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A Transgenic Heavy Chain IgG Mouse Platform as a Source of High Affinity Fully Human Single-Domain Antibodies for Therapeutic Applications

The antibody repertoires of transgenic mice expressing human heavy chain only antibodies (HCAbs) can be retrieved from immune cells after antigen challenge. Compared with genetically modified rodents expressing conventional human antibodies (tetramers consisting of two heavy chains paired with two light chains), there is no chain pairing problem, since each antibody consists of a heavy chain dimer which is solely responsible for antigen binding. HCAbs can be obtained by classical hybridoma fusion, or the generation of phage libraries or eukaryotic cell libraries displaying or secreting HCAbs. Combined transcriptomic/serum proteomic approaches can also be used to determine the repertoire of antibodies, as well as single cell technologies such as the Beacon system that enable capture of immune cells of interest, analysis, and sequencing of antibodies in a short period of time.
Here, we describe a protocol for obtaining monoclonal HCAbs from immunized Harbour transgenic mice through the generation and screening of HEK cell libraries of secreted antibodies. The method can be used routinely and is fast and affordable for everyone. Selected VH regions (single domains) are sequenced and individual HCAbs can be produced and purified from the same expression vector that is used for library generation (hIgG1 Fc). They can also be cloned into other expression plasmids and reformatted to equip them with a particular effector function, modify lifespan in serum, or optimize valency and avidity depending on the specific aim.

Adaptation of an ELISA assay for detection of IgG 2a responses against therapeutic monoclonal antibodies in a mouse immunization model

Biotherapeutic monoclonal antibodies (mAb) play important roles in clinical medicine but their potential to elicit immune responses in patients remains a major issue. In a study designed to investigate the effect of aggregation on immunogenic responses, mice were immunized with two monoclonal antibodies (mAb1 and mAb2). Serum levels of total IgG, IgG1, and IgG2a were measured by ELISA. An anti-mouse IgG2a monoclonal detection antibody cross-reacted with mAb2 but not mAb1, leading to high background when the ELISA plate was coated with mAb2. The problem was solved by use of a goat anti-mouse IgG2a polyclonal antibody that demonstrated the required specificity. IgG2a responses were similar for monomer- or aggregate-coated ELISA plates. The results demonstrate the importance of assessment of the specificity of individual reagents when measuring antibody responses against therapeutic antibodies by ELISA.

IgM to IgG Class Switching Is a Necessary Step for Pemphigus Phenotype Induction in Desmoglein 3-Specific B Cell Receptor Knock-in Mouse

Pemphigus vulgaris is an autoimmune blistering disease caused by IgG targeting desmoglein 3 (Dsg3), an adhesion molecule of keratinocytes. Anti-Dsg3 IgG production is prevented in healthy individuals, but it is unclear how Dsg3-specific B cells are regulated. To clarify the immunological condition regulating Dsg3-specific B cells, a pathogenic anti-Dsg3 Ig (AK23) knock-in mouse was generated. AK23 knock-in B cells developed normally without undergoing deletion or acquiring an anergic phenotype in vivo. The knock-in B cells showed Ca2+ influx upon IgM cross-linking and differentiated into AK23-IgG+ B cells after LPS and IL-4 stimulation in vitro that induced a pemphigus phenotype after adoptive transfer into Rag2 -/- mice. However, the knock-in mouse itself produced AK23-IgM but little IgG without blisters in vivo. Dsg3 immunization and skin inflammation caused AK23-IgG production and a pemphigus phenotype in vivo.
Furthermore, Fcgr2b deficiency or haploinsufficiency spontaneously induced AK23-IgG production and a pemphigus phenotype with poor survival rates in AK23 knock-in mice. To assess Fcgr2b involvement in Ig class-switch efficiency, postswitch transcripts of B cells were quantified and significantly higher in Fcgr2b-/- and Fcgr2b+/- mice than wild-type mice in a gene dose-dependent manner. Finally, RNA sequencing revealed reduced expression of FCGR2B and FcγRIIB-related genes in patient B cells. These results indicated that Dsg3-specific B cells do not spontaneously perform pathogenic class switching in vivo, and pemphigus phenotype induction was prevented under normal conditions. Attenuated FcγRIIB signaling is also one of the drivers for pathogenic class switching and is consistent with immunological features identified from clinical samples. This study unveiled a characteristic immune state silencing autoreactive B cells in mice.

Addition of an Fc-IgG induces receptor clustering and increases the in vitro efficacy and in vivo anti-tumor properties of the thrombospondin-1 type I repeats (3TSR) in a mouse model of advanced stage ovarian cancer

 Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound.
 Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated.
 Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer.
 The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer.

 Identification of a Guinea Pig Fcγ Receptor that Exhibits Enhanced Binding to Afucosylated Human and Mouse IgG

Glyco-engineered recombinant antibodies are currently being developed as the next generation therapeutics to treat human diseases, including cancer, autoimmunity and infection. Antibodies lacking core fucosylation show great increase in affinity for FcγRIIIA, leading to an improved receptor-mediated effector function. While afucosyl human IgG1 exhibits 50-100-fold increase in antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism underlying the anti-cancer effect of some approved therapeutic antibodies, it is not clear whether such glyco-engineered antibodies would find similar use for infectious disease. Due to the species difference, human antibodies may have different binding properties towards corresponding IgG receptors from animals used for modeling infection and intoxication.

Mouse IgG

1265-100 Biovision each 183.6 EUR

Mouse IgG

1265-1000 Biovision each 444 EUR

Mouse IgG

31C-CH1001 Fitzgerald 25 mg Ask for price

Mouse IgG

31-1011 Fitzgerald 1 mg 150 EUR

Mouse IgG

31-1012 Fitzgerald 1 mg 175 EUR

Mouse IgG

31-AM15 Fitzgerald 10 mg 116 EUR

Mouse IgG

31-AM15S Fitzgerald 10 mg 156 EUR

Mouse IgG

31-AM15S-LQ Fitzgerald 20 mg 147 EUR

Mouse IgG

E61I007 EnoGene 1mg 225 EUR

Mouse IgG

IHR-8152-10 ImmunoBioscience 10 mg 47.75 EUR

Mouse IgG

IHR-8152-100 ImmunoBioscience 100 mg 120.05 EUR

Mouse IgG

E-AB-1122-1mg Elabscience Biotech 1mg 70 EUR

Mouse IgG

E-AB-1122-each Elabscience Biotech each Ask for price

Mouse IgG

BA1046 Antagene 5mg 238 EUR

Mouse IgG Fc

31R-1077 Fitzgerald 1 mg 378 EUR

Mouse IgG (Fc)

31-AM15FC Fitzgerald 1 mg 338 EUR

Mouse IgG (HRP)

65C-CH1003 Fitzgerald 1 mg 254 EUR

Mouse IgG (Fab)

31C-CH1022 Fitzgerald 2 mg 346 EUR
During the course of studying a recombinant human IgG1 in neutralizing diphtheria toxin (DT) in Guinea pigs (Cavia porcellus), we identified a previously uncharacterized Guinea pig protein H0VDZ8 from UNIPROT database that shows high sequence homologies to human FcγRIIIA and mouse FcγRIV. This Fcγ receptor, which we named as gpFcγRIV, also demonstrates functional similarity although not to the same extent as the human and mouse counterparts, in that it binds to afucosyl human and mouse IgG much stronger than to the wild type antibodies. Thus, Guinea pigs can be used to compare the efficacies of wild type vs. afucosyl anti-DT human IgG1 in toxin removal and animal protection. Molecular and functional characterization of human FcγRIIIA and mouse FcγRIV equivalents in other species could expand the list of preclinical animal models for testing afucosyl human antibodies in treating various human diseases.
Christopher Miller

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