Humanized Anti-CD79B Antibodies and Immunoconjugates and Methods of Use

• US 20090068202A1
• Filed: 07/15/2008
• Published: 03/12/2009
• Est. Priority Date: 07/16/2007
• Status: Active Grant

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Polson, Andrew, Dennis, Mark, Junutula, Jagath Reddy, Zheng, Bing, Elkins, Kristi, Chen, Yvonne
• Granted Patent: US 9,845,355 B2
• Time in Patent Office: Days
• US Class Current: 424/173.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/39541 against normal tissues, cells
• CPC Class Codes: A61K 39/39558 against tumor tissues, cells, antigens
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 51/1027 against receptors, cell-surface antigens or cell-surface determinants
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/3061 Blood cells
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention is directed to compositions of matter useful for the treatment of hematopoietic tumor in mammals and to methods of using those compositions of matter for the same.

Claims

1. An anti-CD79b antibody comprising:

(a) at least one HVR sequence selected from the group consisting of;

(i) HVR-L1 comprising sequence A1-A16, wherein A1-A16 is KSSQSLLDSDGKTYLN (SEQ ID NO;

     59)

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is LVSKLDS (SEQ ID NO;

     60)

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is WQGTHFPYT (SEQ ID NO;

     61)

(iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWMN (SEQ ID NO;

     62)

(v) HVR-H2 comprising sequence E1-E18, wherein E1-E18 is GMIDPSDSETHYNHIFKD (SEQ ID NO;

     63)

(vi) HVR-H3 comprising sequence F1-F6, wherein F1-F6 is ARNLYL (SEQ ID NO;

     64); and

(b) at least one variant HVR, wherein the variant HVR comprises modification of at least one residue of the sequence depicted in SEQ ID NO;

59, 60, 61, 62, 63 or 64.

2. The antibody of claim 1, wherein Cl in a variant HVR-L3 is F.

3. The antibody of claim 1, wherein C8 in a variant HVR-L3 is F.

4. The antibody of claim 1, wherein the antibody is humanized.

5. The antibody of claim 1, wherein at least a portion of the framework sequence is a human consensus framework sequence.

6. The antibody of claim 1, wherein said modification is substitution, insertion or deletion.

7. The antibody of claim 1, wherein a HVR-L3 variant comprises one substitution in any of the following positions:

C1 (F) And C8 (F).

8. The antibody of claim 1 comprising a HVR-L1 having the sequence of SEQ ID NO:

59.

9. The antibody of claim 1 comprising a HVR-L2 having the sequence of SEQ ID NO:

60.

10. The antibody of claim 1 comprising a HVR-H1 having the sequence of SEQ ID NO:

62.

11. The antibody of claim 1 comprising a HVR-H2 having the sequence of SEQ ID NO:

63.

12. The antibody of claim 1 comprising a HVR-H3 having the sequence of SEQ ID NO:

64.

13. The antibody of claim 1, wherein C1 in a variant HVR-L3 is F.

14. The antibody of claim 1, wherein C8 in a variant HVR-L3 is F.

18. The humanized antibody of claim 13, 14 or 15 wherein the murine antibody is produced by hybridoma cell line deposited with the ATCC as PTA-7712 on Jul. 11, 2006.

25. The antibody of any of claims 13-23 wherein the binding affinity is measured by Biacore or radioimmunoassay.

26. The antibody of claims 1-3 comprising human κ

subgroup 1 consensus framework sequence.

27. The antibody of claims 1-3 comprising heavy chain human subgroup III consensus framework sequence.

28. The antibody of claim 27 wherein the framework sequence comprises a substitution at position 71, 73 and/or 78.

29. The antibody of claim 28 wherein said substitution is R71A, N73T and/or L78A.

31. The antibody of the preceding claims, wherein both the humanized antibody and chimeric antibody are monovalent or bivalent.

32. The antibody of the preceding claims, wherein both the humanized antibody and chimeric antibody comprise a single Fab region linked to an Fc region.

44. The antibody of claim 1, wherein the antibody comprises a light chain variable domain having at least 90% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:

12.

45. The antibody of claim 1, wherein the antibody comprises a heavy chain variable domain having at least 90% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:

16.

46. The antibody of claim 1, wherein the antibody comprises a heavy chain variable domain comprising one, two, three or four framework amino acid sequences selected from SEQ ID NOs:

69, 70, 71 and 72.

47. The antibody of claim 1, wherein the antibody comprises a light chain variable domain comprising one, two, three or four framework amino acid sequences selected from SEQ ID NOs:

65, 66, 67 and 68.

48. The antibody of claim 1, wherein the antibody comprises a heavy chain variable domain comprising one, two, three or four framework amino acid sequences having at least 90% amino acid sequence identity to an amino acid selected from SEQ ID NOs:

69, 70, 71 and 72.

49. The antibody of claim 1, wherein the antibody comprises a light chain variable domain comprising one, two, three or four framework amino acid sequences having at least 90% amino acid sequence identity to an amino acid selected from selected from SEQ ID NOs:

65, 66, 67 and 68.

50. The antibody of claim 44, wherein the antibody comprises a heavy chain variable domain having at least 90% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:

16.

51. The antibody of claim 45, wherein the antibody comprises a light chain variable domain having at least 90% amino acid sequence identity to an amino acid sequence selected from SEQ ID NO:

12.

55. A polynucleotide encoding an antibody of claim 1, 7, 42, 54 or 177.

56. A vector comprising the polynucleotide of claim 55.

57. A host cell comprising the vector of claim 56.

59. The antibody of claim 1, 7, 42, 54 or 177, wherein the antibody binds to an epitope within a region of CD79b from amino acid 29-39 of SEQ ID NO:

2 or amino acid 1-11 of SEQ ID NO;

78.

60. The antibody of claim 1, 7, 42, 54 or 177, wherein the CD79b is expressed on the surface of a cell.

61. The antibody of claim 60, wherein the cell is a B cell.

62. The antibody of claim 60, wherein the B cell is associated with a B cell proliferative disorder.

63. The antibody of claim 62, wherein the B cell proliferative disorder is a cancer.

64. The antibody of claim 63, wherein the B cell proliferative disorder is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

65. The antibody of claim 1, 7, 42, 54 or 177, wherein the antibody is a monoclonal antibody.

66. The antibody of claim 65, wherein the antibody is an antibody fragment selected from a Fab, Fab′

-SH, Fv, scFv or (Fab′

)₂ fragment.

67. The antibody of claim 65, wherein the antibody is humanized.

68. The antibody of claim 1, 7, 42, 54 or 177, wherein the antibody binds to the same epitope as an antibody selected from ATCC as PTA-7712, deposited on Jul. 11, 2006 and an antibody comprising a heavy chain variable domain of SEQ ID NO:

16 and a light chain variable domain of SEQ ID NO;

12.

69. An immunoconjugate comprising an antibody of claim 1, 7, 42, 54 or 177 covalently attached to a cytotoxic agent.

70. The immunoconjugate of claim 69, wherein the cytotoxic agent is selected from a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioactive isotope and a nucleolytic enzyme.

71. The immunoconjugate of claim 70, wherein the immunoconjugate having the formula Ab-(L-D)p, wherein:

(a) Ab is the antibody of claim 1, 7, 42, 54 or 177;

(b) L is a linker;

(c) D is a drug moiety.

72. The immunoconjugate of claim 71, wherein L is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (SMCC), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate (SIAB).

73. The immunoconjugate of claim 71, wherein D is selected from an auristatin and dolostatin.

74. A pharmaceutical composition comprising the immunoconjugate of claim 71 and a pharmaceutically acceptable carrier.

75. A method of inhibiting the growth of a cell that expresses CD79b, said method comprising contacting said cell with an antibody of any of claims 1, 7, 42, 54 or 177, thereby causing an inhibition of growth of said cell.

76. The method of claim 75 wherein said antibody is conjugated to a cytotoxic agent.

77. The method of claim 75 wherein said antibody is conjugated to a growth inhibitory agent.

78. A method of treating a subject having cancer, said method comprising administering to the subject an effective amount of an antibody of any of claims 1, 7, 42, 54 or 177.

79. The method of claim 78 wherein the cancer is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

80. The method of claim 78 wherein said antibody is conjugated to a cytotoxic agent.

81. The method of claim 78 wherein said antibody is conjugated to a growth inhibitory agent.

82. A method of treating a proliferative disorder in a subject, said method comprising administering to the subject an effective amount of an antibody of any of claims 1, 7, 42, 54 or 177.

83. The method of claim 82 wherein said proliferative disorder is cancer.

84. The method of claim 83 wherein said cancer is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

85. The method of claim 82 wherein said antibody is conjugated to a cytotoxic agent.

86. The method of claim 82 wherein said antibody is conjugated to a growth inhibitory agent.

87. The method of inhibiting the growth of a cell, wherein growth of said cell is at least in part dependent upon a growth potentiating effect of CD79b, said method comprising contacting said cell with an effective amount of an antibody of any of claims 1, 7, 42, 54 or 177, thereby inhibiting the growth of said cell.

88. The method of claim 87 wherein said antibody is conjugated to a cytotoxic agent.

89. The method of claim 87, wherein said antibody is conjugated to a growth inhibitory agent.

90. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of CD79b, said method comprising contacting said cell with an effective amount of an antibody of any of claims 1, 7, 42, 54 or 177.

91. The method of claim 90 wherein said tumor is associated with lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

92. The method of claim 90 wherein said antibody is conjugated to a cytotoxic agent.

93. The method of claim 90 wherein said antibody is conjugated to a growth-inhibitory agent.

94. A method of inhibiting B cell proliferation comprising exposing a cell to an immunoconjugate of claim 70 under conditions permissive for binding of the immunoconjugate to CD79b.

95. The method of claim 94, wherein the B cell proliferation is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

96. The method of claim 94, wherein the B cell is a xenograft.

97. The method of claim 94, wherein the exposing takes place in vitro.

98. The method of claim 94, wherein the exposing takes place in vivo.

99. A method of determining the presence of CD79b in a sample suspected of containing CD79b, said method comprising exposing said sample to an antibody of any of claims 1, 7, 42, 54 or 177, and determining binding of said antibody to CD79b in said sample wherein binding of said antibody to CD79b in said sample is indicative of the presence of said protein in said sample.

100. The method of claim 99 wherein the biological sample is from a patient suspected of having a B cell proliferative disorder.

101. The method of claim 100 wherein the B cell proliferative disorder is selected from lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

102. A method of binding an antibody of any of claims 1, 7, 42, 54 or 177 to a cell that expresses CD79b, said method comprising contacting said cell with an antibody of any of claims 1, 7, 42, 54 or 177.

103. The method of claim 102 wherein said antibody is conjugated to a cytotoxic agent.

104. The method of claim 102 wherein said antibody is conjugated to a growth-inhibitory agent.

105. A nucleic acid encoding the antibody of any of claims 1, 7, 42, 54 or 177.

106. A host cell comprising the nucleic acid of claim 105.

107. A composition comprising the antibody of any of claims 1-43.

108. The composition of claim 107, wherein the composition comprises a carrier.

109. The antibody of claims 1, 7, 42, 54 or 177, wherein the antibody is a cysteine engineered antibody comprising one or more free cysteine amino acids, wherein the cysteine engineered antibody is prepared by a process comprising replacing one or more amino acid residues of a parent antibody by a free cysteine amino acid.

110. The antibody of claim 109 wherein the one or more free cysteine amino acids have a thio reactivity value in the range of 0.6 to 1.0.

111. The cysteine engineered antibody of claim 109, wherein the cysteine engineered antibody is more reactive than the parent antibody with a thio-reactive reagent.

112. The cysteine engineered antibody of claim 109 wherein the process further comprises determining the thiol reactivity of the cysteine engineered antibody by reacting the cysteine engineered antibody with a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive than the parent antibody with the thiol-reactive reagent.

113. The cysteine engineered antibody of claim 109 wherein the one or more free cysteine amino acid residues are located in a light chain.

114. The cysteine engineered antibody of claim 109, wherein the antibody is an immunoconjugate comprising the cysteine engineered antibody covalently attached to a cytotoxic agent.

115. The cysteine engineered antibody of claim 114, wherein the cytotoxic agent is selected from a toxin, a chemotherapeutic agent, a drug moiety, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

116. The cysteine engineered antibody of claim 109, wherein the antibody is covalently attached to a capture label, a detection label, or a solid support.

117. The cysteine engineered antibody of claim 116 wherein the antibody is covalently attached to a biotin capture label.

118. The cysteine engineered antibody of claim 116 wherein the antibody is covalently attached to a fluorescent dye detection label.

119. The cysteine engineered antibody of claim 118 wherein the fluorescent dye is selected from a fluorescein type, a rhodamine type, dansyl, Lissamine, a cyanine, a phycoerythrin, Texas Red, and an analog thereof.

120. The cysteine engineered antibody of claim 116 wherein the antibody is covalently attached to a radionuclide detection label selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At, and ²¹³Bi.

121. The cysteine engineered antibody of claim 116 wherein the antibody is covalently attached to a detection label by a chelating ligand.

122. The cysteine engineered antibody of claim 121 wherein the chelating ligand is selected from DOTA, DOTP, DOTMA, DTPA and TETA.

123. The antibody of claims 1, 7, 42, 54 or 177 comprising an albumin binding peptide.

124. The antibody of claim 123, wherein the albumin binding peptide is selected from SEQ ID NOs:

80-84.

125. The antibody of claims 1, 7, 42, 54 or 177 wherein the antibody further comprises a free cysteine amino acid at one or more positions selected from 15, 43, 110, 144, 168 and 205 of the light chain according to Kabat numbering convention and 41, 88, 115, 118, 120, 171, 172, 282, 375, and 400 of the heavy chain according to EU numbering convention.

126. The antibody of claim 125, wherein a cysteine is at position 205 of the light chain.

127. The antibody of claim 125, wherein a cysteine is at position 118 of the heavy chain.

128. The antibody of claim 125, wherein a cysteine is at position 400 of the heavy chain.

129. The antibody of claim 125 wherein the antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, and a humanized antibody.

130. The antibody of claim 125 which is an antibody fragment.

131. The antibody of claim 130 wherein the antibody fragment is a Fab fragment.

132. The antibody of claim 125 which is selected from a chimeric antibody, a human antibody, or a humanized antibody.

133. The antibody of claim 125 which is produced in bacteria.

134. The antibody of claim 125 which is produced in CHO cells.

135. A method of determining the presence of a CD79b protein in a sample suspected of containing said protein, said method comprising exposing said sample to an antibody of claim 120 and determining binding of said antibody to said CD79b protein in said sample, wherein binding of the antibody to said protein is indicative of the presence of said protein in said sample.

136. The method of claim 135 wherein said sample comprises a cell suspected of expressing said CD79b protein.

137. The method of claim 135 wherein said cell is B cell.

138. The method of claim 135 wherein the antibody is covalently attached to a label selected from a fluorescent dye, a radioisotope, biotin, or a metal-complexing ligand.

139. A pharmaceutical formulation comprising the anti-CD79b antibody of claim 125, and a pharmaceutically acceptable diluent, carrier or excipient.

140. The antibody of claim 125 wherein the antibody is covalently attached to an auristatin or a maytansinoid drug moiety whereby an antibody drug conjugate is formed.

141. The antibody-drug conjugate of claim 140 comprising an antibody (Ab), and an auristatin or maytansinoid drug moiety (D) wherein the cysteine engineered antibody is attached through one or more free cysteine amino acids by a linker moiety (L) to D;

the compound having Formula I;

Ab-(L-D)_p 

 

I

where p is 1, 2, 3, or 4.

142. The antibody-drug conjugate compound of claim 141 wherein p is 2.

143. The antibody-drug conjugate compound of claim 141 wherein L has the formula:

-A_a-W_w-Y_y-

where;

A is a Stretcher unit covalently attached to a cysteine thiol of the cysteine engineered antibody (Ab);

a is 0 or 1;

each W is independently an Amino Acid unit;

w is an integer ranging from 0 to 12;

Y is a Spacer unit covalently attached to the drug moiety; and

y is 0, 1 or 2.

144. The antibody-drug conjugate compound of claim 143 having the formula:

145. The antibody-drug conjugate compound of claim 143 wherein WW is valine-citrulline.

146. The antibody-drug conjugate compound of claim 143 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

147. The antibody-drug conjugate compound of claim 143 having the formula:

148. The antibody-drug conjugate compound of claim 147 wherein R¹⁷ is (CH₂)₅ or (CH₂)₂.

149. The antibody-drug conjugate compound of claim 143 having the formula:

150. The antibody-drug conjugate compound of claim 141 wherein L is SMCC, SPP, SPDB or BMPEO.

151. The antibody-drug conjugate compound of claim 141 wherein D is MMAE, having the structure:

152. The antibody-drug conjugate compound of claim 141 wherein D is MMAF, having the structure:

153. The antibody-drug conjugate compound of claim 141 wherein D is DM1 or DM4, having the structures:

154. The antibody-drug conjugate compound of claim 140 wherein the antibody is selected from a monoclonal antibody, a bispecific antibody, a chimeric antibody, a human antibody, a humanized antibody, and an antibody fragment.

155. The antibody-drug conjugate compound of claim 140 wherein the antibody fragment is a Fab fragment.

156. An antibody-drug conjugate compound selected from the structures:

157. The antibody drug conjugate of claim 140 wherein the auristatin is MMAE or MMAF.

158. The antibody drug conjugate of claim 141 wherein L is MC-val-cit-PAB or MC.

159. An assay for detecting B cells comprising:

(a) exposing cells to an antibody-drug conjugate compound of claim 140; and

(b) determining the extent of binding of the antibody-drug conjugate compound to the cells.

160. A method of inhibiting cellular proliferation comprising treating mammalian cancerous B cells in a cell culture medium with an antibody-drug conjugate compound of claim 140, whereby proliferation of the cancerous B cells is inhibited.

161. A pharmaceutical formulation comprising the antibody drug conjugate of claim 140, and a pharmaceutically acceptable diluent, carrier or excipient.

162. A method of treating cancer comprising administering to a patient the pharmaceutical formulation of claim 161.

163. The method of claim 162 wherein the cancer is selected from the group consisting of lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

164. The method of claim 162 wherein the patient is administered a cytotoxic agent in combination with the antibody-drug conjugate compound.

165. An article of manufacture comprising:

the pharmaceutical formulation of claim 160;

a container; and

a package insert or label indicating that the compound can be used to treat cancer characterized by the overexpression of a CD79b polypeptide.

166. The article of manufacture of claim 165 wherein the cancer is selected from the group consisting of lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

167. A method for making an antibody drug conjugate compound comprising an anti-CD79b antibody (Ab) of claim 125, and an auristatin or maytansinoid drug moiety (D) wherein the antibody is attached through the one or more engineered cysteine amino acids by a linker moiety (L) to D;

the compound having Formula I;

Ab-(L-D)_p 

 

I

where p is 1, 2, 3, or 4;

the method comprising the steps of;

(a) reacting an engineered cysteine group of the antibody with a linker reagent to form antibody-linker intermediate Ab-L; and

(b) reacting Ab-L with an activated drug moiety D;

whereby the antibody-drug conjugate is formed;

or comprising the steps of;

(c) reacting a nucleophilic group of a drug moiety with a linker reagent to form drug-linker intermediate D-L; and

(d) reacting D-L with an engineered cysteine group of the antibody;

whereby the antibody-drug conjugate is formed.

168. The method of claim 167 further comprising the step of expressing the antibody in chinese hamster ovary (CHO) cells.

169. The method of claim 167 further comprising the step of treating the expressed antibody with a reducing agent.

170. The method of claim 169 wherein the reducing agent is selected from TCEP and DTT.

171. The method of claim 169 further comprising the step of treating the expressed antibody with an oxidizing agent, after treating with the reducing agent.

172. The method of claim 171 wherein the oxidizing agent is selected from copper sulfate, dehydroascorbic acid, and air.

173. The antibody of claim 125 wherein the antibody comprises a heavy chain sequence having at least 90% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs:

90 or 85.

174. The antibody of claim 125 wherein the antibody comprises a light chain sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:

89 and a heavy chain sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO;

85.

175. The antibody of claim 125 wherein the antibody comprises a light chain sequence having at least 90% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs:

89 or 88.

176. The antibody of claim 125 wherein the antibody comprises a light chain sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:

88 and a heavy chain sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO;

90.

178. A composition comprising the antibody of any of claims 1, 7, 42, 54 or 177.

179. The composition of claim 178, wherein the composition comprises a carrier.

15. A humanized anti-CD79b antibody wherein monovalent affinity of the antibody to human CD79b is substantially the same as monovalent affinity of a murine antibody comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIGS. 8A-B (SEQ ID NO;

     14).

24. The antibody of any of claims 15-23 wherein the binding affinity is expressed as a Kd value.

16. A humanized anti-CD79b antibody wherein monovalent affinity of the antibody to human CD79b is at least 1, 2 or 3-fold greater than monovalent affinity of a murine antibody or chimeric antibody comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIG. 8A B (SEQ ID NO;

     14).

17. A humanized anti-CD79b antibody wherein monovalent affinity of the antibody to human CD79b is at least 1, 2 or 3-fold lower than monovalent affinity of a murine or chimeric antibody comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIGS. 8A-B (SEQ ID NO;

     14).

19. A humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form to human CD79b is substantially the same as the affinity of a murine antibody in its bivalent form and comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIGS. 8A-B (SEQ ID NO;

     14).

20. A humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form to human CD79b is at least 1, 2, or 3-fold greater than the affinity of a murine or chimeric antibody in its bivalent form and comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIGS. 8A-B (SEQ ID NO;

     14).

21. A humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form to human CD79b is at least 1, 2 or 3-fold lower than the affinity of a murine or chimeric antibody in its bivalent form and comprising a light chain and heavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO:

10) and FIGS. 8A-B (SEQ ID NO;

     14).

22. A humanized anti-CD79b antibody wherein the affinity of the antibody in its bivalent form to human CD79b is 2.0 nM.

23. The humanized anti-CD79 antibody of claim 22 wherein the affinity of the antibody in its bivalent form to human CD79b is 2.0 nM+/−

0.53.

30. A humanized anti-CD79b 2F2 antibody wherein the humanized antibody when conjugated to a cytotoxic agent inhibits tumor cell growth.

33. An antibody comprising a heavy chain variable domain comprising HVR1-HC, HVR2-HC and/or HVR3-HC sequence depicted in FIG. 13 (SEQ ID NO:

31-33).

34. The antibody of claim 33, wherein the variable domain comprises FR1-HC, FR2-HC, FR3-HC and/or FR4-HC sequence depicted in FIG. 13 (SEQ ID NO:

27-30).

35. The antibody of claim 33 or 34, wherein the antibody comprises CHI and/or Fc sequence depicted in (SEQ ID NO:

34 and/or

     35).

41. The antibody made by the process of:

(a) culturing a cell expressing an antibody comprising a heavy chain variable domain of any of claims 33-35 and a light chain variable domain of any of claims 36-38; and

(b) isolating the antibody from said cultured cell.

42. An antibody comprising a heavy chain variable domain of any of claims 33-35 and a light chain variable domain of any of claims 36-38.

43. The antibody of claim 42, wherein the antibody is monovalent and comprises an Fc region.

36. An antibody comprising a light chain variable domain comprising HVR1-LC, HVR2-LC and/or HVR3-LC sequence depicted in FIG. 13 (SEQ ID NO:

23-25).

37. The antibody of claim 36, wherein the variable domain comprises FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence depicted in FIG. 13 (SEQ ID NO:

19-22).

38. The antibody of claim 36 or 37, wherein the antibody comprises CL1 sequence depicted in FIG. 13 (SEQ ID NO:

26).

39. The polypeptide comprising the sequence depicted in FIG. 13 (SEQ ID NO:

12).

40. The polypeptide comprising the sequence depicted in FIG. 13 (SEQ ID NO:

16).

52. An antibody that binds to CD79b, wherein the antibody comprises a heavy chain variable domain having a least 90% sequence identity to the amino acid sequence of SEQ ID NO:

16.

53. An antibody that binds to CD79b, wherein the antibody comprises a light chain variable domain having a least 90% sequence identity to the amino acid sequence of SEQ ID NO:

12.

54. An antibody that binds to CD79b, wherein the antibody comprises a heavy chain variable domain having a least 90% sequence identity to the amino acid sequence of SEQ ID NO:

16 and a light chain variable domain having at least 90% sequence identity to the amino acid sequence of SEQ ID NO;

12.

58. A method of making an anti-CD79b antibody, wherein the method comprises (a) culturing a host cell selected from the group comprising a eukaryotic cell and a CHO cell under conditions suitable for expression of the polynucleotide encoding the antibody, and (b) isolating the antibody.

177. An antibody that binds to CD79b, wherein the antibody comprises a heavy chain variable domain having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO:

14 and a light chain variable domain having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NO;

10.