ANTIBODY THAT BINDS ERBB-2 AND ERBB-3
20220348683 · 2022-11-03
Assignee
Inventors
- Cecilia Anna Wilhelmina GEUIJEN (Utrecht, NL)
- Cornelis Adriaan De Kruif (Utrecht, NL)
- Mark Throsby (Utrecht, NL)
- Ton Logtenberg (Utrecht, NL)
- Alexander Berthold Hendrik BAKKER (Utrecht, NL)
Cpc classification
A61K31/436
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K31/185
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
A61K31/4375
HUMAN NECESSITIES
A61P9/04
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/185
HUMAN NECESSITIES
A61K31/436
HUMAN NECESSITIES
A61K31/4375
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
Abstract
The invention relates among others to antibodies comprising a first antigen-binding site that binds Erb B-2 and a second antigen-binding site that binds Erb B-3. The antibodies can typically reduce a ligand-induced receptor function of Erb B-3 on a Erb B-2 and Erb B-3 positive cell. Also described are method for the treatment and use of the antibodies in imaging and in the treatment of subjects having an Erb B-2, Erb B-3 or Erb B-2/3 positive tumor.
Claims
1-35. (canceled)
36. A method for the treatment of a subject having a ErbB-2, ErbB-3 or ErbB-2/ErbB-3 positive tumor or at risk of having said tumor comprising administering to the subject an antibody comprising: a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the antibody can reduce a ligand-induced receptor function of ErbB-3 on a ErbB-2 and ErbB-3 positive cell; a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein said first antigen-binding site binds domain I of ErbB-2 and said second antigen-binding site binds domain III of ErbB-3; a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, wherein the affinity (KD) of said second antigen-binding site for an ErbB-3 positive cell is equal to, or higher than, the affinity of said first antigen-binding site for an ErbB-2 positive cell; two antigen-binding sites that bind ErbB-2, wherein at least one of said antigen-binding sites binds domain I of ErbB-2; or two antigen-binding sites that bind ErbB-3, wherein at least one of said antigen-binding sites binds domain III of ErbB-3.
37-39. (canceled)
40. A method for the treatment of a subject having a ErbB-2, ErbB-3 or ErbB-2/ErbB-3 positive tumor or at risk of having said tumor comprising administering to the subject: a bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3, and one or more compounds selected from the group consisting of an inhibitor of a component of the PI3Kinase pathway, an inhibitor of a component of the MAPK pathway, a microtubuli disrupting drug and an HDAC inhibitor, preferably one or more compounds selected from the group consisting of a tyrosine kinase inhibitor, a PI3Ka inhibitor, an Akt inhibitor, an mTOR inhibitor, an Src inhibitor, vorinostat and paclitaxel.
41-48. (canceled)
49. A method for counteracting the formation of a metastasis in a subject having a ErbB-2, ErbB-3 or ErbB-2/ErbB-3 positive tumor, wherein said ErbB-2, ErbB-3 or ErbB-2/ErbB-3 positive tumor cell has a heregulin expression level that is at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or 95% of the heregulin expression level of BXPC3 or MCF7 cells, comprising administering to the subject a bispecific antibody comprising a first antigen-binding site that binds ErbB-2 and a second antigen-binding site that binds ErbB-3.
50. (canceled)
51. The method of claim 36, wherein said subject has an ErbB-2 or ErbB-2/ErbB-3 positive tumor that has less than 1.000.000 ErbB-2 cell-surface receptors per cell.
52. (canceled)
53. The method of claim 36, wherein said tumor is a breast cancer, gastric cancer, colorectal cancer, colon cancer, gastro-esophageal cancer, esophageal cancer, endometrial cancer, ovarian cancer, liver cancer, lung cancer including non-small cell lung cancer, clear cell sarcoma, salivary gland cancer, head and neck cancer, brain cancer, bladder cancer, pancreatic cancer, prostate cancer, kidney cancer, skin cancer, or melanoma tumor.
54. The method of claim 36, wherein said subject has a cardiac function that is lower than 90% as compared to a healthy cardiac function.
55. The method of claim 54, wherein said cardiac function comprises the Left Ventricular Ejection Fraction (LVEF).
56. The method or antibody for use according to claim 39 or 54-55, wherein said subject suffers from congestive heart failure (CHF), left ventricular dysfunction (LVD) and/or a≥10% decreased Left Ventricular Ejection Fraction (LVEF), and/or wherein said subject has had a myocardial infarction.
57. (canceled)
58. The method of claim 36, wherein the antibody is a bispecific antibody comprising a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of AYYIN (SEQ ID NO:49), RIYPGSGYTSYAQKFQG (SEQ ID NO:50), and PPVYYDSAWFAY (SEQ ID NO:51) and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87; and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences GYYMH (SEQ ID NO:64), WINPNSGGTNYAQKFQG (SEQ ID NO:65), and DHGSRHFWSYWGFDY (SEQ ID NO:66) and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87.
59. The method of claim 36, wherein the antibody is fucosylated in order to enhance antibody dependent cellular cytotoxicity (ADCC).
60. The method of claim 36, wherein the antibody comprises two different immunoglobulin heavy chains with compatible heterodimerization domains.
61. The method of claim 60, wherein the compatible heterodimerization domains are compatible immunoglobulin heavy chain CH3 heterodimerization domains.
62. The method of claim 36, comprising administering to the subject at least one additional therapeutic agent.
63. The method of claim 62, wherein said at least one additional therapeutic agent is selected from afatinib, laptinib, neratinib, BYL719, MK-2206, everolimus, saracatinib, paclitaxel, vorinostat.
64. The method of claim 36, wherein the antibody comprises the light chain variable region comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 87).
65. The method of claim 36, wherein the antibody comprises a first binding arm comprising a heavy chain variable region comprising SEQ ID NO: 48, a second binding arm comprising a heavy chain variable region comprising SEQ ID NO: 63, and both the first and second binding arms comprise a light chain variable region comprising SEQ ID NO: 87.
66. The method of claim 36, wherein the antibody comprises a first binding arm comprising a heavy chain comprising SEQ ID NO: 88, a second binding arm comprising a heavy chain comprising SEQ ID NO: 89, and both the first and second binding arms comprise a light chain comprising SEQ ID NO: 87.
67. The method of claim 36, wherein the tumor is a ErbB-2/ErbB-3 positive tumor.
68. The method of claim 36, wherein the tumor is a ErbB-2 positive tumor.
69. The method of claim 36, wherein the tumor is a ErbB-3 positive tumor.
70. The method of claim 36, wherein the antibody is a bispecific antibody comprising: a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising: the CDR1 sequence of SEQ ID NO:24 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:24, the CDR2 sequence of SEQ ID NO:25 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:25, and the CDR3 sequence of SEQ ID NO:26 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:26, the CDR1 sequence of SEQ ID NO:29 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:29, the CDR2 sequence of SEQ ID NO:30 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:30, and the CDR3 sequence of SEQ ID NO:31 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:31, the CDR1 sequence of SEQ ID NO:34 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:34, the CDR2 sequence of SEQ ID NO:35 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:35, and the CDR3 sequence of SEQ ID NO:36 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:36, the CDR1 sequence of SEQ ID NO:39 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:39, the CDR2 sequence of SEQ ID NO:40 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:40, and the CDR3 sequence of SEQ ID NO:41 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:41, the CDR1 sequence of SEQ ID NO:54 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:54, the CDR2 sequence of SEQ ID NO:55 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:55, and the CDR3 sequence of SEQ ID NO:56 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:56, the CDR1 sequence of SEQ ID NO:117 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:117, the CDR2 sequence of SEQ ID NO:118 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:118, and the CDR3 sequence of SEQ ID NO:119 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:119, or the CDR1 sequence of SEQ ID NO:44 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:44, the CDR2 sequence of SEQ ID NO:45 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:45, and the CDR3 sequence of SEQ ID NO:46 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:46, and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87 or comprising CDR1, CDR2, and CDR3 sequences each having at most two conservative amino acid substitutions relative to the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87; and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising: the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66, the CDR1 sequence of SEQ ID NO:69 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:69, the CDR2 sequence of SEQ ID NO:70 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:70, and the CDR3 sequence of SEQ ID NO:71 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:71, the CDR1 sequence of SEQ ID NO:74 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:74, the CDR2 sequence of SEQ ID NO:75 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:75, and the CDR3 sequence of SEQ ID NO:76 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:76, or the CDR1 sequence of SEQ ID NO:79 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:79, the CDR2 sequence of SEQ ID NO:80 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:80, and the CDR3 sequence of SEQ ID NO:81 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:81, and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87 or comprising CDR1, CDR2, and CDR3 sequences each having at most two conservative amino acid substitutions relative to the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87.
71. The method of claim 36, wherein the antibody is a bispecific antibody comprising: a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:24 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:24, the CDR2 sequence of SEQ ID NO:25 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:25, and the CDR3 sequence of SEQ ID NO:26 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:26, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:29 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:29, the CDR2 sequence of SEQ ID NO:30 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:30, and the CDR3 sequence of SEQ ID NO:31 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:31, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:34 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:34, the CDR2 sequence of SEQ ID NO:35 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:35, and the CDR3 sequence of SEQ ID NO:36 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:36, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:39 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:39, the CDR2 sequence of SEQ ID NO:40 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:40, and the CDR3 sequence of SEQ ID NO:41 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:41, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:44 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:44, the CDR2 sequence of SEQ ID NO:45 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:45, and the CDR3 sequence of SEQ ID NO:46 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:46, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:34 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:34, the CDR2 sequence of SEQ ID NO:35 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:35, and the CDR3 sequence of SEQ ID NO:36 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:36, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:69 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:69, the CDR2 sequence of SEQ ID NO:70 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:70, and the CDR3 sequence of SEQ ID NO:71 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:71; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:39 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:39, the CDR2 sequence of SEQ ID NO:40 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:40, and the CDR3 sequence of SEQ ID NO:41 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:41, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:74 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:74, the CDR2 sequence of SEQ ID NO:75 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:75, and the CDR3 sequence of SEQ ID NO:76 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:76; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:34 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:34, the CDR2 sequence of SEQ ID NO:35 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:35, and the CDR3 sequence of SEQ ID NO:36 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:36, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:74 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:74, the CDR2 sequence of SEQ ID NO:75 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:75, and the CDR3 sequence of SEQ ID NO:76 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:76; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:34 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:34, the CDR2 sequence of SEQ ID NO:35 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:35, and the CDR3 sequence of SEQ ID NO:36 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:36, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:79 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:79, the CDR2 sequence of SEQ ID NO:80 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:80, and the CDR3 sequence of SEQ ID NO:81 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:81; a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:54 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:54, the CDR2 sequence of SEQ ID NO:55 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:55, and the CDR3 sequence of SEQ ID NO:56 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:56, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; or a first binding arm that specifically binds to the extracellular domain of a human ErbB2 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:117 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:117, the CDR2 sequence of SEQ ID NO:118 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:118, and the CDR3 sequence of SEQ ID NO:119 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:119, and a second binding arm that specifically binds to the extracellular domain of a human ErbB3 polypeptide and comprises a heavy chain variable region comprising the CDR1 sequence of SEQ ID NO:64 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:64, the CDR2 sequence of SEQ ID NO:65 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:65, and the CDR3 sequence of SEQ ID NO:66 or a sequence having at most two conservative amino acid substitutions relative to SEQ ID NO:66; and the first binding arm and second binding arm each comprising a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87 or comprising CDR1, CDR2, and CDR3 sequences each having at most two conservative amino acid substitutions relative to the CDR1, CDR2, and CDR3 sequences of a light chain comprising SEQ ID NO: 87.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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a) HER2 crystal structure (PDB #1S78) showing verified PG3958 epitope residues as light gray spheres and surrounding residues (+/− five amino acid residues) as dark gray spheres. b) Solvent exposed surface of epitope region showing verified epitope residues in gray and surrounding residues (+/− five residues) in black. c) Detailed view of epitope region with verified epitope residues in light gray and surrounding residues (+/− five residues) in dark gray. d) Primary amino acid sequence of HER2 PG3958 epitope region indicating verified epitope residues (gray underlined), surrounding residues (black) and distant residues (gray italic, not shown in a, b and c). Figures and analyses were made with Yasara (www.yasara.org).
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a) HER3 crystal structure (PDB #4P59) showing epitope residue Arg 426 in gray spheres and all surface exposed residues within an 11.2 Aradius from Arg 426 in black spheres. b) Solvent exposed surface of epitope region with Arg 426 and distant residues shown in gray and all surface exposed residues within a 11.2 Å radius from Arg 426 shown in black. c) Residues in the epitope region Arg 426 in light gray and surrounding residues (all labeled) in dark gray. Figures and analyses were made with Yasara (www.yasara.org).
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EXAMPLES
[0210] Methods, Materials and Screening for Antibodies
[0211] Cell Lines:
[0212] BxPC-3-luc2 (Perkin Elmer 125058), N87 (ATCC® CRL-5822™), SK-BR-3 (ATCC® HTB-30™), BT-474 (ATCC® HTB-20™), JIMT-1 (DSMZ ACC 589), L929 (Sigma Aldrich 85011425), K562 (DSMZ ACC10), HEK293T (ATCC®-CRL-11268™) CHO-K1(DSMZ ACC110), MCF-7 (DSMZ ACC 115), MDA-MB-468 (#300279-513, Cell line services) SK-OV-3 (ATCC® HTB-77™), MDA-MB-175 (ATCC-HTB-25), MDA-MB-453 (ATCC-HTB-131), MDA-MB-361(ATCC-HTB-27), ZR-75-1 (ATCC-CRL-1500) and MKN-45 (DSMZ ACC409) cell lines were purchased from ATCC, DSMZ or Sigma Aldrich and routinely maintained in growth media supplemented with 10% heat inactivated fetal bovine serum (FBS). HEK293F Freestyle cells were obtained from INVITROGEN® and routinely maintained in 293 FreeStyle medium.
[0213] Generation of Recombinant Human, Chicken, Rat and Swapped Domain Vectors (Cloning of HER)
[0214] Human HER2. Full length Human HER2 was amplified by PCR from cDNA derived from RNA isolated from the breast cancer cell line JIMT-1. The primers used for the amplification of human HER2 were as follows. Forward primer: AAGCTGGCTAGCACCATGGAGCTGGCGGCCTTGTGC (SEQ ID NO: 1). Reversed primer: AATAATTCTAGACTGGCACGTCCAGACCCAGG (SEQ ID NO: 2). The full-length amplified product was digested with NheI and XbaI and subsequently cloned in the corresponding sites of pcDNA3.1 (INVITROGEN®). The sequence was verified by comparison with the NCBI Reference Sequence NM_004448.2. To generate constructs solely expressing the human HER2 extracellular domain (ECD) for transfection and immunization purposes the HER2 transmembrane domain and ECD were PCR amplified and recloned in pVaxl. For transfection purposes another construct was generated in pDisplay by amplifying the HER2 ECD domain, in this construct the HER2 ECD domain is fused to the PDGFR transmembrane domain.
[0215] Human HER3. The full length human cDNA clone of HER3 was obtained from Origene. To generate constructs solely expressing the human HER3 ECD for transfection and immunization purposes the HER3 transmembrane domain and ECD were PCR amplified and recloned in pVaxl. In addition another construct was generated in pVaxl whereby the HER3 ECD domain was fused to the PDGFR transmembrane domain. All sequences were verified by comparison with the NCBI Reference NM_001982.3
[0216] Cynomolgus HER2extracellular domain was PCR amplified from cynomolgus cDNA—Monkey) Normal Colon Tissue (Biochain). The primers used for the amplification of cynomolgus HER2 were as follows: Forward primer: AAGCTGGCTAGCACCATGGAGCTGGCGGCCTGGTAC (SEQ ID NO: 3). Reversed primer: AATAATTCTAGACTGGCACGTCCAGACCCAGG (SEQ ID NO: 4). The full-length amplified product was digested with NheI-XbaI and subsequently cloned in the corresponding sites of pcDNA3.1. The clone was sequenced and aligned with sequences available of rhesus monkeys (XM_002800451) to check correctness of the ErbB-2 clone.
[0217] Cynomolgus HER3extracellular domain was PCR amplified from cynomolgus cDNA—Monkey) Normal Colon Tissue (Biochain). The primers used for the amplification of cynomolgus HER3 were as follows:
TABLE-US-00001 Forward primer: (SEQ ID NO: 5) AAGCTGGCTAGCACCATGAGGGCGAACGGCGCTCTG, Reversed primer: (SEQ ID NO: 6) AATAATTCTAGATTACGTTCTCTGGGCATTAGC.
[0218] The full-length amplified product was digested with NheI-XbaI and subsequently cloned in the corresponding sites of pcDNA3.1. The clone was sequenced and aligned with sequences available of rhesus monkeys (ENSMMUP00000027321) to check correctness of the HER3 clone.
[0219] The chicken HER2sequence was based on the reference sequence NM_001044661.1. Chimeric swapped domain constructs were generated by swapping domains I until IV of the chicken HER2 sequence for the human I domains I until IV. Sequences containing a myc tag were optimized for expression in mammalian cells and synthesized at Geneart.
[0220] The ratHER3sequence was based on the reference sequence NM_001044661.1. Chimeric swapped domain constructs were generated by swapping domains I until IV of the rat HER3 sequence for the human I domains I until IV. Sequences containing a myc tag were optimized for expression in mammalian cells and synthesized at Geneart.
[0221] Generation of HER2 and HER3 over-expressing cell lines To generate cell lines that express high levels of HER3 on the cell surface a mammalian expression vector was generated by excising the full length HER3 by a NotI and KpnI digestion. Subsequently the fragment was cloned in the corresponding sites of the pcDNA3.1(-)/hygro vector. A full length HER2 and HER3 expression vector encoding a neomycin resistance gene was used to generate cell lines that express high levels of HER2 on the cell surface. Prior to transfection the plasmids were linearized by a SSpI and FspI digestion. Both vectors were transfected separately into K562 cells and stable pools were generated following antibiotic selection. The resultant cell lines (K562-HER2 and K562-HER3) expressed high levels of HER2 and HER3 on their cell surface.
[0222] Immunizations
[0223] HER2immunizations. Four different immunization strategies were applied. For cohort #A, six C57B11/6 mice were immunized with 2×10.sup.6 L929 cells transiently transfected with HER2 in 200 μl via intraperitoneal injection. Subsequently, mice were boosted with 20 μg Erbb-2-Fc (RND systems) protein dissolved in 125 μl Titermax Gold via intraperitoneal injection on day 14, followed by boosts with 2×10.sup.6 L929 cells transiently transfected with HER2 in 200 μl on days 28 and 42. For cohort #C, six C57B1/6 mice were immunized with 2×10.sup.6 L929 cells transiently transfected with HER2 via intraperitoneal injection. Subsequently, mice were boosted with 2×10.sup.6 L929 cells transiently transfected with HER2 in 200 μl via intraperitoneal injection on day 14, followed by a protein boosts with 20 μg Erbb-2-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection on day 35 and a final boost with 20 μg Erbb-2-Fc protein dissolved in 200 μl PBS via intraperitoneal injection on day 49. For cohort #E, six C57B1/6 mice were immunized with 20 μg Erbb-2-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection. Subsequently, protein boosts with 20 μg Erbb-2-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection were made at day 14 and 28 and a final boost with 20 μg Erbb-2-Fc protein dissolved in 200 μl PBS via intraperitoneal injection on day 42. For cohort #G, six C57B11/6 mice were immunized by DNA vaccination at Genovac (Freiburg, Germany) according to their protocols. The endotoxin-free provided vectors used for the DNA vaccination encoded the transmembrane and extracellular part of HER2 cloned in pVaxl. Subsequently, DNA boosts were given at day 14, 28 and 66.
[0224] HER7immunizations. Four different immunization strategies were applied. For cohort #B, six (C57B11/6) mice were immunized with 2×10.sup.6 L929 cells transiently transfected with HER3 in 200 μl via intraperitoneal injection. Subsequently, mice were boosted with 2×10.sup.6 L929 cells transiently transfected with HER3 in 200 μl on days 14, 28, 49 and 63. For cohort #D, six C57B11/6 mice were immunized with 2×10.sup.6 L929 cells transiently transfected with HER3 via intraperitoneal injection on day 0, 14 and 28. Subsequently, mice were boosted with 20 μg Erbb-3-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection on day 49 and a final boost with 20 μg Erbb-3-Fc protein dissolved in 200 μl PBS via intraperitoneal injection on day 66. For cohort #F, six C57B1/6 mice were immunized with 20 μg Erbb-3-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection. Subsequently, mice were boosted with 20 μg Erbb-3-Fc protein dissolved in 125 μl Titermax Gold via intraperitoneal injection at day 14 and 28 and a final boost was given with 20 μg Erbb-3-Fc protein dissolved in 200 μl PBS via intraperitoneal injection on day 42. For cohort #H, six C57B1/6 mice were immunized by DNA vaccination at Genovac (Freiburg, Germany) according to their protocols. The endotoxin-free provided vectors used for the DNA vaccination encoded the transmembrane of PDGFR and extracellular part of HER3 cloned in pVaxl. Subsequently, DNA boosts were given at day 14, 28 and 66.
[0225] Determination of Antibody Titers.
[0226] Anti-HER2 titers in the serum from immunized C57B1/6 mice were determined by ELISA against ECD-Erbb-2 protein (Bendermedsystems) and FACS analysis on the HER2 negative K562, the HER2 low expressing cell line MCF-7 and HER2 amplified SKBR-3 and BT-474 cells. Anti-HER3 titers in the serum from immunized C57B11/6 mice were determined by ELISA against Erbb-3-Fc protein and FACS analysis on the HER3 negative K562, the HER2 low expressing cell line MCF-7 and HER2 amplified SKBR-3 and BT-474 cells.
[0227] Serum titers against HER2 and HER3 before sacrificing the animals are described in Table 1 and Table 2 respectively. Animals in all cohorts developed antibody responses against HER2 or HER3.
[0228] Recovery of lymphoid tissue.
[0229] Spleen and draining lymph nodes were removed from all mice vaccinated with DNA (cohorts #G and #H). Single cell suspensions were generated from all tissues and subsequently tissues were lysed in Trizol reagent. From cohorts #A until #F spleens were removed from all mice except for one mouse of cohort #C that died after the first boost. Single cell suspensions were generated from all spleens and the total B cell fraction was isolated using the MACS separation procedure either by CD19 enrichment (cohorts # A, E, F) or depletion of non-B cells (cohorts # B, C, D).
[0230] Generation of Phage Display Libraries from Immunized Mice
[0231] One phage library was built for each mouse. To this end the material from all mice per group (5 or 6 mice per group) was used to prepare phage libraries using the following approach. From each individual mouse RNA was isolated and cDNA was synthesized and VH-family specific PCRs were performed. Subsequently all VH-family PCR products per mouse were purified and the DNA concentration was determined and digested and ligated in a phage-display vector containing the common-light chain to generate a mouse-human chimeric phage library. All phage libraries contained>106 clones with an insert frequency of >85%. Selection of Phages Carrying Fab Fragments Specifically Binding to HER2 and HER3
[0232] Antibody fragments were selected using antibody phage display libraries. Immunized libraries and synthetic libraries (as described in de Kruif et al. Mol. Biol. (1995), 248, 97-105) were used for selections.
[0233] HER2 Phage Selection and Screening
[0234] Phage libraries were rescued with VCS-M13 helper phage (Stratagene) and selected for two rounds in immunotubes (Nunc) coated recombinant protein. In the first round ECD-Erbb-2 protein (Bendermedsystems) was coated onto immunotubes whereas in the second round Erbb-2-Fc (RND systems) was coated onto immunotubes. The immunotubes were blocked with 4% non fat dry milk (ELK). Phage antibody libraries were also blocked with 4% ELK prior to the addition of the phage library to the immunotubes. Incubation with the phage library with the coated protein in the immune tubes was performed for 2 H at room temperature under rotating conditions. Immunotubes were then washed five to ten times with 0.05% Tween-20 in PBS followed by 5 to 10 times in PBS. Bound phages were eluted using 50 mM glycine (pH 2.2) and added to E. coli XL-1 Blue and incubated at 37° C. for phage infection. Subsequently infected bacteria were plated on agar plates containing Ampicillin, tetracyclin and glucose and incubated at 37° C. overnight. After the first round, colonies were scraped off the plates and combined and thereafter rescued and amplified to prepare an enriched first round library. The enriched library was then selected on Erbb-2-Fc (RND systems) using the protocol described above. After the second round selection individual clones were 25 picked and rescued to prepare a phage monoclonal miniprep. Positive phage clones binding Erbb2 were then identified in FACS for binding to the breast cancer cell line BT-474. The VH genes of all Erbb2 specific clones were sequenced. VH gene rearrangements were established with VBASE2 software to identify unique clones. All unique clones were then tested in phage format for binding in FACS to HEK293T cells (negative control), HEK293T cells transiently transfected with ErbB-2 and BT-474 cells.
[0235] HER3 Phage Selection and Screening
[0236] Phage libraries were rescued with VCS-M13 helper phage (Stratagene) and selected for two rounds in immunotubes (Nunc) coated with recombinant protein. In both selection rounds round Erbb-3-Fc (RND systems) was coated onto immunotubes. To overcome a selection bias towards the Fc part of the fusion protein, both selection rounds on Erbb-3-Fc were performed in the presence of 150 g/ml human IgG. The immunotubes were blocked with 4% ELK. Phage antibody libraries were blocked with 4% ELK prior to the addition of the phage library to the immunotubes. Incubation with the phage library was performed for 2 H under rotating conditions. Immunotubes were then washed five to ten times with 0.05% Tween-20 in PBS followed by 5 to 10 times in PBS. Bound phages were eluted using 50 mM glycine (pH 2.2) and added to E. coli XL-1 Blue and incubated for phage infection. Subsequently infected bacteria were plated on agar plates containing Ampicillin, tetracyclin and glucose and incubated at 37° C. overnight. After the first round, colonies were scraped off the plates and combined and phages were rescued and amplified to prepare an enriched first round library. The enriched library was then selected on Erbb-3-Fc (RND systems) using the protocol described above. After the second round selection individual clones were picked and rescued to prepare a phage monoclonal miniprep. Positive phage clones were identified in FACS for binding to the breast cancer cell line BT-474. The VH genes of all positive clones were sequenced. VH gene rearrangements were established with VBASE2 software to identify unique clones. All unique clones were tested in phage format for binding in FACS to K562 cells (negative control), stable K562-HER3 cells and BT-474 cells.
[0237] In total 36 selections were performed on Erbb2 and Erbb3 antigen formats. All selection screening procedures resulted in 89 unique Fab clones directed against HER2 and 137 unique Fab clones directed against HER3. A Fab was considered unique based on its unique HCDR3 sequence, an indication of a unique VDJ recombination event. In some cases clonal variants were obtained, with an identical HCDR3 but differences in the CDR1 and/or CDR2. From the immunized mice libraries clusters of clonal variants containing substitutions in the VH gene reflecting affinity variants were selected.
[0238] Antibody Selection/Characterization
[0239] Generation of Monoclonal Antibodies
[0240] VH genes of unique antibodies, as judged by VH gene sequence and some sequence variants thereof, derived from the immunized mouse phage libraries were cloned in the backbone IgG1 vector. Two different production cell lines were used during the process; HEK293T and 293F Freestyle cells. Adherent HEK293T cells were cultivated in 6-well plates to a confluency of 80%. The cells were transiently transfected with the individual DNA-FUGENE mixture and further cultivated. Seven days after transfection, supernatant was harvested and medium was refreshed. Fourteen days after transfection supernatants were combined and filtrated through 0.22 μM (Sartorius). The sterile supernatant was stored at 4° C. Suspension adapted 293F Freestyle cells were cultivated in T125 flasks at a shaker plateau until a density of 3.0×106 cells/ml. Cells were seeded at a density of 0.3-0.5×106 viable cells/ml in each well of a 24-deep well plate. The cells were transiently transfected with the individual sterile DNA: PEl mixture and further cultivated. Seven days after transfection, supernatant was harvested and filtrated through 0.22 μM (Sartorius). The sterile supernatant was stored at 4° C.
[0241] Generation of Bispecific Antibodies
[0242] Bispecific antibodies were generated using the proprietary CH3 technology to ensure efficient hetero-dimerisation and formation of a bispecific antibody. The CH3 technology uses charge-based point mutations in the CH3 region to allow efficient pairing of two different heavy chain molecules as previously described (PCT/NL2013/050294; published as WO 2013/157954 A1).
[0243] IgG Purification for Functional Screening
[0244] The purification of IgG was performed at small scale (<500 μg), medium scale (<10 mg) and large scale (>10 mg) using affinity chromatography. Small scale purifications were performed under sterile conditions in 24 well filter plates using vacuum filtration. First the pH of the medium was adjusted to pH 8.0 and subsequently the small scale productions were incubated with protein A SEPHAROSE™ CL-4B beads (50% v/v) (Pierce) for 2 H at 25° C. on a shaking platform at 600 rpm (Heidolph plate shaker). Next the beads were harvested by vacuum filtration. Beads were washed twice with PBS pH 7.4. IgG was eluted at pH 3.0 with 0.1 M citrate buffer and the IgG fraction was immediately neutralized by Tris pH 8.0. Buffer exchange was performed by centrifugation using multiscreen ULTRAEL® 10 multiplates MILLIPORE®. The samples ended up in a final buffer of PBS pH 7.4
[0245] Validation of HER2/HER3 Specific IgGs
[0246] Antibodies were tested for binding in FACS to BT-474, HEK293T and HEK293T overexpressing HER2 or HER3. Therefore cells were harvested using trypsin and diluted to 10.sup.6 cells/ml in FACS buffer (PBS/0.5% BSA/0.5 mM EDTA). 1-2×10.sup.5 cells were added to each well in a U-bottom 96 well plate. Cells were centrifuged for 2 minutes at 300 g at 4° C. Supernatant was discarded by inverting plate(s). 50 μl of each IgG sample was added at a concentration of 10 μg/ml and incubated for 1H on ice. Cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. 50 μl diluted 1:100 mouse anti human IgG PE (INVITROGEN®) was added and incubated for 30-60 minutes on ice in the dark. After adding FACS buffer, cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. Cells were analysed on a FACSCanto Flow cytometer in a HTS setting. Binding of the antibodies to cells was assessed by mean fluorescence intensity (MFI).
[0247] To test for non-specific binding reactivity ELISA assays were used. HER2 and HER3 antibodies were tested for reactivity against the antigens fibrinogen, hemoglobulin and tetanus toxin. To test specific binding to HER2 and HER3, the antibodies were tested for binding to purified recombinant extracellular domains of EGFR, HER2, HER3 and HER4. Antigens were coated overnight to MAXISORP™ ELISA plates. Wells of the ELISA plates were blocked with PBS (pH 7.2) containing 5% BSA for 1 hour at 37° C. Selected antibodies were tested in duplo at a concentration of 10 μg/ml diluted in PBS-2% BSA and allowed to bind for 2 hours at 25° C. As a control the procedure was performed simultaneously with an antibody specific for the coated antigens and a negative control antibody. The ELISA plates were washed 5 times with PBS-T (PBS-0.05% v/v Tween 20). Bound IgG was detected with 1:2000 diluted HRP-conjugate (Goat anti-mouse BD) and was allowed to bind for 2 hours at 25° C. The ELISA plates were washed 5 times with PBS-T (PBS-0.05% Tween 20) and bound IgG was detected by means of OD492 nm measurement.
[0248] Epitope Grouping of HER2/HER3 Specific IgGs
[0249] The panel of anti-HER2 antibodies was binned based on their reactivity to the HER2 ECD derived from other species (mouse, chicken) and on their binding to specific domains in the HER2 molecule i.e. domains I, II, III and IV using chimeric constructs.
[0250] The panel of anti-HER3 antibodies was binned based on their reactivity to the HER3 ECD derived from other species (cyno, rat) and on their binding to specific domains in the HER3 molecule i.e. domains I, II, III and IV using chimeric constructs.
[0251] For this purpose CHO-K1 cells were transiently transfected with the relevant constructs using lipofectamin/DNA mixes. In the chimeric swapped domain construct, domains of chicken HER2 or rat HER3 are replaced by the human counterpart. Binding of the specific antibodies was measured by FACS. Expression of the constructs was confirmed using an anti-myc antibody. FACS staining with trastuzumab was included as a control for specific binding to domain IV. Antibodies in each group could be ranked based on the intensity of staining (MFI). The HER2 panel of 65 antibodies could be mapped into seven bins (Table 3). [0252] 1. Domain I specific (25) [0253] 2. Domain II specific (2) [0254] 3. Domain III specific (23) [0255] 4. Domain IV specific (7) [0256] 5. Domain IV specific and cross reactive to mouse (2) [0257] 6. Reactive to all constructs (2) [0258] 7. Only reactive to human HER2 (4)
[0259] Competition with Trastuzumab
[0260] Two antibodies mapped to HER2 domain IV inhibited proliferation of SKBR-3 cells. Both antibodies shared a similar CDR3 except for one amino acid difference. One antibody, PG1849 was investigated for its capacity to compete with trastuzumab in a competition ELISA. In this ELISA Fc-HER2 was coated and incubated with a concentration of 15 μg/ml IgG antibody. After an incubation of 15 minutes phages were allowed to incubate for another hour. Thereafter, phages were detected. Table 4 demonstrates that PG1849 and trastuzumab could bind simultaneously to HER2 since no loss of signal appeared during the ELISA. True competition only was observed when the same phage and antibody were combined in the assay.
[0261] The HER3 panel of 124 antibodies could be mapped into five bins (Table 5): [0262] 1. High Domain III reactivity, rat and mouse reactive and minor reactivity to domain IV (8) [0263] 2. High Domain III reactivity, rat, human and cyno reactive, minor reactivity to domain IV (8) [0264] 3. Only reactivity to rat, cyno and human HER3 (43) [0265] 4. Only reactive to human HER3 (32) [0266] 5. Reactive to all constructs (33)
[0267] Cell Line Proliferation Assays
[0268] SK-BR-3 cells were cultured in DMEM-F/12 supplemented with L-glutamine and 10% heat inactivated FBS. BxPC-3-luc2 cells were cultured in RPMI1640 supplemented with 10% heat inactivated FBS. MCF-7 cells were cultured in RPMI1640 supplemented with 100 μM, NEAA1 mM sodium pyruvate, 4 μg/ml insulin and 10% heat inactivated FBS.
[0269] For the proliferation assay of SK-BR-3 cells, subconfluent cell cultures were washed with PBS, trypsinized and trypsin was inactivated by adding culture medium. Cells were diluted to 6×10.sup.4 cells/ml in culture medium. Antibodies were diluted to concentrations of 10 and 1 μg/ml and added in a volume of 100 μl in 96-well black bottom plates (ABgene AB-0932). Cells were added at density of 6000 cells/well. The cells were cultivated for 3 days at 37° C., 5% CO, in 95% relative humidity. ALAMAR BLUE™ (INVITROGEN®) was added according to the manufacturer's instructions and incubated for 6 hours at 37° C., 5% CO, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation and 590 nm emission wavelength. The extent of growth inhibition was compared to that of the same concentration of trastuzumab (Table 6).
[0270] For the proliferation assay of MCF-7 and BxPC-3-luc2 cells, subconfluent cell cultures were washed with PBS, trypsinized and trypsin was inactivated by adding culture medium. Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 μg/ml Holo Transferrin). MCF-7 cells were diluted to 5×10.sup.4 cells/ml in culture medium. Antibodies were diluted to concentrations of 10 and 1 μg/ml and added in a volume of 100 μl in 96-well black bottom plates (ABgene AB-0932). Cells were added at a density of 5000 cells/well in the presence of 1 ng/ml final concentration human Recombinant Human NRG1-beta 1/HRG1-beta 1 EGF Domain; (396-HB-050 RND). Human NRG1-beta 1/HRG1-beta 1 EGF Domain will hereinafter be referred to as HRG. The cells were cultivated for 5 days at 37° C., 5% CO, in 95% relative humidity. ALAMAR BLUE™ (INVITROGEN®) was added according to the manufacturer's instructions and incubated for 24 hours at 37° C., 5% CO2, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation with 590 nm emission wave length. The extent of growth inhibition was compared to that of the same concentration of #Ab6 (Table 7).
[0271] BxPC-3-luc-2 proliferation assays were used to screen the bispecific antibodies. BxPC-3-luc-2 cells were diluted to 8×10.sup.4 cells/ml in culture medium. Antibodies were diluted to concentrations of 10 and 1 μg/ml and added in a volume of 100 μl in 96-well black bottom plates (ABgene AB-0932). Cells were added at density of 8000 cells/well in the absence or presence of 10 ng/ml final concentration human HRG. The cells were cultivated for 4 days at 37° C., 5% CO, in 95% relative humidity. ALAMAR BLUE™ (INVITROGEN®) was added according to the manufacturer's instructions and incubated for 4 hours at 37° C., 5% CO, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation with 590 nm emission wave length.
[0272] To minimalize edge effects, the outer wells of the 96 well plates were fully filled with PBS.
[0273] Affinity Ranking of HER2 Specific IgGs
[0274] We used the method described by Devash (PNAS, 1990) to rank the antibodies in a limited antigen-ELISA. The use of decreased antigen coating concentrations eliminates observed cross-reactivity reactions and can be used to detect high-affinity/avidity antibodies. Therefore the antigen concentration on the solid support was gradually decreased to investigate the weak immunoreactivities. A serial titration of ECD-Erbb-2 protein starting from 2.5 μg/ml until 0.019 μg/ml was coated overnight to MAXISORP™ ELISA plates. Wells of the ELISA plates were blocked with PBS (pH 7.2) containing 5% BSA for 1 hour at 37° C. Selected antibodies were tested in duplo at a concentration of 10 μg/ml diluted in PBS-2% BSA and allowed to bind for 2 hours at 25° C. As a control the procedure was performed simultaneously with an antibody specific for the coated antigens and a negative control antibody. The ELISA plates were washed 5 times with PBS-T (PBS-0.05% v/v Tween 20). Bound IgG was detected with 1:2000 diluted HRP-conjugate (Goat anti-mouse IgG, BD Biosciences) and was allowed to bind for 2 hours at 25° C. The ELISA plates were washed 5 times with PBS-T (PBS-0.05% Tween 20) and bound IgG was detected by means of OD492 nm measurement. PG1849, PG2916, PG2926, PG2930, PG2971, PG2973, PG3004 and PG3031 were tested in an HER2 antigen titration ELISA (
[0275] Binding of HER2 VH Genes with Various Kappa Light Chains
[0276] To investigate the binding of HER2 VHs derived from different phage display libraries a panel of HER2 antibodies was cloned and expressed in the context of another VK kappa chain, i.e. the VL of MEHD7945A. Produced IgGs were subjected to FACS analysis on K562 cells and stable K562-HER2 cells. VH genes derived from the combinatorial libraries and non-combinatorial libraries are listed in Table 8. The VH chains MF2971, MF3958, MF2916, MF2973, MF3004, MF3025, MF3031 all could be combined with the MEHD7945 Å light chain without loosing significant antigen specificity and binding as observed when combined with the common light chain IGKV1-39. VH chain MF1849 was not able to combine with the variant kappa light chain and retain antigen specificity and binding.
[0277] Other HER2 and HER3 Antibodies
[0278] Antibodies that inhibit the function of HER2 or HER3 are known in the art. Further antibodies were constructed according to published information and expressed in 293F Freestyle cells. The anti-HER2 antibodies pertuzumab and trastuzumab were generated based on the information disclosed in US2006/0212956 A1 (Genentech). The anti-HER3 antibody #Ab6, was based on the information disclosed in WO 2008/100624 (Merrimack Pharmaceuticals, Inc.) and recloned in a IgG1 back bone vector. The information of the 1-53 and U1-59 anti-HER3 antibodies was obtained from U.S. Pat. No. 7,705,103 B2 (U3 Pharma AG). The information of the anti-HER3 LJM716 antibody was obtained from US 2012/0107306. The information for the construction of the two-in-one anti-EGFR anti-HER3 antibody MEHD7945 Å was obtained from WO2010/108127.
[0279] Screening of HER2×HER3 Bispecific Antibodies
[0280] VH from the HER2 and HER3 antibody panel were recloned into the charged engineered vectors such that upon expression of the antibody heavy chains heterodimerization of heavy chains is forced resulting in the generation of bispecific antibodies after transfection. Three different strategies were used in combining HER2 and HER3 arms in bispecific IgG format:
[0281] 1. HER2 (blocking ligand independent growth) xHER3 (blocking ligand independent growth)
[0282] 2. HER2 (blocking ligand independent growth) xHER3 (blocking ligand dependent growth)
[0283] 3. HER2 from different epitope bins×HER3 (blocking ligand dependent growth)
[0284] In some bispecific combinations, antibodies generated in group 2 and 3 overlapped with group 1.
[0285] A total of 495 bispecific antibodies was produced in 24-well format and purified. All antibodies were tested for their capacity to inhibit the proliferation of the HER2
[0286] and HER3-expressing pancreatic BxPC-3-luc-2 cell line (Caliper). The potency of the antibodies was determined in a HRG-dependent and HRG-independent setting in a black and white screening with antibodies being present at a concentration of 10 and 1 μg/ml. Trastuzumab was included as a reference antibody as well as a negative control antibody at the same concentrations. The functional activity of the top 80 HER2×HER3 bispecifics (based on combined inhibition) at 1 μg/ml is shown in
[0287] Antibodies (40 in total) that showed a higher inhibitory activity compared to the positive control antibody were selected, reproduced and purified in a 24-well format and tested again in the black-and-white BxPC-3-luc-2 screen at 10 and 1 μg/ml concentrations. These antibodies were further titrated in HRG-dependent MCF-7 assay and compared against the combination of trastuzumab and pertuzumab (1:1) and a negative control antibody.
[0288] The IC.sub.50 for the bispecific antibodies, monoclonals and comparator antibodies was calculated using non-linear regression analysis with Prism software. Graph pad software lists the IC.sub.50 values of the bispecific antibodies in the MCF-7 assay and their inhibitory activity in the BxPC3 assay for comparison. A panel of 12 HER2×HER3 bispecific antibodies had more potent inhibiting activity compared to trastuzumab+ pertuzumab. In addition the bispecific antibodies were equally or more potent than the parental monoclonal PG3178 (Table 9).
[0289] The bispecific antibodies that inhibited ligand dependent cell growth were composed of HER2 arms in combination with the HER3 arms 3178, 3163, 3099 and 3176. Both the HER2 and HER3 arms of the most potent bispecifics were as a bivalent monoclonal also capable of inhibiting ligand-independent SKBR-3 proliferation (both the HER2 and HER3 arms) (Table 6) or ligand dependent MCF-7 proliferation (HER3 arms) (Table 7). The majority of the potent antibodies was composed of a HER2 arm recognizing domain I in combination with anti-HER3 antibody 3178.
[0290] Inhibition of BxPC-3-Luc2 Tumor Growth
[0291] The antibodies described in Table 9 were tested in a BxPC-3-luc2 pancreatic xenograft model. The BxPC-3-luc2 cell line expresses both HER2 and HER3 and is considered a HER2 low expressing cell line. CB17 SCID female mice, 8-10 weeks old at the beginning of the study were engrafted orthotopically in the pancreas with 1×10.sup.6 tumor cells in 20 μl. To this aim mice were anesthetized and laid on the right side to expose the left side and a 0.5 cm incision was made on the left flank region. The pancreas and spleen were exteriorized and 1×10.sup.6 tumor cells in 20 μl was injected into the sub-capsulary space of the pancreas tail. One week after implantation, bioluminescence (BLI) data were generated. 15 minutes prior to the imaging, all of the mice received i.p. injections of 150 mg/kg Luciferin (D-Luciferin-EF Potassium Salt, Cat. #E6552, Promega). BLI imaging was performed once or twice weekly using the left side view. Outlier animals—based on BLI/tumor volume—were removed and the mice were randomly distributed into groups of 7 mice each. On experimental day 8, the treatment was started. The animals in the antibody treatment group were dosed weekly for 3 consecutive weeks (days 0, 7, 14 and 21) with 30 mg/kg of antibody. At day 0 of the treatment the animals received twice the loading dose, i.e. 60 mg/kg of antibody. The final imaging was carried out at day 31.
[0292] Two BxPC-3-luc2 xenograft models were run with a different panel of bispecific antibodies and parental antibodies In the first BxPC-3-luc2 xenograft model (
[0293] All five bispecific antibodies tested were able to inhibit tumor growth. The mean tumor mass (BLI) of bispecific HER2×HER3 antibody treated animals was similar to that in the animals treated with the combination of trastuzumab+ pertuzumab. (
[0294] Based on the in vivo and in vitro data a bispecific panel of antibodies was selected of which the HER2 arms were composed of MF2971, MF3004, MF1849 and the HER3 arm was composed of MF3178. The MF2971 and MF3004 arm were of mouse origin and were humanized.
[0295] Binding of Bispecific HER2×HER3 Antibody Compared to Parental Monoclonal Antibodies
[0296] Binding of HER2×HER3 bispecific antibodies as compared to their parental counterparts was determined by FACS analysis. A FACS was performed on BxPC-3-luc2 cells and MCF-7 cells with a serial titration of antibodies ranging from 2,5 μg g/ml-0, 01 μg g/ml. The tested antibody panel was composed of the bispecific antibody PB3566 and its parental antibodies the anti-HER3 antibody PG3178 and the anti-HER2 antibody PG3004. The MFI data were plotted and the graphs on both cell lines show that the bispecific PB3566 binds more effectively to both tumor cell lines compared to the anti-HER3 antibody PG3178 and the anti-HER2 antibody PG3004. (
[0297] Humanization of MF2971 and MF3004
[0298] MF2971 and MF3004 were humanized according to technology known in the art. A total of seven humanised/de-immunised variant sequences of MF2971 were expressed, validated and characterised in vitro as monoclonal and in bispecific format combination with the HER3-specific antibody MF3178. The same was done for seven variant sequences of MF3004, which were created by replacing the HCDR3 of MF2971 in the seven MF2971 variants with the HCDR3 of MF3004. The expression, integrity, thermal stability and functional activity of all humanized variants was analysed. Based on production, integrity, stability and functionality integrity, a variant of MF2971 (2971-var2) was chosen as the optimal humanized variant of the VH to be used in a bispecific format with MF3178. This 2971-var2 was renamed MF3958. The bispecific HER2×HER3 combination MF3958×MF3178 resulted in PB4188.
[0299] Large Scale Production, Purification and Analytical Studies of PB4188
[0300] Suspension adapted 293F Freestyle cells were cultivated in Erlenmeyer flasks at a shaker plateau until a density of 3.0×106 cells/ml. Cells were seeded in a 4 L erlen flasks at a density of 0.3-0.5×106 viable cells/ml. The cells were transiently transfected with the individual sterile DNA: PEl mixture and further cultivated. Seven days after transfection, conditioned medium containing bispecific antibody was harvested by low-speed centrifugation, 5 minutes 1000 g, followed by high speed centrifugation, 5 minutes at 4000g. Collected conditioned medium was concentrated over a 5 kDa Satorius hydrosart cassette to about 600 ml and subsequently diafiltrated against 4 L PBS. Antibodies were bound on column to ˜35 ml MabSelectSure XL (11° C.). A-specifically bound proteins were removed by washing the column in reversed flow mode with 150 ml PBS, 150 ml PBS containing 1 M NaCl, 100 ml PBS. The bound antibodies were eluted using 100 mM citrate pH 3.0 in reversed flow mode and 5 ml fractions were collected in 10 ml tubes containing 4 ml 1Tris pH 8.0 for neutralization. The eluted antibodies were further purified by gel-filtration using superdex 200 50/1000. Thepurified antibody was filter-sterilized using a 0.22 μm syringe filter. IgG concentration was determined by OD280 measurement and the protein concentration was calculated based on the amino acid sequence. Protein was tested for aggregation (HPSEC), purity (SDS-PAGE, nMS, IEX and IEF). Protein samples were stored at −80° C.
[0301] IgG Purification for Analytical and Xenograft Studies.
[0302] Medium scale purifications were performed on an AKTA 100 Explorer using HiTrap MabSelect Sure columns and HiTrap desalting columns. Samples were loaded at 5 ml/min. The column was washed with 2 column volumes of PBS. IgG was eluted at pH 3.0 with 0.1 M citrate buffer. Next the sample was desalted and ended up in a final buffer of PBS pH 7.4. IgGs were filtered through a 0.45 μM filter (Sartorius). The IgG concentration was measured using Octet with protein A sensors. Protein was tested for aggregation (HPSEC), purity (SDS-PAGE, nMS, IEX and IEF). Protein samples were stored at −80° C.
[0303] Analytical Characteristics of PB4188
[0304] The PB4188 (MF3958×MF3178) was subjected to analysis by HP-SEC and CIEX-HPLC (TSK gel-STAT 7 μm column, 4.6 mm ID×10 cm L). The analytical profile of PB4188 was in general consistent with the behavior of normal monospecific IgG1, such as the parental HER2 arm PG3958 and the anti-RSV monoclonal control antibody (
[0305] Affinity Determination
[0306] The monovalent binding affinity of PB4188 and PB3448 for recombinant HER2 and HER3 was determined by SPR (BIACORE™ T100). BIACORE™ T100 (GE Healthcare, Uppsala, Sweden) was used to conduct all experiments described. Sensor surface preparation and interaction analyses were performed at 25° C. Buffer and BIACORE™ reagents were purchased from GE Healthcare. ErbB2-Fc and ERbB3-Fc(RND) was coated to the surface of a CM5 sensor chip in potassium acetate buffer (pH5.5) at the target immobilization level of 500 RU. Running buffer was HBS (hepes-buffered saline): 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween-20; 0.2 μm) filter-sterilized. The bispecific antibodies were diluted to 100, 50, 20, 10, 1 and 0.1 nM in HBS and run at high (30 μl/min) flow rate over the antigen-coupled surface of the CM5 sensor chip. With the BIA evaluation software, a curve fitting model for 1:1 monovalent interaction allowed for determination of the HER2 arms affinities (mono-valent interaction), the affinities of the HER2 arms, could be determined. Due to the low-off rate of the HER3 arm the affinity could not be determined. To determine the affinity of the HER3 arm PB4188 was coated to a CM5 sensor chip at the target immobilization level of 500 RU. Her2-Fc and Her3-Fc antigens were diluted to 100, 50, 20, 10, 1 and 0.1 nM in HBS and run at high flow rate (40 μl/min) over the PB4188 surface. To determine the k.sub.on and k.sub.off values, the BIA evaluation software was used in conjunction with a model that takes into account that a monovalent molecule was coated to the sensor chip surface and that the ErbB3-Fc antigen was a bivalent molecule. The affinities of PB4188 and PB3448 are shown in Table 10.
[0307] PB4188 Affinity Determination on Cells
[0308] Binding affinities were also determined via steady state cell affinity measurements using BT-474 and SK-BR-3 cells. Four IgG were analyzed: 1) PB4188 (bispecific HER2×HER3), containing anti-HER2 antibody 3958 and anti-HER3 antibody 3178; 2) PB9215 (bispecific HER3×TT), containing anti-HER3 antibody 3178 and anti-TT (tetanus toxoid) antibody 1337; 3) PB9216 (bispecific HER2×TT), containing anti-HER2 antibody 3958 and anti-TT antibody 1337; 4) HERCEPTIN® (monospecific HER2). The IgG were radioactively labeled with.sup.1251 using IODO-GEN® Precoated Iodonation Tubes (Pierce) and associated instructions. The labeled IgG were diluted to an activity of ˜1-2×10.sup.8 cpm/ml in 25 mM Tris-HCl, 0.4 M NaCl, 0.25% BSA, 5 mM EDTA, 0.05% NaNs. Protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Flow cytometry analysis of the labeled and non-labeled IgG using BT-474 and SK-BR-3 cells showed no or only minor signs of reduction in binding after labeling. Steady state cell affinity measurements were performed as follows. Cells were seeded in 96-well plates and incubated at 4° C. with various concentrations of labeled IgG. Unbound radioactivity was removed after 4 hours and the cell-bound radioactivity was measured using a gamma well counter. Non-specific binding was measured by adding a receptor-blocking concentration (100-fold excess) of unlabeled antibody. Each condition was tested in triplicate and three independent experiments were performed per antibody. KD values were calculated based on a non-linear regression model that compensates for non-specific binding, using Prism 6.Od (GraphPad Software). Graphs including fitted curves are given in
[0309] Anti-Proliferative Activity on HER2 Amplified Breast Cancer Cells
[0310] JIMT-1 in Soft Agar
[0311] PB3448 and PB4188 were tested for their potency to inhibit the growth of the trastuzumab resistant JIMT-1 cells in soft agar. To this aim 96 well suspension cell culture plates were prepared. 100 μL of the soft agar bottom layer (0,6% final concentration in complete medium) was poured and left to solidify. 50 μL of the soft agar top layer (0,4% final concentration) containing 10.000 JIMT-1 cells/well were then added on top, solidified and such 96 well plates incubated overnight at 37° C., 10% CO2. Next day, a negative control antibody, pertuzumab+ trastuzumab (1:1 v/v), PB3448 and PB4188 were added in DMEM medium in a semi-log titration ranging from 10-0,003 μg/ml. Subsequently, the assay was incubated in cell culture incubators for 8 days. Finally, the cells were incubated with Alamar Blue for 3-5 h at 37° C. and fluorescence intensity was determined (excitation: 560 nm; emission: 590 nm). An example of dose dependent inhibition of JIMT-1 proliferation by PB3448 and PB4188 is shown. (
[0312] BT-474 and SKBR-Sin MATRIGEL®
[0313] PB3448 and PB4188 were tested for their potency to inhibit the growth of BT-474 and SKBR-3 cells. The cells were tested at the company Ocello based in Leiden, the Netherlands that grows cells in three dimensional MATRIGEL® and uses principle component analysis to distinguish non-treated cells from treated cells. 2000 SK-BR-3 or 2250 BT474 cells were seeded in 15 μl MATRIGEL® per well of a 384 well plate (Greiner 781091). The next day a semi-log titration ranging from 10 to 0.003 μg/ml of antibodies were added in culture medium in the absence or presence of 5 ng/ml HRG. The test antibodies included a negative control antibody, pertuzumab+ trastuzumab (1:1 v/v), PB3448, PB4188 and the bispecific anti-EGFRxHER3 two-in-one antibody MEHD7945A. In addition a dose-dependent titration of HRG was included as a positive control. Each dose was tested in quadruplicate. Cells were incubated for 7 days in a cell culture incubator at 37° C., 5% CO2. Next, the cells were fixed and actin cytoskeleton of the cells was stained with phalloidin and the nuclei are stained with Hoechst. Next, fluorescent images were taken at different levels through the gel (Z-stack) and the images were superimposed. Abroad range of morphological features were measured (800 in total). Only features that differed between medium and HRG treatments were selected for analysis. Features that were associated with growth, mean spheroid area and nuclei per spheroid were most significantly different between medium and HRG treatments. Both multiparameter and single parameter analyses were made. For single parameter measurements, t-tests were performed to compare treatments (HRG or antibody) to medium. P-values for each point were determined. Principal component analysis (PCA), a method for finding low-dimensional combinations of high-dimensional data that capture most of the variability was used in relation to antibody concentration, to plot the data.
[0314] Superior Anti-Proliferative Activity of PB4188 in the Presence of HRG on HER2 Amplified Breast Cancer Cells
[0315] The activity of PB4188 in the presence of 10 ng/ml HRG on SKBR-3 and BT-474 was compared to a panel of HER2, HER3 antibodies and combinations thereof. The assay was performed in MATRIGEL®, as described above, and morphological features were analyzed. PCA data plotted in
[0316] Superior Anti-Proliferative Activity of PB4188 on HER2 Amplified Gastric Cancer Cells Compared to HER2 and HER3 Monoclonal Antibodies.
[0317] Upregulation of NRG1-P1 is a key resistance mechanism against HER2 targeted therapies (Wilson, 2012). To evaluate whether upregulation of NRG1-31 would interfere with the anti-proliferative potency of PB4188 a panel of antibodies was tested at 100 ng/ml HRG on the N87 (HER2 amplified) gastric cancer cell line. N87 cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS. For the proliferation assay subconfluent cell cultures of N87 cells were washed with PBS trypsinized and trypsin was inactivated by adding culture medium.
[0318] Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 μg/ml Holo Transferrin). Antibodies were diluted in a semi-log titration that varied from 1-0,0001 μg/ml. Cells were added at a density of 10000 cells/well in the presence of 100 ng/ml final concentration of HRG. The cells were cultivated for 3 days at 37° C., 5% CO2, in 95% relative humidity. ALAMAR BLUE™ (INVITROGEN®) was added according to the manufacturer's instructions and incubated for 6 hours at 37° C., 5% CO2, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation with 590 nm emission wavelength. PB4188 showed superior activity over anti-HER2 or anti-HER3 monoclonal antibodies (
[0319] HER2×HER3 Bipecific Antibodies Induce ADCC
[0320] ADCC activity is an important anti-tumour mechanism of action for therapeutic antibodies in cancer. Human monoclonal antibodies directed to the HER family of receptors like cetuximab and trastuzumab induce ADCC. The baseline and enhanced ADCC activity of PB4188 and PB3448 were determined in validated in vitro ADCC assays. Trastuzumab and a negative control antibody were included as control antibodies in the experiment. Whole blood and PBMC fractions were obtained from healthy donors. Each antibody was tested against the HER2 high (SK-BR-3) and HER2 low (MCF-7) expressing target cells. Target cells were loaded with .sup.51Cr (Amersham) and opsonized with the indicated concentrations of antibody. Whole-blood or PBMC fraction were used as effector cells in a 200 μL reaction in RPMI 1640+10% heat inactivated FCS. Cells were incubated together for 4 h, and lysis was estimated by measuring radioactivity in the supernatant using a y-scintillator. Percentage of specific lysis was calculated as follows: (experimental cpm—basal cpm)/(maximal cpm—basal cpm)×100, with maximal lysis determined in the presence of 5% Triton X-100 and basal lysis in the absence of antibody and effectors. As shown in
[0321] HER2×HER3 Bipecific Antibodies Show Higher ADCC Compared to the Combination of Parental Antibodies
[0322] In a different ADCC setup, the ADCC Reporter Bioassay (Promega) was used. The 20 bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor, V158 (high affinity) or F158 (low affinity) variant, and an NFAT response element driving expression of firefly luciferase. The assay was validated by comparing data obtained with the ADCC Reporter Bioassay to the classical .sup.51Cr release assay. The ADCC assays were performed using the Promega ADCC Bioassay kit using 384 white well plates. In this experimental setup SKBR-3 cells were plated at a density of 1000 cells/well in 30 μl assay medium (RPMI with 4% low IgG serum) 20-24H before the bioassay. The next day, the culture medium was removed. Next, a serial dilution of antibodies, PB4188 and its parental anti-HER2 PG3958 and anti-HER3 PG3178 as well as the combination thereof was generated in duplo. 10 μl antibody dilutions were added to the wells. The starting concentration of the antibody was 10 μg/ml and a 10 points semi-log fold serial dilution was generated to provide a full dose-response curve. Finally, 5 μl of ADCC Bioassay effector cells (15000 cells/well, V158) were added. The cells were incubated for 6H at 37° C. Next, 15 μl BIO-Glo luciferase substrate was added and 5 minutes later luminescence was detected in a plate reader. The obtained data are shown in
[0323] ADCC Enhancement of PB4188
[0324] ADCC activity can be enhanced by different techniques, one of them being the removal of fucose. Removal of fucose has resulted in increased anti-tumour activity in several in vivo models [Junttila, 2010]. To maximize PB4188 activity, afucosylation technology was applied (Cheng Liu and Andreia Lee. ADCC Enhancement Technologies for Next Generation Therapeutic Antibody. Antibody therapeutics-Trends in Bio/Pharmaceutical Industry 2009 [13-17]), thereby preventing fucosylation of the N-linked carbohydrate structure in the Fc region. The ADCC potency of afucosylated PB4188 compared to the wildtype PB4188 was determined in an ADCC .sup.51Cr release assay using HER2 low expressing cells (MCF-7) and HER2 amplified cells (SK-BR-3). Both antibodies were applied in a serial dilution and a negative control antibody and trastuzumab were included in the assay.
[0325] Afucosylated PB4188 Shows Superior ADCC Activity with Low Affinity FcγRIII Receptors
[0326] Afucosylated PB4188 activity was tested on ADCC reporter cells containing either the V158 (high affinity) FcγRIIIa receptor variant or the F158 (low affinity) FcγRIIIa receptor variant. A serial titration of antibody, i.e. control antibody, trastuzumab and afucosylated PB4188, was added in combination with ADCC reporter cells harbouring the different FcγRIIIa variants to adherent SK-BR-3 cells. ADCC activity was measured by measuring luciferase activity. Afucosylated PB4188 showed equal activity compared to trastuzumab in combination with the high affinity V158 FcγRIIIa receptor variant. In contrast afucosylated PB4188 displayed superior ADCC activity compared to trastuzumab in combination with the low affinity F158 FcγRIIIa receptor variant. (
[0327] JIMT-1 Xenograft Study
[0328] JIMT-1 human breast carcinoma cells were grown in DMEM containing 10% fetal bovine serum, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, 25 μg/mL gentamicin, and 2 mM glutamine until the time of implantation. At the day of implantation JIMT-1 breast cells were harvested during log phase growth and resuspended in cold PBS. Female CB.17 SCID mice (Charles River) were 8 weeks old on Day 1 of the study and had a body weight range of 16.5 to 20.7 g. Each mouse was injected subcutaneously in the right flank with 5×10.sup.6 tumor cells (0.2 mL cell suspension). The tumors were measured with a caliper in two dimensions to monitor size as the mean volume twice per week. Once tumors had reached approximately 100-150 mm.sup.3 in size animals were enrolled in the efficacy study. Outlier animals-tumor volume—were removed and the mice were randomly distributed into groups of 10 mice each. Mice were injected once weekly (antibody) or daily (lapatinib) for a period of four weeks. Details of the treatment groups are depicted in Table 11.
[0329] Tumor sizes were measured weekly by caliper measurement. The efficacy study revealed that PB4188 at both dosing schedules was equal effective and more potent than lapatinib or the combination pertuzumab and trastuzumab. The data are shown in
[0330] PB4188 can Overcome HRG Mediated Resistance
[0331] Upregulation of NRG1-P1 is a key resistance mechanism against HER2 targeted therapies (Wilson, 2012). PB4188 was tested in comparison to its parental anti-HER3 monoclonal antibody PG3178 in a serial titration in the presence of an increasing concentration of HRG (NRG1-P1 EGF). To this aim N87 cells were cultured in RPMI 1640 supplemented with 10% heat inactivated FBS. For the proliferation assay subconfluent cell cultures of N87 cells were washed with PBS trypsinized and trypsin was inactivated by adding culture medium. Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 μg/ml Holo Transferrin). Antibodies were diluted in a semi-log titration ranging from 1 to 0.0001 μg/ml. Cells were added at a density of 10000 cells/well in the presence an increasing concentration of HRG (0.04-39.5 nM). The cells were cultivated for 3 days at 37° C., 5% CO2, in 95% relative humidity.
[0332] ALAMAR BLUE™ (INVITROGEN®) was added according to the manufacturer's instructions and incubated for 6 hours at 37° C., 5% CO2, in 95% relative humidity in the dark. Fluorescence was measured at 550 nm excitation with 590 nm emission wavelength. PB4188 showed superior activity compared to the parental anti-HER3 monoclonal antibody (
[0333] Hence, in case of an escape mechanism, such as for instance upregulation of NRG1-P1, a bispecific antibody according to the invention is preferred.
[0334] Epitope Mapping of HER2/HER3 Specific IgGs
[0335] Shotgun Mutagenesis Experiments
[0336] Alanine scanning mutagenesis was used to map the epitopes of PG3958 and PG3178 for HER2 and respectively HER3. In the shotgun mutagenesis assay, clones are generated whereby each amino acid residue of the HER2/HER3 extracellular domain (ECD) is substituted for alanine. Next, a cell array was prepared by reverse transfection (patent US2011/0077163A1). Therefore, DNA of each clone was mixed with lipofectamin and the mixture was placed in a dedicated well of a 384 well plate. HEK293T cells were added to each well and expression of protein was measured 24H later. Subsequently, the reactivity of antibodies was measured by immunofluorescent staining leading to binding maps and identification of critical residues for antibody binding. Expression levels of the HER2 and HER3 ECD constructs were verified by FACS analysis using commercially available monoclonal antibodies (R&D mAb 1129 (HER2) and R&D mAb 66223 (HER3)).
HER2
[0337] Binding of monovalent PG3958 Fab to HER2 ECD mutants was tested at a concentration of 0.25 μg/ml in the assay and stringent washing conditions were used (pH 9.0, 350 mM NaCl). This resulted in the identification of three ‘critical’ residues (T144, R166, R181) in HER2 that showed less than 35% residual binding of the PG3958 Fab compared to WT HER2 while retaining control mAb binding. Two residues (P172, G179) that are positioned near the critical residues in the HER2 structure showed significant, but less severe loss of binding and were designated ‘secondary critical’ residues (Table 13 and
[0338] Confirmation Experiments HER2 Epitope
[0339] 15 Constructs encoding Wildtype (WT) HER2 ECD and the HER2 ECD variants listed in Table 13 were expressed in CHO-K1 cells. Three Domain I residues that are surface exposed and structurally near the determined critical residues were selected for further analysis. T164, S180 and D143 point mutations to tyrosine were generated in the HER2 ECD construct and the resulting constructs were also 20 expressed in CHO-K1. The L159 Å HER2 ECD variant was expressed in CHO-K1 cells as control sample.
[0340] The bispecific PG3958×TT antibody tested for binding to the ECD variants in a FACS titration experiment. The anti-HER2 antibody trastuzumab which binds domain IV of HER2 was used to verify HER2 ECD expression at the cell surface. 25 Mean MFI values were plotted and for each curve the AUC was calculated using GraphPad Prism 5 software. WT HER2 binding was used to normalize the data. The FACS data showed that in addition to T144A, R166A, R181A, P172A, G179 Å the mutations T164Y and S180Y resulted in significant reduction in binding of the PG3958×TT antibody (
HER3
[0341] Binding analysis of PG3178 IgG at 0.25 μg/ml to HER3 ECD mutants in FACS resulted in the identification of two so-called ‘critical’ residues (F409, R426) for which mutation to alanine caused substantial loss of binding compared to WT HER3, while binding of the control mAb was retained (Table 14 and
[0342] Confirmation Experiments HER3 Epitope
[0343] CHO-K1 cells were transfected with HER3 ECD mutation constructs (listed in Table 14), WT HER3 ECD and two control constructs (H407 Å and Y424A). PG3178 binding to the HER3 ECD variants was tested in a FACS titration experiment. Two control antibodies, binding Domain I (MM-121) and Domain III (MEHD7945A) of HER3 were included to verify HER3 ECD expression on the cell surface. Mean MFI values were plotted and for each curve the AUC was calculated using GraphPad Prism 5 software. WT HER3 binding was used to normalize the data. The R426 Å mutation was shown to be critical for PG3178 binding whereas the binding to F409 Å could not be confirmed due to loss of cell surface expression (
[0344] PB4188 Activity on Cardiomyocytes In Vitro
[0345] HER2 is involved in growth, repair, and survival of adult cardiomyocytes as part of a signalling network that involves the heregulin receptor complex HER2:HER4. Cardiotoxicity is a known risk factor in HER2 targeting and the frequency of complications is increased when trastuzumab is used in conjunction with anthracyclines thereby inducing cardiac stress. A model system based on human stem cell derived cardiomyocytes was used to test the potential toxicity of PB4188 and benchmark it against trastuzumab and the combination of trastuzumab and pertuzumab in the presence of the anthracyclin doxorubicin. Human stem cell derived cardiomyocytes (Pluriomics BV) were seeded at a concentration of 20.000 well in white flat-bottom assay plates (corning 655098). On day 5 of culture the medium was replaced for glucose and galactose free culture medium supplemented with 10 ng/ml HRG. On day 7 test antibodies were added in combination with doxorubicin (3 μM). Cell viability was assayed on day 9 using the Promega Cell titer Glo assay. The monospecific antibodies were tested at single concentrations of 68 nM whereas PB4188 was tested at three concentrations in the presence of 3 μM doxorubicin.
[0346] PB4188 Binding to Cells with Different HER2 Levels
[0347] The binding of PB4188 in comparison to trastuzumab and the HER3 antibody U1-59 was analyzed by FACS on breast and gastric cancer cell lines expressing different levels of HER2. Cells were considered HER2+++ if they express millions of HER2 copies and/or are HER2 gene amplified. The following cell lines were used: MCF-7 (HER 2+); MDA-MB-468 (HER2+, MKN-45 (HER2+), MDA-MB-175 (HER2+), MDA-MB-453 (HER2++), MDA-MB-361(HER2++), ZR-75-1(HER2++), JIMT-1 (HER2+++), BT-474 (HER2+++), SKBR-3 (HER2+++), SK-OV-3 (HER2+++), N87 (HER2+++). Cells of an exponentially grown culture were harvested by trypsin and diluted to 106 cells/ml in FACS buffer (PBS/0.5% BSA/0.5 mM EDTA). 1-2 105 cells were added to each well in a U-bottom 96 well plate. Cells were centrifuged for 2 minutes at 300 g at 4° C. Supernatant was discarded by inverting plate(s) above, followed by flicking once. 50 μl of each IgG sample was added in a serial dilution from 3.16 ng-10 μg/ml and incubated for 1H on ice. Cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. 50 μl diluted 1:100 mouse anti human IgG gamma PE (INVITROGEN®) was added and incubated for 30-60 minutes on ice in the dark. Cells were centrifuged once, supernatant was removed and cells were washed twice with FACS buffer. Cells were analysed on a FACSCanto Flow cytometer in a HTS setting. The quantity of antibody bound was was assessed by median fluorescence. Data were plotted and the area under the curve (AUC, a cumulative measurement of the median fluorescence intensity) was determined for each antibody per cell line tested (
[0348] From this experiment it is concluded that PB4188 has a higher binding affinity for HER2+++ cells, HER++ cells and HER+ cells as compared to trastuzumab.
[0349] Simultaneous Binding with Trastuzumab
[0350] PB4188 and trastuzumab do not compete for binding to HER2 PB4188 binds domain I of the HER2 protein whereas the binding epitope of trastuzumab is localized in domain IV. To demonstrate that both antibodies do not compete for HER2 binding, a binding assay with HER2 amplified SKBR-3 breast cells was performed. First unlabeled antibody was allowed to bind SKBR-3 at saturating concentrations. Next FITC-labeled PB4188 was added in a titration range and fluorescence was measured by FACS.
[0351] Targeting Domain I of HER2 by a HER2×HER3 Bispecific Molecule can Overcome Heregulin Resistance
[0352] To test whether the orientation of PB4188 on the HER2×HER3 dimer was preferred for inhibiting cell proliferation under HRG stress conditions, bispecific antibodies were generated composed of the 3178 HER3 arm and HER2 arms targeting either domain I, II, III or IV. Two HER2×HER3 bispecific antibodies were generated for each of the HER2 domains I-IV. The HER2 arms included: MF3958 and MF3003 targeting domain I; MF2889 and MF2913 targeting domain II; MF1847 and MF3001 targeting domain III and MF1849 and MF1898 targeting domain IV. Each HER2 Fab arm was combined with the 3178 HER3 Fab arm and tested for their potency to inhibit cell proliferation in the presence of high concentrations of heregulin. Antibody titrations were performed on HER2 low expressing MCF-7 cells and the HER2 overexpresssing N87 and SK-BR-3 cells. Subconfluent cell cultures of N87, SK-BR-3, and MCF-7 cells were washed with PBS trypsinized and trypsin was inactivated by adding culture medium. Cells were washed twice in large volumes of assay medium (RPMI 1640 medium containing 0.05% BSA and 10 μg/ml Holo Transferrin). Antibodies were diluted in a semi-log titration. Cells were added at a density of 10000 cells/well (N87, SKB-BR-3) and 5000 cells/well MCF-7 in the presence the experimentally defined stress concentration of HRG (10 nM SK-BR-3, 100 nM N87 and MCF-7). The cells were cultivated for 3-4 days at 37° C., 5% CO2, in 95% relative humidity. ALAMAR BLUE™ (INVITROGEN®) was added to assess the proliferation. Absorbance was measured at 550 nm excitation with 590 nm emission wave length. In all assays tested, only the bispecific antibodies targeting domain I of HER2 were able to inhibit proliferation in the presence of a high heregulin concentration (
[0353] Drug Combinations with PB4188 In Vitro.
[0354] To investigate the possibility to combine PB4188 with small molecule drugs PB4188 was combined with drugs interfering at different levels of the PI3K or MAPK pathway. Moreover, combination with chemotherapeutic drugs and cyclin inhibitors were tested. Combinations were tested on HER2 overexpressing cells growing in the presence of HRG in MATRIGEL® (SK-BR-3 and BT-474) or in the presence of HRG stress concentrations (N87 and SK-BR-3 as described in proliferation assays). The inhibitory effect of drug combinations was tested by imaging or by measuring proliferation using Alamar Blue as described herein before. First, the EC20 PB4188 and drugs tested was determined. Next, checkerboard titrations were performed with PB4188 and the drugs. Synergies were observed in all cell lines tested with tyrosine kinase inhibitors (afatinib, lapatinib, neratinib), the PI3Ka inhibitor BYL719, the Akt inhibitor MK-2206, the mTOR inhibitor everolimus, the Src inhibitor saracatinib, the microtubuli disrupting drug paclitaxel, and the HDAC inhibitor vorinostat (which is misspelled in
[0355] These data demonstrate that drugs acting on the PI3K pathway are particular effective in combination with PB4188. In addition, combinations with Tyrosine Kinase Inhibitors are effective. Moreover, a combination with the growth and migration/invasion drug saracatinib can be favourable in the metastatic setting.
[0356] PB4188 In Vitro Inhibition of Phosphorylation
[0357] Cells of an exponentially grown culture were harvested and seeded in 6 well plates (3.75×10.sup.6 cells for N87 and 1.5×10.sup.6 cells for SKBR-3) in starvation medium (N87 cells: RPMI-1640, 0.05% BSA, 10 μg/ml Holo-transferrin; SKBR-3 cells: DMEM/F-12, 2 mM L-glutamine, 0.05% BSA, 10 μg/ml Holo-transferrin) and incubated incubated overnight at 37° C., 5% CO2, in 95% relative humidity. The next day, antibodies were added to a final concentration of 5 nM and cells were incubated for one hour at 37° C., 5% CO2, in 95% relative humidity. HRG was then added to a final concentration of 100 ng/ml. After 1, 3, 6 or 24 hours at 37° C., 5% CO2, in 95% relative humidity, plates were placed on ice, cells were washed twice with cold PBS. Subsequently 0.3 ml ice-cold lysis buffer was added (Cell signaling RTK #9803 or IC #7018) and cells were lysed for a minimum of 30 minutes on ice. Next, protein concentrations were measured using BCA (Pierce #23235). Protein concentrations were adjusted to 2 mg/ml with lysis buffer. Next, lysates were applied to PathScan RTK Signaling Antibody Arrays (Cell signaling #7949) or PathScan Intracellular Signaling Antibody Arrays. All incubations were performed with sealed wells on an orbital shaker at room temperature. Lysates (75 μl) were diluted 2 times to 0.8 mg/ml concentration with 75 μl Array Diluent Buffer supplemented with protease inhibitor cocktail and kept on ice. Array wells were blocked with 100 μl Array block buffer for 15 minutes. Block buffer was removed and Lysates were applied to the wells and allowed to incubate for 2 hours. Lysate was aspirated and wells were washed 4 times with 100 μl Wash buffer. Next, 100 μl detection antibody cocktail was added per well and incubated for 1 hour. Antibody cocktail was aspirated and wells were washed 4 times with 100 μl Wash buffer. 75 μl DYLIGHT80™ Streptavidin was added to each well. DYLIGHT80™ Streptavidin was aspirated and wells were washed 4 times with 100 μl Wash buffer. The multi-gasket was removed and slides were washed for 10 seconds in 10 ml in deionized water. Slides were allowed to dry and processed for imaging on an ODYSSEE® Clx. Spot fluorescence intensity was calculated using Image Studio software.
[0358] In N87 and SKBR-3, PB4188 completely blocks AKT phosphorylation during the first 6H of incubation, in contrast to the combination of trastuzumab+ pertuzumab. In addition a strong inhibition is observed in ERK and S6 phosphorylation in contrast to the combination of trastuzumab+ pertuzumab. PB4188 does not inhibit phosphorylation of HER2 (
[0359] Western Blot Analyses
[0360] To verify the phosphorylation inhibition observed in the RTK and intracellular Pathscan arrays Western blots were performed of cells treated with PB4188, the combination pertuzumab and trastuzumab and a control antibody in the presence of HRG stress concentrations. Cells of an exponentially grown culture were harvested and seeded in 10 cm dishes (20×106 cells for N87 and 7×10.sup.6 cells for SKBR-3) in starvation medium (N87 cells: RPMI-1640, 0.05% BSA, 10 μg/ml Holo-transferrin; SKBR-3 cells: DMEM/F-12, 2 mM L-glutamine, 0.05% BSA, 10 μg/ml Holo-transferrin). The next day, antibodies were added to a final concentration of 5 nM and cells were incubated for one hour. HRG was then added to a final concentration of 100 ng/ml. After 1, 3, 6 or 24 hours, dishes were placed on ice, cells were washed twice with cold PBS, transferred to Eppendorf tubes and lysed with 250 μl of RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). Lysis was allowed to proceed for 30 minutes on ice. Cell lysates were centrifuged and supernatants were collected in new Eppendorf tubes. Protein concentration was determined using the BCA method (Pierce). 30 μg of the lysate was separated on a 4-12% Bis-Tris NuPage gel (INVITROGEN®) and proteins on the gel were transferred to a nitrocellulose membrane. Membranes were blocked for one hour with TBS-T containing 5% BSA and stained with the indicated antibodies according to the manufacturer's instructions (Cell Signaling Technology). Membranes were then incubated with a HRP-conjugated secondary antibody, incubated with ECL substrate and subjected to autoradiography using X-ray films (Amersham). All detection antibodies were from Cell Signaling Technology: Phospho-Akt (ser 473) #4060, Total Akt #4691, Phospho-HER2 (Tyr 1221/1222) #2243, Total HER2 #2242, Phospho-HER3 (Tyr 1289) #4791, Total HER3 #4754, Phospho-ERK1/2 (Thr 202/Tyr 204) #4377, Total ERK1/2 #4695, Phospho-S6 RP (Ser 235/236) #2211, Total S6 RP #2217, Goat anti-rabbit HRP-linked #7074.
[0361] The results show that PB4188 shows a prolonged inhibition of HER3 phosphorylation resulting in the inhibition of both the MAPK and PI3 kinase pathway with a profound effect on AKT phosphorylation inhibition (
[0362] PB4188 In Vivo Pharmacodynamics
[0363] Phosphoprotein Analysis by Luminex
[0364] Tumors (100 mm3) of JIMT-1 transplanted mice treated with 2 doses of PB4188 and 4 doses of PB4188 were removed 24H after dosing. Tumors were flash-frozen and processed to powder. Tumor lysates were prepared to a concentration of 50 mg tumor/mL using cold BioRad Lysis Buffer (supplemented with 0.4% BioRad Factor 1, 0.2% BioRad Factor 2, and 2 mM PMSF) to the frozen powder samples, incubated at 4° C. on a rocker for 60 minutes to ensure complete lysis. The samples were centrifuged at 4° C. for 10 minutes at 16000×g, and aliquoted. Total protein was determined using the Biorad DC Protein Assay reagents according to manufacturer's instructions. Luminex Assay: The JIMT-1 tumor lysate samples were processed and analyzed for: Total AKT AKT(Ser473) and AKT(Thr308using commercially available Luminex kits from Millipore (Cat #48-618MAG (Lot No. 2532050), 46-645MAG (Lot No. 46645M-1K). Each sample was tested in duplicate. Dilutions were prepared in sample diluent to load a target of approximately 25 μg protein per well for all total and phosphorylated analyte determinations. The Millipore kits were used according to the manufacturer's specifications.
[0365] Tumors treated with PB4188 showed an increase in Akt expression in comparison to untreated tumors. Phosphorylation of AKT was completely inhibited by PB4188 both after a two-weekly dose as after a four-weekly dose (
[0366] Phosphoprotein Analysis by VeraTag Assay
[0367] Tumors (100 mm.sup.3 or 400 mm.sup.3) of JIMT-1 transplanted mice treated with 1 or 2 doses doses of PB4188 were removed and fixed in 10% neutral buffered formalin. Mice bearing 100 mm.sup.3 tumors were sacrificed 24H after a single PB4188 dose (25 mg/kg) whereas mice bearing 400 mm.sup.3 tumors received 2 weekly dosis of 25 mg/kg and were sacrificed 4H after dosing. Next, samples were paraffin-embedded. Sections of 7 um in thickness were sliced with a microtome (LEICA) and placed on positively charged glass slides (VWR) with serial number labeled. Slides were air-dried for 30 min and then baked in a heated oven set at 60° C. Next samples were processed for different VeraTag analysis. Total HER2 analysis (HT2) according to 25 U.S. patent application Ser. No. 12/340,436, total HER3 analysis (H3T) according to U.S. Pat. No. 8,349,574; U.S. Patent Appl. No. 2013/0071859 and finally HER2-HER3 heterodimer (H23D), HER3pY1289 (H3pY1289) and HER3-PI3 kinase (H3PI3K) according to Int'l Patent Appl. No. PCT/US2014/033208. In both dosing regimens a significant PB4188 mediated reduction in HER2:HER3 dimers became apparent in comparison to untreated controls. There was no difference observed in total HER2, HER3 or phosphorylated HER3 between PB4188 treated tumors and controls. Tumors that were analyzed 4H after PB4188 dosing showed a significant reduction in HER3-p85 (PI3K) compared with untreated controls.
[0368] PB4188 Reduces Cell Cycle Progression in HRG-Stimulated Cancer Cells
[0369] The ability of PB4188 to influence cell cycle progression was investigated in cancer cell lines expressing various protein levels of HER2. HER2+(MCF-7), HER2+++(JIMT-1, SK-BR-3 and N87 cells) cells were seeded in assay medium (MCF-7 cells: RPMI-1640, 0.05% BSA, 10 μg/ml Holo-transferrin, 1 mM sodium pyruvate, MEM NEAA; JIMT-1: DMEM, 0.05% BSA, 10 μg/ml Holo-transferrin; SK-BR-3 cells: DMEM/F-12, 2 mM L-glutamine, 0.05% BSA, 10 μg/ml Holo-transferrin; N87 cells: RPMI-1640, 0.05% BSA, 10 μg/ml Holo-transferrin). Per well of 24-well plate, 300.000 MCF-7, or 400.000N87 or 150.000 SK-BR-3 or 150.000 JIMT-1 or cells seeded in 1 ml assay medium and incubated overnight at 37° C., 5% CO2, in 95% relative humidity. The next day, PB4188 or pertuzumab+ trastuzumab or PG3178 or PG1337 were added to the cells in the presence of a final concentration of HRG of 1 or 100 ng/ml. After 24 hrs (for JIMT-1, N87 or SK-BR-3 cells) or 48 hrs (for MCF-7 cells) incubation at 37° C., 5% CO2, in 95% relative humidity, cells were supplemented with EdU (10 μM final concentration) for 2 hrs before being harvested and stained for EdU incorporation using the Click-iT EdU AlexaFluor488 kit according to the manufacturer instructions (LifeTechnologies, cat.no. C10425). At least 30 min before analyzing the cells by flow cytometry on FACSCanto, cells were incubated with 200 nM FxCycle far red dye (LifeTechnologies, cat.no. F10348) and 100 μg/ml RNAse A (LifeTechnologies, cat.no. 12091-039). Events were acquired in the AlexFluor488 channel (for EdU detection) and in the APC channel (for total DNA stain with the FxCycle dye). Data were analyzed by gating single cells on a FSC-width vs FSC-height scatter plot, and subgating the GO/G1, S and G2M phases of the cell cycle on an APC vs AlexaFluor488 scatter plot, as EdU.sup.negAPC.sup.low, EdU.sup.neg and EdU.sup.negAPC.sup.high populations, respectively.
[0370] Data are represented as the proliferation index calculated by dividing the percentage of cells in the S and G2/M phases by the percentage of cells in the GO/G1 phase.
[0371] PB4188 Induces Receptor Internalization
[0372] Internalization pattern of antibodies was measured using pH-sensitive dyes. This has been described in the art in WO2013134686 A1 where such dyes, when coupled to an antibody, display an increased fluorescence signal when exposed to lower pH. This occurs when the dye-coupled antibodies internalize from the surface of target cells into mildly acidic endosomes (pH 6-6.5) to acidic lysosomes (pH lower than 5.5). To investigate whether PB4188 internalizes in cancer cells, the antibody was coupled to the pH sensor dye with succinimidyl ester reactive group (Promega, cat.no. CS1783A01) according to the manufacturer's instructions. As comparators, anti-HER2 (trastuzumab, pertuzumab, PG3958), anti-HER3 (PG3178, #Ab6) and negative control (anti-tetanus toxin, PG1337) dye labeled antibodies were included. HER2-overexpressing SKBR-3 and N87 cancer cells of an exponentially grown culture were harvested and seeded on 96 well plates (15×103 cells per well) in 100 μl assay medium (N87 cells: RPMI-1640, 0.05% BSA, 10 μg/ml Holo-transferrin; SKBR-3 cells: DMEM/F-12, 2 mM L-glutamine, 0.05% BSA, 10 μg/ml Holo-transferrin) containing 1 ng/ml HRG and incubated overnight at 37° C., 5% CO2, in 95% relative humidity. The next day, 20 μl pH-sensitive dye-labelled antibodies were added to reach a final concentration of 100 nM and cells were incubated overnight at 37° C., 5% CO2, in 95% relative humidity. The next day, cells were harvested by collecting non-adherent cells and trypsinizing adherent cells. After washing cells with FACS buffer (PBS 0.5% BSA 0.1% sodium azide), cells were stained with APC-labelled anti-human IgG (Jackson Immunoresearch, cat.no. 109-136-098, 1:100 dilution). Cells were analyzed by flow cytometry on FACSCanto (BD Biosciences) measuring median fluorescence intensities (MFI) of the PE and APC channels to determine internalization and residual surface binding of antibodies, respectively. Data shown in
[0373] Generation and Characterization of Anti-HER3 Antibody 3178 Variants
[0374] Variants of anti-HER3 antibody MF3178 were designed with the aim to improve antibody properties. Mutations were introduced in the VH gene framework region 1 (FR1), complementarity determining region 1 (CDR1), FR2, CDR2 and/or FR3, while CDR3 and FR4 were not modified. The design included, but was not limited to, mutations that were introduced to remove post-translational modification (PTM) motifs (e.g. by changing the deamidation motif NS to NQ), to reduce surface hydrophobicity (e.g. by changing I to T) or to increase the iso-electric point (pI; e.g. by changing Q to K). All 20 variants (See
[0375] Serum titers of the different cohorts of immunized mice as determined by FACS. D=day of antibody titer determination. Table 1: response against HER2. Table 2: response against HER3. Cell lines used are indicated (MCF7, SKBR3, BT474). The different mice are in the columns
TABLE-US-00002 TABLE 1 anti-HER2 response ErbB2 K562 MCF7 SKBR3 A, D35 236 168 315 148 116 145 5909 5728 6147 5491 4838 4930 67748 C, D42 163 144 154 152 166 2574 3212 2140 2346 2172 15448 E, D35 129 134 152 132 147 157 6214 5542 5625 5634 4812 3905 27730 G, D52 145 129 126 133 163 5752 5088 4268 4899 5240 22769 Average 130.8 Average 194.4 Average D 0 D 0 D 0 5× 654 5× 972.2 5× 10× 1308 10× 1944 10× 20× 2616 20× 3889 20× 30× 3924 30× 5833 30× ErbB2 BT474 A, D35 29537 45315 44737 33508 38355 38707 18928 27240 24784 17659 18713 C, D42 17188 12627 12432 12067 10259 9669 7789 6618 6030 E, D35 19765 26863 26232 19478 13968 22716 17413 19139 18317 16397 12787 G, D52 26157 16726 14633 15783 19413 16640 16424 16959 18633 300.2 Average 241 D 0 1501 5× 1205 3002 10× 2410 6004 20× 4819 9005 30× 7229
TABLE-US-00003 TABLE 2 anti-HER3 response ErbB3 K562 MCF7 B, D56 332 356 453 535 417 645 1630 1236 3251 1401 1297 1814 D, D56 336 445 277 185 319 1159 3260 959 643 2362 F, D35 265 245 249 285 291 262 4370 3985 3445 3428 3579 2718 H, D52 263 289 233 271 242 4083 4239 2970 4167 4584 Average 130 Average 172 D 0 D 0 2.5× 326 2.5× 430 5× 651 5× 859 10× 1303 10× 1718 20× 2605 20× 3437 ErbB3 SKBR3 BT474 B, D56 1666 1100 3072 1199 1268 1503 1675 1204 3393 1380 1295 1725 D, D56 964 2180 721 510 1577 1030 3754 945 584 2042 F, D35 4139 3378 2676 2659 2674 2414 4618 3690 3522 3144 3208 2776 H, D52 5183 4319 3256 5408 5474 6326 4920 4542 6653 6938 Average 200 Average 222 D 0 D 0 2.5× 501 2.5× 556 5× 1002 5× 1112 10× 2004 10× 2223 20× 4008 20× 4446
TABLE-US-00004 TABLE 3 Binning of HER2 antibodies depending on their reactivity with chicken-human-HER2 chimera's and reactivity to mouse HER2. ‘Number’ indicates the number of unique antibodies in each group Group Domain reactivity Number 1 Domain I specific 25 2 Domain II specific 2 3 Domain III specific 23 4 Domain IV specific 7 5 Domain IV specific + murine cross-reactive 2 6 Reactive to all constructs 2 7 Human WT reactive only 4
TABLE-US-00005 TABLE 4 Competition ELISA using IgGs and phage antibodies. Four IgG antibodies are used in the competition assay: two HER2 antibodies recognizing domain IV (trastuzumab and PG1849); one antibody recognizing domain II (PG2971) and one negative control anti- RSV antibody. Loss of signal is observed when the phage and antibody encoded by the same variable region genes are competing; i.e. MF1849 and PG1849 and MF2971 and PG2971. — MF1849 MF2971 MF2708 Trastuzumab 0.046 1.02 1.115 0.044 PG1849 0.043 0.384 1.139 0.041 PG2971 0.042 1.202 0.091 0.042 Anti-RSV mAB 0.044 0.94 1.003 0.047 — 0.045 1.432 1.481 0.038
TABLE-US-00006 TABLE 5 Binning of HER3 antibodies depending on their reactivity with rat-human-HER2 chimera's and reactivity to HER3 and HER3 of other species. ‘Number’ indicates the number of unique antibodies in each group Group Reactivity Number 1 High Domain III reactivity, rat and mouse 8 reactive and minor reactivity to domain IV 2 High Domain III reactivity, rat, human and 8 cyno reactive, minor reactivity to domain IV 3 Reactivity to rat, cyno and human HER3 43 4 Reactive to human HER3 32 5 Reactive to all constructs 33
TABLE-US-00007 TABLE 6 Functional activity of the most potent HER2 monoclonals at 1 μg/ml IgG. Percentage activity compared to reference antibodies, i.e. trastuzumab in SKBR-3 and #Ab6 in MCF-7. For HER2 antibodies the domains of all antibodies except PG2926 were mapped to domains I, III or IV Epitope HER2 PG ID nr Target Bin domain SKBR-3 MCF-7 PG2916 HER2 1 I 58% 30% PG2973 HER2 1 I 49% 58% PG3004 HER2 1 I 49% 56% PG1849 HER2 5 IV 42% 22% PG3025 HER2 1 I 38% 28% PG2971 HER2 1 I 25% 51% PG3031 HER2 1 I 33% 38% PG2926 HER2 7 NA 0% 35% PG2930 HER2 3 III 0% 7%
TABLE-US-00008 TABLE 7 Functional activity of the most potent HER3 monoclonals at 1 μg/ml IgG in a HRG dependent MCF-7 assay. Percentage activity compared to reference antibody #Ab6. Epitope PG ID nr Target group MCF-7 PG3178 HER3 5 162% PG3163 HER3 5 119% PG3176 HER3 5 68% PG3099 HER3 3 ND
TABLE-US-00009 TABLE 8 FACS stainings of HER2 antibodies whereby the HER2 VH is combined with a different light chain than the common light chain indicated in FIG. 16. MFI, indicates Mean Fluorescence Intensity in FACS. The HER2 MF number is indicated in between brackets, HER2 binding clones in the context of the different light chain are indicated in bold. MFI K562 cells MFI PGnumber (neg control) K562 HER2 PG4462 (MF2971) 267 14900 PG4463 (MF3958) 248 15600 PG4474 (MF2916) 254 14700 PG4478 (MF2973) 254 18000 PG4481 (MF3004) 267 16200 PG4482 (MF3025) 299 12000 PG4483 (MF3031) 260 14900 PG4465 (MF1849) 270 249 Anti-HER2 mAb 309 7618 Anti-RSV mAb 263 276
TABLE-US-00010 TABLE 9 Functional activity of lead HER2 x HERS bispecific antibodies (indicated using the PB prefix; each PB comprises an HER2 arm and an HER3 arm as indicated in the table) compared to comparator antibodies in the HRG dependent MCF-7 and BxPCS assays. Based on binding profiles using chimeric constructs HER2 and HERS antibodies could be separated over different bins. For HER2 antibodies the domains all antibodies except PG2926 could be mapped to domains I, III or IV. MCF-7 BxPC3 HER2 HER2 HER3 HER3 IC50 % Name arm domain arm bin (pM} Inhibition PB3441 2926 NA 3178 5 51.7 −24% PB3443 2930 III 3178 5 136 −31% PB3448 1849 IV 3178 5 371 −22% PB3565 2973 I 3178 5 30.9 −19% PB3566 3004 I 3178 5 7.9 −20% PB3567 2971 I 3178 5 46.5 −17% PB3709 3025 I 3178 5 34.5 −19% PB3710 2916 I 3178 5 74.2 −19% PB3883 2971 I 3176 5 113 −19% PB3986 3025 I 3163 5 30.7 −21% PB3990 2971 I 3163 5 13 −18% PB4011 2971 I 3099 3 40.2 ND PB3437 3031 I 3178 5 14 −10% PG3178 NA NA 3178 5 139 −17% #Ab6 504 −7% trastuz. + 352 ND pertuz. trastuzumab 500 −3%
TABLE-US-00011 TABLE 11 JIMT-1 xenograft study treatment groups Regimen 1 Gr. N Agent Vehicle mg/kg Route Schedule .sup. 1.sup.# 10 PBS X — ip qwk × 4 (start on day 1) 2 10 lapatinib — 150 po qd × 28 (start on day 1) 3 10 PB4188 — 2.5 ip qwk × 4 (start on day 1) 4 10 PB4188 — 25 ip qwk × 4 (start on day 1) 5 10 Pertuzumab + — 2.5 ip qwk × 4 Trastuzumab (start on day 1) 6 10 Pertuzumab + — 25 ip qwk × 4 Trastuzumab (start on day 1)
TABLE-US-00012 TABLE 10 Monovalent binding affinities of PB4188 and PB3448 for HER2 and HER3 as measured in BIACORE ™. Both bispecific antibodies share the same HER3 arm. ND, not done. PB KD on Her2 (nM) KD on Her3 (nM) PB3448 5.4* ND PB4188 0.16* 3.9
TABLE-US-00013 TABLE 12 Affinities of .sup.125I-labeled IgG HER2xHER3 IgG (PB4188), HER3xTT (PB9215), HER2xTT (PB9216) and HERCEPTIN ® H(monospecific for HER2), as determined using steady state cell affinity measurements with BT-474 cells and SK-BR-3 cells. Data were obtained from three independent experiments. BT-474 SK-BR-3 HERCEPTIN ® 3.7 ± 0.5 nM 1.3 ± 0.1 nM PB4188 3.2 ± 0.5 nM 2.0 ± 0.4 nM HER2xTT 3.9 ± 0.6 nM 2.3 ± 0.7 nM HER3xTT 0.23 ± 0.08 nM 0.99 ± 0.4 nM
TABLE-US-00014 TABLE 13 The mean binding protein reactivities (and ranges) listed for all critical residues identified. Critical residues involved in PG3958Fab binding were identified as those mutated in clones that were negative for PG3958Fab binding (<35% WT) but positive for the control mAb 1129 binding (>80% WT). Two additional critical residues were identified which did not meet the threshold guidelines, but whose mutation reduced antibody binding by a lesser extent. Residue numbering is that of PDB ID #1S78. PG3958 Fab Control mAb binding % of binding % of HER2 wt binding wt binding Residue Mutation (range) (range) Designation 144 T144A 31.9 (11) 82.1 (13) Critical 166 R166A 32.2 (5) 93.7 (17) Critical 181 R181A 10.1 (5) 98.6 (34) Critical 172 P172A 52.5 (2) 94.9 (24) Secondary 179 G179A 41.7 (18) 87.9 (25) Secondary
TABLE-US-00015 TABLE 14 The mean binding protein reactivities (and ranges) are listed for both critical residues. Critical residues involved in PG3178 binding were identified as those mutated in clones that were negative for PG3178 mAb binding (<20% WT) but positive for the control mAb 66223 binding (>70% WT). Residue numbering is that of PDB ID #4P59. PG3178 Control mAb binding % of binding % of HER3 wt binding wt binding Residue Mutation (range) (range) Designation 409 F409A 16.74 (8) 79.63 (0) Critical 426 R426A 3.17 (5) 93.08 (36) Critical
TABLE-US-00016 TABLE 15 List of exposed residues within 11.2 Å radius of Arg 426 in HER3: Leu 423 L423 Tyr 424 Y424 Asn 425 N425 Gly 427 G427 Gly 452 G452 Arg 453 R453 Tyr 455 Y455 Glu 480 E480 Arg 481 R481 Leu 482 L482 Asp 483 D483 Lys 485 K485
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