Targeting immune checkpoints: how to use natural killer cells for fighting against solid tumors

6.2.1 PB-NK cells

The PB-NK cells are phenotypically mature and highly functional. However, they need blood or leukapheresis from a healthy person who is willing to donate. Even though PB-NK cells are readily available, their low transduction efficiency, coupled with poor expansion, limits their application [212]. However, this source has certain limitations. The process of selecting an appropriate allogeneic NK cell donor is time-consuming, which may cause the patient's treatment to be delayed. The number of peripheral blood mononuclear cells (PBMCs) that can be extracted and the proportion of NK cells contained in the PBMCs vary considerably among donors, all of which can have an impact on the amount of purified NK cells available for transplant. Furthermore, NK cell phenotypes and functions differ between donors (depending on age, sex, weight, and other factors), reducing consistency in the expansion rate and final product of NK cells.

6.2.2 UCB-NK cells

Another interesting source of NK cells, UCB, has a number of advantages, such as its ready availability “off-the-shelf”. UCB collection is easy and harmless to the mother or baby, and they are almost never infected with Epstein-Barr Virus (EBV) or cytomegalovirus (CMV). UCB is easily frozen [213]. The benefit of UCB banks is the ability to select donors with certain HLA types and specific NK receptor profiles. UCB also has fewer T cells than PB, and these cells have a naive phenotype, lowering the risk of GVHD from contaminated T cells infused into patients via an NK cell product infusion [214]. The NK cells derived from the UCB are younger and have a greater potential for proliferating than those derived from the PB [215]. By using feeder cells and cytokines, UCB-NK cells can be expanded and genetically modified to express CARs. Because of their higher proliferative capacity, NK cells from this source are easier to engineer, as demonstrated in the first reported clinical trial of CAR-NK cells [216]. In UCB, NK cells comprise up to 30% of lymphocytes [217], with a higher proportion of CD56^bright cells and hence inferior cytotoxic capabilities [215, 217, 218]. Unexpanded UCB-derived NK cells have some limitations, including availability in low numbers due to the UCB unit's small volume and immature function [217]. UCB-NK cells express lower levels of certain adhesion molecules, along with KIRs, CD16, perforin, and granzyme B, compared to PB-NK cells, as well as higher levels of inhibitory molecules, including NKG2A [219]. One study used Good Manufacturing Practice (GMP)-grade K562-based artificial antigen-presenting cells (aAPCs) that express membrane-bound IL-21 and 4-1BB ligand to develop a GMP-compliant procedure that reliably produces therapeutically relevant amounts of GMP-grade NK cells from a CB unit for the purposes of adoptive immunotherapy [199]. When activated and expanded ex vivo, CB-derived NK cells express the full array of activating and inhibitory receptors, strongly express eomesodermin (Eomes) and T-bet, two factors needed to mature NK cells, and have cytotoxic properties similar to PB-NK cells [220, 221]. However, unlike PBMCs, CAR-NK cells produced from UCBs are not homogeneous, making standardisation challenging [222].

6.2.3 NK cell lines

In most clinical trials with CAR-NK cells, the NK-92 cell line is used because it shows clinical benefits and has minimal side effects, and it is the only one that has been approved for clinical application [223, 224]. It demonstrates unlimited proliferation in vitro and is less sensitive to repeated freeze/thaw cycles [225]. These properties will make it easier and cheaper to manufacture “off-the-shelf” CAR-NK products for clinical use. Furthermore, they are a genetically modifiable NK cell population. In addition, NK-92 cells have higher transduction efficiency than primary NK cells [226, 227]. However, this cell line has some limitations: neither CD16 (are hence unable to trigger ADCC) nor NKp44 are expressed due to their tumor cell line origin, they must be irradiated before infusion and they can't be expanded in vivo, owing to the lethal irradiation required before their infusion [228], rendering them unsuitable for CAR-NK cell therapy. Genetically modifying NK-92 cells to express the high-affinity V158 variant of the FcγRIIIa/CD16a (termed haNK^TM) and endogenous, intracellularly retained IL-2 helped overcome these cytotoxic limitations [224, 229]. NK-92 also lacks some KIRs, with the exception of KIR2DL4 (CD158d), which may promote GVHD [230-233]. A recent study has suggested that the host PBMCs might show cytotoxicity against CAR-transduced NK-92 cells [234]. Moreover, they are EBV-positive and exhibit multiple cytogenetic abnormalities similar to those of NK lymphoma [235]. While repeated infusions of irradiated NK-92 cells are possible and may be utilized to overcome their short-term persistence, such an approach is likely to result in the production of Abs and cellular immunity against the allogeneic cell line, as well as faster rejection with each infusion. Since IL-2 is essential to the growth and survival of these cells in culture, it must be accompanied by any additional stimulation factors [225, 236]. NK-92MI cells were created by genetically engineering NK-92 cells to produce soluble IL-2, thus eliminating the need for exogenous IL-2. In addition to NK-92 cells, other human cell lines have also been evaluated as alternatives, such as NKL, KHYG-1, YTS or NKG [237]. Irradiation reduces the anti-tumor potency of this product when compared to NK cells from other sources, and it may not be clinically relevant.

6.2.4 NK cells derived from stem cells

NK cell products derived from UCB stem cells, hESCs and iPSCs are currently being tested in clinical trials. Because ESCs are more difficult to obtain and their applications raise ethical concerns, they are used less frequently than CB stem cells and iPSCs. It is possible to obtain large numbers of NK cells by differentiating them from CD34⁺ HPCs. This approach provides virtually unlimited numbers of homogeneous NK cells, as well as the advantage of allowing their genetic manipulation over primary NK cells. CD34⁺ HPCs can be isolated from BM, mobilized PB, or UCB, expanded and then differentiated into mature NK cells in a culture system using a cocktail of cytokines [238]. As a result, a more homogeneous and well-defined NK cell product can be produced. Although PB apheresis after granulocyte colony-stimulating factor (G-CSF) stimulation is a well-established alternative to extracting CD34⁺ progenitors from BM, it may influence the NK cell phenotype [239]. It was previously common to use BM CD34⁺ cells for the generation of NK cells, but UCB CD34⁺ cells have become more frequently used as UCB is easier to obtain and contains higher levels of HSCs [240-245]. In this way, various combinations of growth factor and cytokine mixtures, BM stroma cells, and culture media with or without animal or human serum were used in the different studies. oNKord, an allogeneic partial HLA-matched NK cell product derived from UCB CD34⁺ progenitors, has been designated as an orphan medication by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) for treating AML patients who are not candidates for allogeneic stem cell transplantation. CD56⁺CD3⁻ NK cells produced from this process are largely similar to PB-NK cells, expressing activating receptors, and exhibiting potent cytotoxicity against leukemia cells in vitro and in vivo [246]. On the other hand, it has been shown that while CD34⁺ cells from CB can be employed to produce a homogeneous population of CD56⁺ NK cells on a large scale, they do not appear to be as mature as PB-NK cells [238] and have little ADCC activity [247] even after expansion, necessitating additional measures to improve potency for clinical application. In addition, the differentiation process is labor-intensive, requiring frequently changing media. In order to develop clinically applicable NK cell expansion protocols, shorter expansion times and less operator involvement are likely to be preferred.

PSCs, such as ESCs and iPSCs, have the ability to self-renew while maintaining pluripotency and could provide limitless supplies for target cells [248]. It has been established that clinical-scale NK cell generation can be achieved using hESC or iPSC [198, 249, 250]. The use of commonly available hESC/iPSC cell lines is often preferred to the use of stem cells from BM biopsy, G-CSF mobilization, or human embryos [251, 252]. To differentiate these cell sources into NK cells, xenogeneic stromal feeder cell lines [253] or human spin embryoid bodies (EBs) [197, 254, 255] are required, which eliminate xenogeneic cells for more defined NK cell development conditions and can therefore be scaled up clinically. hESC/iPSC were centrifuged to form spin EBs [256], which gave rise to HPCs expressing CD34 and CD45, which were then differentiated into mature NK cells by using a specific cytokine cocktail. The choice of target donor somatic cell type and the reprograming protocol, which includes the nature and combination of genes as well as the method used to deliver transcription factors into somatic cells, have an impact on the pluripotency and differentiation abilities of reprogramed cells in iPS cells [257]. NK cells derived from iPSC/hESC expressed common NK cell markers, such as KIRs, CD16, NKp44, NKp46, NKG2D, and TRAIL, and were cytotoxic against several hematological and solid tumor cells in vitro [198, 249]. NK cells produced from iPSCs or hESCs combine the advantages of PB-NK and NK-92 cells, as they have a similar phenotype to PB-NK cells and are a homogeneous population. NK cells derived from the H9 hESC cell line had a lower allogeneic immunological response, but they had a more mature cytotoxic profile than CB-NK cells [249]. In contrast to PB-NK cells, iPSC-derived NK cells, similar to UCB-NK cells, have an immature phenotype, with lower KIR and CD16 expression and higher NKG2A expression [197, 254, 258]. Because of their unlimited proliferation capacity, iPSCs have emerged as an attractive source of CAR-NK cells [259]. Using iPSCs to produce NK cells allows for more gene modification, repeat dosing, and the generation of standardized products, allowing for more successful treatment of refractory solid tumors [260]. Another advantage of using iPSC-NK products over iPSC-T cell therapies is that iPSC-NK products can be truly “off-the-shelf” because they don't require HLA matching between donors and patients.

There are still obstacles to overcome before iPSC-NK cells can be safely employed to generate CAR-NK for clinical usage. iPSC-derived cells are always capable of malignant transformation and have the potential to be immunogenic, causing effector cell death or even adverse immunological reactions like cytokine release storms. The 5-week biomanufacturing time for iPSC-NK cells, on the other hand, could be a barrier to establishing and maintaining iPSC-NK cell banks, especially if genetic modifications such as CARs are required. It would be useful to shorten and streamline the production process.

6.2.5 Memory-like NK cells

Memory-like NK cells were first discovered as a result of CMV infection. CMV was observed to cause the expansion of an NK cell subpopulation with CD94/NKG2C overexpression as well as decreased expression of PD-1, TIGIT, and NKG2A [261, 262]. When activated, CD56^dim/CD16^bright/CD94/NKG2C cells showed enhanced proliferation, degranulation, and elevated IFN-γ and TNF-α production [263]. In addition to living longer in vivo, CMV-induced adaptive memory NK cells can withstand the suppressive effects of Tregs and MDSCs [264, 265]. Incubation of NK cells with IL-12, IL-15, and IL-18 for 16 hours stimulates the generation of memory-like NK cells with abilities and features comparable to CMV-induced cells [87]. On the other hand, IL-12 has been shown to promote NKG2A expression and inhibit NK cell activation [266]. Cytokine-induced memory-like NK cells demonstrated an excellent safety profile, expanded in vivo, and resulted in remission in 44 percent of evaluable patients with AML in a phase I trial [89]. According to preclinical research, inserting CAR into a memory-like NK cell improves its survival and cytotoxicity [267].

6.2.6 Post-partum placenta

Placenta-derived cells contain a proportion of NK cell progenitors with the ability to develop into NK cells during the maturation and expansion stages. After a 21-day NK culture of placenta-isolated NKs, an average of 1.2 × 10⁹ NK cells with an 80% viability rate was obtained [200]. Derived placental-NK cells are largely similar to UCB-NK cells phenotypically and functionally. The FDA has approved CYNK-001, an allogeneic “off-the-shelf” cell therapy enriched for CD56⁺/CD3⁻ NK cells generated from human placental CD34⁺ cells, as an investigational new drug (IND) for the treatment of glioblastoma (GBM).

6.2.7 CAR-engineered stem cells

A CAR can be expressed by transfecting stem cells with the genetic sequence. It is a newly developed method that allows stem cells to differentiate into CAR-NK cells after being engineered. Stem and progenitor cells can be cryopreserved and have better transduction effectiveness than mature NK cells. Engineered stem cells could potentially produce a cellular product that is “off-the-shelf,” eliminating the necessity for a personalized, patient-specific product. Performing gene engineering or gene editing on a small number of stem cells could reduce the amount of gene-engineering/editing materials, which could be cost-limiting, and allow for the most efficient gene engineering/editing, which could be passed down to subsequent divisions and final cell products. Therefore, there is no need to use viral vectors to transfect NK cells, avoiding high cell mortality from viral infection and uncontrollable variation of gene insertion and expression. Furthermore, the advancement of stem cell-engineered cellular therapy will create new opportunities for product upscaling and banking. In addition, gene-engineering technologies like clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 can be employed to boost the anti-cancer properties of CAR-engineered stem cells. The genetic changes will be evident in differentiated immune cells since these cells are non-differentiated [258, 268]. One study successfully introduced CD19-targeted CARs into CD34⁺ progenitors followed by NK differentiation. Gene transfer had no effect on hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell [269]. In media supplemented with stem cell factor (SCF), bone morphogenetic protein 4 (BMP4), and vascular endothelial growth factor (VEGF), genetically engineered iPSCs can stably differentiate into HPCs. A substantial number of highly homogeneous CAR-NK cells may be produced for therapeutic usage after activation using aAPCs and stimulation with IL-15, IL-7, SCF, and FMS-like tyrosine kinase 3 ligand (FLT-3L) [197, 254, 258]. Only one CAR-engineered iPSC cell is required for this approach to produce a large number of highly homogeneous CAR-NK cell products for therapeutic usage. Li et al. [255] used a CAR genetic sequence to transduce iPSCs. In the xenograft model of ovarian cancer, iPSC-derived CAR-NK cells showed prolonged survival and better anti-tumor efficacy than iPSC-derived CAR-modified T cells and non-CAR-bearing immune cells. In another study, researchers used zinc finger nuclease (ZFN) technology to genetically modify human iPSCs to insert a cDNA encoding an anti-EpCAM-CAR into the adeno-associated virus integration site 1, a “safe harbour” for transgene insertion into the human genome, and then differentiated the modified iPSCs into CAR-expressing iPSC-derived NK (iNK) cells. The CAR-iNK cells' lytic activity against NK cell-resistant, EpCAM-positive cancer cells was validated in an in vitro cytotoxicity experiment but not against EpCAM-positive normal cells, confirming the CAR-iNK cells' retained tolerability towards normal cells [270]. A recent study found that anti-glypican 3 (GPC3) CAR-modified NK/ILC cells made from iPSC had a strong effect against tumors and helped people with ovarian cancer live longer without causing acute systemic toxicity [271]. In a recent phase I trial, iPSC was employed for an “off-the-shelf” CAR-NK product (called FT596) to treat CD19-expressing B cell malignancies (NCT04245722). iPS cell-derived NK cell products that have been genetically modified are being tested in people with different types of cancers (NCT03841110 and NCT04023071). In conclusion, it is not clear currently, which source is optimal for creating CAR-NK cells with the most potent activity.

6.4.1 PD-1

In the study of Guo et al. [287], different chimeric receptors that mediate robust NK cell signaling have been designed using NKG2D. The extracellular domain of the inhibitory receptor PD-1 was used in these NKG2D signaling-based chimeric receptors to counteract the immunological escape mediated by PD-1 ligands in solid tumors. They used the PD-1 ectodomain and NKG2D TM domain as a backbone to rationally design four chimeric receptors by TM protein structure modeling. PNBB, a chimeric receptor that was constructed by fusing the human PD-1 extracellular domain to the hinge region, TM and cytoplasmic domains of human NKG2D and human 4-1BB cytoplasmic domain, showed stable surface expression on NK-92 cells and induced significant in vitro cytotoxicity against various tumor cells. As a result, this strategy now offers a promising method for the rational design of NK cell-tailored chimeric receptors with NKG2D signaling. One study created a novel chimeric costimulatory converting receptor (CCCR) by rationally combining the extracellular domain of PD-1, the TM and cytoplasmic domains of NKG2D, and the cytoplasmic domain of 4-1BB created based on the study of Guo et al. [287]. This NK-tailored CCCR was able to switch the negative PD-1 signal into an activating signal, reversing PD-1's immune suppressive effects. When compared to untransduced NK-92 cells, the CCCR-modified NK-92 (CCCR-NK-92) cells retained their typical NK cell features and displayed enhanced anti-tumor activity against human lung cancer H1299 cells in vitro. The rapid clearance of H1299 cells was due to the significant gasdermin E (GSDME)-dependent pyroptosis caused by CCCR-NK-92 cells. In a lung cancer xenograft model, CCCR-NK-92 cells dramatically inhibited tumor growth. Their results highlighted the promising immunotherapeutic potential of using NK-tailored CCCR-engineered NK-92 cells for the treatment of human lung cancer [288]. Li et al. [289] developed a structure-based rational design approach and created a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D, which consists of the ectodomain of PD-1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D as the second recognition receptor, to assess solid tumor cell killing capacity. Increased PD-1 and NKG2D cell surface expression in NK-92 cells after retroviral DTCR transduction promoted strong cytotoxicity against human gastric cell SGC-7901. TNF-α and TRAIL levels increased significantly after stimulation of the chimeric receptor DTCR, which can cause apoptosis in SGC-7901 cells. DTCR-NK-92 cells had significant anti-tumor activity in the solid tumor cell SGC-7901- bearing mouse model. In another study, a CAR-NK-92 cell line, with an IL-15Rα-sushi/IL-15 complex and a PD-1 signal inverter, was created and named SP (Sushi-IL15-PD1). cDNA fragments of a CAR encoded IL-15Rα-sushi, TM domain of CD8, signal transduction zone of 4-1BB, and CD3ζ, 2A signal peptide, PD1 extracellular region, and IL-15. They showed that CAR expression allowed SP cells to proliferate independently of IL-2 and become more resistant to nutrition-starvation-induced apoptosis. Meanwhile, SP cells were more effective in pancreatic ductal adenocarcinoma (PDAC) cell killing assays than NK-92 in vitro and in vivo, and there was a positive correlation between the killing capability of SP cells and PD-L1 expression in pancreatic cancer cells. The adhesion, responsiveness, degranulation efficiency, targeted delivery of cytotoxic granule content, and cytotoxicity of SP cells were considerably stronger compared to NK-92. Finally, the SP cell line is a promising adoptive immunotherapy cell line that could be useful as an adjuvant treatment for pancreatic cancer, particularly in patients with high PD-L1 expression [290]. In another study, CRISPR/Cas9 was used to develop a highly efficient method for editing the genome of human NK cells to knock out inhibitory signaling molecules. Up to 90% of primary PB-NK cells were efficiently edited by their method. They showed highly efficient knockout of A disintegrin and metalloproteinase-17 (ADAM17) and PD1 (PDCD1) genes that have a functional impact on NK cells and demonstrated that these gene-edited NK cells have considerably improved activity, cytokine production, and cancer cell cytotoxicity. In addition, they were able to expand cells to clinically relevant numbers without losing their activity. They also demonstrated that their CRISPR/Cas9 method can be used to efficiently knock in (KI) genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Their platform represents a feasible method for producing engineered primary NK cells as a universal cancer immunotherapy [291]. They suggested that this simple method for stable gene KI provides a platform for the delivery of other relevant cargo, including CARs and self-stimulating cytokine receptors.

6.4.2 PD-L1

Other researchers discovered that PD-L1 t-haNK cells expressed PD-L1-targeting CAR (composed of an extracellular scFv that binds to PD-L1, a TM domain, and a FcεRIγ intracellular domain for downstream signaling) and CD16, retained the expression of native NK receptors, and had a high content of granzyme and perforin granules. They showed that irradiated PD-L1 t-haNK cells could lyse 20 of the 20 human cancer cell lines studied in vitro, including triple-negative breast cancer (TNBC), lung, urogenital, and stomach cancer cells. The cytotoxicity of PD-L1 t-haNK cells was correlated to the PD-L1 expression of the tumor targets and could be improved by pretreating the targets with IFN-γ. It was found that irradiated PD-L1 t-haNK cells inhibited the growth of engrafted TNBC, lung, and bladder tumors in NSG mice. The combination of PD-L1 t-haNK cells with N-803 (formerly ALT-803), an IL-15 super agonist complexed with IL-15Rα−Sushi-Fc fusion protein and anti-PD-1 Ab resulted in superior tumor growth control of engrafted oral cavity squamous carcinoma tumors in C57BL/6 mice. Also, when cocultured with human PBMCs, PD-L1 t-haNK cells lysed MDSCs preferentially but not other immune cell types. This study provides a rationale for using these cells in clinical trials [292]. haNKs were engineered by Robbins et al. [293] to express a CAR targeting PD-L1. This second-generation CAR included a scFv derived from NANT-601, and an IgG1 mAb targeting PD-L1, along with a CD8 hinge, a CD28 TM domain, and an intracellular FcεR1γ signaling domain. They demonstrated that haNKs engineered to express a PD-L1-CAR killed a panel of human and murine head and neck cancer cells at low effector-to-target ratios in a PD-L1-dependent fashion. Treatment of syngeneic tumors led to CD8 and PD-L1-dependent tumor rejection or growth inhibition and a reduction in myeloid cells endogenously expressing high levels of PD-L1. Also, treatment of xenograft tumors resulted in PD-L1-dependent inhibition of tumor growth. In the PB of patients with head and neck cancer, PD-L1-CAR-haNKs reduced levels of macrophages and other myeloid cells endogenously expressing high PD-L1. Therefore, clinical study of PD-L1-CAR-haNKs is recommended. Another study developed experimental models to investigate mechanisms of T cell escape and demonstrated that resistance to T cell killing can be overcome by adding NK cells engineered to express a CAR targeting PD-L1 developed in two previous studies [292, 293]. In engineered models of tumor heterogeneity, PD-L1-CAR-engineered NK cells (PD-L1 t-haNKs) inhibited the clonal selection of T cell-resistant tumor cells seen with T cell treatment alone in multiple models. T cell treatment of heterogeneous cancer cell populations in vitro and in vivo resulted in IFN-γ release and subsequent PD-L1 upregulation on tumor cells that escaped T cell killing due to defects in antigen processing and presentation, priming escape cell populations for PD-L1 dependent killing by PD-L1 t-haNKs. These findings shed light on the processes underpinning the synergistic anti-cancer effectiveness of T cell-based immunotherapy that result in IFN-γ production, PD-L1 overexpression on T cell escape cells, and the utilization of PD-L1-CAR-engineered NK cells to target and eradicate resistant tumor cell populations [294]. In another study, PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. They generated a new atezolizumab-based scFv and combined it with a standard second-generation CAR backbone comprised of the IgG4 hinge region, CD28 TM and signaling portions, and the CD3ζ signaling domain. PD-L1-CAR effector cells degranulated and produced cytokines in response to PD-L1^high MDA-MB-231 cells but not to PD-L1^low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells have been killed by the PD-L1-CAR cells, but with a delay in the case of PD-L1^low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1-CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1-CAR cells. In tumor xenograft models, PD-L1-CAR-T cells were active not only against MDA-MD-231 and MCF-7-PD-L1, but also against MCF-7-pLVX (PD-L1^low/−) cells. Significantly, PD-L1-CAR cells exhibited potent cytotoxic effects against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and did not respond to the stimulation. Finally, they discovered that HER-2-CAR-T cells stimulate expression of PD-L1 on MCF-7 cells and hence accelerate the functionality of PD-L1-CAR-T cells when used in combination [295].

Combining genetically engineered immune cells with ICIs is an intriguing strategy. A number of studies have shown that co-administration of PD-1/PD-L1 blockers with CAR-T cell therapy enhances anti-tumor activity and prevents T cell exhaustion in preclinical models [296] and patients receiving mesothelin-based CAR-T cell therapy [297]. CAR-T cells were further engineered to produce and secrete an anti-PD-1 scFv for targeting and boosting CAR-T cell activity in order to reduce the systemic effects of ICIs therapy [298]. Cherkassky et al. [299] successfully co-transduced anti-MSLN-CAR-T cells with vectors expressing shRNAs targeting PD-1 genes. As a result of cotransduction with PD-1 shRNAs and subsequent downregulation of PD-1 intrinsic levels, CAR-T cells were able to further expand and kill MSLN/PD-L1-positive cancer cells. Reduced N-linked glycosylation of PD-1 may reduce PD-1 expression and relieve its inhibitory effects on CAR-T cells. Therefore, a preclinical investigation using an adenine base editor (ABE) to decrease PD-1 suppression by changing the glycosylated residue in CAR-T cells downregulated the expression of PD-1 in CAR-T cells and improved cytotoxic functions in vitro and in vivo [300]. These strategies can also be used for CAR-NK cells.

6.4.3 B7-H3 (CD276)

The B7-H3-CAR was generated by Grote et al. [301] by conjugating a B7-H3 clone m851-derived scFv on a second-generation CAR backbone incorporating a CD8 hinge domain, a CD28 TM domain and the cytoplasmic CD28 co-stimulatory as well as CD3ζ signaling domains. NK-92 cells engineered with B7-H3-CAR could eliminate neuroblastoma (NB) cells in vitro specifically and long-term while sparing B7-H3-negative cancer cells. In addition, B7-H3-CAR-NK-92 cells demonstrated enhanced cytotoxicity in a 3D NB spheroid model recapitulating in vivo morphology as well as cell connectivity, polarity, gene expression, and tissue architecture, thus bridging the gap between in vitro and in vivo studies. A multitude of NK effector molecules were produced by B7-H3-CAR-NK-92 cells as well as pro-inflammatory cytokines that stimulated the immune system. A stable surface expression of B7-H3-CAR and cytotoxic effector function was observed for more than six months. Grote et al. [302] also used these CAR-NK-92 cells that target B7-H3, which is abundantly expressed in melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia, and other features of the TME that affect anti-tumor responses. Additionally, CRISPR/Cas9-induced disruption of NKG2A was tested for its potential to enhance the anti-tumor effect of NK-92. These B7-H3-CAR-NK-92 cells had the ability to cytolyze melanoma cell lines while being able to overcome the immunosuppressive effects typically exerted by TME. Cytotoxicity of CAR-NK-92 cells was not further improved by knocking out NKG2A. This study concluded that B7-H3-CAR-NK-92 cells are a promising cellular product for treating melanoma and beyond due to the cytotoxic properties of CARs targeting B7-H3 and the capabilities of an “off-the-shelf” NK-92 cell line unaffected by many negative factors associated with the TME. To improve NK cell functions, one study generated NK-92MI cells carrying anti-B7-H3-CAR by lentiviral transduction. The anti-B7-H3-CAR was comprised of an anti-B7-H3 scFv derived from 8H9 Ab, followed by the CD8 TM region, the intracellular domains of 4-1BB and CD3ζ. The expression of anti-B7-H3-CAR drastically enhanced the cytotoxicity of NK-92MI cells against B7-H3-positive tumor cells. Perforin and granzyme B secretions, as well as CD107a expression, were significantly increased in anti-B7-H3-CAR-NK-92MI cells. Furthermore, anti-B7-H3-CAR-NK-92MI cells efficiently inhibited tumor growth in mouse xenografts of NSCLC and significantly increased mouse survival days when compared to unmodified NK-92MI cells [303].

6.4.4 HLA-G

Another study established a novel CAR strategy comprised of the HLA-G scFv, TM and cytosolic domains of KIR2DS4, the P2A (porcine teschovirus-1 2A) sequence, the full-length DAP12 sequence, and inducible caspase 9 (iC9) (FK506-binding protein 12 fused to caspase-9). HLA-G-CAR-transduced NK cells showed effective cytolysis of breast, brain, pancreatic, and ovarian cancer cells in vitro, as well as decreased xenograft tumor growth with extended median survival in orthotopic mouse models. In tumor coculture assays, the anti-HLA-G scFv moiety increases Syk/Zap70 activation of NK cells, implying a reversal of the HLA-G-mediated immunosuppression and therefore, restoration of native NK cytolytic functions. Tumor expression of HLA-G can be further promoted using low-dose chemotherapy, which when combined with anti-HLA-G-CAR-NK, leads to extensive tumor ablation both in vitro and in vivo. This upregulation of tumor HLA-G involves the inhibition of DNA methyltransferase 1 (DNMT1) and demethylation of transporters associated with antigen processing 1 promoter (TAP-1) to trigger the translocation of HLA-G [304].

6.4.5 Cytokine-induced SH2-containing protein (CIS)

Another study developed a strategy that couples targeting of the CIS protein, a major negative regulator of interleukin IL-15 signaling, by CRISPR/Cas9 gene editing with fourth generation ‘armored’ CAR encoded iC9.CAR19.CD28-zeta-2A-IL-15 in CB-derived NK cells. This combined strategy boosted NK cell effector function via enhancing the Akt/mTORC1 axis and c-MYC signaling and led to increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach enhanced NK cell anti-tumor activity more than either alteration alone, eliminating lymphoma xenografts with no signs of any measurable toxicity. They concluded that targeting a cytokine checkpoint further improves the anti-tumor activity of IL-15 secreting armored CAR-NK cells by enhancing their metabolic fitness and anti-tumor activity. This combined approach could pave the way for the next generation of cancer immunotherapy using NK cells [305]. This strategy could also be used against solid tumors.