antibody rnai conjugate
IMMU has patent on this technolgy.
Also issued today was U.S. Patent 9,492,561 to IBC Pharmaceuticals, Inc., the Company’s majority-owned subsidiary, for the new patent family “Dock-and-Lock (DNL) complexes for delivery of interference RNA.” This new patent concerns compositions for targeted delivery of small interfering RNA (siRNA) to treat a variety of disease states, including cancer, autoimmune inflammatory disease, or infectious disease, and expires on arch 24, 2026.
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Apoptosis of Pancreatic Cancer Using siRNAs Against CD74 and CEACAM6
The siRNAs for CD74 (sc-35023, Santa Cruz Biotechnology, Santa Cruz, Calif.) and CEACAM6 [sense strand 5'-CCGGACAGUUCCAUGUAUAdTdT-3' (SEQ ID NO:82)], are obtained from commercial sources. Sense and antisense siRNAs are dissolved in 30 mM HEPES buffer to a final concentration of 20 .mu.M, heated to 90.degree. C. for 1 min and incubated at 37.degree. C. for 60 min to form duplex siRNA. The duplex siRNA is mixed with E1-L-thP1 and incubated with BxPC-3 (CEACAM6-siRNA) and Capan2 (CD74-siRNA) cells. After 24 h, the changes in the levels of mRNA for the corresponding proteins are determined by real time quantitative PCR analysis. The levels CD74 and CEACAM6 proteins are determined by Western blot analysis and immunohistochemistry. Controls include nonspecific siRNA and the non-targeting DNL complex 20-L-thP1, which contains a humanized anti-CD20 antibody (hA20).
The effects of reduced expression of CD74 and CEACAM6 on the growth of pancreatic cancer cells is determined using the clonogenic assay. About 1.times.10.sup.3BxPC-3 cells are plated and treated with E1-L-thP1 carrying CEACAM6-siRNA. Media is changed every 3-4 days and after 14 days colonies are fixed with 4% para-formaldehyde solution, stained with 0.5 trypan blue and counted. Similar experiments are performed for Capan2 cells using E1-L-thP1 carrying CD74-siRNA. The effect of E1-L-thP1 carrying both CEACAM6- and CD74-siRNAs on inhibiting the growth of BxPC-3 and Capan2 cells is determined. Cell proliferation by the MTS assay is performed.
Two xenograft models are established in female athymic nu/nu mice (5 weeks of age, weighing 18-20 g). The subcutaneous model has BxPC-3 (ATCC No. CRL-1687) and Capan2 (ATCC No. HTB-80) implanted in opposite flanks of each animal with treatment initiated once tumors reach 50 mm.sup.3. The orthotopic model bears only BxPC-3 cells and treatment is started 2 weeks after implantation.
For the subcutaneous model, the efficacy of E1-L-thP1 to deliver a mixture of CEACAM6- and CD74-siRNAs is assessed and compared to that of E1-L-thP1 to deliver CEACAM6-, CD74-, or control siRNA individually. Additional controls are saline and the use of 20-L-thP1 instead of E1-L-thP1 to deliver the specific and control siRNAs. The dosage, schedule, and administration are 150 .mu.g/kg based on siRNA, twice weekly for 6 weeks, and via tail vein injection (Table 4). Cells are expanded in tissue culture, harvested with Trypsin/EDTA, and re-suspended with matrigel (1:1) to deliver 5.times.10.sup.6 cells in 300 .mu.L.
Animals are monitored daily for signs of toxicity and weighed twice weekly. Tumor dimensions are measured weekly and tumor volumes calculated.
The orthotopic model is set up as follows. Briefly, nude mice are anesthetized and a left lateral abdominal incision is made. The spleen and attached pancreas are exteriorized with forceps. Then 50 .mu.L of a BxPC-3 cell suspension (2.times.10.sup.6 cells) is injected into the pancreas. The spleen and pancreas are placed back into the abdominal cavity and the incision closed. Therapy begins two weeks after implantation. Mice are treated systemically with CEACAM6- or control siRNA bound to E1-L-thP1 or 20-L-thP1 with the same dosing schedule and route as the subcutaneous model. Animals are monitored daily and weighed weekly.
TABLE-US-00017 TABLE 4 Subcutaneous model with dual tumors Group (N) Treatment Dose/Schedule Specific Therapy 1 12 E1-L-thP1-CEACAM6- 150 .mu.g/kg i.v. (twice siRNA weekly .times.6) 2 12 E1-L-thP1-CD74- 150 .mu.g/kg i.v. (twice siRNA weekly .times.6) 3 12 E1-L-thP1-CEACAM6- 150 .mu.g/kg each i.v. (twice siRNA + weekly .times.6) E1-L-thP1-CD74-siRNA Controls 4 12 Saline 100 .mu.L i.v. (twice weekly .times.6) 5 12 20-L-thP1-CEACAM6- 150 .mu.g/kg i.v. (twice siRNA weekly .times.6) 6 12 20-L-thP1-CD74- 150 .mu.g/kg i.v. (twice siRNA weekly .times.6) 7 12 E1-L-thP1-control- 150 .mu.g/kg each i.v. (twice siRNA weekly .times.6) 8 12 20-L-thP1-control- 150 .mu.g/kg each i.v. (twice siRNA weekly .times.6)
The results of the study show that both CEACAM6 and CD74 siRNA are internalized into pancreatic cancer cells by the E1-L-thP1 DNL construct and induce apoptosis of pancreatic cancer, while the control DNL construct with non-targeting anti-CD20 antibody is ineffective to induce siRNA uptake or cancer cell death. "
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