afm26 3082 of multiple myeloma - affimed.com · significant progress in multiple myeloma (mm)...
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BackgroundUnmet need• High-dose melphalan plus autologous stem cell transplantation (ASCT) is regarded to be standard of
care in first line treatment of MM in eligible patients but fails to eradicate disease as the majority of patients eventually relapse1.
• Immunotherapy holds the potential to eradicate chemoresistant minimal residual disease (MRD).
• NK cells rapidly recover after ASCT providing the first cytotoxic effector cell population to target MRD following transplantation2.
AFM26• BCMA is universally expressed on myeloma cells and constitutes an attractive target for
immunotherapy of MM3.
• High affinity CD16A engagement potently stimulates NK cell cytotoxicity.
• AFM26 is a first-in-class BCMA-directed NK cell engager.
High avidity engagement of NK cells by AFM26• Bivalent binding to CD16A results in high avidity engagement of NK cells
• High affinity NK cell binding is retained in the presence of patient-derived IgG1 paraprotein
• NK cell engagement by AFM26 is independent of CD16A polymorphism4
AFM26 – Targeting BCMA for NK cell-mediated immunotherapy of multiple myeloma
Thorsten Gantke¹, Uwe Reusch1, Christian Kellner2, Katja Klausz2, Thomas Müller1, Kristina Ellwanger1, Ivica Fucek1, Ute Schniegler-Mattox1, Joachim Koch1, Thomas Valerius2, Roland Repp2, Matthias Peipp2, Martin Treder1#
¹Affimed GmbH, Im Neuenheimer Feld 582, 69120 Heidelberg, Germany; 2Christian-Albrechts-University of Kiel, Division of Stem Cell Transplantation and Immunotherapy, Arnold-Heller Straße 3, 24105 Kiel, Germany
#corresponding author, Disclosures: T.G., U.R., T.M., K.E., I.F., U.M., J.K. and M.T. are employees of Affimed GmbH. C.K., K.K., R.R., T.V. and M.P. received research funding from Affimed GmbH; References: 1) Rajkumar, S.V. Mayo Clin Proc 83, 1142-5 (2008). 2) Jacobs, B. et al. Front Immunol 6, 583 (2015). 3) Carpenter, R.O. et al. Clin Cancer Res 19, 2048-60 (2013). 4) Gantke et al., EACR-AACR-SIC 2017 Special Conference, Abstract No. 575. 5) Seckinger, A. et al. Cancer Cell 31, 396-410 (2017).
Abstract
3082
AbstractSignificant progress in multiple myeloma (MM) treatment has been made in recent years and achieving sustainedminimal residual disease (MRD) negativity now constitutes a major goal of treatment. High dose melphalan combinedwith autologous stem cell transplantation (ASCT) is currently regarded the standard of care in 1st line treatment of MMin eligible patients, but fails to eradicate MRD in the majority of patients ultimately leading to disease relapse.Consequently, additional therapies are needed to target residual disease to delay or potentially prevent relapse.The cell surface antigen B cell maturation antigen (BCMA, CD269) constitutes a highly attractive target antigen forimmunotherapy because of its restricted expression in non-malignant tissue but almost universal expression on MMcells.
AFM26 is a BCMA-targeting tetravalent bispecific antibody that selectively engages natural killer (NK) cells to inducemyeloma cell lysis. Through its high-affinity bivalent binding to CD16A (FcγRIIIa), AFM26 possesses prolonged NK cellretention times, and has been shown to be less prone to interference by high levels of circulating IgG than antibody Fc-domains. Thus, AFM26 can be used to “arm” NK cells for effective killing of tumor cells. AFM26 also induces markedlylower levels of pro-inflammatory cytokines in peripheral blood mononuclear cell (PBMC) cultures in the presence oftarget cells compared to BCMA-directed T cell activating approaches. In contrast to other monoclonal antibodies(mAbs) developed in MM such as daratumumab and elotuzumab, AFM26 does not confer target-independent NK cellactivation or NK cell depletion.
NK cells are promising effectors to target MRD, due to their early reconstitution following ASCT and their ability torapidly destroy malignant cells through direct cytotoxicity. Redirecting NK cells through AFM26 may therefore offer aneffective treatment of MM patients early after ASCT.
Employing a wide range of functional assays, we demonstrate here that AFM26 exhibits potent cytotoxic activityagainst primary myeloma cells using patient (autologous) and donor-derived (allogeneic) NK cells as well as against apanel of high and low BCMA expressing cell lines. In particular, we show that AFM26 induces NK cell cytotoxicitytowards cells expressing almost undetectable surface levels of BCMA, suggesting the potential for broad and deepanti-MM activity. In summary, AFM26 is well-differentiated from other mAbs and a promising therapeutic candidate toaddress the unmet medical need of MRD positivity in MM.
Key results – AFM26:• Induces autologous NK cell-mediated lysis of primary myeloma cells
• Potently kills cells expressing very low levels of BCMA
• Binds with high affinity to NK cells in the presence of patient-derived paraprotein
• Does not induce NK cell depletion compared with daratumumab or elotuzumab
• Effector cell activation is strictly dependent on target cell contact
• Induces only low inflammatory cytokine release in the presence of targets in vitro
Conclusions and Outlook
• AFM26 is a first-in-class BCMA-targeting NK cell engager which effectively induces killing of MM cells.
• Post-HDT/ASCT may provide optimal conditions for NK cell-based immunotherapy.
• AFM26’s anticipated safety profile suggest suitability for early BCMA-directed treatment to target MRD in conjunction with HDT/ASCT.
In vitro cytotoxicity towards primary myeloma cells
Retained activity towards BCMAlow target cells• Low BCMA expression on myeloma cells has
been reported5
• AFM26 induces NK cell-mediated target cell lysis despite very low levels of surface BCMA
AFM26
BCMA
CD16A
1) Antigen binding 2) Immunological synapse
3) Release of Perforins and Granzyme
4) Tumor Cell Lysis
Mechanism of action of AFM26
NK cell binding of AFM26 and comparators in patient MM serum (IgG1 paraprotein)
NK cell surface retention of AFM26
AFM26 does not induce NK cell depletion• AFM26 does not induce depletion of NK cells
in vitro, unlike anti-CD38 (daratumumab) and anti-CS1 (elotuzumab)
• AFM26-mediated NK cell activation is strictly dependent on target cell contact
Antibody-induced lysis of NK cells in vitro
Antibody-induced lysis of NK cells was assessed in vitro using primary human NK cells(calcein-release assay, 4h incubation). Anti-CD38 IgG1 (daratumumab) and anti-CS1(elotuzumab) IgG1 were produced by Affimed.
In vitro cytotoxicity assay
NK cell activation by AFM26 +/- target cells
AFM26 may offer superior safety compared with T cell engagement
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BiTE [ng/mL] Anti-BCMA IgG1 [ng/mL] AFM26 [ng/mL]
IL-4 IL-2 IL-10 IL-6 TNF-a IFN-γ
Cytokine release by human PBMCs induced by increasing concentrations of BCMA-targeting BiTE, anti-BCMA IgG1 andAFM26 in presence of NCI-H929 target cells was quantified in supernatant following 24h incubation (E:T ratio 50:1).
BiTE Anti-BCMA IgG1 AFM26
In vitro cytokine release induced by AFM26 and comparators In vitro target cell lysis induced by AFM26, anti-BCMA BiTE and anti-BCMA IgG1
Induction of in vitro lysis of NCI-H929 target cells by human PBMC (E:T ratio 50:1) by AFM26 was compared with lysis induced by BCMA-targeting BiTE (left panel; 24h incubation) and anti-BCMA IgG1 (right panel, 4h incubation).
AFM26-induced lysis of primary myeloma cells by allogeneic and autologous NK cells
In vitro cytotoxicity assay In vitro cytotoxicity assay
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Potency of AFM26-induced target cell lysis in vitro
Summary of EC50 values (triangles, left axis) of AFM26-induced target cell lysis in in vitro cytotoxicity assays using primary human NK cells and the indicated target cells (4h calcein-release assay, E:T ratio 5:1). Data are mean ± SD. Right axis: Specific antibody binding capacity (SABC) of anti-BCMA ANC3B1
AFM26, non-fucosylated anti-BCMA IgG1 and anti-BCMA IgG1 were titrated on primary human NK cells at 37°C in presence (solid lines) or absence (dashed lines) of plasma containing IgG1 paraproteinobtained from a patient with progressed myeloma (final concentration: 28.8mg/ml IgG1). Surface bound antibody was quantified by detection with BCMA-His/anti-His-FITC and flow cytometry.
Non-fucosylated IgG1
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AFM26
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in p re s e n c e a n d a b s e n c e o f 1 0 m g /m L
p o ly c lo n a l Ig G (3 7 °C )
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Ig G 1 (S 2 3 9 D /I3 3 2 E )
Ig G 1 ( W T )
A F M 26
A B
A) AFM26, Fc-enhanced anti-BCMA IgG1 (S239D/I332E) and anti-BCMA IgG1 were added to primary human NK cells at 100µg/ml, 400µg/ml and 400µg/ml, respectively, before removal of unbound antibodyby washing and incubation in 10ml PBS (2% FCS, 0.1% NaN3) at 37°C. Remaining surface bound antibody was quantified by detection with BCMA-His/anti-His-FITC and flow cytometry. B) AFM26 NK cellsurface retention in presence and absence of 10mg/ml polyclonal IgG (Gammanorm, Octapharma). Remaining surface bound antibody was detected as in A).
KD: 1.5nM
KD: 17.3nM
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M26
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A) In vitro cytotoxicity of healthy donor NK cellstowards primary myeloma cells from apreviously untreated patient (left) and apatient with relapsed myeloma (right) with orwithout increasing concentrations of AFM26,daratumumab or controls EGFRxCD16Abispecific antibody (left) and trastuzumab(right) was assessed in Cr51 release assays (4h,E:T ratio 10:1). B) Autologous lysis of primarymyeloma cells (RRMM) by NK cells in presenceor absence of 0.4nM of the indicatedantibodies.
RRMMBA
NK cell activation in increasing concentrations of AFM26 or without antibody wasmonitored by quantifying the percentage of CD69-positive NK cells following 4hcytotoxicity assay in presence or absence of NCI-H929 cells as targets (E:T ratio 5:1).
In vitro cytotoxicity assay
Cell line Anti-BCMA SABC (ANC3B1) Mean EC50 [pM]
NCI-H929 51,479 21.8
RPMI-8226 14,707 55.1
MM.1S 13,182 12.1
HuNS1 1,438 59.3
ARH77 1,183 79.6
MC/CAR 449 96.5
A n tib o d y c o n c e n tra t io n [p M ]
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a n ti-C S 1 Ig G 1
a n ti-C D 3 8 Ig G 1
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