Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells

AbstractModulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

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Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽

10.3390/hemato2020010 ◽

2021 ◽

Vol 2 (2) ◽

pp. 167-181

Author(s):

Marie Thérèse Rubio ◽

Adèle Dhuyser ◽

Stéphanie Nguyen

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibodies ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Proteasome Inhibitors ◽

Disease Evolution ◽

Cell Functions

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

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Network Pharmacology to Unveil the Biological Basis of Health-Strengthening Herbal Medicine in Cancer Treatment

Cancers ◽

10.3390/cancers10110461 ◽

2018 ◽

Vol 10 (11) ◽

pp. 461 ◽

Cited By ~ 31

Author(s):

Jiahui Zheng ◽

Min Wu ◽

Haiyan Wang ◽

Shasha Li ◽

Xin Wang ◽

...

Keyword(s):

Cancer Treatment ◽

Tumor Cells ◽

High Throughput ◽

Antigen Processing ◽

Killer Cell ◽

Network Pharmacology ◽

Gene Expressions ◽

Antitumor Effects ◽

Biological Basis ◽

Radix Paeoniae Alba

Health-strengthening (Fu-Zheng) herbs is a representative type of traditional Chinese medicine (TCM) widely used for cancer treatment in China, which is in contrast to pathogen eliminating (Qu-Xie) herbs. However, the commonness in the biological basis of health-strengthening herbs remains to be holistically elucidated. In this study, an innovative high-throughput research strategy integrating computational and experimental methods of network pharmacology was proposed, and 22 health-strengthening herbs were selected for the investigation. Additionally, 25 pathogen-eliminating herbs were included for comparison. First, based on network-based, large-scale target prediction, we analyzed the target profiles of 1446 TCM compounds. Next, the actions of 166 compounds on 420 antitumor or immune-related genes were measured using a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS2. Furthermore, the structural information and the antitumor activity of the compounds in health-strengthening and pathogen-eliminating herbs were compared. Using network pharmacology analysis, we discovered that: (1) Functionally, the predicted targets of compounds from health strengthening herbs were enriched in both immune-related and antitumor pathways, similar to those of pathogen eliminating herbs. As a case study, galloylpaeoniflorin, a compound in a health strengthening herb Radix Paeoniae Alba (Bai Shao), was found to exert antitumor effects both in vivo and in vitro. Yet the inhibitory effects of the compounds from pathogen eliminating herbs on tumor cells proliferation as a whole were significantly stronger than those in health-strengthening herbs (p < 0.001). Moreover, the percentage of assay compounds in health-strengthening herbs with the predicted targets enriched in the immune-related pathways (e.g., natural killer cell mediated cytotoxicity and antigen processing and presentation) were significantly higher than that in pathogen-eliminating herbs (p < 0.05). This finding was supported by the immune-enhancing effects of a group of compounds from health-strengthening herbs indicated by differentially expressed genes in the HTS2 results. (2) Compounds in the same herb may exhibit the same or distinguished mechanisms in cancer treatment, which was demonstrated as the compounds influence pathway gene expressions in the same or opposite directions. For example, acetyl ursolic acid and specnuezhenide in a health-strengthening herb Fructus Ligustri lucidi (Nv Zhen Zi) both upregulated gene expressions in T cell receptor signaling pathway. Together, this study suggested greater potentials in tumor immune microenvironment regulation and tumor prevention than in direct killing tumor cells of health-strengthening herbs generally, and provided a systematic strategy for unveiling the commonness in the biological basis of health-strengthening herbs in cancer treatment.

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799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.799 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A834-A834

Author(s):

Xue Yao ◽

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Nk Cell ◽

Killer Cell ◽

Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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Natural killer cell immunotherapy to target stem-like tumor cells

Journal for ImmunoTherapy of Cancer ◽

10.1186/s40425-016-0124-2 ◽

2016 ◽

Vol 4 (1) ◽

Cited By ~ 24

Author(s):

Steven K. Grossenbacher ◽

Robert J. Canter ◽

William J. Murphy

Keyword(s):

Natural Killer Cell ◽

Natural Killer ◽

Tumor Cells ◽

Killer Cell ◽

Cell Immunotherapy

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Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Journal of Visualized Experiments ◽

10.3791/60714 ◽

2020 ◽

Author(s):

Suresh Chava ◽

Suresh Bugide ◽

Romi Gupta ◽

Narendra Wajapeyee

Keyword(s):

Natural Killer Cell ◽

Natural Killer ◽

Tumor Cells ◽

Killer Cell ◽

Hepatic Tumor ◽

And Migration

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Bortezomib Augments Natural Killer Cell Targeting of Stem-Like Tumor Cells

Cancers ◽

10.3390/cancers11010085 ◽

2019 ◽

Vol 11 (1) ◽

pp. 85 ◽

Cited By ~ 9

Author(s):

Jesus Luna ◽

Steven Grossenbacher ◽

Ian Sturgill ◽

Erik Ames ◽

Sean Judge ◽

...

Keyword(s):

Stem Cell ◽

Natural Killer ◽

Tumor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Human Tumor ◽

Growth Delay ◽

Cell Targeting ◽

Immunodeficient Mice

Tumor cells harboring stem-like/cancer stem cell (CSC) properties have been identified and isolated from numerous hematological and solid malignancies. These stem-like tumor cells can persist following conventional cytoreductive therapies, such as chemotherapy and radiotherapy, thereby repopulating the tumor and seeding relapse and/or metastasis. We have previously shown that natural killer (NK) cells preferentially target stem-like tumor cells via non- major histocompatibility complex (MHC) restricted mechanisms. Here, we demonstrated that the proteasome inhibitor, bortezomib, augments NK cell targeting of stem cell-like tumor cells against multiple solid human tumor-derived cancer lines and primary tissue samples. Mechanistically, this was mediated by the upregulation of cell surface NK ligands MHC class I chain-related protein A and B (MICA and MICB) on aldehyde dehydrogenases (ALDH)-positive CSCs. The increased expression of MICA and MICB on CSC targets thereby enhanced NK cell mediated killing in vitro and ex vivo from both human primary tumor and patient-derived xenograft samples. In vivo, the combination of bortezomib and allogeneic NK cell adoptive transfer in immunodeficient mice led to increased elimination of CSCs as well as tumor growth delay of orthotopic glioblastoma tumors. Taken together, our data support the combination bortezomib and NK transfer as a strategy for both CSC targeting and potentially improved outcomes in clinical cancer patients.

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Successful Generation of Cytotoxic T-Cells That Kill CLL Cells Using Heteroclitic Peptides Is Independent of the Native Peptide Binding Affinity to HLA-A*0201.

Blood ◽

10.1182/blood.v106.11.54.54 ◽

2005 ◽

Vol 106 (11) ◽

pp. 54-54 ◽

Cited By ~ 3

Author(s):

Katja Mauerer ◽

Gullu Gorgun ◽

David Zahrieh ◽

John G. Gribben

Keyword(s):

Tumor Cells ◽

Binding Affinity ◽

Mhc Class I ◽

Binding Assay ◽

Cytotoxic T Cells ◽

B Cell Malignancies ◽

Specific Lysis ◽

Native Peptide ◽

Native Peptides ◽

Heteroclitic Peptides

Abstract Targeting immunoglobulin (Ig) framework region (FR) derived peptides offers the advantage of a less patient specific immunotherapeutic strategy in B-cell malignancies. A major limitation of this method is the generally low immunogenicity and low binding affinity of these peptides to MHC class I and class II molecules. Heteroclitic peptide modifications can increase immunogenicity of low binding peptides while leaving T-cell recognition residues intact, and improve ability to generate cytotoxic T cells lines (CTL). It is not known whether such CTLs can still kill tumor cells that express native peptides below a lower threshold of binding affinity. To address this, we sequenced Ig, identified nonameric and decameric peptide sequences that potentially bind to HLA-A*0201 (HLA-A2) that were frequently shared among patients. We used two independent computer prediction analysis tools, determined binding using the T2-binding assay, screened peptide specific CTL responses with an established T-cell expansion system, and assessed cytotoxicity of the CTL lines against native and heteroclitic peptide pulsed APCs and also primary tumor cells from which the native peptides were derived. 34 FR-derived peptides were synthesized, 17 native peptides selected to represent a wide range of predicted binding to HLA-A2, and 17 corresponding heteroclitic counterparts. There was a strong correlation of predicted binding assessed by both scores (Spearmen rho=0.62; p=0.0001) and with respective T2-binding (Spearmen rho=0.66; p<0.0001). Heteroclitic peptides has significantly enhanced predicted binding compared to their native counterparts (Parker Score p<0.00001; Rammensee Score p=0.004, T2-binding p=0.0005) and CTLs generated against heteroclitic peptides had significantly enhanced killing of CD40 activated B-cells pulsed not only with the corresponding heteroclitic peptide (p=0.0003), but also with the native peptide (p=0.04). The binding affinity of low (n=10; FI<0.5) or intermediate binding (n=7; FI>0.6) native peptides did not correlate with specific lysis of peptide pulsed CD40 activated B-cells by CTLs generated against native peptides. Binding affinity of heteroclitic peptides correlated with the ability to induce CTL responses (Spearman rho=0.50; p=0.04). CTLs generated against heteroclitic peptide killed primary CLL cells more effectively than those generated against their native counterparts (p=0.01). Most importantly, the specific lysis of primary tumor cells by successfully generated CTLs was independent of the original binding affinity of the native peptides, both as measured by T2 binding assay (Spearman rho=0.38; p=0.36) and as predicted by the Parker Score (Spearman rho=0.22; p=0.60) or the Rammensee Score (Spearman rho=0.02; p=0.95). Thus, the present study demonstrates recognition of naturally processed Ig-derived peptides even with extremely low binding affinity to MHC class I when higher affinity analogues are used as ‘in vitro’ immunogens. Once CTLs are generated, cytotoxicity appears to be independent of the original binding affinity, suggesting that the rate limiting factor is the ability to generate the immune response, but that once generated, these CTLs have ability to kill tumor cells bearing even very weakly immunogenic peptides. These findings have significant implication for vaccination strategies in B-cell malignancies and warrant in vivo evaluation of this model in CLL.

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The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody

Blood ◽

10.1182/blood-2010-02-271874 ◽

2010 ◽

Vol 116 (13) ◽

pp. 2286-2294 ◽

Cited By ~ 459

Author(s):

Don M. Benson ◽

Courtney E. Bakan ◽

Anjali Mishra ◽

Craig C. Hofmeister ◽

Yvonne Efebera ◽

...

Keyword(s):

Multiple Myeloma ◽

Immune Response ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Killer Cell ◽

Cell Immune Response ◽

Cell Versus

Abstract T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti–PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1+ MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

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