Treating solid tumors is still very difficult. In fact, about 90% of cancer related deaths are caused by solid tumors. A consistent problem has been the inability of monoclonal antibody treatments to reach into and come in contact with the entire tumor. In general, due to toxicity issues, saturating doses of antibodies cannot be used. And, lower doses, particularly of high-affinity antibodies, tend to bind to the outer antigen layer and never reach the inner part of the tumor, localizing at the tumor vasculature. This problem is referred to as the binding site barrier hypothesis. To overcome this problem, researchers from the University at Buffalo used a novel competitive inhibition strategy. In addition, they used a Reichert SR7500DC Surface Plasmon Resonance (2-channel SPR) instrument to help study the problem.1

To treat a solid cancer mass effectively, the treatment needs to be as well distributed into the mass as possible. Researchers refer to the inability of this to happen as the binding site barrier hypothesis. This term has been used for about 30 years and various researchers have tried different strategies to overcome this problem. It is primarily an issue with high affinity antibodies, which bind tightly to outer antigens. And, it is noteworthy that the majority of treatments based on monoclonal antibodies (mAbs) which are approved for use tend to be high affinity.1 So, this is a key issue. Antibody-Drug conjugates (ADCs) or a combination of a targeted antibody linked to an anticancer drug are also known to be affected by the binding site barrier problem with lower ADC concentrations found with increased distance from blood vessels.2 Researchers in this current study determined that competitive inhibition of antibody binding to tumor antigens improved distribution of mAb into solid tumors. They hypothesize that the competitive inhibitor can improve tumor penetration of a high-affinity mAb without impacting the tumor selectivity of the therapeutic mAb. Thus, the competitive inhibitor is acting as an adjuvant to improve response.1


Researchers were interested in seeing improved diffusion of monoclonal antibody-based treatments into tumors. They used SPR to study the interaction between the two antibodies they were using:1 


SR7500DC (2-channel SPR)
 Sensor Chip: Dextran (P/N 13206066, Reichert)
 Temperature:  25 C
 Flow Rate:  25 μL/min
 Target: Trastuzumab
 Analyte: Anti-Trastuzumab single domain antibody 1HE
Running Buffer 10mM phosphate, 150mM NaCl, 0.05% Tween, pH 7.4
 Association Time: 3.5 minutes or 1.5 minutes
Dissociation Time 10 minutes or 10 hours
 Regeneration: 10 mM Glycine pH 2


When just 1HE was administered, it was rapidly eliminated from plasma. But, when trastuzumab was administered with the 1HE, 1HE elimination was much slower.1 SPR (a Reichert 2-channel SR7500DC) was used to determine kinetics for the reaction between 1HE and trastuzumab. Results obtained using either a shorter dissociation (10 minutes) or a much longer dissociation (10 hours) showed that the binding interaction is high affinity with a slow off-rate.1 See selected results below.

In addition to using SPR, researchers also ran a competitive cell-based assay which confirmed that 1HE is a competitive inhibitor of trastuzumab-HER2 binding. When 1HE was dosed with trastuzumab, the combination led to significant increases in how far the trastuzumab permeated from vasculature and also the percentage of the tumor that stained positive for trastuzumab. In addition, when 1HE was combined with a single dosage of T-DM1 (an ADC), NCI-N87 xenograft bearing mice exhibited enhanced T-DM1 efficacy and longer median survival rate.1

Figure 1: Concentrations of 1HE binding to immobilized Trastuzumab range from 1nM to 50nM in the above set of results. The data is fit to a 1:1 binding model with an association rate of 2.6e5 and a dissociation rate of 3.9e-4. Researchers also ran the experiment using concentrations from 10 nM to 35 nM 1HE with a much longer dissociation time of ten hours (see inset for selected view using 1st two hours of dissociation) which yielded a KD of 112 pM and indicated that the dissociation rate was closer to 10-5.



  • Researchers showed that the transient inhibition of trastuzumab-HER2 binding by co-administration of an anti-trastuzumab antibody, 1HE, allowed trastuzumab to bypass the binding site barrier, while retaining the benefit of high-affinity HER2 binding.1
  • The research summarized here focused on the development of an anti-trastuzumab inhibitor, which is timely, because there are already two trastuzumab ADCs that are FDA approved and there is a third in phase III clinical trials.1
  • After results were generated in this study, it was further hypothesized that this competitive inhibition strategy may be applicable to a wide range of high affinity anti-cancer antibody therapies that are on the market and in current development. It may also be a clinically feasible approach to improve the effectiveness of other targeted therapies directed against solid tumors.1