HETERODIMERIZED POLYPEPTIDE

• US 20140199294A1
• Filed: 06/29/2012
• Published: 07/17/2014
• Est. Priority Date: 06/30/2011
• Status: Active Grant

• Current Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Original Assignee: Chugai Seiyaku Kabushiki Kaisha (Roche Holding AG)
• Inventors: Mimoto, Futa, Kuramochi, Taichi, Igawa, Tomoyuki, Katada, Hitoshi, Shiraiwa, Hirotake, Kadono, Shojiro, Kawazoe, Meiri
• Granted Patent: US 9,890,218 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: C07K 1/1075 by covalent attachment of amino acids or peptide residues
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/303 Liver or Pancreas
• CPC Class Codes: C07K 16/36 against blood coagulation factors
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/71 Decreased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The present inventors produced a heterodimerized polypeptide having an Fc region formed from two polypeptides with different amino acid sequences (a first polypeptide and a second polypeptide), and succeeded in producing a heterodimerized polypeptide containing an Fc region with improved Fc region function compared to that of a homodimer in which the Fc region is composed of only the first polypeptide or only the second polypeptide by conventional technology.

Claims

1-78. -78. (canceled)

79. A molecule comprising a heterodimeric Fc region,

wherein the heterodimeric Fc region is a heterodimer of a first polypeptide and a second polypeptide that differs in amino acid sequence from the first polypeptide,

wherein a function of the heterodimeric Fc region is increased compared to said function of one or both of (a) a dimeric Fc region that is a homodimer of the first polypeptide, and (b) a dimeric Fc region that is a homodimer of the second polypeptide,

wherein the increased function is (i) binding affinity for an Fc receptor, or (ii) selectivity of binding to one type of Fc receptor over another type of Fc receptor.

80. The molecule of claim 79, wherein one or both of the first and second polypeptide comprises an IgG1 CH2 domain with one or more mutations, and wherein at least one of the one or more mutations is at an amino acid position selected from the group consisting of

Ala at position 231;

Pro at position 232;

Glu at position 233;

Leu at position 234;

Leu at position 235;

Gly at position 236;

Gly at position 237;

Pro at position 238;

Ser at position 239;

Val at position 240;

Asp at position 265;

Val at position 266;

Ser at position 267;

His at position 268;

Glu at position 269;

Asp at position 270;

Pro at position 271;

Gln at position 295;

Tyr at position 296;

Ser at position 298;

Tyr at position 300;

Ser at position 324;

Asn at position 325;

Lys at position 326;

Ala at position 327;

Leu at position 328;

Pro at position 329;

Ala at position 330;

Pro at position 331;

Ile at position 332;

Glu at position 333;

Lys at position 334;

Thr at position 335;

Ile at position 336; and

Ser at position 337 (all positions by EU numbering).

81. The molecule of claim 79, wherein the increased function is binding affinity for an Fcγ

receptor.

82. The molecule of claim 81, wherein the Fcγ

receptor is human Fcγ

RIa.

83. The molecule of claim 82, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region i of Tables 2-1 and 2-2 of the specification.

84. The molecule of claim 82, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region ii of Tables 2-1, 2-2, and 2-3 of the specification.

85. The molecule of claim 81, wherein the Fcγ

receptor is human Fcγ

RIIa R.

86. The molecule of claim 85, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region i of Tables 3-1 and 3-2 of the specification.

87. The molecule of claim 85, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region ii of Tables 3-1 and 3-2 of the specification.

88. The molecule of claim 81, wherein the Fcγ

receptor is human Fcγ

RIIa H.

89. The molecule of claim 88, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region i of Table 4 of the specification.

90. The molecule of claim 88, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region ii of Table 4 of the specification.

91. The molecule of claim 81, wherein the Fcγ

receptor is human Fcγ

RIIb.

92. The molecule of claim 91, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region i of Table 5 of the specification.

93. The molecule of claim 91, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region ii of Table 5 of the specification.

94. The molecule of claim 81, wherein the Fcγ

receptor is human Fcγ

RIIIa.

95. The molecule of claim 94, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region i of Table 6 of the specification.

96. The molecule of claim 94, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region ii of Table 6 of the specification.

97. The molecule of claim 79, wherein the heterodimer exhibits selectivity of binding to a first Fcγ

receptor versus a second Fcγ

receptor that is increased (a) compared to the selectivity of binding of a homodimer of the first polypeptide to the first Fcγ

receptor versus the second Fcγ

receptor, and (b) compared to the selectivity of binding of a homodimer of the second polypeptide to the first Fcγ

receptor versus the second Fcγ

receptor.

98. The molecule of claim 97, wherein the first Fcγ

receptor is an activating Fcγ

receptor and the second Fcγ

receptor is an inhibitory Fcγ

receptor.

99. The molecule of claim 98, wherein the activating Fcγ

receptor is human Fcγ

RIa.

100. The molecule of claim 99, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region a of Tables 19-1, 19-2, 19-3, and 19-4 of the specification.

101. The molecule of claim 99, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region b of Tables 19-1, 19-2, 19-3, 19-4 and 19-5 of the specification.

102. The molecule of claim 98, wherein the activating Fcγ

receptor is human Fcγ

RIIa R.

103. The molecule of claim 102, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region a of Table 20-1 of the specification.

104. The molecule of claim 102, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region b of Tables 20-1, 20-2, and 20-3 of the specification.

105. The molecule of claim 98, wherein the activating Fcγ

receptor is human Fcγ

RIIa H.

106. The molecule of claim 105, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region a of Table 21-1 of the specification.

107. The molecule of claim 105, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region b of Tables 21-1, 21-2, and 21-3 of the specification.

108. The molecule of claim 98, wherein the activating Fcγ

receptor is human Fcγ

RIIIa.

109. The molecule of claim 108, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region a of Table 22-1 of the specification.

110. The molecule of claim 108, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region b of Tables 22-1, 22-2, and 22-3 of the specification.

111. The molecule of claim 97, wherein the first Fcγ

receptor is an inhibitory Fcγ

receptor and the second Fcγ

receptor is an activating Fcγ

receptor.

112. The molecule of claim 111, wherein the activating Fcγ

receptor is human Fcγ

RIa.

113. The molecule of claim 112, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region c of Tables 23-1 and 23-2 of the specification.

114. The molecule of claim 112, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region d of Tables 23-1 and 23-2 of the specification.

115. The molecule of claim 111, wherein the activating Fcγ

receptor is human Fcγ

RIIa R.

116. The molecule of claim 115, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region c of Table 24-1 of the specification.

117. The molecule of claim 115, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region d of Tables 24-1 and 24-2 of the specification.

118. The molecule of claim 111, wherein the activating Fcγ

receptor is human Fcγ

RIIa H.

119. The molecule of claim 118, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region c of Tables 25-1 and 25-2 of the specification.

120. The molecule of claim 118, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region d of Tables 25-1, 25-2, and 25-3 of the specification.

121. The molecule of claim 111, wherein the activating Fcγ

receptor is human Fcγ

RIIIa.

122. The molecule of claim 121, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region c of Tables 26-1 and 26-2 of the specification.

123. The molecule of claim 121, wherein at least one of the first and second polypeptides comprises one or more amino acid mutations described in Region d of Tables 26-1, 26-2, 26-3, and 26-4 of the specification.

124. The molecule of claim 79, wherein at least one of the first and second polypeptides comprises an IgG1 CH2 domain comprising one or more of the following amino acid substitutions:

substitution of L at position 234 with Y;

substitution of L at position 235 with Y or Q;

substitution of G at position 236 with W;

substitution of S at position 239 with M;

substitution of H at position 268 with D;

substitution of D at position 270 with E;

substitution of S at position 298 with A;

substitution of K at position 326 with D;

substitution of A at position 327 with D;

substitution of L at position 328 with W;

substitution of A at position 330 with M or K;

substitution of K at position 334 with E or L (all positions by EU numbering).

125. The molecule of claim 79, wherein at least one of the first and second polypeptides comprises an IgG1 CH2 domain in which one or more of the following positions is mutated:

Leu at position 234,

Leu at position 235,

Gly at position 236,

Ser at position 239,

His at position 268,

Asp at position 270,

Ser at position 298,

Lys at position 326,

Ala at position 327,

Leu at position 328,

Ala at position 330,

Lys at position 334 (all positions by EU numbering).

126. The molecule of claim 79, wherein the first polypeptide comprises an IgG1 CH2 domain in which one or more of the following positions is mutated:

Leu at position 234,

Leu at position 235,

Gly at position 236,

Ser at position 239,

His at position 268,

Asp at position 270,

Ser at position 298,

Ala at position 327,

Leu at position 328,

Lys at position 334;

and wherein the second polypeptide comprises an IgG1 CH2 domain in which one or more of the following positions is mutated;

Asp at position 270,

Lys at position 326,

Ala at position 330,

Lys at position 334 (all positions by EU numbering).

127. The molecule of claim 79, wherein the first polypeptide comprises an IgG1 CH2 domain comprising one or more of the following amino acid substitutions:

substitution of L at position 234 with Y,

substitution of L at position 235 with Y or Q,

substitution of G at position 236 with W,

substitution of S at position 239 with M,

substitution of H at position 268 with D,

substitution of D at position 270 with E,

substitution of S at position 298 with A,

substitution of A at position 327 with D,

substitution of L at position 328 with W,

substitution of K at position 334 with L;

and wherein the second polypeptide comprises an IgG1 CH2 domain comprising one or more of the following amino acid substitutions;

substitution of D at position 270 with E;

substitution of K at position 326 with D;

substitution of A at position 330 with M or K;

substitution of K at position 334 with E (all positions by EU numbering).

128. The molecule of claim 79, wherein the first polypeptide comprises an IgG1 CH2 domain comprising a set of amino acid mutations selected from sets (i)-(vi):

(i) substitution of L at position 234 with Y,

substitution of L at position 235 with Y,

substitution of G at position 236 with W,

substitution of H at position 268 with D, and

substitution of S at position 298 with A;

(ii) substitution of L at position 234 with Y,

substitution of L at position 235 with Y,

substitution of G at position 236 with W,

substitution of H at position 268 with D,

substitution of D at position 270 with E, and

substitution of S at position 298 with A;

(iii) substitution of L at position 234 with Y,

substitution of L at position 235 with Q,

substitution of G at position 236 with W,

substitution of S at position 239 with M,

substitution of H at position 268 with D,

substitution of D at position 270 with E, and

substitution of S at position 298 with A;

(iv) substitution of L at position 234 with Y,

substitution of L at position 235 with Y,

substitution of G at position 236 with W,

substitution of H at position 268 with D,

substitution of S at position 298 with A, and

substitution of A at position 327 with D;

(v) substitution of L at position 234 with Y,

substitution of L at position 235 with Y,

substitution of G at position 236 with W,

substitution of S at position 239 with M,

substitution of H at position 268 with D,

substitution of S at position 298 with A, and

substitution of A at position 327 with D;

(vi) substitution of L at position 234 with Y,

substitution of L at position 235 with Y,

substitution of G at position 236 with W,

substitution of S at position 239 with M,

substitution of H at position 268 with D,

substitution of S at position 298 with A,

substitution of A at position 327 with D,

substitution of L at position 328 with W, and

substitution of K at position 334 with L;

and the second polypeptide comprises an IgG1 CH2 domain comprising a set of amino acid mutations selected from sets (vii)-(ix);

(vii) substitution of K at position 326 with D,

substitution of A at position 330 with M, and

substitution of K at position 334 with E;

(viii) substitution of D at position 270 with E,

substitution of K at position 326 with D,

substitution of A at position 330 with M, and

substitution of K at position 334 with E;

(ix) substitution of D at position 270 with E,

substitution of K at position 326 with D,

substitution of A at position 330 with K, and

substitution of K at position 334 with E (all positions by EU numbering).

129. The molecule of claim 79, wherein the heterodimer binds with higher affinity to an Fcγ

receptor and/or has increased selectivity of binding to an Fcγ

receptor over another Fcγ

receptor, compared to a homodimer of the first polypeptide.

130. The molecule of claim 129, wherein the heterodimer has increased physicochemical stability compared to a homodimer of the first polypeptide.

131. The molecule of claim 130, wherein the heterodimer has a higher Tm (temperature of thermal denaturation) than a homodimer of the first polypeptide.

132. The molecule of claim 129, wherein the function is increased binding affinity for human Fcγ

RIa.

133. The molecule of claim 132, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations described in Tables 31-1, 31-2, and 31-3 of the specification.

134. The molecule of claim 129, wherein the function is increased binding affinity for human Fcγ

RIIa R.

135. The molecule of claim 134, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations described in Tables 32-1 and 32-2 of the specification.

136. The molecule of claim 129, wherein the function is increased binding affinity for human Fcγ

RIIa H.

137. The molecule of claim 136, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations described in Tables 33-1 and 33-2 of the specification.

138. The molecule of claim 129, wherein the function is increased binding affinity for human Fcγ

RIIb.

139. The molecule of claim 138, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations described in Tables 34-1 and 34-2 of the specification.

140. The molecule of claim 129, wherein the function is increased binding affinity for human Fcγ

RIIIa.

141. The molecule of claim 140, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations described in Tables 35-1 and 35-2 of the specification.

142. The molecule of claim 79, wherein at least one of the first and the second polypeptides comprises one or more amino acid mutations that confer or increase a difference in isoelectric points between the first polypeptide and the second polypeptide.

143. The molecule of claim 142, wherein the first and second polypeptides comprise an IgG1 Fc region with one or more mutations, and wherein the one or more amino acid mutations that confer or increase a difference in isoelectric points between the first and the second polypeptides comprise mutations at one or more of the following positions:

Gly at position 137,

Gly at position 138,

Thr at position 139,

Lys at position 147,

Ser at position 192,

Leu at position 193,

Gln at position 196,

Tyr at position 198,

Ile at position 199,

Asn at position 203,

Lys at position 214,

Val at position 263,

Glu at position 272,

Lys at position 274,

Tyr at position 278,

Lys at position 288,

Lys at position 290,

Gly at position 316,

Lys at position 317,

Lys at position 320,

Lys at position 324,

Thr at position 335,

Ser at position 337,

Lys at position 340,

Leu at position 358,

Lys at position 360,

Gln at position 362,

Ser at position 364,

Ser at position 383,

Asn at position 384,

Gly at position 385,

Gln at position 386,

Pro at position 387,

Asn at position 390,

Val at position 397,

Val at position 422 (by EU numbering).

144. The molecule of claim 142, wherein the first and second polypeptides comprise an IgG1 Fc region with one or more mutations, and wherein the one or more amino acid mutations that confer or increase a difference in isoelectric points between the first and the second polypeptides comprise:

a mutation at one or more of the following positions in the first polypeptide;

Gln at position 196,

Ile at position 199,

Val at position 263,

Glu at position 272,

Gly at position 316,

Leu at position 358,

Ser at position 364,

Ser at position 383,

Pro at position 387,

Val at position 397;

and a mutation at one or more of the following positions in the second polypeptide;

Gly at position 137,

Gly at position 138,

Thr at position 139,

Lys at position 147,

Ser at position 192,

Leu at position 193,

Tyr at position 198,

Ile at position 199,

Asn at position 203,

Lys at position 214,

Lys at position 274,

Tyr at position 278,

Lys at position 288,

Lys at position 290,

Gly at position 316,

Lys at position 317,

Lys at position 320,

Lys at position 324,

Thr at position 335,

Ser at position 337,

Lys at position 340,

Leu at position 358,

Lys at position 360,

Gln at position 362,

Ser at position 383,

Asn at position 384,

Gly at position 385,

Gln at position 386,

Asn at position 390,

Val at position 422 (all positions by EU numbering).

145. The molecule of claim 79, wherein the molecule is an antibody.

146. The molecule of claim 145, wherein the antibody is a bispecific antibody.

147. The molecule of claim 79, wherein the molecule is a peptide Fc fusion protein or a scaffold Fc fusion protein.

148. A pharmaceutical composition comprising the molecule of claim 79 and a medically acceptable carrier.

149. A method of producing the molecule of claim 79, the method comprising:

identifying a parent molecule comprising an Fc region that is a homodimer of the first polypeptide;

producing a mutated version of the first polypeptide, the mutated version being the second polypeptide; and

generating the molecule comprising a heterodimeric Fc region that is a heterodimer of the first polypeptide and the second polypeptide.

150. The method of claim 149, further comprising:

assaying said function of the generated molecule, thereby determining that said function of the generated molecule is increased compared to said function of both (a) a dimeric Fc region that is a homodimer of the first polypeptide, and (b) a dimeric Fc region that is a homodimer of the second polypeptide.

151. A method of producing the molecule of claim 79, the method comprising:

identifying a parent molecule comprising an Fc region that is a homodimer of a starting polypeptide;

producing a first mutated version of the starting polypeptide, the first mutated version being the first polypeptide;

producing a second mutated version of the starting polypeptide, the second mutated version being the second polypeptide; and

generating the molecule comprising a heterodimeric Fc region that is a heterodimer of the first polypeptide and the second polypeptide.

152. The method of claim 151, further comprising:

assaying said function of the generated molecule, thereby determining that said function of the generated molecule is increased (a) compared to said function of a dimeric Fc region that is a homodimer of the first polypeptide, and (b) compared to said function of a dimeric Fc region that is a homodimer of the second polypeptide.