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)