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Potent anti-tumor activity against patient CLL, MM and AML cells by LAVA-051, a bispecific Vγ9Vδ2-T and type 1 NKT cell engager targeting CD1d

Roeland Lameris1, Jurjen M Ruben1,3, Iris de Weerdt2, Rob Roovers3, Arnon P Kater2, Thilo Riedl3, Victoria Iglesias3, Benjamin Winograd3, Ton EP Adang3, Tanja D de Gruijl1, Paul WHI Parren3, Hans J van der Vliet1,3

1Amsterdam UMC, location VUMC, department of Medical Oncology, Cancer Center Amsterdam, Amsterdam, The Netherlands; 2Amsterdam UMC, location AMC, Department of Hematology, Amsterdam, The Netherlands; 3Lava Therapeutics, Utrecht, The Netherlands.

Cancer Center Amsterdam

Vγ9Vδ2-T

Bispecific antibodies that target tumors by engaging innate-like T cell subsets with inherent antitumor activity

Nature Cancer 2020;1:1054

Cancer Center Amsterdam

Bispecific antibodies that target tumors by engaging innate-like T cell subsets with inherent antitumor activity

type 1 NKT

tumor

TCR

• CD1d can be expressed by several hematologic malignancies

• Bispecific Vγ9Vδ2-T cell engager

• Unique ability to also trigger type 1 natural killer T (NKT) cells

• LAVA-051: humanized CD1d-Vδ2 bsVHHVγ9Vδ2-T

CD1d-Vδ2 bsVHH

Nature Cancer 2020;1:1054

Cancer Center Amsterdam

CD1d expressed by CLL and MM cells in the majority of patients, while expression by AML cells is most pronounced on (myelo)monocytic subtypes

tumor

CD1d

Expression of CD1d (median fluorescence index, MFI) on patient B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and acute myeloid leukemia (AML) cells, analysed byflow cytometry.

line of treatment

B-cell CLL

100

CD1d

expre

ssion

(MFI) 102

101

naïve ≥1

line of treatment

MM

100

102

101

naïve ≥2

n=85

n=3

1

n=12n=14

n=7

AML subtype

AML

100

102

101

myelomonocytic

other

monocytic

n=6

n=16

n=28

CD1d

expre

ssion

(MFI)

CD1d

expre

ssion

(MFI)

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CD1d-Vδ2 bsVHH induces type 1 NKT cell and Vγ9Vδ2-T cell degranulation, cytokine production and cytotoxicity towards CD1d positive tumor cells

type 1 NKT Vγ9Vδ2-T

tumor

100

type 1 NKT cellEC50 0.366 nM

Vγ9Vδ2-T cellsEC50 0.004 nM

MM cells

80

60

40

20

0CD

107a

expre

ssion

(%)

NC 10-410-5 10-3 10-2 10-1 100 101 102 103

concentration (nM)

n=5

degranulation

**

ns

ns

10

8

6

4

2

0

ng/m

l

IL-2

ng/m

l

****

****

****

15

12

9

6

3

0

type 1

NKT

type 1

NKT /

Vγ9Vδ2-

TVγ9V

δ2-T

IFN-γ

type 1

NKT

type 1

NKT /

Vγ9Vδ2-

TVγ9V

δ2-T

CD1d-Vδ2 bsVHHnegative control

10

8

6

4

2

0

type 1

NKT

type 1

NKT /

Vγ9Vδ2-

T

Vγ9Vδ2-

T

ng/m

lTNF

***

*

***

n=5

cytokine response

*p<0.05, **p0.01, ***p<0.001, ****p<0.00011

0

type 1

NKT

type 1

NKT /

Vγ9Vδ2-

TVγ9V

δ2-T

**** **** ****100

80

60

40

20

0

spec

ific ly

sis (r

elativ

e %)

AML cells

CD1d-Vδ2 bsVHHnegative control

n=4

cytotoxicity

Expression (%) of CD107a on type I NKT cells, Vγ9Vδ2-T cells or a 1:1 mixture thereof after 4 h coculture with either MM cells(MM.1s.mCherry.CD1d) or AML cells (MOLM-13) ± CD1d-Vδ2 bsVHH (100 nM or concentration range), analysed by flow cytometry.

Cytotoxicity towards AML (MOLM-13) cells after 16 h coculture with type I NKT cells, Vγ9Vδ2-T cells or a mixture (1:1) (E:T ratio of 1:2) ± CD1d-Vδ2 bsVHH (100 nM), quantified by flow cytometry counting beads andexpressed relative to tumor cells only.

Cytotoxicity towards AML (MOLM-13) cells after 16 h coculture with type I NKT cells, Vγ9Vδ2-T cells or a mixture (1:1) (E:T ratio of 1:2) ± CD1d-Vδ2 bsVHH (100 nM), quantified by flow cytometry counting beads and expressed relative to tumor cells only.

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CD1d-Vδ2 bsVHH controls tumor cell growth and triggers expansion of type 1 NKT and Vy9Vd2-T cells

Tumor growth of MOLM-13 and MM.1s.mCherry/luc.CD1d cells after coculture with PBMC ± CD1d-Vδ2 bsVHH (50 nM) (PBMC:target ratio of 10:1). Living cells (7-AAD negative) werequantified at days 4 and 7 by flow cytometry counting beads.

*p<0.05, **p0.01

bsVHH CD1d-Vδ2negative control

ns

ns

4

3

2

1

0

day 4

day 7

grow

th (fo

ld ch

ange

)

MM

**

**

20

15

10

5

0day

4day

7

AML

reduced tumor cell growth

Tumor cell line

type 1 NKT

Peripheral blood mononuclear cells(PBMC)

Vγ9Vδ2-T

leukocyte

CD1d-Vδ2 bsVHH

Cancer Center Amsterdam

Tumor cell line

type 1 NKT

Vγ9Vδ2-T

Expansion factor of type I NKT cells and Vγ9Vδ2-T cells in PBMC ± CD1d-Vδ2 bsVHH (50 nM) after coculture with MM.1s.mCherry/luc.CD1d cells (PBMC:target ratio of 10:1). Living cells (7-AAD negative) were quantified at days 4 and 7 by flow cytometry counting beads.

leukocyte

*p<0.05, **p0.01

CD1d-Vδ2 bsVHHnegative control

day 4

day 7

Vγ9Vδ2-T cell

ns

*

20

15

10

5

0

expa

nsion

(fold

chan

ge)

type 1 NKT cell

day 4

day 7

ns

**

80

60

40

20

0 n=7

effector cell expansion

bsVHH CD1d-Vδ2negative control

ns

ns

4

3

2

1

0

day 4

day 7

grow

th (fo

ld ch

ange

)

MM

**

**

20

15

10

5

0

day 4

day 7

AML

reduced tumor cell growth

CD1d-Vδ2 bsVHH

Peripheral blood mononuclear cells(PBMC)

CD1d-Vδ2 bsVHH controls tumor cell growth and triggers expansion of type 1 NKT and Vy9Vd2-T cells

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Anti-tumor activity of LAVA-051 against patient CLL, MM and AML cells

Expression (%) of CD107a on autologous Vγ9Vδ2-T cells after o/n coculture of patient samples (PBMC (CLL) or bone marrow (MM and AML)) ± CD1d-Vδ2 bsVHH (50 nM), analysed by flow cytometry.

**p0.01

autologous Vγ9Vδ2-T cell degranulation

patient CLL

negativ

e cont

rol

CD1d-Vδ2

bsVHH

**

50

40

30

20

10

0

CD10

7a ex

pres

sion (

%)

patient MM

negativ

e cont

rol

CD1d-Vδ2

bsVHH

**

100

80

60

40

20

0

CD10

7a ex

pres

sion (

%)

patient (myelo)monocytic AML

negativ

e cont

rol

CD1d-Vδ2

bsVHH

**

100

80

60

40

20

0

CD10

7a ex

pres

sion (

%)

n=5 n=6 n=5patient tumor cells

type 1 NKT leukocyte

Patient PBMC or bone marrow cells

Vγ9Vδ2-T

CD1d-Vδ2 bsVHH

Cancer Center Amsterdam

Allogeneic Vγ9Vδ2-T

patient AML

100

Vγ9Vδ2-T cellsEC50 0.001 nM

patient CLL

80

60

40

20

0spec

ific ly

sis (r

elativ

e %)

NC 10-410-5 10-3 10-2 10-1 100 101 102 103

n=4

100

Vγ9Vδ2-T cellsEC50 0.0004 nM

patient AML

80

60

40

20

0spec

ific ly

sis (r

elativ

e %)

NC 10-410-5 10-3 10-2 10-1 100 101 102

103concentration (nM)

n=3patient tumor cells

type 1 NKT leukocyte

**p0.01

cytotoxicity

Cytotoxicity of Vγ9Vδ2-T cells towards patient CLL and patient AML cells after o/n coculture (Vγ9Vδ2-T:PBMC/BM ratio of 1:1) plus negative control (NC) or CD1d-Vδ2 bsVHH (concentration range) quantified by flow cytometry counting beads and expressed relative to tumor cells only.

CD1d-Vδ2 bsVHH

Patient PBMC or bone marrow cells

Anti-tumor activity of LAVA-051 against patient CLL, MM and AML cells

Cancer Center Amsterdam

survi

val (%

)

100

80

60

40

20

040200 60 80 100

days after i.v. tumour

survi

val (%

)

100

80

60

40

20

040200 60 80 100

*

***

days after i.v. tumour

survi

val (%

)

100

80

60

40

20

040200 60 80 100

*

days after i.v. tumour

Survival of NOD scid gamma (NSG) mice after intravenous (i.v.) injection with 2.5*106 MM.1s.mcherry/luc.CD1d cells via the tail vain and treated i.v. with PBS or 107 effector cells (mixed 1:1) at day 7, 14 and 21 ± biweekly i.p. CD1d-Vδ2 bsVHH (100 µg/mouse) (untreated/ CD1d-Vδ2 bsVHH n=6 mice, mixed effector cells ± biweekly CD1d-Vδ2 bsVHH n=8 mice).

untreatedCD1d-Vδ2 bsVHHtype 1 NKT/Vγ9Vδ2-T (1:1 ratio)type 1 NKT/Vγ9Vδ2-T (1:1 ratio) + CD1d-Vδ2 bsVHH

biweekly i.p. CD1d-Vδ2 bsVHH

± i.v. type 1 NKT/Vγ9Vδ2-T

i.v. MM cells

CD1d-Vδ2 bsVHH improves survival in a multiple myeloma mouse model

Cancer Center Amsterdam

100

Bindin

g toV

γ9 (M

F)

LAVA-039

103

102

101

104

-7-14 7days after i.v. infusion

0 1 2 3 4 5 6

LAVA-048

100

Bindin

g toV

γ9 (M

F)

103

102

101

104

0CD

69 ex

pressi

on (%

)

LAVA-039

30

20

10

50

-14days after i.v. infusion

1 2 3 4

LAVA-048

40

0

CD69

expre

ssion

(%)

30

20

10

50

40

0

0.1 mg/kg0.3 mg/kg1.0 mg/kg

i.v. dose

binding to NHP Vγ9Vδ2-T cells

Infusion of a cross-reactive surrogate bispecific engager (LAVA-039) in NHP induces Vγ9Vδ2-T cell activation and is well tolerated

Vγ9Vδ2-T

CD1d+ cells

LAVA-039

Vγ9Vδ2-T

LAVA-048

• No cross-reactivity of LAVA-051 with NHP CD1d and Vγ9Vδ2-T cells

• Surrogate bispecific engagers well tolerated

• No signs of clinical, laboratory or histopathological toxicity

100

Bindin

g toVγ

9 (MF)

LAVA-039

103

102

101

104

-7-14 7days after i.v. infusion

0 1 2 3 4 5 6

LAVA-048

100

Bindin

g toVγ

9 (MF)

103

102

101

104

0CD

69 ex

pressi

on (%

)

LAVA-039

30

20

10

50

-14days after i.v. infusion

1 2 3 4

LAVA-048

40

0

CD69

expre

ssion

(%)

30

20

10

50

40

0

NHP Vγ9Vδ2-T cell activation

0.1 mg/kg0.3 mg/kg1.0 mg/kg

i.v. dose

LAVA-039

-7-14 7days

0 1 2 3 4 5 6

LAVA-0480

IL-6 (

pg m

l-1 ) 400

300

100

500

200

0

IL-6 (

pg m

l-1 ) 400

300

100

500

200

limited and transient IL-6 release

Binding of surrogate molecules to Vγ9Vδ2-T cells over time (single dose administrations, up to 1 mg /kg i.v.), analysed by flow cytometry (MF, median fluorescence).Expression (%) of the activation marker CD69 on Vγ9Vδ2-T cells over time (single dose administrations, up to 1 mg /kg i.v.), analysed by flow cytometry.Plasma IL-6 levels (right) (7 daily doses, up to 1 mg/kg i.v.), analysed by CBA.

0.1 mg/kg0.3 mg/kg1.0 mg/kg

i.v. dose

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Conclusion

• CD1d is expressed on patient CLL, multiple myeloma and AML cells• LAVA-051 demonstrates preclinical activity in vitro, ex vivo

and in vivo models• Surrogate engager well tolerated in NHPs

LAVA-051 planned to enter first-in-human clinical phase 1/2a study in patients with CLL, MM or AML who are refractory to prior therapy

type 1 NKT Vγ9Vδ2-T

tumor