the cd30/cd16a bispecific innate immune cell engager afm13
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The CD30/CD16A bispecific innate immune cell engager AFM13 elicits heterogeneous single-
cell NK cell responses and effectively triggers memory like (ML) NK cellsNancy Marin1, Michelle Becker-Hapak1, Joachim Koch2, Melissa M. Berrien-Elliott1, Mark Foster1, Carly Neal1, Ethan McClain1, Sweta Desai1, Julia A. Wagner1, Timothy Schappe1, Lynne
Marsala1, Pamela Wong1, Martin Treder2, Todd A. Fehniger1
1Washington University School of Medicine, Saint Louis, MO, 2Affimed GmbH, Heidelberg, Germany
Natural killer (NK) cells are crucial effector cells of the innate immune system capable of rapidly
recognizing and eliminating infected, stressed, and malignant cells. NK cells discriminate tumor targets
from healthy cells by integrating activating and inhibitory receptor signals. One barrier to broadly applying
NK cell immunotherapy across many cancer types is inconsistent cancer cell recognition, which may be
overcome by innate cell engagers. AFM13 is a tetravalent, bispecific antibody developed on the ROCK®
platform. AFM13 is characterized by bivalent binding to CD30 and CD16A and has demonstrated a
favorable safety profile and promising efficacy against CD30+ malignancies in clinical studies as
monotherapy and in combination with anti-PD-1. However, our understanding of NK cell functional
responses triggered via AFM13 remain incomplete. Moreover, because adoptively transferred memory-like
(ML) NK cells have demonstrated enhanced anti-tumor activity (Romee R et al., Sci Transl Med, 2016), we
expect that they may be receptive to AFM13-based targeting to enhance target cell recognition. To address
these questions, we analyzed single-cell conventional (cNK) and ML (IL-12/15/18-induced) NK cell
functional responses to NK-resistant CD30+ lymphoma cells +/- AFM13. Primary cNK cells co-incubated
with AFM13-treated Hut-78 cells demonstrated increased IFN-γ, TNF, and degranulation, compared to Hut-
78 cells or Raji (CD30-) targets + AFM13 as a negative control (p < 0.05). To define the single-cell
specificity of NK cell responses to AFM13, similar assays were performed using mass cytometry analysis
of 39 lineage, maturation, activating and inhibitory receptors, and function-relevant NK cell markers. tSNE-
based multidimensional analyses revealed marked distinctions between Hut-78 and AFM13-Hut-78
stimulated cNK cells, due in part to IFN-γ, MIP-1α, CD107a, and CD16. To define the impact of specific
NK receptors, SPADE was used to define highly activated IFN-γ+ NK cell sub-populations. In a
KIR3DL1+ donor, activation was primarily within the KIR3DL1+ subset, consistent with the lack of its
inhibitory ligand on Hut-78 (HLA-Bw4-). In KIR3DL1 negative donors, responding NK cells were
enriched in mature KIR2DL2/L3+ CD57+ NK cells that lacked NKG2A. Additional experiments revealed
that both control and ML NK cells exhibited increased IFN-γ, degranulation, and cytotoxicity with AFM13
(P < 0.01), and AFM13-stimulated ML NK cells exhibited the highest IFN-γ response and killing.
Collectively, these data indicate that AFM13 significantly enhanced NK cell target cell recognition.
However, NK cell activation is still influenced by inhibitory receptor expression, maturation state, and
memory-like differentiation. Thus, these data suggest that the status and repertoire of NK cells in a patient
may offer diagnostic potential for therapeutic response, and the combination of ML NK cells with AFM13
appears to be a promising therapeutic approach.
Abstract
1. AFM13 triggers IFN-γ, TNF and degranulation of conventional (cNK) cells co-
incubated with CD30-expressing tumor cells.
no stim
K562
HuT-78
HuT-78+AFM13
Raji
Raji+AFM13
0
20
40
60
%
IFN
- p
ositi
ve
*
no stim
K562
HuT-78
HuT-78+AFM13
Raji
Raji+AFM13
0
5
10
15
20
%
TN
F p
ositi
ve
*
no stim
K562
HuT-78
HuT-78+AFM13
Raji
Raji+AFM13
0
20
40
60
% C
D10
7a p
ositi
ve
**
2. Functional mass cytometry reveals that AFM13 profoundly alters cNK cell viSNE
maps via activation, cytokine secretion and reveals single cell heterogeneity.
Unstimulated Hut-78 Hut-78 + AFM13
CD107aIFN- TNF Mip-1a CD16
3. Enhanced AFM13 mediated IFN- response correlates with NK maturation statusand absence of KIR-KIR ligand mediated inhibition.
4. ML NK display enhanced cytokine response and superior killing of CD30-expressing tumor cells. AFM13 significantly enhanced ML NK cell functionality.
no stim K562 Hut-78 Hut-78 + AFM130
10
20
30
40
50
%
IF
N-
po
sitiv
e **** **** ********
****
no stim K562 Hut-78Hut-78 + AFM130
20
40
60
80
% C
D1
07a
positiv
e
********
no stim K562 Hut-78 Hut-78 + AFM130
5
10
15
%
TN
F p
ositiv
e
******
Control NKML NK
3:1 1:1 2:10
20
40
60
80
% s
pecific
lysis ***
***
3:1 1:1 2:10
20
40
60
80
3:1 1:1 2:10
20
40
60
80
Control NK
Hut-78
Control NK
Control + AFM13
ML
ML + AFM13
3:1 1:1 2:10
20
40
60
80 ****
****
****
N=4
ML NK Control NK ML NK
Raji (CD30-)
Results Results
Conclusions
- AFM13 significantly enhances NK cell recognition of CD30+ malignancies correlating with superior
NK cell activation.
- ML NK cells exhibit improved tumor recognition and enhanced functionality against tumor cells.
- AFM13 pretreatment of tumor targets potentiates ML NK cell effector functions including cytokine
secretion and cytotoxicity.
- NK cells expression of inhibitory receptors, maturation state and memory-like differentiation influence
AFM13 mediated NK cell response against CD30+ targets
- Superior induction of IFN-, TNF and MIP1-a by AFM13 in conventional and ML NK cells leads to
consider potential bystander effects in the cellular composition and leukocytes recruitment into the tumor
microenvironment.
- Mass Cytometry can be successfully applied to evaluate AFM13-triggered functional responses of
conventional and ML NK cells at single cell resolution
- The combination of ML NK cells with AFM13 appears to be a promising therapeutic approach for
treating CD30+ malignancies.
Unts
HuT-7
8
HuT-7
8 + A
FM13
0
10
20
30
40
50
60
%
IF
N-
po
sitiv
e
*
Unts
HuT-7
8
HuT-7
8 + A
FM13
0
10
20
30
40
% C
D107a p
ositiv
e
**
Unts
HuT-7
8
HuT-7
8 + A
FM13
0
5
10
15
20
25
%
TN
F p
ositiv
eUnts
HuT-7
8
HuT-7
8 + A
FM13
0
20
40
60
80
100
%
MIP
1a
po
sitiv
e
PBMC
K562
Hut-78 +/- AFM13
Raji +/- AFM13+
In vitro stimulation (6h)
Flow cytometry
IFN-
TNF
CD107
Mass Cytometry
Phenotypic panel
Functional panel
IFN-
No Stim K562
CD
56
Hut-78 Hut-78 + AFM13
Mass Cytometry PanelCisplatin, Intercalator-IR, Barcode
CD3, CD8, CD11b, CD14, CD16, CD19, CD45, CD56, CD25, CD127
CD27, CD57, CD62L, CD69, CD137
KIR2DL1/2DS1, KIR2DL2/2DL3, KIR3DL1/3DS1, KIR2DS4, KIR2DL5
CD94, NKG2A, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80
Tim-3, Lag-3, PD-1, CD107a, IFN-γ, Mip-1α, TNF, PDL-1/2
- Explore AFM13 binding to CD16+ monocytes and whether this binding affects their activation and
function.
- Evaluate how AFM13 binding in NK cells modifies phenotype and function of surrounding immune
cells including T cells, monocytes and even NK cells.
- Characterize the immune profile of cytokines and chemokines secreted by conventional and ML NK
cells upon co-incubation with AFM13-coated or non-coated target cells. Evaluate whether they can
differentially induce migration of specific immune cell subsets.
- Evaluate efficacy of AFM13 to induced superior NK cell responses using CD30+ primary tumors as
target cells.
Ongoing Work and Future Directions
- This study was supported in part from research funds provided to Washington University by Affimed.
Funding
KIR3DL1- Donor
KIR2DL2/2DL3 CD57+
tSN
E2
tSNE1
1
2
3
4
5
6
7
8
9
10
Node ID:
tSN
E2
tSNE1
Nodes 2, 3
KIR3DL1
tSN
E2
tSNE1
KIR3DL1+ Donor
1
2
3
4
5
6
7
8
9
10
Node ID:
tSN
E2
tSNE1
Nodes 1, 10Median IFN-
Low High
No Stim. HuT-78 HuT-78+AFM13
Node ID(IFN- high)
Nodes 1, 10
pre-activated
control12-
16h6h
in vitro (rhIL-15)
IL-15
IL-12+
IL-15+
IL-18
differentiation
memory-like
#3 Stimulation
control
wash
#1 Activation #2 Resting
PBMC
NK
K562
Hut-78 +/- AFM13
Raji +/- AFM13
IFN-
CD
56
K562 Hut-78
KIR3DL1
(Inhibitory)
NK cell
Target cell
HLA-Bw4
X
CD56dim
CD56bright
Maturation
KIR
CD57
- Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses
against tumor cells.
- NK cells are frequently deficient or dysfunctional in cancer patients.
- NK cell activation is regulated by the balance of signals received through a diverse group of activating
and inhibitory receptors stochastically expressed on the cell membrane.
- NK cells mediate cytotoxic functions by secreting perforin- and granzyme-containing granules or via
death receptor ligands.
- NK cells also communicate and recruit other immune cells by secreting cytokines (e.g. IFN-, TNF) and
chemokines (e.g. MIP1-α).
- NK cells with memory-like (ML) properties differentiate after a short-term stimulation with IL-12, IL-15
and IL-18 and display enhanced functionality and anti-tumor response.
- AFM13 is a first-in-class tetravalent, bispecific innate immune cell engager characterized by bivalent
binding to CD30 and CD16A (FcgRIIIA).
- AFM13 binding to CD30+ malignancies potentiate NK cells activation and results in enhanced
cytotoxicity and cytokine and chemokine secretion.
- AFM13 has shown promising efficacy data in CD30+ malignancies both as monotherapy and in
combination with anti-PD-1.
- Based on its favorable safety profile, AFM13 is suitable for combination with alternative approaches to
induce NK cells endowed with enhanced ability to recognize and kill tumor targets as potential
immunotherapeutic approaches.
Here, we evaluate the contribution of immune cell engagers along with the enhanced functionality of
ML NK cells in the overall response to tumor targets and explore the mechanisms underlying this
enhanced response.
ML NK cells properties Description
Proliferation Function passed to progeny after cell division
Long-lived > 4 months in vivo in mice, >6 weeks in vivo for human
Non-specific = Flexible Enhanced response to cytokines, activating receptors, or tumor targets
Produce cytokines Increased INF-γ, TNF, MIP1-α production in response to restimulation
Kill target cells Increased granzyme B and killing of leukemia targets
Eliminate tumor cells in vivo Enhanced elimination of murine and human xenograft malignancies in vivo
Rescue “unlicensed” NK cells Response by both licensed and unlicensed NK cells
Ignore inhibitory KIR Response by both KIR ligand matched and mismatched NK cells
Introduction
Median IFN-
Low High
Node ID(IFN- high)
Node ID(IFN- high)
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