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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.

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.