Combination therapies comprising sirp alpha-based chimeric proteins
A technology of chimeric protein and composition, applied in the direction of drug combination, immunoglobulin, peptide/protein composition, etc., can solve the problems of not being able to stimulate TNF receptors, not being able to block checkpoints, etc.
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Embodiment 1
[0561] Example 1: Generation and Characterization of SIRPα-Fc-CD40L
[0562] The extracellular domain (ECD) of SIRPα was fused to the ECD of CD40L via the antibody Fc domain to generate SIRPα ECD -Fc-CD40L ECD chimeric protein. When the human protein was used, the chimeric protein was called hSIRPα-Fc-CD40L; when the murine protein was used, the chimeric protein was called mSIRPα-Fc-CD40L. Typically, the chimeric protein is referred to as SIRPα-Fc-CD40L. In silico structural modeling predicted that each individual domain of the contiguous construct would fold according to the native molecule, suggesting retention of both binding functions ( Figure 3A ,top). Mammalian cells were then transfected with a construct expressing hSIRPα-Fc-CD40L, and the secreted protein was purified from the conditioned medium by affinity chromatography. The purified protein was then analyzed by Western blot for the presence of each individual domain using anti-SIRPα, anti-Fc and anti-CD40L ant...
Embodiment 2
[0564] Example 2: SIRPα-Fc-CD40L functional activity - CD40L domain
[0565] To examine the functional activity of the CD40L domain of SIRPα-Fc-CD40L, a series of in vitro functional assays were developed. First, the relative signaling activity of SIRPα-Fc-CD40L was determined by two different NFκB reporter systems via the canonical and non-canonical NFκB pathways ( Figure 5A with Figure 5B ). These data demonstrate that hSIRPα-Fc-CD40L has similar activity to unilateral hCD40L fusion proteins in both reporter systems. Importantly, in both assays, hSIRPα-Fc-CD40L was present in soluble form and in the absence of Fc receptors or other crosslinkers. On the other hand, CD40 agonist antibodies failed to stimulate NIK / NFκB activity in the absence of accessory cells providing Fc receptor engagement ( Figure 5B ). These data suggest that SIRPα-Fc-CD40L can stimulate CD40 signaling in the absence of crosslinking, possibly due to the hexameric structure of the chimeric protein....
Embodiment 3
[0568] Example 3: Visualization of tumor cells undergoing phagocytosis
[0569] Here, confocal microscopy can be used to visualize tumor cells undergoing phagocytosis by antigen-presenting cells (eg, macrophages and dendritic cells). Here, the combination of SIRPα(CD172a)-Fc-CD40L chimeric protein and antibodies related to antibody-dependent cytotoxicity (such as anti-CD20 antibody) can stimulate antigen-presenting cells to phagocytose tumor cells. Figure 7A Macrophages labeled fluorescently with CD11b are shown ( Figure 7A ) and fluorescently labeled tumor cells stained with FITC ( Figure 7B ). Figure 7C to Figure 7E Confocal microscopy images of fluorescent markers (FITC staining) for tumor cells are each shown. Figure 7F to Figure 7H Each shows tumor cells (FITC staining, Figure 7F ), macrophages (DAPI staining, Figure 7G ) and macrophages (DAPI staining, mosaic images, Figure 7H ) confocal microscopy images of fluorescent markers. By combining confocal image...
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