GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth
Background Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target...
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BMJ Publishing Group
2024-12-01
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| Series: | Journal for ImmunoTherapy of Cancer |
| Online Access: | https://jitc.bmj.com/content/12/12/e008891.full |
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| author | Ana Alvarez de Cienfuegos Lawrence H Cheung Khalid A Mohamedali Michael G Rosenblum Brian Aguirre Cheng-Hsiang Lu Anubhav Chandla Nidhi Kejriwal Lucia Liu Ann M Chan SuYin Kok Sergio Duarte David Casero Madhuri Wadehra |
| author_facet | Ana Alvarez de Cienfuegos Lawrence H Cheung Khalid A Mohamedali Michael G Rosenblum Brian Aguirre Cheng-Hsiang Lu Anubhav Chandla Nidhi Kejriwal Lucia Liu Ann M Chan SuYin Kok Sergio Duarte David Casero Madhuri Wadehra |
| author_sort | Ana Alvarez de Cienfuegos |
| collection | DOAJ |
| description | Background Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), and represents an attractive therapeutic target.Methods We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. We assessed the construct’s GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct’s pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated.Results GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with half-maximal cytotoxicity (IC50) values below 100 nM for most positive lines. Ex vivo stability at 37°C indicated a half-life exceeding 96 hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (t1/2α=18.4 hours) and a much slower terminal clearance rate (t1/2β=73.1 hours). No toxicity was measured in a Chem16 panel between the control and the GrB-Fc-KS49. In vivo, the GrB-Fc-KS49 showed efficacy against a TNBC syngeneic (4T1/FLuc) mouse model, reducing tumor volume and cell proliferation and increasing cell death compared with controls. Treatment using an EMT6 mouse model confirmed these results. In addition to a significant impact on cell proliferation, GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor-infiltrating CD45+ cells and redistribution of tumor-associated macrophages. Transcriptomic analysis of tumors post-treatment confirmed the remodeling of the immune tumor microenvironment by the GrB-Fc-KS49 immunotoxin.Conclusions GrB-Fc-KS49 showed high specificity and cytotoxicity towards EMP2-positive cells. In vivo, it reduced tumor burden and increased the recruitment of immune cells into the tumor, suggesting that GrB-Fc-KS49 is a promising therapeutic candidate against BC. |
| format | Article |
| id | doaj-art-28d45bf51ce5496489d720f40a7911bc |
| institution | Kabale University |
| issn | 2051-1426 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | Journal for ImmunoTherapy of Cancer |
| spelling | doaj-art-28d45bf51ce5496489d720f40a7911bc2025-01-27T07:45:09ZengBMJ Publishing GroupJournal for ImmunoTherapy of Cancer2051-14262024-12-01121210.1136/jitc-2024-008891GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growthAna Alvarez de Cienfuegos0Lawrence H Cheung1Khalid A Mohamedali2Michael G Rosenblum3Brian Aguirre4Cheng-Hsiang Lu5Anubhav Chandla6Nidhi Kejriwal7Lucia Liu8Ann M Chan9SuYin Kok10Sergio Duarte11David Casero12Madhuri Wadehra13Experimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, USAExperimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, USAExperimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, USAExperimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USAWidjaja Inflammatory Bowel Disease Institute, Cedars-Sinai Medical Center, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USADepartment of Surgery, University of California Los Angeles, Los Angeles, California, USAWidjaja Inflammatory Bowel Disease Institute, Cedars-Sinai Medical Center, Los Angeles, California, USADepartment of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USABackground Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer (BC), including triple-negative BC (TNBC), and represents an attractive therapeutic target.Methods We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single-chain antibody tethered through the immunoglobulin G heavy chain (Fc) domain. We assessed the construct’s GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct’s pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated.Results GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with half-maximal cytotoxicity (IC50) values below 100 nM for most positive lines. Ex vivo stability at 37°C indicated a half-life exceeding 96 hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (t1/2α=18.4 hours) and a much slower terminal clearance rate (t1/2β=73.1 hours). No toxicity was measured in a Chem16 panel between the control and the GrB-Fc-KS49. In vivo, the GrB-Fc-KS49 showed efficacy against a TNBC syngeneic (4T1/FLuc) mouse model, reducing tumor volume and cell proliferation and increasing cell death compared with controls. Treatment using an EMT6 mouse model confirmed these results. In addition to a significant impact on cell proliferation, GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor-infiltrating CD45+ cells and redistribution of tumor-associated macrophages. Transcriptomic analysis of tumors post-treatment confirmed the remodeling of the immune tumor microenvironment by the GrB-Fc-KS49 immunotoxin.Conclusions GrB-Fc-KS49 showed high specificity and cytotoxicity towards EMP2-positive cells. In vivo, it reduced tumor burden and increased the recruitment of immune cells into the tumor, suggesting that GrB-Fc-KS49 is a promising therapeutic candidate against BC.https://jitc.bmj.com/content/12/12/e008891.full |
| spellingShingle | Ana Alvarez de Cienfuegos Lawrence H Cheung Khalid A Mohamedali Michael G Rosenblum Brian Aguirre Cheng-Hsiang Lu Anubhav Chandla Nidhi Kejriwal Lucia Liu Ann M Chan SuYin Kok Sergio Duarte David Casero Madhuri Wadehra GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth Journal for ImmunoTherapy of Cancer |
| title | GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| title_full | GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| title_fullStr | GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| title_full_unstemmed | GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| title_short | GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| title_sort | grb fc ks49 an anti emp2 granzyme b fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth |
| url | https://jitc.bmj.com/content/12/12/e008891.full |
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