Enabling the formation of native mAb, Fab' and Fc-conjugates using a bis-disulfide bridging reagent to achieve tunable payload-to-antibody ratios (PARs)

Abstract

Either as full IgGs or as fragments (Fabs, Fc, etc.), antibodies have received tremendous attention in the development of new therapeutics such as antibody-drug conjugates (ADCs). The production of ADCs involves the grafting of active payloads onto an antibody, which is generally enabled by the site-selective modification of native or engineered antibodies via chemical or enzymatic methods. Whatever method is employed, controlling the payload-antibody ratio (PAR) is a challenge in terms of multiple aspects including: (i) obtaining homogeneous protein conjugates; (ii) obtaining unusual PARs (PAR is rarely other than 2, 4 or 8); (iii) using a single method to access a range of different PARs; (iv) applicability to various antibody formats; and (v) flexibility for the production of heterofunctional antibody-conjugates (e.g. attachment of multiple types of payloads). In this article, we report a single pyridazinedione-based trifunctional dual bridging linker that enables, in a two-step procedure (re-bridging/click), the generation of either mAb-, Fab'-, or Fc-conjugates from native mAb, (Fab')₂ or Fc formats, respectively. Fc and (Fab')₂ formats were generated via enzymatic digestion of native mAbs. Whilst the same reduction and re-bridging protocols were applied to all three of the protein formats, the subsequent click reaction(s) employed to graft payload(s) drove the generation of a range of PARs, including heterofunctional PARs. As such, exploiting click reactivity and/or orthogonality afforded mAb-conjugates with PARs of 6, 4, 2 or 4 + 2, and Fab'- and Fc-conjugates with a PAR of 3, 2, 1 or 2 + 1 on-demand. We believe that the homogeneity, novelty and variety in accessible PARs, as well as the applicability to various antibody-conjugate formats enabled by our non-recombinant method could be a suitable tool for antibody-drug conjugates optimisation (optimal PAR value, optimal payloads combination) and boost the development of new antibody therapeutics (Fab'- and Fc-conjugates).

Fig. 1. General principle of the dual re-bridging of two solvent accessible disulfides of a protein with a pyridazinedione-based trifunctional dual bridging linker, enabling introduction of corresponding click handles.

Fig. 2. Convergent synthesis of trifunctional, dual bridging linker 16.

Fig. 3. Application of trifunctional, dual bridging linker 16 to antibody format: (a) reduction, re-bridging and functionalisation of native trastuzumab antibody. Re-bridging with linker 16 allows introduction of four phenylazide and two tetrazine click handles, of which reactivity and orthogonality enables controlled access to various payload–antibody ratios (PARs) depending on the click handle present on reacting payload. * For clarity only the major full antibody species is drawn for Trastu conjugate 17 in part (a), but there is some minor half-antibody species present from intrachain rebridging in the Fc region, as seen in part (b) of the figure. (b) SDS-PAGE analysis of the re-bridging step. L: ladder, lane 1: native trastuzumab, lane 2: trastuzumab conjugate 17, generated via re-bridging of native trastuzumab with linker 16 (densitometry analysis with ImageJ software revealed a ratio full mAb/half mAb of ca. 80 : 20), lane 3: trastuzumab conjugate 17 under reducing condition (high excess TCEP) (densitometry analysis with ImageJ software revealed a ratio full mAb/half mAb of ca. 80 : 20). We also note that the antibody and antibody conjugates appear at higher masses than one would expect relative to the ladder, but that this is more-or-less consistent with the field. (c) Denaturing LC-MS analysis of trastuzumab conjugate 17. Expected mass: 148 205 Da, 74 098 Da (half-antibody); observed mass: 148 203 Da, 74 098 Da.

Fig. 4. Denaturing LC-MS analysis after click reaction of Trastu_[PhN₃]₄_[MeTz]₂17 with (a) BCN-fluorescein to generate an antibody–payload conjugate with a PAR of 6 in fluorescein (Trastu_[fluorescein]₄_[fluorescein]₂) via SPAAC and IEDDA reactions; (b) DBCO-rhodamine to generate an antibody–payload conjugate with a PAR of 4 in rhodamine (Trastu_[rhodamine]₄_[MeTz]₂) via SPAAC reaction; (c) TCO–biotin to generate an antibody–payload conjugate with a PAR of 2 in biotin (Trastu_[PhN₃]₄_[biotin]₂) via IEDDA reaction; (d) DBCO-rhodamine followed by BCN-fluorescein to generate a heterofunctional antibody–payload conjugate with a PAR of 4 in rhodamine and 2 in fluorescein (PAR 4 + 2) (Trastu_[rhodamine]₄_[fluorescein]₂) via sequential SPAAC and IEDDA reactions.

Fig. 5. (a) Reaction scheme of enzymatic (Fab′)₂ production from native mAb, followed by reduction and re-bridging steps. Low electrostatic HC–HC interactions after (Fab′)₂ reduction with TCEP prevents the two Fab′ fragment to be maintained together, promoting intra-molecular re-bridging of single Fab′ formats. (b) SDS-PAGE analysis comparing unmodified (Fab′)₂ and re-bridged (Fab′)₂ under non-reducing or reducing conditions (excess TCEP). L: ladder; lane 1: (Fab′)₂; lane 2: (Fab′)₂ reduced and re-bridged with linker 16 at 4 °C; lane 3: reduced and re-bridged with linker 16 at 37 °C; lane 4: reduced and re-bridged with linker 16 at 60 °C; lane 5: (Fab′)₂ crude after TCEP reduction step, yields HC and LC; lane 6: native (Fab′)₂ analysed under reducing conditions (TCEP), yields HC and LC; lane 7: (Fab′)₂ re-bridged with linker 16 at 4 °C, analysed under reducing conditions (excess TCEP). Densitometry analysis confirmed that no (Fab′)₂ species remained on lanes 2 to 7, and neither HC nor LC were detected on lanes 2, 3, 4 and 7. (c) Denaturing LC-MS analysis of (Fab′)₂ format produced via native mAb pepsin digestion. Even though not detrimental, two extra peaks are observed (97 179 Da and 97 067 Da) that correspond to loss of an extra Leucine on one or both HCs. (d) Denaturing LC-MS analysis of Fab′ format re-bridged with bridging linker 16. HC: heavy chain. LC: might chain.

Fig. 6. Denaturing LC-MS analysis after click reaction of re-bridged Fab′_[PhN₃]₂_[MeTz]₁24 with (a) BCN-fluorescein to generate a Fab′-payload conjugate with a PAR of 3 in fluorescein (Fab′_[fluorescein]₂_[fluorescein]₁) via SPAAC and IEDDA reactions; (b) DBCO-rhodamine to generate a Fab′-payload conjugate with a PAR of 2 in rhodamine (Fab′_[rhodamine]₂_[MeTz]₁) via SPAAC reaction; (c) TCO–biotin to generate a Fab′–payload conjugate with a PAR of 1 in biotin (Fab′_[PhN₃]₂_[biotin]₁) via IEDDA reaction; (d) DBCO-rhodamine followed by BCN-fluorescein to generate a hetero-functional Fab′-payload conjugate with a PAR of 2 in rhodamine and 1 in fluorescein (PAR 2 + 1) (Fab′_[rhodamine]₂_[fluorescein]₁) via sequential SPAAC and IEDDA reactions.

Fig. 7. (a) Reaction scheme of enzymatic Fc production from native mAb, followed by reduction and re-bridging steps. (b) SDS-PAGE analysis comparing unmodified Fc (lane 1) and re-bridged Fc under non-reducing (lane 2) or reducing conditions (excess TCEP, lane 3). L: ladder. Densitometry analysis with ImageJ indicated that the band of Fc conjugate 30 accounted for 89% and 85% of total measured intensity on lanes 2 and 3, respectively. (c) Denaturing LC-MS analysis of Fc format produced via native mAb papain digestion. (d) Denaturing LC-MS analysis of Fc format re-bridged with bridging linker 16.

Fig. 8. Denaturing LC-MS analysis after click reaction of re-bridged Fc_[PhN₃]₂_[MeTz]₁30 with (a) BCN-fluorescein to generate a Fc–payload conjugate with a PAR of 3 in fluorescein (Fc_[fluorescein]₂_[fluorescein]₁) via SPAAC and IEDDA reactions; (b) DBCO-rhodamine to generate a Fc–payload conjugate with a PAR of 2 in rhodamine (Fc_[rhodamine]₂_[MeTz]₁) via SPAAC reaction; (c) TCO–biotin to generate a Fc-payload conjugate with a PAR of 1 in biotin (Fc-[PhN₃]₂_[biotin]₁) via IEDDA reaction; (d) DBCO-rhodamine followed by BCN-fluorescein to generate a hetero-functional Fc–payload conjugate with a PAR of 2 in rhodamine and 1 in fluorescein (PAR 2 + 1) (Fc_[rhodamine]₂_[fluorescein]₁) 34via sequential SPAAC and IEDDA reactions.