May 25, 2024

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How to understand NK Cell Immune Checkpoints?

How to understand NK Cell Immune Checkpoints?



 

How to understand NK Cell Immune Checkpoints?

Human natural killer cells ( NK ) make up 15% of all circulating lymphocytes.

NK cells were discovered in the 1970s and are mainly involved in the killing of infected microorganisms and malignantly transformed allogeneic and autologous cells.

NK cells exhibit antitumor cytotoxicity without prior sensitization and produce cytokines and chemokines that regulate various immune responses.

 

According to the density of CD56 on the cell surface, NK cells are divided into two types: CD56 bright and CD56 dim , which have different phenotypic characteristics.

CD56 bright NK cells have the ability to produce abundant cytokines, while CD56 dim NK cells are more cytotoxic and express more immunoglobulin-like receptors and FcγRIII ( Fcγ receptor III, also known as CD16 ).

 

How to understand NK Cell Immune Checkpoints?

 

 

Both activating and inhibitory receptors are expressed on the surface of NK cells and help NK cells to perform their functions.

MHC-I ( major histocompatibility complex class I ) antigen-specific inhibitory receptors tightly regulate NK cell-mediated cytotoxicity and lymphokine production.

This MHC-I recognition inhibitory receptor forms three families of NK cell surface receptors, namely KIRs ( Killer Cell Immunoglobulin-like Receptors ), LIRs ( Leukocyte Immunoglobulin-like Receptors ) and NKG2A ( Natural Killer Immunoglobulin-like Receptors ) cell family 2 A ).

 

The killing effect of NK cells requires not only the detection of MHC-I molecules on transformed cells through inhibitory receptors, but also the activation of NK cells through activating receptors.

Natural cytotoxicity receptors (NCRs) are a group of natural killer cell surface activating receptors, including NKp46, NKp30 and NKp44.

CD16 ( or FcγRIII ) is also an activating receptor mainly expressed by the CD56 dim NK cell subset and is critical for antibody-dependent cellular cytotoxicity ( ADCC ) of IgG-coated target cells.

 

Tumors evade the immune system by establishing an immunosuppressive tumor microenvironment.

Immune evasion involving NK cells involves multiple mechanisms, one such mechanism being the use of NK cell inhibitory receptors by tumors for immune evasion, known as immune checkpoint inhibition, and has proven to be the most effective and favored therapeutic target .

 

 

 


KIRs


The KIR family ( also known as CD158 ) is a diverse and polymorphic subtype of NK cell receptors, including inhibitory and activating KIRs, each of which recognizes a specific HLA class I homologue (HLA- a , -B or -C ) as a ligand.

The inhibitory KIR 2 DL 1 , KIR 2 DL 2 and KIR 2 DL 3 recognize HLA-C as their ligand, while HLA-B and HLA-A serve as ligands for other KIRs, including the inhibitory KIR 3 DL 1 and KIR 3 DL 2 .

In addition to NK cells, T cell subsets and NKT cells ( invariant natural killer T cells ) also express KIRs. MHC-I molecules ( HLA-A, -B, and -C ) as KIRs and KIRs ligands themselves exhibit a wide range of natural polymorphisms.

The diversity of KIR allelic combinations (a total of 17 KIR genes on chromosome 19q13, 14 ), polymorphisms within each gene, and each KIR-expressing NK cell allow this complex KIR sequence to recognize MHC-I small changes in expression.

 

IPH2101 and lirilumab ( IPH2102/BMS-986015 ), IgG4 monoclonal antibodies targeting KIR 2 DL 1/2/3 NK cell inhibitory receptors, are currently being evaluated in various hematology ( AML, CLL, NHL ) or solid malignancies ( breast cancer and ovarian cancer ) for clinical evaluation and development .

Up to 10 mg/kg of IPH2101 has been reported to remain well tolerated. However, so far, IPH2101 has not achieved satisfactory results as a monotherapy .

In a dose-escalation phase I trial of IPH2101 as monotherapy in RRMM , only 11 ( 34% ) patients achieved a best response with stable disease.

 

The phase II trial of lirilumab was terminated for failing to meet the objective efficacy criteria ( 50% reduction in M ​​protein ) set for patients with MM , with only 1 ( 11% ) and 6 ( 66% ) of a total of 9 patients enrolled Minimal response and stable disease were achieved.

However, lirilumab enhanced elotuzumab-mediated cell killing, and a phase I ( NCT2252263 ) study evaluating the safety of the combination of elotuzumab and lirilumab in patients with multiple myeloma is currently underway.

 

Recently, the third member of this panel of anti-KIR antibodies, IPH4102 , a humanized anti-KIR3DL2 monoclonal antibody, has entered clinical evaluation.

IPH4102, also known as lacutamab, was well tolerated in a Phase I clinical evaluation in relapsed/refractory cutaneous T-cell lymphoma, with the most common adverse reactions including edema, fatigue, and lymphopenia.

Clinical activity was also encouraging, with 16 of 44 patients ( 36% ) achieving an overall response.

Patients with relapsed/refractory cutaneous T-cell lymphoma with Sézary syndrome showed a better clinical response ( 43% ).

Currently, a Phase II clinical trial ( NCT03902184 ) is investigating IPH4102 as a single agent or in combination with chemotherapy in T-cell lymphoma.

 

 

 


LIRs (ILTs)


Leukocyte immunoglobulin-like receptors ( LIRs ) or immunoglobulin-like transcripts ( ILTs ), like KIRs, belong to the Ig superfamily and consist of activating and inhibitory receptors.

Five inhibitory receptors ( LIRB1–5 ) were identified among a total of 11 LIR members .

Many immune cells ( NK, T, B, and myeloid cells including macrophages and dendritic cells ) express these receptors to varying degrees. Among them, LIRB1 ( ILT2 ) and LIRB2 ( ILT4 ) recognize HAL-G as their main ligand in addition to other ligands, leading to immunogenic tolerance.

ILT2 is expressed on natural killer cells ( 36±18% of normal NK cells ), T cells, B cells, monocytes, dendritic cell subsets and myeloid-derived suppressor cells ( MDSCs ), while ILT4 is mainly expressed on myeloid expressed on the cell. Therefore, the interaction of ILT2 and HLA-G can suppress the immune function of NK, T and B cells, thus serving as an immunotherapeutic target.

 

A variety of primary tumors and metastatic malignancies express HLA-G, and it is also considered an indicator of progression and prognosis in various cancers.

Its expression is associated with decreased NK function in various cancers such as hepatocellular carcinoma ( HCC ), ovarian cancer, non-small cell lung cancer ( NSCLC ), glioma and renal cell carcinoma ( RCC ).

In addition, the interaction of membrane surface-expressed HLA-G or soluble HLA-G with ILT2 has been shown to inhibit NK functions, including cytotoxicity, cytokine production, and chemokine secretion.

 

 

 


NGK2A and CD94


NKG2A ( also known as CD159 ) and CD94 are heterodimeric inhibitory receptors of the C-type lectin family that recognize the nonclassical MHC-I molecule HLA-E as a ligand.

CD94-NKG2A and its HLA-E ligands were non-polymorphic. HLA-E*0101 and HLA-E*0103 are the only two HLA-E alleles in the global population.

Nearly 50% of NK cells in peripheral blood express CD94/NKG2A, mainly those NK cells that do not express inhibitory KIRs. CD94/NKG2A was also co-expressed with other inhibitory receptors with different specificities.

In addition, γδ and CD8+ T cells also express CD94/NKG2A. NKG2A and CD94 react with HLA-E expressed on normal cells to inhibit signaling activation, thereby avoiding damage to normal bystander cells.

 

Tumor cells ( hematology and solid tumors ) show upregulation of HLA-E expression in order to avoid killing by NK cells. In various cancers, poor prognosis is associated with HLA-E upregulation.

Blocking the CD94/NKG2A receptor with antibodies could be a therapeutic strategy.

Therefore, an anti-CD94/NKG2A antibody ( IPH2201 Monalizumab ) developed by Natural Pharma has been used in various trials.

The results of in vitro and in vivo studies showed that the application of humanized anti-NKG2A antibody to hematological malignancies is safe and effective. Monalizumab improves NK cell dysfunction in chronic lymphocytic leukemia in vitro.

Monalizumab was well tolerated as monotherapy in gynecological malignancies ( up to 10 mg/kg intravenously or SC ), with no reported DTLs or SAEs.

This ongoing trial of a large pretreatment cohort showed stable disease in 41% of evaluable patients ( n = 128 ).

 

In addition, monalizumab is also being evaluated in combination with durvalumab, cetuximab and ibrutinib.

A preliminary evaluation of the safety and efficacy of monalizumab in combination with cetuximab in previously treated, recurrent and/or metastatic squamous cell carcinoma ( SCC ) of the head and neck showed an ORR ( objective response rate ) of 27.5%, Median PFS ( progression-free survival ) of 5 months and median overall survival ( OS ) of 10 months.

This is an encouraging result ( ORR 12.6%, PFS 2.3 m, OS 5.6 m ) if compared with the historical record of efficacy of cetuximab alone in previous studies .

Adverse effects of combination therapy were similar to those of cetuximab alone . Recent in vivo analyzes have shown that induction of NKG2A on CD8+ T cells hinders the efficacy of vaccine therapy and that blocking NKG2A receptors improves the efficacy of vaccine therapy.

Overall, blocking NKG2A represents an exciting therapeutic approach, and in particular, its combination with other immuno-oncology therapeutics is a way forward and deserves further exploration.

 

 


TIGIT and CD96


TIGIT ( T cell immune receptor with immunoglobulin and ITIM domains ) is an immunosuppressive receptor expressed on NK and T cells, such as activated NK, T, mT ( memory T cells ), fTh ( filter T helper cells ) and regulatory T cells ( Tregs ).

CD96, a member of the same immunoglobulin superfamily, has similar inhibitory effects compared to TIGIT, but has a lower binding affinity to the ligand CD155. CD226 is an activating receptor that competes with TIGIT and CD96 for CD155 binding. CD155 ( mainly ) and CD112 serve as ligands for TIGIT and CD96 binding to suppress T cell and NK cell mediated immunity.

 

Intrinsic expression of TIGIT inhibits NK and CD8+ T cell function, thereby helping tumor ( colorectal cancer ) growth in vivo.

TIGIT was associated with NK cell depletion in tumor-bearing mice and colon cancer patients, and this depletion was restored by its blockade, eliciting robust antitumor immunity.

The presence of NK cells is important for the therapeutic effect of TIGIT and/or PD-L1 blockade or dual blockade of these two checkpoints, because NK cell loss is associated with IFN- γA lower frequency of – or TNF-secreting TILs ( CD8+ ) correlated with a higher frequency of PD-1 expressing TILs ( CD8+ ).

NK cells account for 25-50% of liver lymphocytes, suggesting their importance for liver immunity. In addition, the survival and prognosis of HCC patients were positively correlated with the number of NK cells in blood and tumor tissues.

Tumor progression in HCC patients is associated with dysfunctional tumor-infiltrating NK cells, mainly the CD11b-CD27-NK subset. Sun et al. identified exhausted tumor-infiltrating CD96+ NK cells and found that their expression correlated with poor clinical outcomes in HCC patients.

NK cell exhaustion was reversed when CD96-CD155 interaction or TGF-β1 was blocked.

 

In recent years, more and more attention has been paid to the combined application of checkpoint inhibitors to achieve synergistic effects. Anti-TIGIT plus anti-PD-L1 blockade has been reported to prevent NK cell exhaustion in tumor-bearing mice and colon cancer patients.

On the other hand, anti-CD96 combined with doxorubicin chemotherapy, anti-CTLA-4 or anti-PD-1 showed more effective inhibition of tumor metastasis in three different tumor models. Exhausted NK cells from bladder cancer ( BC ) patients showed upregulation of TIM-3 and TIGIT both in the periphery and in the tumor.

In fact, the role of TIGIT and CD96 in NK cell exhaustion in various cancers is still under investigation and requires further unraveling to determine its potential as monotherapy or in combination with other checkpoints.

 

 


Siglec-7/9


Sialic acid-binding immunoglobulin-like lectins ( Siglecs ) are immunomodulatory sialic acid-binding receptors belonging to the type I lectin family. Siglecs are expressed on a variety of immune cells, including those of lymphoid and myeloid origin. Siglecs exhibit diversity in two properties: expression and specificity for sialic acid-containing ligands.

Most of these Siglecs are inhibitory receptors, such as Siglec-2, Siglec-3, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10 and Siglec-11.

Among the suppressive Siglecs, Siglec-7 and Siglec-9 were reported to be expressed on human NK cells.

Similar to the classical NK cell inhibitory receptors NKG2A/CD94 and KIRs, the inhibitory siglec also contains one or more ITIM and ITIM-like motifs at the C-terminus of its intracellular segment.

After ligation, ITIM is phosphorylated by Src family kinases, recruiting and activating Src homology 2 ( SH2 ) domain proteins, primarily the tyrosine phosphatases SHP1 and SHP2 or suppressor of cytokine signaling 3 proteins ( SOCS3 ) .

 

Siglec-sialic acid interactions are involved in the regulation of immune tolerance and may serve as targets for the induction of antitumor immunity.

Antibody drug conjugates targeting these checkpoint inhibitory antibodies ( anti-Siglec-2; Inotuzumab ozogamicin and anti-Siglec-3; Gemtuzumab ozogamicin ) in combination with cytotoxic agents have been tested clinically. Human NK cells mainly upregulate Siglec-7 and Siglec-9.

Furthermore, in cancer, Siglec-9 is also upregulated by peripheral NK cells, mainly on CD56 dim CD16+ NK cells. Blockade of Siglec-7 and Siglec-9 by Fab fragments can enhance the cytotoxicity of NK cells against tumor cells ( K562 ) in vitro.

 

In an in vivo model of immunodeficient mice transplanted with human NK cells and human tumor cells, tumor cell killing was mediated by inhibition of NK cell sialoglycan dependence.

The Siglec-7-negative phenotype of the developed NK-92MI cell line demonstrated high and sustained cytotoxicity against leukemia cells.

Various remodeling tumor lines ( Siglec-7-rich tumor cell lines ) of breast, brain, colon, liver, or lymphoid tissue showed increased sensitivity to NK cell killing after sialidase treatment .

In vitro fusion of sialidase with HER2-targeting antibody enhances NK cell killing of HER2+ tumor cells. Cleavage of sialic acid ligands, especially those bound by Siglec-7 and Siglec-9, by sialidases enhances NK cell-mediated killing.

This suggests that selective desialylation of tumor cell surface glycoproteins by this antibody-sialidase conjugate could render tumors more susceptible to ADCC.

 

High-affinity Siglec-9 antibody enhances NK cell cytotoxicity by blocking sialic acid expression on tumor target cells.

These antibodies against Siglec-9 also enhanced the antitumor response induced by NKG2A blockade.

Siglec-9 is upregulated on tumor-infiltrating CD8+ T cells in non-small cell lung cancer, ovarian cancer, and colorectal cancer.

Intratumoral effector memory CD8+ T cell subsets in melanoma also showed involvement of Siglec-9 in upregulation and suppression by phosphorylating SHP1.

Targeting the sialoglycan-SAMP/Siglec pathway in vitro and in vivo enhances antitumor immunity. Other inhibitory receptors such as PD-1 are also co-expressed by Siglec-9-expressing T cells, suggesting the possibility of co-suppression.

Siglec-9 is expressed on different types of immune cells, suggesting a multimodal role for Siglec-9.

These data support the idea that anti-Siglec-7 and anti-Siglec-9 blocking antibodies could be developed for cancer immunotherapy and could be used in combination with other immune checkpoint inhibitors.

 

 


LAG-3


LAG-3 ( Lymphocyte Activation Gene-3 ) is also a member of the immunoglobulin superfamily of receptors and is inhibitory.

LAG-3 was found to be upregulated on the surface of activated CD4+ T cells, CD8+ T cells and NK cells.

In addition to these cells, Lag-3 is also expressed on several other immune cells, including TILs, regulatory T cells, iNKT cells, B cells, and DC cells. It recognizes MHC class II molecules, which are similar in structure to CD4 molecules, but bind to MHC-II molecules with greater affinity than CD4.

LSECtin, expressed in the liver and several other tumors, is a member of the DC-SIGN family and has also been described as a potential ligand for LAG-3-expressing immune cells.

LAG-3 is involved in suppressing T cell effector function and is involved in T cell exhaustion.

It also promotes the suppressive activity of regulatory T cells. Blocking LAG-3 has been shown to induce improvements in T cell function.

Relatlimab, an anti-LAG-3 monoclonal antibody, is being studied in several ongoing clinical trials, either alone or in combination with a PD-1 blockade, for various cancers.

LAG-3 and PD-1 have been shown to act synergistically in the regulation of T cell function to promote tumor immune escape.

 

Although LAG-3 is expressed on NK cells, its role in NK cell regulation has not been well established.

Knocking out the LAG-3 gene in a mouse model resulted in the inability of NK cells to kill certain tumors. However, this deletion had no effect on the cytolytic activity of MHC class I mismatches.

Human NK cells, on the other hand, showed the opposite result. Antibodies that block the LAG-3 pathway fail to induce cytotoxicity in human NK cells.

Soluble LAG-3 can bind to MHC-II molecules and has no effect on the killing ability of human NK cells.

In HIV patients, viral control is associated with low expression of LAG-3 on NK cells, as well as other inhibitory molecules.

Wiskott-Aldrich syndrome protein ( WASp ) deficiency is associated with high susceptibility to cancer, most likely due to impaired anticancer capacity of NK cells and DCs.

WASp-knockout NK cells showed a correlation between cellular exhaustion and enhanced expression of NK cell memory LAG-3.

There seems to be an obvious link, however, the direct effects and underlying mechanisms of LAG-3 on NK cell function require further investigation.

In contrast to NK cells, its regulation of NKT ( natural killer T cell ) function has been widely reported. In chronic HIV patients, iNKT cell depletion and reduced IFN-γ production were associated with elevated LAG-3 expression.

The LAG-3 signaling pathway restricts the proliferation of NKT cells by blocking the cell cycle in S phase and downregulating activated CD1d.

 

Soluble recombinant LAG-3-Ig fusion protein Eftilagimod alpha ( IMP321 ) has been used as an immune adjuvant for the prevention of various infections and cancers. It is also used in monotherapy or in combination with chemotherapy for cancer.

In a short-term in vitro test, IMP321 was able to induce NK cells to produce cytokines ( IFN-γ and/or TNF-α ) in healthy individuals ( 52 out of 60 donors ) and to a lesser extent in 21 untreated donors.

Induction of cytokine production in treated metastatic cancer patients . In patients with metastatic RCC, IMP321 induced NK cell activation as monotherapy in a dose-escalation study ( P003 ) .

IMP321 with standard chemotherapy is associated with enhanced NK cell activation over several months in breast cancer patients. Therefore, LAG-3 has the potential to activate T cells and NK cells. Therefore, it can be further investigated as a potential target of checkpoint inhibition.

Furthermore, in a recent study on CIML NK cells, CD56bright , CD16- and CD62L+ NK cells were identified as a dominant subset of cytokine-induced memory-like ( CIML ) NK cells, and persistent expression of NKG2A was involved in the suppression of HLA-E Killing of positive target cells.

CIML-NK cell subsets KIR+ and NKG2C+ express LAG-3, suggesting that CIML-NK cells are potential targets for dual checkpoint inhibition.

 

 

 


TIM-3


The co-inhibitory receptor TIM-3 ( T-cell immunoglobulin and mucin domain 3 ), which recognizes galectin-9 as a ligand, is upregulated in various cancers and chronic infections.

In addition, TIM-3 variable IgV domains have also been reported to bind other ligands, such as HMGB1 ( high mobility histone B1 protein ), Ceacam-1 ( carcinoembryonic antigen cell adhesion molecule 1 ) and PtdSer ( phosphatidylserine ).

TIM-3 expression is diverse and includes several types of immune cells, including CD4+ T cells, CD8+ T cells, regulatory T cells, B cells, NK cells, NKT cells, and myeloid cells. Binding of TIM-3 to its ligand induces immune tolerance by depleting T cells and NK cells.

Upregulation of this pathway is associated with T and NK cell depletion in various chronic infections and cancers, making TIM-3 a negative regulator of T and NK cell immunity. Accordingly, its blockade reversed T-cell or NK-cell dysfunction.

Coexpression of TIM-3 and PD-1 is involved in mediating CD8+ T cell exhaustion in various cancers and chronic viral infections.

Studies have shown that TIM-3 and/or PD-1 blockade reverses T cell exhaustion and slows tumor growth.

Antibodies targeting TIM-3, such as Sym023, Cobolimab, LY3321367, BGB-A425, and MBG453, as well as several anti-PD-1/PD-L1 antibodies, are undergoing clinical studies to determine their efficacy against various cancers.

 

TIM-3 is expressed on NK cells for several reasons. It is considered a marker of maturation, activation and prognosis.

TIM-3 was highly expressed in the resting CD56+/CD3+ NK cell population compared to the CD56+/CD3+ NKT and CD56-/CD3+ T cell populations.

Some mature CD56 dim CD16+ NK cell subsets in healthy adult blood showed TIM-3 expression, while its expression was heterogeneous in immature CD56 bright CD16- NK cell subsets.

In addition, several cytokines ( IL-12, IL-15 and IL-18 ) strongly induce the expression of TIM-3, mainly in immature CD56 bright NK cells.

NK cell activation induced by IL-12 and IL-18 and maturation induced by IL-15 are mainly responsible for the expression of TIM-3 in these cells, recognizing the expression of TIM-3 in NK cells as a factor of activation and differentiation or both Both marks.

Upregulation of TIM-3 in peripheral NK cells has been observed in some cancers, namely gastric cancer, lung adenocarcinoma, advanced melanoma and bladder cancer, leading to NK cell exhaustion.

Increased levels of TIM-3 in tumor-growing NK cells suggest that TIM-3 expression is a prognostic biomarker.

Tumor-infiltrating NK cells from GIST ( gastrointestinal stromal tumor ) and bladder cancer patients also express TIM-3.

Similar to the co-expression of TIM-3 and PD-1 in T cells, exhausted tumor-infiltrating NK cells also showed detectable co-expression in MHC-I-deficient tumors.

However, GIST patients lack PD-1 co-expression in TIM-3+ tumor-infiltrating NK cells.

 

 


CD200R


CD200R is another inhibitory receptor expressed on T, B, NK and myeloid cells. It recognizes CD200 as its ligand.

In addition to being expressed on various tumors, CD200 is also expressed in various normal tissues, such as the central nervous system, retina, hair follicle cells, vascular endothelial cells and thymocytes, as well as activated T, B and DC cells.

CD200 is considered a marker of tumor progression because it is overexpressed in various cancers of hematopoietic and non-hematopoietic origin, and its expression is associated with the worst prognosis. In addition, the expression of CD200 can also be induced in cancer cells.

Indeed, expression of CD200 by cancer cells has no effect on the suppression of anticancer responses by CD200–CD200R signaling.

Therefore, CD200 blockade is a potential therapeutic option that is not limited to the treatment of CD200-expressing tumors.

The inhibitory CD200-CD200R pathway indirectly suppresses T cell effector functions by regulating macrophages and DCs.

Thus, blocking the CD200–CD200R interaction can inhibit tumor growth, supporting antagonistic CD200 or CD200R antibodies as an option for cancer therapy.

Samalizumab ( humanized anti-CD200 antibody ) was well tolerated, with dose-dependent changes in CD4-positive T cells and CD200-positive B-CLL, and moderate Th1 cytokine responses.

 

For NK cells, there is evidence that the CD200–CD200R inhibitory pathway is directly involved in NK cell suppression. In AML patients, overexpression of CD200 suppresses the antitumor response of NK cells, thereby increasing the risk of relapse in these patients.

NK cell subsets in AML patients express CD200R, suggesting that CD200-CD200R interaction suppresses NK cells. Furthermore, antibodies that block CD200 restore NK cell activity in CD200hi patients.

These data suggest that the CD200-CD200R interaction directly leads to the suppression of NK cells in AML patients. This is the only study that directly shows that target cells expressing CD200 are able to suppress the cytotoxic and IFNγ-producing activity of NK cells. Studying the CD200 signaling component of CD200+ melanoma growth and metastasis restriction, Liu et al. found that the number of NK cells was significantly reduced in livers grown from CD200-deficient metastatic tumors.

However, how CD200 deficiency affects local NK responses in the liver remains to be elucidated. This suggests that the CD200-CD200R checkpoint is a good target for checkpoint blockade in hematology and solid tumors.

Since NK cells are associated with other checkpoint receptors ( such as KIR and NKG2A ) in AML and multiple myeloma , combined checkpoint targets can be validated in such patients.

 

 

 


CD47


CD47, also known as integrin-associated protein ( IAP ), is a widely expressed glycoprotein in the immunoglobulin superfamily.

It was first discovered as a leukocyte membrane protein involved in β3 integrin-mediated signal transduction .

It is a transmembrane protein that, in addition to integrins, interacts with thrombospondin-1 ( TSP-1 ) and signal regulatory protein alpha ( SIRPα ).

Among the functions performed by CD47, its association with SIRPα and TSP-1 establishes its role as an inhibitory receptor involved in immune evasion of cancer by inhibiting phagocytosis, antigen presentation, and T/NK cell suppression.

Therefore, targeting this signaling pathway with antibodies has shown potential to treat tumors.

 

CD-47 plays an inhibitory role in NK cell-mediated antiviral or antitumor cytotoxicity. NK cytotoxicity is related to CD47 expression in HNSCC cells.

HNSCC cell lines highly expressing CD47 exhibited lower levels of NK cytotoxicity.

Pretreatment of cells with neutralizing MHC-1 or anti-CD47 antibodies increased NK cell cytotoxicity against HNSCC cell lines.

Both SIRPα and TSP-1 have been implicated in NK cell-mediated cytotoxicity. In immunocompetent syngeneic mice, anti-SIRPα antibody significantly inhibited tumor formation of RCC or melanoma cells by blocking CD47 interaction.

In addition to macrophages and CD8+ T cells, selective depletion of NK cells greatly attenuated the antitumor effect of anti-SIRPα Abs.

However, in vitro, the killing effect of NK cells on tumor cells was not inhibited by the same antibody, suggesting that the CD47 function of NK cells is independent of SIRPα.

Similarly, TSP-1 has also been shown to play a role in inhibiting early proliferation and promoting late expansion of NK cells, but the role of CD47 has not been confirmed.

Therefore, there are still many problems to be solved in this regard. However, attacking CD-47 with antibodies is worth exploring, not only in macrophages, dendritic cells, and T cells, but also in NK cells. Dual blockade of CD47 and PD-L1 has also been investigated and shown to enhance immunotherapy against circulating tumor cells.

The anti-CD47 antibody Magrolimab ( Hu5F9-G4 ) is being tested in multiple phase I and II clinical trials in different combinations with other drugs ( such as rituximab, cetuximab, azacitidine, acalabrutinib, and atezolizumab, etc. ) Research.

 

 


CTLA-4


CTLA-4 ( cytotoxic T lymphocyte-associated antigen 4 ) is a co-inhibitory receptor that can be expressed on a variety of immune cells, such as activated T lymphocytes ( CD4+T cells and CD8+T cells in tumor-bearing mice ), regulatory T cells, tumor-infiltrating NK cells, and spleen Kit+CD11b−NK cells, and induced mouse NK cells under IL-2 stimulation.

CTLA-4 competes with the co-stimulatory receptor CD28 for ligands B7-1 ( CD80 ) and B7-2 ( CD86 ) on cancer cells or antigen-presenting cells.

CTLA-4 is recognized as a negative regulator of T cell activation and a regulator of peripheral T cell tolerance and autoreactivity. Blocking CTLA-4 with an antibody ( ipilimumab ) improves T-cell function in various cancers.

 

Studies have shown that the CTLA-4/CD28/CD80/CD86 pathway is involved in NK cell-mediated cytotoxicity. In vivo, CD28 triggers the proliferation of NK cells, and their cytotoxicity and cytokine secretion have been described in many studies.

The ligands B7-1 and B7-2 on cancer cells also appear to enhance the cytotoxicity of human NK cells.

Similarly, CTLA-4 expressed by NK cells can inhibit the production of IFN-γ by acting on B7-1 of dendritic cells.

In mice, IL-18 produced by tumor cells can induce Kit+CD11b− NK cells with B7-H1-dependent immune clearance.

Kit+CD11b− NK cells have also reported upregulation of CTLA-4; however, its involvement in tumor progression in NK cell-controlled cancers has not been investigated.

These studies reveal an undeniable role for this pathway in NK cell-mediated cytotoxicity. However, CD80-CD28/CTLA-4-mediated co-stimulation is absent in human NK cells.

Costimulation of CD28/B7 has also been denied any important role in CMV-infected peripheral NK cells in mice.

 

How to understand NK Cell Immune Checkpoints?

 

 

CTLA-4+ tumor-infiltrating NK cells are a prospective immunotherapy target based on anti-CTLA-4 monoclonal antibodies. Blocking CTLA-4 might indirectly alleviate suppressed NK cells, as shown in the figure above. Expression of CTLA-4 on T regs is thought to be required for their suppressive function.

Suppression of NK cytotoxicity and increased CTLA-4-positive T regs are associated with poor prognosis in cetuximab-treated head and neck cancer patients .

Ipilimumab, an anti-CTLA-4 monoclonal antibody, leads to the depletion of Tregs , resulting in clinical efficacy in melanoma patients in an Fc-mediated manner, possibly due in part to attenuated inhibition of NK cell toxicity by Tregs.

Ipilimumab can also trigger ADCC through FcyRIIIA on primary NK cells as well as IL-2-activated NK cells and γδT cells in response to CTLA-4 on melanoma cell lines and tissues.

In addition, the interaction of Ipilimumab and CTLA-4-positive melanoma cells also resulted in the release of TNF-α by NK cells. Combination of CTLA-4 blockade and IL-2 immunotherapy delays melanoma growth and prolongs survival, showing synergistic effect.

CTLA-4 blockade increased tumor infiltration by immune cells , including CD8-positive T cells and NK cells , while IL-2 decreased the proportion of tumor-infiltrating NK cell exhaustion and differentiation. Ipilimumab induces IL-2Rα chain expression on NK cell phenotypes, followed by enhanced responses to IL-2 stimulation and cytotoxicity, which correlates with better clinical responses in patients with advanced melanoma.

 

 


PD-1


Programmed death receptor-1 ( PD-1 ) is expressed on various immune cells, including T cells ( CD4+ & CD8+ ), B cells and myeloid cells, NK cells, NKT cells and other innate lymphocytes.

Upregulation of PD-1, PD-L1, and PD-L2 ligands has been reported in various cancers, and their interaction leads to T cell suppression, resulting in immune escape.

High expression of PD-1 on NK cells can be detected in the peripheral blood of about a quarter of healthy people.

However, PD-1 expression on NK cells is upregulated in cancer patients, such as ascites from ovarian cancer patients, peripheral blood from Kaposi’s sarcoma patients, renal cell carcinoma, and multiple myeloma patients.

Peripheral blood NK cells and tumor-infiltrating NK cells were also upregulated in gastrointestinal cancers, such as esophageal, gastric, cholangiocarcinoma, liver and colorectal cancers.

Chronic infections such as HIV (human immunodeficiency virus), HCV ( hepatitis C virus ), HCMV ( human cytomegalovirus ), and Mycobacterium tuberculosis have also shown enhanced NK cell PD-1 expression.

 

PD-1 expression on NK cells is diverse and varies from cancer to cancer. PD-1 is generally lacking in CD56 bright NK cells.

However, CD56 dim NK cells have demonstrated that PD-1 expression is restricted to the NKG2A−KIR+CD57+ phenotype, a fully mature NK cell.

NKG2A−KIR+CD57+ phenotype NK cells are thought to have significantly downregulated activating receptors such as NKp30 and NKp46.

Furthermore, there was a correlation between PD-1 expression and impaired NK cell antitumor activity, while antibody interference with the interaction of PD-1 and PD-L1 resulted in partial repair. Pediatric transplant patients with PTLD also showed altered NK cell function, increased PD-1, and decreased expression of NKp46 and NKG2D.

On the other hand, CD56 bright NK cells express PD-1 in chronic HCV patients. Meanwhile, both types of NK cells ( CD56 bright and CD56 dim NK cells ) showed increased expression of PD-1 in patients with gastrointestinal cancer .

In addition, the newly discovered infiltrating CD3−CD49a+CD56+ NK cells in HCC tissues also showed a large amount of PD-1 expression on their surface, which was associated with reduced survival in HCC patients.

 

In some cancers, upregulation of NK cell PD-1 expression indicates that NK cells are in a dysfunctional state, possibly due to overstimulation of MHC-I-deficient tumor cells.

Comparing PD-1+ NK cells with PD-1-NK cells revealed that PD-1+ NK cells were functionally exhausted, with impaired cytotoxicity and cytokine production, and reduced proliferative capacity.

Blockade with anti-PD-1 mAbs has been shown to restore NK cell function. Mouse tumor cells express PD-1, and anti-PD-1 blockade induces NK cells to generate anti-tumor immune responses.

In vitro, anti-PD-1 antibodies enhanced NK cell-mediated killing of autologous MM cells. PD-1 blockade also promoted the killing effect of mouse NK cells on mouse glioma stem cells.

 

 


B7-H3


B7 homologue 3 protein ( B7-H3 ) is a ligand molecule of the B7-CD28 family, and its receptors may exist on T cells and NK cells.

It seems to inhibit the functions of both T cells and NK cells, but has not been identified confirmed. B7-H3 is thought to simultaneously co-stimulate and co-inhibit to regulate T cell function.

B7-H3 stimulates T cell activation by binding to TLT-2, whereas binding to an unknown receptor leads to co-inhibition of T cells. At the same time, it inhibits NK cells and osteoblasts by activating unknown receptors.

 

B7-H3 has limited expression in various normal tissues including pancreas, liver, small intestine, colon, heart, thymus, spleen, placenta and testis.

However, abnormal expression of B7-H3 can be found in various malignancies associated with poor prognosis, including renal cell carcinoma, breast cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, pancreatic cancer, gallbladder cancer, colorectal cancer, prostate cancer, ovarian cancer Carcinoma, cervical cancer, endometrial cancer, osteosarcoma, neuroblastoma.

Circulating serum B7-H3 levels were significantly higher in patients with lung cancer, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, and glioma than in healthy volunteers. Inhibition of NK cell-mediated cytotoxicity is one of the multiple mechanisms by which B7-H3 expressing cells escape tumors.

Glioma malignancy and reduced survival are associated with B7-H3 expression in tumor and endothelial cells. Both soluble B7-H3 and cell-associated B7-H3 in glioma cell supernatants inhibit natural killer cell-mediated tumor cell lysis. Sensitivity to killing was demonstrated in an in vivo model of B7-H3-silenced glioma cell lines.

The mAb-mediated 4Ig-B7-H3 molecule, identified as a neuroblastoma-associated molecule, masked cell transfectants or freshly isolated neuroblastoma cells and protected them from killing by NK cells.

Similarly, in neuroblastoma among ovarian cystic teratomas, B7-H3 is expressed in addition to abundant HLA class I molecules , suggesting that neuroblastoma cells are protective against NK cell-mediated lysis immune evasion mechanism.

In addition, the expression intensity of receptors such as DNAM-1 ( CD226 ) and CD16 was lower on NK cells isolated from the peritoneal fluid of these patients compared with NK cells from peripheral blood .

The BiKE bispecific antibody targeting B7-H3 can significantly inhibit the growth of tumor cells by inducing natural killer cells in the treatment of NSCLC.

 

A B7-H3 binding Fc-optimized humanized IgG1 monoclonal antibody, Enoblituzumab, is currently being explored. It has been shown to inhibit tumor growth in B7-H3-positive kidney and bladder cancer xenografts. MGA271 is an Fc-optimized humanized monoclonal antibody targeting B7-H3 that has shown safety and antitumor efficacy in several tumor types.

This antitumor activity was attributed to increased T cell clonality in patients. Further characterization of Enoblituzumab , including its pharmacokinetics and kinetics, as well as its safety, dose tolerability, and antitumor activity, against relapsed or refractory solid malignancies positive for B7-H3 receptor expression in younger patients , is being evaluated in an open-label Phase I study ( NCT02982941 ).

Orlotamab ( MGD009, a humanized B7-H3 x CD3 DART® protein ), a bispecific antibody targeting CD3 in addition to B7-H3, was developed by Macrgenenics and is currently in clinical studies ( NCT03406949 ), evaluating Safety and efficacy of this antibody in combination with an anti-PD-1 antibody ( MGA012 ) in relapsed or refractory tumors expressing B7-H3.

Altogether, B7-H3 is a potential candidate for checkpoint-based immunotherapy against T cells and NK cells.

 

 


outlook
Natural killer cells are a unique group of anti-tumor effector cells with functions such as MHC-independent cytotoxicity, cytokine production and immune memory, making them key players in both innate and adaptive immune response systems.

Some cancers are associated with dysfunctional NK cells. Therefore, repairing such NK cells may be a potential option for anti-tumor immunotherapy.

One way this repair is done is by inhibiting immune checkpoints, immune evasion by cancer cells by controlling inhibitory receptors on the surface of immune cells.

Immune checkpoint inhibition is successful in the case of T cells. NK cells have also recently been used for the same purpose.

Immune checkpoint inhibitors targeting these inhibitory receptors on the surface of NK cells, such as monalizumab and lirilumab, have been administered as monotherapy and have shown a favorable safety profile, but have had only modest success in prolonging progression-free survival.

Therefore, combinations of immune checkpoint inhibitors such as CTLA-4 and PD-1 inhibitors can also be tried in the context of NK cells, as anti-PD-1 and anti-PD-L1 inhibitors have also been shown to enhance NK cells mediated cytotoxicity.

Likewise, NKG2A enhanced tumor vaccine-induced CD8 T cell immunity also underscores the potential of combination therapy.

Therefore, combining anti-PD-1 or anti-PD-L1 inhibitors with NK cell-specific checkpoint inhibitors ( such as anti-KIR or anti-NKG2A inhibitors ) can be used for combined immunotherapy based on checkpoint inhibition.

With the addition of new checkpoints such as B7-H3, CD200R, CD47, and Siglecs7/9, combining these checkpoints for a synergistic anti-tumor response is the direction for the future to fully exert the tumor-killing effect of NK cells.

 

 

 

 

 

 

 

 

references:

1. NK Cell-Based Immune Checkpoint Inhibition . Front. Immunol., 13 February 2020

How to understand NK Cell Immune Checkpoints?

(source:internet, reference only)


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