Shively, J. E. | Search bioRxiv

bioRxiv2026

Direct interactions of CEACAM1 and CD36 with LPS and each other

Micellar lipopolysaccharide (mLPS), a major shed product of gram-negative bacteria in the intestine, may result in TLR4-mediated sepsis in the circulation. CEACAM1 and CD36, expressed on both epithelia and immune cells, regulate TLR4 signaling and inflammation, suggesting a shared role in innate immunity. Furthermore, CEACAM1 associates with CD36 on hepatocytes, regulating lipid storage and bile acids (BAs) in the liver, where LPS detoxification occurs. Using E. coli mLPS-Ra as a model and soluble forms sCEACAM1 or sCD36, we assessed their direct binding to mLPS by SPR, SEC, and TEM. SPR derived steady state affinity constants (KD= 1.47 x 10-6 M and KD= 2.23 x 10-6 M) were obtained for mLPS-Ra binding to sCEACAM1 and sCD36, respectively. mLPS-Ra binding to sCEACAM1 and sCD36 was reduced by bile acids in order of sodium deoxycholate > sodium cholate. mLPS exhibited dose dependent binding to sCEACAM and sCD36 by SEC and the isolated complexes formed well defined micelles on TEM. A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36. Antibody-based-proximity ligation assays (PLA) demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic (P< 0.001) and HEK cells (P< 0.018), while biotin-based PLA demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein in HEK cells. Alpha Fold predicted CD36 binding in cis to CEACAM1 in a membrane model, in agreement with the results of the PLAs. Based on the SPR, SEC and TEM binding studies, CEACAM1 and CD36 share a cooperative mLPS binding function distinct from their regulation of TLR4 binding.

biochemistry↗

bioRxiv2019

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell Engaging antibodies) for cellular immunotherapy

Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. Here we show that an intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (db BiTES, 300 kDa). The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of the 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES maintain high in vivo tumor targeting as demonstrated by PET imaging, and redirect dbBiTE coated T-cells (1 microgram/10 million cells) to kill CEA+ target cells both in vitro, and in vivo in CEA transgenic mice.

cancer biology↗