We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKE™) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting.
Bibliographical noteFunding Information:
Funding: This work was supported in part by the US Public Health Service Grant R01-CA72669, P01-CA65493, P01-CA111412, R35 CA197292, R03-CA2-31766, R03-CA2-16114, 2T32HL007062 Hematology Research Training Program T32 at the University of Minnesota, and P30 CA077598 awarded by the NCI and the NIAID, DHHS. It was also supported by the Randy Shaver Cancer Research and Community Fund, the Mayo Partnership Award, the Lion Fund, the Minnesota Ovarian Cancer Alliance, the CETI Translational Award from the Masonic Cancer Center, Minnesota Masonic Charities, Sarcoma Foundation of America, and the Killebrew–Thompson Memorial Fund.
MA-148 (established locally at the University of Minnesota) is a human epithelial high-grade serous ovarian carcinoma cell line. For in vivo experiments, lines were transfected with a luciferase reporter construct using Invitrogen’s Lipofectamine Reagent and selective pressure applied with 10 µg/mL of blasticidin. Ovarian carcinoma cell lines OVCAR5 and OVCAR8 were obtained from the DTP, DCTD Tumor Repository sponsored by the Biological Testing Branch, Developmental Therapeutics Program, National Cancer Institute (NCI), National Institutes of Health (NIH, Frederick, MD, USA). Other cell lines were obtained from the American Type Culture Collection including OVCAR3 (ovarian), C4-2 (prostate), DU145 (prostate), LNCaP (prostate), PC-3 (prostate), A549 (lung), NCI-H322 (lung), NCI-H460 (lung), and Raji cells (Burkitt’s lymphoma). With the exception of Raji cells, used as a negative control, all lines express high levels of B7-H3 (Figure S1A–E). Lines were maintained in RPMI 1640 medium supplemented with 10–20% fetal bovine serum (FBS) and 2 mmol/L L-glutamine. Lines were incubated at a humidified atmosphere containing 5% CO2 at a constant 37 ◦C. When adherent cells were more than 90% confluent, they were passaged using trypsin-EDTA for detachment. For the cell counts a standard hemocytometer was used. Only those cells with a viability >95% were used for the experiments. The sequence for the monoclonal antibody scFv fragment 376.96 was obtained by Dr. Ferrone and used to construct the TriKE.
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- Bispecific antibodies
- Innate immunotherapy
- NK cells
PubMed: MeSH publication types
- Journal Article