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Cell therapy is a kind of medicine aiming to cure disease or alleviate disease symptoms via direct infusion or transplantation of cells, which can be autologous or allogeneic. With several decades’ development and optimization, immuno-oncology cells (such as T cells, nature killer cells, etc.), stem cells (embryonic stem cells, induced pluripotent stem cells, progenitor cells, etc.) or other genetic re-engineered cells have been widely applied for cell therapy. Numerous cell types have been translated into clinical trials and promising cell therapy outcomes have been achieved from Phase I, Phase II and Phase III trials for a great number of diseases.

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In the past several decades, great progress has been made in the field of anti-tumor therapy, but there are also multiple strategies for tumors to evade the recognition and efficient suppression by the immune system. Therefore, a variety of immunotherapeutic targets (Fig. 1) and strategies have been developed to reactivate and reorganize the human immune system, such as CAR-T, TCR-T, CAR-NK, DC-CIK, oncolytic virus and so on.

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Figure 1 Immunosuppressive targets for tumor therapy [1].



CAR-T

Chimeric antigen receptor (CAR)-modified T cells (CAR-T) are T cells genetically engineered to express CAR (Fig. 2A) [2,3], which can specifically recognize their target antigen via the scFv binding domain of CAR, resulting in T cell activation to specifically target and destroy tumor cells [3-5]. To date, four generations of CAR have been developed according to the structure of the endodomain (Fig. 2B) [3]. Due to little ability to generate enough interleukin-2 (IL-2), the 1st generation CAR-T cells (such as Ag-specific CD3ζ (MFEζ)-CAR-T cells, alpha-folate receptor (FR) -CAR-T cells, CE7R-CAR-T cells, scFv(G250)-CAR-T cells, GD2- CAR-T cells and CD10- CAR-T cells) benefitted substantially from the combination of cytokines [6], and were used for the treatment of different tumors [7-10]. However, most of the studies using the 1st generation CAR-T cells did not show very satisfactory results because of the inadequate proliferation, cytotoxicity, and insufficient secreted cytokines in vivo. To overcome these shortcomings, the 2nd generation CAR-T cells were designed by adding intracellular signaling domains from various co-stimulatory protein receptors to the cytoplasmic tail of the CARs, [11-13]. The 2nd generation CAR-T cells, such as scFvCD19-CD137-CD3-CAR-T cells, MOv19-BBζ-CAR-T cells and scFvCD19-CD28-CD3ζ-CAR-T cells displayed better curative effects on B cell malignancies [12,14]. On the basis of the 2nd generation CAR-T cells, the 3rd generation CAR-T cells were produced with the addition of multiple signaling domains, such as CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB, to promote cytokine production and killing ability. The 3rd generation CAR-T cells, such as CD20-CD28-CD137-CD3ζ-CAR-T cells and HER2-CAR-T cells were applied for the treatment of lymphoma and colon cancer, but with no better outcomes than the 2nd generation CAR-T cells, and the reasons need to be further studied [15,16]. Based on the 2nd generation CAR-T cells, the 4th generation CAR-T cells were obtained with the addition of IL-12, and can mediate T cell redirected for universal cytokine-mediated killing (TRUCKs). TRUCK T cells show great outcomes in disease therapy by augmenting T-cell activation, and also activating and recruiting innate immune cells to destroy the antigen-negative cancer cells in the targeted lesion, and can also be used for the therapy of viral infections, metabolic disorders and auto-immune diseases [17]. Overall, these successive generations of CAR-T cells have yielded remarkable efficacy in several types of cancer or tumor therapy, and some of them have been translated into clinical trials with few side effects (Table 1) [18].

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Figure 2 Structures of chimeric antigen receptor (CAR) [3]. (A) CAR structure: The CAR contains antigen recognition domain of thesingle-chain Fragment variant (scFv) derived from an antibody, transmembrane domain and an intracellular T cell activation domain of CD3ζ. (B) Evolution of CAR. ITAM: immunoreceptor tyrosine-based activation motifs. CM1: costimulatory molecule.


Table 1. Examples of CAR T-cell therapies in tumors [5]
AntigenDiseaseIn vitro, in vivo, in preclinical or in clinical trialsNCT/Reference
CD19haematologic malignanciesclinical trials[19-22]
CD20haematologic malignanciesclinical trials[23,24]
TRAIL receptor 1LymphomaIn vitro[25]
KappaLymphomaclinical trialsNCT00881920
CD22follicular lymphoma, non-Hodgkin’s lymphomaclinical trialsNCT02315612
HA-1 HLeukaemiaIn vitro[26]
NKG2DLeukaemiaclinical trialsNCT02203825
FAPB cell chronic lymphocytic leukaemiaclinical trialsNCT01722149
ROR1chronic lymphocytic leukaemiaclinical trialsNCT02194374
CD138multiple myelomaclinical trialsNCT01886976
NY-ESO-1multiple myelomaIn vitro[27]
Lewis Ymultiple myelomaclinical trialsNCT01716364
HER2OsteosarcomaIn vitro[28]
HER2Breast cancerIn vitro[29]
HER2SarcomaClinical trialNCT00902044
HER2Metastatic cancerClinical trialNCT00924287
HER2GlioblastomaClinical trialNCT01109095
HER2Solid tumorsClinical trialNCT01935843
CEAColorectal cancerIn vivo[30]
CEAColorectal cancerClinical trialNCT00673322
CEABreast cancerClinical trialNCT00673829
CEALiver metastasesClinical trialNCT01373047
CEAMetastatic cancersClinical trialNCT01723306
CSPG4Melanoma, breast carcinomaIn vivo[31]
EphA2GlioblastomaIn vivo[32]
FROvarian cancerIn vivo[33]
IL-11RαOsteosarcomaIn vivo[34]
IL-13Rα2GlioblastomaPreclinical trial[35]
IL-13Rα2Malignant gliomaClinical trialNCT02208362
IL-13RGliomaPreclinical trial[36]
CD171NeuroblastomaClinical trialNCT02311621
EGFRAdvanced EGFR-positive solid tumorsClinical trialNCT01869166
EGFRAdvanced gliomaClinical trialNCT02331693


TCR-T

T-cell receptor (TCR)–engineered T-cell therapy (TCR-T) is one potentially powerful treatment that utilizes genetically modified natural T cells to specifically target tumors and destroy tumors with greater potentials [37]. Unlike CARs, TCRs rely on their interactions with peptide-major histocompatibility complex (pMHC), formed by peptide [38,39], generated from intracellular antigen proteolysis, bound with MHC (Fig. 3) [40]. Besides TCR, additional costimulatory or co-inhibitory signals are also required for TCR-T function. For example, CD4 on the surface of helper T cells, which binds to class II MHC complex, and CD8 on the surface of cytotoxic T cells, which binds to class I MHC complex, activates the TCR-T mediated cell destruction [41], while cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are responsible for T cell signaling extinguisher [42]. Given that only ~28% antigens are expressed on the cell surface, it is difficult for most of the antigens to be recognized by CAR [43]. Therefore, compared to CAR-T, TCR-T cell therapy shows remarkable advantages over the destruction of tumor cells with intracellular antigens [44]. To date, TCR-T mediated cell therapy has achieved many promising curative potency in both solid tumors and hematological cancers, and some of them have been applied in clinic trials (Table 2).

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Figure 3 Diagrams of CAR-T and TCR-T-cell therapy [45]. Unlike CAR, TCR is a heterodimer containing 2 different transmembrane polypeptide chains: α chain and β chain. The α chain and β chain each consist of a constant region and a variable region, the former anchoring the chain inside the T-cell surface membrane and the latter recognizing and binding to the antigen presented by MHCs [46].


Table 2. Examples of TCR T-cell therapies in tumors [45]
AntigenDiseasePhase of clinical trialsNCT/Reference
PRAMEUnknownPhase I/IINCT03503968
PRAMEAcute myeloid leukemia, myelodysplastic syndromePhase INCT02743611
MAGE-A3/A6UnknownPhase INCT03139370
UnknownHead and neck squamous cell carcinoma, squamous cell NSCLCPhase INCT03139370
MAGE-A10Non-small cell lung carcinomaPhase INCT02592577
NY-ESO-1Synovial sarcomaPhase I/IINCT01343043
NY-ESO-1Multiple myelomaPhase I/IINCT01892293
NY-ESO-1Multiple myelomaPhase I/IINCT01352286
NY-ESO-1Ovarian cancerPhase I/IINCT01567891
NY-ESO-1MelanomaPhase I/IINCT01350401
NY-ESO-1Non-small cell lung carcinomaPhase I/IINCT02588612
AFPHepatocellular cancerPhase INCT03132792
NY-ESO-1Refractory multiple myelomaPhase INCT03168438
MAGE-A10Urinary bladder cancer, head and neck cancer, melanomaPhase INCT02989064


CAR-T vs TCR-T

CAR-T and TCR-T both are gene-engineered technologies targeted for the destruction of tumors by improving the recognition and destroy potentials of T cells, thus called “T cell receptor redirection” technologies. However, there are also some differences in the two technologies: 1) CAR-T recognizes their target antigen via the scFv binding domain of CAR, while TCR-T relies on the interactions with peptide-major histocompatibility complex (pMHC) and requires co-stimulatory od co-inhibitory signals; 2) CAR-T recognizes and targets cell surface antigens, while TCR-T can target both cell surface antigens and intracellular antigens; 3) CAR-T shows great therapy outcomes in the treatment of hematological cancers but little effects on solid tumors, while TCR-T displays encouraging curative outcomes in the therapy of solid tumors.

CAR-NK

Although CAR-T has been applied in clinic, quite a lot of side effects, such as off-target, cytokine release syndrome (CRS) etc., pose a great restriction in their further clinical application [47]. Based on natural killer (NK) cells, CAR-NK cell therapy is much safer than CAR-T therapy and becomes a good candidate for targeting and combating solid tumors [48,49]. Based on the four generation of CAR, CARs in CAR-NK are designed with the addition of personalized proteins, such as DNAX-activation protein (DAP) 12 and NKG2 member D (NKG2D), which show great anti-tumor activity in acute myeloid leukemia (ALL), osteosarcoma, and prostate tumors [50-52]. Therefore, CAR-NK cells not only have the ability of CAR to specifically recognize antigen-expressing tumors, but also can destroy tumors via NK cell receptors. To date, numerous CAR-NK cell therapies have been used for anti-tumor therapy in clinical trials (Table 3).

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Figure 4 Schematic of CAR-NK cell therapy [53]. Gene engineered natural killer (NK) cells are infused into patients to destroy tumor cells. PBMC, peripheral blood mononuclear cell; UCB, umbilical cord blood; ESCs, human embryonic stem cells; iPSCs, human induced pluripotent stem cells.


Table 3. Examples of CAR-NK-cell therapies in tumors [53]
AntigenDiseaseNK sourcePhase of clinical trialsNCT/Reference
CD7Lymphoma,leukemia,NK-92Phase I/IINCT02742727
CD19Lymphoma,leukemia,NK-92Phase I/IINCT02892695
CD33Acute myeloid leukemiaNK-92Phase I/II (complete)NCT02944162
MUC1Solid tumorsNK-92Phase I/IINCT02839954
NRNon-small cell lung carcinomaNK-92Phase INCT03656705
HER2Glioblastoma multiformeNK-92Phase INCT03383978
CD19B-acute lymphocytic leukaemiaPB-NKPhase IINCT01974479
CD19B-acute lymphocytic leukaemiaPB-NKPhase I (complete)NCT00995137
CD19B-lymphomaUCB-NKPhase I/IINCT03056339


Viral vectors used in CAR-T, TCR-T and CAR-NK

The broad application of gene engineered adoptive cell therapy, such as CAR-T, TCR-T and CAR-NK, in clinical trials for the therapy of cancers led to the development of safety-enhanced self-inactivating (SIN) vectors, such as γ-retroviral (gRV), lentiviral (LV) vectors, adenovirus, and AAV vectors. Scalable manufacturing processes for the production of gRV vectors (Fig. 5) and LV vectors using transfection in closed-system bioreactors in compliance with current good manufacturing practices (cGMP) for clinical applications have been developed and optimized [54,55].

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Figure 5 Clinical manufacturing process flow chart of γ-retroviral vectors based on adherent cells [55].


DC-CIK

Cytokine-induced killer cells (CD3+ CD56+ cells, CIK cells) [56] are non-major histocompatibility complex-restricted natural killer T lymphocytes and exhibit stronger anti-tumor and cytolytic activities than lymphokine-activated killer cells [57-59]. Co-cultured with dendritic cells (DCs) or Ag-DC cells, the selective cytotoxicity and anti-tumor immunity of CIK cells would be significantly enhanced, i.e. DC-CIK therapy [60,61]. Compared with traditional treatment, DC-CIK immunotherapy not only improves the clinical indices, but also shows mild adverse and reduces mortality [62].

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Figure 6 Schematic of DC-CIK cell therapy [63]. Co-cultured dendritic cells (DCs) and cytokine-induced killer cells (CIKs) (DC-CIK) are infused into patients for anti-tumor therapy.


Oncolytic virus

Oncolytic virotherapy is a novel therapeutic modality utilizing oncolytic viruses (such as herpes simplex-, pox-, parvo-, or adenoviruses), which selectively replicate in cancer cells, to directly induce tumor cell lysis or indirectly reactivate human immune system to mediate tumor destruction (Fig. 7) [64]. Through decades’ optimization, a great number of oncolytic virus therapies have been put into clinical trials, such as ONYX-015 (E1B-deleted adenovirus) [65], DNX-2401 (Ad5) [66], CG0070 (Ad5 carrying GM-CSF gene) [67], OBP-301 (Ad5-hTERT-E1A/B) [68], G207 (a conditionally-replicative HSV-1 with ICP34.5 deletion and UL39 disruption) [69], and JX-594 (Pexa-Vec, GM-CSF-enhanced vaccinia virus with TK gene disruption) [67]. Among them, Amgen's T-VEC (a modified HSV-1 with expression of GM-CSF but deletions in ICP34.5 and ICP47 genes) was the first oncolytic virus approved by America Food and Drug Administration (FDA) for the treatment of melanoma in 2015 [70]. Moreover, when combined with the other therapy, such as Anti-PD-1 immunotherapy, oncolytic virus can significantly promote immune recognition of cancer cell as well as T cell infiltration, and lead to high response rate in patients with advanced melanoma [71].

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Figure 7 Oncolytic viruses-induced anti-tumor immunity [64]. Oncolytic viruses mediate direct cancer lysis and indirect activation of anti-tumor immune responses.


Table 4. Examples of oncolytic virotherapy against tumors [64]
Oncolytic virusesDiseaseDescriptionNCT/Reference
AdenovirusHepatic carcinomaGolgi protein 73 (GP73) needed[72]
AdenovirusPancreatic carcinoma, prostate cancerTat-PTD modified hexon and Ad5/35[73]
AdenovirusGastric cancerAcetylcholinesterase (AChE)[74]
AdenovirusGastric cancerOBP-301, telomerase-specific[75]
Herpes simplex virus 2Colon cancerNo virus modification or co-therapies[76]
VacciniaColon cancerViral Thymidine kinase (TK) deficiency[77]
Measles virusHepatocellular carcinoma, colon cancerRetargeted to CD133[78]
Herpes simplex virusOvarian cancerInterleukin−12[79]
ONYX-015 (adenovirus)Malignant mesothelioma cellsEB1 gene deletion[65]
DNX-2401 (adenovirus)Glioblastoma and gliosarcomaNCT02197169
CG0070 (adenovirus)Bladder cancercarrying CM-CSF geneNCT02143804, NCT02365818
OBP-301 (adenovirus)carrying hTERT-E1A/BNCT0229385
HF10 (Herpes simplex virus)Solid superficial malignant tumorsdeletions in neurolatency genes UL43, UL49.5, UL55, and UL56NCT02428036
G207 (Herpes simplex virus)Glioblastomaa conditionally-replicative HSV-1 with ICP34.5 deletion and UL39 disruptionNCT00157703
JX-594 (Vaccinia Virus)Hepatocellular Carcinoma, colorectal cancerGM-CSF-enhanced vaccinia virus with TK gene disruptionNCT02562755
GL-ONC1 (Vaccinia Virus)Head and neck cancerTK-inactivatedNCT01443260
Reolysin (Retrovirus)Metastatic melanoma or pancreatic cancerNCT02514382, NCT02444546
Cavatak™ (Coxsackievirus A21)MelanomaOverexpress ICAM-1 and DAFNCT02565992, NCT02316171


Taken together, different kinds of immune-oncology cell therapy show great variety in their curative effects. Although promising therapeutic outcomes in acute lymphocytic leukemia (ALL) and large B cell lymphomas have been achieved by CAR-T [80,81], little therapeutic effects were acquired in solid tumors as well as tumors with intracellular antigens, and multiple side effects, such as cytokine release syndrome, prohibited the broad application of CAR-T [82-84]. TCR-T displays better effects for the treatment of hematopoietic cancers and solid tumors, but requires MHC and co-stimulatory or co-inhibitory signals to combat tumors [45]. Without the drawback of CAR-T, CAR-NK is safer than CAR-T and presents excellent tumor elimination ability in both hematopoietic cancers and solid tumors via CAR-dependent and NK receptor-dependent mechanisms [53]. DC-CIK immunotherapy shows improved immunologic function, reduced mortality, and mild adverse effects in both hematologic malignancies and solid tumors, and may be suitable for patients that are intolerance to radiotherapy and chemotherapy [62,85]. Without the need of defined antigens included in the vectors, oncolytic viruses show a durable anti-tumor effects by directly infecting and lysing tumor cells in situ and also activating the host immune response to eliminate tumor cells, thus displaying curative effects on both hematopoietic cancers [86,87] and solid tumors [64]. Moreover, these immune-oncology cell therapies can also be combined with programmed cell death protein 1 (PD-1) antibody or PD-1 inhibitor, showing reduced drug toxicity and enhanced the tumor cytotoxicity [88]. For example, CD19-CAR-T cell carrying the single-chain variable fragment (scFv) of antibody against PD-1 exhibited potent therapeutic effects superior to those of conventional CAR-T cells [89]; oncolytic Herpes Simplex Virus Type 2 encoding an antibody against PD-1 presented an enhanced therapeutic efficacy with a durable antitumor response both in the tumor microenvironment and in the systemic immune system [90].



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