Dr. Cristina Eguizabal Argaiz
Cell Therapy, Stem Cells and Tissues Group
Basque Center for Blood Transfusion and Human Tissues
Biocruces Bizkaia Health Research Institute
Galdakao, Bizkaia, SPAIN.
Dr. Lara Herrera del Val
Cell Therapy, Stem Cells and Tissues Group
Basque Center for Blood Transfusion and Human Tissues
Biocruces Bizkaia Health Research
Disclaimer/Conflict of interest statement: none.
Phase contrast image of NK cells in culture, before the transduction with lentiviral vectors (CD19-CAR). Image kindly provided by Sara Martin.
The immunotherapy with CARs has been originally described by using T cells, named CAR-T therapy (1). A CAR is a chimeric receptor construct consisting of an extracellular single-chain variable fragment (scFv) derived from an antibody or a full-length antibody. It is connected to a hinge fragment, which acts as a “spacer” between the extracellular and intracellular part, being usually a CD8α, which enhances responses initiated by TCR; a transmembrane domain, and a CD3ζ chain, or FcR receptor, consisting of an intracellular tyrosine-based activation motif. This was the structure of the first generation CARs (1G). Researchers designed a CAR which included the two-signal model of T-cell activation CD28 co-stimulatory domain along with CD3 ζ ITAM domains. These kinds of constructs constitute second generation CARs (2G). Furthermore, it has also been reported that other co-stimulatory domains, such as 4-1BB, support comparable in vivo improvements to CAR-T cell persistence and function. Nonetheless, CAR-T cell properties could change in regard to these last two domains; CD28- based CARs have direct antitumor efficacy, while 4-1BB-based CARs have long persistence activity. As a result, third generation (3G) CARs have been developed to include two co-stimulatory domains, 4-1BB and CD28 intracellular domains. CARs from 2G or 3G containing the 4-1BB domain have been reported to have greater in vivo expansion and anti-tumor activity compared to CD28 2G CARs. Due to the vast heterogeneity of cancer cells in solid tumors, a fourth generation of CARs (4G), known as TRUCKs (“T cells redirected for antigen-unrestricted cytokine-initiated killing”) were developed, where cytokines are used to armor CARs. These CARs contain an additional modification which consists of an inducible or constitutive expression cassette for a transgenic protein, for example a cytokine, which is released by the CAR-T cell to modulate the T-cell response. As a consequence, an improvement of T cell properties and recruitment of additional immune cells can be achieved.
As CARs seem to be a newer and more effective way to treat cancers in relapse or refractoriness, especially hematological cancers, there are various clinical trials going on. Most of them use T cells as a vehicle for the CARs, with more than 1.000 clinical trials worldwide, being CD19 the most prevalent target.
Furthermore, there is an impossibility to use an allogeneic CAR-T product due to the generation of graft-versus-host-disease (GvHD), whereas autologous products are often limited due to the usual depletion of lymphocytic precursors in oncologic patients. As a result, seeking new cell sources to generate CAR immunotherapeutic products has pointed to NK cells as an attractive model since they exhibit antitumor responses without the need for sensitization.
Natural Killer cells, or NK cells, belong to the innate immune system, providing rapid responses against viral infections and tumors. Usually, the detection of the major histocompatibility complex (MHC) on the surface of the infected cells by the immune cells triggers cytokine release, causing lysis or apoptosis. NK cells have, in fact, a unique ability to recognize stressed cells lacking antibodies and MHC, accelerating an immune reaction. They owe their name “natural killers” to the initial perception that they do not need prior activation to kill cells with no “self” antigens of MHC class I. As malignant cells do not express MHC I markers, T cells cannot destroy them, so NK cells play a key role. NK cells and their functions were described more than 30 years ago, but for the first time in 1975 these cells were described as bigger lymphocytes than B cells and T cells, which contained distinctive cytoplasmic granules. NK cells were characterized as cells which showed a co-stimulatory independent spontaneous cytotoxic capacity, differentiating them functionally from B cells and T cells. In the immune system, NK cells are the third major lymphocyte subset. These large granular cells constitute approximately 10–15% of lymphocytes in the blood. NK cells are able to kill tumor cells and infected cells “naturally”, i.e., in a casual manner that does not need any prior activation and is not limited to the expression of MHC molecules. NK cells are usually defined within the lymphocyte population by a lack of CD3 and expression of CD56, a neural cell adhesion molecule (NCAM).
NK cells can be activated through CD3ζ, resulting in ADCC mediated by CD16 receptor. Thus, the vast majority of CAR constructs used for engineering NK cells contains this signaling component, classically present since 1G CARs. Despite the fact that traditional 2G CARs designed for T cells, that is, CARs containing CD3ζ and CD28 or 4-1BB domains, are functional in NK cells new approaches need to be explored for NK cells. Taking into account other main signaling pathways that activate NK cells, new CAR constructs have been designed, in which signaling domains derived from 2B4, NKG2D, DAP10 or DAP12 have shown some promising results.
Despite the good results of CAR-T cells, there are some expected side-effects. On the one hand, the main and most serious side-effect of CAR-T cell therapy is cytokine-release syndrome (CRS) and its dangerous form, a “cytokine storm”, in which T-cells are massively activated, triggering a cascade of pro-inflammatory cytokines which cause flushing, fever and dyspnea. Although an acute cytokine storm can potentially be lethal, it has been proved that the anti-interleukin-6 receptor antibody tocilizumab is an effective treatment.
Immune effector cell associated neurotoxicity syndrome (ICANS) has also emerged as a serious side-effect after CAR-T cell therapy. On the other hand, as these patients are usually highly medicated, it is not always viable to expand and manufacture their own autologous modified T-cells from lymphocytes, due to the scarce lymphocyte count or the poor state of the cells. Hence, the manufacture of off-the-shelf allogenic CAR-T cells from healthy donors’ lymphocytes is promising in many aspects, although there are some concerns that keep them from use in clinical trials. Allogenic T cells express the human leucocyte antigen (HLA), which can give a mismatch between donor and recipient, leading severe, even lethal GvHD. This leads to a new source of cells, since the less alloreactive T cell subset, such as CD45RA- lymphocytes and Natural Killer (NK) cells, are good candidates because they suppress GvHD by inhibiting activated, alloreactive T cells without causing GvHD themselves. NK lymphocytes constitute an attractive source for CAR-based treatments, owing to their innate ability to kill malignant or infected cells without prior activation or HLA restriction. Moreover, due to NK cells’ shorter lifespan, B cell depletion could be less severe for the patient. Another point to address is that, with a good initial cell product, T cells are easier to expand and less resistant to genetic engineering, thanks to the use of a CD3/CD28 activation kit. Although NK cells appear to be harder to expand and transfect, some groups have achieved impressive fold expansion numbers by co-culturing them with activation beads or modified feeder cells along with good transduction numbers. The mechanism of action of NK cells differs from that of T cells. On one hand, NK cells interact with target cells through activating and
inhibitory receptors, and the outcome is determined by the accumulation of signal strength. If they are activated, they release cytotoxic granules, such as perforin and granzyme, and they secrete a variety of cytokines. Finally, T cells and NK cells are differentially activated. Despite the fact that some of the signal domains are conserved between these two types of lymphocytes, such as CD3 ζ and 4-1BB, other co-stimulatory domains typically present in T cells are absent in an NK cell, such as CD8α and CD28. NK cells can operate through several adapter domains for downstream signaling, such as CD3 ζ, DAP10, DAP12, and FcR chains. While CD3 ζ signaling occurs via CD16, NKp30, and NKp46, FcR chains signalling also occurs via CD16, NKp30 and NKp46. DAP10 mediates signaling through NKG2D whereas DAP12 activates KIRs, NKG2C, and NKp44.
In the 1980s, Israeli researchers expressed chimeric TCR genes including the TCR constant domains united to the variable domain from an antibody, which led to a hypothesis about CAR-T treatments. In 1989, Gideon Gross and Zelig Eshhar developed the first CAR-T cells at the Weizmann Institute, Israel. Some years later, Prof. Carl H. June from the University of Pennsylvania tested genetically modified CAR-Ts in humans for the treatment of hematological cancer and clinical use. Thanks to his work, the first FDA-approved gene therapy in 2017, named Kymriah (Novartis), was developed and commercialized. From 2017 until 2021 several CAR-T products were approved by FDA and commercialized against several B cell hematological cancers, such as, Yescarta and Tecartus (Kite Pharma) and Breyanzi (Juno Therapeuctics).
From academic point of view, the group of Dr. Manel Juan in Hospital Clinic in Barcelona, Spain is pioneer in developing the first academic CD19-CAR-T cells, named ARI-0001 against R/R B cell hematological cancers (NCT03144583; NCT04778579) (2,3,4,5).
Although T cells have typically been used in CAR technology-based therapy, with more than 400 clinical trials targeting CD19 antibody on-going and 3 commercial products, Kymriah, Yescarta, Tecartus and Breyanzi, NK cells are also emerging as one of the new promises in this field. Due to their low infection rate, poor in vivo expansion and short life span, NK cells were not taken into account from the beginning for this kind of therapy.
Nevertheless, newer protocols that enhance viral transduction efficiency and prosperous expansion of these cells have made a space for NK cells in the CAR therapy field (6,7,8,9, 10). Furthermore, allogenic NK cells have a major advantage over allogenic T cells, i.e., they could be used as a “universal” product as they do not cause GvHD as they lack TCR. The interest in using allogenic NK cells for this kind of therapy is increasing, and there are already 13 clinical trials using CD19 CAR-NK cells (NCT03056339, NCT03690310, NCT00995137, NCT01974479, NCT04639739, NCT02892695, NCT04887012, NCT04796675, NCT03824964, NCT05020678, NCT03579927, NCT04796688, NCT02134262), and numerous preclinical studies with NK cells from different sources as vehicles. Allogenic primary NK cells from adult peripheral blood (AB) or umbilical cord blood (CB) could represent feasible, safe, off-the-shelf CAR-cell products to treat various malignancies such as hematological cancers. When studying AB CD19-CAR-NK cells, they not only successfully kill CD19-expressing target cells, but they also retain the function and expression of their native activating receptors, preserving their activity. However, AB NK cells are more variable from donor to donor in number, and they expand and activate less in vitro than CB NK cells. Some studies show higher antitumor activity of CB cells compared with other NK cell sources, which justifies the use of CB derived immunotherapy. Moreover, CB units stored in blood banks could be used for this purpose. CB CAR-NK cells have shown great performance against their target cells, and more flexibility to be expanded. These CB CAR- NK cells are currently being used in an academic Phase I clinical trial at MD Anderson Cancer Center targeting CD19 cells, with great results. CAR-NK cells from cord blood were administered to 11 patients with relapsed or refractory CD19-positive cancers (NHL or CLL). An anti-CD19-CD28-CD3 ζ CAR was used for the transduction and the retroviral vector included an IL-15 gene and a suicidal switch. Seven out of eleven patients achieved a complete remission, exhibiting a significantly higher early expansion of CAR-NK cells compared to the non-responders. There were no reported side-effects associated with the high response rate of the treatment, even in even in KIR-ligand mismatch cases (5/11), and there was no interleukin-6 increase, which proved the safety of the treatment. The published observation of circulating CAR-NK cells by flow cytometry was limited to the first three weeks (11).
Currently, other non-academic clinical trials are ongoing by using CAR-NK cell products against hematological and solid tumors from Nkarta and Fate Therapeutics Company, among others.
Several laboratories worldwide are currently working in the development of new immunotherapies with CAR-NK against hematological and solid tumors. In China numerous groups are conducting clinical trials. In United States, one of the most relevant laboratory is leaded by Dr. Katy Rezvani at M D Anderson (Houston) with positive results in the Phase I clinical trial for treating patients with B cell malignancies (11). In Europe there are some research teams and hospitals working with CAR-NK immunotherapy. For example in Spain, under the Spanish Network of Advanced Therapies (RICORS-TERAV) several groups were developed preclinical studies with CAR-NK cells that actually are translated into academic Phase I clinical trials to treat hematological and solid tumors, such as Dr. Cristina Eguizabal at Basque Center for Blood Transfusion and Human Tissues / Biocruces Bizkaia Health Research Institute; Dr. Joaquin Martinez at Hospital 12 de Octubre (Madrid) and other relevant groups in Hospital Clinic (Barcelona), Hospital Universitario La Paz (Madrid), Banco de Sangre y Tejidos de Cataluña (Barcelona), IDIBELL-ICO, etc.
In order to successfully use these CAR-NK cell products against tumor cells, there are crucial steps that need to be precisely done in GMP conditions, such as purification and culture of NK cells from allogenic blood sample, then CAR transduction, expansion and CAR-NK product cryopreservation and storage. Afterwards the CAR-NK cell product will be ready for the infusion of oncologic patients enrolled in a clinical trial (4,7,9).
Did you know that…the benefits of CAR-NK cells over CAR-T cells augur promising applications in cellular immunotherapy against hematological malignancies as an alternative or combination cell drug? Nevertheless, there are still challenges to be addressed. For instance, due to the heterogeneity of NK cells with various functional features, the selection of appropriate NK cell subsets to specifically expand and arm CAR-NK cells has to be explored. NK cells are known for their transduction difficulty. Although several protocols with retrovirus and lentivirus have been successfully developed, there is still room for new techniques in order to improve NK cell transduction, such as mRNA electroporation or CRISPR/Cas9 technology. As CAR-NK cells are thought to be an “off-the-shelf” product, cryopreservation is also a crucial step. Due to their short persistence in vivo, the NK cell cytolytic effect could also be restricted, but they are probably unable to trigger cytokine storms or on-target/off tumor effects; continuous cytokine support or several infusions of CAR-NK cells may will be needed. Additionally, various NK cell sources have been studied (peripheral blood, cord blood or hiPSCs), and therefore in the future the foremost appropriate one could be used for refractory malignancies. Moreover, the best configuration of CARs to boost the activation, proliferation, cytolytic activity and cytokine secretion of NK cells has not yet been found. Perhaps the future of these CAR constructs for NK cells lies on exploring NK cell activating domains such as NKG2D, DAP10, DAP12 and 2B4 in order to improve their performance. In conclusion, as more evidence from clinical trials is procured within the coming years, CAR-NK cell therapies could provide meaningful progress in tumor immunotherapy. Moreover, CAR-NK therapies in combination with other immunotherapies or even other CAR-T cells may pave a new way for CAR-NK cell-based immunotherapy in the future.
1. Herrera, L. et al. The Race of CAR Therapies: CAR-NK Cells for Fighting B-Cell Hematological Cancers. Cancers (Basel). 13, 5418 (2021).
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4. Castella M et al. Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase I Clinical Trial.Front Immunol. 2020 Mar 20;11:482.
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6. Eguizabal, C. et al. Natural Killer Cells for Cancer Immunotherapy: Pluripotent Stem Cells-Derived NK Cells as an Immunotherapeutic Perspective. Front. Immunol. 5, (2014).
7. Herrera, L. et al. Adult peripheral blood and umbilical cord blood NK cells are good sources for effective CAR therapy against CD19 positive leukemic cells. Sci. Rep. 9, 18729 (2019).
8. Herrera, L. et al. OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors. Front. Immunol. 8, 1–10 (2017).
9. Herrera, L., Juan, M. & Eguizabal, C. Purification, Culture, and CD19‐CAR Lentiviral Transduction of Adult and Umbilical Cord Blood NK Cells. Curr. Protoc. Immunol. 131, 2–13 (2020).
10. Herrera, L. et al. Identifying SARS‐CoV‐2 ‘memory’ NK cells from COVID‐19 convalescent donors for adoptive cell therapy. Immunology 165, 234–249 (2022).
11. Liu E et al. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors.N Engl J Med. 2020 Feb 6;382(6):545-553.