Nat Rev Cancer: The future direction of NK cell therapy

Natural killer cells (NK cells) are a class of innate lymphocytes that have the ability to
recognize and eliminate virus-infected cells and tumor cells.
With the disruptive success of CAR-T cell therapy, interest in the potential of NK cells in anti-tumor immunotherapy has also
increased.

Figure 1 NK cells and other immune cells

Clinically, the first generation of NK cell therapy has achieved promising results, showing encouraging efficacy and good safety profile
.
A review in the top international journal Nature Reviews Cancer (IF=69.
800), authors describe various ways to increase NK cytotoxicity and longevity, meet challenges and seize opportunities, and guide the design of
next-generation NK cell products from clinical development lessons learned.

1.
NK cell source and donor selection

NK cells were discovered in the 70s of the 20th century and are mainly associated with
killing infected microorganisms and malignant transformation of allogeneic and autologous cells.
In humans, NK cells are derived from CD34+ co-lymphatic progenitor cells
.
It is estimated that the half-life of NK cells is about 7~10 days, and they exert key immunomodulatory functions
through interaction with DC cells.

NK cells come from a wide range of sources, including peripheral blood, cord blood, pluripotent stem cells (ESCs and iPSCs), and NK cell lines (such as NK92 cell lines).

All sources are available with clinically meaningful cell doses, suitable for the preparation of CAR-NK, and have transitioned to human studies
.
Each source has advantages and potential challenges
.
CAR-NK cells have been successfully engineered
from different platforms, including cord blood.

Figure 2 Source and donor selection of NK cells

▉ Sources of peripheral blood or cord blood

Primary NK cells can be harvested from peripheral blood (PB-NK cells) or from umbilical cord blood (CB-NK cells).

PB-NK cells, the first successful platform led by Dario Campana to successfully deliver a CAR construct into NK cells in 2005
.
PB-NK cells provide the building blocks
for a variety of products currently in clinical trials.

▉ Induced pluripotent stem cell source

iPSCs are a source of NK cells, which can be efficiently expanded and differentiated in vitro
.
A large number of NK cell products
with a consistent genetic background can be manufactured through iPSCs.
The preparation of iPSCs requires fewer seed cells, can be cultured in large quantities, has low cost, can achieve autologous supply, and has low
immunogenicity.

iPSC-derived NK cells have two drawbacks: first, they express low levels of CD16
.
Second, iPSCs may contain "epigenetic memory," which may affect the development
of specific cell lineages that differ from donor cells.

▉ NK cell line source

NK-92, the first NK cell approved by the U.
S.
FDA for clinical trials, is a homogeneous, immortal, NK lymphoma cell line that can be expanded
ex vitro.
NK-92 cells lack the expression of most KIRs and are therefore unlikely to be inhibited
.

However, irradiation of NK-92-derived cell products is required prior to dosing to the patient, which affects the long-term persistence and overall therapeutic potential
in vivo.
Another drawback is that NK-92 cells lack the ability to
mediate cell killing through ADCC due to lack of CD16 expression.

Fig.
3 Advantages and limitations of NK cells from different sources

2.
How to enhance NK cell function

Fig.
3 Strategies to enhance NK cell specificity

▉ Chimeric antigen receptors

CARs are synthetic fusion proteins that include extracellular antigen recognition domains and intracellular signaling moieties
that trigger cell activation.
CARs can be expressed on immune effector cells to reprogram their specificity
for specific targets.
CAR-T cell therapy was the first to appear
.
Since then, CAR, traditionally designed for T cells, has been used to produce CAR-NK cells, which have been shown to target specific tumors while maintaining an ideal safety profile
.
Intracellular antigens are present in the form of peptide-HLA complexes, detected
by TCR.
Engineered NK cells expressing TCR can detect this peptide
.
TCR-NK cells, which have been shown to elicit anti-tumor responses
.

▉ NK cell adapter

NK cell adaptors, which can guide NK finely to the tumor site, trigger a strong anti-tumor response by triggering activating receptors on NK cells, while binding to the target antigen
on tumor cells.
Development strategies include three or four specific designs that target multiple antigens on the tumor, or promote NK amplification and survival by crosslinking cytokine moieties, with the aim of enhancing anti-tumor effects
.
The use of cell adapters thus represents a simpler and less costly manufacturing process to deliver products
capable of inducing CAR-like activity.

Figure 4 Principles and strategies of CAR design

▉ Cytokines

Cytokines, which can enhance NK cytotoxicity and promote NK cell proliferation
.
Continuous ex vivo stimulation makes NK cells "cytokine addicted" and reduces persistence
when infused with these cells without cytokine support in vivo.
Through genetic engineering, NK cells are modified to produce cytokines, maintain cellular potency, proliferation, and persistence
.
This autocrine maintenance has aroused great interest, and CAR-NK cells are emerging
.

▉ Overcome immunosuppression

Tumors often exhibit abnormal metabolic behavior, resulting in elevated levels of lactate and increased
concentrations of toxic catabolites, adenosine, and reactive oxygen species in the tumor's living environment.
In addition, there are proliferating and dysfunctional vasculature and immunosuppressive cell subsets in the tumor microenvironment (TME), resulting in the inability of various immune effector cells entering TME
.

To overcome immunosuppression, current strategies focus on two areas:

In preclinical studies, the deletion of adenosine A2A receptors in CAR-T cells and CAR-NK cells by gene editing showed anti-tumor efficacy
.

▉ Block immune checkpoints

Tumors can evade immune surveillance (e.
g.
, immune checkpoint involvement), and NK dysfunction
can be reversed with corresponding mAbs.
However, the patient's immune cells are modulated by mAb, but multiple infusions
are required.
With the advancement of gene editing technology, NK cells can be stably modified to enhance NK cell function
.
In addition, after determining CISH (cytokine-induced SH2-containing protein) as a key negative regulatory protein for NK cell function, CAR-NK cells lacking such intracellular cytokine checkpoints were designed to significantly improve
their metabolic adaptability and antitumor activity.

▉ Enhance the transport of NK cells to tumors

The ability of NK cells to enter and penetrate tumors is the key to
effective anti-tumor immunity.
NK cells, similar to other immune cells, are guided to the tumor site
through the dynamic interaction of chemokine receptors and their homologous ligands secreted in TME.
Due to the rapid loss of chemokine expression due to internalization and degradation, the use of genetic engineering to equip NK cells with stable ectopic chemokine receptors is a direction
for the future development of CAR-NK.

3.
Clinical lessons learned

CAR-T cell therapy has allowed some patients to achieve effective treatment
for up to ten years.
NK cells have unique anti-tumor effects, MHC-limited cytotoxicity, cytokine production, and immune memory, making them a key player
in the innate and adaptive immune response system.
Early clinical data suggest that NK cells are well suited for allogeneic use in allogeneic therapeutic settings
.
While the safety results are encouraging, further research is needed to clarify whether allogeneic NK cells are able to evade T cell rejection for long-term persistence
.

Clinical studies have shown that NK cells have safety and efficacy
in the treatment of hematological malignancies.
Importantly, regardless of the approach, NK cell therapy has consistently shown a good safety profile
.
To date, no CRS or GVHD
has been observed.
However, most reported successes are limited to hematologic malignancies, not all patients respond to NK cell therapy, and some eventually relapse
.

In any case, CAR-NK cell therapy is a promising clinical research field with good safety and preliminary efficacy
for some cancer patients.
It is believed that CAR-NK cell therapy may lead to revolutionary advances
in tumor immunotherapy.

Rezvani

Rezvani is a professor
in the Division of Cancer Medicine at the University of Texas MD Anderson Cancer Center.
His laboratory focuses extensively on the protective effects of natural killer cells (NKs) in hematological malignancies and solid tumors, as well as enhancement strategies for various cancer-killing functions
.
Research areas include immunotherapy, immunology, genetics, CAR T cells, CAR-NK cells
.

Current discoveries and research topics in Rezvani's lab include: comprehensive analysis of NK cells and their receptors in cancer after hematopoietic stem cell transplantation using mass spectrometry (CyTOF) and transcriptome analysis; NK cells are engineered to express chimeric antigen receptor (CAR) and cytokine genes to enhance their effector function and persistence; Understand the mechanism of NK immune evasion and target checkpoints using gene-editing tools to enhance NK effector function; CARs and gene-editing tools such as CRISPR are used to enhance the function of
T cells against viral and cancer antigens.

Resources:

[1] Laskowski, T.
J.
, Biederstädt, A.
& Rezvani, K.
Natural killer cells in antitumour adoptive cell immunotherapy.
Nat Rev Cancer (2022).

[2] Maskalenko et al.
, Harnessing natural killer cells for cancer immunotherapy: dispatching the first responders.
 Nature Reviews Drug Discovery,  (2022).

[3] Le Saux and Schvartzman Advanced Materials and Devices for the Regulation and Study of NK Cells.
 Int.
J.
Mol.
Sci.
,  (2019).

[4] Zhang et al.
,.
CAR-NK cells for cancer immunotherapy: from bench to bedside.
 Biomarker Research,(2022)