Evaluating Fc gamma receptors

This article is based on a poster originally authored by Zachary Fagiani, Rebecca L. Rich, Noah T. Ditto, Larry Yu, Michael Friedman, Glory Gao, Spencer Chiang and Jane Liu from ACROBiosystems..

Fc-gamma receptors (FcγRs) are a group of cell surface proteins found on a range of immune cells. These proteins represent the primary cell signaling method between IgG antibodies and our immune systems.

Their close interactions with the human immune system mean that Fc-containing biologics are rapidly becoming a significant and ever-expanding drug class with more than 175 antibody therapeutics already approved or currently undergoing regulatory review.

Antibody therapeutics’ drug discovery and development stage requires careful assessment of FcγR binding in order to ensure the safety and efficacy of the final therapeutic. This includes wild-type FcγR binding, various species cross-reactivity, and Fc mutation impact.

FcγR’s diversity and its associated replicates require numerous studies, resulting in an increase in instrument lab time, use of consumables, and protein/antibody requirements.

A high-throughput panel of FcγR was developed in order to simplify the analytical characterization of potential antibody therapeutics. This was made possible by pairing Carterra’s LSA^XT with ACROBiosystems’ proteins.

This article outlines an example workflow that utilized High-Throughput SPR (HT-SPR) on the LSA^XT in order to characterize trastuzumab (IgG1 mAb) against eleven FcγRs. This approach resulted in highly sensitive, reproducible characterization using a fraction of the time and resources of other methods.

Figure 1. Different types of FcyRs that have different affinities for IgG as well as structures that modulate the immune system. Image Credit: ACROBiosystems

Methods and materials

A number of FcγRs receptors were tested as a binding panel against trastuzumab on both the Biacore 8K and LSA^XT. These are presented in Table 1. Both studies leveraged an identical experimental design comprised of multi-cycle regenerative binding.

Each instrument was run at 22 °C using a combination of streptavidin-coated chips and biotinylated receptors. An assay buffer comprised of Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.01 % Tween-20 was used.

Receptors were diluted to around 1 μg/ml and captured on the streptavidin surface. This process was followed by injections of trastuzumab from 12 nM up to 24 uM. Data was double-referenced in every instance.

Fitting was performed using a steady-state model for more rapid interactions, while a 1:1 Langmuir model was used for slower interactions.

Table 1. FcγRs used from ACROBiosystems. Source: ACROBiosystems

The full set of eleven receptors was captured at a concentration of 1 µg/mL or lower using the SAHC30M sensor chip. The capture level of each biotinylated receptor replicate exhibited a low standard deviation (<15 %), enabling highly comparable replicates.

It should be noted that CD64 was run separately on the 8K using Biacore’s regenerative biotin CAPture chip. This enabled direct comparison with the LSA^XT’s non-regenerative assay format.

Results

Comparison of LSA^XT with Biacore 8K

Figure 4 features binding characterizations for a total of four different FcγRs as measured on the LSA^XT and 8K. Results were found to be generally comparable between the two instruments, with the LSA^XT able to resolve both low and high affinity with peak-to-peak RU variations in the single digits along with low background noise.

High-resolution sensorgrams were acquired, with the LSA^XT simultaneously performing each of the four binding characterizations.

Figure 2. Four FcγRs (CDA-H82E6/CDA-H82E7/CDA-H82E8/CDA-H82E9) were selected to compare the performance of the Biacore 8K and LSA^XT with trastuzumab. Image Credit: ACROBiosystems

Table 2. Association (left) and dissociation (right) constants of selected FcγRs from the LSA FcyRs measured on the Carterra LSA^XT and Biacore 8K. Source: ACROBiosystems

The resulting affinities were found to be highly similar between both instruments. All receptors were analyzed using a 1:1 Langmuir model.

It was observed that affinities were statistically indistinguishable, and dissociation values (K_Ds) were all statistically similar, other than CD64, which likely differed due to variations in chip chemistry.

Uniformity of HT-SPR sensorgrams

Data was found to be uniform across replicate FcγRs on the LSA^XT for both slow and rapid interactions (Figure 3). This highlights how a combination of high-quality reagents and robust assay design is key to ensuring highly confident measurements.

The LSA^XT was demonstrated as being able to generate comparable data to that of the 8K, while requiring considerably less time, consumable supplies, and sample quantity. It was noted that the LSA^XT’s cost per measurement was over 28 times less than that of the 8K.

Figure 3. Binding profiles of trastuzumab against a panel of 11 FcyRs with 8 replicates of each. Image Credit: ACROBiosystems

Conclusion

Carterra’s LSA^XT, working in conjunction with high-quality and consistent FcγRs from ACROBiosystems, further expands the possibility of high-throughput SPR. This advanced setup is able to provide high-resolution sensorgrams and binding kinetics data that are comparable to those of the Biacore 8K.

This article featured a robust, reproducible workflow able to effectively characterize potential antibody therapeutics against a wide-ranging panel of FcγRs. Using this approach, it is possible to quickly capture sensorgrams, kinetic constants, and other data at a fraction of the cost, sample requirements, and lab time of other methods, with no notable loss in data quality.

This method introduces a new level of efficiency into any laboratory and can also be applied to a wide range of characterizations, including drug screening or other receptor panels.

Acknowledgments

Produced from materials originally authored by Zachary Fagiani from Dragonfly Therapeutics; Rebecca L. Rich and Noah T. Ditto from Carterra; and Larry Yu, Michael Friedman, Glory Gao, Spencer Chiang, and Jane Liu from ACROBiosystems.

About ACROBiosystems

ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.

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