U.S. patent application number 15/403145 was filed with the patent office on 2017-08-03 for site-specific labeling methods and molecules produced thereby.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Badry BURSULAYA, Bernhard Hubert GEIERSTANGER, Jan GRUNEWALD. Invention is credited to Badry BURSULAYA, Bernhard Hubert GEIERSTANGER, Jan GRUNEWALD.
Application Number | 20170218085 15/403145 |
Document ID | / |
Family ID | 48652329 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218085 |
Kind Code |
A1 |
GEIERSTANGER; Bernhard Hubert ;
et al. |
August 3, 2017 |
SITE-SPECIFIC LABELING METHODS AND MOLECULES PRODUCED THEREBY
Abstract
The present invention provides methods of site-specific labeling
of antibodies, using proteins having 4'-phosphopantetheinyl
transferase activity that catalyze post-translational modification
of peptide sequences ("peptide tags") incorporated into one or more
specific sites of an antibody of interest. Enzymatic labeling
enables quantitative and irreversible covalent modification of a
specific serine residue within the peptide tags incorporated into
the antibody, and thus creates desirable antibody conjugates.
Inventors: |
GEIERSTANGER; Bernhard Hubert;
(Solana Beach, CA) ; GRUNEWALD; Jan; (San Diego,
CA) ; BURSULAYA; Badry; (Escondito, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GEIERSTANGER; Bernhard Hubert
GRUNEWALD; Jan
BURSULAYA; Badry |
Solana Beach
San Diego
Escondito |
CA
CA
CA |
US
US
US |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Family ID: |
48652329 |
Appl. No.: |
15/403145 |
Filed: |
January 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13906584 |
May 31, 2013 |
9585970 |
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15403145 |
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61777430 |
Mar 12, 2013 |
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61655143 |
Jun 4, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/32 20130101;
C07K 16/00 20130101; C07K 2317/94 20130101; C07K 2318/10 20130101;
C07K 2319/90 20130101; A61K 47/6815 20170801; A61K 47/6851
20170801; A61P 31/00 20180101; C07K 16/30 20130101; A61K 47/6817
20170801; A61K 47/6889 20170801; C07K 2317/24 20130101; A61P 35/00
20180101; A61P 29/00 20180101 |
International
Class: |
C07K 16/32 20060101
C07K016/32 |
Claims
1. A modified antibody or an antigen binding fragment thereof,
which comprises at least one peptide tag that is a substrate of
4'-phosphopantetheinyl transferase, and is located within the
structural loop of said antibody or antigen binding fragment
thereof.
2. The modified antibody or antigen binding fragment thereof of
claim 1, wherein said 4'-phosphopantetheinyl transferase is Sfp,
AcpS, T. maritima PPTase, human PPTase or a mutant or homolog form
thereof that retains the 4'-phosphopantetheinyl transferase
activity.
3. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the peptide tag is selected from the group
consisting of: TABLE-US-00028 GDSLSWLLRLLN, (SEQ ID NO: 1) GDSLSWL,
(SEQ ID NO: 2) GDSLSWLVRCLN, (SEQ ID NO: 3) GDSLSWLLRCLN, (SEQ ID
NO: 4) GDSLSWLVRLLN, (SEQ ID NO: 5) GDSLSWLLRSLN, (SEQ ID NO: 6)
GSQDVLDSLEFIASKLA, (SEQ ID NO: 7) VLDSLEFIASKLA, (SEQ ID NO: 8)
DSLEFIASKLA, (SEQ ID NO: 9) GDSLDMLEWSLM, (SEQ ID NO: 10)
GDSLDMLEWSL, (SEQ ID NO: 11) GDSLDMLEWS, (SEQ ID NO: 12) GDSLDMLEW,
(SEQ ID NO: 13) DSLDMLEW, (SEQ ID NO: 14) GDSLDM, (SEQ ID NO: 15)
LDSVRMMALAAR, (SEQ ID NO: 16) LDSLDMLEWSLR, (SEQ ID NO: 17)
DSLEFIASKL, (SEQ ID NO: 18) DSLEFIASK, (SEQ ID NO: 19) DVLDSLEFI,
(SEQ ID NO: 20) and VLDSLEFIAS. (SEQ ID NO: 21)
4. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the peptide tag is located within the structural
loop of VH, VL, CH1, CH2, CH3, or C.sub.L region of the antibody or
antigen binding fragment thereof.
5. The modified antibody or antigen binding fragment thereof of
claim 1, wherein said the peptide tag is located within the
structural loop of the CH1 region of the antibody or antigen
binding fragment thereof.
6. The modified antibody or antigen binding fragment thereof of
claim 1, wherein said peptide tag is inserted between any two amino
acids that are listed in Table 1.
7. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the peptide tag is inserted between amino acid
residues 2 and 3 of the VH or VL domain, or between amino acid
residue 110 and 111 of the light chain, or between 119 and 120, or
between 120 and 121, or between 135 and 136, or between 136 and
137, or between 138 and 139, or between 164 and 165, or between 165
and 166, or between 194 and 195 of the CH1 domain, or between 388
and 389, or between 445 and 446, or between 446 and 447 of the CH3
domain of a parental antibody or antigen binding fragment
thereof.
8. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the peptide tag is inserted between amino acid
residue 110 and 111 of the light chain, or between 119 and 120, or
between 120 and 121, or between 135 and 136, or between 136 and
137, or between 138 and 139, or between 165 and 166 of the CH1
domain, or between 388 and 389 of the CH3 domain of a parental
antibody or antigen binding fragment thereof.
9. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the peptide tag is grafted between amino acid
residues 62 to 64 or 62 to 65 of the V.sub.H domain, or between
amino acid residues 133 and 138 of the CH1 domain, or between 189
and 195 of the CH1 domain, or between 190 and 197 of the CH1
domain.
10. (canceled)
11. (canceled)
12. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the enzyme having 4'-phosphopantetheinyl
transferase activity is Sfp and the peptide tag is GDSLSWLLRLLN
(SEQ ID NO:1), GDSLSWLVRCLN (SEQ ID NO:3), GDSLSWLLRCLN (SEQ ID
NO:4), GDSLSWLVRLLN (SEQ ID NO:5), GDSLSWLLRSLN (SEQ ID NO:6),
GSQDVLDSLEFIASKLA (SEQ ID NO:7), VLDSLEFIASKLA (SEQ ID NO:8),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), GDSLDMLEWSL
(SEQ ID NO:11), GDSLDMLEWS (SEQ ID NO:12), GDSLDMLEW (SEQ ID
NO:13), DSLDMLEW (SEQ ID NO:14), LDSLDMLEWSLR (SEQ ID NO:17),
DSLEFIASKL (SEQ ID NO:18), DSLEFIASK (SEQ ID NO:19), or DSLEFIAS
(SEQ ID NO:22).
13. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the enzyme having 4'-phosphopantetheinyl
transferase activity is Sfp and the peptide tag is GDSLSWLLRLLN
(SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ ID NO:9),
GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18), or
DSLEFIASK (SEQ ID NO:19).
14. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the antibody or antigen binding fragment thereof
is an isotype selected from IgG, IgM, IgE and IgA.
15. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the antibody or antigen binding fragment thereof
is a subtype of IgG selected from IgG1, IgG2, IgG3 and IgG4.
16. The modified antibody or antigen binding fragment thereof of
claim 1, wherein the antibody or antigen binding fragment thereof
is a human or humanized antibody or antigen binding fragment
thereof.
17. The modified antibody or antigen binding fragment thereof of
claim 16, wherein the antibody or antigen binding fragment thereof
is an anti-HER2 antibody or anti-HER2 antibody fragment.
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. The modified antibody or antigen binding fragment thereof
according to claim 1 for use as a medicament.
41. The modified antibody or antigen binding fragment thereof
according to claim 1 for use in the treatment of cancer, an
inflammatory disease, or an infectious disease.
42. The modified antibody or antigen binding fragment thereof
according to claim 1 for use in the treatment of cancer.
43. (canceled)
44. (canceled)
45. A nucleic acid encoding the modified antibody or antigen
binding fragment thereof of claim 1.
46. A host cell comprising the nucleic acid of claim 45.
47. (canceled)
48. (canceled)
49. (canceled)
50. (canceled)
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to site-specific labeling
process and molecules produced thereby.
BACKGROUND
[0002] Conjugation has been widely used to optimize the properties
of biologically active proteins, such as protein therapies,
antibody drug conjugates (ADCs), vaccines, tissue selective
targeting vehicles, molecular diagnostics, and protein nucleic acid
conjugates. Traditional conjugation method utilizes lysine based
covalent ligation, which makes it difficult to achieve homogeneity
due to the abundance of lysines on the protein's surface.
[0003] Site-specific labeling of proteins can be achieved by
post-translational enzymatic reactions, for example, using human
O.sup.6-alkylguanine-DNA alkyl-transferase (AGT), biotin ligase,
transglutaminase, sortase, cutinase, or 4'-phosphopantetheinyl
transferases for the covalent attachment of a label to a
protein.
[0004] For post-translational enzymatic reactions using human
O.sup.6-alkylguanine-DNA alkyl-transferase, the AGT is fused to a
target protein of interest, followed by the addition of a labeled
O.sup.6-benzylguanine, which is a suicide substrate for the AGT
(Keppler et al., Nat. Biotechnol. 21:86-89, 2003). This approach is
the basis for a technology called SNAP-Tag.TM., which utilizes a
180 amino acid tag (Tirat et al., International Journal of
Biological Macromolecules, 39:66-76, 2006). However, labeling of
proteins using this approach occurs only at the C- or
N-termini.
[0005] For biotin ligation, the enzyme biotin protein ligase (BPL)
attaches biotin to the biotin carrier domain of certain
carboxylases or decarboxylases. BPL catalyzes, in a two-step,
adenosine-5'-triphosphate (ATP)-dependent reaction, the
post-translational formation of an amide bond between the carboxyl
group of biotin and the .epsilon.-amino group of a specific lysine
residue located within a highly conserved Ala-Met-Lys-Met
recognition located motif within the biotin carrier domain (Tirat
et al., International Journal of Biological Macromolecules,
39:66-76, 2006). This approach can be used to create fusion tags at
the C-terminus, the N-terminus or even within the target protein
and is the basis for a technology called BioEase.TM. (72 amino acid
tag) and AviTag.TM. (uses the biotin ligase, BirA and 15-residue
acceptor peptide tag (AP)).
[0006] Transglutaminases catalyze the formation of stable
isopeptidic bonds between the side chains of glutamine (Gln) and
lysine (Lys) with the loss of ammonia, and have been used to label
glutamine side chains in proteins with fluorophores in vitro (Sato
et al., Biochemistry 35:13072-13080, 1996). Also, bacterial and
human tissue transglutaminases (BTGase and TG2) have been used to
catalyze the post-translational modification of different IgG's via
the Lys or Gin side chains located in the IgG heavy chain (Mindt et
al., Bioconjugate Chem. 19:271-278, 2008; Jeger et al., Angew.
Chem. Int. 49:9995-9997, 2010).
[0007] Sortases have been used for C-terminal and N-terminal site
specific modification of proteins, where sortase A catalyzes the
transpeptidation reaction (Antos et al., JACS, 131:10800-10801,
2009).
[0008] Cutinase is a 22-kDa serine esterase that forms a
site-specific covalent adduct with phosphonate ligands that is
resistant to hydrolysis. Cutinases have been used for C-terminal
and N-terminal site specific modification of antibodies followed by
immobilization onto surfaces (Kwon et al., Anal. Chem.
76:5713-5720, 2004; Hodneland et al., Proc. Natl. Acad. Sci.
U.S.A., 99:5048-5052, 2002).
[0009] 4'-Phosphopantetheinylation of acyl carrier proteins (ACPs)
and peptidyl carrier proteins (PCPs) are involved in an essential
post-translational modification that is required to activate
metabolite synthesis by polyketide synthases (PKSs) and
nonribosomal peptide synthetases (NRPSs), respectively (Fischbach
et al., Chem. Rev. 106(8):3468-3496, 2006). The apo to holo
conversion of ACPs and PCPs is catalyzed by 4'-phosphopantetheine
(ppan) transferases, which attach a 4'-phospho-pantetheinyl moiety
of coenzyme A (CoA) to an invariant serine residue of the protein
domains (Lambalot et al., Chem. Biol. 3(11):923-936, 1996). Due to
the comparably small size of the carrier proteins and the ability
of 4'-phosphopantetheinyl transferases to accept functionalized CoA
analogues as substrates, researchers have used carrier proteins as
fusion tags to label target proteins with a variety of small
molecule probes (see, e.g., La Clair et al., Chem. Biol.
11(2):195-201, 2004; Yin et al., J. Am. Chem. Soc.
126(25):7754-7755, 2004). In an effort to further reduce the
carrier protein tag size, Walsh and co-workers used phage display
to identify 8- to 12-residue peptides that are recognized as
efficient substrates by the bacterial 4'-phosphopantetheinyl
transferase Sfp (previously identified as a genetic locus
responsible for surfactin production) and AcpS (Yin et al., Proc.
Natl. Acad. Sci. USA 102(44):15815-15820, 2005; Zhou et al., ACS
Chem. Biol. 2(5):337-346, 2007; Zhou et al., J. Am. Chem. Soc.
130(30):9925-9930, 2008).
[0010] Antibody drug conjugates (ADCs) have been used for the local
delivery of cytotoxic agents in the treatment of cancer (see e.g.,
Lambert, Curr. Opinion In Pharmacology 5:543-549, 2005). ADCs allow
targeted delivery of the drug moiety where maximum efficacy with
minimal toxicity may be achieved. As more ADCs show promising
clinical results, there is an increased need to develop stable
engineered antibodies that provide reactive groups capable of
conjugation to various agents, especially site-specific
conjugations that can generate homogeneous immunoconjugates with a
defined drug-to-antibody ratio for use in cancer therapy.
SUMMARY
[0011] The present invention provides modified antibodies or an
antigen binding fragments thereof, which comprise at least one
peptide tag that is a substrate of 4'-phosphopantetheinyl
transferase, and is located within the structural loop of said
antibodies or antigen binding fragments. The present invention
further provides immunoconjugates comprising such modified
antibodies or antigen binding fragments, and a terminal group. The
present invention also provides methods of making such modified
antibodies, antigen binding fragments, and the immunoconjugates, as
well as methods of using such compositions.
[0012] In some embodiments, the present invention provides modified
antibodies or an antigen binding fragments thereof, which comprise
at least one peptide tag that is a substrate of
4'-phosphopantetheinyl transferase, and is located within the
structural loop of said antibodies or antigen binding fragments,
and wherein the 4'-phosphopantetheinyl transferase is Sfp, AcpS, T.
maritima PPTase, human PPTase or a mutant or homolog form thereof
that retains the 4'-phosphopantetheinyl transferase activity. In
some embodiments, the peptide tag is selected from the group
consisting of: GDSLSWLLRLLN (SEQ ID NO: 1), GDSLSWL (SEQ ID NO: 2),
GDSLSWLVRCLN (SEQ ID NO: 3), GDSLSWLLRCLN (SEQ ID NO: 4),
GDSLSWLVRLLN (SEQ ID NO: 5), GDSLSWLLRSLN (SEQ ID NO: 6),
GSQDVLDSLEFIASKLA (SEQ ID NO: 7), VLDSLEFIASKLA (SEQ ID NO: 8),
DSLEFIASKLA (SEQ ID NO: 9), GDSLDMLEWSLM (SEQ ID NO: 10),
GDSLDMLEWSL (SEQ ID NO: 11), GDSLDMLEWS (SEQ ID NO: 12), GDSLDMLEW
(SEQ ID NO: 13), DSLDMLEW (SEQ ID NO: 14), GDSLDM (SEQ ID NO: 15),
LDSVRMMALAAR (SEQ ID NO: 16), LDSLDMLEWSLR (SEQ ID NO: 17),
DSLEFIASKL (SEQ ID NO: 18), DSLEFIASK (SEQ ID NO: 19), DVLDSLEFI
(SEQ ID NO: 20), and VLDSLEFIAS (SEQ ID NO: 21). The present
invention further provides immunoconjugates comprising such
modified antibodies or antigen binding fragments thereof.
[0013] In some embodiments, The present invention provides modified
antibodies or antigen binding fragments thereof, which comprise at
least one peptide tag that is a substrate of 4'-phosphopantetheinyl
transferase, and is located within the structural loop of VH, VL,
CH1, CH2, CH3, or C.sub.L region of the antibody or antigen binding
fragment thereof. In some embodiments, the peptide tag is inserted
between any two amino acids that are listed in Table 1. In some
embodiments, the present invention provides modified antibodies or
antigen binding fragments comprising at least one peptide tag that
is a substrate of 4'-phosphopantetheinyl transferase, and is
located within the structural loop of the CH1 region of an antibody
or antigen binding fragment thereof. The present invention further
provides immunoconjugates comprising such modified antibodies or
antigen binding fragments thereof.
[0014] In some embodiments, the peptide tag is inserted between
amino acid residues 2 and 3 of the V.sub.H or V.sub.L domain, or
between amino acid residues 63 and 64 of the V.sub.H domain, or
between 64 and 65 of the V.sub.H domain, or between 138 and 139 of
the CH1 domain, or between 197 and 198 of the CH1 domain, or
between 359 and 360 of the CH3 domain, or between 388 and 389 of
the CH3 domain, or after 447 of the CH3 domain of a parental
antibody or antigen binding fragment thereof. The present invention
further provides immunoconjugates comprising such modified
antibodies or antigen binding fragments thereof.
[0015] In some embodiments, the peptide tag is inserted between
amino acid residues 2 and 3 of the VH or VL domain, or between
amino acid residue 110 and 111 of the light chain, or between 119
and 120, or between 120 and 121, or between 135 and 136, or between
136 and 137, or between 138 and 139, or between 164 and 165, or
between 165 and 166, or between 194 and 195 of the CH1 domain, or
between 388 and 389, or between 445 and 446, or between 446 and 447
of the CH3 domain of a parental antibody or antigen binding
fragment thereof. The present invention further provides
immunoconjugates comprising such modified antibodies or antigen
binding fragments thereof.
[0016] In some embodiments, the peptide tag is inserted between
amino acid residue 110 and 111 of the light chain, or between 119
and 120, or between 120 and 121, or between 135 and 136, or between
136 and 137, or between 138 and 139, or between 165 and 166 of the
CH1 domain, or between 388 and 389 of the CH3 domain of a parental
antibody or antigen binding fragment thereof. The present invention
further provides immunoconjugates comprising such modified
antibodies or antigen binding fragments thereof.
[0017] In some embodiments, the peptide tag is grafted between
amino acid residues 62 to 64 or 62 to 65 of the V.sub.H domain, or
between amino acid residues 133 and 138 of the CH1 domain, or
between 189 and 195 of the CH1 domain, or between 190 and 197 of
the CH1 domain. The present invention further provides
immunoconjugates comprising such modified antibodies or antigen
binding fragments thereof.
[0018] In some embodiments, the present invention provides modified
antibodies or an antigen binding fragments comprising SEQ ID NO:
103, SEQ ID NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122,
SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or
SEQ ID NO:141. The present invention further provides
immunoconjugates comprising such modified antibodies or antigen
binding fragments thereof.
[0019] In some embodiments, the present invention provides modified
antibodies or antigen binding fragments comprising SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID
NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129,
SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID
NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158,
SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID
NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257,
SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:277, SEQ ID
NO:348, SEQ ID NO:349, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:359,
SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:373, SEQ ID
NO:374, SEQ ID NO:380, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:387,
or SEQ ID NO:388. The present invention further provides
immunoconjugates comprising such modified antibodies or antigen
binding fragments thereof.
[0020] In some embodiments, the present invention provides modified
antibodies or antigen binding fragments comprising SEQ ID NO:32,
SEQ ID NO:63, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID
NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160,
SEQ ID NO:169, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID
NO:257, SEQ ID NO:259, SEQ ID NO:268, SEQ ID NO:358, SEQ ID NO:359,
SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:374, or SEQ
ID NO:384. The present invention further provides immunoconjugates
comprising such modified antibodies or antigen binding fragments
thereof.
[0021] In one embodiment, the present invention provides modified
antibodies or an antigen binding fragments thereof, which comprise
at least one peptide tag that is a substrate of Sfp, and is located
within the structural loop of said antibodies or antigen binding
fragments, and wherein the peptide tag is GDSLSWLLRLLN (SEQ ID
NO:1), GDSLSWLVRCLN (SEQ ID NO:3), GDSLSWLLRCLN (SEQ ID NO:4),
GDSLSWLVRLLN (SEQ ID NO:5), GDSLSWLLRSLN (SEQ ID NO:6),
GSQDVLDSLEFIASKLA (SEQ ID NO:7), VLDSLEFIASKLA (SEQ ID NO:8),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), GDSLDMLEWSL
(SEQ ID NO:11), GDSLDMLEWS (SEQ ID NO:12), GDSLDMLEW (SEQ ID
NO:13), DSLDMLEW (SEQ ID NO:14), LDSLDMLEWSLR (SEQ ID NO:17),
DSLEFIASKL (SEQ ID NO:18), DSLEFIASK (SEQ ID NO:19), or DSLEFIAS
(SEQ ID NO:22). In another embodiment, the peptide tag is
GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2), DSLEFIASKLA (SEQ
ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL (SEQ ID NO:18),
or DSLEFIASK (SEQ ID NO:19). The present invention further provides
immunoconjugates comprising such modified antibodies or antigen
binding fragments thereof.
[0022] In some embodiments, the modified antibodies or antigen
binding fragments of the invention are an isotype selected from
IgG, IgM, IgE and IgA. In some other embodiments, the modified
antibodies or antigen binding fragments of the invention are a
subtype of IgG selected from IgG1, IgG2, IgG3 and IgG4. In some
embodiments, the modified antibodies or antigen binding fragments
of the invention are a human or humanized antibody or antigen
binding fragment. In a specific embodiment, the modified antibody
or antigen binding fragment of the invention is an anti-HER2
antibody or anti-HER2 antibody fragment. The present invention
further provides immunoconjugates comprising such modified
antibodies or antigen binding fragments thereof.
[0023] The present invention provides nucleic acids encoding the
modified antibodies or antigen binding fragments described herein,
and host cells comprising such nucleic acids.
[0024] The present invention provides immunoconjugates comprising a
modified antibody or an antigen binding fragment thereof, and a
terminal group, wherein the modifice antibody or antigen binding
fragment comprises at least one peptide tag that is a substrate of
4'-phosphopantetheinyl transferase, and is located within the
structural loop of the antibody or antigen binding fragment. In
some embodiments, the modified antibody or antigen binding fragment
further comprises one or more orthogonal conjugation sites. In a
specific embodiment, each orthogonal conjugation site is
independently selected from a substrate of Sfp
4'-phosphopantetheinyl transferase, a substrate of AcpS
4'-phosphopantetheinyl transferase, a lysine, a cysteine, a
tyrosine, a histidine, a formyl glycine, an unnatural amino acid,
pyrrolysine and pyrroline-carboxylysine.
[0025] Another aspect provided herein are immunoconjugates
comprising a modified antibody or antigen binding fragment, and a
terminal group (TG) attached to the peptide tag in the modified
antibody or antigen binding fragment by a linker having the
structure according to Formula (I-b):
##STR00001##
[0026] wherein: [0027] L.sub.1 is a bond, a non-enzymatically
cleavable linker, a non-cleavable linker; an enzymatically
cleavable linker, a photo stable linker or a photo-cleavable
linker; [0028] L.sub.2 is a bond, a non-enzymatically cleavable
linker, a non-cleavable linker; an enzymatically cleavable linker,
a photo stable linker or a photo-cleavable linker; [0029] L.sub.3
is a bond, a non-enzymatically cleavable linker, a non-cleavable
linker; an enzymatically cleavable linker, a photo stable linker or
a photo-cleavable linker; [0030] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker; an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer, [0031] the *
denotes where the 4'-phosphopantetheinyl moiety is attached to the
peptide tag, [0032] and wherein the terminal group is a drug
moiety, an affinity probe, a chelator, a spectroscopic probe, a
radioactive probe, an imaging reagent, a lipid molecule, a
polyethylene glycol, a polymer, a nanoparticle, a quantum dot, a
liposome, a PLGA particle, a polysaccharide, an acetyl group, or a
surface.
[0033] In certain embodiments of such immunoconjugates: [0034]
L.sub.1 is -A.sub.1X.sup.2-- or --X.sup.2--; L.sub.2 is a bond,
-A.sub.2-, or -A.sub.2X.sup.2--; [0035] L.sub.3 is a bond,
-A.sub.3-, or -A.sub.3X.sup.2--; [0036] L.sub.4 is a bond,
-A.sub.4-, -A.sub.4X.sup.2--,
[0036] ##STR00002## ##STR00003## [0037] A.sub.1 is --C(.dbd.O)NH--,
--NHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
(O(CH.sub.2).sub.n).sub.m--, --(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0038] A.sub.2 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NR.sup.4--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--, --(CH.sub.2).sub.nNH--,
--(C(R.sup.4).sub.2).sub.nNH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0038] ##STR00004## [0039] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(.dbd.O)--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
H or
[0039] ##STR00005## [0040] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0041] each X.sup.2 is independently selected from a bond,
##STR00006## ##STR00007## ##STR00008##
[0041] --S--, --S--, --Si(OH).sub.2O--,
##STR00009##
[0042] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0043] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0044] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0045] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0046] R.sup.7 is
independently selected from H, phenyl and pyridine; [0047] R.sup.8
is independently selected from
[0047] ##STR00010## [0048] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0049] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0050] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In other
embodiments of such immunoconjugates: [0051] L.sub.1 is
-A.sub.1X.sup.2-- or --X.sup.2--; [0052] L.sub.2 is a bond,
-A.sub.2-, or -A.sub.2X.sup.2--; [0053] L.sub.3 is a bond,
-A.sub.3-, or -A.sub.3X.sup.2--; [0054] L.sub.4 is a bond,
-A.sub.4-, -A.sub.4X.sup.2--,
[0054] ##STR00011## ##STR00012## [0055] A.sub.1 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2)NHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0056]
A.sub.2 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2)NHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2)--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0056] ##STR00013## [0057] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2)NHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0057] ##STR00014## [0058] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2)--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2)NHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0059]
each X.sup.2 is independently selected from a bond,
##STR00015## ##STR00016## ##STR00017##
[0059] --S--, --Si(OH).sub.2O--,
##STR00018##
[0060] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0061] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0062] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0063] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0064] R.sup.7 is
independently selected from H, phenyl and pyridine; [0065] R.sup.8
is independently selected from
[0065] ##STR00019## [0066] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0067] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0068] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In
certain embodiments of such aforementioned immunoconjugates, the
linker of Formula (I-b) is a linker having the structure according
to Formula (I-c):
##STR00020##
[0068] In other embodiments of such aforementioned
immunoconjugates: [0069] L.sub.1 is -A.sub.1X.sup.2--, where
A.sup.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
[0069] ##STR00021## [0070] L.sub.2 is a bond; L.sub.3 is a bond,
and L.sub.4 is -A.sub.4- wherein A.sub.4 is
--(CH.sub.2).sub.nNHC(.dbd.O)--. In other embodiments of such
aforementioned immunoconjugates: [0071] L.sub.1 is
-A.sub.1X.sup.2--, wherein A.sub.1 is
--C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
[0071] ##STR00022## [0072] L.sub.2 is a bond; L.sub.3 is a bond;
L.sub.4 is -A.sub.4-, wherein A.sub.4 is
--(CH.sub.2).sub.nC(.dbd.O)--. In other embodiments of such
immunoconjugates: [0073] L.sub.1 is -A.sub.1X.sup.2--, wherein
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
[0073] ##STR00023## [0074] L.sub.2 is -A.sub.2-, wherein A.sub.2 is
--(CH.sub.2).sub.nC(.dbd.O; [0075] L.sub.3 is -A.sub.3-, wherein
A.sub.3 is
##STR00024##
[0075] and [0076] L.sub.4 is
##STR00025##
[0076] In other embodiments of such aforementioned
immunoconjugates: [0077] L.sub.1 is a -A.sub.1X.sup.2--, wherein
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; [0078] L.sub.2 is a bond-; L.sub.3
is -A.sub.3-, wherein A.sub.3 is --(CH.sub.2).sub.nC(.dbd.O)--, and
L.sub.4 is a bond. In other embodiments of such aforementioned
immunoconjugates: [0079] L.sub.1 is a -A.sub.1X.sup.2--, wherein
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, X.sup.2 is
--CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH-- and R.sup.4 is
--C(.dbd.O)OH; [0080] L.sub.2 is a bond; L.sub.3 is -A.sub.3-,
wherein A.sub.3 is --(CH.sub.2).sub.nC(.dbd.O)-- and. L.sub.4 is a
bond. In other embodiments of such aforementioned immunoconjugates:
[0081] L.sub.1 is -A.sub.1X.sup.2--, where A.sub.1 is
--C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; [0082] L.sub.2 is a bond; L.sub.3
is a bond, and L.sub.4 is -A.sub.4- wherein A.sub.4 is
--(CH.sub.2).sub.nNHC(.dbd.O)--. In other embodiments of such
aforementioned immunoconjugates: [0083] L.sub.1 is
-A.sub.1X.sup.2--, wherein A.sub.1 is
--C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; [0084] L.sub.2 is a bond; L.sub.3
is a bond; L.sub.4 is -A.sub.4-, wherein A.sub.4 is
--(CH.sub.2).sub.nC(.dbd.O)--. In other embodiments of such
aforementioned immunoconjugates: [0085] L.sub.1 is
-A.sub.1X.sup.2--, wherein A.sub.1 is
--C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; [0086] L.sub.2 is -A.sub.2-,
wherein A.sub.2 is --(CH.sub.2).sub.nC(.dbd.O; [0087] L.sub.3 is
-A.sub.3-, wherein A.sub.3 is
##STR00026##
[0087] and [0088] L.sub.4 is
##STR00027##
[0088] In other embodiments of such aforementioned
immunoconjugates: [0089] L.sub.1 is a -A.sub.1X.sup.2--, wherein
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; [0090] L.sub.2 is a bond-; L.sub.3
is -A.sub.3-, wherein A.sub.3 is --(CH.sub.2).sub.nC(.dbd.O)--, and
L.sub.4 is a bond. In other embodiments of such aforementioned
immunoconjugates: [0091] L.sub.1 is a -A.sub.1X.sup.2--, wherein
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, X.sup.2 is
--CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH-- and R.sup.4 is
--C(.dbd.O)OH; [0092] L.sub.2 is a bond; L.sub.3 is -A.sub.3-,
wherein A.sub.3 is --(CH.sub.2).sub.nC(.dbd.O)-- and. L.sub.4 is a
bond.
[0093] In the embodiments of the aforementioned immunoconjugates
the terminal group is a drug moiety selected from an
anti-inflammatory agent, an anticancer agent, an antifungal agent,
an antibacterial agent, an anti-parasitic agent, an anti-viral
agent and an anesthetic agent. In certain embodiments of such
immunoconjugates the drug moiety is selected from a V-ATPase
inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor,
a microtubule stabilizer, a microtubule destabilizers, an
auristatin, a dolastatin, a maytansinoid, a MetAP (methionine
aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a
DPPIV inhibitor, an inhibitors of phosphoryl transfer reactions in
mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a
CDK9 inhibitor, an EG5 inhibitor, an HDAC inhibitor, a DNA damaging
agent, a DNA alkylating agent, a DNA intercalator, a DNA minor
groove binder, a proteasome inhibitor, and a DHFR inhibitor. In
certain embodiments of such immunoconjugates the spectroscopic
probe is selected from a fluorophore, a chromophore, a quantum dot,
a magnetic probe, a radioactive probe, an imaging reagent, or a
contrast reagent. In certain embodiments of such immunoconjugates
the affinity probe is biotin.
[0094] Another aspect provided herein is the preparation of an
immunoconjugate by a process comprising the steps of: [0095] (a)
providing a modified antibody or antigen binding fragment thereof,
wherein the modified antibody or antigen binding fragment comprises
a peptide tag, and wherein the peptide tag is a substrate of an
enzyme having phosphopantetheinyl transferase activity, and [0096]
(b) labeling the modified antibody or antigen binding fragment with
a terminal group by incubating the modified antibody or antigen
binding fragment thereof with an enzyme having phosphopantetheinyl
transferase activity in the presence of a coenzyme A analog having
the structure of Formula B:
[0096] ##STR00028## [0097] wherein L.sub.1, L.sub.2, L.sub.3,
L.sub.4, R.sub.2 and TG are as defined herein; [0098] thereby the
terminal group is attached to the peptide tag by a linker having
the structure according to Formula (I-b):
[0098] ##STR00029## [0099] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag. In
certain embodiments the compound of Formula (B) is selected
from
##STR00030## ##STR00031##
[0099] Another aspect provided herein is the preparation of an
immunoconjugate by a process comprising the steps of: [0100] (a)
providing a modified antibody or antigen binding fragment thereof,
wherein the a modified antibody or antigen binding fragment
thereof, comprises a peptide tag, and wherein the peptide tag is a
substrate of an enzyme having phosphopantetheinyl transferase
activity; [0101] (b) labeling the modified antibody or antigen
binding fragment thereof, with a terminal group (TG) by [0102] i)
incubating the modified antibody or antigen binding fragment
thereof, with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (D),
[0102] ##STR00032## [0103] thereby attaching an activated
phosphopentathienyl group of Formula (D-a) to the peptide tag,
[0103] ##STR00033## [0104] wherein R.sub.1 is a functional group;
[0105] and [0106] ii) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-a),
[0106] X-L.sub.2-L.sub.3-L.sub.4-TG Formula (II-a) [0107] wherein X
is a group which reacts with functional group R.sub.1, [0108]
wherein: [0109] when X is a thiol, then R.sub.1 is a thiol, a
maleimide or a haloacetamide; or, [0110] when X is a an azide, then
R.sub.1 is an alkyne, a triaryl phosphine, a cyclooctene or an
oxanobornadiene; or, [0111] when X is a a triaryl phosphine, then
R.sub.1 is an azide; or, [0112] when X is a an oxanobornadiene,
then R.sub.1 is an azide; or, [0113] when X is a an alkyne, then
R.sub.1 is an azide; or, [0114] when X is a an alkene, then R.sub.1
is an azide; or, [0115] when X is a a cyclooctene, then R.sub.1 is
a diaryl tetrazine; or, [0116] when X is a a diaryl tetrazine, then
R.sub.1 is a cyclooctene; or, [0117] when X is a a monoaryl
tetrazine, then R.sub.1 is a norbornene; or, [0118] when X is a a
norbornene, then R.sub.1 is a monoaryl tetrazine; or, [0119] when X
is a an aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0120] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0121] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0122] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0123] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0124] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0125] when X is a a maleimide, then R.sub.1 is a thiol;
[0126] thereby the terminal group is attached to the peptide tag by
a linker having the structure according to Formula (III-b):
[0126] ##STR00034## [0127] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein A.sub.1, X.sub.2, L.sub.2, L.sub.3, L.sub.4, R.sub.2 and TG
are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0128] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0129] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) [0130]
i) by incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (E),
[0130] ##STR00035## [0131] thereby attaching an activated
phosphopentathienyl group of Formula (E-a) to the peptide tag,
[0131] ##STR00036## [0132] wherein R.sub.1 is a functional group;
[0133] and [0134] ii) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-c),
[0134] X-L.sub.3-L.sub.4-TG Formula (II-c) [0135] wherein X is a
group which reacts with functional group R.sub.1, [0136] wherein:
[0137] when X is a thiol, then R.sub.1 is a thiol, a maleimide or a
haloacetamide; or, [0138] when X is a an azide, then R.sub.1 is an
alkyne, a triaryl phosphine, a cyclooctene or an oxanobornadiene;
or, [0139] when X is a a triaryl phosphine, then R.sub.1 is an
azide; or, [0140] when X is a an oxanobornadiene, then R.sub.1 is
an azide; or, [0141] when X is a an alkyne, then R.sub.1 is an
azide; or, [0142] when X is a an alkene, then R.sub.1 is an azide;
or, [0143] when X is a a cyclooctene, then R.sub.1 is a diaryl
tetrazine; or, [0144] when X is a a diaryl tetrazine, then R.sub.1
is a cyclooctene; or, [0145] when X is a a monoaryl tetrazine, then
R.sub.1 is a norbornene; or, [0146] when X is a a norbornene, then
R.sub.1 is a monoaryl tetrazine; or, [0147] when X is a an
aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0148] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0149] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0150] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0151] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0152] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0153] when X is a a maleimide, then R.sub.1 is a thiol;
[0154] thereby the terminal group is attached to the peptide tag by
a linker having the structure according to Formula (II-d):
[0154] ##STR00037## [0155] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, A.sub.2, X.sub.2, L.sub.3, L.sub.4, R.sub.2 and TG
are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0156] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0157] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) [0158]
i) by incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (F),
[0158] ##STR00038## [0159] thereby attaching an activated
phosphopentathienyl group of Formula (F-a) to the peptide tag,
[0159] ##STR00039## [0160] wherein R.sub.1 is a functional group;
[0161] and [0162] ii) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-e),
[0162] X-L.sub.4-TG Formula (II-e) [0163] wherein X is a group
which reacts with functional group R.sub.1, [0164] wherein: [0165]
when X is a thiol, then R.sub.1 is a thiol, a maleimide or a
haloacetamide; or, [0166] when X is a an azide, then R.sub.1 is an
alkyne, a triaryl phosphine, a cyclooctene or an oxanobornadiene;
or, [0167] when X is a a triaryl phosphine, then R.sub.1 is an
azide; or, [0168] when X is a an oxanobornadiene, then R.sub.1 is
an azide; or, [0169] when X is a an alkyne, then R.sub.1 is an
azide; or, [0170] when X is a an alkene, then R.sub.1 is an azide;
or, [0171] when X is a a cyclooctene, then R.sub.1 is a diaryl
tetrazine; or, [0172] when X is a a diaryl tetrazine, then R.sub.1
is a cyclooctene; or, [0173] when X is a a monoaryl tetrazine, then
R.sub.1 is a norbornene; or, [0174] when X is a a norbornene, then
R.sub.1 is a monoaryl tetrazine; or, [0175] when X is a an
aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0176] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0177] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0178] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0179] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0180] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0181] when X is a a maleimide, then R.sub.1 is a thiol;
[0182] thereby the terminal group is attached to the peptide tag by
a linker having the structure according to Formula (III-f):
[0182] ##STR00040## [0183] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, L.sub.2, A.sub.3, X.sub.2, L.sub.4, R.sub.2 and TG
are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0184] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0185] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) [0186]
i) by incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (G),
[0186] ##STR00041## [0187] thereby attaching an activated
phosphopentathienyl group of Formula (G-a) to the peptide tag,
[0187] ##STR00042## [0188] wherein R.sub.1 is a functional group;
[0189] and [0190] ii) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-g),
[0190] X-TG Formula (II-g) [0191] wherein X is a group which reacts
with functional group R.sub.1, wherein: [0192] when X is a thiol,
then R.sub.1 is a thiol, a maleimide or a haloacetamide; or, [0193]
when X is a an azide, then R.sub.1 is an alkyne, a triaryl
phosphine, a cyclooctene or an oxanobornadiene; or, [0194] when X
is a a triaryl phosphine, then R.sub.1 is an azide; or, [0195] when
X is a an oxanobornadiene, then R.sub.1 is an azide; or, [0196]
when X is a an alkyne, then R.sub.1 is an azide; or, [0197] when X
is a an alkene, then R.sub.1 is an azide; or, [0198] when X is a a
cyclooctene, then R.sub.1 is a diaryl tetrazine; or, [0199] when X
is a a diaryl tetrazine, then R.sub.1 is a cyclooctene; or, [0200]
when X is a a monoaryl tetrazine, then R.sub.1 is a norbornene; or,
[0201] when X is a a norbornene, then R.sub.1 is a monoaryl
tetrazine; or, [0202] when X is a an aldehyde, then R.sub.1 is a
hydroxylamine or a hydrazine or NH.sub.2--NH--C(.dbd.O)--; or,
[0203] when X is a a ketone, then R.sub.1 is a hydroxylamine or a
hydrazine or NH.sub.2--NH--C(.dbd.O)--; or, [0204] when X is a a
hydroxylamine, then R.sub.1 is an aldehyde or a ketone; or, [0205]
when X is a a hydrazine, then R.sub.1 is an aldehyde or a ketone;
or, [0206] when X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is
an aldehyde or a ketone; or, [0207] when X is a a haloacetamide,
then R.sub.1 is a thiol; or, [0208] when X is a a maleimide, then
R.sub.1 is a thiol; [0209] thereby the terminal group is attached
to the peptide tag by a linker having the structure according to
Formula (III-h):
[0209] ##STR00043## [0210] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, L.sub.2, L.sub.3, A.sub.4, X.sub.2, R.sub.2 and TG
are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0211] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0212] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) by
incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (H),
[0212] ##STR00044## [0213] thereby attaching a protected
phosphopentathienyl group of Formula (H-a) to the peptide tag,
[0213] ##STR00045## [0214] wherein R.sub.1-PG is a protected
functional group R.sub.1; [0215] (c) deprotecting the protected
phosphopentathienyl group to give an activated phosphopentathienyl
group of Formula (D-a) attached to the peptide tag,
[0215] ##STR00046## [0216] wherein R.sub.1 is a functional group;
[0217] and [0218] (d) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(III-a),
[0218] X-L.sub.2-L.sub.3-L.sub.4-TG Formula (II-a) [0219] wherein X
is a group which reacts with functional group R.sub.1, wherein:
[0220] when X is a thiol, then R.sub.1 is a thiol, a maleimide or a
haloacetamide; or, [0221] when X is a an azide, then R.sub.1 is an
alkyne, a triaryl phosphine, a cyclooctene or an oxanobornadiene;
or, [0222] when X is a a triaryl phosphine, then R.sub.1 is an
azide; or, [0223] when X is a an oxanobornadiene, then R.sub.1 is
an azide; or, [0224] when X is a an alkyne, then R.sub.1 is an
azide; or, [0225] when X is a an alkene, then R.sub.1 is an azide;
or, [0226] when X is a a cyclooctene, then R.sub.1 is a diaryl
tetrazine; or, [0227] when X is a a diaryl tetrazine, then R.sub.1
is a cyclooctene; or, [0228] when X is a a monoaryl tetrazine, then
R.sub.1 is a norbornene; or, [0229] when X is a a norbornene, then
R.sub.1 is a monoaryl tetrazine; or, [0230] when X is a an
aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0231] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0232] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0233] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0234] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0235] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0236] when X is a a maleimide, then R.sub.1 is a thiol;
thereby the terminal group is attached to the peptide tag by a
linker having the structure according to Formula (II-b):
[0236] ##STR00047## [0237] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein A.sub.1, X.sub.2, L.sub.2, L.sub.3, L.sub.4, R.sub.2, PG
and TG are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0238] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0239] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) by
incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (J),
[0239] ##STR00048## [0240] thereby attaching a protected
phosphopentathienyl group of Formula (J-a) to the peptide tag,
[0240] ##STR00049## [0241] wherein R.sub.1--PG is a protected
functional group R.sub.1; [0242] (c) deprotecting the protected
phosphopentathienyl group to give an activated phosphopentathienyl
group of Formula (E-a) attached to the peptide tag,
[0242] ##STR00050## [0243] wherein R.sub.1 is a functional group;
[0244] and [0245] (d) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(III-c),
[0245] X-L.sub.3-L.sub.4-TG Formula (II-c) [0246] wherein X is a
group which reacts with functional group R.sub.1, wherein: [0247]
when X is a thiol, then R.sub.1 is a thiol, a maleimide or a
haloacetamide; or, [0248] when X is a an azide, then R.sub.1 is an
alkyne, a triaryl phosphine, a cyclooctene or an oxanobornadiene;
or, [0249] when X is a a triaryl phosphine, then R.sub.1 is an
azide; or, [0250] when X is a an oxanobornadiene, then R.sub.1 is
an azide; or, [0251] when X is a an alkyne, then R.sub.1 is an
azide; or, [0252] when X is a an alkene, then R.sub.1 is an azide;
or, [0253] when X is a a cyclooctene, then R.sub.1 is a diaryl
tetrazine; or, [0254] when X is a a diaryl tetrazine, then R.sub.1
is a cyclooctene; or, [0255] when X is a a monoaryl tetrazine, then
R.sub.1 is a norbornene; or, [0256] when X is a a norbornene, then
R.sub.1 is a monoaryl tetrazine; or, [0257] when X is a an
aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0258] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0259] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0260] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0261] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0262] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0263] when X is a a maleimide, then R.sub.1 is a thiol;
[0264] thereby the terminal group is attached to the peptide tag by
a linker having the structure according to Formula (II-d):
[0264] ##STR00051## [0265] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, A.sub.2, X.sub.2, L.sub.3, L.sub.4, R.sub.2, PG
and TG are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0266] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0267] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) by
incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (K),
[0267] ##STR00052## [0268] thereby attaching a protected
phosphopentathienyl group of Formula (J-a) to the peptide tag,
[0268] ##STR00053## [0269] wherein R.sub.1--PG is a protected
functional group R.sub.1; [0270] (c) deprotecting the protected
phosphopentathienyl group to give an activated phosphopentathienyl
group of Formula (F-a) attached to the peptide tag,
[0270] ##STR00054## [0271] wherein R.sub.1 is a functional group;
[0272] and [0273] (d) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-e),
[0273] X-L.sub.4-TG Formula (II-e) [0274] wherein X is a group
which reacts with functional group R.sub.1, wherein: [0275] when X
is a thiol, then R.sub.1 is a thiol, a maleimide or a
haloacetamide; or, [0276] when X is a an azide, then R.sub.1 is an
alkyne, a triaryl phosphine, a cyclooctene or an oxanobornadiene;
or, [0277] when X is a a triaryl phosphine, then R.sub.1 is an
azide; or, [0278] when X is a an oxanobornadiene, then R.sub.1 is
an azide; or, [0279] when X is a an alkyne, then R.sub.1 is an
azide; or, [0280] when X is a an alkene, then R.sub.1 is an azide;
or, [0281] when X is a a cyclooctene, then R.sub.1 is a diaryl
tetrazine; or, [0282] when X is a a diaryl tetrazine, then R.sub.1
is a cyclooctene; or, [0283] when X is a a monoaryl tetrazine, then
R.sub.1 is a norbornene; or, [0284] when X is a a norbornene, then
R.sub.1 is a monoaryl tetrazine; or, [0285] when X is a an
aldehyde, then R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0286] when X is a a ketone, then
R.sub.1 is a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)--; or, [0287] when X is a a hydroxylamine,
then R.sub.1 is an aldehyde or a ketone; or, [0288] when X is a a
hydrazine, then R.sub.1 is an aldehyde or a ketone; or, [0289] when
X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is an aldehyde or a
ketone; or, [0290] when X is a a haloacetamide, then R.sub.1 is a
thiol; or, [0291] when X is a a maleimide, then R.sub.1 is a thiol;
[0292] thereby the terminal group is attached to the peptide tag by
a linker having the structure according to Formula (III-f):
[0292] ##STR00055## [0293] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, L.sub.2, A.sub.3, X.sub.2, L.sub.4, R.sub.2, PG
and TG are as defined herein. Another aspect provided herein is the
preparation of an immunoconjugate by a process comprising the steps
of: [0294] (a) providing a modified antibody or antigen binding
fragment thereof, wherein the modified antibody or antigen binding
fragment thereof comprises a peptide tag, and wherein the peptide
tag is a substrate of an enzyme having phosphopantetheinyl
transferase activity; [0295] (b) labeling the modified antibody or
antigen binding fragment thereof with a terminal group (TG) by
incubating the modified antibody or antigen binding fragment
thereof with an enzyme having phosphopantetheinyl transferase
activity in the presence of a compound of Formula (L),
[0295] ##STR00056## [0296] thereby attaching a protected
phosphopentathienyl group of Formula (L-a) to the peptide tag,
[0296] ##STR00057## [0297] wherein R.sub.1--PG is a protected
functional group R.sub.1; [0298] (c) deprotecting the protected
phosphopentathienyl group to give an activated phosphopentathienyl
group of Formula (G-a) attached to the peptide tag,
[0298] ##STR00058## [0299] wherein R.sub.1 is a functional group;
[0300] and [0301] (d) reacting the functional group R.sub.1 of the
activated phosphopentathienyl group with a compound of Formula
(II-g),
[0301] X-TG Formula (II-g) [0302] wherein X is a group which reacts
with functional group R.sub.1, wherein: [0303] when X is a thiol,
then R.sub.1 is a thiol, a maleimide or a haloacetamide; or, [0304]
when X is a an azide, then R.sub.1 is an alkyne, a triaryl
phosphine, a cyclooctene or an oxanobornadiene; or, [0305] when X
is a a triaryl phosphine, then R.sub.1 is an azide; or, [0306] when
X is a an oxanobornadiene, then R.sub.1 is an azide; or, [0307]
when X is a an alkyne, then R.sub.1 is an azide; or, [0308] when X
is a an alkene, then R.sub.1 is an azide; or, [0309] when X is a a
cyclooctene, then R.sub.1 is a diaryl tetrazine; or, [0310] when X
is a a diaryl tetrazine, then R.sub.1 is a cyclooctene; or, [0311]
when X is a a monoaryl tetrazine, then R.sub.1 is a norbornene; or,
[0312] when X is a a norbornene, then R.sub.1 is a monoaryl
tetrazine; or, [0313] when X is a an aldehyde, then R.sub.1 is a
hydroxylamine or a hydrazine or NH.sub.2--NH--C(.dbd.O)--; or,
[0314] when X is a a ketone, then R.sub.1 is a hydroxylamine or a
hydrazine or NH.sub.2--NH--C(.dbd.O)--; or, [0315] when X is a a
hydroxylamine, then R.sub.1 is an aldehyde or a ketone; or, [0316]
when X is a a hydrazine, then R.sub.1 is an aldehyde or a ketone;
or, [0317] when X is a NH.sub.2--NH--C(.dbd.O)--, then R.sub.1 is
an aldehyde or a ketone; or, [0318] when X is a a haloacetamide,
then R.sub.1 is a thiol; or, [0319] when X is a a maleimide, then
R.sub.1 is a thiol; [0320] thereby the terminal group is attached
to the peptide tag by a linker having the structure according to
Formula (III-h):
[0320] ##STR00059## [0321] where the * denotes the
phosphopantetheinyl moiety is attached to the peptide tag, and
wherein L.sub.1, L.sub.2, L.sub.3, A.sub.4, X.sub.2, R.sub.2, PG
and TG are as defined herein
[0322] In certain embodiments of the above methods of preparation
[0323] A.sub.1 is --C(.dbd.O)NH--, --NHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0324] L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2--;
[0325] L.sub.3 is a bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0326]
L.sub.4 is a bond, -A.sub.4-, -A.sub.4X.sup.2--,
[0326] ##STR00060## ##STR00061## [0327] A.sub.2 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NR.sup.4--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--, --(CH.sub.2).sub.nNH--,
--(C(R.sup.4).sub.2).sub.nNH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(C-
(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--;
[0327] ##STR00062## [0328] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(.dbd.O)--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--;
[0328] ##STR00063## [0329] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0330] each X.sup.2 is independently selected from a bond,
##STR00064## ##STR00065## ##STR00066##
[0330] --S--, --Si(OH).sub.2O--,
##STR00067##
[0331] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0332] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0333] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0334] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0335] R.sup.7 is
independently selected from H, phenyl and pyridine; [0336] R.sup.8
is independently selected from
[0336] ##STR00068## [0337] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0338] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0339] each m is independently
selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0340] TG is
selected from a drug moiety, an affinity probe, a chelator, a
spectroscopic probe, a radioactive probe, a lipid molecule, a
polyethylene glycol, a polymer, a nanoparticle, a quantum dot, a
liposome, a PLGA particle, and a polysaccharide.
[0341] In other embodiments of the above methods of preparation
[0342] A.sub.1 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0343]
L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0344] L.sub.3
is a bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0345] L.sub.4 is a
bond, -A.sub.4-, or -A.sub.4X.sup.2--;
[0345] ##STR00069## [0346] A.sub.1 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0347]
A.sub.2 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m,
((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0347] ##STR00070## [0348] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0348] ##STR00071## [0349] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0350]
each X.sup.2 is independently selected from a bond,
##STR00072## ##STR00073## ##STR00074##
[0350] --S--, --Si(OH).sub.2O--,
##STR00075##
[0351] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0352] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0353] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0354] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0355] R.sup.7 is
independently selected from H, phenyl and pyridine; [0356] R.sup.8
is independently selected from
[0356] ##STR00076## [0357] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0358] each n is independently selected
from 1, 3, 4, 5, 6, 7, 8 and 9, and [0359] each m is independently
selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. In other embodiments of
the above methods of preparation [0360] L.sub.1 is a bond,
-A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; [0361] L.sub.3 is a
bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0362] L.sub.4 is a bond,
-A.sub.4-, or -A.sub.4X.sup.2--;
[0362] ##STR00077## [0363] A.sub.1 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0364]
A.sub.2 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; or
[0364] ##STR00078## [0365] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0365] ##STR00079## [0366] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0367]
each X.sup.2 is independently selected from a bond,
##STR00080## ##STR00081## ##STR00082##
[0367] --S--, --Si(OH).sub.2O--,
##STR00083##
[0368] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0369] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0370] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0371] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0372] R.sup.7 is
independently selected from H, phenyl and pyridine; [0373] R.sup.8
is independently selected from
[0373] ##STR00084## [0374] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0375] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0376] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
[0377] Another aspect provided herein are conjugated antibodies or
antibody fragment thereof, comprising the modified antibody or
antibody fragment provided herein, wherein a serine residue of the
peptide tag in the modified antibody or antibody fragment thereof
is conjugated to a 4'-phosphopantetheine group having the structure
of Formula (D-a), Formula (E-a), Formula (F-a) or Formula
(G-a):
##STR00085##
wherein: [0378] L.sub.1 is -A.sub.1X.sup.2-- or --X.sup.2--; [0379]
L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0380] L.sub.3
is a bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0381] L.sub.4 is a
bond, -A.sub.4-, -A.sub.4X.sup.2--,
[0381] ##STR00086## ##STR00087## [0382] A.sub.1 is --C(.dbd.O)NH--,
--NHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0383] A.sub.2 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NR.sup.4--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--, --(CH.sub.2).sub.nNH--,
--(C(R.sup.4).sub.2).sub.nNH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0383] ##STR00088## [0384] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(.dbd.O)--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0384] ##STR00089## [0385] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0386] each X.sup.2 is independently selected from a bond,
##STR00090## ##STR00091## ##STR00092##
[0386] --S--, --Si(OH).sub.2O--,
##STR00093##
[0387] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0388] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0389] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0390] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0391] R.sup.7 is
independently selected from H, phenyl and pyridine; [0392] R.sup.9
is independently selected from
[0392] ##STR00094## [0393] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0394] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, [0395] each m is independently
selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0396] R.sup.1 is a
thiol, a maleimide, a haloacetamide, an alkyne, a triaryl
phosphine, a cyclooctene, an oxanobornadiene, an azide, a diaryl
tetrazine, a norbornene, a monoaryl tetrazine, a hydroxylamine, a
hydrazine, NH.sub.2--NH--C(.dbd.O)--, an aldehyde or a ketone.
[0397] In certain embodiments of such conjugated antibodies or
antibody fragments thereof, the 4'-phosphopantetheine group is
##STR00095##
[0398] In certain embodiments of such conjugated antibodies or
antibody fragments thereof, the conjugated serine has a structure
selected from:
##STR00096##
[0399] In other embodiments of such conjugated antibodies or
antibody fragments thereof, the conjugated serine is
##STR00097##
[0400] Another aspect provided herein are conjugated antibodies or
antibody fragment thereof, comprising a modified antibody or
antibody fragment thereof provided herein, wherein a serine residue
of the peptide tag is conjugated to a modified
4'-phosphopantetheine group and the conjugated serine has a
structure selected from:
##STR00098##
wherein [0401] L.sub.1 is -A.sub.1X.sup.2-- or --X.sup.2--; [0402]
L.sub.2 is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0403] L.sub.3
is a bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0404] L.sub.4 is a
bond, -A.sub.4-, -A.sub.4X.sup.2--,
[0404] ##STR00099## ##STR00100## [0405] A.sub.1 is --C(.dbd.O)NH--,
--NHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
(O(CH.sub.2).sub.n).sub.m--, --(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0406] A.sub.2 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NR.sup.4--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--, --(CH.sub.2).sub.nNH--,
--(C(R.sup.4).sub.2).sub.nNH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n,
[0406] ##STR00101## [0407] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--C.sub.2C(.dbd.O)NH--, --(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(.dbd.O)--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0407] ##STR00102## [0408] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0409] each X.sup.2 is independently selected from a bond,
##STR00103## ##STR00104## ##STR00105##
[0409] --S--, --Si(OH).sub.2O--,
##STR00106##
[0410] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0411] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0412] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0413] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0414] R.sup.7 is
independently selected from H, phenyl and pyridine; [0415] R.sup.8
is independently selected from
[0415] ##STR00107## [0416] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0417] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, [0418] each m is independently
selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0419] TG is a drug
moiety, an affinity probe, a chelator, a spectroscopic probe, a
radioactive probe, an imaging reagent, a lipid molecule, a
polyethylene glycol, a polymer, a nanoparticle, a quantum dot, a
liposome, a PLGA particle, a polysaccharide, an acetyl group, or a
surface.
[0420] In certain embodiments of such conjugated antibodies or
antibody fragments thereof, the conjugated serine is
##STR00108##
of such conjugated antibodies or antibody fragments thereof,
X.sub.2 is
##STR00109##
or --(CH.sub.2).sub.nC(.dbd.O)NH--.
[0421] The present invention also provides pharmaceutical
compositions comprising an effective amount of the immunoconjugate
of the invention, or a pharmaceutically acceptable salt thereof,
and a pharmaceutically acceptable diluent, carrier or
excipient.
[0422] The present invention provides a method of treating a
disease, such as cancer, comprising administering to a mammal in
need thereof an effective amount of an immunoconjugate of the
invention. In some embodiments, the present invention provides
immunoconjugates for use as a medicament. In some embodiments, the
present invention provides use of an immunoconjugate in the
manufacture of a medicament for treatment of cancer, autoimmune
diseases, inflammatory diseases, infectious diseases (e.g.,
bacterial, fungus, virus), genetic disorders, cardiovascular
diseases, and/or metabolic diseases.
[0423] The present invention provides methods of producing the
immunoconjugates described herein. In one embodiment, the method
comprises incubating the modified antibody or antibody fragment of
invention, a 4'-phosphopantetheinyl transferase, and a terminal
group linked to CoA under suitable conditions to promote formation
of an immunoconjugate comprising the antibody or antibody fragment
and the terminal group linked together by 4'-phosphopantetheine. In
a specific embodiment, the suitable condition comprises a
temperature between 4.degree. C. to 37.degree. C. and pH 6.5 to pH
9.0.
Definitions
[0424] The terms "alkenyl" or "alkene", as used herein, refer to a
branched or straight chain hydrocarbon having at least one
carbon-carbon double bond. Atoms oriented about the double bond are
in either the cis (Z) or trans (E) conformation. As used herein,
the terms "C.sub.2-C.sub.4alkenyl", "C.sub.2-C.sub.5alkenyl",
"C.sub.2-C.sub.6alkenyl", "C.sub.2-C.sub.7alkenyl",
"C.sub.2-C.sub.8alkenyl", "C.sub.2-C.sub.4alkene",
"C.sub.2-C.sub.5alkene", "C.sub.2-C.sub.6alkene",
"C.sub.2-C.sub.7alkene", and "C.sub.2-C.sub.8alkene" refer to a
branched or straight chain hydrocarbon having at least one
carbon-carbon double bond and containing at least 2, and at most 4,
5, 6, 7 or 8 carbon atoms, respectively. Non-limiting examples of
alkenyl groups, as used herein, include ethenyl, ethane, epropenyl,
propene, allyl (2-propenyl), 2-propene, butenyl, pentenyl, pentene,
hexenyl, heptenyl, heptene, octenyl, nonenyl, nonene, decenyl,
decene and the like. If not otherwise specified, an alkenyl group
generally is a C.sub.2-C.sub.6 alkenyl.
[0425] The terms "alkynyl" or "alkyne", as used herein, refer to a
branched or straight chain hydrocarbon radical having at least one
carbon-carbon triple bond. As used herein, the terms
"C.sub.2-C.sub.4alkynyl", "C.sub.2-C.sub.5alkynyl",
"C.sub.2-C.sub.6alkynyl", "C.sub.2-C.sub.7alkynyl", and
"C.sub.2-C.sub.8alkynyl" refer to a branched or straight chain
hydrocarbon radical having at least one carbon-carbon triple bond
and containing at least 2, and at most 4, 5, 6, 7 or 8 carbon
atoms, respectively. Non-limiting examples of alkynyl groups, as
used herein, include ethynyl, propynyl, butynyl, pentynyl, hexynyl,
heptynyl, octynyl, nonynyl, decynyl and the like. If not otherwise
specified, an alkynyl group generally is a C.sub.2-C.sub.6
alkynyl.
[0426] The term "alkyl," as used herein, refers to a saturated
branched or straight chain hydrocarbon. As used herein, the terms
"C.sub.1-C.sub.3alkyl", "C.sub.1-C.sub.4alkyl",
"C.sub.1-C.sub.5alkyl", "C.sub.1-C.sub.6alkyl",
"C.sub.1-C.sub.7alkyl" or "C.sub.1-C.sub.8alkyl" refer to saturated
branched or straight chain hydrocarbon containing at least 1, and
at most 3, 4, 5, 6, 7 or 8 carbon atoms, respectively. Non-limiting
examples of alkyl groups as used herein include methyl, ethyl,
n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl,
n-pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl and the
like. If not otherwise specified, an alkyl group generally is a
C.sub.1-C.sub.6 alkyl.
[0427] The term "alkoxy," as used herein, refers to the group
--OR.sub.a, where R.sub.a is an alkyl group as defined herein. As
used herein, the terms "C.sub.1-C.sub.3alkoxy",
"C.sub.1-C.sub.4alkoxy", "C.sub.1-C.sub.5alkoxy",
"C-C.sub.6alkoxy", "C.sub.1-C.sub.7alkoxy" and
"C.sub.1-C.sub.8alkoxy" refer to an alkoxy group wherein the alkyl
moiety contains at least 1, and at most 3, 4, 5, 6, 7 or 8, carbon
atoms. Non-limiting examples of alkoxy groups, as used herein,
include methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy,
t-butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy,
decyloxy and the like.
[0428] The term "aryl", as used herein, refers to monocyclic,
bicyclic, and tricyclic ring systems having a total of six to
fourteen ring members, wherein at least one ring in the system is
aromatic. An aryl group also includes one or more aromatic rings
fused to one or more non-aromatic hydrocarbon rings. Non-limiting
examples of aryl groups, as used herein, include phenyl (Ph),
naphthyl, fluorenyl, indenyl, azulenyl, anthracenyl and the like.
An aryl group may contain one or more substituents and thus may be
"optionally substituted". Unless otherwise specified, aryl groups
can have up to four substituents.
[0429] The term "cycloalkyl", as used herein, refers to a saturated
monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic
ring assembly. As used herein, the terms
"C.sub.3-C.sub.5cycloalkyl", "C.sub.3-C.sub.6cycloalkyl",
"C.sub.3-C.sub.7cycloalkyl", "C.sub.3-C.sub.8cycloalkyl,
"C.sub.3-C.sub.9cycloalkyl and "C.sub.3-C.sub.10cycloalkyl refer to
a saturated monocyclic, fused bicyclic, fused tricyclic or bridged
polycyclic ring assembly which contains at least 3, and at most 5,
6, 7, 8, 9 or 10, carbon atoms. Non-limiting examples of cycloalkyl
groups, as used herein, include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl,
cyclodecyl, decahydronaphthalenyl and the like. If not otherwise
specified, a cycloalkyl group generally is a C.sub.3-C.sub.8
cycloalkyl.
[0430] The terms "cycloalkenyl" or "cycloalkene", as used herein,
refers to a monocyclic, fused bicyclic, fused tricyclic or bridged
polycyclic ring assembly having at least one carbon-carbon double
bond. Atoms oriented about the double bond are in either the cis
(Z) or trans (E) conformation. A monocyclic cycloalkene can be
fused to one or two aryl rings. Non-limiting examples of
cycloalkenyl groups, as used herein, include cyclopropenyl,
cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
cyclooctenyl, cyclononenyl, cyclodecenyl, and the like. If not
otherwise specified, a cycloalkenyl group generally is a
C.sub.5-C.sub.8 cycloalkenyl.
[0431] The terms "cycloalkynyl" or "cycloalkyne", as used herein,
refers to a monocyclic, fused bicyclic, fused tricyclic or bridged
polycyclic ring assembly having at least one carbon-carbon triple
bond. A monocyclic cycloalkyne can be fused to one or two aryl
rings. Non-limiting examples of cycloalkynyl groups, as used
herein, include cyclopropynyl, cyclobutynyl, cyclopentynyl,
cyclohexynyl, cycloheptynyl, cyclooctynyl, cyclononynyl,
cyclodecynyl, and the like. If not otherwise specified, a
cycloalkynyl group generally is a C.sub.6-C.sub.8 cycloalkynyl.
[0432] The term "heteroaryl," as used herein, refers to a 5-6
membered heteroaromatic monocyclic ring having 1 to 4 heteroatoms
independently selected from nitrogen, oxygen and sulfur, an 8-10
membered fused bicyclic ring having 1 to 4 heteroatoms
independently selected from nitrogen, oxygen and sulfur as ring
members and where at least one of the rings is aromatic, or a 12-14
membered fused tricyclic ring having 1 to 4 heteroatoms
independently selected from nitrogen, oxygen and sulfur and where
at least one of the rings is aromatic. Such fused bicyclic and
tricyclic ring systems may be fused to one or more aryl,
cycloalkyl, or heterocycloalkyl rings. Non-limiting examples of
heteroaryl groups, as used herein, include 2- or 3-furyl; 1-, 2-,
4-, or 5-imidazolyl; 3-, 4-, or 5-isothiazolyl; 3-, 4-, or
5-isoxazolyl; 2-, 4-, or 5-oxazolyl; 4- or 5-1,2,3-oxadiazolyl; 2-
or 3-pyrazinyl; 1-, 3-, 4-, or 5-pyrazolyl; 3-, 4-, 5- or
6-pyridazinyl; 2-, 3-, or 4-pyridyl; 2-, 4-, 5- or 6-pyrimidinyl;
1-, 2- or 3-pyrrolyl; 1- or 5-tetrazolyl; 2- or
5-1,3,4-thiadiazolyl; 2-, 4-, or 5-thiazolyl; 2- or 3-thienyl; 2-,
4- or 6-1,3,5-triazinyl; 1-, 3- or 5-1,2,4-triazolyl; 1-, 4- or
5-1,2,3-triazolyl; 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl;
1-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-benzo[g]isoquinoline; 2-, 4-,
5-, 6-, or 7-benzoxazolyl; 1-, 2-, 4-, 5-, 6-, or 7-benzimidazolyl;
2-, 4-, 5-, 6-, or 7-benzothiazolyl; 2-, 3-, 4-, 5-, 6-,
7-benzo[b]thienyl; 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-benzo[b]oxepine;
2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl; 1-, 2-, 3-, 4-, 5-, 6-, 7-,
8, or 9-carbazolyl; 3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl; 2-, 4-, or
5-4H-imidazo[4,5-d] thiazolyl; 2-, 3-, 5-, or 6-imidazo[2,1-b]
thiazolyl; 2-, 3-, 6-, or 7-imidazo[1,2-b][1,2,4]triazinyl; 1-, 3-,
4-, 5-, 6-, or 7-indazolyl; 1-, 2-, 3-, 5-, 6-, 7-, or
8-indolizinyl; 1-, 2-, 3-, 4-, 5-, 6-, or 7-indolyl; 1-, 2-, 3-,
4-, 5-, 6- or 7-isoindolyl; 1-, 3-, 4-, 5-, 6-, 7-, or
8-isoquinoliyl; 2-, 3-, 4-, 5-, 6-, or 7-naphthyridinyl; 1-, 2-,
4-, 5-, 6-, 7-, 8-, or 9-perimidinyl; 1-, 2-, 3-, 4-, 6-, 7-, 8-,
9-, or 10-phenanthridinyl; 1-, 2-, 3-, 4- , 5-, 6-, 7-, 8-, 9-, or
10-phenathrolinyl; 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-phenazinyl; 1-,
2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenothiazinyl; 1-, 2-, 3-, 4-,
6-, 7-, 8-, 9-, or 10-phenoxazinyl; 1-, 4-, 5-, 6-, 7-, or
8-phthalazinyl; 2-, 4-, 6-, or 7-pteridinyl; 2-, 6-, 7-, or
8-purinyl; 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or
11-7H-pyrazino[2,3-c]carbazolyl; 2-, 3-, 5-, 6-, or
7-furo[3,2-b]-pyranyl; 1-, 3-, or 5-1H-pyrazolo[4,3-d]-oxazolyl;
2-, 3-, 5-, or 8-pyrazino[2,3-d]pyridazinyl; 1-, 2-, 3-, 4-, 5-, or
8-5H-pyrido[2,3-d]-o-oxazinyl; 1-, 2-, 3-, 4-, 6-, 7-, 8-, or
9-quinolizinyl; 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl; 2-, 3-,
4-, 5-, 6-, 7-, or 8-quinazolinyl; 2-, 3-, 4-, or
5-thieno[2,3-b]furanyl, and 1-, 3-, 6-, 7-, 8-, or
9-furo[3,4-c]cinnolinyl.
[0433] The term "heteroatoms," as used herein, refers to nitrogen
(N), oxygen (O) or sulfur (S) atoms.
[0434] The term "heterocycloalkyl," as used herein refers to a to
saturated 3-8 membered monocyclic hydrocarbon ring structure, a
saturated 6-9 membered fused bicyclic hydrocarbon ring structure,
or a saturated 10-14 membered fused tricyclic hydrocarbon ring
structure, wherein one to four of the ring carbons of the
hydrocarbon ring structure are replaced by one to four groups
independently selected from --O--, --NR--, and --S--, wherein R is
hydrogen, C.sub.1-C.sub.4alkyl or an amino protecting group.
Non-limiting examples of heterocycloalkyl groups, as used herein,
include aziridinyl, aziridin-1-yl, aziridin-2-yl, aziridin-3-yl,
oxiranyl, oxiran-2-yl, oxiran-3-yl, thiiranyl, thiiran-2-yl,
thiiran-3-yl, azetadinyl, azetadin-1-yl, azetadin-2-yl,
azetadin-3-yl, oxetanyl, oxetan-2-yl, oxetan-3-yl, oxetan-4-yl,
thietanyl, thietan-2-yl, thietan-3-yl, thietan-4-yl, pyrrolidinyl,
pyrrolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, pyrrolidin-4-yl,
pyrrolidin-5-yl, tetrahydrofuranyl, tetrahydrofuran-2-yl,
tetrahydrofuran-3-yl, tetrahydrofuran-4-yl, tetrahydrofuran-5-yl,
tetrahydrothienyl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,
tetrahydrothien-4-yl, tetrahydrothien-5-yl, piperidinyl,
piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl,
piperidin-5-yl, piperidin-6-yl, tetrahydropyranyl,
tetrahydropyran-2-yl, tetrahydropyran-3-yl, tetrahydropyran-4-yl,
tetrahydropyran-5-yl, tetrahydropyran-6-yl, tetrahydrothiopyranyl,
tetrahydrothiopyran-2-yl, tetrahydrothiopyran-3-yl,
tetrahydrothiopyran-4-yl, tetrahydrothiopyran-5-yl,
tetrahydrothiopyran-6-yl, piperazinyl, piperazin-1-yl,
piperazin-2-yl, piperazin-3-yl, piperazin-4-yl, piperazin-5-yl,
piperazin-6-yl, morpholinyl, morpholin-2-yl, morpholin-3-yl,
morpholin-4-yl, morpholin-5-yl, morpholin-6-yl, thiomorpholinyl,
thiomorpholin-2-yl, thiomorpholin-3-yl, thiomorpholin-4-yl,
thiomorpholin-5-yl, thiomorpholin-6-yl, oxathianyl, oxathian-2-yl,
oxathian-3-yl, oxathian-5-yl, oxathian-6-yl, dithianyl,
dithian-2-yl, dithian-3-yl, dithian-5-yl, dithian-6-yl, azepanyl,
azepan-1-yl, azepan-2-yl, azepan-3-yl, azepan-4-yl, azepan-5-yl,
azepan-6-yl, azepan-7-yl, oxepanyl, oxepan-2-yl, oxepan-3-yl,
oxepan-4-yl, oxepan-5-yl, oxepan-6-yl, oxepan-7-yl, thiepanyl,
thiepan-2-yl, thiepan-3-yl, thiepan-4-yl, thiepan-5-yl,
thiepan-6-yl, thiepan-7-yl, dioxolanyl, dioxolan-2-yl,
dioxolan-4-yl, dioxolan-5-yl, thioxanyl, thioxan-2-yl,
thioxan-3-yl, thioxan-4-yl, thioxan-5-yl, dithiolanyl,
dithiolan-2-yl, dithiolan-4-yl, dithiolan-5-yl, pyrrolinyl,
pyrrolin-1-yl, pyrrolin-2-yl, pyrrolin-3-yl, pyrrolin-4-yl,
pyrrolin-5-yl, imidazolinyl, imidazolin-1-yl, imidazolin-3-yl,
imidazolin-4-yl, imidazolin-5-yl, imidazolidinyl,
imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-3-yl,
imidazolidin-4-yl, imidazolidin-4-yl, pyrazolinyl, pyrazolin-1-yl,
pyrazolin-3-yl, pyrazolin-4-yl, pyrazolin-5-yl, pyrazolidinyl,
pyrazolidin-1-yl, pyrazolidin-2-yl, pyrazolidin-3-yl,
pyrazolidin-4-yl, pyrazolidin-5-yl, hexahydro-1,4-diazepinyl,
dihydrofuranyldihydropyranyl, 1,2,3,6-tetrahydropyridinyl,
2H-pyranyl, 4H-pyranyl, dihydropyranyl, dihydrothienyl,
dihydrofuranyl, 3-azabicyclo[3.1.0]hexanyl,
3-azabicyclo[4.1.0]heptanyl, pyrrolidinyl-2-one, piperidinyl-3-one
piperidinyl-2-one, piperidinyl-4-one, and 2H-pyrrolyl.
[0435] The term "optionally substituted", as used herein, means
that the referenced group may or may not be substituted with one or
more additional group(s) in place of one or more hydrogen atoms of
the unsubstituted group. The number of such groups that can be
present ranges from one up to the number of hydrogen atoms on the
unsubstituted group. The optional substituents, unless otherwise
specified, are individually and independently selected from alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl,
hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl,
isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl,
perfluoroalkyl, and amino, including mono- and di-substituted amino
groups, and the protected derivatives thereof. Non-limiting
examples of optional substituents include, halo (particularly F, Cl
and Br), --CN, --OR, --R, --NO.sub.2, --C(.dbd.O)R, --OC(.dbd.O)R,
--C(.dbd.O)OR, --OC(.dbd.O)NHR, --C(.dbd.O)N(R).sub.2, --SR--,
--S(.dbd.O)R, --S(.dbd.O).sub.2R, --NHR, --N(R).sub.2,
--NHC(.dbd.O)R, --NRC(.dbd.O)R, --NRC(S)R, NHC(.dbd.O)OR,
--NRCO.sub.2R, --NRC(.dbd.O)N(R).sub.2, --NRC(S)N(R).sub.2,
--NRNRC(.dbd.O)R, --NRNRC(.dbd.O)N(R).sub.2, --NRNRCO.sub.2R,
--C(.dbd.O)NH--, S(.dbd.O).sub.2NHR, --S(.dbd.O).sub.2N(R).sub.2,
--NHS(.dbd.O).sub.2, --NHS(.dbd.O).sub.2R,
--C(.dbd.O).sub.nC(.dbd.O)R, --C(.dbd.O)CH.sub.2C(.dbd.O)R,
--C(S)R, --C(.dbd.O)N(R).sub.2, --C(S)N(R).sub.2,
--OC(.dbd.O)N(R).sub.2, --C(.dbd.O)N(OR)R, --C(NOR)R,
--S(.dbd.O).sub.3R, --NRSO.sub.2N(R).sub.2, --NRSO.sub.2R,
--N(OR)R, --C(.dbd.NH)--N(R).sub.2, --P(.dbd.O).sub.2R,
--PO(R).sub.2, --OPO(R).sub.2, --(CH.sub.2).sub.0-2NHC(.dbd.O)R,
phenyl (Ph) optionally substituted with R, --O(Ph) optionally
substituted with R, --(CH.sub.2).sub.1-2(Ph), optionally
substituted with R, --CH.dbd.CH(Ph), optionally substituted with R,
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, aryl, heteroaryl,
cycloalkyl, heterocycloalkyl, halo-substituted
C.sub.1-C.sub.6alkyl, halo-substituted C.sub.1-C.sub.6alkoxy, where
each R is independently selected from H, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6
alkoxy, aryl, heteroaryl, C.sub.3-8 cycloalkyl, C.sub.3-8
heterocycloalkyl, halo-substituted C.sub.1-C.sub.6alkyl,
halo-substituted C.sub.1-C.sub.6alkoxy; and two R groups on the
same or on adjacent connected atoms can be taken together to form a
5-6 membered ring optionally containing an additional N, O or S as
a ring member. Suitable substituents for alkyl, cycloalkyl, and
heterocycloalkyl groups can further include .dbd.CHR, .dbd.O (oxo)
and .dbd.N--R. Preferred substituents for an aryl or heteroaryl
group are selected from F, Cl, Br, CN, --NR'.sub.2, hydroxy,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 haloalkoxy, C.sub.1-C.sub.4
alkoxy-C.sub.1-C.sub.4alkyl, --COOR', --CONR'.sub.2, --SR', and
--SO.sub.2R', where each R' is H or C.sub.1-C.sub.4 alkyl.
Preferred substituents for an alkyl, cycloalkyl or heterocycloalkyl
group are selected from oxo (.dbd.O), F, Cl, Br, CN, --NR'.sub.2,
hydroxy, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl,
C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 haloalkoxy, C.sub.1-C.sub.4
alkoxy-C.sub.1-C.sub.4alkyl, --COOR', --CONR'.sub.2, --SR', and
--SO.sub.2R', where each R' is H or C.sub.1-C.sub.4 alkyl.
[0436] The term "amino acid" refers to naturally occurring,
synthetic, and unnatural amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally occurring amino acids
are those encoded by the genetic code, as well as those amino acids
that are later modified, e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs
refer to compounds that have the same basic chemical structure as a
naturally occurring amino acid, i.e., an .alpha.-carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R
group, e.g., homoserine, norleucine, methionine sulfoxide,
methionine methyl sulfonium. Such analogs have modified R groups
(e.g., norleucine) or modified peptide backbones, but retain the
same basic chemical structure as a naturally occurring amino acid.
Amino acid mimetics refers to chemical compounds that have a
structure that is different from the general chemical structure of
an amino acid, but that functions in a manner similar to a
naturally occurring amino acid.
[0437] The term "unnatural amino acid", as used herein, is intended
to represent amino acid structures that cannot be generated
biosynthetically in any organism using unmodified or modified genes
from any organism, whether the same or different. In addition, it
is understood that such "unnatural amino acids" require a modified
tRNA and a modified tRNA synthetase (RS) for incorporation into a
protein. These "selected" orthogonal tRNA/RS pair are specific for
the unnatural amino acid and are generated by a selection process
as developed by Schultz et al. (see, e.g., Liu et al., Annu. Rev.
Biochem. 79:413-444, 2010) or a similar procedure. The term
"unnatural amino acid" does not include the natural occurring
22.sup.nd proteinogenic amino acid pyrrolysine (Pyl) as well as its
demethylated analog pyrroline-carboxy-lysine (Pcl), because
incorporation of both residues into proteins is mediated by the
unmodified, naturally occurring pyrrolysyl-tRNA/tRNA synthetase
pair (see, e.g., Ou et al., Proc. Natl. Acad. Sci. USA.
108:10437-10442, 2011).
[0438] The term "antibody" as used herein refers to a polypeptide
of the immunoglobulin family that is capable of binding a
corresponding antigen non-covalently, reversibly, and in a specific
manner. For example, a naturally occurring IgG antibody is a
tetramer comprising at least two heavy (H) chains and two light (L)
chains inter-connected by disulfide bonds. Each heavy chain is
comprised of a heavy chain variable region (abbreviated herein as
V.sub.H) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as VL) and a light chain constant region. The
light chain constant region is comprised of one domain, C.sub.L.
The V.sub.H and V.sub.L regions can be further subdivided into
regions of hypervariability, termed complementarity determining
regions (CDR), interspersed with regions that are more conserved,
termed framework regions (FR). Each V.sub.H and V.sub.L is composed
of three CDRs and four FRs arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, and FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.
[0439] The term "antibody" includes, but is not limited to,
monoclonal antibodies, human antibodies, humanized antibodies,
camelid antibodies, chimeric antibodies, and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention). The antibodies can be of any
isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).
[0440] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used functionally. In this regard, it will be
appreciated that the variable domains of both the light (V.sub.L)
and heavy (V.sub.H) chain portions determine antigen recognition
and specificity. Conversely, the constant domains of the light
chain (C.sub.L) and the heavy chain (CH1, CH2 or CH3) confer
important biological properties such as secretion, transplacental
mobility, Fc receptor binding, complement binding, and the like. By
convention, the numbering of the constant region domains increases
as they become more distal from the antigen binding site or
amino-terminus of the antibody. The N-terminus is a variable region
and at the C-terminus is a constant region; the CH3 and C.sub.L
domains actually comprise the carboxy-terminal domains of the heavy
and light chain, respectively. The term "antigen binding fragment",
as used herein, refers to one or more portions of an antibody that
retain the ability to specifically interact with (e.g., by binding,
steric hindrance, stabilizing/destabilizing, spatial distribution)
an epitope of an antigen. Examples of binding fragments include,
but are not limited to, single-chain Fvs (scFv), disulfide-linked
Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and CH1 domains; a
F(ab).sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; a Fd
fragment consisting of the V.sub.H and CH1 domains; a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody; a dAb fragment (Ward et al., Nature 341:544-546, 1989),
which consists of a V.sub.H domain; and an isolated complementarity
determining region (CDR), or other epitope-binding fragments of an
antibody.
[0441] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv ("scFv"); see, e.g., Bird et al., Science
242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci.
85:5879-5883, 1988). Such single chain antibodies are also intended
to be encompassed within the term "antigen binding fragment." These
antigen binding fragments are obtained using conventional
techniques known to those of skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0442] Antigen binding fragments can also be incorporated into
single domain antibodies, maxibodies, minibodies, nanobodies,
intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv
(see, e.g., Hollinger and Hudson, Nature Biotechnology
23:1126-1136, 2005). Antigen binding fragments can be grafted into
scaffolds based on polypeptides such as fibronectin type III (Fn3)
(see U.S. Pat. No. 6,703,199, which describes fibronectin
polypeptide monobodies). Antigen binding fragments can be
incorporated into single chain molecules comprising a pair of
tandem Fv segments (V.sub.H-CH1-V.sub.H-CH1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and
U.S. Pat. No. 5,641,870).
[0443] The term "monoclonal antibody" or "monoclonal antibody
composition" as used herein refers to polypeptides, including
antibodies and antigen binding fragments that have substantially
identical amino acid sequence or are derived from the same genetic
source. This term also includes preparations of antibody molecules
of single molecular composition. A monoclonal antibody composition
displays a single binding specificity and affinity for a particular
epitope.
[0444] The term "human antibody", as used herein, includes
antibodies having variable regions in which both the framework and
CDR regions are derived from sequences of human origin.
Furthermore, if the antibody contains a constant region, the
constant region also is derived from such human sequences, e.g.,
human germline sequences, or mutated versions of human germline
sequences or antibody containing consensus framework sequences
derived from human framework sequences analysis, for example, as
described in Knappik et al., J. Mol. Biol. 296:57-86, 2000).
[0445] The human antibodies of the invention may include amino acid
residues not encoded by human sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo, or a conservative substitution to promote
stability or manufacturing).
[0446] The term "humanized" antibody, as used herein, refers to an
antibody that retains the reactivity of a non-human antibody while
being less immunogenic in humans. This can be achieved, for
instance, by retaining the non-human CDR regions and replacing the
remaining parts of the antibody with their human counterparts. See,
e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855
(1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen
et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun.,
28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).
[0447] The term "recognize" as used herein refers to an antibody or
antigen binding fragment thereof that finds and interacts (e.g.,
binds) with its epitope, whether that epitope is linear or
conformational. The term "epitope" refers to a site on an antigen
to which an antibody or antigen binding fragment of the invention
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include techniques in the art, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance (see,
e.g., Epitope Mapping Protocols in Methods in Molecular Biology,
Vol. 66, G. E. Morris, Ed. (1996)).
[0448] The term "affinity" as used herein refers to the strength of
interaction between antibody and antigen at single antigenic sites.
Within each antigenic site, the variable region of the antibody
"arm" interacts through weak non-covalent forces with antigen at
numerous sites; the more interactions, the stronger the
affinity.
[0449] The term "isolated antibody" refers to an antibody that is
substantially free of other antibodies having different antigenic
specificities. An isolated antibody that specifically binds to one
antigen may, however, have cross-reactivity to other antigens.
Moreover, an isolated antibody may be substantially free of other
cellular material and/or chemicals.
[0450] The term "conservatively modified variant" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or essentially identical
amino acid sequences, or where the nucleic acid does not encode an
amino acid sequence, to essentially identical sequences. Because of
the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0451] For polypeptide sequences, "conservatively modified
variants" include individual substitutions, deletions or additions
to a polypeptide sequence which result in the substitution of an
amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles of the invention. The following eight groups
contain amino acids that are conservative substitutions for one
another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (VV); 7)
Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
[0452] In some embodiments, the term "conservative sequence
modifications" are used to refer to amino acid modifications that
do not significantly affect or alter the binding characteristics of
the antibody containing the amino acid sequence.
[0453] The term "optimized" as used herein refers to a nucleotide
sequence has been altered to encode an amino acid sequence using
codons that are preferred in the production cell or organism,
generally a eukaryotic cell, for example, a yeast cell, a Pichia
cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary
cell (CHO) or a human cell. The optimized nucleotide sequence is
engineered to retain completely or as much as possible the amino
acid sequence originally encoded by the starting nucleotide
sequence, which is also known as the "parental" sequence.
[0454] The terms "percent identical" or "percent identity," in the
context of two or more nucleic acids or polypeptide sequences,
refers to two or more sequences or subsequences that are the same.
Two sequences are "substantially identical" if two sequences have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99% identity over a specified region, or, when not
specified, over the entire sequence), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection.
Optionally, the identity exists over a region that is at least
about 50 nucleotides (or 10 amino acids) in length, or more
preferably over a region that is 100 to 500 or 1000 or more
nucleotides (or 20, 50, 200 or more amino acids) in length.
[0455] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0456] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. USA 85:2444 (1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Brent et al., Current Protocols in Molecular Biology,
2003).
[0457] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol.
215:403-410, 1990, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a word length (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
word length of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0458] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0459] The percent identity between two amino acid sequences can
also be determined using the algorithm of E. Meyers and W. Miller,
Comput. Appl. Biosci. 4:11-17, 1988) which has been incorporated
into the ALIGN program (version 2.0), using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4. In
addition, the percent identity between two amino acid sequences can
be determined using the Needleman and Wunsch, J. Mol. Biol.
48:444-453, 1970) algorithm which has been incorporated into the
GAP program in the GCG software package (available at www.gcg.com),
using either a Blossom 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2,
3, 4, 5, or 6.
[0460] Other than percentage of sequence identity noted above,
another indication that two nucleic acid sequences or polypeptides
are substantially identical is that the polypeptide encoded by the
first nucleic acid is immunologically cross reactive with the
antibodies raised against the polypeptide encoded by the second
nucleic acid, as described below. Thus, a polypeptide is typically
substantially identical to a second polypeptide, for example, where
the two peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions, as described below. Yet
another indication that two nucleic acid sequences are
substantially identical is that the same primers can be used to
amplify the sequence.
[0461] The term "nucleic acid" is used herein interchangeably with
the term "polynucleotide" and refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0462] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, as detailed below, degenerate codon
substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J.
Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell.
Probes 8:91-98).
[0463] The term "operably linked" in the context of nucleic acids
refers to a functional relationship between two or more
polynucleotide (e.g., DNA) segments. Typically, it refers to the
functional relationship of a transcriptional regulatory sequence to
a transcribed sequence. For example, a promoter or enhancer
sequence is operably linked to a coding sequence if it stimulates
or modulates the transcription of the coding sequence in an
appropriate host cell or other expression system. Generally,
promoter transcriptional regulatory sequences that are operably
linked to a transcribed sequence are physically contiguous to the
transcribed sequence, i.e., they are cis-acting. However, some
transcriptional regulatory sequences, such as enhancers, need not
be physically contiguous or located in close proximity to the
coding sequences whose transcription they enhance.
[0464] The terms "polypeptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer. Unless otherwise indicated, a particular
polypeptide sequence also implicitly encompasses conservatively
modified variants thereof.
[0465] The term "immunoconjugate" or "antibody conjugate" as used
herein refers to the linkage of an antibody or an antigen binding
fragment thereof with another agent, such as a chemotherapeutic
agent, a toxin, an immunotherapeutic agent, an imaging probe, a
spectroscopic probe, and the like. The linkage can be covalent
bonds, or non-covalent interactions such as through electrostatic
forces. Various linkers, known in the art, can be employed in order
to form the immunoconjugate. Additionally, the immunoconjugate can
be provided in the form of a fusion protein that may be expressed
from a polynucleotide encoding the immunoconjugate. As used herein,
"fusion protein" refers to proteins created through the joining of
two or more genes or gene fragments which originally coded for
separate proteins (including peptides and polypeptides).
Translation of the fusion gene results in a single protein with
functional properties derived from each of the original
proteins.
[0466] The term "subject" includes human and non-human animals.
Non-human animals include all vertebrates, e.g., mammals and
non-mammals, such as non-human primates, sheep, dog, cow, chickens,
amphibians, and reptiles. Except when noted, the terms "patient" or
"subject" are used herein interchangeably.
[0467] The term "cytotoxin", or "cytotoxic agent" as used herein,
refer to any agent that is detrimental to the growth and
proliferation of cells and may act to reduce, inhibit, or destroy a
cell or malignancy.
[0468] The term "anti-cancer agent" as used herein refers to any
agent that can be used to treat a cell proliferative disorder such
as cancer, including but not limited to, cytotoxic agents,
chemotherapeutic agents, radiotherapy and radiotherapeutic agents,
targeted anti-cancer agents, and immunotherapeutic agents.
[0469] The term "terminal group (TG)" as used herein refers to a
chemical moiety or a surface that is conjugated to the antibody or
antigen binding fragment of the invention. For example, a terminal
group can be a drug moiety selected from an anti-cancer agent, an
anti-inflammatory agent, an antifungal agent, an antibacterial
agent, an anti-parasitic agent, an anti-viral agent, an anesthetic
agent. In certain embodiments a drug moiety is selected from a
V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor
inhibitor, a microtubule stabilizer, a microtubule destabilizers,
an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine
aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a
DPPIV inhibitor, an inhibitors of phosphoryl transfer reactions in
mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a
CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, an EG5
inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA
alkylating agent, a DNA intercalator, a DNA minor groove binder and
a DHFR inhibitor. Suitable examples include auristatins such as
MMAE and MMAF; calicheamycins such as gamma-calicheamycin; and
maytansinoids such as DM1 and DM4. Methods for attaching each of
these to a linker compatible with the antibodies and method of the
invention are known in the art. See, e.g., Singh et al.,
Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457
(2009). In addition a terminal group can be a biophysical probe, a
fluorophore, a spin label, an infrared probe an affinity probe, a
chelator, a spectroscopic probe, a radioactive probe, a lipid
molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA,
a protein, a peptide, a surface, an antibody, an antibody fragment,
a nanoparticle, a quantum dot, a liposome, a PLGA particle, or a
polysaccharide. In embodiments wherein the terminal group is a
surface, such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0470] "Tumor" refers to neoplastic cell growth and proliferation,
whether malignant or benign, and all pre-cancerous and cancerous
cells and tissues.
[0471] The term "anti-tumor activity" means a reduction in the rate
of tumor cell proliferation, viability, or metastatic activity. A
possible way of showing anti-tumor activity is to show a decline in
growth rate of abnormal cells that arises during therapy or tumor
size stability or reduction. Such activity can be assessed using
accepted in vitro or in vivo tumor models, including but not
limited to xenograft models, allograft models, MMTV models, and
other known models known in the art to investigate anti-tumor
activity.
[0472] The term "malignancy" refers to a non-benign tumor or a
cancer. As used herein, the term "cancer" includes a malignancy
characterized by deregulated or uncontrolled cell growth. Exemplary
cancers include: carcinomas, sarcomas, leukemias, and
lymphomas.
[0473] The term "cancer" includes primary malignant tumors (e.g.,
those whose cells have not migrated to sites in the subject's body
other than the site of the original tumor) and secondary malignant
tumors (e.g., those arising from metastasis, the migration of tumor
cells to secondary sites that are different from the site of the
original tumor).
[0474] The term "insertion" in the context of inserting a peptide
tag into an antibody means the incorporation of a peptide tag
between two specific residues of an antibody. The total number of
residues of the antibody is increased by the number of inserted tag
residues.
[0475] The term "grafting" in the context of incorporating a
peptide tag into an antibody refers to the incorporation of a
peptide tag into an antibody by mutagenesis. For instance, a short
stretch of amino acid residues within a non-CDR loop is substituted
by a peptide sequence. In this case, the total number of residues
of the antibody remains unchanged. In some embodiments, the term
"grafting" also encompasses a combination of substitution and
insertion of peptide tag residues. For example, one part of the
peptide tag is incorporated by substitution of structural loop
residues, while the remaining part is inserted between specific
residues of the non-CDR loop. The total number of residues of the
IgG antibody is increased by a number that is smaller than the
number of tag residues.
BRIEF DESCRIPTION OF THE FIGURES
[0476] FIG. 1. Schematic description of 4'-phosphopantetheinyl
transferase (PPTase)-mediated generation of ADCs.
[0477] FIG. 2A-2B. Design of IgG1 constructs which contain peptide
tags for site-specific antibody labeling via post-translational
4'-phosphopantetheinylation. (A) IgG1 constructs contain peptide
tags (underlined) in the V.sub.H, CH1, and CH3 domains. (B) IgG1
constructs contain peptide tags (underlined) in the CH3, V.sub.L,
and C.sub.L domains. Designed constructs that were successfully
cloned are marked by a plus (+) sign in the left column.
Unsuccessful cloning is indicated by a minus (-) sign. Successfully
cloned constructs are grouped as non-expressors (-) and expressors
(+) (middle column). Expressors which do not show any detectable
Sfp-catalyzed product formation in the presence of CoA-MC-MMAF
substrate (acetyl-CoA substrate was used for SEQ ID NOs: 28, 105,
118, 120, 123, and 126) are marked with a minus (-) sign in the
right column. Very low but detectable formation of the respective
MC-MMAF ADC is indicated with a plus (+) symbol. Significantly more
efficient but non-quantitative MC-MMAF ADC formation is indicated
by a double plus (++) sign. Quantitatively generated MC-MMAF ADCs
with two terminal groups (TGs) are classified with a triple plus
(+++) rating (according to HPLC analysis).
[0478] FIG. 3A-3B. (A) Sequence of CH1 domain, CH2 domain, CH3
domain, and hinge region of the Ig gamma 1 heavy chain. (B)
Sequence of C.sub.L domain of the Ig kappa light chain. Underlined
amino acids are structural loops. Amino acid positions are numbered
according to the EU numbering system as described in Edelman et
al., Proc. Natl. Acad. USA 63:78-85 (1969). X'.sub.1, X'.sub.2,
X'.sub.3, X'.sub.4, X'.sub.5, and X'.sub.6 indicate residues that
are present at allotypic positions within the IgG1 subclass and the
kappa isotype (according to Jefferis et al., MAbs. 1:332-338
(2009)).
[0479] FIG. 4A-4B. (A) Sequence alignment of CH1 domain, CH2
domain, CH3 domain, and hinge region of the four human Ig gamma
subclasses with Trastuzumab. (B) Sequence alignment of C.sub.L
domain with Trastuzumab. Underlined residues belong to structural
loops (see also FIG. 3). Boxed residues indicate allotypic
positions according to Jefferis et al., MAbs. 1:332-338 (2009). For
simplicity, only the allotypic positions within the IgG1 subclass
and the kappa isotype are shown. Protein sequences of the human Ig
gamma subclasses and the human kappa isotype are derived from the
UniProt database (entry numbers P01857, P01859, P01860, P01861, and
P01834).
[0480] FIG. 5A-5H. HPLC characterization of Sfp-catalyzed ADC
formation. (A) HPLC trace confirming the near quantitative
formation of the immunoconjugate
anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360. (B) HPLC trace
confirming the near quantitative formation of the immunoconjugate
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389. (C) HPLC trace
confirming the near quantitative formation of the immunoconjugate
anti-hHER2-HC-V2-DS-ppan-MC-MMAF-LEFIASKLA-Q3. (D) HPLC trace
confirming the quantitative formation of the immunoconjugate
anti-hHER2-HC-V2-GDS-ppan-MC-MMAF-LSWLLRLLN-Q3. (E) HPLC trace
confirming the near quantitative formation of the immunoconjugate
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKL-N389. (F) HPLC trace
confirming the quantitative formation of the immunoconjugate
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389. (G) HPLC trace
confirming the near quantitative formation of the immunoconjugate
mAb2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360. (H) HPLC trace
exemplifying partial formation of the immunoconjugate
anti-hHER2-LC-12-DS-ppan-MC-MMAF-LEFIASKLA-Q3.
[0481] FIG. 6A-6C. Characterization of three trastuzumab
immunoconjugates by analytical size-exclusion chromatography
(AnSEC) exemplifies the formation of monomeric, non-aggregated
ADCs. (A) AnSEC analysis of the immunoconjugate
anti-hHER2-HC-V2-GDS-ppan-MC-MMAF-LSWLLRLLN-Q3. (B) AnSEC analysis
of the immunoconjugate
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389. (C) AnSEC
analysis of the immunoconjugate
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKL-N389.
[0482] FIG. 7. HPLC characterization of unsuccessful labeling of
trastuzumab with incorporation of a peptide tag at a specific
location. HPLC trace indicating no conjugation between
anti-hHER2-HC-S190D-S191-S192L-L193E-G194F-T1951-Q196A-T197S-Y198K-1199L
and CoA-MC-MMAF.
[0483] FIG. 8A-8B. HPLC characterization of the labeling of mixed
grafting/insertion constructs with CoA-MC-MMAF. (A) HPLC trace
indicating partial formation of the immunoconjugate
anti-hHER2-HC-S63-ppan-MC-MMAF-V64L-EFIASKLA-K65. (B) HPLC trace
indicating no formation of the immunoconjugate
anti-hHER2-LC-S76D-S77-ppan-MC-MMAF-L78-EFIASKLA-Q79.
[0484] FIG. 9A-9B. HPLC characterization of fluorophore attachment
to IgGs. (A) HPLC trace confirming the near quantitative formation
of the antibody-fluorophore conjugate
anti-hHER2-HC-P189G-S190D-S191-ppan-maleimidoethylamido-TMR-S192L-L193S-G-
194W-T195L. The extensive overlap between the HPLC traces monitored
at 280 and 555 nm indicates near quantitative fluorophore
conjugation. (B) HPLC trace confirming the near quantitative
formation of the antibody-fluorophore conjugate
anti-hHER2-HC-T359-GDS-ppan-maleimidoethylamido-TMR-LSWLLRLLN-K360.
The extensive overlap between the HPLC traces monitored at 280 and
555 nm indicates near quantitative fluorophore conjugation.
[0485] FIG. 10A-10B. HPLC characterization of antibody labeling
with hydrolyzed maleimido- or bromoacetyl thioether-linked
cytotoxins. (A) HPLC trace confirming the near quantitative
conjugation of maleimide-ring-opened CoA-MC-MMAF to
hHER2-HC-T359-GDSLSWLLRLLN-K360. (B) HPLC trace confirming the near
quantitative conjugation of CoA-Ac-Ahx-MMAF to
hHER2-HC-T359-GDSLSWLLRLLN-K360.
[0486] FIG. 11A-11B. HPLC characterization of antibody labeling
with cytotoxins connected via a cleavable linker. (A) HPLC trace
confirming the near quantitative conjugation of
CoA-MC-Val-Cit-PABC-MMAF to anti-hHER2-HC-T359-GDSLSWLLRLLN-K360.
(B) HPLC trace confirming the near quantitative conjugation of
CoA-MC-Val-Cit-PABC-MMAF to
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389.
[0487] FIG. 12. Optimization of 4'-phosphopantetheinyl transferase
(PPTase)-catalyzed ADC formation as a function of pH. The bar graph
representation shows the amount of generated ADC with a
drug-to-antibody ratio (DAR) of 2 as a function of pH. The data is
based on the HPLC analysis (280 nm) of the reaction of CoA-MC-MMAF
with either anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 or
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 at a pH range of 5.0 to
10.0.
[0488] FIG. 13A-13C. Optimization of conjugation reaction as a
function of Sfp enzyme concentration in 50 mM HEPES buffer (pH 7.5)
containing 2.5 .mu.M antibody, 50 .mu.M CoA-MC-MMAF, and 10 mM
MgCl.sub.2 (37.degree. C., 16 hours). (A) Deconvoluted mass
spectrum showing primarily unconjugated
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 at an Sfp concentration of 0.1
.mu.M. (B) Deconvoluted mass spectrum showing near quantitative ADC
formation of anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 at
an Sfp concentration of 0.25 .mu.M. (C) Deconvoluted mass spectrum
showing near quantitative ADC formation of
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 at an Sfp
concentration of 0.5 .mu.M.
[0489] FIG. 14A-14B. Optimization of enzymatic conjugation reaction
as a function of CoA-MC-MMAF substrate concentration at pH 8.0. (A)
The HPLC traces represent three conjugation reactions with 2.5
.mu.M anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 that contained 2.5 .mu.M
(top trace), 7.5 .mu.M (middle trace), or 25 .mu.M (bottom trace)
of CoA-MC-MMAF. The peak at a retention time of 4.9 min corresponds
to unlabeled antibody (DAR=0), the peak at 5.3 min to mono-labeled
antibody (DAR=1), and the peak at 5.7 min to bi-labeled antibody
(DAR=2). (B) The bar graph representation shows the amount of
generated ADC with a DAR of 2 as a function of CoA-MC-MMAF
substrate concentration. The titration series was performed at an
Sfp enzyme concentration of either 0.25 .mu.M (black bars) or 1.0
.mu.M (white bars).
[0490] FIG. 15A-15C. Thermal stability of peptide-tagged ADCs as
measured by differential scanning fluorometry (DSF) using SYPRO
Orange gel stain. (A) Determination of the thermal stability of the
immunoconjugate anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360.
Two transition temperatures of 68.5 and 81.5 degrees Celsius are
observed by DSF (average of two measurements). (B) Determination of
the thermal stability of the immunoconjugate
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389. Two transition
temperatures of 66.3 and 81.0 degrees Celsius are observed by DSF
(single measurement). (C) Determination of the thermal stability of
unmodified Trastuzumab IgG1 (anti-hHER2) which was used as
reference for comparison with peptide-tagged ADCs. Two transition
temperatures of 69.7 and 81.1 degrees Celsius are observed by DSF
(average of two measurements).
[0491] FIG. 16A-16B. Pharmacokinetic (PK) study of two
peptide-tagged Trastuzumab immunoconjugates. Plasma titers of both
ADCs were determined by capturing the respective immunoconjugates
with plate-absorbed human HER2 (extracellular domains 3-4) followed
by detection with anti-human IgG and anti-MMAF antibodies. (A)
Comparison of plasma titers of
anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 and unmodified
Trastuzumab (anti-hHER2) antibody by ELISA. The plasma titer of the
immunoconjugate exhibits a rapid decay within 4 days. (B)
Comparison of plasma titers of
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 and unmodified
Trastuzumab (anti-hHER2) antibody by ELISA. The plasma titer of the
immunoconjugate closely parallels the control titer of the
unmodified anti-hHER2 antibody within a 14 day period.
[0492] FIG. 17. In vitro cell-killing assay of peptide-tagged
immunoconjugates using the HER2-expressing MDA-231 cell line. Plots
are based on cell viability measurements using the Cell Titer Glo
Luminescent Cell Viability Assay (Promega).
[0493] FIG. 18A-18B. Enzymatic generation of ADCs with a DAR of 4.
(A) ADCs with a DAR of 4 can be generated by incorporating multiple
peptide tags into an antibody, such as the ybbR- and the S6-tags.
(B) HPLC analysis of Sfp-catalyzed conjugation of CoA-MC-MMAF to
Trastuzumab IgG containing an S6 tag in the V.sub.H domain as well
as a ybbR tag in the CH3 domain
(anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389). Room
temperature incubation of 2.5 .mu.M of antibody and 50 .mu.M of CoA
substrate in the presence of 1 .mu.M of Sfp enzyme leads to near
quantitative formation of an ADC with a DAR of 4 (t.sub.R=6.1 min,
bottom trace). The top trace represents the corresponding uncoupled
antibody (DAR=0, t.sub.R=5.2 min).
[0494] FIG. 19A-19F. Pharmacokinetic profiles of peptide-tagged
trastuzumab immunoconjugates displaying high and low AUC IgG
values. Each of the six peptide-tagged ADCs corresponding to SEQ ID
NO:248 (A), SEQ ID NO:33 (B), SEQ ID NO:251 (C), SEQ ID NO:218 (D),
SEQ ID NO:202 (E), and SEQ ID NO:244 (F) was administered
intravenously into three mice at a single dose of 1 mg/kg. After
collection of plasma samples over a time period of 340 hours,
trastuzumab ADC molecules were captured by using the immobilized
extracellular domain of human HER2. Plasma titers were then
determined by two ELISA formats based on either anti-MMAF or
anti-hlgG antibodies. While the first format provides readout on
the concentration of "intact" ADC, the latter format generates a
signal proportional to the concentration of total IgG, comprising
both conjugated and unconjugated trastuzumab molecules. A-C
exemplify PK curves of peptide-tagged MMAF ADCs displaying high AUC
IgG values, whereas D-F show examples of immunoconjugates
exhibiting very low AUC IgG values. In all cases, anti-MMAF and
anti-hlgG titers closely parallel each other indicating negligible
deconjugation of the MMAF payload during the time course of the PK
study.
[0495] FIG. 20. In vivo efficacy study of the ybbR-tagged
trastuzumab ADC anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389
(SEQ ID NO:129) in immune-deficient nude mice implanted with a
human tumor cell line. The xenograft tumor model was performed with
nu/nu mice which were subcutaneously administered with the
HER2-dependent breast cancer cell line MDA-MB231 clone 16. After
the tumor has grown to a size of about 200 mm.sup.3, single doses
of 3 mg/kg (.tangle-solidup.), 5 mg/kg ( ) of the ybbR-tagged ADC
or vehicle alone (-) were intravenously injected into nine mice per
treatment group. The vertical arrow indicates the time point of ADC
administration. Weekly monitoring of tumor growth revealed that
both dose levels resulted in tumor regression demonstrating in vivo
efficacy of the peptide-tagged ADC.
DETAILED DESCRIPTION
[0496] The present invention provides methods of site-specific
labeling of antibodies, using proteins having
4'-phosphopantetheinyl transferase activity ("PPTases") that
catalyze post-translational modification of peptide sequences
("peptide tags") incorporated into one or more specific sites of an
antibody of interest. Enzymatic labeling under ambient reaction
conditions enables quantitative and irreversible covalent
modification of a specific serine residue within the peptide tags
incorporated into the antibody, and thus creates desirable antibody
conjugates.
[0497] Given the broad substrate tolerance of PPTases,
site-specific antibody labeling according to the present invention
can be achieved with a variety of chemically accessible labeling
reagents, such as anti-cancer agents, fluorophores, peptides,
sugars, detergents, polyethylene glycols, immune potentiators,
radio-imaging probes, prodrugs, and other molecules. Furthermore,
PPTases can be used to immobilize peptide-tagged antibodies on
solid support, such as polystyrene nanoparticles and gold surfaces
(see, e.g., Wong et al., Org. Biomol. Chem. 8: 782-787, 2010; Wong
et al., Nanoscale 4:659-666, 2012, for methodology of
immobilization of functional enzymes).
[0498] Accordingly, the present invention provides methods of
preparation of homogeneous immunoconjugates with a defined
drug-to-antibody ratio for use in cancer therapy, and
immunoconjugates prepared thereby, as well as pharmaceutical
compositions comprising these immunoconjugates. The methods of the
instant invention can be used in combination with other conjugation
methods known in the art.
1. Antibody Engineering
Site-Specific Labeling
[0499] A "structural loop" or "non-CDR-loop" according to the
present invention is to be understood in the following manner:
antibodies are made of domains with immunoglobulin folds. In
essence, anti-parallel beta sheets are connected by loops to form a
compressed antiparallel beta barrel. In the variable region, some
of the loops of the domains contribute essentially to the
specificity of the antibody, i.e., the binding to an antigen. These
loops are called "CDR-loops." All other loops of antibody domains
are rather contributing to the structure of the molecule and/or the
effector function. These loops are defined herein as "structural
loops" or "non-CDR-loops."
[0500] The antibodies (e.g., a parent or native antibody,
optionally containing one or more non-naturally occurring amino
acids) of the present invention are numbered according to the EU
numbering system as set forth in Edelman et al., Proc. Natl. Acad.
USA 63:78-85 (1969). Human IgG1 constant region is used as a
representative throughout the application. However, the invention
is not limited to human IgG1; corresponding amino acid positions
can be readily deduced by sequence alignment. For example, FIG. 3
(A) shows IgG1 heavy chain constant region where the structural
loops are underlined, these underlined structural loops can be
readily identified for IgG2, IgG3, and IgG4 as shown in the
sequence alignment of FIG. 4 (A). FIG. 3 (B) shows the light chain
constant region where the structural loops are underlined. For the
light chain constant region, IgG1, IgG2, IgG3 and IgG4 are the
same. Table 1 below lists the amino acid positions in the
structural loop of IgG1, IgG2, IgG3 and IgG4, respectively.
TABLE-US-00001 TABLE 1 Identified Structural Loop Positions (IgG1
according to EU numbering) IgG1 IgG2 IgG3 IgG4 Heavy
119(S)120(T)121(K) 119(S)120(T)121(K) 119(S)120(T)121(K)
119(S)120(T)121(K) Chain 131(S)132(S)133(K) 131(C)132(S)133(R)
131(C)132(S)133(R) 131(C)132(S)133(R) 134(S)135(T)136(S)
134(S)135(T)136(S) 134(S)135(T)136(S) 134(S)135(T)136(S)
137(G)138(G)139(T) 137(E)138(S)139(T) 137(G)138(G)139(T)
137(E)138(S)139(T) 152(E)153(P)154(V) 152(E)153(P)154(V)
152(E)153(P)154(V) 152(E)153(P)154(V) 159(N)160(S)161(G)
159(N)160(S)161(G) 159(N)160(S)161(G) 159(N)160(S)161(G)
162(A)163(L)164(T) 162(A)163(L)164(T) 162(A)163(L)164(T)
162(A)163(L)164(T) 165(S)166(G) 165(S)166(G) 165(S)166(G)
165(S)166(G) 171(P)172(A) 171(P)172(A) 171(P)172(A) 171(P)172(A)
176(S)177(S)178(G) 176(S)177(S)178(G) 176(S)177(S)178(G)
176(S)177(S)178(G) 189(P)190(S)191(S) 189(P)190(S)191(S)
189(P)190(S)191(S) 189(P)190(S)191(S) 192(S)193(L)194(G)
192(N)193(F)194(G) 192(S)193(L)194(G) 192(S)193(L)194(G)
195(T)196(Q)197(T) 195(T)196(Q)197(T) 195(T)196(Q)197(T)
195(T)196(K)197(T) 205(K)206(P)207(S) 205(K)206(P)207(S)
205(K)206(P)207(S) 205(K)206(P)207(S) 208(N) 208(N) 208(N) 208(N)
230(P)231(A)232(P) 227(P)228(A)229(P) 277(P)278(A)279(P)
227(P)228(A)229(P) 233(E)234(L)235(L) 230(P)231(V)232(A)
280(E)281(L)282(L) 230(E)231(F)232(L) 236(G)236(G) 233(G)
283(G)284(G) 233(G)234(G) 244(P)245(P)246(K) 240(P)241(P)242(K)
291(P)292(P)293(K) 241(P)242(P)243(K) 253(I)254(S)255(R)
249(I)250(S)251(R) 300(I)301(S)302(R) 250(I)251(S)252(R)
256(T)257(P)258(E) 252(T)253(P)254(E) 303(T)304(P)305(E)
253(T)254(P)255(E) 267(S)268(H)269(E) 263(S)264(H)265(E)
314(S)315(H)316(E) 264(S)265(Q)266(E) 270(D)271(P)272(E)
266(D)267(P)268(E) 317(D)318(P)319(E) 267(D)268(P)269(E)
280(D)281(G) 276(D)277(G) 327(D)328(G) 277(D)278(G)
285(H)286(N)287(A) 281(H)282(N)283(A) 332(H)333(N)334(A)
282(H)283(N)284(A) 291(P)292(R) 287(P)288(R) 338(P)339(R)
288(P)289(R) 295(Q)296(Y)297(N) 291(Q)292(F)293(N)
342(Q)343(Y)344(N) 292(Q)293(F)294(N) 298(S)299(T) 294(S)295(T)
345(S)346(T) 295(S)296(T) 307(T)308(V)309(L) 303(T)304(V)305(V)
354(T)355(V)356(L) 304(T)305(V)306(L) 310(H)311(Q) 306(H)307(Q)
357(H)358(Q) 307(H)308(Q) 315(N)316(G)317(K) 311(N)312(G)313(K)
362(N)363(G)364(K) 312(N)313(G)314(K) 318(E) 314(E) 365(E) 315(E)
326(K)327(A)328(L) 322(K)323(G)324(L) 373(K)374(A)375(L)
323(K)324(G)325(L) 329(P)330(A)331(P) 325(P)326(A)327(P)
376(P)377(A)378(P) 326(P)327(S)328(S) 339(A)340(K)341(G)
335(T)336(K)337(G) 386(T)387(K)388(G) 336(A)337(K)338(G)
342(Q)343(P)344(R) 338(Q)339(P)340(R) 389(Q)390(P)391(R)
339(Q)340(P)341(R) 345(E) 341(E) 392(E) 342(E) 355(R)356(D/E)
351(R)352(E)353(E) 402(R)403(E)404(E) 352(Q)353(E)354(E)
357(E)358(L/M) 354(M)355(T)356(K) 405(M)406(T)407(K)
355(M)356(T)357(K) 359(T)360(K)361(N) 357(N) 408(N) 358(N)
384(N)385(G) 380(N)381(G) 431(S)432(G) 381(N)382(G)
388(E)389(N)390(N) 384(E)385(N)386(N) 435(E)436(N)437(N)
385(E)386(N)387(N) 394(T)395(P)396(P) 390(T)391(P)392(P)
441(T)442(P)443(P) 391(T)392(P)393(P) 399(D)400(S)401(D)
395(D)396(S)397(D) 446(D)447(S)448(D) 396(D)397(S)398(D) 402(G)
398(G) 449(G) 399(G) 415(S)416(R) 411(S)412(R)413(W)
462(S)463(R)464(W) 412(S)413(R)414(W) 417(W)418(Q)
414(Q)415(Q)416(G) 465(Q)466(Q)467(G) 415(Q)416(E)417(G)
419(Q)420(G) 417(N)418(V) 468(N)469(I) 418(N)419(V) 421(N)422(V)
433(H)434(N)435(H) 429(H)430(N)431(H) 480(H)481(N)482(R)
430(H)431(N)432(H) 442(S)443(L)444(S) 438(S)439(L)440(S)
489(S)490(L)491(S) 439(S)440(L)441(S) 445(P)446(G) 441(P)442(G)
492(P)493(G) 442(L)443(G) Light 109(T)110(V)111(A)
109(T)110(V)111(A) 109(T)110(V)111(A) 109(T)110(V)111(A) Chain
112(A) 112(A) 112(A) 112(A) 119(P)120(P)121(S) 119(P)120(P)121(S)
119(P)120(P)121(S) 119(P)120(P)121(S) 122(D)123(E) 122(D)123(E)
122(D)123(E) 122(D)123(E) 140(Y)141(P)142(R) 140(Y)141(P)142(R)
140(Y)141(P)142(R) 140(Y)141(P)142(R) 143(E)144(A) 143(E)144(A)
143(E)144(A) 143(E)144(A) 151(D)152(N)153(A) 151(D)152(N)153(A)
151(D)152(N)153(A) 151(D)152(N)153(A) 154(L)155(Q)156(S)
154(L)155(Q)156(S) 154(L)155(Q)156(S) 154(L)155(Q)156(S)
161(E)162(S)163(V) 161(E)162(S)163(V) 161(E)162(S)163(V)
161(E)162(S)163(V) 164(T)165(E)166(Q) 164(T)165(E)166(Q)
164(T)165(E)166(Q) 164(T)165(E)166(Q) 167(D)168(S) 167(D)168(S)
167(D)168(S) 167(D)168(S) 197(T)198(H)199(Q) 197(T)198(H)199(Q)
197(T)198(H)199(Q) 197(T)198(H)199(Q) 200(G)201(L)202(S)
200(G)201(L)202(S) 200(G)201(L)202(S) 200(G)201(L)202(S)
203(S)204(P) 203(S)204(P) 203(S)204(P) 203(S)204(P) 207(K)208(S)
207(K)208(S) 207(K)208(S) 207(K)208(S)
[0501] FIG. 3 as well as SEQ ID NOs 25 and 93 represent the
sequences of the Ig kappa light chain constant region and the Ig
gamma-1 heavy chain constant region, respectively. X'.sub.1,
X'.sub.2, X'.sub.3, X'.sub.4, X'.sub.5, and X'.sub.6 in SEQ ID NOs:
25 and 93 indicate residues that are present at allotypic positions
within the IgG1 subclass and the kappa isotype (according to
Jefferis et al., MAbs. 1:332-338 (2009)). X'.sub.1 can be Arg or
Lys, X'.sub.2 can be Asp or Glu, X'.sub.3 can be Leu or Met,
X'.sub.4 can be Ala or Gly, X'.sub.5 can be Val or Ala, and
X'.sub.6 can be Leu or Val.
[0502] Because of the high sequence homology of constant regions of
IgG1, IgG2, IgG3 and IgG4 antibodies, findings of the invention are
not limited to any specific antibodies. In addition, the findings
of the invention are not limited to using PPTases. The positions in
the antibody structural loops identified herein can also be used
for incorporating other peptide tags, which are substrates for
other enzymatic conjugation approaches such as the enzyme biotin
protein ligase (BPL), transglutaminases, and formylglycine forming
enzymes.
[0503] In one aspect, the present invention provides
immunoconjugates comprising a modified antibody or an antigen
binding fragment thereof, and a terminal group, wherein said
modified antibody or antigen binding fragment thereof comprises a
peptide tag that by itself is a substrate of a
4'-phosphopantetheinyl transferase, and wherein said peptide tag is
located within a structural loop, or C- or N-terminus of the
modified antibody or antigen binding fragment thereof. The present
invention also provides modified antibodies or antigen binding
fragments thereof comprising a peptide tag that is a substrate of a
4'-phosphopantetheinyl transferase, and wherein said peptide tag is
located within a structural loop, or C- or N-terminus of the
antibody or antigen binding fragment thereof. In a specific
embodiment, said peptide tag is one or more peptides selected from
those described in Table 2. In one aspect, the peptide tag is
inserted between two amino acids of a structural loop of said
antibody or antigen binding fragment thereof. In another aspect,
the peptide tag is grafted into a structural loop, C- or N-terminus
of said antibody or antigen binding fragment thereof, wherein the
peptide tag replaces one or more amino acids of the parent antibody
or antigen binding fragment thereof. In one aspect, the structural
loop refers to a structural loop located at the CH1, CH2, CH3, or
C.sub.L region of said antibody or antigen binding fragment
thereof. The modified antibody heavy chain and/or light chain (or
antigen binding fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7,
8, or more protein tags in its structural loops. In one aspect, the
modified antibodies or antigen binding fragments contain 2, 4, 6,
8, or more protein tags in its structural loops. In another aspect,
said 4'-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima
PPTase, human PPTase, or a mutant form thereof that retains the
4'-phosphopantetheinyl transferase activity. In one aspect, said
4'-phosphopantetheinyl transferase originates from Homo sapiens,
Bacillus subtilis, Escherichia coli, Thermotoga maritima,
Clostridium thermocellum, as well as any other mammalian, bacterial
or fungal genome. In another aspect, said 4'-phosphopantetheinyl
transferase is a homologous protein to Sfp, AcpS, Tmaritima PPTase,
human PPTase, or a mutant thereof. In one embodiment, said
4'-phosphopantetheinyl transferase is from a thermophilic organism.
In some embodiments, the parental antibody (antibody without
incorporating the peptide tag) is an IgG, IgM, IgE, or IgA
antibody. In some embodiments, the parental antibody is an IgG1
antibody. In some other embodiments, the parental antibody is an
IgG2, IgG3, or IgG4 antibody.
[0504] "A substrate of 4'-phosphopantetheinyl transferase" as used
herein means the structure being described can serve as an acceptor
for a 4'-phosphopantetheine (ppan) or modified ppan group as
illustrated in Scheme Ia herein when contacted with
4'-phosphopantetheinyl transferase and CoA or a CoA analog having a
terminal group attached to it.
[0505] In one aspect, the present invention provides
immunoconjugates comprising a modified antibody or an antigen
binding fragment thereof, and a terminal group, wherein said
modified antibody or antigen binding fragment thereof comprises a
CH1, CH2, CH3, and/or C.sub.L region, and wherein said CH1, CH2,
CH3, and/or C.sub.L region further comprises a peptide tag that by
itself is a substrate of a 4'-phosphopantetheinyl transferase. The
present invention also provides modified antibodies or antigen
binding fragments thereof comprising a CH1, CH2, CH3, and/or
C.sub.L region, and wherein said CH1, CH2, CH3, and/or C.sub.L
region further comprises a peptide tag that is a substrate of a
4'-phosphopantetheinyl transferase. In some embodiments, said
peptide tag is one or more peptides selected from those described
in Table 2. In some embodiments, the peptide tag is inserted
between two amino acids of a structural loop of said antibody or
antigen binding fragment thereof. In some embodiments, the peptide
tag is grafted into a structural loop of said antibody or antigen
binding fragment thereof. The modified antibody heavy chain and/or
light chain (or antigen binding fragment thereof) may contain 1, 2,
3, 4, 5, 6, 7, 8, or more protein tags in its structural loops. In
some embodiments, the modified antibodies or antigen binding
fragments contain 2, 4, 6, 8, or more protein tags in its
structural loops. In some embodiments, said 4'-phosphopantetheinyl
transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a
mutant form thereof that retains the 4'-phosphopantetheinyl
transferase activity. In some embodiments, said
4'-phosphopantetheinyl transferase originates from Homo sapiens,
Bacillus subtilis, Escherichia coli, Thermotoga maritima,
Clostridium thermocellum, as well as any other mammalian, bacterial
or fungal genome. In some embodiments, said 4'-phosphopantetheinyl
transferase is a homologous protein to Sfp, AcpS, Tmaritima PPTase,
or a mutant thereof. In one embodiment, said 4'-phosphopantetheinyl
transferase is from a thermophilic organism. In some embodiments,
the parental antibody is an IgG, IgM, IgE, or IgA antibody. In a
specific embodiment, the parental antibody is an IgG1 antibody. In
some embodiments, the parental antibody is an IgG2, IgG3, or IgG4
antibody.
[0506] As used herein, "retains" activity means the enzyme being
described maintains at least about 10% of the activity of the
reference material, which is the B. subtilis Sfp
4'-phosphopantetheinyl transferase (see, e.g., Quadri et al.,
Biochemistry 37: 1585-1595 (1998)). For example, a different
4'-phosphopantetheinyl transferase or a mutant form of the enzyme
retains at least about 10% of the 4'-phosphopantetheinyl
transferase activity compared to Sfp under identical reaction
conditions, i.e., using the same CoA substrate, the same
peptide-tagged antibody, identical buffer conditions, identical
substrate and enzyme concentrations, the same temperature, and the
same reaction duration.
[0507] In one aspect, the present invention provides
immunoconjugates comprising a modified antibody or an antigen
binding fragment thereof, and a terminal group, wherein said
modified antibody or antigen binding fragment thereof comprises a
peptide tag that by itself is a substrate of a
4'-phosphopantetheinyl transferase, and wherein said peptide tag is
inserted between positions 2 and 3 of the V.sub.H domain, positions
63 and 64 of the V.sub.H domain, positions 64 and 65 of the V.sub.H
domain, positions 138 and 139 of the CH1 domain, positions 197 and
198 of the CH1 domain, positions 359 and 360 of the CH3 domain,
positions 388 and 389 of the CH3 domain, the C-terminus of the CH3
domain (after Lys447), and/or positions 2 and 3 of the V.sub.L
domain of a parental antibody or antigen binding fragment thereof.
In another aspect, the present invention provides immunoconjugates
comprising a modified antibody or antigen binding fragment thereof,
and a terminal group, wherein said modified antibody or antigen
binding fragment thereof comprises a peptide tag that by itself is
a substrate of a 4'-phosphopantetheinyl transferase, and wherein
the peptide tag is inserted between amino acid residues 2 and 3 of
the VH or VL domain, or between amino acid residue 110 and 111 of
the light chain, or between 119 and 120, or between 120 and 121, or
between 135 and 136, or between 136 and 137, or between 138 and
139, or between 164 and 165, or between 165 and 166, or between 194
and 195 of the CH1 domain, or between 388 and 389, or between 445
and 446, or between 446 and 447 of the CH3 domain of a parental
antibody or antigen binding fragment thereof. In some embodiments,
the peptide tag is inserted between amino acid residue 110 and 111
of the light chain, or between 119 and 120, or between 120 and 121,
or between 135 and 136, or between 136 and 137, or between 138 and
139, or between 165 and 166 of the CH1 domain, or between 388 and
389 of the CH3 domain of a parental antibody or antigen binding
fragment thereof,
[0508] In one aspect, the invention provides immunoconjugates
comprising a modified antibody or an antigen binding fragment
thereof, and a terminal group, wherein said modified antibody or
antigen binding fragment thereof comprises SEQ ID NO: 103, SEQ ID
NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or SEQ ID
NO:141. In another aspect, the invention provides immunoconjugates
comprising a modified antibody or an antigen binding fragment
thereof, and a terminal group, wherein said modified antibody or
antigen binding fragment comprises comprises SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:94, SEQ ID NO:95, SEQ
ID NO:96, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:139, SEQ ID NO:149,
SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:248,
SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID
NO:259, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:277, SEQ ID NO:348,
SEQ ID NO:349, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:359, SEQ ID
NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:373, SEQ ID NO:374,
SEQ ID NO:380, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:387, or SEQ
ID NO:388. In some embodiments, the modified antibody or antigen
binding fragment comprises SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:151, SEQ ID NO:152,
SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:169, SEQ ID
NO:250, SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259,
SEQ ID NO:268, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID
NO:365, SEQ ID NO:367, SEQ ID NO:374, or SEQ ID NO:384.
[0509] With respect to the immunoconjugates described herein, in
one aspect, said peptide tag is one or more peptides selected from
those described in Table 2. The modified antibody heavy chain
and/or light chain (or antigen binding fragment thereof) may
contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its
structural loops. In one embodiment, the modified antibodies or
antigen binding fragments contain 2, 4, 6, 8, or more protein tags
in its structural loops. In another embodiment, said
4'-phosphopantetheinyl transferase is Sfp, AcpS, T. maritima
PPTase, human PPTase, or a mutant form thereof that retains the
4'-phosphopantetheinyl transferase activity. In one embodiment,
said 4'-phosphopantetheinyl transferase originates from Homo
sapiens, Bacillus subtilis, Escherichia coli, Thermotoga maritima,
Clostridium thermocellum, as well as any other mammalian, bacterial
or fungal genome. In a specific embodiment, said
4'-phosphopantetheinyl transferase is Sfp and the peptide tag is
selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL
(SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19). In one embodiment,
the parental antibody is an IgG, IgM, IgE, or IgA antibody. In a
specific embodiment, the parental antibody is an IgG1 antibody. In
another specific embodiment, the parental antibody is an IgG2,
IgG3, or IgG4 antibody.
[0510] In another aspect, the present invention provides
immunoconjugates comprising a modified antibody or an antigen
binding fragment thereof, and a terminal group, wherein said
modified antibody or antigen binding fragment thereof comprises a
peptide tag that by itself is a substrate of a
4'-phosphopantetheinyl transferase, and wherein said peptide tag is
grafted into a structural loop, or C- or N-terminus of the antibody
or antigen binding fragment thereof. In a specific embodiment, said
peptide tag is grafted at amino acid positions from 62 to 64 of the
V.sub.H domain (mutations at amino acids 62 and 63, and insertion
of the rest of the peptide tag between amino acids 63 and 64), at
amino acid positions from 62 to 65 of the V.sub.H domain (mutations
at amino acids 62-64, and insertion of the rest of the peptide tag
between amino acids 64 and 65); at amino acid positions from 133 to
139 of the CH1 domain (mutations of amino acids 133-138, and
insertion of the rest of the peptide tag between amino acids
138-139), amino acid positions from 189 to 195 of the CH1 domain,
and/or amino acid positions from 190 to 198 of the CH1 domain
(mutations from amino acids 190-197, and insertion of the rest of
the peptide tag between 197 and 198) of a parental antibody or
antigen binding fragment thereof. In one embodiment, said peptide
tag is one or more peptides selected from those described in Table
2. The modified antibody heavy chain and/or light chain (or antigen
binding fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or
more protein tags in its structural loops. In one embodiment, the
modified antibodies or antigen binding fragments contain 2, 4, 6,
8, or more protein tags in its structural loops. In another
embodiment, said 4'-phosphopantetheinyl transferase is Sfp, AcpS,
T. maritima PPTase, human PPTase, or a mutant form thereof that
retains the 4'-phosphopantetheinyl transferase activity. In one
embodiment, said 4'-phosphopantetheinyl transferase originates from
Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga
maritima, Clostridium thermocellum, as well as any other mammalian,
bacterial or fungal genome. In a specific embodiment, said
4'-phosphopantetheinyl transferase is Sfp and the peptide tag is
selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL
(SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19). In one embodiment,
the parental antibody is an IgG, IgM, IgE, or IgA antibody. In a
specific embodiment, the parental antibody is an IgG1 antibody. In
another specific embodiment, the parental antibody is an IgG2,
IgG3, or IgG4 antibody.
[0511] In another aspect, the present invention provides modified
antibodies or antigen binding fragments thereof comprising a
peptide tag that is a substrate of a 4'-phosphopantetheinyl
transferase, and wherein said peptide tag is inserted between
positions 2 and 3 of the V.sub.H domain, positions 63 and 64 of the
V.sub.H domain, positions 64 and 65 of the V.sub.H domain,
positions 138 and 139 of the CH1 domain, positions 197 and 198 of
the CH1 domain, positions 359 and 360 of the CH3 domain, positions
388 and 389 of the CH3 domain, the C-terminus of the CH3 domain
(after Lys447), and/or positions 2 and 3 of the V.sub.L domain of a
parental antibody or antigen binding fragment thereof. In another
aspect, the peptide tag is inserted between amino acid residues 2
and 3 of the VH or VL domain, or between amino acid residue 110 and
111 of the light chain, or between 119 and 120, or between 120 and
121, or between 135 and 136, or between 136 and 137, or between 138
and 139, or between 164 and 165, or between 165 and 166, or between
194 and 195 of the CH1 domain, or between 388 and 389, or between
445 and 446, or between 446 and 447 of the CH3 domain of a parental
antibody or antigen binding fragment thereof. In some embodiments,
the peptide tag is inserted between amino acid residue 110 and 111
of the light chain, or between 119 and 120, or between 120 and 121,
or between 135 and 136, or between 136 and 137, or between 138 and
139, or between 165 and 166 of the CH1 domain, or between 388 and
389 of the CH3 domain of a parental antibody or antigen binding
fragment thereof.
[0512] In another aspect, the present invention provides a modified
antibody or antigen binding fragment thereof comprising SEQ ID NO:
103, SEQ ID NO: 109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:122,
SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, and/or
SEQ ID NO:141. In another aspect, the present invention provides a
modified antibody or antigen binding fragment thereof comprising
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:32, SEQ ID NO:63, SEQ ID
NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:126, SEQ ID NO:127,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:139, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:157,
SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:168, SEQ ID NO:169, SEQ ID
NO:178, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:256,
SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:267, SEQ ID NO:268, SEQ ID
NO:277, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:356, SEQ ID NO:358,
SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:367, SEQ ID
NO:373, SEQ ID NO:374, SEQ ID NO:380, SEQ ID NO:384, SEQ ID NO:386,
SEQ ID NO:387, or SEQ ID NO:388. In some embodiments, the present
invention provides a modified antibody or antigen binding fragment
thereof comprising SEQ ID NO:32, SEQ ID NO:63, SEQ ID NO:127, SEQ
ID NO:129, SEQ ID NO:132, SEQ ID NO:151, SEQ ID NO:152, SEQ ID
NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:169, SEQ ID NO:250,
SEQ ID NO:251, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:259, SEQ ID
NO:268, SEQ ID NO:358, SEQ ID NO:359, SEQ ID NO:364, SEQ ID NO:365,
SEQ ID NO:367, SEQ ID NO:374, or SEQ ID NO:384.
[0513] In one aspect, said peptide tag is one or more peptides
selected from those described in Table 2. The antibody heavy chain
and/or light chain (or antigen binding fragment thereof) may
contain 1, 2, 3, 4, 5, 6, 7, 8, or more protein tags in its
structural loops. In some embodiments, the antibodies or antigen
binding fragments contain 2, 4, 6, 8, or more protein tags in its
structural loops. In some embodiments, said 4'-phosphopantetheinyl
transferase is Sfp, AcpS, T. maritima PPTase, human PPTase, or a
mutant form thereof that retains the 4'-phosphopantetheinyl
transferase activity. In some embodiments, said
4'-phosphopantetheinyl transferase originates from Homo sapiens,
Bacillus subtilis, Escherichia coli, Thermotoga maritima,
Clostridium thermocellum, as well as any other mammalian, bacterial
or fungal genome. In a specific embodiment, said
4'-phosphopantetheinyl transferase is Sfp and the peptide tag is
selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL
(SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19). In some embodiments,
the parental antibody is an IgG, IgM, IgE, or IgA antibody. In a
specific embodiment, the parental antibody is an IgG1 antibody. In
some embodiments, the parental antibody is an IgG2, IgG3, or IgG4
antibody.
[0514] In another aspect, the present invention provides modified
antibodies or antigen binding fragments thereof comprising a
peptide tag that is a substrate of a 4'-phosphopantetheinyl
transferase, and wherein said peptide tag is grafted into a
structural loop, or C- or N-terminus of the antibody or antigen
binding fragment thereof. In some embodiments, said peptide tag is
grafted at amino acid positions from 62 to 64 of the V.sub.H domain
(mutations at amino acids 62 and 63, and insertion of the rest of
the peptide tag between amino acids 63 and 64), at amino acid
positions from 62 to 65 of the V.sub.H domain (mutations at amino
acids 62-64, and insertion of the rest of the peptide tag between
amino acids 64 and 65); at amino acid positions from 133 to 139 of
the CH1 domain (mutations of amino acids 133-138, and insertion of
the rest of the peptide tag between amino acids 138-139), amino
acid positions from 189 to 195 of the CH1 domain, and/or amino acid
positions from 190 to 198 of the CH1 domain (mutations from amino
acids 190-197, and insertion of the rest of the peptide tag between
197 and 198) of a parental antibody or antigen binding fragment
thereof. In one embodiment, said peptide tag is one or more
peptides selected from those described in Table 2. The modified
antibody heavy chain and/or light chain (or antigen binding
fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more
protein tags in its structural loops. In one embodiment, the
modified antibodies or antigen binding fragments contain 2, 4, 6,
8, or more protein tags in its structural loops. In another
embodiment, said 4'-phosphopantetheinyl transferase is Sfp, AcpS,
T. maritima PPTase, human PPTase, or a mutant form thereof that
retains the 4'-phosphopantetheinyl transferase activity. In one
embodiment, said 4'-phosphopantetheinyl transferase originates from
Homo sapiens, Bacillus subtilis, Escherichia coli, Thermotoga
maritima, Clostridium thermocellum, as well as any other mammalian,
bacterial or fungal genome. In some embodiments, said
4'-phosphopantetheinyl transferase is Sfp and the peptide tag is
selected from GDSLSWLLRLLN (SEQ ID NO:1), GDSLSWL (SEQ ID NO:2),
DSLEFIASKLA (SEQ ID NO:9), GDSLDMLEWSLM (SEQ ID NO:10), DSLEFIASKL
(SEQ ID NO:18), and DSLEFIASK (SEQ ID NO:19). In one embodiment,
the parental antibody is an IgG, IgM, IgE, or IgA antibody. In a
specific embodiment, the parental antibody is an IgG1 antibody. In
some embodiments, the parental antibody is an IgG2, IgG3, or IgG4
antibody.
[0515] In certain aspects, the modified antibodies provided herein
are engineered to contain one or more orthogonal conjugation sites.
Such orthogonal conjugation sites include, but are not limited to,
a substrate of Sfp 4'-phosphopantetheinyl transferase, a substrate
of AcpS 4'-phosphopantetheinyl transferase, T. maritima
4'-phosphopantetheinyl transferase, human 4'-phosphopantetheinyl
transferase, a lysine, a cysteine, a tyrosine, a histidine, an
unnatural amino acid, pyrrolysine and pyrroline-carboxy-lysine. The
orthogonal conjugation sites may also be peptide sequences that can
be enzymatically or chemically modified, e.g., a tetracysteine tag,
a LPXTG-sortase peptide (X is any amino acid), a biotin acceptor
peptide, a CXPXR-aldehyde tag (X is any amino acid), or a His tag.
In certain embodiments, such engineered antibodies are labeled
using the methods of the invention in combination with other
conjugation methods known in the art including, but not limited to,
chemoselective conjugation through cysteine, lysine, histidine,
tyrosine, formyl-glycine, pyrrolysine, pyrroline-carboxylysine and
unnatural amino acids.
[0516] In certain aspects, the enzymes Sfp and AcpS are used for
orthogonal site-specific labeling of the same or two different
labels onto an antibody engineered to contain an S-series peptide
(for example, S1, S2, S3, S4, S5, S6 and S7) and an A-series
peptide (for example, A1, A-1, A-2, A-3, A-4 and A-6) located in
the VH, VL, CH1, CH2, CH3, or C.sub.L region of the antibody (see
also Table 2).
[0517] In other aspects, the enzymes Sfp and AcpS are used for
orthogonal site-specific labeling of two different labels onto an
antibody engineered to contain an ybbR-series peptide (for example,
ybbR11, ybbR12 and ybbR13) and an A-series peptide (for example,
A1, A-1, A-2, A-3, A-4 and A-6) located in the CH1, CH2, CH3, or
C.sub.L region of the antibody.
[0518] In other aspects, the enzymes Sfp or AcpS are used for
orthogonal site-specific labeling onto an antibody engineered to
contain an ybbR-series peptide (for example, ybbR11, ybbR12 and
ybbR13) and an A-series peptide (for example, A1, A-1, A-2, A-3,
A-4 and A-6) located in the VH, VL, CH1, CH2, CH3, or C.sub.L
region of the antibody in combination with other conjugation
methods. Such methods include but are not limited to conjugation to
lysine, cysteine, tyrosine, histidine, formyl glycine, unnatural
amino acids, pyrrolysine and/or pyrroline-carboxy-lysine. Such
methods can be used to attached the same or different labels than
used for the enzymatic conjugation through Sfp or AcpS.
Proteins Having 4'-Phosphopantetheinyl Transferase Activity and
Peptide Substrates
[0519] As used herein, the terms "4'-phosphopantetheinyl
transferase" (PPTases) and "protein having 4'-phosphopantetheinyl
transferase activity" are used interchangeably and refer to any
protein or a fragment thereof, which is capable of transferring a
ppan group from a donor molecule, such as coenzyme A (CoA) or an
analog thereof, to a substrate, such as a peptide tag or an acyl
carrier protein.
[0520] PPTases are enzymes which catalyze post-translational
modification of carrier proteins associated with fatty acid
synthases (FASs), polyketide synthases (PKSs) and nonribosomal
peptide synthetases (NRPSs). These carrier proteins are commonly
referred to as ACP, acyl carrier proteins (FASs and PKSs) or to as
PCP, peptidyl carrier proteins (NRPSs). ACPs and PCPs consist of
about 80 amino acids and are usually integrated as domains in FAS,
PKS, or NRPS multienzyme complexes. In some instances, ACPs and
PCPs are also found as free-standing autonomously folded proteins.
The ACP is essential for fatty acid and polyketide biosynthesis,
because it carries the corresponding metabolic intermediates via
covalent attachment to its flexible ppan prosthetic group. The PCP
carries out the analogous function in nonribosomal peptide
synthesis by transporting peptide intermediates between active
sites in NRPS multienzyme complexes. PPTases have been classified
into three groups, based on sequence and structural similarity and
substrate specificity. Members of the first group of PPTases, for
example, AcpS of Escherichia coli, are about 120 amino acid
residues long, function as homotrimers, and have fairly narrow
substrate specificities limited to, for example, to the acetyl
carrier proteins (ACPs) of type II FAS and PKS systems. Members of
the second group, exemplified by Sfp of Bacillus subtilis or the
human PPTase, function as monomers, and have been reported to have
broad substrate specificities that include carrier proteins
associated with NRPs, FASs and PKSs. (see, e.g., Suo et al., Proc.
Natl. Acad. Sci. USA3 98:99-104, 2001; Quadri et al., Biochem.,
37:1585-95, 1998; Liu et al., Arch. Microbiol, 183:37-44, 2005;
Joshi et al., J. Biol. Chem., 278:33142-33149, 2003). The third
group includes PPTases that are attached covalently to the type I
FASs, such as those associated with the yeast cytosolic FAS. (see,
e.g., Fichtlscherer et al., Eur. J. Biochem., 267:2666-71,
2000).
[0521] According to the present invention, PPTases include
naturally occurring proteins having 4'-phosphopantetheinyl
transferase activity, including but not limited to, AcpS from E.
coli (type I PPTase) and Sfp from B. subtilis (type II PPTase),
integrated PPTase domains (type III PPTase) associated with fatty
acid synthases (FAS) from S. cerevisiae, S. pombe, C. albacans, E.
nidulans, and P. patulum, EntD from E. coli, S. flexneri, S.
typhimurium and S. austin, Psf-1 from B. pumilus, Gsp from B.
brevis, Hetl from Anabaena sp., Lys5 from S. cerevisiae, Lpa-14
from B. subtilis and 0195 from E. coli, PPTase (NP_228501) of T.
maritima MSB8, PPTase (NP_056238) of Homo sapiens, and homologs and
mutants thereof. PPTases of the present invention also include
proteins having 4'-phosphopantetheinyl transferase activity from
species other than the ones described above, as well as those
artificially or recombinantly produced proteins, which are capable
of 4'-phospopantetheinylating a peptide moiety described herein.
Sfp and AcpS represent two classes of 4'-phosphopantetheinyl
transferases that show differences both in their substrate
specificity for the carrier protein domains and in their structures
(Flugal et al., J. Biol. Chem., 275:959-968, 2000; Lambalot et al.,
Chem. Biol., 3:923-936, 1996). The Sfp class of pseudodimeric
PPTases are about 230 residues in size and the crystal structure of
Sfp suggests it has a twofold symmetry with the N- and the
C-terminal halves of the molecule adopting similar folds, with the
active site of the enzyme at the interface (Hodneland et al., Proc.
Natl. Acad. Sci. USA, 99:5048-5052, 2002; Koglin et al., Science,
312:273-276, 2006). In contrast, AcpS is about 120 residues in
length, about half the size of Sfp, and the crystal structures of
AcpS show that the enzyme assembles into trimers and the ACP and
CoA binding sites are formed at the interface between each monomer
(Reuter et al., Embo. J., 18:6823-6831, 1999; Chirgadze et al.,
Embo. J., 19:5281-5287, 2000). It has been reported that Sfp
exhibits a much broader substrate specificity than AcpS in that Sfp
can modify both PCP and ACP domains from nonribosomal peptides
synthetases, polyketide synthases, and fatty acid synthases, while
AcpS modifies only ACP (Flugel et al., J. Biol. Chem., 275:959-968,
2000; Parris et al., Structure, 8:883-895, 2000; Mofid et al., J.
Biol. Chem., 277:17023-17031, 2002).
[0522] ACP and PCP substrates of both kinds of PPTases adopt
similar folds as four-helix bundle proteins with the serine residue
to be modified by the ppan prosthetic group at the top of the
second alpha-helix, which has been shown to play an important role
for interacting with Sfp and AcpS (Hodneland et al., Proc. Natl.
Acad. Sci. USA, 99:5048-5052, 2002; Chirgadze et al., Embo. J.,
19:5281-5287, 2000; Quadri et al., Biochem., 37:1585-1595, 1998; Li
et al., Biochem., 42:4648-4657, 2003). Although there is not an
obvious consensus sequence difference between PCPs and ACPs, the
most significant difference between the two is the electrostatic
surface potential of the carrier proteins, with a neutral protein
surface for PCPs and a negatively charged acidic surface for ACP
domains in FAS and PKS systems (Parris et al., Structure,
8:883-895, 2000).
[0523] Groups of short peptides have been identified as efficient
substrates for PPTases. For example, ybbR13 is an 11 amino acid
residue peptide, which is a substrate of Sfp (J. Yin et al., Proc.
Natl. Acad. Sci. USA, 102:15815-15820, 2005; Z. Zhou et al., ACS
Chem Biol., 2:337-346, 2007; Z. Zhou et al., J. Am. Chem. Soc.,
130: 9925-9930, 2008). The ybbR13 peptide (DSLEFIASKLA) was
isolated from a phage displayed library of the B. subtilis genome
(J. Yin et al., Proc. Natl. Acad. Sci. USA, 102:15815-15820, 2005).
A part of the sequence of the ybbR13 peptide is derived from a B.
subtilis open reading frame, called ybbR, and it includes the
(H/D)S(L/I) tri-peptide sequence at the N-terminus, which is
conserved in known substrates of PPTases such as ACPs, PCPs, and
aryl carrier proteins (ArCPs). The ybbR peptide does not include
the amino acid sequence, DxFFxxLGG at its N-terminus, which is
found to be conserved in PCPs. Modifications and variants of the
ybbR13 peptide have been described which can be used as substrates
in 4'-phosphopantetheinylation reactions for site specific labeling
(J. Yin et al., Proc. Natl. Acad. Sci. USA, 102:15815-15820, 2005).
Additional peptide substrates for PPTases are the S series of
peptides and the A series of peptides, designated as "S" or "A"
based on their reactivity with Sfp or AcpS, respectively (Z. Zhou
et al. ACS Chem Biol., 2:337-346, 2007 and Z. Zhou et al. J. Am.
Chem. Soc., 130:9925-9930, 2008). Exemplary S series of peptides
include, but are not limited to, S6, which is an efficient
substrate for Sfp, and exemplary A series of peptides include, but
are not limited to, A1, which is an efficient substrate for AcpS.
Both S6 and A1 peptides are 12 amino acid residues in length.
[0524] Examples of peptide substrates are listed in Table 2 below.
According to the present invention, these short peptide tags can be
used for the site-specific labeling of target proteins (including
antibodies) in reactions catalyzed by PPTases. Additionally, a
pairing of peptide tags and respective PPTases described herein,
e.g., ybbR13/Sfp or S6/Sfp and A1/AcpS, can also be used for
orthogonal site-specific labeling of one (or multiple) target
proteins, e.g., in cell lysates or on the surface of live
cells.
TABLE-US-00002 TABLE 2 PPTase peptide substrate examples. The
modified serine residue is underlined. SEQ ID Sequence NO: Name
GDSLSWLLRLLN 1 S6 GDSLSWL 2 S6 truncate GDSLSWLVRCLN 3 S1
GDSLSWLLRCLN 4 S2 GDSLSWLVRLLN 5 S3 GDSLSWLLRSLN 6 S7
GSQDVLDSLEFIASKLA 7 Ybbr11 VLDSLEFIASKLA 8 Ybbr12 DSLEFIASKLA 9
Ybbr13 GDSLDMLEWSLM 10 A1 GDSLDMLEWSL 11 A-1 GDSLDMLEWS 12 A-2
GDSLDMLEW 13 A-3 DSLDMLEW 14 A-4 GDSLDM 15 A-6 LDSVRMMALAAR 16 E0
LDSLDMLEWSLR 17 E2 DSLEFIASKL 18 ybbR truncate 1 DSLEFIASK 19 ybbR
truncate 2 DVLDSLEFI 20 ybbR8 VLDSLEFIAS 21 ybbR14
[0525] Accordingly, the present invention provides engineered
antibodies which contain one or more of the peptide tags listed in
Table 2, and methods of labeling such antibodies, e.g., conjugating
with a cytotoxin. The labeling chemistry is illustrated below and
in the Examples.
2. Labeling Chemistry
[0526] The modified antibody or antigen binding fragment thereof
provided herein are site-specifically labeled by post-translational
modification of the short peptide tag (inserted or grafted or
combination thereof) using PPTases or mutants thereof, including,
but not limited to, Sfp, AcpS, human PPTase or T. maritima PPTase.
Such post-translational modifications involve a PPTase catalyzed
reaction between a conserved serine residue in the short peptide
tag and a 4'-phosphopantetheinyl (ppan) group of coenzyme A (CoA)
or a coenzyme A analogue. In this reaction, the ppan prosthetic
group of CoA, or modified ppan prosthetic group of the CoA
analogue, is attached to the short peptide tag through the
formation of a phosphodiester bond with the hydroxyl group of the
conserved serine residue of the short peptide tag which has been
incorporated (i.e. inserted or grafted or combination thereof) into
the antibody. The ppan or modified ppan is linked to a terminal
group (TG) and the formation of the phosphodiester bond thereby
conjugates the terminal group (TG) to the modified antibody or
antigen binding fragment thereof via a linker which includes the
ppan or modified ppan moiety.
[0527] In certain embodiments the modified antibodies or antigen
binding fragment thereof provided herein are labeled by a one-step
method wherein the post-translational modification occurs by
reacting a CoA linked to a terminal group (TG), or a CoA analogue
linked to a terminal group (TG), with the conserved serine of the
short peptide tag engineered into the antibody, as shown in Schemes
(Ia)-(Ic) below. Alternatively, in other embodiments of the
post-translational modification of the modified antibodies or
antigen binding fragment thereof provided herein, the modified
antibodies or antigen binding fragment thereof are labeled by a
two-step method wherein the post-translational modification
involves first reacting an activated CoA or an activated CoA
analogue with the conserved serine of the short peptide tag
engineered into the antibody, followed by reacting a functionalized
terminal group (TG) with the reactive group on the activated CoA or
an activated CoA. Such two-step methods are illustrated in Schemes
(IIa)-(IIf) below. In other embodiments of the post-translational
modification of the modified antibodies or antigen binding fragment
thereof provided herein, the modified antibodies or antigen binding
fragment thereof are labeled by a three-step method, whereinthe
post-translational modification involves first reacting a CoA
having a protected ppan prosthetic group, or a CoA analogue having
protected ppan prosthetic group, with the conserved serine of the
short peptide tag engineered into the antibody, thereby attaching
the CoA or CoA analogue to the antibody. In the second step the
protected ppan prosthetic group is deprotected thereby generating a
reactive functional group on the protected ppan prosthetic group.
In the third step, this reactive functional group is linked to a
terminal group (TG), thereby attaching the terminal group to the
modified antibody or antigen binding fragment thereof. Such
three-step methods are illustrated in Schemes (IIIa)-(IIIf)
below
One-Step Method
[0528] The One-step method used to label the modified antibodies or
antigen binding fragment thereof provided herein is shown in Scheme
(Ia):
##STR00110## [0529] where: [0530] R.sub.2 is H or
--P(.dbd.O)(OH).sub.2; [0531] Linker Unit (LU) is a chemical moiety
that links the terminal group (TG) to the modified ppan prosthetic
group of the CoA analogue and [0532] terminal group (TG) is a drug
moiety selected from an anti-cancer agent, an anti-inflammatory
agent, an antifungal agent, an antibacterial agent, an
anti-parasitic agent, an anti-viral agent, and an anesthetic agent,
a biophysical probe, a fluorophore, an affinity probe, a chelator,
a spectroscopic probe, a radioactive probe, a lipid molecule, a
polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein,
a peptide, an antibody, an antibody fragment, a nanoparticle, a
quantum dot, a liposome, a PLGA particle, a polysaccharide, or a
surface. [0533] In certain embodiments the Linker Unit (LU)
comprises a linker selected from a non-enzymatically cleavable
linker, a non-cleavable linker, an enzymatically cleavable linker,
a photo stable linker, a photo-cleavable linker or any combination
thereof, and the Linker Unit (LU) optionally contains a
self-immolative spacer. [0534] In certain embodiments the Linker
Unit (LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0535]
L.sub.1 is a bond, a non-enzymatically cleavable linker, a
non-cleavable linker, an enzymatically cleavable linker, a photo
stable linker or a photo-cleavable linker; [0536] L.sub.2 is a
bond, a non-enzymatically cleavable linker, a non-cleavable linker,
an enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker; [0537] L.sub.3 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker, and [0538] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer. [0539] In
certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0540] L.sub.1 is a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker; [0541] L.sub.2 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker; [0542] L.sub.3 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker, and [0543] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer. [0544] In
certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0545] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; where: [0546]
A.sub.1 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m, --(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0547] each X.sup.2 is independently selected from a bond,
##STR00111## ##STR00112## ##STR00113##
[0547] --S--, --Si(OH).sub.2O--,
##STR00114##
[0548] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0549] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0550] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0551] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0552] R.sup.7 is
independently selected from H, phenyl and pyridine; [0553] R.sup.8
is independently selected from
[0553] ##STR00115## [0554] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0555] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0556] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0557]
L.sub.2 is a bond, a non-enzymatically cleavable linker, a
non-cleavable linker, an enzymatically cleavable linker, a photo
stable linker or a photo-cleavable linker; [0558] L.sub.3 is a
bond, a non-enzymatically cleavable linker, a non-cleavable linker,
an enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker, and [0559] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer. [0560] In
certain embodiments, L.sub.1 is
C(.dbd.O)--CH.sub.2CH.sub.2--NH--C(.dbd.O)--CH.sub.2CH.sub.2--S--,
so LU is
--C(.dbd.O)--CH.sub.2CH.sub.2--NH--C(.dbd.O)--CH.sub.2CH.sub.2--S-L.su-
b.2-L.sub.3-L.sub.4-. [0561] In certain embodiments the Linker Unit
(LU) is -L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0562] L.sub.1
is a bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; where:
[0563] A.sub.1 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--, --((CH.sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--, or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0564]
each X.sup.2 is independently selected from a bond,
##STR00116## ##STR00117## ##STR00118##
[0564] --S--, --Si(OH).sub.2O--,
##STR00119##
[0565] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0566] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0567] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0568] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0569] R.sup.7 is
independently selected from H, phenyl and pyridine; [0570] R.sup.8
is independently selected from
[0570] ##STR00120## [0571] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0572] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0573] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0574]
L.sub.2 is a bond, a non-enzymatically cleavable linker, a
non-cleavable linker, an enzymatically cleavable linker, a photo
stable linker or a photo-cleavable linker; [0575] L.sub.3 is a
bond, a non-enzymatically cleavable linker, a non-cleavable linker,
an enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker; [0576] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer. [0577] In
certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0578] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; where: [0579]
A.sub.1 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0580]
each X.sup.2 is independently selected from a bond,
##STR00121## ##STR00122## ##STR00123##
[0580] --S--, --Si(OH).sub.2O--,
##STR00124##
[0581] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0582] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0583] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0584] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0585] R.sup.7 is
independently selected from H, phenyl and pyridine; [0586] R.sup.8
is independently selected from
[0586] ##STR00125## [0587] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0588] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0589] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0590]
L.sub.2 is a bond, a non-enzymatically cleavable linker, a
non-cleavable linker, an enzymatically cleavable linker, a photo
stable linker or a photo-cleavable linker; [0591] L.sub.3 is a
bond, a non-enzymatically cleavable linker, a non-cleavable linker,
an enzymatically cleavable linker, a photo stable linker or a
photo-cleavable linker, and [0592] L.sub.4 is a bond, a
non-enzymatically cleavable linker, a non-cleavable linker, an
enzymatically cleavable linker, a photo stable linker, a
photo-cleavable linker or a self-immolative spacer. [0593] In
certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0594] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; where: [0595]
A.sub.1 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; each
X.sup.2 is independently selected from a bond,
##STR00126## ##STR00127## ##STR00128##
[0595] --S--, --Si(OH).sub.2O--,
##STR00129##
[0596] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0597] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0598] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0599] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0600] R.sup.7 is
independently selected from H, phenyl and pyridine; [0601] R.sup.8
is independently selected from
[0601] ##STR00130## [0602] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0603] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0604] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; [0605]
L.sub.2 is a bond, a non-enzymatically cleavable linker or a
non-cleavable linker; [0606] L.sub.3 is a bond, a non-enzymatically
cleavable linker or a non-cleavable linker; [0607] L.sub.4 is a
bond, an enzymatically cleavable linker or a self-immolative
spacer. [0608] In certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0609] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; [0610] L.sub.2
is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0611] L.sub.3 is a
bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0612] L.sub.4 is a bond,
-A.sub.4-, -A.sub.4X.sup.2--,
[0612] ##STR00131## ##STR00132## [0613] A.sub.1 is --C(.dbd.O)NH--,
--NHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0614] A.sub.2 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NR.sup.4--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--, --(CH.sub.2).sub.nNH--,
--(C(R.sup.4).sub.2).sub.nNH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--, (C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0614] ##STR00133## [0615] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--(CH.sub.2).s-
ub.nC(.dbd.O)NH--, --(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--, --(CH.sub.2).sub.nS--,
--(C(R.sup.4).sub.2).sub.nS--, --S(CH.sub.2).sub.n--,
--S(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)NH(CH.sub.2).sub.n---
,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)NH(-
C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mOC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mOC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mC(.dbd.O)--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
[0615] ##STR00134## [0616] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m--,
--(O(C(R.sup.4).sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--(((C(R.sup.4).sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(((C(R.sup.4).sub.2).sub.nO).sub.mC(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--(CH.sub.2).sub.nNHC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)--,
--NHC(.dbd.O)(CH.sub.2).sub.n--,
--NHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--S(C(R.sup.4).sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)NH(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--
-, --C(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)--,
--(C(R.sup.4).sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.n(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(-
R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(C(R.sup.4).sub.2).sub.nNH((C(R.sup.4).sub.2).sub.nO).sub.m(C(R.sup.4).-
sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--, or
--(O(C(R.sup.4).sub.2).sub.n).sub.mNHC(.dbd.O)(C(R.sup.4).sub.2).sub.n-
--; [0617] each X.sup.2 is independently selected from a bond,
##STR00135## ##STR00136## ##STR00137##
[0617] --S--, --Si(OH).sub.2O--,
##STR00138##
[0618] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0619] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0620] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0621] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0622] R.sup.7 is
independently selected from H, phenyl and pyridine; [0623] R.sup.8
is independently selected from
[0623] ##STR00139## [0624] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0625] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0626] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9. [0627] In
certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0628] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; [0629] L.sub.2
is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0630] L.sub.3 is a
bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0631] L.sub.4 is a bond,
-A.sub.4-, -A.sub.4X.sup.2--,
[0631] ##STR00140## [0632] A.sub.1 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --(O(CH.sub.2).sub.n).sub.m,
--((CH.sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--, --NHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0633]
A.sub.2 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.m, --((CH.sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--, --NHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0633] ##STR00141## [0634] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--, --NHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0634] ##STR00142## [0635] A.sub.4-C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nC(.dbd.O)NH--, --NHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--S(CH.sub.2).sub.nC(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--C(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nC(.dbd.O)--,
--(CH.sub.2).sub.n(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0636]
each X.sup.2 is independently selected from a bond,
##STR00143##
[0636] --S--, --Si(OH).sub.2O--,
##STR00144##
[0637] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0638] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0639] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0640] each n
is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and
[0641] each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8
and 9. [0642] In certain embodiments the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-, wherein [0643] L.sub.1 is a
bond, -A.sub.1-, -A.sub.1X.sup.2-- or --X.sup.2--; [0644] L.sub.2
is a bond, -A.sub.2-, or -A.sub.2X.sup.2--; [0645] L.sub.3 is a
bond, -A.sub.3-, or -A.sub.3X.sup.2--; [0646] L.sub.4 is a bond,
-A.sub.4-, -A.sub.4X.sup.2--, H
[0646] ##STR00145## [0647] A.sub.1 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; [0648]
A.sub.2 is --C(.dbd.O)NH--, --C(.dbd.O)NH(CH.sub.2).sub.n--,
--C(.dbd.O)NH(CH.sub.2).sub.nS--, --(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0648] ##STR00146## [0649] A.sub.3 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m--,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n-- or
[0649] ##STR00147## [0650] A.sub.4 is --C(.dbd.O)NH--,
--C(.dbd.O)NH(CH.sub.2).sub.n--, --C(.dbd.O)NH(CH.sub.2).sub.nS--,
--(O(CH.sub.2).sub.n).sub.m,
--((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n--,
--NHC(.dbd.O)(CH.sub.2).sub.n--, --(CH.sub.2).sub.nNHC(.dbd.O)--,
--C(.dbd.O)NH(CH.sub.2).sub.nNHC(.dbd.O)(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nNH((CH.sub.2).sub.nO).sub.m(CH.sub.2).sub.n-- or
--(O(CH.sub.2).sub.n).sub.mNHC(.dbd.O)(CH.sub.2).sub.n--; each
X.sup.2 is independently selected from a bond,
##STR00148## ##STR00149## ##STR00150##
[0650] --S--, --Si(OH).sub.2O--,
##STR00151##
[0651] --CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--,
--CHR.sup.4(CH.sub.2).sub.nNHC(.dbd.O)--, --C(.dbd.O)NH-- and
--NHC(.dbd.O)--; [0652] each R.sup.4 is independently selected from
H, C.sub.1-4alkyl, --C(.dbd.O)OH and --OH, [0653] each R.sup.5 is
independently selected from H, C.sub.1-4alkyl, phenyl or
C.sub.1-4alkyl substituted with 1 to 3 --OH groups; [0654] each
R.sup.6 is independently selected from H, fluoro, benzyloxy
substituted with --C(.dbd.O)OH, benzyl substituted with
--C(.dbd.O)OH, C.sub.1-4alkoxy substituted with --C(.dbd.O)OH and
C.sub.1-4alkyl substituted with --C(.dbd.O)OH; [0655] R.sup.7 is
independently selected from H, phenyl and pyridine; [0656] R.sup.8
is independently selected from
[0656] ##STR00152## [0657] R.sup.9 is independently selected from H
and C.sub.1-6haloalkyl; [0658] each n is independently selected
from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and [0659] each m is
independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.
[0660] In certain embodiments of any of the compounds or methods
described herein, L.sub.1 is
--C(.dbd.O)--NH--CH.sub.2--CH.sub.2--S-[L.sub.2-L.sub.3-L.sub.4-TG].
(Portions of these formulas depicted in brackets such as
[L.sub.2-L.sub.3-L.sub.4-TG] are added to the formula being
described in order to identify which open valence of the formula is
attached to the bracket-enclosed part of the remainder of the
structure.)
[0661] In certain embodiments of any of the compounds or methods
described herein, L.sub.2 is selected from:
##STR00153##
[0662] In certain embodiments of any of the compounds or methods
described herein, L.sub.3 is selected from
--(CH.sub.2).sub.2-6--C(.dbd.O)--[L.sub.4-TG];
--(CH.sub.2).sub.2-6--NH-[L.sub.4-TG];
--(CH.sub.2).sub.2-6--S-[L.sub.4-TG];
--(CH.sub.2).sub.2-6--Z-[L.sub.4-TG]; and
--(CH.sub.2).sub.2-6--Z--C(.dbd.O)--[L.sub.4-TG], where Z is O, NH
or S.
[0663] In certain embodiments of any of the compounds or methods
described herein, L.sub.4 is a bond or a val-cit linker of this
formula:
##STR00154##
When L.sub.4 is a val-cit linker, L.sub.3 is preferably
--(CH.sub.2).sub.2-6--C(.dbd.O)--.
[0664] In certain embodiments of any of the compounds or methods
described herein, TG is a maytansinoid such as DM1 or DM4, or a
dolostatin 10 compound, e.g. auristatins MMAF or MMAE, or a
calicheamicin such as N-acetyl-.gamma.-calicheamicin, or a label or
dye such as rhodamine or tetramethylrhodamine.
[0665] As used herein, a "linker" is any chemical moiety that is
capable of linking an antibody or a fragment thereof to a terminal
group. Linkers can be susceptible to cleavage, such as,
acid-induced cleavage, light-induced cleavage, peptidase-induced
cleavage, esterase-induced cleavage, and disulfide bond cleavage,
at conditions under which the compound or the antibody remains
active. Alternatively, linkers can be substantially resistant to
cleavage. A linker may or may not include a self-immolative
spacer.
[0666] Non-limiting examples of the non-enzymatically cleavable
linkers as used herein to conjugate a terminal group (TG) to the
modified antibodies or antigen binding fragment thereof provided
herein include, acid-labile linkers, linkers containing a disulfide
moiety, linkers containing a triazole moiety, linkers containing a
hydrazine moiety, linkers containing a thioether moiety, linkers
containing a diazo moiety, linkers containing an oxime moiety,
linkers containing an amide moiety and linkers containing an
acetamide moiety.
[0667] Non-limiting examples of the enzymatically cleavable linkers
as used herein to conjugate a terminal group (TG) to the modified
antibodies or antigen binding fragment thereof provided herein
include, but are not limited to, linkers which are cleaved by a
protease, linkers which are cleaved by an amidase, and linkers
which are cleaved by 3-glucuronidase.
[0668] In certain embodiments, such enzyme cleavable linkers are
linkers which are cleaved by cathepsin, including cathepsin Z,
cathepsin B, cathepsin H and cathepsin C. In certain embodiments
the enzymatically cleavable linker is a dipeptide cleaved by
cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin
B, cathepsin H or cathepsin C. In certain embodiments the
enzymatically cleavable linker is a cathepsin B-cleavable peptide
linker. In certain embodiments the enzymatically cleavable linker
is a cathepsin B-cleavable dipeptide linker. In certain embodiments
the enzymatically cleavable linker is a cathepsin B-cleavable
dipeptide linker is valine-citrulline or phenylalanine-lysine.
Other non-limiting examples of the enzymatically cleavable linkers
as used herein conjugate a terminal group (TG) to the modified
antibodies or antigen binding fragment thereof provided herein
include, but are not limited to, linkers which are cleaved by
.beta.-glucuronidase, e.g.,
##STR00155##
See Ducry et al, Bioconjugate Chem, vol. 21(1), 5-13 (2010).
[0669] "Self-immolative spacers" are bifunctional chemical moieties
covalently linked at one termini to a first chemical moiety and at
the other termini to a second chemical moiety, thereby forming a
stable tripartate molecule. Upon cleavage of a bond between the
self-immolative spacer and the first chemical moiety,
self-immolative spacers undergoing rapid and spontaneous
intramolecular reactions and thereby separate from the second
chemical moiety. These intramolecular reactions generally involve
electronic rearrangements such as 1,4, or 1,6, or 1,8 elimination
reactions or cyclizations to form highly favored five- or
six-membered rings. In certain embodiments of the present
invention, the first moiety is an enzyme cleavable linker and this
cleavage results from an enzymatic reaction, while in other
embodiments the first moiety is an acid labile linker and this
cleavage occurs due to a change in pH. As applied to the present
invention, the second moiety is the "Label" group as defined
herein. In certain embodiments, cleavage of the first moiety from
the self-immolative spacer results from cleavage by a proteolytic
enzyme, while in other embodiments it results from cleaved by a
hydrolase. In certain embodiments, cleavage of the first moiety
from the self-immolative spacer results from cleavage by a
cathepsin enzyme.
[0670] In certain embodiments, the enzyme cleavable linker is a
peptide linker and the self-immolative spacer is covalently linked
at one of its ends to the peptide linker and covalently linked at
its other end to a drug moiety. This tripartite molecule is stable
and pharmacologically inactive in the absence of an enzyme, but
which is enzymatically cleavable by enzyme at the bond covalently
linking the spacer moiety and the peptide moiety. The peptide
moiety is cleaved from the tripartate molecule which initiates the
self-immolating character of the spacer moiety, resulting in
spontaneous cleavage of the bond covalently linking the spacer
moiety to the drug moiety, to thereby effect release of the drug in
pharmacologically active form.
[0671] Non-limiting examples of the self-immolative spacer
optionally used in the conjugation of a terminal group (TG) to the
modified antibodies or antigen binding fragment thereof provided
herein include, but are not limited to, moieties which include a
benzyl carbonyl moiety, a benzyl ether moiety, a 4-aminobutyrate
moiety, a hemithioaminal moiety or a N-acylhemithioaminal
moiety.
[0672] Other examples of self-immolative spacers include, but are
not limited to, p-aminobenzyloxycarbonyl groups, aromatic compounds
that are electronically similar to the p-aminobenzyloxycarbonyl
group, such as 2-aminoimidazol-5-methanol derivatives and ortho or
para-aminobenzylacetals. In certain embodiments, self-immolative
spacers used herein which undergo cyclization upon amide bond
hydrolysis, include substituted and unsubstituted 4-aminobutyric
acid amides and 2-aminophenylpropionic acid amides.
[0673] In certain embodiments, the self-immolative spacer is
##STR00156##
while in other embodiments the self-immolative spacer is
##STR00157##
where n is 1 or 2. In other embodiments the self-immolative spacer
is
##STR00158##
where n is 1 or 2. In other embodiments the self-immolative spacer
is
##STR00159##
where n is 1 or 2. In other embodiments the self-immolative spacer
is
##STR00160##
where n is 1 or 2. In other embodiments the self-immolative spacer
is
##STR00161##
where n is 1 or 2.
[0674] Scheme (Ib) illustrates the post-translational modification
of the modified antibodies or antigen binding fragment thereof
provided herein wherein the Linker Unit (LU) is
-L.sub.1-L.sub.2-L.sub.3-L.sub.4-.
##STR00162##
where R.sub.2, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and TG are as
defined herein.
[0675] The CoA analogues of Scheme (Ia) and Scheme (Ib) may be
obtained by total chemical synthesis, however the CoA analogues of
Scheme (Ia) and Scheme (Ib) are preferably obtained by a
chemoenzymatic process wherein pantetheine analogues are chemically
synthesized and then biosynthetically converted into the
corresponding CoA analogue (see Kristine M. Clarke et al., "In Vivo
Reporter Labeling of Proteins via Metabolic Delivery of Coenzyme A
Analogues", J. Am. Chem. Soc., 2005, 127, p. 11234-11235 and Jordan
L. Meier et al., "Synthesis and Evaluation of Bioorthogonal
Pantetheine Analogues for in Vivo Protein Modification", J. Am.
Chem. Soc., 2006, 128, p. 12174-12184). The biosynthetic conversion
for CoA analogues of Scheme (Ia) is shown below:
##STR00163##
while the biosynthetic conversion for CoA analogues of Scheme (Ib)
is shown below:
##STR00164##
where LU, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and TG are as defined
herein.
[0676] In certain embodiments the biosynthetic conversion occurs
"in-vivo", wherein the pantetheine analogue enters a cell from the
surrounding media whereby once inside the cell it is converted by
the CoA enzymatic pathway into the corresponding CoA analogue. In a
specific embodiment, E. coli is used for the biosynthetic
conversion of pantetheine analogues into the corresponding CoA
analogues, wherein the pantetheine analogue enters E. coli from the
surrounding media and once inside the cell the pantetheine analogue
is initially phosphorylated by the pantothenate kinase (PanK or
CoaA) using adenosine-5'-triphosphate (ATP), then adenylated by the
phosphopantetheine adenylyltransferase (PPAT or CoaD) to give the
dephospho-CoA analogue and then further phosphorylated by the
dephosphocoenzyme A kinase (DPCK or CoaE) to yield the CoA
analogue.
[0677] In other embodiments the biosynthetic conversion occurs
"in-vitro", wherein the enzymatic CoA pathway is reconstituted with
the pantetheine analogue, whereby it is converted "in-vitro" by the
reconstituted CoA enzymatic pathway into the corresponding CoA
analogue. In a specific embodiment of "in-vitro" conversion, the
reconstituted CoA enzymatic pathway is the E. coli CoA enzymatic
pathway, wherein the pantetheine analogue is initially
phosphorylated by CoaA and ATP, then adenylated by CoaD to give the
dephospho-CoA analogue and then further phosphorylated by CoaE to
yield the CoA analogue.
[0678] In certain embodiments the Linker Unit (LU) is
--C(.dbd.O)NH(CH.sub.2).sub.2S-L.sub.2-L.sub.3-L.sub.4- and R.sub.2
is --P(.dbd.O)(OH).sub.2, and in such an embodiment the terminal
group is linked to CoA. Scheme (Ic) illustrates the
post-translational modification of the modified antibodies or
antigen binding fragment thereof provided herein for the specific
embodiment wherein the PPTase catalyzes the reaction between the
conserved serine residue in the incorporated short peptide tag and
a terminal group (TG) linked to coenzyme A (CoA):
##STR00165##
where L.sub.2, L.sub.3, L.sub.4 and TG are as defined herein
[0679] In certain embodiments, the modified antibodies or antigen
binding fragment thereof provided herein are site-specifically
labeled by a one-step method as shown in Scheme (Ia), Scheme (Ib)
and Scheme (Ic), wherein a terminal group linked to CoA or a CoA
analogue reacts with the conserved serine of the short peptide tag
engineered into the antibody.
The one step method includes the steps of: [0680] (a) providing a
modified antibody or antigen binding fragment thereof which has
been engineered to contain a small peptide tag, and wherein the
peptide tag is a substrate of an enzyme having
4'-phosphopantetheinyl transferase activity, and [0681] (b)
labeling the modified antibody or antigen binding fragment thereof
with a terminal group by incubating the modified antibody or
antigen binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound having the structure of Formula (A):
[0681] ##STR00166## [0682] wherein: [0683] R.sub.2, Linker Unit
(LU) and TG are as described herein.
[0684] In such One-Step methods using a compound of Formula (A) the
terminal group (TG) is thereby conjugated to the modified antibody
or antigen binding fragment thereof via a linker having the
structure according to Formula (I-a). The linker of Formula (I-a)
is attached to the small peptide tag by a phosphodiester bond
formed between the 4'-phosphopantetheinyl moiety and the hydroxyl
group of the conserved serine residue of the short peptide tag
engineered into the antibody:
##STR00167##
where LU is as defined herein and the * denotes that the
4'-phosphopantetheinyl moiety is attached to the small peptide
tag.
[0685] In certain embodiments, the one step method includes the
steps of: [0686] (a) providing a modified antibody or antigen
binding fragment thereof which has been engineered to contain a
small peptide tag, and wherein the peptide tag is a substrate of an
enzyme having 4'-phosphopantetheinyl transferase activity, and
[0687] (b) labeling the modified antibody or antigen binding
fragment thereof with a terminal group by incubating the modified
antibody or antigen binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound having the structure of Formula (B):
##STR00168##
[0687] where R.sub.2, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and TG are
as defined herein.
[0688] In such One-Step methods using a compound of Formula (B)
described above the terminal group is thereby attached to the
modified antibody or antigen binding fragment thereof via a linker
having the structure according to Formula (I-b). The linker of
Formula (I-b) is attached to the small peptide tag by a
phosphodiester bond formed between the 4'-phosphopantetheinyl
moiety and the hydroxyl group of the conserved serine residue of
the short peptide tag engineered into the antibody:
##STR00169##
where L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are as defined herein
and the * denotes that the 4'-phosphopantetheinyl moiety is
attached to the small peptide tag. In other embodiments, the one
step method includes the steps of: [0689] (a) providing a modified
antibody or antigen binding fragment thereof which has been
engineered to contain a small peptide tag, and wherein the peptide
tag is a substrate of an enzyme having 4'-phosphopantetheinyl
transferase activity, and [0690] (b) labeling the modified antibody
or antigen binding fragment thereof with a terminal group by
incubating the modified antibody or antigen binding fragment
thereof with an enzyme having 4'-phosphopantetheinyl transferase
activity in the presence of a compound having the structure of
Formula (C):
##STR00170##
[0690] where L.sub.2, L.sub.3, L.sub.4 and TG are as defined
herein.
[0691] In such One-Step methods using a compound of Formula (C)
described above the terminal group is thereby attached to the
modified antibody or antigen binding fragment thereof via a linker
having the structure according to Formula (I-c). The linker of
Formula (I-c) is attached to the small peptide tag by a
phosphodiester bond formed between the 4'-phosphopantetheinyl
moiety and the hydroxyl group of the conserved serine residue of
the short peptide tag engineered into the antibody:
##STR00171##
where L.sub.2, L.sub.3 and L.sub.4 are as defined herein and the *
denotes that the 4'-phosphopantetheinyl moiety is attached to the
small peptide tag.
[0692] In certain embodiments of the One-Step Methods described
herein, the modified antibody or antigen binding fragment thereof
is contacted with a compound having the structure of Formula (A),
Formula (B) or Formula (C) and a 4'-phosphopantetheinyl transferase
enzyme that is co-expressed in the same cell as the expressed
modified antibody or antigen binding fragment thereof. In certain
embodiments of the One-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted in the
cell culture media with a compound having the structure of Formula
(A), Formula (B) or Formula (C) and 4'-phosphopantetheinyl
transferase enzyme produced in the same or in another cell. In
certain embodiments of the One-Step Methods described herein, the
4'-phosphopantetheinyl transferase enzyme is immobilized on solid
support. In certain embodiments the solid support is optionally
comprised of a polymer on a bead or a column.
[0693] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is a bond, L.sub.4 is -A.sub.4-, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.4 is
--(CH.sub.2).sub.nNHC(.dbd.O)--, and X.sup.2 is
##STR00172##
[0694] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is a bond, L.sub.4 is -A.sub.4-, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.4 is
--(CH.sub.2).sub.nNHC(.dbd.O)--; X.sup.2 is
##STR00173##
and TG is a fluorescent probe.
[0695] In certain embodiments of the compound of Formula (B) is
##STR00174##
[0696] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is a bond, L.sub.4 is -A.sub.4-, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.4 is
--(CH.sub.2).sub.nC(.dbd.O)--, and X.sup.2 is
##STR00175##
[0697] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is a bond, L.sub.4 is -A.sub.4-, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.4 is
--(CH.sub.2).sub.nC(.dbd.O)--; X.sup.2 is
##STR00176##
and TG is a drug moiety.
[0698] In certain embodiments the compound of Formula (B) is
##STR00177##
[0699] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is -A.sub.2-, L.sub.3 is -A.sub.3-, L.sub.4 is
##STR00178##
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.2 is
--(CH.sub.2).sub.nC(.dbd.O, A.sub.3 is
##STR00179##
and X.sup.2 is
##STR00180##
[0701] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
L.sub.2 is -A.sub.2-, L.sub.3 is -A.sub.3-, L.sub.4 is
##STR00181##
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.2 is
--(CH.sub.2).sub.nC(.dbd.O, A.sub.3 is
##STR00182##
X.sup.2 is
##STR00183##
[0702] and TG is a drug moiety.
[0703] In certain embodiments the compound of Formula (B) is
##STR00184##
[0704] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is a -A.sub.1X.sup.2--,
L.sub.2 is a bond-, L.sub.3 is -A.sub.3-, L.sub.4 is a bond,
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.3 is
--(CH.sub.2).sub.nC(.dbd.O)--, and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--.
[0705] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is a -A.sub.1X.sup.2--,
L.sub.2 is a bond-, L.sub.3 is -A.sub.3-, L.sub.4 is a bond,
A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS--, A.sub.3 is
--(CH.sub.2).sub.nC(.dbd.O)--, X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--, and TG is a drug moiety.
[0706] In certain embodiments the compound of Formula (B) is
##STR00185##
[0707] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is a -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is -A.sub.3-, L.sub.4 is a bond, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS, A.sub.3 is
--(CH.sub.2).sub.nC(.dbd.O)--, X.sup.2 is
--CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--, and R.sup.4 is
--C(.dbd.O)OH.
[0708] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is a -A.sub.1X.sup.2--,
L.sub.2 is a bond, L.sub.3 is -A.sub.3-, L.sub.4 is a bond, A.sub.1
is --C(.dbd.O)NH(CH.sub.2).sub.nS, A.sub.3 is
--(CH.sub.2).sub.nC(.dbd.O)--, X.sup.2 is
--CHR.sup.4(CH.sub.2).sub.nC(.dbd.O)NH--, R.sup.4 is --C(.dbd.O)OH,
and TG is a drug moiety.
[0709] In certain embodiments the compound of Formula (B) is
##STR00186##
[0710] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
where A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; L.sub.2 is a bond; L.sub.3 is a
bond, and L.sub.4 is -A.sub.4- wherein A.sub.4 is
--(CH.sub.2).sub.nNHC(.dbd.O)--.
[0711] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
wherein A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; L.sub.2 is a bond; L.sub.3 is a
bond; L.sub.4 is -A.sub.4-, wherein A.sub.4 is
--(CH.sub.2).sub.nC(.dbd.O)--.
[0712] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is -A.sub.1X.sup.2--,
wherein A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; L.sub.2 is -A.sub.2-, wherein
A.sub.2 is --(CH.sub.2).sub.nC(.dbd.O; L.sub.3 is -A.sub.3-,
wherein A.sub.3 is
##STR00187##
and L.sub.4 is
##STR00188##
[0714] In certain embodiments of methods, compounds and
immunoconjugates provided herein: L.sub.1 is a -A.sub.1X.sup.2--,
wherein A.sub.1 is --C(.dbd.O)NH(CH.sub.2).sub.nS-- and X.sup.2 is
--(CH.sub.2).sub.nC(.dbd.O)NH--; L.sub.2 is a bond-; L.sub.3 is
-A.sub.3-, wherein A.sub.3 is --(CH.sub.2).sub.nC(.dbd.O)--, and
L.sub.4 is a bond.
Two-Step Method
[0715] Alternatively, the modified antibodies or antigen binding
fragment thereof provided herein are site-specifically labeled by a
two-step method, wherein, in the first step the ppan prosthetic
group of CoA, or modified ppan prosthetic group of the CoA
analogue, which contain a functional group (R.sub.1), is attached
to the short peptide tag by a phosphodiester bond formed between
the 4'-phosphopantetheinyl moiety and the hydroxyl group of the
conserved serine residue of the short peptide tag which has been
incorporated into the antibody. In the second step a terminal group
(TG) linked, or directly attached to, a group which is reactive
with the functional group (R.sub.1) is reacted with the functional
group (R.sub.1) on the ppan prosthetic group of CoA, or on the
modified ppan prosthetic group of the CoA analogue, thereby
directly attaching the terminal group to the modified antibody or
antigen binding fragment thereof or attaching the terminal group to
the modified antibody or antigen binding fragment thereof via a
Linker Unit (LU).
[0716] One embodiment of the Two-Step Method is shown in Scheme
(IIa).
##STR00189##
wherein X and a corresponding R.sub.1 are as given below in Table
3, and where R.sub.2, A.sub.1, L.sub.2, X.sub.2, L.sub.3, L.sub.4
and TG are as defined herein:
TABLE-US-00003 TABLE 3 X R.sub.1 a thiol a thiol, a maleimide or a
haloacetamide an azide an alkyne, a triaryl phosphine, a
cyclooctene or an a triaryl phosphine oxanobornadiene an azide an
oxanobornadiene an azide an alkyne an azide an alkene an azide a
cyclooctene a diaryl tetrazine a diaryl tetrazine a cyclooctene a
monoaryl tetrazine a norbornene a norbornene a monoaryl tetrazine
an aldehyde a hydroxylamine or a hydrazine or
NH.sub.2--NH--C(.dbd.O)-- a ketone a hydroxylamine or a hydrazine
or NH.sub.2--NH--C(.dbd.O)-- a hydroxylamine an aldehyde or a
ketone a hydrazine an aldehyde or a ketone
NH.sub.2--NH--C(.dbd.O)-- an aldehyde or a ketone a haloacetamide a
thiol a maleimide a thiol
The alkene, alkyne, triaryl phosphine, cyclooctene,
oxanobornadiene, diaryl tetrazine, monoaryl tetrazine and
norbornene of X and R.sub.1 are optionally substituted. The
Two-Step Method of Scheme (IIa) includes the steps of: [0717] (a)
providing a modified antibody or antigen binding fragment thereof
which has been engineered to contain a peptide tag, and wherein the
peptide tag is a substrate of an enzyme having
4'-phosphopantetheinyl transferase activity; [0718] (b) labeling
the modified antibody or antigen binding fragment thereof by:
[0719] incubating the modified antibody or antigen binding fragment
thereof with an enzyme having 4'-phosphopantetheinyl transferase
activity in the presence of a compound of Formula (D),
[0719] ##STR00190## [0720] thereby attaching an activated
4'-phosphopantetheinyl group of Formula (D-a) to the peptide
tag;
[0720] ##STR00191## [0721] and [0722] (c) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (IIa):
[0722] X-L.sub.2-L.sub.3-L.sub.4-TG Formula (III-a),
where X, R.sub.1, R.sub.2, A.sub.1, L.sub.2, L.sub.3, L.sub.4 and
TG are as defined herein.
[0723] As a result of the Two-Step Method of Scheme (IIa) the
Terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (IIb):
##STR00192##
where A.sub.1, X.sub.2, L.sub.2, L.sub.3 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0724] Another embodiment of the Two-Step Method is shown in Scheme
(IIb).
##STR00193##
where X, R.sub.1, R.sub.2, L.sub.1, A.sub.2, X.sub.2, L.sub.3,
L.sub.4 and TG are as defined herein. The Two-Step Method of Scheme
(IIb) includes the steps of: [0725] (a) providing a modified
antibody or antigen binding fragment thereof which has been
engineered to contain a short peptide tag, and wherein the peptide
tag is a substrate of an enzyme having 4'-phosphopantetheinyl
transferase activity; [0726] (b) labeling the modified antibody or
antigen binding fragment thereof by: [0727] incubating the modified
antibody or antigen binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound of Formula (E),
[0727] ##STR00194## [0728] thereby attaching an activated
4'-phosphopantetheinyl group of Formula (E-a) to the short peptide
tag;
[0728] ##STR00195## [0729] and [0730] (c) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (II-c):
[0730] X-L.sub.3-L.sub.4-TG Formula (III-c),
where X, R.sub.1, R.sub.2, L.sub.1, A.sub.2, L.sub.3, L.sub.4 and
TG are as defined herein.
[0731] As a result of the Two-Step Method of Scheme (IIb) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (II-d):
##STR00196##
where L.sub.1, A.sub.2, X.sub.2, L.sub.3 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0732] Another embodiment of the Two-Step Method is shown in Scheme
(II-c).
##STR00197##
where X, R.sub.1, R.sub.2, L.sub.1, L.sub.2, X.sub.2, A.sub.3,
L.sub.4 and TG are as defined herein. The Two-Step Method of Scheme
(IIc) includes the steps of: [0733] (a) providing a modified
antibody or antigen binding fragment thereof which has been
engineered to contain a short peptide tag, and wherein the peptide
tag is a substrate of an enzyme having 4'-phosphopantetheinyl
transferase activity; [0734] (b) labeling the modified antibody or
antigen binding fragment thereof by: [0735] incubating the modified
antibody or antigen binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound of Formula (F),
[0735] ##STR00198## [0736] thereby attaching an activated
4'-phosphopantetheinyl group of Formula (F-a) to the short peptide
tag;
[0736] ##STR00199## [0737] and [0738] (c) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (IIe):
[0738] X-L.sub.4-TG Formula (II-e),
where X, R.sub.1, R.sub.2, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and
TG are as defined herein.
[0739] As a result of the Two-Step Method of Scheme (IIc) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (III-f):
##STR00200##
where L.sub.1, L.sub.2, A.sub.3, X.sub.2 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0740] Another embodiment of the Two-Step Method is shown in Scheme
(IId).
##STR00201##
where X, R.sub.1, R.sub.2, L.sub.1, L.sub.2, L.sub.3, A.sub.4,
X.sub.2 and TG are as defined herein. The Two-Step Method of Scheme
(IId) includes the steps of: [0741] (a) providing a modified
antibody or antigen binding fragment thereof which has been
engineered to contain a short peptide tag, and wherein the peptide
tag is a substrate of an enzyme having 4'-phosphopantetheinyl
transferase activity; [0742] (b) labeling the modified antibody or
antigen binding fragment thereof by: [0743] incubating the modified
antibody or antigen binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound of Formula (G),
[0743] ##STR00202## [0744] thereby attaching an activated
4'-phosphopantetheinyl of Formula (G-a) to the short peptide
tag;
[0744] ##STR00203## [0745] and [0746] (c) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (II-g):
[0746] X-TG Formula (II-g),
where X, R.sub.1, R.sub.2, L.sub.1, L.sub.2, L.sub.3, L.sub.4 and
TG are as defined herein.
[0747] As a result of the Two-Step Method of Scheme (IId) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (III-h):
##STR00204##
where L.sub.1, L.sub.2, L.sub.3, A.sub.4 and X.sub.2 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0748] In certain embodiments of the Two-Step Methods described
herein, the modified antibody or antigen binding fragment thereof
is contacted with a compound having the structure of Formula (D),
Formula (E), Formula (F) or Formula (G) and a
4'-phosphopantetheinyl transferase enzyme that is co-expressed in
the same cell as the expressed modified antibody or antigen binding
fragment thereof. In certain embodiments of the Two-Step Methods
described herein, the modified antibody or antigen binding fragment
thereof is contacted in the cell culture media with a compound
having the structure of Formula (D), Formula (E), Formula (F) or
Formula (G) and 4'-phosphopantetheinyl transferase enzyme produced
in the same or in another cell. In certain embodiments of the
Two-Step Methods described herein, the 4'-phosphopantetheinyl
transferase enzyme is immobilized on solid support. In certain
embodiments the solid support is optionally comprised of a polymer
on a bead or a column.
[0749] Table 4 shows certain embodiments of the activated
4'-phosphopantetheinyl groups of Formula (D-a) and compounds of
Formula (III-a) used in the Two-step methods and the Three-step
methods described herein and the resulting modified serine located
in the modified antibody or antigen binding fragment thereof. Note
A.sub.1, L.sub.2, L.sup.3, L.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 and TG are as defined herein, and Y is
##STR00205##
TABLE-US-00004 TABLE 4 ##STR00206##
X--L.sub.2--L.sub.3--L.sub.4--TG Formula (IIa) ##STR00207##
##STR00208## HC.ident.C--L.sub.2--L.sub.3--L.sub.4--TG ##STR00209##
##STR00210## HC.ident.C--L.sub.2--L.sub.3--L.sub.4--TG ##STR00211##
##STR00212## N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00213##
##STR00214## N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00215##
##STR00216## NH.sub.2--O--L.sub.2--L.sub.3--L.sub.4--TG
##STR00217## ##STR00218##
NH.sub.2--O--L.sub.2--L.sub.3--L.sub.4--TG ##STR00219##
##STR00220## CH.sub.3C(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG
##STR00221## ##STR00222## HC(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG
##STR00223## ##STR00224## HS--L.sub.2--L.sub.3--L.sub.4--TG
##STR00225## ##STR00226## ##STR00227## ##STR00228## ##STR00229##
##STR00230## ##STR00231## ##STR00232##
HS--L.sub.2--L.sub.3--L.sub.4--TG ##STR00233## ##STR00234##
##STR00235## ##STR00236## ##STR00237##
HS--L.sub.2--L.sub.3--L.sub.4--TG ##STR00238## ##STR00239##
NH.sub.2--NH--C(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG ##STR00240##
##STR00241## NH.sub.2--NH--C(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG
##STR00242## ##STR00243##
R.sub.5C(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG ##STR00244##
##STR00245## HC(.dbd.O)--L.sub.2--L.sub.3--L.sub.4--TG ##STR00246##
##STR00247## HS--L.sub.2--L.sub.3--L.sub.4--TG ##STR00248##
##STR00249## ##STR00250## ##STR00251## ##STR00252##
N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00253## ##STR00254##
##STR00255## ##STR00256## ##STR00257##
N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00258## ##STR00259##
##STR00260## ##STR00261## ##STR00262##
N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00263## ##STR00264##
##STR00265## ##STR00266## ##STR00267## ##STR00268## ##STR00269##
##STR00270## ##STR00271## ##STR00272## ##STR00273## ##STR00274##
##STR00275## ##STR00276## N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG
##STR00277## ##STR00278## ##STR00279## ##STR00280## ##STR00281##
##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286##
##STR00287## ##STR00288## ##STR00289## ##STR00290##
N.sub.3--L.sub.2--L.sub.3--L.sub.4--TG ##STR00291##
[0750] Table 5 shows certain embodiments of the activated
4'-phosphopantetheinyl groups of Formula (E-a) and compounds of
Formula (III-c) used in the Two-step methods and the Three-step
methods described herein and the resulting modified serine located
in the modified antibody or antigen binding fragment thereof. Note
L.sub.1, A.sub.2, L.sub.3, L.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 and TG are as defined herein, and Y is
##STR00292##
TABLE-US-00005 TABLE 5 ##STR00293## X--L.sub.3--L.sub.4--TG Formula
(II-c) ##STR00294## ##STR00295## HC.ident.C--L.sub.3--L.sub.4--TG
##STR00296## ##STR00297## HC.ident.C--L.sub.3--L.sub.4--TG
##STR00298## ##STR00299## N.sub.3--L.sub.3--L.sub.4--TG
##STR00300## ##STR00301## N.sub.3--L.sub.3--L.sub.4--TG
##STR00302## ##STR00303## NH.sub.2--O--L.sub.3--L.sub.4--TG
##STR00304## ##STR00305## NH.sub.2--O--L.sub.3--L.sub.4--TG
##STR00306## ##STR00307## CH.sub.3C(.dbd.O)--L.sub.3--L.sub.4--TG
##STR00308## ##STR00309## HC(.dbd.O)--L.sub.3--L.sub.4--TG
##STR00310## ##STR00311## HS--L.sub.3--L.sub.4--TG ##STR00312##
##STR00313## ##STR00314## ##STR00315## ##STR00316## ##STR00317##
##STR00318## ##STR00319## HS--L.sub.3--L.sub.4--TG ##STR00320##
##STR00321## ##STR00322## ##STR00323## ##STR00324##
HS--L.sub.3--L.sub.4--TG ##STR00325## ##STR00326##
NH.sub.2--NH--C(.dbd.O)--L.sub.3--L.sub.4--TG ##STR00327##
##STR00328## NH.sub.2--NH--C(.dbd.O)--L.sub.3--L.sub.4--TG
##STR00329## ##STR00330## R.sub.5C(.dbd.O)--L.sub.3--L.sub.4--TG
##STR00331## ##STR00332## HC(.dbd.O)--L.sub.3--L.sub.4--TG
##STR00333## ##STR00334## SH--L.sub.3--L.sub.4--TG ##STR00335##
##STR00336## ##STR00337## ##STR00338## ##STR00339##
N.sub.3--L.sub.3--L.sub.4--TG ##STR00340## ##STR00341##
##STR00342## ##STR00343## ##STR00344##
N.sub.3--L.sub.3--L.sub.4--TG ##STR00345## ##STR00346##
##STR00347## ##STR00348## ##STR00349##
N.sub.3--L.sub.3--L.sub.4--TG ##STR00350## ##STR00351##
##STR00352## ##STR00353## ##STR00354## ##STR00355## ##STR00356##
##STR00357## ##STR00358## ##STR00359## ##STR00360## ##STR00361##
##STR00362## ##STR00363## N.sub.3--L.sub.3--L.sub.4--TG
##STR00364## ##STR00365## ##STR00366## ##STR00367## ##STR00368##
##STR00369## ##STR00370## ##STR00371## ##STR00372## ##STR00373##
##STR00374## ##STR00375## ##STR00376## ##STR00377##
N.sub.3--L.sub.3--L.sub.4--TG ##STR00378##
[0751] Table 6 shows certain embodiments of the activated
4'-phosphopantetheinyl groups of Formula (F-a) and compounds of
Formula (II-e) used in the Two-step methods and the Three-step
methods described herein and the resulting modified serine located
in the modified antibody or antigen binding fragment thereof. Note
L.sub.1, L.sub.2, A.sub.3, L.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 and TG are as defined herein, and Y is
##STR00379##
TABLE-US-00006 TABLE 6 ##STR00380## X--L.sub.4--TG Formula (II-e)
##STR00381## ##STR00382## HC.ident.C--L.sub.4--TG ##STR00383##
##STR00384## HC.ident.C--L.sub.4--TG ##STR00385## ##STR00386##
N.sub.3--L.sub.4--TG ##STR00387## ##STR00388## N.sub.3--L.sub.4--TG
##STR00389## ##STR00390## NH.sub.2--O--L.sub.4--TG ##STR00391##
##STR00392## NH.sub.2--O--L.sub.4--TG ##STR00393## ##STR00394##
CH.sub.3C(.dbd.O)--L.sub.4--TG ##STR00395## ##STR00396##
HC(.dbd.O)--L.sub.4--TG ##STR00397## ##STR00398## HS--L.sub.4--TG
##STR00399## ##STR00400## ##STR00401## ##STR00402## ##STR00403##
##STR00404## ##STR00405## ##STR00406## HS--L.sub.4--TG ##STR00407##
##STR00408## ##STR00409## ##STR00410## ##STR00411## HS--L.sub.4--TG
##STR00412## ##STR00413## NH.sub.2--NH--C(.dbd.O)--L.sub.4--TG
##STR00414## ##STR00415## NH.sub.2--NH--C(.dbd.O)--L.sub.4--TG
##STR00416## ##STR00417## R.sub.5C(.dbd.O)--L.sub.4--TG
##STR00418## ##STR00419## HC(.dbd.O)--L.sub.4--TG ##STR00420##
##STR00421## HS--L.sub.4--TG ##STR00422## ##STR00423## ##STR00424##
##STR00425## ##STR00426## N.sub.3--L.sub.4--TG ##STR00427##
##STR00428## ##STR00429## ##STR00430## ##STR00431##
N.sub.3--L.sub.4--TG ##STR00432## ##STR00433## ##STR00434##
##STR00435## ##STR00436## N.sub.3--L.sub.4--TG ##STR00437##
##STR00438## ##STR00439## ##STR00440## ##STR00441## ##STR00442##
##STR00443## ##STR00444## ##STR00445## ##STR00446## ##STR00447##
##STR00448## ##STR00449## ##STR00450## N.sub.3--L.sub.4--TG
##STR00451## ##STR00452## ##STR00453## ##STR00454## ##STR00455##
##STR00456## ##STR00457## ##STR00458## ##STR00459## ##STR00460##
##STR00461## ##STR00462## ##STR00463## ##STR00464##
N.sub.3--L.sub.4--TG ##STR00465##
[0752] Table 7 shows certain embodiments of the activated
4'-phosphopantetheinyl groups of Formula (G-a) and compounds of
Formula (II-g) used in the Two-step methods and the Three-step
methods described herein and the resulting modified serine located
in the modified antibody or antigen binding fragment thereof. Note
L.sub.1, L.sub.2, L.sub.3, A.sub.4, R.sub.5, Re, R.sub.7, R.sub.8
and TG are as defined herein, and Y is
##STR00466##
TABLE-US-00007 TABLE 7 ##STR00467## X--TG Formula (II-g)
##STR00468## ##STR00469## HC.ident.--C--TG ##STR00470##
##STR00471## HC.ident.--C--TG ##STR00472## ##STR00473## N.sub.3--TG
##STR00474## ##STR00475## N.sub.3--TG ##STR00476## ##STR00477##
NH.sub.2--O--TG ##STR00478## ##STR00479## NH.sub.2--O--TG
##STR00480## ##STR00481## CH.sub.3C(.dbd.O)--TG ##STR00482##
##STR00483## HC(.dbd.O)--TG ##STR00484## ##STR00485## HS--TG
##STR00486## ##STR00487## ##STR00488## ##STR00489## ##STR00490##
##STR00491## ##STR00492## ##STR00493## HS--TG ##STR00494##
##STR00495## ##STR00496## ##STR00497## ##STR00498## HS--TG
##STR00499## ##STR00500## NH.sub.2--NH--C(.dbd.O)--TG ##STR00501##
##STR00502## NH.sub.2--NH--C(.dbd.O)--TG ##STR00503## ##STR00504##
R.sub.5C(.dbd.O)--TG ##STR00505## ##STR00506## HC(.dbd.O)--TG
##STR00507## ##STR00508## HS--TG ##STR00509## ##STR00510##
##STR00511## ##STR00512## ##STR00513## N.sub.3--TG ##STR00514##
##STR00515## ##STR00516## ##STR00517## ##STR00518## N.sub.3--TG
##STR00519## ##STR00520## ##STR00521## ##STR00522## ##STR00523##
N.sub.3--TG ##STR00524## ##STR00525## ##STR00526## ##STR00527##
##STR00528## ##STR00529## ##STR00530## ##STR00531## ##STR00532##
##STR00533## ##STR00534## ##STR00535## ##STR00536## ##STR00537##
N.sub.3--TG ##STR00538## ##STR00539## ##STR00540## ##STR00541##
##STR00542## ##STR00543## ##STR00544## ##STR00545## ##STR00546##
##STR00547## ##STR00548## ##STR00549## ##STR00550## ##STR00551##
N.sub.3--TG ##STR00552##
Three-Step Method
[0753] Alternatively, the modified antibodies or antigen binding
fragment thereof provided herein are site-specifically labeled by a
three-step method, wherein, in the first step a protected ppan
prosthetic group of CoA, or a protected modified ppan prosthetic
group of the CoA analogue, is attached to the short peptide tag by
a phosphodiester bond formed between the 4'-phosphopantetheinyl
moiety and the hydroxyl group of the conserved serine residue of
the short peptide tag incorporated into the antibody. In the second
step the protected ppan prosthetic group of CoA, or protected
modified ppan prosthetic group of the CoA analogue, is deprotected;
thereby generating a reactive functional group (R.sub.1). In the
third step a terminal group (TG) linked, or directly attached to, a
group which is reactive with the functional group (R.sub.1) is
reacted with the functional group (R.sub.1) on the ppan prosthetic
group of CoA, or on the modified ppan prosthetic group of the CoA
analogue, thereby directly attaching the terminal group to the
modified antibody or antigen binding fragment thereof or attaching
the terminal group to the modified antibody or antigen binding
fragment thereof via a Linker Unit (LU).
[0754] One embodiment of the Three-Step Method is shown in Scheme
(IIIa).
##STR00553##
wherein X and a corresponding R.sub.1 are as given in Table 3, and
where PG is a protecting group and R.sub.2, A.sub.1, L.sub.2,
X.sub.2, L.sub.3, L.sub.4 and TG are as defined herein. The
Three-Step Method of Scheme (IIIa) includes the steps of: [0755]
(a) providing a modified antibody or antigen binding fragment
thereof which has been engineered to contain a short peptide tag,
and wherein the peptide tag is a substrate of an enzyme having
4'-phosphopantetheinyl transferase activity; [0756] (b) labeling
the modified antibody or antigen binding fragment thereof by:
[0757] incubating the modified antibody or antigen binding fragment
thereof with an enzyme having 4'-phosphopantetheinyl transferase
activity in the presence of a compound of Formula H,
[0757] ##STR00554## [0758] thereby attaching a protected
4'-phosphopantetheinyl group of Formula (H-a) to the short peptide
tag;
[0758] ##STR00555## [0759] (c) deprotecting the protected
4'-phosphopantetheinyl group to give an activated
4'-phosphopantetheinyl group of Formula (D-a)
[0759] ##STR00556## [0760] and [0761] (d) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (IIa):
[0761] X-L.sub.2-L.sub.3-L.sub.4-TG Formula (III-a),
where PG is a protecting group and X, R.sub.1, R.sub.2, A.sub.1,
L.sub.2, L.sub.3, L.sub.4 and TG are as defined herein.
[0762] As a result of the Three-Step Method of Scheme (IIIa) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (IIb):
##STR00557##
where A.sub.1, X.sub.2, L.sub.2, L.sub.3 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0763] Another embodiment of the Three-Step Method is shown in
Scheme (IIIb).
##STR00558##
where PG is a protecting group and X, R.sub.1, R.sub.2, L.sub.1,
A.sub.2, X.sub.2, L.sub.3, L.sub.4 and TG are as defined herein.
The Three-Step Method of Scheme (IIIb) includes the steps of:
[0764] (a) providing a modified antibody or antigen binding
fragment thereof which has been engineered to contain a short
peptide tag, and wherein the peptide tag is a substrate of an
enzyme having 4'-phosphopantetheinyl transferase activity; [0765]
(b) labeling modified antibody or antigen binding fragment thereof
by: [0766] incubating the modified antibody or antigen binding
fragment thereof with an enzyme having 4'-phosphopantetheinyl
transferase activity in the presence of a compound of Formula
(J),
[0766] ##STR00559## [0767] thereby attaching an protected
4'-phosphopantetheinyl group of Formula (I-a) to the short peptide
tag;
[0767] ##STR00560## [0768] (c) deprotecting the protected
4'-phosphopantetheinyl group to give an activated
4'-phosphopantetheinyl group of Formula (E-a)
[0768] ##STR00561## [0769] and [0770] (d) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (II-c):
[0770] X-L.sub.3-L.sub.4-TG Formula (II-c),
where PG is a protecting group and X, R.sub.1, R.sub.2, L.sub.1,
A.sub.2, L.sub.3, L.sub.4 and TG are as defined herein.
[0771] As a result of the Three-Step Method of Scheme (IIIb) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (II-d):
##STR00562##
where L.sub.1, A.sub.2, X.sub.2, L.sub.3 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0772] Another embodiment of the Three-Step Method is shown in
Scheme (IIIc).
##STR00563##
where PG is a protecting group and X, R.sub.1, R.sub.2, L.sub.1,
L.sub.2, X.sub.2, A.sub.3, L.sub.4 and TG are as defined herein.
The Three-Step Method of Scheme (IIIc) includes the steps of:
[0773] (a) providing a modified antibody or antigen binding
fragment thereof which has been engineered to contain a short
peptide tag, and wherein the peptide tag is a substrate of an
enzyme having 4'-phosphopantetheinyl transferase activity; [0774]
(b) labeling the modified antibody or antigen binding fragment
thereof by: [0775] incubating the modified antibody or antigen
binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound of Formula (K),
[0775] ##STR00564## [0776] thereby attaching an protected
4'-phosphopantetheinyl group of Formula (K-a) to the short peptide
tag;
[0776] ##STR00565## [0777] (c) deprotecting the protected
4'-phosphopantetheinyl group to give an activated
4'-phosphopantetheinyl group of Formula (F-a) [0778] and
[0778] ##STR00566## [0779] (d) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (IIe):
[0779] X-L.sub.4-TG Formula (II-e),
where PG is a protecting group and X, R.sub.1, R.sub.2, L.sub.1,
L.sub.2, L.sub.3, L.sub.4 and TG are as defined herein.
[0780] As a result of the Two-Step Method of Scheme (IIIc) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (III-f):
##STR00567##
where L.sub.1, L.sub.2, A.sub.3, X.sub.2 and L.sub.4 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0781] Another embodiment of the Three-Step Method is shown in
Scheme (IIId).
##STR00568##
where PG is a protecting group and X, R.sub.1, R.sub.2, L.sub.1,
L.sub.2, L.sub.3, A.sub.4, X.sub.2 and TG are as defined herein.
The Three-Step Method of Scheme (IIId) includes the steps of:
[0782] (a) providing a modified antibody or antigen binding
fragment thereof which has been engineered to contain a short
peptide tag, and wherein the peptide tag is a substrate of an
enzyme having 4'-phosphopantetheinyl transferase activity; [0783]
(b) labeling the modified antibody or antigen binding fragment
thereof by: [0784] incubating the modified antibody or antigen
binding fragment thereof with an enzyme having
4'-phosphopantetheinyl transferase activity in the presence of a
compound of Formula (L),
[0784] ##STR00569## [0785] thereby attaching an activated
4'-phosphopantetheinyl of Formula (L-a) to the short peptide
tag;
[0785] ##STR00570## [0786] (c) deprotecting the protected
4'-phosphopantetheinyl group to give an activated
4'-phosphopantetheinyl group of Formula (G-a)
[0786] ##STR00571## [0787] and [0788] (d) reacting the activated
4'-phosphopantetheinyl group with a compound of Formula (II-g):
[0788] X-TG Formula (II-g),
where PG is a protecting group X, R.sub.1, R.sub.2, L.sub.1,
L.sub.2, L.sub.3, L.sub.4 and TG are as defined herein.
[0789] As a result of the Three-Step Method of Scheme (IIId) the
terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (III-h):
##STR00572##
where L.sub.1, L.sub.2, L.sub.3, A.sub.4 and X.sub.2 are as defined
herein and the * denotes that the modified 4'-phosphopantetheinyl
moiety is attached to the small peptide tag.
[0790] Scheme (IIIe) shows a certain embodiment of the Three-Step
Method where the modified antibodies or antigen binding fragment
thereof provided herein are site-specifically labeled by a CoA
analogue where the thiol of the 4'-phosphopantetheinyl prosthetic
group is protected. In step 1 the protected CoA analogue reacts
with the conserved serine of the short peptide tag engineered into
the antibody thereby attaching the prosthetic group containing the
protected thiol to the short peptide tag through the formation of a
phosphodiester bond with the hydroxyl group of the conserved serine
residue of the short peptide tag. In the second step the thiol
protecting group is removed and the resulting modified antibody or
antigen binding fragment thereof having a pendant
4'-phosphopantetheinyl group is reacted with a thiol reactive group
linked to a terminal group (TG).
##STR00573##
where X.sub.SH, protecting group (PG), R.sub.2, A.sub.2, L.sub.3,
L.sub.4 and TG are as defined herein.
[0791] Scheme (IIIf) shows a certain embodiment of the Three-Step
Method where the modified antibodies or antigen binding fragment
thereof provided herein are site-specifically labeled using a CoA
where the thiol of the 4'-phosphopantetheinyl prosthetic group is
protected. In step 1 the protected CoA reacts with the conserved
serine of the short peptide tag engineered into the antibody
thereby attaching the prosthetic group containing the protected
thiol to the short peptide tag through the formation of a
phosphodiester bond with the hydroxyl group of the conserved serine
residue of the short peptide tag. In the second step the thiol
protecting group is removed and the resulting modified antibody or
antigen binding fragment thereof having a pendant
4'-phosphopantetheinyl group is reacted with a thiol reactive group
linked to a terminal group (TG).
##STR00574##
where X.sub.SH, protecting group (PG), R.sub.2, A.sub.2, L.sub.3,
L.sub.4 and TG are as defined herein. In the Three-Step Method of
Scheme (IIIe) and Scheme (IIIf), the thiol protecting group
includes, but is not limited to, acetyl, acetamidomethyl, benzyl,
4-methylbenzyl, 4-methoxybenzyl, trityl, methoxytrityl, t-butyl,
t-butylthiol and 3-nitro-2-pyridinesulphenyl. The thiol reactive
group of Scheme (IIIe) and Scheme (IIIf) includes, but is not
limited to, maleimide, a haloacetyl, a haloacetamide, a
pyridyldisulfide and a vinyl sulfone. The Three-Step Method of
Scheme (IIIf) includes the steps of: [0792] (a) providing a
modified antibody or antigen binding fragment thereof which has
been engineered to contain a short peptide tag, and wherein the
peptide tag is a substrate of an enzyme having
4'-phosphopantetheinyl transferase activity; [0793] (b) labeling
the modified antibody or antigen binding fragment thereof by:
[0794] (i) incubating the modified antibody or antigen binding
fragment thereof with an enzyme having 4'-phosphopantetheinyl
transferase activity in the presence of a thiol protected coenzyme
A, thereby attaching the thiol protected prosthetic group of
coenzyme A to the short peptide tag; [0795] (ii) deprotecting the
thiol group thereby forming a 4'-phosphopantetheinyl group having a
pendant thiol, [0796] and [0797] (iii) reacting the pendant thiol
of the 4'-phosphopantetheinyl group with a compound of Formula
(IIIf):
[0797] X.sub.SH-L.sub.2-L.sub.3-L.sub.4-TG Formula (IIIf).
where X.sub.SH is a thiol reactive group including, but not limited
to, a maleimide, a haloacetyl, a haloacetamide, a pyridyldisulfide
and a vinyl sulfone. A.sub.2, L.sub.3, L.sub.4 and TG are as
defined herein. In addition, in the Two-Step Method of Scheme (IIf)
the terminal group is attached to the modified antibody or antigen
binding fragment thereof via a linker having the structure
according to Formula (III-a):
##STR00575##
The * denotes the 4'-phosphopantetheinyl moiety is attached to the
small peptide tag and L.sub.2, L.sub.3, L.sub.4 and TG are as
defined herein. In this embodiment X.sub.2 is a group formed by
reaction of X.sub.SH and the pendant thiol, including, but not
limited to,
##STR00576##
and --S--S--.
[0798] In certain embodiments X.sub.SH-L.sub.2-L.sub.3-L.sub.4-TG
is
##STR00577##
wherein:
[0799] X.sub.1 is a bond, --C(.dbd.O)--, --NH--, --NHC(.dbd.O)--,
--(C(.dbd.O)NH(CH.sub.2).sub.n).sub.m--,
##STR00578##
In other embodiments X.sub.SH-L.sub.2-L.sub.3-L.sub.4-TG is
##STR00579##
[0800] In certain embodiments of the Three-Step Methods described
herein, the modified antibody or antigen binding fragment thereof
is contacted with a compound having the structure of Formula (H),
Formula (J), Formula (K) or Formula (L) and a
4'-phosphopantetheinyl transferase enzyme that is co-expressed in
the same cell as the expressed modified antibody or antigen binding
fragment thereof. In certain embodiments of the Two-Step Methods
described herein, the modified antibody or antigen binding fragment
thereof is contacted in the cell culture media with a compound
having the structure of Formula (H), Formula (J), Formula (K) or
Formula (L) and 4'-phosphopantetheinyl transferase enzyme
co-expressed by the same or another cell. In certain embodiments of
the Two-Step Methods described herein, the 4'-phosphopantetheinyl
transferase enzyme is immobilized on solid support. In certain
embodiments the solid support is optionally comprised of a polymer
on a bead or a column.
[0801] In certain embodiments of the Three-Step Method, the
modified antibody or antigen binding fragment thereof will be
contacted with a 4'-phosphopantetheinyl transferase enzyme that is
coexpressed in the same cell. In certain embodiments of the
Three-Step Method, the thiol protected coenzyme A is
acetyl-coenzyme A. In certain embodiments of the Three-Step Method,
the modified antibody or antigen binding fragment thereof is
contacted in the cell culture media with the thiol protected
coenzyme A and a 4'-phosphopantetheinyl transferase enzyme
co-expressed by the same or another cell. In certain embodiments of
the Three-Step Method, the 4'-phosphopantetheinyl transferase
enzyme is immobilized on solid support. The solid support is
optionally comprised of a polymer on a bead or a column.
[0802] In certain embodiments of the One-Step Method, Two-Step
Methods or the Three-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) and a 4'-phosphopantetheinyl transferase enzyme at
temperatures between 0 and 37 degree Celsius in buffer or media
adjusted to pH values between 3 and 10, preferably between 7 and 9
and most preferably around 8, for reaction times between 5 mins and
48 hours.
[0803] In certain embodiments of the One-Step Method, Two-Step
Methods or the Three-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) in the presence of 4'-phosphopantetheinyl transferase
in solution. In other embodiments of the One-Step Method, Two-Step
Methods or the Three-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) in the presence of 4'-phosphopantetheinyl transferase
in cell media. In certain embodiments of the One-Step Method,
Two-Step Methods or the Three-Step Methods described herein, the
modified antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) in the presence of 4'-phosphopantetheinyl transferase
inside a cell.
[0804] In certain embodiments of the One-Step Method, Two-Step
Methods or the Three-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) in the presence of 4'-phosphopantetheinyl transferase,
wherein the 4'-phosphopantetheinyl transferase is immobilized on a
surface. In certain embodiments the surface is polymer bead.
[0805] In certain embodiments of the One-Step Method, Two-Step
Methods or the Three-Step Methods described herein, the modified
antibody or antigen binding fragment thereof is contacted,
depending on the Method used, with a compound having the structure
of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E),
Formula (F), Formula (G), Formula (H), Formula (J), Formula (K) or
Formula (L) in the presence of 4'-phosphopantetheinyl transferase,
wherein the modified antibody or antigen binding fragment thereof
is immobilized on a surface. In certain embodiments the surface is
polymer bead.
[0806] In certain embodiments, the modified antibody or antigen
binding fragment thereof provided herein are labeled with a
terminal group ("TG")-to-antibody ratio of 1, 2, 3, 4, 5, 6, 7, or
8, wherein the modified antibody or antigen binding fragment
thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 short peptide tags
located in the structural loop of the antibody and where the short
peptide tags are substrates of Sfp 4'-phosphopantetheinyl
transferase, AcpS 4'-phosphopantetheinyl transferase, T. maritima
4'-phosphopantetheinyl transferase, C. thermocellum
4'-phosphopantetheinyl transferase, human 4'-phosphopantetheinyl
transferase, or a mutant form thereof. For example, a
TG-to-antibody ratio of 4 is achieved by conjugating the terminal
group to four copies of inserted S6 tags, or to four copies of
inserted ybbR tags or to four copies of inserted A1 tags, or to a
combination of two copies of inserted S6 tags and two copies of
inserted ybbR tags. In certain embodiments, the modified antibodies
or antigen binding fragment thereof provided herein are labeled
with two different terminal groups using two different peptide tags
and two different 4'-phosphopantetheinyl transferases. By way of
example, two copies of the A1 tag are conjugated to a first
terminal group using the AcpS 4'-phosphopantetheinyl transferase.
Then a second terminal group is attached to two copies of an S6 tag
using the Sfp 4'-phosphopantetheinyl transferase (see, e.g., Zhou
et al., ACS Chem. Biol. 2:337-346, 2007).
[0807] In certain embodiments, the modified antibodies or antigen
binding fragment thereof provided herein are labeled with a
terminal group (TG)-to-antibody ratio (e.g., DAR) of 1, 2, 3, 4, 5,
6, 7, or 8, wherein the modified antibody or antigen binding
fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 short peptide
tags located in the structural loop of the antibody and where the
short peptide tags are substrates of Sfp 4'-phosphopantetheinyl
transferase, AcpS 4'-phosphopantetheinyl transferase, T. maritima
4'-phosphopantetheinyl transferase, C. thermocellum
4'-phosphopantetheinyl transferase, human 4'-phosphopantetheinyl
transferase, or a mutant form thereof. For example, a
TG-to-antibody ratio of 4 is achieved by conjugating a drug moiety
to four copies of inserted S6 tags, or to four copies of inserted
ybbR tags, or to four copies of inserted A1 tags, or to a
combination of two copies of inserted S6 tags and two copies of
inserted ybbR tags. In certain embodiments, the modified antibodies
or antigen binding fragment thereof provided herein are labeled
with two different drug moieties using two different peptide tags
and two different 4'-phosphopantetheinyl transferases. By way of
example, two copies of the A1 tag are conjugated to a first drug
moiety using the AcpS 4'-phosphopantetheinyl transferase. Then a
second drug moiety is attached to two copies of an S6 tag using the
Sfp 4'-phosphopantetheinyl transferase (see, e.g., Zhou et al., ACS
Chem. Biol. 2:337-346, 2007).
3. Further Alteration of the Framework of Fc Region
[0808] The present invention provides site-specific labeled
immunoconjugates. The immunoconjugates of the invention may
comprise modified antibodies or antigen binding fragments thereof
that further comprise modifications to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "back-mutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. To return the framework region sequences to
their germline configuration, the somatic mutations can be
"back-mutated" to the germline sequence by, for example,
site-directed mutagenesis. Such "back-mutated" antibodies are also
intended to be encompassed by the invention.
[0809] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T-cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0810] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below.
[0811] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0812] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half-life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0813] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector functions of the antibody. For
example, one or more amino acids can be replaced with a different
amino acid residue such that the antibody has an altered affinity
for an effector ligand but retains the antigen-binding ability of
the parent antibody. The effector ligand to which affinity is
altered can be, for example, an Fc receptor or the C1 component of
complement. This approach is described in, e.g., U.S. Pat. Nos.
5,624,821 and 5,648,260, both by Winter et al.
[0814] In another embodiment, one or more amino acids selected from
amino acid residues can be replaced with a different amino acid
residue such that the antibody has altered Clq binding and/or
reduced or abolished complement dependent cytotoxicity (CDC). This
approach is described in, e.g., U.S. Pat. No. 6,194,551 by Idusogie
et al.
[0815] In another embodiment, one or more amino acid residues are
altered to thereby alter the ability of the antibody to fix
complement. This approach is described in, e.g., the PCT
Publication WO 94/29351 by Bodmer et al. In a specific embodiment,
one or more amino acids of an antibody or antigen binding fragment
thereof of the present invention are replaced by one or more
allotypic amino acid residues, such as those shown in FIG. 4 for
the IgG1 subclass and the kappa isotype. Allotypic amino acid
residues also include, but are not limited to, the constant region
of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as well
as the constant region of the light chain of the kappa isotype as
described by Jefferis et al., MAbs. 1:332-338 (2009).
[0816] In yet another embodiment, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids. This approach is described in, e.g., the PCT Publication WO
00/42072 by Presta. Moreover, the binding sites on human IgG1 for
Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RII and FcRn have been mapped
and variants with improved binding have been described (see Shields
et al., J. Biol. Chem. 276:6591-6604, 2001).
[0817] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycosylated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
"antigen." Such carbohydrate modifications can be accomplished by,
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.
[0818] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. For example, EP 1,176,195 by Hang et al. describes a
cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase, such that antibodies expressed in such a cell
line exhibit hypofucosylation. PCT Publication WO 03/035835 by
Presta describes a variant CHO cell line, Lecl3 cells, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host
cell (see also Shields et al., (2002) J. Biol. Chem.
277:26733-26740). PCT Publication WO 99/54342 by Umana et al.
describes cell lines engineered to express glycoprotein-modifying
glycosyl transferases (e.g., beta(1,4)-N
acetylglucosaminyltransferase IIII (GnTIII)) such that antibodies
expressed in the engineered cell lines exhibit increased bisecting
GlcNac structures which results in increased ADCC activity of the
antibodies (see also Umana et al., Nat. Biotech. 17:176-180,
1999).
[0819] In another embodiment, the antibody is modified to increase
its biological half-life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half-life, the
antibody can be altered within the CH1 or C.sub.L region to contain
a salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
4. Antibody Conjugates
[0820] The present invention provides site-specific labeling
methods, modified antibodies and antigen binding fragments thereof,
and immunoconjugates prepared accordingly. Using the methods of the
invention, a modified antibody or antigen binding fragments thereof
can be conjugated to a label, such as a drug moiety, e.g., an
anti-cancer agent, an autoimmune treatment agent, an
anti-inflammatory agent, an antifungal agent, an antibacterial
agent, an anti-parasitic agent, an anti-viral agent, or an
anesthetic agent. An antibody or antigen binding fragments can also
be conjugated using several identical or different labeling
moieties combining the methods of the invention with other
conjugation methods.
[0821] In certain embodiments, the terminal group of the
immunoconjugates of the present invention is selected from a
V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor
inhibitor, a microtubule stabilizer, a microtubule destabilizers,
an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine
aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a
DPPIV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl
transfer reactions in mitochondria, a protein synthesis inhibitor,
a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, an EG5
inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA
alkylating agent, a DNA intercalator, a DNA minor groove binder and
a DHFR inhibitor.
[0822] Further, the modified antibodies or antigen binding
fragments of the present invention may be conjugated to a
therapeutic moiety or drug moiety that modifies a given biological
response. Therapeutic moieties or drug moieties are not to be
construed as limited to classical chemical therapeutic agents. For
example, the drug moiety may be a protein, peptide, or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, cholera toxin, or diphtheria toxin, a protein such as
tumor necrosis factor, .alpha.-interferon, 1-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, a cytokine, an apoptotic agent, an anti-angiogenic
agent, or, a biological response modifier such as, for example, a
lymphokine.
[0823] In one embodiment, the modified antibodies or antigen
binding fragments of the present invention are conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Examples of cytotoxin include
but not limited to, taxanes (see, e.g., International (PCT) Patent
Application Nos. WO 01/38318 and PCT/US03/02675), DNA-alkylating
agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs,
duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and
cytotoxic agents comprising a reactive polyethylene glycol moiety
(see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000),
Suzawa et al., Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et
al., J. Antibiot. (Tokyo), 44, 1045-53 (1991), Francisco et al.,
Blood (2003) (electronic publication prior to print publication),
U.S. Pat. Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S.
Patent Application Publication No. 2001/0036923 A1, Pending U.S.
patent application Ser. Nos. 10/024,290 and 10/116,053, and
International (PCT) Patent Application No. WO 01/49698), taxon,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, t. colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents also include, for
example, anti-metabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating
agents (e.g., mechlorethamine, thioepa chloraxnbucil, meiphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine). (See e.g., Seattle Genetics
US20090304721).
[0824] Other examples of therapeutic cytotoxins that can be
conjugated to the modified antibodies or antigen binding fragments
of the invention include duocarmycins, calicheamicins, maytansines
and auristatins, and derivatives thereof. An example of a
calicheamicin antibody conjugate is commercially available
(Mylotarg.TM.; Wyeth-Ayerst).
[0825] For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also
Saito et al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et
al., (2003) Cancer Immunol. Immunother. 52:328-337; Payne, (2003)
Cancer Cell 3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763;
Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs
3:1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev.
53:247-264.
[0826] According to the present invention, modified antibodies or
antigen binding fragments thereof can also be conjugated to a
radioactive isotope to generate cytotoxic radiopharmaceuticals,
referred to as radioimmunoconjugates. Examples of radioactive
isotopes that can be conjugated to antibodies for use
diagnostically or therapeutically include, but are not limited to,
iodine.sup.31, indium.sup.111, yttrium.sup.90, and lutetium.sup.77.
Methods for preparing radioimmunoconjugates are established in the
art. Examples of radioimmunoconjugates are commercially available,
including Zevalin.TM. (DEC Pharmaceuticals) and Bexxar.TM. (Corixa
Pharmaceuticals), and similar methods can be used to prepare
radioimmunoconjugates using the antibodies of the invention. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA) which can be attached to the antibody via a linker molecule.
Such linker molecules are commonly known in the art and described
in Denardo et al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson
et al., (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al.,
(1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by
reference in their entireties.
[0827] The present invention further provides modified antibodies
or antigen binding fragments thereof that specifically bind to an
antigen conjugated to a heterologous protein or polypeptide (or
fragment thereof, preferably to a polypeptide of at least 10, at
least 20, at least 30, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90 or at least 100 amino acids) to
generate fusion proteins. In particular, the invention provides
fusion proteins comprising an antibody fragment described herein
(e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a
V.sub.H domain, a V.sub.H CDR, a V.sub.L domain or a V.sub.L CDR)
and a heterologous protein, polypeptide, or peptide.
[0828] Additional fusion proteins may be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to alter the activities
of antibodies of the invention or fragments thereof (e.g.,
antibodies or fragments thereof with higher affinities and lower
dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793,
5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al.,
(1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends
Biotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol.
287:265-76; and Lorenzo and Blasco, (1998) Biotechniques
24(2):308-313 (each of these patents and publications are hereby
incorporated by reference in its entirety). Antibodies or fragments
thereof, or the encoded antibodies or fragments thereof, may be
altered by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to
recombination. A polynucleotide encoding an antibody or fragment
thereof that specifically binds to an antigen may be recombined
with one or more components, motifs, sections, parts, domains,
fragments, etc. of one or more heterologous molecules.
[0829] Moreover, the modified antibodies or antigen binding
fragments thereof of the present invention can be conjugated to
marker sequences, such as a peptide to facilitate purification. In
preferred embodiments, the marker amino acid sequence is a
hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among
others, many of which are commercially available. As described in
Gentz et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Other peptide tags useful for purification
include, but are not limited to, the hemagglutinin ("HA") tag,
which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the
"FLAG" tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49:
455-465, 2001). According to the present invention, antibodies or
antigen binding fragments can also be conjugated to
tumor-penetrating peptides in order to enhance their efficacy.
[0830] In other embodiments, modified antibodies or antigen binding
fragments of the present invention are conjugated to a diagnostic
or detectable agent. Such immunoconjugates can be useful for
monitoring or prognosing the onset, development, progression and/or
severity of a disease or disorder as part of a clinical testing
procedure, such as determining the efficacy of a particular
therapy. Such diagnosis and detection can accomplished by coupling
the antibody to detectable substances including, but not limited
to, various enzymes, such as, but not limited to, horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; prosthetic groups, such as, but not limited
to, streptavidin/biotin and avidin/biotin; fluorescent materials,
such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405,
Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514,
Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568,
Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647,
Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750,
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as, but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such
as, but not limited to, iodine (.sup.131I, .sup.125I, .sup.123I,
and .sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In, and
.sup.111In), technetium (.sup.99Tc), thallium (.sup.201Ti), gallium
(.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, 47Sc, .sup.186Re, .sup.188Re, 142 Pr,
.sup.105Rh, .sup.97Ru, .sup.68Ge, 57Co, .sup.65Zn, .sup.85Sr,
.sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se,
.sup.64Cu, .sup.113Sn, and .sup.117Sn; and positron emitting metals
using various positron emission tomographies, and non-radioactive
paramagnetic metal ions.
[0831] Modified antibodies or antigen binding fragments of the
invention may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
5. Pharmaceutical Composition
[0832] To prepare pharmaceutical or sterile compositions including
immunoconjugates, the immunoconjugates of the invention are mixed
with a pharmaceutically acceptable carrier or excipient. The
compositions can additionally contain one or more other therapeutic
agents that are suitable for treating or preventing cancer (breast
cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian
cancer, liver cancer, gastric cancer, pancreatic cancer, acute
myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous
cell carcinoma, peripheral nerve sheath tumors schwannoma, head and
neck cancer, bladder cancer, esophageal cancer, Barretts esophageal
cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant
mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate
cancer, benign prostatic hyperplasia (BPH), gynacomastica, and
endometriosis).
[0833] Formulations of therapeutic and diagnostic agents can be
prepared by mixing with physiologically acceptable carriers,
excipients, or stabilizers in the form of, e.g., lyophilized
powders, slurries, aqueous solutions, lotions, or suspensions (see,
e.g., Hardman et al., Goodman and Gilman's The Pharmacological
Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001; Gennaro,
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.),
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
N.Y., 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, N.Y., 1990; Lieberman, et al. (eds.)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.,
1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y., 2000).
[0834] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. In certain embodiments, an administration
regimen maximizes the amount of therapeutic delivered to the
patient consistent with an acceptable level of side effects.
Accordingly, the amount of biologic delivered depends in part on
the particular entity and the severity of the condition being
treated. Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991; Bach
(ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y., 1993; Baert et al., New
Engl. J. Med. 348:601-608, 2003; Milgrom et al., New Engl. J. Med.
341:1966-1973, 1999; Slamon et al., New Engl. J. Med. 344:783-792,
2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619, 2000;
Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al., New
Engl. J. Med. 343:1594-1602, 2000).
[0835] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0836] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors known in the medical
arts.
[0837] Compositions comprising antibodies or fragments thereof of
the invention can be provided by continuous infusion, or by doses
at intervals of, e.g., one day, one week, or 1-7 times per week.
Doses may be provided intravenously, subcutaneously, topically,
orally, nasally, rectally, intramuscular, intracerebrally, or by
inhalation. A specific dose protocol is one involving the maximal
dose or dose frequency that avoids significant undesirable side
effects.
[0838] For the immunoconjugates of the invention, the dosage
administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the
patient's body weight. The dosage may be between 0.0001 mg/kg and
20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg,
0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75
mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg,
0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg,
0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body
weight. The dosage of the antibodies or fragments thereof of the
invention may be calculated using the patient's weight in kilograms
(kg) multiplied by the dose to be administered in mg/kg.
[0839] Doses of the immunoconjugates the invention may be repeated
and the administrations may be separated by at least 1 day, 2 days,
3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75
days, 3 months, or at least 6 months. In a specific embodiment,
does of the immunoconjugates of the invention are repeated every 3
weeks.
[0840] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side effects (see, e.g., Maynard
et al., A Handbook of SOPs for Good Clinical Practice, Interpharm
Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good
Clinical Practice, Urch Publ., London, UK, 2001).
[0841] The route of administration may be by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or by sustained
release systems or an implant (see, e.g., Sidman et al.,
Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res.
15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et
al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al.,
Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos.
6,350,466 and 6,316,024). Where necessary, the composition may also
include a solubilizing agent and a local anesthetic such as
lidocaine to ease pain at the site of the injection. In addition,
pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309,
5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT
Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO
98/31346, and WO 99/66903, each of which is incorporated herein by
reference their entirety.
[0842] A composition of the present invention may also be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Selected routes of administration for the immunoconjugates of the
invention include intravenous, intramuscular, intradermal,
intraperitoneal, subcutaneous, spinal or other parenteral routes of
administration, for example by injection or infusion. Parenteral
administration may represent modes of administration other than
enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion. Alternatively, a composition of the invention can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically. In one embodiment, the immunoconjugates of the invention
is administered by infusion. In another embodiment, the
immunoconjugates of the invention is administered
subcutaneously.
[0843] If the immunoconjugates of the invention are administered in
a controlled release or sustained release system, a pump may be
used to achieve controlled or sustained release (see Langer, supra;
Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al.,
Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574,
1989). Polymeric materials can be used to achieve controlled or
sustained release of the therapies of the invention (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley,
New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol.
Chem. 23:61, 1983; see also Levy et al., Science 228:190, 1985;
During et al., Ann. Neurol. 25:351, 1989; Howard et al., J.
Neurosurg. 7 1:105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No.
5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.
Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT
Publication No. WO 99/20253. Examples of polymers used in sustained
release formulations include, but are not limited to,
poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one
embodiment, the polymer used in a sustained release formulation is
inert, free of leachable impurities, stable on storage, sterile,
and biodegradable. A controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138, 1984).
[0844] Controlled release systems are discussed in the review by
Langer, Science 249:1527-1533, 1990). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more immunoconjugates of the
invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO
91/05548, PCT publication WO 96/20698, Ning et al., Radiotherapy
& Oncology 39:179-189, 1996; Song et al., PDA Journal of
Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et
al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854,
1997; and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact.
Mater. 24:759-760, 1997, each of which is incorporated herein by
reference in their entirety.
[0845] If the immunoconjugates of the invention are administered
topically, they can be formulated in the form of an ointment,
cream, transdermal patch, lotion, gel, shampoo, spray, aerosol,
solution, emulsion, or other form well-known to one of skill in the
art. See, e.g., Remington's Pharmaceutical Sciences and
Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub.
Co., Easton, Pa. (1995). For non-sprayable topical dosage forms,
viscous to semi-solid or solid forms comprising a carrier or one or
more excipients compatible with topical application and having a
dynamic viscosity, in some instances, greater than water are
typically employed. Suitable formulations include, without
limitation, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, and the like, which are, if desired,
sterilized or mixed with auxiliary agents (e.g., preservatives,
stabilizers, wetting agents, buffers, or salts) for influencing
various properties, such as, for example, osmotic pressure. Other
suitable topical dosage forms include sprayable aerosol
preparations wherein the active ingredient, in some instances, in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon) or in a squeeze bottle. Moisturizers or humectants
can also be added to pharmaceutical compositions and dosage forms
if desired. Examples of such additional ingredients are well-known
in the art.
[0846] If the compositions comprising the immunoconjugates are
administered intranasally, it can be formulated in an aerosol form,
spray, mist or in the form of drops. In particular, prophylactic or
therapeutic agents for use according to the present invention can
be conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of
a suitable propellant (e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas). In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges (composed of, e.g.,
gelatin) for use in an inhaler or insufflator may be formulated
containing a powder mix of the compound and a suitable powder base
such as lactose or starch.
[0847] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are known in the art (see, e.g.,
Hardman et al., (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice: A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.). An effective amount of
therapeutic may decrease the symptoms by at least 10%; by at least
20%; at least about 30%; at least 40%, or at least 50%.
[0848] Additional therapies (e.g., prophylactic or therapeutic
agents), which can be administered in combination with the
immunoconjugates of the invention may be administered less than 5
minutes apart, less than 30 minutes apart, 1 hour apart, at about 1
hour apart, at about 1 to about 2 hours apart, at about 2 hours to
about 3 hours apart, at about 3 hours to about 4 hours apart, at
about 4 hours to about 5 hours apart, at about 5 hours to about 6
hours apart, at about 6 hours to about 7 hours apart, at about 7
hours to about 8 hours apart, at about 8 hours to about 9 hours
apart, at about 9 hours to about 10 hours apart, at about 10 hours
to about 11 hours apart, at about 11 hours to about 12 hours apart,
at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24
hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52
hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours
apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or
96 hours to 120 hours apart from the immunoconjugates of the
invention. The two or more therapies may be administered within one
same patient visit.
[0849] In certain embodiments, the immunoconjugates of the
invention can be formulated to ensure proper distribution in vivo.
For example, the blood-brain barrier (BBB) excludes many highly
hydrophilic compounds. To ensure that the therapeutic compounds of
the invention cross the BBB (if desired), they can be formulated,
for example, in liposomes. For methods of manufacturing liposomes,
see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et
al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant
protein A receptor (Briscoe et al., (1995) Am. J. Physiol.
1233:134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090);
see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J.
J. Killion; I. J. Fidler (1994) Immunomethods 4:273.
[0850] The invention provides protocols for the administration of
pharmaceutical composition comprising immunoconjugates of the
invention alone or in combination with other therapies to a subject
in need thereof. The therapies (e.g., prophylactic or therapeutic
agents) of the combination therapies of the present invention can
be administered concomitantly or sequentially to a subject. The
therapy (e.g., prophylactic or therapeutic agents) of the
combination therapies of the present invention can also be
cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time and repeating this
sequential administration, i.e., the cycle, in order to reduce the
development of resistance to one of the therapies (e.g., agents) to
avoid or reduce the side effects of one of the therapies (e.g.,
agents), and/or to improve, the efficacy of the therapies.
[0851] The therapies (e.g., prophylactic or therapeutic agents) of
the combination therapies of the invention can be administered to a
subject concurrently.
[0852] The term "concurrently" is not limited to the administration
of therapies (e.g., prophylactic or therapeutic agents) at exactly
the same time, but rather it is meant that a pharmaceutical
composition comprising antibodies or fragments thereof the
invention are administered to a subject in a sequence and within a
time interval such that the antibodies of the invention can act
together with the other therapy(ies) to provide an increased
benefit than if they were administered otherwise. For example, each
therapy may be administered to a subject at the same time or
sequentially in any order at different points in time; however, if
not administered at the same time, they should be administered
sufficiently close in time so as to provide the desired therapeutic
or prophylactic effect. Each therapy can be administered to a
subject separately, in any appropriate form and by any suitable
route. In various embodiments, the therapies (e.g., prophylactic or
therapeutic agents) are administered to a subject less than 15
minutes, less than 30 minutes, less than 1 hour apart, at about 1
hour apart, at about 1 hour to about 2 hours apart, at about 2
hours to about 3 hours apart, at about 3 hours to about 4 hours
apart, at about 4 hours to about 5 hours apart, at about 5 hours to
about 6 hours apart, at about 6 hours to about 7 hours apart, at
about 7 hours to about 8 hours apart, at about 8 hours to about 9
hours apart, at about 9 hours to about 10 hours apart, at about 10
hours to about 11 hours apart, at about 11 hours to about 12 hours
apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week
apart. In other embodiments, two or more therapies (e.g.,
prophylactic or therapeutic agents) are administered to a within
the same patient visit.
[0853] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
[0854] The invention having been fully described, it is further
illustrated by the following examples and claims, which are
illustrative and are not meant to be further limiting.
Examples
Example 1. Design of Peptide-Tagged IgG Constructs
[0855] Visual inspection of the NMR structure of the
4'-phosphopantetheinyl transferase (PPTase) Sfp (PDB ID: 2GE1,
Koglin et al., (2006) Science 312: 273-276) model with a peptide
substrate reveals that the reactive Ser residue of the S6 tag is
inserted deeply into the enzyme active site and is positioned near
the alpha phosphate of coenzyme A. The peptide substrate adopts a
helix-kink-loop conformation with the Ser residue at the kink.
Based on these observations, several loops on the surface of IgG
antibodies were selected. The selection procedure involved the
following steps. We first built a Trastuzumab homology model using
human IgG1 B12 antibody (PDB ID: 1HZH, Saphire et al., (2001)
Science 293: 1155-1159) as a template. Next, the loops with
significant content of solvent exposed residues were selected and
transformed into S6 tag loops.
[0856] To that end, different strategies were exploited: grafting
of full-length peptide tag, grafting of truncated peptide tag, and
insertions (both truncated and full-length). One example of the
grafting of a full-length ybbR tag is exemplified by the mutant
anti-hHER2-HC-S132D-K133S-S134L-T135E-S136F-G1371-G138A-T139S-A140K-A141L-
-L142A (SEQ ID NO:102), while the Trastuzumab
anti-hHER2-HC-P189G-S190D-S191-S192L-L193S-G194W-T195L (SEQ ID
NO:109) mutant constitutes grafting of a truncated S6 tag. Another
variant of the grafting strategy was employed, for example, in
mutant anti-hHER2-HC-S190G-S191
D-S192-L193-G194S-T195W-Q196L-T197L-RLLN-Y198 (SEQ ID NO:113)
wherein residues S190 and S191 were mutated to glycine and aspartic
acid, respectively, G194 to serine, T195 to tryptophan, Q196 and
T197 to leucine and the truncated S6 tag RLLN was inserted between
L197 and Y198. Alternatively, both truncated and full-length
peptide tags were inserted into loops between antibody
residues.
[0857] Through out the Example section, the peptide-tagged
antibodies are named according to the immunoglobulin heavy or light
chain, which contains the grafted or inserted peptide tag. For
simplicity, the associated unmodified heavy or light chain is not
explicitly mentioned. For example, mAb2-HC-T359-GDSLSWLLRLLN-K360
(SEQ ID NO:148) refers to an IgG1, which comprises the
corresponding peptide-tagged heavy chain and the associated
unmodified kappa light chain anti-hHER2-LC (SEQ ID NO:24) with
X5=Ala and X6=Val. In contrast, the peptide-tagged mAb2 heavy chain
constructs are associated with the unmodified lambda light chain
mAb2-LC (SEQ ID NO:25). As another example,
anti-hHER2-LC-S76D-S77-L78-EFIASKLA-Q79 (SEQ ID NO:30) refers to an
IgG1 antibody containing a peptide-tagged light chain that is
associated with the unmodified Ig gamma 1 heavy chain anti-hHER2-HC
(SEQ ID NO:93) with X1=Lys, X2=Asp, X3=Leu, and X4=Ala. In cases
where the peptide tag(s) is inserted or grafted into the constant
region of the heavy or light chain of an antibody, only sequences
of the constant region are given.
[0858] In all cases, the peptide tag was mapped on the selected
loops in such a way that the reactive Ser residue was at or near
the tip of the loop in order to allow a deeper fit into the active
site of Sfp enzyme. The complexes between IgG and Sfp enzyme were
constructed next and examined for clashes. Those with significant
clashes were rejected and the corresponding loops were excluded
from the selection.
[0859] To systematically insert the S6 and ybbR tag sequences into
structural loops of the constant regions of Trastuzumab IgG1,
insertion sites were chosen both by visual inspection of the
crystal structure of the human IgG1 B12 antibody (PDB ID: 1HZH) as
well as by calculating the solvent-accessible surface area of
residues by using the program ICM from MolSoft LLC.
Example 2. Production of Peptide-Tagged IgG Constructs
[0860] The heavy and light chains of Trastuzumab IgG1 were
transiently expressed in mammalian cells using the pOG expression
vector under the control of a CMV promoter. Peptide tags for
labeling with 4'-phosphopantetheinyl transferases were incorporated
into Trastuzumab IgG1 at various positions by standard molecular
biology methods. All primers used for cloning are listed in Table
8.
[0861] Cell culture and transfection of HEK293F cells was performed
using the PEI method as described previously (see for example
Erbacher et al., J Gene Med., 1: 210-222 (1999)). Briefly, HEK293F
cells were co-transfected with plasmid DNA encoding the heavy and
light chains of Trastuzumab (human kappa isotype). The mammalian
cells were cultured in FreeStyle.TM. 293 Expression Medium at
37.degree. C. under 5% CO.sub.2, and were split to
0.7.times.10.sup.6 cells/ml one day prior to transfection.
Following transfection, the HEK293F cells were cultured for five
days before harvest by centrifugation at 2000.times.g for 30
minutes at 4.degree. C.
[0862] The resulting medium supernatant was filtered through a
0.22-.mu.m-pore-size filter. The filtrate was then loaded at a flow
rate of about 1 mL/min on a protein A affinity column that was
previously equilibrated with 20 column volumes of PBS. After
washing the column with 20 column volumes of PBS, the antibody was
eluted with 5 column volumes of 0.1 M sodium acetate (pH 3.0). The
eluate was immediately neutralized with 10% (v/v) 1 M Tris/HCl (pH
10). Dialysis into PBS was performed using Slide-a-Lyzer dialysis
cassettes with 3.5 or 7.0 kDa molecular weight cut-off
(Pierce).
[0863] The purity of the final product was assessed by SDS-PAGE.
Protein yields were determined by either the Bradford method or by
ultraviolet spectroscopy at 280 nm using an ND-1000 UV-Vis
Spectrophotometer. Protein yields of peptide-tagged Trastuzumab
IgGs are listed in Table 9.
TABLE-US-00008 TABLE 8 DNA sequences of primers used for
constructing recombinant PPTase enzymes and mutants thereof as well
as Trastuzumab IgGs with inserted/grafted peptide-tags (HC, heavy
chain; LC, light chain) SEQ ID Sequence name Sequence NO
anti-hHER2-HC-A118- CTGAGCTGGCTGCTGAGACTGCTGAACAGCACCAAGGGCCCCAGCG
355 GDSLSWLLRLLN-S119
TCTCAGCAGCCAGCTCAGGCTGTCGCCAGCCGAGGAGACGGTGACCAG 356
anti-hHER2-HC-S119-
CTGAGCTGGCTGCTGAGACTGCTGAACACCAAGGGCCCCAGCGTGTTC 357
GDSLSWLLRLLN-T120 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTAGCCGAGGAGACGGTGAC
358 anti-hHER2-HC-T120-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGGGCCCCAGCGTGTTCCC 359
GDSLSWLLRLLN-K121 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTGCTAGCCGAGGAGACGG
360 anti-hHER2-HC-S131-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAAGAGCACCAGCGGCGG 361
GDSLSWLLRLLN-S132 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGGGGCCAGGGGGAAC
362 anti-hHER2-HC-S132-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGAGCACCAGCGGCGGCAC 363
GDSLSWLLRLLN-K133 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTGGGGGCCAGGGG 364
anti-hHER2-HC-K133- CTGAGCTGGCTGCTGAGACTGCTGAACAGCACCAGCGGCGGCACAG
365 GDSLSWLLRLLN-S134
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGCTGCTGGGGGCCAGG 366
anti-hHER2-HC-S134- CTGAGCTGGCTGCTGAGACTGCTGAACACCAGCGGCGGCACAGCC
367 GDSLSWLLRLLN-T135
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCTTGCTGCTGGGGGCC 368
anti-hHER2-HC-T135- CTGAGCTGGCTGCTGAGACTGCTGAACAGCGGCGGCACAGCCGCC
369 GDSLSWLLRLLN-S136
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTGCTCTTGCTGCTGGGGG 370
anti-hHER2-HC-S136- CTGAGCTGGCTGCTGAGACTGCTGAACGGCGGCACAGCCGCCCTG
371 GDSLSWLLRLLN-G137
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGTGCTCTTGCTGCTGGG 372
anti-hHER2-HC-G137- CTGAGCTGGCTGCTGAGACTGCTGAACGGCACAGCCGCCCTGGGC
373 GDSLSWLLRLLN-G138
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCGCTGGTGCTCTTGCTGC 374
anti-hHER2-HC-G138- CTGAGCTGGCTGCTGAGACTGCTGAACACAGCCGCCCTGGGCTGC
375 GDSLSWLLRLLN-T139
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCGCCGCTGGTGCTCTTG 376
anti-hHER2-HC-E152-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCGTGACCGTGTCCTGGAAC 377
GDSLSWLLRLLN-P153 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCGGGGAAGTAGTCCTTCACC
378 anti-hHER2-HC-P153-
CTGAGCTGGCTGCTGAGACTGCTGAACGTGACCGTGTCCTGGAACAGCG 379
GDSLSWLLRLLN-V154 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGCTCGGGGAAGTAGTCCTTC
380 anti-hHER2-HC-N159-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGGAGCCCTGACCTCCG 381
GDSLSWLLRLLN-S160 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTCCAGGACACGGTCACGGG
382 anti-hHER2-HC-S160-
CTGAGCTGGCTGCTGAGACTGCTGAACGGAGCCCTGACCTCCGGCGTGCAC 383
GDSLSWLLRLLN-G161 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGTTCCAGGACACGGTCACG
384 anti-hHER2-HC-G161-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCCTGACCTCCGGCGTG 385 GDSLSWLLRLLN-A162
TCTCAGCAGCCAGCTCAGGCTGTCGCCTCCGCTGTTCCAGGACACGG 386
anti-hHER2-HC-A162- CTGAGCTGGCTGCTGAGACTGCTGAACCTGACCTCCGGCGTGCACAC
387 GDSLSWLLRLLN-L163
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCTCCGCTGTTCCAGGACAC 388
anti-hHER2-HC-L163-
CTGAGCTGGCTGCTGAGACTGCTGAACACCTCCGGCGTGCACACCTTC 389
GDSLSWLLRLLN-T164 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGGCTCCGCTGTTCCAGG
390 anti-hHER2-HC-T164-
CTGAGCTGGCTGCTGAGACTGCTGAACTCCGGCGTGCACACCTTCCC 391
GDSLSWLLRLLN-S165 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCAGGGCTCCGCTGTTCC
392 anti-hHER2-HC-S165-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCGTGCACACCTTCCCCG 393
GDSLSWLLRLLN-G166 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGAGGTCAGGGCTCCGCTG
394 anti-hHER2-HC-P171-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCGTGCTGCAGAGCAGCG 395
GDSLSWLLRLLN-A172 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGAAGGTGTGCACGCCG
396 anti-hHER2-HC-S176-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGGCCTGTACAGCCTGTCC 397
GDSLSWLLRLLN-S177 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCTGCAGCACGGCGGG 398
anti-hHER2-HC-S177-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCCTGTACAGCCTGTCCAGC 399
GDSLSWLLRLLN-G178 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTCTGCAGCACGGCG
400 anti-hHER2-HC-P189-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAGCAGCCTGGGCACCC 401
GDSLSWLLRLLN-S190 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGCACTGTCACCACGCTGG
402 anti-hHER2-HC-S190-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAGCCTGGGCACCCAGAC 403
GDSLSWLLRLLN-S191 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGGCACTGTCACCACGC
404 anti-hHER2-HC-S191-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCCTGGGCACCCAGACCTAC 405
GDSLSWLLRLLN-S192 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTGGGCACTGTCACCAC
406 anti-hHER2-HC-S192-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGGGCACCCAGACCTACATC 407
GDSLSWLLRLLN-L193 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTGCTGGGCACTGTCAC
408 anti-hHER2-HC-L193-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCACCCAGACCTACATCTGC 409
GDSLSWLLRLLN-G194 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGCTGCTGCTGGGCACTG
410 anti-hHER2-HC-G194-
CTGAGCTGGCTGCTGAGACTGCTGAACACCCAGACCTACATCTGCAACGTG 411
GDSLSWLLRLLN-T195 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCCAGGCTGCTGCTGGG 412
anti-hHER2-HC-P189G-S190D-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGACCTACATCTGCAACGTGAAC 413
S191-S192L-L193S-G194W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTGCCCAGGCTGCTGCTG 414 T195L-LRLLN-Q196
anti-hHER2-HC-Q196-
CTGAGCTGGCTGCTGAGACTGCTGAACACCTACATCTGCAACGTGAACCAC 415
GDSLSWLLRLLN-T197 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGGGTGCCCAGGCTGCTG
416 anti-hHER2-HC-K205-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCAGCAACACCAAGGTGGAC 417
GDSLSWLLRLLN-P206
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGTGGTTCACGTTGCAGATGTAGG 418
anti-hHER2-HC-P206-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAACACCAAGGTGGACAAGAAAG 419
GDSLSWLLRLLN-S207 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGCTTGTGGTTCACGTTGCAG
420 anti-hHER2-HC-S207-
CTGAGCTGGCTGCTGAGACTGCTGAACAACACCAAGGTGGACAAGAAAGTGG 421
GDSLSWLLRLLN-N208 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGGCTTGTGGTTCACGTTG
422 anti-hHER2-HC-P230-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCCCAGAGCTGCTGGGC 423 GDSLSWLLRLLN-A231
TCTCAGCAGCCAGCTCAGGCTGTCGCCTGGGCAGGGGGGGCAGGTG 424
anti-hHER2-HC-A231- CTGAGCTGGCTGCTGAGACTGCTGAACCCAGAGCTGCTGGGCGGAC
425 GDSLSWLLRLLN-P232 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCTGGGCAGGGGGGGC
426 anti-hHER2-HC-P232-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGCTGCTGGGCGGACCC 427 GDSLSWLLRLLN-E233
TCTCAGCAGCCAGCTCAGGCTGTCGCCTGGGGCTGGGCAGGGGGG 428
anti-hHER2-HC-E233- CTGAGCTGGCTGCTGAGACTGCTGAACCTGCTGGGCGGACCCTCC
429 GDSLSWLLRLLN-L234 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCTGGGGCTGGGCAGGG
430 anti-hHER2-HC-L234-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGGGCGGACCCTCCGTG 431 GDSLSWLLRLLN-L235
TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGCTCTGGGGCTGGGCAG 432
anti-hHER2-HC-L235- CTGAGCTGGCTGCTGAGACTGCTGAACGGCGGACCCTCCGTGTTCC
433 GDSLSWLLRLLN-G236
TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGCAGCTCTGGGGCTGGG 434
anti-hHER2-HC-G236-
CTGAGCTGGCTGCTGAGACTGCTGAACGGACCCTCCGTGTTCCTGTTCC 435
GDSLSWLLRLLN-G237 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCCAGCAGCTCTGGGGC 436
anti-hHER2-HC-P244-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCAAGCCCAAGGACACCCTG 437
GDSLSWLLRLLN-P245 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGAACAGGAACACGGAGGG
438 anti-hHER2-HC-P245-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGCCCAAGGACACCCTGATGATC 439
GDSLSWLLRLLN-K246 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGGGGAACAGGAACACGG
440 anti-hHER2-HC-I253-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAGGACCCCCGAGGTGAC 441
GDSLSWLLRLLN-S254 TCTCAGCAGCCAGCTCAGGCTGTCGCCGATCATCAGGGTGTCCTTGGGC
442 anti-hHER2-HC-S254-
CTGAGCTGGCTGCTGAGACTGCTGAACAGGACCCCCGAGGTGACCTG 443
GDSLSWLLRLLN-R255
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGATCATCAGGGTGTCCTTGG 444
anti-hHER2-HC-R255- CTGAGCTGGCTGCTGAGACTGCTGAACACCCCCGAGGTGACCTGCG
445 GDSLSWLLRLLN-T256
TCTCAGCAGCCAGCTCAGGCTGTCGCCCCTGCTGATCATCAGGGTGTCC 446
anti-hHER2-HC-T256- CTGAGCTGGCTGCTGAGACTGCTGAACCCCGAGGTGACCTGCGTGG
447 GDSLSWLLRLLN-P257
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCCTGCTGATCATCAGGGTG 448
anti-hHER2-HC-P257-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGGTGACCTGCGTGGTGGTG 449
GDSLSWLLRLLN-E258 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGGTCCTGCTGATCATCAG
450 anti-hHER2-HC-S267-
CTGAGCTGGCTGCTGAGACTGCTGAACCACGAGGACCCAGAGGTGAAGTTC 451
GDSLSWLLRLLN-H268 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCACGTCCACCACCACGC
452 anti-hHER2-HC-H268-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGGACCCAGAGGTGAAGTTCAAC 453
GDSLSWLLRLLN-E269 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTGGCTCACGTCCACCACCAC
454 anti-hHER2-HC-E269-
CTGAGCTGGCTGCTGAGACTGCTGAACGACCCAGAGGTGAAGTTCAACTGG 455
GDSLSWLLRLLN-D270 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCGTGGCTCACGTCCACCAC
456 anti-hHER2-HC-D270-
CTGAGCTGGCTGCTGAGACTGCTGAACCCAGAGGTGAAGTTCAACTGGTAC 457
GDSLSWLLRLLN-P271 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCCTCGTGGCTCACGTCCAC
458 anti-hHER2-HC-P271-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGGTGAAGTTCAACTGGTACGTGG 459
GDSLSWLLRLLN-E272 TCTCAGCAGCCAGCTCAGGCTGTCGCCTGGGTCCTCGTGGCTCACGTC
460 anti-hHER2-HC-D280-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCGTGGAGGTGCACAACGC 461
GDSLSWLLRLLN-G281
TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCCACGTACCAGTTGAACTTCACC 462
anti-hHER2-HC-H285-
CTGAGCTGGCTGCTGAGACTGCTGAACAACGCCAAGACCAAGCCCAGAG 463
GDSLSWLLRLLN-N286 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTGCACCTCCACGCCGTCC
464 anti-hHER2-HC-N286-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCAAGACCAAGCCCAGAGAG 465
GDSLSWLLRLLN-A287 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGTGCACCTCCACGCCGTC
466 anti-hHER2-HC-P291-
CTGAGCTGGCTGCTGAGACTGCTGAACAGAGAGGAGCAGTACAACAGCACC 467
GDSLSWLLRLLN-R292 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGCTTGGTCTTGGCGTTGTG
468 anti-hHER2-HC-T307-
CTGAGCTGGCTGCTGAGACTGCTGAACGTGCTGCACCAGGACTGGCTG 469
GDSLSWLLRLLN-V308 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCAGCACGGACACCACCC
470 anti-hHER2-HC-V308-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGCACCAGGACTGGCTGAAC 471
GDSLSWLLRLLN-L309 TCTCAGCAGCCAGCTCAGGCTGTCGCCCACGGTCAGCACGGACACCAC
472 anti-hHER2-HC-L309-
CTGAGCTGGCTGCTGAGACTGCTGAACCACCAGGACTGGCTGAACGGC 473
GDSLSWLLRLLN-H310 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGCACGGTCAGCACGGACAC
474 anti-hHER2-HC-H310-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGGACTGGCTGAACGGCAAG 475
GDSLSWLLRLLN-Q311 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTGCAGCACGGTCAGCACGG
476 anti-hHER2-HC-N315-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCAAGGAATACAAGTGCAAGGTC 477
GDSLSWLLRLLN-G316 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTCAGCCAGTCCTGGTGCAG
478 anti-hHER2-HC-G316-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGGAATACAAGTGCAAGGTCTCCAAC 479
GDSLSWLLRLLN-K317 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCGTTCAGCCAGTCCTGGTG
480 anti-hHER2-HC-K317-
CTGAGCTGGCTGCTGAGACTGCTGAACGAATACAAGTGCAAGGTCTCCAACAAG 481
GDSLSWLLRLLN-E318 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGCCGTTCAGCCAGTCCTG
482 anti-hHER2-HC-K326-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCCTGCCAGCCCCCATC 483 GDSLSWLLRLLN-A327
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGTTGGAGACCTTGCACTTGTATTC 484
anti-hHER2-HC-A327-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGCCAGCCCCCATCGAAAAG 485
GDSLSWLLRLLN-L328 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCCTTGTTGGAGACCTTGCAC
486 anti-hHER2-HC-L328-
CTGAGCTGGCTGCTGAGACTGCTGAACCCAGCCCCCATCGAAAAGACC 487
GDSLSWLLRLLN-P329 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGGCCTTGTTGGAGACCTTG
488 anti-hHER2-HC-P329-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCCCCATCGAAAAGACCATCAG 489
GDSLSWLLRLLN-A330 TCTCAGCAGCCAGCTCAGGCTGTCGCCTGGCAGGGCCTTGTTGGAGAC
490 anti-hHER2-HC-A330-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCATCGAAAAGACCATCAGCAAG 491
GDSLSWLLRLLN-P331 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCTGGCAGGGCCTTGTTGG
492 anti-hHER2-HC-A339-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGGGCCAGCCACGGGAGC 493
GDSLSWLLRLLN-K340
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCCTTGCTGATGGTCTTTTCGATG 494
anti-hHER2-HC-K340- CTGAGCTGGCTGCTGAGACTGCTGAACGGCCAGCCACGGGAGCCC
495 GDSLSWLLRLLN-G341
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGGCCTTGCTGATGGTCTTTTC 496
anti-hHER2-HC-G341- CTGAGCTGGCTGCTGAGACTGCTGAACCAGCCACGGGAGCCCCAG
497 GDSLSWLLRLLN-Q342
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCCTTGGCCTTGCTGATGGTC 498
anti-hHER2-HC-Q342- CTGAGCTGGCTGCTGAGACTGCTGAACCCACGGGAGCCCCAGGTG
499 GDSLSWLLRLLN-P343
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGGCCCTTGGCCTTGCTGATG 500
anti-hHER2-HC-P343- CTGAGCTGGCTGCTGAGACTGCTGAACCGGGAGCCCCAGGTGTACAC
501 GDSLSWLLRLLN-R344
TCTCAGCAGCCAGCTCAGGCTGTCGCCTGGCTGGCCCTTGGCCTTGC 502
anti-hHER2-HC-R344-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGCCCCAGGTGTACACCCTG 503
GDSLSWLLRLLN-E345 TCTCAGCAGCCAGCTCAGGCTGTCGCCCCGTGGCTGGCCCTTGGC 504
anti-hHER2-HC-R355-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGGAGATGACCAAGAACCAGGTG 505
GDSLSWLLRLLN-E356 TCTCAGCAGCCAGCTCAGGCTGTCGCCCCGGGAGGGGGGCAGGG 506
anti-hHER2-HC-E356-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGATGACCAAGAACCAGGTGTCC 507
GDSLSWLLRLLN-E357 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCCCGGGAGGGGGGCAG 508
anti-hHER2-HC-E357-
CTGAGCTGGCTGCTGAGACTGCTGAACATGACCAAGAACCAGGTGTCCCTG 509
GDSLSWLLRLLN-M358 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCCTCCCGGGAGGGGGG 510
anti-hHER2-HC-M358-
CTGAGCTGGCTGCTGAGACTGCTGAACACCAAGAACCAGGTGTCCCTGAC 511
GDSLSWLLRLLN-T359 TCTCAGCAGCCAGCTCAGGCTGTCGCCCATCTCCTCCCGGGAGGGG
512 anti-hHER2-HC-T359-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGAACCAGGTGTCCCTGACCTG 513
GDSLSWLLRLLN-K360 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCATCTCCTCCCGGGAGG
514 anti-hHER2-HC-K360-
CTGAGCTGGCTGCTGAGACTGCTGAACAACCAGGTGTCCCTGACCTGTC 515
GDSLSWLLRLLN-N361 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGGTCATCTCCTCCCGGGAG
516 anti-hHER2-HC-N384-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCCAGCCCGAGAACAACTAC 517
GDSLSWLLRLLN-G385 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGCTCTCCCACTCCACGGC
518 anti-hHER2-HC-E388-
CTGAGCTGGCTGCTGAGACTGCTGAACAACAACTACAAGACCACACCTCCAG 519
GDSLSWLLRLLN-N389 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCGGGCTGGCCGTTGCTC
520 anti-hHER2-HC-N389-
CTGAGCTGGCTGCTGAGACTGCTGAACAACTACAAGACCACACCTCCAGTGC 521
GDSLSWLLRLLN-N390 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTCTCGGGCTGGCCGTTGC
522 anti-hHER2-HC-T394-
CTGAGCTGGCTGCTGAGACTGCTGAACCCTCCAGTGCTGGACAGCGAC 523
GDSLSWLLRLLN-P395
TCTCAGCAGCCAGCTCAGGCTGTCGCCTGTGGTCTTGTAGTTGTTCTCGGGC 524
anti-hHER2-HC-P395- CTGAGCTGGCTGCTGAGACTGCTGAACCCAGTGCTGGACAGCGACGG
525 GDSLSWLLRLLN-P396
TCTCAGCAGCCAGCTCAGGCTGTCGCCAGGTGTGGTCTTGTAGTTGTTCTCG 526
anti-hHER2-HC-D399-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGACGGCAGCTTCTTCCTG 527
GDSLSWLLRLLN-S400 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCCAGCACTGGAGGTGTGGTC
528 anti-hHER2-HC-S400-
CTGAGCTGGCTGCTGAGACTGCTGAACGACGGCAGCTTCTTCCTGTACAG 529
GDSLSWLLRLLN-D401 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGTCCAGCACTGGAGGTGTG
530 anti-hHER2-HC-D401-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCAGCTTCTTCCTGTACAGCAAG 531
GDSLSWLLRLLN-G402 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCGCTGTCCAGCACTGGAGG
532 anti-hHER2-HC-S415-
CTGAGCTGGCTGCTGAGACTGCTGAACAGGTGGCAGCAGGGCAACGTG 533
GDSLSWLLRLLN-R416 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGACTTGTCCACGGTCAGCTTG
534 anti-hHER2-HC-R416-
CTGAGCTGGCTGCTGAGACTGCTGAACTGGCAGCAGGGCAACGTGTTC 535
GDSLSWLLRLLN-W417 TCTCAGCAGCCAGCTCAGGCTGTCGCCCCTGGACTTGTCCACGGTCAG
536 anti-hHER2-HC-W417-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGCAGGGCAACGTGTTCAGC 537
GDSLSWLLRLLN-Q418 TCTCAGCAGCCAGCTCAGGCTGTCGCCCCACCTGGACTTGTCCACGGTC
538 anti-hHER2-HC-Q418-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGGGCAACGTGTTCAGCTGC 539
GDSLSWLLRLLN-Q419 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGCCACCTGGACTTGTCCAC
540 anti-hHER2-HC-Q419-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCAACGTGTTCAGCTGCAGC 541
GDSLSWLLRLLN-G420 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGCTGCCACCTGGACTTGTC
542 anti-hHER2-HC-G420-
CTGAGCTGGCTGCTGAGACTGCTGAACAACGTGTTCAGCTGCAGCGTGATG 543
GDSLSWLLRLLN-N421 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCCTGCTGCCACCTGGAC
544 anti-hHER2-HC-N421-
CTGAGCTGGCTGCTGAGACTGCTGAACGTGTTCAGCTGCAGCGTGATGC 545
GDSLSWLLRLLN-V422 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGCCCTGCTGCCACCTGG
546 anti-hHER2-HC-H433-
CTGAGCTGGCTGCTGAGACTGCTGAACAACCACTACACCCAGAAGAGCCTG 547
GDSLSWLLRLLN-N434 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTGCAGGGCCTCGTGCATCAC
548 anti-hHER2-HC-N434-
CTGAGCTGGCTGCTGAGACTGCTGAACCACTACACCCAGAAGAGCCTGAG 549
GDSLSWLLRLLN-H435 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGTGCAGGGCCTCGTGCATC
550 anti-hHER2-HC-S442-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGTCCCCCGGCAAGTAATCTAG 551
GDSLSWLLRLLN-L443 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCAGGCTCTTCTGGGTGTAG
552 anti-hHER2-HC-L443-
CTGAGCTGGCTGCTGAGACTGCTGAACTCCCCCGGCAAGTAATCTAGACAC 553
GDSLSWLLRLLN-S444 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGCTCAGGCTCTTCTGGGTG
554 anti-hHER2-HC-S444-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCGGCAAGTAATCTAGACACCTC 555
GDSLSWLLRLLN-P445 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGACAGGCTCAGGCTCTTCTG
556 anti-hHER2-HC-P445-
CTGAGCTGGCTGCTGAGACTGCTGAACGGCAAGTAATCTAGACACCTCAGAC 557
GDSLSWLLRLLN-G446 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGGACAGGCTCAGGCTC
558 anti-hHER2-HC-G446-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGTAATCTAGACACCTCAGACAATCAA 559
GDSLSWLLRLLN-K447 C TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCGGGGGACAGGCTCAG
560 anti-hHER2-HC-A118-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCACCAAGGGCCCCAGCG 561 DSLEFIASKLA-S119
CTTGCTGGCGATGAACTCCAGGCTGTCAGCCGAGGAGACGGTGACCAG 562
anti-hHER2-HC-S119-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCAAGGGCCCCAGCGTGTTC 563
DSLEFIASKLA-T120 CTTGCTGGCGATGAACTCCAGGCTGTCGCTAGCCGAGGAGACGGTGAC
564 anti-hHER2-HC-T120-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGGGCCCCAGCGTGTTCCC 565
DSLEFIASKLA-K121 CTTGCTGGCGATGAACTCCAGGCTGTCGGTGCTAGCCGAGGAGACGG
566 anti-hHER2-HC-S131-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAAGAGCACCAGCGGCGG 567
DSLEFIASKLA-S132 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGGGCCAGGGGGAAC 568
anti-hHER2-HC-S132- CTGGAGTTCATCGCCAGCAAGCTGGCCAAGAGCACCAGCGGCGGCAC
569 DSLEFIASKLA-K133 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGCTGGGGGCCAGGGG
570 anti-hHER2-HC-K133-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCACCAGCGGCGGCACAG 571 DSLEFIASKLA-S134
CTTGCTGGCGATGAACTCCAGGCTGTCCTTGCTGCTGGGGGCCAGG 572
anti-hHER2-HC-S134- CTGGAGTTCATCGCCAGCAAGCTGGCCACCAGCGGCGGCACAGCC
573 DSLEFIASKLA-T135 CTTGCTGGCGATGAACTCCAGGCTGTCGCTCTTGCTGCTGGGGGCC
574 anti-hHER2-HC-T135-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGGCGGCACAGCCGCC 575 DSLEFIASKLA-S136
CTTGCTGGCGATGAACTCCAGGCTGTCGGTGCTCTTGCTGCTGGGGG 576
anti-hHER2-HC-S136- CTGGAGTTCATCGCCAGCAAGCTGGCCGGCGGCACAGCCGCCCTG
577 DSLEFIASKLA-137
CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGTGCTCTTGCTGCTGGG 578
anti-hHER2-HC-G137- CTGGAGTTCATCGCCAGCAAGCTGGCCGGCACAGCCGCCCTGGGC
579 DSLEFIASKLA-G138
CTTGCTGGCGATGAACTCCAGGCTGTCGCCGCTGGTGCTCTTGCTGC 580
anti-hHER2-HC-G138- CTGGAGTTCATCGCCAGCAAGCTGGCCACAGCCGCCCTGGGCTGC
581 DSLEFIASKLA-T139 CTTGCTGGCGATGAACTCCAGGCTGTCGCCGCCGCTGGTGCTCTTG
582 anti-hHER2-HC-E152-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCGTGACCGTGTCCTGGAAC 583
DSLEFIASKLA-P153 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGGGGAAGTAGTCCTTCACC
584 anti-hHER2-HC-P153-
CTGGAGTTCATCGCCAGCAAGCTGGCCGTGACCGTGTCCTGGAACAGCG 585
DSLEFIASKLA-V154 CTTGCTGGCGATGAACTCCAGGCTGTCGGGCTCGGGGAAGTAGTCCTTC
586 anti-hHER2-HC-N159-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGGAGCCCTGACCTCCG 587 DSLEFIASKLA-S160
CTTGCTGGCGATGAACTCCAGGCTGTCGTTCCAGGACACGGTCACGGG 588
anti-hHER2-HC-S160-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGAGCCCTGACCTCCGGCGTGCAC 589
DSLEFIASKLA-G161 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGTTCCAGGACACGGTCACG
590 anti-hHER2-HC-G161-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCCTGACCTCCGGCGTG 591 DSLEFIASKLA-A162
CTTGCTGGCGATGAACTCCAGGCTGTCTCCGCTGTTCCAGGACACGG 592
anti-hHER2-HC-A162- CTGGAGTTCATCGCCAGCAAGCTGGCCCTGACCTCCGGCGTGCACAC
593 DSLEFIASKLA-L163
CTTGCTGGCGATGAACTCCAGGCTGTCGGCTCCGCTGTTCCAGGACAC 594
anti-hHER2-HC-L163-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCTCCGGCGTGCACACCTTC 595
DSLEFIASKLA-T164 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGGCTCCGCTGTTCCAGG
596 anti-hHER2-HC-T164-
CTGGAGTTCATCGCCAGCAAGCTGGCCTCCGGCGTGCACACCTTCCC 597
DSLEFIASKLA-S165 CTTGCTGGCGATGAACTCCAGGCTGTCGGTCAGGGCTCCGCTGTTCC
598 anti-hHER2-HC-S165-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCGTGCACACCTTCCCCG 599 DSLEFIASKLA-G166
CTTGCTGGCGATGAACTCCAGGCTGTCGGAGGTCAGGGCTCCGCTG 600
anti-hHER2-HC-P171- CTGGAGTTCATCGCCAGCAAGCTGGCCGCCGTGCTGCAGAGCAGCG
601 DSLEFIASKLA-A172 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGAAGGTGTGCACGCCG
602 anti-hHER2-HC-S176-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGGCCTGTACAGCCTGTCC 603
DSLEFIASKLA-S177 CTTGCTGGCGATGAACTCCAGGCTGTCGCTCTGCAGCACGGCGGG 604
anti-hHER2-HC-S177-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCCTGTACAGCCTGTCCAGC 605
DSLEFIASKLA-G178 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGCTCTGCAGCACGGCG 606
anti-hHER2-HC-P189- CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAGCAGCCTGGGCACCC
607 DSLEFIASKLA-S190
CTTGCTGGCGATGAACTCCAGGCTGTCGGGCACTGTCACCACGCTGG 608
anti-hHER2-HC-S190- CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAGCCTGGGCACCCAGAC
609 DSLEFIASKLA-S191
CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGCACTGTCACCACGC 610
anti-hHER2-HC-S191-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCCTGGGCACCCAGACCTAC 611
DSLEFIASKLA-S192 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGCTGGGCACTGTCACCAC
612 anti-hHER2-HC-S192-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGGGCACCCAGACCTACATC 613
DSLEFIASKLA-L193 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGCTGCTGGGCACTGTCAC
614 anti-hHER2-HC-L193-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCACCCAGACCTACATCTGC 615
DSLEFIASKLA-G194 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGCTGCTGCTGGGCACTG
616 anti-hHER2-HC-G194-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCCAGACCTACATCTGCAACGTG 617
DSLEFIASKLA-T195 CTTGCTGGCGATGAACTCCAGGCTGTCGCCCAGGCTGCTGCTGGG 618
anti-hHER2-HC-T195-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGACCTACATCTGCAACGTGAAC 619
DSLEFIASKLA-Q196 CTTGCTGGCGATGAACTCCAGGCTGTCGGTGCCCAGGCTGCTGCTG 620
anti-hHER2-HC-Q196-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCTACATCTGCAACGTGAACCAC 621
DSLEFIASKLA-T197 CTTGCTGGCGATGAACTCCAGGCTGTCCTGGGTGCCCAGGCTGCTG 622
anti-hHER2-HC-K205-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCAGCAACACCAAGGTGGAC 623
DSLEFIASKLA-P206
CTTGCTGGCGATGAACTCCAGGCTGTCCTTGTGGTTCACGTTGCAGATGTAGG 624
anti-hHER2-HC-P206-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAACACCAAGGTGGACAAGAAAG 625
DSLEFIASKLA-S207 CTTGCTGGCGATGAACTCCAGGCTGTCGGGCTTGTGGTTCACGTTGCAG
626 anti-hHER2-HC-S207-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACACCAAGGTGGACAAGAAAGTGG 627
DSLEFIASKLA-N208 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGCTTGTGGTTCACGTTG
628 anti-hHER2-HC-P230-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCCCAGAGCTGCTGGGC 629 DSLEFIASKLA-A231
CTTGCTGGCGATGAACTCCAGGCTGTCTGGGCAGGGGGGGCAGGTG 630
anti-hHER2-HC-A231- CTGGAGTTCATCGCCAGCAAGCTGGCCCCAGAGCTGCTGGGCGGAC
631 DSLEFIASKLA-P232 CTTGCTGGCGATGAACTCCAGGCTGTCGGCTGGGCAGGGGGGGC
632 anti-hHER2-HC-P232-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGCTGCTGGGCGGACCC 633 DSLEFIASKLA-E233
CTTGCTGGCGATGAACTCCAGGCTGTCTGGGGCTGGGCAGGGGGG 634
anti-hHER2-HC-E233- CTGGAGTTCATCGCCAGCAAGCTGGCCCTGCTGGGCGGACCCTCC
635 DSLEFIASKLA-L234 CTTGCTGGCGATGAACTCCAGGCTGTCCTCTGGGGCTGGGCAGGG
636 anti-hHER2-HC-L234-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGGGCGGACCCTCCGTG 637 DSLEFIASKLA-L235
CTTGCTGGCGATGAACTCCAGGCTGTCCAGCTCTGGGGCTGGGCAG 638
anti-hHER2-HC-L235- CTGGAGTTCATCGCCAGCAAGCTGGCCGGCGGACCCTCCGTGTTCC
639 DSLEFIASKLA-G236 CTTGCTGGCGATGAACTCCAGGCTGTCCAGCAGCTCTGGGGCTGGG
640 anti-hHER2-HC-G236-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGACCCTCCGTGTTCCTGTTCC 641
DSLEFIASKLA-G237 CTTGCTGGCGATGAACTCCAGGCTGTCGCCCAGCAGCTCTGGGGC
642
anti-hHER2-HC-P244-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCAAGCCCAAGGACACCCTG 643
DSLEFIASKLA-P245 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGAACAGGAACACGGAGGG
644 anti-hHER2-HC-P245-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGCCCAAGGACACCCTGATGATC 645
DSLEFIASKLA-K246 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGGGGAACAGGAACACGG
646 anti-hHER2-HC-I253-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAGGACCCCCGAGGTGAC 647
DSLEFIASKLA-S254 CTTGCTGGCGATGAACTCCAGGCTGTCGATCATCAGGGTGTCCTTGGGC
648 anti-hHER2-HC-S254-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGGACCCCCGAGGTGACCTG 649
DSLEFIASKLA-R255 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGATCATCAGGGTGTCCTTGG
650 anti-hHER2-HC-R255-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCCCCGAGGTGACCTGCG 651 DSLEFIASKLA-T256
CTTGCTGGCGATGAACTCCAGGCTGTCCCTGCTGATCATCAGGGTGTCC 652
anti-hHER2-HC-T256- CTGGAGTTCATCGCCAGCAAGCTGGCCCCCGAGGTGACCTGCGTGG
653 DSLEFIASKLA-P257
CTTGCTGGCGATGAACTCCAGGCTGTCGGTCCTGCTGATCATCAGGGTG 654
anti-hHER2-HC-P257-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGGTGACCTGCGTGGTGGTG 655
DSLEFIASKLA-E258 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGGTCCTGCTGATCATCAG
656 anti-hHER2-HC-S267-
CTGGAGTTCATCGCCAGCAAGCTGGCCCACGAGGACCCAGAGGTGAAGTTC 657
DSLEFIASKLA-H268 CTTGCTGGCGATGAACTCCAGGCTGTCGCTCACGTCCACCACCACGC
658 anti-hHER2-HC-H268-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGGACCCAGAGGTGAAGTTCAAC 659
DSLEFIASKLA-E269 CTTGCTGGCGATGAACTCCAGGCTGTCGTGGCTCACGTCCACCACCAC
660 anti-hHER2-HC-E269-
CTGGAGTTCATCGCCAGCAAGCTGGCCGACCCAGAGGTGAAGTTCAACTGG 661
DSLEFIASKLA-D270 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGTGGCTCACGTCCACCAC
662 anti-hHER2-HC-D270-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCAGAGGTGAAGTTCAACTGGTAC 663
DSLEFIASKLA-P271 CTTGCTGGCGATGAACTCCAGGCTGTCGTCCTCGTGGCTCACGTCCAC
664 anti-hHER2-HC-P271-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGGTGAAGTTCAACTGGTACGTGG 665
DSLEFIASKLA-E272 CTTGCTGGCGATGAACTCCAGGCTGTCTGGGTCCTCGTGGCTCACGTC
666 anti-hHER2-HC-D280-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCGTGGAGGTGCACAACGC 667
DSLEFIASKLA-G281
CTTGCTGGCGATGAACTCCAGGCTGTCGTCCACGTACCAGTTGAACTTCACC 668
anti-hHER2-HC-H285-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACGCCAAGACCAAGCCCAGAG 669
DSLEFIASKLA-N286 CTTGCTGGCGATGAACTCCAGGCTGTCGTGCACCTCCACGCCGTCC 670
anti-hHER2-HC-N286-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCAAGACCAAGCCCAGAGAG 671
DSLEFIASKLA-A287 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGTGCACCTCCACGCCGTC
672 anti-hHER2-HC-P291-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGAGAGGAGCAGTACAACAGCACC 673
DSLEFIASKLA-R292 CTTGCTGGCGATGAACTCCAGGCTGTCGGGCTTGGTCTTGGCGTTGTG
674 anti-hHER2-HC-T307-
CTGGAGTTCATCGCCAGCAAGCTGGCCGTGCTGCACCAGGACTGGCTG 675
DSLEFIASKLA-V308 CTTGCTGGCGATGAACTCCAGGCTGTCGGTCAGCACGGACACCACCC
676 anti-hHER2-HC-V308-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGCACCAGGACTGGCTGAAC 677
DSLEFIASKLA-L309 CTTGCTGGCGATGAACTCCAGGCTGTCCACGGTCAGCACGGACACCAC
678 anti-hHER2-HC-L309-
CTGGAGTTCATCGCCAGCAAGCTGGCCCACCAGGACTGGCTGAACGGC 679
DSLEFIASKLA-H310 CTTGCTGGCGATGAACTCCAGGCTGTCCAGCACGGTCAGCACGGACAC
680 anti-hHER2-HC-H310-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGGACTGGCTGAACGGCAAG 681
DSLEFIASKLA-Q311 CTTGCTGGCGATGAACTCCAGGCTGTCGTGCAGCACGGTCAGCACGG
682 anti-hHER2-HC-N315-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCAAGGAATACAAGTGCAAGGTC 683
DSLEFIASKLA-G316 CTTGCTGGCGATGAACTCCAGGCTGTCGTTCAGCCAGTCCTGGTGCAG
684 anti-hHER2-HC-G316-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGGAATACAAGTGCAAGGTCTCCAAC 685
DSLEFIASKLA-K317 CTTGCTGGCGATGAACTCCAGGCTGTCGCCGTTCAGCCAGTCCTGGTG
686 anti-hHER2-HC-K317-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAATACAAGTGCAAGGTCTCCAACAAG 687
DSLEFIASKLA-E318 CTTGCTGGCGATGAACTCCAGGCTGTCCTTGCCGTTCAGCCAGTCCTG
688 anti-hHER2-HC-K326-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCCTGCCAGCCCCCATC 689 DSLEFIASKLA-A327
CTTGCTGGCGATGAACTCCAGGCTGTCCTTGTTGGAGACCTTGCACTTGTATTC 690
anti-hHER2-HC-A327-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGCCAGCCCCCATCGAAAAG 691
DSLEFIASKLA-L328 CTTGCTGGCGATGAACTCCAGGCTGTCGGCCTTGTTGGAGACCTTGCAC
692 anti-hHER2-HC-L328-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCAGCCCCCATCGAAAAGACC 693
DSLEFIASKLA-P329 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGGCCTTGTTGGAGACCTTG
694 anti-hHER2-HC-P329-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCCCCATCGAAAAGACCATCAG 695
DSLEFIASKLA-A330 CTTGCTGGCGATGAACTCCAGGCTGTCTGGCAGGGCCTTGTTGGAGAC
696 anti-hHER2-HC-A330-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCATCGAAAAGACCATCAGCAAG 697
DSLEFIASKLA-P331 CTTGCTGGCGATGAACTCCAGGCTGTCGGCTGGCAGGGCCTTGTTGG
698 anti-hHER2-HC-A339-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGGGCCAGCCACGGGAGC 699 DSLEFIASKLA-K340
CTTGCTGGCGATGAACTCCAGGCTGTCGGCCTTGCTGATGGTCTTTTCGATG 700
anti-hHER2-HC-K340- CTGGAGTTCATCGCCAGCAAGCTGGCCGGCCAGCCACGGGAGCCC
701 DSLEFIASKLA-G341
CTTGCTGGCGATGAACTCCAGGCTGTCCTTGGCCTTGCTGATGGTCTTTTC 702
anti-hHER2-HC-G341- CTGGAGTTCATCGCCAGCAAGCTGGCCCAGCCACGGGAGCCCCAG
703 DSLEFIASKLA-Q342
CTTGCTGGCGATGAACTCCAGGCTGTCGCCCTTGGCCTTGCTGATGGTC 704
anti-hHER2-HC-Q342- CTGGAGTTCATCGCCAGCAAGCTGGCCCCACGGGAGCCCCAGGTG
705 DSLEFIASKLA-P343
CTTGCTGGCGATGAACTCCAGGCTGTCCTGGCCCTTGGCCTTGCTGATG 706
anti-hHER2-HC-P343- CTGGAGTTCATCGCCAGCAAGCTGGCCCGGGAGCCCCAGGTGTACAC
707 DSLEFIASKLA-R344
CTTGCTGGCGATGAACTCCAGGCTGTCTGGCTGGCCCTTGGCCTTGC 708
anti-hHER2-HC-R344-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGCCCCAGGTGTACACCCTG 709
DSLEFIASKLA-E345 CTTGCTGGCGATGAACTCCAGGCTGTCCCGTGGCTGGCCCTTGGC 710
anti-hHER2-HC-R355-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGGAGATGACCAAGAACCAGGTG 711
DSLEFIASKLA-E356 CTTGCTGGCGATGAACTCCAGGCTGTCCCGGGAGGGGGGCAGGG 712
anti-hHER2-HC-E356-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGATGACCAAGAACCAGGTGTCC 713
DSLEFIASKLA-E357 CTTGCTGGCGATGAACTCCAGGCTGTCCTCCCGGGAGGGGGGCAG 714
anti-hHER2-HC-E357-
CTGGAGTTCATCGCCAGCAAGCTGGCCATGACCAAGAACCAGGTGTCCCTG 715
DSLEFIASKLA-M358 CTTGCTGGCGATGAACTCCAGGCTGTCCTCCTCCCGGGAGGGGGG 716
anti-hHER2-HC-M358-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCAAGAACCAGGTGTCCCTGAC 717
DSLEFIASKLA-T359 CTTGCTGGCGATGAACTCCAGGCTGTCCATCTCCTCCCGGGAGGGG 718
anti-hHER2-HC-T359-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGAACCAGGTGTCCCTGACCTG 719
DSLEFIASKLA-K360 CTTGCTGGCGATGAACTCCAGGCTGTCGGTCATCTCCTCCCGGGAGG
720 anti-hHER2-HC-K360-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACCAGGTGTCCCTGACCTGTC 721
DSLEFIASKLA-N361 CTTGCTGGCGATGAACTCCAGGCTGTCCTTGGTCATCTCCTCCCGGGAG
722 anti-hHER2-HC-N384-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCCAGCCCGAGAACAACTAC 723
DSLEFIASKLA-G385 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGCTCTCCCACTCCACGGC
724 anti-hHER2-HC-E388-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACAACTACAAGACCACACCTCCAG 725
DSLEFIASKLA-N389 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGGGCTGGCCGTTGCTC 726
anti-hHER2-HC-N389-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACTACAAGACCACACCTCCAGTGC 727
DSLEFIASKLA-N390 CTTGCTGGCGATGAACTCCAGGCTGTCGTTCTCGGGCTGGCCGTTGC
728 anti-hHER2-HC-T394-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCTCCAGTGCTGGACAGCGAC 729
DSLEFIASKLA-P395
CTTGCTGGCGATGAACTCCAGGCTGTCTGTGGTCTTGTAGTTGTTCTCGGGC 730
anti-hHER2-HC-P395- CTGGAGTTCATCGCCAGCAAGCTGGCCCCAGTGCTGGACAGCGACGG
731 DSLEFIASKLA-P396
CTTGCTGGCGATGAACTCCAGGCTGTCAGGTGTGGTCTTGTAGTTGTTCTCG 732
anti-hHER2-HC-D399-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGACGGCAGCTTCTTCCTG 733
DSLEFIASKLA-S400 CTTGCTGGCGATGAACTCCAGGCTGTCGTCCAGCACTGGAGGTGTGGTC
734 anti-hHER2-HC-S400-
CTGGAGTTCATCGCCAGCAAGCTGGCCGACGGCAGCTTCTTCCTGTACAG 735
DSLEFIASKLA-D401 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGTCCAGCACTGGAGGTGTG
736 anti-hHER2-HC-D401-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCAGCTTCTTCCTGTACAGCAAG 737
DSLEFIASKLA-G402 CTTGCTGGCGATGAACTCCAGGCTGTCGTCGCTGTCCAGCACTGGAGG
738 anti-hHER2-HC-S415-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGGTGGCAGCAGGGCAACGTG 739
DSLEFIASKLA-R416 CTTGCTGGCGATGAACTCCAGGCTGTCGGACTTGTCCACGGTCAGCTTG
740 anti-hHER2-HC-R416-
CTGGAGTTCATCGCCAGCAAGCTGGCCTGGCAGCAGGGCAACGTGTTC 741
DSLEFIASKLA-W417 CTTGCTGGCGATGAACTCCAGGCTGTCCCTGGACTTGTCCACGGTCAG
742 anti-hHER2-HC-W417-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGCAGGGCAACGTGTTCAGC 743
DSLEFIASKLA-Q418 CTTGCTGGCGATGAACTCCAGGCTGTCCCACCTGGACTTGTCCACGGTC
744 anti-hHER2-HC-Q418-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGGGCAACGTGTTCAGCTGC 745
DSLEFIASKLA-Q419 CTTGCTGGCGATGAACTCCAGGCTGTCCTGCCACCTGGACTTGTCCAC
746 anti-hHER2-HC-Q419-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCAACGTGTTCAGCTGCAGC 747
DSLEFIASKLA-G420 CTTGCTGGCGATGAACTCCAGGCTGTCCTGCTGCCACCTGGACTTGTC
748 anti-hHER2-HC-G420-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACGTGTTCAGCTGCAGCGTGATG 749
DSLEFIASKLA-N421 CTTGCTGGCGATGAACTCCAGGCTGTCGCCCTGCTGCCACCTGGAC 750
anti-hHER2-HC-N421-
CTGGAGTTCATCGCCAGCAAGCTGGCCGTGTTCAGCTGCAGCGTGATGC 751
DSLEFIASKLA-V422 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGCCCTGCTGCCACCTGG
752 anti-hHER2-HC-H433-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACCACTACACCCAGAAGAGCCTG 753
DSLEFIASKLA-N434 CTTGCTGGCGATGAACTCCAGGCTGTCGTGCAGGGCCTCGTGCATCAC
754 anti-hHER2-HC-N434-
CTGGAGTTCATCGCCAGCAAGCTGGCCCACTACACCCAGAAGAGCCTGAG 755
DSLEFIASKLA-H435 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGTGCAGGGCCTCGTGCATC
756 anti-hHER2-HC-S442-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGTCCCCCGGCAAGTAATCTAG 757
DSLEFIASKLA-L443 CTTGCTGGCGATGAACTCCAGGCTGTCGCTCAGGCTCTTCTGGGTGTAG
758 anti-hHER2-HC-L443-
CTGGAGTTCATCGCCAGCAAGCTGGCCTCCCCCGGCAAGTAATCTAGACAC 759
DSLEFIASKLA-S444 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGCTCAGGCTCTTCTGGGTG
760 anti-hHER2-HC-S444-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCGGCAAGTAATCTAGACACCTC 761
DSLEFIASKLA-P445 CTTGCTGGCGATGAACTCCAGGCTGTCGGACAGGCTCAGGCTCTTCTG
762 anti-hHER2-HC-P445-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCAAGTAATCTAGACACCTCAGAC 763
DSLEFIASKLA-G446 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGGACAGGCTCAGGCTC 764
anti-hHER2-HC-G446-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGTAATCTAGACACCTCAGACAATCAA 765
DSLEFIASKLA-K447 C CTTGCTGGCGATGAACTCCAGGCTGTCGCCGGGGGACAGGCTCAG
766 anti-hHER2-LC-T109-
CTGAGCTGGCTGCTGAGACTGCTGAACGTGGCCGCTCCCAGCGTG 767 GDSLSWLLRLLN-V110
TCTCAGCAGCCAGCTCAGGCTGTCGCCCGTTCGTTTGATCTCCACCTTGGT 768
anti-hHER2-LC-V110-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCGCTCCCAGCGTGTTCATC 769
GDSLSWLLRLLN-A111
TCTCAGCAGCCAGCTCAGGCTGTCGCCCACCGTTCGTTTGATCTCCACCTTG 770
anti-hHER2-LC-A111-
CTGAGCTGGCTGCTGAGACTGCTGAACGCTCCCAGCGTGTTCATCTTCC 771
GDSLSWLLRLLN-A112 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCCACCGTTCGTTTGATCTCC
772 anti-hHER2-LC-P119-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCAGCGACGAGCAGCTGAAG 773
GDSLSWLLRLLN-P120 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGAAGATGAACACGCTGGG
774 anti-hHER2-LC-P120-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGACGAGCAGCTGAAGAGC 775
GDSLSWLLRLLN-S121 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGGGGAAGATGAACACGCTG
776 anti-hHER2-LC-S121-
CTGAGCTGGCTGCTGAGACTGCTGAACGACGAGCAGCTGAAGAGCGGC 777
GDSLSWLLRLLN-D122 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGGGGGGAAGATGAACAC
778 anti-hHER2-LC-D122-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGCAGCTGAAGAGCGGCACC 779
GDSLSWLLRLLN-E123 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCGCTGGGGGGGAAGATGAAC
780 anti-hHER2-LC-Y140-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCCGGGAGGCCAAGGTG 781 GDSLSWLLRLLN-P141
TCTCAGCAGCCAGCTCAGGCTGTCGCCGTAGAAGTTGTTCAGCAGGCACAC 782
anti-hHER2-LC-P141- CTGAGCTGGCTGCTGAGACTGCTGAACCGGGAGGCCAAGGTGCAGTG
783 GDSLSWLLRLLN-R142
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGGTAGAAGTTGTTCAGCAGGC 784
anti-hHER2-LC-R142-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGGCCAAGGTGCAGTGGAAG 785
GDSLSWLLRLLN-E143 TCTCAGCAGCCAGCTCAGGCTGTCGCCCCGGGGGTAGAAGTTGTTCAGC
786 anti-hHER2-LC-E143-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCAAGGTGCAGTGGAAGGTG 787
GDSLSWLLRLLN-A144 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCCCGGGGGTAGAAGTTGTTC
788 anti-hHER2-LC-D151-
CTGAGCTGGCTGCTGAGACTGCTGAACAACGCCCTGCAGAGCGGCAAC 789
GDSLSWLLRLLN-N152 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCCACCTTCCACTGCACCTTG
790 anti-hHER2-LC-N152-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCCTGCAGAGCGGCAACAG 791
GDSLSWLLRLLN-A153 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGTCCACCTTCCACTGCACC
792 anti-hHER2-LC-A153-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGCAGAGCGGCAACAGCCAG 793
GDSLSWLLRLLN-L154 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGCGTTGTCCACCTTCCACTG
794 anti-hHER2-LC-L154-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGAGCGGCAACAGCCAGGAG 795
GDSLSWLLRLLN-Q155 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGGCGTTGTCCACCTTCCAC
796 anti-hHER2-LC-Q155-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGGCAACAGCCAGGAGAGC 797
GDSLSWLLRLLN-S156 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGCAGGGCGTTGTCCACCTTC
798 anti-hHER2-LC-E161-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCGTCACCGAGCAGGACAGC 799
GDSLSWLLRLLN-S162 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCCTGGCTGTTGCCGCTCTG
800 anti-hHER2-LC-S162-
CTGAGCTGGCTGCTGAGACTGCTGAACGTCACCGAGCAGGACAGCAAG 801
GDSLSWLLRLLN-V163 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCTCCTGGCTGTTGCCGC
802 anti-hHER2-LC-V163-
CTGAGCTGGCTGCTGAGACTGCTGAACACCGAGCAGGACAGCAAGGAC 803
GDSLSWLLRLLN-T164 TCTCAGCAGCCAGCTCAGGCTGTCGCCGACGCTCTCCTGGCTGTTGCC
804 anti-hHER2-LC-T164-
CTGAGCTGGCTGCTGAGACTGCTGAACGAGCAGGACAGCAAGGACTCC 805
GDSLSWLLRLLN-E165 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTGACGCTCTCCTGGCTGTTG
806 anti-hHER2-LC-E165-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGGACAGCAAGGACTCCACC 807
GDSLSWLLRLLN-Q166 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCGGTGACGCTCTCCTGGC
808 anti-hHER2-LC-Q166-
CTGAGCTGGCTGCTGAGACTGCTGAACGACAGCAAGGACTCCACCTACAG 809
GDSLSWLLRLLN-D167 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGCTCGGTGACGCTCTCCTG
810 anti-hHER2-LC-D167-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCAAGGACTCCACCTACAGCC 811
GDSLSWLLRLLN-S168 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTCCTGCTCGGTGACGCTCTC
812 anti-hHER2-LC-T197-
CTGAGCTGGCTGCTGAGACTGCTGAACCACCAGGGCCTGTCCAGCC 813
GDSLSWLLRLLN-H198 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCACCTCGCAGGCGTACAC
814 anti-hHER2-LC-H198-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGGGCCTGTCCAGCCCC 815 GDSLSWLLRLLN-Q199
TCTCAGCAGCCAGCTCAGGCTGTCGCCGTGGGTCACCTCGCAGGCG 816
anti-hHER2-LC-Q199- CTGAGCTGGCTGCTGAGACTGCTGAACGGCCTGTCCAGCCCCGTG
817 GDSLSWLLRLLN-G200
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTGGTGGGTCACCTCGCAGG 818
anti-hHER2-LC-G200-
CTGAGCTGGCTGCTGAGACTGCTGAACCTGTCCAGCCCCGTGACCAAG 819
GDSLSWLLRLLN-L201 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCCCTGGTGGGTCACCTCG
820 anti-hHER2-LC-L201-
CTGAGCTGGCTGCTGAGACTGCTGAACTCCAGCCCCGTGACCAAGAGC 821
GDSLSWLLRLLN-S202 TCTCAGCAGCCAGCTCAGGCTGTCGCCCAGGCCCTGGTGGGTCACC
822 anti-hHER2-LC-S202-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCCCCGTGACCAAGAGCTTC 823
GDSLSWLLRLLN-S203 TCTCAGCAGCCAGCTCAGGCTGTCGCCGGACAGGCCCTGGTGGGTC
824 anti-hHER2-LC-S203-
CTGAGCTGGCTGCTGAGACTGCTGAACCCCGTGACCAAGAGCTTCAACAG 825
GDSLSWLLRLLN-P204 TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGACAGGCCCTGGTGG
826 anti-hHER2-LC-K207-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCTTCAACAGGGGCGAGTGC 827
GDSLSWLLRLLN-S208 TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGGTCACGGGGCTGGACAG
828 anti-hHER2-LC-T109-
CTGGAGTTCATCGCCAGCAAGCTGGCCGTGGCCGCTCCCAGCGTG 829 DSLEFIASKLA-V110
CTTGCTGGCGATGAACTCCAGGCTGTCCGTTCGTTTGATCTCCACCTTGGT 830
anti-hHER2-LC-V110-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCGCTCCCAGCGTGTTCATC 831
DSLEFIASKLA-A111
CTTGCTGGCGATGAACTCCAGGCTGTCCACCGTTCGTTTGATCTCCACCTTG 832
anti-hHER2-LC-A111-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCTCCCAGCGTGTTCATCTTCC 833
DSLEFIASKLA-A112 CTTGCTGGCGATGAACTCCAGGCTGTCGGCCACCGTTCGTTTGATCTCC
834 anti-hHER2-LC-P119-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCAGCGACGAGCAGCTGAAG 835
DSLEFIASKLA-P120 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGAAGATGAACACGCTGGG
836 anti-hHER2-LC-P120-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGACGAGCAGCTGAAGAGC 837
DSLEFIASKLA-S121 CTTGCTGGCGATGAACTCCAGGCTGTCGGGGGGGAAGATGAACACGCTG
838 anti-hHER2-LC-S121-
CTGGAGTTCATCGCCAGCAAGCTGGCCGACGAGCAGCTGAAGAGCGGC 839
DSLEFIASKLA-D122 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGGGGGAAGATGAACAC
840 anti-hHER2-LC-D122-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGCAGCTGAAGAGCGGCACC 841
DSLEFIASKLA-E123 CTTGCTGGCGATGAACTCCAGGCTGTCGTCGCTGGGGGGGAAGATGAAC
842 anti-hHER2-LC-Y140-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCCGGGAGGCCAAGGTG 843 DSLEFIASKLA-P141
CTTGCTGGCGATGAACTCCAGGCTGTCGTAGAAGTTGTTCAGCAGGCACAC 844
anti-hHER2-LC-P141- CTGGAGTTCATCGCCAGCAAGCTGGCCCGGGAGGCCAAGGTGCAGTG
845 DSLEFIASKLA-R142
CTTGCTGGCGATGAACTCCAGGCTGTCGGGGTAGAAGTTGTTCAGCAGGC 846
anti-hHER2-LC-R142-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGGCCAAGGTGCAGTGGAAG 847
DSLEFIASKLA-E143 CTTGCTGGCGATGAACTCCAGGCTGTCCCGGGGGTAGAAGTTGTTCAGC
848 anti-hHER2-LC-E143-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCAAGGTGCAGTGGAAGGTG 849
DSLEFIASKLA-A144 CTTGCTGGCGATGAACTCCAGGCTGTCCTCCCGGGGGTAGAAGTTGTTC
850 anti-hHER2-LC-D151-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACGCCCTGCAGAGCGGCAAC 851
DSLEFIASKLA-N152 CTTGCTGGCGATGAACTCCAGGCTGTCGTCCACCTTCCACTGCACCTTG
852 anti-hHER2-LC-N152-
CTGGAGTTCATCGCCAGCAAGCTGGCCGCCCTGCAGAGCGGCAACAG 853
DSLEFIASKLA-A153 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGTCCACCTTCCACTGCACC
854 anti-hHER2-LC-A153-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGCAGAGCGGCAACAGCCAG 855
DSLEFIASKLA-L154 CTTGCTGGCGATGAACTCCAGGCTGTCGGCGTTGTCCACCTTCCACTG
856 anti-hHER2-LC-L154-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGAGCGGCAACAGCCAGGAG 857
DSLEFIASKLA-Q155 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGGCGTTGTCCACCTTCCAC
858 anti-hHER2-LC-Q155-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGGCAACAGCCAGGAGAGC 859
DSLEFIASKLA-S156 CTTGCTGGCGATGAACTCCAGGCTGTCCTGCAGGGCGTTGTCCACCTTC
860 anti-hHER2-LC-E161-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCGTCACCGAGCAGGACAGC 861
DSLEFIASKLA-S162 CTTGCTGGCGATGAACTCCAGGCTGTCCTCCTGGCTGTTGCCGCTCTG
862 anti-hHER2-LC-S162-
CTGGAGTTCATCGCCAGCAAGCTGGCCGTCACCGAGCAGGACAGCAAG 863
DSLEFIASKLA-V163 CTTGCTGGCGATGAACTCCAGGCTGTCGCTCTCCTGGCTGTTGCCGC
864 anti-hHER2-LC-V163-
CTGGAGTTCATCGCCAGCAAGCTGGCCACCGAGCAGGACAGCAAGGAC 865
DSLEFIASKLA-T164 CTTGCTGGCGATGAACTCCAGGCTGTCGACGCTCTCCTGGCTGTTGCC
866 anti-hHER2-LC-T164-
CTGGAGTTCATCGCCAGCAAGCTGGCCGAGCAGGACAGCAAGGACTCC 867
DSLEFIASKLA-E165 CTTGCTGGCGATGAACTCCAGGCTGTCGGTGACGCTCTCCTGGCTGTTG
868 anti-hHER2-LC-E165-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGGACAGCAAGGACTCCACC 869
DSLEFIASKLA-Q166 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGGTGACGCTCTCCTGGC
870 anti-hHER2-LC-Q166-
CTGGAGTTCATCGCCAGCAAGCTGGCCGACAGCAAGGACTCCACCTACAG 871
DSLEFIASKLA-D167 CTTGCTGGCGATGAACTCCAGGCTGTCCTGCTCGGTGACGCTCTCCTG
872 anti-hHER2-LC-D167-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCAAGGACTCCACCTACAGCC 873
DSLEFIASKLA-S168 CTTGCTGGCGATGAACTCCAGGCTGTCGTCCTGCTCGGTGACGCTCTC
874 anti-hHER2-LC-T197-
CTGGAGTTCATCGCCAGCAAGCTGGCCCACCAGGGCCTGTCCAGCC 875 DSLEFIASKLA-H198
CTTGCTGGCGATGAACTCCAGGCTGTCGGTCACCTCGCAGGCGTACAC 876
anti-hHER2-LC-H198- CTGGAGTTCATCGCCAGCAAGCTGGCCCAGGGCCTGTCCAGCCCC
877 DSLEFIASKLA-Q199 CTTGCTGGCGATGAACTCCAGGCTGTCGTGGGTCACCTCGCAGGCG
878 anti-hHER2-LC-Q199-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCCTGTCCAGCCCCGTG 879 DSLEFIASKLA-G200
CTTGCTGGCGATGAACTCCAGGCTGTCCTGGTGGGTCACCTCGCAGG 880
anti-hHER2-LC-G200-
CTGGAGTTCATCGCCAGCAAGCTGGCCCTGTCCAGCCCCGTGACCAAG 881
DSLEFIASKLA-L201 CTTGCTGGCGATGAACTCCAGGCTGTCGCCCTGGTGGGTCACCTCG 882
anti-hHER2-LC-L201-
CTGGAGTTCATCGCCAGCAAGCTGGCCTCCAGCCCCGTGACCAAGAGC 883
DSLEFIASKLA-S202 CTTGCTGGCGATGAACTCCAGGCTGTCCAGGCCCTGGTGGGTCACC 884
anti-hHER2-LC-S202-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCCCCGTGACCAAGAGCTTC 885
DSLEFIASKLA-S203 CTTGCTGGCGATGAACTCCAGGCTGTCGGACAGGCCCTGGTGGGTC 886
anti-hHER2-LC-S203-
CTGGAGTTCATCGCCAGCAAGCTGGCCCCCGTGACCAAGAGCTTCAACAG 887
DSLEFIASKLA-P204 CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGACAGGCCCTGGTGG 888
anti-hHER2-LC-K207-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCTTCAACAGGGGCGAGTGC 889
DSLEFIASKLA-S208 CTTGCTGGCGATGAACTCCAGGCTGTCCTTGGTCACGGGGCTGGACAG
890 B. subtilis Sfp pET22b
GAAGGAGATATACATATGAAAATTTATGGGATTTACATGGATCGC 891
GTGGTGGTGGTGGTGGTGCAGCAATTCTTCATAGGAGACCATCG 892 pET22b
CACCACCACCACCACCACTGAG 893
CATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC 894 TEV into B. subtilis
GAGAACCTGTACTTCCAAGGCCACCACCACCACCACCACTGAG 895 Sfp pET22b
GCCTTGGAAGTACAGGTTCTCCAGCAATTCTTCATAGGAGACCATCG 896 B. subtilis Sfp
K28E GTCTTTCATTTCACCAGAGGAGCGCGAAAAATGCCGTCGCT 897
AGCGACGGCATTTTTCGCGCTCCTCTGGTGAAATGAAAGAC 898 B. subtilis Sfp T44E
AAAGAAGATGCTCACCGCGAGCTGCTGGGAGATGTGCTG 899
CAGCACATCTCCCAGCAGCTCGCGGTGAGCATCTTCTTT 900 B. subtilis Sfp C77Y
GCAGGAATATGGCAAACCGTATATTCCAGATCTTCCAGATGC 901
GCATCTGGAAGATCTGGAATATACGGTTTGCCATATTCCTGC 902 E. coli AcpS pET22b
AATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGCAATATTAGGTTTAG 903 GCACG
CAGTGGTGGTGGTGGTGGTGACTTTCAATAATTACCGTGGCACAAGC 904 pET22b
CACCACCACCACCACCACTG 905 ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATT 906
anti-hHER2-HC-V64L-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGGGCCGTTTCACTATAAGCGC 907 EFIASKLA-K65
CTTGCTGGCGATGAACTCCAGGCTATCGGCATATCTAGTATAACCATTCGTAGG 908
anti-hHER2-HC-S63-
GACAGCCTGGAGTTCATCGCCAGCAAGGTCAAGGGCCGTTTCACTATAAGC 909 LEFIASK-V64
CTTGCTGGCGATGAACTCCAGGCTGTCGGCATATCTAGTATAACCATTCGTAGG 910
anti-hHER2-HC-V64L-
GACAGCCTGGAGTTCATCGCCAGCAAGGGCCGTTTCACTATAAGCGCAGAC 911 EFIAS-K65
CTTGCTGGCGATGAACTCCAGGCTGTCGGCATATCTAGTATAACCATTCGTAGG 912
anti-hHER2-LC-S76D-S77-
CTGGAGTTCATCGCCAGCAAGCTGGCCCAGCCGGAAGACTTCGCAACTTATTAC 913
L78-EFIASKLA-Q79 CTTGCTGGCGATGAACTCCAGGCTGTCGATGGTCAGAGTGAAATCCGTCC
914 anti-hHER2-HC-S132G-K133D-
CTGAGCTGGCTGCTGAGACTGCTGAACTGCCTGGTGAAGGACTACTTCC 915
S134-T135L-S136-G137W- TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGGGGGCCAGGGGG
916 G138L-T139L-A140R-A141L- L142-G143N anti-hHER2-HC-K133G-S134D-
CTGAGCTGGCTGCTGAGACTGCTGAACACAGCCGCCCTGGGCTGC 917
T135S-S136L-G137S-G138W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTGGGGGCCAGGG 918 LLRLLN-T139
anti-hHER2-HC-S134G-T135D- GGCGACAGCCTGAGCTGGCTGGCCCTGGGCTGCCTGGTG
919 S136-G137L-G138S-T139W-
CAGCCAGCTCAGGCTGTCGCCCTTGCTGCTGGGGGCCAGG 920 A140L
anti-hHER2-HC-S134G-T135D- CTGAGACTGCTGAACGCCCTGGGCTGCCTGGTG 921
S136-G137L-G138S-T139W- GTTCAGCAGTCTCAGCAGCCAGCTCAGGCTGTCGC 922
A140L-LRLLN-A141 anti-hHER2-HC-T135G-S136D-
CTGAGCTGGCTGCTGAGACTGCTGAACGCCGCCCTGGGCTGCCTG 923
G137S-G138L-T139S-WLLRLLN-
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTCTTGCTGCTGGGGGCC 924 A140
anti-hHER2-HC-T359G-K360D-
GGCGACAGCCTGAGCTGGCTGACCTGTCTGGTGAAGGGCTTC 925
N361S-Q362L-V363S-S364W CAGCCAGCTCAGGCTGTCGCCCATCTCCTCCCGGGAGGGG
926 anti-hHER2-HC-S132G-K133D-
GGCGACAGCCTGAGCTGGCTGACAGCCGCCCTGGGCTGC 927
S134-T135L-S136-G137W-G138L CAGCCAGCTCAGGCTGTCGCCGCTGGGGGCCAGGGGG
928 anti-hHER2-HC-S134G-T135D-
CTGAGCTGGCTGCTGAGACTGCTGAACGTGAAGGACTACTTCCCCGAGC 929
S136-G137L-G138S-T139W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGCTGCTGGGGGCCAGG 930
A140L-A141L-L142R-G143L- C144L-L145N anti-hHER2-HC-L193G-G194D-
GGCGACAGCCTGAGCTGGCTGTGCAACGTGAACCACAAGCCCAG 931
T195S-Q196L-T197S-Y198W- CAGCCAGCTCAGGCTGTCGCCGCTGCTGCTGGGCACTGTC
932 I199L anti-hHER2-HC-L193G-G194D-
CTGAGACTGCTGAACTGCAACGTGAACCACAAGCCCAG 933 T195S-Q196L-T197S-Y198W-
GTTCAGCAGTCTCAGCAGCCAGCTCAGGCTGTCGC 934 I199L-LRLLN-C200
anti-hHER2-HC-L193G-G194D-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGCCCAGCAACACCAAGGTGG 935
T195S-Q196L-T197S-Y198W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCGCTGCTGCTGGGCACTGTC 936
I199L-C200L-N201R-V202L- N203L-H204N anti-hHER2-HC-E357G-M 358D-
GGCGACAGCCTGAGCTGGCTGTCCCTGACCTGTCTGGTGAAGG 937
T359S-K360L-N361S-Q362W- CAGCCAGCTCAGGCTGTCGCCCTCCCGGGAGGGGGGC 938
V363L anti-hHER2-HC-E388-GDSLSWL-
GGCGACAGCCTGAGCTGGCTGAACAACTACAAGACCACACCTCCAG 939 N389
CAGCCAGCTCAGGCTGTCGCCCTCGGGCTGGCCGTTGCTC 940
anti-hHER2-HC-P189G-S190D-
GCGACAGCCTGAGCTGGCTGCAGACCTACATCTGCAACGTGAAC 941
S191-S192L-L193S-G194W- CAGCCAGCTCAGGCTGTCGCCCACTGTCACCACGCTGGACAG
942 T195L anti-hHER2-HC-P189G-S190D-
CTGAGCTGGCTGCTGAGACTGCTGAACAACGTGAACCACAAGCCCAGCAAC 943
S191-S192L-L193S-G194W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCCACTGTCACCACGCTGGACAG 944
T195L-Q196L-T197R-Y198L- I199L-C200N anti-hHER2-ANTI-HHER2-HC-
CTGAGCTGGCTGCTGAGACTGCTGAACTTCCTGTACAGCAAGCTGACCGTG 945
L398G-D399-S400-D401L-
TCTCAGCAGCCAGCTCAGGCTGTCGCCCACTGGAGGTGTGGTCTTGTAG 946
G402S-S403W-F404L-LRLLN- F405 anti-hHER2-HC-P189G-S190D-
CTGAGACTGCTGAACCAGACCTACATCTGCAACGTGAAC 947 S191-S192L-L193S-G194W-
GTTCAGCAGTCTCAGCAGCCAGCTCAGGCTGTCGC 948 T195L-LRLLN-Q196
anti-hHER2-HC-P189D-S190-
CTGGAGTTCATCGCCAGCAAGCTGGCCTGCAACGTGAACCACAAGCCCAG 949
S191L-S192E-L193F-G194I-
CTTGCTGGCGATGAACTCCAGGCTGTCCACTGTCACCACGCTGGACAG 950
T195A-Q196S-T197K-Y198L- I199A anti-hHER2-HC-S190G-S191D-
CTGAGCTGGCTGCTGAGACTGCTGAACTACATCTGCAACGTGAACCACAAGC 951
S192-L193-G194S-T195W-
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGGCACTGTCACCACGCTGG 952
Q196L-T197L-RLLN-Y198 anti-hHER2-HC-S190D-S191-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACGTGAACCACAAGCCCAGCAAC 953
S192L-L193E-G194F-T195I-
CTTGCTGGCGATGAACTCCAGGCTGTCGGGCACTGTCACCACGCTGG 954
Q196A-T197S-Y198K-I199L- C200A anti-hHER2-HC-D413-K414S-
AGCCTGAGCTGGCTGCTGAGACTGCTGTTCAGCTGCAGCGTGATGCACG 955
S415L-R416S-W417-Q418L-
CAGCAGTCTCAGCAGCCAGCTCAGGCTGTCCACGGTCAGCTTGCTGTAC 956
Q419L-G420R-N421L-V422L anti-hHER2-HC-D413-K414S-
AGCCTGGAGTTCATCGCCAGCAAGCTGTTCAGCTGCAGCGTGATGCACG 957
S415L-R416E-W417F-Q418I-
CAGCTTGCTGGCGATGAACTCCAGGCTGTCCACGGTCAGCTTGCTGTAC 958
Q419A-G420S-N421K-V422L anti-hHER2-HC-E382D-S383-
GACAGCCTGGAGTTCATCGCCAACAACTACAAGACCACACCTCCAG 959
N384L-G385E-Q386F-P387I- GGCGATGAACTCCAGGCTGTCCCACTCCACGGCGATGTCGC
960 E388A anti-hHER2-HC-E382D-S383-
GACAGCCTGAGCTGGCTGCTGAACAACTACAAGACCACACCTCCAG 961
N384L-G385S-Q386W-P387L- CAGCAGCCAGCTCAGGCTGTCCCACTCCACGGCGATGTCGC
962 E388L anti-hHER2-HC-V2-
CTGAGCTGGCTGCTGAGACTGCTGAACCAGCTGGTGGAGTCTGGCGG 963 GDSLSWLLRLLN-Q3
TCTCAGCAGCCAGCTCAGGCTGTCGCCAACCTCAGCAGTGGCACCGGG 964
anti-hHER2-LC-I2- CTGAGCTGGCTGCTGAGACTGCTGAACCAGATGACCCAGTCCCCGAGC
965 GDSLSWLLRLLN-Q3
TCTCAGCAGCCAGCTCAGGCTGTCGCCGATATCAGCAGTGGCACCGGG
anti-hHER2-LC-C214-
CTGAGCTGGCTGCTGAGACTGCTGAACTAATCTAGACACCTCAGACAATCAACC 966
GDSLSWLLRLLN TCTCAGCAGCCAGCTCAGGCTGTCGCCGCACTCGCCCCTGTTGAAGC 967
anti-hHER2-LC-I2- CTGGAGTTCATCGCCAGCAAGCTGGCCCAGATGACCCAGTCCCCGAG
968 DSLEFIASKLA-Q3 CTTGCTGGCGATGAACTCCAGGCTGTCGATATCAGCAGTGGCACCGGG
969 anti-hHER2-LC-C214-
CTGGAGTTCATCGCCAGCAAGCTGGCCTAATCTAGACACCTCAGACAATCAACC 970
DSLEFIASKLA CTTGCTGGCGATGAACTCCAGGCTGTCGCACTCGCCCCTGTTGAAGC 971
anti-hHER2-HC-V2- CTGGAGTTCATCGCCAGCAAGCTGGCCCAGCTGGTGGAGTCTGGCGG
972 DSLEFIASKLA-Q3 CTTGCTGGCGATGAACTCCAGGCTGTCAACCTCAGCAGTGGCACCGG
973 anti-hHER2-HC-K447-
CTGAGCTGGCTGCTGAGACTGCTGAACTAATCTAGACACCTCAGACAATCAACC 974
GDSLSWLLRLLN TCTCAGCAGCCAGCTCAGGCTGTCGCCCTTGCCGGGGGACAGGCTC 975
anti-hHER2-HC-K447-
CTGGAGTTCATCGCCAGCAAGCTGGCCTAATCTAGACACCTCAGACAATCAACC 976
DSLEFIASKLA CTTGCTGGCGATGAACTCCAGGCTGTCCTTGCCGGGGGACAGGCTC 977
anti-hHER2-HC-S132D-K133S-
CTGGAGTTCATCGCCAGCAAGCTGGCCGGCTGCCTGGTGAAGGACTAC 978
S134L-T135E-S136F-G137I-
CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGGGCCAGGGGG 979
G138A-T139S-A140K-A141L- L142A anti-hHER2-HC-S190D-S191-
AGCCTGGAGTTCATCGCCAGCAAGCTGTGCAACGTGAACCACAAGCCCAG 980
S192L-L193E-G194F-T195I-
CTTGCTGGCGATGAACTCCAGGCTGTCGGGCACTGTCACCACGCTGG 981
Q196A-T197S-Y198K-I199L anti-hHER2-HC-S191D-S192-
GACAGCCTGGAGTTCATCGCCAGCAAGTGCAACGTGAACCACAAGCCCAG 982
L193-G194E-T195F-Q196I-
CTTGCTGGCGATGAACTCCAGGCTGTCGCTGGGCACTGTCACCACGC 983
T197A-Y198S-I199K anti-hHER2-HC-L398D-D399S-
CTGGAGTTCATCGCCAGCAAGCTGGCCAAGCTGACCGTGGACAAGTCCAG 984
S400L-D401E-G402F-S403I-
CTTGCTGGCGATGAACTCCAGGCTGTCCACTGGAGGTGTGGTCTTGTAG 985
F404A-F405S-L406K-Y407L- S408A anti-hHER2-HC-E388-
GACAGCCTGGAGTTCATCGCCAGCAAGAACAACTACAAGACCACACCTCCAG 986
DSLEFIASK-N389 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGGGCTGGCCGTTGCTC 987
anti-hHER2-HC-E388-
AGCCTGGAGTTCATCGCCAGCAAGCTGAACAACTACAAGACCACACCTCCAG 988
DSLEFIASKL-N389 CTTGCTGGCGATGAACTCCAGGCTGTCCTCGGGCTGGCCGTTGCTC 989
pET22b/TEV GAGAACCTGTACTTCCAAGGCCAC 990
ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC 991 CACCACCACCACCACCACTGAG
992 PPTase_C. TTGGAAGTACAGGTTCTCACGTTCGCAGAGGAATTTACACACTTC 993
thermocellum_pET22b/TEV
TAAGAAGGAGATATACATATGGGTTTTCTGCCGAAAGAGAAAAAG 994 ACP_ C.
GTGGTGGTGGTGGTGGTGGCTATTATTTTTAATATATTCAACGACGTCGC 995
thermocellum_pET22b TAAGAAGGAGATATACATATGTTCGAGAAAGTCCGTAAAATCATTGC
996 ACP_E.coli_pET22b GTGGTGGTGGTGGTGGTGCGCCTGGTGGCCGTTGATGTAATC
997 TAAGAAGGAGATATACATATGAGCACTATCGAAGAACGCGTTAAG 998
anti-hHER2-HC-E388-
CTGGACATGCTGGAGTGGAGCCTGATGAACAACTACAAGACCACACCTCCAG 999
GDSLDMLEWSLM-N389 CCACTCCAGCATGTCCAGGCTGTCGCCCTCGGGCTGGCCGTTGCTC
1000 anti-hHER2-HC-V2-
CTGGACATGCTGGAGTGGAGCCTGATGCAGCTGGTGGAGTCTGGCGG 1001
GDSLDMLEWSLM-Q3 CCACTCCAGCATGTCCAGGCTGTCGCCAACCTCAGCAGTGGCACCGG
1002 mAb2-HC-T359-GDSLSWLLRLLN-
CTGAGCTGGCTGCTGAGACTGCTGAACAAGAACCAGGTCAGCCTGACCTG 1003 K360
TCTCAGCAGCCAGCTCAGGCTGTCGCCGGTCATCTCCTCCCGGGATG 1004
mAb2-HC-E388-GDSLSWLLRLLN-
CTGAGCTGGCTGCTGAGACTGCTGAACAACAACTACAAGACCACGCCTCCC 1005 N389
TCTCAGCAGCCAGCTCAGGCTGTCGCCCTCCGGCTGCCCATTGCTCTC 1006
anti-hHER2-HC-Y296-
CTGAGCTGGCTGCTGAGACTGCTGAACAACAGCACCTACAGGGTGGTGTC 1007
GDSLSWLLRLLN-N297 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTACTGCTCCTCTCTGGGCTTG
anti-hHER2-HC-N297-
CTGAGCTGGCTGCTGAGACTGCTGAACAGCACCTACAGGGTGGTGTCC 1008
GDSLSWLLRLLN-S298 TCTCAGCAGCCAGCTCAGGCTGTCGCCGTTGTACTGCTCCTCTCTGGGC
anti-hHER2-HC-Y296-
CTGGAGTTCATCGCCAGCAAGCTGGCCAACAGCACCTACAGGGTGGTGTC 1009
DSLEFIASKLA-N297 CTTGCTGGCGATGAACTCCAGGCTGTCGTACTGCTCCTCTCTGGGCTTG
anti-hHER2-HC-N297-
CTGGAGTTCATCGCCAGCAAGCTGGCCAGCACCTACAGGGTGGTGTCC 1010
DSLEFIASKLA-S298 CTTGCTGGCGATGAACTCCAGGCTGTCGTTGTACTGCTCCTCTCTGGGC
Tras_HC_S6_i415_S418A GCCCGAGGGCGACGCCCTGAGCTGGCTG 1011
CAGCCAGCTCAGGGCGTCGCCCTCGGGC Human PPTase_N-His6 (PIPE
CATCACCATCACCATCACGTTTTCCCTGCCAAACGGTTCTGC 1012 cloning)
ACGGGCCCTCTAGACTTATGACTTTGTACCATTTCGTATTGGAATTTC pRS_N-His6 (PIPE
cloning) TAAGTCTAGAGGGCCCGTTTAAACC 1013
GTGATGGTGATGGTGATGAGGCTGAGCAGTGGCACCGG Human PPTase_C-His6 (PIPE
GGTGCCACTGCTCAGCCTGTTTTCCCTGCCAAACGGTTCTGC 1014 cloning)
GTGATGGTGATGGTGATGTGACTTTGTACCATTTCGTATTGGAATTTC pRS_C-His6 (PIPE
cloning) CATCACCATCACCATCACTAAGTCTAG 1015 AGGCTGAGCAGTGGCACCGG T.
maritima PPTase ATGATAGTCGGTGTGGGTATTGATG 1016
TTACTCTCCGATGAGGATGTTACC Top = Forward primer Bottom = Reverse
primer
TABLE-US-00009 TABLE 9 Expression yields of Trastuzumab IgGs with
inserted/grafted peptide-tags (HC, heavy chain; LC, light chain).
The values in brackets, ( ) [ ], correspond to antibody yields
after scale-up. Construct (whole antibody tested, the name
represents part of Yield per the HC or LC that contains the peptide
tag, the paired wildtype liter culture/ Expression SEQ chain is not
listed) mg scale/L ID NO
anti-hHER2-HC-S134G-T135D-S136-G137L-G138S-T139W-A140L 26 0.02 105
anti-hHER2-HC-L193G-G194D-T195S-Q196L-T197S-Y198W-I199L 73 0.02 117
anti-hHER2-HC-P189G-S190D-S191-S192L-L193S-G194W-T195L 61 (36) 0.02
(1) 109 anti-hHER2-HC-T359G-K360D-N361S-Q362L-V363S-S364W 43 0.02
123 anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 45 (29) 0.02 (1) 121
mAb2-HC-T359-GDSLSWLLRLLN-K360 78 0.05 148
mAb2-HC-E388-GDSLSWLLRLLN-N389 26 0.05 149
anti-hHER2-HC-E357G-M358D-T359S-K360L-N361S-Q362W-V363L 59 0.02 120
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 39 (13) [16] 0.02 (1) [0.5]
127 anti-hHER2-HC-E388-GDSLSWL-N389 39 0.02 126
anti-hHER2-LC-C214-GDSLSWLLRLLN 42 0.02 28
anti-hHER2-HC-S134G-T135D-S136-G137L-G138S-T139W-A140L- 2 0.05 106
LRLLN-A141 anti-hHER2-HC-K133G-S134D-T135S-S136L-G137S-G138W- 19
0.05 103 LLRLLN-T139
anti-hHER2-HC-P189G-S190D-S191-S192L-L193S-G194W-T195L- 48 0.3 110
LRLLN-Q196 anti-hHER2-HC-S190G-S191D-S192-L193-G194S-T195W-Q196L-
33 0.05 113 T197L-RLLN-Y198 anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3 20
(11) 0.05 (0.5) 94 anti-hHER2-LC-I2-GDSLSWLLRLLN-Q3 3 0.05 26
anti-hHER2-LC-I2-DSLEFIASKLA-Q3 43 (29) 0.05 (0.4) 27
anti-hHER2-LC-C214-DSLEFIASKLA 55 0.05 29
anti-hHER2-HC-V2-DSLEFIASKLA-Q3 34 (20) 0.05 (0.4) 95
anti-hHER2-HC-K447-DSLEFIASKLA 32 0.05 141
anti-hHER2-HC-S132D-K133S-S134L-T135E-S136F-G137I-G138A- 32 0.05
102 T139S-A140K-A141L-L142A
anti-hHER2-HC-S190D-S191-S192L-L193E-G194F-T195I-Q196A- 41 0.05 114
T197S-Y198K-I199L
anti-hHER2-HC-S191D-S192-L193-G194E-T195F-Q196I-T197A- 30 0.05 116
Y198S-I199K
anti-hHER2-HC-L398D-D399S-S400L-D401E-G402F-S403I-F404A- 13 0.05
135 F405S-L406K-Y407L-S408A anti-hHER2-HC-Y296-GDSLSWLLRLLN-N297 23
0.05 143 anti-hHER2-HC-N297-GDSLSWLLRLLN-S298 23 0.05 145
anti-hHER2-HC-Y296-DSLEFIASKLA-N297 21 0.05 144
anti-hHER2-HC-N297-DSLEFIASKLA-S298 23 0.05 146
anti-hHER2-HC-E388-DSLEFIASKLA-N389 36 (15) 0.05 (0.5) 129
anti-hHER2-HC-E388-DSLEFIASKL-N389 35 (20) 0.05 (0.5) 130
anti-hHER2-HC-E388-DSLEFIASK-N389 56 0.05 131
anti-hHER2-HC-T359-DSLEFIASKLA-K360 43 (18) 0.05 (0.5) 122
anti-hHER2-HC-S190D-S191-S192L-L193E-G194F-T195I-Q196A- 19 0.05 115
T197S-Y198K-I199L-C200A
anti-hHER2-HC-P189D-S190-S191L-S192E-L193F-G194I-T195A- 40 0.05 112
Q196S-T197K-Y198L-I199A
anti-hHER2-HC-D413-K414S-S415L-R416E-W417F-Q418I-Q419A- 29 0.05 138
G420S-N421K-V422L
anti-hHER2-HC-E382D-S383-N384L-G385E-Q386F-P387I-E388A 39 0.05 125
anti-hHER2-HC-E382D-S383-N384L-G385S-Q386W-P387L-E388L 33 0.05 124
anti-hHER2-LC-S76D-S77-L78-EFIASKLA-Q79 13 0.05 30
anti-hHER2-HC-S63-LEFIASK-V64 12 0.05 97
anti-hHER2-HC-V64L-EFIAS-K65 23 0.05 98
anti-hHER2-HC-V64L-EFIASKLA-K65 11 0.05 99
anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389 8 (19) 0.05
(0.4) 142 anti-hHER2-HC-V2-GDSLDMLEWSLM-Q3 56 0.05 96
anti-hHER2-HC-E388-GDSLDMLEWSLM-N389 32 0.05 132
Example 3. Production of Sfp 4'-Phosphopantetheinyl Transferase
(PPTase)
[0864] The B. subtilis Sfp PPTase was cloned into the pET22b
expression vector by using the PIPE method (see Klock et al.,
Proteins 71:982-994 (2008)). To allow cleavage of the C-terminal
His.sub.6 tag, a TEV (tobacco etch virus) protease recognition site
was inserted downstream of the Sfp coding sequence. All primers
used for cloning are listed in Table 8.
[0865] Protein expression and purification were performed according
to Yin et al. (see Nat. Protoc. 1:280-285 (2006)) with some minor
modifications. First, a 5 mL LB starter culture was inoculated from
the glycerol stock of E. coli BL21 (DE3) cells harboring the
pET22b/sfp expression plasmid. The culture was grown to saturation
by overnight incubation at 37.degree. C. at 300 rpm. The next day,
the starter culture was used to inoculate 1 L of TB medium (Sigma),
which was agitated at 300 rpm and maintained at 37.degree. C. After
reaching an optical density of 0.5 at 600 nm, the culture was
induced by the addition of IPTG to a final concentration of 1 mM
and the temperature was reduced to 30.degree. C. The culture was
shaken for another 12-16 hours and the bacterial cells were
harvested by centrifugation. Prior to use, the cell pellet was
stored at -20.degree. C.
[0866] To initiate protein purification, the frozen pellet was
thawed for 15 minutes on ice and re-suspended in a buffer
containing 20 mM Tris/HCl (pH 7.9), 0.5 M NaCl, 5 mM imidazole, and
2 U/mL DNase I (3 mL of buffer per g wet weight of cells). Cell
lysis was induced by sonication for 4 min, with intervals of 0.5
sec on and 0.5 sec off. In order to remove insoluble cell debris,
the resulting lysate was centrifuged at 40,000.times.g for 20 min
at 4.degree. C. The His.sub.6-tagged Sfp enzyme was then captured
by the addition of 4 mL of 50% Ni-NTA slurry (Qiagen) to the
cleared lysate. After shaking for 1 hour at 4.degree. C., the
resin-lysate mixture was poured into a disposable column (Bio-Rad).
The settled resin was washed with 25 column volumes of 50 mM
NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole (pH 8.0) and eluted
with 5 column volumes of 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250
mM imidazole (pH 8.0). Purified Sfp enzyme was then dialyzed twice
against 10 mM Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using a
Slide-A-Lyzer Dialysis Cassette (Pierce) with a 3.5 kDa cut-off,
and subsequently concentrated to a final concentration of at least
100 .mu.M using an Amicon Ultra-15 Centrifugal Filter Unit
(Millipore) with a 10 kDa cut-off. Finally, the concentrated enzyme
was aliquoted, flash-frozen in liquid nitrogen, and stored at
-80.degree. C.
[0867] In order to improve the purity of Sfp using reverse Ni-NTA
chromatography, a TEV cleavage site was introduced before the
C-terminal His.sub.6 tag. Ni-NTA purification of this construct was
performed as described above. However, after elution, the Sfp
enzyme was exchanged into TEV cleavage buffer containing 50 mM
Tris/HCl, 50 mM NaCl (pH 8.0). His.sub.6 tag removal was carried
out by digestion with 7% (w/w) TEV protease at 23.degree. C. for 1
hour and then at 4.degree. C. for 16 hours. The TEV-digested Sfp
enzyme was then reloaded onto a Ni-NTA column equilibrated with
1.times.PBS (pH 7.2). The cleaved enzyme was collected from the
column flow-through and from a washing step involving 5 column
volumes of 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole
(pH 8.0). Purified Sfp enzyme was then dialyzed twice against 10 mM
Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using a Slide-A-Lyzer
Dialysis Cassette (Pierce) with a 3.5 kDa cut-off. Following
dialysis, Sfp was concentrated to a final concentration of at least
100 .mu.M using an Amicon Ultra-15 Centrifugal Filter Unit
(Millipore) with a 10 kDa cut-off. Finally, the concentrated enzyme
was aliquoted, flash-frozen in liquid nitrogen, and stored at
-80.degree. C.
[0868] The purity of Sfp was assessed by SDS-PAGE. His.sub.6 tag
removal was verified by LC-MS and Sfp yield was quantified by
ultraviolet spectroscopy at 280 nm (ND-1000 UV-Vis
Spectrophotometer, NanoDrop Technologies, Wilmington, Del.) using a
molar extinction coefficient of 28620 M.sup.-1 cm.sup.-1. 48 mg of
TEV-cleaved Sfp enzyme was obtained per liter culture.
Example 4. Identification and Production of PPTase Homologs and
Mutants
Sfp Mutant R4-4
[0869] Using standard molecular biology methods, we inserted the
following mutations into the B. subtilis Sfp PPTase: Lys28Glu,
Thr44Glu, and Cys77Tyr. The sequences of the oligonucleotides used
for the mutagenesis reactions are listed in Table 8.
[0870] For protein expression, 0.5 L of TB medium was inoculated
with a 5 mL starter culture. The culture was agitated at 300 rpm
and maintained at 37.degree. C. After reaching an optical density
of 0.5 at 600 nm, the culture was induced by the addition of IPTG
to a final concentration of 1 mM and the temperature was reduced to
30.degree. C. The culture was shaken for another 16 hours at 300
rpm and the bacterial cells were harvested by centrifugation (15
min at 3400 rpm). Prior to use, the cell pellet was stored at
-20.degree. C.
[0871] The frozen pellet was thawed for 10 minutes on ice and
re-suspended in a buffer containing 50 mM Tris/HCl (pH 8), 300 mM
NaCl, 10 mM imidazole, 1 U/mL DNase I, and Complete.TM. EDTA-free
protease inhibitor cocktail tablets (Roche) (3 mL of buffer per g
wet weight of cells). Cell lysis was induced by sonication for 3
min on ice, with intervals of 0.5 sec on and 0.5 sec off. After
incubation for another 10 min on ice, the lysate was centrifuged at
40,000.times.g for 30 min at 4.degree. C. The His.sub.6-tagged Sfp
mutant R4-4 was then captured by the addition of 2 mL of 50% Ni-NTA
slurry (Qiagen) to the cleared lysate. After shaking for 1 hour at
4.degree. C., the resin-lysate mixture was poured into a disposable
column (Bio-Rad). The flowthrough was collected and the settled
resin was washed with 50 column volumes of 50 mM Tris, 300 mM NaCl,
20 mM imidazole (pH 8.0) and eluted with 5 column volumes of 50 mM
Tris, 300 mM NaCl, 250 mM imidazole (pH 8.0). After
buffer-exchanging the eluate into TEV protease cleavage buffer
containing 50 mM Tris/HCl, 50 mM NaCl (pH 8.0) using a PD-10
column, His.sub.6 tag removal was carried out by digestion with 7%
(w/w) TEV protease at 23.degree. C. for 1 hour and then at
4.degree. C. for 16 hours.
[0872] The TEV-digested Sfp mutant R4-4 was then reloaded onto a
Ni-NTA column (1 mL bed volume), which was equilibrated with
1.times.PBS (pH 7.2). The cleaved enzyme was collected from the
column flow-through and from a washing step involving 5 column
volumes of 50 mM Tris, 300 mM NaCl, 20 mM imidazole (pH 8.0). The
purified Sfp mutant R4-4 was then buffer-exchanged against 10 mM
Tris/HCl, 1 mM EDTA, 10% glycerol (pH 7.5) using PD-10 columns.
According to Bradford assay using BSA as standard, the enzyme had a
final concentration of 3.1 mg/mL at a final volume of 17 mL, which
corresponds to 105 mg of TEV-cleaved R4-4 mutant per liter culture.
Finally, the enzyme was aliquoted into 100 to 1000 .mu.L fractions,
flash-frozen in liquid nitrogen, and stored at -80.degree. C. The
purity of the enzyme was assessed by SDS-PAGE analysis and
His.sub.6 tag removal was verified by ESI-MS.
AcpS
[0873] Using standard molecular biology methods, we cloned the acpS
gene from E. coli K-12 into a pET22b vector that allows expression
of the recombinant enzyme with a C-terminal His6 tag. The sequences
of the oligonucleotides used for cloning are listed in Table 8.
[0874] Following inoculation from a saturated 5 mL starter culture,
the AcpS enzyme was expressed in 1 L of TB medium. After shaking
the culture at 37.degree. C. with 300 rpm, protein production was
induced by the addition of 1 mM IPTG at an optical density of 0.5
(600 nm). Protein expression was carried out overnight at
30.degree. C. and 300 rpm. The next day, the cells were harvested
by centrifugation at 3400 rpm for 15 min. The cell pellet was
stored at -20.degree. C. prior to protein purification.
[0875] To initiate protein purification, the frozen pellet was
thawed for 10 min on ice and resuspended in buffer (3 mL of buffer
per g wet weight of cells) containing 50 mM Tris/HCl (pH 8), 300 mM
NaCl, 10 mM imidazole, 1 U/mL DNase I, and Complete.TM. EDTA-free
protease inhibitor cocktail tablets (Roche). Cell lysis was
achieved by sonicating the cell suspension on ice for 3 min with
intervals of 0.5 sec on and 0.5 sec off. After another incubation
period of 10 min on ice, the lysate was centrifuged at 40,000 g for
30 min at 4.degree. C. Then 2 mL of 50% Ni-NTA slurry was added to
the cleared lysate and the lysate/resin mixture was shaken for 1
hour at 4.degree. C. The lysate/resin mixture was poured into a
disposable column. After collecting the flowthrough, the Ni-NTA
column was washed with 50 column volumes of buffer containing 50 mM
Tris (pH 8), 300 mM NaCl, and 20 mM imidazole. Elution was
performed with 5 column volumes of buffer containing 50 mM Tris (pH
8), 300 mM NaCl, and 250 mM imidazole. Using a 3.5 kDa cut-off
dialysis cassette (Slide-A-Lyzer, Thermo Scientific), the eluate
was dialyzed overnight into buffer containing 50 mM Tris (pH 8),
and 300 mM NaCl. Precipitated protein was removed by using a 0.45
.mu.m filter (Millipore). After addition of glycerol to a final
concentration of 10% (v/v), the Ni-NTA-purified protein was
flash-frozen in liquid nitrogen and stored at -80.degree. C. (100
and 200 .mu.L aliquots). The purity of AcpS was assessed by
SDS-PAGE and the yield was quantified by Bradford assay using BSA
as standard. About 13 mg of AcpS enzyme was obtained per liter
culture.
T. maritima PPTase
[0876] T. maritima PPTase expression was carried out at a 1 L scale
in native FM medium by inoculation with a 10 mL saturated starter
culture. The 1 L culture was shaken at 300 rpm at a temperature of
37.degree. C. After 2.5 hours, the culture reached an optical
density of 0.5 at 600 nm. Protein production was induced by the
addition of arabinose to a final concentration of 0.1% (w/v) and
the culture was shaken for an additional 4 hours. Cells were
harvested by centrifugation at 4000 rpm for 15 minutes and the cell
pellets were stored at -20.degree. C. Initial purification of T.
maritima PPTase was performed by IMAC (immobilized metal affinity
chromatography) using Ni-NTA agarose resin (Qiagen). Cell pellets
were thawed and resuspended in 60 mL lysis buffer (40 mM Tris
buffer (pH 8.0), 300 mM NaCl, 10 mM Imidazole, 1 mM TCEP). The cell
suspension was sonicated on ice for 1.5 minutes (using 1 sec
pulses) and centrifuged at 15000 rpm for 30 minutes at 5.degree. C.
The cleared lysate was loaded onto a 1.5 mL Ni-NTA column. After
collecting the flowthrough, the column was washed with 5 column
volumes of wash buffer (40 mM Tris buffer (pH 8.0), 300 mM NaCl, 40
mM imidazole, 10% glycerol, 1 mM TCEP). Protein elution was carried
out with 2 column volumes of elution buffer (20 mM Tris buffer (pH
8.0), 150 mM NaCl, 300 mM Imidazole, 1 mM TCEP).
[0877] The Ni-NTA eluate was further purified using a Superdex 75
column (GE Healthcare) connected to an Akta FPLC system.
Size-exclusion chromatography (SEC) was performed at flow rate of 1
mL/min in 10 mM Tris buffer (pH 7.4) supplemented with 1 mM EDTA
and 10% (v/v) glycerol. After analyzing protein-containing
fractions by SDS-PAGE, fractions containing the T. maritima PPTase
were pooled and dialyzed again against the buffer previously used
for SEC. The purified enzyme was then concentrated using an Amicon
Ultra-15 Centrifugal Filter Unit (Millipore) with a 10 kDa cut-off.
Precipitate was removed by centrifugation at 13000 rpm for 2 min
using a table top centrifuge. The concentrated protein (1.0 mg/mL,
48 .mu.M) was aliquoted into 100 .mu.L fractions, flash-frozen in
liquid nitrogen, and stored at minus 80.degree. C. The purity of T.
maritima PPTase was assessed by SDS-PAGE and the yield was
quantified by Bradford assay using BSA as standard. After all
purification steps, 1.4 mg of AcpS enzyme was obtained per liter
culture.
Example 5. Synthesis of Coenzyme A (CoA) Analogs
CoA-Maleimidoethylamido-Tetramethylrhodamine
##STR00580##
[0879] Tetramethylrhodamine-C2-maleimide (5.5 mg, 10.4 .mu.mol)
dissolved in 300 .mu.L of DMSO was added to CoA (10.4 .mu.mol in
150 .mu.L water) in 750 .mu.L of 10.times.PBS buffer and stirred at
23.degree. C. for 1 hour. After the reaction, the reaction mixture
was lyophilized to obtain the crude product, which was purified by
RP-C18 flash chromatography. Fractions of the desired product were
combined and lyophilized to afford
CoA-maleimidoethylamido-tetramethylrhodamine (9.8 mg with 94.4%
purity) as a dark purple powder. ESI-MS calculated for
C.sub.52H.sub.64N.sub.10O.sub.22P.sub.3S [MH].sup.+: 1320.3;
observed: 1320.3.
CoA-maleimidocaproyl (MC)-MMAF
##STR00581##
[0881] MC-MMAF (see Doronina et al., Bioconj. Chem. 17:114-124
(2006)) (36.0 mg, 38.9 mol) dissolved in 1.8 mL of DMSO was added
to CoA (39.0 mol in 312 .mu.L water) in 2.9 mL of 10.times.PBS
buffer and stirred at 23.degree. C. for 1 hour. After the reaction,
the reaction mixture was lyophilized to obtain the crude material,
which was purified by RP-C18 flash chromatography. Fractions of the
desired product were combined and lyophilized to afford CoA-MC-MMAF
(35.5 mg with 97.5% purity) as a white powder. ESI-MS calculated
for C.sub.70H.sub.112N.sub.13O.sub.27P.sub.3S [MH].sup.+: 1691.7;
observed: 1691.2.
CoA-MC-Val-Cit-PABC-MMAF
##STR00582##
[0883] MC-Val-Cit-PABC-MMAF (see Doronina et al., Bioconj. Chem.
17:114-124 (2006)) (5.7 mg, 4.3 mol) dissolved in 300 .mu.L of DMSO
was added to CoA (4.3 mol in 34 .mu.L water) in 2666 .mu.L of
10.times.PBS buffer and stirred at 23.degree. C. for 1 hour. After
the reaction, the reaction mixture was lyophilized to obtain the
crude material, which was purified by RP-C18 flash chromatography.
Fractions of the desired product were combined and lyophilized to
afford CoA-MC-Val-Cit-PABC-MMAF (6.1 mg with 98.0% purity) as a
white powder. ESI-MS calculated for
C.sub.89H.sub.139N.sub.18O.sub.32P.sub.3S [MH.sub.2].sup.2+/2:
1049.4; observed: 1049.4.
CoA-Ac-Ahx-M MAF
##STR00583##
[0885] Bromoacetyl-Ahx-MMAF (see, Alley et al., Bioconj. Chem.
19:759-765 (2008)) (1.3 mg, 1.4 .mu.mol) dissolved in 400 .mu.L of
DMSO was added to CoA (5.4 .mu.mol in 43 .mu.L water) in 3.6 mL of
borate buffer (6.7 mM at pH 8.5) and stirred at 23.degree. C. for
24 hours. After the reaction, the reaction mixture was lyophilized
to obtain the crude material, which was purified by RP-C18 flash
chromatography. Fractions of the desired product were combined and
lyophilized to afford CoA-Ac-Ahx-MMAF (1.1 mg with 96.9% purity) as
a white powder. ESI-MS calculated for
C.sub.68H.sub.112N.sub.13O.sub.26P.sub.3S [MH].sup.+: 1651.7;
observed: 1651.3.
CoA-Open-Ring-MC-MMAF
##STR00584## ##STR00585## ##STR00586##
[0887] CoA-MC-MMAF (5 .mu.mol in 1 mL of water) was added to 9 mL
of 1 M NH.sub.4OH.sub.(aq) and stirred at 23.degree. C. for 30
minutes. After the reaction, the reaction mixture was lyophilized
to obtain the crude material, which was purified by RP-C18 flash
chromatography. Fractions of the desired product were combined and
lyophilized to afford 3.9 mg of maleimide-ring-opened CoA-MC-MMAF
as a mixture of four positional and diastereomeric isomers as shown
in the scheme above (white powder, 96.6% purity). ESI-MS calculated
for C.sub.70H.sub.114N.sub.13O.sub.28P.sub.3S [MH].sup.+: 1709.7;
observed: 1709.2.
Example 6. Labeling of Peptide-Tagged IgGs with CoA Analogs In
Vitro
[0888] To exemplify the single-step conjugation of CoA analogs to
peptide-tagged IgGs in vitro, various peptide-tagged Trastuzumab
constructs were reacted with CoA-MC-MMAF in the presence of Sfp
enzyme. Generally, conjugation reactions were carried out in 50 or
75 mM HEPES or Tris buffer, pH 7.5 or 8.0 supplemented with 10.0 or
12.5 mM MgCl.sub.2. The final concentration of peptide-tagged
Trastuzumab was kept constant at 2.5 .mu.M, which corresponds to
5.0 .mu.M per peptide tag, while the final concentration of the CoA
substrates was usually varied between 40 .mu.M and 100 .mu.M. To
initiate the conjugation reaction, Sfp enzyme was added to give a
final concentration of typically 1 .mu.M. The enzymatic reaction
was allowed to proceed at either 23.degree. C. or 37.degree. C. for
16 hours. After this time period, the reaction progress was
analyzed by ESI-MS and HPLC.
Example 7. Labeling of Insertions
[0889] Nearly quantitative conjugation of CoA-MC-MMAF to six
Trastuzumab antibodies and one 2.sup.nd antibody ("mAb2") against a
different target with inserted S6- or ybbR-tags was accomplished by
incubating reaction mixtures with Sfp as described in Example 6.
HPLC of single-step conjugation reaction mixtures (Table 10) of
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121, FIG. 5A),
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127, FIG. 5B),
anti-hHER2-HC-V2-DSLEFIASKLA-Q3 (SEQ ID NO:95, FIG. 5C),
anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3 (SEQ ID NO:94, FIG. 5D),
anti-hHER2-HC-E388-DSLEFIASKL-N389 (SEQ ID NO:130, FIG. 5E),
anti-hHER2-HC-E388-DSLEFIASKLA-N389 (SEQ ID NO: 129, FIG. 5F), and
mAb2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:148, FIG. 5G) indicate
near complete conversion of the tagged antibodies into an
immunoconjugate with an approximate drug-to-antibody-ratio (DAR) of
2. ESI-MS of reduced conjugate samples suggest site-specific
modification of only the heavy chain as designed. For
anti-hHER2-LC-12-DSLEFIASKLA-Q3 (SEQ ID NO:27, FIG. 5H), HPLC
suggests only partial formation of the immunoconjugate as
significant amounts of unmodified antibody (39%, retention time 4.8
mins) remain and a mixture of DAR=1 (46%, retention time 5.4 mins)
and DAR=2 (16%, retention time 5.9 mins) species is observed.
TABLE-US-00010 TABLE 10 MS and HPLC analysis of conjugation
reactions with inserted tags: Antibody construct (whole antibody
tested, the name represents part of the HC or Expected mass LC that
contains the peptide Observed Expected mass unmodified DAR = 2 SEQ
tag,the paired wildtype chain mass immunoconjugate antibody
according ID NO is not listed) (Da) (Da) (Da) to HPLC 121
anti-hHER2-HC-T359- 51785.20 51791 50525 92% GDSLSWLLRLLN-K360 127
anti-hHER2-HC-E388- 51786.40 51791 50525 97% GDSLSWLLRLLN-N389 95
anti-hHER2-HC-V2- 51588.00 51598 50332 96% DSLEFIASKLA-Q3 142
anti-hHER2-HC-V2- 51780.40 51791 50525 100% GDSLSWLLRLLN-Q3 130
anti-hHER2-HC-E388- 51517.20 51527 50261 94% DSLEFIASKL-N389 129
anti-hHER2-HC-E388- 51588.00 51598 50332 100% DSLEFIASKLA-N389 27
anti-hHER2-LC-I2- 25878.40 25884 24618 16% DSLEFIASKLA-Q3 148
mAb2-HC-T359- 52848.80 52849 51597 95% GDSLSWLLRLLN-K360 (major);
51600.40 (minor)
As shown in FIG. 6, the trastuzumab immunoconjugates (A)
anti-hHER2-HC-V2-GDS-ppan-MC-MMAF-LSWLLRLLN-Q3, (B)
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389, and (C)
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKL-N389 were analyzed by
analytical size-exclusion chromatography (AnSEC) on a Shodex
PROTEIN KW-803 column. In all three cases, the ADCs were monomeric
(no detectable amounts of aggregated material).
Example 8. Labeling of Constructs with Grafted Peptide Tags
[0890] Single-step, in vitro Sfp-catalyzed conjugation of
CoA-MC-MMAF to Trastuzumab antibody with a grafted ybbR tag was
also attempted. The Sfp-catalyzed reaction of the IgG1 construct
anti-hHER2-HC-S190D-S191-S192L-L193E-G194F-T1951-Q196A-T197S-Y198K-1199L
was performed as described in Example 6. HPLC (FIG. 7) and ESI-MS
analysis of the reaction mixture indicate that the immunoconjugate
with MMAF (expected mass conjugate: 50489 Da, expected mass
unmodified antibody: 49223 Da, observed: 49216.8 Da) was not
formed. Other grafted constructs also failed to react and failed to
form immunoconjugates.
Example 9. Labeling of Mixed Grafting/Insertion Constructs
[0891] Single-step, in vitro Sfp-catalyzed conjugation of
CoA-MC-MMAF to Trastuzumab antibodies with grafted/inserted S6- or
ybbR-tags was also attempted. Two Trastuzumab mutants
anti-hHER2-HC-V64L-EFIASKLA-K65 and
anti-hHER2-LC-S76D-S77-L78-EFIASKLA-Q79 were reacted with
CoA-MC-MMAF and Sfp as described in Example 6. While
anti-hHER2-HC-V64L-EFIASKLA-K65 is partially modified as indicated
by HPLC of the reaction mixture (FIG. 8A),
anti-hHER2-LC-S76D-S77-L78-EFIASKLA-Q79 (FIG. 8B) failed to react
under identical conditions (Table 11).
TABLE-US-00011 TABLE 11 ESI-MS results of conjugation reactions
with mixed grafted/inserted tags: Antibody construct (whole
antibody tested, the name represents part of the HC or Expected
mass LC that contains the peptide Observed Expected mass unmodified
DAR = 2 SEQ tag, the paired wildtype chain mass immunoconjugate
antibody according ID NO is not listed) (Da) (Da) (Da) to HPLC 99
anti-hHER2-HC- 51287.20 51297 50031 32% V64L-EFIASKLA-K65 30
anti-hHER2-LC- 24324.80 25597 24331 0% S76D-S77-L78-
EFIASKLA-Q79
Example 10. Labeling with Fluorescent Dyes
[0892] To extend enzymatic antibody labeling beyond the
site-specific attachment of cytotoxins, we demonstrate the
feasibility of Sfp-catalysis to generate antibody-fluorophore
conjugates. This example represents two Sfp-catalyzed conjugations
of CoA-tetramethylrhodamine (CoA-TMR) to Trastuzumab antibodies
with either grafted or inserted S6 tags performed as described in
Example 6. HPLC traces of reaction mixtures were monitored at both
280 nm and 555 nm (FIG. 9). The latter wavelength is near the
absorption maximum of the TMR dye (.about.550 nm). Furthermore, the
data of the deconvoluted mass spectra of the antibody-fluorophore
conjugates is summarized in Table 12.
[0893] For the
anti-hHER2-HC-P189G-S190D-S191-S192L-L193S-G194W-T195L that
contains a truncated grafted S6 tag, conjugation resulted primarily
in the formation of a two-dye per antibody conjugate (FIG. 9A).
Likewise, the anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 with a
full-length S6 tag inserted between residue T359 and K360 showed
predominantly conjugation of two dye molecules to each antibody
(FIG. 9B). The results illustrate the S6 tags can be used for
conjugation of fluorescent labeling of modified antibodies.
TABLE-US-00012 TABLE 12 ESI-MS results of the conjugation reactions
with a fluorescent dye: Antibody construct (whole antibody tested,
the name represents part of the HC or Expected mass Expected mass
LC that contains the peptide Observed fluorophore unmodified SEQ
tag,the paired wildtype chain mass conjugate antibody ID NO is not
listed) (Da) (Da) (Da) 109 anti-hHER2-HC-P189G- 50177.20 50180
49286 S190D-S191-S192L-L193S- G194W-T195L 121 anti-hHER2-HC-T359-
51422.00 51419 50525 GDSLSWLLRLLN-K360
Example 11. Near Quantitative Labeling with Cytotoxins Linked
Through Thioether or Hydrolyzed Maleimide Linkage
[0894] Although not observed for conjugates of the invention,
maleimide-linked payloads may undergo deconjugation in plasma via
maleimide exchange with reactive thiols of albumin, glutathione,
and cysteine (Alley et al., Bioconjugate Chem. 2008, 19, 759-765).
Maleimide-based conjugates can be stabilized through chemical
ring-opening of the maleimidocaproyl linkage (see, Shen et al.,
Nature Biotech. 30:184-189 (2012)). To test this hydrolysis
procedure, the respective ADC of
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 was prepared using
CoA-open-ring-MC-MMAF. Moreover, to test alternative thiol-reactive
chemistries, we attached the MMAF cytotoxin to the terminal thiol
of CoA via an acetamide-based thioether linkage resulting in
CoA-Ac-Ahx-MMAF (see, Alley et al., Bioconj. Chem. 19:759-765
(2008)). The ESI-MS and HPLC results of these enzymatic conjugation
reactions (according to the protocol described in Example 6) are
summarized in Table 13. Near quantitative labeling with DAR=2 was
observed for anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 reacted with
CoA-open-ring-MC-MMAF (FIG. 10A) and
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 reacted with
CoA-Ac-Ahx-MC-MMAF (FIG. 10B).
TABLE-US-00013 TABLE 13 ESI-MS results of the conjugation reactions
with alternative linkers: SEQ ID NO of the antibody (whole antibody
tested, the name represents part of the Expected mass HC or LC that
contains Observed Expected mass unmodified DAR = 2 the peptide tag,
the paired CoA mass immunoconjugate antibody according wildtype
chain is not listed) substrate (Da) (Da) (Da) to HPLC 121
maleimide- 51802.00 51809 50525 85% ring-opened CoA-MC- MMAF 121
CoA-Ac-Ahx- 51742.40 51750 50525 80% MMAF
Example 12. Near Quantitative Labeling with Cytotoxin with
Cleavable Linker
[0895] To demonstrate the labeling of peptide-tagged IgGs with
cytotoxins that are attached via cleavable linkers, we conjugated
CoA-MC-Val-Cit-PABC-MMAF containing the cathepsin B-sensitive
valine-citrulline linker to either
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (FIG. 11A) or
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (FIG. 11B) in the presence of
Sfp. HPLC and ESI-MS results of this single-step enzymatic
conjugation are summarized in Table 14 and indicate near
quantitative labeling with a DAR=2 for both tag positions.
TABLE-US-00014 TABLE 14 ESI-MS results of the conjugation reactions
with CoA-MC-Val-Cit-PABC-MMAF: Expected mass Observed Expected mass
unmodified DAR = 2 SEQ Antibody mass immunoconjugate antibody
according to ID NO construct (Da) (Da) (Da) HPLC 121
anti-hHER2-HC-T359- 52189.60 52196 50525 91% GDSLSWLLRLLN-K360 127
anti-hHER2-HC-E388- 52188.40, 52196 50525 95% GDSLSWLLRLLN-N389
51412.40
Example 13. Optimization of Labeling Reaction as a Function of
pH
[0896] The purpose of this experiment was to determine the optimal
pH range for Sfp-catalyzed conjugation of CoA substrates to
peptide-tagged antibodies. In three experiments, 2.5 .mu.M of
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 or 2.5 .mu.M of
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 were reacted with 10 .mu.M of
CoA-MC-MMAF in the presence of 0.25 .mu.M of Sfp (for
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389) or 1.0 .mu.M of Sfp (for
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360), and the pH was titrated from
pH 5.0 to 10.0. In order to cover this pH range, five buffers were
utilized: 75 mM sodium acetate buffer for pH 5.0; 75 mM MES buffer
for pH 5.5, 6.0, and 6.5; 75 mM HEPES buffer for pH 7.0, 7.5, and
8.0; 75 mM sodium borate buffer for pH 9.0; 75 mM sodium carbonate
buffer for pH 10.0. All buffers were supplemented with 12.5 mM of
MgCl.sub.2 to ensure enzyme activity. The pH titration series was
performed at 23.degree. C. for 25 to 35 min in a volume of 100
.mu.L for each reaction. After quenching the enzymatic reaction by
the addition of 30 .mu.L of 4% (v/v) trifluoroacetic acid (TFA),
reaction mixtures were analyzed by HPLC at 280 nm as summarized in
Table 15.
TABLE-US-00015 TABLE 15 HPLC results of labeling reactions as a
function of pH: Antibody construct (whole antibody tested, the name
represents part of the HC or LC that contains the peptide SEQ tag,
the paired wildtype chain pH ID NO is not listed) value DAR = 0 DAR
= 1 DAR = 2 127 anti-hHER2-HC-E388- 5.0 100%; 100% 0%; 0% 0%; 0%
GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 100% 0% 0%
GDSLSWLLRLLN-K360 127 anti-hHER2-HC-E388- 5.5 100% 0% 0%
GDSLSWLLRLLN-N389 127 anti-hHER2-HC-E388- 6.0 88%; 90% 12%; 10% 0%;
0% GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 100% 0% 0%
GDSLSWLLRLLN-K360 127 anti-hHER2-HC-E388- 6.5 68% 23% 9.2%
GDSLSWLLRLLN-N389 127 anti-hHER2-HC-E388- 7.0 25%; 26% 48%; 44%
28%; 31% GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 77% 23% 0%
GDSLSWLLRLLN-K360 127 anti-hHER2-HC-E388- 7.5 12% 41% 47%
GDSLSWLLRLLN-N389 127 anti-hHER2-HC-E388- 8.0 7.7%; 11% 36%; 36%
56%; 52% GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 52% 37% 11%
GDSLSWLLRLLN-K360 127 anti-hHER2-HC-E388- 9.0 12% 31% 57%
GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 32% 46% 23%
GDSLSWLLRLLN-K360 127 anti-hHER2-HC-E388- 10.0 100% 0% 0%
GDSLSWLLRLLN-N389 121 anti-hHER2-HC-T359- 53% 36% 11%
GDSLSWLLRLLN-K360
[0897] The HPLC results indicate that the pH range 8 to 9 is
optimal for the conjugation of CoA-MC-MMAF to peptide-tagged
Trastuzumab. In this pH range, the lowest amount of uncoupled
antibody (DAR=0) and the highest amount of bi-conjugated ADC
(DAR=2) could be detected by HPLC. Furthermore, plotting the
percentage of ADC with a DAR of 2 against the pH (FIG. 12)
indicates that the pH optimum is independent of the insertion site
of the S6 tag for the two sites tested.
Example 14. Optimization of Labeling Reaction as a Function of
Enzyme Concentration
[0898] To test the amount of Sfp required for efficient enzymatic
conjugation, 2.5 .mu.M of anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ
ID NO: 127) was incubated at 370.degree. C. for 16 hours with 50
.mu.M CoA-MC-MMAF in 50 mM HEPES buffer (pH 7.5) supplemented with
10 mM MgCl.sub.2 in the presence of no Sfp enzyme or 0.1, 0.25,
0.5, 1, 2.5, 5 or 10 .mu.M Sfp enzyme. After 16 hours, aliquots of
the reaction were analyzed by ESI-MS. For Sfp concentrations of 0.1
.mu.M, mainly non-conjugated modified antibody is detectable by
ESI-MS (FIG. 13A). Quantitative conjugation was obtained for all
Sfp concentrations equal (FIG. 13B) or greater than 0.25 .mu.M,
such as 0.5 .mu.M of Sfp (FIG. 13C).
Example 15. Optimization of Labeling Reaction as a Function of CoA
Analog
[0899] To determine the minimal concentration of CoA substrate that
would be required for quantitative labeling of an peptide-tagged
IgG1 antibody, 2.5 .mu.M anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 was
incubated with 0.25 .mu.M or 1.0 .mu.M Sfp in 75 mM Tris buffer (pH
8.0) containing 12.5 mM MgCl.sub.2 and supplemented with
CoA-MC-MMAF at the following concentrations: 2.5, 5, 7.5, 10, 15,
25, and 50 .mu.M. The reaction was allowed to proceed for 13 hours
at 23.degree. C. and then quenched with 30 .mu.L of 4% (v/v)
trifluoroacetic acid (TFA). According to HPLC analysis (FIGS. 14A
and 14B, Table 16), nearly quantitative antibody conjugation was
achieved for all CoA-MC-MMAF concentrations equal or higher than
7.5 .mu.M. The degree of labeling was almost independent on the Sfp
concentration, with 86% DAR 2 species observed at 0.25 .mu.M Sfp
and 92% DAR 2 species observed at 1.0 .mu.M Sfp.
TABLE-US-00016 TABLE 16 HPLC results of labeling reactions as a
function of CoA concentration: Retention time CoA-MC- 4.9 min 5.3
min 5.7 min MMAF (.mu.M) Sfp (.mu.M) DAR = 0 DAR = 1 DAR = 2 50
0.25 3.8% 7.1% 89% 25 0.25 3.7% 7.0% 89% 15 0.25 3.2% 6.9% 90% 10
0.25 3.9% 7.2% 89% 7.5 0.25 5.0% 8.6% 86% 5 0.25 5.7% 20.5% 74% 2.5
0.25 28% 48.3% 24% 50 1.0 -- 7.1% 93% 25 1.0 -- 6.3% 94% 15 1.0 --
5.7% 94% 10 1.0 -- 5.7% 94% 7.5 1.0 1.0% 6.7% 92% 5 1.0 2.8% 24%
73% 2.5 1.0 31% 48% 21%
[0900] To determine the aggregation state of the
anti-hHER2-HC-E388-GDS-MC-MMAF-LSWLLRLLN-N389 immunoconjugate, 5.6
mg of anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (2.5 .mu.M) were reacted
with 40 M CoA-MC-MMAF in the presence of 1 M Sfp in 50 mM HEPES
buffer (pH 7.5) supplemented with 10 mM MgCl.sub.2. After
incubation at 23.degree. C. for 3 days, the reaction mixture was
purified on a Sephacryl 100-HR size-exclusion column (Sigma). After
confirming quantitative conjugation by ESI-MS (observed mass,
51786.40 Da; expected mass immunoconjugate, 51791 Da; expected mass
unmodified antibody, 50525 Da), the quaternary structure of the
respective ADC was analyzed on a Tricorn S200 column (Agilent). The
ADC was primarily monomeric (98%) and contained trace amounts of an
oligomerized species (2%).
Example 16. Thermal Stability of S6 Antibodies and ADCs
[0901] To examine the thermal stability of peptide-tagged
immunoconjugates, purified ADC samples were measured by
differential scanning fluorometry (DSF) (Table 17) or differential
scanning calorimetry (DSC) (Table 18). Samples were diluted to a
final concentration of 0.25 mg/mL (1.67 .mu.M) in PBS, pH 7.4. For
DSF, SYPRO Orange gel stain (Sigma) was added to a final
concentration of 5.times. as the tracer to indicate thermal
unfolding of the ADCs. Samples were heated with 20 fluorescence
scans/degree in a Lightcycler (Roche) instrument. For DSC, thermal
unfolding was monitored by measuring heat capacity as temperature
was increased at a rate of 1 degree Celsius per min in a MicroCal
VP-DSC instrument. Melting temperatures were calculated using in
the respective controller software packages assuming a 3-state
model.
[0902] As described previously (Wakankar et al. Bioconjugate Chem.
2010, 21, 1588-1595), unmodified trastuzumab exhibits two
transitions. The transitions were observed at 69.7 and 81.1 degrees
Celsius by DSF and 72.3 and 81.0 degrees Celsius by DSC. Similar to
the unmodified antibody, most CoA-MC-MMAF immunoconjugates exhibit
two transitions although with different amplitudes (FIG. 15). DSF
and DSC measurements of thermal melting points agree well although
DSF reports a roughly 2 degree lower first transition. Generally,
most engineered, non-conjugated antibodies and the respective
peptide-tagged ADCs show little destabilization as compared to the
wild-type antibody anti-hHER2.
TABLE-US-00017 TABLE 17 Thermal stability as measured by DSF.
.DELTA.Tm values are relative to unmodified anti-hHER2 antibody.
Sample (whole antibody tested, the name represents part of the HC
or LC that SEQ contains the peptide tag, the paired ID NO wildtype
chain is not listed) Tm.sub.1/.degree. C. Tm.sub.2/.degree. C.
.DELTA.Tm.sub.1/.degree. C. .DELTA.Tm.sub.2/.degree. C. 93/25
anti-hHER2 69.7 81.2 94 anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3 70.8 --
1.1 -- 95 anti-hHER2-HC-V2-DSLEFIASKLA-Q3 70.0 78.0 0.3 -3.1 102
anti-hHER2-HC-S132D-K133S-S134L-T135E- 69.3 -- -0.4 --
S136F-G137I-G138A-T139S-A140K-A141L- L142A 103
anti-hHER2-HC-K133G-S134D-T135S-S136L- 68.4 81.0 -1.3 -0.1
G137S-G138W-LLRLLN-T139 109 anti-hHER2-HC-P189G-S190D-S191-S192L-
69.6; 69.3 81.0; 80.6 -0.1; -0.4 -0.1; -0.5 L193S-G194W-T195L 110
anti-hHER2-HC-P189G-S190D-S191-S192L- 69.4 80.7 -0.3 -0.4
L193S-G194W-T195L-LRLLN-Q196 112
anti-hHER2-HC-P189D-S190-S191L-S192E- 69.4 78.6 -0.3 -2.5
L193F-G194I-T195A-Q196S-T197K-Y198L- I199A 113
anti-hHER2-HC-S190G-S191D-S192-L193- 68.1 78.1 -1.7 -3.1
G194S-T195W-Q196L-T197L-RLLN-Y198 114
anti-hHER2-HC-S190D-S191-S192L-L193E- 67.8 -- -1.9 --
G194F-T195I-Q196A-T197S-Y198K-I199L 115
anti-hHER2-HC-S190D-S191-S192L-L193E- 67.1 -- -2.6 --
G194F-T195I-Q196A-T197S-Y198K-I199L- C200A 116
anti-hHER2-HC-S191D-S192-L193-G194E- 69.3 -- -0.4 --
T195F-Q196I-T197A-Y198S-I199K 121
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 70.1; 70.3 81.7; 81.7 0.3; 0.5
0.6; 0.6 anti-hHER2-HC-T359-GDS-ppan-MC-MMAF- 68.4; 68.6 81.5; 81.4
-1.3; -1.2 0.4; 0.3 LSWLLRLLN-K360 122
anti-hHER2-HC-T359-DSLEFIASKLA-K360 70.1 81.7 0.3 0.6 127
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 66.6 81.5 -3.1 0.4
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF- 66.3 81.0 -3.4 -0.1
LSWLLRLLN-N389 anti-hHER2-HC-E388-GDS-ppan-MC-ValCit- 66.7 80.9
-3.0 -0.2 PABC-MMAF-LSWLLRLLN-N389 131
anti-hHER2-HC-E388-DSLEFIASK-N389 69.4 81.6 -0.3 0.5 130
anti-hHER2-HC-E388-DSLEFIASKL-N389 68.9 81.5 -0.9 0.4 129
anti-hHER2-HC-E388-DSLEFIASKLA-N389 69.3 81.6 -0.4 0.5 135
anti-hHER2-HC-L398D-D399S-S400L-D401E- 49.2 81.2 -20.6 0.1
G402F-S403I-F404A-F405S-L406K-Y407L- S408A 141
anti-hHER2-HC-K447-DSLEFIASKLA 70.1 81.0 0.4 -0.1 27
anti-hHER2-LC-I2-DSLEFIASKLA-Q3 70.0 78.8 0.2 -2.3 29
anti-hHER2-LC-C214-DSLEFIASKLA 69.7 80.9 0.0 -0.2
TABLE-US-00018 TABLE 18 Thermal stability as measured by DSC.
.DELTA.Tm values are relative to unmodified anti-hHER2 antibody.
SEQ ID No Tm1/ Tm2/ Tm3/ (antibody) Sample .degree. C. .degree. C.
.degree. C. .DELTA.Tm1/.degree. C. .DELTA.Tm2/.degree. C. 93/25
anti-hHER2 72.3; 72.3 80.9; 81.0 -- 95
anti-hHER2-HC-V2-DSLEFIASKLA- 72.3 77.9 83.3 0.1 -3.1 Q3
anti-hHER2-HC-V2-GDS-ppan-MC- 70.4 82.6 -- -1.9 1.7
MMAF-LSWLLRLLN-Q3 102 anti-hHER2-HC-S132D-K133S- 70.3 76.2 83.3
-2.0 -4.8 S134L-T135E-S136F-G137I-G138A- T139S-A140K-A141L-L142A
103 anti-hHER2-HC-K133G-S134D- 69.7 80.5 -- -2.6 -0.5
T135S-S136L-G137S-G138W- LLRLLN-T139 109 anti-hHER2-HC-P189G-S190D-
72.8; 70.7 80.1; 79.9 -- 0.5; -1.6 -0.9; -1.1
S191-S192L-L193S-G194W-T195L 110 anti-hHER2-HC-P189G-S190D- 71.2
80.0 -- -1.1 -1.0 S191-S192L-L193S-G194W-T195L- LRLLN-Q196 121
anti-hHER2-HC-T359- 70.4; 70.6 80.9; 80.6 -- -1.9; -1.6 -0.1; -0.4
GDSLSWLLRLLN-K360 anti-hHER2-HC-T359-GDS-ppan- 68.7 80.6 -- -3.6
-0.4 MC-MMAF-LSWLLRLLN-K360 anti-hHER2-HC-T359-GDS-ppan- 68.8 80.4
-- -3.5 -0.6 MC-MMAF-LSWLLRLLN-K360 122 anti-hHER2-HC-T359- 71.8
80.9 -- -0.5 -0.1 DSLEFIASKLA-K360 anti-hHER2-HC-E388-GDS-ppan-
67.0 80.2 -- -5.3 -0.8 MC-MMAF-LSWLLRLLN-N389
anti-hHER2-HC-E388-GDS-ppan- 66.0 80.1 -- -6.3 -0.9
MC-ValCit-PABC-MMAF- LSWLLRLLN-N389 131
anti-hHER2-HC-E388-DSLEFIASK- 71.2 80.8 -- -1.0 -0.2 N389 130
anti-hHER2-HC-E388- 70.7 80.8 -- -1.6 -0.2 DSLEFIASKL-N389
anti-hHER2-HC-E388-DS-ppan-MC- 69.9 80.2 -- -2.3 -0.8
MMAF-LEFIASKL-N389 129 anti-hHER2-HC-E388- 71.1 80.8 -- -1.2 -0.2
DSLEFIASKLA-N389 anti-hHER2-HC-E388-DS-ppan-MC- 70.2 80.3 -- -2.1
-0.7 MMAF-LEFIASKLA-N389 135 anti-hHER2-HC-L398D-D399S- -- 81.0 --
-- 0.1 S400L-D401E-G402F-S403I-F404A- F405S-L406K-Y407L-S408A 141
anti-hHER2-HC-K447- 72.7 81.0 -- 0.4 0.1 DSLEFIASKLA 29
anti-hHER2-LC-C214- 71.6 81.1 -- -0.7 0.1 DSLEFIASKLA
Example 17. Pharmacokinetic Properties of Peptide-Tagged ADCs
[0903] To check the in vivo stability of two peptide-tagged
Trastuzumab ADCs with MMAF payload (DAR of 2), we conducted a
pharmacokinetic (PK) study in mice.
Anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 and
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 were injected
i.v. into 3 mice using ADC concentrations of 1.0 mg/kg. 10 samples
were collected at 0.2, 1, 3, 7, 24, 48, 96, 168, 240, and 336
hours. The plasma titers of both ADCs were monitored up to two
weeks using ELISA assays with anti-human IgG as well as anti-MMAF
antibodies and ELISA plates coated with truncated human HER2
(extracellular domains 3-4). The ELISA results were then compared
to PK studies of an unmodified Trastuzumab IgG1. While
anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360 showed a fast
decay in mice in comparison to unmodified trastuzumab,
anti-hHER2-HC-E388-GDS-ppan-MC-MMAF-LSWLLRLLN-N389 exhibited a
serum clearance similar to unmodified trastuzumab over a two week
time period (FIG. 16). For both ADCs, anti-hlgG and anti-MMAF
titers track each other, suggesting that little if any drug is lost
during the in vivo exposure in mice.
Example 18. In Vitro Potency of Peptide-Tagged ADCs
[0904] In vitro cell-killing assays of peptide-tagged ADCs were
carried out with the HER2-expressing MDA-231 cell line. ADCs with
DAR=2 were prepared as described in Example 6 by reacting
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360 (SEQ ID NO:121) and
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389 (SEQ ID NO:127) with
non-cleavable MC-MMAF and cleavable MC-ValCit-PABC-MMAF (Example
12). The in vitro potency of the corresponding ADCs,
anti-hHER2-HC-T359-GDS-ppan-MC-MMAF-LSWLLRLLN-K360,
anti-hHER2-HC-E388-GDS-ppan-MC-M MAF-LSWLLRLLN-N389, anti-hH
ER2-HC-T359-GDS-ppan-MC-ValCit-PABC-MMAF-LSWLLRLLN-K360, and
anti-hHER2-HC-E388-GDS-ppan-MC-ValCit-PABC-MMAF-LSWLLRLLN-N389 were
tested in PC3-31 (high copy number of HER2) and PC3 (low copy
number of HER2) ErbB2 engineered cells. Regarding the PC3-31 cell
line, all peptide-tagged ADCs revealed potent cytotoxic activities
with inhibitory concentrations (IC.sub.50) in the picomolar or
sub-picomolar range. In contrast, the corresponding IC.sub.50
values on PC3 cells were equal or higher than 23 nM. The results
are summarized in Table 19 and FIG. 17 and indicate that all four
conjugates are highly potent ADCs and kill HER2/neu-positive cells
in an antigen-dependent manner.
TABLE-US-00019 TABLE 19 In vitro potency of S6-tag conjugated MMAF
immunoconjugates. IC.sub.50 value ADC PC3-31 cell line PC3 cell
line anti-hHER2-HC-T359-GDS-ppan-MC- 1.9 ng/mL; 13 pM >9000
ng/mL; >60000 pM MMAF-LSWLLRLLN-K360
anti-hHER2-HC-E388-GDS-ppan-MC- 5.8 ng/mL; 39 pM >9000 ng/mL;
>60000 pM MMAF-LSWLLRLLN-N389 anti-hHER2-HC-T359-GDS-ppan-MC-
3.2 ng/mL; 21 pM 3400 ng/mL; 23000 pM
ValCit-PABC-MMAF-LSWLLRLLN-K360 anti-hHER2-HC-E388-GDS-ppan-MC-
0.01 ng/mL; 0.07 pM >9000 ng/mL; >60000 pM
ValCit-PABC-MMAF-LSWLLRLLN-N389
Example 19. Labeling of Peptide-Tagged IgGs with a Cytotoxic CoA
Analog in Cell Culture Media
[0905] The bioorthogonality of PPTase-catalyzed
4'-phosphopantetheinylation enables the site-specific labeling of
peptide-tagged IgGs in complex mixtures such as conditioned medium.
Following the secretion of the peptide-tagged antibody, exogenously
added PPTase (such as Sfp) and drug-CoA substrate (such as
CoA-MC-MMAF) lead to the formation of homogeneous ADCs, which can
be purified in a single step using protein A affinity
chromatography.
[0906] For example, HEK293F cells are transfected with plasmid DNA
coding for IgG1 heavy chain with S6 tag insertion in the CH3 domain
and plasmid DNA coding for unmodified kappa light chain. The 40 mL
HEK293F suspension culture is cultured for five days at 37.degree.
C. After harvesting by centrifugation at 2000 rpm for 10 minutes,
the medium supernatant is supplemented to a final concentration of
40 .mu.M of CoA-MC-MMAF, 10 mM of MgCl.sub.2, and 50 mM of HEPES
(pH 7.5). The medium supernatant is then split into two 20 mL
aliquots. Recombinantly produced Sfp enzyme (5 .mu.M) is added to
one of the aliquots (see Table 20, Experiment #2) and the enzymatic
reaction is allowed to proceed for 24 hours at room
temperature.
TABLE-US-00020 TABLE 20 In-medium labeling scheme. Experiment # 1 2
Addition of CoA-MC-MMAF (40 .mu.M) to X X medium supernatant
Addition of Sfp (5 .mu.M) to medium supernatant X
[0907] Antibody purification is carried out using Protein A
Sepharose Fast Flow columns with 0.25 mL bed volume for each
experiment. After equilibration with PBS, the medium supernatants
are applied to the columns at a flow rate of about 1 mL/min and the
flowthrough is collected. Following washing with 20 column volumes
of PBS, bound antibody is eluted using 6 column volumes of 0.1 M
sodium acetate (pH 3.0) followed by immediate neutralization with 1
M Tris/HCl (pH 10) to reach a final pH of about 8. The purity of
the eluates is assessed by SDS-PAGE analysis and the antibody yield
is determined by the Bradford method. Finally, Sfp-dependent
in-medium ADC formation is confirmed by ESI-MS and HPLC analysis of
the protein A eluates.
Example 20. In Vitro Labeling of Peptide-Tagged IgGs with
Acetyl-CoA and Subsequent Conjugation with a Cytotoxin
[0908] The principle of the preparation of immune conjugates via
acetyl-CoA is a three-step chemoenzymatic conjugation protocol in
which the acetyl moiety serves as a protecting group for the
reactive thiol group of CoA. Furthermore, although PPTases such as
Sfp tolerate large CoA analogs (e.g. peptidyl-CoA) for catalysis,
the catalytic efficiency (k.sub.cat/K.sub.M) is significantly
reduced compared to CoA itself (see, Sieber et al., J. Am. Chem.
Soc. 125: 10862-10866 (2003)). Hence, it is expected that the small
acetyl group ensures similar enzyme kinetics as seen for the native
CoA substrate.
[0909] For example, covalent conjugation of the acetylated ppan
moiety to a peptide-tagged IgG antibody is carried out as described
in Example 6 using acetyl-CoA instead of CoA-MC-MMAF. After
confirming quantitative conjugation by ESI-MS, the conjugate is
dialyzed into Reaction Buffer (0.1 M sodium phosphate (pH 7.2),
0.15 M NaCl). The dialyzed conjugate is concentrated to about 5
mg/mL and supplemented with 10% (v/v) of Deacetylation Solution
containing Reaction Buffer (pH 7.2) with 0.5 M hydroxylamine and 25
mM EDTA. This chemical thioester cleavage reaction is allowed to
proceed for 3 hours at room temperature, followed by
buffer-exchanging the reaction mixture into Reaction Buffer (pH
7.2) supplemented with 10 mM EDTA. After confirmation of
quantitative deacetylation by ESI-MS, the deprotected ppan moiety
is then conjugated with 15 equivalents of thiol-reactive
maleimide-MC-MMAF (0.5 mM) for 1 hour at room temperature. The
reaction is quenched by buffer-exchange into PBS. Finally,
quantitative ADC formation is confirmed by ESI-MS and HPLC
analysis.
Example 21. Labeling of Peptide-Tagged IgGs with Acetyl-CoA in Cell
Culture Media and Subsequent Conjugation with a Cytotoxin
[0910] The bioorthogonality of PPTase-catalyzed generation of
homogeneous ADCs allows the site-specific labeling of IgGs in cell
culture media (see Example 19). Instead of directly attaching the
cytotoxic drug molecule to the antibody, it is also possible to
carry out in-medium labeling with acetyl-CoA for ADC generation via
a three-step chemoenzymatic conjugation process. The small
acetyl-CoA analog allows conjugation reactions with improved
catalytic efficiency (k.sub.cat/K.sub.M) as compared to large
cytotoxic CoA analogs, thereby significantly decreasing the amount
of enzyme needed for quantitative conjugation. Furthermore, for
process development, it would be advantageous to perform labeling
reactions in large culture volumes with non-toxic compounds. The
peptide-tagged IgG conjugated with the acetyl-ppan moiety can be
purified in a single step using protein A affinity chromatography.
In order to prepare the immune conjugate starting from the purified
acetyl-ppan-conjugated antibody, the two subsequent chemical
reactions are carried out as described in Example 20.
Example 22. Labeling of Peptide-Tagged IgGs with Acetyl-CoA or
Bioorthogonal CoA Analogs in Cell Culture Media and Subsequent
Conjugation with a Cytotoxin
[0911] The bioorthogonality of PPTase-catalyzed generation of
homogeneous ADCs also allows the site-specific labeling of IgGs in
cell culture media (see Example 19). Instead of directly attaching
the cytotoxic drug molecule to the antibody, it is also possible to
carry out in-medium labeling with acetyl-CoA for ADC generation via
a three-step chemoenzymatic conjugation process. The small
acetyl-CoA analog allows conjugation reactions with improved
catalytic efficiency (k.sub.cat/K.sub.M) as compared to large
cytotoxic CoA analogs, thereby significantly decreasing the amount
of enzyme needed for quantitative conjugation. Furthermore, for
process development, it would be advantageous to perform labeling
reactions in large culture volumes with non-toxic compounds. The
peptide-tagged IgG conjugated with the acetyl-ppan moiety can be
purified in a single step using protein A affinity chromatography.
In order to prepare the immune conjugate starting from the purified
acetyl-ppan-conjugated antibody, the two subsequent chemical
reactions are carried out as described in Example 20.
[0912] Alternatively, instead of using acetyl-CoA, in-medium
labeling can also be performed with CoA analogs covalently linked
to bioorthogonal groups, such as azido, alkene, alkyne, ketone, or
aldehyde moieties. Following in-medium PPTase catalysis, the
peptide-tagged antibody with the ppan-bound bioorthogonal group is
purified to homogeneity using protein A affinity chromatography. As
the last step of this two-step chemoenzymatic labeling strategy for
ADC preparation, the reaction with the complementary bioorthogonal
group leads to the site-specific attachment of the drug moiety to
the antibody.
Example 23. Production and Properties of ADCs with a DAR of 4
[0913] ADCs with a DAR of 4 can be generated by inserting/grafting
multiple peptide tags into an antibody, which are substrates of the
same enzyme (FIG. 18A). For instance, both the ybbR- and the
S6-tags are recognized as substrates by the PPTase Sfp. Conversely,
labeling of antibodies with multiple different ligands is achieved
by inserting/grafting peptide tags into an antibody, which are
substrates of two different PPTases. For example, the A1 tag is
exclusively recognized by the AcpS PPTase, while the S6 tag is
preferentially modified by the Sfp PPTase. Furthermore,
immunoconjugates with higher DARs (e.g., DARs of 6, 8, 10, 12,
etc.) may be generated by adding additional tags. Enzymatic
conjugation can also be combined with other labeling strategies
such as site-specific conjugation through cysteine, pyrrolysine,
pyrroline-carboxy-lysine, and unnatural amino acids as well as
chemoselective methods such as Lys, Cys or Tyr selective
chemistries.
[0914] In order to prepare homogeneous ADCs with a DAR of 4, two
peptide tags were incorporated into the heavy chain of Trastuzumab
IgG1, namely an S6 tag into the V.sub.H domain and a ybbR tag into
the CH3 domain
(anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389). This
dual-tagged antibody was expressed in HEK293F cells on a 50 mL
scale. Following transfection, the HEK293F cells were cultured for
five days before harvest by centrifugation at 3400 rpm for 15 min.
The resulting medium supernatant was filtered through a
0.22-.mu.m-pore-size filter. Purification was accomplished using a
Protein A Sepharose Fast Flow column (GE Healthcare) with a bed
volume of 0.6 mL, which was equilibrated with 20 column volumes of
PBS. The filtered medium supernatant was loaded at a flow rate of
about 1 mL/min. After washing the column with 20 column volumes of
PBS, the peptide-tagged antibody was eluted with 5 column volumes
of 0.1 M sodium acetate (pH 3.0) followed by immediate
neutralization with 1 M Tris/HCl (pH 10) to a final pH of about 8.
According to the Bradford method, the total yield was 8 mg of
purified antibody per liter culture. The purity of the antibody
construct was assessed by SDS-gel electrophoresis. After
concentration with a 30 kDa cut-off Amicon Ultra Centrifugal Filter
Unit, 2.5 .mu.M
anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388-DSLEFIASKLA-N389 was
incubated with 50 .mu.M CoA-MC-MMAF, 1 .mu.M Sfp, 12.5 mM
MgCl.sub.2, in 75 mM HEPES buffer, pH 7.5, at 23.degree. C. for 16
hours to enzymatically label the dual-tagged antibody with four
drug molecules.
[0915] The deconvoluted mass spectrum of the reduced and
deglycosylated antibody construct confirmed the covalent attachment
of two ppan-MC-MMAF units to each heavy chain of Trastuzumab
(observed mass, 54223.20 Da; expected mass, 54231 Da). Neither
uncoupled (expected mass, 51700 Da) nor mono-labeled species
(expected mass, 52966 Da) were observed by ESI-MS. Near
quantitative conversion to an ADC with a DAR of 4 (95% according to
peak area integration) was further confirmed by HPLC analysis (FIG.
18B).
Example 24. Generation of a Comprehensive Library of Peptide-Tagged
ADCs Using the Protein Expression and Purification Platform
(PEPP)
[0916] Based on the examination of the crystal structure of human
IgG1 B12 antibody as well as surface accessibility calculations
(Example 1), a library of 268 peptide-tagged trastuzumab IgG1
constructs was proposed. Systematic insertion of S6 and ybbR tag
sequences into the constant regions was accomplished by standard
molecular biology methods using the oligonucleotides listed in
Table 8. Sequence confirmed plasmids harboring either the heavy or
light chain genes of trastuzumab were used for transient
co-transfection of 293 Freestyle.TM. cells according to the PEI
method (Meissner et al., 2001). Culturing of each library member in
a volume of 35 mL of Freestyle.TM. expression media (Invitrogen)
for five days at 37.degree. C. under 5% CO.sub.2 was carried out on
the PEPP system (Gonzalez R, Jennings L L, Knuth M, Orth A P, Klock
H E, Ou W, Feuerhelm J, Hull M V, Koesema E, Wang Y, Zhang J, Wu C,
Cho C Y, Su A1, Batalov S, Chen H, Johnson K, Laffitte B, Nguyen D
G, Snyder E Y, Schultz P G, Harris J L, Lesley S A. Proc Natl Acad
Sci USA. 2010, 107(8):3552-7). Following automated cell harvest,
the same system was used to purify the library of peptide-tagged
antibodies by Protein A affinity chromatography. Briefly, after
0.22 .mu.m filtration of the medium supernatant, each filtrate was
loaded onto a Protein A affinity column containing 0.2 mL of
settled resin at an approximate flow rate of 1 mL/min. The column
was then washed with 20 column volumes of PBS followed by elution
with 5 column volumes of 0.1 M sodium acetate, pH 3.0. The eluate
was immediately neutralized with 25% (v/v) of 1 M Tris-HCl (pH
8.0).
[0917] To determine the yield of the Protein A-purified antibodies
(Table 21), protein concentrations of the eluates were measured in
duplicate on a ND-1000 UV-Vis spectrophotometer (NanoDrop
Technologies) at 280 nm using the preset molar extinction
coefficient for IgG molecules. After concentrating the
peptide-tagged antibodies using 30 kDa cut-off Amicon Ultra-0.5
centrifugal filter devices (EMD Millipore), enzyme-catalyzed
conjugation reactions were performed for about 16 hours at
20.degree. C. with 2.5 .mu.M of peptide-tagged antibody, IM of
CoA-MC-MMAF substrate, and 1 .mu.M of Sfp enzyme in Tris-HCl buffer
(75 mM, pH 8.0) supplemented with 12.5 mM of MgCl.sub.2 and 20 mM
of NaCl. The degree of labeling of the peptide-tagged antibodies
was quantified by analytical HPLC on a PLRP-S column (4000 .ANG., 5
M, 50.times.4.6 mm, Agilent Technologies) with a 6-min linear
gradient of 25-50% acetonitrile in water containing 0.1%
trifluoroacetic acid. The corresponding uncoupled antibodies were
used as negative controls (Table 21). After concentrating the
antibody conjugates using Amicon Ultra-4 centrifugal filter devices
(EMD Millipore), the enzymatic reactions were further analyzed by
mass spectrometry on an Agilent 6520 Q-TOF instrument (Agilent
Technologies). Deconvoluted ESI-MS spectra of the reduced and
deglycosylated antibody conjugates were obtained by using 10 .mu.L
of concentrated reaction mixture (Table 21).
[0918] The peptide-tagged ADC constructs were further purified by
Ni-NTA (nickel-nitrilotriacetic acid) chromatography to remove Sfp
enzyme and excess CoA-MC-MMAF substrate. After equilibration of the
Ni-NTA columns (0.2 mL bed volume each) with PBS, the concentrated
conjugation samples were loaded onto the columns at an approximate
flow rate of 1 mL/min. Next, the columns were washed with 20 column
volumes of PBS followed by elution with 5 column volumes of
Tris-HCl buffer (50 mM, pH 8.0) supplemented with 250 mM imidazole
and 300 mM NaCl. According to Bradford assay, the recovery of the
peptide-tagged ADCs averaged 39% of the Protein A-purified starting
material. The PEPP system was then used to buffer-exchange each
sample into PBS using NAP-10 Columns (GE Healthcare). Following
buffer-exchange, the peptide-tagged ADCs were concentrated using
Amicon Ultra-4 centrifugal filter devices (EMD Millipore), and the
concentrations of the conjugates were adjusted by dilution with
PBS. Adjusted to the appropriate concentration, the ADC samples
were further characterized by DSF (differential scanning
fluorimetry), LC90 (LabChip 90), AnSEC (analytical size-exclusion
chromatography), and in vitro potency assays (data not shown).
[0919] Of the originally planned 268 peptide-tagged trastuzumab
antibodies, expression was tested for 183 constructs (68%). The
expression levels exhibit a great variability ranging from 0 to
more than 30 mg of antibody per liter culture (Table 21), with the
average being 16 mg (.+-.8 mg standard deviation) of antibody per
liter culture. Furthermore, the expression levels correlate with
the position of the peptide tag insertion with the 46 light chain
constructs (13.+-.8 mg per liter culture) exhibiting lower average
expressions levels than the 137 heavy chain constructs (17.+-.8 mg
per liter culture). The expression levels also depend on the type
of peptide tag: 95 antibody constructs with ybbR tag insertions on
average show higher expression levels (19.+-.7 mg per liter
culture) than the corresponding 88 constructs with S6 tag
insertions (13.+-.8 mg per liter culture). The opposite trend is
observed for the conjugation efficiencies based on reverse-phase
HPLC analysis: 44 (72%) peptide-tagged constructs with near
quantitative ADC formation (drug-to-antibody ratio >1.9) are
based on insertion of the S6 peptide sequence, while only 17 (28%)
ybbR-tagged antibodies displayed near quantitative conversion to
the corresponding ADC.
[0920] On average, heavy chain constructs were conjugated more
efficiently than peptide insertions in the light chain: 19% (8 out
of 43) of the constructs with peptide tag insertions in the light
chain revealed DARs of at least 1.9 while 40% (53 out of 131) of
the constructs with peptide tag insertions in the heavy chain could
be conjugated with the same efficiency. The best overall
conjugation efficiencies are displayed by peptide tag insertions in
several loop regions of the CH1 domain. Overall, of the 183
expressed peptide-tagged antibodies, conjugation efficiencies of
174 constructs could be determined with 61 (35%) constructs
yielding drug-to-antibody ratios (DARs) of 1.9 or higher.
[0921] Thermostability of the resulting ADCs depends on the site of
peptide tag insertion. For instance, most peptide tag insertions in
the CH2 domain lead to a significant decrease of the lowest
observed thermal transition (Tm1) according to DSF (differential
scanning fluorimetry) measurements as will be illustrated in more
detail in Example 25. Little aggregation or antibody oligomers were
observed for 135 (87%) out of 156 peptide-tagged ADCs that were
examined by analytical size-exclusion chromatography (>90%
monomeric species). The in vitro potency of the peptide-tagged ADCs
correlated as expected with their degree of labeling. Although a
large number of peptide-tagged ADCs with preferred properties can
be generated, the data also illustrate that expression yield,
thermal stability, conjugation efficiency and other properties are
greatly affected by the choice of tag insertion site.
TABLE-US-00021 TABLE 21 ADC preparation and characterization of
material prepared on PEPP system. Anti- Anti- body Expected
Expected body yield.sup.b mass anti- mass Observed SEQ ID ADC
name.sup.a (mg/L) DAR.sup.c body.sup.d (Da) ADC.sup.e (Da)
mass.sup.f (Da) SEQ ID anti-hHER2-HC-A118- 10 2.0 50525.0 51790.5
51792.7 NO: 150 GDS-ppan-MC-MMAF- 51814.6 LSWLLRLLN-S119 SEQ ID
anti-hHER2-HC-S119- 12 2.0 50525.0 51790.5 51792.4 NO: 151
GDS-ppan-MC-MMAF- LSWLLRLLN-T120 SEQ ID anti-hHER2-HC-T120- 11 2.0
50525.0 51790.5 51797.2 NO: 152 GDS-ppan-MC-MMAF- LSWLLRLLN-K121
SEQ ID anti-hHER2-HC-T135- 24 2.0 50525.0 51790.5 51792.8 NO: 157
GDS-ppan-MC-MMAF- LSWLLRLLN-S136 SEQ ID anti-hHER2-HC-S136- 20 2.0
50525.0 51790.5 51792.0 NO: 158 GDS-ppan-MC-MMAF- LSWLLRLLN-G137
SEQ ID anti-hHER2-HC-G138- 14 2.0 50525.0 51790.5 51792.3 NO: 160
GDS-ppan-MC-MMAF- 51814.6 LSWLLRLLN-T139 SEQ ID anti-hHER2-HC-E152-
3 0.2 50525.0 51790.5 50528.4 NO: 161 GDS-ppan-MC-MMAF- 51794.8
LSWLLRLLN-P153 SEQ ID anti-hHER2-HC-P153- 0 N/A 50525.0 51790.5 N/A
NO: 162 GDS-ppan-MC-MMAF- LSWLLRLLN-V154 SEQ ID anti-hHER2-HC-N159-
0 N/A 50525.0 51790.5 N/A NO: 163 GDS-ppan-MC-MMAF- LSWLLRLLN-S160
SEQ ID anti-hHER2-HC-S160- 10 1.4 50525.0 51790.5 51792.0 NO: 164
GDS-ppan-MC-MMAF- LSWLLRLLN-G161 SEQ ID anti-hHER2-HC-G161- 9 1.3
50525.0 51790.5 51798.0 NO: 165 GDS-ppan-MC-MMAF- 50529.2
LSWLLRLLN-A162 SEQ ID anti-hHER2-HC-A162- 15 2.0 50525.0 51790.5
51798.4 NO: 166 GDS-ppan-MC-MMAF- LSWLLRLLN-L163 SEQ ID
anti-hHER2-HC-T164- 22 2.0 50525.0 51790.5 51796.8 NO: 168
GDS-ppan-MC-MMAF- LSWLLRLLN-S165 SEQ ID anti-hHER2-HC-S165- 15 2.0
50525.0 51790.5 51794.4 NO: 169 GDS-ppan-MC-MMAF- LSWLLRLLN-G166
SEQ ID anti-hHER2-HC-P171- 3 N/A 50525.0 51790.5 N/A NO: 170
GDS-ppan-MC-MMAF- LSWLLRLLN-A172 SEQ ID anti-hHER2-HC-S176- 8 1.9
50525.0 51790.5 51791.7 NO: 171 GDS-ppan-MC-MMAF- 51812.9
LSWLLRLLN-S177 SEQ ID anti-hHER2-HC-P189- 24 1.5 50525.0 51790.5
51792.4 NO: 173 GDS-ppan-MC-MMAF- LSWLLRLLN-S190 SEQ ID
anti-hHER2-HC-S191- 21 2.0 50525.0 51790.5 51792.0 NO: 175
GDS-ppan-MC-MMAF- 51814.0 LSWLLRLLN-S192 SEQ ID anti-hHER2-HC-S192-
32 2.0 50525.0 51790.5 51792.0 NO: 176 GDS-ppan-MC-MMAF- 51813.7
LSWLLRLLN-L193 SEQ ID anti-hHER2-HC-L193- 18 2.0 50525.0 51790.5
51791.0 NO: 177 GDS-ppan-MC-MMAF- LSWLLRLLN-G194 SEQ ID
anti-hHER2-HC-G194- 19 2.0 50525.0 51790.5 51796.8 NO: 178
GDS-ppan-MC-MMAF- LSWLLRLLN-T195 SEQ ID anti-hHER2-HC-T195- 17 2.0
50525.0 51790.5 51800.0 NO: 179 GDS-ppan-MC-MMAF- 53918.8
LSWLLRLLN-Q196 SEQ ID anti-hHER2-HC-Q196- 23 1.9 50525.0 51790.5
51791.9 NO: 180 GDS-ppan-MC-MMAF- 51813.5 LSWLLRLLN-T197 SEQ ID
anti-hHER2-HC-K205- 22 0.2 50525.0 51790.5 50526.7 NO: 181
GDS-ppan-MC-MMAF- 51792.6 LSWLLRLLN-P206 50548.6 SEQ ID
anti-hHER2-HC-P206- 25 1.9 50525.0 51790.5 51792.1 NO: 182
GDS-ppan-MC-MMAF- 51813.9 LSWLLRLLN-S207 SEQ ID anti-hHER2-HC-A231-
35 2.0 50525.0 51790.5 51789.5 NO: 185 GDS-ppan-MC-MMAF- 51810.4
LSWLLRLLN-P232 SEQ ID anti-hHER2-HC-E233- 13 1.9 50525.0 51790.5
51789.5 NO: 187 GDS-ppan-MC-MMAF- 51770.4 LSWLLRLLN-L234 51809.6
SEQ ID anti-hHER2-HC-L235- 16 1.9 50525.0 51790.5 51790.1 NO: 189
GDS-ppan-MC-MMAF- 51811.8 LSWLLRLLN-G236 SEQ ID anti-hHER2-HC-P244-
12 0.8 50525.0 51790.5 50522.7 NO: 191 GDS-ppan-MC-MMAF- 51790.6
LSWLLRLLN-P245 50545.4 SEQ ID anti-hHER2-HC-I253- 23 1.9 50525.0
51790.5 51789.0 NO: 193 GDS-ppan-MC-MMAF- 51809.6 LSWLLRLLN-S254
SEQ ID anti-hHER2-HC-S254- 20 2.0 50525.0 51790.5 51789.5 NO: 194
GDS-ppan-MC-MMAF- 51810.5 LSWLLRLLN-R255 SEQ ID anti-hHER2-HC-R255-
25 2.0 50525.0 51790.5 51792.2 NO: 195 GDS-ppan-MC-MMAF- 51814.5
LSWLLRLLN-T256 SEQ ID anti-hHER2-HC-S267- 20 2.0 50525.0 51790.5
51789.2 NO: 198 GDS-ppan-MC-MMAF- 51810.1 LSWLLRLLN-H268 SEQ ID
anti-hHER2-HC-H268- 10 2.0 50525.0 51790.5 51789.6 NO: 199
GDS-ppan-MC-MMAF- 51810.0 LSWLLRLLN-E269 SEQ ID anti-hHER2-HC-E269-
0 N/A 50525.0 51790.5 N/A NO: 200 GDS-ppan-MC-MMAF- LSWLLRLLN-D270
SEQ ID anti-hHER2-HC-D270- 18 2.0 50525.0 51790.5 51789.8 NO: 201
GDS-ppan-MC-MMAF- 51771.0 LSWLLRLLN-P271 51811.2 SEQ ID
anti-hHER2-HC-P271- 8 2.0 50525.0 51790.5 51796.4 NO: 202
GDS-ppan-MC-MMAF- LSWLLRLLN-E272 SEQ ID anti-hHER2-HC-P291- 23 1.8
50525.0 51790.5 51789.8 NO: 206 GDS-ppan-MC-MMAF- 51811.3
LSWLLRLLN-R292 SEQ ID anti-hHER2-HC-T307- 4 n.d. 50525.0 51790.5
51793.6 NO: 207 GDS-ppan-MC-MMAF- 50526.4 LSWLLRLLN-V308 SEQ ID
anti-hHER2-HC-L309- 10 n.d. 50525.0 51790.5 51795.6 NO: 209
GDS-ppan-MC-MMAF- 50530.8 LSWLLRLLN-H310 SEQ ID anti-hHER2-HC-N315-
13 0.9 50525.0 51790.5 51788.9 NO: 211 GDS-ppan-MC-MMAF- 50523.3
LSWLLRLLN-G316 51810.4 SEQ ID anti-hHER2-HC-G316- 7 0.8 50525.0
51790.5 50524.1 NO: 212 GDS-ppan-MC-MMAF- 51789.7 LSWLLRLLN-K317
50545.9 SEQ ID anti-hHER2-HC-A327- 14 0.5 50525.0 51790.5 51789.9
NO: 215 GDS-ppan-MC-MMAF- 50522.7 LSWLLRLLN-L328 SEQ ID
anti-hHER2-HC-L328- 16 1.0 50525.0 51790.5 51789.8 NO: 216
GDS-ppan-MC-MMAF- 50523.2 LSWLLRLLN-P329 51810.9 SEQ ID
anti-hHER2-HC-P329- 18 1.5 50525.0 51790.5 51790.1 NO: 217
GDS-ppan-MC-MMAF- 51811.9 LSWLLRLLN-A330 SEQ ID anti-hHER2-HC-A330-
9 1.7 50525.0 51790.5 51792.4 NO: 218 GDS-ppan-MC-MMAF- 50527.6
LSWLLRLLN-P331 SEQ ID anti-hHER2-HC-K340- 6 1.8 50525.0 51790.5
51792.4 NO: 220 GDS-ppan-MC-MMAF- 51604.8 LSWLLRLLN-G341 SEQ ID
anti-hHER2-HC-G341- 26 1.9 50525.0 51790.5 51790.0 NO: 221
GDS-ppan-MC-MMAF- LSWLLRLLN-Q342 SEQ ID anti-hHER2-HC-Q342- 0 N/A
50525.0 51790.5 N/A NO: 222 GDS-ppan-MC-MMAF- LSWLLRLLN-P343 SEQ ID
anti-hHER2-HC-P343- 14 2.0 50525.0 51790.5 51792.2 NO: 223
GDS-ppan-MC-MMAF- 51809.3 LSWLLRLLN-R344 SEQ ID anti-hHER2-HC-R344-
16 2.0 50525.0 51790.5 51794.4 NO: 224 GDS-ppan-MC-MMAF-
LSWLLRLLN-E345 SEQ ID anti-hHER2-HC-K360- 26 2.0 50525.0 51790.5
51796.8 NO: 229 GDS-ppan-MC-MMAF- LSWLLRLLN-N361 SEQ ID
anti-hHER2-HC-N384- 2 2.0 50525.0 51790.5 51792.8 NO: 230
GDS-ppan-MC-MMAF- LSWLLRLLN-G385 SEQ ID anti-hHER2-HC-E388- 23 2.0
50525.0 51790.5 51794.4 NO: 127 GDS-ppan-MC-MMAF- LSWLLRLLN-N389
SEQ ID anti-hHER2-HC-T394- 3 0.7 50525.0 51790.5 51793.2 NO: 232
GDS-ppan-MC-MMAF- 50525.2 LSWLLRLLN-P395 SEQ ID anti-hHER2-HC-P395-
4 n.d. 50525.0 51790.5 51794.6 NO: 233 GDS-ppan-MC-MMAF- 51773.9
LSWLLRLLN-P396 51820.4 SEQ ID anti-hHER2-HC-D401- 10 0.2 50525.0
51790.5 51793.7 NO: 235 GDS-ppan-MC-MMAF- 51818.2 LSWLLRLLN-G402
SEQ ID anti-hHER2-HC-S415- 5 1.1 50525.0 51790.5 51792.8 NO: 236
GDS-ppan-MC-MMAF- 50526.8 LSWLLRLLN-R416 SEQ ID anti-hHER2-HC-R416-
5 1.7 50525.0 51790.5 51794.1 NO: 237 GDS-ppan-MC-MMAF-
LSWLLRLLN-W417 SEQ ID anti-hHER2-HC-W417- 15 1.4 50525.0 51790.5
51798.8 NO: 238 GDS-ppan-MC-MMAF- 51921.6.sup.g LSWLLRLLN-Q418 SEQ
ID anti-hHER2-HC-Q418- 9 2.0 50525.0 51790.5 51794.4 NO: 239
GDS-ppan-MC-MMAF- LSWLLRLLN-Q419 SEQ ID anti-hHER2-HC-H433- 5 2.0
50525.0 51790.5 51793.6 NO: 243 GDS-ppan-MC-MMAF- 51922.4.sup.g
LSWLLRLLN-N434 51735.6 SEQ ID anti-hHER2-HC-N434- 20 2.0 50525.0
51790.5 51797.6 NO: 244 GDS-ppan-MC-MMAF- 51923.6.sup.g
LSWLLRLLN-H435 SEQ ID anti-hHER2-HC-L443- 24 0.0 50525.0 51790.5
50527.2 NO: 246 GDS-ppan-MC-MMAF- 50547.1 LSWLLRLLN-S444 SEQ ID
anti-hHER2-HC-P445- 10 2.0 50525.0 51790.5 51786.8 NO: 248
GDS-ppan-MC-MMAF- 51915.6.sup.g LSWLLRLLN-G446 51729.6 SEQ ID
anti-hHER2-HC-A118- 18 1.5 50331.8 51597.3 51598.4 NO: 249
DS-ppan-MC-MMAF- 51618.3 LEFIASKLA-S119 SEQ ID anti-hHER2-HC-S119-
15 1.6 50331.8 51597.3 51602.4 NO: 250 DS-ppan-MC-MMAF-
LEFIASKLA-T120 SEQ ID anti-hHER2-HC-T120- 27 2.0 50331.8 51597.3
51600.8 NO: 251 DS-ppan-MC-MMAF- LEFIASKLA-K121 SEQ ID
anti-hHER2-HC-S136- 19 2.0 50331.8 51597.3 51603.2 NO: 257
DS-ppan-MC-MMAF- LEFIASKLA-137 SEQ ID anti-hHER2-HC-G138- 21 2.0
50331.8 51597.3 51601.6 NO: 259 DS-ppan-MC-MMAF- LEFIASKLA-T139 SEQ
ID anti-hHER2-HC-P153- 15 0.1 50331.8 51597.3 50339.6 NO: 261
DS-ppan-MC-MMAF- LEFIASKLA-V154 SEQ ID anti-hHER2-HC-N159- 13 n.d.
50331.8 51597.3 50334.4 NO: 262 DS-ppan-MC-MMAF- 51600.4
LEFIASKLA-S160 SEQ ID anti-hHER2-HC-A162- 16 1.7 50331.8 51597.3
51598.3 NO: 265 DS-ppan-MC-MMAF- 51618.5 LEFIASKLA-L163 SEQ ID
anti-hHER2-HC-T164- 18 1.2 50331.8 51597.3 51597.7 NO: 267
DS-ppan-MC-MMAF- 51616.8 LEFIASKLA-S165 SEQ ID anti-hHER2-HC-S165-
23 1.9 50331.8 51597.3 51595.2 NO: 268 DS-ppan-MC-MMAF-
LEFIASKLA-G166 SEQ ID anti-hHER2-HC-P171- 15 1.0 50331.8 51597.3
50332.9 NO: 269 DS-ppan-MC-MMAF- 50353.8 LEFIASKLA-A172 SEQ ID
anti-hHER2-HC-S176- 13 0.1 50331.8 51597.3 50333.0 NO: 270
DS-ppan-MC-MMAF- 50354.0 LEFIASKLA-S177 SEQ ID anti-hHER2-HC-S190-
23 0.2 50331.8 51597.3 50333.6 NO: 273 DS-ppan-MC-MMAF- 51600.8
LEFIASKLA-S191 SEQ ID anti-hHER2-HC-S191- 24 1.6 50331.8 51597.3
51598.9 NO: 274 DS-ppan-MC-MMAF- 51620.3 LEFIASKLA-S192 SEQ ID
anti-hHER2-HC-S192- 21 2.0 50331.8 51597.3 51598.4 NO: 275
DS-ppan-MC-MMAF- 51618.8 LEFIASKLA-L193 SEQ ID anti-hHER2-HC-G194-
14 1.6 50331.8 51597.3 51599.2
NO: 277 DS-ppan-MC-MMAF- LEFIASKLA-T195 SEQ ID anti-hHER2-HC-T195-
14 1.9 50331.8 51597.3 51599.0 NO: 278 DS-ppan-MC-MMAF- 51617.2
LEFIASKLA-Q196 SEQ ID anti-hHER2-HC-Q196- 21 2.0 50331.8 51597.3
51598.1 NO: 279 DS-ppan-MC-MMAF- 51618.7 LEFIASKLA-T197 SEQ ID
anti-hHER2-HC-K205- 24 0.0 50331.8 51597.3 50327.6 NO: 280
DS-ppan-MC-MMAF- LEFIASKLA-P206 SEQ ID anti-hHER2-HC-P206- 23 0.0
50331.8 51597.3 50333.3 NO: 281 DS-ppan-MC-MMAF- 50354.7
LEFIASKLA-S207 SEQ ID anti-hHER2-HC-E233- 28 0.6 50331.8 51597.3
50330.8 NO: 286 DS-ppan-MC-MMAF- 51596.6 LEFIASKLA-L234 51615.6 SEQ
ID anti-hHER2-HC-L235- 24 2.0 50331.8 51597.3 51596.7 NO: 288
DS-ppan-MC-MMAF- 51617.5 LEFIASKLA-G236 SEQ ID anti-hHER2-HC-G236-
22 1.3 50331.8 51597.3 51598.8 NO: 289 DS-ppan-MC-MMAF- 51620.7
LEFIASKLA-G237 SEQ ID anti-hHER2-HC-P244- 8 1.4 50331.8 51597.3
51596.8 NO: 290 DS-ppan-MC-MMAF- 51614.6 LEFIASKLA-P245 SEQ ID
anti-hHER2-HC-P245- 22 1.0 50331.8 51597.3 50330.6 NO: 291
DS-ppan-MC-MMAF- 51595.9 LEFIASKLA-K246 50351.5 SEQ ID
anti-hHER2-HC-I253-DS- 0 N/A 50331.8 51597.3 N/A NO: 292
ppan-MC-MMAF- LEFIASKLA-S254 SEQ ID anti-hHER2-HC-S254- 24 1.9
50331.8 51597.3 51596.6 NO: 293 DS-ppan-MC-MMAF- 51616.8
LEFIASKLA-R255 SEQ ID anti-hHER2-HC-R255- 21 2.0 50331.8 51597.3
51596.3 NO: 294 DS-ppan-MC-MMAF- 51616.5 LEFIASKLA-T256 SEQ ID
anti-hHER2-HC-P257- 22 1.9 50331.8 51597.3 51596.3 NO: 296
DS-ppan-MC-MMAF- 51616.1 LEFIASKLA-E258 SEQ ID anti-hHER2-HC-S267-
23 0.2 50331.8 51597.3 51596.0 NO: 297 DS-ppan-MC-MMAF- 50330.9
LEFIASKLA-H268 51615.6 SEQ ID anti-hHER2-HC-H268- 22 0.7 50331.8
51597.3 51596.2 NO: 298 DS-ppan-MC-MMAF- 50331.0 LEFIASKLA-E269
51616.8 SEQ ID anti-hHER2-HC-E269- 17 1.8 50331.8 51597.3 51598.7
NO: 299 DS-ppan-MC-MMAF- 51620.0 LEFIASKLA-D270 SEQ ID
anti-hHER2-HC-D270- 26 1.3 50331.8 51597.3 51596.4 NO: 300
DS-ppan-MC-MMAF- 51616.5 LEFIASKLA-P271 SEQ ID anti-hHER2-HC-P271-
22 1.7 50331.8 51597.3 51595.9 NO: 301 DS-ppan-MC-MMAF- 51615.4
LEFIASKLA-E272 SEQ ID anti-hHER2-HC-D280- 4 0.7 50331.8 51597.3
50330.8 NO: 302 DS-ppan-MC-MMAF- 51596.3 LEFIASKLA-G281 50351.7 SEQ
ID anti-hHER2-HC-H285- 25 0.0 50331.8 51597.3 50331.0 NO: 303
DS-ppan-MC-MMAF- 50352.7 LEFIASKLA-N286 SEQ ID anti-hHER2-HC-N286-
20 0.0 50331.8 51597.3 50332.0 NO: 304 DS-ppan-MC-MMAF- 50354.1
LEFIASKLA-A287 SEQ ID anti-hHER2-HC-P291- 21 0.5 50331.8 51597.3
50333.5 NO: 305 DS-ppan-MC-MMAF- 51598.8 LEFIASKLA-R292 51620.0 SEQ
ID anti-hHER2-HC-N315- 15 n.d. 50331.8 51597.3 50331.5 NO: 310
DS-ppan-MC-MMAF- 51596.8 LEFIASKLA-G316 50353.1 SEQ ID
anti-hHER2-HC-G316- 9 1.1 50331.8 51597.3 51596.6 NO: 311
DS-ppan-MC-MMAF- 50331.0 LEFIASKLA-K317 51614.0 SEQ ID
anti-hHER2-HC-K317- 10 0.8 50331.8 51597.3 50330.9 NO: 312
DS-ppan-MC-MMAF- 51596.3 LEFIASKLA-E318 50352.1 SEQ ID
anti-hHER2-HC-K326- 15 0.0 50331.8 51597.3 50330.8 NO: 313
DS-ppan-MC-MMAF- 51597.2 LEFIASKLA-A327 SEQ ID anti-hHER2-HC-A327-
25 0.1 50331.8 51597.3 50333.6 NO: 314 DS-ppan-MC-MMAF- 50355.1
LEFIASKLA-L328 SEQ ID anti-hHER2-HC-L328- 13 1.9 50331.8 51597.3
51598.8 NO: 315 DS-ppan-MC-MMAF- LEFIASKLA-P329 SEQ ID
anti-hHER2-HC-P329- 7 0.9 50331.8 51597.3 51601.6 NO: 316
DS-ppan-MC-MMAF- 50334.8 LEFIASKLA-A330 SEQ ID anti-hHER2-HC-A330-
25 1.8 50331.8 51597.3 51602.4 NO: 317 DS-ppan-MC-MMAF-
LEFIASKLA-P331 SEQ ID anti-hHER2-HC-A339- 25 0.0 50331.8 51597.3
50333.6 NO: 318 DS-ppan-MC-MMAF- LEFIASKLA-K340 SEQ ID
anti-hHER2-HC-K340- 27 0.4 50331.8 51597.3 51600.4 NO: 319
DS-ppan-MC-MMAF- 50333.2 LEFIASKLA-G341 SEQ ID anti-hHER2-HC-G341-
25 0.2 50331.8 51597.3 51599.9 NO: 320 DS-ppan-MC-MMAF- 50334.7
LEFIASKLA-Q342 SEQ ID anti-hHER2-HC-Q342- 28 0.8 50331.8 51597.3
51599.8 NO: 321 DS-ppan-MC-MMAF- 50334.5 LEFIASKLA-P343 SEQ ID
anti-hHER2-HC-P343- 24 1.9 50331.8 51597.3 51599.1 NO: 322
DS-ppan-MC-MMAF- 51615.8 LEFIASKLA-R344 SEQ ID anti-hHER2-HC-R344-
29 1.9 50331.8 51597.3 51600.1 NO: 323 DS-ppan-MC-MMAF- 51616.6
LEFIASKLA-E345 SEQ ID anti-hHER2-HC-E356- 20 0.8 50331.8 51597.3
51600.8 NO: 325 DS-ppan-MC-MMAF- 50335.1 LEFIASKLA-E357 51616.8 SEQ
ID anti-hHER2-HC-M358- 26 0.2 50331.8 51597.3 50333.9 NO: 327
DS-ppan-MC-MMAF- 51599.4 LEFIASKLA-T359 SEQ ID anti-hHER2-HC-K360-
24 0.6 50331.8 51597.3 51599.9 NO: 328 DS-ppan-MC-MMAF- 51615.1
LEFIASKLA-N361 SEQ ID anti-hHER2-HC-N384- 24 0.0 50331.8 51597.3
50334.3 NO: 329 DS-ppan-MC-MMAF- 50354.2 LEFIASKLA-G385 SEQ ID
anti-hHER2-HC-E388- 21 1.9 50331.8 51597.3 51601.2 NO: 129
DS-ppan-MC-MMAF- LEFIASKLA-N389 SEQ ID anti-hHER2-HC-N389- 25 1.6
50331.8 51597.3 51600.1 NO: 330 DS-ppan-MC-MMAF- 51620.9
LEFIASKLA-N390 SEQ ID anti-hHER2-HC-P395- 25 0.0 50331.8 51597.3
50334.4 NO: 332 DS-ppan-MC-MMAF- 50352.8 LEFIASKLA-P396 SEQ ID
anti-hHER2-HC-D399- 11 0.0 50331.8 51597.3 50335.1 NO: 333
DS-ppan-MC-MMAF- 50353.6 LEFIASKLA-S400 SEQ ID anti-hHER2-HC-D401-
23 0.0 50331.8 51597.3 50334.9 NO: 335 DS-ppan-MC-MMAF- 50353.0
LEFIASKLA-G402 SEQ ID anti-hHER2-HC-S415- 21 0.2 50331.8 51597.3
50335.0 NO: 336 DS-ppan-MC-MMAF- 51600.5 LEFIASKLA-R416 SEQ ID
anti-hHER2-HC-R416- 15 1.9 50331.8 51597.3 51599.9 NO: 337
DS-ppan-MC-MMAF- 51615.8 LEFIASKLA-W417 SEQ ID anti-hHER2-HC-W417-
9 0.2 50331.8 51597.3 50334.8 NO: 338 DS-ppan-MC-MMAF- 51599.9
LEFIASKLA-Q418 50353.4 SEQ ID anti-hHER2-HC-Q418- 22 0.5 50331.8
51597.3 51600.5 NO: 339 DS-ppan-MC-MMAF- 50335.2 LEFIASKLA-Q419
51616.7 SEQ ID anti-hHER2-HC-Q419- 21 0.8 50331.8 51597.3 51600.0
NO: 340 DS-ppan-MC-MMAF- 51616.5 LEFIASKLA-G420 SEQ ID
anti-hHER2-HC-G420- 22 1.1 50331.8 51597.3 51599.5 NO: 341
DS-ppan-MC-MMAF- 51616.0 LEFIASKLA-N421 SEQ ID anti-hHER2-HC-N421-
24 1.4 50331.8 51597.3 51600.6 NO: 342 DS-ppan-MC-MMAF- 51614.9
LEFIASKLA-V422 SEQ ID anti-hHER2-HC-H433- 26 0.0 50331.8 51597.3
50334.7 NO: 343 DS-ppan-MC-MMAF- 50276.2 LEFIASKLA-N434 SEQ ID
anti-hHER2-HC-N434- 25 0.6 50331.8 51597.3 51592.4 NO: 344
DS-ppan-MC-MMAF- 50326.8 LEFIASKLA-H435 50268.8 SEQ ID
anti-hHER2-HC-L443- 26 0.0 50331.8 51597.3 50334.5 NO: 346
DS-ppan-MC-MMAF- 50275.8 LEFIASKLA-S444 50353.4 SEQ ID
anti-hHER2-HC-G446- 29 1.8 50331.8 51597.3 51595.2 NO: 349
DS-ppan-MC-MMAF- LEFIASKLA-K447 SEQ ID anti-hHER2-LC-T109- 34 1.4
24811.6 26077.1 26077.8 NO: 31 GDS-ppan-MC-MMAF- 26058.3
LSWLLRLLN-V110 26096.4 SEQ ID anti-hHER2-LC-V110- 5 1.9 24811.6
26077.1 26076.4 NO: 32 GDS-ppan-MC-MMAF- LSWLLRLLN-A111 SEQ ID
anti-hHER2-LC-A111- 13 2.0 24811.6 26077.1 26075.6 NO: 33
GDS-ppan-MC-MMAF- LSWLLRLLN-A112 SEQ ID anti-hHER2-LC-P119- 1 N/A
24811.6 26077.1 N/A NO: 34 GDS-ppan-MC-MMAF- LSWLLRLLN-P120 SEQ ID
anti-hHER2-LC-D122- 1 N/A 24811.6 26077.1 N/A NO: 37
GDS-ppan-MC-MMAF- LSWLLRLLN-E123 SEQ ID anti-hHER2-LC-Y140- 3 0.8
24811.6 26077.1 26077.2 NO: 38 GDS-ppan-MC-MMAF- LSWLLRLLN-P141 SEQ
ID anti-hHER2-LC-P141- 3 0.3 24811.6 26077.1 26076.8 NO: 39
GDS-ppan-MC-MMAF- LSWLLRLLN-R142 SEQ ID anti-hHER2-LC-R142- 5 0.3
24811.6 26077.1 26077.7 NO: 40 GDS-ppan-MC-MMAF- 24811.8
LSWLLRLLN-E143 26097.2 SEQ ID anti-hHER2-LC-E143- 6 0.4 24811.6
26077.1 26075.6 NO: 41 GDS-ppan-MC-MMAF- 26097.6 LSWLLRLLN-A144 SEQ
ID anti-hHER2-LC-D151- 16 0.3 24811.6 26077.1 24811.7 NO: 42
GDS-ppan-MC-MMAF- 26077.3 LSWLLRLLN-N152 24829.7 SEQ ID
anti-hHER2-LC-N152- 5 1.0 24811.6 26077.1 26077.2 NO: 43
GDS-ppan-MC-MMAF- LSWLLRLLN-A153 SEQ ID anti-hHER2-LC-A153- 13 1.9
24811.6 26077.1 26077.7 NO: 44 GDS-ppan-MC-MMAF- 26096.6
LSWLLRLLN-L154 SEQ ID anti-hHER2-LC-L154- 21 1.2 24811.6 26077.1
26078.2 NO: 45 GDS-ppan-MC-MMAF- 26096.9 LSWLLRLLN-Q155 SEQ ID
anti-hHER2-LC-Q155- 14 2.0 24811.6 26077.1 26075.2 NO: 46
GDS-ppan-MC-MMAF- LSWLLRLLN-S156 SEQ ID anti-hHER2-LC-E161- 19 1.9
24811.6 26077.1 26077.6 NO: 47 GDS-ppan-MC-MMAF- 26097.6
LSWLLRLLN-S162 SEQ ID anti-hHER2-LC-S162- 17 0.7 24811.6 26077.1
26077.2 NO: 48 GDS-ppan-MC-MMAF- LSWLLRLLN-V163 SEQ ID
anti-hHER2-LC-T164- 14 0.0 24811.6 26077.1 24810.0 NO: 50
GDS-ppan-MC-MMAF- LSWLLRLLN-E165 SEQ ID anti-hHER2-LC-E165- 0 N/A
24811.6 26077.1 N/A NO: 51 GDS-ppan-MC-MMAF- LSWLLRLLN-Q166 SEQ ID
anti-hHER2-LC-Q166- 17 0.0 24811.6 26077.1 24810.4 NO: 52
GDS-ppan-MC-MMAF- 24832.4 LSWLLRLLN-D167 SEQ ID anti-hHER2-LC-D167-
24 0.7 24811.6 26077.1 26077.4 NO: 53 GDS-ppan-MC-MMAF- 24812.3
LSWLLRLLN-S168 26096.5 SEQ ID anti-hHER2-LC-T197- 8 1.2 24811.6
26077.1 24812.0 NO: 54 GDS-ppan-MC-MMAF- 26077.9 LSWLLRLLN-H198
24831.4 SEQ ID anti-hHER2-LC-Q199- 5 1.9 24811.6 26077.1 26076.0
NO: 56 GDS-ppan-MC-MMAF- LSWLLRLLN-G200 SEQ ID anti-hHER2-LC-S202-
8 2.0 24811.6 26077.1 26077.4 NO: 59 GDS-ppan-MC-MMAF- 26095.9
LSWLLRLLN-S203 SEQ ID anti-hHER2-LC-V110- 15 2.0 24618.4 25883.9
25883.2 NO: 63 DS-ppan-MC-MMAF- LEFIASKLA-A111 SEQ ID
anti-hHER2-LC-A111- 17 1.6 24618.4 25883.9 25881.2 NO: 64
DS-ppan-MC-MMAF- LEFIASKLA-A112 SEQ ID anti-hHER2-LC-P119- 13 0.0
24618.4 25883.9 24618.1 NO: 65 DS-ppan-MC-MMAF- 24637.6
LEFIASKLA-P120 SEQ ID anti-hHER2-LC-P120- 9 0.0 24618.4 25883.9
24617.2 NO: 66 DS-ppan-MC-MMAF- LEFIASKLA-S121
SEQ ID anti-hHER2-LC-S121- 4 0.0 24618.4 25883.9 24616.8 NO: 67
DS-ppan-MC-MMAF- LEFIASKLA-D122 SEQ ID anti-hHER2-LC-D122- 2 0.0
24618.4 25883.9 24616.8 NO: 68 DS-ppan-MC-MMAF- LEFIASKLA-E123 SEQ
ID anti-hHER2-LC-Y140- 5 0.1 24618.4 25883.9 24616.4 NO: 69
DS-ppan-MC-MMAF- LEFIASKLA-P141 SEQ ID anti-hHER2-LC-R142- 13 0.1
24618.4 25883.9 24618.8 NO: 71 DS-ppan-MC-MMAF- 25884.0
LEFIASKLA-E143 24639.3 SEQ ID anti-hHER2-LC-E143- 10 0.0 24618.4
25883.9 24616.8 NO: 72 DS-ppan-MC-MMAF- LEFIASKLA-A144 SEQ ID
anti-hHER2-LC-D151- 17 0.0 24618.4 25883.9 24617.2 NO: 73
DS-ppan-MC-MMAF- LEFIASKLA-N152 SEQ ID anti-hHER2-LC-N152- 17 0.0
24618.4 25883.9 24616.8 NO: 74 DS-ppan-MC-MMAF- LEFIASKLA-A153 SEQ
ID anti-hHER2-LC-A153- 20 1.8 24618.4 25883.9 25882.8 NO: 75
DS-ppan-MC-MMAF- LEFIASKLA-L154 SEQ ID anti-hHER2-LC-L154-DS- 25
0.6 24618.4 25883.9 25884.6 NO: 76 ppan-MC-MMAF- 24618.9
LEFIASKLA-Q155 25904.2 SEQ ID anti-hHER2-LC-Q155- 27 1.1 24618.4
25883.9 25883.9 NO: 77 DS-ppan-MC-MMAF- 24619.0 LEFIASKLA-S156
25903.2 SEQ ID anti-hHER2-LC-S162- 7 0.0 24618.4 25883.9 24616.4
NO: 79 DS-ppan-MC-MMAF- LEFIASKLA-V163 SEQ ID
anti-hHER2-LC-T164-DS- 10 0.0 24618.4 25883.9 24616.4 NO: 81
ppan-MC-MMAF- LEFIASKLA-E165 SEQ ID anti-hHER2-LC-E165- 29 0.0
24618.4 25883.9 24618.9 NO: 82 DS-ppan-MC-MMAF- 24639.4
LEFIASKLA-Q166 SEQ ID anti-hHER2-LC-Q166- 20 0.0 24618.4 25883.9
24617.2 NO: 83 DS-ppan-MC-MMAF- LEFIASKLA-D167 SEQ ID
anti-hHER2-LC-D167- 28 0.0 24618.4 25883.9 24618.8 NO: 84
DS-ppan-MC-MMAF- 24639.0 LEFIASKLA-S168 SEQ ID
anti-hHER2-LC-T197-DS- 5 0.0 24618.4 25883.9 24615.2 NO: 85
ppan-MC-MMAF- LEFIASKLA-H198 SEQ ID anti-hHER2-LC-Q199- 7 0.0
24618.4 25883.9 24617.2 NO: 87 DS-ppan-MC-MMAF- LEFIASKLA-G200 SEQ
ID anti-hHER2-LC-G200- 18 0.2 24618.4 25883.9 24618.8 NO: 88
DS-ppan-MC-MMAF- 25884.4 LEFIASKLA-L201 24638.9 SEQ ID
anti-hHER2-LC-L201-DS- 15 0.8 24618.4 25883.9 25884.0 NO: 89
ppan-MC-MMAF- LEFIASKLA-S202 .sup.aName represents part of the HC
or LC that contains the peptide tag with the attached compound, the
paired wildtype chain is not listed. .sup.bYield of antibody per
liter culture (based on 35 mL cultures) measured after protein A
purification. .sup.cDrug-to-antibody ratio according to HPLC.
.sup.dMass in Dalton as predicted for the antibody. .sup.eMass in
Dalton as predicted for the ADC. .sup.fMass in Dalton as detected
on an Agilent 6520 Q-TOF instrument (Agilent Technologies). Most
prominent observation is listed first. .sup.gObserved mass
corresponds to non-clipped C-terminal lysine residue of heavy
chain. n.d., not determined. The drug-to-antibody ratio could not
be determined accurately be HPLC because of peak overlap. N/A, not
applicable. Conjugation was not attempted or data could not be
obtained because of low yield.
Example 25. Scale Up of Selected Peptide-Tagged ADCs for
Pharmacokinetic (PK) Studies and Further Characterization
[0922] The PEPP system does not provide enough quantities of
peptide-tagged ADCs for PK studies. Subsequently, expression of 39
constructs (Table 22) selected from among the 183 antibodies tested
in Example 24 (Table 21) was scaled up to 200-400 mL culture
volume. Selection criteria for scale-up were high conjugation
efficiency, reasonable expression yield, confirmed in vitro
potency, and low aggregation level as observed for the ADCs
prepared in Example 24.
[0923] After expression of the selected S6/ybbR-tagged antibodies
in Freestyle.TM. expression media (Invitrogen) for five days at
37.degree. C. under 5% CO.sub.2, the cultures were harvested by
centrifugation, and the resulting medium supernatants were passed
through 0.22 m filters (EMD Millipore). Antibody expression was
verified by SDS-PAGE analysis. Next, the filtrates were loaded at a
flowrate of 0.5-1 mL/min onto PBS-equilibrated columns containing
0.5 mL of Protein A resin by using a MINIPULS Evolution peristaltic
pump (Gilson Inc.). After washing the columns with 100-200 column
volumes of PBS, the antibody constructs were eluted with 0.1 M
sodium acetate (pH 3.0) in two 2.5 mL fractions. Both fractions
were immediately neutralized with 25-38% (v/v) of Tris-HCl buffer
(1 M, pH 8.0). In order to determine the yield of the Protein
A-purified antibodies (Table 22), protein concentrations of the
eluates were measured in duplicate on a ND-1000 UV-Vis
Spectrophotometer (NanoDrop Technologies) at 280 nm according to
the preset molar extinction coefficient for IgG molecules. Using
Slide-A-Lyzer Dialysis Cassettes (3.5-7.0 kDa cut-off, Pierce), the
second elution fraction of each construct was dialyzed into PBS for
subsequent thermostability measurements of non-conjugated
antibodies by DSF (Table 23). The first elution fraction of each
peptide-tagged antibody was dialyzed into conjugation buffer (75 mM
Tris-HCl buffer at pH 8.0 supplemented with 20 mM NaCl and 12.5 mM
MgCl.sub.2). After adjusting the antibody concentration to 2.5 M,
conjugation reactions were initiated by addition of CoA-MC-MMAF and
Sfp enzyme to final concentrations of 30-60 M and 1-4 M,
respectively. The enzymatic reaction was allowed to proceed for
about 20 hours at room temperature, before verifying the degree of
labeling by analytical reverse-phase HPLC using the respective
uncoupled antibody as control (Table 22). All conjugation reactions
were analyzed by mass spectrometry on an Agilent 6520 Q-TOF
instrument (Table 22). After confirming near quantitative
conjugation, reaction mixtures were concentrated to a final volume
of 1 mL using 30 kDa cut-off Amicon Ultra centrifugal filter
devices (EMD Millipore). Following removal of precipitate by
centrifugation, Sfp enzyme and excess CoA-MC-MMAF substrate were
removed by SEC (size-exclusion chromatography) on a HiLoad 26/60
Superdex 200 prep grade column (GE Healthcare) in PBS at a flowrate
of 1 mL/min. The purity of the peptide-tagged ADCs after SEC was
assessed by reverse-phase HPLC. After 0.22 .mu.m filtration, the
final yields of the ADCs were determined using triplicate
measurements on a ND-1000 UV-Vis Spectrophotometer (NanoDrop
Technologies) as above (Table 22).
TABLE-US-00022 TABLE 22 ADC production and characterization from
200-400 mL scale-up culture. Anti- Anti- body ADC Expt. Obs. body
yield.sup.b yield.sup.c Monomer.sup.e mass.sup.f mass.sup.g SEQ ID
ADC name.sup.a (mg/L) (mg/L) DAR.sup.d (%) (Da) (Da) SEQ ID
anti-hHER2-HC-S119- 57 31 2.0 97 51790.5 51786.4 NO: 151
GDS-ppan-MC-MMAF- LSWLLRLLN-T120 SEQ ID anti-hHER2-HC-T120- 40 23
2.0 100 51790.5 51796.4 NO: 152 GDS-ppan-MC-MMAF- LSWLLRLLN-K121
SEQ ID anti-hHER2-HC-T135- 41 20 2.0 100 51790.5 51785.2 NO: 157
GDS-ppan-MC-MMAF- LSWLLRLLN-S136 SEQ ID anti-hHER2-HC-S136- 40 20
2.0 100 51790.5 51785.6 NO: 158 GDS-ppan-MC-MMAF- LSWLLRLLN-G137
SEQ ID anti-hHER2-HC-A162- 25 16 2.0 100 51790.5 51791.6 NO: 166
GDS-ppan-MC-MMAF- LSWLLRLLN-L163 SEQ ID anti-hHER2-HC-T164- 32 15
2.0 100 51790.5 51787.6 NO: 168 GDS-ppan-MC-MMAF- LSWLLRLLN-S165
SEQ ID anti-hHER2-HC-S165- 39 21 2.0 100 51790.5 51786.4 NO: 169
GDS-ppan-MC-MMAF- LSWLLRLLN-G166 SEQ ID anti-hHER2-HC-P189- 36 25
2.0 100 51790.5 51792.0 NO: 173 GDS-ppan-MC-MMAF- LSWLLRLLN-S190
SEQ ID anti-hHER2-HC-G194- 35 21 2.0 100 51790.5 51794.8 NO: 178
GDS-ppan-MC-MMAF- LSWLLRLLN-T195 SEQ ID anti-hHER2-HC-T195- 39 21
1.9 100 51790.5 51790.4 NO: 179 GDS-ppan-MC-MMAF- LSWLLRLLN-Q196
SEQ ID anti-hHER2-HC-P271- 9 4 1.9 100 51790.5 51782.0 NO: 202
GDS-ppan-MC-MMAF- LSWLLRLLN-E272 SEQ ID anti-hHER2-HC-A330- 30 14
1.8 100 51790.5 51796.4 NO: 218 GDS-ppan-MC-MMAF- 50526.8.sup.h
LSWLLRLLN-P331 SEQ ID anti-hHER2-HC-K340- 20 9 2.0 100 51790.5
51794.4 NO: 220 GDS-ppan-MC-MMAF- 51918.4.sup.i LSWLLRLLN-G341 SEQ
ID anti-hHER2-HC-G341- 47 26 1.9 100 51790.5 51794.8 NO: 221
GDS-ppan-MC-MMAF- LSWLLRLLN-Q342 SEQ ID anti-hHER2-HC-R344- 37 21
2.0 100 51790.5 51795.6 NO: 224 GDS-ppan-MC-MMAF- LSWLLRLLN-E345
SEQ ID anti-hHER2-HC-K360- 46 21 1.9 100 51790.5 51785.2 NO: 229
GDS-ppan-MC-MMAF- LSWLLRLLN-N361 SEQ ID anti-hHER2-HC-E388- 40 25
2.0 100 51790.5 51792.4 NO: 127 GDS-ppan-MC-MMAF- LSWLLRLLN-N389
SEQ ID anti-hHER2-HC-Q418- 55 26 2.0 100 51790.5 51786.8 NO: 239
GDS-ppan-MC-MMAF- 51914.4.sup.i LSWLLRLLN-Q419 SEQ ID
anti-hHER2-HC-N434- 41 10 1.9 n.d. 51790.5 51785.2 NO: 244
GDS-ppan-MC-MMAF- 51912.8.sup.i LSWLLRLLN-H435 SEQ ID
anti-hHER2-HC-P445- 9 3 1.9 100 51790.5 51783.2 NO: 248
GDS-ppan-MC-MMAF- 51910.8.sup.i LSWLLRLLN-G446 SEQ ID
anti-hHER2-HC-S119- 35 25 1.9 100 51597.3 51591.2 NO: 250
DS-ppan-MC-MMAF- LEFIASKLA-T120 SEQ ID anti-hHER2-HC-T120- 42 24
1.9 100 51597.3 51592.4 NO: 251 DS-ppan-MC-MMAF- LEFIASKLA-K121 SEQ
ID anti-hHER2-HC-S136- 33 20 1.9 100 51597.3 51602.0 NO: 257
DS-ppan-MC-MMAF- LEFIASKLA-137 SEQ ID anti-hHER2-HC-G138- 26 14 1.9
100 51597.3 51592.0 NO: 259 DS-ppan-MC-MMAF- LEFIASKLA-T139 SEQ ID
anti-hHER2-HC-S165- 33 21 1.9 100 51597.3 51595.2 NO: 268
DS-ppan-MC-MMAF- LEFIASKLA-G166 SEQ ID anti-hHER2-HC-G194- 24 14
1.9 100 51597.3 51592.4 NO: 277 DS-ppan-MC-MMAF- LEFIASKLA-T195 SEQ
ID anti-hHER2-HC-L328- 35 22 1.9 100 51597.3 51600.4 NO: 315
DS-ppan-MC-MMAF- LEFIASKLA-P329 SEQ ID anti-hHER2-HC-A330- 20 12
1.8 100 51597.3 51589.2 NO: 317 DS-ppan-MC-MMAF- 50323.6.sup.h
LEFIASKLA-P331 SEQ ID anti-hHER2-HC-E388- 51 28 1.9 100 51597.3
51592.0 NO: 129 DS-ppan-MC-MMAF- LEFIASKLA-N389 SEQ ID
anti-hHER2-HC-G446- 37 23 1.9 100 51597.3 51590.4 NO: 349
DS-ppan-MC-MMAF- LEFIASKLA-K447 SEQ ID anti-hHER2-LC-V110- 8 3 2.0
93 26077.1 26074.8 NO: 32 GDS-ppan-MC-MMAF- LSWLLRLLN-A111 SEQ ID
anti-hHER2-LC-A111- 20 13 2.0 100 26077.1 26073.6 NO: 33
GDS-ppan-MC-MMAF- LSWLLRLLN-A112 SEQ ID anti-hHER2-LC-Q155- 29 19
1.9 100 26077.1 26070.8 NO: 46 GDS-ppan-MC-MMAF- LSWLLRLLN-S156 SEQ
ID anti-hHER2-LC-S162- 9 5 1.9 100 26077.1 26076.0 NO: 48
GDS-ppan-MC-MMAF- LSWLLRLLN-V163 SEQ ID anti-hHER2-LC-Q199- 10 3
1.9 100 26077.1 26074.4 NO: 56 GDS-ppan-MC-MMAF- LSWLLRLLN-G200 SEQ
ID anti-hHER2-LC-V110- 53 30 1.9 100 25883.9 25880.8 NO: 63
DS-ppan-MC-MMAF- LEFIASKLA-A111 SEQ ID anti-hHER2-LC-A111- 12 8 1.9
100 25883.9 25880.4 NO: 64 DS-ppan-MC-MMAF- 25901.2.sup.j
LEFIASKLA-A112 SEQ ID anti-hHER2-LC-A153- 14 7 1.9 100 25883.9
25878.0 NO: 75 DS-ppan-MC-MMAF- LEFIASKLA-L154 SEQ ID
anti-hHER2-LC-L201-DS- 26 15 1.8 100 25883.9 25881.2 NO: 89
ppan-MC-MMAF- LEFIASKLA-S202 .sup.aName represents part of the HC
or LC that contains the peptide tag with the attached compound, the
paired wildtype chain is not listed. .sup.bYield of antibody per
liter culture (based on 200-400 mL cultures) measured after protein
A purification. .sup.cYield of ADC per liter of culture measured
after size-exclusion chromatography. .sup.dDrug-to-antibody ratio
according to HPLC. .sup.eAnalytical size exclusion chromatography
results for ADC (percent of monomer). .sup.fMass in Dalton as
predicted for the ADC. .sup.gMass in Dalton as detected on an
Agilent 6520 Q-TOF instrument (Agilent Technologies). Most
prominent observation is listed first. .sup.hObserved mass
corresponds to non-conjugated antibody. .sup.iObserved mass
corresponds to non-clipped C-terminal lysine residue of heavy
chain. .sup.jObserved mass presumably corresponds to sodium adduct.
n.d., not determined.
[0924] Expression levels of the selected peptide-tagged antibodies
averaged 32 mg per liter of cell culture (ranging from 8 to 57
mg/L) (Table 22) and the final yield of purified ADC averaged 17 mg
per liter of cell culture (ranging from 3 to 31 mg/L) (Table 22).
All ADCs were site-specifically conjugated with two CoA-MC-MMAF
molecules (DAR=1.8 to 2) as verified by HPLC and MS (Table 22). No
aggregation or oligomeric species were detected for 36 of 39 ADCs
prepared (Table 22). All ADCs were more than 93% monomeric as
determined by analytical size exclusion chromatography. The thermal
stability of nonconjugated antibodies and ADCs was characterized by
DSF (Table 23). For wild-type trastuzumab, two DSF thermal melting
transitions (Tm1 and Tm2) were observed at 69.7 and 81.2.degree. C.
For 29 of 39 peptide-tagged antibodies, both transitions were
within less than 3.degree. C. of what was observed for wild-type
trastuzumab. Conjugation of CoA-MC-MMAF had no significant effect
on Tm2 but lowered Tm1 of the ADC by on average 1.degree. C.
relative to the nonconjugated antibody (Table 23). For 11
antibodies (and ADCs), the thermal stability was significantly
reduced relative to wild-type trastuzumab as illustrated by the
difference in Tm1. This transition is attributed to the unfolding
of the CH2 domain of an IgG and indeed most of the antibodies that
are destabilized (SEQ ID NO: 202, 218, 220, 221, 224, 315 and 317)
have the peptide-tag inserted at positions in the CH2 domain. As
stated above, the location of the peptide-tag can significantly
affect the properties of the resulting antibody and ADC.
TABLE-US-00023 TABLE 23 Thermal stability of modified antibodies
and ADCs as determined by differential scanning fluorometry. Anti-
Anti- Ab- Anti- body body ADC ADC WT body Tm1 Tm2 Tm1 Tm2
.DELTA.Tm1.sup.b .DELTA.Tm2.sup.b .DELTA.Tm1.sup.c SEQ ID: ADC
name.sup.a (.degree. C.) (.degree. C.) (.degree. C.) (.degree. C.)
(.degree. C.) (.degree. C.) (.degree. C.) SEQ ID
anti-hHER2-HC-S119- 69.8 t.b. 69.2 t.b. -0.6 t.b. 0.1 NO: 151
GDS-ppan-MC-MMAF- LSWLLRLLN-T120 SEQ ID anti-hHER2-HC-T120- 69.1
t.b. 68.8 t.b. -0.3 t.b. -0.6 NO: 152 GDS-ppan-MC-MMAF-
LSWLLRLLN-K121 SEQ ID anti-hHER2-HC-T135- 67.6 81.3 67.1 81.2 -0.5
-0.1 -2.1 NO: 157 GDS-ppan-MC-MMAF- LSWLLRLLN-S136 SEQ ID
anti-hHER2-HC-S136- 67.9 81.3 67.3 81.3 -0.6 0 -1.8 NO: 158
GDS-ppan-MC-MMAF- LSWLLRLLN-G137 SEQ ID anti-hHER2-HC-A162- 69.3
80.0 68.9 79.8 -0.3 -0.2 -0.4 NO: 166 GDS-ppan-MC-MMAF-
LSWLLRLLN-L163 SEQ ID anti-hHER2-HC-T164- 68.9 80.4 68.8 80.5 -0.2
0.1 -0.8 NO: 168 GDS-ppan-MC-MMAF- LSWLLRLLN-S165 SEQ ID
anti-hHER2-HC-S165- 69.2 80.4 68.8 80.2 -0.4 -0.2 -0.5 NO: 169
GDS-ppan-MC-MMAF- LSWLLRLLN-G166 SEQ ID anti-hHER2-HC-P189- 69.0
80.5 68.3 80.4 -0.7 -0.2 -0.7 NO: 173 GDS-ppan-MC-MMAF-
LSWLLRLLN-S190 SEQ ID anti-hHER2-HC-G194- 68.7 80.8 67.6 80.9 -1.1
0.1 -1.0 NO: 178 GDS-ppan-MC-MMAF- LSWLLRLLN-T195 SEQ ID
anti-hHER2-HC-T195- 69.3 81.1 68.8 80.9 -0.5 -0.1 -0.4 NO: 179
GDS-ppan-MC-MMAF- LSWLLRLLN-Q196 SEQ ID anti-hHER2-HC-P271- 53.4
81.6 51.2 81.2 -2.2 -0.4 -16.3 NO: 202 GDS-ppan-MC-MMAF-
LSWLLRLLN-E272 SEQ ID anti-hHER2-HC-A330- 52.5 81.5 49.0 81.1 -3.5
-0.3 -17.2 NO: 218 GDS-ppan-MC-MMAF- LSWLLRLLN-P331 SEQ ID
anti-hHER2-HC-K340- 65.2 77.3 58.7 81.0 -6.5 3.7 -4.5 NO: 220
GDS-ppan-MC-MMAF- LSWLLRLLN-G341 SEQ ID anti-hHER2-HC-G341- 65.0
76.9 56.0 81.0 -9 4.2 -4.7 NO: 221 GDS-ppan-MC-MMAF- LSWLLRLLN-Q342
SEQ ID anti-hHER2-HC-R344- 58.6 81.4 57.7 81.2 -0.9 -0.2 -11.1 NO:
224 GDS-ppan-MC-MMAF- LSWLLRLLN-E345 SEQ ID anti-hHER2-HC-K360-
70.1 81.7 68.8 81.4 -1.3 -0.3 0.4 NO: 229 GDS-ppan-MC-MMAF-
LSWLLRLLN-N361 SEQ ID anti-hHER2-HC-E388- 66.4 81.3 66.2 80.9 -0.2
-0.4 -3.3 NO: 127 GDS-ppan-MC-MMAF- LSWLLRLLN-N389 SEQ ID
anti-hHER2-HC-Q418- 69.0 81.0 68.3 81.1 -0.7 0.1 -0.7 NO: 239
GDS-ppan-MC-MMAF- LSWLLRLLN-Q419 SEQ ID anti-hHER2-HC-N434- 60.5
81.5 n.d. n.d. n.d. n.d. -9.2 NO: 244 GDS-ppan-MC-MMAF-
LSWLLRLLN-H435 SEQ ID anti-hHER2-HC-P445- 71.8 81.0 69.9 80.5 -1.8
-0.5 2.1 NO: 248 GDS-ppan-MC-MMAF- LSWLLRLLN-G446 SEQ ID
anti-hHER2-HC-S119- 70.1 t.b. 71.2 t.b. 1 t.b. 0.4 NO: 250
DS-ppan-MC-MMAF- LEFIASKLA-T120 SEQ ID anti-hHER2-HC-T120- 70.4
t.b. 70.4 t.b. 0 t.b. 0.7 NO: 251 DS-ppan-MC-MMAF- LEFIASKLA-K121
SEQ ID anti-hHER2-HC-S136- 69.3 80.8 68.3 81.0 -1.1 0.2 -0.4 NO:
257 DS-ppan-MC-MMAF- LEFIASKLA-137 SEQ ID anti-hHER2-HC-G138- 69.3
80.9 68.5 81.2 -0.7 0.2 -0.4 NO: 259 DS-ppan-MC-MMAF-
LEFIASKLA-T139 SEQ ID anti-hHER2-HC-S165- 69.6 80.3 69.2 80.5 -0.4
0.1 -0.1 NO: 268 DS-ppan-MC-MMAF- LEFIASKLA-G166 SEQ ID
anti-hHER2-HC-G194- 69.3 81.1 68.5 80.9 -0.9 -0.1 -0.4 NO: 277
DS-ppan-MC-MMAF- LEFIASKLA-T195 SEQ ID anti-hHER2-HC-L328- 56.9
78.8 50.4 81.0 -6.5 2.1 -12.8 NO: 315 DS-ppan-MC-MMAF-
LEFIASKLA-P329 SEQ ID anti-hHER2-HC-A330- 54.2 81.1 51.3 81.2 -2.9
0.1 -15.5 NO: 317 DS-ppan-MC-MMAF- LEFIASKLA-P331 SEQ ID
anti-hHER2-HC-E388- 69.3 81.5 68.8 81.0 -0.6 -0.5 -0.4 NO: 129
DS-ppan-MC-MMAF- LEFIASKLA-N389 SEQ ID anti-hHER2-HC-G446- 69.9
81.2 69.9 80.9 0 -0.4 0.2 NO: 349 DS-ppan-MC-MMAF- LEFIASKLA-K447
SEQ ID anti-hHER2-LC-V110- 66.9 t.b. 66.3 t.b. -0.6 t.b. -2.8 NO:
32 GDS-ppan-MC-MMAF- LSWLLRLLN-A111 SEQ ID anti-hHER2-LC-A111- 67.3
t.b. 66.0 t.b. -1.3 t.b. -2.4 NO: 33 GDS-ppan-MC-MMAF-
LSWLLRLLN-A112 SEQ ID anti-hHER2-LC-Q155- 69.4 80.0 68.7 79.4 -0.7
-0.6 -0.3 NO: 46 GDS-ppan-MC-MMAF- LSWLLRLLN-S156 SEQ ID
anti-hHER2-LC-S162- 68.5 t.b. 67.3 t.b. -1.2 t.b. -1.2 NO: 48
GDS-ppan-MC-MMAF- LSWLLRLLN-V163 SEQ ID anti-hHER2-LC-Q199- 67.5
t.b. 67.4 t.b. -0.1 t.b. -2.2 NO: 56 GDS-ppan-MC-MMAF-
LSWLLRLLN-G200 SEQ ID anti-hHER2-LC-V110- 69.0 t.b. 67.6 t.b. -1.4
t.b. -0.7 NO: 63 DS-ppan-MC-MMAF- LEFIASKLA-A111 SEQ ID
anti-hHER2-LC-A111- 69.6 t.b. 68.5 t.b. -1.1 t.b. -0.1 NO: 64
DS-ppan-MC-MMAF- LEFIASKLA-A112 SEQ ID anti-hHER2-LC-A153- 69.6
79.7 69.0 79.2 -0.5 -0.5 -0.1 NO: 75 DS-ppan-MC-MMAF-
LEFIASKLA-L154 SEQ ID anti-hHER2-LC-L201- 69.5 75.1 68.7 74.8 -0.8
-0.4 -0.2 NO: 89 DS-ppan-MC-MMAF- LEFIASKLA-S202 .sup.aName
represents part of the HC or LC that contains the peptide tag with
the attached compound, the paired wildtype chain is not listed.
.sup.bTm of ADC minus Tm of antibody. .sup.cTm1 of antibody minus
Tm1 of wild-type trastuzumab (69.7.degree. C.). n.d., Not
determined. Measurement was not performed due to insufficient
sample amounts. t.b., Transition too broad for accurate
determination of Tm2.
[0925] Purified ADCs were further characterized for in vitro
potency against selected cell lines (Table 24) including two
engineered cell lines, MDA-MB231 clone 16 and clone 40, and two
cell lines (JimT1 and HCC1954) that endogenously express the
targeted antigen, human HER2, on the cell surface. MDA-MB231 clone
16 cells stably express .about.500,000 copies of HER2 per cell
while clone 40 expresses only -5000 copies/cell. HCC1954 cells
endogenously express high level (.about.500,000 copies/cell) of
human HER2 on the surface (Clinchy B, Gazdar A, Rabinovsky R,
Yefenof E, Gordon B, Vitetta E S. Breast Cancer Res Treat. (2000)
61:217-228). The JimT1 cell line expresses approximately 80,000
copies of HER2 per cell (Mocanu M-M, Fazekas Z, Petras M, Nagy P,
Sebestyen Z, Isola J, TimBr J, Park J W, Vereb G, Szollosi J.
Cancer Letters (2005) 227: 201-212). The cell proliferation assays
were conducted with Cell-Titer-Glo.TM. (Promega) five days after
cells were incubated with various concentrations of ADCs (Riss et
al., (2004) Assay Drug Dev Technol. 2:51-62) with an automated
system (Melnick et al., (2006) Proc Natl Acad Sci USA.
103:3153-3158). Trastuzumab peptide-tagged-MMAF ADCs specifically
killed MDA-MB231 clone 16, HCC1954 and JimT1 cells (Table 24) but
not MDA-MB231 clone 40 cells. IC.sub.50 values of the trastuzumab
peptide-tagged-MMAF ADCs averaged around 0.24 .mu.M, 0.9 nM and 2.9
nM for MDA-MB231 clone 16, HCC1954 and JimT1 cells, respectively
(Table 24), consistent with the different HER2 expression
levels.
TABLE-US-00024 TABLE 24 In vitro potency of anti-HER2 ADCs.
IC.sub.50 cell killing concentrations are reported for several HER2
positive cell lines..sup.b MDA- Anti- MB-231 body HCC1954 JimT1
clone 16 SEQ ID ADC name.sup.a IC.sub.50 (.mu.M) IC.sub.50 (.mu.M)
IC.sub.50 (.mu.M) SEQ ID anti-hHER2-HC-S119- 1.94E-04 5.10E-04
6.82E-04 NO: 151 GDS-ppan-MC-MMAF- LSWLLRLLN-T120 SEQ ID
anti-hHER2-HC-T120- 1.69E-04 7.53E-04 7.02E-04 NO: 152
GDS-ppan-MC-MMAF- LSWLLRLLN-K121 SEQ ID anti-hHER2-HC-T135-
1.36E-04 2.57E-04 3.10E-04 NO: 157 GDS-ppan-MC-MMAF- LSWLLRLLN-S136
SEQ ID anti-hHER2-HC-S136- 1.64E-04 2.43E-04 3.05E-04 NO: 158
GDS-ppan-MC-MMAF- LSWLLRLLN-G137 SEQ ID anti-hHER2-HC-A162-
1.55E-04 8.66E-04 3.31E-04 NO: 166 GDS-ppan-MC-MMAF- LSWLLRLLN-L163
SEQ ID anti-hHER2-HC-T164- 1.89E-04 5.36E-04 4.69E-04 NO: 168
GDS-ppan-MC-MMAF- LSWLLRLLN-S165 SEQ ID anti-hHER2-HC-S165-
1.69E-04 6.19E-04 4.00E-04 NO: 169 GDS-ppan-MC-MMAF- LSWLLRLLN-G166
SEQ ID anti-hHER2-HC-P189- 1.47E-04 2.69E-04 2.86E-04 NO: 173
GDS-ppan-MC-MMAF- LSWLLRLLN-S190 SEQ ID anti-hHER2-HC-G194-
1.03E-04 1.33E-03 3.56E-04 NO: 178 GDS-ppan-MC-MMAF- LSWLLRLLN-T195
SEQ ID anti-hHER2-HC-T195- 1.42E-04 3.00E-04 2.79E-04 NO: 179
GDS-ppan-MC-MMAF- LSWLLRLLN-Q196 SEQ ID anti-hHER2-HC-P271-
1.33E-04 4.50E-04 6.75E-04 NO: 202 GDS-ppan-MC-MMAF- LSWLLRLLN-E272
SEQ ID anti-hHER2-HC-A330- 9.68E-05 3.18E-04 4.66E-04 NO: 218
GDS-ppan-MC-MMAF- LSWLLRLLN-P331 SEQ ID anti-hHER2-HC-K340-
3.76E-04 5.55E-04 3.08E-04 NO: 220 GDS-ppan-MC-MMAF- LSWLLRLLN-G341
SEQ ID anti-hHER2-HC-G341- 7.21E-05 3.58E-04 7.82E-04 NO: 221
GDS-ppan-MC-MMAF- LSWLLRLLN-Q342 SEQ ID anti-hHER2-HC-R344-
2.13E-03 4.47E-04 3.21E-04 NO: 224 GDS-ppan-MC-MMAF- LSWLLRLLN-E345
SEQ ID anti-hHER2-HC-K360- 1.80E-04 1.31E-03 7.57E-04 NO: 229
GDS-ppan-MC-MMAF- LSWLLRLLN-N361 SEQ ID anti-hHER2-HC-E388-
1.57E-04 4.21E-04 5.42E-04 NO: 127 GDS-ppan-MC-MMAF- LSWLLRLLN-N389
SEQ ID anti-hHER2-HC-Q418- 2.48E-04 1.24E-03 7.31E-04 NO: 239
GDS-ppan-MC-MMAF- LSWLLRLLN-Q419 SEQ ID anti-hHER2-HC-N434- n.d.
n.d. n.d. NO: 244 GDS-ppan-MC-MMAF- LSWLLRLLN-H435 SEQ ID
anti-hHER2-HC-P445- 7.42E-05 3.84E-03 7.44E-04 NO: 248
GDS-ppan-MC-MMAF- LSWLLRLLN-G446 SEQ ID anti-hHER2-HC-S119-
1.80E-04 3.46E-04 3.21E-04 NO: 250 DS-ppan-MC-MMAF- LEFIASKLA-T120
SEQ ID anti-hHER2-HC-T120- 1.98E-04 4.59E-04 3.94E-04 NO: 251
DS-ppan-MC-MMAF- LEFIASKLA-K121 SEQ ID anti-hHER2-HC-S136- 6.48E-05
3.95E-04 2.62E-04 NO: 257 DS-ppan-MC-MMAF- LEFIASKLA-137 SEQ ID
anti-hHER2-HC-G138- 1.58E-04 3.33E-02 3.21E-04 NO: 259
DS-ppan-MC-MMAF- LEFIASKLA-T139 SEQ ID anti-hHER2-HC-S165- 1.65E-04
4.07E-04 3.79E-04 NO: 268 DS-ppan-MC-MMAF- LEFIASKLA-G166 SEQ ID
anti-hHER2-HC-G194- 1.22E-04 6.48E-04 1.83E-04 NO: 277
DS-ppan-MC-MMAF- LEFIASKLA-T195 SEQ ID anti-hHER2-HC-L328- 1.37E-04
2.79E-04 1.15E-03 NO: 315 DS-ppan-MC-MMAF- LEFIASKLA-P329 SEQ ID
anti-hHER2-HC-A330- 4.09E-04 2.24E-02 2.85E-04 NO: 317
DS-ppan-MC-MMAF- LEFIASKLA-P331 SEQ ID anti-hHER2-HC-E388- 2.26E-04
1.83E-03 3.12E-04 NO: 129 DS-ppan-MC-MMAF- LEFIASKLA-N389 SEQ ID
anti-hHER2-HC-G446- 2.12E-04 6.82E-04 7.77E-04 NO: 349
DS-ppan-MC-MMAF- LEFIASKLA-K447 SEQ ID anti-hHER2-LC-V110- 2.31E-04
4.14E-04 5.18E-04 NO: 32 GDS-ppan-MC-MMAF- LSWLLRLLN-A111 SEQ ID
anti-hHER2-LC-A111- 1.95E-04 1.15E-02 5.05E-04 NO: 33
GDS-ppan-MC-MMAF- LSWLLRLLN-A112 SEQ ID anti-hHER2-LC-Q155-
1.43E-04 5.47E-04 3.70E-04 NO: 46 GDS-ppan-MC-MMAF- LSWLLRLLN-S156
SEQ ID anti-hHER2-LC-S162- 2.67E-04 8.13E-04 7.14E-04 NO: 48
GDS-ppan-MC-MMAF- LSWLLRLLN-V163 SEQ ID anti-hHER2-LC-Q199-
1.92E-04 9.21E-04 4.77E-04 NO: 56 GDS-ppan-MC-MMAF- LSWLLRLLN-G200
SEQ ID anti-hHER2-LC-V110- 3.97E-04 4.62E-04 2.77E-04 NO: 63
DS-ppan-MC-MMAF- LEFIASKLA-A111 SEQ ID anti-hHER2-LC-A111- 1.59E-04
6.32E-04 1.68E-02 NO: 64 DS-ppan-MC-MMAF- LEFIASKLA-A112 SEQ ID
anti-hHER2-LC-A153- 1.80E-04 2.03E-02 2.60E-04 NO: 75
DS-ppan-MC-MMAF- LEFIASKLA-L154 SEQ ID anti-hHER2-LC-L201- 4.25E-04
3.86E-04 4.74E-04 NO: 89 DS-ppan-MC-MMAF- LEFIASKLA-S202 .sup.aName
represents part of the HC or LC that contains the peptide tag with
the attached compound, the paired wildtype chain is not listed.
.sup.bNo cell killing was observed for HER2 negative cells at the
highest concentration measured (33 nM). n.d., not determined.
[0926] Good pharmacokinetic properties are essential for in vivo
efficacy of ADCs (Hamblett, et al., Clin Cancer Res., 10:7063-7070
(2004); Alley et al., Bioconjug. Chem. 19:759-765 (2008)). The
conjugation of a CoA-MC-MMAF molecule to an antibody can negatively
affect its biophysical properties resulting in rapid clearance and
dramatically reduced in vivo efficacy of the corresponding ADC
(Hamblett et al., 2004). To evaluate the effects of conjugation
site on in vivo clearance and ADC in vivo stability,
pharmacokinetic (PK) studies were performed in non-tumor bearing
mice with all 39 peptide-tagged trastuzumab ADCs (Table 25).
[0927] Each peptide-tagged MMAF ADC was injected intravenously into
three mice at a single dose of 1 mg/kg. Nine plasma samples were
then collected over a time course of 340 hours before plasma titers
of the ADCs were determined by ELISA. The ELISA assay uses the
immobilized extracellular domain of human HER2 for capturing
trastuzumab ADC molecules from plasma samples. Following the
capture step of this assay, an anti-MMAF antibody is used to
exclusively measure the plasma concentration of the "intact"
trastuzumab MMAF conjugate. In a second ELISA experiment, an
anti-hlgG antibody generates a signal indicating the plasma
concentration of both conjugated and unconjugated trastuzumab
molecules. If no payload deconjugation of the ADC occurs in vivo,
both anti-MMAF and anti-hlgG ELISAs are expected to provide
identical readouts on ADC plasma concentration. However, in the
case of payload loss in vivo, the anti-MMAF ELISA is expected to
produce a lower signal than the anti-hlgG ELISA. The comparison of
both ELISA signals therefore allows the quantification of payload
deconjugation during the in vivo exposure of the respective ADC.
The interpretation of the PK data is based on standard curves that
were generated with the same ADCs as used for intravenous injection
into mice.
[0928] The area-under-the-plasma-concentration-versus-time-curve
(AUC) is an important pharmacokinetic parameter that can be used to
determine the total clearance and bioavailability of the
administered biotherapeutic agent. For each peptide-tagged MMAF
ADC, two characteristic AUC values, AUC hlgG and AUC MMAF, were
obtained by the anti-hlgG and anti-MMAF ELISA experiments,
respectively. Table 25 summarizes the AUC hlgG and AUC MMAF values
as well as their respective ratios of the 39 tested peptide-tagged
ADCs. The obtained AUC hlgG values span over a wide range with the
highest value of 28334 nM*hr being about 20-fold higher than the
lowest value of 1362 nM*hr, with the average being 16275 nM*hr.
FIG. 19 A-C exemplifies PK curves of three peptide-tagged MMAF ADCs
displaying high AUC hlgG values (ADC of SEQ ID NO:248, 28334 nM*hr;
ADC of SEQ ID NO:33, 21011 nM*hr; ADC of SEQ ID NO:251, 21689
nM*hr). On the contrary, PK curves of three constructs showing low
AUC hlgG values (ADC of SEQ ID NO:218, 1362 nM*hr; ADC of SEQ ID
NO:202, 1757 nM*hr; ADC of SEQ ID NO:244, 2378 nM*hr) are
illustrated in FIG. 19 D-F. Despite the great variation of AUC hlgG
values, both anti-hlgG and anti-MMAF titers track each other,
suggesting that little if any payload deconjugation occurred in
vivo. Moreover, the ratios between AUC MMAF and AUC hlgG values of
all 39 tested peptide-tagged ADCs average at 1.0.+-.0.1
(AUC(MMAF)/AUC(hlgG), see Table 25) suggesting that the
maleimide-based linkage between the MC-MMAF and the terminal thiol
of the 4'-phosphopantetheine (ppan) moiety remained stable in
circulation over the time course of the PK experiment. Likewise,
these results also indicate a high in vivo stability of the
phosphodiester-based linkage between the ppan prosthetic group and
the serine residue of the inserted S6/ybbR peptide tag.
[0929] The rapid clearance observed for some of the peptide-tagged
ADCs may be attributed to the insertion of an S6 or ybbR peptide
sequence into specific regions of the IgG1 molecule. This putative
relationship between tag insertion site and pharmacokinetic profile
is exemplified by the two peptide-tagged MMAF ADCs of SEQ ID NO:218
and SEQ ID NO:202, which display the lowest AUC hlgG values of 1362
nM*hr and 1757 nM*hr, respectively. Both ADCs contain S6 tag
insertions in the CH2 domain of the heavy chain. In addition to the
instability in murine circulation, these ADCs also exhibit the
lowest and third lowest thermostabilities of the 39 tested samples
of the PK study. According to DSF measurements, the corresponding
ADCs display Tm1s of 49.0.degree. C. (ADC of SEQ ID NO:218) and
51.2.degree. C. (ADC of SEQ ID NO:202), resulting in a decrease of
20.7.degree. C. and 18.5.degree. C., respectively, in comparison to
wild-type trastuzumab having a Tm1 of 69.7.degree. C. In contrast,
the 16 ADCs with the highest AUC hlgG values (18406-28334 nM*hr)
display Tm1 values which are not more than 3.7.degree. C. below the
Tm1 of wild-type trastuzumab, suggesting a possible correlation
between pharmacokinetics and thermostability of ADCs. Moreover, ten
of these 16 ADCs contain S6 or ybbR tags in loop regions of the CH1
domain of the heavy chain. As mentioned above, peptide tag
insertions at these favorable sites also display the best overall
conjugation efficiencies, making them preferred candidates for ADC
production. These include antibodies with heavy chain insertions
between S119-T120, T120-K121, T135-S136, S136-G137, G138-T139,
S165-G166, and E388-N389 (CH3 domain) corresponding to SEQ ID
numbers 151, 250, 152, 251, 157, 256, 158, 257, 160, 259, 169, 268,
126, 127, 128, 129, 130, 131, 132, 149, and 356.
TABLE-US-00025 TABLE 25 Pharmacokinetics data. Anti- AUC.sup.b
AUC.sup.c body hIgG MMAF AUC(MMAF)/ SEQ ID ADC name.sup.a (nM*hr)
(nM*hr) AUC(hIgG) SEQ ID anti-hHER2-HC-S119- 22485 21693 1.0 NO:
151 GDS-ppan-MC-MMAF- LSWLLRLLN-T120 SEQ ID anti-hHER2-HC-T120-
13880 12542 0.9 NO: 152 GDS-ppan-MC-MMAF- LSWLLRLLN-K121 SEQ ID
anti-hHER2-HC-T135- 21494 16931 0.8 NO: 157 GDS-ppan-MC-MMAF-
LSWLLRLLN-S136 SEQ ID anti-hHER2-HC-S136- 22833 23533 1.0 NO: 158
GDS-ppan-MC-MMAF- LSWLLRLLN-G137 SEQ ID anti-hHER2-HC-A162- 11178
10981 1.0 NO: 166 GDS-ppan-MC-MMAF- LSWLLRLLN-L163 SEQ ID
anti-hHER2-HC-T164- 20916 22125 1.1 NO: 168 GDS-ppan-MC-MMAF-
LSWLLRLLN-S165 SEQ ID anti-hHER2-HC-S165- 23242 21304 0.9 NO: 169
GDS-ppan-MC-MMAF- LSWLLRLLN-G166 SEQ ID anti-hHER2-HC-P189- 8922
8840 1.0 NO: 173 GDS-ppan-MC-MMAF- LSWLLRLLN-S190 SEQ ID
anti-hHER2-HC-G194- 20702 18593 0.9 NO: 178 GDS-ppan-MC-MMAF-
LSWLLRLLN-T195 SEQ ID anti-hHER2-HC-T195- 16083 17465 1.1 NO: 179
GDS-ppan-MC-MMAF- LSWLLRLLN-Q196 SEQ ID anti-hHER2-HC-P271- 1757
1550 0.9 NO: 202 GDS-ppan-MC-MMAF- LSWLLRLLN-E272 SEQ ID
anti-hHER2-HC-A330- 1362 1768 1.3 NO: 218 GDS-ppan-MC-MMAF-
LSWLLRLLN-P331 SEQ ID anti-hHER2-HC-K340- 17396 16060 0.9 NO: 220
GDS-ppan-MC-MMAF- LSWLLRLLN-G341 SEQ ID anti-hHER2-HC-G341- 9214
10336 1.1 NO: 221 GDS-ppan-MC-MMAF- LSWLLRLLN-Q342 SEQ ID
anti-hHER2-HC-R344- 15196 16061 1.1 NO: 224 GDS-ppan-MC-MMAF-
LSWLLRLLN-E345 SEQ ID anti-hHER2-HC-K360- 7867 8209 1.0 NO: 229
GDS-ppan-MC-MMAF- LSWLLRLLN-N361 SEQ ID anti-hHER2-HC-E388- 14224
14887 1.0 NO: 127 GDS-ppan-MC-MMAF- LSWLLRLLN-N389 SEQ ID
anti-hHER2-HC-Q418- 8561 6136 0.7 NO: 239 GDS-ppan-MC-MMAF-
LSWLLRLLN-Q419 SEQ ID anti-hHER2-HC-N434- 2378 2249 0.9 NO: 244
GDS-ppan-MC-MMAF- LSWLLRLLN-H435 SEQ ID anti-hHER2-HC-P445- 28334
24130 0.9 NO: 248 GDS-ppan-MC-MMAF- LSWLLRLLN-G446 SEQ ID
anti-hHER2-HC-S119- 22854 24551 1.1 NO: 250 DS-ppan-MC-MMAF-
LEFIASKLA-T120 SEQ ID anti-hHER2-HC-T120- 21689 19734 0.9 NO: 251
DS-ppan-MC-MMAF- LEFIASKLA-K121 SEQ ID anti-hHER2-HC-S136- 27232
24064 0.9 NO: 257 DS-ppan-MC-MMAF- LEFIASKLA-137 SEQ ID
anti-hHER2-HC-G138- 17184 15404 0.9 NO: 259 DS-ppan-MC-MMAF-
LEFIASKLA-T139 SEQ ID anti-hHER2-HC-S165- 12794 13854 1.1 NO: 268
DS-ppan-MC-MMAF- LEFIASKLA-G166 SEQ ID anti-hHER2-HC-G194- 20659
21603 1.0 NO: 277 DS-ppan-MC-MMAF- LEFIASKLA-T195 SEQ ID
anti-hHER2-HC-L328- 7590 8039 1.1 NO: 315 DS-ppan-MC-MMAF-
LEFIASKLA-P329 SEQ ID anti-hHER2-HC-A330- 12960 14302 1.1 NO: 317
DS-ppan-MC-MMAF- LEFIASKLA-P331 SEQ ID anti-hHER2-HC-E388- 21023
21257 1.0 NO: 129 DS-ppan-MC-MMAF- LEFIASKLA-N389 SEQ ID
anti-hHER2-HC-G446- 20329 16452 0.8 NO: 349 DS-ppan-MC-MMAF-
LEFIASKLA-K447 SEQ ID anti-hHER2-LC-V110- 17358 18734 1.1 NO: 32
GDS-ppan-MC-MMAF- LSWLLRLLN-A111 SEQ ID anti-hHER2-LC-A111- 21011
20711 1.0 NO: 33 GDS-ppan-MC-MMAF- LSWLLRLLN-A112 SEQ ID
anti-hHER2-LC-Q155- 15444 17657 1.1 NO: 46 GDS-ppan-MC-MMAF-
LSWLLRLLN-S156 SEQ ID anti-hHER2-LC-S162- 11348 11645 1.0 NO: 48
GDS-ppan-MC-MMAF- LSWLLRLLN-V163 SEQ ID anti-hHER2-LC-Q199- 16832
17973 1.1 NO: 56 GDS-ppan-MC-MMAF- LSWLLRLLN-G200 SEQ ID
anti-hHER2-LC-V110- 20373 24757 1.2 NO: 63 DS-ppan-MC-MMAF-
LEFIASKLA-A111 SEQ ID anti-hHER2-LC-A111- 16092 16196 1.0 NO: 64
DS-ppan-MC-MMAF- LEFIASKLA-A112 SEQ ID anti-hHER2-LC-A153- 18406
19496 1.1 NO: 75 DS-ppan-MC-MMAF- LEFIASKLA-L154 SEQ ID
anti-hHER2-LC-L201- 17223 15036 0.9 NO: 89 DS-ppan-MC-MMAF-
LEFIASKLA-S202 .sup.aName represents part of the HC or LC that
contains the peptide tag with the attached compound, the paired
wildtype chain is not listed. .sup.bArea-under-the-curve measured
by anti-human IgG ELISA. .sup.cArea-under-the-curve measured by
anti-MMAF ELISA.
Example 26. Labeling of Peptide-Tagged IgGs with a Co-Expressed
4'-Phospho-Pantetheinyl Transferase in Culture Medium
[0930] In order to streamline the process of preparing ADCs,
enzymatic labeling of peptide-tagged antibodies with co-expressed
4'-phosphopantetheinyl transferase (PPTase) was carried out in
Freestyle.TM. expression media (Invitrogen). In addition to
reducing the number of purification steps, co-expression of the
PPTase during antibody production could circumvent problems
associated with the addition and the removal of a recombinantly
produced version of such an enzyme. As a proof-of-concept, AcpS
PPTase from E. coli was used to site-specifically conjugate an
A1-tagged antibody with acetyl coenzyme A (acetyl CoA) in culture
medium.
[0931] To facilitate co-expression, the gene encoding the AcpS
PPTase was cloned into the mammalian expression vector pRS, which
appends the N-terminal signal sequence MKTFILLLVVVLLLWVIFLLPGATA
(SEQ ID NO: 355). The construct, pRS-AcpS, also adds a C-terminal
His.sub.6 tag to the recombinant enzyme. To co-express the
A1-tagged antibody mAb2-HC-E388-GDSLDMLEWSLM-N389 (SEQ ID NO:356),
an oligonucleotide fragment encoding the 12-amino-acid A1 peptide
sequence was inserted into the heavy chain gene of the antibody
mAb2-HC in the mammalian expression vector pM4, resulting in the
construct pM4-A1. This plasmid also co-expresses the corresponding
light chain under the CMV promoter. Using the PEI method (Meissner
et al., 2001), 293 Freestyle.TM. cells were transiently transfected
with a 1:1 mixture of the recombinant expression plasmids pM4-A1
and pRS-AcpS, and cultured in 50 mL of Freestyle.TM. expression
media (Invitrogen) for five days at 37.degree. C. under 5%
CO.sub.2. Next, the cell culture was harvested by centrifugation at
3,000 rpm for 10 min and passed through a 0.22 m filter. To mimic
the higher concentrations that can be expected with production cell
lines, the filtrate was concentrated about 30-fold using a 30 kDa
cut-off Amicon Ultra centrifugal filter unit (EMD Millipore). After
removing precipitate by centrifugation at 20,800.times.g for 1 min,
900 .mu.L of the concentrate was supplemented with 100 .mu.L of
10-fold reaction buffer (pH 8.8) containing 750 mM of Tris-HCl and
100 mM of MgCl.sub.2. The labeling reaction was then initiated by
adding 20 .mu.L of 25 mM acetyl CoA (Sigma-Aldrich) to 480 .mu.L of
the concentrate, yielding a final concentration of 1 mM of acetyl
CoA substrate (Exp. #1). To test whether labeling of the A1-tagged
antibody can occur in the absence of exogenously added acetyl CoA
substrate, the remaining 500 .mu.L of the concentrate were left
untreated (Exp. #2). The two reaction mixtures were incubated for
approximately 16 h at 37.degree. C.
[0932] To determine the degree of labeling of the A1-tagged
antibody as well as to quantify expression levels of both enzyme
and antibody, the reaction mixtures were purified by Protein A and
Ni-NTA affinity chromatography. Each reaction was diluted two-fold
with PBS, and applied to Protein A-Sepharose columns (0.5 mL bed
volume, GE Healthcare) at an approximate flowrate of 1 mL/min. The
column flowthrough was directly applied to PBS-equilibrated IMAC
columns filled with 0.5 mL of Ni-NTA Agarose (Qiagen). Protein A
and Ni-NTA affinity columns were washed with 20 column volumes of
50 mM of Tris-HCl buffer (pH 8) supplemented with 300 mM of NaCl
and 20 mM of imidazole. His.sub.6-tagged AcpS enzyme was eluted
from the Ni-NTA affinity columns with 5 column volumes of 50 mM of
Tris-HCl buffer (pH 8) containing 300 mM of NaCl and 250 mM of
imidazole. Likewise, the A1-tagged antibody was eluted from the
Protein A affinity columns with 5 column volumes of 0.1 M sodium
acetate buffer (pH 3.0) followed by immediate neutralization with 1
M of Tris-HCl buffer (pH 10).
[0933] SDS-PAGE and ESI-MS confirmed elution of A1-tagged antibody
and AcpS enzyme, respectively. UV-Vis and Bradford measurements
indicated that approximately 0.3 to 0.4 mg of A1-tagged antibody
and 0.08 to 0.2 mg of AcpS enzyme were recovered (Table 26). This
suggests an antibody concentration of approximately 4-5 .mu.M
(0.6-0.8 mg/mL) during the labeling reactions in cell culture
medium.
TABLE-US-00026 TABLE 26 Expression yields of
mAb2-HC-E388-GDSLDMLEWSLM-N389 and AcpS PPTase as well as mass
spectrometric evaluation of enzymatic labeling in cell culture
medium. Yield of Yield of mAb2-HC-E388- AcpS GDSLDMLEWSLM- Observed
Expected PPTase N389 mass mass Exp. (mg) (mg) (Da) (Da).sup.a Exp.
0.08 0.3 51926.79 Uncoupled, 51589.2 #1 Coupled, 51971.6 Exp. 0.2
0.4 51586.41 Coupled and #2 deacetylated, 51929.6 .sup.aExpected
masses are shown for pyroglutamic acid formation of the N-terminal
glutamine residue of the heavy chain after signal peptide
cleavage.
[0934] The antibody sample of Exp. #1 was reduced and
deglycosylated followed by mass spectrometric analysis on an
Agilent 6520 Q-TOF instrument (Agilent Technologies). The
corresponding sample of Exp. #2 served as a control in order to
rule out potential "in-medium" labeling of the A1-tagged antibody
via an endogenous source of CoA or an analog thereof. As shown in
Table 26, quantitative conjugation of the A1-tagged antibody was
observed in the presence of 1 mM of acetyl CoA substrate (Exp. #1).
In contrast, no detectable formation of the antibody conjugate was
found when acetyl CoA was omitted (Exp. #2), thereby excluding the
presence of significant amounts of CoA or one of its analogs in the
cell-culture medium. Notably, the antibody conjugate of Exp. #1
completely lacks the acetyl group of the acetyl CoA substrate which
indicates hydrolysis of the thioester bond by nucleophilic
components in the conditioned cell-culture medium. The formation of
a free thiol group after deacetylation in cell-culture medium could
enable a two-step preparation of peptide-tagged ADCs. Following
Protein A purification, the antibody with the in situ generated
free thiol group could be reacted with a maleimide-toxin conjugate
to afford the corresponding ADC in the second step. In summary, the
experiment demonstrates that a peptide-tagged antibody can be
quantitatively labeled with a supplemented CoA analog in 30-fold
concentrated cell-culture medium via PPTase catalysis.
Example 27. In Vivo Efficacy Assessment of a ybbR-Tagged
Trastuzumab MMAF ADC
[0935] The in vivo efficacy of the ybbR-tagged trastuzumab ADC
anti-hHER2-HC-E388-DS-ppan-MC-MMAF-LEFIASKLA-N389 (SEQ ID NO:129)
was assessed by using a xenograft tumor model, which is based on
the implantation of a human tumor cell line into immune-deficient
nude mice. As described previously (Sausville and Burger, 2006),
studies with such tumor xenograft mice have provided valuable
insights into the in vivo efficacy of anti-cancer reagents.
Specifically, the in vivo efficacy study was carried out with nu/nu
mice that were subcutaneously injected with MDA-MB231 clone 16
cells (Morton and Houghton, 2007). This cell line was chosen based
on previous in vitro potency assays revealing its high sensitivity
to the aforementioned ybbR-tagged MMAF ADC in an antigen dependent
manner (see Table 24). After the tumor reached a size of about 200
mm.sup.3, the ybbR-tagged MMAF ADC was intravenously injected in a
single dose at either 5 mg/kg or 3 mg/kg, with each treatment group
comprising nine mice. After administering the antibody-drug
conjugate, the tumor growth was monitored weekly. As shown in FIG.
20, i.v. administration of the ybbR-tagged MMAF ADC caused tumor
regression at both dose levels. Furthermore, the treatment of the
mice with the ADC was well tolerated with no weight loss observed
in any of the treatment groups. The effective regression of
MDA-MB231 clone 16 tumors at single doses as low as 3 mg/kg
demonstrates that the ybbR-tagged ADC is efficacious in a
HER2-dependent tumor mouse model. All animal studies were conducted
in accordance with the Guide for the Care and Use of Laboratory
Animals (NIH publication; National Academy Press, 8.sup.th edition,
2001).
TABLE-US-00027 Informal Sequence Listing SEQ ID NO: 1 GDSLSWLLRLLN
SEQ ID NO: 2 GDSLSWL SEQ ID NO: 3 GDSLSWLVRCLN SEQ ID NO: 4
GDSLSWLLRCLN SEQ ID NO: 5 GDSLSWLVRLLN SEQ ID NO: 6 GDSLSWLLRSLN
SEQ ID NO: 7 GSQDVLDSLEFIASKLA SEQ ID NO: 8 VLDSLEFIASKLA SEQ ID
NO: 9 DSLEFIASKLA SEQ ID NO: 10 GDSLDMLEWSLM SEQ ID NO: 11
GDSLDMLEWSL SEQ ID NO: 12 GDSLDMLEWS SEQ ID NO: 13 GDSLDMLEW SEQ ID
NO: 14 DSLDMLEW SEQ ID NO: 15 GDSLDM SEQ ID NO: 16 LDSVRMMALAAR SEQ
ID NO: 17 LDSLDMLEWSLR SEQ ID NO: 18 DSLEFIASKL SEQ ID NO: 19
DSLEFIASK SEQ ID NO: 20 DVLDSLEFI SEQ ID NO: 21 VLDSLEFIAS SEQ ID
NO: 22, Homo sapiens PPTase (with N-terminal His6 tag after signal
peptide cleavage):
QPHHHHHHVFPAKRFCLVPSMEGVRWAFSCGTWLPSRAEWLLAVRSIQPEEKERIGQ
FVFARDAKAAMAGRLMIRKLVAEKLNIPWNHIRLQRTAKGKPVLAKDSSNPYPNFNFNI
SHQGDYAVLAAEPELQVGIDIMKTSFPGRGSIPEFFHIMKRKFTNKEWETIRSFKDEWT
QLDMFYRNWALKESFIKAIGVGLGFELQRLEFDLSPLNLDIGQVYKETRLFLDGEEEKE
WAFEESKIDEHHFVAVALRKPDGSRHQDVPSQDDSKPTQRQFTILNFNDLMSSAVPMT
PEDPSFWDCFCFTEEIPIRNGTKS SEQ ID NO: 23, Homo sapiens PPTase (with
C-terminal His6 tag after signal peptide cleavage):
QPVFPAKRFCLVPSMEGVRWAFSCGTWLPSRAEWLLAVRSIQPEEKERIGQFVFARD
AKAAMAGRLMIRKLVAEKLNIPWNHIRLQRTAKGKPVLAKDSSNPYPNFNFNISHQGDY
AVLAAEPELQVGIDIMKTSFPGRGSIPEFFHIMKRKFTNKEWETIRSFKDEWTQLDMFY
RNWALKESFIKAIGVGLGFELQRLEFDLSPLNLDIGQVYKETRLFLDGEEEKEWAFEES
KIDEHHFVAVALRKPDGSRHQDVPSQDDSKPTQRQFTILNFNDLMSSAVPMTPEDPSF
WDCFCFTEEIPIRNGTKSHHHHHH IgG sequences after signal peptide removal
(according to SignalP 3.0 Server, Technical University of Denmark).
Light chain constructs: SEQ ID NO: 24, Ig kappa light chain C
region: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNX5LQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKX6YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25,
mAb2-LC:
PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ
SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 26,
anti-hHER2-LC-I2-GDSLSWLLRLLN-Q3:
DIGDSLSWLLRLLNQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKL
LIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27,
anti-hHER2-LC-I2-DSLEFIASKLA-Q3:
DIDSLEFIASKLAQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI
YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 28,
anti-hHER2-LC-C214-GDSLSWLLRLLN:
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGDSLSWLLRL LN SEQ
ID NO: 29, anti-hHER2-LC-C214-DSLEFIASKLA:
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDSLEFIASKLA SEQ ID
NO: 30, anti-hHER2-LC-576D-S77-L78-EFIASKLA-Q79:
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVP
SRFSGSRSGTDFTLTIDSLEFIASKLAQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 31,
anti-hHER2-LC-T109-GDSLSWLLRLLN-V110
TGDSLSWLLRLLNVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 32, anti-hHER2-LC-V110-GDSLSWLLRLLN-A111
TVGDSLSWLLRLLNAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 33, anti-hHER2-LC-A111-GDSLSWLLRLLN-A112
TVAGDSLSWLLRLLNAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 34, anti-hHER2-LC-P119-GDSLSWLLRLLN-P120
TVAAPSVFIFPGDSLSWLLRLLNPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 35, anti-hHER2-LC-P120-GDSLSWLLRLLN-S121
TVAAPSVFIFPPGDSLSWLLRLLNSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 4 SEQ
ID NO: 36, anti-hHER2-LC-S121-GDSLSWLLRLLN-D122
TVAAPSVFIFPPSGDSLSWLLRLLNDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 37, anti-hHER2-LC-D122-GDSLSWLLRLLN-E123
TVAAPSVFIFPPSDGDSLSWLLRLLNEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 38, anti-hHER2-LC-Y140-GDSLSWLLRLLN-P141
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYGDSLSWLLRLLNPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 39, anti-hHER2-LC-P141-GDSLSWLLRLLN-R142
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPGDSLSWLLRLLNREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 40, anti-hHER2-LC-R142-GDSLSWLLRLLN-E143
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRGDSLSWLLRLLNEAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 41, anti-hHER2-LC-E143-GDSLSWLLRLLN-A144
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREGDSLSWLLRLLNAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 42, anti-hHER2-LC-D151-GDSLSWLLRLLN-N152
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDGDSLSWLLRLLNNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 43, anti-hHER2-LC-N152-GDSLSWLLRLLN-A153
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNGDSLSWLLRLLNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 44, anti-hHER2-LC-A153-GDSLSWLLRLLN-L154
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAGDSLSWLLRLLNL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 45, anti-hHER2-LC-L154-GDSLSWLLRLLN-Q155
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALGDSLSWLLRLLN
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
SEQ ID NO: 46, anti-hHER2-LC-Q155-GDSLSWLLRLLN-S156
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQGDSLSWLLRLL
NSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 47, anti-hHER2-LC-E161-GDSLSWLLRLLN-S162
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEGDSLS
WLLRLLNSVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 48, anti-hHER2-LC-S162-GDSLSWLLRLLN-V163
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESGDSL
SWLLRLLNVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 49, anti-hHER2-LC-V163-GDSLSWLLRLLN-T164
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVGDS
LSWLLRLLNTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C SEQ
ID NO: 50, anti-hHER2-LC-T164-GDSLSWLLRLLN-E165
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTGD
SLSWLLRLLNEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 51, anti-hHER2-LC-E165-GDSLSWLLRLLN-Q166
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEG
DSLSWLLRLLNQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 52, anti-hHER2-LC-Q166-GDSLSWLLRLLN-D167
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
GDSLSWLLRLLNDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 53, anti-hHER2-LC-D167-GDSLSWLLRLLN-S168
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DGDSLSWLLRLLNSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 54, anti-hHER2-LC-T197-GDSLSWLLRLLN-H198
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTGDSLSWLLRLLNHQGLSSPVTKSFNRG EC SEQ
ID NO: 55, anti-hHER2-LC-H198-GDSLSWLLRLLN-Q199
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHGDSLSWLLRLLNQGLSSPVTKSFNRG EC SEQ
ID NO: 56, anti-hHER2-LC-Q199-GDSLSWLLRLLN-G200 6
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGDSLSWLLRLLNGLSSPVTKSFNRG EC SEQ
ID NO: 57, anti-hHER2-LC-G200-GDSLSWLLRLLN-L201
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGGDSLSWLLRLLNLSSPVTKSFNRG EC SEQ
ID NO: 58, anti-hHER2-LC-L201-GDSLSWLLRLLN-S202
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLGDSLSWLLRLLNSSPVTKSFNRG EC SEQ
ID NO: 59, anti-hHER2-LC-S202-GDSLSWLLRLLN-S203
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSCDSLSWLLRLLNSPVTKSFNRG EC SEQ
ID NO: 60, anti-hHER2-LC-S203-GDSLSWLLRLLN-P204
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSGDSLSWLLRLLNPVTKSFNRG EC SEQ
ID NO: 61, anti-hHER2-LC-K207-GDSLSWLLRLLN-S208
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKGDSLSWLLRLLNSFNRG EC SEQ
ID NO: 62, anti-hHER2-LC-T109-DSLEFIASKLA-V110
TDSLEFIASKLAVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 63, anti-hHER2-LC-V110-DSLEFIASKLA-A111
TVDSLEFIASKLAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 64, anti-hHER2-LC-A111-DSLEFIASKLA-A112
TVADSLEFIASKLAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 65, anti-hHER2-LC-P119-DSLEFIASKLA-P120
TVAAPSVFIFPDSLEFIASKLAPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 66, anti-hHER2-LC-P120-DSLEFIASKLA-S121
TVAAPSVFIFPPDSLEFIASKLASDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 67, anti-hHER2-LC-S121-DSLEFIASKLA-D122
TVAAPSVFIFPPSDSLEFIASKLADEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 68, anti-hHER2-LC-D122-DSLEFIASKLA-E123
TVAAPSVFIFPPSDDSLEFIASKLAEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 69, anti-hHER2-LC-Y140-DSLEFIASKLA-P141
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYDSLEFIASKLAPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 70, anti-hHER2-LC-P141-DSLEFIASKLA-R142
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPDSLEFIASKLAREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 71, anti-hHER2-LC-R142-DSLEFIASKLA-E143
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRDSLEFIASKLAEAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 72, anti-hHER2-LC-E143-DSLEFIASKLA-A144
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREDSLEFIASKLAAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 73, anti-hHER2-LC-D151-DSLEFIASKLA-N152
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDDSLEFIASKLANALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 74, anti-hHER2-LC-N152-DSLEFIASKLA-A153
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNDSLEFIASKLAALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 75, anti-hHER2-LC-A153-DSLEFIASKLA-L154
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNADSLEFIASKLALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 76, anti-hHER2-LC-L154-DSLEFIASKLA-Q155
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALDSLEFIASKLAQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 77, anti-hHER2-LC-Q155-DSLEFIASKLA-S156
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQDSLEFIASKLAS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 78, anti-hHER2-LC-E161-DSLEFIASKLA-S162
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQEDSLEFI
ASKLASVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 79, anti-hHER2-LC-S162-DSLEFIASKLA-V163
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESDSLE
FIASKLAVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 80, anti-hHER2-LC-V163-DSLEFIASKLA-T164
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVDSL
EFIASKLATEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 81, anti-hHER2-LC-T164-DSLEFIASKLA-E165
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTDS
LEFIASKLAEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 82, anti-hHER2-LC-E165-DSLEFIASKLA-Q166
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTED
SLEFIASKLAQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 83, anti-hHER2-LC-Q166-DSLEFIASKLA-D167
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSLEFIASKLADSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 84, anti-hHER2-LC-D167-DSLEFIASKLA-S168
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DDSLEFIASKLASKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID
NO: 85, anti-hHER2-LC-T197-DSLEFIASKLA-H198
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTDSLEFIASKLAHQGLSSPVTKSFNRGEC SEQ ID
NO: 86, anti-hHER2-LC-H198-DSLEFIASKLA-Q199
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHDSLEFIASKLAQGLSSPVTKSFNRGEC SEQ ID
NO: 87, anti-hHER2-LC-Q199-DSLEFIASKLA-G200
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQDSLEFIASKLAGLSSPVTKSFNRGEC SEQ ID
NO: 88, anti-hHER2-LC-G200-DSLEFIASKLA-L201
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGDSLEFIASKLALSSPVTKSFNRGEC SEQ ID
NO: 89, anti-hHER2-LC-L201-DSLEFIASKLA-S202
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLDSLEFIASKLASSPVTKSFNRGEC SEQ ID
NO: 90, anti-hHER2-LC-S202-DSLEFIASKLA-S203
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSDSLEFIASKLASPVTKSFNRGEC SEQ ID
NO: 91, anti-hHER2-LC-S203-DSLEFIASKLA-P204
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSDSLEFIASKLAPVTKSFNRGEC SEQ ID
NO: 92, anti-hHER2-LC-K207-DSLEFIASKLA-S208
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKDSLEFIASKLASFNRGEC Heavy
chain constructs: SEQ ID NO: 93, Ig gamma-1 heavy chain C region:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKX1NEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRX2EX3TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEX4LHNHYTQKSLSLSPG SEQ ID NO: 94,
anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3:
EVGDSLSWLLRLLNQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE
WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG
FYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:
95, anti-hHER2-HC-V2-DSLEFIASKLA-Q3:
EVDSLEFIASKLAQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW
VARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY
AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:
96, anti-hHER2-HC-V2-GDSLDMLEWSLM-Q3:
EVGDSLDMLEWSLMQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL
EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD
GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO:
97, anti-hHER2-HC-563-LEFIASK-V64:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSLEFIASKVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY
WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 98,
anti-hHER2-HC-V64L-EFIAS-K65:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSLEFIASKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 99,
anti-hHER2-HC-V64L-EFIASKLA-K65:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT
RYADSLEFIASKLAKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMD
YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 100,
anti-hHER2-HC-S132G-K133D-S134-T135L-S136- G137W-G138L:
STKGPSVFPLAPSGDSLSWLTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 101,
anti-hHER2-HC-S132G-K133D-S134-T135L-S136-
G137W-G138LT139L-A140R-A141L-L142-G143N:
STKGPSVFPLAPSGDSLSWLLRLLNCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 102,
anti-hHER2-HC-S132D-K133S-S134L-T135E-
S136F-G137I-G138AT139S-A140K-A141L-L142A:
STKGPSVFPLAPSDSLEFIASKLAGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 103,
anti-hHER2-HC-K133G-S134D-T135S-S136L- G137S-G138WLLRLLN-T139:
STKGPSVFPLAPSSGDSLSWLLRLLNTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 104,
anti-hHER2-HC-S134G-T135D-G137L-G1385- WLLRLLN-A141:
STKGPSVFPLAPSSKGDSLSWLALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 105,
anti-hHER2-HC-S134G-T135D-S136-G137L- G1385-T139W-A140L:
STKGPSVFPLAPSSKGDSLSWLALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 106,
anti-hHER2-HC-Sl34G-T135D-S136-G137L- G1385-T139W-A140LLRLLN-A141:
STKGPSVFPLAPSSKGDSLSWLLRLLNALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 107,
anti-hHER2-HC-Sl34G-T135D-S136-G137L-
G1385-T139W-A140LA141L-L142R-G143L-C144L-L145N:
STKGPSVFPLAPSSKGDSLSWLLRLLNVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 108,
anti-hHER2-HC-T135G-S136D-G137S-G138L- T1395-WLLRLLNA140:
STKGPSVFPLAPSSKSGDSLSWLLRLLNAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 109,
anti-hHER2-HC-P189G-S190D-S191-S192L- L193S-G194W-T195L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVGDSLSWLQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 110,
anti-hHER2-HC-P189G-S190D-S191-S192L- L193S-G194W-T195LLRLLN-Q196:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVGDSLSWLLRLLNQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 111,
anti-hHER2-HC-P189G-S190D-S191-S192L-
L193S-G194W-T195LQ196L-T197R-Y198L-1199L-C200N:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVGDSLSWLLRLLNNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 112,
anti-hHER2-HC-P189D-S190-S191L-S192E-
L193F-G194I-T195AQ196S-T197K-Y198L-I199A:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVDSLEFIASKLACNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 113,
anti-hHER2-HC-S190G-S191D-S192-L193-
G194S-T195W-Q196LT197L-RLLN-Y198:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPGDSLSWLLRLLNYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 114,
anti-hHER2-HC-S190D-S191-S192L-L193E-
G194F-T195I-Q196AT197S-Y198K-1199L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPDSLEFIASKLCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 115,
anti-hHER2-HC-S190D-S191-S192L-L193E-
G194F-T195I-Q196AT197S-Y198K-I199L-C200A:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPDSLEFIASKLANVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 116,
anti-hHER2-HC-S191D-S192-L193-G194E- T195F-Q196I-T197AY198S-I199K:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSDSLEFIASKCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 117,
anti-hHER2-HC-L193G-G194D-T1955-Q196L- T1975-Y198W-I199L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSGDSLSWLCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 118,
anti-hHER2-HC-Ll93G-G 194D-T1955-
Q196L-T197S-Y198W-I199LLRLLN-C200:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSGDSLSWLLRLLNCNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 119,
anti-hHER2-HC-L193G-G194D-T1955-Q196L-
T1975-Y198W-I199LC200L-N201R-V202L-N203L-H204N:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSGDSLSWLLRLLNKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 120,
anti-hHER2-HC-E357G-M358D-T359S-K360L- N361S-Q362W-V363L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREGDSLSWLSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 121,
anti-hHER2-HC-T359-GDSLSWLLRLLN-K360:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTGDSLSWLLRLLNKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 122,
anti-hHER2-HC-T359-DSLEFIASKLA-K360:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTDSLEFIASKLAKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 123,
anti-hHER2-HC-T359G-K360D-N361S-Q362L- V3635-S364W:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMGDSLSWLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 124,
anti-hHER2-HC-E382D-S383-N384L-G3855- Q386W-P387L-E388L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWDSLSWLLNNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 125,
anti-hHER2-HC-E382D-S383-N384L-G385E- Q386F-P387I-E388A:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWDSLEFIANNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 126,
anti-hHER2-HC-E388-GDSLSWL-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDSLSWLNNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 127,
anti-hHER2-HC-E388-GDSLSWLLRLLN-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDSLSWLLRLLNNNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 128,
anti-hHER2-HC-E388-GDALSWLLRLLN-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDALSWLLRLLNNNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 129,
anti-hHER2-HC-E388-DSLEFIASKLA-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDSLEFIASKLANNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 130,
anti-hHER2-HC-E388-DSLEFIASKL-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDSLEFIASKLNNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 131,
anti-hHER2-HC-E388-DSLEFIASK-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDSLEFIASKNNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 132,
anti-hHER2-HC-E388-GDSLDMLEWSLM-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDSLDMLEWSLMNNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 133,
anti-hHER2-HC-L398G-D401L-G402S-S403W- F404L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVGDSLSWLFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 134,
anti-hHER2-HC-L398G-D399-S400-D401L- G402S-5403W-F404LLRLLN-F405:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVGDSLSWLLRLLNF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 135,
anti-hHER2-HC-L398D-D399S-S400L-D401E-
G402F-S403I-F404AF405S-L406K-Y407L-S408A:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVDSLEFIASKLAKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 136,
anti-hHER2-HC-S400-GDSLSWLLRLLN-D401:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSGDSLSWLLRL
LNDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 137,
anti-hHER2-HC-D413-K414S-S415L-R4165-
W417-Q418L-Q419LG420R-N421L-V422L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDSLSWLLRLLFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 138,
anti-hHER2-HC-D413-K414S-S415L-R416E-
W417F-Q4181-Q419AG420S-N421K-V422L:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDSLEFIASKLFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 139,
anti-hHER2-HC-G446-GDSLSWLLRLLN-K447:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDSLSWLLRLLN SEQ ID NO: 140,
anti-hHER2-HC-K447-GDSLSWLLRLLN:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGDSLSWLLRLLN SEQ ID NO: 141,
anti-hHER2-HC-K447-DSLEFIASKLA:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDSLEFIASKLA SEQ ID NO: 142,
anti-hHER2-HC-V2-GDSLSWLLRLLN-Q3-E388- DSLEFIASKLA-N389:
EVGDSLSWLLRLLNQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE
WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDG
FYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDS
LEFIASKLANNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPG SEQ ID NO: 143, anti-hHER2-HC-Y296-GDSLSWLLRLLN-N297:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYGDSLSWLLRLLNNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 144,
anti-hHER2-HC-Y296-DSLEFIASKLA-N297:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYDSLEFIASKLANSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 145,
anti-hHER2-HC-N297-CDSLSWLLRLLN-S298:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNGDSLSWLLRLLNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 146,
anti-hHER2-HC-N297-DSLEFIASKLA-S298:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNDSLEFIASKLASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 147,
mAb2-HC: STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 148,
mAb2-HC-T359-CDSLSWLLRLLN-K360:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTGDSLSWLLRLLNKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 149,
mAb2-HC-E388-GDSLSWLLRLLN-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDSLSWLLRLLNNNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 150,
anti-hHER2-HC-A118-GDSLSWLLRLLN-S119
GDSLSWLLRLLNSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 151,
anti-hHER2-HC-S119-GDSLSWLLRLLN-T120
SGDSLSWLLRLLNTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 152,
anti-hHER2-HC-T120-GDSLSWLLRLLN-K121
STGDSLSWLLRLLNKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 153,
anti-hHER2-HC-S131-GDSLSWLLRLLN-S132
STKGPSVFPLAPSGDSLSWLLRLLNSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 154,
anti-hHER2-HC-S132-GDSLSWLLRLLN-K133
STKGPSVFPLAPSSGDSLSWLLRLLNKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 155,
anti-hHER2-HC-K133-GDSLSWLLRLLN-S134
STKGPSVFPLAPSSKGDSLSWLLRLLNSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 156,
anti-hHER2-HC-S134-GDSLSWLLRLLN-T135
STKGPSVFPLAPSSKSGDSLSWLLRLLNTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 157,
anti-hHER2-HC-T135-GDSLSWLLRLLN-S136
STKGPSVFPLAPSSKSTGDSLSWLLRLLNSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 158,
anti-hHER2-HC-S136-GDSLSWLLRLLN-G137
STKGPSVFPLAPSSKSTSGDSLSWLLRLLNGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 159,
anti-hHER2-HC-G137-GDSLSWLLRLLN-G138
STKGPSVFPLAPSSKSTSGGDSLSWLLRLLNGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 160,
anti-hHER2-HC-G138-GDSLSWLLRLLN-T139
STKGPSVFPLAPSSKSTSGGGDSLSWLLRLLNTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 161,
anti-hHER2-HC-E152-GDSLSWLLRLLN-P153
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEGDSLSWLLRLLNPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 162,
anti-hHER2-HC-P153-GDSLSWLLRLLN-V154
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPGDSLSWLLRLLNVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 163,
anti-hHER2-HC-N159-GDSLSWLLRLLN-S160
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNGDSLSWLLRLLNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 164,
anti-hHER2-HC-S160-GDSLSWLLRLLN-G161
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGDSLSWLLRLLNGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 165,
anti-hHER2-HC-G161-GDSLSWLLRLLN-A162
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGGDSLSWLLRLLNALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 166,
anti-hHER2-HC-A162-GDSLSWLLRLLN-L163
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAGDSLSWLLRLLNLT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 167,
anti-hHER2-HC-L163-GDSLSWLLRLLN-T164
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALGDSLSWLLRLLNT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 168,
anti-hHER2-HC-T164-GDSLSWLLRLLN-S165
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTGDSLSWLLRLLN
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 169,
anti-hHER2-HC-S165-GDSLSWLLRLLN-G166
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGDSLSWLLRLL
NGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 170,
anti-hHER2-HC-P171-GDSLSWLLRLLN-A172
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPGDSLS
WLLRLLNAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 171,
anti-hHER2-HC-S176-GDSLSWLLRLLN-S177
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
GDSLSWLLRLLNSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 172,
anti-hHER2-HC-S177-GDSLSWLLRLLN-G178
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGDSLSWLLRLLNGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 173,
anti-hHER2-HC-P189-GDSLSWLLRLLN-S190
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPGDSLSWLLRLLNSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 174,
anti-hHER2-HC-S190-GDSLSWLLRLLN-S191
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSGDSLSWLLRLLNSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 175,
anti-hHER2-HC-S191-GDSLSWLLRLLN-S192
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSGDSLSWLLRLLNSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 176,
anti-hHER2-HC-S192-GDSLSWLLRLLN-L193
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSGDSLSWLLRLLNLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 177,
anti-hHER2-HC-L193-GDSLSWLLRLLN-G194
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGDSLSWLLRLLNGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 178,
anti-hHER2-HC-G194-GDSLSWLLRLLN-T195
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGGDSLSWLLRLLNTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 179,
anti-hHER2-HC-T195-GDSLSWLLRLLN-Q196
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTGDSLSWLLRLLNQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 180,
anti-hHER2-HC-Q196-GDSLSWLLRLLN-T197
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQGDSLSWLLRLLNTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 181,
anti-hHER2-HC-K205-GDSLSWLLRLLN-P206
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKGDSLSWLLRLLNPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 182,
anti-hHER2-HC-P206-GDSLSWLLRLLN-S207
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPGDSLSWLLRLLNSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 183,
anti-hHER2-HC-S207-GDSLSWLLRLLN-N208
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSGDSLSWLLRLLNNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 184,
anti-hHER2-HC-P230-GDSLSWLLRLLN-A231
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPG DSL
SWLLRLLNAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 185,
anti-hHER2-HC-A231-GDSLSWLLRLLN-P232
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAGDS
LSWLLRLLNPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 186,
anti-hHER2-HC-P232-GDSLSWLLRLLN-E233
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPGD
SLSWLLRLLNELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 187,
anti-hHER2-HC-E233-GDSLSWLLRLLN-L234
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEG
DSLSWLLRLLNLLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 188,
anti-hHER2-HC-L234-GDSLSWLLRLLN-L235
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELG
DSLSWLLRLLNLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 189,
anti-hHER2-HC-L235-GDSLSWLLRLLN-G236
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GDSLSWLLRLLNGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 190,
anti-hHER2-HC-G236-GDSLSWLLRLLN-G237
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGDSLSWLLRLLNGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 191,
anti-hHER2-HC-P244-GDSLSWLLRLLN-P245
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPGDSLSWLLRLLNPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 192,
anti-hHER2-HC-P245-GDSLSWLLRLLN-K246
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPGDSLSWLLRLLNKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 193,
anti-hHER2-HC-I253-GDSLSWLLRLLN-S254
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMIGDSLSWLLRLLNSRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 194,
anti-hHER2-HC-S254-GDSLSWLLRLLN-R255
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISGDSLSWLLRLLNRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 195,
anti-hHER2-HC-R255-GDSLSWLLRLLN-T256
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRGDSLSWLLRLLNTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 196,
anti-hHER2-HC-T256-CDSLSWLLRLLN-P257
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTGDSLSWLLRLLNPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 197,
anti-hHER2-HC-P257-GDSLSWLLRLLN-E258
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPGDSLSWLLRLLNEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 198,
anti-hHER2-HC-S267-GDSLSWLLRLLN-H268
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSGDSLSWLLRLLNHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 199,
anti-hHER2-HC-H268-GDSLSWLLRLLN-E269
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHGDSLSWLLRLLNEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 200,
anti-hHER2-HC-E269-GDSLSWLLRLLN-D270
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEGDSLSWLLRLLNDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 201,
anti-hHER2-HC-D270-GDSLSWLLRLLN-P271
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDGDSLSWLLRLLNPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 202,
anti-hHER2-HC-P271-GDSLSWLLRLLN-E272
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPGDSLSWLLRLLNEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 203,
anti-hHER2-HC-D280-GDSLSWLLRLLN-G281
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGDSLSWLLRLLNG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 204,
anti-hHER2-HC-H285-GDSLSWLLRLLN-N286
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHGDSLSWLL
RLLNNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 205,
anti-hHER2-HC-N286-GDSLSWLLRLLN-A287
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNGDSLSW
LLRLLNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 206,
anti-hHER2-HC-P291-GDSLSWLLRLLN-R292
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPGD
SLSWLLRLLNREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 207,
anti-hHER2-HC-T307-CDSLSWLLRLLN-V308
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTGDSLSWLLRLLNVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 208,
anti-hHER2-HC-V308-GDSLSWLLRLLN-L309
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVGDSLSWLLRLLNLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 209,
anti-hHER2-HC-L309-GDSLSWLLRLLN-H310
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLGDSLSWLLRLLNHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 210,
anti-hHER2-HC-H310-GDSLSWLLRLLN-Q311
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHGDSLSWLLRLLNQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 211,
anti-hHER2-HC-N315-GDSLSWLLRLLN-G316
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGDSLSWLLRLLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 212,
anti-hHER2-HC-G316-GDSLSWLLRLLN-K317
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGGDSLSWLLRLLNKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 213,
anti-hHER2-HC-K317-GDSLSWLLRLLN-E318
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKGDSLSWLLRLLNEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 214,
anti-hHER2-HC-K326-GDSLSWLLRLLN-A327
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGDSLSWLLRLLNALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 215,
anti-hHER2-HC-A327-GDSLSWLLRLLN-L328
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAGDSLSWLLRLLNLPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 216,
anti-hHER2-HC-L328-GDSLSWLLRLLN-P329
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGDSLSWLLRLLNPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 217,
anti-hHER2-HC-P329-GDSLSWLLRLLN-A330
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPGDSLSWLLRLLNAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 218,
anti-hHER2-HC-A330-GDSLSWLLRLLN-P331
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAGDSLSWLLRLLNPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 219,
anti-hHER2-HC-A339-GDSLSWLLRLLN-K340
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGDSLSWLLRLLNK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 220,
anti-hHER2-HC-K340-GDSLSWLLRLLN-G341
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGDSLSWLLRLLN
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 221,
anti-hHER2-HC-G341-GDSLSWLLRLLN-Q342
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGGDSLSWLLRLL
NQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 222, anti-hHER2-HC-Q342-GDSLSWLLRLLN-P343
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQGDSLSWLLRL
LNPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 223,
anti-hHER2-HC-P343-GDSLSWLLRLLN-R344
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPGDSLSWLL
RLLNREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 224,
anti-hHER2-HC-R344-GDSLSWLLRLLN-E345
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRGDSLSWL
LRLLNEPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 225,
anti-hHER2-HC-R355-GDSLSWLLRLLN-E356
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRGDSLSWLLRLLNEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 226,
anti-hHER2-HC-E356-GDSLSWLLRLLN-E357
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREGDSLSWLLRLLNEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 227,
anti-hHER2-HC-E357-GDSLSWLLRLLN-M358
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEGDSLSWLLRLLNMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 228,
anti-hHER2-HC-M358-GDSLSWLLRLLN-T359
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMGDSLSWLLRLLNTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 229,
anti-hHER2-HC-K360-GDSLSWLLRLLN-N361
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKGDSLSWLLRLLNNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 230,
anti-hHER2-HC-N384-GDSLSWLLRLLN-G385
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGDSLSWLLRLLNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 231,
anti-hHER2-HC-N389-GDSLSWLLRLLN-N390
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENGDSLSWLLRLLNNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 232,
anti-hHER2-HC-T394-GDSLSWLLRLLN-P395
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTGDSLSWLLRLLNPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 233,
anti-hHER2-HC-P395-GDSLSWLLRLLN-P396
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPGDSLSWLLRLLNPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 234,
anti-hHER2-HC-D399-GDSLSWLLRLLN-S400
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDGDSLSWLLRLL
NSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 235,
anti-hHER2-HC-D401-GDSLSWLLRLLN-G402
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGDSLSWLLR
LLNGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 236,
anti-hHER2-HC-S415-GDSLSWLLRLLN-R416
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSGDSLSWLLRLLNRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 237,
anti-hHER2-HC-R416-GDSLSWLLRLLN-W417
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRGDSLSWLLRLLNWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 238,
anti-hHER2-HC-W417-GDSLSWLLRLLN-Q418
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWGDSLSWLLRLLNQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 239,
anti-hHER2-HC-Q418-GDSLSWLLRLLN-Q419
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQGDSLSWLLRLLNQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 240,
anti-hHER2-HC-Q419-GDSLSWLLRLLN-G420
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGDSLSWLLRLLNGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 241,
anti-hHER2-HC-G420-GDSLSWLLRLLN-N421
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGGDSLSWLLRLLNNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 242,
anti-hHER2-HC-N421-GDSLSWLLRLLN-V422
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNGDSLSWLLRLLNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 243,
anti-hHER2-HC-H433-CDSLSWLLRLLN-N434
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHGDSLSWLLRLLNNHYTQKSLSLSPG SEQ ID NO: 244,
anti-hHER2-HC-N434-CDSLSWLLRLLN-H435
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNGDSLSWLLRLLNHYTQKSLSLSPG SEQ ID NO: 245,
anti-hHER2-HC-5442-GDSLSWLLRLLN-L443
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSGDSLSWLLRLLNLSPG SEQ ID NO: 246,
anti-hHER2-HC-L443-GDSLSWLLRLLN-S444
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLGDSLSWLLRLLNSPG SEQ ID NO: 247,
anti-hHER2-HC-S444-CDSLSWLLRLLN-P445
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSGDSLSWLLRLLNPG SEQ ID NO: 248,
anti-hHER2-HC-P445-GDSLSWLLRLLN-G446
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGDSLSWLLRLLNG SEQ ID NO: 249,
anti-hHER2-HC-A118-DSLEFIASKLA-S119
DSLEFIASKLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 250,
anti-hHER2-HC-S119-DSLEFIASKLA-T120
SDSLEFIASKLATKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 251,
anti-hHER2-HC-T120-DSLEFIASKLA-K121
STDSLEFIASKLAKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 252,
anti-hHER2-HC-S131-DSLEFIASKLA-S132
STKGPSVFPLAPSDSLEFIASKLASKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 253,
anti-hHER2-HC-S132-DSLEFIASKLA-K133
STKGPSVFPLAPSSDSLEFIASKLAKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 254,
anti-hHER2-HC-K133-DSLEFIASKLA-S134
STKGPSVFPLAPSSKDSLEFIASKLASTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 255,
anti-hHER2-HC-S134-DSLEFIASKLA-T135
STKGPSVFPLAPSSKSDSLEFIASKLATSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 256,
anti-hHER2-HC-T135-DSLEFIASKLA-S136
STKGPSVFPLAPSSKSTDSLEFIASKLASGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 257,
anti-hHER2-HC-S136-DSLEFIASKLA-G137
STKGPSVFPLAPSSKSTSDSLEFIASKLAGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 258,
anti-hHER2-HC-G137-DSLEFIASKLA-G138
STKGPSVFPLAPSSKSTSGDSLEFIASKLAGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 259,
anti-hHER2-HC-G138-DSLEFIASKLA-T139
STKGPSVFPLAPSSKSTSGGDSLEFIASKLATAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 260,
anti-hHER2-HC-E152-DSLEFIASKLA-P153
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEDSLEFIASKLAPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 261,
anti-hHER2-HC-P153-DSLEFIASKLA-V154
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPDSLEFIASKLAVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 262,
anti-hHER2-HC-N159-DSLEFIASKLA-S160
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNDSLEFIASKLASGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 263,
anti-hHER2-HC-S160-DSLEFIASKLA-G161
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSDSLEFIASKLAGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 264,
anti-hHER2-HC-G161-DSLEFIASKLA-A162
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGDSLEFIASKLAALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 265,
anti-hHER2-HC-A162-DSLEFIASKLA-L163
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGADSLEFIASKLALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 266,
anti-hHER2-HC-L163-DSLEFIASKLA-T164
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALDSLEFIASKLATSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 267,
anti-hHER2-HC-T164-DSLEFIASKLA-S165
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTDSLEFIASKLASG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 268,
anti-hHER2-HC-S165-DSLEFIASKLA-G166
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSDSLEFIASKLAG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 269,
anti-hHER2-HC-P171-DSLEFIASKLA-A172
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPDSLEFI
ASKLAAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 270,
anti-hHER2-HC-S176-DSLEFIASKLA-S177
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
DSLEFIASKLASGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 271,
anti-hHER2-HC-S177-DSLEFIASKLA-G178
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SDSLEFIASKLAGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 272,
anti-hHER2-HC-P189-DSLEFIASKLA-S190
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPDSLEFIASKLASSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 273,
anti-hHER2-HC-S190-DSLEFIASKLA-S191
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSDSLEFIASKLASSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 274,
anti-hHER2-HC-S191-DSLEFIASKLA-S192
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSDSLEFIASKLASLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 275,
anti-hHER2-HC-S192-DSLEFIASKLA-L193
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSDSLEFIASKLALGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 276,
anti-hHER2-HC-L193-DSLEFIASKLA-G194
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLDSLEFIASKLAGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 277,
anti-hHER2-HC-G194-DSLEFIASKLA-T195
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGDSLEFIASKLATQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 278,
anti-hHER2-HC-T195-DSLEFIASKLA-Q196
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTDSLEFIASKLAQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 279,
anti-hHER2-HC-Q196-DSLEFIASKLA-T197
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQDSLEFIASKLATYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 280, anti-hHER2-HC-K205-DSLEFIASKLA-P206
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKDSLEFIASKLAPSNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 281,
anti-hHER2-HC-P206-DSLEFIASKLA-S207
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPDSLEFIASKLASNTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 282,
anti-hHER2-HC-S207-DSLEFIASKLA-N208
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSDSLEFIASKLANTKVDKKVEPKSCDKTHT
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 283,
anti-hHER2-HC-P230-DSLEFIASKLA-A231
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPDSLEF
IASKLAAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 284,
anti-hHER2-HC-A231-DSLEFIASKLA-P232
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPADSLE
FIASKLAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 285,
anti-hHER2-HC-P232-DSLEFIASKLA-E233
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPDSL
EFIASKLAELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 286,
anti-hHER2-HC-E233-DSLEFIASKLA-L234
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPED
SLEFIASKLALLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 287,
anti-hHER2-HC-L234-DSLEFIASKLA-L235
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELD
SLEFIASKLALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 288,
anti-hHER2-HC-L235-DSLEFIASKLA-G236
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
DSLEFIASKLAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 289,
anti-hHER2-HC-G236-DSLEFIASKLA-G237
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GDSLEFIASKLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 290,
anti-hHER2-HC-P244-DSLEFIASKLA-P245
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPDSLEFIASKLAPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 291,
anti-hHER2-HC-P245-DSLEFIASKLA-K246
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPDSLEFIASKLAKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 292,
anti-hHER2-HC-I253-DSLEFIASKLA-S254
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMIDSLEFIASKLASRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 293,
anti-hHER2-HC-5254-DSLEFIASKLA-R255
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISDSLEFIASKLARTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 294,
anti-hHER2-HC-R255-DSLEFIASKLA-T256
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRDSLEFIASKLATPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 295,
anti-hHER2-HC-T256-DSLEFIASKLA-P257
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTDSLEFIASKLAPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 296,
anti-hHER2-HC-P257-DSLEFIASKLA-E258
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPDSLEFIASKLAEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 297,
anti-hHER2-HC-S267-DSLEFIASKLA-H268
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSDSLEFIASKLAHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 298,
anti-hHER2-HC-H268-DSLEFIASKLA-E269
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHDSLEFIASKLAEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 299,
anti-hHER2-HC-E269-DSLEFIASKLA-D270
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDSLEFIASKLADPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 300,
anti-hHER2-HC-D270-DSLEFIASKLA-P271
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDDSLEFIASKLAPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 301,
anti-hHER2-HC-P271-DSLEFIASKLA-E272
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPDSLEFIASKLAEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 302,
anti-hHER2-HC-D280-DSLEFIASKLA-G281
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDDSLEFIASKLAGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 303,
anti-hHER2-HC-H285-DSLEFIASKLA-N286
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHDSLEFIASK
LANAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 304,
anti-hHER2-HC-N286-DSLEFIASKLA-A287
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNDSLEFIAS
KLAAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 305,
anti-hHER2-HC-P291-DSLEFIASKLA-R292
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPDS
LEFIASKLAREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 306,
anti-hHER2-HC-T307-DSLEFIASKLA-V308
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTDSLEFIASKLAVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 307,
anti-hHER2-HC-V308-DSLEFIASKLA-L309
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVDSLEFIASKLALHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 308,
anti-hHER2-HC-L309-DSLEFIASKLA-H310
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLDSLEFIASKLAHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 309,
anti-hHER2-HC-H310-DSLEFIASKLA-Q311
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHDSLEFIASKLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 310,
anti-hHER2-HC-N315-DSLEFIASKLA-G316
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNDSLEFIASKLAGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 311,
anti-hHER2-HC-G316-DSLEFIASKLA-K317
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGDSLEFIASKLAKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 312,
anti-hHER2-HC-K317-DSLEFIASKLA-E318
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKDSLEFIASKLAEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 313,
anti-hHER2-HC-K326-DSLEFIASKLA-A327
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKDSLEFIASKLAALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 314,
anti-hHER2-HC-A327-DSLEFIASKLA-L328
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKADSLEFIASKLALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 315,
anti-hHER2-HC-L328-DSLEFIASKLA-P329
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALDSLEFIASKLAPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 316,
anti-hHER2-HC-P329-DSLEFIASKLA-A330
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPDSLEFIASKLAAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 317,
anti-hHER2-HC-A330-DSLEFIASKLA-P331
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPADSLEFIASKLAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 318,
anti-hHER2-HC-A339-DSLEFIASKLA-K340
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKADSLEFIASKLAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 319,
anti-hHER2-HC-K340-DSLEFIASKLA-G341
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKDSLEFIASKLAGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 320,
anti-hHER2-HC-G341-DSLEFIASKLA-Q342
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGDSLEFIASKLAQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 321,
anti-hHER2-HC-Q342-DSLEFIASKLA-P343
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQDSLEFIASKLA
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 322,
anti-hHER2-HC-P343-DSLEFIASKLA-R344
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPDSLEFIASKL
AREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 323,
anti-hHER2-HC-R344-DSLEFIASKLA-E345
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRDSLEFIAS
KLAEPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 324,
anti-hHER2-HC-R355-DSLEFIASKLA-E356
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSRDSLEFIASKLAEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 325,
anti-hHER2-HC-E356-DSLEFIASKLA-E357
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREDSLEFIASKLAEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 326,
anti-hHER2-HC-E357-DSLEFIASKLA-M358
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEDSLEFIASKLAMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 327,
anti-hHER2-HC-M358-DSLEFIASKLA-T359
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMDSLEFIASKLATKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG SEQ ID NO: 328,
anti-hHER2-HC-K360-DSLEFIASKLA-N361
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKDSLEFIASKLANQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 329,
anti-hHER2-HC-N384-DSLEFIASKLA-G385
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNDSLEFIASKLAGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 330,
anti-hHER2-HC-N389-DSLEFIASKLA-N390
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENDSLEFIASKLANYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 331,
anti-hHER2-HC-T394-DSLEFIASKLA-P395
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTDSLEFIASKLAPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 332,
anti-hHER2-HC-P395-DSLEFIASKLA-P396
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPDSLEFIASKLAPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 333,
anti-hHER2-HC-D399-DSLEFIASKLA-S400
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDDSLEFIASKLAS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 334,
anti-hHER2-HC-S400-DSLEFIASKLA-D401
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDSLEFIASKLA
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 335,
anti-hHER2-HC-D401-DSLEFIASKLA-G402
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDDSLEFIASKL
AGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 336,
anti-hHER2-HC-5415-DSLEFIASKLA-R416
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSDSLEFIASKLARWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 337,
anti-hHER2-HC-R416-DSLEFIASKLA-W417
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRDSLEFIASKLAWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 338,
anti-hHER2-HC-W417-DSLEFIASKLA-Q418
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWDSLEFIASKLAQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 339,
anti-hHER2-HC-Q418-DSLEFIASKLA-Q419
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQDSLEFIASKLAQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 340,
anti-hHER2-HC-Q419-DSLEFIASKLA-G420
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQDSLEFIASKLAGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 341,
anti-hHER2-HC-G420-DSLEFIASKLA-N421
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGDSLEFIASKLANVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 342,
anti-hHER2-HC-N421-DSLEFIASKLA-V422
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNDSLEFIASKLAVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 343,
anti-hHER2-HC-H433-DSLEFIASKLA-N434
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHDSLEFIASKLANHYTQKSLSLSPG SEQ ID NO: 344,
anti-hHER2-HC-N434-DSLEFIASKLA-H435
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNDSLEFIASKLAHYTQKSLSLSPG SEQ ID NO: 345,
anti-hHER2-HC-5442-DSLEFIASKLA-L443
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSDSLEFIASKLALSPG SEQ ID NO: 346,
anti-hHER2-HC-L443-DSLEFIASKLA-5444
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLDSLEFIASKLASPG SEQ ID NO: 347,
anti-hHER2-HC-S444-DSLEFIASKLA-P445
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSDSLEFIASKLAPG SEQ ID NO: 348,
anti-hHER2-HC-P445-DSLEFIASKLA-G446
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPDSLEFIASKLAG SEQ ID NO: 349,
anti-hHER2-HC-G446-DSLEFIASKLA-K447
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGDSLEFIASKLA PPTases: SEQ ID NO:
350, Sfp enzyme (with TEV protease cleavage site):
MKIYGIYMDRPLSQEENERFMSFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQY
QLDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVICAFDSQPIGIDIEKTKPISLEIAKR
FFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSI
ELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELLENLYFQGHHHHHH SEQ ID
NO: 351, Sfp enzyme (without TEV protease cleavage site):
MKIYGIYMDRPLSQEENERFMSFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQY
QLDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVICAFDSQPIGIDIEKTKPISLEIAKR
FFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSI
ELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELLHHHHHH SEQ ID NO: 352,
Sfp mutant R4-4 (with TEV protease cleavage site):
MKIYGIYMDRPLSQEENERFMSFISPEEREKCRRFYHKEDAHRELLGDVLVRSVISRQY
QLDKSDIRFSTQEYGKPYIPDLPDAHFNISHSGRWVICAFDSQPIGIDIEKTKPISLEIAKR
FFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSI
ELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELLENLYFQGHHHHHH SEQ ID
NO: 353, AcpS enzyme:
MAILGLGTDIVEIARIEAVIARSGDRLARRVLSDNEWAIWKTHHQPVRFLAKRFAVKEAA
AKAFGTGIRNGLAFNQFEVFNDELGKPRLRLWGEALKLAEKLGVANMHVTLADERHYA
CATVIIESHHHHHH SEQ ID NO: 354, Thermotoga maritima PPTase:
GSDKIHHHHHHMIVGVGIDVLEVERVPEKFAERILGESEKRLFLTRKRRREFIAGRFALK
EAFFKALGTGLNGHSFTDVEFLESNGKPVLCVHKDFGFFNYAHVSLSHDRFAVALVVLE
KRKGDIIVEGDESFLRKRFEVLERSVEGWEIETSLPPFTLKKLLESSGCRLVRYGNILIGE SEQ
ID NO: 355, Signal peptide for protein secretion
MKTFILLLWVLLLWVIFLLPGATA SEQ ID NO: 356,
mAb2-HC-E388-GDSLDMLEWSLM-N389:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEGDSLDMLEWSLMNNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 357,
anti-hHER2-HC-A118-GDSLDMLEWSLM-S119
GDSLDMLEWSLMSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 358,
anti-hHER2-HC-S119-GDSLDMLEWSLM-T120
SGDSLDMLEWSLMTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 359,
anti-hHER2-HC-T120-GDSLDMLEWSLM-K121
STGDSLDMLEWSLMKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 360,
anti-hHER2-HC-S131-GDSLDMLEWSLM-S132
STKGPSVFPLAPSGDSLDMLEWSLMSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 361,
anti-hHER2-HC-S132-GDSLDMLEWSLM-K133
STKGPSVFPLAPSSGDSLDMLEWSLMKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 362,
anti-hHER2-HC-K133-GDSLDMLEWSLM-S134
STKGPSVFPLAPSSKGDSLDMLEWSLMSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 363,
anti-hHER2-HC-S134-GDSLDMLEWSLM-T135
STKGPSVFPLAPSSKSGDSLDMLEWSLMTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 364,
anti-hHER2-HC-T135-GDSLDMLEWSLM-S136
STKGPSVFPLAPSSKSTGDSLDMLEWSLMSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 365,
anti-hHER2-HC-S136-GDSLDMLEWSLM-G137
STKGPSVFPLAPSSKSTSGDSLDMLEWSLMGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 366,
anti-hHER2-HC-G137-GDSLDMLEWSLM-G138
STKGPSVFPLAPSSKSTSGGDSLDMLEWSLMGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 367,
anti-hHER2-HC-G138-GDSLDMLEWSLM-T139
STKGPSVFPLAPSSKSTSGGGDSLDMLEWSLMTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 368,
anti-hHER2-HC-N159-GDSLDMLEWSLM-S160
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNGDSLDMLEWSLMSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 369,
anti-hHER2-HC-S160-GDSLDMLEWSLM-G161
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGDSLDMLEWSLMGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 370,
anti-hHER2-HC-G161-GDSLDMLEWSLM-A162
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGGDSLDMLEWSLMAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 371,
anti-hHER2-HC-A162-GDSLDMLEWSLM-L163
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAGDSLDMLEWSLML
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 372,
anti-hHER2-HC-L163-GDSLDMLEWSLM-T164
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALGDSLDMLEWSLM
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 373,
anti-hHER2-HC-T164-GDSLDMLEWSLM-S165
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTGDSLDMLEWSL
MSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 374,
anti-hHER2-HC-S165-GDSLDMLEWSLM-G166
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGDSLDMLEWSL
MGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 375,
anti-hHER2-HC-P189-GDSLDMLEWSLM-S190
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPGDSLDMLEWSLMSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 376,
anti-hHER2-HC-S190-GDSLDMLEWSLM-S191
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSGDSLDMLEWSLMSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 377,
anti-hHER2-HC-S191-GDSLDMLEWSLM-S192
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSGDSLDMLEWSLMSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 378,
anti-hHER2-HC-S192-GDSLDMLEWSLM-L193
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSGDSLDMLEWSLMLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 379,
anti-hHER2-HC-L193-GDSLDMLEWSLMN-G194
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGDSLDMLEWSLMNGTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 380,
anti-hHER2-HC-G194-GDSLDMLEWSLMN-T195
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGGDSLDMLEWSLMNTQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 381,
anti-hHER2-HC-T195-GDSLDMLEWSLMN-Q196
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTGDSLDMLEWSLMNQTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 382,
anti-hHER2-HC-Q196-GDSLDMLEWSLMN-T197
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQGDSLDMLEWSLMNTYICNVNHKPSNTKVDKKVEPKSCD
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 383,
anti-hHER2-LC-T109-GDSLDMLEWSLM-V110
TGDSLDMLEWSLMVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 384, anti-hHER2-LC-V110-GDSLDMLEWSLM-A111
TVGDSLDMLEWSLMAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 385, anti-hHER2-LC-A111-GDSLDMLEWSLM-A112
TVAGDSLDMLEWSLMAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ
ID NO: 386, anti-hHER2-HC-P445-GDSLDMLEWSLM-G446
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGDSLDMLEWSLMG SEQ ID NO: 387,
anti-hHER2-HC-G446-GDSLDMLEWSLM-K447:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGDSLDMLEWSLM SEQ ID NO: 388,
anti-hHER2-LC-I2-GDSLDMLEWSLM-Q3:
DIGDSLDMLEWSLMQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK
LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV
EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170218085A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170218085A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References