An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies

Abstract

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Fig. 1. Isolation of Fc variants with improved FcRn binding.

a Screening strategy for isolating Fc variants using the bacterial display. b Schematic diagram displaying the expression cassettes of the mutagenized Fc libraries (Library-F and Library-E). c Solid ribbon structure showing the mutation sites for the isolated Fc variants. The mutations identified for PFc29 (left) and PFc41 (right) are overlaid on the structure of the Fc region of the human IgG crystal structure (PDB: 1HZH). d The dot plot shows the improved hFcRn binding of the Fc variants relative to a wild-type Fc at pH 6.0 and pH 7.4. The x-axis and y-axis of the dot plot indicate the fold improvement of hFcRn binding affinity (K_D) for the Fc variants at pH 6.0 and the fold improvement of hFcRn binding signal (RU_max) for the Fc variants at pH 7.4 relative to a wild-type Fc, respectively. The Fc variants and wild-type Fc were introduced into trastuzumab, and K_D and RU_max were measured using an SPR analysis.

Fig. 2. Pharmacokinetic profile analysis of trastuzumab-Fc variants and aflibercept-Fc variants.

Serum concentration vs. time profiles for the trastuzumab-Fc variants (5 mg/kg, n = 5) a and aflibercept-Fc variants (4 mg/kg, n = 5) b in hFcRn transgenic mice and trastuzumab-Fc variants (6 mg/kg, n = 2) c in cynomolgus monkeys. Data were presented as the mean values and standard errors.

Fig. 3. Molecular interpretation of the improved PFc29 binding to hFcRn at pH 6.0.

a The combinatorial effects of the two mutations (Q311R and M428L) of PFc29 on hFcRn binding. For the FACS analysis, E. coli spheroplasts displaying an Fc variant (wild-type Fc, Fc-428L, Fc-Q311R, or Fc-PFc29) were labeled with 40 nM hFcRn-SA-Alexa 488 at pH 6.0 and pH 7.4. The mean fluorescence intensities (MFIs) resulting from the FACS analysis are represented as a bar graph. Error bars indicate the standard deviations calculated from triplicate samples. b Superposition of two complex model structures, hFcRn (green)/Fc-M428L (blue) and hFcRn (yellow)/wild-type Fc (white). To examine how the M428L mutation affects the distance between the Fc region and hFcRn, the two complex models (hFcRn/Fc-M428L and hFcRn/wild-type Fc) are superposed based on hFcRn C^α, and a zoomed-in view showing the interaction between hFcRn and the 250-helix region (residues K246–M252) is outlined with a thick line box. The bidirectional arrow in the box indicates the structural distance between Fc-M428L (blue) and wild-type Fc (gray), and the side chain of the M428L residue of Fc-428L is shown in stick representation (purple). c Molecular complex model of PFc29/hFcRn. PFc29, hFcRn α-chain, and β2-microglobulin are colored cyan, yellow, and green, respectively. A contact area close to Q311R is indicated by a box, and electrostatic interactions (Q311R_PFc29–E138_hFcRn or Q311R_PFc29–E139_hFcRn) are represented by dashed lines.

Fig. 4. Effects of Fc variants on effector functions of IgG antibodies.

The ADCC activities of trastuzumab-YTE a, trastuzumab-LS b, and trastuzumab-PFc29 c were compared with that of trastuzumab. Luminescence signals resulting from trastuzumab-Fc variants engaging with target cells (SKBR-3) and effector cells (FcRIIIa-158V-expressing engineered Jurkat) were measured. d–f The CDC activities of rituximab-YTE d, rituximab-LS e, and rituximab-PFc29 f were analyzed using normal human complements and Raji cells as target cells. Rituximab-containing wild-type Fc was used as the control.

Fig. 5. Analysis of the binding of Fc ligands (hFcRn, C1q, and hFcγRIIIa) and effector functions (ADCC and CDC) when the two mutations of PFc29 were combined with effector function-silencing mutations (LALA, LALAPG, or TL).

a ELISA binding signals (OD₄₅₀) upon binding rituximab-PFc29, rituximab-PFc29-LALA, rituximab-PFc29-LALAPG, rituximab-PFc29-TL, and rituximab-PFc29-LALATL to hFcRn at two pH conditions (pH 6.0 and pH 7.4) are represented as a bar graph. Errors bars indicate the standard deviations calculated from duplicate samples. b ELISA binding signals (OD₄₅₀) upon binding rituximab-PFc29, rituximab-PFc29-LALA, rituximab-PFc29-LALAPG, rituximab-PFc29-TL, and rituximab-PFc29-LALATL to C1q are represented as a graph. c, d SPR sensorgrams showing the binding of rituximab-Fc variants to hFcγRIIIa. Interactions between hFcγRIIIa and rituximab-PFc29 c and rituximab-PFc29-TL d were analyzed using an SPR instrument. e ADCC activities of rituximab-PFc29-TL and rituximab-PFc29. Raji cells and engineered Jurkat cells expressing FcγRIIIa-158V were used as target cells and effector cells, respectively. f CDC activities of rituximab-PFc29-TL and rituximab-PFc29. Cytotoxicity was measured using Raji cells and normal human complements.

Fig. 6. Analysis of T-cell activation induced by rituximab-PFc29.

The immunogenicity of the rituximab-Fc variants was examined by measuring the proliferation of CD4+ or CD8+ T cells. a, b Scatter plots representing the proliferation stimulation index (SI) values of CD4+ (a) and CD8+ (b) T cells from four healthy donors upon incubation with rituximab, rituximab-PFc29, or anti-CD3/CD28 antibody. Red lines indicate the mean values, and black lines represent standard deviations. The black dashed line indicates statistical significance at SI ≥2.0 and response at SI ≥2.0, which was considered positive in this study.