Crystal structures of heterodimeric Fc domains

Patent metadata

Field	Value
Publication number	US-9732155-B2
Application number	US-201314439532-A
Country	US
Kind code	B2
Filing date	Oct 31, 2013
Priority date	Nov 4, 2011
Publication date	Aug 15, 2017
Grant date	Aug 15, 2017

Abstract

Disclosed are the atomic coordinates of compositions comprising Fc heterodimer proteins in crystalline form derived from high resolution x-ray diffraction. Further disclosed are systems and methods for using all or a portion of these atomic coordinates to identify and design improved Fc heterodimer proteins. Further disclosed are compositions comprising a mixture of (i) a solubilized Fc heterodimer protein and (ii) a mother liquor solution. The mother liquor solution comprises between 2% and 10% (v/v) ethylene glycol, between 10% and 25% (w/v) polyethylene glycol having an average molecular weight of between 2000 Daltons and 10000 Daltons, and between 0.05 M and 0.40 M ammonium iodide. Further disclosed are systems and methods of identifying a mutation which promotes heterodimeric Fc chain pair formation in which structure based modeling is performed to identify a candidate mutation to an Fc chain using all or a portion of the disclosed three-dimensional atomic coordinates.

First claim

What is claimed:

1. A composition comprising an Fc heterodimer protein in crystalline form, wherein:
said Fc heterodimer protein comprises the amino acid sequences set forth in (i) SEQ ID NOS:2 and 3 or (ii) SEQ ID NOS:4 and 5;
said crystal is in space group P2 1 2 1 2 1 ; and
said crystal has unit cell dimensions a=49±2 Å, b=75±2 Å, c=149±2 Å, α=β=γ=90°.

2. The composition of claim 1 , wherein said Fc heterodimer protein comprises the amino acid sequences set forth in SEQ ID NOS:2 and 3 and has a three dimensional structure characterized by the atomic coordinates of (i) chains A and B of FIG. 27 or (ii) chains a and b of FIG. 27 .

3. The composition of claim 1 , wherein said Fc heterodimer protein comprises the amino acid sequences set forth in SEQ ID NOS:4 and 5 and has a three dimensional structure characterized by the atomic coordinates of (i) chains A and B of FIG. 26 or (ii) chains a and b of FIG. 26 .

4. The composition of claim 1 , wherein said Fc heterodimer protein comprises the amino acid sequences set forth in SEQ ID NOS: 2 and 3 forming a CH3 domain interface, and wherein the Fc heterodimer protein provides complementary hydrophobic and electrostatic surfaces, created by residues 366, 392, 394 of SEQ ID NO: 2 and residues 351, 405, 407 of SEQ ID NO: 3, at the CH3 domain interface with opposite surface complementarity to corresponding wild type Fc interface surfaces.

5. The composition of claim 1 , wherein said Fc heterodimer protein comprises the amino acid sequences set forth in SEQ ID NOS: 4 and 5 forming a CH3 domain interface, and wherein the Fc heterodimer protein provides complementary hydrophobic and electrostatic surfaces, created by residues 366, 392, 394 of SEQ ID NO: 4 and residues 351, 405, 407 of SEQ ID NO: 5, at the CH3 domain interface with distinct surface complementarity relative to the corresponding wild type Fc interface surfaces.

6. The composition of claim 1 , wherein said Fc heterodimer protein comprises a D399-K409 salt bridge.

7. The composition of claim 4 , wherein said CH3 domain interface is formed in two orientations and said crystalline form of said Fc heterodimer protein comprises a 50:50 mixture of the two orientations.

8. A method of obtaining the composition of claim 1 , comprising the steps of:
a) producing and purifying said Fc heterodimer protein; and
b) subjecting the purified Fc heterodimer protein of step a) to conditions which promote crystallization, thereby obtaining the Fc heterodimer protein in crystalline form.

9. The method of claim 8 , wherein the conditions which promote crystallization comprise mixing the purified Fc heterodimer protein with a mother liquor solution, wherein the mother liquor solution comprises between 2% and 10% (v/v) ethylene glycol, between 10% and 25% (w/v) polyethylene glycol having an average molecular weight of between 2000 Daltons and 10000 Daltons, and between 0.05 M and 0.40 M ammonium iodide.

10. The method of claim 9 , wherein the mother liquor solution comprises 5% (v/v) ethylene glycol, 18% (w/v) polyethylene glycol having an average molecular weight of 3350 Daltons, and 0.15 M ammonium iodide.

11. The method of claim 9 , wherein the purified Fc heterodimer protein is mixed with a first aliquot of the mother liquor solution and suspended over a second aliquot of the mother liquor in a hanging drop method.

12. The method of claim 11 , wherein the purified Fc heterodimer protein is mixed with a first aliquot of the mother liquor solution in a 2:1 ratio.

13. The method of claim 11 , wherein the purified Fc heterodimer protein is incubated at a temperature of between 15° C. and 25° C. after the mixing.

14. A crystallizable composition comprising a mixture of (i) a solubilized Fc heterodimer protein comprising the amino acid sequence set forth in (a) SEQ ID NOS: 2 and 3 or (b) SEQ ID NOS: 4 and 5 and (ii) a mother liquor solution, wherein the mother liquor solution comprises between 2% and 10% (v/v) ethylene glycol, between 10% and 25% (w/v) polyethylene glycol having an average molecular weight of between 2000 Daltons and 10000 Daltons, and between 0.05 M and 0.40 M ammonium iodide.

15. The crystallizable composition of claim 14 , wherein the mother liquor solution comprises 5% (v/v) ethylene glycol, 18% (w/v) polyethylene glycol having an average molecular weight of 3350 Daltons, and 0.15 M ammonium iodide.

Assignees

• Zymeworks Inc

Inventors

• Spreter Von Kreudenstein Thomas
• Dixit Surjit Bhimarao
• Lario Paula Irene
• Escobar-Cabrera Eric
• Boulanger Martin J
• Suits Michael D L

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P37/02

  Immunomodulators · CPC title

• C07K16/468

  Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies · CPC title

• C07K2317/524

  CH2 domain · CPC title

• C07K2317/526

  CH3 domain · CPC title