Anti-Claudin-2 Antibody-Drug Conjugates for the Treatment of Colorectal Cancer Liver Metastasis.

We have demonstrated that Claudin-2 is required for colorectal cancer (CRC) liver metastasis. Expression of Claudin-2 in primary CRC is associated with poor survival and is highly expressed in liver metastases. Claudin-2 also promotes breast cancer liver metastasis by enabling seeding and cancer cell survival. These observations support Claudin-2 as a potential therapeutic target for managing patients with liver metastases. Antibody-drug conjugates (ADCs) are promising anti-tumor therapeutics that combine the specific targeting ability of monoclonal antibodies with the potent cell killing activity of cytotoxic drugs. Here we report the generation of twenty-eight anti-Claudin-2 antibodies for which the binding specificities, the cross-reactivity with Claudin family members and the cross-species reactivity were assessed by flow cytometry analysis. Multiple drug conjugates were tested and PNU was selected for conjugation with anti-Claudin-2 antibodies binding either extracellular loop 1 or extracellular loop 2. Anti-Claudin-2 ADCs were efficiently internalized and effective at killing Claudin-2-expressing CRC cancer cells in vitro. Importantly, PNU-conjugated-anti-Claudin-2 ADCs impaired the development of replacement type CRC liver metastases in vivo, using established CRC cell lines and patient-derived xenograft (PDX) models of CRC liver metastases. Our results suggest that the development of ADCs targeting Claudin-2 is a promising therapeutic strategy for managing CRC liver-metastatic patients that present with replacement type liver metastases.

Influence of variant-specific mutations, temperature and pH on conformations of a large set of SARS-CoV-2 spike trimer vaccine antigen candidates.

SARS-CoV-2 subunit vaccines continue to be the focus of intense clinical development worldwide. Protein antigens in these vaccines most commonly consist of the spike ectodomain fused to a heterologous trimerization sequence, designed to mimic the compact, prefusion conformation of the spike on the virus surface. Since 2020, we have produced dozens of such constructs in CHO cells, consisting of spike variants with different mutations fused to different trimerization sequences. This set of constructs displayed notable conformational heterogeneity, with two distinct trimer species consistently detected by analytical size exclusion chromatography. A recent report showed that spike ectodomain fusion constructs can adopt an alternative trimer conformation consisting of loosely associated ectodomain protomers. Here, we applied multiple biophysical and immunological techniques to demonstrate that this alternative conformation is formed to a significant extent by several SARS-CoV-2 variant spike proteins. We have also examined the influence of temperature and pH, which can induce inter-conversion of the two forms. The substantial structural differences between these trimer types may impact their performance as vaccine antigens.

A glyco-engineering approach for site-specific conjugation to Fab glycans.

Effective processes for synthesizing antibody-drug conjugates (ADCs) require: 1) site-specific incorporation of the payload to avoid interference with binding to the target epitope, 2) optimal drug/antibody ratio to achieve sufficient potency while avoiding aggregation or solubility problems, and 3) a homogeneous product to facilitate approval by regulatory agencies. In conventional ADCs, the drug molecules are chemically attached randomly to antibody surface residues (typically Lys or Cys), which can interfere with epitope binding and targeting, and lead to overall product heterogeneity, long-term colloidal instability and unfavorable pharmacokinetics. Here, we present a more controlled process for generating ADCs where drug is specifically conjugated to only Fab N-linked glycans in a narrow ratio range through functionalized sialic acids. Using a bacterial sialytransferase, we incorporated N-azidoacetylneuraminic acid (Neu5NAz) into the Fab glycan of cetuximab. Since only about 20% of human IgG1 have a Fab glycan, we extended the application of this approach by using molecular modeling to introduce N-glycosylation sites in the Fab constant region of other therapeutic monoclonal antibodies. We used trastuzumab as a model for the incorporation of Neu5NAz in the novel Fab glycans that we designed. ADCs were generated by clicking the incorporated Neu5NAz with monomethyl auristatin E (MMAE) attached to a self-immolative linker terminated with dibenzocyclooctyne (DBCO). Through this process, we obtained cetuximab-MMAE and trastuzumab-MMAE with drug/antibody ratios in the range of 1.3 to 2.5. We confirmed that these ADCs still bind their targets efficiently and are as potent in cytotoxicity assays as control ADCs obtained by standard conjugation protocols. The site-directed conjugation to Fab glycans has the additional benefit of avoiding potential interference with effector functions that depend on Fc glycan structure.

Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography.

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

AlbuCORE: an albumin-based molecular scaffold for multivalent biologics design.

As biologics have become a mainstay in the development of novel therapies, protein engineering tools to expand on their structural advantages, namely specificity, affinity, and valency are of interest. Antibodies have dominated this field as the preferred scaffold for biologics development while there has been limited exploration into the use of albumin with its unique physiological characteristics as a platform for biologics design. There has been a great deal of interest to create bispecific and more complex multivalent molecules to build on the advantages offered by protein-based therapeutics relative to small molecules. Here, we explore the use of human serum albumin (HSA) as a scaffold for the design of multispecific biologics. In particular, we describe a structure-guided approach to the design of split HSA molecules we refer to as AlbuCORE, that effectively and spontaneously forms a native albumin-like molecule, but in a heterodimeric state upon co-expression. We show that the split AlbuCORE designs allow the creation of novel fusion entities with unique alternate geometries. We also show that, apart from these AlbuCORE fusion entities, there is an opportunity to explore their albumin-like small hydrophobic molecule carrying capacity as a drug conjugate in these designs.

Reaction dynamics of ATP hydrolysis catalyzed by P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug-drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ(18)O4-labeled ATP, no positional isotope exchange is detectable at the bridging ß-phosphorus-O-γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three (18)O/two (18)O/one (18)O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO4(2-) (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states.

Coagulation factor binding orientation and dimerization may influence infectivity of adenovirus-coagulation factor complexes.

Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to hexon and its apparently poor capacity for supporting virus cell entry, we analyzed the HAdv5-FVII complex by using high-resolution cryo-electron microscopy (cryo-EM) followed by molecular dynamic flexible fitting (MDFF) simulations. The results indicate that although hexon amino acids T423, E424, and T425, identified earlier as critical for FX binding, are also involved in mediating binding of FVII, the FVII GLA domain sits within the surface-exposed hexon trimer depression in a different orientation from that found for FX. Furthermore, we found that when bound to hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparan sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. In contrast, earlier cryo-EM studies of the Ad-FX interaction showed no evidence of dimer formation. Dimerization of FVII bound to Ad may be a contributing mechanistic factor for the differential infectivity of Ad-FX and Ad-FVII complexes, despite high-affinity binding of both these coagulation factors to the virus.

Enzymatic detoxication, conformational selection, and the role of molten globule active sites.

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning "induced fit" versus "conformational selection" has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1-1 (GSTA1-1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1-1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that "local" molten globule behavior optimizes detoxication enzymes.

Coagulation factor X activates innate immunity to human species C adenovirus.

Although coagulation factors play a role in host defense for "living fossils" such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding-ablated virus failed to activate a distinct network of nuclear factor κB-dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling. Our study implicates host factor "decoration" of the virus as a mechanism to trigger an innate immune sensor that responds to a misplacement of coagulation FX from the blood into intracellular macrophage compartments upon virus entry into the cell.

Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and dynamics.

To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ((5F)W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that (5F)W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when (5F)W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each (5F)W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates.

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHISNLC and aHISTPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.

Ensemble perspective for catalytic promiscuity: calorimetric analysis of the active site conformational landscape of a detoxification enzyme.

Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. However, catalytic promiscuity is a poorly understood property, and no thermodynamic treatment has described the conformational landscape of promiscuous versus substrate-specific enzymes. Here, two structurally similar glutathione transferase (GST, glutathione S-transferase) isoforms with high specificity or high promiscuity are compared. Differential scanning calorimetry (DSC) indicates a reversible low temperature transition for the promiscuous GSTA1-1 that is not observed with substrate-specific GSTA4-4. This transition is assigned to rearrangement of the C terminus at the active site of GSTA1-1 based on the effects of ligands and mutations. Near-UV and far-UV circular dichroism indicate that this transition is due to repacking of tertiary contacts with the remainder of the subunit, rather than "unfolding" of the C terminus per se. Analysis of the DSC data using a modified Landau theory indicates that the local conformational landscape of the active site of GSTA1-1 is smooth, with barrierless transitions between states. The partition function of the C-terminal states is a broad unimodal distribution at all temperatures within this DSC transition. In contrast, the remainder of the GSTA1-1 subunit and the GSTA4-4 protein exhibit folded and unfolded macrostates with a significant energy barrier separating them. Their partition function includes a sharp unimodal distribution of states only at temperatures that yield either folded or unfolded macrostates. At intermediate temperatures the partition function includes a bimodal distribution. The barrierless rearrangement of the GSTA1-1 active site within a local smooth energy landscape suggests a thermodynamic basis for catalytic promiscuity.

Light chain somatic mutations change thermodynamics of binding and water coordination in the HyHEL-10 family of antibodies.

Thermodynamic and structural studies addressed the increased affinity due to L-chain somatic mutations in the HyHEL-10 family of affinity matured IgG antibodies, using ITC, SPR with van't Hoff analysis, and X-ray crystallography. When compared to the parental antibody H26L26, the H26L10 and H26L8 chimeras binding to lysozyme showed an increase in favorable DeltaG(o) of -1.2+/-0.1 kcal mol(-1) and -1.3+/-0.1 kcal mol(-1), respectively. Increase in affinity of the H26L10 chimera was due to a net increase in favorable enthalpy change with little difference in change in entropy compared to H26L26. The H26L8 chimera exhibited the greatest increase in favorable enthalpy but also showed an increase in unfavorable entropy change, with the result being that the affinities of both chimeras were essentially equivalent. Site-directed L-chain mutants identified the shared somatic mutation S30G as the dominant contributor to increasing affinity to lysozyme. This mutation was not influenced by H-chain somatic mutations. Residue 30L is at the periphery of the binding interface and S30G effects an increase in hydrophobicity and decrease in H-bonding ability and size, but does not make any new energetically important antigen contacts. A new 1.2-A structure of the H10L10-HEL complex showed changes in the pattern of both inter- and intra-molecular water bridging with no other significant structural alterations near the binding interface compared to the H26L26-HEL complex. These results highlight the necessity for investigating both the structure and the thermodynamics associated with introduced mutations, in order to better assess and understand their impact on binding. Furthermore, it provides an important example of how backbone flexibility and water-bridging may favorably influence the thermodynamics of an antibody-antigen interaction.

Association energetics of cross-reactive and specific antibodies.

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG(1) antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.

Fluorescence based structural analysis of tryptophan analogue-AMP formation in single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase.

The symmetrical dimer structure of tryptophanyl-tRNA synthetase is similar to that of tyrosyl-tRNA synthetase whose binding behavior and structural details have been elucidated in detail. The structure of both subunits after forming the intermediate tryptophanyl-AMP has important implications for the binding of the cognate tRNA(Trp). Single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase have been constructed and expressed and used to probe structural changes in different domains of the enzyme in both subunits. Substrate titrations using the Trp analogues 4-fluorotryptophan and 7-azatryptophan in the presence of ATP to form the corresponding aminoacyl-adenylate reveal significant structural changes occurring throughout the active subunit in regions not confined to the active site. Changes in environment around the specific Trp residues were monitored using UV absorbance and steady-state fluorescence measurements. When titrated with 4-fluorotryptophan, both Trp 91 and Trp 290 fluorescence is quenched (49 and 22%, respectively) when one subunit has formed Trp-AMP. The fluorescence of Trp 48 is enhanced 19%. No further change in signal was observed after a 1:1 dimer/L-4FW-AMP complex ratio had been established. Using an anion-exchange filter binding assay with radiolabeled l-Trp as a substrate, binding to only one subunit was observed under nonsaturating conditions. This agrees with the results of the assay using 7-azatryptophan as a substrate. The observed changes extend to the unfilled subunit where a similar structure is believed to form after one subunit has formed tryptophan-AMP. Movement in the regions of the enzyme containing Trp 290 and Trp 91 suggests a mechanism for cross-subunit communication involving the helical backbone and dimer interface containing these two residues.