Car-NK Cell Therapy Targets - In-depth Overview

1. What is Car-NK therapy?

Technical Background:

Human natural killer cells (NK) make up 15% of all circulating lymphocytes. NK cells were discovered in the 1970s and are mainly involved in the killing of infected microorganisms and malignantly transformed allogeneic and autologous cells. NK cells exhibit antitumor cytotoxicity without prior sensitization and produce cytokines and chemokines that regulate various immune responses. Human peripheral blood NK cells can be divided into two categories: CD56bright and CD56dim. CD56brightNK cells are generally considered low-cytotoxic cytokine-producing cells, while CD56dimNK cells are potentially cytotoxic. Since NK cells can recognize and decompose tumor cells, the field of NK cell-based tumor immunotherapy has reached an exciting juncture.

Mechanism of action of CAR-NK cell therapy

CAR-NK is an enhanced version of NK cells, which means that NK cells are genetically modified with a chimeric antigen receptor (CAR), endowing NK cells with the ability to target and recognize tumor cells, and inject them into the human body after in vitro expansion to achieve the effect of tumor treatment . Chimeric antigen receptors can significantly improve the specificity of NK cell efficacy, which is similar to the construction of CAR-T. The CAR structure includes an extracellular recognition domain (such as scFv) to recognize tumor-specific antigens, a transmembrane domain and an intracellular signaling domain (CD3ζ chain). The modified immune cells are like missiles equipped with infrared guidance function, which can identify tumor cells and achieve precise killing.

In addition to inhibiting cancer cells by recognizing tumor surface antigens through single-chain antibodies, NK cells can also inhibit cancer cells by recognizing various ligands through a variety of receptors, such as natural cytotoxicity receptors (NKp46, NKp44 and NKp30), NKG2D and DNAM-1 (CD226). These NK cell receptors often recognize stress-inducing ligands expressed on immune cells or tumor cells under chronic therapeutic stress. Furthermore, NK cells induce antibody-dependent cytotoxicity through FcγRIII (CD16). Therefore, CAR-NK cells can inhibit cancer cells through CAR-dependent and NK cell receptor-dependent pathways, thereby eliminating tumor antigen-positive cancer cells or cancer cells expressing NK cell receptor ligands.

Therapeutic Scope of CAR-NK Cell Therapy

As an emerging immune cell therapy, CAR-NK cell therapy is currently mainly used in the treatment of malignant tumors. The following is the therapeutic range of CAR-NK cell therapy in different cancer types:

1. Hematological malignancies: CAR-NK cell therapy has made significant progress in hematological malignancies. Especially in the treatment of diseases such as acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma, CAR-NK cell therapy has shown potential therapeutic effect.
2. Solid tumors: Although CAR-NK cell therapy has been relatively less studied in the treatment of solid tumors, there are some preliminary studies showing its potential in the treatment of solid tumors. CAR-NK cell therapy is being explored for the treatment of solid tumors such as breast cancer, lung cancer, gastrointestinal tumors, liver cancer, and ovarian cancer.
3. Important viral infections: In addition to cancer treatment, CAR-NK cell therapy is also being studied for the treatment of important viral infections. For example, CAR-NK cell therapy against human immunodeficiency virus (HIV) is being investigated to find new ways to combat this virus.

It should be noted that CAR-NK cell therapy is still in the clinical research stage and still faces some challenges and limitations in the treatment of different cancer types. The scope and results of clinical studies will further determine the therapeutic scope of CAR-NK cell therapy. Before considering the use of CAR-NK cell therapy, patients should consult a medical professional to understand its scope of application and potential efficacy.

Advantages of CAR-NK Therapy

(1). High security

CAR-NK cell immunotherapy has a better safety profile than CAR-T cell immunotherapy. One of the major side effects of CAR-T cell immunotherapy is the on-target effect due to the persistence of CAR-T cells. On the contrary, due to the short lifespan of CAR-NK cells, they disappear after functioning, which prevents the activated cells from killing normal tissues due to their existence in the body for too long. In addition, NK cells do not secrete IL-1, IL6 and other inflammatory factors, but produce IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF), which makes the pro-inflammatory cytokines secreted by CAR-T cells such as INF The risk of cytokine storm (CRS) induced by -γ, TGF-α, IL-1 and IL-6 factors is reduced. At the same time, the infusion of allogeneic NK cells was well tolerated and did not cause graft-versus-host disease (GVHD) and obvious toxicity.

(2). Broad-spectrum anti-cancer

NK cells do not require tumor-specific recognition and are not limited by the inhibitory activity of the major histocompatibility complex (MHC) on the cell surface. In addition to inhibiting cancer cells by recognizing tumor surface antigens through single-chain antibodies, NK cells can also inhibit cancer cells by recognizing various ligands through a variety of receptors, such as natural cytotoxicity receptors (NKp46, NKp44 and NKp30), NKG2D and DNAM-1 (CD226). These NK cell receptors often recognize stress-inducing ligands expressed on immune cells or tumor cells under chronic therapeutic stress. In addition, NK cells can also clear tumor cells through the ADCC mechanism mediated by CD16. Clinical trials have shown that CAR-T cells cannot eliminate highly heterogeneous cancer cells, but CAR-NK cells can effectively kill residual tumor cells that may change their phenotype after long-term treatment.

(3). Good curative effect on solid tumors

A major obstacle to current CAR therapy is the lack of efficacy against solid tumors due to poor tumor perfusion, TAA heterogeneity, and the concomitant immunosuppressive tumor microenvironment (TME) of solid tumors. Due to the expression of PD-1 in the tumor microenvironment, the function of CAR-T cells is inhibited, but the level of PD-1 secreted by NK cells is low, and the immunosuppressive effect is less.

(4). Easy to prepare

NK cells are very abundant in clinical samples and can be generated from peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (HESC), induced pluripotent stem cells (IPSC), and NK-92 cell lines. Among them, the preparation of iPSC requires fewer seed cells, can be cultured in large quantities, has low cost, can realize autologous supply, and has low immunogenicity, while NK92 cell line can proliferate indefinitely and is less sensitive to freeze-thaw.

(5). Can be used as a general-purpose (Off-the-Shelf) product.

Based on the high safety of NK cells, a great value of NK cell therapy is that it can be used as a universal ready-to-use product, which is suitable for different individuals, can be frozen for a long time, can provide timely treatment for critically ill patients, and can effectively reduce production costs and Terminal selling price, thereby reducing the pressure on patients' out-of-pocket payments and medical insurance payments.

2. The process of CAR-NK cell therapy

The process of Car-NK cell therapy usually includes the following steps:

1. Collection of natural killer cells (NK cells): First, natural killer cells (NK cells) are collected from the blood or bone marrow of the patient or a donor. These cells have a natural ability to recognize and attack cancer cells.
2. Isolation and activation of NK cells: The collected cells will be processed in the laboratory, using specific techniques to isolate NK cells, and activate their anti-cancer ability through activation methods, such as virus infection or chemical treatment.
3. Cultivation and expansion of NK cells: Activated NK cells will be put into the culture medium and cultured and expanded under laboratory conditions to increase the number of cells. This process usually takes a certain amount of time to ensure that the number and quality of cells meet the treatment requirements.
4. Transformation of NK cells: In the process of cell culture, specific receptors (usually chimeric antigen receptor CAR) can be introduced into NK cells by genetic engineering technology, so that they have stronger recognition of cancer cells and attack capabilities.
5. Verification and quality control: The modified NK cells will undergo a series of verification and quality control steps to ensure that their function, purity and safety meet the therapeutic requirements.
6. Infusion to patients: The modified NK cells that pass the quality control will be infused to patients and enter the patient's body through intravenous injection. These cells then go on to seek out and attack cancer cells in order to achieve the therapeutic effect.

It should be noted that the specific process of Car-NK cell therapy may vary depending on the treatment center and patient condition. The treatment plan may vary for each patient. Therefore, patients need to discuss their condition and treatment plan with their doctor in detail before undergoing treatment.

3. Structure and Generations of CAR-NK Cell

Functional CAR (Chimeric Antigen Receptor) molecules usually consist of the following parts on NK cells:

1. Antigen Recognition Domain: The Antigen Recognition Domain is a key component of the CAR molecule, which is responsible for recognizing and binding the target antigen. The antigen recognition region is usually constructed from a single-chain antibody (scFv), which can be highly specific and used to recognize specific antigens on the surface of cancer cells.
2. Signaling Domain: The signaling domain is the activation part of the CAR molecule, which is responsible for transmitting signals and activating the anti-cancer function of NK cells. Common signaling domains include the CD3ζ chain, which is associated with NK cell activation, and co-stimulatory molecules such as CD28 or 4-1BB (CD137), which can provide additional stimulatory signals to enhance NK cell activity and persistence.
3. Transmembrane Domain: The transmembrane domain is located in the transmembrane region of the CAR molecule, responsible for connecting the antigen recognition region and the signal transduction region together, and firmly embedding the CAR molecule into the cell membrane of NK cells.

These three parts together constitute the functional structure of CAR molecules on NK cells. The antigen recognition region enables CAR-NK cells to recognize and bind specific antigens, while the signal transduction region activates the anti-cancer function of NK cells, thereby achieving the attack and elimination of cancer cells. The membrane junction region plays the role of connecting and stabilizing CAR molecules on the cell membrane.

NK cells (natural killer cells) are an important immune cell in the body, not only related to anti-tumor, anti-viral infection and immune regulation, but also related to the occurrence of hypersensitivity and autoimmune diseases. And also experienced the development of four generations of CAR-NK.

First-generation CAR-NK cells: The first-generation CAR-NK cells are the earliest CAR-NK cell designs, and they are mainly composed of antigen recognition regions (usually single-chain antibodies) and activation signal transduction regions (such as CD3ζ chains). These CAR-NK cells can recognize and attack specific antigens and activate the killing function of NK cells. However, the first-generation CAR-NK cells have limited killing effect, short survival time, and are susceptible to immunosuppressive mechanisms.

Second-generation CAR-NK cells: The second-generation CAR-NK cells are improved on the basis of the first-generation CAR-NK cells. In addition to the antigen recognition region and the activation signal transduction region, the second-generation CAR-NK cells also introduced co-stimulatory molecules, such as CD28 or 4-1BB (CD137). These co-stimulatory molecules provide additional activation signals to enhance the proliferation, survival and killing effects of CAR-NK cells. Compared with the first-generation CAR-NK cells, the second-generation CAR-NK cells have significantly improved anti-tumor effects.

Third-generation CAR-NK cells: Third-generation CAR-NK cells have further improved the design of second-generation CAR-NK cells. In addition to the antigen recognition region, activation signal transduction region and co-stimulatory molecules, the third-generation CAR-NK cells introduced additional co-stimulatory molecules or cytokines, such as a combination of CD28 and 4-1BB. These additional co-stimulatory molecules can further enhance the killing activity and survival ability of CAR-NK cells, and improve the anti-tumor effect. The third-generation CAR-NK cells have made some progress in preclinical and clinical research.

Fourth-generation CAR-NK cells: The fourth-generation CAR-NK cells are research and exploration based on the third-generation CAR-NK cells. The concept of fourth-generation CAR-NK cells is still under development, but some possible improved features include enhanced persistence and survival, tunable on-off mechanisms, and specific adaptations to the tumor microenvironment. These improvements are aimed at improving the therapeutic efficacy and safety of CAR-NK cells.

4. Targets of CAR-NK Cell Therapy

Hematological malignancy target protein

CD19	CD7	CD5
FLT3	BCMA	CD33
IL-3R alpha/CD123	Syndecan-1/CD138	CD20
CD22	SLAMF7/CD319	CD38
CD4

Solid tumor target protein

HER2	EGFR	c-MET
EpCAM	NKG2D	CEACAM5
CD73	B7-H3	Glypican 3/GPC3
Mesothelin	PSMA	ROBO1
IL3RA/CD123	CXCR4/CD184	PD-L1
MUC1	FLOR1	CD147

CD19

CD19 is a cell surface antigen, which is mainly expressed on the surface of B lymphocytes in the human body. CD19 is an important regulator of B cell development and activation, and plays an important role in the immune response of B cells.

In oncology, CD19 has been widely studied and applied, especially in the treatment of B cell malignancies. Because CD19 is highly expressed in a variety of B-cell tumors, it has become an important therapeutic target.

CAR-NK cell therapy utilizes engineered NK cells to express CAR (chimeric antigen receptor), enabling them to recognize and kill CD19-expressing cancer cells. CAR usually consists of an antigen recognition region, an activation signal transduction region, and co-stimulatory signal molecules.

In CAR-NK cell therapy, CD19, as a target antigen, is used as a marker for CAR-NK cells to recognize and attack B-cell tumor cells. When CAR-NK cells interact with CD19-positive cancer cells, the antigen recognition region of CAR will recognize and bind to the CD19 surface antigen, activating the killing mechanism of CAR-NK cells. Activated CAR-NK cells release cytotoxic granules and cytokines, leading to apoptosis and death of target cancer cells.

CD19 is widely expressed in B-cell malignancies and thus becomes an important target for CAR-NK cell therapy. Through the application of CAR-NK cells, CD19-positive cancer cells can be targeted to kill, so as to achieve the therapeutic effect. This therapy could provide a new treatment option for malignant B-cell tumors and has shown potential therapeutic activity and safety in several clinical studies.

CD20

CD20 is a B cell-specific surface antigen widely present on the surface of B lymphocytes. In oncology, CD20 is widely studied and used as a target for immunotherapy, especially in the treatment of non-Hodgkin's lymphoma (NHL).

CD20 is highly expressed in non-Hodgkin's lymphoma and thus considered an ideal target. Immunotherapy targeting CD20 can selectively identify and attack CD20-positive cancer cells, thereby achieving therapeutic effects on tumors.

The principle of Car-NK cell therapy targeting CD20 is to introduce specific chimeric antigen receptor (CAR) into natural killer cells (NK cells), so that they have the ability to recognize and attack CD20-positive tumor cells. CAR-NK cells recognize CD20 through the external domain on the CAR, and kill CD20-positive tumor cells by activating intracellular anti-tumor mechanisms, such as releasing cytotoxic substances and inducing tumor cell apoptosis.

As a target of immunotherapy, CD20 has been proven to have significant efficacy in the treatment of non-Hodgkin's lymphoma. Specific anti-CD20 antibodies, such as rituximab (Rituximab), have been widely used in the treatment of non-Hodgkin's lymphoma. By introducing CAR into NK cells so that they have CD20-specific recognition and attack capabilities, CAR-NK cell therapy provides a new potential option for the treatment of non-Hodgkin's lymphoma.

HER2

HER2 (Human Epidermal Growth Factor Receptor 2) is a member of the epidermal growth factor receptor family, also known as ErbB2. It is a membrane receptor protein widely expressed in a variety of tissues and cell types, including breast, stomach, ovary and lung.

In oncology, overexpression or gene amplification of HER2 is associated with the development and progression of various malignancies, especially breast cancer. HER2-positive breast cancer refers to the abnormal increase in the expression level of HER2 in breast cancer cells. About 20-25% of breast cancer patients are HER2-positive.

Car-NK cell therapy can target HER2. By introducing specific chimeric antigen receptor (CAR) into natural killer cells (NK cells), it has the ability to recognize and attack HER2-positive tumor cells. CAR-NK cells bind to HER2 through the external domain on the CAR, and kill HER2-positive tumor cells through mechanisms such as releasing cytotoxic substances and inducing tumor cell apoptosis.

In the treatment of breast cancer, HER2-targeted therapy has been widely used, including rituximab (Trastuzumab) and other anti-HER2 antibody drugs. The application of CAR-NK cell therapy targeting HER2 provides a new treatment option with the advantages of strong targeting and significant cytotoxic effect.

EGFR

EGFR (Epidermal Growth Factor Receptor) is an epidermal growth factor receptor, also known as HER1 (Human Epidermal Growth Factor Receptor 1). It is a membrane receptor protein widely expressed in various tissues and cell types, including lung, head and neck, breast, colorectal, etc.

EGFR plays an important role in the occurrence and development of tumors. In some cancers, overexpression or mutation of EGFR is related to tumor proliferation, invasion and metastasis, so it becomes one of the important targets for treatment.

Car-NK cell therapy can target EGFR for treatment. By introducing specific chimeric antigen receptor (CAR) into natural killer cells (NK cells), it has the ability to recognize and attack EGFR-positive tumor cells. CAR-NK cells bind to EGFR through the external domain on the CAR, and kill EGFR-positive tumor cells through mechanisms such as releasing cytotoxic substances and inducing tumor cell apoptosis.

EGFR-targeted therapy has been widely used clinically. For example, drugs that inhibit EGFR activity, such as Gefitinib and Erlotinib, have been approved for the treatment of EGFR-mutant tumors such as lung cancer. The application of Car-NK cell therapy targeting EGFR provides a new treatment option, which has the advantages of strong pertinence and significant cytotoxic effect.

5. CAR-NK Cell Therapy Clinical Trial and Side Effect

Car-NK cell therapy is currently undergoing several clinical trials to evaluate its safety and efficacy in the treatment of different types of cancer. Here are some examples of clinical trials of Car-NK cell therapy:

1. Multiple myeloma (MM): CAR-NK cell therapy targeting CD38 has entered the clinical trial stage. These trials are designed to evaluate the efficacy of CAR-NK cell therapy in patients with relapsed/refractory multiple myeloma.
2. Acute lymphoblastic leukemia (ALL): CAR-NK cell therapy targeting CD19 is undergoing clinical trials. These trials are designed to evaluate the efficacy of CAR-NK cell therapy in patients with relapsed/refractory ALL, especially those who cannot receive CAR-T cell therapy or have relapsed after undergoing CAR-T cell therapy.
3. Chronic lymphocytic leukemia (CLL): CAR-NK cell therapy targeting CD19 and CD20 targets is undergoing clinical trials. These trials are designed to evaluate the efficacy of CAR-NK cell therapy in patients with relapsed/refractory CLL.
4. Non-Hodgkin's lymphoma (NHL): CAR-NK cell therapy targeting CD20 and CD19 targets is undergoing clinical trials. These trials are designed to evaluate the efficacy of CAR-NK cell therapy in patients with relapsed/refractory NHL.

In addition, other types of cancer, such as breast cancer, lung cancer, colorectal cancer, etc., are also undergoing clinical trials of CAR-NK cell therapy targeting different targets.

But in the same way, Car-NK cell therapy as an immune cell therapy is not perfect and can cause some side effects, the following are some possible side effects:

1. Cytokine Release Syndrome (CRS): In Car-NK cell therapy, activated NK cells will release a large number of cytokines, such as tumor necrosis factor (TNF-α), interferon gamma (IFN-γ ), etc., leading to overactivation of the immune system. This can trigger symptoms such as fever, chills, low blood pressure, shortness of breath and, in severe cases, a threat to heart and lung function.
2. Hematological side effects: Car-NK cell therapy may cause some hematological side effects, such as anemia, thrombocytopenia, and leukopenia. These side effects can lead to fatigue, easy bleeding, and an increased risk of infection.
3. Allergic reactions: Car-NK cell therapy may trigger allergic reactions, including severe symptoms such as rashes, urticaria, shortness of breath, and anaphylactic shock. Hypersensitivity reactions may be related to components in the media used in NK cell therapy or to foreign proteins in engineered cells.
4. Neurotoxicity: Some patients may experience side effects related to the nervous system, such as headache, increased intracranial pressure, ataxia and convulsions.

It should be noted that each individual may respond differently to the side effects of Car-NK cell therapy, and the occurrence and severity of side effects may depend on a variety of factors, including the patient's overall health and specific details of the treatment regimen. When patients receive this therapy, doctors and treatment teams closely monitor and manage side effects to ensure patient safety.

References:

[1] Burger MC, Zhang C, Harter PN, et al. CAR-Engineered NK Cells for the Treatment of Glioblastoma: Turning Innate Effectors Into Precision Tools for Cancer Immunotherapy. Front Immunol. 2019;10:2683. Published 2019 Nov 14. doi:10.3389/fimmu.2019.02683 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868035/

[2] Wang W, Jiang J, Wu C. CAR-NK for tumor immunotherapy: Clinical transformation and future prospects. Cancer Lett. 2020;472:175-180. doi:10.1016/j.canlet.2019.11.033 https://pubmed.ncbi.nlm.nih.gov/31790761/

[3] Marofi F, Saleh MM, Rahman HS, et al. CAR-engineered NK cells; a promising therapeutic option for treatment of hematological malignancies. Stem Cell Res Ther. 2021;12(1):374. Published 2021 Jul 2. doi:10.1186/s13287-021-02462-y https://pubmed.ncbi.nlm.nih.gov/34215336/

[4] Pollock NI, Grandis JR. HER2 as a therapeutic target in head and neck squamous cell carcinoma. Clin Cancer Res. 2015;21(3):526-533. doi:10.1158/1078-0432.CCR-14-1432 https://pubmed.ncbi.nlm.nih.gov/25424855/

[5] Cantor AJ, Shah NH, Kuriyan J. Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites. Proc Natl Acad Sci U S A. 2018;115(31):E7303-E7312. doi:10.1073/pnas.1803598115 https://pubmed.ncbi.nlm.nih.gov/30012625/