Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Activity of murine surrogate antibodies for durvalumab and tremelimumab lacking effector function and the ability to deplete regulatory T cells in mouse models of cancer.

Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.

Generating therapeutic monoclonal antibodies to complex multi-spanning membrane targets: Overcoming the antigen challenge and enabling discovery strategies.

Complex integral membrane proteins, which are embedded in the cell surface lipid bilayer by multiple transmembrane spanning helices, encompass families of proteins which are important target classes for drug discovery. These protein families include G protein-coupled receptors, ion channels and transporters. Although these proteins have typically been targeted by small molecule drugs and peptides, the high specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. However, it remains the case that isolation of antibodies with desired pharmacological function(s) has proven difficult due to technical challenges in preparing membrane protein antigens suitable to support antibody drug discovery. In this review recent progress in defining strategies for generation of membrane protein antigens is outlined. We also highlight antibody isolation strategies which have generated antibodies which bind the membrane protein and modulate the protein function.

Preclinical development of a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo.

There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

Therapeutic Monoclonal Antibodies to Complex Membrane Protein Targets: Antigen Generation and Antibody Discovery Strategies.

Cell surface membrane proteins comprise a wide array of structurally and functionally diverse proteins involved in a variety of important physiological and homeostatic processes. Complex integral membrane proteins, which are embedded in the lipid bilayer by multiple transmembrane-spanning helices, are represented by families of proteins that are important target classes for drug discovery. Such protein families include G-protein-coupled receptors, ion channels and transporters. Although these targets have typically been the domain of small-molecule drugs, the exquisite specificity of monoclonal antibodies offers a significant opportunity to selectively modulate these target proteins. Nevertheless, the isolation of antibodies with desired pharmacological functions has proved difficult because of technical challenges in preparing membrane protein antigens for antibody drug discovery. In this review, we describe recent progress in defining strategies for the generation of membrane protein antigens. We also describe antibody-isolation strategies that identify antibodies that bind the membrane protein and modulate protein function.

Identification of optimal protein binders through the use of large genetically encoded display libraries.

The use of large genetically encoded binder libraries in co-operation with display technologies has matured over the past 25 years, and is now one of the primary methods used for selection of protein binders. Display technology has proven to be a robust and versatile method for generating binders to almost any antigen of interest. The evolution of this technology beyond antibody phage display has opened up new aspects for the concept of designer biologics. The ability to construct large populations of eukaryotic cells, including mammalian cells, where each cell expresses an individual antibody, peptide or engineered protein has added great value in identifying binders with desired properties. Here we review the evolution of display technology and highlight how it is being used today to generate binders with exquisite specificity, selectivity, affinity and developability characteristics.

Genetic methods of antibody generation and their use in immunohistochemistry.

Genetic methods of antibody generation offer a highly tuneable tool for the production of target specific reagents suitable for a wide range of applications, including immunohistochemistry. The direct linkage between binder phenotype and genotype enables the rapid identification and manipulation of specific binders into formats suitable for highly specific and sensitive detection of targets from soluble proteins to individual components of multi-protein structures within the context of living tissues. Here we review the types of genetic methods employed and binder formats available and demonstrate how mining huge combinatorial repertoires of binders can deliver diverse and exquisitely sensitive tools for the use in immunohistochemistry. Finally, we offer a perspective on how this approach might be further refined to routinely deliver binders for specific use in immunohistochemical studies.

Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity.

Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC.

Spatial configuration of hepatitis E virus antigenic domain.

Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis. When an HEV capsid protein containing a 52-amino-acid deletion at the C terminus and a 111-amino-acid deletion at the N terminus is expressed in insect cells, the recombinant HEV capsid protein can self-assemble into a T=1 virus-like particle (VLP) that retains the antigenicity of the native HEV virion. In this study, we used cryoelectron microscopy and image reconstruction to show that anti-HEV monoclonal antibodies bind to the protruding domain of the capsid protein at the lateral side of the spikes. Molecular docking of the HEV VLP crystal structure revealed that Fab224 covered three surface loops of the recombinant truncated second open reading frame (ORF2) protein (PORF2) at the top part of the spike. We also determined the structure of a chimeric HEV VLP and located the inserted B-cell tag, an epitope of 11 amino acids coupled to the C-terminal end of the recombinant ORF2 protein. The binding site of Fab224 appeared to be distinct from the location of the inserted B-cell tag, suggesting that the chimeric VLP could elicit immunity against both HEV and an inserted foreign epitope. Therefore, the T=1 HEV VLP is a novel delivery system for displaying foreign epitopes at the VLP surface in order to induce antibodies against both HEV and the inserted epitope.

Application of phage display to high throughput antibody generation and characterization.

We have created a high quality phage display library containing over 1010 human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.

A simple vector system to improve performance and utilisation of recombinant antibodies.

BACKGROUND: Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. RESULTS: We have developed a simple vector system for expression, dimerisation and detection of recombinant antibody fragments in the form of single chain Fvs (scFvs). Expression is driven by the T7 RNA polymerase promoter in conjunction with the inducible lysogen strain BL21 (DE3). The system is compatible with a simple auto-induction culture system for scFv production. As an alternative to periplasmic expression, expression directly in the cytoplasm of a mutant strain with a more oxidising cytoplasmic environment (Origami 2 (DE3)) was investigated and found to be inferior to periplasmic expression in BL21 (DE3) cells. The effect on yield and binding activity of fusing scFvs to the N terminus of maltose binding protein (a solubility enhancing partner), bacterial alkaline phosphatase (a naturally dimeric enzymatic reporter molecule), or the addition of a free C-terminal cysteine was determined. Fusion of scFvs to the N-terminus of maltose binding protein increased scFv yield but binding activity of the scFv was compromised. In contrast, fusion to the N-terminus of bacterial alkaline phosphatase led to an improved performance. Alkaline phosphatase provides a convenient tag allowing direct enzymatic detection of scFv fusions within crude extracts without the need for secondary reagents. Alkaline phosphatase also drives dimerisation of the scFv leading to an improvement in performance compared to monovalent constructs. This is illustrated by ELISA, western blot and immunohistochemistry. CONCLUSION: Nine scFv expression vectors have been generated and tested. Three vectors showed utility for expression of functional scFv fragments. One vector, pSANG14-3F, produces scFv-alkaline phosphatase fusion molecules which offers a simple, convenient and sensitive way of determining the reactivity of recombinant antibody fragments in a variety of common assay systems.

Human monoclonal antibodies that react with the E2 glycoprotein of hepatitis C virus and possess neutralizing activity.

Active and/or passive immunoprophylaxis against hepatitis C virus (HCV) remain unachieved goals. Monoclonal antibodies might provide one approach to protection. We derived human monoclonal antibodies from the bone marrow of a patient with a well-controlled HCV infection of 22 years duration. Five distinct antibodies reactive with the E2 glycoprotein of the homologous 1a strain of HCV were recovered as antigen-binding fragments (FAbs). They demonstrated affinity constants as high as 2 nanomolar. "Neutralization of binding" titers paralleled the affinity constants. All five FAbs reacted with soluble E2 protein only in nonreducing gels, indicating that the relevant epitopes were conformational. The FAbs could be divided into two groups, based on competition analysis. Three of the FAbs neutralized the infectivity of pseudotyped virus particles (pp) bearing the envelope glycoproteins of the homologous HCV strain (genotype 1a). The three FAbs also neutralized genotype 1b pp and one also neutralized genotype 2a pp. In conclusion, one or more of these monoclonal antibodies may be useful in preventing infections by HCV belonging to genotype 1 or 2, the most medically important genotypes worldwide.

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus.

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.

Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine.

In order to obtain monoclonal antibodies that might have prophylactic applications and to understand better the immune response to hepatitis E virus (HEV), we used phage display to isolate chimpanzee antibodies to HEV. The panning antigen was an two open reading frame (ORF2) recombinant protein that elicits a broadly protective immune response in vaccinated monkeys. Two major antigenic sites were identified on the ORF2 protein: one site was not accessible on the surface of infectious virions but the other site was accessible to antibodies and was recognized specifically by antibodies that neutralize the virus.

Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells.

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.