April 12, 2024

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How to overcome tumor resistance mechanisms in CAR-NK cell therapy?

How to overcome tumor resistance mechanisms in CAR-NK cell therapy?


How to overcome tumor resistance mechanisms in CAR-NK cell therapy?

In the past decade, autologous CAR-T therapy has revolutionized the treatment paradigm of hematological tumors.

Currently, 8 different CAR-T therapies have been marketed globally for the treatment of multiple myeloma ( MM ) and CD19+ B-cell malignancies.

However, despite the clinical success of CAR-T in relapsed and refractory hematological tumors, it still faces some challenges, such as cumbersome and high manufacturing cost and high toxicity, and relapse and drug resistance in many patients.


What followed was CAR-NK cell immunotherapy, emerging as a safer, faster, and more cost-effective approach without the severe toxicity of CAR-T cells.

Due to the unique biological properties and multiple mechanisms of action of NK cells, CAR-NK cell therapy is a strong clinical candidate.


Numerous preclinical studies of CAR-NK have been demonstrated to be effective in cancer therapy, especially in the treatment of hematological malignancies.

To date, 31 clinical trials have explored 11 different CAR targets, including CD19, CD20, CD22, NKG2D, CD33, and BCMA, among others.

Impressive responses of bicistronic CD19-CD28-ζCAR/IL-15 UCB NK cells in chronic lymphocytic leukemia (CLL) and refractory and relapsed lymphoma ( NCT03056339 ) ( ORR: 73 %; CR: 64% ).


Despite its multiple advantages, CAR-NK therapy still faces many challenges that may induce drug resistance and affect its efficacy.

Therefore, we need to focus on these mechanisms, especially the CAR-NK dysfunction that causes hematological tumors to evade immune surveillance, so as to develop effective strategies to overcome CAR-NK functional exhaustion and migration barriers and ensure the persistence of CAR-NK effects.




Manufacturing Conditions: Key Factors in CAR-NK Therapeutic Efficacy

One of the major problems with adoptive cell therapy is the need for large numbers of potentially proliferative potentiated effector cells for optimal clinical response.

Therefore, optimizing the source, cytokine priming and expansion protocols can guarantee the cytotoxicity and in vivo persistence of CAR-NK cells.


Cytokine initiation and amplification

The most common sources used to develop CAR-NK therapeutic platforms include NK cells from PB or UCB, NK cell lines such as NK92 , and stem cell-derived NK cells from IPSCs, HESCs, or CD34+ hematopoietic stem cells.


Regarding NK cell priming and expansion strategies, most are based on the use of soluble cytokines and artificial antigen-presenting cells ( aAPCs ) with membrane-bound molecules such as cytokines and/or costimulatory ligands .

The common gamma chain cytokines IL-2, IL-7, IL-15 and IL-21, and others such as IL-12 or IL-18, were used alone or in combination.

For example, the use of feeder cells genetically modified to express membrane-bound IL-15 or IL-21 and 4-1BBL can greatly increase the expansion fold while maintaining the cytotoxic potential of NK and CAR-NK cells.


Furthermore, since systemic administration of cytokines themselves may have undesired effects, recent engineering approaches have focused on in situ delivery and utilization of cytokine signaling to prolong the persistence of NK/CAR-NK cells while maintaining their optimized functions .

For example, an IL-15-secreting CD19 CAR-UCB-NK cell that showed enhanced cytotoxicity in vitro and persistence of CAR-NK in a phase I/II clinical trial without the patient’s Elevated levels of systemic IL-15 ( NCT03579927 ).


Suicidal tendencies reduce NK cell efficacy

Expansion of NK cells in vitro may lead to an undesirable phenomenon of “suicide”, in which cells recognize receptors or ligands on the surface of other homogeneous cells and trigger cytotoxic activity against them.

Several mechanisms may lead to cannibalism during NK or CAR-NK cell expansion.


Of these, the well-known Fas/FasL axis is one of the most relevant mechanisms.

FasL-mediated cytotoxicity plays a critical role in NK cell function because when it binds to the receptor Fas in target cells, it triggers caspase-dependent apoptosis.

Fas can also be expressed by NK cells as a homeostatic mechanism to inhibit NK cell activity, termed activation-induced cell death ( AICD ).

It has been reported that its expression may be abnormally increased during NK cell expansion, especially when cultured after IL-2, IL-15, or in the presence of specific feeder cells such as K562-mIL21, leading to cannibalism.


Another receptor that may lead to cannibalism among NK cells is NKG2D. NKG2D is a natural receptor, mainly expressed by NK, CD8+T and γδT cells, that recognizes a variety of stress-inducing ligands.

There is a growing body of data describing the expression of NKG2D-L in activated NK cells, but its origin and impact on NK cell function remain controversial.


In CAR-NK cells, cannibalism may also occur due to CAR ligand/antigen recognition.

Such as CD38 CAR-NK cells, since NK cells naturally express CD38 and in the presence of IL-2 or engineered feeder cells, its expression can be upregulated during in vitro expansion.

Therefore, NK cells may be recognized and destroyed by anti-CD38 CAR.


T cell allogeneic rejection

Recognition and rejection of donor NK cells by the host immune system may potentially reduce the persistence of allogeneic CAR-NK cells in the clinical setting.

The primary effector responsible for these mechanisms are alloreactive T cells, which recognize non-self HLA molecules on allogeneic NK cells.


Lymphoid chemotherapy induces a transient reduction of the host immune system, thereby improving adoptive cell engraftment.

In addition to reducing T and NK cells, lymphopenia drugs reduce cell populations with immunosuppressive properties, such as Treg and myeloid-derived suppressor cells ( MDSCs ), creating a more favorable microenvironment for adoptive cell expansion.


How to overcome tumor resistance mechanisms in CAR-NK cell therapy?



Currently, other strategies are being developed to prevent host system rejection.

As in human PB-NK cells, HLA class I expression was disrupted by targeting the β-2-microglobulin gene ( β2M ) to circumvent CD8+ T cell alloreactivity.



Overcoming aging: enhancing CAR-NK immune efficacy

In the absence of cytokine support, NK cells have a shorter in vivo lifespan, reducing non-tumor-targeted toxicity and tumorigenic risk, but also narrowing the therapeutic window. In vivo persistence and proliferation of NK cells following adoptive transfer have been shown to correlate with clinical response.

Therefore, early relapse may be caused due to low in vivo persistence of CAR-NK cells.

In addition, the short lifespan also limits the in vitro proliferation and expansion of NK cells during manufacturing, making it difficult to obtain sufficient cell numbers and shortening the time to optimize NK cells through genetic engineering.

Therefore, prolonging lifespan can improve the efficacy of CAR-NK cells.


Unlike T cells, which can persist for months or even years, the lifespan of human NKs is not well-defined, varies between subpopulations, and can be manipulated in vitro.

In vivo, mature NK cells require constant cytokine support, without which they remain in circulation for only 1-2 weeks.

When CAR-NKs were engineered to express IL-15, survival was up to 68 days.

However, despite allowing lifespan modification through exogenous cytokine or HLA matching, NKs and CAR-NKs are short-lived cells affected by senescence, which inevitably occurs in vitro and does not persist for long in patients.


Cellular senescence is associated with loss of proliferative capacity and functional defects, characterized by shortening of telomeres, detection of double-strand breaks in genomic DNA, activation of repair mechanisms, and arrest of the cell cycle.

Among the factors involved in the control of NK cell lifespan, telomere length is crucial.

For adoptive NK cell therapy, telomere length depends on the NK source or the chosen activation/expansion method. For example, iPSC-derived NK cells had much longer telomere lengths than cells expanded from PB.

In addition, IL-2, IL-15, and IL-21 have all been shown to upregulate telomerase activity in NK cells, thereby preventing telomere loss and allowing cells to replicate for extended periods of time.

Ectopic expression of hTERT by genetic engineering may be an effective strategy to enhance the persistence of CAR-NK cells, thereby enhancing their therapeutic potential, in which the maintenance of telomere length and replication capacity correlates with antitumor efficiency.




The tumor microenvironment: a stumbling block limiting the efficacy of CAR-NK therapy

NK cells can be induced in the tumor microenvironment into a reversible state of exhaustion characterized by impaired effector function, altered phenotype, and upregulated expression of tumor-associated immune checkpoints.

In addition to this, patient NK cells and infused CAR-NK cells are also subject to an unfavorable environment in the tumor niche for the production of immunosuppressive cells and soluble cytokines, thereby suppressing NK cells.


How to overcome tumor resistance mechanisms in CAR-NK cell therapy?



Depletion-related immune checkpoints

Several studies have shown that expanded NK cells increase PD-1 expression, and IFN-γ produced by NK cells increases PD-L1 expression in a mouse model of lung cancer.

A Phase II clinical trial ( NCT04847466 ) of PD-L1 CAR-NK cells in combination with pembrolizumab and N-803 is currently underway in gastric and head and neck cancers.


Similar to NKG2A, NK cell expansion in vitro upregulates the expression of other depletion receptors, such as TIM-3 and TIGIT.

Blockade of TIM-3 or TIGIT in preclinical studies increases the cytotoxic potency of NK cells against solid and hematological malignancies, and inhibitory antibodies ( such as NCT04623216, NCT03489343, NCT04150965, NCT04354246 and NCT05289492 are currently being tested in multiple clinical trials) ).

Furthermore, unlike T cells, the expression levels and inhibitory correlations of other receptors such as LAG-3 or CTLA-4 in NK cells remain unclear.


In conclusion, not all immune checkpoints were induced at the same level in ex vivo-expanded NK cells, nor were they similarly correlated in regulating NK cell antitumor activity.

The balance of activating and inhibitory signals regulates NK cell function, therefore, more efforts are needed to assess the impact of each immune checkpoint expression in the presence of CAR stimulation to guide strategies to improve CAR-NK therapy.


Other inhibitors in the TME

Soluble factors from the TME may enhance NK cell suppression, such as soluble NKG2D-L ( sNKG2D-1 ) produced by proteolytic shedding, which reduces NKG2D expression and attenuates the antitumor efficacy of NK cells.

CARs designed to recognize the same MICA/B domains have shown anti-leukemia efficacy in iPSC-derived NK cells.


In addition, other soluble factors, such as interleukins, enzymes, and metabolites, are present in the TME of most cancers, affecting the effectiveness of NK cells.

Most of these are released not only by tumor cells but also by immunosuppressive cells co-located in the tumor niche.

High concentrations of other inhibitory cytokines, such as IL-6, IL-10 and TGF-β, are widely present in hematological tumors.

CAR-NK cell therapy has been enhanced by knocking down TGF-α receptor expression or adding small-molecule receptor kinase inhibitors in engineered NK cells.


In addition to soluble factors, immunosuppressive cells present in the TME promote tumor proliferation through direct contact or release soluble factors, while reducing NK cell function.

Such as Treg, Breg, MDSC and tumor-associated macrophages ( TAM ), mainly M2 phenotype.

Hypoxia and metabolic factors, such as nutrient deficiency and acidity, also create an unfavorable microenvironment that impairs the antitumor activity of NK cells.




Chemotaxis: affecting NK cell migration and homing

One of the challenges of CAR-NK immunotherapy lies in the limited transport and homing ability to reach the BM and LNs .

Clinical studies have shown that improved adoptive infusion of NK cells into the bone marrow is associated with better disease control in patients with AML .

Multiple strategies to maintain and / or enhance chemokine or adhesion receptor expression in CAR-NK cells are currently being explored in preclinical models to improve their migration and homing.


CXCR4, CXCR3, CCR3, CCR5, and CX3CR1 are the major chemokine receptors expressed by the NK population, which contribute to the response to chemokines present in the TME.

Considering the high levels of chemokines found in the TME of hematological malignancies, modification of the expression of chemokine receptors in adoptively transferred NK cells appears to be an advantageous strategy.

The expression level of CXCR4 in UCB-derived NK cells was higher than that in PB-NK cells, indicating better homing ability of BM.

Modification of CXCR4-overexpressing CD19-CAR-NK cells by bicistronic lentiviral transduction increased migration to CD19+ tumor cells more than twofold compared with huCAR19 NK cells.


In addition to strategies to modulate chemokine receptor expression, BM homing can be enhanced by promoting the interaction of NK cells with adhesion molecules such as E-selectin.

For example, treatment of NK-92MI cells with human fucosyltransferase 6 ( FUT6 ) and GDP fucose yielded the cell surface E-selectin ligand sialyl Lewis X ( sLeX ) to improve migration to the BM and increase exposure to B lymphocytes killing of tumor cells.




Gene Editing: Designing CAR-NK 2.0

The application of gene editing holds great promise for improving the efficiency and persistence of NK cells.

To this end, different studies have focused on identifying negative regulators that can modulate immune function and enhance NK and CAR-NK potency.


How to overcome tumor resistance mechanisms in CAR-NK cell therapy?



One of the main strategies to improve CAR-NK persistence is to target suppressive immune checkpoints, such as the PD-1/PD-L1 axis, and indeed, PD-1 knockdown in NK cells increases cytotoxicity in ovarian cancer xenograft models.

Antitumor activity. TIM-3 is another checkpoint receptor expressed by NK cells, and TIM-3 knockout of NK cells has improved cytotoxicity in vitro.

A similar CRISPR/Cas9-based strategy has been applied to the Siglec-7 receptor, which, when bound to certain sialylated glycans expressed on tumor cells, triggers NK inhibition.


To avoid NK cell depletion, targeting cytokine-related immune checkpoints is another interesting approach.

Several proteins including inhibitors of cytokine signaling ( SOCS1–7 and CIS ) downregulate cytokine signaling through the JAK/STAT pathway in NK cells, and their disruption increases NK cell activity and persistence.

From different studies it has been shown that knockdown of CISH in NK cells increases their cytotoxic properties and even improves their metabolic fitness.


One of the biggest challenges of CAR therapy, especially in solid tumors, is the immunosuppressive microenvironment created around the tumor.

Several research groups have successfully edited NK cells to disrupt TGFβ-R2 in NK and CAR-NK cells, making them resistant to TGF-β inhibition in vitro and thus enhancing tumor control of tumors.


Elimination of CAR-targeted receptors in CAR-NK cells is critical for the development of effective immunotherapy products.

For example, CD70 is upregulated in NK cells, leading to cannibalism. By depleting CD70 in NK cells using CRISPR/Cas9, suicidal-prone cells were obtained without affecting their cytotoxic potency.

Following a similar strategy, CD38 knockdown in CD38-CAR-NK cells has been shown to result in reduced cannibalism-induced cell death and a stronger cytotoxic response of CAR-NK cells to AML primary cells.


Finally, gene editing can be used to regulate NK cell migration.

It has been shown that disrupting CCR5 using gene editing alters NK cell migration in vivo, which reduces transport to the liver and increases BM homing.

This approach promises to redirect CAR-NK cells and increase their antitumor efficacy.

Similar strategies can also be used to target other chemokine receptors on the surface of CAR-NKs and redirect them to the tumor site.





Summary: How to overcome tumor resistance mechanisms in CAR-NK cell therapy?

Advances and advancements in the field of NK cell immunobiology have laid the foundation for better and more novel immunotherapies, and the excellent antitumor effects of NK cells have made them the focus of cellular immunotherapy.

CAR-NK cell therapy is a promising area of ​​clinical research. Compared with CAR-T cells, CAR-NK cells have their own unique advantages, but still face some challenges.

These challenges include cell persistence, overcoming the immunosuppressive microenvironment, and tumor homing.

It is believed that solving these problems, based on the excellent anti-tumor lineage of NK cells, is very likely to bring new breakthroughs to tumor treatment under the arm of CAR modification.







1. Front Immunol. 2022; 13: 953849.

How to overcome tumor resistance mechanisms in CAR-NK cell therapy?

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