Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

PURPOSE: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. RESULTS: After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. CONCLUSIONS: We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction.

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating "knob" and "hole" heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric "knob" and "hole" Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K(D) of <1 nM. Homodimers of the "knob" and "hole" mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines.

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization.

The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.

Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain.

LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca(2+)-coordinating Asp310. The library was panned in Ca(2+)-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K(d) value of 71 nM versus 935 nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca(2+)-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca(2+). EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca(2+)-independent fashion. The findings indicate that randomization of an important Ca(2+)-chelating residue in conjunction with "selection pressure" applied by Ca(2+)-free phage selection conditions can yield variants with an alternatively stabilized Ca(2+) loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca(2+)-binding EGF domain subfamily.

Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab')(2)-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

Antagonists induce a conformational change in cIAP1 that promotes autoubiquitination.

Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.

Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target.

Monoclonal antibodies have therapeutic potential for treating diseases of the central nervous system, but their accumulation in the brain is limited by the blood-brain barrier (BBB). Here, we show that reducing the affinity of an antibody for the transferrin receptor (TfR) enhances receptor-mediated transcytosis of the anti-TfR antibody across the BBB into the mouse brain where it reaches therapeutically relevant concentrations. Anti-TfR antibodies that bind with high affinity to TfR remain associated with the BBB, whereas lower-affinity anti-TfR antibody variants are released from the BBB into the brain and show a broad distribution 24 hours after dosing. We designed a bispecific antibody that binds with low affinity to TfR and with high affinity to the enzyme ß-secretase (BACE1), which processes amyloid precursor protein into amyloid-ß (Aß) peptides including those associated with Alzheimer's disease. Compared to monospecific anti-BACE1 antibody, the bispecific antibody accumulated in the mouse brain and led to a greater reduction in brain Aß after a single systemic dose. TfR-facilitated transcytosis of this bispecific antibody across the BBB may enhance its potency as an anti-BACE1 therapy for treating Alzheimer's disease.

TMEFF2 is a PDGF-AA binding protein with methylation-associated gene silencing in multiple cancer types including glioma.

BACKGROUND: TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA-stimulated fibroblast proliferation in a dose-dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers.

X chromosome-linked inhibitor of apoptosis regulates cell death induction by proapoptotic receptor agonists.

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-alpha signaling. By contrast, blockade of tumor necrosis factor-alpha does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.

Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection.

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Crystal structure of procaspase-1 zymogen domain reveals insight into inflammatory caspase autoactivation.

One key event in inflammatory signaling is the activation of the initiator caspase, procaspase-1. Presented here is the crystal structure of the procaspase-1 zymogen without its caspase recruitment domain solved to 2.05 A. Although the isolated domain is monomeric in solution, the protein appeared dimeric in crystals. The loop arrangements in the dimer provide insight into the first autoproteolytic events that occur during activation by oligomerization. Additionally, in contrast to other caspases, we demonstrate that autoproteolysis at the second cleavage site, Asp316, is necessary for conversion to a stable dimer in solution. Critical elements of secondary structure are revealed in the crystal structure that explain why a dimeric protein is favored after proteolysis at this aspartic acid. Dimer stabilization is concurrent with a 130-fold increase in kcat, the sole contributing kinetic factor to an activated and efficient mediator of inflammation.

High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate.

CD19 and CD21 (CR2) are co-receptors found on B-cells and various B-cell lymphomas, including non-Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization-dependent antibody-drug conjugates [such as antibody-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) (MCC-DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B-cell lines. Anti-CD21 antibodies were not sufficiently internalized even in the highest CD21-expressing Raji cells, resulting in lack of efficacy with anti-CD21-MCC-DM1 conjugates. Anti-CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21-expressing Raji, ARH77 and primary B-cells only very slowly internalized anti-CD19 antibodies, while CD21-negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin-coated vesicles followed by lysosomal delivery. Anti-CD19-MCC-DM1 caused greater cytotoxicity in the faster anti-CD19-internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti-CD19 uptake and decreased anti-CD19-MCC-DM1 efficacy, suggesting that CD21-negative tumours should respond better to such anti-CD19 conjugates. This may have possible clinical implications, as anti-CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B-cell lymphoma patients lack CD21 expression.

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding.

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens.

The complement system serves an important role in clearance of pathogens, immune complexes, and apoptotic cells present in the circulation. Complement fragments deposited on the particle surface serve as targets for complement receptors present on phagocytic cells. Although Kupffer cells, the liver resident macrophages, play a dominant role in clearing particles in circulation, complement receptors involved in this process have yet to be identified. Here we report the identification and characterization of a Complement Receptor of the Immunoglobulin superfamily, CRIg, that binds complement fragments C3b and iC3b. CRIg expression on Kupffer cells is required for efficient binding and phagocytosis of complement C3-opsonized particles. In turn, Kupffer cells from CRIg-deficient mice are unable to efficiently clear C3-opsonized pathogens in the circulation, resulting in increased infection and mortality of the host. CRIg therefore represents a dominant component of the phagocytic system responsible for rapid clearance of C3-opsonized particles from the circulation.

Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity.

Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.

Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.