NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo

Daniel A. Vallera; Soldano Ferrone; Behiye Kodal; Peter Hinderlie; Laura Bendzick; Brianna Ettestad; Caroline Hallstrom; Nicholas A. Zorko; Arpit Rao; Naomi Fujioka; Charles J. Ryan; Melissa A. Geller; Jeffrey S. Miller; Martin Felices

doi:10.3390/cancers12092659

NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo

Daniel A. Vallera, Soldano Ferrone, Behiye Kodal, Peter Hinderlie, Laura Bendzick, Brianna Ettestad, Caroline Hallstrom, Nicholas A. Zorko, Arpit Rao, Naomi Fujioka, Charles J. Ryan, Melissa A. Geller, Jeffrey S. Miller, Martin Felices

Research output: Contribution to journal › Article › peer-review

53 Scopus citations

Abstract

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.

Original language	English (US)
Article number	2659
Pages (from-to)	1-18
Number of pages	18
Journal	Cancers
Volume	12
Issue number	9
DOIs	https://doi.org/10.3390/cancers12092659
State	Published - Sep 2020

Bibliographical note

Funding 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.

Funding Information:
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.

Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

ADCC
Bispecific antibodies
Carcinoma
IL-15
Innate immunotherapy
NK cells
TriKEs

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.3390/cancers12092659

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Cite this

Vallera, D. A., Ferrone, S., Kodal, B., Hinderlie, P., Bendzick, L., Ettestad, B., Hallstrom, C., Zorko, N. A., Rao, A., Fujioka, N., Ryan, C. J., Geller, M. A., Miller, J. S., & Felices, M. (2020). NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo. Cancers, 12(9), 1-18. Article 2659. https://doi.org/10.3390/cancers12092659

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abstract = "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{\texttrademark}) 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.",

keywords = "ADCC, Bispecific antibodies, Carcinoma, IL-15, Innate immunotherapy, NK cells, TriKEs",

author = "Vallera, {Daniel A.} and Soldano Ferrone and Behiye Kodal and Peter Hinderlie and Laura Bendzick and Brianna Ettestad and Caroline Hallstrom and Zorko, {Nicholas A.} and Arpit Rao and Naomi Fujioka and Ryan, {Charles J.} and Geller, {Melissa A.} and Miller, {Jeffrey S.} and Martin Felices",

note = "Funding 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. Funding Information: 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{\textquoteright}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{\textquoteright}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. Publisher Copyright: {\textcopyright} 2020 by the authors. Licensee MDPI, Basel, Switzerland.",

year = "2020",

month = sep,

doi = "10.3390/cancers12092659",

language = "English (US)",

volume = "12",

pages = "1--18",

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T1 - NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo

AU - Vallera, Daniel A.

AU - Ferrone, Soldano

AU - Kodal, Behiye

AU - Hinderlie, Peter

AU - Bendzick, Laura

AU - Ettestad, Brianna

AU - Hallstrom, Caroline

AU - Zorko, Nicholas A.

AU - Rao, Arpit

AU - Fujioka, Naomi

AU - Ryan, Charles J.

AU - Geller, Melissa A.

AU - Miller, Jeffrey S.

AU - Felices, Martin

N1 - Funding 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. Funding Information: 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. Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

PY - 2020/9

Y1 - 2020/9

N2 - 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.

AB - 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.

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KW - Innate immunotherapy

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AN - SCOPUS:85091057168

SN - 2072-6694

VL - 12

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JO - Cancers

JF - Cancers

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ER -

NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo

Abstract

Bibliographical note

Keywords

UN SDGs

Access

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IVIS 100 In Vivo Imaging System

Small Animal Imaging

University Imaging Centers

Cite this

NK-cell-mediated targeting of various solid tumors using a B7-H3 tri-specific killer engager in vitro and in vivo

Abstract

Bibliographical note

Keywords

UN SDGs

Access

OpenUrl availability

Other files and links

Fingerprint

Equipment

IVIS 100 In Vivo Imaging System

Small Animal Imaging

University Imaging Centers

Cite this