Methods of identifying agents that block MCD28 cleavage by MMPS

A method for producing an agent for decreasing soluble CD28 (sCD28) levels, treating cancer, or improving PD-1 and/or PD-L1 based immunotherapy, the method comprising:

• screening agents for their ability to bind to a CD28 extracellular domain or fragment thereof and selecting at least one agent that binds, testing an ability of said at least one 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;
• or
• culturing a host cell comprising one or more vectors comprising a nucleic acid sequence encoding an agent, wherein the nucleic acid sequence is that of an agent that was selected by:
• i. screening agents for their ability to that bind to a CD28 extracellular domain or fragment thereof and selecting at least one agent that binds;
• ii. testing an ability of said at least one selected agent to block cleavage of mCD28 by MMP-2, MMP-13 or both;
• and
• iii. selecting at least one agent that blocks cleavage of mCD28 by MMP-2, MMP-13 or both;
• wherein said agent is selected from an antibody, a Fab fragment, a single chain antibody, a DARPin and a single domain antibody;
• and
• wherein said at least one assay comprises:
• contacting cells expressing said mCD28 with said agent in the presence of said MMP-2, MMP-13 or both and measuring sCD28 levels produced or uncleaved mCD28 levels on said cells, wherein a decrease is sCD28 levels or an increase in uncleaved mCD28 levels as compared to levels from/on cells in the presence of said MMP-2, MMP-13 or both in the absence of said agent indicates the agent blocks cleavage of mCD28 by MMP-2, MMP-13 or both;
• thereby producing an agent for decreasing soluble CD28 (sCD28) levels, treating cancer, or improving PD-1 and/or PD-L1 based immunotherapy.