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PDF file - 60K, Lack of competition between bsFab and human IgG for FcγRIIIa binding.

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Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen–positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies. Mol Cancer Ther; 12(8); 1481–91. ©2013 AACR.

HER2 over-expression in breast cancer correlates with reduced survival. Anti-idiotypic antibodies (Abs) that closely mimic HER2 could play a crucial role in the development of effective therapeutic vaccines. Here, we show that an anti-idiotypic single domain antibody (sdAb) 1HE isolated from a library generated from a Trastuzumab F(ab')(2)-immunized llama, closely mimics HER2. SdAb 1HE shows high affinity for Trastuzumab F(ab')(2), selectively inhibits HER2 binding to Trastuzumab F(ab')(2), and sera from sdAb 1HE-immunized BALB/c mice contain anti-HER2 antibodies that inhibit viability of HER2-positive cells. These results indicate that sdAb 1HE could be used as an anti-idiotype-based vaccine to boost immunity in patients bearing HER2-positive tumours.

A 2.9-kb fragment of the Pasteurella multocida (Pm) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pm, p25 is not related to known bacterial porins.

International audience; Single-domain antibodies (sdAbs), which occur naturally in camelids, are endowed with many characteristics that make them attractive candidates as building blocks to create new antibody-related therapeutic molecules. In this study, we isolated from an immunized llama several high-affinity sdAbs directed against human carcinoembryonic antigen (CEA), a heavily glycosylated tumor-associated molecule expressed in a variety of cancers. These llama sdAbs bind a different epitope from those defined by current murine mAbs, as shown by binding competition experiments using immunofluorescence and surface plasmon resonance. Flow cytometry analysis shows that they bind strongly to CEA-positive tumor cells but show no cross-reaction toward nonspecific cross-reacting antigen, a highly CEA-related molecule expressed on human granulocytes. When injected into mice xenografted with a human CEA-positive tumor, up to 2% of the injected dose of one of these sdAbs was found in the tumor, despite rapid clearance of this 15 kDa protein, demonstrating its high potential as a targeting moiety. The single-domain nature of these new anti-CEA IgG fragments should facilitate the design of new molecules for immunotherapy or diagnosis of CEA-positive tumors.

FcgammaRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcgammaRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcgammaRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcgammaRIIIB extra-cellular domains. These sdAbs bind FcgammaRIIIA(+) NK cells and FcgammaRIIIB(+) polymorphonuclear cells, but not FcgammaRI(+) or FcgammaRII(+) cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcgammaRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcgammaRIII with a K(D) in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcgammaRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-gamma production, respectively. These anti-FcgammaRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcgammaRIII killer cells to target and destroy tumor cells.

Single-domain antibodies specific to methotrexate (MTX) were obtained after immunization of one llama (Llama glama). Specific VHH domains (V-D-J-REGION) were selected by panning from an immune-llama library using phage display technology. The antibody fragments specific to MTX were purified from Escherichia coli (C41 strain) periplasm by immobilized metal affinity chromatography with an expression level of around 10mg/L. A single band around 16,000Da corresponding to VHH fragments was found after analysis by SDS-PAGE and Western blotting, while competition ELISA demonstrated selective binding to soluble MTX. Surface plasmon resonance (SPR) analysis showed that anti-MTX VHH domains had affinities in the nanomolar range (29-515nM) to MTX-serum albumin conjugates. The genes encoding anti-MTX VHH were found by IMGT/V-QUEST to be similar to the previously reported llama and human IGHV germline genes. The V-D and D-J junction rearrangements in the seven anti-MTX CDR3 sequences indicate that they were originated from three distinct progenitor B cells. Our results demonstrate that camelid single-domain antibodies are capable of high affinity binding to low molecular weight hydrosoluble haptens. Furthermore, these anti-MTX VHH give new insights on how the antigen binding repertoire of llama single-domain antibody can provide combining sites to haptens in the absence of a VL. This type of single-domain antibodies offers advantages compared to murine recombinant antibodies in terms of production rate and sequence similarity to the human IGHV3 subgroup genes.