Unique epitope-antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display.

Carbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX's enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody-epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX's IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX's entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody-epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO2, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO2 by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

Delta agonist hydroxy bioisosteres: the discovery of 3-((1-benzylpiperidin-4-yl){4-[(diethylamino)carbonyl]phenyl}amino)benzamide with improved delta agonist activity and in vitro metabolic stability.

We have investigated phenol replacements in a series of diaryl amino piperidine delta opioid agonists. From this study we have demonstrated that the hydroxy functional group can be replaced with a primary amide group, giving enhanced activity at the delta receptor, increased selectivity versus mu and kappa as well as improved in vitro metabolic stability.

New scaffolds in the development of mu opioid-receptor ligands.

A new class of mu selective receptor antagonists has been developed using a combinatorial approach based on previously reported Dmt-Tic dipeptide ligands. Modified tetrahydroisoquinoline (Tiq) residues were reacted with different electrophiles in order to create novel molecules that would mimic the original dipeptide. A specific class of thioureas bearing basic pyrrolidine residues were shown to give good binding affinities. Further alkylation of the pyrrolidine ring with benzyl derivatives also proved to increase the mu binding affinity. In addition, it was demonstrated that mu binding was enhanced by the presence of polar groups around the benzyl ring having hydrogen-bonding character (donor/acceptor). This new class of ligands represents a novel scaffold in the development of opioid analogues.

Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs.

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.