Engineering a pure and stable heterodimeric IgA for the development of multispecific therapeutics

Figures & data

Figure 1. Steric and electrostatic in silico design strategies to drive IgA Fc heterodimer formation and OAA format used to test designs in vitro. (a) Cartoon depictions of steric and electrostatic negative and positive design concepts for in silico modeling of mutations to drive heterodimerization of an IgA Fc (left). Examples of structural models of steric and electrostatic designs in an IgA CH3 predicted to favor heterodimer formation of the Fc (right). Cartoon depiction of concepts are overlayed on the structural models for clarity. (b) Example of in silico knowledge-based and physics-based metrics used to rank favorable steric and electrostatic designs (single design shown). (c) Antibody formats used to test heterodimeric designs in vitro. Mutations favoring formation of a heterodimeric IgA Fc assemble into a OAA species that can be resolved by size from homodimeric Fc-only and full-sized antibody species. The OAA used is composed of anti-Her2 IgG1 Fab, IgG1/IgA2 hybrid hinge and IgA2m1 Fc.

Table 1. Summary of purification results and CH3 thermal stability of heterodimeric IgA Fc steric designs.

Design	Chain A Mutations	Chain B Mutations	Post-affinity Purification			Post prepSEC Purification
			HPLC-SEC OAA purity (%)	CE-SDS OAA purity (%)	Total yield (mg/L culture)	HPLC-SEC OAA purity (%)	CE-SDS OAA purity (%)	OAA yield (mg/L culture)	CH3 Tm (°C)**
Wild-type	NA	NA	52	49	324	91	92	76	74.2
Steric 1	A412Y_T414L	L396T_W398L_I416L	49	36	148	98	93	36	71.1
Steric 2	A412Y_T414Y	L396T_W398L_I416L	65	55	240	97	96	76	55.0
Steric 3	A412F_T414Y	L396V_W398L_I416L	91	89	328	100	98	136	65.9
Steric 4	L370M_A412F_T414W	W398L	5	3.7	60	NA	NA	NA	NA
Steric 5	A412Y_T414M	L396V_W398L_I416L	NA	NA	NA	NA	NA	NA	NA
Steric 6 *	A412F_T414Y	L396V_W398T_I416L	96	92	320	100	97	100	71.9
Steric 7	T366V_A412F_T414Y	L396V_W398T_I416L	82	31	130	100	99	52	69.2
Steric 8	T366L_A412F_T414Y	L396V_W398T_I416L	75	97	370	100	95	140	67.6
Steric 9	T366I_A412F_T414Y	L396V_W398T_I416L	87	97	390	100	95	210	69.0
Steric 10 *	A412F_T414Y	L352F_L396V_W398T_I416L	72	88	370	100	85	82	72.0
Steric 11 *	H349Y_A412F_T414Y	H350Y_L396V_W398T_ I416L	74	95	440	100	93	71	73.6

*Lead variants. **Tm of CH3 domain determined by DSC. NA = Not applicable, variants did not express in sufficient quantity.

Figure 2. Lead steric designs 6, 10, and 11 drive formation of a heterodimeric IgA Fc as determined by formation of OAA species in vitro and possess wild-type IgA-like thermal stability. (a) Non-reducing CE-SDS profiles of IgA Fc OAA variants after affinity purification. Data are shown for variants comprising a wild-type IgA CH3 and those containing heterodimer-driving mutations from steric designs 1–4 and 6–11. (b) Analysis of IgA OAA variants by analytical SEC after affinity purification. Data is shown for a wild-type IgA Fc (left) as well as steric design 6 (right). (c) Overlay of DSC thermograms measured for SEC-purified wild-type IgA Fc (black) and heterodimeric IgA Fc containing mutations from steric designs 2 (red), 3 (orange), 6 (yellow), 10 (light green), and 11 (green) OAA variants. The first and second transitions are attributed to CH3 and CH2 + Fab unfolding, respectively.

Figure 3. The crystal structure of heterodimeric IgA Fc steric design 6 was solved in complex with S. aureus SSL7 (PDB ID: 7TTZ). The heterodimeric IgA Fc has high structural homology with the predicted in silico model and differs from wild-type IgA Fc only at the CH3:CH3 interface. (a) Cartoon representation of the heterodimeric IgA Fc (steric 6) in pale cyan and pale green in complex with two S. aureus SSL7 monomers in pale orange and pale yellow (PDB ID: 7TTZ). (b) Superimposition of the heterodimeric IgA Fc (steric 6) crystal structure (pale blue and pale green) and wild-type IgA Fc (PDB ID: 2QEJ; gray) illustrates the structural homology between heterodimeric and homodimeric IgA CH2 and CH3 domain structure. The peptide backbone is drawn as ribbon and mutated residues and equivalent wild-type residues in the CH3:CH3 interface are drawn as sticks. (c) Cartoon representation of heterodimeric IgA (steric 6) CH3:CH3 interface with mutated residues drawn as sticks (right) compared to wild-type homodimeric IgA Fc (PDB ID: 2QEJ) interface (left) and the predicted in silico model (middle).

Figure 4. IgA heterodimeric Fc variants retain binding to the FcαRI. (a) Surface plasmon resonance sensorgrams for wild-type IgA OAA (WT) (top, apparent K_D = 11 nM), IgA OAA FcαRI-KO 1x (middle, K_D = 273 nM), and heterodimeric IgA Fc (steric 6) OAA (bottom, apparent K_D = 23 nM) measured at FcαRI ligand density of 14 RU, fit using a 1:1 Langmuir model. (b) Dependence of apparent K_D of binding of IgA OAA variants to FcαRI on ligand density of FcαRI immobilized on the SPR sensor chip. Data for a wild-type IgA OAA, steric heterodimeric designs 6, 10, 11 as well as a single sided FcαRI-binding-deficient IgA Fc based on heterodimeric design 6 (IgA OAA FcαRI-KO 1x) is shown.

Figure 5. Applications of a heterodimeric IgA Fc in the development of IgA multispecifics and interrogation of IgA biology. The heterodimeric IgA Fc designs described herein (center) can be used for a variety of applications. These include engineering of an asymmetric IgA Fc, for example to introduce a FcRn-binding motif to increase the half-life of IgA while preserving binding to FcαRI (top left). A heterodimeric IgA can also be used to create multispecific therapeutics with a variety of formats, ranging from OAAs, bispecifics and biparatopics to multispecific multimers (top right) which can be used to target the mucosa when bound to the secretory compartment (bottom left). Heterodimeric IgAs can furthermore be used as a tool to interrogate fundamental questions surrounding IgA biology such as the effect of FcαRI binding valency on the activation of effector cells such as neutrophils (bottom right).