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T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3\u03b5, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell\u2013mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished Fc\u03b3R and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex\u00a0vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19\u20134-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1182\/blood.2023023381","type":"journal-article","created":{"date-parts":[[2024,3,4]],"date-time":"2024-03-04T17:27:50Z","timestamp":1709573270000},"page":"2152-2165","update-policy":"https:\/\/doi.org\/10.1182\/blood.2019cm0000","source":"Crossref","is-referenced-by-count":44,"title":["CD19-CD28: an affinity-optimized CD28 agonist for\u00a0combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy"],"prefix":"10.1182","volume":"143","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7455-0609","authenticated-orcid":false,"given":"Johannes","family":"Sam","sequence":"first","affiliation":[{"name":"1Roche Innovation Center Zurich, Roche Pharma Research and Early Development, Schlieren, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Hofer","sequence":"additional","affiliation":[{"name":"1Roche Innovation 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