NK (Natural Killer) cell-mediated adoptive immunotherapy has gained attention in hematology due to the progressing knowledge of NK cell receptor structure, biology and function. Today, challanges related to NK cell expansion and persistence in vivo as well as low cytotoxicity have been mostly overcome by pioneering trials that focused on harnessing NK cell functions. Recent technology advancements in gene delivery, gene editing and chimeric antigen receptor (CARs) have made it possible to generate genetically modified NK cells that enhance the anti-tumor efficacy and represent suitable 'off-the-shelf' products with fewer side effects. The recent advanced in NK cell biology along with current approaches for potentiating NK cell proliferation and activity was highlighted, redirecting NK cells using CARs and optimizing the procedure to manufacture clinical-grade NK and CAR NK cells for adoptive immunotherapy.
Immunobiology and immunotherapy of hematological malignancies have captured great interest in recent years. NK (Natural Killer) cells are components of the innate system that identify and kill tumor- and virus-infected cells in a major histocompatibility complex (MHC) unrestricted fashion. Unlike T cells, which recognize through an antigen-specific T-cell receptor (TCR) and express receptors encoded by rearranging genes, NK cells have activating and inhibitory receptors (killer immunoglobulin receptors, or KIRs) that ligate MHC molecules . Tumor cells down-regulate or lose the MHC class I expression and become susceptible to lysis by NK cells. Several activating NK cell receptors and co-stimulatory molecules recognize tumors . NK cells also exhibit antigen-dependent cellular cytotoxicity (ADCC) by detecting antibodies on tumor cells through the low-affinity Fcγ CD16 receptor .
NK cells have become an attractive modality in adoptive immunotherapy during the last two decades due to the growing research about NK cell biology that has elucidated the insufficient anti-tumor effect and expansion. Initially, trials examined the ex vivo activated and expanded primary peripheral blood (PB) NK cells or NK cell lines (e.g., NK-92) in autologous and allogeneic settings . Umbilical cord blood (UCB)-derived NK cells are demonstrated to be younger, recover better after cryopreservation and have stronger proliferation potential. Manufacturing NK cell-based immunotherapies from induced pluripotent stem cells (iPSCs) has prevented long production times while maintaining “off-the-shelf” capabilities .
NK-cell mediated antitumor immunotherapy can be enhanced by checkpoint blockade, bi- and tri-specific killer engagers (BIKEs and TriKEs), anti-KIR monoclonal antibodies and chimeric antigen receptor (CAR)-engineered NK cells (CAR-NK cells). Cytokines play essential roles in NK cell expansion and potentiating NK cell therapy products . Genetic modifications have further improved the specificity, strength and efficacy of NK cell-based immunotherapies. Today, the optimal time for NK cell infusions has not been determined. Non-modified or modified NK cells can be used as maintenance therapy after chemotherapy or can be combined with autologous or allogeneic stem cell transplantation.
NK cells have historically been considered as “naturally” cytotoxic cells with limited life span and proliferative capacity, but recent research indicates that NK cells also require priming of various factors such as IL-15, IL-2, IL-12 or IL-18 for maximum effector function . Early clinical trials showed that administration of exogenous IL-2 facilitated NK cell expansion and persistence . IL-15 plays a role in NK cell development and promotes NK cell survival through expression of anti-apoptotic factor Bcl-2 . Miller and colleagues showed that IL-15 has superior activity to IL-2 for in vivo NK cell persistence .
NK cells not only function in innate immunity but also obtain immunological memory like T and B cells in adaptive immunity. Memory-like NK cells develop following infection with, for example, human cytomegalovirus (CMV) and respond to a cytokine cocktail (IL-12, IL-15 and IL-18) . The memory-like response was correlated with the expression of CD94, NKG2A and CD69 and a lack of CD57 and KIR in CD56-dim NK cells . When NK cells are stimulated with cytokines, immunomodulator-semaphoring 7A (SEMA 7A) is upregulated on NK cells, maintaining increased functionality .
|Patients||Donor/NK Cell Source||NK Cell Expansion Method||Conditioning Regimen Prior to NK Infusion||Adverse Event/Toxicity||Response||Reference|
|4 Follicular Lymphoma, 5 Diffuse Large B Cell Lymphoma||Autologous/PBMC||IL-2 and IL-15 stimulation||None||None||CR in 7/9, median F/U: 44 months|||
|9 AML||Allogeneic/PBMC||IL2, IL-12, IL-15, and IL-18 stimulation, CD3 depletion, CD56-positive selection||Flu + Cy||N/A||ORR 55%, CR 45%|||
|4 AML, 1 CML||Haploidentical/PBMC||CD3 depletion, CD56 enrichment||None *||None||2/5 patients donor chimerism|||
|19 AML||Haploidentical/PBMC||CD3 depletion, IL-2 stimulation||Flu + Cy||Pleural effusion in 1 patient||CR in 5/19|||
|10 AML||Haploidentical/PBMC||CD3-depletion, CD56-enrichment, IL-2 stimulation||Flu + Cy||None||CR 100%|||
|41 hematological malignancies||Haploidentical/PBMC||CD3-depletion, IL-15, IL-21 stimulation||None *||None||Significant reduction of leukemia progression 46% vs. 74% (historical cohort)|||
|29 lymphoma||Autologous/PBMC||Ex vivo IL-2 stimulation||None||None||No change in outcome compared to historical controls|||
|41 AML||Haploidentical/PBMC||CD3-depletion, IL-15, IL-21 and hydrocortisone stimulation||None *||Grade 2 to 4 aGVHD 28%, cGVHD 30%,fever 73%||CR 57%, 3-year leukemia progression 75%|||
|6 B cell NHL||Allogeneic/PBMC||CD3-depletion, IL-2 stimulation||Flu + Cy + R||None||4/6 clinical response|||
|7 AML||“Off-the-shelf”/NK-92||IL2 stimulation||None||None||1 blast reduction, 2 SD|||
|26 AML||Haploidentical/PBMC||CD19 and CD3 depletion, rhIL15 stimulation||Flu + Cy||CRS in 56% of patients, neurologic toxicity in 5/9 patients||CR: 40%|||
|8 AML, 5 CML||Haploidentical/PBMC||CD3-depletion K562 Clone9.mbIL21 feeder cells||None *||aGVHD grade 1–2 54%||CR: 11/13 median F/U: 14.7 months|||
|9 AML||“Off-the-shelf“/iPSC||IL2 stimulation||Flu + Cy||3 patients Grade 3 febrile neutropenia||4/9 CR|||
|11 B cell NHL||“Off-the-shelf“/iPSC||IL 2 stimulation||Flu + Cy||None||8/11 had objective response, CR median F/U: 5.2 months|||
|3 AML||“Off-the-shelf“/iPSC||IL2 stimulation||Flu + Cy||None||1/3 CR|||
|14 B cell NHL||“Off-the-shelf“/iPSC||IL2 stimulation||Flu + Cy + R||None||10/14 patients achieved objective response, 7 CR|||
|10 AML||Allogeneic/UCB||CD34+ selection||Flu + Cy||None||4/10 disease free|||
|12 MM||Allogeneic/UCB||CD3 depletion, K562-9.mbIL21, IL-2 stimulation||Lenalidomide/melphalan||None||10 patients achieved at least VGPR, Median F/U 21 months|||
|Target||Tumor Type||NK Cell Source||Structure of CAR Constructs||References|
|CD19||B-cell leukemia||NK-92 cell line||Anti CD19 scFv + CD3ζ|||
|CD19||B-cell leukemia||Peripheral blood||Anti CD19 scFv + 41BB-CD3ζ|||
|CD19||B-cell malignancies||NK-92||Anti-CD19 scFV + CD3ζ, CD28 + CD3ζ or CD13 + CD3ζ|||
|CD19||B-cell malignancies||Cord blood||Anti-CD19 scFv + 4-1BB + CD3ζ + iCasp9 + IL-15|||
|CD19||B-cell malignancies||Peripheral blood||Anti CD19 scFv + 41BB + CD28 + CD3ζ|||
|CD20||B-cell malignancies||Peripheral blood||Anti CD19 scFv + 41BB-CD3ζ|||
|CD20||Burkitt lymphoma||Peripheral blood||Anti CD19 scFv + 41BB-CD3ζ + IL15|||
|CD138||Multiple myeloma||NK-92MI||Anti CD19 scFv + CD3ζ|||
|CS-1||Multiple myeloma||NK-92||Anti CD19 scFv + CD28 + CD3ζ|||
|CD5||T-cell malignancies||NK-92||Anti CD19 scFv + 41BB + CD28 + CD3ζ|||
|Antigen Target||Tumor||NK Cell Source||Structure of the CAR Construct||Phase of the Study||ClinicalTrials.Gov Identifier # (Number)|
|CD22||B lymphoma||Unknown||Anti-CD22 + CD244||I||NCT03692767|
|CD19||B lymphoma||NK-92||Anti-C19 + CD244||I||NCT03690310|
|CD19/CD22||B lymphoma||Unknown||Anti-CD19/22 + CD244||I||NCT03824964|
|BCMA||Multiple myeloma||NK-92||Anti-BCMA + CD8αTM-4-1BB-CD3ζ||I/II||NCT03940833|
|CD19||B-lymphoid malignancies||Cord blood NK cells||Anti-CD19 + CD28-CD3ζ||I/II||NCT03056339|
|CD33||Acute myeloid leukemia||NK-92||Anti-CD33 + CD28-4-1BB-CD3ζ||I/II||NCT02944162|
|CD7||T-cell leukemia/lymphoma||NK-92||Anti-CD7 + CD28-4-1BB-CD3ζ||I/II||NCT02742727|
|CD19||B-cell malignancies||NK-92||Anti-CD19 + CD28-4-1BB-CD3ζ||I/II||NCT02892695|
|CD19||B lymphoma||iPS-derived NK cells||Anti-CD19 + CD244||I||NCT03824951|
|CD19||B-cell leukemia||Peripheral blood||Anti-CD19 + CD8αΤΜ + 4-1ΒΒ + CD3ζ||I||NCT00995137|