Interest in natural killer (NK) cells has escalated as large players drum up collaborations to bring NK-driven programs and tools into their portfolios. In December, Sanofi deepened its commitment to antibody-based NK cell engagers, expanding an ongoing partnership with Marseille-based Innate Pharma. A few months earlier, Sanofi also turned to Scribe Therapeutics’ CRISPR platform to develop off-the shelf NK cell therapies for oncology, paying $25 million up front in a deal that could be worth $1 billion. Other deals and partnerships involving AbbVie, Bristol Myers Squibb, Gilead Sciences, Merck, Cambridge, UK-based AstraZeneca and Tokyo-based Takeda added to the momentum. Most programs are, however, at an early stage — and developers, for the most part, are still seeking signs of efficacy and refining protocols to manufacture the cells at scale.

Natural killer cells attack a tumor cell. Credit: Science Photo Library / Alamy Stock Photo

NK cells are important for immunosurveillance. Their role, as part of the innate immune system, is to kill virally infected cells and eliminate early signs of cancer. As immunotherapies, NK cells — both natural and engineered — are a safer and more user-friendly alternative to T cells (Table 1). “The major advantage of NK cells is they’ve never killed anybody, unlike CAR-T cells,” says Lewis Lanier of the University of California, San Francisco, and the Parker Institute of Cancer Immunotherapy, who has pioneered research into the NK cell biology over the past 40 years. For reasons that are not fully understood, cytokine release syndrome, if it occurs during NK cell therapy, tends to be much milder. “They interact differently with the myeloid cells that seem to be the main drivers of cytokine release syndrome,” says Katy Rezvani of the MD Anderson Cancer Center, who was first to demonstrate the clinical utility of chimeric antigen receptor (CAR)-NK therapies.

Table 1 Selected NK cell therapies in clinical development

What’s more, NK cell therapies are highly suitable for allogeneic approaches, as they do not cause graft-versus-host disease. Their cell-killing functions do not involve the human leukocyte antigen-based self/non-self recognition mechanism that drives cytotoxic T cell activity, so NK cells from an unrelated donor do not attack host tissues. Instead, NK cell activity is controlled through an array of inhibitory and activating interactions with receptors and ligands expressed on healthy and stressed cells.

Beyond cell-based therapies, investors have committed vast amounts of cash to finance a whole slew of antibody-like NK cell engagers and bispecific T cell engagers against defined antigens (Table 2). Innate Pharma’s collaboration with Sanofi on an NK cell engager platform was recently strengthened by the big pharma’s in-licensing of a preclinical multi-specific NK cell engager targeting the immune checkpoint B7H3 and the activating NK cell receptors CD16a and NKp46. And Scribe’s CasX editing tools will also be taken up by Sanofi to bolster its existing K-NK cell platform, which it gained when it acquired Kiadis in 2020. “The two areas of focus are to increase in vivo persistence and improve infiltration into solid tumors,” says Valeria Fantin, global head of oncology research at Sanofi. Despite their potential advantages, NK cell therapies have lagged behind T cell therapy counterparts. The reason is the NK cells themselves: it has proven challenging to expand, cultivate and genetically modify them at industrial scale. “Even to this day, to get the numbers that you need for clinical trials, most people end up using feeder cells, like we do,” Rezvani says. These display activating ligands that boost NK cell proliferation but are irradiated and do not replicate.

Table 2 Selected NK cell-directed antibodies in clinical development

Sanofi’s K-NK platform is an exception: instead of using intact cells, it employs nanoparticles bearing activating receptors to provide the stimulus. Cryopreservation without effects on NK cell activity after thawing, which is essential for truly off-the-shelf therapies, has also taken time to achieve. Another manufacturing issue for developers is the need to supply exogenous cytokines, such as interleukin (IL)-15, to expand the cells and ensure they persist. “T cells make their own growth factors, and they can clonally expand because they’re making their own gas,” Lanier says. “NK cells can’t make their own gas.” Some developers have circumvented this issue by engineering NK cells to do so, but excessive activation can, says Lanier, result in desensitization. “If we just soak NK cells in IL-15, it will kind of burn them out.”

Developers are also adding features, such as CARs, which confer antigen specificities that the cells would otherwise lack. “That’s the trade-off,” says Lanier. We can genetically make them more and more like T cells. At some point, the tox will come along as baggage.”

Bispecific engagers are also designed to activate patients’ endogenous NK cells in a tumor-targeted way, but they face a different hurdle: patients must have enough healthy NK cells for them to work. That may explain why Affimed’s NK cell engager molecule, AFM13, had limited effects as monotherapy, demonstrating far more promising activity when combined with an unmodified NK cell therapy. AFM13, a tetravalent, bispecific NK cell engager, targets both the antibody constant fragment (Fc) domain receptor CD16a (also called FcγRIIIa) and CD30, a receptor overexpressed in certain lymphomas. The molecule is designed to direct NK cells to train an antibody-dependent cellular cytotoxicity response against CD30-expressing cancer cells. At a recent American Society of Hematology meeting in New Orleans, Affimed reported that 32% of 108 patients treated on an open-label study attained an objective response, with median responses lasting just 2.3 months. But a subset of patients — about 20% — did experience extended progression-free survival. “This means that about 80% of patients do not have enough functional NK cells for long-term remission,” says Affimed’s chief medical officer, Andreas Harstrick.

In contrast, MD Anderson reported at the same meeting an objective response rate of 94% and a complete response rate of 71% in 35 patients with Hodgkin or non-Hodgkin lymphoma (NHL) who received optimal doses of AFM13 plus unmodified NK cells isolated from umbilical cord blood. Of the 24 patients who completed at least six months of follow-up during the ongoing phase 1/2 trial, 63% were still in complete response. “The data look much more encouraging,” Harstrick says.

The protocol employed by MD Anderson, however, requires freshly prepared NK cells, which limits the therapy’s utility. To overcome this constraint, Affimed has teamed up with Artiva Biotherapeutics, which can produce industrial quantities of NK cells. From a single unit of cord blood, it can generate about 8,000 doses, each containing 1 billion cryopreserved NK cells. A screening step preselects NK cells with two genetic features that improve their downstream performance: they encode a high-affinity variant of the CD16 receptor and possess a KIR-B haplotype, the more active of the two general NK cell genotypes. After two expansion and activation steps, the resulting NK cells then undergo a quality control process to maintain batch-to-batch consistency.

Artiva’s AB-101, an unmodified allogeneic NK cell therapy, is already undergoing a phase 1/2 trial, in combination with the CD20 inhibitor rituximab, in patients with B cell NHL, but the combination with AFM13 will require a separate investigational new drug approval from the US Food and Drug Administration before it can be tested in patients. That trial will be designed to enable registration, should the partners detect a similar efficacy signal to that obtained on the MD Anderson study.

An alternative to pairing an NK cell engager with NK cell therapy is to combine the two in a CAR-NK cell. Rezvani’s group has led the way in establishing clinical proof of concept for this approach, with an IL-15-armed, CD19-directed CAR-NK therapy, which elicited responses in 8 of 11 patients (73%) with relapsed or refractory CD19-positive NHL or chronic lymphocytic leukemia. Seven of the 8 responders had complete remission at a median follow-up of 13.8 months. Now called TAK-007, the program is in the hands of Takeda, which is conducting a multicenter trial in patients with B cell NHL.

Rezvani’s team is about to move two more NK cell therapies into clinical trials. One, in development for glioblastoma, comprises NK cells edited with CRISPR–Cas9 to no longer express the genes encoding the cytokine TGF-β, which plays an immunosuppressive role in glioblastoma, and the glucocorticoid receptor, which mediates the lymphotoxic effects on the transplanted cells of glucocorticoids administered to patients who develop edema. The second therapy comprises a CAR-NK that targets CD70, which is aberrantly expressed in a wide range of hematologic malignancies and solid tumors. Artiva is also developing its own CAR-NK therapy for human epidermal growth factor receptor 2-positive (HER2+) solid tumors; it will shortly enter the clinic.

Innate Pharma has, so far, eschewed pairing with cells, instead combining its NK cell engagers with a variety of antibodies, such as programmed death ligand 1 (PD-L1) inhibitors, or combining multiple targeting moieties in a single molecule. A CD20-targeting molecule will be its first tetraspecific NK cell engager to enter the clinic, with a trial in patients with B cell malignancies getting underway later this year. The construct also targets the activating receptors NKp46 and CD16a and signals via the pro-inflammatory IL-2 pathway by engaging the intermediate-affinity dimeric IL-2 receptor (which contains the IL-2 receptor β (CD122) and γ (CD132) chains). The goal is to trigger the formation of what the company calls a “cytolytic synapse” between activated NK cells and the cancer cells expressing the appropriate target. “The cornerstone of our technology is NKp46,” says Yannis Morel, executive vice president, product portfolio strategy and business development at Innate. It is, he says, “a lineage-defining molecule” for NK cells. “In contrast with the others, it is very stably expressed on the surface of NK cells within the tumor microenvironment.”

Whether NK-directed therapies will tackle solid tumors any more successfully than their T cell counterparts remains an open question. The challenge of getting enough active NK cells into the tumor microenvironment and then overcoming the immunosuppressive milieu once they are in is the same for both cell-based or antibody-based therapies. “I know no of reason why an NK cell would work better against a solid tumor than a T cell. There’s just no basis for that,” says Lanier. NK cells infiltrate tumors the same way that T cells do, he says, by following a “trail of breadcrumbs” or chemokine signals emanating from the tumor.

Once inside, the differences are apparent: T cells become activated and proliferate when they engage tumor antigens, whereas NK cells do not expand in the same way. On the other hand, CAR-NK cells retain both non-specific killing capabilities as well as their more targeted functions. Artiva CEO Fred Aslan says the company is “intrigued” by this additional antigen-agnostic mechanism as it may help to overcome tumor immune escape due to loss of antigens such as HER2. At their present stage of development, NK cell-based therapies generate more questions than answers about their potential clinical utility, but many of the uncertainties will be clarified in the next couple of years.