Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies.

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their V regions are functional as single domains, forming the smallest active antibody fragment. These V regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human V regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals.

Strategies to Obtain Diverse and Specific Human Monoclonal Antibodies From Transgenic Animals.

Techniques to obtain large quantities of antigen-specific monoclonal antibodies (mAbs) were first established in the 1970s when Georges Köhler and César Milstein immortalized antibody-producing mouse B-lymphocytes by fusion with myeloma cells (http://www.whatisbiotechnology.org/exhibitions/milstein).

Antigen-specific single B cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies.

Background: There is an ever-increasing need of monoclonal antibodies (mAbs) for biomedical applications and fully human binders are particularly desirable due to their reduced immunogenicity in patients. We have applied a strategy for the isolation of antigen-specific B cells using tetramerized proteins and single-cell sorting followed by reconstruction of human mAbs by RT-PCR and expression cloning.

Results: This strategy, using human peripheral blood B cells, enabled the production of low affinity human mAbs against major histocompatibility complex molecules loaded with peptides (pMHC).

Human antibody production in transgenic animals.

Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful.

Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences.

High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igκ/Igλ loci bearing the rat CH region.

Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery.

Human monoclonal antibodies to HIV-1 gp140 from mice bearing YAC-based human immunoglobulin transloci.

Mice carrying human immunoglobulin transloci were immunised with HIV-1 gp140 antigen to gain insight into the range and nature of human monoclonal antibodies (mAbs) that can be elicited from such humanised mice. Using five-feature mice that harbour YAC-based germline-configuration human IgM, Igκ and Igλ transloci in a mouse background disrupted for endogenous mouse IgH and Igκ expression, gp140-specific human IgM mAbs were readily elicited following serial immunisation. These mAbs were converted to human IgG1 format and were found to bind diverse epitopes within gp140, exhibiting high functional affinity for the antigen-typically in the nanomolar or sub-nanomolar range.

Characterization of immunoglobulin heavy chain knockout rats.

The rat is a species frequently used in immunological studies but, until now, there were no models with introduced gene-specific mutations. In a recent study, we described for the first time the generation of novel rat lines with targeted mutations using zinc-finger nucleases. In this study, we compare immune development in two Ig heavy-chain KO lines; one with truncated Cμ and a new line with removed JH segments.

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice.

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal.

Removal of the BiP-retention domain in Cmicro permits surface deposition and developmental progression without L-chain.

Nascent, full length, immunoglobulin (Ig) heavy (H)-chains are post-translationally associated with H-chain-binding protein (BiP or GRP78) in the endoplasmic reticulum (ER). The first constant (C) domain, CH1 of a C gene (Cmu, Cgamma, Calpha), is important for this interaction. The contact is released upon BiP replacement by conventional Ig light (L)-chain (kappa or lambda).

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice.

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies.

Heavy-chain-only antibody expression and B-cell development in the mouse.

Antibodies expressed in mice, humans, and most mammals consist of paired heavy (H) and light (L) chains. Cellular release of single H-chains without L-chains is prevented by chaperone association and retention in the endoplasmic reticulum. Consequently, H-chain-only antibodies are absent, except in pathological conditions known as Heavy Chain Disease, where they occur in mutated form.

Expression of a dromedary heavy chain-only antibody and B cell development in the mouse.

In mature B cells of mice and most mammals, cellular release of single H chain Abs without L chains is prevented by H chain association with Ig-specific chaperons in the endoplasmic reticulum. In precursor B cells, however, surface expression of mu-H chain in the absence of surrogate and conventional L chain has been identified. Despite this, Ag-specific single H chain Ig repertoires, using mu-, gamma-, epsilon-, or alpha-H chains found in conventional Abs, are not produced.

The two-domain hypothesis in Beckwith-Wiedemann syndrome: autonomous imprinting of the telomeric domain of the distal chromosome 7 cluster.

A large cluster of imprinted genes is located on the mouse distal chromosome 7. This cluster is well conserved in humans and its dysregulation results in the overgrowth- and tumour-associated Beckwith-Wiedemann syndrome. Two imprinting centres (IC1 and IC2) controlling different sets of genes have been identified in the cluster, raising the hypothesis that the cluster is divided into two functionally independent domains.

Silencing of the immunoglobulin heavy chain locus by removal of all eight constant-region genes in a 200-kb region.

Silencing or removal of individual C (constant)-region genes and/or adjacent control sequences did not generate fully deficient Ig (immunoglobulin)- mice. A reason is that different C genes share many functional tasks and most importantly are individually capable of ensuring lymphocyte differentiation. Nevertheless, incomplete arrests in B-cell development were found, most pronounced at the onset of H-chain expression.

Heavy-chain only antibodies derived from dromedary are secreted and displayed by mouse B cells.

Whereas functional heavy (H)-chain antibodies devoid of light (L)- chains account for about half of the circulating immunoglobulins in Camelidae, H-chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen-recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H-chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen-egg lysozyme (HEL). This IgG2a H-chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H- or L-chains.

Block in development at the pre-B-II to immature B cell stage in mice without Ig kappa and Ig lambda light chain.

Silencing individual C (constant region) lambda genes in a kappa(-/-) background reduces mature B cell levels, and L chain-deficient (lambda(-/-)kappa(-/-)) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g.