A novel monoclonal antibody against human thymic stromal lymphopoietin for the treatment of TSLP-mediated diseases.

Introduction: Thymic stromal lymphopoietin (TSLP) is a master regulator of allergic inflammation against pathogens at barrier surfaces of the lung, skin, and gut. However, aberrant TSLP activity is implicated in various allergic, chronic inflammation and autoimmune diseases and cancers. Biologics drugs neutralizing excess TSLP activity represented by tezepelumab have been approved for severe asthma and are being evaluated for the treatments of other TSLP-mediated diseases.

The process using a synthetic library that generates multiple diverse human single domain antibodies.

Background: Single domain antibodies (sdAbs) possess unique characteristics that make them highly effective for developing complex therapeutics.

Methods: Our process uses a fully synthetic phage display library to generate single domain antibodies that can bind to disease relevant antigen conformations. A human IGHV3 family scaffold makes up the phage display libraries, and these VHO libraries are applied to diverse phage biopannings against target antigens.

A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma.

T-cell-mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein-coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells.

Selection and maturation of antibodies by phage display through fusion to pIX.

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries.

Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage.

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment.

De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII.

Generation, characterization and biological activity of CCL2 (MCP-1/JE) and CCL12 (MCP-5) specific antibodies.

The human CCL2 chemokine is implicated in many chronic inflammatory conditions. In the mouse, there are two CCL2 homologues, CCL2 (MCP-1/JE) and CCL12 (MCP-5). Both are potent monocyte chemoattractants and bind to and activate the same receptor, CCR2.

Isolation of human anti-idiotypic antibodies by phage display for clinical immune response assays.

The clinical development of therapeutic proteins requires assays that measure the pharmacokinetic (PK) profile of, and the potential immune response (IR) to, the protein agent. Each assay requires reagents that are highly specific for the therapeutic protein. For therapeutic monoclonal antibodies, anti-CDR-specific, or anti-idiotypic (anti-id), antibodies are an ideal class of reagents suitable for these assays because of their high specificity and affinity to the drug antibody.

Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies.

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity.

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized.

Chimeric receptors of the human C3a receptor and C5a receptor (CD88).

Chimeras were generated between the human anaphylatoxin C3a and C5a receptors (C3aR and C5aR, respectively) to define the structural requirements for ligand binding and discrimination. Chimeric receptors were generated by systematically exchanging between the two receptors four receptor modules (the N terminus, transmembrane regions 1 to 4, the second extracellular loop, and transmembrane region 5 to the C terminus). The mutants were transiently expressed in HEK-293 cells (with or without Galpha-16) and analyzed for cell surface expression, binding of C3a and C5a, and functional responsiveness (calcium mobilization) toward C3a, C5a, and a C3a as well as a C5a analogue peptide.

Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor.

The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.

Evidence that the receptor for C4a is distinct from the C3a receptor.

The cDNAs encoding the human (hC3aR) and mouse C3a receptors (mC3aR) were functionally expressed in RBL-2H3 cells. A calcium mobilization assay was utilized to assess the biologic activity of human anaphylatoxins, and C3a synthetic peptide agonists on hC3aR and mC3aR cells and this activity was compared to the activity of the anaphylatoxins on human neutrophils. Both hC3aR and mC3aR cells responded in a concentration-dependent manner with a robust calcium mobilization response to C3a with 50% effective concentrations (EC50s) of 0.

The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes.

The pathophysiological relevance of the complement split product C3a as a proinflammatory mediator is still ill defined. The expression pattern of the human C3a receptor (C3aR) can provide important clues for the role of this anaphylatoxin in inflammation. There is strong evidence for C3aR expression on basophils, and eosinophils, but additionally, only on tumor cell lines of leukemic or hepatic origin.

The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization, and functional expression.

The anaphylatoxin C3a receptor (C3aR) is unique among the family of G protein-coupled receptors in possessing an unusually large predicted second extracellular loop. To isolate the mouse C3aR, a probe derived from this extracellular loop was used to screen a mouse brain cDNA library. A 3.

Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning.

We isolated a large panel of human Abs directed against the respiratory syncytial virus (RSV) Ag from combinatorial phage display libraries. Following initial differentiation of the Fabs by BstNI restriction patterns, DNA sequence analysis revealed 10 different classes of VH paired with more than 35 different VL genes. All the Fabs bound with high affinity to the F Ag.

Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.

The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions.

Neutralizing murine monoclonal antibodies to human IL-5 isolated from hybridomas and a filamentous phage Fab display library.

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rIL-5. Spleens from two mice were used to generate hybridomas.

Isolation of neutralizing anti-C5a monoclonal antibodies from a filamentous phage monovalent Fab display library.

A panel of mAbs against the activated complement component C5a was obtained from a filamentous phage M13-Fab display library generated from mice immunized with human rC5a. Fabs isolated from the library after iterative selection vs rC5a bound to both rC5a and purified C5. To isolate Fabs specific for neoepitopes expressed on C5a but not on the native complement component C5 the library was rescreened in a competitive manner.