Advances in natural killer (NK) cell sources, amplification techniques, and GMP-compliant manufacturing methods

Natural killer (NK) cells, as a natural immune cell with the ability to directly kill malignant cells, are increasingly considered to be a promising immunotherapy tool, and various clinical trials

In order to obtain a large number of high-purity and highly cytotoxic NK cells, several strategies

Figure 1.

In vitro expansion of PBMC-derived NK cells

PBMCs are one of the main sources of NK cells, with the advantages of relatively easy collection, easy expansion in vitro, and non-toxic side effects

Figure 2.

Cytokines are key components in maintaining NK cell survival and supporting NK cell proliferation in vitro without the need for trophoblasts

In addition to strategies using cytokines, other options were explored, including irradiated tumor cells, allogeneic PBMCs, and lymphoblast derivatives as feeder cells to promote the expansion of NK cells in vitro

In addition, tumor cell membrane granules are also an effective substitute for tumor cell line expansion of NK cells, which not only maintains the activation efficiency of membrane receptors that induce NK cell expansion, but also avoids the risk

At present, PB-NK cells can be expanded up to 15 weeks in vitro, and the maximum yield of amplified PB-NK cells can reach 1×10¹³, however, a better amplification effect can be achieved

Recently, in order to achieve the production of GMP-compliant genetically engineered NK cells and promote the clinical application of CAR NK cells, a growing number of studies have used the CliniMACS Prodigy device to integrate multiple steps

In vitro expansion of NK cells derived from cord blood

There are two different ways to obtain large numbers of NK cells

Single or simple combinations of cytokines such as IL-2, IL-5, and IL-21 have been used to expand UCB-NK cells

Genetic modification of CB-NK cells allows for better in vivo persistence, mainly focused on the TGFβR and IL-15 signaling pathways

Netherlands-based Glycostem Therapeutics is the world's first GMP-compliant platform to build a fully enclosed system for the production of allogeneic NK cells from fresh cord blood stem cells

Expansion of NK cell lines

NK-92, the first NK cell-based immunotherapy approved by the FDA for clinical trials, is a homogeneous, immortalized NK lymphoma cell line that can be expanded in vitro to obtain large numbers of cells

After the establishment of NK-92 cells, culture
in α-MEM with bovine serum, horse serum, hydrocortisone, inositol, folic acid, and IL-2.

Cells double in about 24 hours and can continue to proliferate in vitro for more than 18 months, with the culture medium
being changed approximately twice a week.

However, because the amplification system contains animal-derived components, it is not suitable as a clinical-grade amplification system
.

NK-92 cells can be easily expanded in culture due to their dependence on IL-2 proliferation, and proliferation of GMP-compliant NK-92 cells in air vent bags, cell vials, or bioreactors is direct
.

Researchers have optimized and validated processes
related to the production of NK-92 cells that meet GMP standards for transgenic CAR expression.

GMP grade X-Vivo 10 culture solution with human plasma and IL-2 can effectively amplify NK-92 cells and maintain specific cytotoxicity and stable CAR expression over a long period of time (>98%)
.

A large number of NK-92 cell cultures were stored in VueLife 750-C1 bags pre-filled with X-Vivo 10 culture solution and then transported over long distances, maintaining a high killing rate (>90%) and a high survival rate (>90%)
for K562 cells.

Expand NK cells from iPSCs or ESCs

In addition to obtaining NK cells from peripheral blood and cord blood or using NK cell lines, NK cells can be induced from PSCs, enabling ready-made production
.

Studies have shown that PSC-derived NK cells not only have the advantages of primary NK cells and NK cell lines, but also do not have their problems
.

PB-NK and UCB-NK cells are readily available, but the number and purity of expanded NK cells is greatly influenced by the donor, and the products are often heterogeneous
.

Cells of the NK cell line are homogeneous and it is often necessary to inhibit the proliferation of such cells in the treatment of patients, but this also affects anti-tumor activity
.

Although studies have shown that NK cells differentiated from PSCs exhibit immature phenotypes, such as higher levels of NKG2A expression, PSCs can be induced to differentiate into iPSC-NK cells similar to primitive NK cells, such as iPSC-NK cells
expressing multiple NK receptors (NKG2D, TRAIL, CD16, etc.
).

In summary, NK cells derived from iPSCs or embryonic stem cells (ESC) have attracted widespread attention
due to their ability to provide homogeneous, highly cytotoxic NK cell products.

It is currently believed that the differentiation of PSCs into NK cells is carried out in two stages, the first stage of PSCs differentiation into hematopoietic progenitor cells (HPC) and the second stage of HPC differentiation into NK cells (Figure 3
).

The incubation time of this differentiation experiment is about 5-8 weeks, and its purity > 90%.

Figure 3.
Differentiation process of PSCs into NK cells

Studies have shown that PSC-NK cells in the differentiation phase have only a small amount of expansion, about 5-40 times
.

Therefore, it is necessary to take additional measures to expand NK cells to obtain enough NK cells for preclinical or clinical treatment
.

Currently, feeder systems are mainly used to expand PSC-NK cells, including transgenic, irradiated K562 cells and irradiated PBMCs
.

Transgenic iPSCs can maintain good pluripotency
.

IPSC or ESC-derived NK cells provide a convenient platform
for gene manipulation in the PSC stage.

A series of studies have shown that modifications to IL-15 and CIS can improve the proliferative capacity
of PSC-NK cells by reducing cytokine requirements.

Fate Treateutics is a company
focused on applying iPSCs to immune cell therapy.

The company's GMP-compliant manufacturing facilities are designed
to produce off-the-shelf cell products.

Fate Treateutics' FT516 product is the first genetically engineered iPSC-derived NK cell therapy product to receive FDA approval for clinical trials
.

To date, in addition to the progress made by Fate Treateutics, GMP production of NK cell products derived from iPSCs has been very limited, as most studies related to iPSCs are still in the scientific research phase
.

Summary

Immunotherapy based on natural killer (NK) cells is a promising way to treat a variety of cancers
.

Unlike T cells, NK cells recognize target cells through mechanisms that are not dependent on the primary histocompatibility complex (MHC) and can directly kill the cells
without sensitization.

In order to obtain a large number of high-purity and highly cytotoxic NK cells, a variety of strategies
have been developed.

Different sources also provide a rich diversity of options for the design of NK cell therapy strategies to meet the needs of different patient populations and disease indications
.

In order to further improve the clinical treatment effect of NK cells, genetic modification of NK cells is developing
rapidly.

Due to the relatively high safety and high feasibility of off-the-shelf production, genetically modified NK cells will become the next generation of cancer immunotherapies
with great potential.

References

1.
Advances in NK cell production.

2.
Natural killer cells in antitumour adoptive cell immunotherapy.

3.
Successful Transfer of Umbilical Cord Blood CD34 Hematopoietic Stem and Progenitor derived NK Cells in Older Acute Myeloid Leukemia Patients.

4.
Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors.