Natural killer cells in clinical development as non-engineered, engineered, and combination therapies

Abstract

Natural killer (NK) cells are unique immune effectors able to kill cancer cells by direct recognition of surface ligands, without prior sensitization. Allogeneic NK transfer is a highly valuable treatment option for cancer and has recently emerged with hundreds of clinical trials paving the way to finally achieve market authorization. Advantages of NK cell therapies include the use of allogenic cell sources, off-the-shelf availability, and no risk of graft-versus-host disease (GvHD). Allogeneic NK cell therapies have reached the clinical stage as ex vivo expanded and differentiated non-engineered cells, as chimeric antigen receptor (CAR)-engineered or CD16-engineered products, or as combination therapies with antibodies, priming agents, and other drugs. This review summarizes the recent clinical status of allogeneic NK cell-based therapies for the treatment of hematological and solid tumors, discussing the main characteristics of the different cell sources used for NK product development, their use in cell manufacturing processes, the engineering methods and strategies adopted for genetically modified products, and the chosen approaches for combination therapies. A comparative analysis between NK-based non-engineered, engineered, and combination therapies is presented, examining the choices made by product developers regarding the NK cell source and the targeted tumor indications, for both solid and hematological cancers. Clinical trial outcomes are discussed and, when available, assessed in comparison with preclinical data. Regulatory challenges for product approval are reviewed, highlighting the lack of specificity of requirements and standardization between products. Additionally, the competitive landscape and business field is presented. This review offers a comprehensive overview of the effort driven by biotech and pharmaceutical companies and by academic centers to bring NK cell therapies to pivotal clinical trial stages and to market authorization.

Keywords: Adoptive cell therapy; Allogeneic; CAR-NK cells; Cancer; Combination therapy; GMP manufacturing; Genetic engineering; Immunotherapy; NK cell therapies; Natural killer cells; Off-the-shelf.

Fig. 1

Overview of the NK cell therapy field. A Total number of NK cell-based clinical trials per year of initiation. Since 1997, a total of N = 420 clinical trials with NK cells have been initiated, the majority of which between 2016 and 2021. Most are Phase I and Phase I/II studies, N = 165 and N = 144, respectively. N = 92 studies are Phase II. As the field has just recently emerged, Phase II/III, III and IV trials are few (N = 2, N = 3, N = 3, respectively). For N = 11 trials, the clinical phase is unknown. B Geographical location of companies and institutions developing NK cell therapies. Color scales are representative of the number per country. Data sources: ClinicalTrials.gov, search terms: “NK cell,” “NK cell therapy,” “Natural killer cells” (text); GlobalData.com, drug descriptor: “Natural Killer (NK) Cell Immunotherapy” (drop-down menu), or filter: “Natural Killer cells” (text). The search is limited to December 2021. Clinical studies not based on administration of NK cells to patients or evaluating the infusion of mixed immune cell populations were excluded

Fig. 2

Statistics of NK cell cancer therapy with non-engineered and engineered NK cells used as monotherapy or in combination therapies. The pie charts present the major features of NK cell products reaching the clinical stage until December 2021, as reported on ClinicalTrials.gov. Data are presented for non-engineered NK cell products (based on N = 36 clinical trials, initiated after March 2017), engineered NK cell products (N = 53 trials), non-engineered (N = 62 trials) and engineered (N = 34 trials) NK cell combination therapies. For every pie, numbers indicate the percentage of the whole taken by each slice. A Cell sources used as starting material for the manufacturing of NK cell products, divided by category: peripheral blood-derived NK cells (PB-NK), umbilical cord blood-derived NK cells (UCB-NK), hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs) and NK cell lines (NK-92). Undisclosed sources are labeled as “Unspecified.” B Classification of the cancer indication targeted in clinical trials, divided between hematological malignancies and solid tumors. C and D Breakdown of the types of hematological malignancies (C) and solid tumors (D) indications most frequently targeted by NK cell therapeutics. Undisclosed indications are labeled as “Unspecified.” In B, C and D, one trial can include more than one indication. AML: acute myeloid leukemia; CML: chronic myeloid leukemia; MM: multiple myeloma; MDS: myelodysplastic syndromes; B-ALL: B cell acute lymphoblastic leukemia; CLL: chronic lymphocytic leukemia; NHL: non-Hodgkin lymphoma; HL: Hodgkin lymphoma

Fig. 3

Natural killer cell therapy manufacturing process pipeline. From left to right: NK cells are isolated from peripheral blood (PB-NK), from umbilical cord blood (UCB-NK), derived from cord blood CD34-positive hematopoietic stem cells (HSCs), from induced hematopoietic stem cells (iPSCs), or from in vitro propagated cell lines (NK-92). Cell expansion (and differentiation) is achieved ex vivo by culture in suitable medium, optionally supplemented with serum, or in presence of feeder cells. Cytokines, antibodies, and other small molecules are added to support cell expansion and maturation. Manufacturing platforms include cell culture flasks, bags, and static and dynamic bioreactors. Once the appropriate number of cells with the desired phenotype is obtained, NK cells are harvested and collected in the final formulation before infusion into patients. Fresh products must be transported to the patient and administered shortly after collection. Cryopreserved products can be stored in appropriate conditions and be delivered to the patient as needed, for on-site thawing and true “off-the-shelf” administration

Fig. 4

Overview of engineered NK cell therapies. From left to right: different NK cell sources (PB-NK, UCB-NK, HSC-derived NK, iPSC-derived NK, NK-92) are engineered to express the desired surface protein(s) by means of non-viral electroporation with mRNA (left) or of transduction with γ-retro or lentiviral vectors and stable integration in the host genome (right). Engineered NK cell therapies can be divided in categories, according to the targeting molecule: (1) antibody-derived single-chain variable fragment (scFv-engineered) for tumor antigen binding, (2) NK cell receptor (receptor-engineered) for ligand binding, and (3) Fc receptor CD16 (158 V) high-affinity non-cleavable hnCD16 variant (CD16-engineered) to enhance antibody-dependent cell cytotoxicity (ADCC). Co-expression of scFv and hnCD16 is also reported. Additional modifications of engineered NK cell products under clinical evaluation enhance NK cell persistence in vivo by expression of membrane-bound IL-15 or of IL-15/IL-15 receptor fusion protein IL-15/IL-15Ra, prevent fratricide by CD38 KO, or increase safety through induction of apoptosis by administration of Rimiducid

Fig. 5

Statistics of engineered NK cell therapies. The pie charts present the types of engineered products under clinical evaluation, as reported on ClinicalTrials.gov until December 2021 (N = 53 trials). For every pie, numbers indicate the percentage of the whole taken by each slice. A Targeting molecules expressed on the surface of engineered NK cells: scFv-engineered chimeric antigen receptor (CAR)-NK, receptor-engineered CAR-NK, CD16-engineered NK and CD16-engineered and scFv-CAR-NK. B Breakdown of the surface tumor antigens targeted by scFv-engineered CAR-NK therapies. C Breakdown of the surface tumor antigens targeted by monoclonal antibodies used in combination with CD16-engineered NK cells. Undisclosed targets are labeled as “Unknown.” In B and C, one trial can have more than one target, belonging to more than one category

Fig. 6

Overview of combination non-engineered and engineered NK cell therapies. From top left: in combination with adoptive cell therapy, NK cells are administered with stem cells from a healthy donor. Monoclonal antibodies are combined with NK cells to enhance tumor targeting via antibody-dependent cell cytotoxicity (ADCC) through endogenous CD16 or engineered high-affinity non-cleavable hnCD16 (158 V) on NK cell surface, or as checkpoint inhibitors to block inhibitory receptors on the surface of NK cells (such as programmed cell death protein 1, PD-1) or to block inhibitory ligands on the surface of target cancer cells (such as programmed death-ligand 1, PD-L1). NK cell priming agents, such as IL-15 or IL-15 and IL-15 receptor fusion protein IL-15/IL-15Ra, stimulate NK activation in vivo. NK cell engagers (NKCE) are synthetic molecules built from fragments of monoclonal antibodies engaging simultaneously a tumor-associated antigen (TAA) on the cancer cells and CD16 on the NK cells (e.g., AFM13). Molecular inhibitors act on target cancer cells, inducing apoptosis via inhibition of the proteasome (Bortezomib) or via inhibition of tyrosine kinases (e.g., Imatinib)

Fig. 7

Statistics of combination non-engineered and engineered NK cell therapies. The pie charts present the types of combination therapies under clinical evaluation, as reported on ClinicalTrials.gov until December 2021, with non-engineered NK cells (N = 62 clinical trials) and with engineered NK cells (N = 34 trials). For every pie, numbers indicate the percentage of the whole taken by each slice. A Types of combinations with non-engineered and engineered NK cells: NK cell priming agents, adoptive cell therapy, antibodies, co-stimulation, molecular inhibitors, NK cell engagers; multiple combinations indicate the use of more than one of these categories. B Breakdown of the surface tumor antigens targeted by monoclonal antibodies used in combination with engineered and non-engineered NK cells. In B, on trial can have more than one target