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 | 60 | 65 | 70 | 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.