SITC 2021: Actimab-A with CD47 Immunotherapy in AML

Figure 2. (A) Binding of

120

100

80

60

40

20

0

Anti-CD33actinium-225 targeted radioimmunotherapy enhances the biologic activity of anti-CD47 antibody immunotherapy in preclinical models

of acute myeloid leukemia

Denis Beckford-Vera, Sagarika Pachhal, Emily Greer, Jason Li, Caroline Jennings, Jesse Hwang, Qing Liang, Mary Chen, Eileen M. Geoghegan, Helen Kotanides, and Dale L. Ludwig

Actinium Pharmaceuticals, New York, NY USA

BACKGROUND	RESULTS

Abstract # 590

Actimab-A, the anti-CD33 antibody lintuzumab armed with the radioisotope Actinium-225 (225Ac, α-emitter, 10 days half-life), has demonstrated single agent antileukemic effects in patients

225Ac-CD33-ARC Binds CD33 Expressing AML Cells and Induces Cytotoxicity

A.	CD33 Binding Flow Cytometry	B.	CD47 Expression in AML Cell Lines
CD33 Binding ELISA

Combination of 225Ac-CD33-ARC and anti-CD47 Antibody Increases Survival in AML Model

A.

with relapsed or refractory acute myeloid leukemia (AML)1. Up-regulation of CD47, a macrophage checkpoint that suppresses phagocytosis, is one mechanism by which myeloid malignancies such as AML can evade targeting by the innate immune response. Therapeutic blocking antibodies against this pathway have shown early clinical promise. We hypothesized that Actimab-A will enhance phagocytosis in AML cells by specifically upregulating calreticulin (CRT), a pro-phagocytic signal. Moreover, we hypothesized that combination of the anti-CD33 antibody radioconjugate (CD33 ARC) and CD47 blocking antibody could act in synergy to

OD (450 nm)

0.8anti-CD33

225Ac-CD33-ARC

0.6 177Lu-CD33-ARC

IgG

0.4

0.2

0.0 10-6 10-5 10-4 10-3 10-2 10-1 100 101 102 103

	4000	IgG (100 μg/mL)		6×10	5
Fluorescence		anti-CD33 (100 μg/mL)	Fluorescence
3000	225Ac-CD33-ARC (100 μg/mL)
		4×105
2000
		2×105
Mean			Mean
1000
	0	MV-4-11		0
	HL60

IgG

CD47

U937

HL60

MV-4-11

	100		225Ac-CD33-ARC (80 nCi) + CD47 Combo
		100
of Survival	75	of Survival	75
Probability	50	Probability	50
25	25

enhance therapeutic outcomes in AML compared to single agent. In this study, we examined, for

μg/mL

225Ac-CD33-ARC (80 nCi)

the first time, the potential mechanistic benefit of combining the anti-CD33 ARC armed with 225Ac or Lutetium-177 (177Lu, β-emitter, 6.6 days half-life) and a CD47 blocking antibody, using in vitro and in vivo human AML preclinical models.

1Blood. 2011;118(21):768.

Proposed Mechanism of Action

A.

B.

C.	Cytotoxicity (XTT Assay)

HL-60

U937

% Cell viability

1

10

100

1000

225Ac-CD33-ARC (nCi/mL)

		Cytotoxicity ( Propidium Iodide)
	100				MV-4-11
viability					U937
75				HL-60
% Cell	50
25
	0	1	10	100	1000
	0.1

225Ac-CD33-ARC (nCi/mL)

																	0													***P = 0.0006
0
10	20	30	40	50	60	70	80	10	20	30	40	50	60	70	80
							Days Post-Treatment												Days Post-Treatment

	Vehicle	225Ac-CD33-ARC (25 nCi)	225Ac-CD33-ARC (25 nCi)+anti-CD47
	anti-CD47	225Ac-CD33-ARC (80 nCi)	225Ac-CD33-ARC (80 nCi)+anti-CD47
B.			C.	1012
+/- SEM (%)	20		(photon/sec)
15		1011
10		1010
5

225Ac- and 177Lu- Lintuzumab (CD33-ARC) to recombinant human CD33 protein in ELISA (EC50= 0.007, 0.019 and 0.029 µg/mL for anti-CD33,225Ac-CD33-ARC and 177Lu-CD33-ARC, respectively) and human AML cell lines by flow cytometry. (B) CD47 expression on AML cells. (C) Dose dependent cytotoxicity of 225Ac-CD33-ARC in AML cell lines treated for 96 hours is shown as % cell viability relative to untreated cells.

225Ac-CD33-ARC Induces an Increase in Cell Surface Calreticulin

WeightChange	0	Signal +/SEM-	109
-5	108
-10	107

Anti-CD33-ARC = Y

Y = Anti-CD47

Figure 1. (A) Schematic of radioisotope armed Lintuzumab (anti- CD33 ARC) (B) Model showing mechanism of cancer cell targeted ARC therapy and potential benefit of combining with CD47 blocking antibody to promote enhanced AML cell killing

	20000			Control		20000
Fluorescence				100 nCi/mL (72h)	Fluorescence	✱	✱
	15000	✱	✱	200 nCi/mL (72h)		15000
Mean	10000				Mean	10000
5000				5000
	0					0
		MV-4-11	HL-60			U937	MV-4-11

Control

100 nCi/mL (96h)

MeanBody	-15
-20				Vehicle vs 225Ac-CD33-ARC (25 nCi)****	106
			225Ac-CD33-ARC (80 nCi) vs Combo**	MeanBLI
	-25	20	30	40	50	60	70	80	105	10	20	30	40	50	60	70	80	90	100
	10	0
			Days Post-Treatment						Days Post-Treatment
						Vehicle		225Ac-CD33-ARC (25 nCi)	225Ac-CD33-ARC (25 nCi)+anti-CD47

anti-CD47

225Ac-CD33-ARC (80 nCi)

225Ac-CD33-ARC (80 nCi)+anti-CD47

Figure 5. Efficacy study of 225Ac-CD33 ARC, anti-CD47 Ab and the combination in a MV-4-11 AML disseminated tumor model in NSG mice (n = 7 per group). ARC single dose was administered on day 1, and anti-CD47 Ab (10 mg/kg) on day 1, 4, and 10. (A) Survival graph of single agent and combination treatment (B) Body weight change in

Figure 3. Cell surface calreticulin (CRT) levels detected by flow cytometry in AML cells treated with indicated dose of 225Ac-CD33-ARC (left: 72 hour,

right: 96 hour, control: untreated cells). Statistical analysis was performed using Two-Way ANOVA (*p < 0.05).

response to treatments (C) Mean bioluminescence (BLI) signal of AML progression. Statistical analysis was performed on GraphPad Prism 9.2 using Two-Way ANOVA (**p< 0.01, ***p<0.001 and ****p < 0.0001).

METHODS

225Ac-CD33-ARC and anti-CD47 Antibody Combination Enhances Phagocytosis

CONCLUSIONS

Radiochemistry

In Vitro

In Vivo

•	Radiolabeling	•	Binding ELISA	• Single agents and
•	Characterization	•	Binding Flow	combination
		• CD47 Expression	efficacy study

• Cytotoxicity

		MV-4-11
	70
Phagocytosis	60	✱✱
✱✱
50	✱✱
40
30
20
10
%
	0

	U937
70
60	✱✱✱✱
✱✱✱✱
50	✱✱✱
40
30
20
10
0

	HL60
70	✱✱✱✱
60	✱✱✱
✱✱✱✱
50
40
30
20
10
0

IgG

CD47

ARC (100 nCi/mL)

ARC (100 nCi/mL)+CD47

Our findings represent the first proof-of-concept studies evaluating a CD33 ARC and anti-CD47 Ab blocking agent combination in AML.

§	CD33 ARC induces targeted cytotoxicity of AML cells and an increase in cell surface CRT in vitro.
§ Combining CD33 ARC and anti-CD47 Ab treatment results in enhanced pro-phagocytic innate immune response in
	vitro and significantly increased survival in an AML disseminated tumor model in vivo compared to each single agent
	therapy.
§	Additional preclinical studies, including investigating 177Lu-CD33-ARC in AML, are ongoing.

•	Cell Surface CRT
•	Phagocytosis

Figure 4. Combination of 225Ac-CD33-ARC and anti-CD47 enhances phagocytosis of AML cells. Target cells were treated with 225Ac-CD33-ARC for 96 hours. The cells were labeled with DiD and cocultured for 2 hours in the presence of anti-CD47 (1 µg/ml) with human macrophages labeled with DiO. The percentage of phagocytosis was measured by flow cytometry (macrophages DiO+/DiD+). Statistical analysis was performed using One-Way ANOVA (*p < 0.05, **p< 0.01, ***p<0.001 and ****p < 0.0001).

§ These observations of potential therapeutic benefit in AML by the combination of an CD33 ARC and anti-CD47

Ab warrant further preclinical exploration to support clinical translation of this approach.

Society for Immunotherapy of Cancer (SITC) 36th Annual Meeting Washington, D.C. November 10-14, 2021