Effects of IL-21, KIR Blockade, and CD137 Agonism on the...
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Introduction N N N Elotuzumab is a humanized IgG1 monoclonal antibody (mAb) targeted against Signaling Lymphocytic Activation Molecule (SLAMF7, also known as CS1), a glycoprotein highly expressed on myeloma and natural killer (NK) cells, but not on normal tissues. 1 —— — Through direct activation and engagement of NK cells, elotuzumab selectively targets and kills SLAMF7 expressing myeloma cells with minimal effects on normal tissue. 2 N N Elotuzumab has demonstrated clinical activity in relapsed/refractory MM in combination with lenalidomide and dexamethasone. 3 Additionally, elotuzumab combined with bortezomib has shown additive effects in a pre-clinical model. 4 N N NK cell activity can be enhanced by stimulating activating NK receptors and by antagonizing inhibitory NK receptors. N N Further opportunities to enhance NK-mediated elotuzumab activity include combination with: —— — IL-21: a pleiotropic cytokine that augments the ability of NK cells to mediate antibody-dependent cellular cytotoxicity (ADCC). 5 —— — CD137 agonism: in combination with tumor-specific antibodies, agonist antibodies to CD137 enhance NK cell ADCC in vitro and inhibit tumor growth in a mouse tumor model. 6 —— — KIR antagonism: ligand engagement of inhibitory killer immunoglobulin-like receptors (KIRs) reduces NK cell activity. Lirilumab is a mAb that binds and blocks ligand interactions of inhibitory KIR2DL1/2/3 and thereby enhances ADCC. 7 Methods N N Antibodies and recombinant proteins: —— — Humanized anti-human SLAMF7 mAb, IgG1 (elotuzumab). —— — Human anti-human CD137 mAb, IgG4 (urelumab). —— — Rat anti-mouse CD137, IgG2a (clone 1D8). —— — Human anti-KIR mAb, IgG4, recognizes KIR2DL1/L2/L3/S1/S2 (lirilumab). —— — Recombinant human and mouse IL-21. N N Cells and cell lines: —— — OPM-2, a human myeloma cell line that expresses high levels of surface SLAMF7, was cultured in standard conditions. —— — Peripheral blood mononuclear cells (PBMC) and bone-marrow mononuclear cells (BM-MNCs) were isolated from healthy donors. —— — Human peripheral blood lymphocytes (hPBLs) were the non-adherent cells isolated from PBMC. N N Mice —— — IL-21 studies: SCID.CB1 Hr CRL mice (Charles River). —— — Anti-CD137 studies: female severe combined immunodeficient (SCID) mice (Harlan), maintained under pathogen-free conditions at BMS Animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. —— — Anti-KIR studies: NOD.CB17-Prkdc scid /NcrCrl and Rag1KO Tg KIR2DL3 on a C57BL/6 background (Innate Pharma). Experiments using these mice were approved by the Innate Pharma ethics committee. —— — Experiments were performed according to the European directive for animal experimentation. References 1. Hsi ED et al. Clin Cancer Res. 2008;14:2775–84. 2. Collins SM et al. Cancer Immunol Immunother. 2013;62:1841–9. 3. Lonial S et al. J Clin Oncol. 2012;30:1953–9. 4. van Rhee F et al. Mol Cancer Ther. 2009;8:2616–24. 5. Roda JM et al. J Immunol. 2006;177:120–9. 6. Houot R et al. Oncoimmunology. 2012;1:957–8. 7. Binyamin L et al. J Immunol. 2008;180:6392–401. Acknowledgments This study was funded by Bristol-Myers Squibb. Professional medical writing support and editorial assistance was provided by Kate Rees, PhD, and Nicole Draghi, PhD, of Caudex Medical, funded by Bristol-Myers Squibb. Disclosures M Robbins, M Jure-Kunkel, and R Graziano are employees and share owners of Bristol-Myers Squibb. G Dito, H Zhang, and N Bezman are employees of Bristol-Myers Squibb. P Andre is an employee of Innate Pharma. Conclusions N N IL-21 increased elotuzumab-mediated NK cell activation and myeloma cell-killing in vitro, but showed no enhancement of elotuzumab activity in a xenograft tumor model. N N CD137 agonism minimally enhanced elotuzumab-mediated myeloma cell-killing in vitro, but synergized with elotuzumab to mediate potent anti-tumor activity in a xenograft tumor model. N N Blocking the inhibitory KIR pathway with lirilumab augmented elotuzumab mediated cell-killing in vitro and synergized with elotuzumab to mediate potent anti-tumor activity in the KIR2DL3 transgenic and RAG deficient mouse model. N N These studies provide pre-clinical rationale for targeting NK cell receptors to augment elotuzumab activity in MM. N N A Phase I study of elotuzumab in combination with lirilumab or urelumab, in patients with MM (NCT2252263), is currently in development. N N IL-21 does not enhance elotuzumab activity in vivo. —— — In mice with established OPM-2 xenograft tumors, elotuzumab- induced ADCC, measured via tumor volume, did not increase with the addition of IL-21 (Figure 3a, b). Urelumab N N CD137 expression on NK cells is upregulated in the presence of elotuzumab (Figure 4). N N NK cells, co-cultured with OPM-2 cells and treated with elotuzumab showed an increase in NK cell degranulation, as measured by CD107a expression. When the anti-CD137 mAb, urelumab, was added, cells showed a minimal further increase in degranulation (Figure 5). N N In mice with established OPM-2 xenograft tumors, treatment with anti-mCD137 plus elotuzumab led to complete regressions in 6/8 mice compared with those treated with only buffer, anti-mCD137 or elotuzumab alone (Figure 6). N N Myeloma tumor model —— — Xenograft tumors were established via subcutaneous injection of OPM-2 tumor cells mixed 1:1 with Matrigel™ (BD Biosciences) into the hind flank of mice. —— — At 8 days post transplantation, mice were randomized by tumor volume. Elotuzumab, IL-21, and/or anti-mCD137 were administered by intra-peritoneal injection; anti-KIR mAb was administered intravenously. —— — Tumor growth was measured by Fowler Electronic Digital Caliper biweekly. Mice were euthanized when the tumors reached 2000 mm 3 . N N CD25 and CD54 expression —— — hPBLs were co-cultured with OPM-2 cells and treated with hIL-21 (0.56 nM) plus increasing concentrations of elotuzumab. Expression on CD56 + CD3 – NK cells was determined by flow cytometry. N N NK cell ADCC —— — PBMC were cultured with or without IL-21 (10 ng/mL) and plated with the respective antibodies. —— — OPM-2 killing was measured by a europium (Eu) release assay. BATDA-labelled OPM-2 cells were incubated with PBMCs for 2 hours. TDA released from OPM-2 cells formed a highly fluorescent chelate (EuTDA), which correlated with lysed cells. N N CD137 expression —— — Purified NK cells from PBMC were cultured with elotuzumab (10 μg/mL) or hIgG1 (10 μg/mL) for 24 hours and stained with anti-CD56 and anti-CD137 antibodies. N N NK cell degranulation —— — PBMCs were pre-treated with elotuzumab and/or anti-CD137 mAb and cultured with OPM-2 targets. —— — BM-MNCs were pre-treated with lirilumab (30 μg/mL) and then cultured with OPM-2 targets and increasing doses of elotuzumab. —— — NK cells were stained with anti-CD107a to measure activation. Results IL-21 N N IL-21 augments elotuzumab-mediated NK cell activation in vitro. —— — In a co-culture of hPBLs with OPM-2 cells, incubation with IL-21 and elotuzumab resulted in an increase in CD56 + CD3 – NK cells positive for CD25 or CD54. This effect was enhanced after 24 hours in the combined presence of elotuzumab and IL-21 (Figure 2a, b). —— — In a fluorescent cytotoxicity assay, elotuzumab-induced ADCC was increased by IL-21, when compared with cells incubated with human IgG1 or elotuzumab alone (Figure 2c). Lirilumab N N Lirilumab enhances elotuzumab-mediated NK cell degranulation in vitro. —— — In BM-MNCs co-cultured with OPM-2 targets, NK degranulation, as measured by CD107a expression, was augmented in cells treated with the elotuzumab plus the anti-KIR mAb lirilumab (Figure 7). N N Lirilumab enhances elotuzumab activity in vivo. —— — In mice with established OPM-2 xenograft tumors, animals treated with elotuzumab plus lirilumab showed a decrease in tumor volume compared with control mice and those treated with elotuzumab alone (Figure 8). Effects of IL-21, KIR Blockade, and CD137 Agonism on the Non-Clinical Activity of Elotuzumab 4717 Michael Robbins 1 , Maria Jure-Kunkel 1 , Gennaro Dito 1 , Pascale Andre 2 , Hui-fen Zhang 1 , Natalie Bezman 1 , Robert F. Graziano 1 1 Bristol-Myers Squibb, Princeton, NJ, USA; 2 Innate Pharma, Marseille, France 56th ASH Annual Meeting and Exposition; San Francisco, CA; December 6–9, 2014 12 10 8 6 4 2 0 % CD137 positive on NK A PBMC only PBMC+hlgG1 PBMC+Elo Donor 1 400 350 300 200 250 150 100 50 0 CD137 MFI on NK PBMC only PBMC+hlgG1 PBMC+Elo 300 250 200 150 100 50 0 CD137 MFI on NK PBMC only PBMC+hlgG1 PBMC+Elo 6 5 4 3 2 1 0 % CD137 positive on NK B PBMC only PBMC+hlgG1 PBMC+Elo Donor 2 Purified NK cells isolated from PBMC of healthy donors were cultured with elotuzumab (10 μg/mL) or hlgG1 isotype (10 μg/mL) for 24 hours. Cells were stained with anti-CD56 and anti-CD137 antibodies. Elo, elotuzumab; NK, natural killer; PBMC, peripheral blood mononuclear cells. Figure 4. CD137 expression on PBMC cultured with elotuzumab NK cells purified from peripheral blood of healthy donors were cultured in medium only, with OPM-2 1:1, OPM-2 + elotuzumab (10 μg/mL), OPM-2 + aCD137 (10 μg/mL) or OPM-2 with both elotuzumab and a CD137. The NK cells were then stained with anti-CD107a antibody to detect NK cell degranulation. CD107a + NK cells CD107a + NK cells NK cells OPM-2 Elo aCD137 + + + + + + – + + + + – + + – – + – – – + + + + + + – + + + + – + + – – + – – – NK cells OPM-2 Elo aCD137 Donor 1 NK:OPM-2 = 1:1 Donor 2 NK:OPM-2 = 1:1 20 A 18 16 14 12 10 8 6 4 2 0 20 B 18 16 14 12 10 8 6 4 2 0 Figure 5. In vitro effects of urelumab, a CD137 receptor agonist, on elotuzumab-mediated NK cell degranulation 3000 Buffer control, d8 0 500 1000 1500 2000 2500 250 750 1250 1750 2250 2750 8 10 13 15 18 20 23 25 28 30 33 35 38 40 43 Days post implant Tumor volume (mm 3 ) A Severe combined immunodeficient female mice with established OPM-2 xenograft tumors (n=8, per treatment group) were administered intra-peritoneal injections on Day 8 of either buffer alone, anti-mCD137 (5 mg/kg), elotuzumab, or anti-mCD137 plus elotuzumab (0.5 mg/kg). Data represent tumor volume measured in individual animals that had tumor growth. 3000 Anti-mCD137, 100 μg, d8 0 500 1000 1500 2000 2500 250 750 1250 1750 2250 2750 8 10 13 15 18 20 23 25 28 30 33 35 38 40 43 Tumor volume (mm 3 ) B 3000 Elotuzumab, 10 μg, d8 0 500 1000 1500 2000 2500 250 750 1250 1750 2250 2750 5 10 15 20 25 30 35 40 45 50 60 55 65 75 70 80 85 Days post implant Tumor volume (mm 3 ) C 3000 Anti-mCD137, 100 μg, d8 + elotuzumab, 10 μg, d8 500 1000 1500 2000 2500 250 750 1250 1750 2250 2750 5 10 15 20 25 30 35 40 45 50 60 55 65 75 70 80 85 Days post implant Days post implant Tumor volume (mm 3 ) D Figure 6. In vivo effects of CD137 activation by anti-mCD137 on elotuzumab activity in a xenograft tumor model 0 10 20 30 40 % CD107a + NK cells No Ab IgG1 IgG1 + IgG4 IgG1 + KIR aKIR aKIR aKIR aKIR – – – – 0.001 0.01 0.1 1 A No Ab IgG1 IgG1 + IgG4 IgG1 + KIR aKIR aKIR aKIR aKIR – – – – 0.001 OPM-2 0.01 0.1 1 Elo μg/mL 0 20 40 60 80 B % CD107a + NK cells No Ab BM-MNCs (n=3 donors) were pre-treated with lirilumab (30 μg/mL). After 24 hours, BM-MNC were grown with or without OPM-2 targets and elotuzumab. Figure shows results from one donor; data are representative of all three donors. After 5 hours, the percentage of CD56 + CD3 – NK cells staining positive for CD107a was measured. Ab, antibody; Elo, elotuzumab; KIR, killer immunoglobulin-like receptor; NK, natural killer. No Ab Elo+KIR Elo IgG1+KIR IgG1+IgG4 IgG1 No Ab Figure 7. In vitro effects of KIR blockade (lirilumab) on elotuzumab-mediated NK cell degranulation hPBLs were co-cultured with OPM-2 cells and treated with hIL-21 plus elotuzumab. Cells were then stained for CD56 + CD3 – NK cells positive for (A) CD25 or (B) CD54. (C) PBMC were cultured overnight with or without IL-21 (10 ng/mL) and then plated with hlgG1 or elotuzumab. BADTA-labelled OPM-2 cells were then added to the plates and the amount of cell lysis was measured after 2 hours. hPBL, human peripheral blood lymphocyte; mAb, monoclonal antibody; NK, natural killer; PBMC, peripheral blood mononuclear cells. 40 30 20 10 0 40 30 20 10 0 % CD25 + NK cells IL-21 OPM-2 IL-21 OPM-2 Elotuzumab (μg/mL) Elotuzumab (μg/mL) A B % CD54 + NK cells 75 50 25 0 C mAb (μg/mL) % killing elotuzumab hlgG1 elotuzumab+IL-21 hlgG1+IL-21 0.1 1 10 - - IgG1 IgG1 0.2 0.2 2 2 20 20 - - IgG1 IgG1 0.2 0.2 2 2 20 20 Figure 2. In vitro effects of IL-21 on elotuzumab activity –10 0 10 20 30 Days post treatment 0 200 400 600 800 1000 Tumor volume (mm 3 ) Mean G1 hIgG1 G2 elotuzumab G3 mIL-21 G4 mIL-21+ hIgG1 G5 mIL-21+ elotuzumab Mice with established OPM-2 xenograft tumours were treated with hIgG1 (0.5 mg/kg; Days 21, 24, 28, 31), elotuzumab (0.5 mg/kg; Days 21, 24, 28, 31) and/or mIL-21 (50 μg/mouse; Days 21, 23, 25, 28, 30, 32). A –10 0 10 20 30 Days post treatment 0 1000 2000 3000 Tumor volume (mm 3 ) Median B Figure 3. In vivo effects of IL-21 on elotuzumab activity in xenograft tumor model C D B 4000 Days (post cell injection) hulgG1 + diluant (control) hulgG1 + lirilumab End of treatment End of experiment D54 End of treatment End of experiment D54 End of treatment End of experiment D54 End of treatment End of experiment D54 0 0 10 20 30 40 50 60 1000 2000 3000 Days (post cell injection) Tumor volume (mm 3 ) A 0 10 Days (post cell injection) 20 30 40 50 60 Days (post cell injection) Tumor volume (mm 3 ) 4000 Days (post cell injection) 0 0 10 20 30 40 50 60 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 Days (post cell injection) Tumor volume (mm 3 ) Days (post cell injection) Tumor volume (mm 3 ) 0 10 20 30 40 50 60 Days (post cell injection) Elotuzumab + diluant Combo Mice with established OPM-2 xenograft tumors (n=10, per treatment group) were treated with (A) control IgG (0.5 mg/kg), (B) elotuzumab (0.5 mg/kg, biweekly from Day 11), (C) IgG1 plus liriumab (IPH2102, 15 mg/kg, Days 11 and 24), or (D) elotuzumab plus liriumab. Data represent tumor volume for individual animals that had tumor growth. KIR, killer immunoglobulin-like receptor. Figure 8. In vivo effects of KIR blockade (liriumab) on elotuzumab activity in xenograft tumor model Elotuzumab other KIR2DL 1/2/3 other CD137 IL21R CD16 Myeloma cell HLAC other Antagonist to inhibitory signals: – Lirilumab (anti-KIR) SLAMF7 Agonist to activating signals: – Elotuzumab (anti-SLAMF7) • CD16 – BMS-982470 (IL21) – Urelumab (anti-CD137) Activating receptors NK cell NK, natural killer. Inhibitory receptors SLAMF7 Figure 1. Potential approaches for enhancing elotuzumab activity
Transcript of Effects of IL-21, KIR Blockade, and CD137 Agonism on the...
IntroductionNN NNElotuzumab is a humanized IgG1 monoclonal antibody (mAb) targeted
against Signaling Lymphocytic Activation Molecule (SLAMF7, also known as CS1), a glycoprotein highly expressed on myeloma and natural killer (NK) cells, but not on normal tissues.1
——— Through direct activation and engagement of NK cells, elotuzumab selectively targets and kills SLAMF7 expressing myeloma cells with minimal effects on normal tissue.2
NN Elotuzumab has demonstrated clinical activity in relapsed/refractory MM in combination with lenalidomide and dexamethasone.3 Additionally, elotuzumab combined with bortezomib has shown additive effects in a pre-clinical model.4
NN NK cell activity can be enhanced by stimulating activating NK receptors and by antagonizing inhibitory NK receptors.
NN Further opportunities to enhance NK-mediated elotuzumab activity include combination with:
——— IL-21: a pleiotropic cytokine that augments the ability of NK cells to mediate antibody-dependent cellular cytotoxicity (ADCC).5
——— CD137 agonism: in combination with tumor-specific antibodies, agonist antibodies to CD137 enhance NK cell ADCC in vitro and inhibit tumor growth in a mouse tumor model.6
——— KIR antagonism: ligand engagement of inhibitory killer immunoglobulin-like receptors (KIRs) reduces NK cell activity. Lirilumab is a mAb that binds and blocks ligand interactions of inhibitory KIR2DL1/2/3 and thereby enhances ADCC.7
MethodsNN Antibodies and recombinant proteins:
——— Humanized anti-human SLAMF7 mAb, IgG1 (elotuzumab).——— Human anti-human CD137 mAb, IgG4 (urelumab).——— Rat anti-mouse CD137, IgG2a (clone 1D8). ——— Human anti-KIR mAb, IgG4, recognizes KIR2DL1/L2/L3/S1/S2 (lirilumab).——— Recombinant human and mouse IL-21.
NN Cells and cell lines:——— OPM-2, a human myeloma cell line that expresses high levels of
surface SLAMF7, was cultured in standard conditions.——— Peripheral blood mononuclear cells (PBMC) and bone-marrow
mononuclear cells (BM-MNCs) were isolated from healthy donors.——— Human peripheral blood lymphocytes (hPBLs) were the
non-adherent cells isolated from PBMC.
NN Mice——— IL-21 studies: SCID.CB1 Hr CRL mice (Charles River).——— Anti-CD137 studies: female severe combined immunodeficient
(SCID) mice (Harlan), maintained under pathogen-free conditions at BMS Animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.
——— Anti-KIR studies: NOD.CB17-Prkdcscid/NcrCrl and Rag1KO Tg KIR2DL3 on a C57BL/6 background (Innate Pharma). Experiments using these mice were approved by the Innate Pharma ethics committee.
——— Experiments were performed according to the European directive for animal experimentation.
References1. Hsi ED et al. Clin Cancer Res. 2008;14:2775–84.2. Collins SM et al. Cancer Immunol Immunother. 2013;62:1841–9.3. Lonial S et al. J Clin Oncol. 2012;30:1953–9.4. van Rhee F et al. Mol Cancer Ther. 2009;8:2616–24.5. Roda JM et al. J Immunol. 2006;177:120–9.6. Houot R et al. Oncoimmunology. 2012;1:957–8.7. Binyamin L et al. J Immunol. 2008;180:6392–401.
AcknowledgmentsThis study was funded by Bristol-Myers Squibb. Professional medical writing support and editorial assistance was provided by Kate Rees, PhD, and Nicole Draghi, PhD, of Caudex Medical, funded by Bristol-Myers Squibb.
DisclosuresM Robbins, M Jure-Kunkel, and R Graziano are employees and share owners of Bristol-Myers Squibb. G Dito, H Zhang, and N Bezman are employees of Bristol-Myers Squibb. P Andre is an employee of Innate Pharma.
ConclusionsNN IL-21 increased elotuzumab-mediated NK cell activation and
myeloma cell-killing in vitro, but showed no enhancement of elotuzumab activity in a xenograft tumor model.
NN CD137 agonism minimally enhanced elotuzumab-mediated myeloma cell-killing in vitro, but synergized with elotuzumab to mediate potent anti-tumor activity in a xenograft tumor model.
NN Blocking the inhibitory KIR pathway with lirilumab augmented elotuzumab mediated cell-killing in vitro and synergized with elotuzumab to mediate potent anti-tumor activity in the KIR2DL3 transgenic and RAG deficient mouse model.
NN These studies provide pre-clinical rationale for targeting NK cell receptors to augment elotuzumab activity in MM.
NN A Phase I study of elotuzumab in combination with lirilumab or urelumab, in patients with MM (NCT2252263), is currently in development.
NN IL-21 does not enhance elotuzumab activity in vivo.
——— In mice with established OPM-2 xenograft tumors, elotuzumab-
induced ADCC, measured via tumor volume, did not increase
with the addition of IL-21 (Figure 3a, b).
Urelumab
NN CD137 expression on NK cells is upregulated in the presence of elotuzumab (Figure 4).
NN NK cells, co-cultured with OPM-2 cells and treated with elotuzumab showed an increase in NK cell degranulation, as measured by CD107a expression. When the anti-CD137 mAb, urelumab, was added, cells showed a minimal further increase in degranulation (Figure 5).
NN In mice with established OPM-2 xenograft tumors, treatment with anti-mCD137 plus elotuzumab led to complete regressions in 6/8 mice compared with those treated with only buffer, anti-mCD137 or elotuzumab alone (Figure 6).
NN Myeloma tumor model
——— Xenograft tumors were established via subcutaneous injection of OPM-2 tumor cells mixed 1:1 with Matrigel™ (BD Biosciences) into the hind flank of mice.
——— At 8 days post transplantation, mice were randomized by tumor volume. Elotuzumab, IL-21, and/or anti-mCD137 were administered by intra-peritoneal injection; anti-KIR mAb was administered intravenously.
——— Tumor growth was measured by Fowler Electronic Digital Caliper biweekly. Mice were euthanized when the tumors reached 2000 mm3.
NN CD25 and CD54 expression
——— hPBLs were co-cultured with OPM-2 cells and treated with hIL-21 (0.56 nM) plus increasing concentrations of elotuzumab. Expression on CD56+CD3– NK cells was determined by flow cytometry.
NN NK cell ADCC
——— PBMC were cultured with or without IL-21 (10 ng/mL) and plated with the respective antibodies.
——— OPM-2 killing was measured by a europium (Eu) release assay. BATDA-labelled OPM-2 cells were incubated with PBMCs for 2 hours. TDA released from OPM-2 cells formed a highly fluorescent chelate (EuTDA), which correlated with lysed cells.
NN CD137 expression
——— Purified NK cells from PBMC were cultured with elotuzumab (10 μg/mL) or hIgG1 (10 μg/mL) for 24 hours and stained with anti-CD56 and anti-CD137 antibodies.
NN NK cell degranulation
——— PBMCs were pre-treated with elotuzumab and/or anti-CD137 mAb and cultured with OPM-2 targets.
——— BM-MNCs were pre-treated with lirilumab (30 μg/mL) and then cultured with OPM-2 targets and increasing doses of elotuzumab.
——— NK cells were stained with anti-CD107a to measure activation.
Results
IL-21
NN IL-21 augments elotuzumab-mediated NK cell activation in vitro.
——— In a co-culture of hPBLs with OPM-2 cells, incubation with IL-21 and elotuzumab resulted in an increase in CD56+CD3– NK cells positive for CD25 or CD54. This effect was enhanced after 24 hours in the combined presence of elotuzumab and IL-21 (Figure 2a, b).
——— In a fluorescent cytotoxicity assay, elotuzumab-induced ADCC was increased by IL-21, when compared with cells incubated with human IgG1 or elotuzumab alone (Figure 2c).
Lirilumab
NN Lirilumab enhances elotuzumab-mediated NK cell degranulation in vitro.
——— In BM-MNCs co-cultured with OPM-2 targets, NK degranulation, as measured by CD107a expression, was augmented in cells treated with the elotuzumab plus the anti-KIR mAb lirilumab (Figure 7).
NN Lirilumab enhances elotuzumab activity in vivo.
——— In mice with established OPM-2 xenograft tumors, animals treated with elotuzumab plus lirilumab showed a decrease in tumor volume compared with control mice and those treated with elotuzumab alone (Figure 8).
Effects of IL-21, KIR Blockade, and CD137 Agonism on the Non-Clinical Activity of Elotuzumab
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Michael Robbins1, Maria Jure-Kunkel1, Gennaro Dito1, Pascale Andre2, Hui-fen Zhang1, Natalie Bezman1, Robert F. Graziano1
1Bristol-Myers Squibb, Princeton, NJ, USA; 2Innate Pharma, Marseille, France
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Figure 4. CD137 expression on PBMC cultured with elotuzumab
NK cells purified from peripheral blood of healthy donors were cultured in medium only, with OPM-2 1:1, OPM-2 + elotuzumab (10 µg/mL), OPM-2 + aCD137 (10�µg/mL) or OPM-2 with both elotuzumab and a CD137. The NK cells were then stained with anti-CD107a antibody to detect NK cell degranulation.
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BM-MNCs (n=3 donors) were pre-treated with lirilumab (30 µg/mL). After 24 hours, BM-MNC were grown with or without OPM-2 targets and elotuzumab. Figure shows results from one donor; data are representative of all three donors. After 5 hours, the percentage of CD56+CD3– NK cells staining positive for CD107a was measured.Ab, antibody; Elo, elotuzumab; KIR, killer immunoglobulin-like receptor; NK, natural killer.
No Ab Elo+KIREloIgG1+KIRIgG1+IgG4IgG1No Ab
Figure 7. In vitro effects of KIR blockade (lirilumab) on elotuzumab-mediated NK cell degranulation
hPBLs were co-cultured with OPM-2 cells and treated with hIL-21 plus elotuzumab. Cells were then stained for CD56+CD3– NK cells positive for (A) CD25 or (B) CD54. (C) PBMC were cultured overnight with or without IL-21 (10 ng/mL) and then plated with hlgG1 or elotuzumab. BADTA-labelled OPM-2 cells were then added to the plates and the amount of cell lysis was measured after 2 hours.hPBL, human peripheral blood lymphocyte; mAb, monoclonal antibody; NK, natural killer; PBMC, peripheral blood mononuclear cells.
40
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% C
D25
+ N
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ells
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ells
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mAb (µg/mL)
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illin
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elotuzumabhlgG1
elotuzumab+IL-21hlgG1+IL-21
0.1 1 10
- -IgG
1IgG
10.2 0.22 220 20
- -IgG
1IgG
10.2 0.22 220 20
Figure 2. In vitro effects of IL-21 on elotuzumab activity
–10 0 10 20 30
Days post treatment
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m3 )
Mean G1 hIgG1G2 elotuzumabG3 mIL-21G4 mIL-21+ hIgG1G5 mIL-21+ elotuzumab
Mice with established OPM-2 xenograft tumours were treated with hIgG1 (0.5 mg/kg; Days 21, 24, 28, 31), elotuzumab (0.5 mg/kg; Days 21, 24, 28, 31) and/or mIL-21 (50 µg/mouse; Days 21, 23, 25, 28, 30, 32).
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Figure 3. In vivo effects of IL-21 on elotuzumab activity in xenograft tumor model
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4000
Days (post cell injection)
hulgG1 + diluant (control)
hulgG1 + lirilumab
End of treatment
End of experiment
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End of treatment
End of experiment
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End of treatment
End of experiment
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End of treatment
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olum
e (m
m3 )
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Days (post cell injection)
Elotuzumab + diluant
Combo
Mice with established OPM-2 xenograft tumors (n=10, per treatment group) were treated with (A) control IgG (0.5 mg/kg), (B) elotuzumab (0.5 mg/kg, biweekly from Day 11), (C) IgG1 plus liriumab (IPH2102, 15 mg/kg, Days 11 and 24), or (D) elotuzumab plus liriumab. Data represent tumor volume for individual animals that had tumor growth.KIR, killer immunoglobulin-like receptor.
Figure 8. In vivo effects of KIR blockade (liriumab) on elotuzumab activity in xenograft tumor model
Elotuzumab
othe
r
KIR2D
L 1/
2/3
othe
r
CD137
IL21
RCD16
Myeloma cell
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AC
othe
r
Antagonist to inhibitory signals:– Lirilumab (anti-KIR)
SL
AM
F7 Agonist to activating signals:
– Elotuzumab (anti-SLAMF7) • CD16– BMS-982470 (IL21)– Urelumab (anti-CD137)
Activating receptors
NK cellNK, natural killer.
Inhibitory receptors
SL
AM
F7
Figure 1. Potential approaches for enhancing elotuzumab activity
