Source: eMedClub

Compared with CAR-T cells, CAR-NK cells have attracted much attention because of their multiple killing mechanism, higher safety and a wide range of sources. Current studies have proved their safety and feasibility. NK cell-mediated immunotherapy has become a convenient and effective method for cancer therapy.

Different from the killing mechanism of T cells, NK cells can initiate the killing process only by recognizing the target cells. The killing activity of NK cells is regulated by both inhibitory and activating receptors on the cell surface. When the balance between activation and inhibition is broken, NK cells will perform corresponding functions.

When enough inhibitory ligands are expressed on the surface of target cells, the function of NK cells is inhibited, while when the expression of active ligands on the surface of target cells is up-regulated, or when the expression level of inhibitory ligands on the surface of target cells is down-regulated, the killing function of NK cells is activated.

NK cells mainly kill target cells through three mechanisms: 1. Directly kill target cells by releasing cytoplasmic particles containing perforin and granzyme. The release of cytokines, such as IFN- γ and TNF- α, induces tumor cell apoptosis by interacting with corresponding receptors on the tumor cell surface, and 3.Fc receptor CD16 binds to the Fc segment of the antibody, which can stimulate and mediate antibody-dependent cytotoxicity (ADCC) to kill cells.

Mechanism of tumor cytotoxicity mediated by NK cells

Induced pluripotent stem cells (iPSC) were formed by introducing transcription factors into cells by Shinya Yamanaka in 2006. after combining with NK cells, the derived iPSC-NK has a more mature phenotype and effective cytolytic activity, and iPSC provides a homogenous CAR-NK cell population that can be expanded to clinical scale. As a result, CAR-iNK cells have become an iconic "existing" product for cancer immunotherapy.

iProduction of PSC-derived CAR-NK cells

There are the following strategies to enhance the function of iPSC-NK cells

Fusion builders enhance security

IL-15 is a necessary condition for the survival of NK cells, but in the process of NK cell culture, it will lead to the expansion of peripheral NK cells and induce the expression of IFN- γ, which leads to systemic toxicity. Therefore, the researchers take advantage of the easy genetic modification of iPSC, introduce specific fusion constructors (IL-15RF) into iPSC, and further enhance the function of NK cells by modifying iPSC, thus enhancing the toxicity and persistence of iPSC-NK cells.

Introducing CD16a to improve Survival rate

NK cell-mediated ADCC is regulated by the receptor CD16a. The receptor CD16a with high affinity and unable to cleavage is introduced into iPSC to improve the resistance of iPSC-NK cells to activation-induced CD16a cleavage and ADCC effect, and significantly improve the survival rate of malignant hematological diseases and solid tumor xenotransplantation models.

Gene editing means

As a bifunctional extracellular enzyme, CD38 has both cyclase and hydrolase activity, which widely acts on the surface of many kinds of immune cells. Through specific gene editing, NK cells have strong innate immune function, ADCC, persistence and metabolic and transcriptional characteristics related to adaptive NK cells, showing significant anti-tumor activity. All kinds of evidence are supporting medical institutions at home and abroad to further develop iPSC-NK cells for tumor immunotherapy.

How to further improve the tumor targeting and cytotoxicity of iPSC-NK cells by using CAR engineering

CAR-iNK cell therapy

Chimeric antigen receptors can recognize specific antigens on tumor cells and activate downstream signal transduction, which has been developed to further improve the cytotoxic function and anti-tumor effect of NK cells. while avoiding risk, CAR-iNK cell therapy also retains the anti-tumor activity of CAR-T cell therapy. In the case of xenotransplantation, the release of cytokines is less, and the overall anti-tumor activity in vivo and in vitro is higher. The clinical efficacy of CAR-iNK cells has greatly encouraged researchers at home and abroad. Clinical trials of CAR-iNK cells from various sources have been carried out. It is expected that CAR-iNK cell therapy will bring immune cell therapy into a new era.

Construction of iPSC cells with CAR for immunotherapy

iPSC can be genetically engineered (CAR-iPSCs) with CAR. At present, most CAR structures used in NK cells are designed to enhance the activation and proliferation of T cells. in order to optimize the function of these NK cells, a foreign research institute designed and screened a NK cell-specific CAR structure (NK-CAR). Compared with CAR-T cells, NK-CAR modified iPSC-NK (NK-CAR-iNK) cells can effectively inhibit growth and prolong survival time in ovarian cancer xenotransplantation model, and avoid weight loss, organ injury or cytokine release syndrome. These results show that NK-CAR-iNK cell therapy is safer and more effective than traditional CAR-T therapy. Therefore, the appropriate CAR construction plays an important role in improving the anti-tumor ability of CAR-iNK cells.

CAR-iNK cells under Multifunctional equipment

In addition to using CAR to design iPSC, many research institutions are also trying other directions of genetic modification to further improve the function of CAR-iNK cells. A foreign institution has created a triple genetically modified CAR-iNK (iDuo-NK) cell to show a lasting response in lymphoma and leukemia. In order to further explore the application of CAR-iNK cells in multiple myeloma, the agency also designed a tetraploid gene-edited CAR-iNK cell, called iDuo-MM-CAR-NK cell. IDuo-MM-CAR-NK cells showed sustained and lasting anti-tumor effect in vivo and in vitro, and its anti-tumor effect was similar to that of CAR-T cells targeting BCMA, but there were no complications such as GVHD. More importantly, when combined with Isatuximab (anti-CD38 monoclonal antibody), iDuo-MM-CAR-NK cells maximized the activity of ADCC and had a significant anti-tumor effect on MM.

Recently, Century Therapeutics has developed a CAR-iNK cell product that has been genetically modified six times to improve durability, functionality and safety. Phase I clinical trials of this CAR-iNK cell therapy for recurrent or refractory differentiated CD19-positive B-cell malignant tumors are being recruited to evaluate its safety, pharmacokinetics and preliminary efficacy.

Challenges in clinical application of CAR-iNK cell therapy

The production of iPSC in accordance with cGMP is a necessary condition to meet the clinical standards. First, somatic cells used to derive iPSC were obtained and tested for related infectious disease pathogens or diseases (RCDAs). Secondly, CAR-iNK cells must be produced through robust, repeatable and cGMP-compliant manufacturing processes.

Strategies for improving the anti-tumor activity of CAR-iNK cells in solid tumors

In recent years, many preclinical and clinical studies have proved the feasibility and effectiveness of CAR-iNK cells in hematological malignant tumors. However, the efficacy of CAR-modified immune cells in solid tumors is hindered by tumor antigen heterogeneity, limited tumor invasion and immunosuppressive tumor microenvironment (TME). In the past few years, several methods have been developed to improve the expansion, persistence, resistance to TME and metabolic adaptability of CAR-T/NK cells. These methods can also be used in CAR-iNK cell therapy.

Improve the expansion and persistence of CAR-iNT cells

As an important cytokine in the proliferation and maintenance of NK cells, IL-15 has been widely used to improve the persistence and cytotoxicity of CAR-iNK cells. In addition to IL-15, memory-like [ML] NK cells induced by IL-12 and IL-18 cytokines also showed enhanced anti-tumor activity and improvement in vivo. In addition, IL-21 plays an important role in the proliferation and maturation of NK cells. The researchers also found that knocking out HLA-I and II from iPSC can evade the recognition of allogeneic T and NK cells, thus promoting the sustainability of iPSC-based regenerative medicine applications.

Strategies to enhance the activity of CAR-iNK cells

Enhance the infiltration of CAR-iNK cells in solid tumors

The insufficient infiltration of CAR-iNK cells in solid tumors is an important factor that limits the therapeutic effect. The researchers refer to the strategy that CAR-T cells secrete matrix-degrading enzymes such as heparanase to promote tumor invasion and anti-tumor activity of CAR-T cells, which can be used in CAR-iNK cell therapy to enhance its delivery to different solid tumors.

Increase the tumor target of CAR-iNK cells to overcome tumor heterogeneity

Antigen loss and tumor heterogeneity are important factors leading to cancer recurrence. Targeting multiple antigens to cancer cells is one of the methods to prevent antigen loss after cell therapy in CAR. In addition to the combination therapy with CAR-T cells targeting different antigens, many institutions have developed bispecific CAR. For example, the multiple antigen initiation and killing recognition pathway mediated by synthetic Notch (synNotch) receptors developed abroad can achieve complete and controllable tumor cell killing by targeting homogenous but non-absolute tumor specific antigens. These techniques may provide a similar scheme for CAR-iNK cells to successfully target heterogeneous tumors while minimizing toxicity inside and outside the tumor.

Genetically engineered CAR-iNK cells overcome immunosuppressive tumor microenvironment (TME)

CAR-iNK cells must face adverse metabolic conditions including hypoxia, acidic pH and hypoglycemia, as well as immunosuppressive cells that may impair the function of NK cells. Transforming growth factor β (TGF- β), as one of the most important immunosuppressive cytokines, usually damages the function of NK cells in many ways under hypoxia. CAR-iNK cells with TGF- β antibodies or inhibitors are expected to improve the resistance of NK cells to immunosuppressive TME. In addition to TGF- β, adenosine is another key immunosuppressive metabolite, which limits the activation of cytotoxic lymphocytes and impairs the anti-tumor immune response. Ablation of adenosine signal transduction or administration of A2A adenosine receptor (A2AR) antagonist can enhance anti-tumor immunity mediated by NK cells. Therefore, blocking or switching these immunosuppressive signaling pathways in CAR-iNK cells is helpful to overcome immunosuppressive TME.

Regulation of metabolic fitness of CAR-iNK cells and prevention of its depletion

Cell metabolism plays a key role in regulating the function of NK cells. The researchers improve the metabolic fitness by activating the mTOR signal pathway, thereby enhancing the expansion and anti-tumor function of iPSC-NK cells. In addition, the up-regulation of inhibitory receptors also contributes to the immune tolerance of NK cells, but these strategies to improve the function of CAR-iNK cells need to be further verified in clinical trials.

Application of CAR-iNK cell therapy in combined therapy

CAR-iNK cells can be used in combination with TKIs, radiotherapy and monoclonal antibody to further enhance their anti-tumor effect.

Summary

The success of CAR-mediated immunotherapy in hematological malignant tumors and some solid tumors has promoted the development of CAR-iNK cells for cancer immunotherapy. The advantages of iPSC provide many opportunities to enhance the function of CAR-modified iPSC-derived NK cells, making it a sharp knife against hematological malignant tumors and solid tumors. In addition, iPSC comes from a wide range of sources, and the proliferation potential of reprogramming in vitro is considerable, which significantly reduces the production cost and makes CAR-iNK cells more beneficial to clinical application.

Reference: https://www.biospace.com