Methods of identifying agents that block MCD28 cleavage by MMPS
Inventors
HAKIM, MOTTI • FRIDMAN-DROR, Anna • Mandel, Ilana • Ben-Moshe, Tehila • Sapir, Yair • Shulman, Avidor
Assignees
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Publication Number
US-12612459-B2
Publication Date
2026-04-28
Expiration Date
Abstract
Methods of decreasing shedding of CD28, decreasing soluble CD28 levels, treating cancer and improving immunotherapies comprising inhibiting matrix metalloproteases are provided. Methods of producing agents for performance of the methods of the invention are also provided.
Core Innovation
The invention concerns a method for producing an agent for decreasing soluble CD28 (sCD28) levels and for treating cancer or improving PD-1 and/or PD-L1 based immunotherapy. The method screens agents for their ability to bind to a CD28 extracellular domain or fragment thereof and selects agents based on their ability to block cleavage of membranal CD28 (mCD28) by MMP-2, MMP-13 or both.
The method includes testing selected agents in at least one assay by contacting cells expressing mCD28 with the selected agent in the presence of MMP-2, MMP-13 or both, and measuring sCD28 levels produced or uncleaved mCD28 levels. A decrease in sCD28 levels or an increase in uncleaved mCD28 levels, compared with levels from cells in the presence of MMP-2, MMP-13 or both in the absence of the agent, indicates that the agent blocks cleavage of mCD28 by MMP-2, MMP-13 or both. The selected agent thereby is produced for decreasing sCD28 levels.
The disclosed agent formats include an antibody, a Fab fragment, a single chain antibody, a DARPin and a single domain antibody. The dependent claim refinements further include screening for binding to CD28 stalk domain features and cleavage-site related sequences within the CD28 stalk domain, and selecting agents that do not substantially agonize or antagonize mCD28 downstream signaling.
Claims Coverage
The claim coverage includes one independent claim directed to producing an agent that decreases sCD28 levels by selecting CD28-binding agents that block mCD28 cleavage by MMP-2 and/or MMP-13 using at least one assay. Dependent claims add refinements for CD28 region/sequence targeting, signaling selectivity, cleavage inhibition specificity, and agent classes.
Screening agents that bind CD28 and select cleavage blockers against MMP-2 and/or MMP-13
Screening agents for their ability to bind to a CD28 extracellular domain or fragment thereof and selecting at least one agent that binds; testing the ability of the selected agent to block cleavage of membranal CD28 (mCD28) by MMP-2, MMP-13 or both by at least one assay; and selecting at least one agent that blocks cleavage of mCD28 by MMP-2, MMP-13 or both, thereby producing an agent for decreasing sCD28 levels, treating cancer, or improving PD-1 and/or PD-L1 based immunotherapy.
Cell-based assay readout selecting agents that decrease sCD28 or increase uncleaved mCD28
Using at least one assay comprising contacting cells expressing mCD28 with the agent in the presence of MMP-2, MMP-13 or both and measuring sCD28 levels produced or uncleaved mCD28 levels on the cells, where a decrease in sCD28 levels or an increase in uncleaved mCD28 levels compared to levels from cells in the presence of MMP-2, MMP-13 or both in the absence of the agent indicates that the agent blocks cleavage of mCD28 by MMP-2, MMP-13 or both.
Binding selection to CD28 stalk domain features and cleavage-site sequences
Screening agents for their ability to bind to a CD28 extracellular domain or fragment thereof and selecting at least one agent that binds, wherein the binding includes binding to the CD28 stalk domain, including MMP-2 and/or MMP-13 cleavage sites within the CD28 stalk domain or PSPL (SEQ ID NO: 15) within the CD28 stalk domain.
Signaling selectivity constraint that does not substantially agonize or antagonize mCD28
Testing an obtained agent for its ability to block cleavage of mCD28 by MMP-2, MMP-13 or both and further assaying downstream signaling with the obtained agent, selecting an agent that does not substantially agonize or antagonize mCD28 signaling.
Selecting agents in defined biologic classes
Selecting that the agent is selected from an antibody, a Fab fragment, a single chain antibody, a DARPin and a single domain antibody.
Separate selection for cleavage blockade specifically against MMP-2 and/or MMP-13
Testing an agent for its ability to block cleavage of mCD28 by MMP-2 and selecting at least one agent that blocks this cleavage, or testing an agent for its ability to block cleavage of mCD28 by MMP-13 and selecting at least one agent that blocks this cleavage, with producing steps including culturing a host cell with vectors encoding the selected agent(s).
The inventive coverage is directed to producing an sCD28-decreasing agent by selecting CD28-binding agents that block mCD28 cleavage by MMP-2 and/or MMP-13 using a cell-based assay measuring sCD28 decrease or mCD28 uncleaved increase. Coverage further emphasizes CD28 stalk-domain or cleavage-site binding, signaling selectivity, defined biologic agent classes, and separate selection against MMP-2 versus MMP-13 mediated cleavage.
Stated Advantages
Decreases soluble CD28 (sCD28) levels.
Treats cancer.
Improves PD-1 and/or PD-L1 based immunotherapy.
Documented Applications
Treating cancer.
Improving PD-1 and/or PD-L1 based immunotherapy.
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