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TRANSCRIPT
The CD30/CD16A innate cell engager AFM13 elicits polyfunctional NK cell
responses effectively triggering memory-like (ML) NK cells against CD30+ targets
Nancy Marin1, Melissa M. Berrien-Elliott1, Michelle Becker-Hapak1, Martin Treder2, Mark Foster1, Carly Neal1, Ethan
McClain1, Sweta Desai1, Julia A. Wagner1, Timothy Schappe1, Lynne Marsala1, Pamela Wong1, Joachim Koch2, Todd A.
Fehniger1.
1Washington University School of Medicine, Saint Louis, MO; 2Affimed GmbH, Heidelberg, Germany
Natural killer (NK) cells are crucial innate immune effector cells that rapidly recognize and eliminate
infected, stressed and malignant cells. One barrier to broadly applying NK cell therapy across many
cancer types is inconsistent cancer cell recognition, which may be overcome by immune cell
engagers. AFM13 is a tetravalent bispecific antibody based on the ROCK® platform characterized by
bispecific tetravalent binding to CD30 and CD16A, with clinical efficacy in CD30+ malignancies.
Additionally, adoptively transferred memory-like (ML) NK cells have demonstrated enhanced anti-
tumor activity that may be receptive to AFM13-based targeting to enhance target cell recognition.
However, our understanding of NK cell phenotype of functional responses triggered via AFM13
remains incomplete. To address these questions, we analyzed healthy donor-derived conventional
(cNK) and ML (IL-12/15/18-induced) NK cell functional responses to CD30+ lymphoma cells treated
with AFM13. Primary cNK cells co-incubated with AFM13-labeled Hut-78 cells demonstrated
increased IFN-g, TNF, and degranulation, compared to Hut-78 cells or Raji (CD30-) targets + AFM13
as a negative control (p<0.01). ML NK cells also displayed enhanced cytokine production and killing
of CD30+ tumor targets when co-incubated with Hut-78+AFM13 (p<0.01). To define the single-cell
specificity of cNK cell responses to AFM13, similar assays were performed using mass cytometry
assessing 39 lineage, maturation, activating and inhibitory receptors, and function-relevant NK cell
markers. We then used CITRUS to define the NK cell subsets associated with increased IFN-γ,
MIP1α, CD107a (SAM, FDR<0.05). CITRUS-based clustering divided the NK cell populations into
two main groups that associated with increased effector responses. Based on back-gating, we
determined that both mature (NKG2A-CD57+) and immature (NKG2A+ CD57-) subsets displayed
enhanced IFN-g (p<0.05), Mip1a (p<0.0001), and CD107a (p<0.0001) in the presence of Hut-
78+AFM13 compared to Hut-78+AFM12 (CD19/CD16A bispecific innate immune cell engager,
negative control), with mature cells producing the most IFN-g and MIP1a compared to immature
AFM13 triggered NK cells (p < 0.05), or NK cells triggered with AFM12 (p < 0.001). AFM13 also
resulted in increased frequencies of IFN-g+CD107a+MIP1α+ multifunctional cells, in both mature and
immature subsets, compared to AFM12 (p<0.05). Finally, within the NK cells stimulated with Hut-
78+AFM13, we observed significantly increased CD57, KIR2DL1, KIR2DL2/2DL3, KIR3DL1
(p<0.01), NKp30, NKp44, and NKp80 (p<0.01) in the IFN-g producing cells compared to IFN-g-
negative cells. Overall, AFM13 enhanced the magnitude and quality of NK cell responses against
lymphoma targets. Collectively, these data indicate that target cell recognition of NK cells can be
significantly enhanced by AFM13, yet influenced by inhibitory receptors expression, maturation state,
and memory-like differentiation.
Results
Abstract
-Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses
against tumor cells. However, they are frequently deficient or dysfunctional in cancer patients.
-Strategies to enhance NK cell functionality and tumor targeting are required to fully harness their
anti-tumor potential.
Introduction
Methods
Uns
tim
K562
Hut
-78
Hut
-78+
AFM13 R
aji
Raj
i+AFM
13
0
20
40
60
%
IF
N-g
po
sitiv
e
*
Uns
tim
K562
Hut
-78
Hut
-78+
AFM13 R
aji
Raj
i+AFM
13
0
5
10
15
20
%
TN
F p
osi
tive
*
Uns
tim
K562
Hut
-78
Hut
-78+
AFM13 R
aji
Raj
i+AFM
13
0
20
40
60
% C
D107a p
osi
tive **
Figure 1. AFM13 binding on CD30+ tumors triggers cytokine secretion and degranulation of cNK cells
from normal donors. Frequency of IFN-g, TNF and CD107 positive NK cells induced after stimulation with
AFM13-prelabeled Hut-78 tumor cells. AFM13 triggering does not modify NK cell cytokine secretion or
degranulation when Raji cells were used as targets. Bars represent Mean + SEM. One-way ANOVA. n=3-5
donors. Data of 3 independent experiments. *p<0.05, **p<0.01
Figure 4. AFM13 binding triggers memory like responses with enhanced cytotoxicity against CD30+
tumors. Control and ML NK cells stimulated with AFM13-labeled Hut-78 cells show increased frequency of
IFN-g, TNF and CD107 positive cells compared to control NK cells. AFM13 labeling (closed symbols)
increased specific killing of Hut-78 cells by control but mainly ML NK cells compared to unlabeled (open
symbols) tumor targets. F) Lack of specific killing of Raji cells demonstrated specificity of AFM13-mediated
enhanced NK responses against CD30+ tumor targets. Two-way ANOVA. Bars represent Mean + SEM.
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n=10.
0
20
40
60
80
%
IF
N-g
po
sitiv
e
****
*
********
Unstim
AFM12 AFM13
+ Hut-78
-5
0
5
10
15
20
25
% T
NF
Po
sitiv
e
***
*
Unstim
AFM12 AFM13
+ Hut-78
0
20
40
60
80
100
% M
IP1a
po
sitiv
e **
Unstim
AFM12 AFM13
+ Hut-78
****
0
20
40
60
% C
D107
positiv
e
*****
**
Unstim
AFM12 AFM13
+ Hut-78
tSN
E2
tSNE1
tSN
E2
tSNE1
tSN
E2
tSNE1
tSN
E2
tSNE1
tSN
E2
tSNE1
3:1 1:1 2:10
20
40
60
80
% S
pecific
Lysis
****
****
****
3:1 1:1 2:10
20
40
60
80
% S
pecific
Lysis
****
****
****
****
0
10
20
30
40
50
% IF
N-g
positiv
e ****
****
****
****
Unstim K562
HUT-78
+AFM-130
20
40
60
80
% C
D107a p
ositiv
e
********
Unstim K562
HUT-78
+AFM-130
3
6
9
12
% T
NF
positiv
e
******
Unstim K562
HUT-78
+AFM-13
Conclusions AFM13 significantly enhances NK cell recognition of CD30+ malignancies correlating with
increased NK cell activation
NK cell expression of inhibitory receptors, maturation state and memory-like differentiation
influence AFM13-mediated NK cell response against CD30+ targets
AFM13 pretreatment of tumor targets potentiates ML NK cell effector functions including cytokine
secretion and cytotoxicity
AFM13 pretreatment trigger polyfunctional responses in NK cells compared to AFM12
pretreatment
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
Figure 2. Functional mass cytometry reveals that AFM13 profoundly alters cNK cell viSNE maps via
activation and cytokine secretion. A) viSNE plot showing the expression of IFN-g, TNF, CD107, MIP1a and
CD16 on CD56+ cells. B) Frequency of IFN-g, TNF, MIP1a and CD107 positive NK cells in unstimulated, and
cNK cells stimulated with Hut-78 cells alone or AFM12 or AFM13-labeled Hut-78 cells. One-way ANOVA. Error
bars represent Mean + SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. n=10 donors
AFM13, a first-in-class tetravalent, bispecific
innate immune cell engager is characterized
by tetravalent binding to CD30 and CD16A
(FcgRIIIA).
-AFM13 binding to CD30+ malignancies
potentiate NK cells activation resulting in
enhanced cytotoxicity and cytokine secretion.
NK cells with memory-like (ML) properties
differentiated after a short-term stimulation with
IL-12, IL- 15 and IL-18 display enhanced
functionality and anti-tumor response.
What is the contribution of immune cell engagers along with the enhanced functionality of
ML NK cells in the overall response to tumor targets? What are the mechanisms underlying
this enhanced response?
Enhanced cytokine secretion
Enhanced cytotoxicity
Results
A
B
Figure 3. AFM13 triggering increases cytokine and chemokine secretion as well as degranulation of
both immature and mature cNK cells. A) Representative SPADE analysis showing phenotype of IFN-g
producing cells in response to AFM13 triggering. Mature KIR+ A), CD57+ B) as well as immature
(NKG2A/CD94+) C) cNK cells display robust IFN-g response upon stimulation with AFM13-labeled Hut-78
cells. Numbers next to each node represent the node ID and color indicates median IFN-g expression for each
node. Nodes positive for IFN-g are depicted over their own tSNE map. D) Expression of maturation markers in
the top 2 nodes positive for IFN-g across the donors. E) Pie charts showing cNK cells stimulated with AFM12
or AFM13-labelled Hut-78 cells single or multiple producers of IFN-g, TNF, MIP1a and CD107. Bars represent
Mean + SEM. n=10.
Media
n E
xpre
ssio
n
(Arc
sin
h)
IFNg
TNF
Mip
1a
CD10
7
0.0
0.5
1.0
1.5
KIR
2DL1
/DS1
KIR
2DL2/
2DL3
CD57
NKG2A
CD94
0.0
0.7
1.4
2.1
2.8
3.5
IFN-ghigh nodes
This study was partially supported with research funds provided to Washington University by Affimed
1+
2+
3+
4+
AFM12(Total=34.2%)
AFM13(Total=58.5%)
D E