{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T17:56:10Z","timestamp":1784829370065,"version":"3.55.0"},"reference-count":57,"publisher":"BMJ","issue":"12","license":[{"start":{"date-parts":[[2024,12,21]],"date-time":"2024-12-21T00:00:00Z","timestamp":1734739200000},"content-version":"unspecified","delay-in-days":20,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"Clayton Foundation","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"DOI":"10.13039\/100006108","name":"NIH\/National Center for Advancing Translational Sciences","doi-asserted-by":"crossref","award":["UL1TR000124"],"award-info":[{"award-number":["UL1TR000124"]}],"id":[{"id":"10.13039\/100006108","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Iris Cantor-UCLA Women\u2019s Health Center\/CTSI","award":["N\/A"],"award-info":[{"award-number":["N\/A"]}]},{"name":"NCI","award":["R21 CA163971"],"award-info":[{"award-number":["R21 CA163971"]}]}],"content-domain":{"domain":["bmj.com"],"crossmark-restriction":true},"short-container-title":["J Immunother Cancer"],"accepted":{"date-parts":[[2024,11,25]]},"published-print":{"date-parts":[[2024,12]]},"abstract":"<jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>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\u2019s GrB enzymatic activity, anti-EMP2 binding affinity, and cytotoxicity against a panel of BC cells. The construct\u2019s pharmacokinetics (PK), toxicity profile, and in vivo efficacy were also evaluated.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>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 (IC<jats:sub>50<\/jats:sub>) values below 100\u2009nM for most positive lines. Ex vivo stability at 37\u00b0C indicated a half-life exceeding 96\u2009hours while in vivo PK indicated a biexponential plasma clearance, with a moderate initial clearance (t<jats:sub>1\/2<\/jats:sub>\u03b1=18.4\u2009hours) and a much slower terminal clearance rate (t<jats:sub>1\/2<\/jats:sub>\u03b2=73.1\u2009hours). 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\/<jats:sub>FLuc<\/jats:sub>) 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\u2009and 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.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jitc-2024-008891","type":"journal-article","created":{"date-parts":[[2024,12,22]],"date-time":"2024-12-22T09:40:41Z","timestamp":1734860441000},"page":"e008891","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":2,"title":["GrB-Fc-KS49, an anti-EMP2 granzyme B fusion protein therapeutic alters immune cell infiltration and suppresses breast cancer growth"],"prefix":"10.1136","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8249-5304","authenticated-orcid":false,"given":"Khalid A","family":"Mohamedali","sequence":"first","affiliation":[{"name":"Experimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, 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Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucia","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann M","family":"Chan","sequence":"additional","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lawrence H","family":"Cheung","sequence":"additional","affiliation":[{"name":"Experimental Therapeutics, University of Texas MD Anderson Cancer Center Division of Cancer Medicine, Houston, Texas, 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