ENGINEERED FC

Abstract

Antigen-binding molecules comprising an Fc region comprising a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) one or more of: A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, L at the position corresponding to position 330, K at the position corresponding to position 345, and G at the position corresponding to position 430 are disclosed. Also discloses are constituent polypeptides of such Fc regions, nucleic acids encoding such antigen- binding molecules and polypeptides, compositions comprising such antigen-binding molecules, polypeptides and nucleic acids, and methods using the same.

Claims

1. An antigen-binding molecule, optionally isolated, comprising an Fc region, the Fc region comprising a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) one or more of: A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, L at the position corresponding to position 330, K at the position corresponding to position 345, and G at the position corresponding to position 430.

2. The antigen binding molecule according to claim 1, wherein the Fc region comprises a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, and L at the position corresponding to position 330; or A at the position corresponding to position 236, D at the position corresponding to position 239, and E at the position corresponding to position 332; or A at the position corresponding to position 236, and D at the position corresponding to position 239; or K at the position corresponding to position 345, and G at the position corresponding to position 430.

3. The antigen binding molecule according to claim 1 or claim 2, wherein the Fc region comprises a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) A at the position corresponding to position 236, D at the position corresponding to position 239, E at the position corresponding to position 332, and L at the position corresponding to position 330.

4. The antigen binding molecule according to claim 1 or claim 2, wherein the Fc region comprises a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) A at the position corresponding to position 236, D at the position corresponding to position 239, and E at the position corresponding to position 332.

5. The antigen binding molecule according to claim 1 or claim 2, wherein the Fc region comprises a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) A at the position corresponding to position 236, and D at the position corresponding to position 239.

6. The antigen binding molecule according to claim 1 or claim 2, wherein the Fc region comprises a polypeptide having: (i) C at the position corresponding to position 242, and C at the position corresponding to position 334, and (ii) K at the position corresponding to position 345, and G at the position corresponding to position 430.

7. The antigen binding molecule according to any one of claims 1 to 6, wherein the Fc region comprises a polypeptide comprising an amino acid sequence having at least 60% sequence identity to SEQ ID NO:39, 38, 37, 41, 22, 21, 20 or 24.

8. A polypeptide, optionally isolated, comprising: an amino acid sequence having at least 60% sequence identity to SEQ ID NO:31 or 6, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) one or more of: A at position 9, D at position 12, L at position 103, E at position 105, K at position 118, and G at position 203.

9. The polypeptide according to claim 8, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) A at position 9, D at position 12, L at position 103, and E at position 105; or A at position 9, D at position 12, and E at position 105; or A at position 9, and D at position 12; or K at position 118, and G at position 203.

10. The polypeptide according to claim 8 or claim 9, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) A at position 9, D at position 12, L at position 103, and E at position 105.

11. The polypeptide according to claim 8 or claim 9, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) A at position 9, D at position 12, and E at position 105.

12. The polypeptide according to claim 8 or claim 9, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) A at position 9, and D at position 12.

13. The polypeptide according to claim 8 or claim 9, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:31 or 6: (i) C at position 15, and C at position 107, and (ii) K at position 118, and G at position 203.

14. A polypeptide, optionally isolated, comprising the amino acid sequence of SEQ ID NO:39, 38, 37, 41, 22, 21, 20 or 24.

15. An Fc region, optionally isolated, comprising a polypeptide according to any one of claims 8 to 14.

16. An antigen-binding molecule, optionally isolated, comprising a polypeptide according to any one of claims 7 to 13, or an Fc region according to claim 14.

17. A nucleic acid, or a plurality of nucleic acids, optionally isolated, encoding an antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, or an Fc region according to claim 15.

18. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 17.

19. A cell comprising an antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, an Fc region according to claim 15, a nucleic acid or a plurality of nucleic acids according to claim 17, or an expression vector or a plurality of expression vectors according to claim 18.

20. A method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids according to claim 17, or an expression vector or a plurality of expression vectors according to claim 18, under conditions suitable for expression of the antigen-binding molecule, polypeptide or Fc region from the nucleic acid(s) or expression vector(s).

21. A composition comprising an antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, an Fc region according to claim 15, a nucleic acid or a plurality of nucleic acids according to claim 17, an expression vector or a plurality of expression vectors according to claim 18, or a cell according to claim 19.

22. An antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, an Fc region according to claim 15, a nucleic acid or a plurality of nucleic acids according to claim 17, an expression vector or a plurality of expression vectors according to claim 18, a cell according to claim 19, or a composition according to claim 21 for use in a method of medical treatment or prophylaxis.

23. An antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, an Fc region according to claim 15, a nucleic acid or a plurality of nucleic acids according to claim 17, an expression vector or a plurality of expression vectors according to claim 18, a cell according to claim 19, or a composition according to claim 21, for use in a method of treatment or prevention of a cancer, an infectious disease or an autoimmune disease.

24. A method, optionally an in vitro method, of killing cells expressing a target antigen, comprising contacting cells expressing the target antigen with an antigen-binding molecule according to any one of claim 1 to 7 or 16, a polypeptide according to any one of claims 8 to 14, an Fc region according to claim 15, a cell according to claim 19, or a composition according to claim 21.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0282] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.

[0283] FIGS. 1A to 1E. Graphs showing the first derivative of the raw data obtained by Differential Scanning Fluorimetry analysis of thermostability of antigen-binding molecules comprising different Fc regions. (1A) shows data for WT Fc and LCKC Fc, (1B) shows data for GASD Fc and GASD_LCKC Fc, (1C) shows data for GASDIE Fc and GASDIE_LCKC Fc, (1D) shows data for GASDALIE Fc and GASDALIE_LCKC Fc, and (1E) shows data for EKEG Fc and EKEG_LCKC Fc.

[0284] FIGS. 2A and 2B. Table and bar chart summarising the data shown in FIGS. 1A to 1E. (2A) summarises data for all molecules. (2B) provides a graphical representation in the Tm shift in ° C. relative to WT Fc (WT IgG1) for LCKC Fc (LCKC), GASDIE_LCKC Fc (GASDIE-LCKC) and GASDIE Fc (GASDIE) format antigen-binding molecules.

[0285] FIGS. 3A to 3J. Sensorgrams showing the binding of antigen-binding molecules comprising different Fc regions to human FcγRIIIa-158V. (3A) shows data for WT Fc, (3B) shows data for GASD Fc, (3C) shows data for GASDIE Fc, (3D) shows data for LCKC Fc, (3E) shows data for GASD_LCKC Fc, (3F) shows data for GASDIE_LCKC Fc, (3G) shows data for GASDALIE Fc, (3H) shows data for EKEG Fc, (3I) shows data for GASDALIE_LCKC Fc, and (3J) shows data for EKEG_LCKC Fc.

[0286] FIG. 4. Table summarising the data shown in FIGS. 3A to 3J.

[0287] FIGS. 5A to 5J. Sensorgrams showing the binding of antigen-binding molecules comprising different Fc regions to human Fcγ receptors. (5A) shows data for binding of WT Fc to hFcγRIIIa-158F, (5B) shows data for binding of WT Fc to hFcγRIIIa-158V, (5C) shows data for binding of WT Fc to hFcγRIIa-167H, (5D) shows data for binding of GASDALIE_LCKC Fc to hFcγRIIIa-158F, (5E) shows data for binding of GASDALIE_LCKC Fc to hFcγRIIIa-158V, (5F) shows data for binding of GASDALIE_LCKC Fc to hFcγRIIa-167H, (5G) shows data for binding of WT Fc to hFcγRIIa-167R, (5H) shows data for binding of WT Fc to hFcγRIIb, (5I) shows data for binding of GASDALIE_LCKC Fc to hFcγRIIa-167R, and (5J) shows data for binding of GASDALIE_LCKC Fc to hFcγRIIb.

[0288] FIGS. 6A to 6F. Sensorgrams showing the binding of antigen-binding molecules comprising different Fc regions to mouse Fcγ receptors. (6A) shows data for binding of WT Fc to mFcγRIV, (6B) shows data for binding of WT Fc to mFcγRIII, (6C) shows data for binding of WT Fc to mFcγRIIb, (6D) shows data for binding of GASDALIE_LCKC Fc to mFcγRIV, (6E) shows data for binding of GASDALIE_LCKC Fc to mFcγRIII, and (6F) shows data for binding of GASDALIE_LCKC Fc to mFcγRIIb.

[0289] FIGS. 7A to 7D. Sensorgrams showing the binding of antigen-binding molecules comprising different Fc regions to human (h) and mouse (m) FcRn receptors. (7A) shows data for binding of WT Fc to hFcRn, (7B) shows data for binding of WT Fc to mFcRn, (7C) shows data for binding of GASDALIE_LCKC Fc to hFcRn, and (7D) shows data for binding of GASDALIE_LCKC Fc to mFcRn.

[0290] FIG. 8. Table summarising the data shown in FIGS. 5A to 5J, 6A to 6F and 7A to 7D.

[0291] FIGS. 9A and 9B. Graph and bar chart showing ADCC mediated by antigen-binding molecules comprising different Fc regions to target antigen-expressing cells, as determined by LDH release assay. (9A) shows ADCC activity of WT Fc or GASDALIE_LCKC Fc regions to target antigen-expressing cells. EC50 values are shown. (9B) shows relative ADCC activity of WT Fc, GASDALIE_LCKC Fc or N297Q Fc to target antigen-expressing cells.

[0292] FIG. 10. Graph showing the results of analysis of tumour volume over time in an A549 cell-line derived mouse model of lung adenocarcinoma. Antigen-binding molecules comprising WT Fc or GASDALIE_LCKC Fc were administered IP, biweekly at 25 mg/kg for a total of 6 weeks. A control treatment group received an equal volume of PBS (vehicle).

EXAMPLES

Example 1

Preparation of Antigen-Binding Molecules Comprising Engineered Fc Regions

[0293] The inventors prepared antigen-binding molecules comprising heavy chains including amino acid substitutions to positions in the CH2 and/or CH3 regions, to investigate the consequence of the substitutions on Fc effector functions.

[0294] Antigen-binding molecules were prepared comprising: (i) light chains comprising the light chain variable region (VL) of an antibody specific for HER3, and the constant region light chain (Cκ), and (ii) heavy chains comprising the heavy chain variable region (VH) of the antibody specific for HER3, and human immunoglobulin G 1 (G1m3 allotype) heavy chain constant region 1 (CH1), hinge region, heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3).

[0295] The CH2 and CH3 regions were either unsubstituted, or were provided with combinations of substitutions, as follows:

TABLE-US-00002 Substitutions relative Amino acid Amino acid Amino acid Fc region to human IGHG1 sequence of sequence of sequence of designation (G1m3 allotype) CH2 region CH3 region CH2-CH3 region Wildtype (WT) — SEQ ID NO: 4 SEQ ID NO: 30 SEQ ID NO: 31 GASD G236A, S293D SEQ ID NO: 8 SEQ ID NO: 30 SEQ ID NO: 33 GASDIE G236A, S293D, SEQ ID NO: 9 SEQ ID NO: 30 SEQ ID NO: 34 I332E GASDALIE G236A, S293D, SEQ ID NO: 10 SEQ ID NO: 30 SEQ ID NO: 35 A330L, I332E EKEG E345K, E430G SEQ ID NO: 4 SEQ ID NO: 32 SEQ ID NO: 40 LCKC L242C, K334C SEQ ID NO: 11 SEQ ID NO: 30 SEQ ID NO: 36 GASD_LCKC G236A, S293D, SEQ ID NO: 12 SEQ ID NO: 30 SEQ ID NO: 37 L242C, K334C GASDIE_LCKC G236A, S293D, SEQ ID NO: 13 SEQ ID NO: 30 SEQ ID NO: 38 I332E, L242C, K334C GASDALIE_LCKC G236A, S293D, SEQ ID NO: 14 SEQ ID NO: 30 SEQ ID NO: 39 A330L, I332E, L242C, K334C EKEG_LCKC E345K, E430G, SEQ ID NO: 11 SEQ ID NO: 32 SEQ ID NO: 41 L242C, K334C

[0296] Antigen-binding molecules were expressed using either 1) Expi293 Transient Expression System Kit (Life Technologies, USA), or 2) HEK293-6E Transient Expression System (CNRC-NRC, Canada) following the manufacturer's instructions.

[0297] 1) Expi293 Transient Expression System:

[0298] Cell Line Maintenance:

[0299] HEK293F cells (Expi293F) were obtained from Life Technologies, Inc (USA). Cells were cultured in serum-free, protein-free, chemically defined medium (Expi293 Expression Medium, Thermo Fisher, USA), supplemented with 50 IU/ml penicillin and 50 μg/ml streptomycine (Gibco, USA) at 37° C., in 8% CO.sub.2 and 80% humidified incubators with shaking platform.

[0300] Transfection:

[0301] Expi293F cells were transfected with expression plasmids encoding the heavy and light chains using ExpiFectamine 293 Reagent kit (Gibco, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by spinning down the culture, cell pellets were re-suspended in fresh media without antibiotics at 1 day before transfection. On the day of transfection, 2.5×10.sup.6/ml of viable cells were seeded in shaker flasks for each transfection. DNA-ExpiFectamine complexes were formed in serum-reduced medium, Opti-MEM (Gibco, USA), for 25 min at room temperature before being added to the cells. Enhancers were added to the transfected cells at 16-18 h post transfection. An equal amount of media was topped up to the transfectants at day 4 post-transfection to prevent cell aggregation. Transfectants were harvested at day 7 by centrifugation at 4000×g for 15 min, and filtered through 0.22 μm sterile filter units.

[0302] 2) HEK6293-6E Transient Expression System:

[0303] Cell Line Maintenance:

[0304] HEK293-6E cells were obtained from National Research Council Canada. Cells were cultured in serum-free, rotein-free, chemically defined Freestyle F17 Medium (Invitrogen, USA), supplemented with 0.1% Kolliphor-P188 and 4 mM L-Glutamine (Gibco, USA) and 25 μg/ml G-418 at 37° C., in 5% CO.sub.2 and 80% humidified incubators with shaking platform.

[0305] Transfection:

[0306] HEK293-6E cells were transfected with expression plasmids encoding the heavy and light chains using PElpro™ (Polyplus, USA) according to its manufacturer's protocol. Briefly, cells at maintenance were subjected to a media exchange to remove antibiotics by centrifugation, cell pellets were re-suspended with fresh media without antibiotics at 1 day before transfection. On the day of transfection, 1.5-2×10.sup.6 cells/ml of viable cells were seeded in shaker flasks for each transfection. DNA and PElpro™ were mixed to a ratio of 1:1 and the complexes were allowed to form in F17 medium for 5 min at RT before adding to the cells. 0.5% (w/v) of Tryptone N1 was fed to transfectants at 24-48 h post transfection. Transfectants were harvested at day 6-7 by centrifugation at 4000×g for 15 min and the supernatant was filtered through 0.22 μm sterile filter units.

[0307] Affinity purification, buffer exchange and storage:

[0308] Antigen-binding molecules secreted by the transfected cells into the culture supernatant were purified using liquid chromatography system AKTA Start (GE Healthcare, UK). Specifically, supernatants were loaded onto HiTrap Protein G column (GE Healthcare, UK) at a binding rate of 5 ml/min, followed by washing the column with 10 column volumes of washing buffer (20 mM sodium phosphate, pH 7.0). Bound mAbs were eluted with elution buffer (0.1 M glycine, pH 2.7) and the eluents were fractionated to collection tubes which contain appropriate amount of neutralization buffer (1 M Tris, pH 9). Neutralised elution buffer containing purified mAb were exchanged into PBS using 30K MWCO protein concentrators (Thermo Fisher, USA) or 3.5K MWCO dialysis cassettes (Thermo Fisher, USA). Monoclonal antibodies were sterilized by passing through 0.22 μm filter, aliquoted and snap-frozen in -−0° C. for storage.

Example 2

Analysis of Thermostability of Antigen-Binding Molecules Comprising Engineered Fc regions by Differential Scanning Fluorimetry

[0309] Thermostability of the antigen-binding molecules prepared as described in Example 1 was evaluated by Differential Scanning Fluorimetry.

[0310] Briefly, triplicate reaction mixes of antibodies at 0.2 mg/mL and SYPRO Orange dye (ThermoFisher) were prepared in 25 μL of PBS, transferred to wells of MicroAmp Optical 96-Well Reaction Plates (ThermoFisher), and sealed with MicroAmp Optical Adhesive Film (ThermoFisher). Melting curves were run in a 7500 fast Real-Time PCR system (Applied Biosystems) selecting TAMRA as reporter and ROX as passive reference. The thermal profile included an initial step of 2 min at 25° C. and a final step of 2 min at 99° C., with a ramp rate of 1.2%. The first derivative of the raw data was plotted as a function of temperature to obtain the derivative melting curves.

[0311] Melting temperatures (Tm) for unpairing of the heavy chains were determined from the peaks of the derivative curves.

[0312] The results are shown in FIGS. 1A to 1E.

[0313] Tm values for unpairing of the light chains from the heavy chains in the Fab regions of the antigen-binding molecules were also determined.

[0314] The results of the Differential Scanning Fluorimetry experiments are summarised in the table shown in FIG. 2A.

[0315] The introduction of the LCKC substitutions stabilised the Fc region of all the engineered variants tested (i.e. GASD, GASDIE, GASDALIE, EKEG), but destabilised the WT Fc region. LCKC increased the Tm of the engineered Fc variants between 9.9° C. and 23.2° C. LCKC decreased the Tm of the WT Fc by 8.5° C.

[0316] The rank order of the thermostability of the engineered Fc variants lacking the LCKC substitutions was as follows: WT (69.7° C.)>GASD (63.6° C.)>EKEG (60.3° C.)>GASDALIE (48.1° C.)≈GASDIE (40.0° C.).

[0317] The rank order of the thermostability of the engineered Fc variants comprising the LCKC substitutions was as follows: GASD_LCKC (75.9° C.)>EKEG_LCKC (70.2° C.)>GASDALIE_LCKC (63.3≈GASDIE_LCKC (63.2° C.)>WT_LCKC (61.2° C.).

[0318] The change in thermostability of the engineered Fc variants with the LCKC substitutions relative to the Fc WT was as follows: GASD_LCKC (+6.2° C.)>EKEG_LCKC (+0.5° C.)>GASDALIE_LCKC (−6.4° C.)≥GASDIE_LCKC (−6.5° C.).

[0319] Introduction of the LCKC substitutions did not affect significantly the thermostability of the Fab region.

Example 3

Analysis of Affinity of Antigen-Binding Molecules Comprising Engineered Fc Regions for Human Fc Receptor FcγRIIIa-158V

[0320] The antigen-binding molecules prepared as described in Example 1 were evaluated for binding to human Fc receptor FcγRIIIa comprising the polymorphism 158V, by Biolayer Interferometry (BLI) using a Pall ForteBio Octet Red384 system.

[0321] Anti-Penta-HIS (HIS1K) biosensors were purchased from Forte Bio (18-5120), and were incubated for 60 sec in PBS buffer (pH 7.2) to obtain the first baseline, and were subsequently loaded for 120 sec with histidine-tagged human FcγRIIIa-158V in PBS pH 7.2. After loading, biosensors were incubated for 60 sec in PBS buffer (pH 7.2) to obtain the second baseline, followed by incubation for 60 sec with a dilution series of the test antigen-binding molecules at concentrations ranging from 15.6 nM to 500 nM in PBS pH 7.2, to obtain association curves. Finally, the biosensors were incubated for 120 sec in PBS pH 7.2 to obtain dissociation curves.

[0322] Kinetic and affinity constants were calculated by global fitting of the association and dissociation data to a 1:1 binding model.

[0323] The results are shown in FIGS. 3A to 3J, and are summarized in the table shown in FIG. 4.

[0324] GASD, GASDIE, GASDALIE and EKEG Fc variants displayed increased binding affinity to human FcγRIIIa-158V as compared to WT Fc.

[0325] The rank order of affinities was as follows: GASDALIE≈GASDIE>GASD≈EKEG>WT.

[0326] The observed increase in binding affinity for the engineered Fc variants appeared to mainly be as a consequence of a decrease in the dissociation kinetics.

[0327] Introduction of LCKC substitutions did not significantly modify the binding affinity of the WT Fc or GASD, GASDIE, GASDALIE and EKEG Fc variants to human FcγRIIIa-158V.

[0328] The rank order of affinities was the same as for the molecules lacking the LCKC substitutions (i.e. GASDALIE_LCKC≈GASDIE_LCKC>GASD_LCKC≈EKEG_LCKC>WT_LCKC).

Example 4

Analysis of Affinity of Antigen-Binding Molecules Comprising Engineered Fc Regions for Human and Mouse Fc Receptors

[0329] The antigen-binding molecules WT and GASDALIE_LCKC prepared as described in Example 1 were analysed by Biolayer Interferometry (BLI) using a Pall ForteBio Octet Red384 system, for binding to: [0330] human Fc receptors: hFcγRIIIa-158F, hFcγRIIIa-158V, hFcγRIIa-167H, hFcγRIIa-167R, hFcγRIIb and hFcRn; and [0331] mouse Fc receptors: mFcγRIV (orthologue of hFcγRIIIa), mFcγRIII (orthologue of hFcγRIIa), mFcγRIIb (orthologue of hFcγRIIb) and mFcRn.

[0332] Anti-Penta-HIS (HIS1K) biosensors were incubated for 60 sec in PBS buffer to obtain the first baseline, and were subsequently loaded for 120 sec with histidine-tagged Fc receptors in PBS. After loading, biosensors were incubated for 60 sec in PBS buffer (pH 7.2 for Fcγ receptors and pH 5.8 for FcRn) to obtain the second baseline, followed by incubation for 60 sec with a dilution series of the test antigen-binding molecules in PBS (pH 7.2 for Fcγ receptors and pH 5.8 for FcRn), at concentrations ranging from 125 nM to 4000 nM (for experiments investigating binding to Fcγ receptors), or 75 nM to 1000 nM (for experiments investigating binding to FcRn receptors), to obtain association curves. Finally, the biosensors were incubated for 120 sec in PBS (pH 7.2 for Fcγ receptors and pH 5.8 for FcRn) to obtain dissociation curves.

[0333] PBS pH 7.2 was used for experiments investigating binding to Fcγ receptors, and PBS pH 5.8 was used for experiments investigating binding to FcRn receptors. Kinetic and affinity constants were calculated by global fitting of the association and dissociation data to a 1:1 binding model.

[0334] The results are shown in FIGS. 5A to 5J, FIGS. 6A to 6F, 7A to 7D and are summarized in the table shown in FIG. 8.

[0335] The GASDALIE_LCKC variant Fc displayed increased affinity to the activatory Fcγ receptors and FcRn receptors relative to WT Fc: hFcγRIIIa-158F, hFcγRIIIa-158V, hFcγRIIa-167H and hFcγRIIa-167R, hFcRn, mFcγRIV and mFcRn.

[0336] The GASDALIE_LCKC variant Fc displayed decreased affinity to the inhibitory Fcγ receptor hFcγRIIb relative to WT Fc.

[0337] The GASDALIE_LCKC variant Fc did not differ significantly relative to WT Fc in its affinity for binding to mFcγRIII and mFcγRIIb. Overall, the GASDALIE_LCKC variant Fc displayed increased affinity for human and mouse activatory Fcγ receptors and FcRn, and increased selectivity for human activatory Fcγ receptors as compared to human inhibitory Fcγ receptors.

Example 5

Analysis of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of Antigen-Binding Molecules Comprising Engineered Fc Regions

[0338] The antigen-binding molecules WT and GASDALIE_LCKC prepared as described in Example 1 were analysed for their ability to cause ADCC to cells expressing the target antigen (HER3), in an in vitro assay.

[0339] HEK 293 T cells stably transfected with constructs encoding human HER3 were used as target cells in the assay (expression of HER3 at the cell surface was confirmed by analysis by flow cytometry using a HER3-specific antibody).

[0340] Briefly, target cells were plated in wells of 96 well U-bottom plates at a density of 20,000 cells/well. Cells were incubated with WT or GASDALIE_LCKC antigen-binding molecules in a dilution series with final concentrations ranging from 50,000 ng/ml to 0.18 ng/ml (50,000 ng/ml, 8,333 ng/ml, 1,389 ng/ml, 231 ng/ml, 38.6 ng/ml, 6.4 ng/ml, 1.1 ng/ml and 0.18 ng/ml), or were left untreated. The cells were incubated at 37° C. and at 5% CO.sub.2 for 30 min.

[0341] Effector cells (Human Natural Killer Cell Line NoGFPCD16.NK92; 176V) were subsequently added to the wells at a density of 60,000 cells/well (i.e. the effector:target cell ratio was 3:1).

[0342] The following control conditions were included: target cell maximal LDH release (contained target cells only), spontaneous release (contained target cells and effectors cells, in the absence of antigen-binding molecules) and background (cell culture media only).

[0343] Plates were span down and incubated at 37° C. and at 5% CO2 for 21 hours. LDH Release Assay was performed using the Pierce LDH Cytotoxicity Assay Kit. 10 μl of Lysis Buffer (10×) was added to target cell maximal LDH release control wells, and incubated at 37° C. and 5% CO.sub.2 for 20 min. After incubation, plates were span down, and 50 ml of the supernatant was transferred to clear flat-bottom 96-well plates. Reactions were started by addition of 50 μl of LDH substrate-containing assay mix to the supernatants, and reactions were incubated at 37° C. for 30 min. Reactions were stopped by addition of 50 μl of stop solution, and absorbance at 490 nm and 680 nm was recorded using a BioTek Synergy HT microplate reader.

[0344] Absorbance from test samples were corrected to values obtained for background and spontaneous release control conditions, and percent cytotoxicity was calculated relative to target cell maximal LDH release control, and plotted as a function of antibody concentration. EC50 values (ng/ml) were calculated.

[0345] The results are shown in FIGS. 9A and 9B. Antigen-binding molecules comprising both WT Fc and GASDALIE_LCKC Fc elicited concentration-dependent ADCC to cells expressing the target antigen. Antigen-binding molecules comprising GASDALIE_LCKC Fc had an increased maximum cytotoxicity as compared to antigen-binding molecules comprising WT Fc, and were potent having a 6-fold decrease in EC50 relative to WT (FIG. 9B).

Example 6

Analysis of Tumor Growth Inhibition In Vivo by Antigen-Binding Molecules Comprising Engineered Fc Regions

[0346] Female NCr nude mice approximately 6-8 weeks old were purchased from In Vivos (Singapore). Animals were housed under specific pathogen-free conditions and were treated in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

[0347] Ectopic tumors were established by subcutaneous injection of 5×10.sup.6 A549 cells (HER3-expressing lung cancer cells) into the right flank. Mice were administered biweekly with IP injection of 25 mg/kg of the antigen-binding molecule comprising WT-Fc (n=6), or the antigen-binding molecule comprising the GASDALIE_LCKC substitutions (n=6), for a total of 6 weeks. A control treatment group received an equal volume of PBS (vehicle; n=8).

[0348] Tumor volumes were measured 3 times a week using a digital caliper and calculated using the formula [L×W2/2]. Study End point was considered to have been reaches once the tumors of the control arm measured >1.5 cm in length.

[0349] The results are shown in FIG. 10. The antigen-binding molecule comprising the GASDALIE_LCKC substitutions was found to be significantly more potent at inhibiting tumor growth than the antigen-binding molecule comprising WT-Fc.

Example 7

Analysis of Possible Sequence Liabilities/Immunogenic Sequences Introduced by GASDALIE LCKC Substitutions

[0350] The inventors next investigated whether introducing the GASDALIE_LCKC substitutions into humanized IgG1 antibody trastuzumab influenced properties relevant to antibody production, or use in therapy.

[0351] The inventors investigated whether the GASDALIE_LCKC substitutions were predicted to affect N-glycosylation, O-glycosylation, C-mannosylation, Asn deamidation, solubility and immunogenicity by in silico analysis of the amino acid sequences of the constituent polypeptides of trastuzumab, and the trastuzumab variant comprising GASDALIE_LCKC substitutions.

[0352] The introduction of GASDALIE_LCKC substitutions into the Fc region of trastuzumab was predicted not to affect N-glycosylation, O-glycosylation, C-mannosylation, Asn deamidation, nor to introduce any immunogenic peptides, and was predicted to result in a 2% increase in solubility.

Example 8

Conclusion

[0353] The antigen-binding molecules comprising GASDALIE_LCKC Fc were demonstrated to be provided with the following combination of advantageous properties relative to WT Fc: [0354] (i) increased affinity for activatory Fcγ receptors(>12 times increase in binding to human FcγRIIIa activatory receptors, and similar binding to murine FcγRIV activatory receptors); [0355] (ii) increased affinity for the neonatal Fc receptor FcRn; [0356] (iii) decreased affinity for inhibitory Fcγ receptors; [0357] (iv) increased selectivity for activatory Fcγ receptors vs. inhibitory Fcγ receptors [0358] (v) similar thermostability [0359] (vi) increased ADCC activity in vitro (6 times increase) [0360] (vii) improved tumor growth inhibition in vivo; [0361] (viii) no additional sequence liabilities/immunogenicity.