A novel human anti-TIGIT monoclonal antibody with excellent function in eliciting NK cell-mediated antitumor immunity

MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.

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The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.1133 ◽

1995 ◽

Vol 181 (3) ◽

pp. 1133-1144 ◽

Cited By ~ 359

Author(s):

J E Gumperz ◽

V Litwin ◽

J H Phillips ◽

L L Lanier ◽

P Parham

Keyword(s):

Monoclonal Antibody ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Inhibitory Effect ◽

Glycosylation Site ◽

Class I ◽

Target Cells ◽

Cell Clones

Although inhibition of natural killer (NK) cell-mediated lysis by the class I HLA molecules of target cells is an established phenomenon, knowledge of the features of class I molecules which induce this effect remains rudimentary. Using class I alleles HLA-B*1502 and B*1513 which differ only at residues 77-83 which define the Bw4 and Bw6 serological epitopes, we tested the hypothesis that the presence of the Bw4 epitope on class I molecules determines recognition by NKB1+ NK cells. HLA-B*1513 possesses the Bw4 epitope, whereas B*1502 has the Bw6 epitope. Lysis by NKB1+ NK cell clones of transfected target cells expressing B*1513 as the only HLA-A, -B, or -C molecule was inhibited, whereas killing of transfectants expressing B*1502 was not. Addition of an an anti-NKB1 monoclonal antibody reconstituted lysis of the targets expressing B*1513, but did not affect killing of targets bearing B*1502. The inhibitory effect of B*1513 could be similarly prevented by the addition of an anti-class I monoclonal antibody. These results show that the presence of the Bw4 epitope influences recognition of HLA-B molecules by NK cells that express NKB1, and suggest that the NKB1 molecule may act as a receptor for Bw4+ HLA-B alleles. Sequences outside of the Bw4 region must also affect recognition by NKB1+ NK cells, because lysis of transfectants expressing HLA-A*2403 or A*2501, which possess the Bw4 epitope but are in other ways substantially different from HLA-B molecules, was not increased by addition of the anti-NKB1 antibody. Asparagine 86, the single site of N-linked glycosylation on class I molecules, is in close proximity to the Bw4/Bw6 region. The glycosylation site of the Bw4-positive molecule B*5801 was mutated, and the mutant molecules tested for inhibition of NKB1+ NK cells. Inhibition that could be reversed by addition of the anti-NKB1 monoclonal antibody was observed, showing the presence of the carbohydrate moiety is not essential for class I recognition by NKB1+ NK cell clones.

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Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells

Blood ◽

10.1182/blood-2009-02-206532 ◽

2009 ◽

Vol 114 (13) ◽

pp. 2667-2677 ◽

Cited By ~ 234

Author(s):

Francois Romagné ◽

Pascale André ◽

Pieter Spee ◽

Stefan Zahn ◽

Nicolas Anfossi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Natural Killer ◽

Tumor Cells ◽

Nk Cell ◽

Hla Class I ◽

Therapeutic Antibody ◽

Class I ◽

Therapeutic Window ◽

Target Cells ◽

Term Survival

Abstract Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

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Dual enhancement of T and NK cell function by pulsatile inhibition of SHIP1 improves antitumor immunity and survival

Science Signaling ◽

10.1126/scisignal.aam5353 ◽

2017 ◽

Vol 10 (500) ◽

pp. eaam5353 ◽

Cited By ~ 19

Author(s):

Matthew Gumbleton ◽

Raki Sudan ◽

Sandra Fernandes ◽

Robert W. Engelman ◽

Christopher M. Russo ◽

...

Keyword(s):

Antitumor Immunity ◽

Cell Function ◽

Nk Cell

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Study On the Molecular Mechanisms of Cytotoxicity of NK Cells against RPMI 8226 Cells.

Blood ◽

10.1182/blood.v114.22.4745.4745 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4745-4745

Author(s):

Xiuju Wang ◽

Songmei Yin ◽

Liping Ma ◽

Danian Nie ◽

Shangfeng Xie ◽

...

Keyword(s):

Monoclonal Antibody ◽

Healthy Volunteers ◽

Nk Cells ◽

Molecular Mechanisms ◽

Nk Cell ◽

Conflicts Of Interest ◽

Control Cell ◽

Killing Activity ◽

Rpmi 8226 ◽

Hla Molecules

Abstract Abstract 4745 To observe how did the allogeneic NK cells kill human multiple myeloma cell RPMI-8266 and the immune mechanisms why RPMI-8266 cells escape from the NK cytotoxicity. Methods Detect the cytotoxicity of NK cells against RPMI 8226 cells by LDH release assay at different effect-to-target cell ratios in vitro, K562, high-sensitive with NK cells,was control cell. Test the expression of MHC-± chain-related molecules (MICA/B), human cytomegalovirus glycoprotein UL16-binding protein (ULBP1-3) and HLA -± MHC molecules on K562 and RPMI-8266 cell by flow cytometry. Detect mRNA level of MICA / B and ULBP1 ∼ 3 in K562, RPMI-266, and KIR genotyping of NK cell from 9 cases of healthy volunteers' by PCR method. As the E:T was 20:1, we used AMO-1, BMO-1, M295, M310, M551 and W6/32 mAb to block the effect of cell-surface protein MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA-± in K562 and RPMI-8266 cell respectively, then observe the change of cytotoxicity of NK cells on K562 and RPMI-8266 cell. Results When the E:T was 5:1, cytotoxicity of NK cells on K562 and RPMI-8266 were (29.52 ± 0.27)% and (2.15 ± 0.32)% respectively; As the E:T was 10:1, the cytotoxicity were (36.37 ± 0.78) % and (5.26 ± 0.84)%; As the E:T was 20:1, they were (59.57 ± 1.05)% and (7.63 ± 1.05)%; As the E:T was 40:1, they were (70.64 ± 1.34)% and (10.18 ± 1.53)%. There wre significantly difference of the cytotoxicity of NK cell between on RPMI-8266 and K562 at all E:T ratio(P <0.05); As for different E:T ratio, the cytotoxicity of NK cell on 8266 cells were statistic difference among them (P <0.05). In K562 cell, we detect MICA / B and ULBP 1 ∼ 3 mRNA and protein, but no HLA -± molecules; RPMI-8266 cell expressed MICA / B and ULBP 1 ∼ 3 and HLA -± molecule, but no MICA / B and ULBP 1 ∼ 3. The genotype of KIR were mismatch between RPMI-8266 cell and NK cells from 9 cases of healthy volunteers. The E:T set as 20:1, we used monoclonal antibodies AMO-1, BMO-1, M295, M310 and M551, respectively, to block the effect of MICA, MICB, ULBP1, ULBP2 and ULBP3, killing activity of NK cell on K562 cells significantly decreased, respectively, (40.82 ± 1.47)%, (43.26 ± 2.41)%, (45.42 ± 1.58)%, (50.74 ± 2.16)%, (41.72 ± 1.66)%, compared with the previous block, they were significantly different (P <0.05); The killing activity of NK cell on RPMI-8266 did not significant change after bloking by all monoclonal antibody, they were (5.81 ± 0.72)%, (7.83 ± 0.91)%, (6.77 ± 0.82)%, (8.25 ± 1.46)%, (6.42 ± 0.87)% respectively (P> 0.05); But as we bloked HLA -± molecules with W6/32 monoclonal antibody, the cytotoxicity of NK cell on RPMI-8266 increased to (48.77 ± 4.61)%, there were significant differences between before and after bloking (P <0.05). Conclusion For 8266 cell, the mechanisms of immune escape from NK killing effect may be related to high expression of HLA -± molecule, and did not express the NKG2D ligands MICA / B and ULBP1 ∼ 3 molecules. Disclosures: No relevant conflicts of interest to declare.

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Combination Therapy of a Defucosylated Anti-HM1.24 Monoclonal Antibody Plus Lenalidomide Significantly Enhances Myeloma Cell Cytotoxicity Via Antibody-Dependent Cellular Cytotoxicity

Blood ◽

10.1182/blood.v118.21.1833.1833 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1833-1833

Author(s):

Takeshi Harada ◽

Shuji Ozaki ◽

Asuka Oda ◽

Hiroe Amou ◽

Shiro Fujii ◽

...

Keyword(s):

Monoclonal Antibody ◽

Combination Therapy ◽

Mononuclear Cells ◽

Plasma Cells ◽

Nk Cell ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Adcc Activity ◽

Effector Cells ◽

Rpmi 8226

Abstract Abstract 1833 Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. During the last decade, treatment of MM has been improved by incorporating bortezomib, thalidomide, and lenalidomide (LEN) into conventional cytotoxic and transplantation regimens in newly diagnosed and relapsed/refractory MM patients. However, MM still remains incurable despite the implementation of these new treatment options, so more efficacious therapies are needed to further improve the prognosis of MM. Monoclonal antibody (mAb)-based immunotherapy has recently become an alternative strategy for treatment of cancers. Our previous studies have shown that HM1.24 (CD317) is selectively expressed on terminally differentiated normal and neoplastic plasma cells and, moreover, expressed on the side population of MM cells that represents MM cancer stem cells. We have previously generated a humanized mAb (AHM) specific to HM1.24 for the treatment of MM. AHM carries an Fc region derived from human IgG1-k and exhibits the ability to induce antibody-dependent cellular cytotoxicity (ADCC) against human MM cells in the presence of human effector cells. To improve the efficacy of AHM, we have developed a defucosylated mAb (YB-AHM) with a higher affinity to Fc gamma RIII. LEN is a structural analog of thalidomide with more potent immunomodulatory activities. Several studies have shown that LEN activates NK cell function and enhances NK cell-mediated lysis of both MM cell lines and patient MM cells in vitro. Here, we evaluated the efficacy of combination therapy of YB-AHM and LEN. First, we investigated whether LEN stimulates the expression of HM1.24 on MM cells. LEN alone did not affect HM1.24 expression, but in the presence of peripheral blood mononuclear cells (PBMCs) LEN augmented the expression of HM1.24 in MM cell lines and primary MM cells. In PBMCs, expression levels of CD56 increased after stimulation with LEN. These results suggest that LEN might augment the ADCC activity by enhancing HM1.24 antigen and NK activity. Next, we evaluated ADCC activity of YB-AHM against RPMI 8226 cells by using flow cytometric PKH-26 assay. When we used PBMCs from healthy donors (n=5) as effectors, ADCC activity of YB-AHM was increased in an E:T ratio-dependent manner. Importantly, YB-AHM induced significantly higher ADCC activity compared with AHM (24±6% vs 11±7%, p<0.05; mAb, 100 ng/mL; E:T ratio, 10). Treatment of PBMCs with LEN (3 micro M for 2 days) slightly enhanced ADCC activity of AHM (12±5%) and YB-AHM (30±6%). In PBMCs from MM patients (n=11), YB-AHM induced ADCC activity (36±15%) that was further enhanced by treatment with LEN (45±15%). To evaluate the efficacy of this combination therapy in a more physiological manner, we assessed the efficacy of YB-AHM using total bone marrow mononuclear cells (BMMCs) from MM patients that contained both MM cells and effector cells. BMMCs were stimulated with LEN (3 micro M) for 2 days and further incubated with YB-AHM for 24 hours. Cytotoxicity was evaluated by the number of CD38-positive MM cells in total BMMCs using flow cytometry. YB-AHM plus LEN significantly reduced the number of MM cells (10.3%) compared to YB-AHM alone (21.6%) in patient No.1. Finally, RPMI 8226 cells were co-cultured with YB-AHM and LEN-stimulated PBMCs from MM patients, and MM colony formation was examined using methylcellulose assay. Colony formation of RPMI 8226 was significantly suppressed by YB-AHM and LEN-stimulated PBMCs compared to control (14±8 vs 49±10 colonies, p<0.01), suggesting that this combination therapy can target MM cancer stem cells. Thus, these results indicate that combining defucosylated HM1.24 mAb with immunomodulatory drugs provides a novel therapeutic strategy in patients with MM. Disclosures: No relevant conflicts of interest to declare.

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Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽

10.1182/blood.v118.21.1865.1865 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1865-1865

Author(s):

Inger S. Nijhof ◽

Michel de Weers ◽

Pascale Andre ◽

Berris van Kessel ◽

Henk M. Lokhorst ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Nk Cells ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Nk Cell ◽

Human Monoclonal Antibody ◽

Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

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CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽

10.1182/blood.v120.21.3598.3598 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3598-3598 ◽

Cited By ~ 4

Author(s):

Samantha J. Busfield ◽

Mark Biondo ◽

Mae Wong ◽

Hayley S. Ramshaw ◽

Erwin M Lee ◽

...

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Mouse Model ◽

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Cell Leukemia ◽

Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

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