Enhancement of the anti-tumor effects of CD47 blockade in solid tumors by combination with targeted radioimmunotherapy

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

Abstract # 589

BACKGROUND

One mechanism that tumors use to escape immunosurveillance is the overexpression of CD47, which inhibits the macrophage mediated phagocytosis pathway. Although blockade

225Ac/177Lu-HER2-ARCs Bind HER2 Expressing Tumor Cells

RESULTS

Combination of HER2-ARC and anti-CD47 Antibody Slows Tumor Xenograft Growth In Vivo

of the CD47-SIRPα axis is a promising approach to enhance tumor targeted phagocytosis, anti-CD47 monotherapies have not shown meaningful responses in clinical studies of solid tumors. Combination cancer therapies aim to increase the probability of response in settings of resistance by combining drugs with different mechanisms of action. Antibody radioconjugates (ARCs) specifically target and deliver therapeutic radiation directly to cancer cells. We rationalized that the immunogenic and cytotoxic properties of ARCs will

Binding (ELISA)

Binding (Flow Cytometry)

1.0

IgG

60000

SKOV3

BT474

(450ODnm)

10-6

10-5

10-4

10

-310-210-1100 101 102 10

FluorescenceMean

FluorescenceMean

60000

3

0.8

Anti-HER2

225Ac-HER2-ARC

0.6

177Lu-HER2-ARC

40000

40000

0.4

0.2

20000

20000

0.0

μg/mL

0

0

IgG Anti-HER2

225Ac-HER2-ARC

177Lu-HER2-ARC

(mm3)

2500

+/-SEM

2000

1500

Voume

1000

Tumor

500

Mean

0

30

35

40

45

50

55

60

65

70

75

25

Days Post-Treatment

(%)

20

ChangeWeightBody+/- SEM

15

SurvivalofProbability

10

5

0

-5

-10

Mean

-15

-20

40

50

60

70

30

Days Post-Treatment

100

75

50

25

0

30

35

40

45

50

55

60

65

70

75

80

25

Days Post-Treatment

upregulate calreticulin (CRT), a pro-phagocytic signal, thereby synergizing with CD47 blocking therapies to enhance phagocytosis and antitumor activity. Here for the first time, we

Figure 2. Binding of 225Ac- and 177Lu- HER2-ARC to recombinant human HER2 protein in ELISA (EC50 = 0.056, 0.056 and 0.093 µg/mL for HER2, 225Ac-HER2-ARC and 177Lu-HER2-ARC) and human tumor cell lines by flow cytometry.

Vehicle

225Ac-HER2-ARC

225Ac-HER2-ARC+anti-CD47

anti-CD47

177Lu-HER2-ARC

177Lu-HER2-ARC+anti-CD47

demonstrate the combination benefit of anti-HER2 (Trastuzumab) specific targeting ARC and a CD47 blocking antibody to enhance therapeutic efficacy in preclinical solid tumor

CD47 Expression in HER2 Cells Before and After HER2 ARC Treatment

225Ac

177Lu

models.

Proposed Mechanism of Action

A.

B.

C.

A.

Mean Fluorescence

IgG CD47

200000

150000

100000

5000

4000

3000

2000

1000

0

SKOV3

BT474

B.

Control

200 nCi/mL (48h)

400 nCi/mL (48h)

Fluorescence

400000

Fluorescence

15000

300000

10000

200000

5000

Mean

Mean

100000

0

0

SKOV3

BT474

Control

200 nCi/mL (72h)

400 nCi/mL (72h)

SKOV3

BT474

Control

100 nCi/mL (72h)

Fluorescence

400000

300000

200000

Mean

100000

0

BT474

(mm3)

2500

2000

+/-SEM

1500

Voume

1000

Tumor

500

225Ac-HER2-ARC vs Combo ****P < 0.0001

Mean

Anti-CD47 vs Combo ****P < 0.0001

(mm3)

2500

2000

+/-SEM

1500

Voume

1000

Tumor

500

177Lu-HER2-ARC vs Combo **P < 0.001

Mean

Anti-CD47 vs Combo ****P < 0.0001

Anti-CD47 vs Combo **** P< 0.0001

Figure 3. (A) CD47 expression on human tumor cell lines (B) CD47 expression in response to 225Ac-HER2-ARC treatment as determined by flow cytometry. Control: untreated cells.

225Ac-HER2-ARC Induces an Increase in Cell Surface CRT and Cytotoxicity

0

30

35

40

45

50

55

60

65

70

75

25

Days Post-Treatment

100

0

30

35

40

45

50

55

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75

25

Days Post-Treatment

100

Anti-HER2 ARC = Y

Y= Anti-CD47

Figure 1. (A) Schematic of radioisotope armed Trastuzumab (anti-HER2 ARC) (B) Illustration of alpha versus beta radioisotope biological properties (C) Model showing mechanism of cancer cell targeted ARC therapy and potential benefit of combining with CD47

A.

CRT expression (Flow cytometry)

Fluorescence

15000

Control

200 nCi/mL (72h)

10000

400 nCi/mL (72h)

Mean

5000

0

SKOV3

BT474

B.

XTT

(%)

120

100

Viability

80

60

40

Cell

20

0

4

8

16

32

64

128

2

225Ac-HER2-ARC (nCi/mL)

Cytotoxicity

XTT

(%)

120

100

Viability

80

60

40

Cell

20

0

10-2

10-1

100 101 102 103 104

105

177Lu-HER2-ARC (nCi/mL)

MTS

(%)

100

80

Viability

60

40

Cell

20

0

100

101

102

103

104

105

10-1

225Ac-HER2-ARC (nCi/mL)

of Survival

75

Probability

25

50

0

30

35

40

45

50

55

60

65

70

75

80

25

Days Post-Treatment

of Survival

75

Probability

25

50

0

30

40

50

60

70

80

Days Post-Treatment

blocking antibody to promote enhanced tumor cell killing

METHODS

Figure 4. (A) Cell surface calreticulin (CRT) levels detected by flow cytometry in tumor cells treated with indicated dose of 225Ac-HER2-ARC for 3 hours followed by media change and incubation for 72 hours. (B) Dose dependent cytotoxicity of 225Ac- and 177Lu- HER2-ARCs in SKOV3 cells treated for 3 hours (XTT) or 12 hours (MTS) is shown as % cell viability relative to untreated control. (Two-Way ANOVA, *p < 0.05).

225Ac-HER2-ARC and anti-CD47 Antibody Combination Enhances Phagocytosis

Figure 6. Antitumor efficacy study of 225Ac- and 177Lu-HER2-ARCs and anti-CD47 combination and single agents in NSG mice (n = 8 per group) bearing human ovarian carcinoma (SKOV-3). A single dose of 225Ac-HER2-ARC (25 nCi) or 177Lu-HER2-ARC (25 µCi) was administered on day 0 and the anti-CD47 (10 mg/kg) agent was administered on day 0, 4 and 10. Tumor volume, body weight and survival was monitored. Statistical analysis was performed on GraphPad Prism 9.2 using Two-Way ANOVA to compare mean tumor volumes.

Radiochemistry

In Vitro

In Vivo

Radiolabeling

Binding ELISA

• Single agents and

Characterization

Binding Flow

combination efficacy

CD47 Expression

study

A.

ARC Treatment (48h)

✱✱✱✱

✱✱✱

Phagocytosis

50

✱✱

40

30

20

%

10

0

aCD47

ARC

ARC+

IgG

aCD47

ARC Treatment (72h)

80

✱✱

% Phagocytosis

ns

60

40

20

0

aCD47

ARC

ARC+

IgG

aCD47

B.

ARC Treatment (48h)

20

✱✱✱✱

✱✱✱✱

Phagocytosis

✱✱✱

15

10

5

%

0

aCD47

ARC

ARC+

IgG

aCD47

ARC Treatment (72h)

% Phagocytosis

40

✱✱✱

✱✱

30

✱✱✱

20

10

0

aCD47

ARC

ARC+

IgG

aCD47

CONCLUSIONS

Here for the first time, we demonstrate enhanced therapeutic efficacy between an anti-HER2 ARC and CD47

blocking antibody combination in a preclinical solid tumor model.

The finding suggests that ARC mediated upregulation of CRT potentiates the pro-phagocytic signal and

synergizes with the anti-CD47 mode of action thereby enhancing antitumor immune response.

Cell Surface CRT

Cytotoxicity

Phagocytosis

Figure 5. (A) Combination of 225Ac-HER2-ARC and anti-CD47 enhances phagocytosis of tumor cells (A) SKOV3 and (B) BT747. Target cells were treated with 225Ac-HER2-ARC for 48 or 72 hours. The cells were labeled with DiD and cocultured for 2 hours in the presence of anti-CD47 (10 µg/ml) with human macrophages labeled with DiO. The percentage of phagocytosis was measured by flow cytometry (macrophages DiO+/DiD+). Statistical analysis was on GraphPad Prism 9.2 using One-Way ANOVA (*p < 0.05, **p< 0.01, ***p<0.001 and ****p < 0.0001).

Additional preclinical studies in other tumor models are ongoing.

This combination mechanism provides a very promising strategy to improve therapeutic responses in patients

harboring solid tumors and warrants further preclinical evaluation.

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

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Actinium Pharmaceuticals Inc. published this content on 12 November 2021 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 12 November 2021 12:10:30 UTC.