Sanofi has developed a simpler way to create site-specific antibody-to-mannose-6-phosphate (M6P) conjugates that eliminates the need for enzyme remodeling. This more straightforward method makes M6P antibody conjugations a more favorable, biologics-based option to achieve targeted protein degradation in lysosomes. Ultimately, these antibody conjugates may enhance the efficacy of therapeutic monoclonal antibodies.
Coupling the M6P glycan to an antibody makes it easier for the entire molecule to be taken up by the cells and to target the lysosome, thus helping the payload to reach its target.
But, creating the M6P antibody conjugate has its share of challenges. The N-glycans are buried inside the CH2 cavity. Unless they are remodeled, the glycans are simple and truncated, which makes them unsuitable for conjugation. Typically, that is addressed by using enzyme modification to replace parts of the N-glycans with a functionalized glycan or sugar nucleotide that has a conjugatable handle.
There is a simpler solution, however, according to Kaori Mukai, PhD, principal scientist at Sanofi, and colleagues, writing in a recent paper. The scientists have created a way to develop antibody (M6P) conjugates without enzyme modification in vitro. The resulting conjugates show good consistency and low aggregation.
Two-step process
For biologics manufacturers, “Our method is relatively simple, with only two major steps in the process and without any need for other enzymes or reagents,” Mukai tells GEN. “The enzyme conjugates using similar process have been generated at large scale in manufacturing.”
The scientists used oxime chemistry in a process that resembles an enzyme conjugation method the team previously developed. Rather than coupling the high-affinity synthetic M6P glycan known as bisM6P through nonspecific conjugation, the Sanofi team coupled bisM6P to sialic acid residues of an Fc-engineered antibody NNAS mutant to eliminate the antibody’s effector functions.
The team accomplished this by “switching the native glycosylation site from position 297 to 298, converting non-sialylated structure to highly-sialylated N-glycans,” they wrote in a recent paper. Then “the sialic acid of the glycans [that were] attached to Asn298 in the engineered antibody was conjugated to bisM6P without chemoenzymatic modification.”
Experiments showed the “mainly homogenous” conjugate that resulted from this process internalized the targeted soluble proteins—in this case, human tumor necrosis factor (TNF)—in cancer cell lines as well as normal human immune cells including basophils, neutrophils, B cells, and monocytes. “TNF in cell culture media was significantly depleted after the cells were incubated with the M6P-conjugated antibody,” they report.
To achieve these results, “only one copy of the glycan is required for the antibody-mediated TNF internalization.” Examinations of multiple antibodies with this mutation showed the antibodies were comparable to the parental antibody in terms of antigen binding, titer, stability, FcRn binding, and pharmacokinetics.
As this method advances, “There is always a need for further optimization with antibody conjugation,” Mukai says.
