Company Logo
Case Studies.  Photo a lab benches.

Ion Channels

Target: Human P2X3

Background: P2X3 is a ligand-gated ion channel that is important for peripheral pain responses. P2X3 antagonists may be therapeutic for neuropathic pain, although to date small molecules have not demonstrated sufficient specificity to be effective. A highly specific and antagonistic P2X3 mAb therefore has potential therapeutic value.

Design Goals: The desired antibodies should: (1) bind human P2X3; (2) act as antagonists to block signaling through the receptor; and (3) should not bind other members of the P2X family. In addition, cross reactivity to P2X3 of other species, including rat and mouse, would facilitate evaluation of the antibodies in animal models of disease.

Strategy: Chickens were immunized with P2X3 presented on viral lipoparticles (VLPs, Integral Molecular, Inc.) that were produced from an overexpressing cell line. VLPs contained native conformation P2X3 as well as other naturally occurring membrane components. To avoid recovering mAbs with these off-target specificities, null VLPs were coated onto beads for a negative control. A different color bead coated with P2X3 VLPs was used as a positive indicator. Only GEMs showing antibodies binding to the P2X3 beads and not the null VLP beads were recovered for antibody gene amplification.

Results: The first cohort of recovered antibodies included over 25 unique sequence mAbs that were confirmed to bind specifically to P2X3 lipoparticles and not to null lipoparticles (in ELISA). Further study of the mAb panel revealed that some clones cross-reacted to other members of the P2X family, and many of them bound to rat P2X3. Biosensor analysis demonstrated that most mAbs were subnanomolar in Kd. Some members of the panel were shown to have an antagonistic effect on signaling by ligand through the P2X3 receptor.

Receptor specificity of P2X3

P2X3 mAbs can bind conformational epitopes

P2X3 mAbs are strong binders

Scientific Graph.
Scientific Graph.
Scientific Graph.
Scientific Graph.
Scientific Graph.
Scientific Graph.

4/8 P2X3 mAbs inhibit receptor function

Scientific Graph.
Scientific Graph.
Scientific Graph.
Scientific Graph.

return to the top

GPCRs

Target: Undisclosed GPCR

Background: GPCRs, are notoriously difficult targets for antibody campaigns because they occur in low density on the cell surface and are very unstable when purified away from the cellular membrane, presenting a challenge to obtaining sufficient amounts of immunogen in which native epitopes are maintained for antibody recognition.

Strategy: Chickens were immunized with the N-term domain of human GPCR and/or plasmid DNA containing a full-length expression cassette for human or murine GPCR. During this campaign, GEMs were prepared with both beads and cells. When beads were used, GPCR-Fc or Fc was coated onto white or blue beads, respectively. This approach allowed for the immediate elimination of any clones binding the Fc portion of the immunogen. CHO cells expressing GPCR were also used in the GEMs, with target specificity controlled for by the inclusion of parental CHO cells labeled with an alternative dye. Even though stable CHO lines were available expressing each of four species GPCR (human, cyno, rat, mouse), we generally opted for use of the CHO cell line expressing murine GPCR since we considered it more likely to identify pan-species cross reactive antibodies in an animal that was immunized with the human GPCR.

Results: Hundreds of GPCR-specific unique-sequence mAbs were recovered, with around half being confirmed to be cross-reactive to cyno and rodent versions of GPCR. Of the cross-reactive clones, ~75% were found to be antagonistic in cell-based cAMP assays. Affinities ranged from 0.009nM – 212nM, with a median of 0.7nM. Notably, epitope binning experiments demonstrated that the chicken mAb panel revealed 4 additional and previously unknown epitopes (relative to a pre-existing murine mAb panel).

Chicken mAbs define novel epitope clusters A, B, C and D on human GPCR protein. Rodent mAbs were only found to bind cluster R.

return to the top

Pro-Apoptotic

Target: DR4 / DR5

Background: The TRAIL receptors R1 and R2, also known as DR4 and DR5, respectively, are members of the TNF receptor family. Binding of the ligand TRAIL can induce apoptosis in a variety of cell types and agonistic antibodies to these receptors are also pro-apoptotic. Other TRAIL receptors such as R3 are considered decoy receptors since they lack the intracellular signaling domain and antagonize TRAIL induced apoptosis.

Design Goals: We aimed to find potent agonistic antibodies that could signal through both DR4 and DR5 but were not inhibited by decoy receptors of the TNFR superfamily. Since DR4 and DR5 share only 63% identity at the protein level we anticipated that finding mAbs meeting these criteria would be challenging.

Strategy: Immunize with DR4 or DR4 + DR5, and screen lymphocytes in GEMs with either a bead + cell assay or dual bead assay. For the former, DR4 protein was conjugated onto undyed beads and co-incorporated into GEMs along with a DR4 expressing cancer cell line. For the latter, DR4 protein was conjugated to green beads and DR5 protein to blue beads. For the bioactivity screen, GEMs were selected that contain both a green signal (showing the reporter cells had picked up the apoptosis indicator Caspase-3 FITC) and a red signal (coming from a secondary antibody detecting IgY on the beads). For screening for DR4 + DR5 dual reactive clones, co-localization of the red secondary antibody with intrinsic bead dye was assessed. With mono-reactive clones (either DR4 or DR5 specific) the red signal was seen on either the green beads (DR4) or blue beads (DR5), but not both.

Results: Recombinant antibodies were evaluated for binding in ELISA to DR4 and DR5, and all hits were sequenced. Twenty-seven unique sequence clones were re-transfected and confirmed in ELISA with DR4 and/or DR5 specificity. These clones were also evaluated for pro-apoptotic activity in a single point assay at 10µg/ml. In this cohort we confirmed 6 clones to be DR4/5+, but only three showed significant pro-apoptotic activity. These three clones were chosen for EC50 analysis in the apoptosis assay, and they ranged between 236-703 ng/ml, showing improved potency relative to that of Lexatumumab, a clinical-grade DR5 antibody with an EC50 of 830ng/ml on Jurkat cells.

GEM screen for pro-apoptotic DR4 reactive antibodies.

In vitro apoptosis on target cells using recombinant mAbs recovered from GEM screen.

GEM screen for dual-reactive DR4 + DR5 antibodies (single reactivity in left panel, double in right).

Scientific image. Scientific image.

ELISA screen confirming dual-reactive recombinant mAbs recovered from GEM screen.

return to the top

Conserved Epitopes

Target: Brain-derived neurotrophic factor (BDNF)

Background: BDNF acts on certain neurons of the central nervous system and the peripheral nervous system, helping to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses. The phenotype for BDNF knockout mice can be severe, including postnatal lethality. Other traits include sensory neuron losses that affect coordination, balance, hearing, taste, and breathing. Knockout mice also exhibit cerebellar abnormalities and an increase in the number of sympathetic neurons. Since there is a very high level of homology between human and murine BDNF (~97% AA identity), mice do not typically raise a titer when immunized. Chickens, on the other hand, have a lower homology (~91% identity), allowing them to recognize and respond to human BDNF. This is also true of engineered chickens expressing human sequence antibodies.

Strategy: Wild-type and HuMab chickens were immunized with human BDNF protein using standard dosing and adjuvants. Titers were monitored bi-weekly and spleens harvested when titer began to plateau. GEMs were harvested on the basis of simple binding to BDNF on beads.

Results: BDNF titer came up quickly in both types of birds (wild-type and HuMab) and with similar strength. Responses were similar to other, less conserved targets. A small panel of mAbs were generated from each genotype and evaluated in potency of blocking BDNF interaction with one of its receptors (TrkB), as compared to a benchmark antibody. For both genotypes mAbs were obtained that matched or bettered the potency of the benchmark clone.

return to the top