What are the advantages of CAR-NK cell immunotherapy?

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What are the advantages of CAR-NK cell immunotherapy?

Because NK cells can recognize and decompose tumor cells, the field of tumor immunotherapy based on NK cells has reached an exciting juncture.

As a receptor protein, the chimeric antigen receptor (CAR) gives immune cells new capabilities to target specific antigen proteins.

The latest research shows that CAR-expressing NK cells may overcome some of the defects of CAR-T cells and show significant anti-tumor effects. CAR-NK cells show broad prospects in tumor immunotherapy.

Research overview of CAR-NK

The number of CAR-NK preclinical studies is increasing year by year, which is reflected in the increasing number of research papers on CAR-NK every year.

What are the advantages of CAR-NK cell immunotherapy?

In addition, in terms of research targets, Her2 is the most commonly used target for solid tumors, and the CD19 antigen is the most common in hematological tumors.

In studies using primary NK cells, 65% of people are studying B-cell malignancies, and CD19 is the most popular target. Interestingly, in the study using the NK cell line, the study of solid tumors is more than twice as much as hematological malignancies.

What are the advantages of CAR-NK cell immunotherapy?

CAR structure design

The functional CAR molecule expressed on NK cells consists of three parts: the extracellular domain, the transmembrane domain, and the intracellular signal domain.

The extracellular domain consists of a signal peptide and an antigen-recognizing single-chain antibody fragment ( scFv ).

A hinge region connects this structure to the transmembrane region, and it also connects to the intracellular domain containing the activation signal in the cell.

Successful CAR design is achieved through a combination of careful design and functional testing.

What are the advantages of CAR-NK cell immunotherapy?

Vector backbone and promoter

The vector backbone contains all the elements required to express CAR, such as promoters, polyA signals and transcriptional regulatory fragments.

The choice of promoter directly affects the expression level of the transgene.

At present, there is only one report on the comparison of CAR expression and function of different promoters in NK cell lines, and there is no comparative data on primary NK cells.

Based on this single report, the optimal promoter for CAR-NK cells has not been determined yet.

Current reports on CAR-NK cells show that a variety of promoters are used to drive the expression of CAR, whether it is derived from cell lines or primary NK cells.

In primary CAR-NK and CAR-NK cell lines, viral promoters ( CMV, MPSV, MMLV, SFFV, etc. ) are more commonly used than constitutively active promoters ( such as EF1α, CMV and PGK ).

Signal peptide

There is huge heterogeneity in signal peptides, which directly translates into different levels of protein secretion efficiency.

For CAR-NK and CAR-T cells, there is no comparative study to determine the best signal peptide. At present, CD8a-SP is the most commonly used signal peptide sequence of primary NK cells ( 16%, 71% of the studies are not published ) and the immunoglobulin heavy chain or light chain signal peptide of NK cell lines ( 29% ).

Single chain antibody

The single-chain antibody fragment is the tumor antigen binding domain of CAR, and this domain will determine the specificity and function of CAR-NK cells.

Since single-chain antibodies are not the natural form of antibodies, the order of heavy and light chains is artificially determined.

So far, for CAR-NK designs, most prefer the VH-VL direction rather than the VL-VH direction. Fujiwara et al. proved that the order of heavy chain and light chain does not affect the expression level of anti-KDR CAR on T cells.

In addition, cells can be equipped with multiple single-chain antibodies to expand the antigen recognition ability of CAR effector cells.

Here, there are many choices: CARs can be transduced with a two-element vector to induce the expression of two CAR structures; or two single-chain antibodies can be fused in one structure to produce a “single-handled” CAR of tandem single-chain antibodies .

Although these technologies have been used to produce CAR-T cells, CAR-NK cells are still unknown.

At present, most clinical CAR-T cell trials use single-chain antibodies derived from mouse antibodies, which increases the risk of host-versus graft disease with anti-mouse IgG cells.

This problem can be avoided by humanization or screening of fully human antibodies .

Unfortunately, due to the chimeric nature of these CAR receptors, even humanized single-chain antibodies may induce an idiotypic immune response against the host.

Fortunately, in the limited number of CAR-NK clinical trials so far, no major side effects related to the anti-CAR immune response have been found.

Connection zone

The linking region between the heavy chain and the light chain helps to stabilize the conformation of the single-chain antibody.

Too short will lead to the formation of multimers, and too long may lead to hydrolysis or reduce the association between the VH and VL domains.

For CAR-NK cells, the multimer of the pentapeptide GGGGS is the most widely used, usually 3 repeats.

Another linker designed to enhance proteolytic stability is the Whitlow “218” linker: GSTGSGSKPGSGEGSTKG.

At present, although most CAR-NK studies do not provide connection details, in the existing research reports, 22 have used G4S connections and 2 have used 218 connections.

Hinge area

The hinge region is the CAR extracellular structural region that connects the single-chain antibody unit and the transmembrane domain.

It usually maintains the stability required for robust CAR expression and activity in effector cells.

Most CAR-NK constructions use derivatives of CD8α or CD28 extracellular domains or IgG-based hinge regions.

What are the advantages of CAR-NK cell immunotherapy?

The type and length of the hinge area have an important influence on the functional activities of CAR.

However, most of the information currently comes from the CAR-T field, and whether it can be directly transformed into CAR-NK remains to be proven.

In a direct comparison between the CD28 and CD8α hinge regions, it is found that CD28 is more likely to promote the dimerization of CAR molecules.

Therefore, the CAR in the CD28 hinge region produces stronger activation stimuli. Although this may be beneficial, it can also cause more serious side effects.

IgG-based hinge regions are also widely used in CAR structures.

One of the main advantages based on the IgG hinge region is the flexibility of the structure, which usually consists of the FC part of IgG1 or IgG4 or the CH2/CH3 domain of the Fc part.

The length of the hinge region can be adjusted to suit antigen recognition, but studies have found that the shorter the spacer region, the higher the production of cytokines, the faster the proliferation of CAR-T cells, and the better the persistence and anti-tumor effect in vivo.

For CAR-NK cells, most studies have adopted the CD8α hinge region in primary NK cells ( 16/35 ) and CAR-NK cell lines ( 41/72 ).

Other hinge regions used include CD28, IgG Fc domain, and DAP12.

Transmembrane domain

The transmembrane ( TM ) domain connects the extracellular domain of CAR and the intracellular activation signal domain. The most commonly used TM part of CAR-NK comes from CD3ζ, CD8 and CD28, but others such as NKG2D, 2B4, and DNAM1 are also used.

The choice of TM domain affects the activation degree of CAR structure in cell function. The molecules normally expressed on NK cells such as DNAM-1, 2B4 and NKG2D TM will cause more CD107a degranulation and higher cytotoxicity. Therefore, the specific source of TM will determine the activity of CAR-NK.

An important aspect of the TM domain is that the optimal TM region should follow the natural protein orientation ( N-terminal to C-terminal sequence ) of transmembrane proteins on T cells or NK cells . Although NKG2D is a powerful NK cell activator, natural NKG2D has a C-terminal to N-terminal transmembrane region.

At present, CD8α and CD28 modified TM are the most common in primary CAR-NK cells, and CD28 is the preferred TM region for CAR-NK cell lines.

CAR-NK activation signal

The number of intracellular activation signals of CAR determines which “generation” of CAR it belongs to.

The first generation of CAR-NK cells, like CAR-T cells, only contains CD3ζ signal. The second and third generation CAR-NKs carry one and two additional costimulatory signals, respectively.

The costimulatory molecules are usually derived from the CD28 family ( CD28 and ICOS ), TNFR family ( 4-1BB, OX40 and CD27 ) or SLAM related Receptor family ( 2B4 ). So far, the only published CAR-NK clinical trials have adopted the second-generation CAR-NK construction, which enhances the activity by adding IL-15 expression and inducing Caspase9.

At present, most CAR structures rely on the CD3ζ chain signal domain.

Strong activation signals are important for inducing effective anti-tumor responses, but they may also lead to rapid depletion of effector cells. Therefore, the combination of costimulatory domains can be used to calibrate the desired immune cell response.

Compared with CARs based on 4-1BB, CARs based on CD28 showed faster effector characteristics and induced higher levels of IFN-γ, granzyme B, and TNF-α.

However, this strong costimulatory signal can also cause activation-induced cell death ( AICD ).

In comparison, 4-1BB-CD3ζ signal preferentially induces memory-related genes and sustained anti-tumor activity. The reason may be that the 4-1BB domain improves T cell exhaustion caused by the CD28 domain.

As shown in the figure above, in the study of CAR-NK cell lines and primary CAR-NK cells, CD3ζ is almost universally used as the main activation domain, about half of which carry an additional activation domain, usually with 4-1BB or CD28 .

As for the third-generation structure, the combination of CD28/4-1BB/CD3ζ is the most commonly used.

CAR transfection or transduction vector

With the advancement of gene modification technology, many methods have been used to produce CAR-NK.

The two main methods are viral transduction ( using lentivirus or retrovirus ), or transfection of naked plasmid DNA, transposase DNA-mediated integration, and mRNA electrotransduction.

Lentivirus

Lentivirus can efficiently transduce periodic and acyclic cells, and has been widely used in the field of gene therapy.

So far, 14 studies on primary CAR-NK cells and 44 studies on CAR-NK cell lines have successfully used lentivirus as a vector.

In preclinical studies, 21 studies used second-generation viruses, and 6 studies used third-generation lentiviruses to generate CAR-expressing NK cell lines ( 17 unknowns ).

In the primary CAR-NK cell study, 5 studies used the third-generation lentivirus, and 7 studies used the second-generation lentivirus vector ( 2 unknown ).

Retroviral

For decades, retroviruses have been used as gene therapy vectors. So far, 20 studies using CAR-NK cell lines and 15 studies using primary NK cells have applied retroviruses.

In a recent phase I clinical trial, CD19 CAR-NK cells transduced by retroviruses were used to treat CD19+ non-Hodgkin’s lymphoma and chronic lymphocytic leukemia.

In this study, 73% of patients responded, and 7 of 8 patients achieved complete remission.

In addition, at all dose levels, CAR-NK has a rapid response within 30 days after infusion.

After a year of follow-up, expanded CAR-NK cells can still be detected. After infusion, the copy number of CAR-NK DNA in peripheral blood remained stable for up to one year.

These results indicate for the first time that retrovirus-transduced CAR-NK cells can survive in the body for a long time.

Different types of retroviruses are used to generate CAR-NK cells. Compared with gamma retrovirus and lentivirus, RD114α retrovirus has higher transduction efficiency in primary NK cells.

Although the use of different retroviruses can achieve long-term stable CAR expression in NK cells, the safety of the retroviral system is still a concern, especially when compared with safer lentiviruses.

mRNA electroporation

Electroporation of CAR-encoded mRNA is a fast, effective but short-lived method. So far, 9 studies on CAR-NK cell lines and 11 primary CAR-NK cells have used mRNA electroporation.

Generally speaking, the mRNA transfection efficiency of amplified or activated NK cells is much higher than that of freshly isolated NK cells.

Since mRNA synthesis complies with GMP regulations, and electroporation can be performed in a clean room, it is feasible to generate GMP-compliant CAR-NK by mRNA electroporation.

However, the main disadvantage of this method is that the window for CAR expression is short: After electroporation, CAR-NK cells should be infused back into the patient within 7 days.

Sleeping Beauty Transposon

Transposon-based systems can efficiently introduce CAR transgenes at predetermined locations, which is an important advantage that traditional methods do not have.

Transposons are mainly introduced into NK cells through electroporation, and then integrated into the host genome through transposase enzymes.

There are two studies using the transposon system to generate CAR-NK cells: one using NK-92-MI cells, and another study transfecting the transposon into iPSC cells and then differentiating them into NK cells.

After enrichment, anti-mesothelin CARs were stably expressed on iPSC-derived NK cells and played a role in mouse models of ovarian cancer.

CRISPR/Cas9

CRISPR/Cas9 is a powerful genetic modification technology that relies on the introduction of Cas9 protein and guide RNA into NK cells.

Initially, this technique was used in primary NK cells to destroy the CD38 gene, aiming to prevent NK cells from cannibalism when used in combination with daratumumab ( anti-CD38 ), because CD38 is found in NK cells, multiple myeloma and AML cells There are expressions on it.

Recently, CRISPR/Cas9 has been used to introduce new genes. In some studies using HDR templates, NK cells expanded with K562-mIL-21 achieved a knock-in efficiency of more than 75%.

However, in fresh NK cells, the knock-in efficiency is only 3-16%.

In general, the CRISPR/Cas9 strategy is a promising technology, which can be used to precisely delete, repair or introduce specific genes, and is expected to produce powerful anti-tumor NK cells.

Advantages of CAR-NK cell immunotherapy

First of all, in clinical applications, CAR-NK cell immunotherapy is safer than CAR-T cell immunotherapy, and the safety of NK cells has been verified in some clinical fields.

For example, some phase I/II trials have shown that allogeneic NK cell infusion is well tolerated and will not cause GVHD and obvious toxicity.

Therefore, NK cells are a more adaptable CAR carrier, not just autologous cells.

One of the main side effects of CAR-T cell immunotherapy is the targeting effect due to the continuous existence of CAR-T cells.

On the contrary, CAR-NK cells have a short lifespan and hardly produce targeting effects.

In addition, the types of cytokines produced by NK cells are very different from those produced by T lymphocytes.

Active NK cells usually produce IFN-γ and granulo-macrophage colony stimulating factor ( GM-CSF ), while CAR-T cells are usually induced by secreting pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 Cytokine storm.

Second, in addition to inhibiting cancer cells by recognizing tumor surface antigens by single-chain antibodies, NK cells can also inhibit cancer cells by recognizing various ligands through a variety of receptors, such as natural cytotoxic receptors ( NKp46, NKp44, and NKp30 ) , NKG2D and DNAM-1 ( CD226 ).

These NK cell receptors generally recognize stress-inducing ligands expressed on tumor cells under the stress of immune cells or long-term treatment.

In addition, NK cells induce antibody-dependent cytotoxicity through FcγRIII ( CD16 ).

Therefore, CAR-NK cells can inhibit cancer cells through CAR-dependent and NK cell receptor-dependent pathways, thereby eliminating tumor antigen-positive cancer cells or cancer cells expressing NK cell receptor ligands.

Clinical trials have shown that CAR-T cells cannot eliminate highly heterogeneous cancer cells, but CAR-NK cells can effectively kill residual tumor cells that may change their phenotype after long-term treatment.

Finally, NK cells are very abundant in clinical samples and can be produced from peripheral blood ( PB ), umbilical cord blood ( UCB ), human embryonic stem cells ( HESC ), induced pluripotent stem cells ( IPSC ) and even NK-92 cell lines.

NK-92 cells provide a uniform cell population and can be easily expanded under appropriate culture conditions for a wide range of clinical applications.

However, due to its tumor cell line origin, it must be irradiated before infusion.

In contrast, active PB-NK cells express a wide range of receptors and can be used without radiation, which allows them to be produced in the body.

NK cells derived from iPSCs or hESCs combine the advantages of PB-NK and NK-92 cells, show a phenotype similar to PB-NK cells, and are a homogeneous population.

More importantly, by using non-viral transgenic methods, CAR can be easily expressed in human embryonic stem cells and/or iPSC-derived NK cells.

The clinical research of CAR-NK cell immunotherapy

Hematoma

Preclinical studies have shown that CD19-CAR-NK cells have a high response rate to hematological tumors and are easy to manufacture.

Compared with the current CAR-T cell immunotherapy, this is a huge improvement. CD19-CAR modified NK cells have more obvious advantages than CAR-T, and are expected to show better anti-tumor effects.

In addition to CD19, CAR-NK cell clinical research for lymphoma and leukemia also targets CD7 ( NCT02742727 ) and CD33 ( NCT02944162 ).

Currently, several CAR-NK cell clinical trials for hematological malignancies are underway.

Solid tumor

A major obstacle to CAR treatment is the lack of efficacy for solid tumors. This is due to poor tumor perfusion, TAA heterogeneity, and the immunosuppressive tumor microenvironment ( TME ) associated with solid tumors .

CAR-NK therapy attempts to solve these problems, and some clinical trials have been applied to the treatment of solid malignant tumors.

The most widely advanced CAR-NK therapy in solid tumors includes checkpoint molecular programmed cell death protein-1 ligand ( PD-L1 ) and common tumor-associated antigens HER2 and MUC1 as targets.

PD-L1 is up-regulated in several cancer types of TME and immunosuppressive cells. A new NK-92 cell line is designed to target PD-L1, ER-retained IL-2 and high-affinity CD16 CAR, called PD-L1 targeting haNK ( t-haNK ).

Exciting preclinical data shows that these cells have specific anti-tumor effects on 15 tumor cell lines in vitro, and have strong anti-tumor effects on triple-negative breast cancer, bladder tumors and lung cancer in vivo.

The QUITL3.064 phase I clinical trial ( NCT04050709 ) is currently underway, and PD-L1 t-haNK combined with other drugs to evaluate the safety and effectiveness of patients with locally advanced or metastatic pancreatic cancer is also underway ( NCT0439099 ).

HER2 is overexpressed in several cancers, such as breast cancer, gastric cancer, esophageal cancer, ovarian cancer, and endometrial cancer.

HER2 is also expressed in 80% of glioblastomas and is associated with low survival rates. Therefore, HER2 is a very attractive target for CAR therapy.

HER2-CAR-T treatment of glioblastoma has shown that these cells are transferred to the tumor, but the high-efficiency T cells in TME lead to rapid selection of antigen-losing variants.

ErbB2-NK-92/5.28z CAR-NK will maintain the effector function in the case of loss of potential antigen, so that the cytotoxic function of adoptively transferred cells can be restored to the cytotoxic function of baseline NK cells.

This HER2-CAR-NK treatment is in a 3+3 dose escalation phase I clinical trial ( CAR2BRAIN; NCT0338978 ).

MUC1 is a highly glycosylated transmembrane glycoprotein expressed on ductal epithelial cells, and its expression on human B cells and T cells is controversial.

MUC1 is overexpressed in many cancer types, making it a suitable target for CAR-T therapy.

However, CAR-T therapy has proven to be unsuccessful because of the immunosuppressive TME.

In order to avoid TME-mediated immune suppression, CAR-NK cells are designed to express MUC1-CAR with CD28 and CD137 signal domains and a truncated PD-1 peptide ( MUC1-CAR-NK-92 ). These cells can kill MUC1 target cells in vitro and in vivo.

Recently completed a phase I clinical trial ( NCT02839954 ) to evaluate the safety and effectiveness of Muc1-CAR-NK-92 in patients with Muc1-positive relapsed or refractory solid tumors.

Among 13 patients with PD-L1 and MUC1 positive tumors, 3 withdrew, 9 with stable disease, and 1 patient with progressive disease.

The main observation index is to determine the toxicity of Muc1-CAR-NK-92 cells. It is worth noting that no patients in the trial showed signs of cytokine storm or bone marrow suppression.

Obstacles to clinical application of CAR-NK cells

Expansion of NK cells in vitro is the first obstacle to CAR-NK cell immunotherapy.

The number of NK cells from a single donor is insufficient for treatment, which makes the expansion and activation of NK cells very critical.

This production process usually takes two to three weeks to culture NK cells and certain cytokines ( IL-2 or in combination with IL-15 or anti-CD3 monoclonal antibodies ).

Although irradiated K562-mb15-4-1BBL cells as feed can improve cell growth during the proliferation of NK cells, the availability of the number of donor cells is still an obstacle.

In addition, T cells must be completely eliminated to prevent GVHD. Therefore, how to obtain enough NK cells is still a challenge.

Choosing an appropriate method to transform CAR into NK cells is the key to CAR-NK cell immunotherapy.

So far, both viral and non-viral vectors have been used to transform CAR. Although the transfection efficiency of retroviral vector is very high, it may cause insertion mutation, carcinogenesis and other adverse reactions.

Although the lentiviral vector shows a low incidence of insertion mutation, its transfection efficiency of peripheral blood NK cells is as low as 20%.

Transfection of mRNA into CAR-NK cells is also considered a safe and practical method of transfection.

Studies have shown that in a xenograft tumor model, the receptor expression level exceeds 80% after 24 hours of electroporation using the mRNA method, and the mRNA-transfected NK cells show obvious cytotoxicity.

Recent studies have shown that transfection of mRNA can effectively avoid the “targeted non-tumor” toxicity, an important limiting factor in the clinical application of CAR-modified immunotherapy.

However, the anti-tumor effect of CAR-NK cells transfected with mRNA by electroporation will be temporary, because the expression level of CARs will not exceed 3 days.

A new generation of CAR-NK

Some research groups are exploring different mechanisms to effectively combat antigen loss and immunosuppressive TME, which will improve the treatment life and efficacy of CAR-NK treatment strategies, and may become the backbone of the next generation of CAR-NK treatments.

In order to avoid antigen escape, Mitwasi and his colleagues developed a universal CAR ( UniCAR ) with a switch to improve safety and controllability.

This technology was first verified on CAR-T cells targeting E5B9, which is a peptide epitope of the nuclear antigen La-SS/B.

Since this protein is not found on the cell surface, CAR-T must be directed to the tumor through a bispecific molecule called the target module ( TM ).

TMs are usually fused to an anti-TAA single-chain antibody with an E5B9 peptide epitope.

Different TMs can be used in combination to target multiple TAAs at the same time to induce the heterogeneity of CARs reactive peptides without the risk of targeting effects.

Mitwasi et al. developed a UniCAR-expressing NK-92 cell line and a TM in which the E5B9 epitope is linked to an anti-GD2 monoclonal antibody on the IgG4 backbone.

GD2 is highly expressed in a variety of human tumors and is one of the immunotherapy targets for neuroblastoma.

UniCAR NK-92, which targets neuroblastoma and melanoma cells expressing GD2, exhibits anti-tumor cytotoxicity in vitro and is ready for further clinical development.

Another challenge facing CAR treatment is TME. In order to avoid immunosuppressive TME, Wang et al. constructed NK-92 cells expressing a modified CAR ( TGFβRII-NKG2D, called NK-92-TN ).

Essentially, this fusion converts the signal delivered by TGF-β produced by the immunosuppressant TME into an activation signal.

NK-92-TN cells cultured in vitro are resistant to TGF-β inhibition, without down-regulation of NKG2D, the killing ability and IFN-γ production increase after TGF-β co-culture.

Although the in vitro experimental data looks promising, in vivo, the anti-tumor activity of NK-92-TN is not significant, and the reduction in end-point tumor weight is small. In this regard, more in-depth research is needed.

Outlook

Natural killer cells are a unique group of anti-tumor effector cells that have cytotoxicity, cytokine production and immune memory functions that are not restricted by MHC, making them a key role in the innate and adaptive immune response system.

CAR-NK cell therapy is a promising field of clinical research, which has good safety and preliminary efficacy for some cancer patients.

Compared with CAR-T cells, CAR-NK cells have their own unique advantages, but they still face some challenges.

These challenges include improving cell proliferation, making the activation of cytotoxicity more effective, and finally finding the best way to rebuild NK cells.

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

References:

1.CAR-expressing NK cells for cancer therapy: a new hope. BiosciTrends. 2020 Sep 6.

2. Natural Born Killers: NK Cells in Cancer Therapy. Cancers (Basel). 2020 Jul 31;12(8):2131

3.Exploring the NK cell platform for cancer immunotherapy. Nat RevClin Oncol. 2020 Sep 15.

4. Ch imeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. JHematol Onc ol. 2021; 14: 73.

What are the advantages of CAR-NK cell immunotherapy?

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What are the advantages of CAR-NK cell immunotherapy?

What are the advantages of CAR-NK cell immunotherapy?

Research overview of CAR-NK