NextCure : ASH 2021 – A Novel LAIR-1 Antibody Target Acute Myeloid Leukemia (AML) Stem Cells

A Novel LAIR-1 Antibody Selectively Targets Acute Myeloid Leukemia

(AML) Stem Cells

Tae Kon Kim2, Rustin Lovewell1, Ana Paucarmayta1, Linjie Tian1, Karla Maloveste1, Junshik Hong2, Carly Hedgepath2, Kwang Woon Kim2, Ron Copeland1, Thomas O'Neill1, Linda Liu1, Sol Langermann1, Dallas B Flies1,

Shashank J. Patel1, and Han Myint1

1NextCure Inc., Beltsville, MD, USA; 2Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA.

Background: Significant unmet need exists for AML patients that are not responsive to standard-of-care (SOC) treatments. In addition, SOC patients usually relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs). Therefore, identification of unique strategies to preferentially target LSCs, promote immune responses to AML and prevent relapse are highly sought after. AML LSCs and blast cells are characterized by the higher expression of leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR-1).LAIR-1 is a checkpoint receptor on T cells and myeloid cells that delimits immune cell activation when binding to endogenous collagen ligands. However, it has been demonstrated that LAIR-1 has differential activity in AML cells and may sustain AML survival signals since downregulation of LAIR-1 promotes AML cell death. We developed a LAIR-1 monoclonal antibody (mAb), termed NC525, that disrupts LAIR-1 mediated survival signaling and preferentially targets and kills LAIR-1 expressing AML LSCs and blast cells.

LAIR-1 Overexpression on AML blast and stem cells

A				LAIR-1 Expression
			Blood	mRNA (AML subtypes)
		AML		Surface protein
		Patients	Bone Marrow
B			FAB Classification	Molecular Mutations
		14
mRNA	RSEM)	12
10
2
LAIR1-	(log

LAIR-1 mAb blocks LAIR-1 signaling and induces antibody dependent

cytotoxicity and phagocytosis

LAIR-1-Mediated LSC and Blast Survival

Multimodal Anti-leukemic Activity

1. Inhibition of survival signal

3. ADCP

LAIR-1

C1Q

Collagen

LAIR-1 mAb

2. ADCC

LAIR-1 mAb blocks binding of natural ligand collagen and signaling

A		B	activityreporter cellsGFP+)		LAIR-1 signaling blockade
			100				Ctrl IgG
				50
			LAIR-1 (%			LAIR-1 mAb	IC50=0.25nM
LAIR-1	LAIR-1	LAIR ECD fused	0	-1	0	1	2
Ligand	mAb	to CD3ζ		-2
				mAb concentration (log µg/ml)

Figure 2. A. Schematic illustration of LAIR-1-TCRζ reporter assay using UT-140NFκB-GFP cell line. B. LAIR-1 mAb blocks COLA1-mediatedLAIR-1 signaling activity in a dose-dependent manner.

LAIR-1 mAb induces ADCC mediated AML killing

	LAIR-1 mAb inhibits colony formation of AML LSCs
	Ctrl IgG	LAIR-1 mAb	colonyof# unitsforming	15	✱	60	✱	LAIR-1 mAb
AML
			10		40		Ctrl IgG
					5		20
					0		0
	CFU-E	BFU-E	CFU-GEMM	CFU-G	CFU-GM	CFU-M		ns	LAIR-1
	LAIR-1								colonyof# formingunits	200
Normal HSPCs								mAb
							150
mAb								Ctrl IgG
										100
	Ctrl									50
										0

Figure 4. Effect of LAIR-1 mAb on the colony forming capacity of LSCs derived from two AML patients and HSPCs from a healthy donor using MethocultTM method.

LAIR-1 mAb eliminates AML cells via ADCP

A		CDX Model		B	Ex vivo		C	In vitro ADCP
MV411-Luc				cytometry spleen	Ctrl IgG		Mouse BMM MV411-Luc
	cells		Treatment			mAb			+
			2X / week
NSG mice	0	Day	35	Flow of	0 25	50 75 100	Time	Ctrl IgG LAIR-1 mAb
						% of Dead MV411 cells	(min)
Leukemiagrowth	6	Ctrl IgG	40	IFImage ofspleen	αLAIR-1	Ctrl IgG	30
flux(Total×10p/s)8	10	20 30	mCD45- mouse immune cells
	4	LAIR-1 mAb
	2							60
	0					hCD45 - MV411
	Time post inoculation (d)		Nuclear stain			MV411 engulfment

Figure 5. A. In-vivoCDX model of implanting MV411-Luccells into NSG mice and treating with LAIR-1mAb showed potent anti-leukemicactivity even without transfer of human PBMCs. B. Flow cytometry and immunofluorescence analyses of spleens harvested on day 35 from CDX model. Arrows indicate MV411 cells undergoing phagocytosis. C. LAIR-1mAb induces ADCP activity in an in-vitrophagocytosis assay utilizing Cell-Traceviolet labelled mouse bone marrow macrophages (BMM) cocultured with MV411-Luccells.

8

M0 M1 M2 M3 M4 M5 M6 M7 UD

NPM1

DNMT3A 11q3 FLT3 TP53

C

Amp

BM Aspirates

Patient #A

Patient #B

Patient #C

HSPC

Ctrl IgG

LSC

LAIR-1 mab

ADCC mediated cell lysis (%)	40		MOLT4			Ctrl IgG
					LAIR-1 mAb
20
0
	-2	-1	0	1	2	3
	mAb concentration (log µg/ml)

	20		MV411		Ctrl IgG
ADCC mediated cell lysis (%)					LAIR-1 mab
10
0	-1	0	1	2	3
	-2
	mAb concentration (log µg/ml)

LAIR-1 mAb restricts AML progression in PDX models

BM aspirates	Leukemiagrowth CD33+humancells)	0	PDX-donor #1			0	PDX-donor #2
treatment
		60						100		Ctrl IgG
NSG-SGM3		40						80		LAIR-1 mAb
mice								60
6		20						40
Weekly								20
12	(%	6	7	8	9	10	11	5	10	15
Time post inoculation (w)	Time post inoculation (w)
Week

Figure 1. A. Schematic illustration of study design to determine LAIR-1expression on AML blasts and leukemic stem cells. B. LAIR-1mRNA expression in AML subtypes defined by FAB classification, and mutation status. UD denotes undetermined. C.

Figure 3. LAIR-1mAb induces antibody dependent cytotoxicity on MOLT4 and MV411 cell lines in a dose-dependentmanner in primary PBMC: leukemic cell coculture assay.

Figure 6. NSG-SGM3mice were irradiated and implanted with BM aspirates from two AML patients. Mice were treated with mAbs on weekly basis from week 6 of BM implantation.

LAIR-1 protein expression on the cell surface of leukemic stem cells (LSCs; CD34+CD38-CD90-CD45RA+/- or CD34-CD117+CD244+/-) vs hematopoietic stem and progenitor cells (HSPCs; CD34+CD38-CD90+CD99-) derived from bone marrow aspirates, and leukemic blasts from the peripheral blood of AML patients.

Conclusion: High expression of LAIR-1 on leukemic cells and its role in mediating survival signaling in LSCs makes it an attractive therapeutic target against AML. To this end, we developed a novel immunomedicine NC525, a LAIR-1 mAb, that potently eradicates LSCs and leukemic blasts while having a minimal effect on healthy HSPCs. We show that LAIR-1 mAb exerts its anti-tumor activity via disruption of survival signals in leukemic cells and Fc-mediated effector functions including ADCC and ADCP.