1.3.1 Progress of Target Tumor Cells
Chimeric antigen receptor (CAR) is the core component of CAR-T, which endows T cells with the ability to recognize tumor antigens in a HLA-independent manner and enables them to recognize more extensive target antigens than natural T cell surface receptor (TCR).A basic CAR includes a tumor-associated antigen (TAA) binding domain (usually from the scFv fragment of antigen-binding region of the monoclonal antibody), an extracellular hinge domain, a transmembrane domain and an intracellular signal domain [3].
The activation of T cells mediated by the first generation of CAR is accomplished by the tyrosine activation motif on CD3ζ chain or FcεRIγ.CD3ζ chain can provide “signal I” for T cell activation,cytolysis, regulation of IL-2 secretion and anti-tumor activity in vivo.However, the anti-tumor activity of the first generation of CAR modified T cells is limited in vivo, and the decreased proliferation of T cells ultimately leads to apoptosis.The second generation of CAR adds a new costimulatory signal in the intracellular region, which enlarges the original “signal Ⅰ” derived from TCR/CD3 complex.Many studies have shown that compared with the first generation of CAR,the second generation of CAR carrying “signal Ⅱ” has the same antigen specificity, increased T cell proliferation and cytokine secretion, enhanced secretion of anti-apoptotic proteins, and delayed cell death.The ubiquitously used costimulatory molecule is CD28, which have been gradually been replaced with CD137 (4-1BB).In addition, an idea of using NK cell receptor CD244 has also been proposed to promote sustained activation and proliferation of CAR-T cells [4].
In order to further improve the design of CAR, many studies began to focus on the development of the third generation of CAR, including not only “signal Ⅰ”, “signal Ⅱ”, but also additional costimulatory signals.Studies using different targets and costimulatory signals were conducted to compare the results of the second and third generations of CAR and obtained quite encouraging experimental results.Combination of CD28 and 4-1BB costimulatory signaling domains is to construct a CAR specific for prostate-specific membrane antigen (PSMA), and then induce the strongest PI3K/Akt activation and Bcl-XL expression in vitro, and the least apoptosis in transduced peripheral blood CD8+ T cells [5].To target a different tumor marker, MUC1, a CAR is designed for containing a fused CD28/OX40/CD3ζ endo-domain, the engineered CAT-T cells and upon MUC1 stimulation can secrete proinflammatory cytokines indicative of both type-1 (IFN-γ) and Th17(IL-17) differentiation in vitro [6].It was noteworthy that IL-17 has been known as tissue destructive cytokine in autoimmune disease animal models, although its anti-tumor effect is still to be elucidated.
It is still uncertain which design is better between the second generation and the third generation of CAR.Additionally, the second and third generations of CAR have their own on-going clinical trials in the US, China and Europe, and the development and outcome of these clinical trials are being closely watched [4].
Immunotherapy with CAR-T cells has achieved tremendous successes in treatment of hematological malignancies.Two CD19-targeting CAR-T cell products, Kymriah from the Novartis(East Hanover, NJ USA) and Yescarta from the Kite Pharma (Santa Monica, CA USA), have been approved by the Unite States Food and Drug Administration (US FDA) for treating B cell acute lymphoblastic leukemia (B-ALL) and diffusing large B-cell lymphoma (DLBCL), respectively [7].However, due to intricacies of solid tumors and their locations in the human body, treatment of solid tumors with CAR-T cells is facing multiple obstacles, such as the hostile tumor microenvironment, on-tumor/off-tumor toxicities, and undesired antigen specificity.Many strategies and approaches have been tried to overcome these obstacles, including arming CAR-T cells with knock-out of PD-1 expression or secretion of cytokines/chemokines and using CAR-T cells in combination with other treatments.Despite these efforts, there are still no CAR-T cells clinically approved for solid tumor treatment so far.Encouragingly and optimistically, in this landscape, more than forty clinical trials in treatment of solid tumors by CAR-T cells have been registered in China alone [4, 8, 9].
As innate immune cells, natural killer (NK) cells are unique and play pivotal functions in cancer immune surveillance.NK cells can eliminate a variety of abnormal or stressed cells without prior sensitization, and even preferentially kill stem-like cells or cancer stem cells.Upon forming immune synapses with target cells, NK cells release preformed cytolytic granules, including perforin, and granzymes, of which function is to induce cell lysis.Several studies have successfully exploited adoptive transfer of NK cells against various tumors, especially hematological malignancies [10].(https://www.daowen.com)
Adoptive transfer of autologous NK cells expanded ex-vivo for treatment patients with lymphoma,colon cancer, breast cancer and lung cancer have been tested in a range of clinical trials.Only very limited antitumor effect was observed.The major reason was that the inhibitory receptors on autologous NK cells matched self MHC class I presented on tumor cells, and this self-recognition signals subsequently inhibited the activation of NK cells.Besides, autologous NK cells derived from cancer patients were actual in an immune suppression state with impaired functions, making these cells difficult to exhibit antitumor capability.The first piece of evidence showing that NK cells had a clinical benefit was reported in 2002.It has been confirmed that donor vs recipient NK cell alloreactivity, which was mainly resulted from KIR ligand incompatibility, could avoid relapse and graft rejection without GVHD in AML patients receiving HLA (Human leukocyte antigens)mismatch donor hematopoietic transplantation.Later, this strategy was used in adoptive cellular immunotherapy of ex vivo activated allogeneic KIR/KIR ligand mismatched NK cells derived from PBMC into AML patients.Subsequently, alloreactive PBMC derived NK cells have been widely investigated as an immunotherapy in clinical trials of hematologic malignancies, as well as in trials of solid tumors including melanoma, breast cancer, ovarian cancer, neuroblastoma, renal cell carcinoma, colorectal cancer, and hepatocellular cancer [11, 12].
Target-activated NK-92 (ta-NK) cells, another engineered variant of NK-92 cells modified with CARs, have been designed to target TAAs-expressing tumor cells.The stable CAR expression and activity of these cells have been demonstrated by HER2.taNK (HER2-specific target activated NK),a cell line that is now being tested in patients with recurrent HER2-positive Glioblastoma(NCT03383978).Unlike immunostimulatory strategies which involve the delivery of molecules to assist NK cells, genetic modification strategies induce changes in the genetics of NK cells directly,leading to far-reaching and sustained changes to the cells.Among them, genetic modification of NK cells with CAR constructs has drawn increasing attention.CAR-NK cells can be produced from different sources of NK cells including primary NK cells, NK cell lines, and HPSCs.CAR-NK has adopted the basic structural framework of CAR-T, that is, chimeric antigen receptors mainly composed of extracellular, hinge, transmembrane and intracellular domains, as well as the transfection methods.The extracellular domain can bind tightly to tumor-associated antigens expressed on the surface of tumor cells, which determines the specificity of CAR structures.Single-chain variable fragments (ScFvs) are the most currently used ectodomains for CARs.The hinge domain is the connecting sequence between the extracellular domain to the transmembrane domain, which endowed CAR with adequate orientation and flexibility to bind to tumor antigens and are expected to impact the CAR-NK activities.The transmembrane domain lies between the hinge and the intracellular signaling domain, including CD3ζ, HLA-A2, or CD28 molecules.The structure of the intracellular signaling domain determines the intensity of the CAR-NK activation signal, which contains the immunoreceptor tyrosine-activated motifs (ITAMs).The majorities of current CAR endo-domains contain an activation region derived from CD3ζ, which is the most classical intracellular domain including three ITAMs.In order to increase the proliferation and cytotoxicity of CAR modified effector cells, co-stimulatory protein receptors such as CD28, 4-1BB,CD134, ICOS are added to the cytoplasmic tail [10, 13, 14].
The first CAR used in NK cells is a CD4-CD3ζ (CD4ζ) fusion receptor [15].The CD4ζ chimeric receptor is biochemically and functionally active and can guide human NK cells efficiently to kill either HIV-infected CD4+T cells or NK-resistant tumor cells expressing gp120 in vitro, indicating CAR structure can be successfully expressed on NK cells, leading to efficient retargeting.Subsequently, more attempts were taken to enhance the anti-tumor ability of NK cells [15].CAR-NK cells have been evaluated for the treatment of hematological cancers and solid tumors in preclinical studies with numerous ideal results.Based on these findings, the clinical translation of CARNK cells has witnessed significant interests.Although CAR-NK therapy is still under clinical evaluation, NK cells possess several advantages over T cells in being engineered to express CARs and used for cancer treatment.First, NK cells are easy to be isolated and have a relatively short lifespan.Therefore, the risk of overexpansion of transferred CAR-NK cells in patients is relatively low.Second, the cytokines secreted by NK cells mainly include IFN-γ and GM-CSF, which are relatively safer than those released by activated CAR-T cells, e.g., TNF-α and IL-6.In particular,the proinflammatory cytokines produced by CART cells may cause life-threatening cytokine release syndrome (CRS), a most common and severe side effect of CAR-T therapy.Third,CAR-NK cells could trigger the lysis of target cells in both CAR-dependent and CAR-independent manners, which further potentiating their killing activity.In addition, CAR-NK therapy is expected to be less expensive, considering that NK cells can be derived from PBMCs, NK cell lines and hPSCs.However, T cells used for CAR-T therapy are required to be autologous [10, 15, 16].
Currently, cancer immunotherapy strategies are dominated by immune cells.However, there are limitations when using immune cells directly for the treatment of cancers.For instance, immune cells can penetrate hardly into the solid tumor, leading to unsatisfactory therapeutic effects.Moreover, the cost for producing, preserving and transporting clinical grade living immune cells is high, posing a challenge to wide applications in clinics.In recent years, EVs, a nano-sized vesicle naturally secreted by many different types of cells including NK cells, have gradually been proposed and studied, providing a new cell-free immunotherapy avenue (Figure 1.1) [10].
Figure 1.1 V arious NK cell-based immunotherapy approaches.(A) Administration of stimulatory cytokines and antibodies to patients triggers activation and expansion of the autologous NK cells and enhance their cytotoxicity.(i) Cytokine.“Super” agonist of IL-2 improves its affinity for IL-2/15Rβ.Arrow width indicates expected intensity of IL-2 signaling.The “super” agonist of IL-15 mimics the physiological trans-presentation of IL-15 to NK cells without the involvement of antigen-presenting cells.(ii) Antibodies.Binding of CD16 to the Fc portion of TAA mAbs leads to NK cells activation and ADCC.Application of BiKE or TriKE target to CD16 or NKG2D (on NK cells) and tumor antigens promotes the formation of immune synapses between NK cells and tumor cells.mAbs against inhibitory receptors on NK cells facilitate NK cytotoxicity.(B) Adoptive transfer of NK cells.(i) NK cells obtained from PBMCs, NK cell lines or hPSCs can be infused into patients directly or in conjunction with immune stimulants.(ii) NK cells are designed to express CAR, which are then allowed to expand ex vivo before being transfused back into the patient.(C) Infusion of engineered NK cells and NK/CAR-NK cell derived EVs.Culture medium of expanded NK cells or CAR NK cells can be exploited to isolate EVs and then infused into the patient.TAA, Tumor-associated antigen; PD-L1,Programmed death ligand-1; ECM, Extracellular matrix.(Please scan the QR code on the Preface to get original color figures.)
Source: Hu W, Wang G, Huang D, Sui M, Xu Y.“Cancer Immunotherapy Based on Natural Killer Cells:Current Progress and New Opportunities”.FrontImmunol, 2019, 10:1205.