Structurally Defined αMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma

Abstract

Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single-domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC-II and rendered "sortase-ready" for the introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.

Keywords: antibody-drug conjugates; antitumor agents; imaging agents; nanobodies; sortase.

Figure 1

Synthesis and characterization of structurally defined VHH7-DM1 conjugate. a) Preparation of VHH7-DM1 conjugate. Conditions: i) DMF, r.t. 2 h; ii) DMF/PBS = 4/1 (v/v), 18 °C, 16 h; iii. Srt A (pentamutant), 10mM CaCl₂, 50 mM Tris, pH = 7.4, 12 °C, 2~4 h; b) LC-MS analysis of VHH7-DM1; c) SDS-PAGE analysis of VHH7-DM1 conjugate (15% gel, InstantBlue, faster mobility of VHH7-DM1 was due to the cyclic structure of DM1, LC-MS profile attached in Fig. S1c).

Figure 2

In vitro characterization of VHH7 conjugates. a) Half maximal effective binding of VHH7-AF647 to murine lymphoma A20 cells. 5×10⁵ cells were incubated with increasing concentration of VHH7-AF647 at 4 °C for 1 h, then washed 3 times and analyzed by flow cytometry. b) Internalization of commercial antibody anti-I-A/E-AF488 (M5/114.15.2) and VHH7-AF647. Equal molar amount of VHH-AF647 and anti-I-A/E-AF488 were premixed and added to cells in poly-L-lysine coated imaging chamber at final concentration of 50 nM. After 5 min, cells were washed with ice-cold PBS, then fixed and mounted for confocal microscopy. c) In vitro cytoxocity of VHH7-DM1 conjugate on MHC-II positive (A20) and negative cell lines (Hela) (n=3, bars, means ± SD).

Figure 3

Non-invasive NIR imaging of A20 lymphoma by VHH7-AF647 conjugate. (color scale: full range, minimum to maximum). a) Balb/c with subcutaneous A20 (left, middle) and MHC-II KO (right) were injected with 40 µg VHH7-AF647 or irrelevant Enhancer-AF647 conjugate as denoted, then imaged on IVIS. Pictures show 5 h p.i. (black dashed circle highlights tumor burden); b) Mouse with subcutaneous A20 was dissected 16 h p.i. of VHH7-AF647 to show clear tumor targeting; c) Healthy (left) and A20 intravenously inoculated (right, 4 weeks p.i.) Balb/c were imaged 5 h p.i. of VHH7-AF647; d) Mouse with metastatic A20 was dissected 5 h p.i. of VHH7-AF647 injection to show clear tumor targeting on lung, liver and spleen; e) Organs from metastatic and healthy mice (panel c) were imaged in the same view; f) Two Balb/c mice with disseminated A20 lymphoma (4 weeks after inoculation) were imaged 24 h p.i. of VHH7-AF647; g) Mice from panel f were dissected to show targeting at tumor sites. h) Comparable doses (6400 RFU) of fluorescently labeled VHH7-AF647 and α-I-A/E-AF647 were injected into mice bearing subcutaneous tumors. The tumors were removed and analyzed by FACS. Shift in mean fluorescence intensity: 92K(I-A/E) to 135K (VHH7).

Figure 4

In vivo efficiency of VHH7-DM1 conjugate in treating A20 lymphoma. a) VHH7-DM1 conjugate inhibited tumor growth in a localized model. 9 Balb/c were inoculated with 2.5 × 10⁶ A20 cells subcutaneously then randomized into 3 groups at day 10 when tumor burdens became measurable by caliper. Starting at day 10, samples were given i.v. at dose of 125 µg/mice (5 mg/kg for 25 g mice) and followed every other day for a total of 5 injections. Tumor volume (V) was used to evaluate tumor size using a modified ellipsoid formula: V = (width)² × length / 2 (Bars, means ± SD, n = 3). Experiment end point was defined as when the largest single tumor size exceeded 2000 mm³. b) VHH7-DM1 protected mice from tumor metastasis in a disseminated model. 15 Balb/c were inoculated with 1.5 × 10⁶ A20 subcutaneously, then randomized into 3 groups. Injection started at day 2 with a dose of 125 µg/mice and followed every other day for a total of 4 injections. Mice were sacrificed on day 27, and the number of metastatic foci on liver was counted (Bars, means ± SD, n = 5).