NK Cell Immunotherapy Rising for Cancer Treatment

Natural Killer (NK) Cells +
mRNAVaccines and T-Cell Receptors (TCR)
This is the second part of a 2 part presentation

Natural Killer (NK) Cells
Video: NK Cell killing a Cancer Cell
NK Cells Wikipedia

Components of the Innate Immune System
From https://kids.frontiersin.org/articles/10.3389/frym.2021.609074 2021

NK cells
From https://en.wikipedia.org/wiki/Natural_killer_cell 2021

Types of NK cells
From https://en.wikipedia.org/wiki/Natural_killer_cell 2021
NK cells can be classified as CD56bright or CD56dim.[19][20][3] CD56bright NK cells are similar
to T helper cells in exerting their influence by releasing cytokines.[20] CD56bright NK cells
constitute the majority of NK cells, being found in bone marrow, secondary lymphoid
tissue, liver, and skin.[3] CD56dim NK cells are primarily found in the peripheral blood,[3] and
are characterized by their cell killing ability.[20] CD56dim NK cells are always CD16 positive
(CD16 is the key mediator of antibody-dependent cellular cytotoxicity (ADCC).
[20] CD56bright can transition into CD56dim by acquiring CD16.[3]

NK cells can eliminate virus-infected cells via CD16-mediated ADCC.[21] All coronavirus
disease 2019 (COVID-19) patients show depleted CD56bright NK cells, but CD56dim is only
depleted in patients with severe COVID-19.[21]

Humoral Immunity
Cytokine

NK cell-based cancer immunotherapy: from basic
biology to clinical development
From https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-01014-w 2021
Natural killer (NK) cells are an essential part of tumor immunosurveillance, evidenced by higher cancer
susceptibility and metastasis in association with diminished NK activity in mouse models and clinical
studies [1,2,3]. Using an array of germline-encoded surface receptors, NK cells are able to recognize and
rapidly act against malignant cells without prior sensitization. Upon activation, NK cells release cytotoxic
granules containing perforin and granzymes to directly lyse tumor cells, in a similar fashion to activated
cytotoxic T cells. NK cells are also potent producers of chemokines and cytokines such as interferon
gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) and thereby are essential in modulating adaptive
immune responses. Due to their innate ability to eliminate tumor cells, NK cell-based immunotherapies
against cancer have been investigated for decades. Early clinical trials have demonstrated the overall
safety of NK cell infusion, even in the allogeneic setting [4,5,6,7]. The feasibility of utilizing allogeneic NK
cells, the established safety profiles, and the fast-acting nature of NK cells largely have led to the
emerging effort to develop “off-the-shelf” NK cell-based cancer immunotherapy. However, there are many
challenges to overcome, such as difficulty to meet clinical-grade ex vivo expansion, limited in vivo
persistence, limited infiltration to solid tumors, and tumor editing to evade NK cell activity. Various
strategies are being employed to overcome these challenges to improve the efficacy of NK cell-based
therapy, such as ex vivo pre-conditioning with cytokines and/or small molecular drugs, engineering an
“off-the-shelf” or iPSC-differentiated chimeric antigen receptor (CAR)-NK. There has been an explosion of
NK-based immunotherapies in pre-clinical development and clinical development. Herein, we will provide
an updated overview of the emerging endeavors for developing NK cell-based cancer immunotherapy
from pre-clinical conceptual development, clinical grade expansion, and ongoing clinical development.

Innate IPH4102 is now Lacutumab)
From https://www.innate-pharma.com/products/lacutamab 2021
Fc Receptor

Innate IPH4102
Background: KIR3DL2 is consistently expressed in all subtypes of Cutaneous
T-cell Lymphomas (CTCL), irrespectively of disease clinical stage, with the
greatest expression in Sézary Syndrome (SS) and transformed Mycosis
Fungoides (MF), two subsets with high unmet need. KIR3DL2 belongs to the
killer immunoglobulin (Ig)-like receptor (KIRs) family expressed on minor
populations of NK, CD8 and CD4 T cells. IPH4102 is a first-in-class anti-
KIR3DL2 monoclonal antibody (mAb). It depletes selectively KIR3DL2-
expressing cells. Its modes of action include Antibody-Dependent Cell-
Cytoxicity (ADCC) and –Phagocytosis (ADCP). IPH4102 has potent efficacy in
non-clinical models, in particular ex vivo autologous assays using primary CTCL
cells
http://ascopubs.org/doi/abs/10.1200/JCO.2016.34.15_suppl.TPS2591 2016

Innate IPH4102 (cont)

http://ascopubs.org/doi/abs/10.1200/JCO.2016.34.15_suppl.TPS2591 2016

The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer
From https://www.frontiersin.org/articles/10.3389/fimmu.2015.00578/full 2015
Although experience has shown that adoptive immunotherapy with allogeneic NK cells maybe more efficacious
than with autologous NK cells, to date, their long-term antitumor benefits have been modest (3). Expansion and
persistence of NK cells following infusion appear to be the main determinants of clinical response (50–52, 70),
thus underscoring the importance of identifying ways to enhance their persistence and antitumor activity. It is
likely that the combination of high-dose lymphodepleting chemotherapy with additional modifications (such as
Treg depletion, in vivo administration of cytokines, such as IL-15 or enhancement of CD16-mediated antigen
targeting) may maximize NK persistence and efficacy.

In addition, the possibility of third-party “off-the-shelf” products with partially HLA-matched NK cells from CB,
third-party donors, or NK cell lines allow the advantage of unlimited sources of cells to improve the practicality of
cell therapy. With increasing focus on genetically modifying NK cells to redirect their specificity or engager-
modified NK cells, it is likely that NK cells will move to the forefront of cancer therapy over the next few years.

NK Cells and Lacutamab (IPH4102)
From https://www.innate-pharma.com/science/innate-immunity-nk-cells 2020

Innate IPH4102 (Lacutamab)
“KIR3DL2 is an inhibitory receptor of the KIR family, expressed by
approximately 65% of patients across all CTCL subtypes and expressed by
up to 85% of certain aggressive CTCL subtypes, in particular, Sézary
syndrome and transformed mycosis fungoides (tMF). KIR3DL2 has a restricted
expression on normal tissues.”
From https://www.innate-pharma.com/science/publications-presentations 2019
Video

Human iPSC-Derived Natural Killer Cells Engineered with
Chimeric Antigen Receptors Enhance Antitumor Activity
From https://www.cell.com/action/showPdf?pii=S1934-5909%2818%2930284-4 2018

iNKT Cells
From: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343663/ 2018
Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells,
are a distinct population of T cells that express an invariant aβ T-cell receptor
(TCR) and a number of cell surface molecules in common with natural killer (NK)
cells. Although iNKT cells are rare in the human blood pool, comprising just
0.01-1% of peripheral blood mononuclear cells (PBMCs), they are
important immunoregulatory cells rapidly producing large amounts of cytokines
that can influence other immune cells.

NK Cell Therapy:A Rising Star in Cancer Treatment

NK Cell Therapy:A Rising Star in Cancer Treatment (cont)

The Molecular Mechanism of Natural Killer Cells Function and Its
Importance in Cancer Immunotherapy
Natural killer (NK) cells are innate immune cells that show strong cytolytic function against physiologically stressed cells such as tumor
cells and virus-infected cells. NK cells show a broad array of tissue distribution and phenotypic variability. NK cells express several
activating and inhibitory receptors that recognize the altered expression of proteins on target cells and control the cytolytic function. NK
cells have been used in several clinical trials to control tumor growth. However, the results are encouraging only in hematological
malignancies but not very promising in solid tumors. Increasing evidence suggests that tumor microenvironment regulate the phenotype
and function of NK cells. In this review, we discussed the NK cell phenotypes and its eﬀector function and impact of the tumor
microenvironment on eﬀector and cytolytic function of NK cells. We also summarized various NK cell-based immunotherapeutic strategies
used in the past and the possibilities to improve the function of NK cell for the better clinical outcome.

Natural killer (NK) cells are a group of innate immune cells that show spontaneous cytolytic activity against cells under stress such as
tumor cells and virus-infected cells. After activation, NK cells also secrete several cytokines such as interferon-γ (IFN-γ), tumor necrosis
factor-α (TNF-α), granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, and
CXCL8) that can modulate the function of other innate and adaptive immune cells. NK cells are identified as CD3−NK1.1+ cells in C57BL/6,
FVB/N, and NZB strains of mice. BALB/c, CBA/J, AKR, C3H, DBA/1, DBA/2, NOD, SJL, and 129 strains of mice do not express NK1.1
and NK cells in these mice can be identified as CD3−CD49b+ cells. NK cells in human are identified as CD3−CD56+ cells. They represent 2–
7% of lymphocytes in mouse peripheral blood (PB) and 5–15% of human peripheral blood mononuclear cells (PBMCs). NK cells are
present in the skin, gut, liver, lung, uterus, kidney, joints, and breast under physiological conditions. NK cells constitute about 20–30% of
total hepatic lymphocytes and 10% of lymphocytes in healthy human liver and lung, respectively (1). The specific subset of NK cell is
reported to control the development at the fetal-maternal interface during the first trimester of the pregnancy, and it constitutes about 50–
90% of total lymphoid cells in the uterus (2, 3). These uterine NK cells secrete IL-8, vascular endothelial growth factor (VEGF), stromal
cell-derived factor-1, and interferon gamma-inducible protein-10 (IP-10) which help in tissue building, remodeling, and angiogenesis (4).
NK cells in human placenta do not show killer activity but assist in establishing immunosuppression and tolerance to fetus allograft.
Similar to T and B cells, NK cells also develop from common lymphoid progenitor cells (5). Although bone marrow is the primary site of
NK cell development (6), they can also develop in the liver and thymus (7). The development of NK cells progresses through various
stages of maturation, expansion, and acquisition of specific receptors. All NK receptors are germ-line encoded and independent of RAG-
mediated recombination (8). Multiple factors such as cell-intrinsic signals (transcription factors) and external signals (cytokines and growth
factors) govern the development of NK cells. NK cells constitute the major component of an innate immune system and play the crucial
role in shaping the early immune response to viral infection and tumors and also in organ transplantation (9). In this review, we discussed
what are inhibitory and activating molecules present on NK cells and how they control NK cell function, how do NK cell function in the
tumor microenvironment, use of NK cell as adoptive cellular therapy to control cancer and what are strategies to improve NK cell
antitumor function.

Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer Immunotherapy
From https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628161/ 2019
Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their potential
for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like receptor (KIR)
mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK cells are platforms for
genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting off-target activation. Current
clinical studies have demonstrated the safety and efficacy of allogeneic NK cell adoptive transfer therapies as a means
for treatment of hematologic malignancies and, to a lesser extent, solid tumors. However, challenges associated with
sourcing allogeneic NK cells have given rise to controversy over the contribution of NK cells to graft-versus-host
disease (GvHD). Specifically, blood-derived NK cell infusions contain contaminating T cells, whose activation with NK-
stimulating cytokines has been known to lead to heightened release of proinflammatory cytokines and trigger the onset
of GvHD in vivo. NK cells sourced from cell lines and stem cells lack contaminating T cells, but can also lack many
phenotypic characteristics of mature NK cells. Here, we discuss the available published evidence for the varying roles
of NK cells in GvHD and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.
In recent years, results from clinical studies have demonstrated safety and efficacy of allogeneic infusions of natural killer
(NK) cells for immunotherapy of hematological malignancies and solid tumors [1]. NK cells are innate immune effectors
whose anti-tumor activity is regulated by a complex interplay of a large variety of inhibitory and activating receptors [2].
These inhibitory receptors, which include killer immunoglobulin-like receptors (KIRs) and CD94/NKG2A, are able to
recognize major histocompatibility complex (MHC) class I molecules determined by human leukocyte antigen (HLA) HLA-
A, HLA-B, HLA-C or HLA-E allotypes [3]. Encoded by genes on different chromosomes, this allows for donor and
recipient mismatching between KIRs and their ligands, allowing control of NK cell activation in immune responses and
their alloreactivity as allogeneic effectors.

The use of NK cells in allogeneic immunotherapy benefits from these cells’ short persistence, their assumed role in the
depletion of alloreactive T cells, and their alloreactivity induced by the mismatch between KIR receptors and their ligands
on target cells [4]. In addition, alloreactive NK cells do not express inhibitory receptors specific for HLA-class I alleles on
target cells [5,6]. Allogeneic NK cells have shown clinical benefits against a number of cancers, particularly against acute
myeloid leukemia (AML), after both hematopoietic stem cell transplantation (HSCT) and allogeneic infusions of isolated
NK cells [7]. Allogeneic NK cells from healthy donors have the advantage of being fully functional. In allogeneic HSCT
settings, donor T cells are responsible for contributing to graft-versus-host disease (GvHD) and graft-versus-tumor (GvT)
responses [8]. NK cells, on the other hand, are thought to mediate GvT effects in the presence or absence of donor T
cells with a limited induction of GvHD [9] and have been used in settings of T cell-depleted or T cell replete HSCT.
Sources of allogeneic NK cells include peripheral blood, cord blood, and bone marrow [10].

Present and Future of Allogeneic Natural Killer Cell Therapy
From https://www.researchgate.net/publication/278788824_Present_and_Future_of_Allogeneic_Natural_Killer_Cell_Therapy 2015

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore
used for cancer therapy. Although many early investigators used autologous NK cells, including
lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte
antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of
allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched
allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells
from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK
cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and
to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion.
In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to
maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell
therapy and its future directions.

Comparison of autogeneic and allogeneic natural killer cells
immunotherapy on the clinical outcome of recurrent breast cancer
In the present study, we aimed to compare the clinical outcome of autogeneic and allogeneic natural killer (NK) cells immunotherapy
for the treatment of recurrent breast cancer. Between July 2016 and February 2017, 36 patients who met the enrollment criteria were
randomly assigned to two groups: autogeneic NK cells immunotherapy group (group I, n=18) and allogeneic NK cells immunotherapy
group (group II, n=18). The clinical efficacy, quality of life, immune function, circulating tumor cell (CTC) level, and other related
indicators were evaluated. We found that allogeneic NK cells immunotherapy has better clinical efficacy than autogeneic therapy.
Moreover, allogeneic NK cells therapy improves the quality of life, reduces the number of CTCs, reduces carcinoembryonic antigen
and cancer antigen 15-3 (CA15-3) expression, and significantly enhances immune function. To our knowledge, this is the first clinical
trial to compare the clinical outcome of autogeneic and allogeneic NK cells immunotherapy for recurrent breast cancer.

There are two types of adoptive NK cells treatment: autogeneic and allogeneic; however, not all cancer patients exhibit clinical effects
after autogeneic NK cells treatment.18,19 The killer cell immunoglobulin-like receptors (KIRs) present on NK cells prevent them from
killing tumor cells that express similar major histocompatibility complex class I (MHC-I) molecules. Hence, in recent years, several
studies have assessed the feasibility of NK cells allograft (rather than autogeneic cells) as an adoptive treatment for cancer. The
clinical trial assessing the use of unrelated donor allogeneic NK cells treatment has indicated that there are no side effects in the
recipients.20,21

Comparative evaluation of autogeneic and allogeneic NK cells immunotherapy in patients with recurrent breast cancer is not well
documented. Therefore, the purpose of this study was to compare the therapeutic efficacy of autogeneic and allogeneic NK cells
immunotherapy in patients with recurrent breast cancer.

In the present study, we prospectively compared the clinical outcomes of autogeneic and allogeneic NK cells immunotherapy in
patients with recurrent breast cancer in order to obtain information regarding which type of NK cells immunotherapy can improve
patients’ clinical outcomes. We found that allogeneic NK cells therapy showed better outcomes than autogeneic NK cells therapy with
regard to improving the antitumor effect and enhancing the immune function of patients. The increase in the total number of T cells
and NK cells observed after allogeneic NK cells therapy may be related to the improvement of cellular immunity and prevention of
apoptosis of T cells.40 Immunocytokines can induce tumor-specific T cells selectively and activate NK cells to sites of tumor. The
increase in the expression of Th1 cytokines may be related to the activation of NK cells.41 Therefore, allogeneic NK cells therapy can
improve the body’s immunosuppression status by promoting the immunocytokines functions. Moreover, allogeneic NK cells can
markedly decrease the levels of CTCs, CEA, and CA15A. Our previous studies have shown that the CTC level is a robust biomarker of
the effects of immunotherapy and its decrease may be related to tumor shrinkage.42,43 Therefore, the decrease in CTC level observed
in the present study may reflect the efficacy of treatment. Furthermore, the clinical efficacy and QOL of patients treated with
allogeneic NK cells therapy were markedly improved compared to those treated with autogeneic NK cells therapy. On
the other hand, the postoperative adverse effects were minimal. Thus, observations indicate that allogeneic NK cells
therapy is more beneficial for recurrent breast cancer.

Major Histocompatibility Complex (MHC)
From https://en.wikipedia.org/wiki/Major_histocompatibility_complex 2021
The major histocompatibility complex (MHC) is a large locus on vertebrate DNA containing a set of closely
linked polymorphic genes that code for cell surface proteins essential for the adaptive immune system. These cell
surface proteins are called MHC molecules.

This locus got its name because it was discovered via the study of transplanted tissue compatibility.[1] Later
studies revealed that tissue rejection due to incompatibility is an experimental artifact masking the real function of
MHC molecules: binding an antigen derived from self-proteins, or from pathogens, and bringing the antigen
presentation to the cell surface for recognition by the appropriate T-cells.[2] MHC molecules mediate the
interactions of leukocytes, also called white blood cells (WBCs), with other leukocytes or with body cells. The
MHC determines donor compatibility for organ transplant, as well as one's susceptibility to autoimmune diseases.

In a cell, protein molecules of the host's own phenotype or of other biologic entities are continually synthesized
and degraded. Each MHC molecule on the cell surface displays a small peptide (a molecular fraction of a protein)
called an epitope.[3] The presented self-antigens prevent an organism's immune system from targeting its own
cells. The presentation of pathogen-derived proteins results in the elimination of the infected cell by the immune
system.

Diversity of an individual's self-antigen presentation, mediated by MHC self-antigens, is attained in at least three
ways: (1) an organism's MHC repertoire is polygenic (via multiple, interacting genes); (2) MHC expression
is codominant (from both sets of inherited alleles); (3) MHC gene variants are highly polymorphic (diversely varying
from organism to organism within a species).[4] Sexual selection has been observed in male mice making mate
choices of females with diﬀerent MHCs and thus demonstrating sexual selection.[5] Also, at least for MHC I
presentation, there has been evidence of antigenic peptide splicing, which can combine peptides from diﬀerent
proteins, vastly increasing antigen diversity

NK Cells and Cancer
From https://escholarship.org/content/qt5t11r9h0/qt5t11r9h0.pdf 2016

NK cells for cancer immunotherapy
From https://www.nature.com/articles/s41573-019-0052-1 2020

Natural killer cells plus high-dose chemotherapy, HSCT
show promise for B-cell lymphoma
From https://tinyurl.com/p3n2yk 2021

Innate IPH4102 and NK Cells
From https://cancerres.aacrjournals.org/content/74/21/6060 2014
Our results show that IPH4102 is able to recruit human NK cells or human
macrophages as effectors to mediate ADCC and ADCP, against the Sézary cell line HuT
78 with a level of efficacy comparable with that of alemtuzumab. However, IPH4102 did
not promote CDC in our experimental conditions.
KIR3DL2 has been reported as a relevant marker for skin-resident and leukemic cells in tMF
and Sézary syndrome (20–22, 24, 26, 27). Using the newly generated anti-KIR3DL2 mAb
clone 13E4, that exhibits more specificity and sensitivity than previously available reagents,
we analyzed the largest cohort of Sézary patients ever gathered in a single study (42
subjects). We confirmed that KIR3DL2 is homogenously expressed on most Sézary patients'
tumor cells in peripheral blood. Furthermore, we corroborated the excellent correlation
between TCR-Vβ clonality and KIR3DL2 expression. This substantial dataset supports the
relevance of KIR3DL2 as a marker to improve advanced CTCL diagnosis and strengthen the
rationale to target KIR3DL2 in these patients.
The present study describes the results of the preclinical efficacy studies of IPH4102, the
first-in-class humanized anti-KIR3DL2 mAb selected for the development of advanced
CTCL patients treatment. This report also provides the largest dataset of KIR3DL2
expression on Sézary syndrome patient samples, generated with novel staining reagents.

Antibody-Directed Therapies: Toward a Durable and Tolerable Treatment Platform for CTCL

Potentiation of Natural Killer Cells for Cancer Immunotherapy: A Review of Literature

Potentiation of Natural Killer Cells for Cancer Immunotherapy: A Review of Literature (cont)

Exploiting Human NK Cells in Tumor Therapy

Exploiting Human NK Cells in Tumor Therapy (cont)

Exploiting Human NK Cells in Tumor Therapy

Therapeutic Antibodies to KIR3DL2 and Other Target Antigens on CTCL

Therapeutic Antibodies to KIR3DL2 and Other Target Antigens on CTCL(cont)

Enhancing a Natural Killer: Modification of NK Cells for Cancer Immunotherapy
Natural killer (NK) cells are potent innate immune system effector lymphocytes armed
with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in
tumor surveillance, they are fast becoming a next-generation tool for adoptive
immunotherapy. Many strategies are being employed to increase their number and
improve their ability to overcome cancer resistance and the immunosuppressive tumor
microenvironment. These include the use of cytokines and synthetic compounds to
bolster propagation and killing capacity, targeting immune-function checkpoints,
addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic
ablation of inhibitory molecules. The next generation of NK cell products will ideally be
readily available as an “off-the-shelf” product and stem cell derived to enable potentially
unlimited supply. However, several considerations regarding NK cell source, genetic
modification and scale up first need addressing. Understanding NK cell biology and
interaction within specific tumor contexts will help identify necessary NK cell
modifications and relevant choice of NK cell source. Further enhancement of
manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become
key components of multifaceted therapeutic strategies for cancer.

https://www.mdpi.com/2073-4409/10/5/1058/review_report 2021

NK Cell Engagers (NKCE)
From https://onlinelibrary.wiley.com/doi/full/10.1002/eji.202048953 2021
NK cells are immune effector cells that can naturally discriminate between healthy and
malignant cells, a property that makes them ideal candidates for inclusion in the
therapeutic arsenal against cancer. Beyond cell therapies, such as CAR NK cells, which
have demonstrated the efficacy of NK cells for controlling hematological malignancies,
NKCEs represent a new class of synthetic molecules developed to promote endogenous
NK cell antitumor activity. Their chemistry and manufacturing profiles are compatible with
industrial development and this strategy may provide cost-effective and widely accessible
off-the-shelf solutions, when compared to cellular therapies. However, combination of
NKCE with cell therapy seems also an interesting alternative to CAR therapy by arming
effector cells transferred in patients without the need of genetic modifications. Patient
infusion with cord blood-derived allogenic NK cells precomplexed with the innate cell
engager AFM13 is under clinical investigation (NCT04074746). NKCEs represent,
therefore, promising candidates for the next generation of anticancer immunotherapies.

A phase 1b study of AFM13 in combination with pembrolizumab in
patients with relapsed or refractory Hodgkin lymphoma
From https://ashpublications.org/blood/article/136/21/2401/461621/A-phase-1b-study-of-AFM13-in-combination-with 2020
In relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-
programmed death-1 inhibitor pembrolizumab have demonstrated eﬃcacy as monotherapy
and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific
antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody,
designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural
killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have
demonstrated signs of eﬃcacy as monotherapy for patients with R/R HL and the combination
of AFM13 with pembrolizumab represents a rational new treatment modality.
AFM13, a first-in-class innate cell engager, is in clinical development for treatment of CD30+
lymphomas including R/R HL and peripheral T-cell lymphoma. Developed by the fit-for-purpose
ROCK platform that generates customizable antibodies, AFM13 is a CD16A/CD30 tetravalent,
bispecific antibody stimulating innate immune cells, such as natural killer (NK) cells and
macrophages.12,13 AFM13 binds CD16A on innate cells and binds CD30 on HL cells, acting as a
bridge to recruit and activate innate immune cells in close proximity to tumor cells.14-16 The
activating receptor CD16A on NK cells facilitates antibody-dependent cell-mediated cytotoxicity
(ADCC) and is the only activating receptor triggering the cytotoxic activity of naïve human NK
cells.15 Research suggests macrophages are also engaged by AFM13, contributing to the innate
immune response.17 AFM13, as the most clinically advanced innate immune cell engager, was
first studied in HL patients as monotherapy in a dose-escalating phase 1 clinical study for
patients with R/R HL.18 AFM13 treatment was safe, well tolerated, and resulted in objective
tumor responses in multiple patients.18 In this study, AFM13 demonstrated significant NK cell
activation and a decrease of soluble CD30 in peripheral blood as well as activity in HL patients
who received prior BV.18

NK Cells
Dietary components modulate tumoricidal activity of NK cells by three
distinct processes including receptor-ligand interactions, the release
of cytokines, and the secretion of lytic enzymes

Expansion of allogeneic NK cells with efficient antibody-dependent cell cytotoxicity
against multiple tumor

Non-Genetically Improving the Natural Cytotoxicity of Natural Killer (NK) Cells

Cancer Immunotherapy Based on Natural Killer Cells: Current Progress and New Opportunities

NK Cells
The enhancement of NK cell function can be accomplished in a variety of ways. For the purposes of this
review, we break down the strategies into three major categories:

1. Targeting inhibitory signaling pathways and negative regulators of NK cell activating signaling pathways, 
2. Manipulation of inhibitory/activating receptors expressed by NK cells, 
3. Cytokine-mediated activation and expansion of NK cells. This review will highlight the scientific progress
in these 3 areas and discuss how these different strategies are currently impacting NK cell-mediated
immunotherapy.
NK cells are not an abundant cell type in human blood, making it difficult to extract enough NK cells
from a healthy donor through leukapheresis for adoptive immunotherapy into a patient. Thus, in order
to make NK cell-based immunotherapy more effective, it is often necessary for NK cells to be
expanded ex vivo before infusion. The most efficient way to expand NK cells ex vivo is through
cytokine stimulation. IL-2, IL-15, IL-12, IL-18, IL-21, TGFβ, IL-10, and type I IFNs are examples of
cytokines that affect NK cell development, maturation, proliferation, and activation.10
The administration of anti-PD-1 antibody (Pembrolizumab) has been shown to enhance NK cell-
mediated cytotoxicity against multiple myeloma.76 Pembrolizumab enhances the interaction between
patient-derived NK cells and myeloma cells expressing PD-L1, which was associated with increased
production of granzyme B and IFNγ. In addition, lenalidomide (standard of care treatment) and
Pembrolizumab displayed synergy in NK cell-mediated cytotoxicity against PD-L1+ myeloma cells

NK Cells
Car-NK: Chimeric antigen receptors (CARs) are engineered fusion proteins that consist of an extracellular
antigen binding domain (scFv) fused to one or more intracellular activating signaling domains
Many strategies and technologies have been developed to improve the safety and therapeutic efficacy in
CAR-based immunotherapy. However, many challenges remain for CAR-NK therapy, such as the ex vivo
expansion and persistence of CAR-modified primary NK cells and low transduction efficiency.
The initial reluctance to use NK cells for CAR therapy was largely due to the uncertainty of whether NK cells
could migrate and penetrate the tumor microenvironment.60 In addition, NK cells display limited persistence
in vivo, which may be desirable for safety but could make the therapy less efficacious. Despite these
concerns, human primary NK cells and the human NK-92 cell line have been successfully transduced to
express CARs against both hematological cancers and solid tumors in preclinical and in clinical trials.

NK Cells
Conclusion: NK cells represent a promising target for treatment of tumors and
chronic infections. Although the effector function of NK cells overlap with CD8+ T
cells, they respond to different stimuli and complement the activity of CD8+ T cells,
especially in settings where CD8+ T cells are no longer effective. However, NK cell
responses alone are often suboptimal to control tumor growth or viral infections. In
addition, NK cells rapidly adjust to their environment and can display signs of
exhaustion after chronic activation, making it difficult to sustain their effector
function. Thus, strategies that involve the enhancement of NK cell activity are
necessary to fully harness their therapeutic potential. In this review, we have
discussed the different strategies that can be employed to boost the function of NK
cells. Manipulation of signaling pathways is an attractive approach, but the research
thus far is limited to mouse models and in vitro human NK cell activation. Most of the
strategies currently being tested at the bedside involve the manipulation of cell
surface receptors and cytokines to enhance the activity of NK cells in neoplastic
settings. Although none of the strategies are yet fully optimized and effective, the
development of novel inhibitors of signaling pathways (e.g., DGK and Cbl) and the
clever combination of cytokines and receptor/ligand pairs will likely improve the
effectiveness of NK cell-based immunotherapy.

NK Cells Research
From: https://med.stanford.edu/sunwoo-lab.html#research 2021
Our laboratory’s overarching goal is to understand how NK cells, in the broader context of the
host’s immune system, protect against developing and metastasizing tumor cells, specifically,
cancer stem cells, and to understand why this system fails in patients with cancer. Significant
heterogeneity of immune potency between individuals with these malignancies has been
observed but not explained. We are particularly interested in the questions of how and why the
immune system can respond to and control malignant cells in some contexts but not in others.
Clarity of the underlying basis for these differences would potentially explain why certain
individuals are more susceptible to cancer, lead to better screening strategies, and ultimately
provide much needed insight into how the host immune system can be manipulated to control
cancer.
Another major focus of our laboratory is to decipher the developmental programs of NK cells. In
many patients afflicted with cancer, the NK cells from those individuals do not respond to typical
NK cell stimuli. A more complete understanding of NK cell development may ultimately reveal
potential ways by which malignancies render NK cells dysfunctional. We are particularly interested
in understanding the transcriptional regulation of NK cell development and differentiation from stem
and progenitor cells. The goal is to further our understanding of the molecular basis underlying NK
cell development and maturation, which will in turn provide much needed insight into disorders
associated with NK cell defects. In addition, it will potentially provide an understanding of how the
development and differentiation of these special lymphocytes can be modulated for therapeutic
purposes.

NK Cells in the Treatment of Hematological Malignancies
From: https://www.mdpi.com/2077-0383/8/10/1557/htm 2019

CD56bright natural killer (NK) cells: an important NK cell subset
Human natural killer (NK) cells can be subdivided into diﬀerent populations based on the relative expression of the surface
markers CD16 and CD56. The two major subsets are CD56bright CD16dim/− and CD56dim CD16+, respectively. In this review,
we will focus on the CD56bright NK cell subset. These cells are numerically in the minority in peripheral blood but
constitute the majority of NK cells in secondary lymphoid tissues. They are abundant cytokine producers but are only
weakly cytotoxic before activation. Recent data suggest that under certain conditions, they have immunoregulatory
properties, and that they are probably immediate precursors of CD56dim NK cells. CD56bright NK cell percentages are
expanded or reduced in a certain number of diseases, but the significance of these variations is not yet clear.

Natural killer (NK) cells have been the focus of interest of immunologists for almost two decades. The increasing
knowledge of NK cell biology acquired throughout this period has led to a paradigm shift – for a long time NK cells were
considered merely as relatively primitive killers but they are now seen not only as bona fide actors in innate immunity but
also as important cells that shape and influence adaptive immune responses and are more and more being endorsed with
an immunoregulatory role. However, NK cells are not a homogeneous cell population and several subtypes exist in both
CD56bright NK cells are currently extensively investigated and are no longer considered as just a minor subpopulation
among total NK cells. As a result of their production of diﬀerent cytokines, they might be important in early immune
responses and in the shaping of the adaptive response (IFN-γ) as well as playing a role of regulatory NK cells (IL-10). This
last point clearly deserves further studies.

Another interesting and emerging concept is the observation of increases or reductions, respectively, in the percentages
of CD56bright NK cells in various diseases. Why are these cells expanded in several clinical conditions? What are the
mechanisms leading to the expansion? One might suppose that CD56dim NK cells have a high turnover under these
conditions and have to be replaced, and consequently their precursor cells (CD56bright) are released in high numbers from
the bone marrow and/or the LN. On the other hand, CD56bright NK cells and their cytokine production might be important
on their own in certain diseases and they would therefore selectively expand. Are these expansions a consequence of or
a predisposing factor of the disease? Are they beneficial or deleterious for the host? The same questions of course also
arise regarding the reductions or the absence of CD56bright NK cells.

Rapid progress in this field can be expected, and soon we will know much more about the true relevance of the CD56bright
NK cell population in human health and disease.

From: https://www.frontiersin.org/articles/10.3389/fimmu.2017.00699/full 2017
Human CD56dimCD16dim Cells As an Individualized Natural Killer Cell Subset
Human natural killer (NK) cells can be subdivided in several subpopulations on the basis of the relative expression of the
adhesion molecule CD56 and the activating receptor CD16. Whereas blood CD56brightCD16dim/− NK cells are classically viewed
as immature precursors and cytokine producers, the larger CD56dimCD16bright subset is considered as the most cytotoxic one.
In peripheral blood of healthy donors, we noticed the existence of a population of CD56dimCD16dim NK cells that was
frequently higher in number than the CD56bright subsets and even expanded in occasional control donors but also in
transporter associated with antigen processing-deficient patients, two familial hemophagocytic lymphohistiocytosis type II
patients, and several common variable immunodeficiency patients. This population was detected but globally reduced in a
longitudinal cohort of 18 HIV-1-infected individuals. Phenotypically, the new subset contained a high percentage of relatively
immature cells, as reflected by a significantly stronger representation of NKG2A+ and CD57− cells compared to their
CD56dimCD16bright counterparts. The phenotype of the CD56dimCD16dim population was differentially affected by HIV-1 infection
as compared to the other NK cell subsets and only partly restored to normal by antiretroviral therapy. From the functional point
of view, sorted CD56dimCD16dim cells degranulated more than CD56dimCD16bright cells but less than CD56dimCD16− NK cells.
The population was also identified in various organs of immunodeficient mice with a human immune system (“humanized”
mice) reconstituted from human cord blood stem cells. In conclusion, the CD56dimCD16dim NK cell subpopulation displays
distinct phenotypic and functional features. It remains to be clarified if these cells are the immediate precursors of the
CD56dimCD16bright subset or placed somewhere else in the NK cell differentiation and maturation pathway.

Highly cytotoxic natural killer cells are associated with poor prognosis in patients with
cutaneous T-cell lymphoma
Previous studies have demonstrated defects in cell-mediated immunity in CTCL
patients, including altered cytokine profiles and impaired neutrophil function, which
lead to a high incidence of recurrent bacterial and viral infections as a result of
decreased Th1-mediated immunity.4-9 It has also been reported that natural killer
(NK)–cell function is decreased in CTCL patients,10-14 which could contribute to an
overall decrease in the innate immune response to both neoplastic cells and viral or
bacterial pathogens. Previous groups have reported that NK cells from SS patients
are capable of responding to activation ex vivo, indicating the potential for
development of immune-based therapeutics.15

Although MF patients often have a prolonged indolent clinical course of disease that
requires localized treatment, there are few eﬀective treatments for the successful
management of patients with SS. Because of the lack of success with traditional
chemotherapeutic approaches, novel immune-based therapeutics are being
developed for use in a multitude of hematologic diseases, including CTCL.4,16-18
Understanding the immune microenvironment in patients with CTCL will be critical to
the successful design of targeted therapies for their disease.

Previous studies by our group and by others have shown increased expression of
interleukin-15 (IL-15) in malignant CD4+ T cells in CTCL patients.19 IL-15 acts through
a trimeric IL-15R complex to enhance NK-cell maturation and function.20-22 Indeed, in
a first-in-human phase 1 trial in patients with refractory solid cancer tumors, IL-15
treatment induced profound expansion of circulating NK cells (NCT01885897).23
Considering that IL-15 is produced by malignant cells in CTCL, we sought to study
the possible eﬀect of chronically elevated IL-15 on NK-cell function in CTCL patients.
In this study, we show that NK-cell activity is significantly enhanced in CTCL, and
strikingly, higher NK-cell numbers are associated with increased mortality.

From: https://pubmed.ncbi.nlm.nih.gov/34768814/ 2021
The Biological Role and Therapeutic Potential of NK Cells in Hematological and Solid Tumors

NK Cells from Cord Blood
Natural killer (NK) cells are a predominant part of innate immune cells and
play a crucial role in anti-cancer immunity. NK cells can kill target cells
nonspecifically, and their recognition of target cells is not restricted by the
major histocompatibility complex. NK cells also fight against tumor cells
independently of antibodies and prior activation. Of note, umbilical cord
blood (UCB) is a rich source of NK cells. Immunotherapies based on UCB-
derived NK cells are becoming increasingly researched, and the
investigations are producing encouraging results. In recent years, non-
modified and modified UCB-derived NK cells have been successfully
developed to fight against tumor cells. Herein, UCB-derived NK cell-
based immunotherapy is a potential strategy for the treatment of cancer in
the future. In this review, we focus on discussing the biological
characteristics of UCB-derived NK cells and their application prospects in
anti-tumor immunotherapy, including the latest preclinical and clinical
researches.

Cord-Blood Natural Killer Cell-Based Immunotherapy for Cancer
Fate Therapeutics iPSC

NKarta Allogeneic NK Cells

Cord Blood

Killer Cell Engagers
From: https://onlinelibrary.wiley.com/doi/full/10.1002/eji.202048953 2021
Immuno-oncology is revolutionizing the treatment of cancers, by inducing the recognition and
elimination of tumor cells by the immune system. Recent advances have focused on generating
or unleashing tumor antigen-specific T-cell responses, leading to alternative treatment
paradigms for many cancers. Despite these successes, the clinical benefit has been limited to a
subset of patients and certain tumor types, highlighting the need for alternative strategies. One
innovative approach is to broaden and amplify antitumoral immune responses by targeting
innate immunity. Particularly, the aim has been to develop new antibody formats capable of
stimulating the antitumor activity of innate immune cells, boosting not only their direct role in
tumor elimination, but also their function in eliciting multicellular immune responses ultimately
resulting in long-lasting tumor control by adaptive immunity. This review covers the development
of a new class of synthetic molecules, natural killer cell engagers (NKCEs), which are built from
fragments of monoclonal antibodies (mAbs) and are designed to harness the immune functions
of NK cells in cancer. As currently shown in preclinical studies and clinical trials, NKCEs are
promising candidates for the next generation of tumor immunotherapies.
NK cells are immune effector cells that can naturally discriminate between healthy and malignant cells,
a property that makes them ideal candidates for inclusion in the therapeutic arsenal against cancer.
Beyond cell therapies, such as CAR NK cells, which have demonstrated the efficacy of NK cells for
controlling hematological malignancies, NKCEs represent a new class of synthetic molecules
developed to promote endogenous NK cell antitumor activity. Their chemistry and manufacturing
profiles are compatible with industrial development and this strategy may provide cost-effective and
widely accessible off-the-shelf solutions, when compared to cellular therapies. However, combination
of NKCE with cell therapy seems also an interesting alternative to CAR therapy by arming effector cells
transferred in patients without the need of genetic modifications. Patient infusion with cord blood-
derived allogenic NK cells precomplexed with the innate cell engager AFM13 is under clinical
investigation (NCT04074746). NKCEs represent, therefore, promising candidates for the next
generation of anticancer immunotherapies.

DragonFly TriNKets
From: https://www.dragonflytx.com/platform 2021

ROCK NK Engagers
From https://www.affimed.com/rock-platform/innate-cell-engagers/ 2021

TriKEs and BiKEs join CARs on the cancer immunotherapy highway

Exploring the NK cell platform for cancer immunotherapy
From: https://www.nature.com/articles/s41571-020-0426-7 2020
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system
that are capable of killing virally infected and/or cancerous cells. Nearly 20 years
ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment
approach for patients with advanced-stage leukaemia. Subsequently, the field of
NK cell-based cancer therapy has grown exponentially and currently constitutes a
major area of immunotherapy innovation. In general, the development of NK cell-
directed therapies has two main focal points: optimizing the source of therapeutic
NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence
in vivo. A wide variety of sources of therapeutic NK cells are currently being
tested clinically, including haploidentical NK cells, umbilical cord blood NK
cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-
induced memory-like NK cells and chimeric antigen receptor NK cells. A
plethora of methods to augment the cytotoxicity and longevity of NK cells are
also under clinical investigation, including cytokine-based agents, NK cell-
engager molecules and immune-checkpoint inhibitors. In this Review, we
highlight the variety of ways in which diverse NK cell products and their
auxiliary therapeutics are being leveraged to target human cancers. We also
identify future avenues for NK cell therapy research.

Exploring the NK cell platform for cancer immunotherapy (cont)
Key points
• Natural killer (NK) cell-based therapies are emerging as safe and efficacious treatments for some cancers. 
• Generally, the two main considerations relating to NK cell therapies are the choice of NK cell source and the
method of in vivo enhancement of NK cell function; determining approaches to optimize both of these
aspects is of high clinical interest. 
• Therapeutic NK cells include haploidentical NK cells, chimeric antigen receptor NK cells, stem cell-derived
NK cells, umbilical cord blood NK cells, NK cell lines, adaptive NK cells and cytokine-induced memory-
like NK cells. 
• Auxiliary methods for enhancing the therapeutic activity of NK cells in vivo include cytokine-based agents,
NK cell-engager molecules (such as TriKEs, ROCK engagers, NKCEs and TriNKETs) and immune-
checkpoint inhibitors. 
• Potential advantages that NK cell therapies have over T cell therapies include more manageable safety
profiles and fewer graft restrictions (for example, no requirement for autologous cells, providing
opportunities for off-the-shelf products). 
• NK cell therapies remain subject to important immunosuppressive barriers in the tumour microenvironment;
the future success of these therapies will require a better understanding of how these suppressive factors
operate and how they can be overcome.

Mechanisms of NK cell dysfunction in the tumor
microenvironment and current clinical approaches to harness
NK cell potential for immunotherapy
From: https://tinyurl.com/3a5uhhwu 2020

Mechanisms of NK cell dysfunction in the tumor
microenvironment and current clinical approaches to harness
NK cell potential for immunotherapy (cont)
From: https://tinyurl.com/3a5uhhwu 2020

Short-course IL-15 given as a continuous infusion led to a massive expansion of
effective NK cells: implications for combination therapy with antitumor antibodies
From: https://pubmed.ncbi.nlm.nih.gov/33883258/ 2021
Background: Full application of cytokines as oncoimmunotherapeutics requires
identification of optimal regimens. Our initial eﬀort with intravenous bolus
recombinant human interleukin-15 (rhIL-15) was limited by postinfusional reactions.
Subcutaneous injection and continuous intravenous infusion for 10 days (CIV-10)
provided rhIL-15 with less toxicity with CIV-10 giving the best increases in CD8+
lymphocytes and natural killer (NK) cells. To ease rhIL-15 administration, we
shortened time of infusion. Treatment with rhIL-15 at a dose of 3-5 µg/kg as a 5-day
continuous intravenous infusion (CIV-5) had no dose-limiting toxicities while eﬀector
cell stimulation was comparable to the CIV-10 regimen.
Conclusions: IL-15 administered as CIV-5 substantially expanded NK cells with increased
cytotoxic functions. Tumor-targeting monoclonal antibodies dependent on ADCC as their
mechanism of action including alemtuzumab, obinutuzumab, avelumab, and
mogamulizumab could benefit from those NK cell expansions and provide a promising
therapeutic strategy.
Results: Impressive expansions of NK cells were seen at all dose levels (mean 34-fold),
including CD56bright NK cells (mean 144-fold for 4 µg/kg), as well as an increase in CD8+ T cells
(mean 3.38-fold). At 5 µg/kg/day, there were no dose-limiting toxicities but pulmonary capillary
leak and slower patient recovery. This led to our choice of the 4 µg/kg as CIV-5 dose for further
testing. Cytolytic capacity of CD56bright and CD56dim NK cells was increased by interleukin-15
assayed by antibody-dependent cellular cytotoxicity (ADCC), natural cytotoxicity and natural
killer group 2D-mediated cytotoxicity. The best response was stable disease.

Cancer Immunotherapy Based on Natural Killer Cells: Current Progress and New
Opportunities
Cancer immunotherapy has been firmly established as a new milestone for cancer therapy, with the
development of multiple immune cells as therapeutic tools. Natural killer (NK) cells are innate immune cells
endowed with potent cytolytic activity against tumors, and meanwhile act as regulatory cells for the immune
system. The efficacy of NK cell-mediated immunotherapy can be enhanced by immune stimulants such as
cytokines and antibodies, and adoptive transfer of activated NK cells expanded ex vivo. In addition, NK cells
can arm themselves with chimeric antigen receptors (CARs), which may greatly enhance their anti-tumor
activity. Most recently, extracellular vesicles (EVs) derived from NK cells show promising anti-tumor effects in
preclinical studies. Herein, we carefully review the current progress in these NK cell-based
immunotherapeutic strategies (NK cells combined with stimulants, adoptive transfer of NK cells, CAR-NK
cells, and NK EVs) for the treatment of cancers, and discussed the challenges and opportunities for opening
a new horizon for cancer immunotherapy.
Cancer immunotherapy, which works by activating the body's own immune system, has become an increasingly
important treatment option for cancers. In recent years, successes in anti-tumor treatments with antibodies and
cell-based immunotherapy has become landmark events in the history of tumor treatment (1, 2). As innate immune
cells, natural killer (NK) cells are unique and play pivotal functions in cancer immune surveillance. NK cells can
eliminate a variety of abnormal or stressed cells without prior sensitization, and even preferentially kill stem-like
cells or cancer stem cells (3–5). Upon forming immune synapses with target cells, NK cells release preformed
cytolytic granules, including perforin, and granzymes, of which function is to induce cell lysis. Several studies have
successfully exploited adoptive transfer of NK cells against various tumors, especially hematological malignancies.
In order to overcome the above problems many strategies have been explored, either by adding immune
stimulants to produce synergistic effects, adoptive transfer of NK cells expanded in vitro, or by genetically
modifying NK cells themselves to be stronger and more resilient. In addition, nano-vesicle structures
secreted by NK cells known as extracellular vesicles (EVs) come into the spotlight for their applications in
cancer therapies.

Cancer Immunotherapy Based on Natural Killer Cells: Current Progress and New Opportunities

From: https://www.researchgate.net/publication/
6389170_Prospects_for_the_use_of_NK_cells_in_immunotherapy_of_human_cancer 2007
Prospects for the use of NK Cells in immunotherapy of human cancers
Note this article is from 2007!

Highly cytotoxic natural killer cells are associated with poor prognosis in patients with
cutaneous T-cell lymphoma
The reason for this counterintuitive finding is not known; however, we speculate that although NK
cells are maintained in a hyperactive state in CTCL patients, malignant cell recognition is impaired. It
is also possible that the ability of NK cells to form an effective immune synapse and polarize actin
and cytolytic granules is altered in the microenvironment of CTCL patients. In support of this theory,
a key mediator of NK-cell polarization, phosphatase and tensin homolog (PTEN), is significantly
overexpressed in NK cells from CTCL patients in the RNA sequencing analysis (data not shown). We
have previously demonstrated a role for PTEN in the organization of the components of the
immunologic synapse and the appropriate convergence of cytolytic granules.41 It is also possible
that the process of NK-cell recognition of malignant CD4+ T cells is altered in CTCL patients.
Previous studies by Bouaziz et al15 suggest that NK cells are potentially able to be activated to kill
autologous CTCL cells, which suggests that malignant CD4+ T cells are susceptible to NK-cell
killing, but additional mechanisms of inhibition such as the ones discussed above prevent this in
CTCL patients. Although further investigation of multiple facets of NK-cell recognition is warranted,
it is clear that the overall immunosuppressive microenvironment in CTCL patients contributes to
insufficiency of patient NK cells to effectively control CTCL progression.
The absolute number of NK cells in peripheral blood was evaluated in CTCL patients and compared
with that in normal donors (n = 51). There was no statistical difference in absolute number of NK
cells when all patients with CTCL were included (Figure 1A); however, SS patients had on average
57.4% fewer NK cells compared with normal donors (supplemental Figure 1). We then evaluated
the association between absolute NK-cell counts and overall survival. NK-cell counts were
significantly associated with overall survival (P = .041; Figure 1B). To evaluate NK-cell function, NK
cells were purified from fresh peripheral blood (Figure 1C) and evaluated for cytotoxic function
against K562 target cells.24 CTCL patients had significantly higher levels of NK-cell cytotoxicity
compared with normal donors (Figure 1D). Although these findings differ from those in previous
reports, earlier work did not use NK cells isolated from fresh peripheral blood,10-12 evaluate frozen
samples, or use cytokine stimulation.14

NK cell-based cancer immunotherapy: from basic biology to clinical development
From: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-01014-w 2021
Natural killer (NK) cell is a specialized immune effector cell type that plays a critical role in immune activation
against abnormal cells. Different from events required for T cell activation, NK cell activation is governed by the
interaction of NK receptors with target cells, independent of antigen processing and presentation. Due to
relatively unsophisticated cues for activation, NK cell has gained significant attention in the field of cancer
immunotherapy. Many efforts are emerging for developing and engineering NK cell-based cancer
immunotherapy. In this review, we provide our current understandings of NK cell biology, ongoing pre-clinical and
clinical development of NK cell-based therapies and discuss the progress, challenges, and future perspectives.
Natural killer (NK) cells are an essential part of tumor immunosurveillance, evidenced by higher cancer
susceptibility and metastasis in association with diminished NK activity in mouse models and clinical studies
[1,2,3]. Using an array of germline-encoded surface receptors, NK cells are able to recognize and rapidly act
against malignant cells without prior sensitization. Upon activation, NK cells release cytotoxic granules containing
perforin and granzymes to directly lyse tumor cells, in a similar fashion to activated cytotoxic T cells. NK cells are
also potent producers of chemokines and cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor
alpha (TNF-α) and thereby are essential in modulating adaptive immune responses. Due to their innate ability to
eliminate tumor cells, NK cell-based immunotherapies against cancer have been investigated for decades. Early
clinical trials have demonstrated the overall safety of NK cell infusion, even in the allogeneic setting [4,5,6,7]. The
feasibility of utilizing allogeneic NK cells, the established safety profiles, and the fast-acting nature of NK cells
largely have led to the emerging effort to develop “off-the-shelf” NK cell-based cancer immunotherapy. However,
there are many challenges to overcome, such as difficulty to meet clinical-grade ex vivo expansion, limited in vivo
persistence, limited infiltration to solid tumors, and tumor editing to evade NK cell activity. Various strategies are
being employed to overcome these challenges to improve the efficacy of NK cell-based therapy, such as ex vivo
pre-conditioning with cytokines and/or small molecular drugs, engineering an “off-the-shelf” or iPSC-
differentiated chimeric antigen receptor (CAR)-NK. There has been an explosion of NK-based immunotherapies in
pre-clinical development and clinical development. Herein, we will provide an updated overview of the emerging
endeavors for developing NK cell-based cancer immunotherapy from pre-clinical conceptual development,
clinical grade expansion, and ongoing clinical development.

NK cell-based cancer immunotherapy: from basic biology to clinical development (cont)
From: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-01014-w 2021
Table 1 NK cell receptors and their ligands in human
Full size table
Table 2 CAR-NK cells that have been evaluated preclinically
Full size table
Table 3 Comparison of commonly used allogeneic NK cell sources
Full size table
Table 4 Summary of NK expansion and activation strategies
Full size table
Table 5 Completed and ongoing clinical trial of NK cell-based therapy for hematological malignancies
Full size table
Table 6 Completed and ongoing clinical trial of NK cell-based therapy for solid tumors
Full size table

Natural Born Killers: NK Cells in Cancer Therapy

NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in
Cancer Immunotherapy

“UniCAR”-modified off-the-shelf NK-92 cells for
targeting of GD2-expressing tumour cells

The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer
Immunotherapy: Recent Innovations and Future Developments

Harnessing the Power of Natural Killer Cells for Cancer Immunotherapy
From: https://www.allcells.com/harnessing-the-power-of-natural-killer-cells-for-cancer-immunotherapy/ 2021

Next generation natural killer cells for cancer immunotherapy: the promise of
genetic engineering
From:https://pubmed.ncbi.nlm.nih.gov/29605760/ 2018
Recent advances in the field of cellular therapy have focused on autologous T cells
engineered to express a chimeric antigen receptor (CAR) against tumor antigens.
Remarkable responses have been observed in patients receiving autologous CD19-
redirected T cells for the treatment of B-lymphoid malignancies. However, the generation
of autologous products for each patient is logistically challenging and expensive.
Extensive research eﬀorts are ongoing to generate an oﬀ-the-shelf cellular product for
the treatment of cancer patients. Natural killer (NK) cells are attractive contenders since
they have potent anti-tumor activity, and their safety in the allogeneic setting expands
the cell sources for NK cell therapy beyond an autologous one. In this review, we discuss
advantages and limitations of NK cellular therapy, and novel genetic engineering
strategies that may be applied to overcome some of the limitations. Next-generation
engineered NK cells are showing great promise in the preclinical setting and it is likely
that in the next few years CAR-engineered NK cells will be incorporated into the current
armamentarium of cell-based cancer therapeutics.

Harnessing Innate Immunity in Cancer Therapy
From https://www.nature.com/articles/s41586-019-1593-5.epdf 2019
New therapies that promote antitumour immunity have been recently developed. Most of
these immunomodulatory approaches have focused on enhancing T-cell responses, either
by targeting inhibitory pathways with immune checkpoint inhibitors, or by targeting
activating pathways, as with chimeric antigen receptor T cells or bispecific antibodies.
Although these therapies have led to unprecedented successes, only a minority of patients
with cancer benefit from these treatments, highlighting the need to identify new cells and
molecules that could be exploited in the next generation of immunotherapy. Given the
crucial role of innate immune responses in immunity, harnessing these responses opens
up new possibilities for long-lasting, multilayered tumour control.

Several studies are currently underway using CAR non-T cells. In particular, given the absence of
graft-versus-host disease following the injection of allogenic NK cells, infusions of oﬀ-the-shelf
cord-blood-derived CAR NK cells are being tested in clinical trials against several types of
leukaemia after chemotherapy158. In addition, CAR NK cells derived from human induced
pluripotent stem cells have been generated; these cells displayed antitumour activity at least as
high as that of CAR T cells, but with lower toxicity, in preclinical models159. Finally, CAR
macrophages are also being generated, based on the rationale that monocytes and macrophages
are actively recruited to solid tumours, and that engineered CAR macrophages can be polarized
towards an antitumour macrophage phenotype (M1), enhancing the activation and recruitment of
immune cells, such as T cells (https://carismatx.com).

Present and future of allogeneic natural killer cell therapy
Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are
therefore used for cancer therapy. Although many early investigators used autologous NK cells, including
lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human
leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor
effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-
mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover,
allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The
use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells
with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without
delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and
recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present
state of allogeneic NK cell therapy and its future directions.
Antitumor activity of allogeneic NK cells was first observed in a setting of HLA-haploidentical HSCT. Allogeneic
NK cell therapy was tried mostly using HLA-haploidentical NK cells with or without allogeneic HSCT and,
recently, allogeneic NK cells from unrelated, random donors have been used in a non-HSCT setting. The
efficacy of allogeneic NK cell therapy can be enhanced by optimal donor selection in terms of the KIR genotype
of donors and donor KIR-recipient MHC incompatibility. Furthermore, efficacy can be increased by genetic
modification of NK cells and optimized therapeutic regimens. In the future, allogeneic NK cell therapy can be an
effective therapeutic modality for cancer treatment.

Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer
Immunotherapy
Natural killer (NK) cells are attractive within adoptive transfer settings in cancer immunotherapy due to their
potential for allogeneic use; their alloreactivity is enhanced under conditions of killer immunoglobulin-like
receptor (KIR) mismatch with human leukocyte antigen (HLA) ligands on cancer cells. In addition to this, NK
cells are platforms for genetic modification, and proliferate in vivo for a shorter time relative to T cells, limiting
oﬀ-target activation. Current clinical studies have demonstrated the safety and eﬃcacy of allogeneic NK cell
adoptive transfer therapies as a means for treatment of hematologic malignancies and, to a lesser extent, solid
tumors. However, challenges associated with sourcing allogeneic NK cells have given rise to controversy over
the contribution of NK cells to graft-versus-host disease (GvHD). Specifically, blood-derived NK cell infusions
contain contaminating T cells, whose activation with NK-stimulating cytokines has been known to lead to
heightened release of proinflammatory cytokines and trigger the onset of GvHD in vivo. NK cells sourced from
cell lines and stem cells lack contaminating T cells, but can also lack many phenotypic characteristics of
mature NK cells. Here, we discuss the available published evidence for the varying roles of NK cells in GvHD
and, more broadly, their use in allogeneic adoptive transfer settings to treat various cancers.
Adoptive transfer of autologous NK cells has been carried out to treat a number of diseases, including
various solid tumors clinically (Table 2). However, autologous infusions of NK cells have failed to show a
sustained anti-tumor response, despite demonstrated safety [72,73,74,75]. Combination with chemotherapy
has, nonetheless, shown somewhat more promising results in patients with colon carcinoma [76]. Similarly, a
number of clinical studies have demonstrated the safety of infused allogeneic NK cells to treat both
hematologic malignancies and solid tumors [1]. These studies utilize allogeneic NK cell products that include
in vitro cytokine and feeder cell expanded NK cells, non-expanded cytokine-activated NK cells, and
cytokine-induced memory-like NK cells, which are generated after a pre-activation period with combinations
of the cytokines interleukin (IL)-12, IL-15, and IL-18 and have the ability to functionally persist long-term in
vivo [77].

Natural Killer Cells as Allogeneic Effectors in Adoptive Cancer
Immunotherapy

The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer
Immunotherapy: Recent Innovations and Future Developments
Natural killer (NK) cells are critical immune effector cells in the fight against cancer. As NK cells in cancer patients are
highly dysfunctional and reduced in number, adoptive transfer of large numbers of cytolytic NK cells and their potential to
induce relevant antitumor responses are widely explored in cancer immunotherapy. Early studies from autologous NK cells
have failed to demonstrate significant clinical benefit. In this review, the clinical benefits of adoptively transferred
allogeneic NK cells in a transplant and non-transplant setting are compared and discussed in the context of relevant NK
cell platforms that are being developed and optimized by various biotech industries with a special focus on augmenting
NK cell functions.
From this literature review, we conclude that adoptive transfer of allogeneic NK cells in a non-transplant setting is safe
and shows early signs of clinical efficacy against hematological and certain solid tumors. Current data are mostly
based on Phase I clinical trials, and hence it is still too early to get an overall picture of NK cell alloreactivity in different
kinds of cancer. Most of the clinical studies conducted so far have used primary NK cells but with limited efficacy,
pointing to the need to improve the functionality of these NK cells after their transfer to patients. The growing
opportunities to augment NK cell functions have attracted several biotech companies to invest in NK cell research,
spearheading NK therapy development with different innovative approaches. This review also stresses the need for
combining adoptive transfer of allogeneic NK cells with NK function-augmenting products to achieve a maximum anti-
tumor effect. As NK cells are safe to infuse, the use of CAR-NK cells may be instrumental in providing a much safer
but still very effective platform, to bring CAR-based therapies to broader clinical applications. It may also facilitate
effective tumor targeting of NK cells. oNKord® and iPSC-derived NK cells could serve as alternative allogeneic
platforms to develop CAR-NK products, besides NK cell lines. In a solid tumor setting, NK cells are challenged by
several factors that affect their homing and penetration into the tumor tissues. Moreover, they should achieve and
maintain an activated effector state, even in the face of immune suppressive conditions, that are prevalent in patients
with cancer. To overcome these bottlenecks in NK therapy of solid tumors, a plethora of creative solutions are being
pursued by numerous research labs as well as by biotech companies in clinical or close to clinical phase. Strategies to
enhance NK cell functions from leading NK cell products are summarized in Figure Figure2.2. With all these exciting
developments, NK cells are set to make a considerable impact on the future treatment of patients with hematological
as well as with solid tumors.

Developmental and Functional Control of Natural Killer Cells by Cytokines
Natural killer (NK) cells are effective in combating infections and tumors and as such are tempting for adoptive transfer
therapy. However, they are not homogeneous but can be divided into three main subsets, including cytotoxic, tolerant,
and regulatory NK cells, with disparate phenotypes and functions in diverse tissues. The development and functions of
such NK cells are controlled by various cytokines, such as fms-like tyrosine kinase 3 ligand (FL), kit ligand (KL),
interleukin (IL)-3, IL-10, IL-12, IL-18, transforming growth factor-β, and common-γ chain family cytokines, which
operate at different stages by regulating distinct signaling pathways. Nevertheless, the specific roles of each cytokine
that regulates NK cell development or that shapes different NK cell functions remain unclear. In this review, we attempt
to describe the characteristics of each cytokine and the existing protocols to expand NK cells using different
combinations of cytokines and feeder cells. A comprehensive understanding of the role of cytokines in NK cell
development and function will aid the generation of better efficacy for adoptive NK cell treatment.

Natural killer (NK) cells were first identified as “natural killer cells” in the mid-1970s and were characterized by
their vital roles in controlling cancer and viral infection (1–3). They are widely distributed in diverse tissues, such
as the peripheral blood (PB), spleen, lungs, liver, and uterus (4). In human PB, NK cells are primarily divided into
two subtypes: CD3−CD56dimCD16+ and CD3−CD56brightCD16− cells. CD56dim NK cells have potent cytotoxicity
and high CD16 expression, allowing them to induce antibody-dependent cell-mediated cytotoxicity (ADCC)
toward target cells, whereas CD56bright NK cells are best known for producing diverse types of cytokines (5–7).
Different from PB NK cells, NK cells in diverse tissues have distinct phenotypes. Through experimental
parabiosis (8), researchers have found that, with the exception of circulating NK cells, the identification of several
markers, such as CD69, CD103, and CD49a, can affirm the phenotype of tissue-resident NK cells in the liver,
skin, and uterus (4, 9–14). Functions of NK cells vary depending on the cellular microenvironment, mainly due to
the cytokine signals of various tissues. For instance, NK cells can regulate the outcome of pregnancy (15, 16)
through the regulation of transforming growth factor (TGF)-β and interleukin (IL)-15 in the uterus (17–19) or
tolerate plentiful food-derived antigens or bacterial products through the regulation of abundant TGF-β and IL-10
in the liver (20–23) (Figure 1).

Developmental and Functional Control of Natural Killer Cells by Cytokines (cont)
. Cytokine regulation of natural killer (NK) cell expansion and cytotoxicity. Genetically modified K562 cells and IL-2 or IL-2 and OKT-3 without feeder
cells, applied for the expansion of primary NK cells, can generate significant amounts of functional NK cells. The differentiation and expansion of NK
cells from CD34+ HSCs are regulated by early activating cytokines, such as FL, KL, and IL-7, to promote HSC proliferation and differentiation, as well
as by cytokines to activate NK cells, such as IL-15, IL-12, IL-21, and IGF-1. To improve NK cell survival or antitumor function, relative signals, such as
the expression of mbIL-15 or preactivation with IL-12/15/18, strengthen activating or block inhibitory signals. These are vital for improving NK cell
efficacy in adoptive cell therapy. Abbreviations: PBMC, peripheral blood mononuclear cell; CBMC, cord blood mononuclear cell; BMMC, bone marrow
mononuclear cell; IL, interleukin; mbIL-15, membrane-bound IL-15; 4-1BBL, 4-1BB ligand; IGF-1, insulin-like growth factor 1; NKG2D, natural killer
group 2D; DAP10, DNAX-activating protein 10; KIR, killer cell immunoglobulin-like receptors; NKG2A, natural killer group 2A.

Natural killer cells, which are derived and expanded from autologous or allogeneic blood samples, can be
applied in adoptive therapy. However, the low concentration or absence of cytokines in the body has often
limited NK cell persistence postinfusion. To improve in vivo expansion, the Dario Campana group linked the
human IL15 gene to the gene encoding the transmembrane domain of CD8α (mbIL15) (131). The mbIL-15-NK
cells can survive and proliferate in vitro or in vivo without exogenous cytokines. They have superior cytotoxicity
against solid tumors and leukemia cells in vitro and against leukemia cells in xenograft models, indicating that
the expression of mbIL15 may improve the postinfusion cytotoxic capacity of NK cells. Similarly, we have noted
that IL-15 can induce prolonged NK cell antitumor eﬀects after cytokine withdrawal, which suggests that IL-15
can be widely used in adoptive NK cell therapy (168). Moreover, the super-agonist IL-15-IL-15Rα-Sushi-Fc
fusion protein (ALT-803) potently stimulates NK cell cytotoxic activity than native IL-15 (286) and has been used
in the clinical trail to evaluate its safety and eﬃcacy (NCT02099539). Additionally, preactivation of NK cells with
IL-12/15/18 can induce memory-like NK cells with enhanced cytotoxicity toward tumors. The cells have been
used in the clinical treatment with AML patients (NCT01898793). IL-12 and IL-21 can promote NK cell
maturation with improved functions, which are good candidates to be applied in NK cell adoptive therapy (189,
190, 212)

Chimeric antigen receptor (CAR)-modified NK cells display a new possibility for the application of adoptive NK cell-based
therapy (287). Preclinical studies to utilize CAR-expressing NK cells targeting CD19 or CD20 in B cell leukemia show
effective killing toward tumor cells (267). In addition, CD19-CAR NK cells have been applied in treating B-ALL
(NCT01974479) or ALL and CLL (NCT03056339) in clinical trails. To improve the efficacy of CAR-NK cells, the efforts to
add genes that can elicit IL-15 production or other activating signals are now underway (288). However, new strategies still
need to be developed to overcome the low transfection efficiency of NK cells.

Negative regulators can be treated as immune-checkpoints to shape immune responses. KIR and NKG2A are
well-studied immune-checkpoints of NK cells, which can be blocked to gain better NK cell efficacy (289, 290)
(Figure 2). The combination of anti-KIR mAbs lirilumab and lenalidomide has been used in a Phase I clinical trial
(NCT01217203) with multiple myeloma patients. However, the outcomes need further study. Furthermore, anti-
NKG2A antibody has also been applied in multiple clinical trials for patients with chronic lymphocytic leukemia
(NCT02557516), squamous cell carcinoma of the head and neck (NCT02643550), gynecologic malignancies
(NCT02459301), and squamous cell carcinoma of the oral cavity (NCT02331875). Other strategies are developing
to upregulate activating signals that can significantly prolong antitumor activity of NK cells, such as retroviral
transduction of NKG2D-DAP10-CD3ζ in NK cells (291) (Figure 2). The design of bi-specific antibodies that link the
antigens on tumor cells, such as CD33, CD20, and CD19, together with CD16 on NK cells direct NK cells toward
tumors and elicit efficient tumor cell killing(292). Additionally, the tri-specific antibody that integrates IL-15 in the
existing bi-specific antibody further promotes NK cell activation to facilitate NK cell cytotoxicity (293). Overall, the
developments to improve the efficacy of NK cell adoptive therapy are ongoing and may result in broader clinical
applications in the near future.
Conclusion
The development and functional maturation of NK cells are controlled by diverse cytokines. Different cytokine
cocktails are needed for distinct NK cell developmental stages that are guided by the expression pattern of
relative cytokine receptors. NK cells are heterogeneous and can be divided into cytotoxic, tolerant and regulatory
NK cells. They distribute throughout the body in different tissues and can be shaped by their specific tissue
environment via diverse combinations of cytokines. Given a robust understanding of each cytokine in NK cell
development and function, NK cells can be differentiated and expanded in vitro to generate sufficient numbers for
clinical treatment. NK cells derived from primary NK cells mainly require cytokines to promote NK cell expansion
and function, such as IL-2, IL-12, and IL-15. Cells from HSC differentiation need cytokines to promote the survival
and proliferation of HSCs, such as FL, KL, and IL-3, and to specify differentiation to NK cells with high cytotoxicity,
such as IL-15, IL-2, IL-12, and IL-21. The application of IL-15 or IL-12/15/18 can further enhance NK cell
cytotoxicity to induce greater efficacy for adoptive transfer therapy. Overall, understanding the primary roles and
modes of action of each cytokine is critical to apply them more effectively in the clinic.

From: https://www.glycostem.com/onkord 2021
Glycostem’s oNKord
Glycostem harvests the power of allogeneic Natural Killer (NK) cells. NK cells arise from blood forming stem cells
and are an important part of the body's innate immune system. NK cells are the new star in the domain of cellular
immunotherapy, due to their tightly regulated “natural killing" properties, caused by a shift of balance between
activating and inhibitory cellular signals and antibody-dependent cellular cytotoxicity (ADCC). NK cells play an
important role in control and even cure of both solid and hematological malignancies, like acute myeloid leukemia
(AML) and multiple myeloma (MM). NK cells can be used as a stand-alone or add-on therapy allowing multiple
treatment options. An allogeneic (partial mismatch) approach provides an excellent basis for effective treatment.
In allogeneic stem cell transplantation (allo-SCT), NK cells have been shown to mediate graft-versus-leukemia
(GVL) immunity towards recipient tumor cells without attacking recipients’ normal tissues, which would otherwise
lead to graft-versus-host disease (GVHD).

Our in-house and GMP-compliant platform technology enables a closed system cell culture process for the
expansion of CD34+ hematopoietic stem and progenitor cells and further differentiation into fully functional, high-
quality NK cells. This strategy allows us to generate highly pure, T cell devoid products without the use of any
contaminating feeder cells. These cells are the basis of both our oNKord® and CAR-NK therapies. The technology
platform is based on the use of closed bioreactor systems in combination with a proprietary synthetic, feeder cell-
free, cell culture medium and a patented combination of growth factors. This enables off-the-shelf, safe and low
production cost products.

Glycostem invents novel treatment options for hematological indications and solid tumors with the company's
first-generation Natural Killer (NK) cell-based immunotherapy product: oNKord® NK cells play an important role in
control and even cure of both solid and hematological malignancies, like acute myeloid leukemia (AML) and
multiple myeloma (MM). In allogeneic stem cell transplantation, NK cells have been shown to mediate graft-
versus-leukemia immunity towards recipient tumor cells without attacking normal tissues, preventing graft-
versus-host disease that is one of the risks when undergoing CAR-T treatment. In contrast to T cells, NK cells
also have a natural killing mechanism recognizing MHC class I-negative targets, which are of great importance for
inducing tumor rejection and which are tolerated by T cells. NK cells can be used as a standalone and add-on
therapy allowing multiple treatment options. oNKord® received an orphan drug designation for AML from both the
EMA (2014) and FDA (2016).
Also https://www.glycostem.com/car-nk

From: https://www.glycostem.com/science-and-technology 2021
Glycostem’s oNKord

From: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/monoclonal-antibody 2021
Monoclonal Antibodies
A type of protein made in the laboratory that can bind to substances in the body, including cancer cells.
There are many kinds of monoclonal antibodies. A monoclonal antibody is made so that it binds to only
one substance. Monoclonal antibodies are being used to treat some types of cancer. They can be used
alone or to carry drugs, toxins, or radioactive substances directly to cancer cells.
From https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/monoclonal-antibodies

From: https://fatetherapeutics.com/about-us/our-cells-of-interest/ 2021
Fate Therapeutics
Human induced pluripotent stem cells (iPSCs) have the unique capacity to be indefinitely expanded and
differentiated in culture into any type of cell in the body. The groundbreaking discovery that fully
differentiated human cells can be induced to a pluripotent state through the expression of certain genes
was recognized with the award of the 2012 Nobel Prize in Science and Medicine. iPSCs represent an ideal
cell source for creating cell therapy product candidates that are well-defined, uniform in composition, have
a consistent and dose-dependent pharmacology profile, and can be delivered off-the-shelf for the
treatment of large numbers of patients.

From: https://ashpublications.org/blood/article/135/6/399/429991/Pluripotent-stem-cell-derived-NK-cells-with-high 2021
Fate Therapeutics 2021

From: https://ashpublications.org/blood/article/135/6/399/429991/Pluripotent-stem-cell-derived-NK-cells-with-high 2021
Fate Therapeutics (cont0

From: https://fatetherapeutics.com/pipeline/immuno-oncology-candidates/ft516/ 2021
Fate Therapeutics FT516

Cord-Blood Natural Killer Cell-Based Immunotherapy for Cancer

Human leukocyte antigen (HLA)
From: https://en.wikipedia.org/wiki/Human_leukocyte_antigen2021
The human leukocyte antigen (HLA) system or complex is a complex of genes on chromosome 6 in humans
which encode cell-surface proteins responsible for the regulation of the immune system.[1] The HLA system is
also known as the human version of the major histocompatibility complex (MHC) found in many animals.[2]

HLAs corresponding to MHC class I (A, B, and C), all of which are the HLA Class1 group, present peptides
from inside the cell. For example, if the cell is infected by a virus, the HLA system brings fragments of the
virus to the surface of the cell so that the cell can be destroyed by the immune system. These peptides are
produced from digested proteins that are broken down in the proteasomes. In general, these particular
peptides are small polymers, of about 8-10 amino acids in length.[4] Foreign antigens presented by MHC class
I attract T-lymphocytes called killer T-cells (also referred to as CD8-positive or cytotoxic T-cells) that destroy
cells. Some new work has proposed that antigens longer than 10 amino acids, 11-14 amino acids, can be
presented on MHC I eliciting a cytotoxic T cell response.[5] MHC class I proteins associate with β2-
microglobulin, which unlike the HLA proteins is encoded by a gene on chromosome 15.

Determinants of Antileukemia Effects of Allogeneic NK Cells
From: https://tinyurl.com/px8tk6x5 2004
This article is from 2004!

Allogenic Natural Killer Cells for Refractory Lymphoma
We reported that IL-2 activated autologous NK cells can induce, but not maintain durable remissions
in lymphoma patients. We hypothesized that allogeneic NK cells may overcome class I MHC-
mediated inhibition of NK cell killing. In a pilot study we evaluated infusion of haploidentical donor NK
cells for anti-tumor eﬃcacy. Six patients with advanced B-cell non-Hodgkin lymphoma (NHL) received
rituximab, cyclophosphamide, and fludarabine as immunosupression to permit homeostatic NK cell
expansion, followed by CD3-depleted NK cell enriched cell products followed by subcutaneous IL-2
administration (10×106 units every other day × 6 doses). At 2 months, four patients showed an
objective clinical response. We observed early donor cell persistence in 2 patients (blood and in
tumor-bearing node), but this was not detectable beyond 7 days. All patients demonstrated
substantial increases in host regulatory T cells (Treg) after NK cell and IL-2 therapy (180±80 cells/μl vs
baseline: 58±24 cells/μl, p=0.04) which may have limited donor cell expansion in vivo. These findings
suggest safety and feasibility of allogeneic NK cell therapy in patients with lymphoma; however host
Treg and inadequate immunodepletion may contribute to a hostile milieu for NK cell survival and
expansion. Cell therapy trials should incorporate novel strategies to limit Treg expansion.

Safety and Eﬃcacy of Allogeneic NK Cell Infusions in Patients With

Relapsed/Refractory AML and High Risk MDS
From: https://healthtree.org/aml/community/clinical-trials/NCT04901416 2021
This study involves the use of an investigational cell therapy known as DVX201. DVX201
is an investigational cell therapy that contains a type of white blood cell called natural
killer (NK) cells. NK cells are a normal part of your immune system and have a lifespan of
only about two weeks. They are called natural killer cells because they have the natural
ability to identify and kill cells in the body that are abnormal, like cancer cells or virally
infected cells. But fighting cancer can also lead to exhaustion and abnormal function of
NK cells. It can also result in a significant decrease in the number of NK cells in the
blood, making it more diﬃcult for the immune system to control the disease. We believe
that infusion of healthy, functional NK cells into patients with AML or MDS may boost the
immune system and help by killing cancer cells that remain after chemotherapy. DVX201
is an investigational NK cell therapy that may provide a rapid and temporary source of
healthy NK cells that are better able to fight those cancer cells.

Sponsor: Deverra Therapeutics

Natural Killer Cell Therapy:A New Treatment Paradigm for Solid Tumors
In treatments of solid tumors, adoptive transfer of ex vivo expanded natural killer (NK) cells has
dawned as a new paradigm. Compared with cytotoxic T lymphocytes, NK cells take a unique
position targeting tumor cells that evade the host immune surveillance by down-regulating self-
antigen presentation. Recent findings highlighted that NK cells can even target cancer stem
cells. The efficacy of allogeneic NK cells has been widely investigated in the treatment of
hematologic malignancies. In solid tumors, both autologous and allogeneic NK cells have
demonstrated potential efficacy. In allogeneic NK cell therapy, the mismatch between the killer
cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) can be harnessed to
increase the antitumor activity. However, the allogeneic NK cells cause more adverse events
and can be rejected by the host immune system after repeated injections. In this regard, the
autologous NK cell therapy is safer. This article reviews the published results of clinical trials and
discusses strategies to enhance the efficacy of the NK cell therapy. The difference in
immunophenotype of the ex vivo expanded NK cells resulted from different culture methods
may affect the final efficacy. Furthermore, currently available standard anticancer therapy,
molecularly targeted agents, and checkpoint inhibitors may directly or indirectly enhance the
efficacy of NK cell therapy. A recent study discovered that NK cell specific genetic defects are
closely associated with the tumor immune microenvironment that determines clinical outcomes.
This finding warrants future investigations to find the implication of NK cell specific genetic
defects in cancer development and treatment, and NK cell deficiency syndrome should be
revisited to enhance our understanding. Overall, it is clear that NK cell therapy is safe and
promises a new paradigm for the treatment of solid tumors.

NK Cell Immunotherapy Rising for Cancer Treatment

NK Cell Immunotherapy Rising for Cancer Treatment

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