her-2 T cells with PD-1 blockade, which indicates that blocking PD-1 immunosuppressed signaling can potently enhance CAR-T cell therapy [81], researches began to focus on this theoretical combination in clinical application and improve it continuously. Most studies added PD-1 blockade in CAR-T cell therapy. For example, a study for the treatment of prostate cancer found that adoptive hPSMA-CAR-T cell immunotherapy was enhanced when combined with PD-1 blockade, although the treatment response was of comparatively short duration, implying a sub-optimal regimen [83]. For the treatment of hematological malignancies, in a clinical trial on 11 patients with Bcell NHL, the combination of CD19 CAR-T and nivolumab was feasible and safe and mediated potent anti-lymphoma activity. Not only all toxicities were manageable and reversible, but also, of 11 patients, 9 (81.8%) patients achieved an objective response, and 5 (45.5%) patients achieved CR [90]. Wang et al. also reported a case of refractory follicular lymphoma (FL) treatment, providing well-documented evidence in such disease. After 6 cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) therapy, the patient was diagnosed with refractory FL and enrolled in a clinical trial. After the infusion of CD19 CAR-T cells, a reduced dose of nivolumab was administrated. Until now, the patient maintained CR for 16 months and encouragingly, a decrease dose of PD-1 blockade reduced the adverse effects and simultaneously ensured its efficacy [80]. Furthermore, engineered CAR-T cells with anti-PD-1 self-secreting and silencing PD-1 expression redefines their combination. In 2016, Suarez et al. used a single bicistronic lentiviral vector to develop a new anti‑carbonic anhydrase IX-targeted CAR-T that consists of anti-PD-1 antibodies at the tumor site. Compared with CAR-T cell therapy alone in a humanized mouse model of metastatic clear cell renal cell carcinoma, more tumor growth diminished, and tumor weight reduced even further [91]. Subsequently, the treatment of lung carcinoma, the study measured IFN-γ production, T-cell proliferation, expansion and

4.4. Summary and further exploration Both CAR-T cell therapy and PD-1 blockade have achieved remarkable therapeutic efficacy as a standalone strategy in the treatment of hematological malignancies, regardless of their respective toxicities and limitations. Some studies found it synergistic to combine the two novel immunotherapies that the activation of CAR-T cells would be augmented by immune checkpoint blockade, such as, PD-1 blockade. To verify and better utilize this combined therapy, many preclinical models and clinical trials have been applied. No matter in which type the two novel immunotherapies combine, incorporating PD-1 blockade and CAR-T together or building PD-1 blocking in CAR-T cells, both of them are proven effective in treating refractory and relapsed patients. However, more explorations need to be conducted. Although PD-1 blockade has been approved in the treatment of hematological malignancies, and CAR-T cell therapy has greater efficacy in patients of hematological neoplasms, the clinical application of their combination is still conducted more in solid tumors and B-cell lymphomas. For T-cell lymphomas, lacking optimal treatment and with many patients resistant to existing therapy [92], new strategies are on urgent need. Secondly, the inner mechanism of combination might need further study. Recently, a study suggested that PD-1 silencing would impair the anti-tumor potential of CAR-T cells because it inhibited T cell proliferation [93], which was in conflict with what we have known. And then, when CAR-T cell therapy and PD-1 blockade are combined, most studies focus on the results of employing PD-1 blockade for CAR-T on CAR-T sides, that is, that most subjects accepted CAR-T cell therapy and then PD-1 blockade. If the treating sequence is changed, conditions and corresponding mechanism remain unclear. In addition, a number of immune checkpoint blockades, like cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) blockade, and targets of CAR-T cell therapy may be more effective in contributing this potent combination. Therefore, great 6

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1, 2

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· iPD1 CD19 eCAR T cells · Fludarabine and cyclophosphamide Pembrolizumab

This work was supported by The National Natural Science Foundation is a great foundation of China, (grant no. 8157010926) and Medical Science Academy and the hospital belong to Zhengzhou University. Declaration of Competing Interest The authors declare no conflicts of interests. Acknowledgements The work was supported by the oncology department of the First Affiliated Hospital of Zhengzhou University and the Medical Science Academy of Zhengzhou University, and I would like to show great gratitude to them all.

Phase I/II study of pembrolizumab in patents failing to respond to or relapsing after anti-CD19 CAR-T cell therapy for relapsed or refractory CD19+ lymphomas NCT02650999

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The table refers to ClinicalTrial.gov. Abbreviations: NA: not appliable; MM: multiple myeloma.

Safety and efficacy of iPD1 CD19 eCAR T cells in relapsed or refractory B-cell lymphoma NCT03208556

NCT03287817

MC-19PD1 CAR-T in relapsed or refractory B cell lymphoma Cytoplasmic activated PD-1 CAR-T cells in refractory/relapsed B cell Lymphoma CD19 CAR and PD-1 knockout engineered T cells for CD19 positive malignant B-cell derived leukemia and lymphoma CD19/22 CAR T cells (AUTO3) for the treatment of diffuse large B cell lymphoma NCT03932955 NCT03540303 NCT03298828

· Lymphoma · Relapsed NHL · ALL · Burkitt lymphoma · DLBCL · Relapsed/refractory DLBCL · Relapsed or refractory B-cell lymphoma · CD19+ DLBCL · FL · MCL

Safety and efficiency study of CD19-PD1-CART cell in relapsed/refractory B cell lymphoma Safety and efficiency study of BCMA-PD1-CART cells in relapsed/refractory multiple myeloma Study of PD-1 inhibitors after CD30.CAR T cell therapy in relapsed/refractory Hodgkin Lymphoma NCT04163302 NCT04162119 NCT04134325

· Lymphoma, B-cell · MM · Relapsed/refractory HL

NA

· CD19-specific chimeric antigen receptor T cell with PD1 knockout · CD19-PD1-CART cell · BCMA-PD1-CART cell · Nivolumab · Pembrolizumab · MC-19PD1 CAR-T cells · CAR19 T cells carrying cytoplasmic activated PD-1 · CD19 CAR and PD-1 knock out engineered T-cells · CD19 CAR T-cells · AUTO3 Quikin CD19-CART in patients with r/r B-cell lymphoma NCT04213469

· B-cell lymphoma

Phase Interventions Title

Targeted diseases

efforts and a large number of painstaking researches are indispensable, and we await that the improvement of this rational combination will provide a new outlook for the treatment of hematological malignancies. Funding

NCT number

Table 3 Ongoing clinical trials of combination therapy for hematological malignancies.

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