Design,Synthesis And Antitumor Activity Of Rhamnose Conjugated Shiga Toxin B Subunit

Gb3（Globotriaosylceramide）is a tumor-associated carbohydrate antigen that abnormally expresses in various tumors including colorectal cancer,rendering it as a potential tumor marker for targeted-therapy.However,currently no effective strategy has been reported for Gb3-targeted cancer therapy.Shiga toxin B subunit（StxB）is a natural Gb3-binding protein,and it has been widely used as a targetting ligand for Gb3-targeted tumor imaging and drug delivery.Antibody-recruiting molecule is a type of bifunctional chimeric molecule consisting of recruiting ligand for endogenous antibodies and targeting ligands for specific antigens,which mediates tumor immounotherapy by recruiting endogenous antibodies to attack target cells.Inspired by the mechanisms of antibody recruiting molecule,we here desinged and prepared a series of novel Gb3-targeted antibody recruiting molecules which used StxB as the targeting ligand and rhamnose as the recruiting ligand.We studied the structure-activity relationship of the conjugates by determining the affinity,specificity,and antibody recruitment ability.Finally,we assessed the antitumor activity of the conjugates in vitro and in vivo using cancer cells and mouse model.The main conclusions are as follows:（1）Recombinant Stx1B and Stx2B fused with Myc tag,Sortase A tag and His tag,with the yields of 20 mg·L^-1and 18 mg·L^-1,respectively;Rhamnose derivative containing triglycine and a PEG linker rhamnose-（PEG）_n-GGG（n=1,3 and 6）were chemically synthesized;Six StxB-Rhamnose conguates were prepared by coupling the rhamnose derivatives with Stx1B or Stx2B through the Srt A enzyme-mediated site-specific ligation,which were named 1B-n R（n=1,3 and 6）and 2B-n R（n=1,3 and 6）.（2）Particle size distribution and Gb3 binding analysis showed that all StxB-Rhamnose conjugates maintained the pentameric composition of StxB and the affinity to Gb3.Cell-based Gb3 targeting assay and rhamnose antibody-recruitment assay showed that all StxB-Rhamnose conjugates could selectively recognize tumor cells and recruit anti-rhamnose antibodies.1B-3R and 2B-1R showed the best targeting abilitiy,while 1B-6R and 2B-6R showed the best antibody recruitment ability.（3）In vitro tumor cell killing experiments showed that all conjugates can mediate antibody-dependent cell-mediated cytotoxicity（ADCC）and complement-dependent cytotoxicity（CDC）to different degrees.1B-3R and 2B-3R showed the highest ADCC activity of 40.9%and 46%.1B-3R and 2B-3R showed the CDC activities of 67.1%and 77.6%.（4）The conjugate 1B-3R showed a significant tumor inhibitory effect in a murine xenograft model with inhibition rate of 49.1%;Histochemical staining analysis demonstrated that 1B-3R was generally safe as it had no significant toxicity to major organs of the tested mice.