October 3, 2022

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What are the current Immune checkpoints of NK cells in tumor treatment?

What are the current Immune checkpoints of NK cells in tumor treatment?



 

What are the current Immune checkpoints of NK cells in tumor treatment?



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

Discovered in the 1970s, NK cells are primarily associated with killing infected microorganisms and malignantly transformed allogeneic and autologous cells.

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

 

According to the density of CD56 on the cell surface, NK cells were 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 ).

 

What are the current Immune checkpoints of NK cells in tumor treatment?

 

Both activating and inhibitory receptors are expressed on the surface of NK cells and help NK cells perform their functions. MHC-I ( major histocompatibility complex class I ) antigen-specific inhibitory receptors closely 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 ) 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, critical for antibody-dependent cytotoxicity ( ADCC ) of IgG-coated target cells.

 

Tumors evade the immune system by establishing an immunosuppressive tumor microenvironment. Immune evasion involving NK cells includes multiple mechanisms, and the use of NK cell inhibitory receptors for immune evasion by tumors is one such mechanism, known as immune checkpoint inhibition, and has been shown to be the most effective and popular therapeutic target. 

 

 

 

 

KIRs


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

Inhibitory KIR 2 DL 1 , KIR 2 DL 2 and KIR 2 DL 3 recognize HLA-C as their ligands, while HLA-B and HLA-A serve as ligands for other KIRs, including inhibitory KIR 3 DL 1 and KIR 3DL2 . _ _ In addition to NK cells, a subset of T cells and NKT cells ( invariant natural killer T cells ) also express KIR. The MHC-I molecules ( HLA-A, -B, and -C ) as KIRs and ligands for KIRs themselves exhibit a wide range of natural polymorphisms.

The diversity of KIR allelic combinations ( there are 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 ) are IgG4 monoclonal antibodies directed against the KIR 2 DL 1/2/3 NK cell inhibitory receptor and are currently being tested in various hematological ( AML, CLL, NHL ) or solid malignancies ( breast cancer and ovarian cancer ) for clinical evaluation and development .

Up to 10 mg/kg of IPH2101 was reported to be still 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 the best response with stable disease.

 

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

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

 

Recently, the third member of this group 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 overall efficacy achieved in 16 of 44 patients ( 36% ).

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 for the treatment of 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 out of 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 major 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 predominantly expressed in myeloid expressed on cells.

Therefore, the interaction of ILT2 and HLA-G can inhibit the immune function of NK, T and B cells, thus serving as an immunotherapy target.

 

Various primary and metastatic malignancies express HLA-G, which is also considered an indicator of the progression and prognosis of 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 ).

Furthermore, 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 non-canonical MHC-I molecule HLA-E as a ligand.

CD94-NKG2A and its HLA-E ligand 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 KIR.

CD94/NKG2A is also co-expressed with other inhibitory receptors with different specificities. In addition, γδ and cd8+ T cells also expressed 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 ( hematological and solid tumors ) exhibit upregulation of HLA-E expression in order to avoid killing by NK cells. In various cancers, poor prognosis is associated with HLA-E upregulation.

Blockade of CD94/NKG2A receptors with antibodies may serve as a therapeutic strategy. Therefore, an anti-CD94/NKG2A antibody ( IPH2201 Monalizumab ) developed by Natural Pharmaceuticals has been used in various trials.

The results of in vitro and in vivo studies indicate that the humanized anti-NKG2A antibody is safe and effective for the application of hematological malignancies. In vitro, monalizumab ameliorated NK cell dysfunction in chronic lymphocytic leukemia.

Monalizumab was well tolerated as monotherapy for gynecologic malignancies ( up to 10 mg/kg intravenously or SC ), and no DTLs or SAEs were reported. This ongoing large conditioning cohort trial showed stable disease in 41% of evaluable patients ( 128 ).

 

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

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

This is an encouraging result if compared with the historical record of efficacy of cetuximab alone in previous studies ( ORR 12.6%, PFS 2.3 m, OS 5.6 m ). Adverse effects of combination therapy were similar to those of cetuximab alone .

Recent in vivo analyses suggest that induction of CD8+ T cells by NKG2A 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 the way forward and warrants further exploration.

 

 

 

TIGIT and CD96


TIGIT ( T cell immunoreceptor 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 ( filtered T cells) vesicular helper T cells ) and regulatory T cells ( Tregs ).

Compared with TIGIT, CD96, a member of the same immunoglobulin superfamily, has a similar inhibitory effect but has a lower binding affinity to the ligand CD155.

CD226 is an activating receptor that competes with TIGIT and CD96 for binding to CD155.

CD155 ( primarily ) and CD112 act 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 tumors ( colorectal cancer ) grow 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, as NK cell loss is associated with IFN- γ- or TNF-secreted TIL ( CD8+ ) frequency was associated with higher frequency of PD-1 expression of TIL ( CD8+ ).

NK cells make up 25-50% of liver lymphocytes, suggesting their importance for hepatic 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 dysfunction of tumor-infiltrating NK cells, mainly the CD11b-CD27-NK subset. Sun et al. found exhausted tumor-infiltrating CD96+ NK cells and found that their expression was associated with poor clinical outcomes in HCC patients.

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

 

In recent years, the combined application of checkpoint inhibitors has received more and more attention to achieve synergistic effects.

Anti-TIGIT plus anti-PD-L1 blockade has been reported to prevent NK cell exhaustion in tumor-bearing mice and patients with colon cancer.

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 show upregulation of TIM-3 and TIGIT both in the periphery and in the tumor.

Indeed, the roles of TIGIT and CD96 in NK cell exhaustion in various cancers are still under investigation, and further insights are needed to determine their 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 that belong to the type I lectin family.

Siglecs are expressed on a variety of immune cells, including those of lymphoid and myeloid origin. Siglecs show 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 suppressive Siglec, 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, inhibitory siglecs also contain one or more ITIM and ITIM-like motifs at the C-terminus of their intracellular segment.

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

 

The Siglec-sialic acid interaction is involved in the regulation of immune tolerance and may serve as a target for the induction of antitumor immunity.

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

Furthermore, in cancer, peripheral NK cells also upregulate Siglec-9, mainly on cd56 dim cd16+ NK cells. Blockade of Siglec-7 and Siglec-9 by Fab fragments enhanced the cytotoxicity of NK cells against tumor cells ( K562 ) in vitro.

In an in vivo model of immunodeficient mice engrafted with human NK cells and human tumor cells, tumor cell killing is mediated by sialoglycan-dependent inhibition of NK cells.

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

Various remodeled tumor lines ( Siglec-7-enriched tumor cell lines ) of breast, brain, colon, liver, or lymphoid tissue have increased susceptibility to NK cell killing following sialidase treatment . In vitro fusion of sialidase with HER2-targeting antibody enhances the killing of NK cells against HER2+ tumor cells.

Cleavage of sialic acid ligands, particularly Siglec-7 and Siglec-9 bound ligands, by sialidase enhances NK cell-mediated killing.

This suggests that selective desialylation of tumor cell surface glycoproteins by this antibody-sialidase conjugate could make 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 show that Siglec-9 is involved in upregulation and repression by phosphorylating SHP1.

Targeting the sialoglycan-SAMP/Siglec pathway in vitro and in vivo enhances antitumor immunity.

Other inhibitory receptors such as PD-1 were also co-expressed by Siglec-9-expressing T cells, suggesting the possibility of co-inhibition. Siglec-9 is expressed on different types of immune cells, suggesting a multimodal effect of 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 up-regulated on the surface of activated CD4+ T cells, CD8+ T cells and NK cells. In addition to these cells, Lag-3 is 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 is similar in structure to CD4 molecules, but binds to MHC-II molecules with greater affinity than CD4.

Expressed in the liver and several other tumors, LSECtin 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 currently being studied in several ongoing clinical trials, either alone or in combination with PD-1 blockers, for a variety of cancers. LAG-3 and PD-1 show a synergistic role 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 fully demonstrated.

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 results.

Antibodies blocking the LAG-3 pathway failed 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 and other inhibitory molecules on NK cells.

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

WASp-knockout NK cells showed cell exhaustion associated with enhanced NK cell memory LAG-3 expression.

There appears to be an obvious association, however, the direct effect and underlying mechanism of LAG-3 on NK cell function requires further investigation.

Compared with 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 are associated with elevated LAG-3 expression.

The LAG-3 signaling pathway restricts NKT cell proliferation by blocking S-phase cell cycle 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 cancer monotherapy or in combination with chemotherapy.

In a short-term in vitro assay, 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 renal cancer, IMP321 induced NK cell activation as monotherapy in a dose escalation study ( P003 ) .

IMP321 from 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 for checkpoint inhibition.

Furthermore, in a recent study of CIML NK cells, CD56 bright , CD16- and CD62L+ NK cells were identified as the dominant subpopulation of cytokine-induced memory-like ( CIML ) NK cells, and sustained expression of NKG2A is involved in suppressing 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 ) recognizes galectin-9 as a ligand and is upregulated in various cancers and chronic infections.

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

Expression of TIM-3 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 ligands induces immune tolerance by depleting T cells and NK cells. Upregulation of this pathway is associated with depletion of T and NK cells in various chronic infections and cancers, making TIM-3 a negative regulator of T and NK cell immunity.

Correspondingly, its blocking effect reversed T cell or NK cell dysfunction. Co-expression 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 depletion and slows tumor growth.

Antibodies against 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 in various cancers.

 

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

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

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

Furthermore, several cytokines ( IL-12, IL-15 and IL-18 ) strongly induced the expression of TIM-3, mainly in immature CD56 bright NK cells. IL-12 and IL-18-induced NK cell activation and IL-15-induced maturation are the main reasons for TIM-3 expression in these cells, identifying TIM-3 expression in NK cells as a function 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 patients with GIST ( gastrointestinal stromal tumor ) and bladder cancer 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, and 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 on 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 induce cancer cells. Indeed, expression of CD200 by cancer cells had no effect on the inhibition 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 inhibits the effector function of T cells by regulating macrophages and DC cells.

Therefore, 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 the suppression of NK cells.

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 from AML patients express CD200R, suggesting that CD200-CD200R interaction suppresses NK cells. Furthermore, in CD200hi patients, CD200-blocking antibodies restored NK cell activity.

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 capable of suppressing NK cell cytotoxicity and IFNγ-producing activity.

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 CD200-deficient metastatic tumor-growing livers.

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

Since NK cells associate with other checkpoint receptors in AML and multiple myeloma, such as KIR and NKG2A , combined checkpoint targets could 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-regulated protein alpha ( SIRPα ).

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

Therefore, targeting this signaling pathway with antibodies shows a potential ability to treat tumors.

 

CD-47 plays an inhibitory role in NK cell-mediated antiviral or antitumor cytotoxicity. NK cytotoxicity was associated with CD47 expression in HNSCC cells.

HNSCC cell lines with high expression of CD47 showed 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 are involved in NK cell-mediated cytotoxicity.

In immunocompetent syngeneic mice, anti-SIRPα antibodies significantly inhibited RCC or melanoma cell tumorigenesis by blocking the interaction of CD47. In addition to macrophages and CD8+ T cells, selective depletion of NK cells greatly attenuated the antitumor effect of anti-SIRPαAbs.

However, in vitro, the killing of tumor cells by NK 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 suppressing early NK cell proliferation and promoting late expansion, but the role of CD47 has not been demonstrated.

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 immunotherapeutic effects on circulating tumor cells.

The anti-CD47 antibody Magrolimab ( Hu5F9-G4 ) is in multiple Phase I and Phase II clinical trials in various combinations with other drugs such as rituximab, cetuximab, azacitidine, acalabrutinib and atezolizumab, among others Research.

 

 

 

CTLA-4


CTLA-4 ( Cytotoxic T lymphocyte-associated antigen 4 ) is a co-inhibitory receptor 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 upon IL-2 stimulation.

CTLA-4 competes with the co-stimulatory receptor CD28 for the 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 induces Kit+CD11b−NK cells with B7-H1-dependent immune clearance. Kit+CD11b−NK cells also report CTLA-4 upregulation; 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 was absent in human NK cells. Costimulation of CD28/B7 was also denied to play any important role in CMV-infected peripheral NK cells in mice.

 

What are the current Immune checkpoints of NK cells in tumor treatment?

 

 

CTLA-4+ tumor-infiltrating NK cells are a prospective immunotherapeutic target based on anti-CTLA-4 monoclonal antibodies.

As shown above, blocking CTLA-4 may indirectly alleviate suppressed NK cells. Expression of CTLA-4 on T regs is thought to be required for its suppressive function.

In cetuximab-treated head and neck cancer patients, suppression of NK cytotoxicity and increased CTLA-4-positive T regs were associated with poor prognosis.

Ipilimumab , an anti-CTLA-4 monoclonal antibody that causes depletion of Tregs , produces clinical efficacy in melanoma patients in an Fc-mediated manner, possibly due in part to attenuated Tregs inhibition of NK cytotoxicity.

Ipilimumab also triggers ADCC effects by reacting FcyRIIIA on primary NK cells as well as IL-2-activated NK cells and γδ T cells with 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 from NK cells.

Combination of CTLA-4 blockade and IL-2 immunotherapy can delay melanoma growth and prolong survival, showing a synergistic effect.

CTLA-4 blockade increased tumor infiltration of immune cells, including CD8-positive T cells and NK cells , whereas IL-2 decreased the proportion of tumor-infiltrating NK cells depleted and differentiated.

Ipilimumab induces IL-2Rα chain expression on the NK cell phenotype and subsequently enhances the response to IL-2 stimulation and cytotoxicity, which is associated 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 leading to immune escape.

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

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

Peripheral blood NK cells and tumor-infiltrating NK cells were similarly upregulated in gastrointestinal cancers such as esophagus, gastric, bile duct, liver, and colorectal cancers.

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

 

The expression of PD-1 on NK cells is diverse and varies by cancer. PD-1 expression 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 antitumor activity of NK cells, while antibody interference with the interaction between PD-1 and PD-L1 resulted in partial repair.

Transplanted patients with PTLD children also showed altered NK cell function, increased PD-1, and decreased NKp46 and NKG2D expression.

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

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

 

In some cancers, upregulation of NK cell PD-1 expression suggests a dysfunctional state of NK cells, possibly due to overstimulation of tumor cells lacking MHC-I.

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

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

In vitro, anti-PD-1 antibody 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 homolog 3 protein ( B7-H3 ) is a ligand molecule of the B7-CD28 family, its receptor may be present on T cells and NK cells, it seems to inhibit the functions of both T cells and NK cells, but has not been confirmed. B7-H3 is thought to regulate T cell function by both co-stimulation and co-suppression.

B7-H3 stimulates T cell activation by binding to TLT-2, whereas binding to an unknown receptor results in co-suppression 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 malignant tumors 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 cancer, cervical cancer, endometrial cancer, osteosarcoma, neuroblastoma.

Circulating serum B7-H3 levels were significantly higher in patients with lung, 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 evade tumors.

The malignant degree and decreased survival rate of gliomas are related to the expression of B7-H3 in tumor and endothelial cells.

Both soluble B7-H3 and cell-bound B7-H3 in glioma cell supernatants inhibited natural killer cell-mediated lysis of tumor cells.

Sensitivity to killing was demonstrated in an in vivo model of a B7-H3-silenced glioma cell line.

The monoclonal antibody-mediated 4Ig-B7-H3 molecule was identified as a neuroblastoma-associated molecule that masks cell transfectants or newly isolated neuroblastoma cells from being killed by NK cells.

Similarly, in neuroblastoma in ovarian cystic teratoma, 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.

Furthermore, 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 .

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

 

The 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 transplants.

MGA271 is an Fc-optimized humanized monoclonal antibody targeting B7-H3 that has demonstrated safety and antitumor efficacy in several tumor types.

This antitumor activity is attributed to the increased clonality of T cells in patients. Further characterization of enoblituzumab , including its pharmacokinetics and kinetics, as well as its safety, dose-tolerability, and antitumor activity against B7-H3 receptor-positive relapsed or refractory solid malignancies in young 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 study ( NCT03406949 ), evaluating Safety and efficacy of this antibody in combination with an anti-PD-1 antibody ( MGA012 ) in the treatment of B7-H3-expressing relapsed or refractory tumors.

In conclusion, 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 antitumor effector cells with functions such as MHC-independent cytotoxicity, cytokine production, and immune memory, making them key players in the innate and adaptive immune response systems. Dysfunctional NK cells are associated with the development of some cancers.

Therefore, repairing such NK cells may be a potential option for antitumor immunotherapy.

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

Immune checkpoint inhibition was successful in the context of T cells. NK cells have recently been used for the same purpose.

Immune checkpoint inhibitors that target 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 with 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 enhances tumor vaccine-induced CD8 T-cell immunity, underscoring 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 combination 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 antitumor response is the future direction for the full use of NK cells to kill tumors.

 

 

 

 

references:

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

What are the current Immune checkpoints of NK cells in tumor treatment?

(source:internet, reference only)


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