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CDCs show a strong immunomodulatory activity and...
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Saravana Kanagavelu 1 , Jennifer Johnson 1 , Hocine Rachid 2 , Chris Sakoda 1 , Liang Li 1 , Sharon Vaturi 1 , Mario Fournier 3 , Rachel Smith 1 , Linda Marban 1 , Reem Al-Daccak 2 , Luis Rodriguez-Borlado 1 1 Capricor Therapeutics, 8700 Beverly Boulevard, Davis Building, Los Angeles, CA 90048; 2 HLA et Medicine, Hôpital Saint Louis, Paris, France; 3 Cedars-Sinai Medical Center, Los Angeles, CA C ONCLUSIONS R EFERENCES 1. Tseliou, E., Fouad, J., Reich, H., Slipczuk, L., de Couto, G., Aminzadeh, M., Middleton, R., Valle, J., Weixin, L. and Marban, E. (2015). Fibroblasts Rendered Antifibrotic, Antiapoptotic, and Angiogenic by Priming With Cardiosphere-Derived Extracellular Membrane Vesicles. J. Am. Coll. 66(6): 599-611. 2. Jefferies, J., Byrne, B., Taylor, M., Lima, J., Venkatesh, B., Ostovaneh, M., Ruckdeschel Smith, R., Malliaras, K., Fedor, B., Rudy, J., Pogoda, J., Marbán, L., Ascheim, D., Marbán, E., Victor, R. D. Cardiac and skeletal muscle improvements after cell therapy in the open-label Phase I/II randomized prospective Halt cardiOmyopathy ProgrEssion (HOPE) trial of Duchenne muscular dystrophy. Manuscript submitted for publication. Action Duchenne International Conference Birmingham (UK), November 10-12, 2017 I NTRODUCTION Figure 2. CDC mechanism of action. CDCs secrete EVs containing bioactive molecules able to regulate different responses involved in tissue regeneration. Cardiosphere-Derived Cells (CAP-1002) Immunomodulation Macrophages Anti-apoptotic Proliferative Cardiomyocytes Angiogenic Vessels Antifibrotic Fibroblasts Regenerative Progenitor cells CAP - 1002 is Immunomodulatory and Regenerative Systemic Therapeutic Effects with IC Delivery in HOPE I S UMMARY -4 -2 0 2 4 10 20 30 40 y = -3.474x + 22.98 R 2 = 0.9996 Efficiency = 94.02 Log (cells in qPCR) Ct Alu Mouse model: C57BL/10 (8-12 weeks old) CDCs: human CDCs, 150,000 cells Mouse model: SCID (8-12 weeks old) CDCs: human CDCs, 150,000 cells CDCs show a strong immunomodulatory activity and improve muscle physiology when systemically delivered. The immunomodulatory and regenerative properties of CDCs have been shown effective with in vitro and in vivo models. Biodistribution of human CDCs was measured in mice using qPCR for human-specific Alu sequence. 10min and 24h after CDC injection, most cells were found in the lungs (Fig. 7b). Less than 5% of cells present in the lungs at 24h in SCID mice remained 1wk after administration. No significant amount of CDCs remained 3wk after injection (Fig. 7c). Figure 7. A) DNA was isolated from CDCs and qPCR was run on serial dilutions. Standard curves of CDC DNA spiked into mouse tissue DNA were prepared for each tissue to be tested for CDC biodistribution in mice. B) CDC biodistribution in WT mice 10 minutes and 24 hours after injection of 150,000 human CDCs. C) CDC biodistribution in SCID mice 24 hours, 1 week, and 3 weeks after CDC injection. A B C B Phenotypic analysis showed that CDCs have a low immunogenic expression profile. CDCs limited the in vitro proliferation of activated human CD4 and CD8 T lymphocytes. Mdx mice (DMD mouse model) intravenously treated with CDCs showed a dose dependent increase of exercise capability and an improvement in muscle physiology when compared with placebo treated mice. Histological analysis also showed a reduced fibrosis in mdx mice treated with CDCs when compared with control mice. CDCs accumulate preferentially in the lungs 10 min and 24h after systemic delivery. We also evaluated clearance of CDCs 1 and 3 weeks after systemic delivery in SCID mice. A B IODISTRIBUTION AND C LEARANCE CAP - 1002 Manufacturing Process CAP-1002 is an allogeneic cell therapy product based on the isolation and expansion of Cardiosphere-Derived Cells (CDCs). • CDCs have a low immunogenic profile and a strong immunomodulation of T cells that can limit the inflammatory process observed in DMD patients. • 150,000 – 250,000 CDCs given intravenously are effective in increasing exercise capacity and diaphragm muscle function of mdx mice 3 – 6 weeks after injection. • Most cells are initially trapped in the lungs, with small redistribution of cells to the heart within 1 week of injection • Cells are cleared within 3 weeks after injection. • These findings support clinical evaluation of systemic administration and repeat dosing of CAP-1002 in patients with DMD. Figure 1. A) Explant-derived cells (EDCs) are produced from donor heart tissue and expanded to produce a master cell bank (MCB). B) Cardiospheres are formed from EDCs. C) CDCs are produced by culturing the cells obtained after seeding the cardiospheres. Figure 3. Performance of the upper limb (PUL) test was used to measure the effects on skeletal muscle function in HOPE I. A) Test description showing shoulder, middle, and distal level tests. B) Results from middle and distal levels in patients treated with CDCs (red) and placebo (blue). Adapted from Tseliou, E. et al. J Am Coll Cardiol. 2015; 66(6):599–611. Medium PHA CDC 5x10 9 EV 0 20 40 60 80 % proliferating cells CD4+ CD8+ ** * D HLA I 96% HLA II 3.6% Human Leukocyte Antigens (HLA) CD80 3.6% CD86 57.3% Co-Stimulatory CD274 (PD-L1) 45.8% CD275 (ICOS-L) 9.4% Co-Stimulatory/Regulatory CD4+ T cells CD8+ T cells 11% PHA + CDC 55% PHA 0.1% medium 28% 72% 0.1% CFSE E FFICACY WITH IV A DMINISTRATION Mouse model: mdx mice (8-10 months old) CDCs: C57BL/10 (syngeneic) Figure 6. Efficacy of CDCs in mdx mice with IV administration. A) Study timeline. Mice received treatment in Week 0, exercise every week, and were sacrificed in week 6. B) Exercise capacity of mice given escalating doses of CDCs. C) Isolated diaphragm muscle function. D) Masson’s Trichrome staining of heart tissue sections 6 weeks after CDC injection. A dose-dependent improvement in exercise capacity (Fig. 6b) and isolated diaphragm muscle function (Fig. 6c) was observed 6 weeks after CDC treatment. Histological analysis of hearts reveal lower collagen deposition (Fig. 6d). PBS (n=10) Study Groups 75K (n=4) 250K (n=4) 150K (n=8) A Ex. Muscle Function Histology Time (Weeks) -1 0 4 5 6 Ex. Echo Ex. Ex. Echo Ex. 1 2 3 Ex. Ex. Ex. B C CDC I MMUNOLOGY Figure 5. Immune modulation of PHA- induced CD4+ and CD8+ T cells proliferation by CDC. Results are mean percentage values ± SEM from 2 different donors each done in triplicates. Figure 4. Expression of immune molecules involved in T immune response Dose Escalation Efficacy D CDCs show a low immunogenic phenotype with expression of class I HLA and CD86 co-stimulatory antigen and the immuno-regulatory marker PD-L1 (Fig. 4). CDCs limit proliferation of in vitro activated human T lymphocytes. Donor heart MCB Explant creation Cardiospheres CDCs Allogeneic CDC product EDC outgrowth CDC Expansion MCB A B C
Transcript of CDCs show a strong immunomodulatory activity and...
Saravana Kanagavelu1, Jennifer Johnson1, Hocine Rachid2, Chris Sakoda1, Liang Li1, Sharon Vaturi1, Mario Fournier3, Rachel Smith1, Linda Marban1, Reem Al-Daccak2, Luis Rodriguez-Borlado1
1 Capricor Therapeutics, 8700 Beverly Boulevard, Davis Building, Los Angeles, CA 90048; 2 HLA et Medicine, Hôpital Saint Louis, Paris, France; 3Cedars-Sinai Medical Center, Los Angeles, CA
CONCLUSIONS
REFERENCES1. Tseliou, E., Fouad, J., Reich, H., Slipczuk, L., de Couto, G., Aminzadeh, M., Middleton, R., Valle,
J., Weixin, L. and Marban, E. (2015). Fibroblasts Rendered Antifibrotic, Antiapoptotic, andAngiogenic by Priming With Cardiosphere-Derived Extracellular Membrane Vesicles. J. Am. Coll.66(6): 599-611.
2. Jefferies, J., Byrne, B., Taylor, M., Lima, J., Venkatesh, B., Ostovaneh, M., Ruckdeschel Smith,R., Malliaras, K., Fedor, B., Rudy, J., Pogoda, J., Marbán, L., Ascheim, D., Marbán, E., Victor, R.D. Cardiac and skeletal muscle improvements after cell therapy in the open-label Phase I/IIrandomized prospective Halt cardiOmyopathy ProgrEssion (HOPE) trial of Duchenne musculardystrophy. Manuscript submitted for publication. ActionDuchenneInternationalConference
Birmingham(UK),November10-12,2017
INTRODUCTION
Figure 2. CDC mechanism of action. CDCs secrete EVs containing bioactive moleculesable to regulate different responses involved in tissue regeneration.
Cardiosphere-Derived Cells(CAP-1002)
Immunomodulation
Macrophages
Anti-apoptoticProliferative
Cardiomyocytes
Angiogenic
Vessels
Antifibrotic
Fibroblasts
Regenerative
Progenitor cells
CAP-1002 is Immunomodulatory and Regenerative
Systemic Therapeutic Effects with IC Delivery in HOPE I
SUMMARY
-4 -2 0 2 4
10
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y = -3.474x + 22.98R2 = 0.9996Efficiency = 94.02
Log (cells in qPCR)
Ct A
lu
Mouse model: C57BL/10 (8-12 weeks old)CDCs: human CDCs, 150,000 cells
Mouse model: SCID (8-12 weeks old)CDCs: human CDCs, 150,000 cells
CDCsshowastrongimmunomodulatoryactivityandimprovemusclephysiologywhensystemicallydelivered.
The immunomodulatory and regenerative properties of CDCs havebeen shown effective with in vitro and in vivo models.
Biodistribution of human CDCs was measured in mice using qPCR forhuman-specific Alu sequence. 10min and 24h after CDC injection,most cells were found in the lungs (Fig. 7b). Less than 5% of cellspresent in the lungs at 24h in SCID mice remained 1wk afteradministration. No significant amount of CDCs remained 3wk afterinjection (Fig. 7c).
Figure 7. A) DNA was isolated from CDCs and qPCR was run on serial dilutions.Standard curves of CDC DNA spiked into mouse tissue DNA were prepared for eachtissue to be tested for CDC biodistribution in mice. B) CDC biodistribution in WT mice10 minutes and 24 hours after injection of 150,000 human CDCs. C) CDCbiodistribution in SCID mice 24 hours, 1 week, and 3 weeks after CDC injection.
A B
C
B
Phenotypic analysis showed that CDCs have a low immunogenicexpression profile. CDCs limited the in vitro proliferation ofactivated human CD4 and CD8 T lymphocytes.Mdx mice (DMD mouse model) intravenously treated with CDCsshowed a dose dependent increase of exercise capability and animprovement in muscle physiology when compared with placebotreated mice. Histological analysis also showed a reduced fibrosis inmdx mice treated with CDCs when compared with control mice.CDCs accumulate preferentially in the lungs 10 min and 24h aftersystemic delivery. We also evaluated clearance of CDCs 1 and 3weeks after systemic delivery in SCID mice.
A
BIODISTRIBUTION AND CLEARANCE
CAP-1002 Manufacturing ProcessCAP-1002 is an allogeneic cell therapy product based on the isolationand expansion of Cardiosphere-Derived Cells (CDCs).
• CDCs have a low immunogenic profile and a strongimmunomodulation of T cells that can limit the inflammatoryprocess observed in DMD patients.
• 150,000 – 250,000 CDCs given intravenously are effective inincreasing exercise capacity and diaphragm muscle function ofmdx mice 3 – 6 weeks after injection.
• Most cells are initially trapped in the lungs, with smallredistribution of cells to the heart within 1 week of injection
• Cells are cleared within 3 weeks after injection.• These findings support clinical evaluation of systemic
administration and repeat dosing of CAP-1002 in patients withDMD.
Figure 1. A) Explant-derived cells (EDCs) are produced from donor heart tissue andexpanded to produce a master cell bank (MCB). B) Cardiospheres are formed from EDCs.C) CDCs are produced by culturing the cells obtained after seeding the cardiospheres.
Figure 3. Performance of the upper limb (PUL) test was used to measure the effects onskeletal muscle function in HOPE I. A) Test description showing shoulder, middle, anddistal level tests. B) Results from middle and distal levels in patients treated with CDCs(red) and placebo (blue).
AdaptedfromTseliou, E.etal.JAmColl Cardiol.2015;66(6):599–611.
Mediu
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**
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D
HLA I96%
HLA II3.6%
Human Leukocyte Antigens (HLA)
CD803.6%
CD8657.3%
Co-Stimulatory
CD274 (PD-L1)45.8%
CD275 (ICOS-L)9.4%
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CFSE
EFFICACY WITH IV ADMINISTRATION
Mouse model: mdx mice (8-10 months old)CDCs: C57BL/10 (syngeneic)
Figure 6. Efficacy of CDCs in mdx mice with IV administration. A) Study timeline.Mice received treatment in Week 0, exercise every week, and were sacrificed in week 6.B) Exercise capacity of mice given escalating doses of CDCs. C) Isolated diaphragmmuscle function. D) Masson’s Trichrome staining of heart tissue sections 6 weeks afterCDC injection.
A dose-dependent improvement in exercise capacity (Fig. 6b) andisolated diaphragm muscle function (Fig. 6c) was observed 6 weeksafter CDC treatment. Histological analysis of hearts reveal lowercollagen deposition (Fig. 6d).
PBS (n=10)
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CDC IMMUNOLOGY
Figure 5. Immune modulation of PHA- induced CD4+ and CD8+ T cells proliferationby CDC. Results are mean percentage values ± SEM from 2 different donors each donein triplicates.
Figure 4. Expression of immune molecules involved in T immune response
Dose Escalation Efficacy
D
CDCs show a low immunogenic phenotype with expression of class IHLA and CD86 co-stimulatory antigen and the immuno-regulatorymarker PD-L1 (Fig. 4). CDCs limit proliferation of in vitro activatedhuman T lymphocytes.
Donorheart MCBExplantcreation
Cardiospheres CDCs AllogeneicCDCproduct
EDCoutgrowth
CDCExpansionMCB
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