Manipulation of Cell-Type Selective Antibody Internalization by a Guide-Effector Bispecific Design

Abstract

Cell-type-specific intracellular payload delivery is desired for antibody-based-targeted therapy development. However, tumor-specific internalizing antigens are rare to find, and even rarer for those that are expressed at uniformly high levels. We constructed a bispecific antibody that is composed of a rapidly internalizing antibody binding to a tumor-associated antigen, ephrin receptor A2 (EphA2), and a noninternalizing antibody binding to a highly expressed tumor-associated antigen, activated leukocyte cell adhesion molecule (ALCAM). We found that the overall internalization property of the bispecific is profoundly impacted by the relative surface expression level (antigen density ratio) of EphA2 versus ALCAM. When the EphA2-to-ALCAM ratio is greater than a threshold level (1:5), the amount of the bispecific taken into the tumor cell exceeds what is achieved by either the monoclonal internalizing antibody or a mixture of the two antibodies, showing a bispecificdependent amplification effect where a small amount of the internalizing antigen EphA2 induces internalization of a larger amount of the noninternalizing antigen ALCAM. When the ratio is below the threshold, EphA2 can be rendered noninternalizing by the presence of excess ALCAM on the same cell surface. We constructed a bispecific antibody-drug conjugate (ADC) based on the above bispecific design and found that the bispecific ADC is more potent than monospecific ADCs in tumor cell killing both in vitro and in vivo. Thus, the internalizing property of a cell surface antigen can be manipulated in either direction by a neighboring antigen, and this phenomenon can be exploited for therapeutic targeting.

Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study

“…That excludes the efficient recycling back to the membrane, which has for instance been described for HER2-targeted ADC Kadcyla [30]. This is in line with other studies, including one study in which surface depletion of the fast internalizing target antigen is observed [28]. This may also be an explanation for the stabilization of the intracellular accumulation we observed.…”

“…An interesting observation is that addition of a slow internalizing sdAb to a fastinternalizing sdAb led to an even faster internalizing sdAb construct, biparatopic 4A10-3G7. In a recent study, a fast internalizing single-chain variable fragment (scFv) fused to a non-binding antibody was rendered slow internalizing by substitution of the non-binding antibody for a slow-internalizing antibody [28]. Based on this example, one could expect that the biparatopic sdAb construct would internalize slower than 4A10 alone.…”

The Clinical and Theranostic Values of Activated Leukocyte Cell Adhesion Molecule (ALCAM)/CD166 in Human Solid Cancers

“…A recent report by Lee and colleagues [140] has shown that using ADC (antibodydrug conjugate) approaches by producing a bispecific designed antibody conjugate of anti-EphA2 and anti-ALCAM was able to trigger a rapid internalisation of the conjugate at the particular ratio. An interesting observation is that when the ADC conjugated with a toxic drum (MMAF, Monomethyl auristatin F), the ADC-drug produced some impressive toxicity in cells that expressed a high level of ALCAM [140]. This is an interesting approach, although the overall impact of targeting/internalising ALCAM itself on the fate of a cell and potentially in the body yet have to be fully assessed.…”

Beyond the Barrier: Targeted Radionuclide Therapy in Brain Tumors and Metastases

“…This can either be a biologically active cytotoxic drug, or a cytotoxic radionuclide. A potential downside of using cytotoxic drugs is that they need to be internalized by the cancer cell and intracellularly released before they can perform their biological function on quickly dividing cells only [17], whereas utilizing targeted radionuclide therapy (TRNT) allows this radiopharmaceutical to exert its anti-tumor effect when bound to the cell surface by inducing DNA damage through radioactive decay. Depending on the travel distance of the emitted cytotoxic particle, more than one cancer cell can be targeted per decay event.…”