U.S. patent application number 17/580970 was filed with the patent office on 2022-08-18 for antigen binding molecules comprising a tnf family ligand trimer.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Maria Amann, Peter Bruenker, Christina Claus, Claudia Ferrara Koller, Sandra Grau-Richards, Christian Klein, Viktor Levitski, Ekkehard Moessner, Joerg Thomas Regula, Pablo Umana.
Application Number | 20220259326 17/580970 |
Document ID | / |
Family ID | 1000006300304 |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220259326 |
Kind Code |
A1 |
Amann; Maria ; et
al. |
August 18, 2022 |
ANTIGEN BINDING MOLECULES COMPRISING A TNF FAMILY LIGAND TRIMER
Abstract
The invention relates to novel TNF family ligand
trimer-containing antigen binding molecules comprising (a) at least
one moiety capable of specific binding to a target cell antigen and
(b) a first and a second polypeptide that are linked to each other
by a disulfide bond, characterized in that the first polypeptide
comprises two ectodomains of a TNF ligand family member or
fragments thereof that are connected to each other by a peptide
linker and in that the second polypeptide comprises only one
ectodomain of said TNF ligand family member or a fragment
thereof.
Inventors: |
Amann; Maria; (Duebendorf,
CH) ; Bruenker; Peter; (Hittnau, CH) ; Claus;
Christina; (Ennetbaden, CH) ; Ferrara Koller;
Claudia; (Zug, CH) ; Grau-Richards; Sandra;
(Birmensdorf, CH) ; Klein; Christian; (Bonstetten,
CH) ; Levitski; Viktor; (Schlieren, CH) ;
Moessner; Ekkehard; (Kreuzlingen, CH) ; Regula; Joerg
Thomas; (Muenchen, DE) ; Umana; Pablo;
(Wollerau, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000006300304 |
Appl. No.: |
17/580970 |
Filed: |
January 21, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16522412 |
Jul 25, 2019 |
11267903 |
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17580970 |
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15067024 |
Mar 10, 2016 |
10392445 |
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16522412 |
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PCT/EP2015/076528 |
Nov 13, 2015 |
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15067024 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70575 20130101;
C07K 16/2815 20130101; C07K 2317/526 20130101; C07K 2317/524
20130101; C07K 2317/565 20130101; C07K 2317/60 20130101; A61K
39/395 20130101; C07K 2319/30 20130101; C07K 2317/522 20130101;
C07K 2317/33 20130101; C07K 16/40 20130101; C07K 2319/33 20130101;
C07K 14/70596 20130101; A61K 38/00 20130101; C07K 2317/35 20130101;
C07K 16/3007 20130101; C07K 2317/92 20130101; C07K 16/2896
20130101; C07K 16/2803 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 16/28 20060101 C07K016/28; C07K 14/705 20060101
C07K014/705; A61K 39/395 20060101 A61K039/395; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 2014 |
EP |
14193260.8 |
Sep 3, 2015 |
EP |
15183736.6 |
Oct 2, 2015 |
EP |
15188142.2 |
Claims
1. A TNF family ligand trimer-containing antigen binding molecule
comprising (a) at least one moiety capable of specific binding to a
target cell antigen and (b) a first and a second polypeptide that
are linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises two ectodomains of a TNF ligand family member or a
fragment thereof that are connected to each other by a peptide
linker and in that the second polypeptide comprises only one
ectodomain of said TNF ligand family member or a fragment
thereof.
2. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, further comprising (c) an Fc domain composed of a first
and a second subunit capable of stable association.
3. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, comprising (a) at least one moiety capable of specific
binding to a target cell antigen and (b) a first and a second
polypeptide that are linked to each other by a disulfide bond,
wherein the antigen binding molecule is characterized in that (i)
the first polypeptide contains a CH1 or CL domain and the second
polypeptide contains a CL or CH1 domain, respectively, wherein the
second polypeptide is linked to the first polypeptide by a
disulfide bond between the CH1 and CL domain, and wherein the first
polypeptide comprises two ectodomains of a TNF ligand family member
or fragments thereof that are connected to each other and to the
CH1 or CL domain by a peptide linker and wherein the second
polypeptide comprises one ectodomain of said TNF ligand family
member or a fragment thereof connected via a peptide linker to the
CL or CH1 domain of said polypeptide, or (ii) the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises only one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide, or (iii) the first polypeptide
contains a VH-CL or a VL-CH1 domain and the second polypeptide
contains a VL-CH1 domain or a VH-CL domain, respectively, wherein
the second polypeptide is linked to the first polypeptide by a
disulfide bond between the CH1 and CL domain, and wherein the first
polypeptide comprises two ectodomains of a TNF ligand family member
or fragments thereof that are connected to each other and to to VH
or VL by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to VL or VH of said
polypeptide.
4. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, wherein the TNF ligand family member costimulates human
T-cell activation.
5. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, wherein the TNF ligand family member is selected from
4-1BBL and OX40L.
6. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, wherein the TNF ligand family member is 4-1BBL.
7. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, wherein the ectodomain of a TNF ligand family member
comprises the amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:96, SEQ ID NO: 373, SEQ ID NO:374 and SEQ ID NO:375,
particularly the amino acid sequence of SEQ ID NO:1 or SEQ ID
NO:96.
8. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, wherein the ectodomain of a TNF ligand family member
comprises the amino acid sequence of SEQ ID NO:96.
9. The TNF family ligand trimer-containing antigen binding molecule
of claim 1, comprising (a) at least one moiety capable of specific
binding to a target cell antigen and (b) a first and a second
polypeptide that are linked to each other by a disulfide bond,
wherein the antigen binding molecule is characterized in that the
first polypeptide comprises the amino acid sequence selected from
the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98 and
SEQ ID NO:99 and in that the second polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:96, SEQ ID NO:3 and SEQ ID NO:4.
10. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, comprising (a) at least one moiety capable of
specific binding to a target cell antigen, and (b) a first
polypeptide containing a CH1 or CL domain and a second polypeptide
containing a CL or CH1 domain, respectively, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the CH1 or CL domain by a
peptide linker and in that the second polypeptide comprises only
one ectodomain of said TNF ligand family member or a fragment
thereof connected via a peptide linker to the CL or CH1 domain of
said polypeptide.
11. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the moiety capable of specific binding
to a target cell antigen is selected from the group consisting of
an antibody fragment, a Fab molecule, a crossover Fab molecule, a
single chain Fab molecule, a Fv molecule, a scFv molecule, a single
domain antibody, an aVH and a scaffold antigen binding protein.
12. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the molecule comprises one moiety
capable of specific binding to a target cell antigen.
13. The TNF family ligand trimer-containing antigen binding
molecule claim 1, wherein the moiety capable of specific binding to
a target cell antigen is a Fab molecule capable of specific binding
to a target cell antigen.
14. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the target cell antigen is selected
from the group consisting of Fibroblast Activation Protein (FAP),
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),
Epidermal Growth Factor Receptor (EGFR), Carcinoembryonic Antigen
(CEA), CD19, CD20 and CD33.
15. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the target cell antigen is Fibroblast
Activation Protein (FAP).
16. The TNF family ligand trimer-containing antigen binding
molecule of claim 15, wherein the moiety capable of specific
binding to FAP comprises a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:100,
(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:8 or
SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid sequence
of SEQ ID NO:9 or SEQ ID NO:102, and a VL domain comprising (iv)
CDR-L1 comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID
NO:103, (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:11 or SEQ ID NO:104, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:12 or SEQ ID NO:105.
17. The TNF family ligand trimer-containing antigen binding
molecule of claim 15, wherein the moiety capable of specific
binding to FAP comprises a variable heavy chain comprising an amino
acid sequence of SEQ ID NO:16 and a variable light chain comprising
an amino acid sequence of SEQ ID NO:17 or wherein the moiety
capable of specific binding to FAP comprises a variable heavy chain
comprising an amino acid sequence of SEQ ID NO:106 and a variable
light chain comprising an amino acid sequence of SEQ ID NO:107.
18. The TNF family ligand trimer-containing antigen binding
molecule of claim 2, wherein the Fc domain is an IgG, particularly
an IgG1 Fc domain or an IgG4 Fc domain.
19. The TNF family ligand trimer-containing antigen binding
molecule of claim 2, wherein the Fc domain is an IgG1 Fc domain
comprising the amino acid substitutions at positions 234 and 235
(EU numbering) and/or 329 (EU numbering).
20. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
first peptide comprising two ectodomains of a TNF ligand family
member or fragments thereof connected to each other by a first
peptide linker fused at its C-terminus by a second peptide linker
to a second heavy or light chain, and a second peptide comprising
one ectodomain of said TNF ligand family member fused at its
C-terminus by a third peptide linker to a second light or heavy
chain, respectively.
21. The TNF family ligand trimer-containing antigen binding
molecule of claim 20, wherein the first peptide comprising two
ectodomains of a TNF ligand family member or fragments thereof
connected to each other by a first peptide linker is fused at its
C-terminus by a second peptide linker to a CH1 domain that is part
of a heavy chain, and the second peptide comprising one ectodomain
of said TNF ligand family member or a fragment thereof is fused at
its C-terminus by a third peptide linker to a CL domain that is
part of a light chain.
22. The TNF family ligand trimer-containing antigen binding
molecule of claim 20, wherein the first peptide comprising two
ectodomains of a TNF ligand family member or fragments thereof
connected to each other by a first peptide linker is fused at its
C-terminus by a second peptide linker to a CL domain that is part
of a heavy chain, and the second peptide comprising one ectodomain
of said TNF ligand family member or a fragment thereof is fused at
its C-terminus by a third peptide linker to a CH1 domain that is
part of a light chain.
23. The TNF family ligand trimer-containing antigen binding
molecule of claim 20, wherein the first peptide comprising two
ectodomains of a TNF ligand family member or fragments thereof
connected to each other by a first peptide linker is fused at its
C-terminus by a second peptide linker to a VH domain that is part
of a heavy chain, and the second peptide comprising one ectodomain
of said TNF ligand family member or a fragment thereof is fused at
its C-terminus by a third peptide linker to a VL domain that is
part of a light chain.
24. The TNF family ligand trimer-containing antigen binding
molecule of claim 22, wherein in the CL domain adjacent to the TNF
ligand family member the amino acid at position 123 (EU numbering)
has been replaced by arginine (R) and the amino acid at position
124 (EU numbering) has been substituted by lysine (K), and wherein
in the CH1 domain adjacent to the TNF ligand family member the
amino acids at position 147 (EU numbering) and at position 213 (EU
numbering) have been substituted by glutamic acid (E).
25. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(a) a first heavy chain and a first light chain, both comprising a
Fab molecule capable of specific binding to a target cell antigen,
(b) a second heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID
NO:98 and SEQ ID NO:99, and a second light chain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ ID NO:4.
26. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(a) a first heavy chain and a first light chain, both comprising a
Fab molecule capable of specific binding to a target cell antigen,
(b) a second heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID
NO:98 and SEQ ID NO:99, and a second light chain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ ID NO:4.
27. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:107, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:14, SEQ ID
NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light
chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
28. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:107, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:115, SEQ
ID NO:117, SEQ ID NO:119 and SEQ ID NO:173, and (iii) a second
light chain comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120 and
SEQ ID NO:174.
29. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, comprising (a) at least one moiety capable of
specific binding to a target cell antigen, and (b) a first and a
second polypeptide that are linked to each other by a disulfide
bond, wherein the antigen binding molecule is characterized in that
the first polypeptide contains a CH3 domain and the second
polypeptide contains a CH3 domain, respectively, and wherein the
first polypeptide comprises two ectodomains of a TNF ligand family
member or fragments thereof that are connected to each other and to
the C-terminus of the CH3 domain by a peptide linker and wherein
the second polypeptide comprises one ectodomain of said TNF ligand
family member or a fragment thereof connected via a peptide linker
to C-terminus of the CH3 domain of said polypeptide.
30. The TNF family ligand trimer-containing antigen binding
molecule of claim 29, comprising two moieties capable of specific
binding to a target cell antigen.
31. The TNF family ligand trimer-containing antigen binding
molecule of claim 29, wherein the antigen binding molecule
comprises (i) a first heavy chain comprising the amino acid
sequence of SEQ ID NO:121, a second heavy chain comprising the
amino acid sequence of SEQ ID NO:122, and two light chains
comprising the amino acid sequence of SEQ ID NO:19, or (ii) a first
heavy chain comprising the amino acid sequence of SEQ ID NO:123, a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:124, and two light chains comprising the amino acid sequence of
SEQ ID NO:125, or (iii) a first heavy chain comprising the amino
acid sequence of SEQ ID NO:126, a second heavy chain comprising the
amino acid sequence of SEQ ID NO:127, and two light chains
comprising the amino acid sequence of SEQ ID NO:125.
32. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the target cell antigen is CD19.
33. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the moiety capable of specific binding
to CD19 comprises a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:195 or SEQ ID NO:252, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:196 or SEQ ID
NO:253, and (iii) CDR-H3 comprising the amino acid sequence of SEQ
ID NO:197 or SEQ ID NO:254, and a VL domain comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:198 or SEQ ID
NO:249, (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:199 or SEQ ID NO:250, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:200 or SEQ ID NO:251.
34. The TNF family ligand trimer-containing antigen binding
molecule of claim 32, wherein the moiety capable of specific
binding to CD19 comprises a variable heavy chain comprising an
amino acid sequence of SEQ ID NO:201 and a variable light chain
comprising an amino acid sequence of SEQ ID NO:202 or wherein the
moiety capable of specific binding to CD19 comprises a variable
heavy chain comprising an amino acid sequence of SEQ ID NO:357 and
a variable light chain comprising an amino acid sequence of SEQ ID
NO:358.
35. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:201 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:202 or a first heavy chain comprising the VH domain
comprising the amino acid sequence of SEQ ID NO:357 and a first
light chain comprising the VL domain comprising the amino acid
sequence of SEQ ID NO:358, (ii) a second heavy chain comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:14, SEQ ID NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a
second light chain comprising the amino acid sequence of SEQ ID
NO:15, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID
NO:114.
36. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:201 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:202 or a first heavy chain comprising the VH domain
comprising the amino acid sequence of SEQ ID NO:357 and a first
light chain comprising the VL domain comprising the amino acid
sequence of SEQ ID NO:358, (ii) a second heavy chain comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:115, SEQ ID NO:117, SEQ ID NO:119 and SEQ ID NO:173, and (iii) a
second light chain comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120
and SEQ ID NO:174.
37. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:209, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:210, and two light chains comprising the amino acid
sequence of SEQ ID NO:206, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:213, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:214, and two light
chains comprising the amino acid sequence of SEQ ID NO:206, or
(iii) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:309, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:310, and two light chains comprising the amino acid
sequence of SEQ ID NO:279, or (iv) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:313, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:314, and two light
chains comprising the amino acid sequence of SEQ ID NO:279.
38. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the target cell antigen is CEA.
39. The TNF family ligand trimer-containing antigen binding
molecule of claim 38, wherein the moiety capable of specific
binding to CEA comprises a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:321, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:322, and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:323, and a
VL domain comprising (iv) CDR-L1 comprising the amino acid sequence
of SEQ ID NO:324, (v) CDR-L2 comprising the amino acid sequence of
SEQ ID NO:325, and (vi) CDR-L3 comprising the amino acid sequence
of SEQ ID NO:326.
40. The TNF family ligand trimer-containing antigen binding
molecule of claim 38, wherein the moiety capable of specific
binding to CEA comprises a variable heavy chain comprising an amino
acid sequence of SEQ ID NO:329 and a variable light chain
comprising an amino acid sequence of SEQ ID NO:330.
41. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:329 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:330, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:14, SEQ ID
NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light
chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
42. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:329 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:330, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:115, SEQ
ID NO:117, SEQ ID NO:119 and SEQ ID NO:173, and (iii) a second
light chain comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120 and
SEQ ID NO:174.
43. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:337, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:338, and two light chains comprising the amino acid
sequence of SEQ ID NO:334, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:341, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:342, and two light
chains comprising the amino acid sequence of SEQ ID NO:334.
44. The TNF family ligand trimer-containing antigen binding
molecule of claim 1, wherein the TNF ligand family member is
OX40L.
45. The TNF family ligand trimer-containing antigen binding
molecule of claim 44, wherein the ectodomain of a TNF ligand family
member comprises the amino acid sequence of SEQ ID NO:53 or SEQ ID
NO:54, particularly the amino acid sequence of SEQ ID NO:53.
46. The TNF family ligand trimer-containing antigen binding
molecule of claim 44, comprising (a) at least one moiety capable of
specific binding to a target cell antigen and (b) a first and a
second polypeptide that are linked to each other by a disulfide
bond, wherein the antigen binding molecule is characterized in that
the first polypeptide comprises the amino acid sequence of SEQ ID
NO:371 or SEQ ID:372 and in that the second polypeptide comprises
the amino acid sequence of SEQ ID NO:53 or SEQ ID NO:54.
47. The TNF family ligand trimer-containing antigen binding
molecule of claim 44, wherein the target cell antigen is Fibroblast
Activation Protein (FAP) and the moiety capable of specific binding
to FAP comprises a VH domain comprising (i) CDR-H1 comprising the
amino acid sequence of SEQ ID NO:7 or SEQ ID NO:100, (ii) CDR-H2
comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:101,
and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:9
or SEQ ID NO:102, and a VL domain comprising (iv) CDR-L1 comprising
the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:103, (v)
CDR-L2 comprising the amino acid sequence of SEQ ID NO:11 or SEQ ID
NO:104, and (vi) CDR-L3 comprising the amino acid sequence of SEQ
ID NO:12 or SEQ ID NO:105.
48. The TNF family ligand trimer-containing antigen binding
molecule of claim 44, wherein the antigen binding molecule
comprises (i) a first heavy chain comprising the VH domain
comprising the amino acid sequence of SEQ ID NO:16 and a first
light chain comprising the VL domain comprising the amino acid
sequence of SEQ ID NO:17 or a first heavy chain comprising the VH
domain comprising the amino acid sequence of SEQ ID NO:106 and a
first light chain comprising the VL domain comprising the amino
acid sequence of SEQ ID NO:107, (ii) a second heavy chain
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:355, and (iii) a second light chain
comprising the amino acid sequence of SEQ ID NO:356.
49. An isolated polynucleotide encoding the TNF family ligand
trimer-containing antigen binding molecule of claim 1.
50. A vector, particularly an expression vector, comprising the
isolated polynucleotide of claim 49.
51. A host cell comprising the isolated polynucleotide of claim 49
or the vector of claim 50.
52. A method for producing the TNF family ligand trimer-containing
antigen binding molecule of claim 1, comprising the steps of (i)
culturing the host cell of claim 51 under conditions suitable for
expression of the antigen binding molecule, and (ii) recovering the
antigen binding molecule.
53. A pharmaceutical composition comprising the TNF family ligand
trimer-containing antigen binding molecule of claim 1 and at least
one pharmaceutically acceptable excipient.
54. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising a TNF family ligand trimer-containing
antigen binding molecule of claim 1 in a pharmaceutically
acceptable form.
55. The method of claim 54, wherein said disease is cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 16/522,412, filed Jul. 25, 2019, which is a
divisional application of U.S. patent application Ser. No.
15/067,024, filed Mar. 10, 2016, now U.S. Pat. No. 10,392,445,
which is a continuation of International Patent Application No.
PCT/EP2015/076528, filed Nov. 13, 2015, which claims the benefit of
and priority to European Patent Application No. 14193260.8, now
withdrawn, filed Nov. 14, 2014, European Patent Application No.
15183736.6, now abandoned, filed Sep. 3, 2015, and European Patent
Application No. 15188142.2, now abandoned, filed Oct. 2, 2015, each
of which are incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Jan. 6,
2022 is named P32429-US-4_SL.txt and is 872,652 bytes in size.
FIELD OF THE INVENTION
[0003] The invention relates to novel TNF family ligand
trimer-containing antigen binding molecules comprising (a) at least
one moiety capable of specific binding to a target cell antigen and
(b) a first and a second polypeptide that are linked to each other
by a disulfide bond, wherein the antigen binding molecules are
characterized in that the first polypeptide comprises two
ectodomains of a TNF ligand family member or two fragments thereof
that are connected to each other by a peptide linker and in that
the second polypeptide comprises only one ectodomain of said TNF
ligand family member or a fragment thereof. The invention further
relates to methods of producing these molecules and to methods of
using the same.
BACKGROUND
[0004] Ligands interacting with molecules of the TNF (tumor
necrosis factor) receptor superfamily have pivotal roles in the
organization and function of the immune system. While regulating
normal functions such as immune responses, hematopoiesis and
morphogenesis, the TNF family ligands (also called cytokines) play
a role in tumorgenesis, transplant rejection, septic shock, viral
replication, bone resorption, rheumatoid arthritis and diabetes
(Aggarwal, 2003). The TNF ligand family comprises 18 genes encoding
19 type II (i.e. intracellular N terminus and extracellular
C-terminus) transmembrane proteins, characterized by the presence
of a conserved C-terminal domain coined the `TNF homology domain`
(THD). This domain is responsible for receptor binding and is thus
critical for the biological activity of the TNF ligand family
members. The sequence identity between family members is
.about.20-30% (Bodmer, 2002). Members of the TNF ligand family
exert their biological function as self-assembling, noncovalent
trimers (Banner et al, Cell 1993, 73, 431-445). Thus, the TNF
family ligands form a trimer that is able to bind to and to
activate the corresponding receptors of TNFR superfamily.
[0005] 4-1BB (CD137), a member of the TNF receptor superfamily, has
been first identified as a molecule whose expression is induced by
T-cell activation (Kwon and Weissman, 1989). Subsequent studies
demonstrated expression of 4-1BB in T- and B-lymphocytes (Snell et
al., 2011; Zhang et al., 2010), NK-cells (Lin et al., 2008),
NKT-cells (Kim et al., 2008), monocytes (Kienzle and von Kempis,
2000; Schwarz et al., 1995), neutrophils (Heinisch et al., 2000),
mast (Nishimoto et al., 2005) and dendritic cells as well as cells
of non-hematopoietic origin such as endothelial and smooth muscle
cells (Broll et al., 2001; Olofsson et al., 2008). Expression of
4-1BB in different cell types is mostly inducible and driven by
various stimulatory signals, such as T-cell receptor (TCR) or
B-cell receptor triggering, as well as signaling induced through
co-stimulatory molecules or receptors of pro-inflammatory cytokines
(Diehl et al., 2002; von Kempis et al., 1997; Zhang et al.,
2010).
[0006] Expression of 4-1BB ligand (4-1BBL or CD137L) is more
restricted and is observed on professional antigen presenting cells
(APC) such as B-cells, dendritic cells (DCs) and macrophages.
Inducible expression of 4-1BBL is characteristic for T-cells,
including both and .gamma..delta. T-cell subsets, and endothelial
cells (reviewed in Shao and Schwarz, 2011).
[0007] CD137 signaling is known to stimulate IFN.gamma. secretion
and proliferation of NK cells (Buechele et al., 2012; Lin et al.,
2008; Melero et al., 1998) as well as to promote DC activation as
indicated by their increased survival and capacity to secret
cytokines and upregulate co-stimulatory molecules (Choi et al.,
2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137
is best characterized as a co-stimulatory molecule which modulates
TCR-induced activation in both the CD4+ and CD8+ subsets of
T-cells. In combination with TCR triggering, agonistic
4-1BB-specific antibodies enhance proliferation of T-cells,
stimulate lymphokine secretion and decrease sensitivity of
T-lymphocytes to activation-induced cells death (reviewed in
(reviewed in Snell et al., 2011).
[0008] In line with these co-stimulatory effects of 4-1BB
antibodies on T-cells in vitro, their administration to tumor
bearing mice leads to potent anti-tumor effects in many
experimental tumor models (Melero et al., 1997; Narazaki et al.,
2010). However, 4-1BB usually exhibits its potency as an anti-tumor
agent only when administered in combination with other
immunomodulatory compounds (Curran et al., 2011; Guo et al., 2013;
Morales-Kastresana et al., 2013; Teng et al., 2009; Wei et al.,
2013), chemotherapeutic reagents (Ju et al., 2008; Kim et al.,
2009), tumor-specific vaccination (Cuadros et al., 2005; Lee et
al., 2011) or radiotherapy (Shi and Siemann, 2006). In vivo
depletion experiments demonstrated that CD8+ T-cells play the most
critical role in anti-tumoral effect of 4-1BB-specific antibodies.
However, depending on the tumor model or combination therapy, which
includes anti-4-1BB, contributions of other types of cells such as
DCs, NK-cells or CD4+ T-cells have been reported (Melero et al.,
1997; Murillo et al., 2009; Narazaki et al., 2010; Stagg et al.,
2011).
[0009] In addition to their direct effects on different lymphocyte
subsets, 4-1BB agonists can also induce infiltration and retention
of activated T-cells in the tumor through 4-1BB-mediated
upregulation of intercellular adhesion molecule 1 (ICAM1) and
vascular cell adhesion molecule 1 (VCAM1) on tumor vascular
endothelium (Palazon et al., 2011).
[0010] 4-1BB triggering may also reverse the state of T-cell anergy
induced by exposure to soluble antigen that may contribute to
disruption of immunological tolerance in the tumor
micro-environment or during chronic infections (Wilcox et al.,
2004).
[0011] It appears that the immunomodulatory properties of 4-1BB
agonistic antibodies in vivo require the presence of the wild type
Fc-portion on the antibody molecule thereby implicating Fc-receptor
binding as an important event required for the pharmacological
activity of such reagents as has been described for agonistic
antibodies specific to other apoptosis-inducing or immunomodulatory
members of the TNFR-superfamily (Li and Ravetch, 2011; Teng et al.,
2009). However, systemic administration of 4-1BB-specific agonistic
antibodies with the functionally active Fc domain also induces
expansion of CD8+ T-cells associated with liver toxicity (Dubrot et
al., 2010) that is diminished or significantly ameliorated in the
absence of functional Fc-receptors in mice. In human clinical
trials (ClinicalTrials.gov, NCT00309023), Fc-competent 4-1BB
agonistic antibodies (BMS-663513) administered once every three
weeks for 12 weeks induced stabilization of the disease in patients
with melanoma, ovarian or renal cell carcinoma. However, the same
antibody given in another trial (NCT00612664) caused grade 4
hepatitis leading to termination of the trial (Simeone and
Ascierto, 2012).
[0012] Collectively, the available pre-clinical and clinical data
clearly demonstrate that there is a high clinical need for
effective 4-1BB agonists. However, new generation drug candidates
should not only effectively engage 4-1BB on the surface of
hematopoietic and endothelial cells but also be capable of
achieving that through mechanisms other than binding to
Fc-receptors in order to avoid uncontrollable side effects. The
latter may be accomplished through preferential binding to and
oligomerization on tumor-specific or tumor-associated moieties.
[0013] Fusion proteins composed of one extracellular domain of a
4-1BB ligand and a single chain antibody fragment (Mueller et al.,
2008; Hornig et al., 2012) or a single 4-1BB ligand fused to the
C-terminus of a heavy chain (Zhang et al, 2007) have been made. WO
2010/010051 discloses the generation of fusion proteins that
consist of three TNF ligand ectodomains linked to each other and
fused to an antibody part.
[0014] However, there is still a need of new antigen binding
molecules that combine a moiety capable of preferred binding to
tumor-specific or tumor-associated targets with a moiety capable of
forming a costimulatory TNF ligand trimer and that have sufficient
stability to be pharmaceutically useful. The antigen binding
molecules of the present invention comprise both and surprisingly
they provide a trimeric and thus biologically active TNF ligand,
although one of the trimerizing TNF ligand ectodomains is located
on another polypeptide than the other two TNF ligand ectodomains of
the molecule.
SUMMARY OF THE INVENTION
[0015] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises two ectodomains of a TNF ligand family member or two
fragments thereof that are connected to each other by a peptide
linker and in that the second polypeptide comprises only one
ectodomain of said TNF ligand family member or a fragment
thereof.
[0016] In a particular aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first and a second polypeptide that are linked
to each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises only one ectodomain of said
TNF ligand family member or a fragment thereof, and (c) an Fc
domain composed of a first and a second subunit capable of stable
association.
[0017] In a further aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that [0018] (i) the first
polypeptide contains a CH1 or CL domain and the second polypeptide
contains a CL or CH1 domain, respectively, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the first polypeptide
comprises two ectodomains of a TNF ligand family member or
fragments thereof that are connected to each other and to the CH1
or CL domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to the CL or CH1
domain of said polypeptide, or [0019] (ii) the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises only one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide, or [0020] (iii) the first
polypeptide contains a VH-CL or a VL-CH1 domain and the second
polypeptide contains a VL-CH1 domain or a VH-CL domain,
respectively, wherein the second polypeptide is linked to the first
polypeptide by a disulfide bond between the CH1 and CL domain, and
wherein the first polypeptide comprises two ectodomains of a TNF
ligand family member or fragments thereof that are connected to
each other and to to VH or VL by a peptide linker and wherein the
second polypeptide comprises one ectodomain of said TNF ligand
family member or a fragment thereof connected via a peptide linker
to VL or VH of said polypeptide.
[0021] In a particular aspect, the TNF ligand family member is one
that costimulates human T-cell activation. Thus, the TNF family
ligand trimer-containing antigen binding molecule comprises (a) at
least one moiety capable of specific binding to a target cell
antigen and (b) a first and a second polypeptide that are linked to
each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises only one ectodomain of said
TNF ligand family member or a fragment thereof, wherein the TNF
ligand family member costimulates human T-cell activation. More
particularly, the TNF ligand family member is selected from 4-1BBL
and OX40L.
[0022] In one aspect, the TNF ligand family member is 4-1BBL.
[0023] In a further aspect, the ectodomain of a TNF ligand family
member comprises the amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:96, SEQ ID NO: 373, SEQ ID NO:374 and SEQ ID NO:375,
particularly the amino acid sequence of SEQ ID NO:1 or SEQ ID
NO:96.
[0024] In another aspect, the ectodomain of a TNF ligand family
member or fragment thereof comprises the amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4 and SEQ ID NO:96, particularly the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:96. More particularly, the
ectodomain of a TNF ligand family member comprises the amino acid
sequence of SEQ ID NO:96.
[0025] In a further aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence selected from the group
consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID
NO:99 and in that the second polypeptide comprises the amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:96, SEQ ID NO:3 and SEQ ID NO:4.
[0026] In one aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:5 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:6.
[0027] In a further aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:5 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:183.
[0028] In yet a further aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:97 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:184 or SEQ ID NO:185.
[0029] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CH1 or CL domain
and a second polypeptide containing a CL or CH1 domain,
respectively, wherein the second polypeptide is linked to the first
polypeptide by a disulfide bond between the CH1 and CL domain, and
wherein the antigen binding molecule is characterized in that the
first polypeptide comprises two ectodomains of a TNF ligand family
member or two fragments thereof that are connected to each other
and to the CH1 or CL domain by a peptide linker and in that the
second polypeptide comprises only one ectodomain of said TNF ligand
family member or a fragment thereof connected by a peptide linker
to the CL or CH1 domain of said polypeptide.
[0030] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CH1 domain and a
second polypeptide containing a CL domain, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the CH1 domain by a peptide
linker and in that the second polypeptide comprises one ectodomain
of said TNF ligand family member or a fragment thereof connected
via a peptide linker to the CL domain of said polypeptide.
[0031] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CL domain and a
second polypeptide containing a CH1 domain, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the CL domain by a peptide
linker and in that the second polypeptide comprises one ectodomain
of said TNF ligand family member or a fragment thereof connected
via a peptide linker to the CH1 domain of said polypeptide.
[0032] In a further aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule as defined herein
before, wherein the moiety capable of specific binding to a target
cell antigen is selected from the group consisting of an antibody,
an antibody fragment and a scaffold antigen binding protein.
[0033] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule as defined herein
before, wherein the moiety capable of specific binding to a target
cell antigen is an antibody fragment.
[0034] In particular, the moiety capable of specific binding to a
target cell antigen is selected from the group consisting of an
antibody fragment, a Fab molecule, a crossover Fab molecule, a
single chain Fab molecule, a Fv molecule, a scFv molecule, a single
domain antibody, an aVH and a scaffold antigen binding protein.
[0035] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule as defined herein
before, wherein the moiety capable of specific binding to a target
cell antigen is a scaffold antigen binding protein.
[0036] In a particular aspect, the invention is concerned with a
TNF family ligand trimer-containing antigen binding molecule as
defined above, wherein the moiety capable of specific binding to a
target cell antigen is a Fab molecule capable of specific binding
to a target cell antigen.
[0037] The invention provides a TNF family ligand trimer-containing
antigen binding molecule that comprises at least one moiety capable
of specific binding to a target cell antigen. In a particular
aspect, the TNF family ligand trimer-containing antigen binding
molecule comprises one moiety capable of specific binding to a
target cell antigen. In another aspect, the invention provides a
TNF family ligand trimer-containing antigen binding molecule
comprising two moieties capable of specific binding to a target
cell antigen.
[0038] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the target cell antigen is selected from the group
consisting of Fibroblast Activation Protein (FAP), Carcinoembryonic
Antigen (CEA), Melanoma-associated Chondroitin Sulfate Proteoglycan
(MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20 and
CD33.
[0039] In a particular aspect, the target cell antigen is
Fibroblast Activation Protein (FAP).
[0040] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to FAP comprises a VH domain comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7 or SEQ
ID NO:100, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:8 or SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:102, and a VL domain
comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:10 or SEQ ID NO:103, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:11 or SEQ ID NO:104, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:12 or SEQ ID
NO:105.
[0041] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to FAP comprises a VH domain comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7, (ii)
CDR-H2 comprising the amino acid sequence of SEQ ID NO:8 and (iii)
CDR-H3 comprising the amino acid sequence of SEQ ID NO:9, and a VL
domain comprising (iv) CDR-L1 comprising the amino acid sequence of
SEQ ID NO:10, (v) CDR-L2 comprising the amino acid sequence of SEQ
ID NO:11 and (vi) CDR-L3 comprising the amino acid sequence of SEQ
ID NO:12.
[0042] In a particular aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, wherein the
moiety capable of specific binding to FAP comprises a VH domain
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:100, (ii) CDR-H2 comprising the amino acid sequence SEQ ID
NO:101, and (iii) CDR-H3 comprising the amino acid sequence of SEQ
ID NO:102, and a VL domain comprising (iv) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:103, (v) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:104, and (vi) CDR-L3 comprising
the amino acid sequence of SEQ ID NO:105.
[0043] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule as defined herein
before, wherein the moiety capable of specific binding to FAP
comprises a variable heavy chain comprising an amino acid sequence
of SEQ ID NO:16 and a variable light chain comprising an amino acid
sequence of SEQ ID NO:17 or wherein the moiety capable of specific
binding to FAP comprises a variable heavy chain comprising an amino
acid sequence of SEQ ID NO:106 and a variable light chain
comprising an amino acid sequence of SEQ ID NO:107.
[0044] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule according to the
invention, wherein a peptide comprising two ectodomains of a TNF
ligand family member or fragments thereof connected to each other
by a first peptide linker is fused at its C-terminus to the CH1 or
CL domain of a heavy chain by a second peptide linker and wherein
one ectodomain of said TNF ligand family member or a fragment
thereof is fused at the its C-terminus the CL or CH1 domain on a
light chain by a third peptide linker.
[0045] In a particular aspect, the invention relates to a TNF
family ligand trimer-containing antigen binding molecule as defined
above, wherein the peptide linker is (G.sub.4S).sub.2, i.e. a
peptide linker of SEQ ID NO:13. In one aspect, the first peptide
linker is (G.sub.4S).sub.2 (SEQ ID NO:13), the second peptide
linker is GSPGSSSSGS (SEQ ID NO:57) and the third peptide linker is
(G.sub.4S).sub.2 (SEQ ID NO:13). In another aspect, the first, the
second and the third peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13).
[0046] The invention is further concerned with a TNF family ligand
trimer-containing antigen binding molecule as defined herein
before, comprising an Fc domain composed of a first and a second
subunit capable of stable association.
[0047] In particular, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprising (c) an Fc
domain composed of a first and a second subunit capable of stable
association further comprises (a) a Fab molecule capable of
specific binding to a target cell antigen, wherein the Fab heavy
chain is fused at the C-terminus to the N-terminus of a CH2 domain
in the Fc domain.
[0048] In a further aspect, the Fc domain is an IgG, particularly
an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc
domain is an IgG1 Fc domain. In a particular aspect, the Fc domain
comprises a modification promoting the association of the first and
second subunit of the Fc domain.
[0049] In another aspect, the invention is concerned with a TNF
family ligand trimer-containing antigen binding molecule as defined
herein before, comprising
(c) an Fc domain composed of a first and a second subunit capable
of stable association, wherein the Fc domain comprises one or more
amino acid substitution that reduces binding to an Fc receptor, in
particular towards Fc.gamma. receptor.
[0050] In particular, the Fc domain comprises amino acid
substitutions at positions 234 and 235 (EU numbering) and/or 329
(EU numbering) of the IgG heavy chains. More particularly, provided
is a trimeric TNF family ligand-containing antigen binding molecule
according to the invention which comprises an IgG1 Fc domain with
the amino acid substitutions L234A, L235A and P329G (EU
numbering).
[0051] In a further aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, wherein the
antigen binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
first peptide comprising two ectodomains of a TNF ligand family
member or fragments thereof connected to each other by a first
peptide linker fused at its C-terminus by a second peptide linker
to a second heavy or light chain, and a second peptide comprising
one ectodomain of said TNF ligand family member fused at its
C-terminus by a third peptide linker to a second light or heavy
chain, respectively.
[0052] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the first
peptide comprising two ectodomains of a TNF ligand family member or
fragments thereof connected to each other by a first peptide linker
is fused at its C-terminus by a second peptide linker to a CH1
domain that is part of a heavy chain,
and the second peptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof is fused at its C-terminus by a
third peptide linker to a CL domain that is part of a light
chain.
[0053] In yet another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the first
peptide comprising two ectodomains of a TNF ligand family member or
fragments thereof connected to each other by a first peptide linker
is fused at its C-terminus by a second peptide linker to a CL
domain that is part of a heavy chain,
and the second peptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof is fused at its C-terminus by a
third peptide linker to a CH1 domain that is part of a light
chain.
[0054] In a further aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, wherein the
first peptide comprising two ectodomains of a TNF ligand family
member or fragments thereof connected to each other by a first
peptide linker is fused at its C-terminus by a second peptide
linker to a VH domain that is part of a heavy chain,
and the second peptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof is fused at its C-terminus by a
third peptide linker to a VL domain that is part of a light
chain.
[0055] Provided is further a TNF family ligand trimer-containing
antigen binding molecule, wherein in the CL domain adjacent to the
TNF ligand family member the amino acid at position 123 (EU
numbering) has been replaced by arginine (R) and the amino acid at
position 124 (EU numbering) has been substituted by lysine (K), and
wherein in the CH1 domain adjacent to the TNF ligand family member
the amino acids at position 147 (EU numbering) and at position 213
(EU numbering) have been substituted by glutamic acid (E).
[0056] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule as described herein
before, wherein the antigen binding molecule comprises
(a) a first heavy chain and a first light chain, both comprising a
Fab molecule capable of specific binding to a target cell antigen,
(b) a second heavy chain comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID
NO:98 and SEQ ID NO:99, and a second light chain comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ ID NO:4.
[0057] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
(a) a Fab molecule capable of specific binding to FAP, and (b) a
second heavy chain comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98 and
SEQ ID NO:99, and a second light chain comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:96, SEQ ID NO:3 and SEQ ID NO:4.
[0058] In a particular aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising a
moiety capable of specific binding to FAP. In one aspect, provided
is a TNF family ligand trimer-containing antigen binding molecule,
wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:107, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:14, SEQ ID
NO:108, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO. 115, SEQ ID NO:139
and SEQ ID NO:148, and (iii) a second light chain comprising the
amino acid sequence of SEQ ID NO:15, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:114 and SEQ ID NO:115.
[0059] In another aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, wherein the
antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of
[0060] SEQ ID NO:107,
(ii) a second heavy chain comprising the amino acid sequence
selected from the group consisting of SEQ ID NO:115, SEQ ID NO:117,
SEQ ID NO:119 and SEQ ID NO:173, and (iii) a second light chain
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120 and SEQ
ID NO:174.
[0061] In yet another aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide.
[0062] In particular, such a TNF family ligand trimer-containing
antigen binding molecule comprises two moieties capable of specific
binding to a target cell antigen.
[0063] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising two moieties
capable of specific binding to FAP. In particular, provided is a
TNF family ligand trimer-containing antigen binding molecule as
described herein before comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:121, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:122, and two light chains comprising the amino acid
sequence of SEQ ID NO:19, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:123, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:124, and two light
chains comprising the amino acid sequence of SEQ ID NO:125, or
(iii) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:126, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:127, and two light chains comprising the amino acid
sequence of SEQ ID NO:125.
[0064] In another particular aspect, the invention provides a TNF
family ligand trimer-containing antigen binding molecule as
described herein before, wherein the target cell antigen is
CD19.
[0065] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CD19 comprises a VH domain
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:195 or SEQ ID NO:252, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:196 or SEQ ID NO:253, and (iii) CDR-H3
comprising the amino acid sequence of SEQ ID NO:197 or SEQ ID
NO:254, and a VL domain comprising (iv) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:198 or SEQ ID NO:249, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:199 or SEQ ID
NO:250, and (vi) CDR-L3 comprising the amino acid sequence of SEQ
ID NO:200 or SEQ ID NO:251.
[0066] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CD19 comprises a variable heavy
chain comprising an amino acid sequence of SEQ ID NO:201 and a
variable light chain comprising an amino acid sequence of SEQ ID
NO:202 or wherein the moiety capable of specific binding to FAP
comprises a variable heavy chain comprising an amino acid sequence
of SEQ ID NO:357 and a variable light chain comprising an amino
acid sequence of SEQ ID NO:358.
[0067] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:201 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:202 or a first heavy chain comprising the VH domain
comprising the amino acid sequence of SEQ ID NO:357 and a first
light chain comprising the VL domain comprising the amino acid
sequence of SEQ ID NO:358, (ii) a second heavy chain comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:14, SEQ ID NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a
second light chain comprising the amino acid sequence of SEQ ID
NO:15, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID
NO:114.
[0068] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:201 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:202 or a first heavy chain comprising the VH domain
comprising the amino acid sequence of SEQ ID NO:357 and a first
light chain comprising the VL domain comprising the amino acid
sequence of SEQ ID NO:358, (ii) a second heavy chain comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:115, SEQ ID NO:117, SEQ ID NO:119 and SEQ ID NO:173, and (iii) a
second light chain comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120
and SEQ ID NO:174.
[0069] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising two moieties
capable of specific binding to CD19. In particular, provided is a
TNF family ligand trimer-containing antigen binding molecule of any
one of claims 1 to 14, 29, 30 and 32 to 34, wherein the antigen
binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:209, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:210, and two light chains comprising the amino acid
sequence of SEQ ID NO:206, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:213, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:214, and two light
chains comprising the amino acid sequence of SEQ ID NO:206, or
(iii) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:309, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:310, and two light chains comprising the amino acid
sequence of SEQ ID NO:279, or (iv) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:313, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:314, and two light
chains comprising the amino acid sequence of SEQ ID NO:279.
[0070] In another particular aspect, the invention provides a TNF
family ligand trimer-containing antigen binding molecule as
described herein before, wherein the target cell antigen is
CEA.
[0071] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CEA comprises a VH domain comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:321,
(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:322,
and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID
NO:323, and a VL domain comprising (iv) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:324, (v) CDR-L2 comprising the amino
acid sequence of SEQ ID NO:325, and (vi) CDR-L3 comprising the
amino acid sequence of SEQ ID NO:326.
[0072] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CEA comprises a variable heavy chain
comprising an amino acid sequence of SEQ ID NO:329 and a variable
light chain comprising an amino acid sequence of SEQ ID NO:330.
[0073] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule as described herein
before, wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:329 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:330, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:14, SEQ ID
NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light
chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
[0074] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:329 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:330, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:115, SEQ
ID NO:117, SEQ ID NO:119 and SEQ ID NO:173, and (iii) a second
light chain comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120 and
SEQ ID NO:174.
[0075] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising two moieties
capable of specific binding to CEA. Particularly, provided is a TNF
family ligand trimer-containing antigen binding molecule, wherein
the antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:337, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:338, and two light chains comprising the amino acid
sequence of SEQ ID NO:334, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:341, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:342, and two light
chains comprising the amino acid sequence of SEQ ID NO:334.
[0076] In a further aspect, provided is aTNF family ligand
trimer-containing antigen binding molecule as described herein
before, wherein the TNF ligand family member is OX40L. In one
aspect, provided is TNF family ligand trimer-containing antigen
binding molecule, wherein the ectodomain of a TNF ligand family
member comprises the amino acid sequence of SEQ ID NO:53 or SEQ ID
NO:54, particularly the amino acid sequence of SEQ ID NO:53.
[0077] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of any one of claims 1
to 5, 10 to 24, 29, 30, 32 to 34, 38 to 40, 44 and 45,
comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:371 or SEQ ID:372
and in that the second polypeptide comprises the amino acid
sequence of SEQ ID NO:53 or SEQ ID NO:54.
[0078] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the target cell
antigen is Fibroblast Activation Protein (FAP) and the moiety
capable of specific binding to FAP comprises a VH domain comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7 or SEQ
ID NO:100, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:8 or SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:102, and a VL domain
comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:10 or SEQ ID NO:103, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:11 or SEQ ID NO:104, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:12 or SEQ ID
NO:105.
[0079] Particularly, provided is a TNF family ligand
trimer-containing antigen binding molecule as described herein,
wherein the antigen binding molecule comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:107, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:355, and
(iii) a second light chain comprising the amino acid sequence of
SEQ ID NO:356.
[0080] According to another aspect of the invention, there is
provided an isolated polynucleotide encoding a TNF family ligand
trimer-containing antigen binding molecule as defined herein
before. The invention further provides a vector, particularly an
expression vector, comprising the isolated polynucleotide of the
invention and a host cell comprising the isolated polynucleotide or
the vector of the invention. In some embodiments the host cell is a
eukaryotic cell, particularly a mammalian cell.
[0081] In another aspect, provided is a method for producing the
TNF family ligand trimer-containing antigen binding molecule of the
invention, comprising the steps of (i) culturing the host cell of
the invention under conditions suitable for expression of the
antigen binding molecule, and (ii) recovering the antigen binding
molecule. The invention also encompasses a TNF family ligand
trimer-containing antigen binding molecule produced by the method
of the invention.
[0082] The invention further provides a pharmaceutical composition
comprising the TNF family ligand trimer-containing antigen binding
molecule of the invention and at least one pharmaceutically
acceptable excipient.
[0083] Also encompassed by the invention is the TNF family ligand
trimer-containing antigen binding molecule of the invention, or the
pharmaceutical composition of the invention, for use as a
medicament. In one aspect is provided the TNF family ligand
trimer-containing antigen binding molecule of the invention, or the
pharmaceutical composition of the invention, for use in the
treatment of a disease in an individual in need thereof. In a
specific embodiment, provided is the TNF family ligand
trimer-containing antigen binding molecule of the invention, or the
pharmaceutical composition of the invention, for use in the
treatment of cancer.
[0084] Also provided is the use of the TNF family ligand
trimer-containing antigen binding molecule of the invention for the
manufacture of a medicament for the treatment of a disease in an
individual in need thereof, in particular for the manufacture of a
medicament for the treatment of cancer, as well as a method of
treating a disease in an individual, comprising administering to
said individual a therapeutically effective amount of a composition
comprising the TNF family ligand trimer-containing antigen binding
molecule of the invention in a pharmaceutically acceptable form. In
a specific embodiment, the disease is cancer. In any of the above
embodiments the individual is preferably a mammal, particularly a
human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIGS. 1A to 1D show the components for the assembly of split
trimeric human 4-1BB ligands including linker GGGGSGGGGS (SEQ ID
NO:13). FIG. 1A shows the dimeric ligand that is fused at the
C-terminus to a human CH1 or CL domain or a VL or VH domain and
FIG. 1B shows the monomeric ligand fused to human CL or CH1 domain
or a VL or VH domain. FIG. 1C shows the dimeric ligand that is
fused at the N-terminus to a human CH3 domain and FIG. 1D shows the
monomeric ligand fused at the N-terminus to a human CH3 domain.
[0086] FIGS. 2A to 2J show the 4-1BBL-trimer-containing antigen
binding molecules Constructs 1.1 to 1.10 of the invention. The
preparation and production of these constructs is described in
Example 1. The VH and VL domains are those of anti-FAP antibody
28H1, the thick black point stands for the knob-into-hole
modification. * symbolizes amino acid modifications in the CH1 and
CL domain (so-called charged residues).
[0087] FIGS. 3A to 3C show the components for the assembly of split
trimeric murine 4-1BB ligands including linker GGGGSGGGGS (SEQ ID
NO:13). FIG. 3A shows the dimeric ligand that is fused at the
C-terminus to murine CL domain and FIG. 3B shows the monomeric
ligand fused at the C-terminus to murine CH1 domain. Components for
the assembly of FAP targeted split trimeric murine 4-1BB ligand.
FIG. 3C shows the assembled murine 4-1BBL-trimer-containing antigen
binding molecules as described in more detail in Example 1.3.
[0088] FIGS. 4A to 4F show the 4-1BBL-trimer-containing antigen
binding molecules Constructs 2.1 to 2.6 of the invention. The
preparation and production of these constructs is described in
Example 2. The VH and VL domains are those of anti-FAP antibody
4B9, the thick black point stands for the knob-into-hole
modification. * symbolizes amino acid modifications in the CH1 and
CL domain (so-called charged residues).
[0089] FIGS. 5A and 5B show the "untargeted" variants of Constructs
1.1 and 1.2 comprising a DP47 Fab molecule instead of the anti-FAP
Fab molecule. The molecules are named Control A and Control B,
respectively. The preparation is described in Example 1.4. FIG. 5C
is a drawing of the monomeric 4-1BB Fc(kih) construct as prepared
in Example 3.
[0090] FIGS. 6A-1 to 6C-2 relate to the binding of FAP-targeted
4-1BB ligand trimer-containing Fc(kih) fusion antigen binding
molecule (FAP split 4-1BBL trimer, filled circles) or DP-47
untargeted 4-1BB ligand trimer-containing Fc(kih) fusion antigen
binding molecule (DP47 split 4-1BBL trimer, open circles) to
resting (naive) or activated human PMBCs. Specifically, the binding
to resting (naive) or activated human CD8+ T cells is shown in
FIGS. 6A-1 and 6A-2, to resting (naive) or activated human CD4+ T
cells in FIGS. 6B-1 and 6B-2 and to resting (naive) or activated
human NK cells in FIGS. 6-C1 and 6-C2. Shown is the binding as
Median of fluorescence intensity (MFI) of red macrophytic algae
Phycoerythrin (R-PE)-labeled anti-human IgG Fc.gamma.-specific goat
IgG F(ab')2 fragment which is used as secondary detection antibody.
MFI was measured by flow cytometry and baseline corrected by
subtracting the WI of the blank control.
[0091] FIGS. 7A-1 to 7B-4 show the binding of different
FAP-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs to human 4-1BB expressing T cells from PHA-L and
Proleukin pre-activated and anti-human CD3/anti-human CD28
re-activated human PBMCs. Binding was detected with
R-Phycoerythrin-fluorochrome conjugated anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment. Shown is the median
of fluorescence intensity (MFI) versus the concentration of tested
Constructs 1.1 to 1.10 of Example 1. For a better display the
binding curves are split in four different blots with Construct 1.1
(monovalent FAP-targeted split trimeric human 4-1BB ligand Fc
(kih)) and Control B (monovalent untargeted split trimeric human
4-1BB ligand Fc (kih) with CH-CL cross and charged residues) as
comparison curves. Binding was monitored on CD3+CD8+ T cells (FIGS.
7A-1 to 7A-4) and CD3+CD4+ T cells (FIGS. 7B-1 to 7B-4). The 4-1BB
expression level on CD8 T cells is normally higher than on CD4 T
cells. All versions bind with a quite similar affinity to human
4-1BB.
[0092] FIGS. 8A-1 to 8B-4 show the binding of different
FAP-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs to CD4+ or CD8+ T cells from fresh PBMCs (FIGS.
8A-1 to 8A-4) or to human 4-1BB expressing PHA-L and Proleukin
pre-activated and anti-human CD3/anti-human CD28 re-activated human
PBMCs (FIGS. 8B-1 to 8B-4). Binding was detected with
R-Phycoerythrin-fluorochrome conjugated anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment. Shown is the median
of fluorescence intensity (MFI) versus the concentration of tested
Constructs 2.1, 2.3, 2.4, 2.5 and 2.6 of Example 2 and control
molecules Control B,
[0093] Control C, Control E and Control F. For a better display the
binding curves are split in two different blots with construct 2.1
(monovalent FAP-targeted split trimeric human 4-1BB ligand Fc
(kih)) and control B (monovalent untargeted split trimeric human
4-1BB ligand Fc (kih) with CH-CL cross and charged residues) as
comparison curves. Binding was monitored on CD45+CD3+CD8+ T cells
(blots on the bottom) and CD45+CD3+CD4+ T cells (blots on the top).
The 4-1BB expression level on CD8 T cells is normally higher than
on CD4 T cells. All constructs bind with a quite similar affinity
to human 4-1BB, whereas the bivalent construct 2.3 and its
untargeted control C show a lower MFI. This can be due to sterical
hindrance of 4-1BB-binding and/or less detection due to the 2nd
detection antibody induced by the Fc-conjugated split 4-1BB
ligand.
[0094] FIGS. 9A and 9B show the binding of FAP-targeted 4-1BB
ligand trimer-containing Fc(kih) fusion antigen binding molecules
(FAP split 4-1BBL trimer, filled circles) or DP47 untargeted 4-1BB
ligand trimer-containing Fc(kih) fusion antigen binding molecules
(DP47 split 4-1BBL trimer; open circles) to activated mouse
splenocytes. In particular, the binding to activated mouse CD4+ T
cells is shown in FIG. 9A and to activated mouse CD8+ T cells in
FIG. 9B. An anti-mouse CD137-specific human IgG1 P329G LALA
antibody (clone Lob12.3) was used as positive control (Triangles).
The binding is characterized by plotting the MFI of R-PE-labeled
anti-human IgG Fc.gamma.-specific goat IgG F(ab')2 Fragment that is
Used as Secondary detection antibody versus the concentration in nM
of the tested split 4-1BBL trimer constructs. MFI was measured by
flow cytometry and baseline corrected by subtracting the MFI of the
blank control.
[0095] FIGS. 10A and 10B show the binding of 4-1BB ligand
trimer-containing Fc(kih) fusion antigen binding molecules (filled
circles: FAP-targeted 4-1BB ligand trimer-containing Fc(kih) fusion
antigen binding molecule Construct 1.1, open circles: DP47
untargeted 4-1BB ligand trimer-containing Fc(kih) fusion antigen
binding molecule Control A to fibroblast activation protein
(FAP)-expressing human melanoma (FIG. 10A) MV-3 cell line and (FIG.
10B) WM-266-4 cell line. The binding is characterized by plotting
the MFI of R-PE-labeled anti-human IgG Fc.gamma.-specific goat IgG
F(ab')2 fragment that is used as secondary detection antibody
versus the concentration in nM of tested split 4-1BBL trimer
constructs. MFI was measured by flow cytometry and baseline
corrected by subtracting the MFI of the blank control.
[0096] FIGS. 11A-1 to 11B-2 show the binding of different
FAP-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs to human-FAP expressing human melanoma MV-3 cells
(FIGS. 11A-1 to 11A-4) and/or NIH/3T3-huFAP clone 39 transfected
mouse embryonic fibroblast cells (FIGS. 11B-1 and 11B-2). Binding
was detected with R-Phycoerythrin-fluorochrome or
fluorescein-fluorochrome conjugated anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragments. Shown is the median
of fluorescence intensity (MFI) versus the concentration of tested
constructs. For a better display binding curves were distributed to
four (FIGS. 11A-1 to 11A-4) or two blots (FIGS. 11B-1 and 11B-2),
whereas construct 1.1 (monovalent FAP-targeted split trimeric human
4-1BB ligand Fc (kih)) is used a comparison curve. All constructs
bind with a similar affinity to human FAP except the bivalent
FAP-targeted constructs (constructs 1.5, 1.7 and 1.8). They showed
a tendency to have lower EC50 values and lower median fluorescence
intensity. This can be explained with their bivalent targeting
(higher avidity, less molecules can bind at the same time due to
occupancy of two epitopes resulting in a lower MFI). Structural
differences may also explain the difference between Construct 1.8
(complete bivalent targeting) and Constructs 1.5 and 1.7 (only
partial bivalent targeting).
[0097] FIGS. 12A-1 to 12B-2 show the binding of different
FAP-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) Constructs 2.1, 2.3, 2.4, 2.5 and 2.6 to human-FAP expressing
human melanoma MV-3 cells (FIGS. 12A-1 and 12A-2) and WM-266-4
cells (FIGS. 12B-1 and 12B-2). Binding was detected with
R-Phycoerythrin-fluorochrome conjugated anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragments. Shown is the median
of fluorescence intensity (MFI) versus the concentration of tested
contructs. For a better display binding curves were distributed to
two blots, whereas construct 2.1 (monovalent FAP-targeted split
trimeric human 4-1BB ligand Fc (kih)) is used as comparison curve.
All constructs bind with a similar affinity to human FAP except the
bivalent FAP-targeted construct 2.3. It has a tendency to show
lower EC50 values and lower median fluorescence intensity. This can
be explained with its bivalent targeting, which results in higher
avidity but less occupancy or FAP molecules on the cell surface
resulting in a lower MFI.
[0098] FIGS. 13A-1 to 13B-2 show the binding of different
FAP-targeted or untargeted split trimeric mouse 4-1BB ligand Fc
(kih) constructs to CD4+ or CD8+ T cells from fresh splenocytes
(FIGS. 13A-1 and 13A-2) or to mouse 4-1BB expressing anti-mouse
CD3/anti-mouse CD28 monoclonal agonistic antibodies activated mouse
splenocytes (FIGS. 13B-1 and 13B-2). Binding was detected with
FITC-fluorochrome conjugated anti-mouse IgG Fc.gamma.-specific goat
IgG F(ab')2 fragment. Shown is the median of fluorescence intensity
(MFI) versus the concentration of tested constructs. Binding was
monitored on CD3+CD8+ T cells (left blot) and CD3+CD4+ T cells
(right blot). The 4-1BB expression level on CD8 T cells is normally
higher than on CD4 T cells. All constructs bind with a quite
similar affinity to mouse 4-1BB.
[0099] FIGS. 14A and 14B show the binding of different FAP-targeted
or untargeted split trimeric mouse 4-1BB ligand Fc (kih) constructs
to human FAP expressing tumor cells. Binding was detected with
FITC-fluorochrome conjugated anti-mouse IgG Fc.gamma.-specific goat
IgG F(ab')2 fragment. Shown is the median of fluorescence intensity
(MFI) versus the concentration of tested constructs. Binding was
monitored on MV-3 cells (FIG. 14A) and WM-266-4 cells (FIG. 14B).
FAP-targeted split trimeric mouse 4-1BB ligand Fc (kih) constructs
M.1 and M.2 bind with a quite similar affinity to FAP.
[0100] FIGS. 15A and 15B show a scheme that illustrates the general
principal of the NFkB activity assay described in Example 6.1 using
a reporter cell line. Shown is the activation assay set up with
human 4-1BB expressing HeLa reporter cell line. A crosslinking of
4-1BB expressed on the reporter cells induces NF.kappa.B activation
and NF.kappa.B-mediated Luciferase expression. After lysis of the
cells Luciferase can catalyze the oxidation of Luciferin to
Oxyluciferin. This chemical reaction correlates positively with the
strength of NF.kappa.B-mediated luciferase expression and can be
measured by the strength of light emission (units of released
light). The ratio of FAP-expressing tumor cells to the reporter
cell line HeLa-huCD137-NFkB-luc was 5 to 1.
[0101] FIGS. 16A to 16C show that the activation of the NFkB
signaling pathway by FAP-targeted 4-1BB ligand trimer-containing
Fc(kih) fusion antigen binding molecule (Construct 1.1) is strictly
dependent on its binding to FAP-expressing target cells. Human
CD137 expressing NFkB reporter HeLa cells were co-cultured with the
indicated tumor cells exhibiting different levels of cell surface
FAP expression. Luciferase activity was assessed as described in
Example 6.1 after culturing cells in the absence or presence of
4-1BBL-containing molecules at the indicated concentrations for 6
hours. Filled circles refer to Construct 1.1. Open circles refer to
DP47 untargeted 4-1BB ligand trimer-containing Fc(kih) fusion
antigen binding molecule (Control A). Cell line NIH/3T3-human FAP
clone 39 was used as target cells in 16A; 16B shows the activation
with MV3 cell line as target cells and 16C with WM-266-4 cell line
as target cells. Activity is characterized by blotting the units of
released light (URL) measured during 0.5 s versus the concentration
in nM of tested split 4-1BBL trimer constructs. URLs are emitted
due to luciferase-mediated oxidation of luciferin to
oxyluciferin.
[0102] FIGS. 17A-1 to 17C-4 shows the NF.kappa.B-activation-induced
Luciferase expression and activity as measured with the assay
described in Example 6.1. Counts of released light per seconds
(CPS) are measured for 0.5 s/well and plotted against the used
concentration of FAP-targeted or untargeted split trimeric human
4-1BB ligand Fc (kih) constructs. Human 4-1BB-expressing
HeLa-reporter cells were incubated for 6 h in the absence (FIGS.
17A-1 to 17A-4) or presence of crosslinking human-FAP expressing
human melanoma cell line MV-3 (FIGS. 17B-1 to 17B-4) or WM-266-4
(FIGS. 17C-1 to 17C-4). CPS were measured and blotted against the
concentrations of different FAP-targeted or untargeted split
trimeric human 4-1BB ligand Fc (kih) Constructs. The cell ratio is
one human 4-1BB-expressing HeLa reporter cell to five tumor cells.
For better display, activation curves were split to four different
display-blots with construct 1.1 (monovalent FAP-targeted split
trimeric human 4-1BB ligand Fc (kih)) and control B (monovalent
untargeted split trimeric human 4-1BB ligand Fc (kih) with CH-CL
cross and charged residues) as comparison curves. FIGS. 17A-1 to
17D-4 show the activation without crosslinking FAP-expressing tumor
cells, FIGS. 17B-1 to 17B-4 show the activation in the presence of
crosslinking FAP-expressing MV-3 tumor cells and FIGS. 17C-1 to
17C-4 show the activation in the presence of crosslinking
FAP-expressing WM-266-4 tumor cells.
[0103] FIGS. 18A to 18F show the NF.kappa.B-activation-induced
Luciferase expression and activity as measured for the constructs
of Example 2. Units of released light (URL) are measured for 0.5
s/well and plotted against the used concentration of FAP-targeted
or untargeted split trimeric human 4-1BB ligand Fc (kih)
constructs. Human 4-1BB-expressing HeLa-reporter cells were
incubated for 6 h in the absence or presence of crosslinking
human-FAP expressing human melanoma cell line MV-3 or WM-266-4.
URLs were measured and blotted against the concentrations of
different FAP-targeted or untargeted split trimeric human 4-1BB
ligand Fc (kih) constructs 2.1, 2.3, 2.4, 2.5 and 2.6 and Controls
B, C, E and F. The cell ratio is one 4-1BB-expressing HeLa reporter
cell to five tumor cells. For better display activation curves were
split to two different display-blots with construct 2.1 (monovalent
FAP-targeted split trimeric human 4-1BB ligand Fc (kih)).
[0104] FIGS. 19A and 19B show the activation assay set up with
cynomolgus monkey 4-1BB expressing T293-HEK reporter cell line. A
crosslinking of cynomolgus monkey 4-1BB expressed on the reporter
cells induces NF.kappa.B activation and NF.kappa.B-mediated
Luciferase expression. After lysis of the cells Luciferase can
catalyze the oxidation of Luciferin to Oxyluciferin. This chemical
reaction correlates positively with the strength of
NF.kappa.B-mediated luciferase expression and can be measured by
the strength of light emission (units of released light).
[0105] FIGS. 20A to 20F show the NF.kappa.B-activation-induced
Luciferase expression and activity. Units of released light (URL)
are measured for 0.5 s/well and plotted against the used
concentration of FAP-targeted or untargeted split trimeric human
4-1BB ligand Fc (kih) constructs. Cynomolgus monkey
4-1BB-expressing T293-HEK-reporter cells were incubated for 6 h in
the absence or presence of crosslinking human-FAP expressing human
melanoma cell line MV-3 or WM-266-4. URLs were measured and blotted
against the concentrations of different FAP-targeted or untargeted
split trimeric human 4-1BB ligand Fc (kih) constructs. The cell
ratio is one 4-1BB-expressing T293-HEK reporter cell to five MV-3
or two WM-266-4 cells. For better display activation curves were
split to two different blots with Construct 2.1 as comparison
curve.
[0106] FIGS. 21A and 21B show a scheme illustrating the principal
of the T-cell activation assay described in Example 6.3. Shown is
the schematic assay activation set up with HLA-A2-NLV-specific CD8
T cells and NLV-pulsed HLA-A2+ FAP+ human melanoma cell line MV-3
in the presence of different titrated concentration of FAP-targeted
or untargeted split trimeric human 4-1BB ligand Fc (kih)
constructs. Cells were incubated for 28 h, the last 4 h in the
presence of monesin-containing Golgi-Stop. The ratio of
NLV-specific CD8 T cells to MV-3 tumor cells is 1:8.
[0107] FIGS. 22A-1 to 22E-3 and 23A-1 to 23E-3 relate to the
Activation assay with HLA-A2-NLV-specific CD8 T cells and
NLV-pulsed HLA-A2+ FAP+ human melanoma cell line MV-3 in the
presence of different titrated concentration of different
FAP-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs as prepared in Example 1. For better display
expression curves were split to several different display-blots
with Construct 1.1 (monovalent FAP-targeted split trimeric human
4-1BB ligand Fc (kih)) and Control B (monovalent untargeted split
trimeric human 4-1BB ligand Fc (kih)) as comparison curves. Results
were obtained in four independent similar experiments and show that
prolonged IFN.gamma. secretion and CD137 expression of NLV-specific
CD8+ T cells is strictly dependent on simultaneous activation of
T-cells via recognition of NLV-HLA-A2 complexes (signal 1) and
4-1BB-triggering by FAP-targeted human split 4-1BBL (signal 2). The
effect of 4-1BB upregulation is shown in graphs of FIGS. 22A-1 to
22E-3, whereas the effect of INF.gamma. expression of CD8+ T cells
is presented in graphs of FIGS. 23A-1 to 23E-3. Shown is always the
frequency in percentage of positive cells in the total CD8+ T cell
population. All FAP-targeted variants induced a similar activation
improvement of NLV-peptide activated CD8 T cells shown in FIGS.
22A-1 to 22E-3 as 4-1BB-upregulation (positive feedback loop) and
in FIGS. 23A-1 to 23E-3 as IFN.gamma. expression after 24 h of
stimulation. Differences of curves lie in the range of normal error
deviation and are not significant.
[0108] FIGS. 24A-1 to 24B-3 and 25A-1 to 25B-3 refer to the
Activation assay with HLA-A2-NLV-specific CD8 T cells and
NLV-pulsed HLA-A2+ FAP+ human melanoma cell line MV-3 in the
presence of titrated concentration of different FAP-targeted or
untargeted split trimeric human 4-1BB ligand Fc (kih) constructs of
Example 2. For better display expression curves were split to two
different display-blots with Construct 2.1 (monovalent FAP-targeted
split trimeric human 4-1BB ligand Fc (kih)) and Control B as
comparison curves. All FAP-targeted split trimeric human 4-1BB
ligand Fc (kih) constructs show a similar activation improvement of
HLA-A2-NLV-peptide specific CD8 T cells shown in FIGS. 24A-1 to
24B-3 as 4-1BB-upregulation (positive feedback loop) and in FIGS.
25A-1 to 25B-3 as IFN.gamma. expression after 24 h of stimulation.
Differences of curves lie in the range of normal error deviation
and are not significant.
[0109] FIGS. 26A and 26B show a scheme illustrating the experiment
as described in Example 6.4.
[0110] FIG. 27 shows the induction of CD8+ T cell proliferation.
Shown is the frequency of proliferating CD8+ T cells versus the
concentration of tested constructs.
[0111] FIG. 28A relates to the single dose PK experiment of
Construct 1.2 and Control B in healthy NOG mice. Shown is the
decline in Construct concentration over the time. FIG. 28B shows
the results of the single dose PK experiment of Constructs 2.1,
2.3, Control B and Control C in tumor bearing NOG mice humaniced
with stem cells. FIG. 28C relates to the single dose PK experiment
comparing Construct 2.1 and 2.3 in healthy NOG mice.
[0112] FIGS. 29A to 29D show components for the assembly of split
trimeric human 4-1BB ligands including linker GGGGSGGGGS (SEQ ID
NO:13). FIG. 29A shows the dimeric ligand that is fused at the
C-terminus to a human CL domain with mutations E123R and Q124K
(charged residues) and FIG. 29B shows the monomeric 4-1BB ligand
fused to human CH1 domain with mutations K147E and K213E (charged
residues). Components for the assembly of bivalent CD19-targeted
split trimeric human 4-1BB ligand (71-254) antigen binding molecule
(construct 3.3). FIG. 29C shows the dimeric ligand being fused to
the C-terminus of human IgG1 Fc hole chain. FIG. 29D shows the
monomeric ligand being fused to the C-terminus of human IgG1 Fc
knob chain.
[0113] FIGS. 30A to 30F show the CD19-targeted
4-1BBL-trimer-containing antigen binding molecules Constructs 3.1
to 3.6 of the invention. The preparation and production of these
constructs is described in Example 3. The VH and VL domains are
those of anti-CD19 antibody 8B8-018, the thick black point stands
for the knob-into-hole modification. * symbolizes amino acid
modifications in the CH1 and CL domain (so-called charged
residues).
[0114] FIGS. 31A-1 to 31A-2 illustrate the randomization strategy
for the CDR regions of the parental clone 8B8. Shown are the
variable domains of the parental clone 8B8 and the CDR regions
(boxed) according to the numbering of Kabat. (X) (SEQ ID NO:202;
SEQ ID NO:201) represents the randomized positions. FIGS. 31B-1 to
31B-2 show the schematic description of the library generation
strategies. Shown is the PCR amplification and cloning strategy
used for the generation of the 8B8-based library with A) randomized
CDR1 and CDR2 regions in the light and heavy chain or B) randomized
CDR1 and CDR3 regions in the light and CDR3 region in the heavy
chain. Respective enzymes used for cloning into the phagemide are
indicated.
[0115] FIGS. 32A and 32B show the alignment of the parental
anti-CD19 clone 8B8 (SEQ ID NO:202; SEQ ID NO:201) with the
selected affinity-matured binders. Shown are the sequences of clone
8B8 and all selected affinity-matured binders. CDRs of both heavy
and light chains are framed (SEQ ID NOs:231-272).
[0116] FIGS. 33A to 33H relate to the SPR analysis of the parental
8B8 clone and its affinity-matured variants. Shown are the
sensorgrams of clone 8B8 and its affinity-matured derivatives that
are devoid of the LCDR1 N27d and N28 hotspots.
[0117] FIG. 34 illustrates the setup of the assay measuring
Simultaneous binding of CD19 targeted trimeric split 4-1BBL to
hu4-1BB and huCD19 (Example 8.2).
[0118] FIGS. 35A-35F show simultaneous binding of the CD19 targeted
trimeric 4-1BBL FC fusion antigen binding molecules Constructs 3.1,
3.3, 3.4, 3.5, 3.6 and 4.4 (Analyte 1) to immobilized human 4-1BB
and human CD19 (Analyte 2).
[0119] FIGS. 36A-1 to 36B-3 show the binding of different
CD19-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs to 4-1BB-expressing CD4 and CD8 T cells of PHA-L
and Proleukin pre-activated and anti-human CD3/anti-human CD28
re-activated human PBMCs. Binding was detected with
R-Phycoerythrin-fluorochrome conjugated anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment. Shown is the median
of fluorescence intensity (MFI) versus the concentration of tested
constructs. For a better display the binding curves are split in
three different blots with construct 3.4 and control F (Isotype
control huIgG1 P329G LALA) as comparison curves. Binding was
monitored on CD45+CD3+CD8+ T cells (FIGS. 36A-1 to 36A-3) and
CD45+CD3+CD4+ T cells (FIGS. 36B-1 to 36B-3). The 4-1BB expression
level on CD8 T cells is normally higher than on CD4 T cells. All
constructs bind with a quite similar affinity to human 4-1BB.
[0120] FIGS. 37A-1 to 37D-3 show the binding of CD19-targeted or
untargeted split trimeric human 4-1BB ligand Fc (kih) antigen
binding molecules to human-CD19 expressing B cell lymphoma cell
lines: diffuse large non-Hodgkin B cell lymphoma SU-DHL-8 (37A-1 to
37A-3), acute B cell precursor lymphoid leukemia Nalm6 (37B-1 to
37B-3), diffuse large cell lymphoblast lymphoma Toledo (37C-1 to
37C-3) and diffuse large B cell lymphoma OCI-Ly18 (37D-1 to 37D-3).
Binding was detected with R-Phycoerythrin-fluorochrome conjugated
anti-human IgG Fc.gamma.-specific goat IgG F(ab')2 fragments. Shown
is the median of fluorescence intensity (MFI) versus the
concentration of tested constructs. For a better display the
binding curves are split in three different blots with construct
3.4 and control F (Isotype control huIgG1 P329G LALA) as comparison
curves. All constructs bind with a quite similar affinity to human
CD19.
[0121] FIGS. 38A to 38C relate to NF.kappa.B-activation-induced
Luciferase expression and activity of CD19-targeted or untargeted
split trimeric human 4-1BB ligand Fc (kih) antigen binding
molecules. Units of released light (URL) are measured for 0.5
s/well and plotted against the used concentration of CD19-targeted
or untargeted split trimeric human 4-1BB ligand Fc (kih) constructs
3.1 and 3.3 and control molecules B and C. Human 4-1BB-expressing
HeLa-reporter cells were incubated for 7.5 h in the absence
presence of crosslinking human-CD19 expressing SU-DHL-8 or Pfeiffer
cells. URLs were measured and blotted against the concentrations of
different CD19-targeted or untargeted split trimeric human 4-1BB
ligand Fc (kih) constructs. The cell ratio is one 4-1BB-expressing
HeLa reporter cell to 2.5 or five tumor cells.
[0122] FIG. 39 shows the binding of different humanized variants of
T84.66 IgG on CEA-expressing human gastric adenocarcinoma cells.
Based on the data humanized variant 1 was selected for including it
into CEA-targeted trimeric human 4-1BB ligand Fc (kih) antigen
binding molecules.
[0123] FIGS. 40A to 40F show the CEA targeted
4-1BBL-trimer-containing antigen binding molecules Constructs 5.1
to 5.6 of the invention. The preparation and production of these
constructs is described in Example 11. The VH and VL domains are
those of anti-CEA antibody T84.66-LCHA, the thick black point
stands for the knob-into-hole modification. * symbolizes amino acid
modifications in the CH1 and CL domain (so-called charged
residues).
[0124] FIG. 41A shows a schematic description of human NA3B3A2-avi
His, the antigen used to assess binding of CEA-targeted trimeric
split 4-1BBL Fc (kih) antigen binding molecules. FIG. 41B
illustrates the setup of the assay measuring simultaneous binding
of CEA-targeted trimeric split 4-1BBL to hu4-1BB and human NA3B3A2
(Example 12.1).
[0125] FIGS. 42A to 42D show simultaneous binding of the CEA
targeted trimeric 4-1BBL Fc fusion antigen binding molecules
Constructs 5.4, 5.6, 5.7 and 5.8 (Analyte 1) to immobilized human
4-1BB and human NA3B3A2 (Analyte 2).
[0126] FIGS. 43A to 43D show binding of different CEA-targeted or
untargeted split trimeric human 4-1BB ligand Fc (kih) constructs to
4-1BB-expressing CD4 and CD8 T cells of PHA-L and Proleukin
pre-activated and anti-human CD3/anti-human CD28 re-activated human
PBMCs. Binding was detected with R-Phycoerythrin-fluorochrome
conjugated anti-human IgG Fc.gamma.-specific goat IgG F(ab')2
fragment. Shown is the median of fluorescence intensity (MFI)
versus the concentration of tested constructs. For a better display
the binding curves are split in two different blots with construct
5.4 and control F (Isotype control huIgG1 P329G LALA) as comparison
curves. Binding was monitored on CD45+CD3+CD8+ T cells (blots on
the bottom) and CD45+CD3+CD4+ T cells (blots on the top). The 4-1BB
expression level on CD8 T cells is normally higher than on CD4 T
cells. All constructs bind with quite similar affinity to human
4-1BB.
[0127] FIGS. 44A and 44B show the binding of CEA-targeted or
untargeted split trimeric human 4-1BB ligand Fc (kih) constructs to
human-CEA expressing human gastric cell line MKN-45 (44A) and human
colorectal adenocarcinoma cells line LS180 (right 44B). Binding was
detected with R-Phycoerythrin-fluorochrome conjugated anti-human
IgG Fc.gamma.-specific goat IgG F(ab') 2 fragments. Shown is the
median of fluorescence intensity (MFI) versus the concentration of
tested constructs.
[0128] FIGS. 45A to 45D relate to NF.kappa.B-activation-induced
Luciferase expression and activity of CEA-targeted or untargeted
split trimeric human 4-1BB ligand Fc (kih) antigen binding
molecules. Units of released light (URL) are measured for 0.5
s/well and blotted against the used concentration of CEA-targeted
or untargeted split trimeric human 4-1BB ligand Fc (kih) constructs
5.4, 5.6, 5.7 and 5.8 and control molecules. Human 4-1BB-expressing
HeLa-reporter cells were incubated for 6 h in the absence or
presence of crosslinking human-CEA expressing human gastric cancer
cell line MKN-45. The cell ratio is one 4-1BB-expressing HeLa
reporter cell to three tumor cells.
[0129] FIGS. 46A and 46B show the components for the assembly of
monovalent FAP targeted split trimeric human OX40 ligand (construct
6.1) including linker GGGGSGGGGS (SEQ ID NO:13). FIG. 46A relates
to dimeric ligand fused to human IgG1-CL domain, FIG. 46B relates
to monomeric ligand fused to human IgG1-CH1 domain. FIG. 46C shows
the FAP targeted OX40L-trimer-containing antigen binding molecule
Construct 6.1. In FIG. 46D is shown the DP47 "untargeted" human
IgG1 PGLALA (control F).
[0130] FIG. 47A shows the binding of FAP targeted split trimeric
human OX40L to FAP positive WM-266-4 cells. WM-266-4 cells express
high levels of human fibroblast activation protein (huFAP). Only
FAP targeted OX40 ligand Fc (kih) constructs (filled square) but
not control F (filled diamond) bound to WM-266-4 cells. Shown is
the binding as median of fluorescence intensity (MFI) of
Fluorescein isothiocyanate (FITC)-labeled anti-human IgG
Fc.gamma.-specific goat IgG F(ab')2 fragment which is used as
secondary detection antibody. MFI was measured by flow cytometry.
The x-axis shows the concentration of antibody constructs. FIG. 47B
shows the binding of FAP targeted OX40 ligand Fc (kih) construct to
human FAP human OX40 negative A549 NucLight.TM. Red cells. FAP
targeted OX40 ligand Fc (kih) construct showed no binding to OX40
negative FAP negative A549 tumor cells. Shown is the binding as
median of fluorescence intensity (MFI) of FITC labeled anti-human
IgG Fc.gamma.-specific goat IgG F(ab')2 fragment which is used as
secondary detection antibody. MFI was measured by flow cytometry
and baseline corrected by subtracting the MFI of the blank
control.
[0131] FIGS. 48A-1 to 48A-2 show the binding of FAP-OX40L to
resting and activated human CD4 T cells. OX40 is not expressed on
resting human CD4 T cells (left side). In the absence of human OX40
expressing cells no binding was observed (48A-1). After activation
of human PBMCs OX40 is up-regulated on CD4+ T cells (48A-2).
FAP-OX40L bound to OX40+ activated CD4 T cells. Shown is the
binding as median of fluorescence intensity (MFI) of FITC labeled
anti-human IgG Fc.gamma.-specific goat IgG F(ab')2 fragment which
is used as secondary detection antibody. MFI was measured by flow
cytometry and baseline corrected by subtracting the MFI of the
blank control. The x-axis shows the concentration of antibody
constructs. FIGS. 48B-1 and 48B-2 show that OX40 is not expressed
on resting human CD8 T cells (left side). In the absence of human
OX40 expressing cells no binding was observed (left graphs). After
activation of human PBMCs OX40 is up-regulated on CD8+ T cells
(right side). OX40 expression on human CD8+ T cells is lower than
on CD4+ T cells and varies between donors and time points.
Expression of OX40 was low on the depicted CD8 T cells. FAP-OX40L
bound to OX40+ activated CD8 T cells. Shown is the binding as
median of fluorescence intensity (MFI) of FITC labeled anti-human
IgG Fc.gamma.-specific goat IgG F(ab')2 fragment which is used as
secondary detection antibody. MFI was measured by flow cytometry
and baseline corrected by subtracting the MFI of the blank control.
The x-axis shows the concentration of antibody constructs.
[0132] In FIGS. 49A and 49B, the activation of NF.kappa.B signaling
pathway by the FAP targeted split trimeric human OX40L antigen
binding molecule (FAP-OX40L) in HeLa_hOx40_NFkB_Luc1 reporter cells
is demonstrated. Shown is the activation with (49B) or without
(49A) crosslinking by secondary antibody. The reporter cells were
cultured for 5 hours in the presence of FAP-OX40L at the indicated
concentrations with or without crosslinking secondary poly-clonal
anti-huIgG1 Fc.gamma.-specific goat IgG F(ab)2 fragment in a 1:2
ratio. Luciferase activity was assessed as described in Example
6.1. Activity is characterized by blotting the units of released
light (URL) measured during 0.5 s versus the concentration in nM of
tested construct. URLs are emitted due to luciferase-mediated
oxidation of luciferin to oxyluciferin.
[0133] FIG. 50A shows the activation of NF.kappa.B by FAP-OX40L in
HeLa_hOx40_NFkB_Luc1 reporter cells in the presence of FAP positive
cells. Shown is the activation of NF.kappa.B signaling pathway in
the reporter cells by FAP-OX40L in the presence of low FAP
expressing NIH-3T3 human FAP cells (ratio 3 FAP+ tumor cells to 1
reporter cell). The NF.kappa.B-mediated luciferase activity was
characterized by blotting the units of released light (URL),
measured during 0.5 s, versus the concentration in nM of tested
compounds. URLs are emitted due to luciferase-mediated oxidation of
luciferin to oxyluciferin. Values are baseline corrected by
subtracting the URLs of the blank control. For a better comparison
the area under the curve of the respective blotted dose-response
curves were quantified as a marker for the agonistic capacity of
each construct. The comparison is illustrated in FIG. 50B. The area
was calculated using GraphPad Prism. Values are baseline corrected
by subtracting the value of the blank control.
[0134] FIGS. 51A to 51D show the OX40 mediated costimulation of
suboptimally TCR triggered resting human PBMC (Example 15.5).
Hyper-crosslinking of FAP-OX40L by the present NIH/3T3-huFAP clone
39 cells strongly promoted survival and proliferation in human CD4
and CD8 T cells. Shown is the event count of vital CD4+(51A and
51C) and CD8+(51B and 51D) T cells. Baseline values of samples
containing only the anti-human CD3 (clone V9, huIgG1), resting
human PBMC and NIH/3T3-huFAP clone 39 were subtracted. Thus the
enhancing effect of OX40 co-stimulation but not the effect of
suboptimal anti-CD3 stimulation per se is shown here. In FIGS. 51A
to 51D on the bottom the rescue of suboptimal TCR stimulation of
resting human PBMC with cell surface immobilized
FAP-OX40L--Proliferation is shown.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0135] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as generally used in the art to
which this invention belongs. For purposes of interpreting this
specification, the following definitions will apply and whenever
appropriate, terms used in the singular will also include the
plural and vice versa.
[0136] As used herein, the term "antigen binding molecule" refers
in its broadest sense to a molecule that specifically binds an
antigenic determinant. Examples of antigen binding molecules are
antibodies, antibody fragments and scaffold antigen binding
proteins.
[0137] As used herein, the term "moiety capable of specific binding
to a target cell antigen" refers to a polypeptide molecule that
specifically binds to an antigenic determinant. In one aspect, the
antigen binding moiety is able to activate signaling through its
target cell antigen. In a particular aspect, the antigen binding
moiety is able to direct the entity to which it is attached (e.g.
the TNF family ligand trimer) to a target site, for example to a
specific type of tumor cell or tumor stroma bearing the antigenic
determinant. Moieties capable of specific binding to a target cell
antigen include antibodies and fragments thereof as further defined
herein. In addition, moieties capable of specific binding to a
target cell antigen include scaffold antigen binding proteins as
further defined herein, e.g. binding domains which are based on
designed repeat proteins or designed repeat domains (see e.g. WO
2002/020565).
[0138] In relation to an antibody or fragment thereof, the term
"moiety capable of specific binding to a target cell antigen"
refers to the part of the molecule that comprises the area which
specifically binds to and is complementary to part or all of an
antigen. A moiety capable of specific antigen binding may be
provided, for example, by one or more antibody variable domains
(also called antibody variable regions). Particularly, a moiety
capable of specific antigen binding comprises an antibody light
chain variable region (VL) and an antibody heavy chain variable
region (VH).
[0139] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, monospecific and
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments so long as they exhibit the desired
antigen-binding activity.
[0140] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g. containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen.
[0141] The term "monospecific" antibody as used herein denotes an
antibody that has one or more binding sites each of which bind to
the same epitope of the same antigen. The term "bispecific" means
that the antigen binding molecule is able to specifically bind to
at least two distinct antigenic determinants. Typically, a
bispecific antigen binding molecule comprises two antigen binding
sites, each of which is specific for a different antigenic
determinant. In certain embodiments the bispecific antigen binding
molecule is capable of simultaneously binding two antigenic
determinants, particularly two antigenic determinants expressed on
two distinct cells.
[0142] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in an
antigen binding molecule. As such, the terms "bivalent",
"tetravalent", and "hexavalent" denote the presence of two binding
sites, four binding sites, and six binding sites, respectively, in
an antigen binding molecule.
[0143] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure. "Native antibodies" refer to naturally
occurring immunoglobulin molecules with varying structures. For
example, native IgG-class antibodies are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a light
chain constant domain (CL), also called a light chain constant
region. The heavy chain of an antibody may be assigned to one of
five types, called .alpha. (IgA), .delta. (IgD), .epsilon. (IgE),
.gamma. (IgG), or .mu. (IgM), some of which may be further divided
into subtypes, e.g. .gamma.1 (IgG1), .gamma.2 (IgG2), .gamma.3
(IgG3), .gamma.4 (IgG4), .alpha.1 (IgA1) and .alpha.2 (IgA2). The
light chain of an antibody may be assigned to one of two types,
called kappa (.kappa.) and lambda (.lamda.), based on the amino
acid sequence of its constant domain.
[0144] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab
fragments; linear antibodies; single-chain antibody molecules (e.g.
scFv); and single domain antibodies. For a review of certain
antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003).
For a review of scFv fragments, see e.g. Pluckthun, in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also
WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with
two antigen-binding sites that may be bivalent or bispecific, see,
for example, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9,
129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90,
6444-6448 (1993). Triabodies and tetrabodies are also described in
Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies
are antibody fragments comprising all or a portion of the heavy
chain variable domain or all or a portion of the light chain
variable domain of an antibody. In certain embodiments, a
single-domain antibody is a human single-domain antibody (Domantis,
Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1).
Antibody fragments can be made by various techniques, including but
not limited to proteolytic digestion of an intact antibody as well
as production by recombinant host cells (e.g. E. coli or phage), as
described herein.
[0145] Papain digestion of intact antibodies produces two identical
antigen-binding fragments, called "Fab" fragments containing each
the heavy- and light-chain variable domains and also the constant
domain of the light chain and the first constant domain (CH1) of
the heavy chain. As used herein, Thus, the term "Fab fragment"
refers to an antibody fragment comprising a light chain fragment
comprising a VL domain and a constant domain of a light chain (CL),
and a VH domain and a first constant domain (CH1) of a heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteins from the antibody hinge region.
Fab'-SH are Fab' fragments in which the cysteine residue(s) of the
constant domains bear a free thiol group. Pepsin treatment yields
an F(ab').sub.2 fragment that has two antigen-combining sites (two
Fab fragments) and a part of the Fc region.
[0146] The term "cross-Fab fragment" or "xFab fragment" or
"crossover Fab fragment" refers to a Fab fragment, wherein either
the variable regions or the constant regions of the heavy and light
chain are exchanged. Two different chain compositions of a
crossover Fab molecule are possible and comprised in the bispecific
antibodies of the invention: On the one hand, the variable regions
of the Fab heavy and light chain are exchanged, i.e. the crossover
Fab molecule comprises a peptide chain composed of the light chain
variable region (VL) and the heavy chain constant region (CH1), and
a peptide chain composed of the heavy chain variable region (VH)
and the light chain constant region (CL). This crossover Fab
molecule is also referred to as CrossFab .sub.(VLVH). On the other
hand, when the constant regions of the Fab heavy and light chain
are exchanged, the crossover Fab molecule comprises a peptide chain
composed of the heavy chain variable region (VH) and the light
chain constant region (CL), and a peptide chain composed of the
light chain variable region (VL) and the heavy chain constant
region (CH1). This crossover Fab molecule is also referred to as
CrossFab .sub.(CLCH1).
[0147] A "single chain Fab fragment" or "scFab" is a polypeptide
consisting of an antibody heavy chain variable domain (VH), an
antibody constant domain 1 (CH1), an antibody light chain variable
domain (VL), an antibody light chain constant domain (CL) and a
linker, wherein said antibody domains and said linker have one of
the following orders in N-terminal to C-terminal direction: a)
VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1
or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide
of at least 30 amino acids, preferably between 32 and 50 amino
acids. Said single chain Fab fragments are stabilized via the
natural disulfide bond between the CL domain and the CH1 domain. In
addition, these single chain Fab molecules might be further
stabilized by generation of interchain disulfide bonds via
insertion of cysteine residues (e.g. position 44 in the variable
heavy chain and position 100 in the variable light chain according
to Kabat numbering).
[0148] A "crossover single chain Fab fragment" or "x-scFab" is a is
a polypeptide consisting of an antibody heavy chain variable domain
(VH), an antibody constant domain 1 (CH1), an antibody light chain
variable domain (VL), an antibody light chain constant domain (CL)
and a linker, wherein said antibody domains and said linker have
one of the following orders in N-terminal to C-terminal direction:
a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and
VL form together an antigen-binding site which binds specifically
to an antigen and wherein said linker is a polypeptide of at least
30 amino acids. In addition, these x-scFab molecules might be
further stabilized by generation of interchain disulfide bonds via
insertion of cysteine residues (e.g. position 44 in the variable
heavy chain and position 100 in the variable light chain according
to Kabat numbering).
[0149] A "single-chain variable fragment (scFv)" is a fusion
protein of the variable regions of the heavy (V.sub.H) and light
chains (V.sub.L) of an antibody, connected with a short linker
peptide of ten to about 25 amino acids. The linker is usually rich
in glycine for flexibility, as well as serine or threonine for
solubility, and can either connect the N-terminus of the V.sub.H
with the C-terminus of the V.sub.L, or vice versa. This protein
retains the specificity of the original antibody, despite removal
of the constant regions and the introduction of the linker. scFv
antibodies are, e.g. described in Houston, J. S., Methods in
Enzymol. 203 (1991) 46-96). In addition, antibody fragments
comprise single chain polypeptides having the characteristics of a
VH domain, namely being able to assemble together with a VL domain,
or of a VL domain, namely being able to assemble together with a VH
domain to a functional antigen binding site and thereby providing
the antigen binding property of full length antibodies.
[0150] "Scaffold antigen binding proteins" are known in the art,
for example, fibronectin and designed ankyrin repeat proteins
(DARPINs.RTM.) have been used as alternative scaffolds for
antigen-binding domains, see, e.g., Gebauer and Skerra, Engineered
protein scaffolds as next-generation antibody therapeutics. Curr
Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new
generation of protein therapeutics. Drug Discovery Today 13:
695-701 (2008). In one aspect of the invention, a scaffold antigen
binding protein is selected from the group consisting of CTLA-4
(Evibody), Lipocalins (ANTICALIN.RTM.), a Protein A-derived
molecule such as Z-domain of Protein A (AFFIBODY.RTM.), an A-domain
(Avimer/Maxibody), a serum transferrin (trans-body); a designed
ankyrin repeat protein (DARPIN.RTM.), a variable domain of antibody
light chain or heavy chain (single-domain antibody, sdAb), a
variable domain of antibody heavy chain (nanobody, aVH), VNAR
fragments, a fibronectin (AdNectin), a C-type lectin domain
(Tetranectin); a variable domain of a new antigen receptor
beta-lactamase (VNAR fragments), a human gamma-crystallin or
ubiquitin (AFFILIN.RTM. molecules); a kunitz type domain of human
protease inhibitors, microbodies such as the proteins from the
knottin family, peptide aptamers and fibronectin (adnectin).
[0151] CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a
CD28-family receptor expressed on mainly CD4+ T-cells. Its
extracellular domain has a variable domain-like Ig fold. Loops
corresponding to CDRs of antibodies can be substituted with
heterologous sequence to confer different binding properties.
CTLA-4 molecules engineered to have different binding specificities
are also known as Evibodies (e.g. U.S. Pat. No. 7,166,697B1).
Evibodies are around the same size as the isolated variable region
of an antibody (e.g. a domain antibody). For further details see
Journal of Immunological Methods 248 (1-2), 31-45 (2001).
[0152] Lipocalins are a family of extracellular proteins which
transport small hydrophobic molecules such as steroids, bilins,
retinoids and lipids. They have a rigid beta-sheet secondary
structure with a number of loops at the open end of the conical
structure which can be engineered to bind to different target
antigens. ANTICALINs.RTM. are between 160-180 amino acids in size,
and are derived from lipocalins. For further details see Biochim
Biophys Acta 1482: 337-350 (2000), U.S. Pat. No. 7,250,297B1 and
US20070224633 (U.S. Pat. No. 7,585,940B2).
[0153] An AFFIBODY.RTM. is a scaffold derived from Protein A of
Staphylococcus aureus which can be engineered to bind to antigen.
The domain consists of a three-helical bundle of approximately 58
amino acids. Libraries have been generated by randomization of
surface residues. For further details see Protein Eng. Des. Sel.
17, 455-462 (2004) and EP 1641818A1.
[0154] Avimers are multidomain proteins derived from the A-domain
scaffold family. The native domains of approximately 35 amino acids
adopt a defined disulfide bonded structure. Diversity is generated
by shuffling of the natural variation exhibited by the family of
A-domains. For further details see Nature Biotechnology 23(12),
1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),
909-917 (June 2007).
[0155] A transferrin is a monomeric serum transport glycoprotein.
Transferrins can be engineered to bind different target antigens by
insertion of peptide sequences in a permissive surface loop.
Examples of engineered transferrin scaffolds include the
Trans-body. For further details see J. Biol. Chem 274, 24066-24073
(1999).
[0156] Designed Ankyrin Repeat Proteins (DARPINs.RTM.) are derived
from Ankyrin which is a family of proteins that mediate attachment
of integral membrane proteins to the cytoskeleton. A single ankyrin
repeat is a 33 residue motif consisting of two alpha-helices and a
beta-turn. They can be engineered to bind different target antigens
by randomizing residues in the first alpha-helix and a beta-turn of
each repeat. Their binding interface can be increased by increasing
the number of modules (a method of affinity maturation). For
further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4),
1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and
US20040132028A1 (U.S. Pat. No. 7,417,130B2).
[0157] A single-domain antibody is an antibody fragment consisting
of a single monomeric variable antibody domain. The first single
domain were derived from the variable domain of the antibody heavy
chain from camelids (nanobodies or V.sub.HH fragments).
Furthermore, the term single-domain antibody includes an autonomous
human heavy chain variable domain (aVH) or VNAR fragments derived
from sharks.
[0158] Fibronectin is a scaffold which can be engineered to bind to
antigen. Adnectins consists of a backbone of the natural amino acid
sequence of the 10th domain of the 15 repeating units of human
fibronectin type III (FN3). Three loops at one end of the
.beta.-sandwich can be engineered to enable an Adnectin to
specifically recognize a therapeutic target of interest. For
further details see Protein Eng. Des. Sel. 18, 435-444 (2005),
US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.
[0159] Peptide aptamers are combinatorial recognition molecules
that consist of a constant scaffold protein, typically thioredoxin
(TrxA) which contains a constrained variable peptide loop inserted
at the active site. For further details see Expert Opin. Biol.
Ther. 5, 783-797 (2005).
[0160] Microbodies are derived from naturally occurring
microproteins of 25-50 amino acids in length which contain 3-4
cysteine bridges--examples of microproteins include KalataBI and
conotoxin and knottins. The microproteins have a loop which can
beengineered to include upto 25 amino acids without affecting the
overall fold of the microprotein. For further details of engineered
knottin domains, see WO2008098796.
[0161] An "antigen binding molecule that binds to the same epitope"
as a reference molecule refers to an antigen binding molecule that
blocks binding of the reference molecule to its antigen in a
competition assay by 50% or more, and conversely, the reference
molecule blocks binding of the antigen binding molecule to its
antigen in a competition assay by 50% or more.
[0162] The term "antigen binding domain" refers to the part of an
antigen binding molecule that comprises the area which specifically
binds to and is complementary to part or all of an antigen. Where
an antigen is large, an antigen binding molecule may only bind to a
particular part of the antigen, which part is termed an epitope. An
antigen binding domain may be provided by, for example, one or more
variable domains (also called variable regions). Preferably, an
antigen binding domain comprises an antibody light chain variable
region (VL) and an antibody heavy chain variable region (VH).
[0163] As used herein, the term "antigenic determinant" is
synonymous with "antigen" and "epitope," and refers to a site (e.g.
a contiguous stretch of amino acids or a conformational
configuration made up of different regions of non-contiguous amino
acids) on a polypeptide macromolecule to which an antigen binding
moiety binds, forming an antigen binding moiety-antigen complex.
Useful antigenic determinants can be found, for example, on the
surfaces of tumor cells, on the surfaces of virus-infected cells,
on the surfaces of other diseased cells, on the surface of immune
cells, free in blood serum, and/or in the extracellular matrix
(ECM). The proteins useful as antigens herein can be any native
form the proteins from any vertebrate source, including mammals
such as primates (e.g. humans) and rodents (e.g. mice and rats),
unless otherwise indicated. In a particular embodiment the antigen
is a human protein. Where reference is made to a specific protein
herein, the term encompasses the "full-length", unprocessed protein
as well as any form of the protein that results from processing in
the cell. The term also encompasses naturally occurring variants of
the protein, e.g. splice variants or allelic variants.
[0164] By "specific binding" is meant that the binding is selective
for the antigen and can be discriminated from unwanted or
non-specific interactions. The ability of an antigen binding
molecule to bind to a specific antigen can be measured either
through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to one of skill in the art, e.g. Surface
Plasmon Resonance (SPR) technique (analyzed on a BIACORE.RTM.
instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
In one embodiment, the extent of binding of an antigen binding
molecule to an unrelated protein is less than about 10% of the
binding of the antigen binding molecule to the antigen as measured,
e.g. by SPR. In certain embodiments, an molecule that binds to the
antigen has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g.
from 10.sup.-8M to 10.sup.-13M, e.g. from 10' M to 10.sup.-13
M).
[0165] "Affinity" or "binding affinity" refers to the strength of
the sum total of non-covalent interactions between a single binding
site of a molecule (e.g. an antibody) and its binding partner (e.g.
an antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g. antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd), which
is the ratio of dissociation and association rate constants (koff
and kon, respectively). Thus, equivalent affinities may comprise
different rate constants, as long as the ratio of the rate
constants remains the same. Affinity can be measured by common
methods known in the art, including those described herein. A
particular method for measuring affinity is Surface Plasmon
Resonance (SPR).
[0166] A "target cell antigen" as used herein refers to an
antigenic determinant presented on the surface of a target cell,
for example a cell in a tumor such as a cancer cell or a cell of
the tumor stroma. In certain embodiments, the target cell antigen
is an antigen on the surface of a tumor cell. In one embodiment,
target cell antigen is selected from the group consisting of
Fibroblast Activation Protein (FAP), Carcinoembryonic Antigen
(CEA), Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),
Epidermal Growth Factor Receptor (EGFR), CD19, CD20 and CD33. In
particular, the target cell antigen is Fibroblast Activation
Protein (FAP).
[0167] The term "Fibroblast activation protein (FAP)", also known
as Prolyl endopeptidase FAP or Seprase (EC 3.4.21), refers to any
native FAP from any vertebrate source, including mammals such as
primates (e.g. humans) non-human primates (e.g. cynomolgus monkeys)
and rodents (e.g. mice and rats), unless otherwise indicated. The
term encompasses "full-length," unprocessed FAP as well as any form
of FAP which results from processing in the cell. The term also
encompasses naturally occurring variants of FAP, e.g., splice
variants or allelic variants. In one embodiment, the antigen
binding molecule of the invention is capable of specific binding to
human, mouse and/or cynomolgus FAP. The amino acid sequence of
human FAP is shown in UniProt accession no. Q12884 (version 149,
SEQ ID NO:20), or NCBI RefSeq NP 004451.2. The extracellular domain
(ECD) of human FAP extends from amino acid position 26 to 760. The
amino acid and nucleotide sequences of a His-tagged human FAP ECD
is shown in SEQ ID NOs 15 and 16, respectively. The amino acid
sequence of mouse FAP is shown in UniProt accession no. P97321
(version 126, SEQ ID NO:23), or NCBI RefSeq NP 032012.1. The
extracellular domain (ECD) of mouse FAP extends from amino acid
position 26 to 761. SEQ ID NOs 24 and 25 show the amino acid and
nucleotide sequences, respectively, of a His-tagged mouse FAP ECD.
SEQ ID NOs 26 and 27 show the amino acid and nucleotide sequences,
respectively, of a His-tagged cynomolgus FAP ECD. Preferably, an
anti-FAP binding molecule of the invention binds to the
extracellular domain of FAP. Exemplary anti-FAP binding molecules
are described in International Patent Application No. WO
2012/020006 A2.
[0168] The term "Carcinoembroynic antigen (CEA)", also known as
Carcinoembryonic antigen-related cell adhesion molecule 5
(CEACAM5), refers to any native CEA from any vertebrate source,
including mammals such as primates (e.g. humans) non-human primates
(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless
otherwise indicated. The amino acid sequence of human CEA is shown
in UniProt accession no. P06731 (version 151, SEQ ID NO:28). CEA
has long been identified as a tumor-associated antigen (Gold and
Freedman, J Exp Med., 121:439-462, 1965; Berinstein N. L., J Clin
Oncol., 20:2197-2207, 2002). Originally classified as a protein
expressed only in fetal tissue, CEA has now been identified in
several normal adult tissues. These tissues are primarily
epithelial in origin, including cells of the gastrointestinal,
respiratory, and urogential tracts, and cells of colon, cervix,
sweat glands, and prostate (Nap et al., Tumour Biol.,
9(2-3):145-53, 1988; Nap et al., Cancer Res., 52(8):2329-23339,
1992). Tumors of epithelial origin, as well as their metastases,
contain CEA as a tumor associated antigen. While the presence of
CEA itself does not indicate transformation to a cancerous cell,
the distribution of CEA is indicative. In normal tissue, CEA is
generally expressed on the apical surface of the cell (Hammarstrom
S., Semin Cancer Biol. 9(2):67-81 (1999)), making it inaccessible
to antibody in the blood stream. In contrast to normal tissue, CEA
tends to be expressed over the entire surface of cancerous cells
(Hammarstrom S., Semin Cancer Biol. 9(2):67-81 (1999)). This change
of expression pattern makes CEA accessible to antibody binding in
cancerous cells. In addition, CEA expression increases in cancerous
cells. Furthermore, increased CEA expression promotes increased
intercellular adhesions, which may lead to metastasis (Marshall J.,
Semin Oncol., 30 (a Suppl. 8):30-6, 2003). The prevalence of CEA
expression in various tumor entities is generally very high. In
concordance with published data, own analyses performed in tissue
samples confirmed its high prevalence, with approximately 95% in
colorectal carcinoma (CRC), 90% in pancreatic cancer, 80% in
gastric cancer, 60% in non-small cell lung cancer (NSCLC, where it
is co-expressed with HER3), and 40% in breast cancer; low
expression was found in small cell lung cancer and
glioblastoma.
[0169] CEA is readily cleaved from the cell surface and shed into
the blood stream from tumors, either directly or via the
lymphatics. Because of this property, the level of serum CEA has
been used as a clinical marker for diagnosis of cancers and
screening for recurrence of cancers, particularly colorectal cancer
(Goldenberg D M., The International Journal of Biological Markers,
7:183-188, 1992; Chau I., et al., J Clin Oncol., 22:1420-1429,
2004; Flamini et al., Clin Cancer Res; 12(23):6985-6988, 2006).
[0170] The term "Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP)", also known as Chondroitin Sulfate
Proteoglycan 4 (CSPG4) refers to any native MCSP from any
vertebrate source, including mammals such as primates (e.g. humans)
non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice
and rats), unless otherwise indicated. The amino acid sequence of
human MCSP is shown in UniProt accession no. Q6UVK1 (version 103,
SEQ ID NO:29). The term "Epidermal Growth Factor Receptor (EGFR)",
also named Proto-oncogene c-ErbB-1 or Receptor tyrosine-protein
kinase erbB-1, refers to any native EGFR from any vertebrate
source, including mammals such as primates (e.g. humans) non-human
primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and
rats), unless otherwise indicated. The amino acid sequence of human
EGFR is shown in UniProt accession no. P00533 (version 211, SEQ ID
NO:30).
[0171] The term "CD19" refers to B-lymphocyte antigen CD19, also
known as B-lymphocyte surface antigen B4 or T-cell surface antigen
Leu-12 and includes any native CD19 from any vertebrate source,
including mammals such as primates (e.g. humans) non-human primates
(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless
otherwise indicated. The amino acid sequence of human CD19 is shown
in Uniprot accession no. P15391 (version 160, SEQ ID NO:31). The
term encompasses "full-length" unprocessed human CD19 as well as
any form of human CD19 that results from processing in the cell as
long as the antibody as reported herein binds thereto. CD19 is a
structurally distinct cell surface receptor expressed on the
surface of human B cells, including, but not limited to, pre-B
cells, B cells in early development {i.e., immature B cells),
mature B cells through terminal differentiation into plasma cells,
and malignant B cells. CD19 is expressed by most pre-B acute
lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B cell
chronic lymphocytic leukemias (CLL), pro-lymphocytic leukemias,
hairy cell leukemias, common acute lymphocytic leukemias, and some
Null-acute lymphoblastic leukemias. The expression of CD19 on
plasma cells further suggests it may be expressed on differentiated
B cell tumors such as multiple myeloma. Therefore, the CD19 antigen
is a target for immunotherapy in the treatment of non-Hodgkin's
lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic
leukemia.
[0172] "CD20" refers to B-lymphocyte antigen CD20, also known as
membrane-spanning 4-domains subfamily A member 1 (MS4A1),
B-lymphocyte surface antigen B1 or Leukocyte surface antigen
Leu-16, and includes any native CD20 from any vertebrate source,
including mammals such as primates (e.g. humans) non-human primates
(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless
otherwise indicated. The amino acid sequence of human CD20 is shown
in Uniprot accession no. P11836 (version 149, SEQ ID NO:32). "CD33"
refers to Myeloid cell surface antigen CD33, also known as SIGLEC3
or gp67, and includes any native CD33 from any vertebrate source,
including mammals such as primates (e.g. humans) non-human primates
(e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless
otherwise indicated. The amino acid sequence of human CD33 is shown
in Uniprot accession no. P20138 (version 157, SEQ ID NO:33).
[0173] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antigen binding molecule to antigen. The variable
domains of the heavy chain and light chain (VH and VL,
respectively) of a native antibody generally have similar
structures, with each domain comprising four conserved framework
regions (FRs) and three hypervariable regions (HVRs). See, e.g.,
Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page
91 (2007). A single VH or VL domain may be sufficient to confer
antigen-binding specificity.
[0174] The term "hypervariable region" or "HVR," as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) Hypervariable regions (HVRs) are also referred to as
complementarity determining regions (CDRs), and these terms are
used herein interchangeably in reference to portions of the
variable region that form the antigen binding regions. This
particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, "Sequences of Proteins of Immunological
Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917
(1987), where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table A as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00001 TABLE A CDR Definitions.sup.1 CDR Kabat Chothia
AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1Numbering of all CDR definitions in Table A is according to
the numbering conventions set forth by KABAT et al. (see below).
.sup.2"AbM" with a lowercase "b" as used in Table A refers to the
CDRs as defined by Oxford Molecular`s "AbM" antibody modeling
software.
[0175] Kabat et al. also defined a numbering system for variable
region sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable region sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an antibody variable region are according to
the Kabat numbering system.
[0176] With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0177] As used herein, the term "affinity matured" in the context
of antigen binding molecules (e.g., antibodies) refers to an
antigen binding molecule that is derived from a reference antigen
binding molecule, e.g., by mutation, binds to the same antigen,
preferably binds to the same epitope, as the reference antibody;
and has a higher affinity for the antigen than that of the
reference antigen binding molecule. Affinity maturation generally
involves modification of one or more amino acid residues in one or
more CDRs of the antigen binding molecule. Typically, the affinity
matured antigen binding molecule binds to the same epitope as the
initial reference antigen binding molecule.
[0178] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0179] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0180] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0181] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g. IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu. respectively.
[0182] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization. Other forms of "humanized antibodies" encompassed by
the present invention are those in which the constant region has
been additionally modified or changed from that of the original
antibody to generate the properties according to the invention,
especially in regard to C1q binding and/or Fc receptor (FcR)
binding.
[0183] A "human" antibody is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0184] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an antibody heavy chain that contains at
least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. An IgG Fc region
comprises an IgG CH2 and an IgG CH3 domain. The "CH2 domain" of a
human IgG Fc region usually extends from an amino acid residue at
about position 231 to an amino acid residue at about position 340.
In one embodiment, a carbohydrate chain is attached to the CH2
domain. The CH2 domain herein may be a native sequence CH2 domain
or variant CH2 domain. The "CH3 domain" comprises the stretch of
residues C-terminal to a CH2 domain in an Fc region (i.e. from an
amino acid residue at about position 341 to an amino acid residue
at about position 447 of an IgG). The CH3 region herein may be a
native sequence CH3 domain or a variant CH3 domain (e.g. a CH3
domain with an introduced "protuberance" ("knob") in one chain
thereof and a corresponding introduced "cavity" ("hole") in the
other chain thereof; see U.S. Pat. No. 5,821,333, expressly
incorporated herein by reference). Such variant CH3 domains may be
used to promote heterodimerization of two non-identical antibody
heavy chains as herein described. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0185] The "knob-into-hole" technology is described e.g. in U.S.
Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine). The protuberance and
cavity can be made by altering the nucleic acid encoding the
polypeptides, e.g. by site-specific mutagenesis, or by peptide
synthesis. In a specific embodiment a knob modification comprises
the amino acid substitution T366W in one of the two subunits of the
Fc domain, and the hole modification comprises the amino acid
substitutions T366S, L368A and Y407V in the other one of the two
subunits of the Fc domain. In a further specific embodiment, the
subunit of the Fc domain comprising the knob modification
additionally comprises the amino acid substitution S354C, and the
subunit of the Fc domain comprising the hole modification
additionally comprises the amino acid substitution Y349C.
Introduction of these two cysteine residues results in the
formation of a disulfide bridge between the two subunits of the Fc
region, thus further stabilizing the dimer (Carter, J Immunol
Methods 248, 7-15 (2001)). The numbering is according to EU index
of Kabat et al, Sequences of Proteins of Immunological Interest,
5th Ed. Public Health Service, National Institutes of Health,
Bethesda, Md., 1991.
[0186] A "region equivalent to the Fc region of an immunoglobulin"
is intended to include naturally occurring allelic variants of the
Fc region of an immunoglobulin as well as variants having
alterations which produce substitutions, additions, or deletions
but which do not decrease substantially the ability of the
immunoglobulin to mediate effector functions (such as
antibody-dependent cellular cytotoxicity). For example, one or more
amino acids can be deleted from the N-terminus or C-terminus of the
Fc region of an immunoglobulin without substantial loss of
biological function. Such variants can be selected according to
general rules known in the art so as to have minimal effect on
activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10
(1990)).
[0187] The term "effector functions" refers to those biological
activities attributable to the Fc region of an antibody, which vary
with the antibody isotype. Examples of antibody effector functions
include: C1q binding and complement dependent cytotoxicity (CDC),
Fc receptor binding, antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells, down regulation of cell surface receptors (e.g. B
cell receptor), and B cell activation.
[0188] An "activating Fc receptor" is an Fc receptor that following
engagement by an Fc region of an antibody elicits signaling events
that stimulate the receptor-bearing cell to perform effector
functions. Activating Fc receptors include Fc.gamma.RIIIa (CD16a),
Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI (CD89). A
particular activating Fc receptor is human Fc.gamma.RIIIa (see
UniProt accession no. P08637, version 141).
[0189] The term "TNF ligand family member" or "TNF family ligand"
refers to a proinflammatory cytokine. Cytokines in general, and in
particular the members of the TNF ligand family, play a crucial
role in the stimulation and coordination of the immune system. At
present, nineteen cyctokines have been identified as members of the
TNF (tumour necrosis factor) ligand superfamily on the basis of
sequence, functional, and structural similarities. All these
ligands are type II transmembrane proteins with a C-terminal
extracellular domain (ectodomain), N-terminal intracellular domain
and a single transmembrane domain. The C-terminal extracellular
domain, known as TNF homology domain (THD), has 20-30% amino acid
identity between the superfamily members and is responsible for
binding to the receptor. The TNF ectodomain is also responsible for
the TNF ligands to form trimeric complexes that are recognized by
their specific receptors.
[0190] Members of the TNF ligand family are selected from the group
consisting of Lymphotoxin a (also known as LTA or TNFSF1), TNF
(also known as TNFSF2), LT.beta. (also known as TNFSF3), OX40L
(also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL
(also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70
or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1BBL (also
known as TNFSF9), TRAIL (also known as APO2L, CD253 or TNFSF10),
RANKL (also known as CD254 or TNFSF11), TWEAK (also known as
TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also known
as CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A
(also known as VEGI or TNFSF15), GITRL (also known as TNFSF18),
EDA-A1 (also known as ectodysplasin A1) and EDA-A2 (also known as
ectodysplasin A2). The term refers to any native TNF family ligand
from any vertebrate source, including mammals such as primates
(e.g. humans), non-human primates (e.g. cynomolgus monkeys) and
rodents (e.g. mice and rats), unless otherwise indicated. In
specific embodiments of the invention, the TNF ligand family member
is selected from the group consisting of OX40L, FasL, CD27L, TRAIL,
4-1BBL, CD40L and GITRL. In a particular embodiment, the TNF ligand
family member is selected from 4-1BBL and OX40L.
[0191] Further information, in particular sequences, of the TNF
ligand family members may be obtained from publically accessible
databases such as Uniprot. For instance, the human TNF ligands have
the following amino acid sequences: human Lymphotoxin a (UniProt
accession no. P01374, SEQ ID NO:34), human TNF (UniProt accession
no. P01375, SEQ ID NO:35), human Lymphotoxin .beta. (UniProt
accession no. Q06643, SEQ ID NO:36), human OX40L (UniProt accession
no. P23510, SEQ ID NO:37), human CD40L (UniProt accession no.
P29965, SEQ ID NO:38), human FasL (UniProt accession no. P48023,
SEQ ID NO:39), human CD27L (UniProt accession no. P32970, SEQ ID
NO:40), human CD30L (UniProt accession no. P32971, SEQ ID NO:41),
4-1BBL (UniProt accession no. P41273, SEQ ID NO:42), TRAIL (UniProt
accession no. P50591, SEQ ID NO:43), RANKL (UniProt accession no.
O14788, SEQ ID NO:44), TWEAK (UniProt accession no. O43508, SEQ ID
NO:45), APRIL (UniProt accession no. O75888, SEQ ID NO:46), BAFF
(UniProt accession no. Q9Y275, SEQ ID NO:47), LIGHT (UniProt
accession no. O43557, SEQ ID NO:48), TL1A (UniProt accession no.
O95150, SEQ ID NO:49), GITRL (UniProt accession no. Q9UNG2, SEQ ID
NO:50) and ectodysplasin A (UniProt accession no. Q92838, SEQ ID
NO:51).
[0192] An "ectodomain" is the domain of a membrane protein that
extends into the extracellular space (i.e. the space outside the
target cell). Ectodomains are usually the parts of proteins that
initiate contact with surfaces, which leads to signal transduction.
The ectodomain of TNF ligand family member as defined herein thus
refers to the part of the TNF ligand protein that extends into the
extracellular space (the extracellular domain), but also includes
shorter parts or fragments thereof that are responsible for the
trimerization and for the binding to the corresponding TNF
receptor. The term "ectodomain of a TNF ligand family member or a
fragment thereof" thus refers to the extracellular domain of the
TNF ligand family member that forms the extracellular domain or to
parts thereof that are still able to bind to the receptor (receptor
binding domain).
[0193] The term "costimulatory TNF ligand family member" or
"costimulatory TNF family ligand" refers to a subgroup of TNF
ligand family members, which are able to costimulate proliferation
and cytokine production of T-cells. These TNF family ligands can
costimulate TCR signals upon interaction with their corresponding
TNF receptors and the interaction with their receptors leads to
recruitment of TNFR-associated factors (TRAF), which initiate
signalling cascades that result in T-cell activation. Costimulatory
TNF family ligands are selected from the group consisting of
4-1BBL, OX40L, GITRL, CD70, CD30L and LIGHT, more particularly the
costimulatory TNF ligand family member is selected from 4-1BBL and
OX40L.
[0194] As described herein before, 4-1BBL is a type II
transmembrane protein and one member of the TNF ligand family.
Complete or full length 4-1BBL having the amino acid sequence of
SEQ ID NO:42 has been described to form trimers on the surface of
cells. The formation of trimers is enabled by specific motives of
the ectodomain of 4-1BBL. Said motives are designated herein as
"trimerization region". The amino acids 50-254 of the human 4-1BBL
sequence (SEQ ID NO:52) form the extracellular domain of 4-1BBL,
but even fragments thereof are able to form the trimers. In
specific embodiments of the invention, the term "ectodomain of
4-1BBL or a fragment thereof" refers to a polypeptide having an
amino acid sequence selected from SEQ ID NO:4 (amino acids 52-254
of human 4-1BBL), SEQ ID NO:1 (amino acids 71-254 of human 4-1BBL),
SEQ ID NO:3 (amino acids 80-254 of human 4-1BBL) and SEQ ID NO:2
(amino acids 85-254 of human 4-1BBL) or a polypeptide having an
amino acid sequence selected from SEQ ID NO:96 (amino acids 71-248
of human 4-1BBL), SEQ ID NO:375 (amino acids 52-248 of human
4-1BBL), SEQ ID NO:374 (amino acids 80-248 of human 4-1BBL) and SEQ
ID NO:373 (amino acids 85-248 of human 4-1BBL), but also other
fragments of the ectodomain capable of trimerization are included
herein.
[0195] As described herein before, OX40L is another type II
transmembrane protein and a further member of the TNF ligand
family. Complete or full length human OX40L has the amino acid
sequence of SEQ ID NO:37. The amino acids 51-183 of the human OX40L
sequence (SEQ ID NO:53) form the extracellular domain of OX40L, but
even fragments thereof that are able to form the trimers. In
specific embodiments of the invention, the term "ectodomain of
OX40L or a fragment thereof" refers to a polypeptide having an
amino acid sequence selected from SEQ ID NO:53 (amino acids 51-183
of human OX40L) or SEQ ID NO:54 (amino acids 52-183 of human
OX40L), but also other fragments of the ectodomain capable of
trimerization are included herein.
[0196] The term "peptide linker" refers to a peptide comprising one
or more amino acids, typically about 2 to 20 amino acids. Peptide
linkers are known in the art or are described herein. Suitable,
non-immunogenic linker peptides are, for example, (G.sub.4S).sub.n
(SEQ ID NO: 390), (SG.sub.4).sub.n (SEQ ID NO: 391) or
G.sub.4(SG.sub.4).sub.n (SEQ ID NO: 392) peptide linkers, wherein
"n" is generally a number between 1 and 10, typically between 1 and
4, in particular 2, i.e. the peptides selected from the group
consisting of GGGGS (SEQ ID NO:128), GGGGSGGGGS (SEQ ID NO:13),
SGGGGSGGGG (SEQ ID NO:55) and GGGGSGGGGSGGGG (SEQ ID NO:56), but
also include the sequences GSPGSSSSGS (SEQ ID NO:57), GSGSGSGS (SEQ
ID NO:394), GSGSGNGS (SEQ ID NO:59), GGSGSGSG (SEQ ID NO:60),
GGSGSG (SEQ ID NO:61), GGSG (SEQ ID NO:62), GGSGNGSG (SEQ ID
NO:63), GGNGSGSG (SEQ ID NO:64) and GGNGSG (SEQ ID NO:65). Peptide
linkers of particular interest are (G.sub.4S).sub.1 or GGGGS (SEQ
ID NO:128), (G.sub.4S).sub.2 or GGGGSGGGGS (SEQ ID NO:13) and
GSPGSSSSGS (SEQ ID NO:57), more particularly (G.sub.4S).sub.2 or
GGGGSGGGGS (SEQ ID NO:13) and GSPGSSSSGS (SEQ ID NO:57).
[0197] The term "amino acid" as used within this application
denotes the group of naturally occurring carboxy .alpha.-amino
acids comprising alanine (three letter code: ala, one letter code:
A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D),
cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),
glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine
(leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe,
F), proline (pro, P), serine (ser, S), threonine (thr, T),
tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).
[0198] A "single chain fusion protein" as used herein refers to a
single chain polypeptide composed of one or two ectodomains of said
TNF ligand family member fused to a part of antigen binding moiety
or Fc part. The fusion may occur by directly linking the N or
C-terminal amino acid of the antigen binding moiety via a peptide
linker to the C- or N-terminal amino acid of the ectodomain of said
TNF ligand family member.
[0199] By "fused" or "connected" is meant that the components (e.g.
a polypeptide and an ectodomain of said TNF ligand family member)
are linked by peptide bonds, either directly or via one or more
peptide linkers.
[0200] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide (protein) sequence is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the reference polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN. SAWI or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for aligning sequences, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc., and
the source code has been filed with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087. The ALIGN-2
program is publicly available from Genentech, Inc., South San
Francisco, Calif., or may be compiled from the source code. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, including digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary. In
situations where ALIGN-2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino
acid sequence A to, with, or against a given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with, or against a given amino acid sequence B) is calculated
as follows:
100 times the fraction X/Y
[0201] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0202] In certain embodiments, amino acid sequence variants of the
TNF ligand trimer-containing antigen binding molecules provided
herein are contemplated. For example, it may be desirable to
improve the binding affinity and/or other biological properties of
the TNF ligand trimer-containing antigen binding molecules. Amino
acid sequence variants of the TNF ligand trimer-containing antigen
binding molecules may be prepared by introducing appropriate
modifications into the nucleotide sequence encoding the molecules,
or by peptide synthesis. Such modifications include, for example,
deletions from, and/or insertions into and/or substitutions of
residues within the amino acid sequences of the antibody. Any
combination of deletion, insertion, and substitution can be made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics, e.g., antigen-binding. Sites
of interest for substitutional mutagenesis include the HVRs and
Framework (FRs). Conservative substitutions are provided in Table B
under the heading "Preferred Substitutions" and further described
below in reference to amino acid side chain classes (1) to (6).
Amino acid substitutions may be introduced into the molecule of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00002 TABLE B Original Preferred Residue Exemplary
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu, Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0203] Amino acids may be grouped according to common side-chain
properties: [0204] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; [0205] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0206] (3) acidic: Asp, Glu; [0207] (4) basic: His, Lys, Arg;
[0208] (5) residues that influence chain orientation: Gly, Pro;
[0209] (6) aromatic: Trp, Tyr, Phe.
[0210] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0211] The term "amino acid sequence variants" includes substantial
variants wherein there are amino acid substitutions in one or more
hypervariable region residues of a parent antigen binding molecule
(e.g. a humanized or human antibody). Generally, the resulting
variant(s) selected for further study will have modifications
(e.g., improvements) in certain biological properties (e.g.,
increased affinity, reduced immunogenicity) relative to the parent
antigen binding molecule and/or will have substantially retained
certain biological properties of the parent antigen binding
molecule. An exemplary substitutional variant is an affinity
matured antibody, which may be conveniently generated, e.g., using
phage display-based affinity maturation techniques such as those
described herein. Briefly, one or more HVR residues are mutated and
the variant antigen binding molecules displayed on phage and
screened for a particular biological activity (e.g. binding
affinity). In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antigen
binding molecule to bind antigen. For example, conservative
alterations (e.g., conservative substitutions as provided herein)
that do not substantially reduce binding affinity may be made in
HVRs. A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as Arg, Asp, His, Lys,
and Glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antigen binding molecule complex to identify contact points
between the antibody and antigen. Such contact residues and
neighboring residues may be targeted or eliminated as candidates
for substitution. Variants may be screened to determine whether
they contain the desired properties.
[0212] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include TNF family ligand
trimer-containing antigen binding molecule with an N-terminal
methionyl residue. Other insertional variants of the molecule
include the fusion to the N- or C-terminus to a polypeptide which
increases the serum half-life of the TNF ligand trimer-containing
antigen binding molecules.
[0213] In certain embodiments, the TNF family ligand
trimer-containing antigen binding molecules provided herein are
altered to increase or decrease the extent to which the antibody is
glycosylated. Glycosylation variants of the molecules may be
conveniently obtained by altering the amino acid sequence such that
one or more glycosylation sites is created or removed. Where the
TNF ligand trimer-containing antigen binding molecule comprises an
Fc region, the carbohydrate attached thereto may be altered. Native
antibodies produced by mammalian cells typically comprise a
branched, biantennary oligosaccharide that is generally attached by
an N-linkage to Asn297 of the CH2 domain of the Fc region. See,
e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GlcNAc), galactose, and sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in TNF family ligand trimer-containing antigen
binding molecule may be made in order to create variants with
certain improved properties. In one aspect, variants of TNF family
ligand trimer-containing antigen binding molecules are provided
having a carbohydrate structure that lacks fucose attached
(directly or indirectly) to an Fc region. Such fucosylation
variants may have improved ADCC function, see e.g. US Patent
Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Further variants of the TNF family
ligand trimer-containing antigen binding molecules of the invention
include those with bisected oligosaccharides, e.g., in which a
biantennary oligosaccharide attached to the Fc region is bisected
by GlcNAc. Such variants may have reduced fucosylation and/or
improved ADCC function., see for example WO 2003/011878
(Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and
US 2005/0123546 (Umana et al., U.S. Pat. No. 9,296,820B2). Variants
with at least one galactose residue in the oligosaccharide attached
to the Fc region are also provided. Such antibody variants may have
improved CDC function and are described, e.g., in WO 1997/30087
(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju,
S.).
[0214] In certain embodiments, it may be desirable to create
cysteine engineered variants of the TNF family ligand
trimer-containing antigen binding molecule of the invention, e.g.,
"thioMAbs," in which one or more residues of the molecule are
substituted with cysteine residues. In particular embodiments, the
substituted residues occur at accessible sites of the molecule. By
substituting those residues with cysteine, reactive thiol groups
are thereby positioned at accessible sites of the antibody and may
be used to conjugate the antibody to other moieties, such as drug
moieties or linker-drug moieties, to create an immunoconjugate. In
certain embodiments, any one or more of the following residues may
be substituted with cysteine: V205 (Kabat numbering) of the light
chain; A118 (EU numbering) of the heavy chain; and 5400 (EU
numbering) of the heavy chain Fc region. Cysteine engineered
antigen binding molecules may be generated as described, e.g., in
U.S. Pat. No. 7,521,541.
[0215] In certain aspects, the TNF family ligand trimer-containing
antigen binding molecules provided herein may be further modified
to contain additional non-proteinaceous moieties that are known in
the art and readily available. The moieties suitable for
derivatization of the antibody include but are not limited to water
soluble polymers. Non-limiting examples of water soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the bispecific antibody derivative
will be used in a therapy under defined conditions, etc. In another
aspect, conjugates of an antibody and non-proteinaceous moiety that
may be selectively heated by exposure to radiation are provided. In
one embodiment, the non-proteinaceous moiety is a carbon nanotube
(Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)
11600-11605). The radiation may be of any wavelength, and includes,
but is not limited to, wavelengths that do not harm ordinary cells,
but which heat the non-proteinaceous moiety to a temperature at
which cells proximal to the antibody-non-proteinaceous moiety are
killed.
[0216] In another aspect, immunoconjugates of the TNF family ligand
trimer-containing antigen binding molecules provided herein maybe
obtained. An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0217] The term "polynucleotide" refers to an isolated nucleic acid
molecule or construct, e.g. messenger RNA (mRNA), virally-derived
RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.
an amide bond, such as found in peptide nucleic acids (PNA). The
term "nucleic acid molecule" refers to any one or more nucleic acid
segments, e.g. DNA or RNA fragments, present in a
polynucleotide.
[0218] By "isolated" nucleic acid molecule or polynucleotide is
intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide encoding a polypeptide contained in a vector is
considered isolated for the purposes of the present invention.
Further examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. An
isolated polynucleotide includes a polynucleotide molecule
contained in cells that ordinarily contain the polynucleotide
molecule, but the polynucleotide molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the present
invention, as well as positive and negative strand forms, and
double-stranded forms. Isolated polynucleotides or nucleic acids
according to the present invention further include such molecules
produced synthetically. In addition, a polynucleotide or a nucleic
acid may be or may include a regulatory element such as a promoter,
ribosome binding site, or a transcription terminator.
[0219] By a nucleic acid or polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference
nucleotide sequence of the present invention, it is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence. As a practical matter, whether any particular
polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence of the present
invention can be determined conventionally using known computer
programs, such as the ones discussed above for polypeptides (e.g.
ALIGN-2).
[0220] The term "expression cassette" refers to a polynucleotide
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
Typically, the recombinant expression cassette portion of an
expression vector includes, among other sequences, a nucleic acid
sequence to be transcribed and a promoter. In certain embodiments,
the expression cassette of the invention comprises polynucleotide
sequences that encode bispecific antigen binding molecules of the
invention or fragments thereof.
[0221] The term "vector" or "expression vector" is synonymous with
"expression construct" and refers to a DNA molecule that is used to
introduce and direct the expression of a specific gene to which it
is operably associated in a target cell. The term includes the
vector as a self-replicating nucleic acid structure as well as the
vector incorporated into the genome of a host cell into which it
has been introduced. The expression vector of the present invention
comprises an expression cassette. Expression vectors allow
transcription of large amounts of stable mRNA. Once the expression
vector is inside the target cell, the ribonucleic acid molecule or
protein that is encoded by the gene is produced by the cellular
transcription and/or translation machinery. In one embodiment, the
expression vector of the invention comprises an expression cassette
that comprises polynucleotide sequences that encode bispecific
antigen binding molecules of the invention or fragments
thereof.
[0222] The terms "host cell", "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. A host cell is any
type of cellular system that can be used to generate the bispecific
antigen binding molecules of the present invention. Host cells
include cultured cells, e.g. mammalian cultured cells, such as CHO
cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63
mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells,
yeast cells, insect cells, and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue.
[0223] An "effective amount" of an agent refers to the amount that
is necessary to result in a physiological change in the cell or
tissue to which it is administered.
[0224] A "therapeutically effective amount" of an agent, e.g. a
pharmaceutical composition, refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of an agent for example eliminates, decreases, delays,
minimizes or prevents adverse effects of a disease.
[0225] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g. humans and non-human
primates such as monkeys), rabbits, and rodents (e.g. mice and
rats). Particularly, the individual or subject is a human.
[0226] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0227] A "pharmaceutically acceptable excipient" refers to an
ingredient in a pharmaceutical composition, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable excipient includes, but is not limited to, a buffer, a
stabilizer, or a preservative.
[0228] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0229] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, the molecules
of the invention are used to delay development of a disease or to
slow the progression of a disease.
[0230] The term "cancer" as used herein refers to proliferative
diseases, such as lymphomas, carcinoma, lymphoma, blastoma,
sarcoma, leukemia, lymphocytic leukemias, lung cancer, non-small
cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone
cancer, pancreatic cancer, skin cancer, cancer of the head or neck,
cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,
rectal cancer, cancer of the anal region, stomach cancer, gastric
cancer, colorectal cancer (CRC), pancreatic cancer, breast cancer,
triple-negative breast cancer, uterine cancer, carcinoma of the
fallopian tubes, carcinoma of the endometrium, carcinoma of the
cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's
Disease, cancer of the esophagus, cancer of the small intestine,
cancer of the endocrine system, cancer of the thyroid gland, cancer
of the parathyroid gland, cancer of the adrenal gland, sarcoma of
soft tissue, cancer of the urethra, cancer of the penis, prostate
cancer, cancer of the bladder, cancer of the kidney or ureter,
renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,
hepatocellular cancer, biliary cancer, neoplasms of the central
nervous system (CNS), spinal axis tumors, brain stem glioma,
glioblastoma multiforme, astrocytomas, schwanomas, ependymonas,
medulloblastomas, meningiomas, squamous cell carcinomas, pituitary
adenoma and Ewings sarcoma, melanoma, multiple myeloma, B-cell
cancer (lymphoma), chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), hairy cell leukemia, chronic
myeloblastic leukemia, including refractory versions of any of the
above cancers, or a combination of one or more of the above
cancers.
[0231] TNF family ligand trimer-containing antigen binding
molecules of the invention
[0232] The invention provides novel TNF family ligand
trimer-containing antigen binding molecules with particularly
advantageous properties such as producibility, stability, binding
affinity, biological activity, targeting efficiency and reduced
toxicity.
[0233] In a first aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises two ectodomains of a TNF ligand family member or two
fragments thereof that are connected to each other by a peptide
linker and in that the second polypeptide comprises only one
ectodomain of said TNF ligand family member or a fragment
thereof.
[0234] In a particular aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first and a second polypeptide that are linked
to each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises only one ectodomain of said
TNF ligand family member or a fragment thereof, and (c) an Fc
domain composed of a first and a second subunit capable of stable
association.
[0235] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule comprises (a) at least
one moiety capable of specific binding to a target cell antigen and
(b) a first and a second polypeptide that are linked to each other
by a disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises two
ectodomains of a TNF ligand family member or two fragments thereof
that are connected to each other by a peptide linker and in that
the second polypeptide comprises only one ectodomain of said TNF
ligand family member or a fragment thereof, wherein the TNF ligand
family member is costimulates human T-cell activation.
[0236] In another particular aspect, the TNF family ligand
trimer-containing antigen binding molecule comprises (a) at least
one moiety capable of specific binding to a target cell antigen
and
(b) a first and a second polypeptide that are linked to each other
by a disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises two
ectodomains of a TNF ligand family member or two fragments thereof
that are connected to each other by a peptide linker and in that
the second polypeptide comprises only one ectodomain of said TNF
ligand family member or a fragment thereof, wherein the ectodomains
of a TNF ligand family member are identical in all instances.
[0237] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of claim 1,
comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that [0238] (i) the first
polypeptide contains a CH1 or CL domain and the second polypeptide
contains a CL or CH1 domain, respectively, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the first polypeptide
comprises two ectodomains of a TNF ligand family member or
fragments thereof that are connected to each other and to the CH1
or CL domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to the CL or CH1
domain of said polypeptide, or [0239] (ii) the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises only one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide, or [0240] (iii) the first
polypeptide contains a VH-CL or a VL-CH1 domain and the second
polypeptide contains a VL-CH1 domain or a VH-CL domain,
respectively, wherein the second polypeptide is linked to the first
polypeptide by a disulfide bond between the CH1 and CL domain, and
wherein the first polypeptide comprises two ectodomains of a TNF
ligand family member or fragments thereof that are connected to
each other and to to VH or VL by a peptide linker and wherein the
second polypeptide comprises one ectodomain of said TNF ligand
family member or a fragment thereof connected via a peptide linker
to VL or VH of said polypeptide.
[0241] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule comprises a TNF ligand
family member that costimulates human T-cell activation which is
selected from 4-1BBL and OX40L. More particularly, the TNF ligand
family member is 4-1BBL.
[0242] In another aspect, wherein the ectodomain of a TNF ligand
family member comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:96, SEQ ID NO: 373, SEQ ID NO:374 and SEQ ID
NO:375, particularly the amino acid sequence of SEQ ID NO:1 or SEQ
ID NO:96. In one aspect, the ectodomain of a TNF ligand family
member or fragment thereof comprises the amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2,
SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:96, particularly the amino
acid sequence of SEQ ID NO:1 or SEQ ID NO:96. In a particular
aspect, the ectodomain of a TNF ligand family member or fragment
thereof comprises the amino acid sequence of SEQ ID NO:96.
[0243] In a further aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence selected from the group
consisting of SEQ ID NO:5, SEQ ID NO:97, SEQ ID NO:98 and SEQ ID
NO:99 and in that the second polypeptide comprises the amino acid
sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:96, SEQ ID NO:3 and SEQ ID NO:4. In a particular aspect, the
first polypeptide comprises the amino acid sequence of SEQ ID NO:97
and the second polypeptide comprises the amino acid sequence of SEQ
ID NO:96.
[0244] In one aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:5 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:6.
[0245] In a further aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:5 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:183.
[0246] In yet a further aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:97 and in that the
second polypeptide comprises the amino acid sequence of SEQ ID
NO:184 or SEQ ID NO:185.
[0247] In another aspect, the TNF ligand family member is OX40L. In
a particular aspect, provided is TNF family ligand
trimer-containing antigen binding molecule, wherein the ectodomain
of a TNF ligand family member comprises the amino acid sequence of
SEQ ID NO:53 or SEQ ID NO:54, particularly the amino acid sequence
of SEQ ID NO:53.
[0248] In one aspect, the invention relates to a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises the amino acid sequence of SEQ ID NO:371 or SEQ ID:372
and in that the second polypeptide comprises the amino acid
sequence of SEQ ID NO:53 or SEQ ID NO:54, respectively.
[0249] In one aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CH1 or CL domain
and a second polypeptide containing a CL or CH1 domain,
respectively, wherein the second polypeptide is linked to the first
polypeptide by a disulfide bond between the CH1 and CL domain, and
wherein the antigen binding molecule is characterized in that the
first polypeptide comprises two ectodomains of a TNF ligand family
member or fragments thereof that are connected to each other and to
the CH1 or CL domain by a peptide linker and in that the second
polypeptide comprises only one ectodomain of said TNF ligand family
member or a fragment thereof connected via a peptide linker to the
CL or CH1 domain of said polypeptide.
[0250] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CH1 domain and a
second polypeptide containing a CL domain, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the CH1 domain by a peptide
linker and in that the second polypeptide comprises one ectodomain
of said TNF ligand family member or a fragment thereof connected
via a peptide linker to the CL domain of said polypeptide.
[0251] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first polypeptide containing a CL domain and a
second polypeptide containing a CH1 domain, wherein the second
polypeptide is linked to the first polypeptide by a disulfide bond
between the CH1 and CL domain, and wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the CL domain by a peptide
linker and in that the second polypeptide comprises one ectodomain
of said TNF ligand family member or a fragment thereof connected
via a peptide linker to the CH1 domain of said polypeptide.
[0252] In another aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) one moiety capable of specific binding to a target cell antigen
and (b) a first and a second polypeptide that are linked to each
other by a disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises two
ectodomains of a TNF ligand family member or two fragments thereof
that are connected to each other by a peptide linker and in that
the second polypeptide comprises only one ectodomain of said TNF
ligand family member or a fragment thereof.
[0253] In yet another aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) more than one moiety capable of specific binding to a target
cell antigen and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
comprises two ectodomains of a TNF ligand family member or two
fragments thereof that are connected to each other by a peptide
linker and in that the second polypeptide comprises only one
ectodomain of said TNF ligand family member or a fragment thereof
connected via a peptide linker to said polypeptide.
[0254] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) two moities capable of specific binding to a target cell
antigen and (b) a first and a second polypeptide that are linked to
each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises only one ectodomain of said
TNF ligand family member or a fragment thereof.
[0255] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond,
[0256] wherein the antigen binding molecule is characterized in
that the first polypeptide contains a CH3 domain and the second
polypeptide contains a CH3 domain, respectively, and wherein the
first polypeptide comprises two ectodomains of a TNF ligand family
member or fragments thereof that are connected to each other and to
the C-terminus of the CH3 domain by a peptide linker and wherein
the second polypeptide comprises one ectodomain of said TNF ligand
family member or a fragment thereof connected via a peptide linker
to C-terminus of the CH3 domain of said polypeptide. Particularly,
such TNF family ligand trimer-containing antigen binding molecule
comprises two moieties capable of specific binding to a target cell
antigen.
[0257] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) two moities capable of specific binding to a target cell
antigen and (b) a first and a second polypeptide that are linked to
each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises one ectodomain of said TNF
ligand family member or a fragment thereof, wherein the two
moieties capable of specific binding to a target cell antigen bind
to two different target cell antigens.
[0258] In a further aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule as defined herein
before, wherein the moiety capable of specific binding to a target
cell antigen is selected from the group consisting of an antibody,
an antibody fragment and a scaffold antigen binding protein.
[0259] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule as described herein
before, wherein the moiety capable of specific binding to a target
cell antigen is selected from the group consisting of an antibody
fragment, a Fab molecule, a crossover Fab molecule, a single chain
Fab molecule, a Fv molecule, a scFv molecule, a single domain
antibody, an aVH and a scaffold antigen binding protein. In one
aspect, the moiety capable of specific binding to a target cell
antigen is an aVH or a scaffold antigen binding protein. In one
aspect, the moiety capable of specific binding to a target cell
antigen is a scaffold antigen binding protein capable of specific
binding to a target cell antigen.
[0260] In particular, the TNF family ligand trimer-containing
antigen binding molecule comprises one or two moieties capable of
specific binding to a target cell antigen.
[0261] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to a target cell antigen is a Fab
molecule or a crossover Fab molecule capable of specific binding to
a target cell antigen. In particular, the moiety capable of
specific binding to a target cell antigen is a Fab capable of
specific binding to a target cell antigen.
[0262] Furthermore, provided is TNF family ligand trimer-containing
antigen binding molecule as described herein, wherein the target
cell antigen is selected from the group consisting of Fibroblast
Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),
Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.
[0263] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule according to the
invention, wherein a peptide comprising two ectodomains of a TNF
ligand family member or fragments thereof connected to each other
by a first peptide linker is fused at its C-terminus to the CH1
domain of a heavy chain by a second peptide linker and wherein one
ectodomain of said TNF ligand family member or a fragment thereof
is fused at the its C-terminus to the CL domain on a light chain by
a third peptide linker.
[0264] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule according to the
invention, wherein a peptide comprising two ectodomains of a TNF
ligand family member or fragments thereof connected to each other
by a first peptide linker is fused at its C-terminus to the CL
domain of a heavy chain by a second peptide linker and wherein one
ectodomain of said TNF ligand family member or a fragment thereof
is fused at the its C-terminus to the CH1 domain on a light chain
by a third peptide linker.
[0265] In a further aspect, the invention is concerned with a TNF
family ligand trimer-containing antigen binding molecule according
to the invention, wherein a peptide comprising two ectodomains of a
TNF ligand family member or fragments thereof connected to each
other by a first peptide linker is fused at its C-terminus to the
CL domain of a light chain by a second peptide linker and wherein
one ectodomain of said TNF ligand family member or a fragment
thereof is fused at the its C-terminus to the CH1 domain of the
heavy chain by a third peptide linker.
[0266] In a particular aspect, the invention relates to a TNF
family ligand trimer-containing antigen binding molecule as defined
above, wherein the peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13). In one aspect, the first peptide linker is (G.sub.4S).sub.2
(SEQ ID NO:13), the second peptide linker is GSPGSSSSGS (SEQ ID
NO:57) and the third peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13). In particular, the invention relates to a TNF ligand
trimer-containing antigen binding molecule as defined above,
wherein the first peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13), the second peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13), and the third peptide linker is (G.sub.4S).sub.2 (SEQ ID
NO:13).
[0267] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule as defined herein before comprises an Fc
domain composed of a first and a second subunit capable of stable
association.
[0268] In particular, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises (a) a Fab
molecule capable of specific binding to a target cell antigen,
wherein the Fab heavy chain is fused at the C-terminus to the
N-terminus of a CH2 domain in the Fc domain and (c) an Fc domain
composed of a first and a second subunit capable of stable
association.
[0269] In a further aspect, the Fc domain is an IgG, particularly
an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc
domain is an IgG1 Fc domain. In a particular aspect, the Fc domain
comprises a modification promoting the association of the first and
second subunit of the Fc domain.
[0270] Fc domain modifications reducing Fc receptor binding and/or
effector function
[0271] The Fc domain of the TNF family ligand trimer-containing
antigen binding molecules of the invention consists of a pair of
polypeptide chains comprising heavy chain domains of an
immunoglobulin molecule. For example, the Fc domain of an
immunoglobulin G (IgG) molecule is a dimer, each subunit of which
comprises the CH2 and CH3 IgG heavy chain constant domains. The two
subunits of the Fc domain are capable of stable association with
each other.
[0272] The Fc domain confers favorable pharmacokinetic properties
to the antigen binding molecules of the invention, including a long
serum half-life which contributes to good accumulation in the
target tissue and a favorable tissue-blood distribution ratio. At
the same time it may, however, lead to undesirable targeting of the
bispecific antibodies of the invention to cells expressing Fc
receptors rather than to the preferred antigen-bearing cells.
Accordingly, in particular aspects, the Fc domain of the TNF family
ligand trimer-containing antigen binding molecule of the invention
exhibits reduced binding affinity to an Fc receptor and/or reduced
effector function, as compared to a native IgG1 Fc domain. In one
aspect, the Fc does not substantially bind to an Fc receptor and/or
does not induce effector function. In a particular aspect the Fc
receptor is an Fc.gamma. receptor. In one aspect, the Fc receptor
is a human Fc receptor. In a specific aspect, the Fc receptor is an
activating human Fc.gamma. receptor, more specifically human
Fc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa, most specifically
human Fc.gamma.RIIIa. In one aspect, the Fc domain does not induce
effector function. The reduced effector function can include, but
is not limited to, one or more of the following: reduced complement
dependent cytotoxicity (CDC), reduced antibody-dependent
cell-mediated cytotoxicity (ADCC), reduced antibody-dependent
cellular phagocytosis (ADCP), reduced cytokine secretion, reduced
immune complex-mediated antigen uptake by antigen-presenting cells,
reduced binding to NK cells, reduced binding to macrophages,
reduced binding to monocytes, reduced binding to polymorphonuclear
cells, reduced direct signaling inducing apoptosis, reduced
dendritic cell maturation, or reduced T cell priming.
[0273] In certain aspects, one or more amino acid modifications may
be introduced into the Fc region of a TNF family ligand
trimer-containing antigen binding molecule provided herein, thereby
generating an Fc region variant. The Fc region variant may comprise
a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4
Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions.
[0274] In a particular aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, (b) a first and a second polypeptide that are linked
to each other by a disulfide bond, wherein the antigen binding
molecule is characterized in that the first polypeptide comprises
two ectodomains of a TNF ligand family member or two fragments
thereof that are connected to each other by a peptide linker and in
that the second polypeptide comprises only one ectodomain of said
TNF ligand family member or a fragment thereof, and (c) an Fc
domain composed of a first and a second subunit capable of stable
association, wherein the Fc domain comprises one or more amino acid
substitution that reduces binding to an Fc receptor, in particular
towards Fc.gamma. receptor.
[0275] In one aspect, the Fc domain of the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises one or more amino acid mutation that reduces the binding
affinity of the Fc domain to an Fc receptor and/or effector
function. Typically, the same one or more amino acid mutation is
present in each of the two subunits of the Fc domain. In
particular, the Fc domain comprises an amino acid substitution at a
position of E233, L234, L235, N297, P331 and P329 (EU numbering).
In particular, the Fc domain comprises amino acid substitutions at
positions 234 and 235 (EU numbering) and/or 329 (EU numbering) of
the IgG heavy chains. More particularly, provided is a trimeric TNF
family ligand-containing antigen binding molecule according to the
invention which comprises an Fc domain with the amino acid
substitutions L234A, L235A and P329G ("P329G LALA", EU numbering)
in the IgG heavy chains. The amino acid substitutions L234A and
L235A refer to the so-called LALA mutation. The "P329G LALA"
combination of amino acid substitutions almost completely abolishes
Fc.gamma. receptor binding of a human IgG1 Fc domain and is
described in International Patent Appl. Publ. No. WO 2012/130831 A1
which also describes methods of preparing such mutant Fc domains
and methods for determining its properties such as Fc receptor
binding or effector functions. "EU numbering" refers to the
numbering according to EU index of Kabat et al, Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md., 1991.
[0276] Fc domains with reduced Fc receptor binding and/or effector
function also include those with substitution of one or more of Fc
domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.
6,737,056). Such Fc mutants include Fc mutants with substitutions
at two or more of amino acid positions 265, 269, 270, 297 and 327,
including the so-called "DANA" Fc mutant with substitution of
residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
[0277] In another aspect, the Fc domain is an IgG4 Fc domain. IgG4
antibodies exhibit reduced binding affinity to Fc receptors and
reduced effector functions as compared to IgG1 antibodies. In a
more specific aspect, the Fc domain is an IgG4 Fc domain comprising
an amino acid substitution at position 5228 (Kabat numbering),
particularly the amino acid substitution S228P. In a more specific
aspect, the Fc domain is an IgG4 Fc domain comprising amino acid
substitutions L235E and S228P and P329G (EU numbering). Such IgG4
Fc domain mutants and their Fc.gamma. receptor binding properties
are also described in WO 2012/130831.
[0278] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0279] Binding to Fc receptors can be easily determined e.g. by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIACORE.RTM. instrument (GE Healthcare),
and Fc receptors such as may be obtained by recombinant expression.
A suitable such binding assay is described herein. Alternatively,
binding affinity of Fc domains or cell activating bispecific
antigen binding molecules comprising an Fc domain for Fc receptors
may be evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor.
[0280] Effector function of an Fc domain, or bispecific antibodies
of the invention comprising an Fc domain, can be measured by
methods known in the art. A suitable assay for measuring ADCC is
described herein. Other examples of in vitro assays to assess ADCC
activity of a molecule of interest are described in U.S. Pat. No.
5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063
(1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502
(1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166,
1351-1361 (1987). Alternatively, non-radioactive assays methods may
be employed (see, for example, ACTI.TM. non-radioactive
cytotoxicity assay for flow cytometry (CellTechnology, Inc.
Mountain View, Calif.); and CytoTox 96.RTM. non-radioactive
cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.
in a animal model such as that disclosed in Clynes et al., Proc
Natl Acad Sci USA 95, 652-656 (1998).
[0281] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the bispecific antibodies of the invention is able to bind
C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISA
in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et
al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103,
2738-2743 (2004)).
[0282] In a particular aspect, the Fc domain comprises a
modification promoting the association of the first and second
subunit of the Fc domain.
[0283] Fc domain modifications promoting heterodimerization
[0284] In one aspect, the TNF family ligand trimer-containing
antigen binding molecules of the invention comprise (a) at least
one moiety capable of specific binding to a target cell antigen,
(b) a first and a second polypeptide that are linked to each other
by a disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises two
ectodomains of a TNF ligand family member or two fragments thereof
that are connected to each other by a peptide linker and in that
the second polypeptide comprises only one ectodomain of said TNF
ligand family member or a fragment thereof, and (c) an Fc domain
composed of a first and a second subunit capable of stable
association, wherein the Fc domain comprises one or more amino acid
substitution that reduces binding to an Fc receptor, in particular
towards Fc.gamma. receptor. Thus, they comprise different moieties,
fused to one or the other of the two subunits of the Fc domain that
are typically comprised in two non-identical polypeptide chains
("heavy chains"). Recombinant co-expression of these polypeptides
and subsequent dimerization leads to several possible combinations
of the two polypeptides. To improve the yield and purity of the TNF
family ligand trimer-containing antigen binding molecules in
recombinant production, it will thus be advantageous to introduce
in the Fc domain of the TNF family ligand trimer-containing antigen
binding molecules of the invention a modification promoting the
association of the desired polypeptides.
[0285] Accordingly, the Fc domain of the TNF family ligand
trimer-containing antigen binding molecules of the invention
comprises a modification promoting the association of the first and
the second subunit of the Fc domain. The site of most extensive
protein-protein interaction between the two subunits of a human IgG
Fc domain is in the CH3 domain of the Fc domain. Thus, said
modification is particularly in the CH3 domain of the Fc
domain.
[0286] In a specific aspect, said modification is a so-called
"knob-into-hole" modification, comprising a "knob" modification in
one of the two subunits of the Fc domain and a "hole" modification
in the other one of the two subunits of the Fc domain. Thus, in a
particular aspect, the invention relates to a TNF family ligand
trimer-containing antigen binding molecule as described herein
before which comprises an IgG molecule, wherein the Fc part of the
first heavy chain comprises a first dimerization module and the Fc
part of the second heavy chain comprises a second dimerization
module allowing a heterodimerization of the two heavy chains of the
IgG molecule and the first dimerization module comprises knobs and
the second dimerization module comprises holes according to the
knob into hole technology.
[0287] The knob-into-hole technology is described e.g. in U.S. Pat.
Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g. alanine or threonine).
[0288] Accordingly, in a particular aspect, in the CH3 domain of
the first subunit of the Fc domain of the TNF family ligand
trimer-containing antigen binding molecules of the invention an
amino acid residue is replaced with an amino acid residue having a
larger side chain volume, thereby generating a protuberance within
the CH3 domain of the first subunit which is positionable in a
cavity within the CH3 domain of the second subunit, and in the CH3
domain of the second subunit of the Fc domain an amino acid residue
is replaced with an amino acid residue having a smaller side chain
volume, thereby generating a cavity within the CH3 domain of the
second subunit within which the protuberance within the CH3 domain
of the first subunit is positionable.
[0289] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g. by site-specific
mutagenesis, or by peptide synthesis.
[0290] In a specific aspect, in the CH3 domain of the first subunit
of the Fc domain the threonine residue at position 366 is replaced
with a tryptophan residue (T366W), and in the CH3 domain of the
second subunit of the Fc domain the tyrosine residue at position
407 is replaced with a valine residue (Y407V). More particularly,
in the second subunit of the Fc domain additionally the threonine
residue at position 366 is replaced with a serine residue (T366S)
and the leucine residue at position 368 is replaced with an alanine
residue (L368A). More particularly, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C), and in the second subunit of the
Fc domain additionally the tyrosine residue at position 349 is
replaced by a cysteine residue (Y349C). The introduction of these
two cysteine residues results in the formation of a disulfide
bridge between the two subunits of the Fc domain. The disulfide
bridge further stabilizes the dimer (Carter, J Immunol Methods 248,
7-15 (2001)).
[0291] In an alternative aspect, a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g. as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable.
[0292] Modifications in the CH1/CL domains
[0293] To further improve correct pairing, the TNF family ligand
trimer-containing antigen binding molecules can contain different
charged amino acid substitutions (so-called "charged residues").
These modifications are introduced in the crossed or non-crossed
CH1 and CL domains. In a particular aspect, the invention relates
to a TNF family ligand trimer-containing antigen binding molecule,
wherein in one of CL domains the amino acid at position 123 (EU
numbering) has been replaced by arginine (R) and the amino acid at
position 124 (EU numbering) has been substituted by lysine (K) and
wherein in one of the CH1 domains the amino acids at position 147
(EU numbering) and at position 213 (EU numbering) have been
substituted by glutamic acid (E).
[0294] More particularly, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, wherein in the
CL domain adjacent to the TNF ligand family member the amino acid
at position 123 (EU numbering) has been replaced by arginine (R)
and the amino acid at position 124 (EU numbering) has been
substituted by lysine (K), and wherein in the CH1 domain adjacent
to the TNF ligand family member the amino acids at position 147 (EU
numbering) and at position 213 (EU numbering) have been substituted
by glutamic acid (E).
[0295] Particular TNF family ligand trimer-containing antigen
binding molecules
[0296] In another aspect, the invention provides a TNF family
ligand trimer-containing antigen binding molecule, wherein the
antigen binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
first peptide comprising two ectodomains of a TNF ligand family
member or fragments thereof connected to each other by a first
peptide linker fused at its C-terminus by a second peptide linker
to a second heavy or light chain, and a second peptide comprising
one ectodomain of said TNF ligand family member fused at its
C-terminus by a third peptide linker to a second light or heavy
chain, respectively.
[0297] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the first
peptide comprising two ectodomains of a TNF ligand family member or
fragments thereof connected to each other by a first peptide linker
is fused at its C-terminus by a second peptide linker to a CH1
domain that is part of a heavy chain, and the second peptide
comprising one ectodomain of said TNF ligand family member or a
fragment thereof is fused at its C-terminus by a third peptide
linker to a CL domain that is part of a light chain.
[0298] In yet another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the first
peptide comprising two ectodomains of a TNF ligand family member or
fragments thereof connected to each other by a first peptide linker
is fused at its C-terminus by a second peptide linker to a CL
domain that is part of a heavy chain,
and the second peptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof that is fused at its C-terminus
by a third peptide linker to a CH1 domain that is part of a light
chain.
[0299] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the first
peptide comprising two ectodomains of a TNF ligand family member or
fragments thereof connected to each other by a first peptide linker
is fused at its C-terminus by a second peptide linker to a VH
domain that is part of a heavy chain,
and the second peptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof is fused at its C-terminus by a
third peptide linker to a VL domain that is part of a light
chain.
[0300] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule of claims 21 to 23,
wherein in the CL domain adjacent to the TNF ligand family member
the amino acid at position 123 (EU numbering) has been replaced by
arginine (R) and the amino acid at position 124 (EU numbering) has
been substituted by lysine (K), and wherein in the CH1 domain
adjacent to the TNF ligand family member the amino acids at
position 147 (EU numbering) and at position 213 (EU numbering) have
been substituted by glutamic acid (E). These modifications lead to
so-called charged residues with advantageous properties that avoid
undesired effects such as for example mispairing.
[0301] Furthermore, provided is TNF family ligand trimer-containing
antigen binding molecule as described herein, wherein the target
cell antigen is selected from the group consisting of Fibroblast
Activation Protein (FAP), Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),
Carcinoembryonic Antigen (CEA), CD19, CD20 and CD33.
[0302] TNF family ligand trimer-containing antigen binding
molecules, wherein the target cell antigen is FAP
[0303] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the target cell
antigen is Fibroblast Activation Protein (FAP).
[0304] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to FAP comprises a VH domain comprising
(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:7 or SEQ
ID NO:100, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:8 or SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:9 or SEQ ID NO:102, and a VL domain
comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID
NO:10 or SEQ ID NO:103, (v) CDR-L2 comprising the amino acid
sequence of SEQ ID NO:11 or SEQ ID NO:104, and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:12 or SEQ ID
NO:105.
[0305] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the moiety capable of specific binding to a target cell
antigen is a Fab molecule capable of specific binding to FAP and
comprises a VH domain comprising (i) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:7, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:8 and (iii) CDR-H3 comprising the amino acid
sequence of SEQ ID NO:9, and a VL domain comprising (iv) CDR-L1
comprising the amino acid sequence of SEQ ID NO:10, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:11 and (vi) CDR-L3
comprising the amino acid sequence of SEQ ID NO:12.
[0306] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the moiety capable of specific binding to a target cell
antigen is a Fab molecule capable of specific binding to FAP and
comprises a VH domain comprising (i) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:100, (ii) CDR-H2 comprising the amino
acid sequence of SEQ ID NO:101 and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:102, and a VL domain comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:103,
(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:104 and
(vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:105.
[0307] In a further aspect, the moiety capable of specific binding
to FAP comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:16 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:17.
[0308] In another aspect, the moiety capable of specific binding to
FAP comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:106 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:107.
[0309] In one aspect, the moiety capable of specific binding to FAP
comprises a variable heavy chain comprising an amino acid sequence
of SEQ ID NO:16 and a variable light chain comprising an amino acid
sequence of SEQ ID NO:17 or a variable heavy chain comprising an
amino acid sequence of SEQ ID NO:106 and a variable light chain
comprising an amino acid sequence of SEQ ID NO:107.
[0310] In a particular aspect, the moiety capable of specific
binding to FAP comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:16 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:17. In
another particular aspect, the moiety capable of specific binding
to FAP comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:106 and a light chain variable region
comprising the amino acid sequence of SEQ ID NO:107. In a specific
aspect, the moiety capable of specific binding to FAP comprises a
VH domain consisting of amino acid sequence of SEQ ID NO:106 and a
VL domain consisting of the amino acid sequence of SEQ ID
NO:107.
[0311] In a further aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:16 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:17, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99 and in that the second
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ
ID NO:4.
[0312] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:16 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:17, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the the amino
acid sequence of SEQ ID NO:97 and the second polypeptide comprises
the amino acid sequence of SEQ ID NO:96.
[0313] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:106 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:107, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99 and in that the second
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ
ID NO:4.
[0314] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:106 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:107, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the the amino
acid sequence of SEQ ID NO:97 and the second polypeptide comprises
the amino acid sequence of SEQ ID NO:96.
[0315] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker that is fused at its C-terminus by a second
peptide linker to a CH1 domain, and a second light chain comprising
one ectodomain of said TNF ligand family member or a fragment
thereof is fused at its C-terminus by a third peptide linker to a
CL domain, and wherein the antigen binding molecule comprises (i) a
first heavy chain comprising the VH domain comprising the amino
acid sequence of SEQ ID NO:16 and a first light chain comprising
the VL domain comprising the amino acid sequence of SEQ ID NO:17 or
a first heavy chain comprising the VH domain comprising the amino
acid sequence of SEQ ID NO:106 and a first light chain comprising
the VL domain comprising the amino acid sequence of SEQ ID NO:107,
(ii) a second heavy chain comprising the amino acid sequence
selected from the group consisting of SEQ ID NO:14, SEQ ID NO:108,
SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light chain
comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:109,
SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
[0316] In a further particular aspect, provided is a TNF family
ligand trimer-containing antigen binding molecule, and wherein the
antigen binding molecule comprises a first heavy chain and a first
light chain, both comprising a Fab molecule capable of specific
binding to a target cell antigen,
a second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker is fused at its C-terminus by a second peptide
linker to a CL domain, and a second light chain comprising one
ectodomain of said TNF ligand family member or a fragment thereof
that is fused at its C-terminus by a third peptide linker to a CH1
domain, and wherein the molecule comprises (i) a first heavy chain
comprising the VH domain comprising the amino acid sequence of SEQ
ID NO:16 and a first light chain comprising the VL domain
comprising the amino acid sequence of SEQ ID NO:17 or a first heavy
chain comprising the VH domain comprising the amino acid sequence
of SEQ ID NO:106 and a first light chain comprising the VL domain
comprising the amino acid sequence of SEQ ID NO:107, (ii) a second
heavy chain comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119 and
SEQ ID NO:173, and (iii) a second light chain comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:116,
SEQ ID NO:118, SEQ ID NO:120 and SEQ ID NO:174.
[0317] More particularly, provided is a TNF family ligand
trimer-containing antigen binding molecule comprising
(a) a first heavy chain and a first light chain, both comprising a
Fab molecule capable of specific binding to a target cell antigen,
wherein the first heavy chain comprises the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and the first light chain
comprises the VL domain comprising the amino acid sequence of SEQ
ID NO:107, and (b) a second heavy chain comprising two ectodomains
of a TNF ligand family member or fragments thereof connected to
each other by a first peptide linker is fused at its C-terminus by
a second peptide linker to a CL domain, and a second light chain
comprising one ectodomain of said TNF ligand family member or a
fragment thereof that is fused at its C-terminus by a third peptide
linker to a CH1 domain, wherein the second heavy chain comprises
the amino acid sequence of SEQ ID NO:119 or SEQ ID NO:173, and the
second light chain comprises the amino acid sequence of SEQ ID
NO:120 or SEQ ID NO:174. In particular, the second heavy chain
comprises the amino acid sequence of SEQ ID NO:119 and the second
light chain comprises the amino acid sequence of SEQ ID NO:120.
[0318] Furthermore, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide.
[0319] In a particular aspect, such a TNF family ligand
trimer-containing antigen binding molecule comprises two moieties
capable of specific binding to a target cell antigen.
[0320] More particular, such TNF family ligand trimer-containing
antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:121, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:122, and two light chains comprising the amino acid
sequence of SEQ ID NO:19, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:123, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:124, and two light
chains comprising the amino acid sequence of SEQ ID NO:125, or
(iii) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:126, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:127, and two light chains comprising the amino acid
sequence of SEQ ID NO:125.
[0321] In a further aspect, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, selected from
the group consisting of:
[0322] a) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:14 and a second
light chain comprising the amino acid sequence of SEQ ID NO:15;
[0323] b) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:115 and a second
light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0324] c) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:135, a first light chain
comprising the amino acid sequence of SEQ ID NO:136, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:108 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:109;
[0325] d) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:139 and a second
light chain comprising the amino acid sequence of SEQ ID
NO:140;
[0326] e) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:19, a first heavy chain comprising
the amino acid sequence of SEQ ID NO:121 and a second heavy chain
comprising the amino acid sequence of SEQ ID NO:122;
[0327] f) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:108 and a second
light chain comprising the amino acid sequence of SEQ ID
NO:110;
[0328] g) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:145, a first light chain
comprising the amino acid sequence of SEQ ID NO:19, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:115 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0329] h) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:148 and a second
light chain comprising the amino acid sequence of SEQ ID
NO:149;
[0330] i) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:111 and a second
light chain comprising the amino acid sequence of SEQ ID NO:112;
and
[0331] j) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:18, a first light chain comprising
the amino acid sequence of SEQ ID NO:19, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:113 and a second
light chain comprising the amino acid sequence of SEQ ID
NO:114.
[0332] In another aspect, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, selected from
the group consisting of:
[0333] a) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:164, a first light chain
comprising the amino acid sequence of SEQ ID NO:125, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:115 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0334] b) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:164, a first light chain
comprising the amino acid sequence of SEQ ID NO:125, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:117 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:118;
[0335] c) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:125, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:123 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:124;
[0336] d) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:164, a first light chain
comprising the amino acid sequence of SEQ ID NO:125, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:119 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:120;
[0337] e) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:164, a first light chain
comprising the amino acid sequence of SEQ ID NO:125, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:173 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:174; and
[0338] f) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:125, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:126 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:127.
[0339] In particular, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising a first heavy
chain comprising the amino acid sequence of SEQ ID NO:164, a first
light chain comprising the amino acid sequence of SEQ ID NO:125, a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:119 and a second light chain comprising the amino acid sequence
of SEQ ID NO:120.
[0340] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the TNF ligand
family member is OX40L and wherein the target cell antigen is
Fibroblast Activation Protein (FAP) and the moiety capable of
specific binding to FAP comprises a VH domain comprising (i) CDR-H1
comprising the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:100,
(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:8 or
SEQ ID NO:101, and (iii) CDR-H3 comprising the amino acid sequence
of SEQ ID NO:9 or SEQ ID NO:102, and a VL domain comprising (iv)
CDR-L1 comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID
NO:103, (v) CDR-L2 comprising the amino acid sequence of SEQ ID
NO:11 or SEQ ID NO:104, and (vi) CDR-L3 comprising the amino acid
sequence of SEQ ID NO:12 or SEQ ID NO:105.
[0341] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of comprises
(i) a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:16 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:17 or a first heavy chain comprising the VH domain comprising
the amino acid sequence of SEQ ID NO:106 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:107, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:355, and
(iii) a second light chain comprising the amino acid sequence of
SEQ ID NO:356.
[0342] TNF family ligand trimer-containing antigen binding
molecules, wherein the target cell antigen is CD19
[0343] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the target cell
antigen is CD19.
[0344] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CD19 comprises a VH domain
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:195 or SEQ ID NO:252, (ii) CDR-H2 comprising the amino acid
sequence of SEQ ID NO:196 or SEQ ID NO:253, and (iii) CDR-H3
comprising the amino acid sequence of SEQ ID NO:197 or SEQ ID
NO:254, and a VL domain comprising (iv) CDR-L1 comprising the amino
acid sequence of SEQ ID NO:198 or SEQ ID NO:249, (v) CDR-L2
comprising the amino acid sequence of SEQ ID NO:199 or SEQ ID
NO:250, and (vi) CDR-L3 comprising the amino acid sequence of SEQ
ID NO:200 or SEQ ID NO:251.
[0345] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the moiety capable of specific binding to a target cell
antigen is a Fab molecule capable of specific binding to CD19 and
comprises a VH domain comprising (i) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:195, (ii) CDR-H2 comprising the amino
acid sequence of SEQ ID NO:196 and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:197, and a VL domain comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:198,
(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:199 and
(vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:200.
[0346] In a further aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the moiety capable of specific binding to a target cell
antigen is a Fab molecule capable of specific binding to CD19 and
comprises a VH domain comprising (i) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:252, (ii) CDR-H2 comprising the amino
acid sequence of SEQ ID NO:253 and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:254, and a VL domain comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:249,
(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:250 and
(vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:251.
[0347] In another aspect, the moiety capable of specific binding to
CD19 comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:201 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:202.
[0348] In a further aspect, the moiety capable of specific binding
to CD19 comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:357 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:358.
[0349] In one aspect, the moiety capable of specific binding to
CD19 comprises a variable heavy chain comprising an amino acid
sequence of SEQ ID NO:201 and a variable light chain comprising an
amino acid sequence of SEQ ID NO:202 or wherein the moiety capable
of specific binding to CD19 comprises a variable heavy chain
comprising an amino acid sequence of SEQ ID NO:357 and a variable
light chain comprising an amino acid sequence of SEQ ID NO:358.
[0350] In a particular aspect, the moiety capable of specific
binding to CD19 comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:201 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:202. In
another particular aspect, the moiety capable of specific binding
to CD19 comprises a heavy chain variable region comprising the
amino acid sequence of SEQ ID NO:357 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:358.
[0351] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:201 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:202, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99 and in that the second
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ
ID NO:4.
[0352] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:201 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:202, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the the amino
acid sequence of SEQ ID NO:97 and the second polypeptide comprises
the amino acid sequence of SEQ ID NO:96.
[0353] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:357 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:358, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99 and in that the second
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ
ID NO:4.
[0354] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:357 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:358, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the the amino
acid sequence of SEQ ID NO:97 and the second polypeptide comprises
the amino acid sequence of SEQ ID NO:96.
[0355] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker that is fused at its C-terminus by a second
peptide linker to a CH1 domain, and a second light chain comprising
one ectodomain of said TNF ligand family member or a fragment
thereof is fused at its C-terminus by a third peptide linker to a
CL domain, and wherein the antigen binding molecule comprises (i) a
first heavy chain comprising the VH domain comprising the amino
acid sequence of SEQ ID NO:201 and a first light chain comprising
the VL domain comprising the amino acid sequence of SEQ ID NO:202
or a first heavy chain comprising the VH domain comprising the
amino acid sequence of SEQ ID NO:357 and a first light chain
comprising the VL domain comprising the amino acid sequence of SEQ
ID NO:358, (ii) a second heavy chain comprising the amino acid
sequence selected from the group consisting of SEQ ID NO:14, SEQ ID
NO:108, SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light
chain comprising the amino acid sequence of SEQ ID NO:15, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
[0356] In a further particular aspect, provided is a TNF family
ligand trimer-containing antigen binding molecule, and wherein the
antigen binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker is fused at its C-terminus by a second peptide
linker to a CL domain, and a second light chain comprising one
ectodomain of said TNF ligand family member or a fragment thereof
that is fused at its C-terminus by a third peptide linker to a CH1
domain, and wherein the molecule comprises (i) a first heavy chain
comprising the VH domain comprising the amino acid sequence of SEQ
ID NO:201 and a first light chain comprising the VL domain
comprising the amino acid sequence of SEQ ID NO:202 or a first
heavy chain comprising the VH domain comprising the amino acid
sequence of SEQ ID NO:357 and a first light chain comprising the VL
domain comprising the amino acid sequence of SEQ ID NO:358, (ii) a
second heavy chain comprising the amino acid sequence selected from
the group consisting of SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119
and SEQ ID NO:173, and (iii) a second light chain comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:116, SEQ ID NO:118, SEQ ID NO:120 and SEQ ID NO:174.
[0357] Furthermore, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide.
[0358] In a particular aspect, such a TNF family ligand
trimer-containing antigen binding molecule comprises two moieties
capable of specific binding to a target cell antigen.
[0359] More particular, such TNF family ligand trimer-containing
antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:209, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:210, and two light chains comprising the amino acid
sequence of SEQ ID NO:206, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:213, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:214, and two light
chains comprising the amino acid sequence of SEQ ID NO:206, or
(iii) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:309, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:310, and two light chains comprising the amino acid
sequence of SEQ ID NO:279, or (iv) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:313, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:314, and two light
chains comprising the amino acid sequence of SEQ ID NO:279.
[0360] In a further aspect, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, selected from
the group consisting of:
[0361] a) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:205, a first light chain
comprising the amino acid sequence of SEQ ID NO:206, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:115 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0362] b) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:205, a first light chain
comprising the amino acid sequence of SEQ ID NO:206, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:117 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:118;
[0363] c) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:206, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:209 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:210;
[0364] d) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:205, a first light chain
comprising the amino acid sequence of SEQ ID NO:206, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:119 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:120;
[0365] e) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:205, a first light chain
comprising the amino acid sequence of SEQ ID NO:206, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:173 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:174; and
[0366] f) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:206, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:213 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:214.
[0367] In particular, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising a first heavy
chain comprising the amino acid sequence of SEQ ID NO:205, a first
light chain comprising the amino acid sequence of SEQ ID NO:206, a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:119 and a second light chain comprising the amino acid sequence
of SEQ ID NO:120.
[0368] In another aspect, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, selected from
the group consisting of:
[0369] a) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:357, a first light chain
comprising the amino acid sequence of SEQ ID NO:358, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:115 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0370] b) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:357, a first light chain
comprising the amino acid sequence of SEQ ID NO:358, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:117 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:118;
[0371] c) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:358, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:209 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:210;
[0372] d) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:357, a first light chain
comprising the amino acid sequence of SEQ ID NO:358, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:119 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:120;
[0373] e) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:357, a first light chain
comprising the amino acid sequence of SEQ ID NO:358, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:173 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:174; and
[0374] f) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:358, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:213 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:214.
[0375] In particular, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising a first heavy
chain comprising the amino acid sequence of SEQ ID NO:357, a first
light chain comprising the amino acid sequence of SEQ ID NO:358, a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:119 and a second light chain comprising the amino acid sequence
of SEQ ID NO:120.
[0376] TNF family ligand trimer-containing antigen binding
molecules, wherein the target cell antigen is CEA.
[0377] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the target cell
antigen is CEA.
[0378] In one aspect, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, wherein the moiety
capable of specific binding to CD19 comprises a VH domain
comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID
NO:321, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID
NO:322, and (iii) CDR-H3 comprising the amino acid sequence of SEQ
ID NO:323, and a VL domain comprising (iv) CDR-L1 comprising the
amino acid sequence of SEQ ID NO:324, (v) CDR-L2 comprising the
amino acid sequence of SEQ ID NO:325, and (vi) CDR-L3 comprising
the amino acid sequence of SEQ ID NO:326.
[0379] In a particular aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule of the invention,
wherein the moiety capable of specific binding to a target cell
antigen is a Fab molecule capable of specific binding to CEA and
comprises a VH domain comprising (i) CDR-H1 comprising the amino
acid sequence of SEQ ID NO:321, (ii) CDR-H2 comprising the amino
acid sequence of SEQ ID NO:322 and (iii) CDR-H3 comprising the
amino acid sequence of SEQ ID NO:323, and a VL domain comprising
(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:324,
(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:325 and
(vi) CDR-L3 comprising the amino acid sequence of SEQ ID
NO:326.
[0380] In a further aspect, the moiety capable of specific binding
to CEA comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:327 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:328.
[0381] In one aspect, the moiety capable of specific binding to CEA
comprises a variable heavy chain comprising an amino acid sequence
of SEQ ID NO:327 and a variable light chain comprising an amino
acid sequence of SEQ ID NO:328.
[0382] In a further aspect, the moiety capable of specific binding
to CEA comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO:329 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:330.
[0383] In one aspect, the moiety capable of specific binding to CEA
comprises a variable heavy chain comprising an amino acid sequence
of SEQ ID NO:329 and a variable light chain comprising an amino
acid sequence of SEQ ID NO:330.
[0384] In another aspect, the TNF family ligand trimer-containing
antigen binding molecule of the invention comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:329 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:330, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the amino
acid sequence selected from the group consisting of SEQ ID NO:5,
SEQ ID NO:97, SEQ ID NO:98 and SEQ ID NO:99 and in that the second
polypeptide comprises the amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:96, SEQ ID NO:3 and SEQ
ID NO:4.
[0385] In a particular aspect, the TNF family ligand
trimer-containing antigen binding molecule of the invention
comprises
(a) at least one moiety capable of specific binding to a target
cell antigen comprising a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO:329 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO:330, and (b)
a first and a second polypeptide that are linked to each other by a
disulfide bond, wherein the antigen binding molecule is
characterized in that the first polypeptide comprises the the amino
acid sequence of SEQ ID NO:97 and the second polypeptide comprises
the amino acid sequence of SEQ ID NO:96.
[0386] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule, wherein the antigen
binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker that is fused at its C-terminus by a second
peptide linker to a CH1 domain, and a second light chain comprising
one ectodomain of said TNF ligand family member or a fragment
thereof is fused at its C-terminus by a third peptide linker to a
CL domain, and wherein the antigen binding molecule comprises (i) a
first heavy chain comprising the VH domain comprising the amino
acid sequence of SEQ ID NO:329 and a first light chain comprising
the VL domain comprising the amino acid sequence of SEQ ID NO:330,
(ii) a second heavy chain comprising the amino acid sequence
selected from the group consisting of SEQ ID NO:14, SEQ ID NO:108,
SEQ ID NO:111 and SEQ ID NO:113, and (iii) a second light chain
comprising the amino acid sequence of SEQ ID NO:15, SEQ ID NO:109,
SEQ ID NO:110, SEQ ID NO:112 and SEQ ID NO:114.
[0387] In a further particular aspect, provided is a TNF family
ligand trimer-containing antigen binding molecule, and wherein the
antigen binding molecule comprises
a first heavy chain and a first light chain, both comprising a Fab
molecule capable of specific binding to a target cell antigen, a
second heavy chain comprising two ectodomains of a TNF ligand
family member or fragments thereof connected to each other by a
first peptide linker is fused at its C-terminus by a second peptide
linker to a CL domain, and a second light chain comprising one
ectodomain of said TNF ligand family member or a fragment thereof
that is fused at its C-terminus by a third peptide linker to a CH1
domain, and wherein the molecule comprises i) a first heavy chain
comprising the VH domain comprising the amino acid sequence of SEQ
ID NO:329 and a first light chain comprising the VL domain
comprising the amino acid sequence of SEQ ID NO:330, (ii) a second
heavy chain comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119 and
SEQ ID NO:173, and (iii) a second light chain comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:116,
SEQ ID NO:118, SEQ ID NO:120 and SEQ ID NO:174.
[0388] Furthermore, the invention provides a TNF family ligand
trimer-containing antigen binding molecule, comprising
(a) at least one moiety capable of specific binding to a target
cell antigen, and (b) a first and a second polypeptide that are
linked to each other by a disulfide bond, wherein the antigen
binding molecule is characterized in that the first polypeptide
contains a CH3 domain and the second polypeptide contains a CH3
domain, respectively, and wherein the first polypeptide comprises
two ectodomains of a TNF ligand family member or fragments thereof
that are connected to each other and to the C-terminus of the CH3
domain by a peptide linker and wherein the second polypeptide
comprises one ectodomain of said TNF ligand family member or a
fragment thereof connected via a peptide linker to C-terminus of
the CH3 domain of said polypeptide.
[0389] In a particular aspect, such a TNF family ligand
trimer-containing antigen binding molecule comprises two moieties
capable of specific binding to a target cell antigen.
[0390] More particular, such TNF family ligand trimer-containing
antigen binding molecule comprises
(i) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:337, a second heavy chain comprising the amino acid sequence
of SEQ ID NO:338, and two light chains comprising the amino acid
sequence of SEQ ID NO:334, or (ii) a first heavy chain comprising
the amino acid sequence of SEQ ID NO:341, a second heavy chain
comprising the amino acid sequence of SEQ ID NO:342, and two light
chains comprising the amino acid sequence of SEQ ID NO:334.
[0391] In a further aspect, the invention relates to a TNF family
ligand trimer-containing antigen binding molecule, selected from
the group consisting of:
[0392] a) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:333, a first light chain
comprising the amino acid sequence of SEQ ID NO:334, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:115 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:116;
[0393] b) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:333, a first light chain
comprising the amino acid sequence of SEQ ID NO:334, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:117 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:118;
[0394] c) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:334, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:337 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:338;
[0395] d) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:333, a first light chain
comprising the amino acid sequence of SEQ ID NO:334, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:119 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:120;
[0396] e) a molecule comprising a first heavy chain comprising the
amino acid sequence of SEQ ID NO:333, a first light chain
comprising the amino acid sequence of SEQ ID NO:334, a second heavy
chain comprising the amino acid sequence of SEQ ID NO:173 and a
second light chain comprising the amino acid sequence of SEQ ID
NO:174; and
[0397] f) a molecule comprising two light chains comprising the
amino acid sequence of SEQ ID NO:334, a first heavy chain
comprising the amino acid sequence of SEQ ID NO:341 and a second
heavy chain comprising the amino acid sequence of SEQ ID
NO:342.
[0398] In particular, the invention provides a TNF family ligand
trimer-containing antigen binding molecule comprising a first heavy
chain comprising the amino acid sequence of SEQ ID NO:333, a first
light chain comprising the amino acid sequence of SEQ ID NO:334, a
second heavy chain comprising the amino acid sequence of SEQ ID
NO:119 and a second light chain comprising the amino acid sequence
of SEQ ID NO:120.
[0399] Polynucleotides
[0400] The invention further provides isolated polynucleotides
encoding a TNF family ligand trimer-containing antigen binding
molecule as described herein or a fragment thereof.
[0401] The isolated polynucleotides encoding TNF ligand
trimer-containing antigen binding molecules of the invention may be
expressed as a single polynucleotide that encodes the entire
antigen binding molecule or as multiple (e.g., two or more)
polynucleotides that are co-expressed. Polypeptides encoded by
polynucleotides that are co-expressed may associate through, e.g.,
disulfide bonds or other means to form a functional antigen binding
molecule. For example, the light chain portion of an immunoglobulin
may be encoded by a separate polynucleotide from the heavy chain
portion of the immunoglobulin. When co-expressed, the heavy chain
polypeptides will associate with the light chain polypeptides to
form the immunoglobulin.
[0402] In some aspects, the isolated polynucleotide encodes the
entire TNF family ligand trimer-containing antigen binding molecule
according to the invention as described herein. In particular, the
isolated polynucleotide encodes a polypeptide comprised in the TNF
family ligand trimer-containing antigen binding molecule according
to the invention as described herein.
[0403] In one aspect, the present invention is directed to an
isolated polynucleotide encoding a TNF family ligand
trimer-containing antigen binding molecule, wherein the
polynucleotide comprises (a) a sequence that encodes a moiety
capable of specific binding to a target cell antigen, (b) a
sequence that encodes a polypeptide comprising two ectodomains of a
TNF ligand family member or two fragments thereof that are
connected to each other by a peptide linker and (c) a sequence that
encodes a polypeptide comprising one ectodomain of said TNF ligand
family member or a fragment thereof.
[0404] In another aspect, provided is an isolated polynucleotide
encoding a 4-1BB ligand trimer-containing antigen binding molecule,
wherein the polynucleotide comprises (a) a sequence that encodes a
moiety capable of specific binding to a target cell antigen, (b) a
sequence that encodes a polypeptide comprising two ectodomains of
4-1BBL or two fragments thereof that are connected to each other by
a peptide linker and (c) a sequence that encodes a polypeptide
comprising one ectodomain of 4-1BBL or a fragment thereof.
[0405] In a further aspect, the invention is directed to an
isolated polynucleotide comprising a sequence that encodes a
polypeptide comprising two 4-1BBL fragments comprising an amino
acid sequence that is at least about 90%, 95%, 98% or 100%
identical to an amino acid sequence shown in SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:96, and to a
polynucleotide comprising a sequence that encodes a polypeptide
comprising one 4-1BBL fragment comprising an amino acid sequence
that is at least about 90%, 95%, 98% or 100% identical to an amino
acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4 or SEQ ID NO:96.
[0406] Furthermore, provided is an isolated polynucleotide encoding
a OX40 ligand trimer-containing antigen binding molecule, wherein
the polynucleotide comprises (a) a sequence that encodes a moiety
capable of specific binding to a target cell antigen, (b) a
sequence that encodes a polypeptide comprising two ectodomains of
OX40L or two fragments thereof that are connected to each other by
a peptide linker and (c) a sequence that encodes a polypeptide
comprising one ectodomain of OX40L or a fragment thereof.
[0407] In another aspect, the invention is directed to an isolated
polynucleotide comprising a sequence that encodes a polypeptide
comprising two 4-1BBL fragments comprising an amino acid sequence
that is at least about 90%, 95%, 98% or 100% identical to an amino
acid sequence shown in SEQ ID NO:53 or SEQ ID NO:54, and to a
polynucleotide comprising a sequence that encodes a polypeptide
comprising one 4-1BBL fragment comprising an amino acid sequence
that is at least about 90%, 95%, 98% or 100% identical to an amino
acid sequence shown in SEQ ID NO:53 or SEQ ID NO:54.
[0408] In further aspects, the invention relates to the
polynucleotides comprising a sequence that is at least about 90%,
95%, 98% or 100% identical to the specific cDNA sequences disclosed
herein. In a particular aspect, the invention relates to a
polynucleotide comprising a sequence that is identical to one of
the specific cDNA sequences disclosed herein.
[0409] In other aspects, the nucleic acid molecule comprises or
consists of a nucleotide sequence that encodes an amino acid
sequence as set forth in any one of SEQ ID NOs: 5, 6, 97, 98, 99,
183, 184 or 185. In a further aspect, the nucleic acid molecule
comprises or consists of a nucleotide sequence that encodes an
amino acid sequence as set forth in any one of SEQ ID NOs:14, 15,
108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 173
or 174.
[0410] In still other aspects, the nucleic acid molecule comprises
or consists of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 66, 67, 68, 69, 129, 130, 131, 132, 133,
134, 137, 138, 141, 142, 143, 144, 146, 147, 150, 151, 152, 153,
162, 163, 165, 166, 167, 168, 169, 170, 171, 172, 175, 176, 177,
178, 203, 204, 207, 208, 211, 212, 215, 216, 273, 274, 277, 278,
281, 282, 285, 286, 289, 290, 293, 294, 297, 298, 301, 302, 305,
307, 308, 311, 312, 315, 316, 331, 332, 335, 336, 339, 340, 343,
344, 347, 348, 353 or 354.
[0411] In certain aspects, the polynucleotide or nucleic acid is
DNA. In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
RNA of the present invention may be single stranded or double
stranded.
[0412] Recombinant Methods
[0413] TNF family ligand trimer-containing antigen binding
molecules of the invention may be obtained, for example, by
solid-state peptide synthesis (e.g. Merrifield solid phase
synthesis) or recombinant production. For recombinant production
one or more polynucleotide encoding the TNF family ligand
trimer-containing antigen binding molecule or polypeptide fragments
thereof, e.g., as described above, is isolated and inserted into
one or more vectors for further cloning and/or expression in a host
cell. Such polynucleotide may be readily isolated and sequenced
using conventional procedures. In one aspect of the invention, a
vector, preferably an expression vector, comprising one or more of
the polynucleotides of the invention is provided. Methods which are
well known to those skilled in the art can be used to construct
expression vectors containing the coding sequence of the TNF family
ligand trimer-containing antigen binding molecule (fragment) along
with appropriate transcriptional/translational control signals.
These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and
Wiley Interscience, N.Y. (1989). The expression vector can be part
of a plasmid, virus, or may be a nucleic acid fragment. The
expression vector includes an expression cassette into which the
polynucleotide encoding the TNF family ligand trimer-containing
antigen binding molecule or polypeptide fragments thereof (i.e. the
coding region) is cloned in operable association with a promoter
and/or other transcription or translation control elements. As used
herein, a "coding region" is a portion of nucleic acid which
consists of codons translated into amino acids. Although a "stop
codon" (TAG, TGA, or TAA) is not translated into an amino acid, it
may be considered to be part of a coding region, if present, but
any flanking sequences, for example promoters, ribosome binding
sites, transcriptional terminators, introns, 5' and 3' untranslated
regions, and the like, are not part of a coding region. Two or more
coding regions can be present in a single polynucleotide construct,
e.g. on a single vector, or in separate polynucleotide constructs,
e.g. on separate (different) vectors. Furthermore, any vector may
contain a single coding region, or may comprise two or more coding
regions, e.g. a vector of the present invention may encode one or
more polypeptides, which are post- or co-translationally separated
into the final proteins via proteolytic cleavage. In addition, a
vector, polynucleotide, or nucleic acid of the invention may encode
heterologous coding regions, either fused or unfused to a
polynucleotide encoding the TNF family ligand trimer-containing
antigen binding molecule of the invention or polypeptide fragments
thereof, or variants or derivatives thereof. Heterologous coding
regions include without limitation specialized elements or motifs,
such as a secretory signal peptide or a heterologous functional
domain. An operable association is when a coding region for a gene
product, e.g. a polypeptide, is associated with one or more
regulatory sequences in such a way as to place expression of the
gene product under the influence or control of the regulatory
sequence(s). Two DNA fragments (such as a polypeptide coding region
and a promoter associated therewith) are "operably associated" if
induction of promoter function results in the transcription of mRNA
encoding the desired gene product and if the nature of the linkage
between the two DNA fragments does not interfere with the ability
of the expression regulatory sequences to direct the expression of
the gene product or interfere with the ability of the DNA template
to be transcribed. Thus, a promoter region would be operably
associated with a nucleic acid encoding a polypeptide if the
promoter was capable of effecting transcription of that nucleic
acid. The promoter may be a cell-specific promoter that directs
substantial transcription of the DNA only in predetermined cells.
Other transcription control elements, besides a promoter, for
example enhancers, operators, repressors, and transcription
termination signals, can be operably associated with the
polynucleotide to direct cell-specific transcription.
[0414] Suitable promoters and other transcription control regions
are disclosed herein. A variety of transcription control regions
are known to those skilled in the art. These include, without
limitation, transcription control regions, which function in
vertebrate cells, such as, but not limited to, promoter and
enhancer segments from cytomegaloviruses (e.g. the immediate early
promoter, in conjunction with intron-A), simian virus 40 (e.g. the
early promoter), and retroviruses (such as, e.g. Rous sarcoma
virus). Other transcription control regions include those derived
from vertebrate genes such as actin, heat shock protein, bovine
growth hormone and rabbit a-globin, as well as other sequences
capable of controlling gene expression in eukaryotic cells.
Additional suitable transcription control regions include
tissue-specific promoters and enhancers as well as inducible
promoters (e.g. promoters inducible tetracyclins). Similarly, a
variety of translation control elements are known to those of
ordinary skill in the art. These include, but are not limited to
ribosome binding sites, translation initiation and termination
codons, and elements derived from viral systems (particularly an
internal ribosome entry site, or IRES, also referred to as a CITE
sequence). The expression cassette may also include other features
such as an origin of replication, and/or chromosome integration
elements such as retroviral long terminal repeats (LTRs), or
adeno-associated viral (AAV) inverted terminal repeats (ITRs).
[0415] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. For example, if secretion of the TNF family
ligand trimer-containing antigen binding molecule or polypeptide
fragments thereof is desired, DNA encoding a signal sequence may be
placed upstream of the nucleic acid encoding a TNF family ligand
trimer-containing antigen binding molecule of the invention or
polypeptide fragments thereof. According to the signal hypothesis,
proteins secreted by mammalian cells have a signal peptide or
secretory leader sequence which is cleaved from the mature protein
once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Those of ordinary skill
in the art are aware that polypeptides secreted by vertebrate cells
generally have a signal peptide fused to the N-terminus of the
polypeptide, which is cleaved from the translated polypeptide to
produce a secreted or "mature" form of the polypeptide. In certain
embodiments, the native signal peptide, e.g. an immunoglobulin
heavy chain or light chain signal peptide is used, or a functional
derivative of that sequence that retains the ability to direct the
secretion of the polypeptide that is operably associated with it.
Alternatively, a heterologous mammalian signal peptide, or a
functional derivative thereof, may be used. For example, the
wild-type leader sequence may be substituted with the leader
sequence of human tissue plasminogen activator (TPA) or mouse
.beta.-glucuronidase.
[0416] DNA encoding a short protein sequence that could be used to
facilitate later purification (e.g. a histidine tag) or assist in
labeling the fusion protein may be included within or at the ends
of the polynucleotide encoding a TNF family ligand
trimer-containing antigen binding molecule of the invention or
polypeptide fragments thereof.
[0417] In a further aspect of the invention, a host cell comprising
one or more polynucleotides of the invention is provided. In
certain embodiments a host cell comprising one or more vectors of
the invention is provided. The polynucleotides and vectors may
incorporate any of the features, singly or in combination,
described herein in relation to polynucleotides and vectors,
respectively. In one aspect, a host cell comprises (e.g. has been
transformed or transfected with) a vector comprising a
polynucleotide that encodes (part of) a TNF family ligand
trimer-containing antigen binding molecule of the invention of the
invention. As used herein, the term "host cell" refers to any kind
of cellular system which can be engineered to generate the fusion
proteins of the invention or fragments thereof. Host cells suitable
for replicating and for supporting expression of antigen binding
molecules are well known in the art. Such cells may be transfected
or transduced as appropriate with the particular expression vector
and large quantities of vector containing cells can be grown for
seeding large scale fermenters to obtain sufficient quantities of
the antigen binding molecule for clinical applications. Suitable
host cells include prokaryotic microorganisms, such as E. coli, or
various eukaryotic cells, such as Chinese hamster ovary cells
(CHO), insect cells, or the like. For example, polypeptides may be
produced in bacteria in particular when glycosylation is not
needed. After expression, the polypeptide may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified. In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for polypeptide-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized", resulting in
the production of a polypeptide with a partially or fully human
glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414
(2004), and Li et al., Nat Biotech 24, 210-215 (2006).
[0418] Suitable host cells for the expression of (glycosylated)
polypeptides are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells. Plant
cell cultures can also be utilized as hosts. See e.g. U.S. Pat.
Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants). Vertebrate cells may also be used as hosts. For
example, mammalian cell lines that are adapted to grow in
suspension may be useful. Other examples of useful mammalian host
cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);
human embryonic kidney line (293 or 293T cells as described, e.g.,
in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney
cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in
Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1),
African green monkey kidney cells (VERO-76), human cervical
carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat
liver cells (BRL 3A), human lung cells (W138), human liver cells
(Hep G.sub.2), mouse mammary tumor cells (MMT 060562), TRI cells
(as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383,
44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian
host cell lines include Chinese hamster ovary (CHO) cells,
including dhfr- CHO cells (Urlaub et al., Proc Natl Acad Sci USA
77, 4216 (1980)); and myeloma cell lines such as YO, NSO, P3X63 and
Sp2/0. For a review of certain mammalian host cell lines suitable
for protein production, see, e.g., Yazaki and Wu, Methods in
Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,
Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured
cells, e.g., mammalian cultured cells, yeast cells, insect cells,
bacterial cells and plant cells, to name only a few, but also cells
comprised within a transgenic animal, transgenic plant or cultured
plant or animal tissue. In one embodiment, the host cell is a
eukaryotic cell, preferably a mammalian cell, such as a Chinese
Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a
lymphoid cell (e.g., YO, NSO, Sp20 cell). Standard technologies are
known in the art to express foreign genes in these systems. Cells
expressing a polypeptide comprising either the heavy or the light
chain of an immunoglobulin, may be engineered so as to also express
the other of the immunoglobulin chains such that the expressed
product is an immunoglobulin that has both a heavy and a light
chain.
[0419] In one aspect, a method of producing a TNF family ligand
trimer-containing antigen binding molecule of the invention or
polypeptide fragments thereof is provided, wherein the method
comprises culturing a host cell comprising polynucleotides encoding
the TNF family ligand trimer-containing antigen binding molecule of
the invention or polypeptide fragments thereof, as provided herein,
under conditions suitable for expression of the TNF family ligand
trimer-containing antigen binding molecule of the invention or
polypeptide fragments thereof, and recovering the TNF family ligand
trimer-containing antigen binding molecule of the invention or
polypeptide fragments thereof from the host cell (or host cell
culture medium).
[0420] In the TNF family ligand trimer-containing antigen binding
molecule of the invention, the components (at least one moiety
capable of specific binding to a target cell antigen, one
polypeptide comprising two ectodomains of a TNF ligand family
member or fragments thereof and a polypeptide comprising one
ectodomain of said TNF family ligand family member or a fragment
thereof) are not genetically fused to each other. The polypeptides
are designed such that its components (two ectodomains of a TNF
ligand family member or fragments thereof and other components such
as CH or CL) are fused to each other directly or through a linker
sequence. The composition and length of the linker may be
determined in accordance with methods well known in the art and may
be tested for efficacy. Examples of linker sequences between
different components of the antigen binding molecules of the
invention are found in the sequences provided herein. Additional
sequences may also be included to incorporate a cleavage site to
separate the individual components of the fusion protein if
desired, for example an endopeptidase recognition sequence.
[0421] In certain embodiments the moieties capable of specific
binding to a target cell antigen (e.g. Fab fragments) forming part
of the antigen binding molecule comprise at least an immunoglobulin
variable region capable of binding to an antigen. Variable regions
can form part of and be derived from naturally or non-naturally
occurring antibodies and fragments thereof. Methods to produce
polyclonal antibodies and monoclonal antibodies are well known in
the art (see e.g. Harlow and Lane, "Antibodies, a laboratory
manual", Cold Spring Harbor Laboratory, 1988). Non-naturally
occurring antibodies can be constructed using solid phase-peptide
synthesis, can be produced recombinantly (e.g. as described in U.S.
Pat. No. 4,186,567) or can be obtained, for example, by screening
combinatorial libraries comprising variable heavy chains and
variable light chains (see e.g. U.S. Pat. No. 5,969,108 to
McCafferty).
[0422] Any animal species of immunoglobulin can be used in the
invention. Non-limiting immunoglobulins useful in the present
invention can be of murine, primate, or human origin. If the fusion
protein is intended for human use, a chimeric form of
immunoglobulin may be used wherein the constant regions of the
immunoglobulin are from a human. A humanized or fully human form of
the immunoglobulin can also be prepared in accordance with methods
well known in the art (see e. g. U.S. Pat. No. 5,565,332 to
Winter). Humanization may be achieved by various methods including,
but not limited to (a) grafting the non-human (e.g., donor
antibody) CDRs onto human (e.g. recipient antibody) framework and
constant regions with or without retention of critical framework
residues (e.g. those that are important for retaining good antigen
binding affinity or antibody functions), (b) grafting only the
non-human specificity-determining regions (SDRs or a-CDRs; the
residues critical for the antibody-antigen interaction) onto human
framework and constant regions, or (c) transplanting the entire
non-human variable domains, but "cloaking" them with a human-like
section by replacement of surface residues. Humanized antibodies
and methods of making them are reviewed, e.g., in Almagro and
Fransson, Front Biosci 13, 1619-1633 (2008), and are further
described, e.g., in Riechmann et al., Nature 332, 323-329 (1988);
Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S.
Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et
al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad
Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92
(1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan,
Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36,
25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol
28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al.,
Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn
et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83,
252-260 (2000) (describing the "guided selection" approach to FR
shuffling). Particular immunoglobulins according to the invention
are human immunoglobulins. Human antibodies and human variable
regions can be produced using various techniques known in the art.
Human antibodies are described generally in van Dijk and van de
Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin
Immunol 20, 450-459 (2008). Human variable regions can form part of
and be derived from human monoclonal antibodies made by the
hybridoma method (see e.g. Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)). Human antibodies and human variable regions may also
be prepared by administering an immunogen to a transgenic animal
that has been modified to produce intact human antibodies or intact
antibodies with human variable regions in response to antigenic
challenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005).
Human antibodies and human variable regions may also be generated
by isolating Fv clone variable region sequences selected from
human-derived phage display libraries (see e.g., Hoogenboom et al.
in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed.,
Human Press, Totowa, N.J., 2001); and McCafferty et al., Nature
348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage
typically display antibody fragments, either as single-chain Fv
(scFv) fragments or as Fab fragments.
[0423] In certain aspects, the moieties capable of specific binding
to a target cell antigen (e.g. Fab fragments) comprised in the
antigen binding molecules of the present invention are engineered
to have enhanced binding affinity according to, for example, the
methods disclosed in PCT publication WO 2012/020006 (see Examples
relating to affinity maturation) or U.S. Pat. Appl. Publ. No.
2004/0132066 (U.S. Pat. No. 7,432,063B2). The ability of the
antigen binding molecules of the invention to bind to a specific
antigenic determinant can be measured either through an
enzyme-linked immunosorbent assay (ELISA) or other techniques
familiar to one of skill in the art, e.g. surface plasmon resonance
technique (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
Competition assays may be used to identify an antigen binding
molecule that competes with a reference antibody for binding to a
particular antigen. In certain embodiments, such a competing
antigen binding molecule binds to the same epitope (e.g. a linear
or a conformational epitope) that is bound by the reference antigen
binding molecule. Detailed exemplary methods for mapping an epitope
to which an antigen binding molecule binds are provided in Morris
(1996) "Epitope Mapping Protocols", in Methods in Molecular Biology
vol. 66 (Humana Press, Totowa, N.J.). In an exemplary competition
assay, immobilized antigen is incubated in a solution comprising a
first labeled antigen binding molecule that binds to the antigen
and a second unlabeled antigen binding molecule that is being
tested for its ability to compete with the first antigen binding
molecule for binding to the antigen. The second antigen binding
molecule may be present in a hybridoma supernatant. As a control,
immobilized antigen is incubated in a solution comprising the first
labeled antigen binding molecule but not the second unlabeled
antigen binding molecule. After incubation under conditions
permissive for binding of the first antibody to the antigen, excess
unbound antibody is removed, and the amount of label associated
with immobilized antigen is measured. If the amount of label
associated with immobilized antigen is substantially reduced in the
test sample relative to the control sample, then that indicates
that the second antigen binding molecule is competing with the
first antigen binding molecule for binding to the antigen. See
Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
[0424] TNF ligand trimer-containing antigen binding molecules of
the invention prepared as described herein may be purified by
art-known techniques such as high performance liquid
chromatography, ion exchange chromatography, gel electrophoresis,
affinity chromatography, size exclusion chromatography, and the
like. The actual conditions used to purify a particular protein
will depend, in part, on factors such as net charge,
hydrophobicity, hydrophilicity etc., and will be apparent to those
having skill in the art. For affinity chromatography purification
an antibody, ligand, receptor or antigen can be used to which the
TNF ligand trimer-containing antigen binding molecule binds. For
example, for affinity chromatography purification of fusion
proteins of the invention, a matrix with protein A or protein G may
be used. Sequential Protein A or G affinity chromatography and size
exclusion chromatography can be used to isolate an antigen binding
molecule essentially as described in the Examples. The purity of
the TNF ligand trimer-containing antigen binding molecule or
fragments thereof can be determined by any of a variety of
well-known analytical methods including gel electrophoresis, high
pressure liquid chromatography, and the like. For example, the TNF
ligand trimer-containing antigen binding molecules expressed as
described in the Examples were shown to be intact and properly
assembled as demonstrated by reducing and non-reducing
SDS-PAGE.
[0425] Assays
[0426] The antigen binding molecules provided herein may be
identified, screened for, or characterized for their
physical/chemical properties and/or biological activities by
various assays known in the art.
[0427] 1. Affinity Assays
[0428] The affinity of the TNF family ligand trimer-containing
antigen binding molecule provided herein for the corresponding TNF
receptor can be determined in accordance with the methods set forth
in the Examples by surface plasmon resonance (SPR), using standard
instrumentation such as a BIACORE.RTM. instrument (GE Healthcare),
and receptors or target proteins such as may be obtained by
recombinant expression. The affinity of the TNF family ligand
trimer-containing antigen binding molecule for the target cell
antigen can also be determined by surface plasmon resonance (SPR),
using standard instrumentation such as a BIACORE.RTM. instrument
(GE Healthcare), and receptors or target proteins such as may be
obtained by recombinant expression. A specific illustrative and
exemplary embodiment for measuring binding affinity is described in
Example 4. According to one aspect, K.sub.D is measured by surface
plasmon resonance using a BIACORE.RTM. T100 machine (GE Healthcare)
at 25.degree. C.
[0429] 2. Binding Assays and Other Assays
[0430] Binding of the TNF family ligand trimer-containing antigen
binding molecule provided herein to the corresponding receptor
expressing cells may be evaluated using cell lines expressing the
particular receptor or target antigen, for example by flow
cytometry (FACS). In one aspect, fresh peripheral blood mononuclear
cells (PBMCs) expressing the TNF receptor are used in the binding
assay. These cells are used directly after isolation (naive PMBCs)
or after stimulation (activated PMBCs). In another aspect,
activated mouse splenocytes (expressing the TNF receptor molecule)
were used to demonstrate the binding of the TNF family ligand
trimer-containing antigen binding molecule of the invention to the
corresponding TNF receptor expressing cells.
[0431] In a further aspect, cancer cell lines expressing the target
cell antigen, for example FAP, were used to demonstrate the binding
of the antigen binding molecules to the target cell antigen.
[0432] In another aspect, competition assays may be used to
identify an antigen binding molecule that competes with a specific
antibody or antigen binding molecule for binding to the target or
TNF receptor, respectively. In certain embodiments, such a
competing antigen binding molecule binds to the same epitope (e.g.,
a linear or a conformational epitope) that is bound by a specific
anti-target antibody or a specific anti-TNF receptor antibody.
Detailed exemplary methods for mapping an epitope to which an
antibody binds are provided in Morris (1996) "Epitope Mapping
Protocols," in Methods in Molecular Biology vol. 66 (Humana Press,
Totowa, N.J.).
[0433] 3. Activity Assays
[0434] In one aspect, assays are provided for identifying TNF
family ligand trimer-containing antigen binding molecules that bind
to a specific target cell antigen and to a specific TNF receptor
having biological activity. Biological activity may include, e.g.,
agonistic signalling through the TNF receptor on cells expressing
the target cell antigen. TNF family ligand trimer-containing
antigen binding molecules identified by the assays as having such
biological activity in vitro are also provided.
[0435] In certain aspects, a TNF family ligand trimer-containing
antigen binding molecule of the invention is tested for such
biological activity. Assays for detecting the biological activity
of the molecules of the invention are those described in Example 6.
Furthermore, assays for detecting cell lysis (e.g. by measurement
of LDH release), induced apoptosis kinetics (e.g. by measurement of
Caspase 3/7 activity) or apoptosis (e.g. using the TUNEL assay) are
well known in the art. In addition the biological activity of such
complexes can be assessed by evaluating their effects on survival,
proliferation and lymphokine secretion of various lymphocyte
subsets such as NK cells, NKT-cells or .gamma..delta. T-cells or
assessing their capacity to modulate phenotype and function of
antigen presenting cells such as dendritic cells,
monocytes/macrophages or B-cells.
[0436] Pharmaceutical Compositions, Formulations and Routes of
Administration
[0437] In a further aspect, the invention provides pharmaceutical
compositions comprising any of the TNF family ligand
trimer-containing antigen binding molecules provided herein, e.g.,
for use in any of the below therapeutic methods. In one embodiment,
a pharmaceutical composition comprises any of the TNF family ligand
trimer-containing antigen binding molecules provided herein and at
least one pharmaceutically acceptable excipient. In another
embodiment, a pharmaceutical composition comprises any of the TNF
family ligand trimer-containing antigen binding molecules provided
herein and at least one additional therapeutic agent, e.g., as
described below.
[0438] Pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of one or more TNF
family ligand trimer-containing antigen binding molecules dissolved
or dispersed in a pharmaceutically acceptable excipient. The
phrases "pharmaceutical or pharmacologically acceptable" refers to
molecular entities and compositions that are generally non-toxic to
recipients at the dosages and concentrations employed, i.e. do not
produce an adverse, allergic or other untoward reaction when
administered to an animal, such as, for example, a human, as
appropriate. The preparation of a pharmaceutical composition that
contains at least one TNF family ligand trimer-containing antigen
binding molecule and optionally an additional active ingredient
will be known to those of skill in the art in light of the present
disclosure, as exemplified by Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, incorporated herein by
reference. In particular, the compositions are lyophilized
formulations or aqueous solutions. As used herein,
"pharmaceutically acceptable excipient" includes any and all
solvents, buffers, dispersion media, coatings, surfactants,
antioxidants, preservatives (e.g. antibacterial agents, antifungal
agents), isotonic agents, salts, stabilizers and combinations
thereof, as would be known to one of ordinary skill in the art.
[0439] Parenteral compositions include those designed for
administration by injection, e.g. subcutaneous, intradermal,
intralesional, intravenous, intraarterial intramuscular,
intrathecal or intraperitoneal injection. For injection, the TNF
family ligand trimer-containing antigen binding molecules of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. The solution may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the fusion proteins may be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. Sterile injectable solutions are
prepared by incorporating the fusion proteins of the invention in
the required amount in the appropriate solvent with various of the
other ingredients enumerated below, as required. Sterility may be
readily accomplished, e.g., by filtration through sterile
filtration membranes. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a
sterile vehicle which contains the basic dispersion medium and/or
the other ingredients. In the case of sterile powders for the
preparation of sterile injectable solutions, suspensions or
emulsion, the preferred methods of preparation are vacuum-drying or
freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered liquid medium thereof. The liquid medium should be
suitably buffered if necessary and the liquid diluent first
rendered isotonic prior to injection with sufficient saline or
glucose. The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less that 0.5 ng/mg protein. Suitable
pharmaceutically acceptable excipients include, but are not limited
to: buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Aqueous injection suspensions may
contain compounds which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, dextran, or the
like. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl cleats or
triglycerides, or liposomes.
[0440] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing
Company, 1990). Sustained-release preparations may be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
e.g. films, or microcapsules. In particular embodiments, prolonged
absorption of an injectable composition can be brought about by the
use in the compositions of agents delaying absorption, such as, for
example, aluminum monostearate, gelatin or combinations thereof
[0441] Exemplary pharmaceutically acceptable excipients herein
further include insterstitial drug dispersion agents such as
soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for
example, human soluble PH-20 hyaluronidase glycoproteins, such as
rHuPH20 (HYLENEX.RTM., Baxter International, Inc.). Certain
exemplary sHASEGPs and methods of use, including rHuPH20, are
described in US Patent Publication Nos. 2005/0260186 (U.S. Pat. No.
7,871,607B2) and 2006/0104968. In one aspect, a sHASEGP is combined
with one or more additional glycosaminoglycanases such as
chondroitinases.
[0442] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the
latter formulations including a histidine-acetate buffer.
[0443] In addition to the compositions described previously, the
fusion proteins may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the fusion proteins may be formulated
with suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0444] Pharmaceutical compositions comprising the fusion proteins
of the invention may be manufactured by means of conventional
mixing, dissolving, emulsifying, encapsulating, entrapping or
lyophilizing processes. Pharmaceutical compositions may be
formulated in conventional manner using one or more physiologically
acceptable carriers, diluents, excipients or auxiliaries which
facilitate processing of the proteins into preparations that can be
used pharmaceutically. Proper formulation is dependent upon the
route of administration chosen.
[0445] The TNF family ligand trimer-containing antigen binding
molecules may be formulated into a composition in a free acid or
base, neutral or salt form. Pharmaceutically acceptable salts are
salts that substantially retain the biological activity of the free
acid or base. These include the acid addition salts, e.g. those
formed with the free amino groups of a proteinaceous composition,
or which are formed with inorganic acids such as for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric or mandelic acid. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides; or such organic bases as isopropylamine,
trimethylamine, histidine or procaine. Pharmaceutical salts tend to
be more soluble in aqueous and other protic solvents than are the
corresponding free base forms.
[0446] The composition herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0447] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
[0448] Therapeutic Methods and Compositions
[0449] Any of the TNF family ligand trimer-containing antigen
binding molecules provided herein may be used in therapeutic
methods.
[0450] For use in therapeutic methods, TNF family ligand
trimer-containing antigen binding molecules of the invention can be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical
practitioners.
[0451] In one aspect, TNF family ligand trimer-containing antigen
binding molecules of the invention for use as a medicament are
provided. In further aspects, TNF family ligand trimer-containing
antigen binding molecules of the invention for use in treating a
disease, in particular for use in the treatment of cancer, are
provided. In certain aspects, TNF family ligand trimer-containing
antigen binding molecules of the invention for use in a method of
treatment are provided. In one aspect, the invention provides a TNF
family ligand trimer-containing antigen binding molecule as
described herein for use in the treatment of a disease in an
individual in need thereof. In certain aspects, the invention
provides a TNF family ligand trimer-containing antigen binding
molecule for use in a method of treating an individual having a
disease comprising administering to the individual a
therapeutically effective amount of the fusion protein. In certain
aspects, the disease to be treated is cancer. Examples of cancers
include solid tumors, bladder cancer, renal cell carcinoma, brain
cancer, head and neck cancer, pancreatic cancer, lung cancer,
breast cancer, ovarian cancer, uterine cancer, cervical cancer,
endometrial cancer, esophageal cancer, colon cancer, colorectal
cancer, rectal cancer, gastric cancer, prostate cancer, blood
cancer, skin cancer, squamous cell carcinoma, bone cancer, and
kidney cancer, melanoma, B-cell lymphoma, B-cell leukemia,
non-Hodgkin lymphoma and acute lymphoblastic leukemia. Thus, a TNF
family ligand trimer-containing antigen binding molecule as
described herein for use in the treatment of cancer is provided.
The subject, patient, or "individual" in need of treatment is
typically a mammal, more specifically a human.
[0452] In another aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule as described herein for
use in the treatment of infectious diseases, in particular for the
treatment of viral infections. In a further aspect, provided is a
TNF family ligand trimer-containing antigen binding molecule as
described herein for use in the treatment of autoimmune diseases
such as for example Lupus disease.
[0453] In one aspect, provided is a TNF family ligand
trimer-containing antigen binding molecule according to the
invention for use in treating head and neck squamous cell carcinoma
(HNSCC), breast cancer, colorectal cancer (CRC), pancreatic cancer
(PAC), gastric cancer, non-small-cell lung carcinoma (NSCLC) and
Mesothelioma, wherein the target cell antigen is FAP.
[0454] In a further aspect, the invention relates to the use of a
TNF family ligand trimer-containing antigen binding molecule in the
manufacture or preparation of a medicament for the treatment of a
disease in an individual in need thereof. In one aspect, the
medicament is for use in a method of treating a disease comprising
administering to an individual having the disease a therapeutically
effective amount of the medicament. In certain embodiments the
disease to be treated is a proliferative disorder, particularly
cancer. Thus, in one aspect, the invention relates to the use of a
TNF family ligand trimer-containing antigen binding molecule of the
invention in the manufacture or preparation of a medicament for the
treatment of cancer. Examples of cancers include solid tumors,
bladder cancer, renal cell carcinoma, brain cancer, head and neck
cancer, pancreatic cancer, lung cancer, breast cancer, ovarian
cancer, uterine cancer, cervical cancer, endometrial cancer,
esophageal cancer, colon cancer, colorectal cancer, rectal cancer,
gastric cancer, prostate cancer, blood cancer, skin cancer,
squamous cell carcinoma, bone cancer, and kidney cancer, melanoma,
B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma and acute
lymphoblastic leukemia. Other cell proliferation disorders that can
be treated using a TNF family ligand trimer-containing antigen
binding molecule of the present invention include, but are not
limited to neoplasms located in the: abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), eye, head and neck, nervous system (central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,
thoracic region, and urogenital system. Also included are
pre-cancerous conditions or lesions and cancer metastases. In
certain embodiments the cancer is chosen from the group consisting
of renal cell cancer, skin cancer, lung cancer, colorectal cancer,
breast cancer, brain cancer, head and neck cancer. A skilled
artisan may recognize that in some cases the TNF family ligand
trimer-containing antigen binding molecule may not provide a cure
but may only provide partial benefit. In some aspects, a
physiological change having some benefit is also considered
therapeutically beneficial. Thus, in some aspects, an amount of TNF
family ligand trimer-containing antigen binding molecule that
provides a physiological change is considered an "effective amount"
or a "therapeutically effective amount".
[0455] In a further aspect, the invention relates to the use of a
TNF family ligand trimer-containing antigen binding molecule as
described herein in the manufacture or preparation of a medicament
for the treatment of infectious diseases, in particular for the
treatment of viral infections or for the treatment of autoimmune
diseases, for example Lupus disease.
[0456] In a further aspect, the invention provides a method for
treating a disease in an individual, comprising administering to
said individual a therapeutically effective amount of a TNF family
ligand trimer-containing antigen binding molecule of the invention.
In one aspect a composition is administered to said individual,
comprising a fusion protein of the invention in a pharmaceutically
acceptable form. In certain aspects, the disease to be treated is a
proliferative disorder. In a particular aspect, the disease is
cancer. In another aspect, the disease is an infectious disease or
an autoimmune disease. In certain aspects, the method further
comprises administering to the individual a therapeutically
effective amount of at least one additional therapeutic agent, e.g.
an anti-cancer agent if the disease to be treated is cancer. An
"individual" according to any of the above embodiments may be a
mammal, preferably a human.
[0457] For the prevention or treatment of disease, the appropriate
dosage of a TNF family ligand trimer-containing antigen binding
molecule of the invention (when used alone or in combination with
one or more other additional therapeutic agents) will depend on the
type of disease to be treated, the route of administration, the
body weight of the patient, the type of fusion protein, the
severity and course of the disease, whether the fusion protein is
administered for preventive or therapeutic purposes, previous or
concurrent therapeutic interventions, the patient's clinical
history and response to the fusion protein, and the discretion of
the attending physician. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0458] The TNF family ligand trimer-containing antigen binding
molecule is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of TNF family ligand trimer-containing antigen binding molecule can
be an initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. One typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment would generally
be sustained until a desired suppression of disease symptoms
occurs. One exemplary dosage of the fusion protein would be in the
range from about 0.005 mg/kg to about 10 mg/kg. In other examples,
a dose may also comprise from about 1 .mu.g/kg body weight, about 5
.mu.g/kg body weight, about 10 .mu.g/kg body weight, about 50
.mu.g/kg body weight, about 100 .mu.g/kg body weight, about 200
.mu.g/kg body weight, about 350 .mu.g/kg body weight, about 500
.mu.g/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body
weight, about 10 mg/kg body weight, about 50 mg/kg body weight,
about 100 mg/kg body weight, about 200 mg/kg body weight, about 350
mg/kg body weight, about 500 mg/kg body weight, to about 1000 mg/kg
body weight or more per administration, and any range derivable
therein. In examples of a derivable range from the numbers listed
herein, a range of about 5 mg/kg body weight to about 100 mg/kg
body weight, about 5 .mu.g/kg body weight to about 500 mg/kg body
weight etc., can be administered, based on the numbers described
above. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0
mg/kg or 10 mg/kg (or any combination thereof) may be administered
to the patient. Such doses may be administered intermittently, e.g.
every week or every three weeks (e.g. such that the patient
receives from about two to about twenty, or e.g. about six doses of
the fusion protein). An initial higher loading dose, followed by
one or more lower doses may be administered. However, other dosage
regimens may be useful. The progress of this therapy is easily
monitored by conventional techniques and assays.
[0459] The TNF family ligand trimer-containing antigen binding
molecules of the invention will generally be used in an amount
effective to achieve the intended purpose. For use to treat or
prevent a disease condition, the TNF family ligand
trimer-containing antigen binding molecules of the invention, or
pharmaceutical compositions thereof, are administered or applied in
a therapeutically effective amount. Determination of a
therapeutically effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0460] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays, such as cell
culture assays. A dose can then be formulated in animal models to
achieve a circulating concentration range that includes the
IC.sub.50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans.
[0461] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0462] Dosage amount and interval may be adjusted individually to
provide plasma levels of the TNF family ligand trimer-containing
antigen binding molecules which are sufficient to maintain
therapeutic effect. Usual patient dosages for administration by
injection range from about 0.1 to 50 mg/kg/day, typically from
about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels
may be achieved by administering multiple doses each day. Levels in
plasma may be measured, for example, by HPLC.
[0463] In cases of local administration or selective uptake, the
effective local concentration of the TNF family ligand
trimer-containing antigen binding molecule may not be related to
plasma concentration. One skilled in the art will be able to
optimize therapeutically effective local dosages without undue
experimentation.
[0464] A therapeutically effective dose of the TNF family ligand
trimer-containing antigen binding molecules described herein will
generally provide therapeutic benefit without causing substantial
toxicity. Toxicity and therapeutic efficacy of a fusion protein can
be determined by standard pharmaceutical procedures in cell culture
or experimental animals. Cell culture assays and animal studies can
be used to determine the LD.sub.50 (the dose lethal to 50% of a
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of a population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which can be
expressed as the ratio LD.sub.50/ED.sub.50. TNF family ligand
trimer-containing antigen binding molecules that exhibit large
therapeutic indices are preferred. In one embodiment, the TNF
family ligand trimer-containing antigen binding molecule according
to the present invention exhibits a high therapeutic index. The
data obtained from cell culture assays and animal studies can be
used in formulating a range of dosages suitable for use in humans.
The dosage lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon a
variety of factors, e.g., the dosage form employed, the route of
administration utilized, the condition of the subject, and the
like. The exact formulation, route of administration and dosage can
be chosen by the individual physician in view of the patient's
condition (see, e.g., Fingl et al., 1975, in: The Pharmacological
Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by
reference in its entirety).
[0465] The attending physician for patients treated with fusion
proteins of the invention would know how and when to terminate,
interrupt, or adjust administration due to toxicity, organ
dysfunction, and the like. Conversely, the attending physician
would also know to adjust treatment to higher levels if the
clinical response were not adequate (precluding toxicity). The
magnitude of an administered dose in the management of the disorder
of interest will vary with the severity of the condition to be
treated, with the route of administration, and the like. The
severity of the condition may, for example, be evaluated, in part,
by standard prognostic evaluation methods. Further, the dose and
perhaps dose frequency will also vary according to the age, body
weight, and response of the individual patient.
[0466] Other Agents and Treatments
[0467] The TNF family ligand trimer-containing antigen binding
molecules of the invention may be administered in combination with
one or more other agents in therapy. For instance, a fusion protein
of the invention may be co-administered with at least one
additional therapeutic agent. The term "therapeutic agent"
encompasses any agent that can be administered for treating a
symptom or disease in an individual in need of such treatment. Such
additional therapeutic agent may comprise any active ingredients
suitable for the particular indication being treated, preferably
those with complementary activities that do not adversely affect
each other. In certain embodiments, an additional therapeutic agent
is another anti-cancer agent.
[0468] Such other agents are suitably present in combination in
amounts that are effective for the purpose intended. The effective
amount of such other agents depends on the amount of fusion protein
used, the type of disorder or treatment, and other factors
discussed above. The TNF family ligand trimer-containing antigen
binding molecules are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99%
of the dosages described herein, or in any dosage and by any route
that is empirically/clinically determined to be appropriate.
[0469] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate compositions), and separate administration,
in which case, administration of the TNF family ligand
trimer-containing antigen binding molecule of the invention can
occur prior to, simultaneously, and/or following, administration of
the additional therapeutic agent and/or adjuvant.
[0470] Articles of Manufacture
[0471] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper that is
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a TNF ligand trimer-containing antigen
binding molecule of the invention.
[0472] The label or package insert indicates that the composition
is used for treating the condition of choice. Moreover, the article
of manufacture may comprise (a) a first container with a
composition contained therein, wherein the composition comprises a
TNF ligand trimer-containing antigen binding molecule of the
invention; and (b) a second container with a composition contained
therein, wherein the composition comprises a further cytotoxic or
otherwise therapeutic agent. The article of manufacture in this
embodiment of the invention may further comprise a package insert
indicating that the compositions can be used to treat a particular
condition.
[0473] Alternatively, or additionally, the article of manufacture
may further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
TABLE-US-00003 TABLE C (Sequences): SEQ ID NO: Name Sequence 1
Human (hu) 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254)
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSE 2 hu 4-1BBL (85-254)
LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL
TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEA
RNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQ LTQGATVLGLFRVTPEIPAGLPSPRSE 3
hu 4-1BBL (80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL
AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPP
ASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR
HAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 4 hu 4-1BBL (52-254)
PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLR
QGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL
SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSG
SVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSA
FGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEIPAGLPSPRSE 5
dimeric hu 4-1BBL (71-254) REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP
connected by (G.sub.4S).sub.2 linker
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP
SPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGM
FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK
EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSL
ALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSE 6
monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254)
plus (G.sub.4S).sub.2 linker LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSEGGGGSGGGGS 7 FAP(28H1)
CDR-H1 SHAMS 8 FAP(28H1) CDR-H2 AIWASGEQYYADSVKG 9 FAP(28H1) CDR-H3
GWLGNFDY 10 FAP(28H1) CDR-L1 RASQSVSRSYLA 11 FAP(28H1) CDR-L2
GASTRAT 12 FAP(28H1) CDR-L3 QQGQVIPPT 13 (G.sub.4S).sub.2
GGGGSGGGGS 14 dimeric hu 4-1BBL (71-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254)-CH1 Fc knob chain
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP
SPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGM
FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK
EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSL
ALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV
LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAA
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQP
REPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 monomeric hu 4-1BBL (71-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254)-CL
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP
SPRSEGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 16 FAP(28H1) VH
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHA MSWVRQAPGKGLEWVSAIWASGEQYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GWLGNFDYWGQGTLVTVSS 17 FAP(28H1)
VL EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLA
WYQQKPGQAPRLLIIGASTRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQGQVIPPTFGQGT KVEIK 18 anti-FAP(28H1) Fc hole
see Table 2 chain 19 anti-FAP(28H1) light chain see Table 2 20
Human (hu) FAP UniProt no. Q12884 21 hu FAP ectodomain + poly-
RPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS lys-tag + his.sub.6-tag
GQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNA
SNYGLSPDRQFVYLESDYSKLWRYSYTATYY1YDLS
NGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYL
KQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATK
YALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYP
RTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVP
AMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL
SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVST
PVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITS
GKWEAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGS
YPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCY
GPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKE
EIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGG
PCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAF
QGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDE
KRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSS
WEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAE
YFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQV
DFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFS LSDGKKKKKKGHHHHHH 22 nucleotide
sequence CGCCCTTCAAGAGTTCATAACTCTGAAGAAAATAC hu FAP ectodomain +
poly- AATGAGAGCACTCACACTGAAGGATATTTTAAATG lys-tag + his.sub.6-tag
GAACATTTTCTTATAAAACATTTTTTCCAAACTGGA
TTTCAGGACAAGAATATCTTCATCAATCTGCAGAT
AACAATATAGTACTTTATAATATTGAAACAGGACA
ATCATATACCATTTTGAGTAATAGAACCATGAAAA
GTGTGAATGCTTCAAATTACGGCTTATCACCTGAT
CGGCAATTTGTATATCTAGAAAGTGATTATTCAAA
GCTTTGGAGATACTCTTACACAGCAACATATTACA
TCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA
AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGG
TCGCCTGTTGGGAGTAAATTAGCATATGTCTATCA
AAACAATATCTATTTGAAACAAAGACCAGGAGAT
CCACCTTTTCAAATAACATTTAATGGAAGAGAAAA
TAAAATATTTAATGGAATCCCAGACTGGGTTTATG
AAGAGGAAATGCTTGCTACAAAATATGCTCTCTGG
TGGTCTCCTAATGGAAAATTTTTGGCATATGCGGA
ATTTAATGATACGGATATACCAGTTATTGCCTATTC
CTATTATGGCGATGAACAATATCCTAGAACAATAA
ATATTCCATACCCAAAGGCTGGAGCTAAGAATCCC
GTTGTTCGGATATTTATTATCGATACCACTTACCCT
GCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGC
AATGATAGCCTCAAGTGATTATTATTTCAGTTGGC
TCACGTGGGTTACTGATGAACGAGTATGTTTGCAG
TGGCTAAAAAGAGTCCAGAATGTTTCGGTCCTGTC
TATATGTGACTTCAGGGAAGACTGGCAGACATGGG
ATTGTCCAAAGACCCAGGAGCATATAGAAGAAAG
CAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAA
CACCAGTTTTCAGCTATGATGCCATTTCGTACTACA
AAATATTTAGTGACAAGGATGGCTACAAACATATT
CACTATATCAAAGACACTGTGGAAAATGCTATTCA
AATTACAAGTGGCAAGTGGGAGGCCATAAATATA
TTCAGAGTAACACAGGATTCACTGTTTTATTCTAG
CAATGAATTTGAAGAATACCCTGGAAGAAGAAAC
ATCTACAGAATTAGCATTGGAAGCTATCCTCCAAG
CAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAA
GGTGCCAATATTACACAGCAAGTTTCAGCGACTAC
GCCAAGTACTATGCACTTGTCTGCTACGGCCCAGG
CATCCCCATTTCCACCCTTCATGATGGACGCACTG
ATCAAGAAATTAAAATCCTGGAAGAAAACAAGGA
ATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTA
AAGAGGAAATTAAGAAACTTGAAGTAGATGAAAT
TACTTTATGGTACAAGATGATTCTTCCTCCTCAATT
TGACAGATCAAAGAAGTATCCCTTGCTAATTCAAG
TGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCT
GTATTTGCTGTTAATTGGATATCTTATCTTGCAAGT
AAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCG
AGGAACAGCTTTCCAAGGTGACAAACTCCTCTATG
CAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAA
GACCAGATTACAGCTGTCAGAAAATTCATAGAAAT
GGGTTTCATTGATGAAAAAAGAATAGCCATATGGG
GCTGGTCCTATGGAGGATACGTTTCATCACTGGCC
CTTGCATCTGGAACTGGTCTTTTCAAATGTGGTATA
GCAGTGGCTCCAGTCTCCAGCTGGGAATATTACGC
GTCTGTCTACACAGAGAGATTCATGGGTCTCCCAA
CAAAGGATGATAATCTTGAGCACTATAAGAATTCA
ACTGTGATGGCAAGAGCAGAATATTTCAGAAATGT
AGACTATCTTCTCATCCACGGAACAGCAGATGATA
ATGTGCACTTTCAAAACTCAGCACAGATTGCTAAA
GCTCTGGTTAATGCACAAGTGGATTTCCAGGCAAT
GTGGTACTCTGACCAGAACCACGGCTTATCCGGCC
TGTCCACGAACCACTTATACACCCACATGACCCAC
TTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGCAA
AAAGAAAAAGAAAAAGGGCCACCACCATCACCAT CAC 23 mouse FAP UniProt no.
P97321 24 Murine FAP RPSRVYKPEGNTKRALTLKDILNGTFSYKTYFPNWIS
ectodomain + poly-lys- EQEYLHQSEDDNIVFYNIETRESYIILSNSTMKSVNAT tag +
his.sub.6-tag DYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLQN
GEFVRGYELPRPIQYLCWSPVGSKLAYVYQNNIYLK
QRPGDPPFQITYTGRENRIFNGIPDWVYEEEMLATKY
ALWWSPDGKFLAYVEFNDSDIPIIAYSYYGDGQYPR
TINIPYPKAGAKNPVVRVFIVDTTYPHHVGPMEVPVP
EMIASSDYYFSWLTWVSSERVCLQWLKRVQNVSVL
SICDFREDWHAWECPKNQEHVEESRTGWAGGFFVST
PAFSQDATSYYKIFSDKDGYKHIHYIKDTVENAIQITS
GKWEAIYIFRVTQDSLFYSSNEFEGYPGRRNIYRISIG
NSPPSKKCVTCHLRKERCQYYTASFSYKAKYYALVC
YGPGLPISTLHDGRTDQEIQVLEENKELENSLRNIQLP
KVEIKKLKDGGLTFWYKMILPPQFDRSKKYPLLIQVY
GGPCSQSVKSVFAVNWITYLASKEGIVIALVDGRGTA
FQGDKFLHAVYRKLGVYEVEDQLTAVRKFIEMGFID
EERIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVS
SWEYYASIYSERFMGLPTKDDNLEHYKNSTVMARA
EYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQV
DFQAMWYSDQNHGILSGRSQNHLYTHMTHFLKQCF SLSDGKKKKKKGHHHHHH 25
nucleotide sequence CGTCCCTCAAGAGTTTACAAACCTGAAGGAAACAC Murine FAP
AAAGAGAGCTCTTACCTTGAAGGATATTTTAAATG ectodomain + poly-lys-
GAACATTCTCATATAAAACATATTTTCCCAACTGG tag + his.sub.6-tag
ATTTCAGAACAAGAATATCTTCATCAATCTGAGGA
TGATAACATAGTATTTTATAATATTGAAACAAGAG
AATCATATATCATTTTGAGTAATAGCACCATGAAA
AGTGTGAATGCTACAGATTATGGTTTGTCACCTGA
TCGGCAATTTGTGTATCTAGAAAGTGATTATTCAA
AGCTCTGGCGATATTCATACACAGCGACATACTAC
ATCTACGACCTTCAGAATGGGGAATTTGTAAGAGG
ATACGAGCTCCCTCGTCCAATTCAGTATCTATGCT
GGTCGCCTGTTGGGAGTAAATTAGCATATGTATAT
CAAAACAATATTTATTTGAAACAAAGACCAGGAG
ATCCACCTTTTCAAATAACTTATACTGGAAGAGAA
AATAGAATATTTAATGGAATACCAGACTGGGTTTA
TGAAGAGGAAATGCTTGCCACAAAATATGCTCTTT
GGTGGTCTCCAGATGGAAAATTTTTGGCATATGTA
GAATTTAATGATTCAGATATACCAATTATTGCCTA
TTCTTATTATGGTGATGGACAGTATCCTAGAACTA
TAAATATTCCATATCCAAAGGCTGGGGCTAAGAAT
CCGGTTGTTCGTGTTTTTATTGTTGACACCACCTAC
CCTCACCACGTGGGCCCAATGGAAGTGCCAGTTCC
AGAAATGATAGCCTCAAGTGACTATTATTTCAGCT
GGCTCACATGGGTGTCCAGTGAACGAGTATGCTTG
CAGTGGCTAAAAAGAGTGCAGAATGTCTCAGTCCT
GTCTATATGTGATTTCAGGGAAGACTGGCATGCAT
GGGAATGTCCAAAGAACCAGGAGCATGTAGAAGA
AAGCAGAACAGGATGGGCTGGTGGATTCTTTGTTT
CGACACCAGCTTTTAGCCAGGATGCCACTTCTTAC
TACAAAATATTTAGCGACAAGGATGGTTACAAACA
TATTCACTACATCAAAGACACTGTGGAAAATGCTA
TTCAAATTACAAGTGGCAAGTGGGAGGCCATATAT
ATATTCCGCGTAACACAGGATTCACTGTTTTATTCT
AGCAATGAATTTGAAGGTTACCCTGGAAGAAGAA
ACATCTACAGAATTAGCATTGGAAACTCTCCTCCG
AGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGA
AAGGTGCCAATATTACACAGCAAGTTTCAGCTACA
AAGCCAAGTACTATGCACTCGTCTGCTATGGCCCT
GGCCTCCCCATTTCCACCCTCCATGATGGCCGCAC
AGACCAAGAAATACAAGTATTAGAAGAAAACAAA
GAACTGGAAAATTCTCTGAGAAATATCCAGCTGCC
TAAAGTGGAGATTAAGAAGCTCAAAGACGGGGGA
CTGACTTTCTGGTACAAGATGATTCTGCCTCCTCAG
TTTGACAGATCAAAGAAGTACCCTTTGCTAATTCA
AGTGTATGGTGGTCCTTGTAGCCAGAGTGTTAAGT
CTGTGTTTGCTGTTAATTGGATAACTTATCTCGCAA
GTAAGGAGGGGATAGTCATTGCCCTGGTAGATGGT
CGGGGCACTGCTTTCCAAGGTGACAAATTCCTGCA
TGCCGTGTATCGAAAACTGGGTGTATATGAAGTTG
AGGACCAGCTCACAGCTGTCAGAAAATTCATAGA
AATGGGTTTCATTGATGAAGAAAGAATAGCCATAT
GGGGCTGGTCCTACGGAGGTTATGTTTCATCCCTG
GCCCTTGCATCTGGAACTGGTCTTTTCAAATGTGG
CATAGCAGTGGCTCCAGTCTCCAGCTGGGAATATT
ACGCATCTATCTACTCAGAGAGATTCATGGGCCTC
CCAACAAAGGACGACAATCTCGAACACTATAAAA
ATTCAACTGTGATGGCAAGAGCAGAATATTTCAGA
AATGTAGACTATCTTCTCATCCACGGAACAGCAGA
TGATAATGTGCACTTTCAGAACTCAGCACAGATTG
CTAAAGCTTTGGTTAATGCACAAGTGGATTTCCAG
GCGATGTGGTACTCTGACCAGAACCATGGTATATT
ATCTGGGCGCTCCCAGAATCATTTATATACCCACA
TGACGCACTTCCTCAAGCAATGCTTTTCTTTATCAG
ACGGCAAAAAGAAAAAGAAAAAGGGCCACCACCA TCACCATCAC 26 Cynomolgus FAP
RPPRVHNSEENTMRALTLKDILNGTFSYKTFFPNWIS ectodomain + poly-lys-
GQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNA tag + his.sub.6-tag
SNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLS
NGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYL
KQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATK
YALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYP
RTINIPYPKAGAKNPFVRIFIIDTTYPAYVGPQEVPVP
AMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVL
SICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVST
PVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITS
GKWEAINIFRVTQDSLFYSSNEFEDYPGRRNIYRISIG
SYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVC
YGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLP
KEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVY
GGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGT
AFQGDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFI
DEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAP
VSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMA
RAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNA
QVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQ CFSLSDGKKKKKKGHHHHHH 27
nucleotide sequence CGCCCTCCAAGAGTTCATAACTCTGAAGAAAATAC Cynomolgus
FAP AATGAGAGCACTCACACTGAAGGATATTTTAAATG ectodomain + poly-lys-
GGACATTTTCTTATAAAACATTTTTTCCAAACTGGA tag + his.sub.6-tag
TTTCAGGACAAGAATATCTTCATCAATCTGCAGAT
AACAATATAGTACTTTATAATATTGAAACAGGACA
ATCATATACCATTTTGAGTAACAGAACCATGAAAA
GTGTGAATGCTTCAAATTATGGCTTATCACCTGAT
CGGCAATTTGTATATCTAGAAAGTGATTATTCAAA
GCTTTGGAGATACTCTTACACAGCAACATATTACA
TCTATGACCTTAGCAATGGAGAATTTGTAAGAGGA
AATGAGCTTCCTCGTCCAATTCAGTATTTATGCTGG
TCGCCTGTTGGGAGTAAATTAGCATATGTCTATCA
AAACAATATCTATTTGAAACAAAGACCAGGAGAT
CCACCTTTTCAAATAACATTTAATGGAAGAGAAAA
TAAAATATTTAATGGAATCCCAGACTGGGTTTATG
AAGAGGAAATGCTTGCTACAAAATATGCTCTCTGG
TGGTCTCCTAATGGAAAATTTTTGGCATATGCGGA
ATTTAATGATACAGATATACCAGTTATTGCCTATTC
CTATTATGGCGATGAACAATATCCCAGAACAATAA
ATATTCCATACCCAAAGGCCGGAGCTAAGAATCCT
TTTGTTCGGATATTTATTATCGATACCACTTACCCT
GCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGC
AATGATAGCCTCAAGTGATTATTATTTCAGTTGGC
TCACGTGGGTTACTGATGAACGAGTATGTTTGCAG
TGGCTAAAAAGAGTCCAGAATGTTTCGGTCTTGTC
TATATGTGATTTCAGGGAAGACTGGCAGACATGGG
ATTGTCCAAAGACCCAGGAGCATATAGAAGAAAG
CAGAACTGGATGGGCTGGTGGATTCTTTGTTTCAA
CACCAGTTTTCAGCTATGATGCCATTTCATACTACA
AAATATTTAGTGACAAGGATGGCTACAAACATATT
CACTATATCAAAGACACTGTGGAAAATGCTATTCA
AATTACAAGTGGCAAGTGGGAGGCCATAAATATA
TTCAGAGTAACACAGGATTCACTGTTTTATTCTAG
CAATGAATTTGAAGATTACCCTGGAAGAAGAAAC
ATCTACAGAATTAGCATTGGAAGCTATCCTCCAAG
CAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAA
GGTGCCAATATTACACAGCAAGTTTCAGCGACTAC
GCCAAGTACTATGCACTTGTCTGCTATGGCCCAGG
CATCCCCATTTCCACCCTTCATGACGGACGCACTG
ATCAAGAAATTAAAATCCTGGAAGAAAACAAGGA
ATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTA
AAGAGGAAATTAAGAAACTTGAAGTAGATGAAAT
TACTTTATGGTACAAGATGATTCTTCCTCCTCAATT
TGACAGATCAAAGAAGTATCCCTTGCTAATTCAAG
TGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTCT
GTATTTGCTGTTAATTGGATATCTTATCTTGCAAGT
AAGGAAGGGATGGTCATTGCCTTGGTGGATGGTCG
GGGAACAGCTTTCCAAGGTGACAAACTCCTGTATG
CAGTGTATCGAAAGCTGGGTGTTTATGAAGTTGAA
GACCAGATTACAGCTGTCAGAAAATTCATAGAAAT
GGGTTTCATTGATGAAAAAAGAATAGCCATATGGG
GCTGGTCCTATGGAGGATATGTTTCATCACTGGCC
CTTGCATCTGGAACTGGTCTTTTCAAATGTGGGAT
AGCAGTGGCTCCAGTCTCCAGCTGGGAATATTACG
CGTCTGTCTACACAGAGAGATTCATGGGTCTCCCA
ACAAAGGATGATAATCTTGAGCACTATAAGAATTC
AACTGTGATGGCAAGAGCAGAATATTTCAGAAAT
GTAGACTATCTTCTCATCCACGGAACAGCAGATGA
TAATGTGCACTTTCAAAACTCAGCACAGATTGCTA
AAGCTCTGGTTAATGCACAAGTGGATTTCCAGGCA
ATGTGGTACTCTGACCAGAACCACGGCTTATCCGG
CCTGTCCACGAACCACTTATACACCCACATGACCC
ACTTCCTAAAGCAGTGTTTCTCTTTGTCAGACGGC
AAAAAGAAAAAGAAAAAGGGCCACCACCATCACC ATCAC 28 human CEA UniProt no.
P06731 29 human MCSP UniProt no. Q6UVK1 30 human EGFR UniProt no.
P00533 31 human CD19 UniProt no. P15391 32 human CD20 Uniprot no.
P11836 33 human CD33 UniProt no. P20138 34 human Lymphotoxin
.alpha. UniProt no. P01374 35 human TNF UniProt no. P01375 36 human
Lymphotoxin .beta. UniProt no. Q06643 37 human OX40L UniProt no.
P23510 38 human CD40L UniProt no. P29965 39 human FasL UniProt no.
P48023 40 human CD27L UniProt no. P32970 41 human CD30L UniProt no.
P32971 42 human 4-1BBL UniProt no. P41273 43 human TRAIL UniProt
no. P50591 44 human RANKL UniProt no. O14788 45 human TWEAK UniProt
no. O43508 46 human APRIL UniProt no. O75888 47 human BAFF UniProt
no. Q9Y275 48 human LIGHT UniProt no. O43557 49 human TL1A UniProt
no. O95150 50 human GITRL UniProt no. Q9UNG2 51 human ectodysplasin
A UniProt no. Q92838 52 hu 4-1BBL (50-254)
ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLL
DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLT
GGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE
GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEAR
NSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQL TQGATVLGLFRVTPEIPAGLPSPRSE 53
hu OX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMK
VQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEP
LFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL DDFHVNGGELILIHQNPGEFCVL 54 hu
OX40L (52-183) VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKV
QNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPL
FQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLD DFHVNGGELILIHQNPGEFCVL 55
Peptide linker (SG.sub.4).sub.2 SGGGGSGGGG 56 Peptide linker
G.sub.4(SG.sub.4).sub.2 GGGGSGGGGSGGGG 57 Peptide linker GSPGSSSSGS
58 Peptide linker (G.sub.4S).sub.4 GGGGSGGGGSGGGGSGGGGS 59 Peptide
linker GSGSGNGS 60 Peptide linker GGSGSGSG 61 Peptide linker GGSGSG
62 Peptide linker GGSG 63 Peptide linker GGSGNGSG 64 Peptide linker
GGNGSGSG 65 Peptide linker GGNGSG 66 nucleotide sequence See Table
2 dimeric hu 4-1BBL (71- 254)-CH1 Fc knob chain 67 nucleotide
sequence See Table 2 monomeric hu 4-1BBL (71- 254)-CL1 68
nucleotide sequence See Table 2 anti-FAP(28H1) Fc hole chain 69
nucleotide sequence See Table 2 anti-FAP (28H1) light chain 70
Murine (mu) 4-1BBL UniProt no. Q3U1Z9-1 71 nucleotide sequence See
Table 13 dimeric mu 4-1BBL (104- 309, C137, 160, 246S)-CH1 Fc knob
chain 72 nucleotide sequence See Table 13 monomeric mu 4-1BBL
(104-309, C137, 160, 246S)- CL 73 nucleotide sequence See Table 13
anti-FAP Fc KK chain 74 nucleotide sequence See Table 13 anti-FAP
light chain 75 dimeric mu 4-1BBL (104- See Table 13 309, C137, 160,
246S)-CL
Fc DD chain 76 monomeric mu 4-1BBL See Table 13 (104-309, C137,
160, 246S)- CL1 77 anti-FAP Fc KK chain See Table 13 78 anti-FAP
light chain See Table 13 79 nucleotide sequence DP47 See Table 18
Fc-hole chain 80 nucleotide sequence DP47 See Table 18 light chain
81 DP47 Fc-hole chain See Table 18 82 DP47 light chain See Table 18
83 Human 4-1BB Fc(kih) See Table 31 84 Cynomolgus 4-1BB Fc(kih) See
Table 31 85 Murine 4-1BB Fc(kih) See Table 31 86 nucleotide
sequence See Table 32 Fc hole chain 87 nucleotide sequence See
Table 32 Human 4-1BB Fc(kih) 88 nucleotide sequence See Table 32
Cynomolgus 4-1BB Fc(kih) 89 nucleotide sequence See Table 32 Murine
4-1BB Fc(kih) 90 Fc hole chain See Table 32 91 Human 4-1BB Fc(kih)
See Table 32 92 Cynomolgus 4-1BB Fc(kih) See Table 32 93 Murine
4-1BB Fc(kih) See Table 32 94 nucleotide sequence See Table 33
Human 4-1BB His 95 Human 4-1BB His See Table 33 96 Human (hu)
4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 248)
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 97 dimeric hu 4-1BBL (71-248)
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP connected by (G.sub.4S).sub.2
linker LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLG
GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVA
QNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL
VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP
LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLH
LSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV TPEIPAGL 98 dimeric hu 4-1BBL
(80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL connected by
(G.sub.4S).sub.2 linker AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPP
ASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR
HAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSG
GGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQ
LELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALT
VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHT
EARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 99 dimeric hu 4-1BBL (52-254)
PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLR connected by (G.sub.4S).sub.2
linker QGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL
SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSG
SVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSA
FGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG
ATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSPWAV
SGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMF
AQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKE
DTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSL
ALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSE 100
FAP(4B9) CDR-H1 SYAMS 101 FAP(4B9) CDR-H2 AIIGSGASTYYADSVKG 102
FAP(4B9) CDR-H3 GWFGGFNY 103 FAP(4B9) CDR-L1 RASQSVTSSYLA 104
FAP(4B9) CDR-L2 VGSRRAT 105 FAP(4B9) CDR-L3 QQGIMLPPT 106 FAP(4B9)
VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA
MSWVRQAPGKGLEWVSAIIGSGASTYYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
GWFGGFNYWGQGTLVTVSS 107 FAP(4B9) VL
EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLA
WYQQKPGQAPRLLINVGSRRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQGIMLPPTFGQGT KVEIK 108 dimeric hu 4-1BBL (71-
see Table 4 254)-CH1* Fc knob chain 109 monomeric hu 4-1BBL (71-
see Table 4 254)-CL* 110 monomeric hu 4-1BBL (71- see Table 7
254)-(G.sub.4S)1-CL* 111 dimeric hu 4-1BBL (52- see Table 10
254)-CH1* Fc knob chain 112 monomeric hu 4-1BBL (52- see Table 10
254)-CL* 113 dimeric hu 4-1BBL (80- see Table 11 254)-CH1* Fc knob
chain 114 monomeric hu 4-1BBL (80- see Table 11 254)-CL* 115
dimeric hu 4-1BBL (71- see Table 3 254)-CL* Fc knob chain 116
monomeric hu 4-1BBL (71- see Table 3 254)-CH1* 117 dimeric hu
4-1BBL (71- see Table 22 254)-CL Fc knob chain 118 monomeric hu
4-1BBL (71- see Table 22 254)-CH1 119 dimeric hu 4-1BBL (71- see
Table 24 248)-CL* Fc knob chain 120 monomeric hu 4-1BBL (71- see
Table 24 248)-CH1* 121 anti-FAP (28H1) Fc hole see Table 6 chain
fused to dimeric 4- 1BBL (71-254) 122 anti-FAP (28H1) Fc knob see
Table 6 chain fused to monomeric 4- 1BBL (71-254) 123 anti-FAP
(4B9) Fc hole see Table 23 chain fused to dimeric 4- 1BBL (71-254)
124 anti-FAP (4B9) Fc knob see Table 23 chain fused to monomeric 4-
1BBL (71-254) 125 anti-FAP (4B9) light chain see Table 21 126
anti-FAP (4B9) Fc hole see Table 26 chain fused to dimeric 4- 1BBL
(71-248) 127 anti-FAP (4B9) Fc knob see Table 26 chain fused to
monomeric 4- 1BBL (71-248) 128 Peptide linker GGGGS 129 nucleotide
sequence dimeric see Table 3 hu 4-1BBL (71-254)-CL* Fc knob chain
130 nucleotide sequence see Table 3 monomeric hu 4-1BBL (71-
254)-CH1* 131 nucleotide sequence dimeric see Table 4 hu 4-1BBL
(71-254)- CH1* Fc knob chain 132 nucleotide sequence see Table 4
monomeric hu 4-1BBL (71- 254)-CL* 133 nucleotide sequence anti- see
Table 4 FAP (28H1) (VHCL) Fc hole chain 134 nucleotide sequence
anti- see Table 4 FAP (28H1) (VLCH1) light chain 135 anti-FAP
(VHCL) (28H1) see Table 4 Fc hole chain 136 anti-FAP (VLCH1) (28H1)
see Table 4 light chain 137 nucleotide sequence see Table 5
monomeric hu 4-1BBL (71- 254)-CH1* Fc knob chain 138 nucleotide
sequence dimeric see Table 5 hu 4-1BBL (71-254)-CL* 139 monomeric
hu 4-1BBL (71- see Table 5 254)-CL* Fc knob chain 140 dimeric hu
4-1BBL (71-254)- see Table 5 CL* 141 nucleotide sequence anti- see
Table 6 FAP (28H1) Fc hole chain fused to dimeric hu 4-1BBL
(71-254) 142 nucleotide sequence anti- see Table 6 FAP (28H1) Fc
knob chain fused to monomeric hu 4- 1BBL (71-254) 143 nucleotide
sequence see Table 7 monomeric hu 4-1BBL (71-
254)-(G.sub.4S).sub.1-CL* 144 nucleotide sequence [anti- see Table
8 FAP (28H1)].sub.2 Fc hole chain 145 [anti-FAP (28H1)].sub.2 Fc
hole see Table 8 chain 146 nucleotide sequence dimeric see Table 9
hu 4-1BBL (71-254)-FAP (VHCL*) Fc knob chain
147 nucleotide sequence see Table 9 monomeric hu 4-1BBL (71-
254)-FAP (VLCH1*) 148 dimeric hu 4-1BBL (71-254)- see Table 9 FAP
(VHCL*) Fc knob chain 149 monomeric hu 4-1BBL (71- see Table 9
254)-FAP (VLCH1*) 150 nucleotide sequence dimeric see Table 10 hu
4-1BBL (52-254)- CH1* Fc knob chain 151 nucleotide sequence see
Table 10 Monomeric hu 4-1BBL (52- 254)-CL* 152 nucleotide sequence
dimeric see Table 11 hu 4-1BBL (80-254)- CH1* Fc knob chain 153
nucleotide sequence see Table 11 Monomeric hu 4-1BBL (80- 254)-CL*
154 nucleotide sequence DP47 see Table 14 FC KK chain 155
nucleotide sequence DP47 see Table 14 light chain 156 DP47 FC KK
chain see Table 14 157 DP47 light chain see Table 14 158 nucleotide
sequence dimeric see Table 15 mu 4-1BBL (104-309, C160S)-CL Fc DD
chain 159 nucleotide sequence see Table 15 monomeric murine 4-1BBL
(104-309, C160S)-CH1 160 dimeric mu 4-1BBL (104- see Table 15 309,
C160S)-CL Fc DD chain 161 monomeric murine 4-1BBL see Table 15
(104-309, C160S)-CH1 162 nucleotide sequence see Table 21 anti-FAP
(4B9) Fc hole chain 163 nucleotide sequence see Table 21 anti-FAP
(4B9) light chain 164 anti-FAP (4B9) Fc hole see Table 21 chain 165
nucleotide sequence dimeric see Table 22 hu 4-1BBL (71-254)-CL Fc
knob chain 166 nucleotide sequence see Table 22 monomeric hu 4-1BBL
(71- 254)-CH1 167 nucleotide sequence anti- see Table 23 FAP (4B9)
Fc hole chain fused to dimeric hu 4-1BBL (71-254) 168 nucleotide
sequence anti- see Table 23 FAP (4B9) Fc knob chain fused to
monomeric hu 4- 1BBL (71-254) 169 nucleotide sequence dimeric see
Table 24 hu 4-1BBL (71-248)-CL* Fc knob chain 170 nucleotide
sequence see Table 24 monomeric hu 4-1BBL (71-248)-CH1* 171
nucleotide sequence dimeric see Table 25 hu 4-1BBL (71-248)-CL Fc
knob chain 172 nucleotide sequence see Table 25 monomeric hu 4-1BBL
(71-248)-CH1 173 Dimeric hu 4-1BBL (71- see Table 25 248)-CL Fc
knob chain 174 Monomeric hu see Table 25 4-1BBL (71-248)-CH1 175
nucleotide sequence anti- see Table 26 FAP (4B9) Fc hole chain
fused to dimeric hu 4-1BBL (71-248) 176 nucleotide sequence anti-
see Table 26 FAP (4B9) Fc knob chain fused to monomeric hu 4- 1BBL
(71-248) 177 nucleotide sequence DP47 see Table 27 Fc hole chain
fused to dimeric hu 4-1BBL (71-254) 178 nucleotide sequence DP47
see Table 27 Fc knob chain fused to monomeric hu 4-1BBL (71- 254)
179 DP47 Fc hole chain fused to see Table 27 dimeric hu 4-1BBL
(71-254) 180 DP47 Fc knob chain fused see Table 27 to monomeric hu
4-1BBL (71-254) 181 nucleotide sequence DP47 see Table 29 heavy
chain (hu IgG1 PGLALA) 182 DP47 heavy chain (hu IgG1 see Table 29
PGLALA) 183 monomeric hu 4-1BBL (71-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 254) plus (G.sub.4S).sub.1
linker LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSEGGGGS 184 monomeric hu
4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 248) plus
(G.sub.4S).sub.2 linker LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLG GGGSGGGGS 185 monomeric hu
4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP 248) plus
(G.sub.4S).sub.1 linker LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY
YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAA
ALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLG GGGS 186 Nucleotide sequence
see Table 43 human CD19 antigen Fc knob chain avi tag 187
Polypeptide sequence see Table 43 human CD19 antigen Fc knob chain
avi tag 188 Nucleotide sequence see Table 43 cynomolgus CD19
antigen Fc knob chain avi tag 189 Polypeptide sequence see Table 43
cynomolgus CD19 antigen Fc knob chain avi tag 190 humanized CD19
(8B8) NSNGNT HVR-L1 191 humanized CD19 (8B8) KFNG HVR-H2 192
humanized CD19 (8B8) TEKFQGRVTM var.1 to 9 HVR-H2 193 humanized
CD19 (8B8) LENPNGNT var.5 HVR-L1 194 humanized CD19 (8B8) LENPSGNT
var.9 HVR-L1 195 CD19 (8B8-018) CDR-H1 DYIMH 196 CD19 (8B8-018)
CDR-H2 YINPYNDGSKYTEKFQG 197 CD19 (8B8-018) CDR-H3 GTYYYGSALFDY 198
CD19 (8B8-018) CDR-L1 KSSQSLENPNGNTYLN 199 CD19 (8B8-018) CDR-L2
RVSKRFS 200 CD19 (8B8-018) CDR-L3 LQLTHVPYT 201 CD19 (8B8-018) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ALFDYWGQGTTVTVSS 202 CD19
(8B8-018) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLTHVPYTFGQGTKLEIK 203 Nucleotide sequence anti-
see Table 47 CD19(8B8-018) Fc hole chain 204 Nucleotide sequence
anti- see Table 47 CD19(8B8-018) light chain 205 anti-CD19(8B8-018)
Fc hole see Table 47 chain 206 anti-CD19(8B8-018) light see Table
47 chain 207 Nucleotide sequence anti- see Table 49 CD19(8B8-018)
Fc hole dimeric ligand chain 208 Nucleotide sequence anti- see
Table 49 CD19(8B8-018) Fc knob monomeric ligand 209
anti-CD19(8B8-018) Fc hole see Table 49 dimeric ligand chain 210
anti-CD19(8B8-018) Fc see Table 49 knob monomeric ligand 211
Nucleotide sequence anti- see Table 52 CD19(8B8-018) Fc hole
dimeric ligand (71-248) chain 212 Nucleotide sequence anti- see
Table 52 CD19(8B8-018) Fc knob monomeric (71-248) ligand 213
anti-CD19(8B8-018) Fc hole see Table 52 dimeric ligand (71-248)
chain 214 anti-CD19(8B8-018) Fc see Table 52 knob monomeric
(71-248) ligand
215 Nucleotide sequence CD19 GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGT
(8B8) VH Parental clone AAAGCCTGGGGCTTCAGTGAAGATGGCCTGCAAG
GCTTCTGGATACACATTCACTGACTATATTATGCA
CTGGGTGAAGCAGAAGACTGGGCAGGGCCTTGAG
TGGATTGGATATATTAATCCTTACAATGATGGTTCT
AAGTACACTGAGAAGTTCAACGGCAAGGCCACAC
TGACTTCAGACAAATCTTCCATCACAGCCTACATG
GAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGT
CTATTACTGTGCAAGAGGGACCTATTATTATGGTA
GCGCCCTCTTTGACTACTGGGGCCAAGGCACCACT CTCACAGTCTCCTCG 216 Nucleotide
sequence CD19 GATGCTGTGATGACCCAAACTCCACTCTCCCTGCC (8B8) VL Parental
clone TGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCA
GGTCTAGTCAGAGCCTTGAAAACAGTAATGGAAA
CACCTATTTGAACTGGTACCTCCAGAAACCAGGCC
AGTCTCCACAACTCCTGATCTACAGGGTTTCCAAA
CGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAG
TGGATCAGGGACAGATTTCACACTGAAAATCAGCA
GAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGC
CTACAACTTACACATGTCCCGTACACGTTCGGAGG GGGGACCAAGCTGGAAATAAAA 217 CD19
L1 reverse random see Table 53 218 CD19 L2 forward random see Table
53 219 CD19 H1 reverse random see Table 53 220 CD19 H2 forward
random see Table 53 221 CD19 H3 reverse constant see Table 53 222
LMB3 see Table 53 223 D19 L1 forward constant see Table 54 224 CD19
L3 reverse random see Table 54 225 CD19 L3 forward constant see
Table 54 226 CD19 H3 reverse random see Table 54 227 Nucleotide
sequence SNAP GGCCGCCGCTAGCGGCATCGACTACAAGGACGAC tag human CD19
ECD- GATGACAAGGCCGGCATCGATGCCATCATGGACA PDGFR
AAGACTGCGAAATGAAGCGCACCACCCTGGATAG
CCCTCTGGGCAAGCTGGAACTGTCTGGGTGCGAAC
AGGGCCTGCACGAGATCAAGCTGCTGGGCAAAGG
AACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCC
CAGCCGCCGTGCTGGGCGGACCAGAGCCACTGAT
GCAGGCCACCGCCTGGCTCAACGCCTACTTTCACC
AGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCC
CTGCACCACCCAGTGTTCCAGCAGGAGAGCTTTAC
CCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGA
AGTTCGGAGAGGTCATCAGCTACCAGCAGCTGGCC
GCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGT
GAAAACCGCCCTGAGCGGAAATCCCGTGCCCATTC
TGATCCCCTGCCACCGGGTGGTGTCTAGCTCTGGC
GCCGTGGGGGGCTACGAGGGCGGGCTCGCCGTGA
AAGAGTGGCTGCTGGCCCACGAGGGCCACAGACT
GGGCAAGCCTGGGCTGGGTGATATCCCCGAGGAA
CCCCTGGTCGTGAAGGTGGAAGAGGGCGACAATG
CCGTGCTGCAGTGCCTGAAGGGCACCTCCGATGGC
CCTACCCAGCAGCTGACCTGGTCCAGAGAGAGCCC
CCTGAAGCCCTTCCTGAAGCTGTCTCTGGGCCTGC
CTGGCCTGGGCATCCATATGAGGCCTCTGGCCATC
TGGCTGTTCATCTTCAACGTGTCCCAGCAGATGGG
CGGCTTCTACCTGTGTCAGCCTGGCCCCCCATCTG
AGAAGGCTTGGCAGCCTGGCTGGACCGTGAACGT
GGAAGGATCCGGCGAGCTGTTCCGGTGGAACGTGT
CCGATCTGGGCGGCCTGGGATGCGGCCTGAAGAA
CAGATCTAGCGAGGGCCCCAGCAGCCCCAGCGGC
AAACTGATGAGCCCCAAGCTGTACGTGTGGGCCAA
GGACAGACCCGAGATCTGGGAGGGCGAGCCTCCT
TGCCTGCCCCCTAGAGACAGCCTGAACCAGAGCCT
GAGCCAGGACCTGACAATGGCCCCTGGCAGCACA
CTGTGGCTGAGCTGTGGCGTGCCACCCGACTCTGT
GTCTAGAGGCCCTCTGAGCTGGACCCACGTGCACC
CTAAGGGCCCTAAGAGCCTGCTGAGCCTGGAACTG
AAGGACGACAGGCCCGCCAGAGATATGTGGGTCA
TGGAAACCGGCCTGCTGCTGCCTAGAGCCACAGCC
CAGGATGCCGGCAAGTACTACTGCCACAGAGGCA
ACCTGACCATGAGCTTCCACCTGGAAATCACCGCC
AGACCCGTGCTGTGGCACTGGCTGCTGAGAACAGG
CGGCTGGAAGGTCGACGAACAAAAACTCATCTCA
GAAGAGGATCTGAATGCTGTGGGCCAGGACACGC
AGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTT
AAGGTGGTGGTGATCTCAGCCATCCTGGCCCTGGT
GGTGCTCACCATCATCTCCCTTATCATCCTCATCAT GCTTTGGCAGAAGAAGCCACGT 228
Nucleotide sequence SNAP CCGGCCGCCGCTAGCGGCATCGACTACAAGGACG tag
cynomolgus CD19 ACGATGACAAGGCCGGCATCGATGCCATCATGGA ECD-PDGFR
CAAAGACTGCGAAATGAAGCGCACCACCCTGGAT
AGCCCTCTGGGCAAGCTGGAACTGTCTGGGTGCGA
ACAGGGCCTGCACGAGATCAAGCTGCTGGGCAAA
GGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGC
CCCAGCCGCCGTGCTGGGCGGACCAGAGCCACTG
ATGCAGGCCACCGCCTGGCTCAACGCCTACTTTCA
CCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAG
CCCTGCACCACCCAGTGTTCCAGCAGGAGAGCTTT
ACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGT
GAAGTTCGGAGAGGTCATCAGCTACCAGCAGCTG
GCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGC
CGTGAAAACCGCCCTGAGCGGAAATCCCGTGCCCA
TTCTGATCCCCTGCCACCGGGTGGTGTCTAGCTCTG
GCGCCGTGGGGGGCTACGAGGGCGGGCTCGCCGT
GAAAGAGTGGCTGCTGGCCCACGAGGGCCACAGA
CTGGGCAAGCCTGGGCTGGGTGATATCCCCCAGGA
ACCCCTGGTCGTGAAGGTGGAAGAGGGCGACAAT
GCCGTGCTCCAGTGTCTCGAGGGCACCTCCGATGG
CCCTACACAGCAGCTCGTGTGGTGCAGAGACAGCC
CCTTCGAGCCCTTCCTGAACCTGTCTCTGGGCCTGC
CTGGCATGGGCATCAGAATGGGCCCTCTGGGCATC
TGGCTGCTGATCTTCAACGTGTCCAACCAGACCGG
CGGCTTCTACCTGTGTCAGCCTGGCCTGCCAAGCG
AGAAGGCTTGGCAGCCTGGATGGACCGTGTCCGTG
GAAGGATCTGGCGAGCTGTTCCGGTGGAACGTGTC
CGATCTGGGCGGCCTGGGATGCGGCCTGAAGAAC
AGAAGCAGCGAGGGCCCTAGCAGCCCCAGCGGCA
AGCTGAATAGCAGCCAGCTGTACGTGTGGGCCAA
GGACAGACCCGAGATGTGGGAGGGCGAGCCTGTG
TGTGGCCCCCCTAGAGATAGCCTGAACCAGAGCCT
GAGCCAGGACCTGACAATGGCCCCTGGCAGCACA
CTGTGGCTGAGCTGTGGCGTGCCACCCGACTCTGT
GTCCAGAGGCCCTCTGAGCTGGACACACGTGCGGC
CTAAGGGCCCTAAGAGCAGCCTGCTGAGCCTGGA
ACTGAAGGACGACCGGCCCGACCGGGATATGTGG
GTGGTGGATACAGGCCTGCTGCTGACCAGAGCCAC
AGCCCAGGATGCCGGCAAGTACTACTGCCACAGA
GGCAACTGGACCAAGAGCTTTTACCTGGAAATCAC
CGCCAGACCCGCCCTGTGGCACTGGCTGCTGAGAA
TCGGAGGCTGGAAGGTCGACGAGCAGAAGCTGAT
CTCCGAAGAGGACCTGAACGCCGTGGGCCAGGAT
ACCCAGGAAGTGATCGTGGTGCCCCACAGCCTGCC
CTTCAAGGTGGTCGTGATCAGCGCCATTCTGGCCC
TGGTGGTGCTGACCATCATCAGCCTGATCATCCTG ATTATGCTGTGGCAGAAAAAGCCCCGC 229
Polypeptide sequence SNAP PAAASGIDYKDDDDKAGIDAIMDKDCEMKRTTLDSP tag
human CD19 ECD- LGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAA PDGFR
VLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHP
VFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGN
PAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEG
GLAVKEWLLAHEGHRLGKPGLGDIPEEPLVVKVEEG
DNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLG
LPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSE
KAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKN
RSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLP
PRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPL
SWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLL
LPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWH
WLLRTGGWKVDEQKLISEEDLNAVGQDTQEVIVVP
HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR 230 Polypeptide sequence SNAP
PAAASGIDYKDDDDKAGIDAIMDKDCEMKRTTLDSP tag cynomolgus CD19
LGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAA ECD-PDGFR
VLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHP
VFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGN
PAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEG
GLAVKEWLLAHEGHRLGKPGLGDIPQEPLVVKVEEG
DNAVLQCLEGTSDGPTQQLVWCRDSPFEPFLNLSLG
LPGMGIRMGPLGIWLLIFNVSNQTGGFYLCQPGLPSE
KAWQPGWTVSVEGSGELFRWNVSDLGGLGCGLKN
RSSEGPSSPSGKLNSSQLYVWAKDRPEMWEGEPVCG
PPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGP
LSWTHVRPKGPKSSLLSLELKDDRPDRDMWVVDTG
LLLTRATAQDAGKYYCHRGNWTKSFYLEITARPAL
WHWLLRIGGWKVDEQKLISEEDLNAVGQDTQEVIV
VPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR 231 CD19 (8B8-5H09) CDR-L1
see Table 56 232 CD19 (8B8-5H09) CDR-L2 see Table 56 233 CD19
(8B8-5H09) CDR-L3 see Table 56 234 CD19 (8B8-5H09) CDR-H1 see Table
57 235 CD19 (8B8-5H09) CDR-H2 see Table 57 236 CD19 (8B8-5H09)
CDR-H3 see Table 57 237 CD19 (8B8-7H07) CDR-L1 see Table 56 238
CD19 (8B8-7H07) CDR-L2 see Table 56 239 CD19 (8B8-7H07) CDR-L3 see
Table 56 240 CD19 (8B8-7H07) CDR-H1 see Table 57 241 CD19
(8B8-7H07) CDR-H2 see Table 57 242 CD19 (8B8-7H07) CDR-H3 see Table
57 243 CD19 (8B8-2B03) CDR-L1 see Table 56 244 CD19 (8B8-2B03)
CDR-L2 see Table 56 245 CD19 (8B8-2B03) CDR-L3 see Table 56 246
CD19 (8B8-2B03) CDR-H1 see Table 57 247 CD19 (8B8-2B03) CDR-H2 see
Table 57 248 CD19 (8B8-2B03) CDR-H3 see Table 57 249 CD19
(8B8-2B11) CDR-L1 see Table 56 250 CD19 (8B8-2B11) CDR-L2 see Table
56 251 CD19 (8B8-2B11) CDR-L3 see Table 56 252 CD19 (8B8-2B11)
CDR-H1 see Table 57 253 CD19 (8B8-2B11) CDR-H2 see Table 57 254
CD19 (8B8-2B11) CDR-H3 see Table 57 255 CD19 (8B8-5A07) CDR-L1 see
Table 56 256 CD19 (8B8-5A07) CDR-L2 see Table 56 257 CD19
(8B8-5A07) CDR-L3 see Table 56 258 CD19 (8B8-5A07) CDR-H1 see Table
57 259 CD19 (8B8-5A07) CDR-H2 see Table 57 260 CD19 (8B8-5A07)
CDR-H3 see Table 57 261 CD19 (8B8-5B08) CDR-L1 see Table 56 262
CD19 (8B8-5B08) CDR-L2 see Table 56 263 CD19 (8B8-5B08) CDR-L3 see
Table 56 264 CD19 (8B8-5B08) CDR-H1 see Table 57 265 CD19
(8B8-5B08) CDR-H2 see Table 57 266 CD19 (8B8-5B08) CDR-H3 see Table
57 267 CD19 (8B8-5D08) CDR-L1 see Table 56 268 CD19 (8B8-5D08)
CDR-L2 see Table 56 269 CD19 (8B8-5D08) CDR-L3 see Table 56 270
CD19 (8B8-5D08) CDR-H1 see Table 57
271 CD19 (8B8-5D08) CDR-H2 see Table 57 272 CD19 (8B8-5D08) CDR-H3
see Table 57 273 nucleotide sequence CD19 see Table 58 (8B8)
parental light chain 274 nucleotide sequence CD19 see Table 58
(8B8) parental heavy chain 275 CD19 (8B8) parental light see Table
58 chain 276 CD19 (8B8) parental heavy see Table 58 chain 277
nucleotide sequence CD19 see Table 59 (8B8-2B11) light chain 278
nucleotide sequence CD19 see Table 59 (8B8-2B11) heavy chain 279
CD19 (8B8-2B11) light see Table 59 chain 280 CD19 (8B8-2B11) heavy
see Table 59 chain 281 nucleotide sequence CD19 see Table 59
(8B8-7H07) light chain 282 nucleotide sequence CD19 see Table 59
(8B8-7H07) heavy chain 283 CD19 (8B8-7H07) light see Table 59 chain
284 CD19 (8B8-7H07) heavy see Table 59 chain 285 nucleotide
sequence CD19 see Table 59 (8B8-2B03) light chain 286 nucleotide
sequence CD19 see Table 59 (8B8-2B03) heavy chain 287 CD19
(8B8-2B03) light see Table 59 chain 288 CD19 (8B8-2B03) heavy see
Table 59 chain 289 nucleotide sequence CD19 see Table 59 (8B8-5A07)
light chain 290 nucleotide sequence CD19 see Table 59 (8B8-5A07)
heavy chain 291 CD19 (8B8-5A07) light see Table 59 chain 292 CD19
(8B8-5A07) heavy see Table 59 chain 293 nucleotide sequence CD19
see Table 59 (8B8-5D08) light chain 294 nucleotide sequence CD19
see Table 59 (8B8-5D08) heavy chain 295 CD19 (8B8-5D08) light see
Table 59 chain 296 CD19 (8B8-5D08) heavy see Table 59 chain 297
nucleotide sequence CD19 see Table 59 (8B8-5B08) light chain 298
nucleotide sequence CD19 see Table 59 (8B8-5B08) heavy chain 299
CD19 (8B8-5B08) light see Table 59 chain 300 CD19 (8B8-5B08) heavy
see Table 59 chain 301 nucleotide sequence CD19 see Table 59
(8B8-5H09) light chain 302 nucleotide sequence CD19 see Table 59
(8B8-5H09) heavy chain 303 CD19 (8B8-5H09) light see Table 59 chain
304 CD19 (8B8-5H09) heavy see Table 59 chain 305 Nucleotide
sequence anti- see Table 62 CD19(8B8-2B11) Fc hole chain 306
anti-CD19(8B8-2B11) Fc see Table 62 hole chain 307 Nucleotide
sequence anti- see Table 64 CD19(8B8-2B11) Fc hole dimeric ligand
chain 308 Nucleotide sequence anti- see Table 64 CD19(8B8-2B11) Fc
knob monomeric ligand 309 anti-CD19(8B8-2B11) Fc see Table 64 hole
dimeric ligand chain 310 anti-CD19(8B8-2B11) Fc see Table 64 knob
monomeric ligand 311 Nucleotide sequence anti- see Table 67
CD19(8B8-2B11) Fc hole dimeric ligand (71-248) chain 312 Nucleotide
sequence anti- see Table 67 CD19(8B8-2B11) Fc knob monomeric
(71-248) ligand 313 anti-CD19(8B8-2B11) Fc see Table 67 hole
dimeric ligand (71-248) chain 314 anti-CD19(8B8-2B11) Fc see Table
67 knob monomeric (71-248) ligand 315 nucleotide sequence see Table
72 CD19(8B8-018) heavy chain (huIgG1 PGLALA) 316 CD19(8B8-018)
heavy chain see Table 72 (huIgG1 PGLALA) 317 anti-mu CEA T84.66
MKCSWVIFFL MAVVTGVNSE VQLQQSGAEL VH VEPGASVKLS CTASGFNIKD
TYMHWVKQRP EQGLEWIGRI DPANGNSKYV PKFQGKATIT ADTSSNTAYL QLTSLTSEDT
AVYYCAPFGY YVSDYAMAYW GQGTSVTVSS 318 anti-mu CEA T84.66 METDTLLLWV
LLLWVPGSTG DIVLTQSPAS VL LAVSLGQRAT MSCRAGESVD IFGVGFLHWY
QQKPGQPPKL LIYRASNLES GIPVRFSGTG SRTDFTLIID PVEADDVATY YCQQTNEDPY
TFGGGTKLEI K 319 IGHV1-69*08 TAAGGGGCTT CCTAGTCCTA AGGCTGAGGA IMGT
Acc No. Z14309 AGGGATCCTG GTTTAGTTAA AGAGGATTTT ATTCACCCCT
GTGTCCTCTC CACAGGTGTC CAGTCCCAGG TCCAGCTGGT GCAATCTGGG GCTGAGGTGA
AGAAGCCTGG GTCCTCGGTG AAGGTCTCCT GCAAGGCTTC TGGAGGCACC TTCAGCAGCT
ATACTATCAG CTGGGTGCGA CAGGCCCCTG GACAAGGGCT TGAGTGGATG GGAAGGATCA
TCCCTATCCT TGGTACAGCA AACTACGCAC AGAAGTTCCA GGGCAGAGTC ACGATTACCG
CGGACAAATC CACGAGCACA GCCTACATGG AGCTGAGCAG CCTGAGATCT GAGGACACGG
CCGTGTATTA CTGTGCGAGA GA 320 IGKV3-11*01 CTGCAGCTGG AAGCTCAGCT
CCCACCCAGC IMGT Acc No. TGCTTTGCAT GTCCCTCCCA GCTGCCCTAC CTTCCAGAGC
CCATATCAAT GCCTGTGTCA GAGCCCTGGG GAGGAACTGC TCAGTTAGGA CCCAGAGGGA
ACCATGGAAG CCCCAGCTCA GCTTCTCTTC CTCCTGCTAC TCTGGCTCCC AGGTGAGGGG
AACATGAGGT GGTTTTGCAC ATTAGTGAAA ACTCTTGCCA CCTCTGCTCA GCAAGAAATA
TAATTAAAAT TCAAAGTATA TCAACAATTT TGGCTCTACT CAAAGACAGT TGGTTTGATC
TTGATTACAT GAGTGCATTT CTGTTTTATT TCCAATTTCA GATACCACCG GAGAAATTGT
GTTGACACAG TCTCCAGCCA CCCTGTCTTT GTCTCCAGGG GAAAGAGCCA CCCTCTCCTG
CAGGGCCAGT CAGAGTGTTA GCAGCTACTT AGCCTGGTAC CAACAGAAAC CTGGCCAGGC
TCCCAGGCTC CTCATCTATG ATGCATCCAA CAGGGCCACT GGCATCCCAG CCAGGTTCAG
TGGCAGTGGG TCTGGGACAG ACTTCACTCT CACCATCAGC AGCCTAGAGC CTGAAGATTT
TGCAGTTTAT TACTGTCAGC AGCGTAGCAA CTGGCCTCCC ACAGTGATTC CACATGAAAC
AAAAACCCCA ACAAGACCAT CAGTGTTTAC TAGATTATTA TACCAGCTGC TTCCTTTACA
GACAGCTAGT GGGGTGGCCA CTCAGTGTTA GCATCTCAGC TCTATTTGGC CATTTTGGAG
TTCAAGT 321 CEA CDR-H1 see Table 81 322 CEA CDR-H2 see Table 81 323
CEA CDR-H3 see Table 81 324 CEA CDR-L1 see Table 81 325 CEA CDR-L2
see Table 81 326 CEA CDR-L3 see Table 81 327 Parental CEA binder VH
see Table 81 328 Parental CEA binder VL see Table 81 329 Humanized
CEA binder VH see Table 81 330 Humanized CEA binder VL see Table 81
331 Nucleotide sequence anti- see Table 82 CEA(T84.66-LCHA) Fc hole
chain 332 Nucleotide sequence anti- see Table 82 CEA(T84.66-LCHA)
light chain 333 anti-CEA (T84.66-LCHA) see Table 82 Fc hole chain
334 anti-CEA (T84.66-LCHA) see Table 82 light chain 335 Nucleotide
sequence anti- see Table 84 CEA(T84.66-LCHA) Fc hole dimeric ligand
chain 336 Nucleotide sequence anti- see Table 84 CEA(T84.66-LCHA)
Fc knob monomeric ligand 337 anti-CEA(T84.66-LCHA) see Table 84 Fc
hole dimeric ligand chain 338 anti-CEA(T84.66-LCHA) see Table 84 Fc
knob monomeric ligand 339 Nucleotide sequence anti- see Table 87
CEA (T84.66-LCHA) Fc
hole dimeric ligand (71-248) chain 340 Nucleotide sequence anti-
see Table 87 CEA (T84.66-LCHA) Fc knob monomeric (71-248) ligand
341 anti-CEA (T84.66-LCHA) see Table 87 Fc hole dimeric ligand (71-
248) chain 342 anti-CEA (T84.66-LCHA) see Table 87 Fc knob
monomeric (71- 248) ligand 343 Nucleotide sequence anti- see Table
88 CEA(T84.66) Fc hole chain 344 Nucleotide sequence anti- see
Table 88 CEA(T84.66) light chain 345 anti-CEA (T84.66) Fc hole see
Table 88 chain 346 anti-CEA (T84.66) light see Table 88 chain 347
Nucleotide sequence anti- see Table 89 CEA(T84.66) Fc hole dimeric
ligand chain 348 Nucleotide sequence anti- see Table 89 CEA(T84.66)
Fc knob monomeric ligand 349 anti-CEA(T84.66) Fc hole see Table 89
dimeric ligand chain 350 anti-CEA(T84.66) Fc knob see Table 89
monomeric ligand 351 nucleotide sequence hu see Table 92
NA3B3A2-avi His 352 human NA3B3A2-avi-His see Table 92 353
Nucleotide sequence see Table 97 Dimeric hu OX40L (51-183)- CL* Fc
knob chain 354 Nucleotide sequence see Table 97 Monomeric hu OX40L
(51-183)-CH1* 355 Dimeric hu OX40L (51-183)- see Table 97 CL* Fc
knob chain 356 Monomeric hu see Table 97 OX40L (51-183)-CH1* 357
CD19 (8B8-2B11) VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGP QLFDYWGQGTTVTVSS 358 CD19
(8B8-2B11) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLLEDPYTFGQGTKLEIK 359 CD19 (8B8-7H07) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ELFDYWGQGTTVTVSS 360 CD19
(8B8-7H07) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQATHIPYTFGQGTKLEIK 361 CD19 (8B8-2B03) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYITH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGP DLFDYWGQGTTVTVSS 362 CD19
(8B8-2B03) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLTHVPYTFGQGXKLEIK 363 CD19 (8B8-5A07) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ALFDYWGQGTTVTVSS 364 CD19
(8B8-5A07) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQPGHYPGTFGQGTKLEIK 365 CD19 (8B8-5D08) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ELFDYWGQGTTVTVSS 366 CD19
(8B8-5D08) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLTHEPYTFGQGTKLEIK 367 CD19 (8B8-5B08) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGP QLFDYWGQGTTVTVSS 368 CD19
(8B8-5B08) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLDSYPNTFGQGTKLEIK 369 CD19 (8B8-5H09) VH
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMH
WVRQAPGQGLEWMGYINPYNDGSKYTEKFQGRVT
MTSDTSISTAYMELSRLRSDDTAVYYCARGTYYYGS ALFDYWGQGTTVTVSS 370 CD19
(8B8-5H09) VL DIVMTQTPLSLSVTPGQPASISCKSSQSLESSTGNTYL
NWYLQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTD
FTLKISRVEAEDVGVYYCLQLIDYPVTFGQGTKLEIK 371 dimeric huOX40L (51-183)
QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMK connected by (G.sub.4S).sub.2
linker VQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEP
LFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL
DDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVS
HRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQN
NSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQ
LKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDF HVNGGELILIHQNPGEFCVL 372
dimeric huOX40L (52-183) VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKV
connected by (G.sub.4S).sub.2 linker
QNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPL
FQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLD
DFHVNGGELILIHQNPGEFCVLGGGGSGGGGSVSHR
YPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNS
VIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLK
KVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHV NGGELILIHQNPGEFCVL 373 hu
4-1BBL (85-248) LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL
TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEA
RNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQ LTQGATVLGLFRVTPEIPAGL 374 hu
4-1BBL (80-248) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL
AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPP
ASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEIPAGL 375
hu 4-1BBL (52-248) PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLR
QGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL
SYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSG
SVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSA
FGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQG ATVLGLFRVTPEIPAGL
[0474] General information regarding the nucleotide sequences of
human immunoglobulins light and heavy chains is given in: Kabat, E.
A., et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991). Amino acids of antibody chains are numbered
and referred to according to the EU numbering systems according to
Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological
Interest, 5th ed., Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)) as defined above.
EXAMPLES
[0475] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
[0476] Recombinant DNA Techniques
[0477] Standard methods were used to manipulate DNA as described in
Sambrook et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions. General information regarding the
nucleotide sequences of human immunoglobulin light and heavy chains
is given in: Kabat, E. A. et al., (1991) Sequences of Proteins of
Immunological Interest, Fifth Ed., NIH Publication No 91-3242.
[0478] DNA Sequencing
[0479] DNA sequences were determined by double strand
sequencing.
[0480] Gene Synthesis
[0481] Desired gene segments were either generated by PCR using
appropriate templates or were synthesized by Geneart AG
(Regensburg, Germany) from synthetic oligonucleotides and PCR
products by automated gene synthesis. In cases where no exact gene
sequence was available, oligonucleotide primers were designed based
on sequences from closest homologues and the genes were isolated by
RT-PCR from RNA originating from the appropriate tissue. The gene
segments flanked by singular restriction endonuclease cleavage
sites were cloned into standard cloning/sequencing vectors. The
plasmid DNA was purified from transformed bacteria and
concentration determined by UV spectroscopy. The DNA sequence of
the subcloned gene fragments was confirmed by DNA sequencing. Gene
segments were designed with suitable restriction sites to allow
sub-cloning into the respective expression vectors. All constructs
were designed with a 5'-end DNA sequence coding for a leader
peptide which targets proteins for secretion in eukaryotic
cells.
[0482] Cell Culture Techniques
[0483] Standard cell culture techniques were used as described in
Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso,
M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M.
(eds.), John Wiley & Sons, Inc.
[0484] Protein Purification
[0485] Proteins were purified from filtered cell culture
supernatants referring to standard protocols. In brief, antibodies
were applied to a Protein A Sepharose column (GE healthcare) and
washed with PBS. Elution of antibodies was achieved at pH 2.8
followed by immediate neutralization of the sample. Aggregated
protein was separated from monomeric antibodies by size exclusion
chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM
Histidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions were
pooled, concentrated (if required) using e.g., a MILLIPORE Amicon
Ultra (30 MWCO) centrifugal concentrator, frozen and stored at
-20.degree. C. or -80.degree. C. Part of the samples were provided
for subsequent protein analytics and analytical characterization
e.g. by SDS-PAGE, size exclusion chromatography (SEC) or mass
spectrometry.
[0486] SDS-PAGE
[0487] The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) was used
according to the manufacturer's instruction. In particular, 10% or
4-12% NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels (pH 6.4) and a
NuPAGE.RTM. MES (reduced gels, with NuPAGE.RTM. Antioxidant running
buffer additive) or MOPS (non-reduced gels) running buffer was
used.
[0488] Analytical Size Exclusion Chromatography
[0489] Size exclusion chromatography (SEC) for the determination of
the aggregation and oligomeric state of antibodies was performed by
HPLC chromatography. Briefly, Protein A purified antibodies were
applied to a Tosoh TSKGEL.RTM. G3000SW column in 300 mM NaCl, 50 mM
KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, pH 7.5 on an Agilent HPLC 1100
system or to a Superdex 200 column (GE Healthcare) in 2.times.PBS
on a Dionex HPLC-System. The eluted protein was quantified by UV
absorbance and integration of peak areas. BioRad Gel Filtration
Standard 151-1901 served as a standard.
[0490] Mass Spectrometry
[0491] This section describes the characterization of the
multispecific antibodies with VH/VL exchange (VH/VL CrossMabs) with
emphasis on their correct assembly. The expected primary structures
were analyzed by electrospray ionization mass spectrometry (ESI-MS)
of the deglycosylated intact CrossMabs and deglycosylated/plasmin
digested or alternatively deglycosylated/limited LysC digested
CrossMabs.
[0492] The VH/VL CrossMabs were deglycosylated with N-Glycosidase F
in a phosphate or Tris buffer at 37.degree. C. for up to 17 h at a
protein concentration of 1 mg/ml. The plasmin or limited LysC
(Roche) digestions were performed with 100 .mu.s deglycosylated
VH/VL CrossMabs in a Tris buffer pH 8 at room temperature for 120
hours and at 37.degree. C. for 40 min, respectively. Prior to mass
spectrometry the samples were desalted via HPLC on a Sephadex
G.sub.25 column (GE Healthcare). The total mass was determined via
ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped
with a TriVersa NanoMate source (Advion).
[0493] Determination of binding and binding affinity of
multispecific antibodies to the respective antigens using surface
plasmon resonance (SPR) (BIACORE.RTM.)
[0494] Binding of the generated antibodies to the respective
antigens is investigated by surface plasmon resonance using a
BIACORE.RTM. instrument (GE Healthcare Biosciences AB, Uppsala,
Sweden). Briefly, for affinity measurements Goat-Anti-Human IgG,
JIR 109-005-098 antibodies are immobilized on a CM5 chip via amine
coupling for presentation of the antibodies against the respective
antigen. Binding is measured in HBS buffer (HBS-P (10 mM HEPES, 150
mM NaCl, 0.005% Tween 20, ph 7.4), 25.degree. C. (or alternatively
at 37.degree. C.). Antigen (R&D Systems or in house purified)
was added in various concentrations in solution. Association was
measured by an antigen injection of 80 seconds to 3 minutes;
dissociation was measured by washing the chip surface with HBS
buffer for 3-10 minutes and a KD value was estimated using a 1:1
Langmuir binding model. Negative control data (e.g. buffer curves)
are subtracted from sample curves for correction of system
intrinsic baseline drift and for noise signal reduction. The
respective Biacore Evaluation Software is used for analysis of
sensorgrams and for calculation of affinity data.
Example 1
[0495] 1.1 Preparation of Targeted Human 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0496] Different fragments of the DNA sequence encoding part of the
ectodomain (amino acids 71-254, 52-254 and 80-254) of human 4-1BB
ligand were synthetized according to the P41273 sequence of Uniprot
database (SEQ ID NO:42).
[0497] As components for the assembly of a TNF ligand
trimer-containing antigen binding molecule a polypeptide comprising
two ectodomains of 4-1BB ligand, separated by (G.sub.4S).sub.2 (SEQ
ID NO:13) linkers, and fused to the human IgG1-CH1 or CL domain,
was cloned as depicted in FIG. 1A (human 4-1BB ligand,
(G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB ligand,
(G.sub.4S).sub.2 (SEQ ID NO:13) connector, human CH1 or CL) or as
depicted in FIG. 1C (human CH3, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand).
[0498] A polypeptide comprising one ectodomain of 4-1BB ligand and
fused to the human IgG1-CL or CH1 domain, was cloned as described
in FIG. 1B (human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human CL or CH1) or as depicted in FIG. 1D (human CH3,
(G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB ligand).
[0499] The polypeptides were subcloned in frame with the human IgG1
heavy chain CH2 and CH3 domains with optional peptide linkers, for
example for construct 1 the polypeptide encoding the dimeric 4-1BB
ligand fused to a human CH1 domain was subcloned in frame with the
human IgG1 heavy chain CH2 and CH3 domains on the knob (Merchant,
Zhu et al. 1998) using a linker (G.sub.4S).sub.2 of SEQ ID NO:13 or
GSPGSSSSGS of SEQ ID NO:57.
[0500] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
i.e. 28H1, were subcloned in frame with either the constant heavy
chain of the hole (Carter, J. Immunol. Methods (2001), 248, 7-15)
or the constant light chain of human IgG1. The generation and
preparation of the FAP binders is described in WO 2012/020006 A2,
which is incorporated herein by reference.
[0501] Table 1 summarizes the characteristics of the constructs
produced. The constructs 1 to 10 differ in their geometry, valency
for FAP, 4-1BB ligand ectodomain, crossover of the CH1 and CL
domain (CrossMab technology), mutations in the CH1 and CL domains
and different peptide linkers in the polypeptide comprising one
ectodomain of 4-1BB ligand (monomeric 4-1BBL chain).
TABLE-US-00004 TABLE 1 Characteristics of produced TNF ligand
trimer-containing antigen binding molecules (FAP split 4-1BBL
trimers) Crossed Linker to 4- Valency for FAP 4-1BBL CH1-CL Charged
1BBL in Construct FAP binder ectodomain domains residues light
chain 1.1 monovalent 28H1 71-254 no no (G.sub.4S).sub.2 (SEQ ID
NO:13) 1.2 monovalent 28H1 71-254 yes yes (G.sub.4S).sub.2 (Ligand)
(Ligand) (SEQ ID NO:13) 1.3 monovalent 28H1 71-254 yes yes
(G.sub.4S).sub.2 (FAP Fab) (Ligand) (SEQ ID NO:13) 1.4 monovalent
28H1 71-254 no yes (G.sub.4S).sub.2 (Ligand) (SEQ ID NO:13) 1.5
bivalent 28H1 71-254 no no (G.sub.4S).sub.2 (SEQ ID NO:13) 1.6
monovalent 28H1 71-254 no yes (G.sub.4S).sub.1 (Ligand) (SEQ ID
NO:128) 1.7 bivalent 28H1 71-254 yes yes (G.sub.4S).sub.2 (Ligand)
(Ligand) (SEQ ID NO:13) 1.8 bivalent 28H1 71-254 yes yes
(G.sub.4S).sub.2 (FAP Fab (Ligand) (SEQ ID fused to NO:13) Ligand)
1.9 monovalent 28H1 52-254 no yes (G.sub.4S).sub.2 (Ligand) (SEQ ID
NO:13) 1.10 monovalent 28H1 80-254 no yes (G.sub.4S).sub.2 (Ligand)
(SEQ ID NO:13)
[0502] In order to avoid mispairing, in most of the constructs one
pair of CH1 and CL domains was replaced by each other (domain
crossover) as described in WO 2009/080253 A1.
[0503] To further improve correct pairing, different charged amino
acid substitutions were introduced in the crossed or non-crossed
CH1 and CL domains as charged residues in constructs 2 to 4 and 6
to 10. In the human CL domain the mutations E123R and Q124K were
introduced, whereas the mutations K147E and K213E were cloned into
the human CH1 domain.
[0504] For all constructs the knobs into holes heterodimerization
technology was used with the S354C/T366W mutations in the CH3
domain of the knob chain and the corresponding
Y349C/T366S/L368A/Y407V mutations in the CH3 domain of the hole
chain (Carter, J Immunol Methods 248, 7-15 (2001)).
[0505] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in International Patent Appl. Publ. No. WO 2012/130831
A1.
[0506] For example, in construct 1 the combination of the ligand-Fc
knob chain containing the S354C/T366W mutations in the first CH3
domain, with the targeted anti-FAP-Fc hole chain containing the
Y349C/T366S/L368A/Y407V mutations in the second CH3 domain allows
generation of a heterodimer, which includes an assembled trimeric
4-1BB ligand and a FAP binding Fab (FIG. 2A, Construct 1.1).
[0507] Table 2 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion antigen binding molecule (Construct 1.1).
TABLE-US-00005 TABLE 2 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.1 SEQ ID NO:
Description Sequence 66 Dimeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG
1BBL (71-254)- ATGATCCTGCTGGACTGCTGGACCT CH1 Fc knob
GCGGCAGGGCATGTTTGCTCAGCTG chain GTGGCCCAGAACGTGCTGCTGATCG
ATGGCCCCCTGTCCTGGTACAGCGA TCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGG ACACCAAAGAACTGGTGGTGGCCAA
GGCCGGCGTGTACTACGTGTTCTTT CAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTC TCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTC TGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCAGGCTGC
TGCACCTGTCTGCCGGCCAGAGGCT GGGAGTGCATCTGCACACAGAGGCC
AGGGCTAGACACGCCTGGCAGCTGA CACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCA GCCGGCCTGCCTTCTCCAAGAAGCG
AAGGCGGAGGCGGATCTGGCGGCGG AGGATCTAGAGAGGGACCCGAACTG
TCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGC
CCAGCTGGTGGCTCAGAATGTGCTG CTGATTGACGGACCTCTGAGCTGGT
ACTCCGACCCAGGGCTGGCAGGGGT GTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACAGCTGG AACTGAGGCGGGTGGTGGCTGGGGA
GGGCTCAGGATCTGTGTCCCTGGCT CTGCATCTGCAGCCACTGCGCTCTG
CTGCTGGCGCAGCTGCACTGGCTCT GACTGTGGACCTGCCACCAGCCTCT
AGCGAGGCCAGAAACAGCGCCTTCG GGTTCCAAGGACGCCTGCTGCATCT
GAGCGCCGGACAGCGCCTGGGAGTG CATCTGCATACTGAAGCCAGAGCCC
GGCATGCTTGGCAGCTGACTCAGGG GGCAACTGTGCTGGGACTGTTTCGC
GTGACACCTGAGATCCCTGCCGGAC TGCCAAGCCCTAGATCAGAAGGGGG
CGGAGGAAGCGGAGGGGGAGGAAGT GCTAGCACCAAGGGCCCTAGCGTGT
TCCCTCTGGCCCCTAGCAGCAAGAG CACAAGTGGAGGAACAGCCGCCCTG
GGCTGCCTGGTCAAGGACTACTTCC CCGAGCCCGTGACCGTGTCCTGGAA
TTCTGGCGCCCTGACAAGCGGCGTG CACACATTTCCAGCCGTGCTGCAGA
GCAGCGGCCTGTACTCTCTGAGCAG CGTCGTGACCGTGCCCTCTAGCTCT
CTGGGCACCCAGACCTACATCTGCA ACGTGAACCACAAGCCCAGCAACAC
CAAAGTGGACAAGAAGGTGGAACCC AAGAGCTGCGACAAGACCCACACCT
GTCCCCCTTGCCCTGCCCCTGAAGC TGCTGGTGGCCCTTCCGTGTTCCTG
TTCCCCCCAAAGCCCAAGGACACCC TGATGATCAGCCGGACCCCCGAAGT
GACCTGCGTGGTGGTCGATGTGTCC CACGAGGACCCTGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAAGT GCACAATGCCAAGACCAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACC GTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCGGCGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAG
CCCCGAGAACCACAGGTGTACACCC TGCCCCCATGCCGGGATGAGCTGAC
CAAGAACCAGGTCAGCCTGTGGTGC CTGGTCAAAGGCTTCTATCCCAGCG
ACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAG
ACCACGCCTCCCGTGCTGGACTCCG ACGGCTCCTTCTTCCTCTACAGCAA
GCTCACCGTGGACAAGAGCAGGTGG CAGCAGGGGAACGTCTTCTCATGCT
CCGTGATGCATGAGGCTCTGCACAA CCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA
67 Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG 1BBL (71-254)-
ATGATCCTGCTGGACTGCTGGACCT CL GCGGCAGGGCATGTTTGCTCAGCTG
GTGGCCCAGAACGTGCTGCTGATCG ATGGCCCCCTGTCCTGGTACAGCGA
TCCTGGACTGGCTGGCGTGTCACTG ACAGGCGGCCTGAGCTACAAAGAGG
ACACCAAAGAACTGGTGGTGGCCAA GGCCGGCGTGTACTACGTGTTCTTT
CAGCTGGAACTGCGGAGAGTGGTGG CCGGCGAAGGATCTGGCTCTGTGTC
TCTGGCCCTGCATCTGCAGCCTCTG AGAAGCGCTGCTGGCGCTGCAGCTC
TGGCACTGACAGTGGATCTGCCTCC TGCCAGCTCCGAGGCCCGGAATAGC
GCATTTGGGTTTCAAGGCAGGCTGC TGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCC AGGGCTAGACACGCCTGGCAGCTGA
CACAGGGCGCTACAGTGCTGGGCCT GTTCAGAGTGACCCCCGAGATTCCA
GCCGGCCTGCCTTCTCCAAGAAGCG AAGGCGGAGGCGGATCTGGCGGCGG
AGGATCTCGTACGGTGGCTGCACCA TCTGTCTTCATCTTCCCGCCATCTG
ATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCA
ATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCA
CCTACAGCCTCAGCAGCACCCTGAC GCTGAGCAAAGCAGACTACGAGAAA
CACAAAGTCTACGCCTGCGAAGTCA CCCATCAGGGCCTGAGCTCGCCCGT
CACAAAGAGCTTCAACAGGGGAGAG TGT 68 anti-FAP Fc hole
GAAGTGCAGCTGCTGGAATCCGGCG chain GAGGCCTGGTGCAGCCTGGCGGATC
TCTGAGACTGTCCTGCGCCGCCTCC GGCTTCACCTTCTCCTCCCACGCCA
TGTCCTGGGTCCGACAGGCTCCTGG CAAAGGCCTGGAATGGGTGTCCGCC
ATCTGGGCCTCCGGCGAGCAGTACT ACGCCGACTCTGTGAAGGGCCGGTT
CACCATCTCCCGGGACAACTCCAAG AACACCCTGTACCTGCAGATGAACT
CCCTGCGGGCCGAGGACACCGCCGT GTACTACTGTGCCAAGGGCTGGCTG
GGCAACTTCGACTACTGGGGACAGG GCACCCTGGTCACCGTGTCCAGCGC
TAGCACCAAGGGCCCCTCCGTGTTC CCCCTGGCCCCCAGCAGCAAGAGCA
CCAGCGGCGGCACAGCCGCTCTGGG CTGCCTGGTCAAGGACTACTTCCCC
GAGCCCGTGACCGTGTCCTGGAACA GCGGAGCCCTGACCTCCGGCGTGCA
CACCTTCCCCGCCGTGCTGCAGAGT TCTGGCCTGTATAGCCTGAGCAGCG
TGGTCACCGTGCCTTCTAGCAGCCT GGGCACCCAGACCTACATCTGCAAC
GTGAACCACAAGCCCAGCAACACCA AGGTGGACAAGAAGGTGGAGCCCAA
GAGCTGCGACAAAACTCACACATGC CCACCGTGCCCAGCACCTGAAGCTG
CAGGGGGACCGTCAGTCTTCCTCTT CCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCA CATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGA GGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAA GCCCTCGGCGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTGCACCCTG
CCCCCATCCCGGGATGAGCTGACCA AGAACCAGGTCAGCCTCTCGTGCGC
AGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCGTGAGCAAGC TCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCC GTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 69 anti-FAP light
GAGATCGTGCTGACCCAGTCCCCCG chain GCACCCTGTCTCTGAGCCCTGGCGA
GAGAGCCACCCTGTCCTGCAGAGCC TCCCAGTCCGTGTCCCGGTCCTACC
TCGCCTGGTATCAGCAGAAGCCCGG CCAGGCCCCTCGGCTGCTGATCATC
GGCGCCTCTACCAGAGCCACCGGCA TCCCTGACCGGTTCTCCGGCTCTGG
CTCCGGCACCGACTTCACCCTGACC ATCTCCCGGCTGGAACCCGAGGACT
TCGCCGTGTACTACTGCCAGCAGGG CCAGGTCATCCCTCCCACCTTTGGC
CAGGGCACCAAGGTGGAAATCAAGC GTACGGTGGCTGCACCATCTGTCTT
CATCTTCCCGCCATCTGATGAGCAG TTGAAATCTGGAACTGCCTCTGTTG
TGTGCCTGCTGAATAACTTCTATCC CAGAGAGGCCAAAGTACAGTGGAAG
GTGGATAACGCCCTCCAATCGGGTA ACTCCCAGGAGAGTGTCACAGAGCA
GGACAGCAAGGACAGCACCTACAGC CTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGA GCTTCAACAGGGGAGAGTGT 14 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)- VAQNVLLIDGPLSWYSDPGLAGVSL
CH1 Fc knob TGGLSYKEDTKELVVAKAGVYYVFF chain
QLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIP
AGLPSPRSEGGGGSGGGGSREGPEL SPDDPAGLLDLRQGMFAQLVAQNVL
LIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPC
RDELTKNQVSLWCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 15 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)- VAQNVLLIDGPLSWYSDPGLAGVSL
CL1 TGGLSYKEDTKELVVAKAGVYYVFF QLELRRVVAGEGSGSVSLALHLQPL
RSAAGAAALALTVDLPPASSEARNS AFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTOGATVLGLFRVTPEIP AGLPSPRSEGGGGSGGGGSRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGE C 18
anti-FAP(28Hl) EVQLLESGGGLVQPGGSLRLSCAAS Fc hole chain
GFTFSSHAMSWVRQAPGKGLEWVSA IWASGEQYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKGWL GNFDYWGQGTLVTVSSASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTC
PPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNK
ALGAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK 19 anti-FAP (28H1) EIVLTQSPGTLSLSPGERATLSCRA
light chain SQSVSRSYLAWYQQKPGQAPRLLII GASTRATGIPDRFSGSGSGTDFTLT
ISRLEPEDFAVYYCQQGQVIPPTFG QGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC
[0508] Table 3 shows the cDNA and amino acid sequences of acid
sequences of the monovalent FAP-targeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecule (FIG.
2B, Construct 1.2) with CH1-CL crossover and charged residues.
TABLE-US-00006 TABLE 3 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.2 SEQ ID NO:
Description Sequence 129 Dimeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG
1BBL (71-254)- ATGATCCTGCTGGACTGCTGGACCT CL* Fc knob
GCGGCAGGGCATGTTTGCTCAGCTG chain GTGGCCCAGAACGTGCTGCTGATCG
ATGGCCCCCTGTCCTGGTACAGCGA TCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGG ACACCAAAGAACTGGTGGTGGCCAA
GGCCGGCGTGTACTACGTGTTCTTT CAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTC TCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTC TGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCAGGCTGC
TGCACCTGTCTGCCGGCCAGAGGCT GGGAGTGCATCTGCACACAGAGGCC
AGGGCTAGACACGCCTGGCAGCTGA CACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCA GCCGGCCTGCCTTCTCCAAGAAGCG
AAGGCGGAGGCGGATCTGGCGGCGG AGGATCTAGAGAGGGACCCGAACTG
TCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGC
CCAGCTGGTGGCTCAGAATGTGCTG CTGATTGACGGACCTCTGAGCTGGT
ACTCCGACCCAGGGCTGGCAGGGGT GTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACTGGTGG TGGCTAAAGCTGGGGTGTACTATGT
GTTTTTTCAGCTGGAACTGAGGCGG GTGGTGGCTGGGGAGGGCTCAGGAT
CTGTGTCCCTGGCTCTGCATCTGCA GCCACTGCGCTCTGCTGCTGGCGCA
GCTGCACTGGCTCTGACTGTGGACC TGCCACCAGCCTCTAGCGAGGCCAG
AAACAGCGCCTTCGGGTTCCAAGGA CGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATAC TGAAGCCAGAGCCCGGCATGCTTGG
CAGCTGACTCAGGGGGCAACTGTGC TGGGACTGTTTCGCGTGACACCTGA
GATCCCTGCCGGACTGCCAAGCCCT AGATCAGAAGGGGGCGGAGGTTCCG
GAGGGGGAGGATCTCGTACGGTGGC TGCACCATCTGTCTTTATCTTCCCA
CCCAGCGACCGGAAGCTGAAGTCTG GCACAGCCAGCGTCGTGTGCCTGCT
GAATAACTTCTACCCCCGCGAGGCC AAGGTGCAGTGGAAGGTGGACAATG
CCCTGCAGAGCGGCAACAGCCAGGA AAGCGTGACCGAGCAGGACAGCAAG
GACTCCACCTACAGCCTGAGCAGCA CCCTGACCCTGAGCAAGGCCGACTA
CGAGAAGCACAAGGTGTACGCCTGC GAAGTGACCCACCAGGGCCTGTCTA
GCCCCGTGACCAAGAGCTTCAACCG GGGCGAGTGCGACAAGACCCACACC
TGTCCTCCATGCCCTGCCCCTGAAG CTGCTGGCGGCCCTAGCGTGTTCCT
GTTCCCCCCAAAGCCCAAGGACACC CTGATGATCAGCCGGACCCCTGAAG
TGACCTGCGTGGTGGTGGATGTGTC CCACGAGGACCCTGAAGTGAAGTTC
AATTGGTACGTGGACGGCGTGGAAG TGCACAATGCCAAGACCAAGCCGCG
GGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCC
TGCACCAGGACTGGCTGAATGGCAA GGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCGGCGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACC CTGCCCCCATGCCGGGATGAGCTGA
CCAAGAACCAGGTCAGCCTGTGGTG CCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAA
GACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCA
AGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGC
TCCGTGATGCATGAGGCTCTGCACA ACCACTACACGCAGAAGAGCCTCTC
CCTGTCTCCGGGTAAA 130 Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG 1BBL
(71-254) ATGATCCTGCTGGACTGCTGGACCT CH1* GCGGCAGGGCATGTTTGCTCAGCTG
GTGGCCCAGAACGTGCTGCTGATCG ATGGCCCCCTGTCCTGGTACAGCGA
TCCTGGACTGGCTGGCGTGTCACTG ACAGGCGGCCTGAGCTACAAAGAGG
ACACCAAAGAACTGGTGGTGGCCAA GGCCGGCGTGTACTACGTGTTCTTT
CAGCTGGAACTGCGGAGAGTGGTGG CCGGCGAAGGATCTGGCTCTGTGTC
TCTGGCCCTGCATCTGCAGCCTCTG AGAAGCGCT GCTGGCGCTGCAGCTCTGGCTCTGA
CAGTGGATCTGCCTCCTGCCAGCTC CGAGGCCCGGAATAGCGCATTTGGG
TTTCAAGGCCGGCTGCTGCACCTGT CTGCCGGCCAGAGACTGGGAGTGCA
TCTGCACACAGAGGCCAGAGCCAGG CACGCCTGGCAGCTGACACAGGGCG
CTACAGTGCTGGGCCTGTTCAGAGT GACCCCCGAGATTCCTGCCGGCCTG
CCTAGCCCTAGATCTGAAGGCGGCG GAGGTTCCGGAGGCGGAGGATCTGC
TAGCACAAAGGGCCCCAGCGTGTTC CCTCTGGCCCCTAGCAGCAAGAGCA
CATCTGGCGGAACAGCCGCCCTGGG CTGCCTGGTGGAAGATTACTTCCCC
GAGCCCGTGACCGTGTCCTGGAATT CTGGCGCCCTGACAAGCGGCGTGCA
CACCTTTCCAGCCGTGCTGCAGAGC AGCGGCCTGTACTCTCTGAGCAGCG
TCGTGACAGTGCCCAGCAGCTCTCT GGGCACCCAGACCTACATCTGCAAC
GTGAACCACAAGCCCAGCAACACCA AGGTGGACGAGAAGGTGGAACCCAA GTCCTGC 68
anti-FAP Fc hole See Table 2 chain 69 anti-FAP light See Table 2
chain 115 Dimeric hu 4- REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)-
VAQNVLLIDGPLSWYSDPGLAGVSL CL* Fc knob TGGLSYKEDTKELVVAKAGVYYVFF
chain QLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIP
AGLPSPRSEGGGGSGGGGSREGPEL SPDDPAGLLDLRQGMFAQLVAQNVL
LIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSRTVAAPSVFIFP PSDRKLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGECDKTHT CPPCPAPEAAGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYT
LPPCRDELTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 116 Monomeric hu
4- REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)-
VAQNVLLIDGPLSWYSDPGLAGVSL CH1* TGGLSYKEDTKELVVAKAGVYYVFF
QLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIP
AGLPSPRSEGGGGSGGGGSASTKGP SVFPLAPSSKSTSGGTAALGCLVED
YFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDEKVEPKSC 18 anti-FAP(28H1) See Table 2 Fc hole chain
19 anti-FAP (28H1) See Table 2 light chain
[0509] Table 4 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.3 (FIG. 2C) (FAP split trimer with
CH1-CL crossover in anti-FAP Fab and charged residues on the 4-1BBL
containing chains).
TABLE-US-00007 TABLE 4 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc(kih) fusion molecule Construct 1.3 SEQ ID NO:
Description Sequence 131 Dimeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG
1BBL (71-254)- ATGATCCTGCTGGACTGCTGGACCT CH1* Fc knob
GCGGCAGGGCATGTTTGCTCAGCTG chain GTGGCCCAGAACGTGCTGCTGATCG
ATGGCCCCCTGTCCTGGTACAGCGA TCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGG ACACCAAAGAACTGGTGGTGGCCAA
GGCCGGCGTGTACTACGTGTTCTTT CAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTC TCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTC TGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCAGGCTGC
TGCACCTGTCTGCCGGCCAGAGGCT GGGAGTGCATCTGCACACAGAGGCC
AGGGCTAGACACGCCTGGCAGCTGA CACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCA GCCGGCCTGCCTTCTCCAAGAAGCG
AAGGCGGAGGCGGATCTGGCGGCGG AGGATCTAGAGAGGGACCCGAACTG
TCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGC
CCAGCTGGTGGCTCAGAATGTGCTG CTGATTGACGGACCTCTGAGCTGGT
ACTCCGACCCAGGGCTGGCAGGGGT GTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACTGGTGG TGGCTAAAGCTGGGGTGTACTATGT
GTTTTTTCAGCTGGAACTGAGGCGG GTGGTGGCTGGGGAGGGCTCAGGAT
CTGTGTCCCTGGCTCTGCATCTGCA GCCACTGCGCTCTGCTGCTGGCGCA
GCTGCACTGGCTCTGACTGTGGACC TGCCACCAGCCTCTAGCGAGGCCAG
AAACAGCGCCTTCGGGTTCCAAGGA CGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATAC TGAAGCCAGAGCCCGGCATGCTTGG
CAGCTGACTCAGGGGGCAACTGTGC TGGGACTGTTTCGCGTGACACCTGA
GATCCCTGCCGGACTGCCAAGCCCT AGATCAGAAGGGGGCGGAGGAAGCG
GAGGGGGAGGAAGTGCTAGCACCAA GGGCCCCTCCGTGTTCCCCCTGGCC
CCCAGCAGCAAGAGCACCAGCGGCG GCACAGCCGCTCTGGGCTGCCTGGT
CGAGGACTACTTCCCCGAGCCCGTG ACCGTGTCCTGGAACAGCGGAGCCC
TGACCTCCGGCGTGCACACCTTCCC CGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCG TGCCTTCTAGCAGCCTGGGCACCCA
GACCTACATCTGCAACGTGAACCAC AAGCCCAGCAACACCAAGGTGGACG
AGAAGGTGGAGCCCAAGAGCTGCGA CAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAAGCTGCAGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCA AGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGC GTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCGGC
GCCCCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATGC CGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGTGGTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAA CGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGG TAAA 132
Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG 1BBL (71-254)-
ATGATCCTGCTGGACTGCTGGACCT CL* GCGGCAGGGCATGTTTGCTCAGCTG
GTGGCCCAGAACGTGCTGCTGATCG ATGGCCCCCTGTCCTGGTACAGCGA
TCCTGGACTGGCTGGCGTGTCACTG ACAGGCGGCCTGAGCTACAAAGAGG
ACACCAAAGAACTGGTGGTGGCCAA GGCCGGCGTGTACTACGTGTTCTTT
CAGCTGGAACTGCGGAGAGTGGTGG CCGGCGAAGGATCTGGCTCTGTGTC
TCTGGCCCTGCATCTGCAGCCTCTG AGAAGCGCTGCTGGCGCTGCAGCTC
TGGCACTGACAGTGGATCTGCCTCC TGCCAGCTCCGAGGCCCGGAATAGC
GCATTTGGGTTTCAAGGCAGGCTGC TGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCC AGGGCTAGACACGCCTGGCAGCTGA
CACAGGGCGCTACAGTGCTGGGCCT GTTCAGAGTGACCCCCGAGATTCCA
GCCGGCCTGCCTTCTCCAAGAAGCG AAGGCGGAGGCGGATCTGGCGGCGG
AGGATCTCGTACGGTGGCTGCACCA TCTGTCTTCATCTTCCCGCCATCTG
ATCGGAAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTAC AGTGGAAGGTGGATAACGCCCTCCA
ATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCA
CCTACAGCCTCAGCAGCACCCTGAC GCTGAGCAAAGCAGACTACGAGAAA
CACAAAGTCTACGCCTGCGAAGTCA CCCATCAGGGCCTGAGCTCGCCCGT
CACAAAGAGCTTCAACAGGGGAGAG TGT 133 anti-FAP (VHCL)
GAAGTGCAGCTGCTGGAATCCGGCG (28H1) Fc hole GAGGCCTGGTGCAGCCTGGCGGATC
chain TCTGAGACTGTCCTGCGCCGCCTCC GGCTTCACCTTCTCCTCCCACGCCA
TGTCCTGGGTCCGACAGGCTCCTGG CAAAGGCCTGGAATGGGTGTCCGCC
ATCTGGGCCTCCGGCGAGCAGTACT ACGCCGACTCTGTGAAGGGCCGGTT
CACCATCTCCCGGGACAACTCCAAG AACACCCTGTACCTGCAGATGAACT
CCCTGCGGGCCGAGGACACCGCCGT GTACTACTGTGCCAAGGGCTGGCTG
GGCAACTTCGACTACTGGGGACAGG GCACCCTGGTCACCGTGTCCAGCGC
TAGCGTGGCCGCTCCCAGCGTGTTC ATCTTCCCACCCAGCGACGAGCAGC
TGAAGTCCGGCACAGCCAGCGTGGT GTGCCTGCTGAACAACTTCTACCCC
CGCGAGGCCAAGGTGCAGTGGAAGG TGGACAACGCCCTGCAGAGCGGCAA
CAGCCAGGAATCCGTGACCGAGCAG GACAGCAAGGACTCCACCTACAGCC
TGAGCAGCACCCTGACCCTGAGCAA GGCCGACTACGAGAAGCACAAGGTG
TACGCCTGCGAAGTGACCCACCAGG GCCTGTCCAGCCCCGTGACCAAGAG
CTTCAACCGGGGCGAGTGCGACAAG ACCCACACCTGTCCCCCTTGCCCTG
CCCCTGAAGCTGCTGGTGGCCCTTC CGTGTTCCTGTTCCCCCCAAAGCCC
AAGGACACCCTGATGATCAGCCGGA CCCCCGAAGTGACCTGCGTGGTGGT
CGATGTGTCCCACGAGGACCCTGAA GTGAAGTTCAATTGGTACGTGGACG
GCGTGGAAGTGCACAATGCCAAGAC CAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCGGCGCCC
CCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAG
GTGTGCACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAG
CCTCTCGTGCGCAGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAA CAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCC TCGTGAGCAAGCTCACCGTGGACAA
GAGCAGGTGGCAGCAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCTCCGGGTAAA 134 anti-FAP
GAGATCGTGCTGACCCAGTCTCCCG (VLCH1) (28H1) GCACCCTGAGCCTGAGCCCTGGCGA
light chain GAGAGCCACCCTGAGCTGCAGAGCC AGCCAGAGCGTGAGCCGGAGCTACC
TGGCCTGGTATCAGCAGAAGCCCGG CCAGGCCCCCAGACTGCTGATCATC
GGCGCCAGCACCCGGGCCACCGGCA TCCCCGATAGATTCAGCGGCAGCGG
CTCCGGCACCGACTTCACCCTGACC ATCAGCCGGCTGGAACCCGAGGACT
TCGCCGTGTACTACTGCCAGCAGGG CCAGGTGATCCCCCCCACCTTCGGC
CAGGGCACCAAGGTGGAAATCAAGA GCAGCGCTTCCACCAAAGGCCCTTC
CGTGTTTCCTCTGGCTCCTAGCTCC AAGTCCACCTCTGGAGGCACCGCTG
CTCTCGGATGCCTCGTGAAGGATTA TTTTCCTGAGCCTGTGACAGTGTCC
TGGAATAGCGGAGCACTGACCTCTG GAGTGCATACTTTCCCCGCTGTGCT
GCAGTCCTCTGGACTGTACAGCCTG AGCAGCGTGGTGACAGTGCCCAGCA
GCAGCCTGGGCACCCAGACCTACAT CTGCAACGTGAACCACAAGCCCAGC
AACACCAAGGTGGACAAGAAGGTGG AACCCAAGTCTTGT 108 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)- VAQNVLLIDGPLSWYSDPGLAGVSL
CHI* Fc knob TGGLSYKEDTKELVVAKAGVYYVFF chain
QLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIP
AGLPSPRSEGGGGSGGGGSREGPEL SPDDPAGLLDLRQGMFAQLVAQNVL
LIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSASTKGPSVFPLA PSSKSTSGGTAALGCLVEDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDEKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPC
RDELTKNQVSLWCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 109 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)- VAQNVLLIDGPLSWYSDPGLAGVSL
CL* TGGLSYKEDTKELVVAKAGVYYVFF QLELRRVVAGEGSGSVSLALHLQPL
RSAAGAAALALTVDLPPASSEARNS AFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSEGGGGSGGGGSRTVAAP
SVFIFPPSDRKLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGE C 135 anti-FAP
(VHCL) EVQLLESGGGLVQPGGSLRLSCAAS (28H1) Fc hole
GFTFSSHAMSWVRQAPGKGLEWVSA chain IWASGEQYYADSVKGRFTISRDNSK
NTLYLQMNSLRAEDTAVYYCAKGWL GNFDYWGQGTLVTVSSASVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGECDK
THTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCK
VSNKALGAPIEKTISKAKGQPREPQ VCTLPPSRDELTKNQVSLSCAVKGF
YPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLVSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTOKSLSLSPGK 136 anti-FAP EIVLTQSPGTLSLSPGERATLSCRA
(VLCH1) (28H1) SQSVSRSYLAWYQQKPGQAPRLLII light chain
GASTRATGIPDRFSGSGSGTDFTLT ISRLEPEDFAVYYCQQGQVIPPTFG
QGTKVEIKSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSC
[0510] Table 5 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.4 (FIG. 2D) (FAP split trimer with
anti-FAP Fab, monomeric 4-1BB ligand fused to CH1-knob chain and
charged residues on the 4-1BBL containing chains).
TABLE-US-00008 TABLE 5 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.4 SEQ ID NO:
Description Sequence 137 Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG
1BBL (71-254)- ATGATCCTGCTGGACTGCTGGACCT CH1* Fc knob
GCGGCAGGGCATGTTTGCTCAGCTG chain GTGGCCCAGAACGTGCTGCTGATCG
ATGGCCCCCTGTCCTGGTACAGCGA TCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGG ACACCAAAGAACTGGTGGTGGCCAA
GGCCGGCGTGTACTACGTGTTCTTT CAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTC TCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTC TGGCTCTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCCGGCTGC
TGCACCTGTCTGCCGGCCAGAGACT GGGAGTGCATCTGCACACAGAGGCC
AGAGCCAGGCACGCCTGGCAGCTGA CACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCT GCCGGCCTGCCTAGCCCTAGATCTG
AAGGCGGCGGAGGTTCCGGAGGCGG AGGATCTGCTAGCACCAAGGGCCCC
TCCGTGTTCCCCCTGGCCCCCAGCA GCAAGAGCACCAGCGGCGGCACAGC
CGCTCTGGGCTGCCTGGTCGAGGAC TACTTCCCCGAGCCCGTGACCGTGT
CCTGGAACAGCGGAGCCCTGACCTC CGGCGTGCACACCTTCCCCGCCGTG
CTGCAGAGTTCTGGCCTGTATAGCC TGAGCAGCGTGGTCACCGTGCCTTC
TAGCAGCCTGGGCACCCAGACCTAC ATCTGCAACGTGAACCACAAGCCCA
GCAACACCAAGGTGGACGAGAAGGT GGAGCCCAAGAGCTGCGACAAAACT
CACACATGCCCACCGTGCCCAGCAC CTGAAGCTGCAGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGA CGTGAGCCACGAAGACCCTGAGGTC
AAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGCCCTCGGCGCCCCCA TCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATGCCGGGATG
AGCTGACCAAGAACCAGGTCAGCCT GTGGTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACGCAGAAGAG
CCTCTCCCTGTCTCCGGGTAAA 138 Dimeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCG
1BBL (71-254)- ATGATCCTGCTGGACTGCTGGACCT CL*
GCGGCAGGGCATGTTTGCTCAGCTG GTGGCCCAGAACGTGCTGCTGATCG
ATGGCCCCCTGTCCTGGTACAGCGA TCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGG ACACCAAAGAACTGGTGGTGGCCAA
GGCCGGCGTGTACTACGTGTTCTTT CAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTC TCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTC TGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGC GCATTTGGGTTTCAAGGCAGGCTGC
TGCACCTGTCTGCCGGCCAGAGGCT GGGAGTGCATCTGCACACAGAGGCC
AGGGCTAGACACGCCTGGCAGCTGA CACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCA GCCGGCCTGCCTTCTCCAAGAAGCG
AAGGCGGAGGCGGATCTGGCGGCGG AGGATCTAGAGAGGGACCCGAACTG
TCCCCTGACGATCCAGCCGGGCTGC TGGATCTGAGACAGGGAATGTTCGC
CCAGCTGGTGGCTCAGAATGTGCTG CTGATTGACGGACCTCTGAGCTGGT
ACTCCGACCCAGGGCTGGCAGGGGT GTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACTGGTGG TGGCTAAAGCTGGGGTGTACTATGT
GTTTTTTCAGCTGGAACTGAGGCGG GTGGTGGCTGGGGAGGGCTCAGGAT
CTGTGTCCCTGGCTCTGCATCTGCA GCCACTGCGCTCTGCTGCTGGCGCA
GCTGCACTGGCTCTGACTGTGGACC TGCCACCAGCCTCTAGCGAGGCCAG
AAACAGCGCCTTCGGGTTCCAAGGA CGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATAC TGAAGCCAGAGCCCGGCATGCTTGG
CAGCTGACTCAGGGGGCAACTGTGC TGGGACTGTTTCGCGTGACACCTGA
GATCCCTGCCGGACTGCCAAGCCCT AGATCAGAAGGGGGCGGAGGTTCCG
GAGGGGGAGGATCTCGTACGGTGGC TGCACCATCTGTCTTCATCTTCCCG
CCATCTGATCGGAAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCT
GAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGA GAGTGTCACAGAGCAGGACAGCAAG
GACAGCACCTACAGCCTCAGCAGCA CCCTGACGCTGAGCAAAGCAGACTA
CGAGAAACACAAAGTCTACGCCTGC GAAGTCACCCATCAGGGCCTGAGCT
CGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGT 68 anti-FAP (28H1) see Table 2
Fc hole chain 69 anti-FAP (28H1) See Table 2 light chain 139
Monomeric hu 4- REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)-
VAQNVLLIDGPLSWYSDPGLAGVSL CL* Fc knob TGGLSYKEDTKELVVAKAGVYYVFF
chain QLELRRVVAGEGSGSVSLALHLQPL RSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIP
AGLPSPRSEGGGGSGGGGSREGPEL SPDDPAGLLDLRQGMFAQLVAQNVL
LIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRR
VVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSASTKGPSVFPLA PSSKSTSGGTAALGCLVEDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDEKVEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALG APIEKTISKAKGQPREPQVYTLPPC
RDELTKNQVSLWCLVKGFYPSDLAV EWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 140 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQL 1BBL (71-254)- VAQNVLLIDGPLSWYSDPGLAGVSL
CL* TGGLSYKEDTKELVVAKAGVYYVFF QLELRRVVAGEGSGSVSLALHLQPL
RSAAGAAALALTVDLPPASSEARNS AFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSEGGGGSGGGGSREGPEL
SPDDPAGLLDLRQGMFAQLVAQNVL LIDGPLSWYSDPGLAGVSLTGGLSY
KEDTKELWAKAGVYYVFFQLELRRW AGEGSGSVSLALHLQPLRSAAGAAA
LALTVDLPPASSEARNSAFGFQGRL LHLSAGQRLGVHLHTEARARHAWQL
TQGATVLGLFRVTPEIPAGLPSPRS EGGGGSGGGGSRTVAAPSVFIFPPS
DRKLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 18 anti-FAP (28H1) see
Table 2 Fc hole chain 19 anti-FAP (28H1) see Table 2 light
chain
[0511] Table 6 shows the cDNA and amino acid sequences of the
bivalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.5 (FIG. 2E) (FAP split trimer with 2
anti-FAP Fabs, dimeric and monomeric 4-1BB ligand fused at the
C-terminus of each heavy chain, respectively).
TABLE-US-00009 TABLE 6 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.5 SEQ ID NO:
Description Sequence 141 anti-FAP (28H1)
GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA Fc hole chain
GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG fused to dimeric
CTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA hu 4-1BBL
GGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTG (71-254)
GGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGG
GCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCC
TGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC
GCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTC
GACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAG
CGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCC
CAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCG
TGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCAC
ACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGC
CTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGC
ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG
CAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT
GCGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCA
AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAA
GAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC
ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCG
GAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTG
AGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAG
GGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTG
ATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTG
GCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGA
GGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGT
ACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGG
CCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATC
TGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGG
CACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCC
GGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACC
TGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACA
GAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGG
CGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT
TCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAG
GCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAA
CTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGA
CAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTG
CTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGG
CTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAA
GAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGT
GTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGT
GGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCA
TCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACT
GGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGC
CAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGC
ATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCAT
ACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCA
GGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGA
GATCCCTGCCGGACTGCCAAGCCCTAGATCAGAA 142 anti-FAP (28H1)
GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA Fc knob chain
GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG fused to
CTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA monomeric hu
GGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTG 4-1BBL (71-254)
GGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGG
GCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCC
TGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC
GCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTC
GACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAG
CGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC
CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC
CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC
ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCA
AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCA
AGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCT
ACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGC
CAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCT
GGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGAC
CGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCA
GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTAC
ACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAG
CTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGG
CAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTG
CTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGA
CTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAA
AGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCG
TGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGG
TGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGC
ATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTC
TGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGG
CCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGC
ACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCAC
ACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACA
GGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCG
AGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAA 69 anti-FAP (28H1) see Table 2
light chain 121 anti-FAP (28H1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA Fc hole chain
PGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYL fused to dimeric
QMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSAS hu 4-1BBL (71-254)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEG
GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNV
LLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV
YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 122 anti-FAP (28H1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA Fc knob chain
PGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYL fused to
QMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSAS monomeric hu
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS 4-1BBL (71-254)
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN
QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 19 anti-FAP (28H1) see
Table 2 light chain
[0512] Table 7 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.6 (FIG. 2F) (FAP split trimer with
anti-FAP Fab, monomeric 4-1BB ligand fused to CL* via a
(G.sub.4S)-linker).
TABLE-US-00010 TABLE 7 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.6 SEQ ID NO:
Description Sequence 131 Dimeric hu 4- see Table 4 1BBL (71-254)-
CH1* Fc knob chain 143 Monomeric hu 4-
AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-254)-
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT (G.sub.45).sub.1- CL*
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCT
GCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCT
CCAAGAAGCGAAGGCGGAGGCGGATCTCGTACGGTGGC
TGCACCATCTGTCTTCATCTTCCCGCCATCTGATCGGAA
GTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG
TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGT
GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT
CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGA
AACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC
CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGT 68 anti-FAP (28H1) see
Table 2 Fc hole chain 69 anti-FAP (28H1) see Table 2 light chain
108 Dimeric hu 4- see Table 4 1BBL (71-254)- CH1* Fc knob chain 110
Monomeric hu 4- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL
(71-254)- SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR
(G.sub.4S).sub.1- CL* RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLPSPRSEGGGGSRTVAAPSVFIFP
PSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 18
anti-FAP (28H1) see Table 2 Fc hole chain 19 anti-FAP (28H1) see
Table 2 light chain
[0513] Table 8 shows the cDNA and amino acid sequences of the
bivalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.7 (FIG. 2G) (FAP split trimer with
double anti-FAP on the N-terminus of Fc hole chain and charged
residues on crossed CH1 and CL fused to 4-1BB ligands).
TABLE-US-00011 TABLE 8 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.7 SEQ ID NO:
Description Sequence 129 Dimeric hu 4- see Table 3 1BBL (71-254)-
CL* Fc knob chain 130 Monomeric hu 4- see Table 3 1BBL (71-254)-
CH1* 144 [anti-FAP GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA
(28H1)].sub.2 Fc hole GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG chain
CTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA
GGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTG
GGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGG
GCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCC
TGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC
GCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTC
GACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAG
CGCTAGCACAAAGGGACCTAGCGTGTTCCCCCTGGCCCC
CAGCAGCAAGTCTACATCTGGCGGAACAGCCGCCCTGG
GCTGCCTCGTGAAGGACTACTTTCCCGAGCCCGTGACCG
TGTCCTGGAACTCTGGCGCTCTGACAAGCGGCGTGCACA
CCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTC
TGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGC
ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAG
CAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCT
GCGACGGCGGAGGGGGATCTGGCGGCGGAGGATCCGAA
GTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCAGCC
TGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGGCTT
CACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACAGGC
TCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTGGGC
CTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGGGCC
GGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGT
ACCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCC
GTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTCGAC
TACTGGGGACAGGGCACCCTGGTCACCGTGTCCAGCGCT
AGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGC
AGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTG
CCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTC
CTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTT
CCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAG
CAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCA
GACCTACATCTGCAACGTGAACCACAAGCCCAGCAACA
CCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACA
GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT
GCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCC
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT
CCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC
TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA
69 anti-FAP (28H1) see Table 2 light chain 115 Dimeric hu 4- see
Table 3 1BBL (71-254)- CL* Fc knob chain 116 Monomeric hu 4- see
Table 3 1BBL (71-254)- CH1* 145 [anti-FAP
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA (28H1)].sub.2 Fc hole
PGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYL chain
QMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGG
GLVQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWV
SAIWASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKGWLGNFDYWGQGTLVTVSSASTKGPSVFPL
APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
GAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 19 anti-FAP (28H1) see Table
2 light chain
[0514] Table 9 shows the cDNA and amino acid sequences of the
bivalent FAP-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 1.8 (FIG. 2H) (FAP split trimer with
4-1BB ligands fused to anti-FAP CrossFab, with charged residues, on
knob chain).
TABLE-US-00012 TABLE 9 Sequences of FAP-targeted human 4-1BB ligand
trimer containing Fc (kih) fusion molecule Construct 1.8 SEQ ID NO:
Description Sequence 146 Dimeric hu 4-
AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-254)-
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT FAP (VHCL*) Fc
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG knob chain
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCT
GCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCT
CCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAG
GATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCA
GCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCA
GCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCT
GAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCC
TGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAA
CTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTT
CAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTC
AGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCG
CTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGA
CCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCT
TCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCC
CGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTG
GGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTG
CCAAGCCCTAGATCAGAAGGGGGCGGAGGTTCCGGAGG
CGGAGGATCTGAGGTGCAGCTGCTGGAATCCGGCGGAG
GCCTGGTGCAGCCTGGCGGATCTCTGAGACTGTCCTGCG
CCGCCTCCGGCTTCACCTTCTCCTCCCACGCCATGTCCTG
GGTCCGACAGGCTCCTGGCAAAGGCCTGGAATGGGTGT
CCGCCATCTGGGCCTCCGGCGAGCAGTACTACGCCGACT
CTGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCA
AGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCC
GAGGACACCGCCGTGTACTACTGTGCCAAGGGCTGGCT
GGGCAACTTCGACTACTGGGGCCAGGGCACCCTGGTCA
CCGTGTCCAGCGCTAGCGTGGCTGCACCATCTGTCTTTA
TCTTCCCACCCAGCGACCGGAAGCTGAAGTCTGGCACA
GCCAGCGTCGTGTGCCTGCTGAATAACTTCTACCCCCGC
GAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCA
GAGCGGCAACAGCCAGGAAAGCGTGACCGAGCAGGAC
AGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGAC
CCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACG
CCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGA
CCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCAC
ACCTGTCCTCCATGCCCTGCCCCTGAAGCTGCTGGCGGC
CCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACC
CTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGGTG
GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAAT
TGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGAC
CAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG
TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC
CATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTG
TGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT
GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA 147
Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL
(71-254)- ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT FAP (VLCH1*)
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCT
GCTGGCGCTGCAGCTCTGGCTCTGACAGTGGATCTGCCT
CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCCGGCTGCTGCACCTGTCTGCCGGCCAGAGACT
GGGAGTGCATCTGCACACAGAGGCCAGAGCCAGGCACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCTGCCGGCCTGCCTAGC
CCTAGATCTGAAGGCGGCGGAGGTTCCGGAGGCGGAGG
ATCTGAGATCGTGCTGACCCAGTCTCCCGGCACCCTGAG
CCTGAGCCCTGGCGAGAGAGCCACCCTGAGCTGCAGAG
CCAGCCAGAGCGTGAGCCGGAGCTACCTGGCCTGGTAT
CAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCAT
CGGCGCCAGCACCCGGGCCACCGGCATCCCCGATAGAT
TCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCA
TCAGCCGGCTGGAACCCGAGGACTTCGCCGTGTACTACT
GCCAGCAGGGCCAGGTGATCCCCCCCACCTTCGGCCAG
GGCACCAAGGTGGAAATCAAGTCCTCTGCTAGCACAAA
GGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAG
CACATCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGG
AAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATT
CTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCG
TGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCG
TGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACA
TCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTG GACGAGAAGGTGGAACCCAAGTCCTGC
68 anti-FAP (28H1) see Table 2 Fc hole chain 69 anti-FAP (28H1) see
Table 2 light chain 148 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-254)-
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR FAP (VHCL*) Fc
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS knob chain
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPEL
SPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA
GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV
LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSEVQLLESGGGL
VQPGGSLRLSCAASGFTFSSHAMSWVRQAPGKGLEWVSAI
WASGEQYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT
AVYYCAKGWLGNFDYWGQGTLVTVSSASVAAPSVFIFPPS
DRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
ALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 149 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-254)-
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR FAP (VLCH1*)
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSEIVLTQS
PGTLSLSPGERATLSCRASQSVSRSYLAWYQQKPGQAPRL
LIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ
QGQVIPPTFGQGTKVEIKSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSC 18 anti-FAP (28H1) see
Table 2 Fc hole chain 19 anti-FAP (28H1) see Table 2 light
chain
[0515] Table 10 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand (52-254) trimer-containing Fc
(kih) fusion molecule Construct 1.9 (FIG. 2I) (FAP split trimer
with 4-1BBL ectodomain amino acids 52-254 and charged residues on
ligand chains).
TABLE-US-00013 TABLE 10 Sequences of FAP-targeted human 4-1BB
ligand trimer containing Fc (kih) fusion molecule Construct 1.9 SEQ
ID NO: Description Sequence 150 Dimeric hu 4-
CCTTGGGCTGTGTCTGGCGCTAGAGCCTCTCCTGGATCT 1BBL (52-254)-
GCCGCCAGCCCCAGACTGAGAGAGGGACCTGAGCTGAG CH1* Fc knob
CCCCGATGATCCTGCCGGACTGCTGGATCTGAGACAGG chain
GCATGTTCGCCCAGCTGGTGGCCCAGAACGTGCTGCTGA
TCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGG
CTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG
GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTA
CTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGC
CGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCT
GCAGCCCCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGG
CACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCC
GGAATAGCGCATTTGGGTTTCAAGGCAGACTGCTGCACC
TGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACA
GAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGG
CGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT
TCCAGCCGGACTGCCCAGCCCTAGATCTGAAGGCGGCG
GAGGAAGCGGAGGCGGAGGATCCCCTTGGGCTGTGTCT
GGCGCTAGAGCCTCTCCTGGATCTGCCGCCAGCCCCAGA
CTGAGAGAGGGACCTGAGCTGAGCCCCGATGATCCTGC
CGGACTGCTGGACCTGCGGCAGGGAATGTTCGCTCAGCT
GGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGTC
CTGGTACTCCGACCCTGGCCTGGCAGGGGTGTCCCTGAC
TGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGG
TGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGC
TGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGA
TCTGTGTCCCTGGCTCTGCATCTGCAGCCTCTGCGCTCTG
CTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGC
CACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGG
TTCCAAGGACGGCTGCTGCATCTGAGCGCCGGACAGCG
CCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGC
ATGCTTGGCAGCTGACCCAGGGGGCAACTGTGCTGGGA
CTGTTTCGCGTGACACCTGAGATCCCCGCTGGCCTGCCT
AGCCCAAGAAGTGAAGGGGGAGGCGGATCTGGCGGAG
GGGGATCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCC
TGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCC
GCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCC
GTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG
CGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCT
GTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAG
CCTGGGCACCCAGACCTACATCTGCAACGTGAACCACA
AGCCCAGCAACACCAAGGTGGACGAGAAGGTGGAGCCC
AAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCC
AGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTT
CCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCAC
CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCT
GACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC
AATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 151 Monomeric hu 4-
CCTTGGGCTGTGTCTGGCGCTAGAGCCTCTCCTGGATCT 1BBL (52-254)-
GCCGCCAGCCCCAGACTGAGAGAGGGACCTGAGCTGAG CL*
CCCCGATGATCCTGCCGGACTGCTGGATCTGAGACAGG
GCATGTTCGCCCAGCTGGTGGCCCAGAACGTGCTGCTGA
TCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGG
CTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG
GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTA
CTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGC
CGGCGAGGGATCTGGATCTGTGTCTCTGGCCCTGCATCT
GCAGCCCCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGG
CACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCC
GGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACC
TGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACA
GAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGG
CGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGAT
TCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAG
GCGGATCTGGCGGCGGAGGATCTCGTACGGTGGCTGCA
CCATCTGTCTTCATCTTCCCGCCATCTGATCGGAAGTTGA
AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT
TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT
AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCAC
AGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCA
GCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG
CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 68 anti-FAP (28H1) see Table
2 Fc hole chain 69 anti-FAP (28H1) see Table 2 light chain 111
Dimeric hu 4- PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGM 1BBL
(52-254)- FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK CH1* Fc knob
ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLR chain
SAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQR
LGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSPWAVSGARASPGSAASPRLREGPELSP
DDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG
VSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE
GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAF
GFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLG
LFRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPSVFPLAPS
SKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
EKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP
IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 112 Monomeric hu 4-
PWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGM 1BBL (52-254)-
FAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTK CL*
ELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLR
SAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQR
LGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSP
RSEGGGGSGGGGSRTVAAPSVFIFPPSDRKLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 18 anti-FAP (28H1) see
Table 2 Fc hole chain 19 anti-FAP (28H1) see Table 2 light
chain
[0516] Table 11 shows the cDNA and amino acid sequences of the
monovalent FAP-targeted 4-1BB ligand (80-254) trimer-containing Fc
(kih) fusion molecule Construct 1.10 (FIG. 2J) (FAP split trimer
with 4-1BBL ectodomain amino acids 80-254 and charged residues on
ligand chains).
TABLE-US-00014 TABLE 11 Sequences of FAP-targeted human 4-1BB
ligand trimer containing Fc (kih) fusion molecule Construct 10 SEQ
ID NO: Description Sequence 152 Dimeric hu 4-
GATCCTGCCGGCCTGCTGGATCTGCGGCAGGGAATGTTT 1BBL (80-254)-
GCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGG CH1* Fc knob
CCCCCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGT chain
GTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCA
AAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTG
TTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAA
GGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCC
CTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGAC
AGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAG
CGCATTTGGGTTTCAAGGCAGACTGCTGCACCTGTCTGC
CGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCA
GGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACA
GTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCC
GGACTGCCCAGCCCTAGATCTGAAGGCGGCGGAGGAAG
CGGAGGCGGAGGATCCGACCCAGCTGGACTGCTGGACC
TGCGGCAGGGAATGTTCGCTCAGCTGGTGGCTCAGAATG
TGCTGCTGATTGACGGACCTCTGTCCTGGTACTCCGACC
CTGGCCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCT
ACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCT
GGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGG
GTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCT
CTGCATCTGCAGCCTCTGCGCTCTGCTGCTGGCGCAGCT
GCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGC
GAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGGCT
GCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATC
TGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTG
ACCCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACA
CCTGAGATCCCCGCTGGCCTGCCTAGCCCAAGAAGTGA
AGGGGGAGGCGGATCTGGCGGAGGGGGATCTGCTAGCA
CCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCA
AGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTG
GTCGAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGG
AACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCC
GCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGC
GTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACC
TACATCTGCAACGTGAACCACAAGCCCAGCAACACCAA
GGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG
ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG
TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCC
TGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGA
GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCTCCGGGTAAA 153
Monomeric hu 4- GATCCTGCCGGCCTGCTGGATCTGCGGCAGGGAATGTTT 1BBL
(80-254)- GCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGG CL*
CCCCCTGAGCTGGTACAGCGATCCTGGACTGGCTGGCGT
GTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCA
AAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTG
TTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAA
GGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCC
CTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGAC
AGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAG
CGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGC
CGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCA
GGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACA
GTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCC
GGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATC
TGGCGGCGGAGGATCTCGTACGGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATCGGAAGTTGAAATCTGG
AACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCC
AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGG
ACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG
ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTA
CGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT
CACAAAGAGCTTCAACAGGGGAGAGTGT 68 anti-FAP (28H1) see Table 2 Fc hole
chain 69 anti-FAP (28H1) see Table 2 light chain 113 Dimeric hu 4-
DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGV 1BBL (80-254)-
SLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGS CH1* Fc knob
GSVSLALHLQPLRSAAGAAALALTVDLPPAS SEARNSAFGF chain
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF
RVTPEIPAGLPSPRSEGGGGSGGGGSDPAGLLDLRQGMFA
QLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL
VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSA
AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLG
VHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE
GGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVE
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV
PSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPC
PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY
TLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 114
Monomeric hu 4- DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGV 1BBL
(80-254)- SLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGS CL*
GSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF
RVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPSVFIFPPSDR
KLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC 18 anti-FAP
(28H1) see Table 2 Fc hole chain 19 anti-FAP (28H1) see Table 2
light chain
[0517] 1.2 Production of FAP (28H1) Targeted Split Trimeric 4-1BB
Ligand Fc Fusion Constructs
[0518] The targeted TNF ligand trimer-containing Fc (kih) fusion
antigen binding molecule encoding sequences were cloned into a
plasmid vector, which drives expression of the insert from an MPSV
promoter and contains a synthetic polyA sequence located at the 3'
end of the CDS. In addition, the vector contains an EBV OriP
sequence for episomal maintenance of the plasmid.
[0519] The targeted TNF ligand trimer-containing Fc (kih) fusion
antigen binding molecule was produced by co-transfecting
HEK293-EBNA cells with the mammalian expression vectors using
polyethylenimine. The cells were transfected with the corresponding
expression vectors at a 1:1:1:1 ratio (e.g. "vector dimeric
ligand-(CH1 or CL)-knob chain": "vector monomeric ligand fusion-(CL
or CH1)": "vector anti-FAP Fab-hole heavy chain": "vector anti-FAP
light chain") for the Constructs 1, 2, 3, 4, 6, 7, 8, 9, 10. For
the bivalent Construct 5, a 1:1:1 ratio ("vector hole heavy chain":
"vector knob heavy chain": "vector anti-FAP light chain") was
used.
[0520] For production in 500 mL shake flasks, 300 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 10 minutes at 210.times.g,
and the supernatant was replaced by 20 mL pre-warmed CD CHO medium.
Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CD
CHO medium. After addition of 540 .mu.L PEI, the solution was
vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere. After the incubation, 160 mL of Excell medium
supplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM
valproic acid was added and cells were cultured for 24 hours. One
day after transfection 12% Feed 7 and Glucose (final concentration
3 g/L) were added. After culturing for 7 days, the supernatant was
collected by centrifugation for 30-40 minutes at 400.times.g. The
solution was sterile filtered (0.22 .mu.m filter), supplemented
with sodium azide to a final concentration of 0.01% (w/v), and kept
at 4.degree. C.
[0521] The targeted TNF ligand trimer-containing Fc (kih) fusion
antigen binding molecule was purified from cell culture
supernatants by affinity chromatography using Protein A, followed
by size exclusion chromatography. For affinity chromatography, the
supernatant was loaded on a MABSELECT SURE.RTM. column (CV=5-15 mL,
resin from GE Healthcare) equilibrated with 20 mM sodium phosphate,
20 mM sodium citrate buffer (pH 7.5). Unbound protein was removed
by washing with at least 6 column volumes of the same buffer. The
bound protein was eluted using either a linear gradient (20 CV) or
a step elution (8 CV) with 20 mM sodium citrate, 100 mM Sodium
chloride, 100 mM Glycine buffer (pH 3.0). For the linear gradient
an additional 4 column volumes step elution was applied.
[0522] The pH of collected fractions was adjusted by adding 1/10
(v/v) of 0.5M sodium phosphate, pH 8.0. The protein was
concentrated prior to loading on a HILOAD.RTM. Superdex 200 column
(GE Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium
chloride, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20) solution of pH
6.0.
[0523] The protein concentration was determined by measuring the
optical density (OD) at 280 nm, using a molar extinction
coefficient calculated on the basis of the amino acid sequence.
Purity and molecular weight of the targeted TNF ligand
trimer-containing Fc (kih) fusion antigen binding molecule was
analyzed by SDS-PAGE in the presence and absence of a reducing
agent (5 mM 1,4-dithiotreitol) and staining with Coomassie
SIMPLYBLUE.TM. SafeStain (Invitrogen USA) or CE-SDS using Caliper
LabChip GXII (Perkin Elmer). The aggregate content of samples was
analyzed using a TSKGEL.RTM. G3000 SW XL analytical size-exclusion
column (Tosoh) equilibrated in 25 mM K.sub.2HPO.sub.4, 125 mM NaCl,
200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN.sub.3, pH 6.7
running buffer at 25.degree. C.
[0524] Table 12 summarizes the yield and final monomer content of
the FAP-targeted 4-1BBL trimer-containing Fc (kih) fusion antigen
binding molecules.
TABLE-US-00015 TABLE 12 Biochemical Analysis of the FAP
(28H1)-targeted 4-1BBL trimer-containing Fc (kih) fusion antigen
binding molecules Monomer Yield [%] Construct [mg/l] (SEC)
Construct 1.1 12.7 95 Construct 1.2 25.2 97 Construct 1.3 22 92
Construct 1.4 14.2 99 Construct 1.5 14 99 Construct 1.6 12 98
Construct 1.7 3.4 99 Construct 1.8 5.4 98 Construct 1.9 11.2 98
Construct 1.10 19.8 99
[0525] 1.3 Preparation of Targeted Murine 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0526] Similarly to targeted human 4-1BB ligand trimer-containing
Fc fusion antigen binding molecules, murine FAP-targeted 4-1BBL
trimer-containing Fc fusion antigen binding molecules were
prepared.
[0527] The DNA sequence encoding part of the ectodomain (amino
acids 104-309) of murine 4-1BB ligand was synthetized according to
the Q3U1Z9-1 sequence of Uniprot database (SEQ ID NO:70). For
Construct M.1 the cysteines at positions 137, 160 and 246 were
mutated to Serine by standard PCR methods, whereas for Construct
M.2 the cysteine at position 160 was mutated to Serine (C160S).
[0528] The murine ligand was assembled as described for the human
4-1BBL and as depicted in FIGS. 3A and 3B. The dimeric 4-1BBL,
separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, was fused to
the murine IgG1-CL domain (FIG. 3A) and the monomeric 4-1BBL was
fused to murine IgG1-CH domain (FIG. 3B). The polypeptide encoding
the dimeric 4-1BB ligand fused to murine CL domain was subcloned in
frame with the murine IgG1 heavy chain CH2 and CH3 domains to build
the Constructs as depicted in FIG. 3C.
[0529] For the murine constructs, mutations Lys392Asp and Lys409Asp
(DD) were introduced in the heavy chain containing the murine
4-1BBL and mutations Glu356Lys and Asp399Lys (KK) were introduced
in the heavy chain containing the anti-FAP Fab to obtain asymmetric
molecules (Gunasekaran K. et al, J Biol. Chem., 2010, Jun. 18;
285(25):19637-46).
[0530] Mutations Asp265Ala and Pro329Gly (DAPG) were introduced in
the constant region of the heavy chains to abrogate binding to Fc
gamma receptors.
[0531] Table 13 shows, respectively, the cDNA and amino acid
sequences of the FAP-targeted murine 4-1BB ligand trimer-containing
Fc fusion antigen binding molecule Construct M.1.
TABLE-US-00016 TABLE 13 Sequences of FAP-targeted murine Construct
M.1 SEQ ID NO: Description Sequence 71 Dimeric murine
AGAACCGAGCCCAGACCCGCCCTGACCATCACCACCAG 4-1BBL (104-309,
CCCTAACCTGGGCACCAGAGAGAACAACGCCGACCAAG C137, 160, 246S)-
TGACCCCCGTGTCCCACATCGGCAGCCCCAATACCACAC CL Fc DD chain
AGCAGGGCAGCCCTGTGTTCGCCAAGCTGCTGGCCAAG
AACCAGGCCAGCCTGAGCAACACCACCCTGAACTGGCA
CAGCCAGGATGGCGCCGGAAGCAGCTATCTGAGCCAGG
GCCTGAGATACGAAGAGGACAAGAAAGAACTGGTGGTG
GACAGCCCTGGCCTGTACTACGTGTTCCTGGAACTGAAG
CTGAGCCCCACCTTCACCAACACCGGCCACAAGGTGCA
GGGCTGGGTGTCACTGGTGCTGCAGGCCAAACCCCAGG
TGGACGACTTCGACAACCTGGCCCTGACCGTGGAACTGT
TCCCCAGCAGCATGGAAAACAAGCTGGTGGATCGGAGC
TGGTCCCAGCTTCTGCTGCTGAAGGCCGGACACAGACTG
AGCGTGGGCCTGAGGGCTTATCTGCACGGCGCCCAGGA
CGCCTACAGAGACTGGGAGCTGAGCTACCCCAACACAA
CCAGCTTCGGCCTGTTCCTCGTGAAGCCCGACAACCCTT
GGGAAGGCGGCGGAGGATCTGGCGGAGGCGGATCTAGA
ACAGAGCCTCGGCCTGCCCTGACAATTACCACATCCCCC
AATCTGGGCACCCGGGAAAACAATGCAGATCAAGTGAC
ACCTGTGTCTCATATTGGCTCCCCAAACACTACCCAGCA
GGGCTCCCCCGTGTTTGCTAAACTGCTGGCTAAAAATCA
GGCCTCCCTGTCTAACACAACACTGAACTGGCACTCCCA
GGACGGCGCTGGCAGCTCTTACCTGAGTCAGGGACTGC
GCTATGAGGAAGATAAGAAAGAACTGGTGGTGGATTCC
CCCGGACTGTACTATGTGTTTCTGGAACTGAAACTGTCC
CCTACCTTTACAAATACCGGGCACAAAGTGCAGGGATG
GGTGTCCCTGGTGCTGCAGGCTAAGCCTCAGGTGGACGA
TTTTGATAATCTGGCTCTGACAGTGGAACTGTTTCCTAG
CAGCATGGAAAACAAGCTGGTGGACAGAAGCTGGTCCC
AGCTCCTGCTGCTGAAGGCCGGACACAGACTGAGCGTG
GGCCTGAGAGCCTATCTGCACGGCGCCCAGGACGCCTA
CAGAGACTGGGAGCTGAGCTACCCCAACACAACCAGCT
TCGGCCTGTTCCTCGTGAAGCCCGACAACCCTTGGGAAG
GCGGCGGAGGATCTGGCGGAGGCGGATCCAGAGCTGAT
GCTGCCCCTACCGTGTCCATCTTCCCACCCAGCAGCGAG
CAGCTGACATCTGGGGGAGCTAGCGTCGTGTGCTTCCTG
AACAACTTCTACCCCAAGGACATCAACGTGAAGTGGAA
GATCGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATA
GCTGGACCGACCAGGACAGCAAGGACTCCACCTACAGC
ATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGA
GCGGCACAACAGCTACACATGCGAGGCCACCCACAAGA
CCAGCACCAGCCCCATCGTGAAGTCCTTCAACCGGAAC
GAGTGCGTGCCCAGAGACTGCGGCTGCAAGCCTTGCAT
CTGCACCGTGCCTGAGGTGTCCAGCGTGTTCATCTTCCC
ACCCAAGCCCAAGGACGTGCTGACCATCACCCTGACAC
CCAAAGTGACCTGCGTGGTGGTGGCCATCAGCAAGGAT
GACCCCGAGGTGCAGTTCAGTTGGTTCGTGGACGACGTG
GAAGTGCACACCGCTCAGACCAAGCCCAGAGAGGAACA
GATCAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCAT
CATGCACCAGGACTGGCTGAACGGCAAAGAATTCAAGT
GCAGAGTGAACAGCGCCGCCTTTGGCGCCCCTATCGAG
AAAACCATCTCCAAGACCAAGGGCAGACCCAAGGCCCC
CCAGGTGTACACAATCCCCCCACCCAAAGAACAGATGG
CCAAGGACAAGGTGTCCCTGACCTGCATGATCACCAATT
TCTTCCCAGAGGATATCACCGTGGAATGGCAGTGGAAC
GGCCAGCCCGCCGAGAACTACGACAACACCCAGCCTAT
CATGGACACCGACGGCTCCTACTTCGTGTACAGCGACCT
GAACGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCT
TCACCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACC
ACACCGAGAAGTCCCTGTCCCACAGCCCTGGCAAG 72 Monomeric
AGAACCGAGCCCAGACCCGCCCTGACCATCACCACCAG murine 4-1BBL
CCCTAACCTGGGCACCAGAGAGAACAACGCCGACCAAG (104-309,
TGACCCCCGTGTCCCACATCGGCAGCCCCAATACCACAC C137, 160, 246S)-
AGCAGGGCAGCCCTGTGTTCGCCAAGCTGCTGGCCAAG CL
AACCAGGCCAGCCTGAGCAACACCACCCTGAACTGGCA
CAGCCAGGATGGCGCCGGAAGCAGCTATCTGAGCCAGG
GCCTGAGATACGAAGAGGACAAGAAAGAACTGGTGGTG
GACAGCCCTGGCCTGTACTACGTGTTCCTGGAACTGAAG
CTGAGCCCCACCTTCACCAACACCGGCCACAAGGTGCA
GGGCTGGGTGTCACTGGTGCTGCAGGCCAAACCCCAGG
TGGACGACTTCGACAACCTGGCCCTGACCGTGGAACTGT
TCCCCAGCAGCATGGAAAACAAGCTGGTGGATCGGAGC
TGGTCCCAGCTTCTGCTGCTGAAGGCCGGACACAGACTG
AGCGTGGGCCTGAGGGCCTATCTGCATGGCGCCCAGGA
CGCCTACAGAGACTGGGAGCTGAGCTACCCCAACACAA
CCAGCTTCGGCCTGTTCCTCGTGAAGCCCGACAACCCTT
GGGAAGGCGGCGGAGGCTCCGGAGGAGGCGGAAGCGC
TAAGACCACCCCCCCCAGCGTGTACCCTCTGGCCCCTGG
ATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCT
GCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGA
CCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACC
TTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGC
AGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCA
GACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCA
CCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC 73 anti-FAP (28H1)
GAAGTGCAGCTGCTGGAATCCGGCGGAGGCCTGGTGCA Fc KK heavy
GCCTGGCGGATCTCTGAGACTGTCCTGCGCCGCCTCCGG chain
CTTCACCTTCTCCTCCCACGCCATGTCCTGGGTCCGACA
GGCTCCTGGCAAAGGCCTGGAATGGGTGTCCGCCATCTG
GGCCTCCGGCGAGCAGTACTACGCCGACTCTGTGAAGG
GCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCC
TGTACCTGCAGATGAACTCCCTGCGGGCCGAGGACACC
GCCGTGTACTACTGTGCCAAGGGCTGGCTGGGCAACTTC
GACTACTGGGGACAGGGCACCCTGGTCACCGTGTCCAG
CGCTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCC
TGGATCTGCCGCCCAGACCAACAGCATGGTGACCCTGG
GCTGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCG
TGACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCAC
ACCTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTG
AGCAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAG
CCAGACAGTGACCTGCAACGTGGCCCACCCTGCCAGCA
GCACCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC
GGCTGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCC
AGCGTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTG
ACCATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTG
GCCATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTG
GTTTGTGGACGACGTGGAGGTGCACACAGCCCAGACAA
AGCCCCGGGAGGAACAGATCAACAGCACCTTCAGAAGC
GTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAAC
GGCAAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTT
CGGCGCCCCCATCGAGAAAACCATCAGCAAGACCAAGG
GCAGACCCAAGGCCCCCCAGGTGTACACCATCCCCCCA
CCCAAAAAACAGATGGCCAAGGACAAGGTGTCCCTGAC
CTGCATGATCACCAACTTTTTCCCCGAGGACATCACCGT
GGAGTGGCAGTGGAATGGCCAGCCCGCCGAGAACTACA
AGAACACCCAGCCCATCATGAAGACCGACGGCAGCTAC
TTCGTGTACAGCAAGCTGAACGTGCAGAAGTCCAACTG
GGAGGCCGGCAACACCTTCACCTGTAGCGTGCTGCACG
AGGGCCTGCACAACCACCACACCGAGAAGTCCCTGAGC CACTCCCCCGGCAAG 74 anti-FAP
(28H1) GAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCTCTG light chain
AGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTC
CCAGTCCGTGTCCCGGTCCTACCTCGCCTGGTATCAGCA
GAAGCCCGGCCAGGCCCCTCGGCTGCTGATCATCGGCG
CCTCTACCAGAGCCACCGGCATCCCTGACCGGTTCTCCG
GCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCC
GGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGC
AGGGCCAGGTCATCCCTCCCACCTTTGGCCAGGGCACCA
AGGTGGAAATCAAGCGTGCCGATGCTGCACCAACTGTA
TCGATTTTCCCACCATCCAGTGAGCAGTTAACATCTGGA
GGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCC
AAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGA
ACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGG
ACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTC
ACGTTGACCAAGGACGAGTATGAACGACATAACAGCTA
TACCTGTGAGGCCACTCACAAGACATCAACTTCACCCAT
TGTCAAGAGCTTCAACAGGAATGAGTGT 75 Dimeric murine
RTEPRPALTITTSPNLGTRENNADQVTPVSHIGSPNTTQQGS 4-1BBL (104-309,
PVFAKLLAKNQASLSNTTLNWHSQDGAGSSYLSQGLRYEE C137, 160, 246S)
DKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVL CL Fc DD chain
QAKPQVDDFDNLALTVELFPSSMENKLVDRSWSQLLLLKA
GHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKP
DNPWEGGGGSGGGGSRTEPRPALTITTSPNLGTRENNADQ
VTPVSHIGSPNTTQQGSPVFAKLLAKNQASLSNTTLNWHS
QDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSP
TFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPSS
MENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYR
DWELSYPNTTSFGLFLVKPDNPWEGGGGSGGGGSRADAA
PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE
RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT
CEATHKTSTSPIVKSFNRNECVPRDCGCKPCICTVPEVSSVF
IFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDV
EVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCR
VNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV
SLTCMITNFFPEDITVEWQWNGQPAENYDNTQPIMDTDGS
YFVYSDLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLS HSPGK 76 Monomeric
RTEPRPALTITTSPNLGTRENNADQVTPVSHIGSPNTTQQGS murine 4-1BBL
PVFAKLLAKNQASLSNTTLNWHSQDGAGSSYLSQGLRYEE (104-309,
DKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVL C137, 160, 246S)-
QAKPQVDDFDNLALTVELFPSSMENKLVDRSWSQLLLLKA CL
GHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKP
DNPWEGGGGSGGGGSAKTTPPSVYPLAPGSAAQTNSMVT
LGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTL
SSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC 77 anti-FAP (28H1)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSHAMSWVRQA Fc KK chain
PGKGLEWVSAIWASGEQYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKGWLGNFDYWGQGTLVTVSSAK
TTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWN
SGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCN
VAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPK
DVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQ
TKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFG
APIEKTISKTKGRPKAPQVYTIPPPKKQMAKDKVSLTCMIT
NFFPEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKL
NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 78 anti-FAP (28H1)
EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKP light chain
GQAPRLLIIGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQGQVIPPTFGQGTKVEIKRADAAPTVSIFPPSSEQ
LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT
DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC
[0532] Table 14 shows, respectively, the cDNA and amino acid
sequences of the untargeted (DP47) murine 4-1 BB ligand
trimer-containing Fc fusion antigen binding molecule Control
M.1.
TABLE-US-00017 TABLE 14 Sequences of untargeted murine Control M.1
SEQ ID NO: Description Sequence 71 Dimeric murine See Table 13
4-1BBL (104-309, C137, 160, 246S)- CL Fc DD chain 72 Monomeric See
Table 13 murine 4-1BBL (104-309, C137, 160, 246S)- CH1 154 DP47 Fc
KK GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA chain
GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGG
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA
GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGA
AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGA
CTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGCG
CTAAGACCACCCCCCCTAGCGTGTACCCTCTGGCCCCTG
GATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGC
TGCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTG
ACCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACAC
CTTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAG
CAGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCA
GACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCA
CCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGCGGC
TGCAAGCCCTGCATCTGCACCGTGCCCGAGGTGTCCAGC
GTGTTCATCTTCCCACCCAAGCCCAAGGACGTGCTGACC
ATCACCCTGACCCCCAAAGTGACCTGCGTGGTGGTGGCC
ATCAGCAAGGACGACCCCGAGGTGCAGTTCTCTTGGTTT
GTGGACGACGTGGAGGTGCACACAGCCCAGACAAAGCC
CCGGGAGGAACAGATCAACAGCACCTTCAGAAGCGTGT
CCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGC
AAAGAATTCAAGTGCAGAGTGAACAGCGCCGCCTTCGG
CGCCCCCATCGAGAAAACCATCAGCAAGACCAAGGGCA
GACCCAAGGCCCCCCAGGTGTACACCATCCCCCCACCCA
AAAAACAGATGGCCAAGGACAAGGTGTCCCTGACCTGC
ATGATCACCAACTTTTTCCCCGAGGACATCACCGTGGAG
TGGCAGTGGAATGGCCAGCCCGCCGAGAACTACAAGAA
CACCCAGCCCATCATGAAGACCGACGGCAGCTACTTCGT
GTACAGCAAGCTGAACGTGCAGAAGTCCAACTGGGAGG
CCGGCAACACCTTCACCTGTAGCGTGCTGCACGAGGGCC
TGCACAACCACCACACCGAGAAGTCCCTGAGCCACTCC CCCGGCAAG 155 DP47 light
chain GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTG
TCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGT
CAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAG
AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGG
CAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAG
ACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCA
GTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCA
AAGTGGAAATCAAACGTGCCGATGCTGCACCAACTGTA
TCGATTTTCCCACCATCCAGTGAGCAGTTAACATCTGGA
GGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCC
AAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGA
ACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGG
ACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTC
ACGTTGACCAAGGACGAGTATGAACGACATAACAGCTA
TACCTGTGAGGCCACTCACAAGACATCAACTTCACCCAT
TGTCAAGAGCTTCAACAGGAATGAGTGT 75 Dimeric murine see Table 13 4-1BBL
(104-309, C137, 160, 246S)- CL Fc DD chain 76 Monomeric See Table
13 murine 4-1BBL (104-309, C137, 160, 246S)- CH1 156 DP47 Fc KK
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA chain
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSAKTTP
PSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGS
LSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAH
PASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL
TITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKP
REEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPI
EKTISKTKGRPKAPQVYTIPPPKKQMAKDKVSLTCMITNFF
PEDITVEWQWNGQPAENYKNTQPIMKTDGSYFVYSKLNV
QKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 157 DP47 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYGSSPLTFGQGTKVEIKRADAAPTVSIFPPSSEQ
LTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT
DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC
[0533] Table 15 shows the cDNA and amino acid sequences of the
FAP-targeted murine 4-1BB ligand trimer-containing Fc fusion
antigen binding molecule Construct M.2.
TABLE-US-00018 TABLE 15 Sequences of FAP-targeted murine Construct
M.2 SEQ ID NO: Description Sequence 158 Dimeric murine
AGAACCGAGCCCAGACCCGCCCTGACCATCACCACCAG 4-1BBL (104-
CCCTAACCTGGGCACCAGAGAGAACAACGCCGACCAAG 309, C160S) - CL
TGACCCCCGTGTCCCACATCGGCTGCCCCAATACCACAC Fc DD chain
AGCAGGGCAGCCCTGTGTTCGCCAAGCTGCTGGCCAAG
AACCAGGCCAGCCTGAGCAACACCACCCTGAACTGGCA
CAGCCAGGATGGCGCCGGAAGCAGCTATCTGAGCCAGG
GCCTGAGATACGAAGAGGACAAGAAAGAACTGGTGGTG
GACAGCCCTGGCCTGTACTACGTGTTCCTGGAACTGAAG
CTGAGCCCCACCTTCACCAACACCGGCCACAAGGTGCA
GGGCTGGGTGTCACTGGTGCTGCAGGCCAAACCCCAGG
TGGACGACTTCGACAACCTGGCCCTGACCGTGGAACTGT
TCCCCTGCAGCATGGAAAACAAGCTGGTGGATCGGAGC
TGGTCCCAGCTTCTGCTGCTGAAGGCCGGACACAGACTG
AGCGTGGGCCTGAGGGCTTATCTGCACGGCGCCCAGGA
CGCCTACAGAGACTGGGAGCTGAGCTACCCCAACACAA
CCAGCTTCGGCCTGTTCCTCGTGAAGCCCGACAACCCTT
GGGAAGGCGGCGGAGGCTCCGGAGGAGGCGGATCTAGA
ACAGAGCCTCGGCCTGCCCTGACAATTACCACATCCCCC
AATCTGGGCACCCGGGAAAACAATGCAGATCAAGTGAC
ACCTGTGTCTCATATTGGGTGCCCCAACACTACCCAGCA
GGGGTCCCCAGTGTTTGCTAAACTGCTGGCTAAAAATCA
GGCCTCCCTGTCTAACACAACACTGAATTGGCATAGTCA
GGACGGGGCTGGCAGCAGCTACCTGTCTCAGGGACTGC
GCTATGAGGAAGATAAGAAAGAACTGGTGGTGGATTCC
CCCGGACTGTACTATGTGTTTCTGGAACTGAAACTGTCC
CCTACCTTTACAAATACCGGGCACAAAGTGCAGGGATG
GGTGTCCCTGGTGCTGCAGGCTAAGCCTCAGGTGGACGA
TTTTGATAATCTGGCTCTGACAGTGGAACTGTTTCCTTGC
TCTATGGAAAACAAACTGGTGGACCGCTCTTGGAGCCA
GTTGCTGCTGCTGAAAGCTGGCCACCGGCTGTCTGTGGG
ACTGAGAGCATACCTGCATGGGGCACAGGATGCCTACC
GGGATTGGGAACTGTCCTACCCTAACACTACTTCCTTCG
GACTGTTCCTCGTGAAACCTGATAATCCCTGGGAGGGCG
GAGGCGGAAGTGGCGGAGGGGGATCCAGAGCTGATGCT
GCCCCTACCGTGTCCATCTTCCCACCCAGCAGCGAGCAG
CTGACATCTGGGGGAGCTAGCGTCGTGTGCTTCCTGAAC
AACTTCTACCCCAAGGACATCAACGTGAAGTGGAAGAT
CGACGGCAGCGAGCGGCAGAACGGCGTGCTGAATAGCT
GGACCGACCAGGACAGCAAGGACTCCACCTACAGCATG
AGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGCG
GCACAACAGCTACACATGCGAGGCCACCCACAAGACCA
GCACCAGCCCCATCGTGAAGTCCTTCAACCGGAACGAG
TGCGTGCCCAGAGACTGCGGCTGCAAGCCTTGCATCTGC
ACCGTGCCTGAGGTGTCCAGCGTGTTCATCTTCCCACCC
AAGCCCAAGGACGTGCTGACCATCACCCTGACACCCAA
AGTGACCTGCGTGGTGGTGGCCATCAGCAAGGATGACC
CCGAGGTGCAGTTCAGTTGGTTCGTGGACGACGTGGAA
GTGCACACCGCTCAGACCAAGCCCAGAGAGGAACAGAT
CAACAGCACCTTCAGAAGCGTGTCCGAGCTGCCCATCAT
GCACCAGGACTGGCTGAACGGCAAAGAATTCAAGTGCA
GAGTGAACAGCGCCGCCTTTGGCGCCCCTATCGAGAAA
ACCATCTCCAAGACCAAGGGCAGACCCAAGGCCCCCCA
GGTGTACACAATCCCCCCACCCAAAGAACAGATGGCCA
AGGACAAGGTGTCCCTGACCTGCATGATCACCAATTTCT
TCCCAGAGGATATCACCGTGGAATGGCAGTGGAACGGC
CAGCCCGCCGAGAACTACGACAACACCCAGCCTATCAT
GGACACCGACGGCTCCTACTTCGTGTACAGCGACCTGAA
CGTGCAGAAGTCCAACTGGGAGGCCGGCAACACCTTCA
CCTGTAGCGTGCTGCACGAGGGCCTGCACAACCACCAC
ACCGAGAAGTCCCTGTCCCACAGCCCTGGCAAG 159 Monomeric
AGAACCGAGCCCAGACCCGCCCTGACCATCACCACCAG murine 4-1BBL
CCCTAACCTGGGCACCAGAGAGAACAACGCCGACCAAG (104-309, C160S) -
TGACCCCCGTGTCCCACATCGGCTGCCCCAATACCACAC CH1
AGCAGGGCAGCCCTGTGTTCGCCAAGCTGCTGGCCAAG
AACCAGGCCAGCCTGAGCAACACCACCCTGAACTGGCA
CAGCCAGGATGGCGCCGGAAGCAGCTATCTGAGCCAGG
GCCTGAGATACGAAGAGGACAAGAAAGAACTGGTGGTG
GACAGCCCTGGCCTGTACTACGTGTTCCTGGAACTGAAG
CTGAGCCCCACCTTCACCAACACCGGCCACAAGGTGCA
GGGCTGGGTGTCACTGGTGCTGCAGGCCAAACCCCAGG
TGGACGACTTCGACAACCTGGCCCTGACCGTGGAACTGT
TCCCCTGCAGCATGGAAAACAAGCTGGTGGATCGGAGC
TGGTCCCAGCTTCTGCTGCTGAAGGCCGGACACAGACTG
AGCGTGGGCCTGAGGGCTTATCTGCACGGCGCCCAGGA
CGCCTACAGAGACTGGGAGCTGAGCTACCCCAACACAA
CCAGCTTCGGCCTGTTCCTCGTGAAGCCCGACAACCCTT
GGGAAGGCGGCGGAGGCTCCGGAGGAGGCGGAAGCGC
TAAGACCACCCCCCCCAGCGTGTACCCTCTGGCCCCTGG
ATCTGCCGCCCAGACCAACAGCATGGTGACCCTGGGCT
GCCTGGTGAAGGGCTACTTCCCCGAGCCTGTGACCGTGA
CCTGGAACAGCGGCAGCCTGAGCAGCGGCGTGCACACC
TTTCCAGCCGTGCTGCAGAGCGACCTGTACACCCTGAGC
AGCTCCGTGACCGTGCCTAGCAGCACCTGGCCCAGCCA
GACAGTGACCTGCAACGTGGCCCACCCTGCCAGCAGCA
CCAAGGTGGACAAGAAAATCGTGCCCCGGGACTGC 73 anti-FAP (28H1) see Table 13
Fc KK chain 74 anti-FAP (28H1) see Table 13 light chain 160 Dimeric
murine RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGS 4-1BBL (104-
PVFAKLLAKNQASLSNTTLNWHSQDGAGSSYLSQGLRYEE 309, C 160 S) - CL
DKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVL Fc DD chain
QAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLK
AGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVK
PDNPWEGGGGSGGGGSRTEPRPALTITTSPNLGTRENNAD
QVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLSNTTLNWH
SQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLS
PTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCS
MENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYR
DWELSYPNTTSFGLFLVKPDNPWEGGGGSGGGGSRADAA
PTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE
RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT
CEATHKTSTSPIVKSFNRNECVPRDCGCKPCICTVPEVSSVF
IFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDV
EVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCR
VNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKV
SLTCMITNFFPEDITVEWQWNGQPAENYDNTQPIMDTDGS
YFVYSDLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLS HSPGK 161 Monomeric
RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGS murine 4-1BBL
PVFAKLLAKNQASLSNTTLNWHSQDGAGSSYLSQGLRYEE (104-309, C160S) -
DKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVL CH1
QAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLK
AGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVK
PDNPWEGGGGSGGGGSAKTTPPSVYPLAPGSAAQTNSMV
TLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYT
LSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC 77 anti-FAP (28H1) see
Table 13 Fc KK chain 78 anti-FAP (28H1) see Table 13 light
chain
[0534] Table 16 shows the cDNA and amino acid sequences of the
DP47-untargeted murine 4-1BB ligand trimer-containing Fc fusion
antigen binding molecule Construct Control M.2.
TABLE-US-00019 TABLE 16 Sequences of FAP-targeted murine Control
M.2 SEQ ID NO: Description Sequence 158 Dimeric mu 4-1BBL see Table
15 (104-309, C160S)- CL Fc DD chain 159 Monomeric mu 4- see Table
15 1BBL (104-309, C160S)-CH1 154 DP47 Fc KK chain see Table 14 155
DP47 light chain see Table 14 160 Dimeric mu 4-1BBL see Table 15
(104-309, C160S)- CL Fc DD chain 161 Monomeric mu 4- see Table 15
1BBL (104-309, C160S)-CH1 156 DP47 Fc KK chain see Table 14 157
DP47 light chain see Table 14
[0535] The murine 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules were produced and purified as described herein
before for the human 4-1BBL constructs.
[0536] Table 17 summarizes the yield and final monomer content of
the FAP-targeted and untargeted murine 4-1BBL trimer-containing Fc
fusion antigen binding molecule.
TABLE-US-00020 TABLE 17 Summary of the production of the
FAP-targeted and untargeted murine 4-1BBL trimer-containing Fc
fusion antigen binding molecules Monomer Yield [%] Construct [mg/l]
(SEC) Construct M.1 2.6 95 Control M.2 2.3 96 Construct M.2 8.5 98
Control M.2 8.1 97
[0537] 1.4 Preparation and Purification of Untargeted Human 4-1BB
Ligand Trimer-Containing Fc Fusion Antigen Binding Molecules
(Control Molecules)
[0538] The control molecules were prepared as described above for
the FAP-targeted Constructs 1 and 2, with the only difference that
the anti-FAP binder (VH-VL) was replaced by a germline control,
termed DP47, not binding to the antigen. The control is an
untargeted monovalent split trimeric human 4-1BB ligand Fc (kih)
(Control A, FIG. 5A) and for Control B, the construct also contains
a CH-CL crossover with charged residues (FIG. 5B). The variable
region of heavy and light chain DNA sequences of the FAP binder
were replaced with those of the germline control (DP47) and
subcloned in frame with either the constant heavy chain of the hole
or the constant light chain of human IgG1.
[0539] The untargeted 4-1BB ligand trimer-containing Fc (kih)
fusion antigen binding molecules were produced as described above
for the FAP-targeted constructs. The cells were transfected with
the corresponding expression vectors at a 1:1:1:1 ratio ("vector
dimeric ligand-CH1 or CL*-knob chain": "vector monomeric ligand
fusion-CL or CH1*": "vector DP47 Fab-hole chain": "vector DP47
light chain").
[0540] Table 18 shows, respectively, the cDNA and amino acid
sequences of the DP47-untargeted 4-1BB ligand trimer-containing Fc
(kih) fusion antigen binding molecule Control A.
TABLE-US-00021 TABLE 18 Sequences of DP47 untargeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecule (DP47
split 4-1BBL trimer) Control A SEQ ID NO: Description Sequence 66
Dimeric hu 4- See Table 2 1BBL (71-254) - CH1 Fc knob chain 67
Monomeric hu see Table 2 4-1BBL (71-254) - CL 79 DP47 Fc hole
GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA chain
GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGG
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA
GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGA
AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGA
CTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGC
TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC
CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA
ACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
AGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA
ACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA 80 DP47 light chain
GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTG
TCTCCAGGGGAAAGAGCCACCCTCTCTTGCAGGGCCAGT
CAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAG
AAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGG
CAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAG
ACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCA
GTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCA
AAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCT
TCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGT
14 Dimeric hu 4- See Table 2 1BBL (71-254) - CH1 Fc knob chain 15
Monomeric hu 4- See Table 2 1BBL (71-254) - CL 81 DP47 Fc hole
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA chain
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVS
LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 82 DP47 light chain
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC
[0541] Table 19 shows the cDNA and amino acid sequences of the
DP47-untargeted 4-1BB ligand trimer-containing Fc (kih) fusion
antigen binding molecule with CH1-CL crossover and charged residues
in the 4-1BB ligand containing arms (Control B).
TABLE-US-00022 TABLE 19 Sequences of DP47 untargeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecule (DP47
split 4-1BBL trimer) Control B SEQ ID NO: Description Sequence 96
Dimeric hu 4-1BBL see Table 3 (71-254)-CL* Fc knob chain 97
Monomeric hu 4-1BBL see Table 3 (71-254)-CH1* 79 DP47 Fc hole chain
see Table 18 80 DP47 light chain see Table 18 98 Dimeric hu 4-1BBL
see Table 3 (71-254)-CL* Fc knob chain 99 Monomeric hu 4-1BBL see
Table 3 (71-254)-CH1* 81 DP47 Fc hole chain see Table 18 82 DP47
light chain see Table 18
[0542] Table 20 summarizes the yield and final monomer content of
the DP47 untargeted 4-1BB ligand trimer-containing Fc (kih) fusion
antigen binding molecules.
TABLE-US-00023 TABLE 20 Production Characteristics of DP47
untargeted 4-1BBL trimer-containing Fc (kih) fusion antigen binding
molecules (Control molecules) Monomer [%] Yield LC/MS Construct
(SEC) [mg/l] (non red) Control A 97 3.7 Theoretical*: 179069.7 Da
Experimental: 179116.2 Da *without terminal lysines Control B 99
15.4
Example 2
[0543] 2.1 Preparation of FAP (4B9) Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0544] Different fragments of the DNA sequence encoding part of the
ectodomain (amino acid 71-254 and 71-248) of human 4-1BB ligand
were synthetized according to the P41273 sequence of Uniprot
database (SEQ ID NO:42).
[0545] 2.1.1 Preparation of Monovalent FAP (4B9) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 2.1)
[0546] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
1A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL.
[0547] A polypeptide containing one ectodomain of 4-1BB ligand
(71-254) and fused to the human IgG1-CH1 domain, was cloned as
described in FIG. 1B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0548] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998)
using a linker (G.sub.4S).sub.2 (SEQ ID NO:13), or alternatively,
GSPGSSSSGS (SEQ ID NO:57).
[0549] To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL the mutations E123R and Q124K were introduced. In the
monomeric 4-1BB ligand fused to human CH1, the mutations K147E and
K213E were cloned into the human CH1 domain.
[0550] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole or the constant light chain of human IgG1.
[0551] The generation and preparation of the FAP binders is
described in WO 2012/020006 A2, which is incorporated herein by
reference.
[0552] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in International Patent Appl. Publ. No. WO
2012/130831.
[0553] For all constructs the knobs into hole heterodimerization
technology was used with the S354C/T366W mutations in the knob
chain and the corresponding Y349C/T366S/L368A/Y407V mutations in
the hole chain.
[0554] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-FAP-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a FAP binding Fab (FIG. 4A, Construct 2.1).
[0555] Table 21 shows the cDNA and amino acid sequences of the
monovalent FAP (4B9)-human 4-1BB ligand (71-254) Fc (kih) fusion
antigen binding molecule containing CH1-CL crossover and charged
residues (Construct 2.1).
TABLE-US-00024 TABLE 21 Sequences of monovalent FAP(4B9)-targeted
human 4-1BB ligand (71-254) containing Fc (kih) fusion molecule
Construct 2.1 SEQ ID NO: Description Sequence 129 Dimeric hu 4- see
Table 3 1BBL (71-254) - CL* Fc knob chain 130 Monomeric hu see
Table 3 4-1BBL (71-254) - CH1* 162 anti-FAP (4B9)
GAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA Fc hole chain
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC
AGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC
ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG
AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAA
CACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGC
GGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGT
GTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCG
CTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCG
TGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCA
AGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAG
CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 163 anti-FAP (4B9)
GAGATCGTGCTGACCCAGTCCCCCGGCACCCTGTCTCTG light chain
AGCCCTGGCGAGAGAGCCACCCTGTCCTGCAGAGCCTC
CCAGTCCGTGACCTCCTCCTACCTCGCCTGGTATCAGCA
GAAGCCCGGCCAGGCCCCTCGGCTGCTGATCAACGTGG
GCAGTCGGAGAGCCACCGGCATCCCTGACCGGTTCTCCG
GCTCTGGCTCCGGCACCGACTTCACCCTGACCATCTCCC
GGCTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGC
AGGGCATCATGCTGCCCCCCACCTTTGGCCAGGGCACCA
AGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCT
TCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC
GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA CAAAGAGCTTCAACAGGGGAGAGTGT
115 Dimeric hu 4- see Table 3 1BBL (71-254) - CL* Fc knob chain 116
Monomeric hu see Table 3 4-1BBL (71-254) - CH1* 164 anti-FAP (4B9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA Fc hole chain
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 125 anti-FAP (4B9)
EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKP light chain
GQAPRLLINVGSRRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQGIMLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC
[0556] 2.1.2 Preparation of Monovalent FAP (4B9) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains without Charged Residues
(Construct 2.2)
[0557] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
1A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL.
[0558] A polypeptide containing one ectodomain of 4-1BB ligand
(71-254) and fused to the human IgG1-CH1 domain, was cloned as
described in FIG. 1B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH1.
[0559] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998)
using a linker (G.sub.4S).sub.2 (SEQ ID NO:13) or, alternatively,
GSPGSSSSGS (SEQ ID NO:57).
[0560] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole or the constant light chain of human IgG1.
[0561] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors (WO 2012/130831).
[0562] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-FAP-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a FAP binding Fab (FIG. 4B, Construct 2.2).
[0563] Table 22 shows the cDNA and amino acid sequences of the
monovalent FAP (4B9)-human 4-1BB ligand (71-254) Fc (kih) fusion
antigen binding molecule containing CH1-CL crossover without
charged residues (Construct 2.2).
TABLE-US-00025 TABLE 22 Sequences of monovalent FAP(4B9)-targeted
human 4-1BB ligand (71-254) containing Fc (kih) fusion molecule
Construct 2.2 SEQ ID NO: Description Sequence 165 Dimeric hu 4-
AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-254) -
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT CL Fc knob chain
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCT
GCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCT
CCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAG
GATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCA
GCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCA
GCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCT
GAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCC
TGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAA
CTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTT
CAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTC
AGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCG
CTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGA
CCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCT
TCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCC
CGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTG
GGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTG
CCAAGCCCTAGATCAGAAGGGGGCGGAGGTTCCGGAGG
GGGAGGATCTCGTACGGTGGCCGCTCCCTCCGTGTTTAT
CTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGC
CTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGA
AGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGT
CCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCC
AAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTG
TCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGC
GAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA
GAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCT
GTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTT
CTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA
TGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTG
GATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGG
TACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAA
GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG
TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGG
GCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
GCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGG
TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA
GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC
AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAA 166 Monomeric
hu 4- AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-254) -
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT CH1
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCT
GCTGGCGCTGCAGCTCTGGCTCTGACAGTGGATCTGCCT
CCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCCGGCTGCTGCACCTGTCTGCCGGCCAGAGACT
GGGAGTGCATCTGCACACAGAGGCCAGAGCCAGGCACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCTGCCGGCCTGCCTAGC
CCTAGATCTGAAGGCGGCGGAGGTTCCGGAGGCGGAGG
ATCTGCTAGCACCAAAGGCCCTTCCGTGTTTCCTCTGGC
TCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCT
CGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGAC
AGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGC
ATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACA
GCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTG
GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCC
CAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGT CTTGT 162 anti-FAP (4B9) see
Table 21 Fc hole chain 163 anti-FAP (4B9) see Table 21 light chain
117 Dimeric hu 4- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL
(71-254) - SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CL Fc knob
chain RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPEL
SPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA
GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNS
AFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV
LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVS
LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 118 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-254) -
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CH1
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC 164
anti-FAP (4B9) see Table 21 Fc hole chain 125 anti-FAP (4B9) see
Table 21 light chain
[0564] 2.1.3 Preparation of Bivalent FAP (4B9) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with the Dimeric and Monomeric 4-1BB Ligands Fused at the
C-Terminus of Each Heavy Chain (Construct 2.3)
[0565] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 1C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 1D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0566] The polypeptide encoding the dimeric 4-1BB ligand was
subcloned in frame at the C-terminus of human IgG1 heavy chain CH2
and CH3 domains on the hole (Merchant, Zhu et al. 1998) using a
(G.sub.4S).sub.2 (SEQ ID NO:13) connector. The polypeptide encoding
the monomeric 4-1BB ligand was subcloned in frame at the C-terminus
of human IgG1 heavy chain CH2 and CH3 domains on the knob
(Merchant, Zhu et al. 1998) using a (G.sub.4S).sub.2 (SEQ ID NO:13)
connector.
[0567] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole, the knob or the constant light chain of human
IgG1.
[0568] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831.
[0569] Combination of the anti-FAP huIgG1 hole dimeric ligand chain
containing the Y349C/T366S/L368A/Y407V mutations, the anti-FAP
huIgG1 knob monomeric ligand chain containing the S354C/T366W
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
two FAP binding Fabs (FIG. 4C, Construct 2.3)
[0570] Table 23 shows the cDNA and amino acid sequences of the
bivalent FAP (4B9)-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 2.3 (FAP split trimer with 2 anti-FAP
Fabs, dimeric and monomeric 4-1BB ligand fused at the C-terminus of
each heavy chain, respectively).
TABLE-US-00026 TABLE 23 Sequences of bivalent FAP(4B9)-targeted
human 4-1BB ligand (71-254) containing Fc (kih) fusion molecule
Construct 2.3 SEQ ID NO: Description Sequence 167 anti-FAP (4B9)
GAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA Fc hole chain
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG fused to dimeric
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC hu 4-1BBL (71-
AGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC 254)
ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG
AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAA
CACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGC
GGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGT
GTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCG
CTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCG
TGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCA
AGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAG
CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGG
CGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCT
GAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTG
CGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTG
CTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCT
GGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTA
CAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCG
GCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAG
TGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCC
TGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAG
CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCG
AGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTG
CTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTG
CACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGAC
ACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCC
CCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAA
GGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGG
ACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGA
TCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGA
ATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCG
ACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTG
TCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAA
AGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAG
GCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCT
GGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGC
AGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTC
TAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGAC
GCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTG
CATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCA
GCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGT
GACACCTGAGATCCCTGCCGGACTGCCAAGCCCTAGATC AGAA 168 anti-FAP (4B9)
GAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA Fc knob chain
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG fused to
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC monomeric hu 4-
AGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC 1BBL (71-254)
ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG
AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAA
CACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGC
GGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGT
GTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCT
GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC
TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA
GCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCA
AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG
TGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTG
ACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGC
TTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAA
CGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTG
TGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAAC
TGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTG
TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCA
CTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAG
GCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCC
TGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCT
GCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGT
GCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCC
TGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTA
CAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCG
GCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAG
TGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCC
TGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAG
CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCG
AGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTG
CTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTG
CACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGAC
ACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCC
CCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAA 163 anti-FAP (4B9) see Table
21 light chain 123 anti-FAP (4B9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA Fc hole chain
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL fused to dimeric
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS hu 4-1BBL (71-
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS 254)
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEG
GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNV
LLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV
YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 124 anti-FAP (4B9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA Fc knob chain
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL fused to
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS monomeric hu 4-
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS 1BBL (71-254)
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN
QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 125 anti-FAP (4B9) see
Table 21 light chain
[0571] 2.1.4 Preparation of Monovalent FAP (4B9) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 2.4)
[0572] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
1A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL. A polypeptide containing one ectodomain of 4-1BB ligand
(71-248) and fused to the human IgG1-CH domain, was cloned as
described in FIG. 1B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0573] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998)
using a linker (G.sub.4S).sub.2 (SEQ ID NO:13) or, alternatively,
GSPGSSSSGS (SEQ ID NO:57). To improve correct pairing the following
mutations have been introduced in the crossed CH-CL. In the dimeric
4-1BB ligand fused to human CL, E123R and Q124K. In the monomeric
4-1BB ligand fused to human CH1, K147E and K213E.
[0574] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole or the constant light chain of human IgG1.
[0575] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831.
[0576] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-FAP-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a FAP binding Fab (FIG. 4D, Construct 2.4).
[0577] Table 24 shows the cDNA and amino acid sequences of the
monovalent FAP (4B9)-human 4-1BB ligand (71-248) Fc (kih) fusion
antigen binding molecule containing CH1-CL crossover with charged
residues (Construct 2.4).
TABLE-US-00027 TABLE 24 Sequences of monovalent FAP(4B9)-targeted
human 4-1BB ligand (71-248) containing Fc (kih) fusion molecule
Construct 2.4 SEQ ID NO: Description Sequence 169 Dimeric hu 4-
AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-248) -
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT CL* Fc knob
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG chain
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCT
GCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGG
CGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCG
AGCTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGA
GACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTG
CTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCA
GGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGG
GGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGT
GGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCT
GCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAG
CACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCG
AGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTG
CTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCT
GCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGA
CTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACAC
CTGAGATCCCCGCTGGACTGGGCGGAGGCGGTTCCGGA
GGGGGAGGATCTCGTACGGTGGCTGCACCATCTGTCTTT
ATCTTCCCACCCAGCGACCGGAAGCTGAAGTCTGGCAC
AGCCAGCGTCGTGTGCCTGCTGAATAACTTCTACCCCCG
CGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGC
AGAGCGGCAACAGCCAGGAAAGCGTGACCGAGCAGGA
CAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGA
CCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTAC
GCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTG
ACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCA
CACCTGTCCTCCATGCCCTGCCCCTGAAGCTGCTGGCGG
CCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACAC
CCTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGGT
GGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAG
ACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT
GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG
CCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC
CCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCC
TGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCG
CCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC
TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGG
TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 170
Monomeric hu 4- AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL
(71-248) - ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT CH1*
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCT
GCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGG
CGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACAAAGG
GCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCA
CATCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGGAA
GATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCT
GGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTG
CTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTG
ACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATC
TGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGA CGAGAAGGTGGAACCCAAGTCCTGC
162 anti-FAP (4B9) see Table 21 Fc hole chain 163 anti-FAP (4B9)
see Table 21 light chain 119 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-248) -
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CL* Fc knob
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS chain
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPA
GLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG
GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV
SLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV
TPEIPAGLGGGGSGGGGSRTVAAPSVFIFPPSDRKLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR
GECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI
SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 120 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-248) -
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CH1*
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLGGGGSGGGGSASTKGPSVFPLA
PSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DEKVEPKSC 164 anti-FAP
(4B9) see Table 21 Fc hole chain 125 anti-FAP (4B9) see Table 21
light chain
[0578] 2.1.5 Preparation of Monovalent FAP (4B9) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains without Charged Residues
(Construct 2.5)
[0579] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
1A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL. A polypeptide containing one ectodomain of 4-1BB ligand
(71-248) and fused to the human IgG1-CH domain, was cloned as
described in FIG. 1B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0580] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998)
using a linker (G.sub.4S).sub.2 (SEQ ID NO:13) or, alternatively,
GSPGSSSSGS (SEQ ID NO:57).
[0581] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole or the constant light chain of human IgG1.
[0582] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831.
[0583] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-FAP-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a FAP binding Fab (FIG. 4E, Construct 2.5)
[0584] Table 25 shows the cDNA and amino acid sequences of the
monovalent FAP (4B9)-human 4-1BB ligand (71-248) Fc (kih) fusion
antigen binding molecule containing CH1-CL crossover without
charged residues (Construct 2.5).
TABLE-US-00028 TABLE 25 Sequences of monovalent FAP(4B9)-targeted
human 4-1BB ligand (71-248) containing Fc (kih) fusion molecule
Construct 2.5 SEQ ID NO: Description Sequence 171 nucleotide
AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG sequence dimeric
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT hu 4-1BBL (71-
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG 248) - CL Fc
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG knob chain
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCT
GCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGG
CGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCG
AGCTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGA
GACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTG
CTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCA
GGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTAC
AAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGG
GGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGT
GGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCT
GCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAG
CACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCG
AGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTG
CTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCT
GCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGA
CTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACAC
CTGAGATCCCCGCTGGACTGGGCGGAGGCGGTTCCGGA
GGGGGAGGATCTCGTACGGTGGCCGCTCCCTCCGTGTTT
ATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACC
GCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGG
GAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCA
GTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACT
CCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACAC
TGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCT
GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACAC
CTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCC
TTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCT
GATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGG
TGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATT
GGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACC
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC
TCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC
ATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGT
GGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA
GGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 172 Monomeric
hu 4- AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGG 1BBL (71-248) -
ACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGT CH1
GGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGG
CGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGG
TGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG
AACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCT
GTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCT
GCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCT
CCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTT
CAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCT
GGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACG
CCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGG
CGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACCAAAG
GCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCAC
CTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGA
TTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGG
AGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCT
GCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGAC
AGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCT
GCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGAC AAGAAGGTGGAACCCAAGTCTTGT 162
anti-FAP (4B9) see Table 21 Fc hole chain 163 anti-FAP (4B9) see
Table 21 light chain 173 Dimeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-248) -
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CL Fc knob chain
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPA
GLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTG
GLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV
SLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQG
RLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRV
TPEIPAGLGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR
GECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI
SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 174 Monomeric hu 4-
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWY 1BBL (71-248) -
SDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR CH1
RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLT
QGATVLGLFRVTPEIPAGLGGGGSGGGGSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC 164 anti-FAP
(4B9) see Table 21 Fc hole chain 125 anti-FAP (4B9) see Table 21
light chain
[0585] 2.1.6 Preparation of Bivalent FAP (4B9) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with the Dimeric and Monomeric 4-1BB Ligands Fused at the
C-Terminus of Each Heavy Chain (Construct 2.6)
[0586] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 1C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-248) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 1D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0587] The polypeptide encoding the dimeric 4-1BB ligand was
subcloned in frame at the C-terminus of human IgG1 heavy chain CH2
and CH3 domains on the hole (Merchant, Zhu et al. 1998) using a
(G.sub.4S).sub.2 (SEQ ID NO:13) connector. The polypeptide encoding
the monomeric 4-1BB ligand was subcloned in frame at the C-terminus
of human IgG1 heavy chain CH2 and CH3 domains on the knob
(Merchant, Zhu et al. 1998) using a (G.sub.4S).sub.2 (SEQ ID NO:13)
connector.
[0588] The variable region of heavy and light chain DNA sequences
encoding a binder specific for fibroblast activation protein (FAP),
clone 4B9, were subcloned in frame with either the constant heavy
chain of the hole, the knob or the constant light chain of human
IgG1.
[0589] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831.
[0590] Combination of the anti-FAP huIgG1 hole dimeric ligand chain
containing the Y349C/T366S/L368A/Y407V mutations, the anti-FAP
huIgG1 knob monomeric ligand chain containing the S354C/T366W
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
two FAP binding Fabs (FIG. 4F, Construct 2.6).
[0591] Table 26 shows the cDNA and amino acid sequences of the
bivalent FAP (4B9)-targeted 4-1BB ligand trimer-containing Fc (kih)
fusion molecule Construct 2.6 (FAP split trimer with 2 anti-FAP
Fabs, dimeric and monomeric 4-1BB ligand fused at the C-terminus of
each heavy chain, respectively).
TABLE-US-00029 TABLE 26 Sequences of bivalent FAP(4B9)-targeted
human 4-1BB ligand (71-248) containing Fc (kih) fusion molecule
Construct 2.6 SEQ ID NO: Description Sequence 175 nucleotide
GAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA sequence of anti-
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG FAP (4B9) Fc
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC hole chain fused
AGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC to dimeric hu 4-
ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG 1BBL (71-248)
AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAA
CACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGC
GGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGT
GTCCAGCGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCT
GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCG
CTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCG
TGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGC
CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAA
GCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCA
AGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCA
GCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTC
CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA
AGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG
AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGG
CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAG
CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGG
CGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCT
GAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTG
CGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTG
CTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCT
GGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTA
CAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCG
GCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAG
TGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCC
TGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAG
CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCG
AGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTG
CTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTG
CACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGAC
ACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCC
CCGAGATTCCAGCCGGCCTGGGCGGAGGCGGATCTGGC
GGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGA
CGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGT
TCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACG
GACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGG
GTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATAC
AAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATG
TGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGG
AGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGC
CACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGA
CTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAAC
AGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGC
GCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGC
CAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAA
CTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCTG CCGGACTG 176 nucleotide
GAGGTGCAGCTGCTCGAAAGCGGCGGAGGACTGGTGCA sequence anti-
GCCTGGCGGCAGCCTGAGACTGTCTTGCGCCGCCAGCG FAP (4B9) Fc
GCTTCACCTTCAGCAGCTACGCCATGAGCTGGGTCCGCC knob chain fused
AGGCCCCTGGCAAGGGACTGGAATGGGTGTCCGCCATC to monomeric hu
ATCGGCTCTGGCGCCAGCACCTACTACGCCGACAGCGTG 4-1BBL (71-248)
AAGGGCCGGTTCACCATCAGCCGGGACAACAGCAAGAA
CACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTACTGCGCCAAGGGATGGTTCGGC
GGCTTCAACTACTGGGGACAGGGCACCCTGGTCACAGT
GTCCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCT
GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC
TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAA
GCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCA
AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG
CACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCC
CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT
GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC
AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG
TGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTG
ACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGC
TTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAA
CGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTG
TGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAAC
TGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTG
TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCA
CTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAG
GCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCC
TGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCT
GCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGT
GCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCC
TGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTA
CAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCG
GCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAG
TGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCC
TGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAG
CTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCG
AGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTG
CTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTG
CACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGAC
ACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCC CCGAGATTCCAGCCGGCCTG 163
anti-FAP (4B9) see Table 21 light chain 126 anti-FAP (4B9)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA Fc hole chain
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL fused to dimeric
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS hu 4-1BBL (71-
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS 248)
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSG
GGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF
QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDL
PPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHA WQLTQGATVLGLFRVTPEIPAGL
127 anti-FAP (4B9) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA Fc knob
chain PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL fused to
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS monomeric hu 4-
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS 1BBL (71-248)
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN
QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 125 anti-FAP (4B9) see Table 21
light chain
[0592] 2.2 Preparation of Untargeted Human 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules (Control
Molecules)
[0593] Further control molecules were prepared as described in
Example 1.4 above for Control A and B. A bivalent variant Control C
was prepared in analogy to the bivalent Construct 2.3 and 2.6 and a
monovalent variant Control E was prepared in analogy to Construct
2.5 (containing a 4-1BB ligand (71-248) trimer), with the only
difference that the anti-FAP binder (VH-VL) was replaced by a
germline control, termed DP47, not binding to the antigen.
[0594] Table 27 shows the cDNA and amino acid sequences of the
bivalent DP47-untargeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion molecule Control C. Table 28 shows the cDNA and amino
acid sequences of the monovalent DP47-untargeted split trimeric
4-1BB ligand (71-248) Fc (kih) fusion molecule Control E.
TABLE-US-00030 TABLE 27 Sequences of bivalent DP47-untargeted human
4-1BB ligand (71-254) containing Fc (kih) fusion molecule Control C
SEQ ID NO: Description Sequence 177 nucleotide
GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA sequence DP47
GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGG Fc hole chain
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA fused to dimeric
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA hu 4-1BBL (71-
GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGA 254)
AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGA
CTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGC
TAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAG
CAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGT
CCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACC
TTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTG
AGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACC
CAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAA CACCAAGGTGGACAAGAAGGTGGAGCC
CAAGAGCTGCG ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
GCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA
GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC
CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA
CCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA
GAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAA
GCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGC
CCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGC
ATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATC
GATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCT
GGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGA
CACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACT
ACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCG
GCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGC
AGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCAC
TGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGA
ATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGT
CTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAG
GCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGC
TACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCC
AGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCG
GATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTG
TCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAG
GGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTG
ATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTG
GCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGA
AGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGT
ACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGG
CTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATC
TGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGG
CTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCA
GAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATC
TGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACT
GAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGG
GGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGAT
CCCTGCCGGACTGCCAAGCCCTAGATCAGAA 178 nucleotide
GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA sequence DP47
GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGG Fc knob chain
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA fused to
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA monomeric hu 4-
GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGA 1BBL (71-254)
AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGA
CTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGC
TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC
CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA
ACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
AGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGA
ACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACC
CCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAG
CCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGA
CAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGT
GGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCT
GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACC
CAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGG
AAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGA
GCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGG
GCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGA
TCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGG
CTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG
GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTA
CTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGC
CGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCT
GCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGC
ACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCG
GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCT
GTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAG
AGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGC
GCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATT
CCAGCCGGCCTGCCTTCTCCAAGAAGCGAA 80 nucleotide see Table 18 sequence
DP47 light chain 179 DP47 Fc hole
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA chain fused to
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL dimeric hu 4-
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS 1BBL (71-254)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKN
QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEG
GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNV
LLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV
YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 180 DP47 Fc knob
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA chain fused to
PGKGLEWVSAIIGSGASTYYADSVKGRFTISRDNSKNTLYL monomeric hu 4-
QMNSLRAEDTAVYYCAKGWFGGFNYWGQGTLVTVSSAS 1BBL (71-254)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK
CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKN
QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQ
LVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELV
VAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA
GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV
HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 82 DP47 light chain see
Table 18
TABLE-US-00031 TABLE 28 Sequences of monovalent untargeted human
4-1BB ligand (71-248) containing Fc (kih) fusion molecule Control E
SEQ ID NO: Description Sequence 171 Dimeric hu 4-1BBL (71-248)- see
Table 25 CL Fc knob chain 172 Monomeric hu 4-1BBL see Table 25
(71-248)-CH1 79 DP47 Fc hole chain see Table 18 80 DP47 light chain
see Table 18 173 Dimeric hu 4-1BBL (71-248)- see Table 25 CL Fc
knob chain 174 Monomeric hu 4-1BBL see Table 25 (71-248)-CH1 81
DP47 Fc hole chain see Table 18 82 DP47 light chain see Table
18
[0595] 2.3 Preparation of Untargeted Human IgG1 as Control F
[0596] An additional control molecule used in the assays was an
untargeted DP47, germline control, human IgG1, containing the
Pro329Gly, Leu234Ala and Leu235Ala mutations, to abrogate binding
to Fc gamma receptors according to the method described in
International Patent Appl. Publ. No. WO 2012/130831).
[0597] Table 29 shows the cDNA and amino acid sequences of the cDNA
and amino acid sequences of the untargeted DP47 huIgG1 PGLALA
(Control F).
TABLE-US-00032 TABLE 29 Sequences of untargeted DP47 huIgG1
(Control F) SEQ ID NO: Description Sequence 181 nucleotide
GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACA sequence DP47
GCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGG heavy chain (hu
ATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCA IgG1 PGLALA)
GGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA
GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGA
AGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC
ACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGA
CACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGA
CTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGC
TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCAC
CCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCA
ACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
AGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA 80 DP47 light chain see Table 18 182
DP47 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA (hu IgG1
PGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYL PGLALA)
QMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK 82 DP47 light chain see
Table 18
[0598] 2.4 Production of Monovalent and Bivalent FAP (4B9) Targeted
Split Trimeric 4-1BB Ligand Fc Fusion Constructs and Control
Molecules
[0599] The targeted and untargeted split trimeric 4-1BB ligand Fc
(kih) fusion encoding sequences were cloned into a plasmid vector,
which drives expression of the insert from an MPSV promoter and
contains a synthetic polyA sequence located at the 3' end of the
CDS. In addition, the vector contains an EBV OriP sequence for
episomal maintenance of the plasmid.
[0600] The split trimeric 4-1BB ligand Fc (kih) fusion was produced
by co-transfecting HEK293-EBNA cells with the mammalian expression
vectors using polyethylenimine. The cells were transfected with the
corresponding expression vectors. For Constructs 2.1, 2.2., 2.4 and
2.5 and corresponding control molecules, a 1:1:1:1 ratio
(e.g."vector dimeric ligand-CL-knob chain": "vector monomeric
ligand fusion-CH1": "vector anti-FAP Fab-hole chain": "vector
anti-FAP light chain") was used. For Constructs 2.3 and 2.6 and its
control molecule, a 1:1:1 ratio ("vector huIgG1 Fc hole dimeric
ligand chain": "vector huIgG1 Fc knob monomeric ligand chain":
"vector anti-FAP light chain") was taken. Human IgGs, used as
control in the assay, were produced as for the bispecific
constructs (for transfection only a vector for light and a vector
for heavy chain were used.
[0601] For production in 500 mL shake flasks, 300 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 10 minutes at 210.times.g,
and the supernatant was replaced by 20 mL pre-warmed CD CHO medium.
Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CD
CHO medium. After addition of 540 .mu.L PEI, the solution was
vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere. After the incubation, 160 mL of Excell medium
supplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM
valproic acid was added and cells were cultured for 24 hours. One
day after transfection 12% Feed 7 and Glucose (final conc. 3 g/L)
were added. After culturing for 7 days, the supernatant was
collected by centrifugation for 30-40 minutes at least 400.times.g.
The solution was sterile filtered (0.22 .mu.m filter), supplemented
with sodium azide to a final concentration of 0.01% (w/v), and kept
at 4.degree. C.
[0602] The targeted and untargeted TNF ligand trimer-containing Fc
(kih) fusion antigen binding molecules and the human IgG1 were
purified from cell culture supernatants by affinity chromatography
using Protein A, followed by size exclusion chromatography. For
affinity chromatography, the supernatant was loaded on a MAB SELECT
SURE.RTM. column (CV=5-15 mL, resin from GE Healthcare)
equilibrated with Sodium Phosphate (20 mM), Sodium Citrate (20 mM)
buffer (pH 7.5). Unbound protein was removed by washing with at
least 6 column volumes of the same buffer. The bound protein was
eluted using either a linear gradient (20 CV) or a step elution (8
CV) with 20 mM sodium citrate, 100 mM Sodium chloride, 100 mM
Glycine buffer (pH 3.0). For the linear gradient an additional 4
column volumes step elution was applied.
[0603] The pH of collected fractions was adjusted by adding 1/10
(v/v) of 0.5M sodium phosphate, pH8.0. The protein was concentrated
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium
chloride, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20) solution of pH
6.0.
[0604] The protein concentration was determined by measuring the
optical density (OD) at 280 nm, using a molar extinction
coefficient calculated on the basis of the amino acid sequence.
Purity and molecular weight of the targeted trimeric 4-1BB ligand
Fc (kih) fusion was analyzed by SDS-PAGE in the presence and
absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining
with Coomassie SIMPLYBLUE.TM. SafeStain (Invitrogen USA) or CE-SDS
using Caliper LabChip GXII (Perkin Elmer). The aggregate content of
samples was analyzed using a TSKGEL.RTM. G3000 SW XL analytical
size-exclusion column (Tosoh) equilibrated in 25 mM
K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride,
0.02% (w/v) NaN3, pH 6.7 running buffer at 25.degree. C.
[0605] Table 30 summarizes the yield and final monomer content of
the FAP (4B9) targeted and untargeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecules and
control molecules.
TABLE-US-00033 TABLE 30 Biochemical analysis of FAP (4B9) targeted
and untargeted 4-1BB ligand trimer-containing Fc (kih) fusion
antigen binding molecules and control molecules Monomer [%] Yield
Construct (SEC) [mg/l] Construct 2.1 95 15.8 Construct 2.3 97 11.5
Construct 2.4 97 14.1 Construct 2.5 100 16.5 Control C (bivalent)
98 12.6 Control E (monovalent) 93 4.1 Control F (germline DP47
human IgG1 100 50 PGLALA
Example 3
Preparation, Purification and Characterization of 4-1BB
[0606] DNA sequences encoding the ectodomains of human, mouse or
cynomolgus 4-1BB (Table 31) were subcloned in frame with the human
IgG1 heavy chain CH2 and CH3 domains on the knob (Merchant et al.,
1998). An AcTEV protease cleavage site was introduced between an
antigen ectodomain and the Fc of human IgG1. An Avi tag for
directed biotinylation was introduced at the C-terminus of the
antigen-Fc knob. Combination of the antigen-Fc knob chain
containing the S354C/T366W mutations, with a Fc hole chain
containing the Y349C/T366S/L368A/Y407V mutations allows generation
of a heterodimer which includes a single copy of 4-1BB ectodomain
containing chain, thus creating a monomeric form of Fc-linked
antigen (FIG. 5C). Table 32 shows the cDNA and amino acid sequences
of the antigen Fc-fusion constructs.
TABLE-US-00034 TABLE 31 Amino acid numbering of antigen ectodomains
(ECD) and their origin SEQ ID NO: Construct Origin ECD 83 human
4-1BB ECD Synthetized according to Q07011 aa 24-186 84 cynomolgus
4-1BB ECD isolated from cynomolgus blood aa 24-186 85 murine 4-1BB
ECD Synthetized according to P20334 aa 24-187
TABLE-US-00035 TABLE 32 cDNA and Amino acid sequences of monomeric
antigen Fc(kih) fusion molecules SEQ ID NO: Antigen Sequence 86
Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA sequence
ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA Fc hole chain
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGT
CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG
AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG
TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA
ACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA
CTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC
AGAAGAGCCTCTCCCTGTCTCCGGGTAAA 87 Nucleotide
CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCACCTTC sequence
TGCGACAACAACCGGAACCAGATCTGCAGCCCCTGCCC human 4-1BB
CCCCAACAGCTTCAGCTCTGCCGGCGGACAGCGGACCT antigen Fc knob
GCGACATCTGCAGACAGTGCAAGGGCGTGTTCAGAACC chain
CGGAAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGA
CTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAG
CATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGA
CCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTC
AACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAA
CTGTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGGCA
CCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCT
GATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCC
CCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAA
CAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGAC
AAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTC
CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCC
AAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA
TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT
CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCA
GGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC
GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTC
CGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA
AGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGA
ACGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 88 Nucleotide
TTGCAGGATCTGTGTAGTAACTGCCCAGCTGGTACATTC sequence
TGTGATAATAACAGGAGTCAGATTTGCAGTCCCTGTCCT cynomolgus 4-
CCAAATAGTTTCTCCAGCGCAGGTGGACAAAGGACCTGT 1BB antigen
GACATATGCAGGCAGTGTAAAGGTGTTTTCAAGACCAG Fc knob chain
GAAGGAGTGTTCCTCCACCAGCAATGCAGAGTGTGACT
GCATTTCAGGGTATCACTGCCTGGGGGCAGAGTGCAGC
ATGTGTGAACAGGATTGTAAACAAGGTCAAGAATTGAC
AAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAA
TGACCAGAAACGTGGCATCTGTCGCCCCTGGACAAACT
GTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGA
AGGAGAGGGACGTGGTCTGCGGACCATCTCCAGCCGAC
CTCTCTCCAGGAGCATCCTCTGCGACCCCGCCTGCCCCT
GCGAGAGAGCCAGGACACTCTCCGCAGGTCGACGAACA
GTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT
GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA
AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA
CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG
ACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC
CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGA
GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA
GAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAA
CGACATCTTCGAGGCCCAGAAGATTGAATGGCACGAG 89 murine 4-1BB
GTGCAGAACAGCTGCGACAACTGCCAGCCCGGCACCTT antigen Fc knob
CTGCCGGAAGTACAACCCCGTGTGCAAGAGCTGCCCCC chain
CCAGCACCTTCAGCAGCATCGGCGGCCAGCCCAACTGC
AACATCTGCAGAGTGTGCGCCGGCTACTTCCGGTTCAAG
AAGTTCTGCAGCAGCACCCACAACGCCGAGTGCGAGTG
CATCGAGGGCTTCCACTGCCTGGGCCCCCAGTGCACCAG
ATGCGAGAAGGACTGCAGACCCGGCCAGGAACTGACCA
AGCAGGGCTGTAAGACCTGCAGCCTGGGCACCTTCAAC
GACCAGAACGGGACCGGCGTGTGCCGGCCTTGGACCAA
TTGCAGCCTGGACGGGAGAAGCGTGCTGAAAACCGGCA
CCACCGAGAAGGACGTCGTGTGCGGCCCTCCCGTGGTGT
CCTTCAGCCCTAGCACCACCATCAGCGTGACCCCTGAAG
GCGGCCCTGGCGGACACTCTCTGCAGGTCCTGGTCGACG
AACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAG
ACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC
ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA
GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC
ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC
CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGT
CTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAA
CCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA
GAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCT
GAACGACATCTTCGAGGCCCAGAAGATTGAATGGCACG AG 90 Fc hole chain
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK
91 human 4-1BB LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDI antigen Fc
knob CRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQ chain
DCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDG
KSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHS
PQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV
SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKSGGLNDIFEAQKIEWHE 92
cynomolgus 4- LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQRTCDIC 1BB
antigen RQCKGVFKTRKECSSTSNAECDCISGYHCLGAECSMCEQD Fc knob chain
CKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGK
SVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSP
QVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVS
LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKSGGLNDIFEAQKIEWHE 93
murine 4-1BB VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPNCNIC antigen Fc
knob RVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCTRCEKDC chain
RPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGR
SVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQ
VLVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQV
SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKSGGLNDIFEAQKIEWHE
[0607] All 4-1BB-Fc-fusion molecule encoding sequences were cloned
into a plasmid vector, which drives expression of the insert from
an MPSV promoter and contains a synthetic polyA signal sequence
located at the 3' end of the CDS. In addition, the vector contains
an EBV OriP sequence for episomal maintenance of the plasmid.
[0608] For preparation of the biotinylated monomeric antigen/Fc
fusion molecules, exponentially growing suspension HEK293 EBNA
cells were co-transfected with three vectors encoding the two
components of fusion protein (knob and hole chains) as well as
BirA, an enzyme necessary for the biotinylation reaction. The
corresponding vectors were used at a 2:1:0.05 ratio ("antigen
ECD-AcTEV-Fc knob": "Fc hole": "BirA").
[0609] For protein production in 500 ml shake flasks, 400 million
HEK293 EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 5 minutes at 210 g, and the
supernatant was replaced by pre-warmed CD CHO medium. Expression
vectors were resuspended in 20 mL of CD CHO medium containing 200
.mu.g of vector DNA. After addition of 540 .mu.L of
polyethylenimine (PEI), the solution was vortexed for 15 seconds
and incubated for 10 minutes at room temperature. Afterwards, cells
were mixed with the DNA/PEI solution, transferred to a 500 mL shake
flask and incubated for 3 hours at 37.degree. C. in an incubator
with a 5% CO.sub.2 atmosphere. After the incubation, 160 mL of F17
medium was added and cells were cultured for 24 hours. The
production medium was supplemented with 5 .mu.M kifunensine. One
day after transfection, 1 mM valproic acid and 7% Feed 1 with
supplements were added to the culture. After 7 days of culturing,
the cell supernatant was collected by spinning down cells for 15
min at 210 g. The solution was sterile filtered (0.22 .mu.m
filter), supplemented with sodium azide to a final concentration of
0.01% (w/v), and kept at 4.degree. C.
[0610] Secreted proteins were purified from cell culture
supernatants by affinity chromatography using Protein A, followed
by size exclusion chromatography. For affinity chromatography, the
supernatant was loaded on a HITRAP.RTM. ProteinA HP column (CV=5
mL, GE Healthcare) equilibrated with 40 mL 20 mM sodium phosphate,
20 mM sodium citrate pH 7.5. Unbound protein was removed by washing
with at least 10 column volumes of 20 mM sodium phosphate, 20 mM
sodium citrate, 0.5 M sodium chloride containing buffer (pH 7.5).
The bound protein was eluted using a linear pH-gradient of sodium
chloride (from 0 to 500 mM) created over 20 column volumes of 20 mM
sodium citrate, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20), pH 3.0.
The column was then washed with 10 column volumes of 20 mM sodium
citrate, 500 mM sodium chloride, 0.01% (v/v) TWEEN.RTM. 20
(polysorbate 20), pH 3.0.
[0611] The pH of collected fractions was adjusted by adding 1/40
(v/v) of 2M Tris, pH8.0. The protein was concentrated and filtered
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 2 mM MOPS, 150 mM sodium chloride,
0.02% (w/v) sodium azide solution of pH 7.4.
[0612] For affinity determination to the human receptor, the
ectodomain of human 4-1BB was also subcloned in frame with an avi
(SEQ ID NO: 376; GLNDIFEAQKIEWHE) and a hexahistidine tag (SEQ ID
NO: 393).
[0613] Protein production was performed as described above for the
Fc-fusion protein. Secreted proteins were purified from cell
culture supernatants by chelating chromatography, followed by size
exclusion chromatography. The first chromatographic step was
performed on a Ni-NTA SUPERFLOW.TM. Cartridge (5 ml, Qiagen)
equilibrated in 20 mM sodium phosphate, 500 nM sodium chloride,
pH7.4. Elution was performed by applying a gradient over 12 column
volume from 5% to 45% of elution buffer (20 mM sodium phosphate,
500 nM sodium chloride, 500 mM Imidazole, pH7.4). The protein was
concentrated and filtered prior to loading on a HILOAD.RTM.
Superdex 75 column (GE Healthcare) equilibrated with 2 mM MOPS, 150
mM sodium chloride, 0.02% (w/v) sodium azide solution of pH 7.4
(Table 33).
TABLE-US-00036 TABLE 33 Sequences of monomeric human 4-1BB His
molecule SEQ ID NO: antigen Sequence 94 nucleotide
CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCACCTTCTG sequence
CGACAACAACCGGAACCAGATCTGCAGCCCCTGCCCCCCC human
AACAGCTTCAGCTCTGCCGGCGGACAGCGGACCTGCGACA 4-1BB His
TCTGCAGACAGTGCAAGGGCGTGTTCAGAACCCGGAAAGA
GTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCC
GGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGC
AGGACTGCAAGCAGGGCCAGGAACTGACCAAGAAGGGCT
GCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCG
GGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGC
AAGAGCGTGCTGGTCAACGGCACCAAAGAACGGGACGTCG
TGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGC
AGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTC
TCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCAG
GCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCA
CGAGGCTCGAGCTCACCACCATCACCATCAC 95 human
LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR 4-1BB His
QCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCK
QGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLV
NGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQL
YFQGGSGLNDIFEAQKIEWHEARAHHHHHH
Example 4
Biochemical Characterization of FAP-Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecule by Surface
Plasmon Resonance
[0614] The binding of FAP-targeted 4-1BB ligand trimer-containing
Fc (kih) fusion antigen binding molecules to recombinant 4-1BB was
assessed by surface plasmon resonance (SPR). All SPR experiments
were performed on a BIACORE.RTM. instrument T100 at 25.degree. C.
with HBS-EP as a running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl,
3 mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).
[0615] The avidity of the interaction between the FAP-targeted or
untargeted 4-1BB ligand trimer-containing Fc (kih) fusion antigen
binding molecules and recombinant 4-1BB (human, cyno and murine)
was determined as described below. The data demonstrated that both
targeted 4-1BB ligand trimer-containing Fc (kih) fusion antigen
binding molecules as well as untargeted DP47 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecules bind
with comparable avidities to human and cynomolgus 4-1BB but
negligibly to the mouse homolog.
[0616] Recombinant biotinylated human, cynomolgus and murine 4-1BB
Fc(kih) fusion molecules were directly coupled on a SA chip using
the standard coupling instruction (Biacore, Freiburg/Germany). The
immobilization level was about 30 RU. FAP-targeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecules, or the
DP47 untargeted controls, were passed at a concentration range from
0.39 to 200 nM with a flow of 30 .mu.L/minutes through the flow
cells over 120 seconds. The dissociation was monitored for 180
seconds. Bulk refractive index differences were corrected for by
subtracting the response obtained on a reference empty flow
cell.
[0617] For affinity measurement, direct coupling of around 7200
resonance units (RU) of an anti-human Fc specific antibody was
performed on a CM5 chip at pH 5.0 using the standard amine coupling
kit (GE Healthcare). FAP-targeted or untargeted 4-1BB ligand
trimer-containing Fc (kih) fusion antigen binding molecules, at 50
nM were captured with a flow rate of 30 .mu.l/min for 60 sec on
flow cell 2. A dilution series (1.95 to 1000 nM) of human
4-1BB-avi-His was passed on both flow cells at 30 .mu.l/min for 180
sec to record the association phase. The dissociation phase was
monitored for 180 s and triggered by switching from the sample
solution to HBS-EP. The chip surface was regenerated after every
cycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1.
Bulk refractive index differences were corrected for by subtracting
the response obtained on reference flow cell 1. For the interaction
between the 4-1BB ligand trimer-containing Fc (kih) fusion antigen
binding molecules and hu4-1BB avi His, the affinity constants were
derived from the rate constants by fitting to a 1:1 Langmuir
binding curve using the Biaeval software (GE Healthcare).
TABLE-US-00037 TABLE 34 Fittings to 1:1 Langmuir binding and
Affinity constants Ligand Analyte ka (1/Ms) kd (1/s) KD (M) FAP
split 4-1BBL hu4-1BB 4.8E+04 2.6E-02 5.5E-07 trimer (Construct 1.1)
DP47 split 4-1BBL hu4-1BB 6.2E+04 3.3E-02 5.2E-07 trimer (Control
A)
Example 5
Functional Characterization of the Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0618] 5.1. Binding on Naive Versus Activated Human PMBCs of the
FAP-Targeted 4-1BB Ligand Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecules
[0619] Buffy coats were obtained from the Zurich blood donation
center. To isolate fresh peripheral blood mononuclear cells (PBMCs)
the buffy coat was diluted with the same volume of DPBS (Gibco by
Life Technologies, Cat. No. 14190 326). 50 mL polypropylene
centrifuge tubes (TPP, Cat.-No. 91050) were supplied with 15 mL
HISTOPAQUE.RTM. reagent 1077 (SIGMA Life Science, Cat.-No. 10771,
polysucrose and sodium diatrizoate, adjusted to a density of 1.077
g/mL) and the diluted buffy coat solution was layered above the
HISTOPAQUE.RTM. reagent 1077. The tubes were centrifuged for 30 min
at 400.times.g. PBMCs were then collected from the interface,
washed three times with DPBS and resuspended in T cell medium
consisting of RPMI 1640 medium (Gibco by Life Technology, Cat. No.
42401-042) supplied with 10% Fetal Bovine Serum (FBS, Gibco by Life
Technology, Cat. No. 16000-044, Lot 941273, gamma-irradiated,
mycoplasma-free and heat inactivated at 56.degree. C. for 35 min),
1% (v/v) GlutaMAX-I (GIBCO by Life Technologies, Cat. No. 35050
038), 1 mM Sodium Pyruvate (SIGMA, Cat. No. S8636), 1% (v/v) MEM
non-essential amino acids (SIGMA, Cat.-No. M7145) and 50 .mu.M
.beta.-Mercaptoethanol (SIGMA, M3148).
[0620] PBMCs were used directly after isolation or stimulated to
induce 4-1BB expression at the cell surface of T and NK cells by
culturing for 4 days in T cell medium supplemented with 200 U/mL
Proleukin (Novartis Pharma Schweiz AG, CHCLB-P-476-700-10340) and 2
ug/mL PHA-L (SIGMA Cat.-No. L2769) in a 6-well tissue culture plate
and then 1 day in a 6-well tissue culture plate coated with 10
ug/mL anti-human CD3 (clone OKT3, BioLegend, Cat.-No. 317315) and 2
ug/mL anti-human CD28 (clone CD28.2, BioLegend, Cat.-No.: 302928)
in T cell medium at 37.degree. C. and 5% CO.sub.2.
[0621] To determine binding of 4-1BBL trimer-containing Fc fusion
antigen binding molecules to human PBMCs, 0.1.times.10.sup.6 naive
or activated PBMCs were added to each well of a round-bottom
suspension cell 96-well plates (Greiner bio-one, cellstar, Cat. No.
650185). Plates were centrifuged 4 minutes with 400.times.g and at
4.degree. C. Supernatant was discarded. Afterwards cells were
stained in 100 .mu.L/well DPBS containing 1:1000 diluted
LIVE/DEAD.RTM. Fixable Blue Dead Cell Stain Kit, for UV excitation
(Life Technologies, Molecular Probes, L-23105) or Fixable Viability
Dye eF660 (eBioscience 65-0864-18) or LIVE/DEAD.RTM. Fixable Green
Dead Cell Stain Kit (Life Technologies, Molecular Probes, L-23101)
for 30 minutes at 4.degree. C. in the dark. If DAPI was used as
Live/Death stain, this staining step was skipped. Cells were washed
once with 200 .mu.L cold FACS buffer (DPBS supplied with 2% (v/v)
FBS, 5 mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM sodium azide
(Sigma-Aldrich S2002).
[0622] Next, 50 .mu.L/well of 4.degree. C. cold FACS buffer
containing different titrated concentrations of 4-1BBL
trimer-containing Fc fusion antigen binding molecules were added
and cells were incubated for 120 minutes at 4.degree. C., washed
four times with 200 .mu.L/well 4.degree. C. FACS buffer and
resuspended. Cells were further stained with 50 .mu.L/well of
4.degree. C. cold FACS buffer containing 0.67 .mu.g/mL anti-human
CD3-PerCP-Cy5.5 (clone UCHT1, mouse IgG1.kappa., BioLegend,
Cat.-No. 300430) or 0.16 .mu.L anti-human CD3-PE/Cy7 (clone SP34-2,
mouse IgG1 .kappa., BD Pharmingen, Cat.-No. 557749, Lot 33324597),
0.67 .mu.g/mL anti-human CD45-AF488 (clone HI30, mouse IgG1.kappa.,
BioLegend, Cat.-No. 304017) or 0.12 .mu.g/mL anti-human CD56-FITC
(clone NCAM16.2, mouse IgG2b.kappa., BD Pharmingen, Cat.-No.
345811) or 1 .mu.L anti-human CD56-APC (clone B159, mouse IgG1
.kappa., BD Pharmingen, Cat.-No. 555518, Lot 3098894), 0.25
.mu.g/mL anti-human CD4-BV421 (clone RPA-T4, mouse IgG1.kappa.,
BioLegend, Cat.-No. 300532) or 0.23 .mu.g/mL anti-human CD4-BV421
(clone OKT4, mouse IgG2b.kappa., BioLegend, Cat.-No. 317434), 0.25
.mu.L anti-human CD8a-APC (clone RPA-T8, mouse IgG1.kappa., BD
Pharmingen, Cat.-No. 555369) or 0.67 .mu.L anti-human CD8a-APC/Cy7
(clone RPA-T8, mouse IgG1.kappa., BioLegend, Cat.-No. 301016) or
0.83 ng/mL anti-human CD8a-BV711 (clone RPA-T8, mouse IgG1.kappa.,
BD Pharmingen, Cat.-No. 301044) and 5 .mu.g/mL PE-conjugated
AffiniPure anti-human IgG Fc.gamma.-fragment-specific goat IgG
F(ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109 116 098 or
109 116 170). Cells were washed twice with FACS-buffer. If cells
were stained with fixable viability dyes, they were fixed with 50
.mu.L/well DPBS containing 1% formaldehyde (Sigma, HT501320-9.5L).
Cells were resuspended in FACS buffer and acquired the next or the
same day using a 5-laser LSR-FORTESSA.RTM. (BD Bioscience with DIVA
software) or 3-laser Miltenyi Quant Analyzer 10 (Mitenyi Biotec)
and Flow Jo (FlowJo X 10.0.7). If DAPI staining was used to detect
dead cells, they were resuspended in 80 .mu.L/well FACS buffer
containing 0.2 .mu.g/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598)
and acquired the same day using a 5-laser LSR-FORTESSA.RTM. (BD
Bioscience with DIVA software).
[0623] As shown in FIGS. 6A-1 to 6C-2, both FAP-targeted or
untargeted 4-1BB ligand trimer-containing Fc fusion antigen binding
molecules did not bind to resting human CD4+ T cells and showed no
detectable binding to resting CD8+ T cells and NK cells. In
contrast, both constructs bound strongly to activated NK, CD8+ or
CD4+ T cells, although the latter showed approximately 10 fold
lower intensity of specific fluorescence as compared to the NK
cells and 20 fold decreased intensity of specific fluorescence as
compared to CD8+ T cells.
[0624] FIGS. 7A-1 to 7A-4 and 7B-1 to 7B-4 show the binding of
Constructs 1.1 to 1.10 as prepared in Example 1 on 4-1BB-expressing
activated human CD3+CD8+ T cells and 4-1BB-expressing activated
human CD3+CD4+ T cells, respectively. Table 35 shows the EC.sub.50
values as measured for Constructs 1.1 to 1.10.
TABLE-US-00038 TABLE 35 Binding on activated human CD3+ CD8+ T
cells and CD3+ CD4+ T cells EC.sub.50 [nM] EC.sub.50 [nM] Construct
4-1BB.sup.+CD8.sup.+ 4-1BB.sup.+CD4.sup.+ Control B 0.11 16.21 1.1
0.43 4.99 1.2 0.18 20.79 1.3 0.07 2.82 1.4 0.19 0.34 1.5 0.17 2.67
1.6 0.19 0.95 1.7 0.26 16.47 1.8 0.14 2.77 1.9 0.18 12.92 1.10 0.12
0.3
[0625] FIGS. 8A-1 to 8A-4 and 8B-1 to 8B-4 show the binding of
Constructs 2.1, 2.3, 2.4, 2.5 and 2.6 as prepared in Example 2 on
CD4+ and CD8+ from fresh human blood and on activated
4-1BB-expressing CD4+ T cells and CD8+ T cells, respectively. Gates
were set on living CD45+CD3+CD4+ or CD45+CD3+CD8+ T cells and MFI
of PE-conjugated AffiniPure anti-human IgG IgG
Fc.gamma.-fragment-specific goat F(ab')2 fragment were blotted
against the titrated concentration of targeted split trimeric 4-1BB
ligand Fc fusion variants. Table 36 shows the EC.sub.50 values as
measured for Constructs 2.1, 2.3, 2.4, 2.5 and 2.6.
TABLE-US-00039 TABLE 36 Binding on activated 4-1BB-expressing CD4+
T cells and CD8 + T cells EC.sub.50 [nM] EC.sub.50 [nM] Construct
4-1BB.sup.+CD8.sup.+ 4-1BB.sup.+CD4.sup.+ Control B 0.36 0.42
Control C 0.39 0.41 Control E 0.57 0.76 2.1 0.21 0.24 2.3 0.44 0.3
2.4 0.3 0.38 2.5 0.35 0.68 2.6 0.33 0.24
5.2 Binding of FAP-Targeted 4-1BB Ligand Trimer-Containing Fc
Fusion Antigen Binding Molecule to Activated Mouse Splenocytes
[0626] Mouse spleens were collected in 3 mL PBS and a single cell
suspension was generated using gentle MACS tubes (Miltenyi Biotec
Cat.-No. 130-096-334) and gentleMACS Octo Dissociator (Miltenyi
Biotec). Afterwards splenocytes were filtered through a 30 .mu.m
Pre-Separation Filters (Miltenyi Biotec Cat.-No. 130-041-407) and
centrifuged for 7 min at 350.times.g and 4.degree. C. Supernatant
was aspirated and cells were resuspended in RPMI 1640 medium
supplied with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 1 mM
Sodium-Pyruvate, 1% (v/v) MEM non-essential amino acids, 50 .mu.M
.beta.-Mercaptoethanol and 10% Penicillin-Streptomycin (SIGMA,
Cat.-No. P4333). 10.sup.6 cells/mL were cultured for 2 days in a
6-well tissue culture plate coated with 10 .mu.g/mL anti-mouse CD3c
Armenian Hamster IgG (clone 145-2C11, BioLegend, Cat.-No. 100331)
and 2 .mu.g/mL anti-mouse CD28 Syrian Hamster IgG (clone 37.51,
BioLegend, Cat.-No. 102102).
[0627] Activated mouse splenocytes were harvested, washed in DPBS,
counted and 0.1.times.10.sup.6 cells were transferred to each well
of a 96 U-bottom non-tissue culture treated well plate. Supernatant
was removed and cells were stained in 100 uL/well DPBS containing
1:5000 diluted Fixable Viability Dye eF660 (Bioscience, Cat-No.
65-0864-18) for 30 min at 4.degree. C. Cells were washed with PBS
and stained in 50 uL FACS buffer containing different concentration
of FAP-targeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules (FAP split 4-1BBL trimer), untargeted 4-1BB
ligand trimer-containing Fc fusion antigen binding molecules (DP47
split 4-1BBL trimer) or anti-mouse CD137 human IgG1 P329G LALA mAb
(clone Lob.12.3, BioXcell Catalog #: BE0169). Cells were incubated
for 120 min at 4.degree. C. Cells were washed four times with FACS
buffer and stained in 50 FACS buffer containing 10 .mu.g/mL
purified anti-mouse CD16/CD32 rat IgG-Fc-Block (BD Pharmingen,
Cat.-No. 553142 clone 2.4G2), 5 .mu.g/mL anti-mouse CD8b rat
IgG2b.kappa.-FITC (BioLegend, Cat.-No. 126606, clone YTS156.7.7),
0.67 .mu.g/mL anti-mouse CD3 rat IgG2b.kappa.-APC-Cy7 (BioLegend,
Cat.-No. 100222, clone 17A2), 0.67 .mu.g/mL anti-mouse CD4 rat
IgG2b.kappa.-PE-Cy7 (BioLegend, Cat.-No. 100422, clone GK1.5), 2
.mu.g/mL anti-mouse NK1.1 Mouse (C3H x BALB/c)
IgG2a.kappa.-PerCp-Cy5.5 (BioLegend, Cat.-No. 108728, clone PK136)
and 10 .mu.g/mL PE-conjugated AffiniPure polyclonal F(ab')2
Fragment goat anti-human IgG, Fey fragment specific, minimal
cross-reactive to bovine mouse and rabbit serum proteins (Jackson
ImmunoResearch, Cat.-No. 109-116-170) for 30 min at 4.degree. C.
Cells were washed twice with 200 .mu.L/well cold FACS buffer. Cells
were fixed with 50 DPBS containing 1% formaldehyde. Cells were
resuspended in FACS-buffer and acquired the next day using a
5-laser LSR-FORTESSA.RTM. (BD Bioscience with DIVA software).
[0628] As shown in FIGS. 9A and 9B, FAP-targeted hu4-1BB ligand
trimer-containing Fc fusion antigen binding molecules (FAP split
hu4-1BBL trimer) and untargeted hu4-1BB ligand trimer-containing Fc
fusion antigen binding molecules (DP47 split hu4-1BBL trimer) do
not bind to mouse 4-1BB. Therefore activity cannot be tested in
immune competent mice. For in vivo mode of action studies either
humanized mouse models in immune incompetent mice or surrogates
containing mouse 4-1BBL trimers as shown in FIGS. 3A to 3C have to
be used.
5.3 Binding to FAP-Expressing Tumor Cells
[0629] For binding assays on FAP expressing cells, the human
melanoma cell line MV-3 (see Ruiter et al., Int. J. Cancer 1991,
48(1), 85-91), WM-266-4 (ATTC CRL-1676) or NIH/3T3-huFAP clone 39
cell line were used. To generate the latter cell line, NIH/3T3
cells were transfected with human FAP (NIH/3T3-huFAP clone 39). The
cells were generated by transfection of mouse embryonic fibroblast
NIH/3T3 cells (ATCC CRL-1658) with the expression pETR4921 plasmid
encoding human FAP under a CMV promoter. Cells were maintained in
the presence of 1.5 .mu.g/mL puromycin (InvivoGen, Cat.-No.:
ant-pr-5). 0.1.times.10.sup.6 of FAP expressing tumor cells were
added to each well of a round-bottom suspension cell 96-well plates
(Greiner bio-one, cellstar, Cat.-No. 650185). Cells were washed
once with 200 .mu.L DPBS and pellets were resuspended. 100
.mu.L/well of 4.degree. C. cold DPBS buffer containing 1:5000
diluted Fixable Viability Dye EFLUOR.RTM. 450 (eBioscience,
Cat.-No. 65-0863-18) or Fixable Viability Dye EFLUOR.RTM. 660
(eBioscience, Cat.-No. 65-0864-18) were added and plates were
incubated for 30 minutes at 4.degree. C. Cells were washed once
with 200 .mu.L 4.degree. C. cold DPBS buffer and resuspended in 50
.mu.L/well of 4.degree. C. cold FACS buffer (DPBS supplied with 2%
(v/v) FBS, 5 mM EDTA pH 8 (Amresco, Cat.-No. E177) and 7.5 mM
Sodium azide (Sigma-Aldrich 52002) containing different
concentrations of titrated 4-1BBL trimer-containing Fc fusion
antigen binding molecules, followed by incubation for 1 hour at
4.degree. C. After washing four times with with 200 .mu.L/well,
cells were stained with 50 .mu.L/well of 4.degree. C. cold FACS
buffer containing 30 .mu.g/mL FITC-conjugated AffiniPure anti-human
IgG Fc.gamma.-fragment-specific goat F(ab') 2 fragment (Jackson
ImmunoResearch, Cat.-No. 109-096-098) or 5 .mu.g/mL PE-conjugated
AffiniPure anti-human IgG Fg.gamma.-fragment-specific goat F(ab')2
fragment (Jackson ImmunoResearch, Cat. No. 109-116-098 or
109-116-170) for 30 minutes at 4.degree. C. Cells were washed twice
with 200 .mu.L 4.degree. C. FACS buffer and then resuspended in 50
.mu.L/well DPBS containing 1% formaldehyde. The same or the next
day cells were resuspended in 100 .mu.L FACS-buffer and acquired
using 5-laser LSR-FORTESSA.RTM. (BD Bioscience with DIVA software)
or 3-laser Miltenyi Quant Analyzer 10 (Mitenyi Biotec) and Flow Jo
(FlowJo X 10.0.7).
[0630] As shown in FIGS. 10A and 10B, the FAP-targeted 4-1BB ligand
trimer-containing Fc(kih) fusion antigen binding molecule (FAP
split 4-1BBL trimer) Construct 1.1, but not the untargeted,
DP47-Fab-containing construct (DP47 split 4-1BBL trimer) Control A,
efficiently bound to human fibroblast activation protein
(FAP)-expressing melanoma (10A) MV-3 cells or (10B) WM-266-4
cells.
[0631] FIGS. 11A-1 to 11A-4 shows the binding of Constructs 1.1 to
1.10 as prepared in Example 1 to human-FAP expressing human
melanoma MV-3 cells and in FIGS. 11B-1 and 11B-2 the binding of
Construct 1.1., 1.2, 1.3 and 1.5 to human FAP expressing
NIH/3T3-huFAP clone 39 transfected mouse embryonic fibroblast cells
is presented. Table 37 shows the EC.sub.50 values as measured for
Constructs 1.1 to 1.10.
TABLE-US-00040 TABLE 37 Binding to human FAP-expressing tumor cells
EC.sub.50 [nM] EC.sub.50 [nM] Construct FAP.sup.+ MV-3 NIH/3T3-hu
FAP 1.1 4.14 12.2 1.2 5.36 9.35 1.3 -- 14.97 1.4 5.13 -- 1.5 0.53
10.06 1.6 8.16 -- 1.7 4.09 -- 1.8 2.79 -- 1.9 4.22 -- 1.10 4.31
--
[0632] FIGS. 12A-1 to 12B-2 shows the binding of different FAP
(4B9)-targeted or untargeted split trimeric human 4-1BB ligand Fc
(kih) constructs to human-FAP expressing human melanoma MV-3 cells
(FIGS. 12A-1 and 12A-2) and WM-266-4 cells (FIGS. 12B-1 and 12B-2).
The constructs 2.1, 2.3, 2.4, 2.5 and 2.6 were prepared as
described in Example 2 and Controls were prepared as described
herein before. Gates were set on living tumor cells and MFI of
PE-conjugated AffiniPure anti-human IgG Fc.gamma.-fragment-specific
goat F(ab')2 fragment were blotted against the titrated
concentration of targeted split trimeric 4-1BB ligand Fc fusion
constructs. Table 38 shows the EC.sub.50 values as measured.
TABLE-US-00041 TABLE 38 Binding to human FAP-expressing tumor cells
EC.sub.50 [nM] EC.sub.50 [nM] Construct FAP.sup.+ MV-3 FAP.sup.+
WM-266-4 2.1 1.66 0.99 2.3 0.53 0.42 2.4 0.83 0.59 2.5 1.66 1.2
[0633] 5.4 Functional Characterization of the Murine Targeted 4-1BB
Ligand Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecules
[0634] 5.4.1 Binding to Activated Mouse Splenocytes
[0635] Mouse spleens were collected in 3 mL PBS and a single cell
suspension was generated using gentle MACS tubes (Miltenyi Biotec
Cat.-No. 130-096-334) and gentleMACS Octo Dissociator (Miltenyi
Biotec). Afterwards splenocytes were filtered through 30 .mu.m
Pre-Separation Filters (Miltenyi Biotec Cat.-No. 130-041-407) and
centrifuged for 7 min at 350.times.g and 4.degree. C. Supernatant
was aspirated and cells were resuspended in RPMI 1640 medium
supplied with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 1 mM
Sodium-Pyruvate, 1% (v/v) MEM non-essential amino acids, 50 .mu.M
.beta.-Mercaptoethanol.
[0636] For binding on fresh mouse splenocytes cells were used
directly. To induce mouse 4-1BB expression on T cells, mouse
splenocytes were activated as following: 10.sup.6 cells/mL were
cultured for 2 days in a 6-well tissue culture plate coated with 10
.mu.g/mL anti-mouse CD3c Armenian Hamster IgG (clone 145-2C11,
BioLegend, Cat.-No. 100331) and 2 .mu.g/mL anti-mouse CD28 Syrian
Hamster IgG (clone 37.51, BioLegend, Cat.-No. 102102).
[0637] Fresh mouse splenocytes or activated mouse splenocytes were
collected, washed in DPBS (Gibco life technologies, Cat.-No.
14190-136), counted and 0.1.times.10.sup.6 cells were transferred
to each well of a 96 U-bottom non-tissue culture treated well plate
(Greiner bio-one, cell star, Cat.-No. 650185). Supernatant was
removed and cells were stained in 100 uL/well 4.degree. C. cold
DPBS containing 1:1000 diluted LIVE/DEAD.RTM. Fixable Aqua Dead
Cell Stain Kit (Life Technologies, L34957) for 30 min at 4.degree.
C. Cells were washed with cold DPBS and stained in 50 uL/well cold
FACS buffer (DPBS supplied with 2% (v/v) FBS, 5 mM EDTA pH8
(Amresco, Cat. No. E177) and 7.5 mM Sodium azide (Sigma-Aldrich
S2002)) containing different concentration of mouse 4-1BB ligand
trimer-containing Fc(kih) fusion molecules or mouse IgG1 Isotype
control (BioLegend, Cat.-No. 400153, clone MOPC-21). Cells were
incubated for 120 min at 4.degree. C., washed four times with cold
DPBS and stained in 50 .mu.L/well cold FACS buffer containing 30
.mu.g/mL FITC-conjugated anti-mouse IgG Fc-gamma-specific goat IgG
F(ab')2 (Jackson Immunoresearch, Cat.-No. 115-096-071) for 30 min
at 4.degree. C. Afterwards cells were washed twice with cold DPBS
and stained with 50 .mu.L/well FACS buffer supplied with 10
.mu.g/mL purified anti-mouse CD16/CD32 rat IgG-Fc-Block (BD
Pharmingen, Cat.-No. 553142 clone 2.4G2), 0.67 .mu.g/mL anti-mouse
CD8a-APC-Cy7 (BioLegend, Cat.-No. 100714, clone 53-6.7), 0.67
.mu.g/mL anti-mouse CD3c-PerCP-Cy5.5 (BioLegend, Cat.-No. 100328,
clone 145-2C11), 0.67 .mu.g/mL anti-mouse CD4 rat
IgG2b.kappa.-PE-Cy7 (BioLegend, Cat.-No. 100422, clone GK1.5) for
30 min at 4.degree. C. Cells were washed twice with 200 .mu.L/well
cold DPBS, fixed with 50 .mu.L/well DPBS containing 1% Formaldehyde
and resuspended in FACS-buffer. Cells were acquired using 3-laser
MACSQuant Analyzer 10 (Miltenyi Biotech) and Flow Jo v10.0.7
(FlowJo LLC). Gates were set on CD3.sup.+CD8.sup.+ or
CD3.sup.+CD4.sup.+ T cells and the median florescence intensity
(MFI) of FITC-conjugated anti-mouse IgG Fc-gamma-specific goat IgG
F(ab')2 was analyzed and normalized by the subtraction of the MFI
of the blank control (no addition of mouse 4-1BB ligand
trimer-containing Fc(kih) fusion molecule). The MFI was blotted
against the concentration of used mouse 4-1BB ligand
trimer-containing Fc(kih) fusion molecules to display the binding
to mouse 4-1BB cell-bound molecule.
[0638] As can be seen in FIGS. 13A-1 to 13B-2, the murine 4-1BBL
Constructs M.1 and M.2 as well as corresponding control molecules
Control M.1 and Control M.2 bind with a quite similar affinity to
mouse 4-1BB. Table 39 shows the EC50 values as measured for
Constructs M.1 and M.2 and the control molecules.
TABLE-US-00042 TABLE 39 Binding on activated 4-1BB-expressing CD4+
T cells and CD8 + T cells EC.sub.50 [nM] EC.sub.50 [nM] Construct
4-1BB.sup.+CD8.sup.+ 4-1BB.sup.+CD4.sup.+ Control M.1 0.95 0.74 M.1
0.87 0.52 Control M.2 0.78 0.6 M.2 0.54 0.42
[0639] 5.4.2 Binding on FAP-Expressing Tumor Cells
[0640] For binding assays on FAP expressing cells, the human
melanoma cell line MV-3 (see Ruiter et al., Int. J. Cancer 1991,
48(1), 85-91) and WM-266-4 (ATTC CRL-1676) were used (anti-FAP
specific clone 28H1 is mouse/human-crossreactive).
0.1.times.10.sup.6 of FAP expressing tumor cells were added to each
well of a round-bottom suspension cell 96-well plates (Greiner
bio-one, cellstar, Cat.-No. 650185). Cells were washed once with
200 .mu.L cold DPBS and pellets were resuspended in 100 .mu.L/well
of 4.degree. C. cold DPBS buffer containing 1:1000 diluted
LIVE/DEAD.RTM. Fixable Aqua Dead Cell Stain Kit (Life Technologies,
L34957) and incubated for 30 min at 4.degree. C. Cells were washed
once with 200 .mu.L cold DPBS buffer and resuspended in 50
.mu.L/well of cold FACS buffer (DPBS supplied with 2% (v/v) FBS, 5
mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM Sodium azide
(Sigma-Aldrich S2002)) containing murine 4-1BB ligand
trimer-containing Fc(kih) fusion molecules at a series of
concentrations followed by incubation for 1 hour at 4.degree. C.
After washing four times with 200 .mu.L DPBS/well, cells were
stained with 50 .mu.L/well of 4.degree. C. cold FACS buffer
containing 30 .mu.g/mL FITC-conjugated anti-mouse IgG
Fc-gamma-specific goat IgG F(ab')2 (Jackson Immunoresearch,
Cat.-No. 115-096-071) for 30 min at 4.degree. C. Cells were washed
twice with 200 .mu.L/well cold DPBS buffer, fixed with 50
.mu.L/well DPBS containing 1% Formaldehyde and resuspended in
FACS-buffer. Cells were acquired using 3-laser MACSQuant Analyzer
10 (Miltenyi Biotech) and Flow Jo v10.0.7 (FlowJo LLC). Gates were
set on living cells and the median florescence intensity (MFI) of
FITC-conjugated anti-mouse IgG Fc-gamma-specific goat IgG F(ab')2
was analyzed and normalized by the subtraction of the MFI of the
blank control (no addition of mouse 4-1BB ligand trimer-containing
Fc(kih) fusion molecule). The MFI was blotted against the
concentration of used murine 4-1BB ligand trimer-containing Fc(kih)
fusion molecules to display the binding to murine 4-1BB cell-bound
molecule. As expected, the murine 4-1BBL constructs M.1 and M.2
bind with a quite similar affinity to FAP whereas the control
molecules do not bind.
[0641] FIGS. 14 and 14B shows the binding of the FAP-targeted or
untargeted split trimeric murine 4-1BB ligand Fc (kih) Constructs
M.1 and M.2 to human-FAP expressing human melanoma MV-3 cells (FIG.
14A) and WM-266-4 cells (FIG. 14B). Table 40 shows the EC.sub.50
values as measured.
TABLE-US-00043 TABLE 40 Binding to human FAP-expressing tumor cells
EC.sub.50 [nM] EC.sub.50 [nM] Construct FAP.sup.+ MV-3 FAP.sup.+
WM-266-4 M.1 7.26 5.14 M.2 6.9 5.63
Example 6
Biological Activity of the Targeted 4-1BB Ligand Trimer-Containing
Fc Fusion Antigen Binding Molecules
[0642] 6.1. NF-.kappa.B Activation in HeLa Cells Expressing Human
4-1BB
[0643] Generation of HeLa Cells Expressing Human 4-1BB and
NF-.kappa.B-Luciferase
[0644] The cervix carcinoma cell line HeLa (ATCC CCL-2) was
transduced with a plasmid based on the expression vector pETR10829,
which contains the sequence of human 4-1BB (Uniprot accession
Q07011) under control of a CMV-promoter and a puromycin resistance
gene. Cells were cultured in DMEM medium supplemented with 10%
(v/v) FBS, 1% (v/v) GlutaMAX-I and 3 .mu.g/mL Puromycin.
[0645] 4-1BB-transduced HeLa cells were tested for 4-1BB expression
by flow cytometry: 0.2.times.10.sup.6 living cells were resuspended
in 100 .mu.L FACS buffer containing 0.1 .mu.s PerCP/Cy5.5
conjugated anti-human 4-1BB mouse IgG1.kappa.clone 4B4-1 (BioLegend
Cat.-No. 309814) or its isotype control (PerCP/Cy5.5 conjugated
mouse IgG1.kappa.isotype control antibody clone MOPC-21, BioLegend
Cat.-No. 400150) and incubated for 30 minutes at 4.degree. C. Cells
were washed twice with FACS buffer, resuspended in 300 .mu.L FACS
buffer containing 0.06 .mu.g DAPI (Santa Cruz Biotec, Cat. No.
Sc-3598) and acquired using a 5-laser LSR-FORTESSA.RTM. (BD
Bioscience, DIVA software). Limited dilutions were performed to
generate single clones as described: human-4-1BB-transduced HeLa
cells were resuspended in medium to a density of 10, 5 and 2.5
cells/ml and 200 .mu.l of cell suspensions were transferred to
round bottom tissue-culture treated 96-well plates (6 plates/cell
concentration, TPP Cat.-No. 92697). Single clones were harvested,
expanded and tested for 4-1BB expression as described above. The
clone with the highest expression of 4-1BB (clone 5) was chosen for
subsequent transfection with the NF-.kappa.B-luciferase
expression-vector 5495p Tranlucent HygB. The vector confers
transfected cells both with resistance to Hygromycin B and capacity
to express luciferase under control of NF-kB-response element (back
bone vector Panomics, Cat.-No. LR0051 with introduced HyB
resistance). Human-4-1BB HeLa clone 5 cells were cultured to 70%
confluence. 50 .mu.g (40 .mu.L) linearized (restriction enzymes
AseI and SalI) 5495p Tranlucent HygB expression vector were added
to a sterile 0.4 cm Gene Pulser/MicroPulser Cuvette (Biorad,
Cat.-No, 165-2081). 2.5.times.10.sup.6 human-4-1BB HeLa clone 5
cells in 400 .mu.l supplement-free DMEM medium were added and mixed
carefully with the plasmid solution. Transfection of cells was
performed using a Gene Pulser Xcell total system (Biorad, Cat-No.
165-2660) under the following settings: exponential pulse,
capacitance 500 g, voltage 160 V, resistance 09. Immediately after
the pulse transfected cells were transferred to a 75 cm.sup.2
tissue culture flask (TPP, Cat.-No. 90075) with 15 mL 37.degree. C.
warm DMEM medium supplied with 10% (v/v) FBS and 1% (v/v)
GlutaMAX-I. Next day, culture medium containing 3 .mu.g/mL
Puromycin and 200 .mu.g/mL Hygromycin B (Roche, Cat.-No.
10843555001) was added. Surviving cells were expanded and limited
dilution was performed as described above to generate single
clones.
[0646] Clones were tested for 4-1BB expression as described above
and for NF-.kappa.B-Luciferase activity as following: Clones were
harvested in selection medium and counted using a Cell Counter
Vi-cell xr 2.03 (Beckman Coulter, Cat.-No. 731050). Cells were set
to a cell density of 0.33.times.10.sup.6 cells/mL and 150 .mu.L of
this cell suspension were transferred to each well of a sterile
white 96-well flat bottom tissue culture plate with lid (greiner
bio-one, Cat.-No. 655083) and--as a control--to normal 96-well flat
bottom tissue culture plate (TPP Cat.-No. 92096) to test survival
and cell density the next day. Cells were incubated at 37.degree.
C. and 5% CO.sub.2 overnight. The next day 50 .mu.L of medium
containing different concentrations of recombinant human tumor
necrosis factor alpha (rhTNF-.alpha., PeproTech, Cat.-No. 300-01A)
were added to each well of a 96-well plate resulting in final
concentration of rhTNF-.alpha. of 100, 50, 25, 12.5, 6.25 and 0
ng/well. Cells were incubated for 6 hours at 37.degree. C. and 5%
CO.sub.2 and then washed three times with 200 .mu.L/well DPBS.
Reporter Lysis Buffer (Promega, Cat-No: E3971) was added to each
well (40 .mu.l) and the plates were stored over night at
-20.degree. C. The next day frozen cell plates and Detection Buffer
(Luciferase 1000 Assay System, Promega, Cat.-No. E4550) were thawed
to room temperature. 100 uL of detection buffer were added to each
well and the plate was measured as fast as possible using a
SpectraMax M5/M5e microplate reader and the SoftMax Pro Software
(Molecular Devices). Measured units of released light for 500
ms/well (URLs) above control (no rhTNF-.alpha. added) were taken as
luciferase activity. The NF-.kappa.B-luc-4-1BB-HeLa clone 26
exhibiting the highest luciferase activity and a considerable level
of 4-1BB-expression and was chosen for further use.
[0647] NF-.kappa.B Activation in Hela Cells Expressing Human 4-1BB
Co-Cultured with FAP-Expressing Tumor Cells
[0648] NF-.kappa.B-luciferase human-4-1BB HeLa cells were harvested
and resuspended in DMEM medium supplied with 10% (v/v) FBS and 1%
(v/v) GlutaMAX-I to a concentration of 0.2.times.10.sup.6 cells/ml.
100 .mu.l (2.times.10.sup.4 cells) of this cell suspension were
transferred to each well of a sterile white 96-well flat bottom
tissue culture plate with lid (greiner bio-one, Cat. No. 655083)
and the plate were incubated at 37.degree. C. and 5% CO.sub.2
overnight. The next day 50 .mu.L of medium containing titrated
concentrations of FAP-targeted 4-1BB ligand trimer-containing Fc
fusion antigen binding molecules (FAP split 4-1BBL trimer) or
DP47-untargeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules (DP47 split 4-1BBL trimer) were added.
FAP-expressing tumor cells (MV3, WM-266-4 or NIH/3T3-huFAP clone
39) were resuspended in DMEM medium supplied with 10% (v/v) FBS and
1% (v/v) GlutaMAX-I to a concentration of 2.times.10.sup.6
cells/ml.
[0649] Suspension of FAP-expressing tumor cell (50 final ratio 1:5)
or only medium were added to each well and plates were incubated
for 6 hours at 37.degree. C. and 5% CO.sub.2. Cells were washed two
times with 200 .mu.L/well DPBS. 40 .mu.l freshly prepared Reporter
Lysis Buffer (Promega, Cat-No: E3971) were added to each well and
the plate were stored over night at -20.degree. C. The next day
frozen cell plate and Detection Buffer (Luciferase 1000 Assay
System, Promega, Cat. No. E4550) were thawed at room temperature.
100 .mu.L of detection buffer were added to each well and
luciferase activity was measured as fast as possible using a
SpectraMax M5/M5e microplate reader and a SoftMax Pro Software
(Molecular Devices) counting light emission in URL (units of
released light for 0.5s/well) or Victor3 1420 multilabel counter
plate reader (Perkin Elmer) and the Perkin Elmer 2030 Manager
Software counting light emission as counts per seconds (CPS) and
blotted against the concentration of tested constructs.
[0650] FAP-targeted 4-1BB ligand trimer-containing Fc fusion
antigen binding molecule (FAP split 4-1BBL trimer) triggered
activation of the NF.kappa.B signaling pathway in the reporter cell
line in the presence of FAP-expressing tumor cells. In contrast,
the untargeted variant of the same molecule failed to trigger such
an effect at any of the tested concentrations (FIGS. 16A to 16C).
This activity of targeted 4-1BBL was strictly dependent on the
expression of FAP at the cell surface of tumor cells as no NF-kB
activation could be detected upon culturing of the NF-kB reporter
cell line with FAP-negative tumor cells even in the presence of
FAP-targeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecule. The activities as measured for Constructs 1.1 to
1.10 are shown in FIGS. 17A-1 to 17C-4 and the data as measured for
Constructs 2.1, 2.4 and 2.5 are presented in FIGS. 18A to 18F.
[0651] 6.2. NF.kappa.B Activation in HEK T293 Cells Expressing
Cynomolgus Monkey 4-1BB
[0652] Generation of HEK T293 Cells Expressing Cynomolgus Monkey
4-1BB and NF.kappa.B-Luciferase
[0653] For the production of viral-like particles (VLP) the Human
Embryonic Kidney (HEK) T293/17 (ATCC CRL-11268) was transfected
using Lipofectamine.RTM. LTX Reagent with PLUS.TM. Reagent (Life
Technologies, Cat.-No. 15338100) with the vector pETR14372 encoding
a NF.kappa.B-luciferase-IRIS-GFP reporter gene cassette
(NF.kappa.B-luc-GFP) accordingly to the manufacture's protocol. 6
hours later DMEM supplied with 10% FBS medium replacement was
performed and VLP were harvested 4 days later. Fresh HEK 293T cells
were transduced at a confluency of 70-80% with the produced
pETR14372-VLP and 4 .mu.g/mL polybrene. Cells were cultured for 24
h and a medium exchange was performed. The transduced HEK T293/17
cells were harvested and a limited dilution of 1 cell/well was
performed to screen for stable single clones. The single clones
were stimulated with 25 ng/mL TNF-.alpha. (PeproTech Inc. Cat.-No.
300-01A) in the medium and were screened for a positive GFP signal
over time using the Incuyte Zoom Fluorescence Microscope System
(Essen Bioscience). After GFP signal recording cells were tested
for luciferase activity using the NANO GLO.RTM. Luciferase Kit
(Promega, N1120) accordingly to the manufacture's protocol.
Luciferase activity was measured using Victor3 1420 multilabel
counter plate reader (Perkin Elmer) and the Perkin Elmer 2030
Manager Software. Light emission was counted in counts per seconds
(CPS) for 0.5 sec/well. The clone 61 showed the highest expression
of GFP and Luciferase after TNF-.alpha. activation and was further
used for the reporter cell line generation.
[0654] As described above, new VLP were produced using the vector
pETR14879 encoding cynomolgus monkey 4-1BB and a puromycine
resistance and the HEK 293T NF.kappa.B-fluc-GFP clone 61 cell line
was transduced at a confluency of 70-80% with the produced
pETR14879-VLP and 4 .mu.g/mL polybrene. Cells were cultured for 24
h and a medium exchange was performed. Four days after transduction
the cells were stained with PE-conjugated anti-human
cynomolgus-crossreactive 4-1BB antibody (mouse IgG1.kappa., clone
MOPC-21, BioLegend, Cat.-No. 309804) in DPBS containing 1% FBS,
were sorted by FACS (ARIA, BD) and seeded with 5 cells/well in DMEM
supplied with 10% FBS medium containing 1 .mu.g/mL Puromycine
(InvivoGen, Cat.-No. ant-pr). Growing clones were tested as
described for GFP and Luciferase activity after TNF-.alpha.
stimulation and for high cynomolgus monkey 4-1BB expression by flow
cytometry. Double positive clones were chosen and tested for
Luficerase activity in the presence of monovalent FAP-targeted
Construct 2.1 or Control B and FAP-expressing MV-3 or WM-266-4
cells. HEK T293/17-NF-.kappa.B-luc-GFP-cy4-1BB expressing Clone
61-13 was chosen to be used for all further experiments.
[0655] NF.kappa.B Activation of HEK T293/17 Reporter Cells
Expressing Cynomolgus Monkey 4-1BB Co-Cultured with FAP-Expressing
Tumor Cells
[0656] HEK T293/17-NF.kappa.B-luc-GFP-cy4-1BB expressing Clone
61-13 cells were harvested and resuspended in DMEM medium supplied
with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I to a concentration of
0.2.times.10.sup.6 cells/mL. 100 .mu.l (2.times.10.sup.4 cells) of
this cell suspension were transferred to each well of a sterile
white 96-well flat bottom tissue culture plate with lid (greiner
bio-one, Cat. No. 655083) and the plate were incubated at
37.degree. C. and 5% CO.sub.2 overnight. The next day 50 .mu.L of
medium containing different titrated concentrations of FAP-targeted
or untargeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules were added. FAP-expressing tumor cells (MV3 and
WM-266-4) were resuspended in medium to a concentration of
2.times.10.sup.6 cells/ml. Suspension of FAP-expressing tumor cell
(50 .mu.l) was added to each well and plates were incubated for 6
hours at 37.degree. C. and 5% CO.sub.2. The principle of the assay
is shown in FIGS. 19A and 19B. After incubation cells were washed
three times with 200 .mu.L/well DPBS. 40 .mu.l freshly prepared
Reporter Lysis Buffer (Promega, Cat-No: E3971) were added to each
well and plates were stored over night at -20.degree. C. The next
day frozen cell plates and detection buffer (Luciferase 1000 Assay
System, Promega, Cat. No. E4550) were thawed to room temperature.
100 .mu.L of detection buffer were added to each well and
luciferase activity was measured as fast as possible using
SpectraMax M5/M5e (Molecular Devices) microplate reader (500 ms
integration time, no filter collecting all wavelength). Light
emission was counted in units of released light (URL) for 0.5
sec/well and blotted against the concentration of tested
FAP-targeted or untargeted 4-1BB ligand trimer-containing Fc fusion
antigen binding molecules. The results for Constructs of Example 2
are shown in FIGS. 20A to 20F.
[0657] 6.3 Antigen-Specific CD8+ T Cell-Based Assay
[0658] Isolation and Culture of Antigen-Specific CD8 T Cells
[0659] Fresh blood was obtained from a HLA-A2+ CMV-infected
volunteer. PBMCs were isolated as described above. CD8 T cells were
purified from PBMCs using a negative selection human CD8 T cell
isolation Kit according to manufacturer's recommendations (Miltenyi
Biotec, Cat. No. 130-094-156). Ten million of isolated CD8 T cells
were resuspended in 1 mL sterile DPBS supplemented with 1% (v/v)
FBS along with 50 .mu.L of PE-labeled HLA-A2-pentamer containing
the CMV-derived NLVPMVATV peptide (SEQ ID NO: 377) (ProImmune, Cat.
No. F008-2B) and incubated for 10 min at room temperature. Cells
were washed twice with 3 mL sterile DPBS supplied with 1% (v/v)
FBS. Cells were resuspended in 1 mL cells DPBS supplied with 1%
(v/v) FBS containing 1 .mu.g/mL anti-human CD8-FITC (clone LT8,
Abcam, Cat. No. Ab28010) and incubated for 30 minutes at 4.degree.
C. Cells were washed twice, resuspended to a concentration of
5.times.10.sup.6 cells/mL in DPBS supplied with 1% (v/v) FBS, and
filtrated through a 30 .mu.m pre-separation nylon-net cell strainer
(Miltenyi Biotec, Cat. No. 130-041-407). NLV-peptide-specific CD8+
T cells were isolated by FACS sorting using an ARIA cell sorter (BD
Bioscience with DIVA software) with the following settings: 100
.mu.m nozzle and purity sort mask. Sorted cells were collected in a
15 ml polypropylene centrifuge tube (TPP, Cat. No. 91015)
containing 5 ml RPMI 1640 medium supplied with 10% (v/v) FBS, 1%
(v/v) GlutaMAX-I and 400 U/mL Proleukin. Sorted cells were
centrifuged for 7 minutes at 350.times.g at room temperature and
resuspended in same medium to a concentration of
0.53.times.10.sup.6 cells/mL. 100 .mu.L/well of this cell
suspension were added to each well of a previously prepared feeder
plate.
[0660] PHA-L-activated irradiated allogeneic feeder cells were
prepared from PBMCs as previously described (Levitsky et al., 1998)
and distributed to 96 well culture plates at 2.times.10.sup.5
feeder cells per well.
[0661] After one day of culturing 100 .mu.L medium/well were
removed from well containing sorted CD8+ T-cells and replaced by
new RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v)
GlutaMAX-I and 400 U/mL Proleukin, this was repeated during culture
on a regular basis (every 2-4 days). As soon as cells start to
proliferate, they were transferred to 24-well flat-bottom tissue
culture plate (TPP, 92024). Cells were expanded/split and
reactivated with new feeder cell preparation on a regular
basis.
[0662] Activation Assay of Antigen-Specific CD8+ T Cells
[0663] MV3 cells were harvested and washed with DPBS and
2.times.10.sup.7 cells were resuspended in 250 .mu.L C diluent of
the PKH-26 Red Fluorescence Cell linker Kit (Sigma, Cat.-No.
PKH26GL). 1 .mu.L PKH26-Red-stain solution was diluted with 250
.mu.L C diluent and added to the suspension of MV3 cells which were
then incubated for 5 min at room temperature in the dark. This was
followed by addition of 0.5 mL FBS and cells were incubated for 1
minute and washed once with T cell medium consisting of RPMI 1640
medium supplemented with 10% (v/v) FBS, 1% (v/v) GlutaMAX-I, 1 mM
Sodium-Pyruvate, 1% (v/v) MEM non-essential amino acids and 50
.mu.M .beta.-Mercaptoethanol. 1.times.10.sup.6 MV3 cells/mL were
resuspended in T cell medium and separated into three tubes.
Synthetic NLVPMVATV peptide (SEQ ID NO: 377) (obtained from think
peptides) was added to a final concentration of 1.times.10.sup.-9 M
or 1.times.10.sup.-8M and cells were incubated for 90 min. MV3
cells were washed once with T cell medium and resuspended to a
density of 0.5.times.10.sup.6 cells/mL, distributed (100
.mu.L/well) to a 96-well round bottom cell-suspension plate
(Greiner bio-one, cellstar, Cat.-No. 650185) and incubated over
night at 37.degree. C. and 5% CO.sub.2. The principle of the assay
is shown in FIGS. 21A and 21B.
[0664] The next day, 50 .mu.L/well T cell medium containing
different titrated concentrations of targeted 4-1BB ligand
trimer-containing Fc fusion antigen binding molecules were added.
NLV-specific CD8 T cells were harvested, CFDA-SE
(5(6)-Carboxyfluoresceindiacetate-N-succinimidylester,
SIGMA-Aldrich, Cat.-No. 21888-25MG-F) was added to a final
concentration of 40 nM and cells were incubated under rotation for
15 min at 37.degree. C. Labeling was stopped by adding FBS, cells
were washed and resuspended in T cell medium to a final
concentration of 0.125.times.10.sup.6 cells/mL. 50 .mu.L of this
CFSE-labeled CD8 T cell suspension were added to each well (final
E:T ratio=1:8). Cell plates were incubated for 24 h, 50 .mu.L/well
were removed and 50 .mu.L T cell medium containing 2.64 .mu.L/mL
Golgi stop (Protein Transport Inhibitor containing Monesin, BD
Bioscience, Cat.-No. 554724) were added to each well (final
concentration 0.66 .mu.L/mL). Cells were incubated for 4 h and then
plates were washed with 200 .mu.L/well DPBS and stained with 100
.mu.L/well 4.degree. C. DPBS containing 1:5000 diluted Fixable
Viability Dye-eF450 (eBioscience, Cat.-No. 65-0864) for 30 minutes
at 4.degree. C. Cell plates were washed with 200 .mu.L/well DPBS
followed by staining with fluorescent dye-conjugated antibodies:
anti-human CD137-PerCP/Cy5.5 (clone 4B4-1, mouse IgG1.kappa.,
BioLegend, Cat.-No. 309814), anti-human CD8-BV605 (clone RPA-T8,
mouse IgG1.kappa., BioLegend, Cat.-No. 301012) or 0.67 .mu.g/mL
anti-human CD8a-APC/Cy7 (clone RPA-T8, mouse IgG1.kappa.,
BioLegend, Cat.-No. 301016) and anti-human CD25 PE/Cy7 (clone BC96,
mouse IgG1.kappa., BioLegend, Cat.-No. 302612). After incubation
for 30 min at 4.degree. C., cells were washed twice with 200
.mu.L/well FACS buffer, resuspended in 50 .mu.L/well freshly
prepared FoxP3 Fix/Perm buffer (eBioscience Cat.-No. 00-5123 and
00-5223) and incubated for 30 min at 4.degree. C. Plates were
washed twice with 200 .mu.L/well Perm-Buffer (DPBS supplied with 2%
(v/v) FBS, 1% (w/v) saponin (Sigma Life Science, 57900) and 1%
(w/v) sodium azide (Sigma-Aldrich, 52002) and stained with 50
.mu.L/well Perm-Buffer (eBioscience, Cat.-No. 00-8333-56)
containing 0.25 .mu.g/mL anti-human IFN.gamma.-APC (clone B27,
mouse IgG1K, BioLegend, Cat.-No. 506510) or 0.33 .mu.s/mL
anti-human IFN.gamma.-BV510 (clone 4S.B3, mouse IgG1K, BioLegend,
Cat.-No. 502543). Plates were incubated for 1 h at 4.degree. C. and
washed twice with 200 .mu.L/well Perm-Buffer. For fixation, 50
.mu.L/well DPBS containing 1% formaldehyde were added. The same or
the next day, cells were resuspended in 100 .mu.L/well FACS buffer
and acquired using a 5-laser FORTESSA.RTM. flow cytometer (BD
Bioscience with DIVA software) or 3-laser Miltenyi Quant Analyzer
10 (Miltenyi Biotec) and Flow Jo (FlowJo X 10.0.7).
[0665] As shown in FIGS. 22A-1 to 22E-3 and FIGS. 23A-1 to 23E-3
for Constructs 1.1 to 1.10 and in FIGS. 24A-1 to 24B-3 and 25A-1 to
25B-3 for Constructs 2.1, 2.3 and 2.4, antigen-specific CD8+ T
cells, but not unstimulated controls, exhibited increased levels of
surface 4-1BB expression in the presence of FAP-targeted 4-1BB
ligand trimer-containing Fc fusion antigen binding molecule (FAP
split 4-1BBL trimer). This effect of 4-1BBL was dose dependent and
required FAP-targeting as addition of the untargeted control
molecule did not affect the level of 4-1BB expression. Furthermore,
T-cells activated at the higher peptide concentration
(1.times.10.sup.-8M) showed sustained secretion of INF.gamma. in
the presence of FAP-targeted 4-1BB ligand trimer-containing Fc
fusion antigen binding molecule (FAP split 4-1BBL trimer).
Collectively, these data demonstrate that the antigen-targeted
4-1BB ligand trimer-containing Fc fusion antigen binding molecule
modulates the surface phenotype and responsiveness of antigen
specific T-cells in a targeting dependent manner.
[0666] 6.4 Comparison of Cell-Targeted and Untargeted Mouse 4-1BBL
Fc Fusion Antigen Binding Molecules
[0667] Targeted and untargeted mouse 4-1BB ligand trimer-containing
Fc fusion antigen binding molecules (FAP split mouse 4-1BBL trimer
and DP47 split mouse 4-1BBL trimer) were prepared as described in
Example 1.3.
[0668] To compare the bioactivity of cell-targeted and untargeted
mouse 4-1BB ligand trimer-containing Fc fusion antigen binding
molecules, Proliferation Dye EFLUOR.RTM. 670-labeled (eBioscience,
Cat.-No. 65-0840-90) or CellTrace Violet Cell Proliferation
dye-labeled (Cell tracer, Cat.-No. C34557) fresh mouse splenocytes
were cocultured for 3-4 days in 96 well tissue culture U-bottom
plates (TTP, Cat.-No. 92097) with adherent 50 Gy irradiated
NIH/3T3-huFAP clone 39 cells (generation see 5.3) in RPMI 1640
medium (Gibco, Cat.-No. 42401-042) supplied with 10% (v/v) FBS, 1%
(v/v) GlutaMAX-I, 1 mM Sodium-Pyruvate, 1% (v/v) MEM non-essential
amino acids and 50 .mu.M .beta.-Mercaptoethanolin the presence of
0.5 .mu.g/mL anti-mouse CD3 Syrian hamster IgG (clone 145-2C11, BD,
Cat.-No. 553057) and the indicated drug candidate molecule added at
a range of concentrations (FIGS. 26A and 26B). After three or four
days, cells were washed with FACS buffer and stained for 30 min at
4.degree. C. in 25 uL FACS buffer/well containing anti-mouse CD8
ratIgG2a-BV711 (BioLegend, Cat.-No. 100747, clone 53-6.7,) and
anti-mouse CD4 ratIgG2a-BV421 (BioLegend, Cat.-No. 100544, clone
RM4-5) and 0.67 .mu.g/mL anti-mouse CD137 (4-1BB) Syrian hamster
IgG-PE (BioLegend, Cat.-No. 106106, clone 17B5) and anti-mouse
CD25-PErCP-Cy5.5 ratIgG2b (BioLegend, Cat.-No. 1019112). Cells were
washed and incubated for 1 h at room temperature in prepared
Fix/Perm Buffer (Foxp3/Transcription Factor Staining Buffer Set,
eBioscience, Cat.-Ni. 00-5523-00). Cells were washed twice with
freshly prepared Perm buffer and co-stained with 25 .mu.L/well
Perm-buffer containing fluorescently-labeled antibodies against the
cytotoxic lineage transcription factor Eomes, i.e. anti-mouse Eomes
ratIgG2a-ALEXA FLUOR.RTM. 488 (eBioscience, Cat.-No. 534875, clone
Dan11mag) and--if CD137 was not stained--against the cytotoxic
effector molecule granzyme B, i.e. anti-mouse ratIgG2a granzyme
B-PE (eBioscience, Cat.-No. 128822, clone 16G6) for 1 h at room
temperature. Cells were then washed twice, resuspended in FACS
buffer and acquired using laser FORTESSA.RTM. flow cytometer (BD
Bioscience with DIVA software) or the 3-laser MACSQuant Analyzer 10
(Miltenyi Biotech) and Flow Jo v10.0.7 (FlowJo LLC). Gates were set
on living CD8+ T cells and CD4+ T cells and the frequency of
proliferating cells was determined as well as the expression levels
of CD25, Eomes and granzyme B or CD137. The proliferation frequency
and frequencies and MFIs of activation markers were blotted against
the concentration of used mouse 4-1BB ligand trimer-containing
Fc(kih) fusion molecules to display the functional activity. As can
be seen in FIG. 27, an increase in proliferating CD8+ T cells could
be observed for Constructs M.1 and M.2.
[0669] 6.5 Liver Changes in Mice Treated with Anti-Murine 4-1BB
Antibody Lob 12.3 (muIgG1 Wt) or with Construct M.2
[0670] C57BL/6 mice bearing MC38-muFAP (murine colorectal cancer
model) s.c. were treated once per week for 3 weeks with agonistic
anti-murine 4-1BB antibodies targeted to FAP (Efficacy Study
020-GA1401: "Experiment to show efficacy of 4-1BB targeted therapy
in combination with a-PD-L1 in MC38-muFAP s.c. model in C57B6
mice."). Antibodies used were Lob 12.3 muIgG1 Wt (with "wildtype"
Fc, clone Lob 12.3 from BioXcell Catalog #: BE0169) or Construct
M.2 with DAPG mutation (inactive Fc). The two antibodies were
administered once weekly for three consecutive weeks. Four
animals/group were sacrificed 7 days after last treatment and
livers examined microscopically.
[0671] Liver changes were observed only in animals receiving Lob
12.3 muIgG1 Wt, consisting in foci of hepatocellular degeneration
with accumulation of F4/80 positive macrophages and a lower amount
of mixed population of inflammatory cells (mainly lymphocytes)
frequently showing a vasocentric distribution. Occasionally single
cell necrosis of hepatocytes, and perivascular mononuclear cell
infiltrates in portal spaces were noted. No treatment related
findings were observed in the liver of animals receiving Construct
M.2 (Table 41).
TABLE-US-00044 TABLE 41 Incidence of Histopathogical Findings (n =
4/group) Lob 12.3 Construct Treatment Vehicle muIgG1 Wt M.2 Foci of
hepatocellular degeneration with -- 4 -- macrophages and
inflammatory cells Perivascular inflammatory cells infiltrates -- 4
-- Single cell necrosis -- 4 --
[0672] Hepatitis, attributed to crosslinking by Fc.gamma.Rs in the
liver, has been observed in patients treated with Urelumab
BMS-663513 (Ascierto P. A. et al. 2010) and in mouse using the
mouse surrogate. The absence of liver findings in animals treated
with an antibody with inactive Fc support this hypothesis.
[0673] 6.6 Determination of Pharmacokinetic Parameters of Human
4-1BB Ligand Trimer-Containing Fc Fusion Antigen Binding
Molecules
[0674] In order to test if the human 4-1BB ligand trimer-containing
Fc fusion antigen binding molecules of the invention are suitable
for pharmaceutical use, the pharmacokinetic parameters (PK data)
such as clearance, volume of distribution or elimination half-time
(t.sub.1/2) in mice were determined. Thus, the following
experiments were carried out:
Experiment A: Single Dose PK of Construct 1.2 and Control B in
Healthy NOG Mice
[0675] NOG female mice at an average age of 8 to 10 weeks at start
of experiment (purchased from Taconic, SOPF facility) were
maintained under specific-pathogen-free condition with daily cycles
of 12 h light/12 h darkness according to committed guidelines
(GV-Solas; Felasa; TierschG). Experimental study protocol was
reviewed and approved by local government (P 2011128). After
arrival animals were maintained for one week to get accustomed to
new environment and for observation. Continuous health monitoring
was carried out on regular basis.
[0676] A single dose pharmacokinetic study (SDPK) was performed to
evaluate exposure of Construct 1.2 and Control B. An i.v. bolus
administration of 2.5 mg/kg was administered to NOG mice and blood
samples were taken at selected time points for pharmacokinetic
evaluation. Mouse serum samples were analyzed by ELISA.
Biotinylated human 4-1BB, test samples, Digoxygenin labelled
anti-huCH1 antibody and anti-Digoxygenin detection antibody (POD)
were added stepwise to a 96-well streptavidin-coated microtiter
plate and incubated after every step for 1h at room temperature.
The plate was washed three times after each step to remove unbound
substances. Finally, the peroxidase-bound complex was visualized by
adding ABTS substrate solution to form a colored reaction product.
The reaction product intensity which was photometrically determined
at 405 nm (with reference wavelength at 490 nm) is proportional to
the analyte concentration in the serum sample. The calibration
range of the standard curve for the constructs was 0.156 to 10
ng/ml, where 3 ng/ml is the lower limit of quantification (LLOQ).
FIG. 28A shows the decrease in concentration over the time as
observed in this experiment.
Experiment B: Single Dose PK of Constructs 2.1, 2.3, Control B and
Control C in Tumor Bearing NOG Mice Humaniced with Stem Cells
[0677] A single dose pharmacokinetic study (SDPK) was performed to
evaluate exposure of Construct 2.1, 2.3, Control B and Control C.
NSG female mice transferred with human stem cells were delivered by
Jackson Laboratories. Mice were maintained under
specific-pathogen-free condition with daily cycles of 12 h light/12
h darkness according to committed guidelines (GV-Solas; Felasa;
TierschG). Experimental study protocol was reviewed and approved by
local government (ZH193-2014). After arrival animals were
maintained for one week to get accustomed to new environment and
for observation. Continuous health monitoring was carried out on
regular basis.
[0678] Human MKN45 cells (human gastric carcinoma) were originally
obtained from ATCC and after expansion deposited in the Glycart
internal cell bank. Cells were cultured in DMEM containing 10% FCS.
Cells were cultured at 37.degree. C. in a water-saturated
atmosphere at 5% CO.sub.2. In vitro passage 9 was used for
subcutaneous injection, at a viability of 97%. Human fibroblasts
NIH-3T3 were engineered at Roche Nutley to express human FAP. Clone
39 was used at an in vitro passage number 12 and at a viability of
98%. 50 microliters cell suspension (1.times.106 MKN45
cells+1.times.106 3T3-huFAP) mixed with 50 microliters Matrigel
were injected subcutaneously in the flank of anaesthetized mice. An
i.v. bolus administration of 10 mg/kg was administered to humaniced
mice when tumor reached an average size of 190 mm.sup.3. Blood
samples were taken at selected time points for pharmacokinetic
evaluation. Mouse serum samples were analyzed by ELISA.
Biotinylated human 4-1BB, test samples, Digoxygenin labelled
anti-huCH1 antibody and anti-Digoxygenin detection antibody (POD)
were added stepwise to a 96-well streptavidin-coated microtiter
plate and incubated after every step for 1h at room temperature.
The plate was washed three times after each step to remove unbound
substances. Finally, the peroxidase-bound complex is visualized by
adding ABTS substrate solution to form a colored reaction product.
The reaction product intensity which was photometrically determined
at 405 nm (with reference wavelength at 490 nm) is proportional to
the analyte concentration in the serum sample. The calibration
range of the standard curve for the constructs was 0.156 to 10
ng/ml, where 3 ng/ml is the lower limit of quantification (LLOQ).
FIG. 28B shows the decrease in concentration of the constructs over
the time as observed in this experiment.
Experiment C: Single Dose PK of Construct 2.1 and 2.3 in Healthy
NOG Mice
[0679] NOG female mice at an average ager of 8-10 weeks at start of
experiment (purchased from Taconic, SOPF facility) were maintained
under specific-pathogen-free condition with daily cycles of 12 h
light/12 h darkness according to committed guidelines (GV-Solas;
Felasa; TierschG). Experimental study protocol was reviewed and
approved by local government (P 2011128). After arrival animals
were maintained for one week to get accustomed to new environment
and for observation. Continuous health monitoring was carried out
on regular basis.
[0680] A single dose pharmacokinetic study (SDPK) was performed to
evaluate exposure of Construct 2.1 and 2.3. An i.v. bolus
administration of 2.5 mg/kg was administered to NOG mice and blood
samples were taken at selected time points for pharmacokinetic
evaluation. Mouse serum samples were analyzed by ELISA.
Biotinylated human 4-1BB, test samples, Digoxygenin labelled
anti-huCH1 antibody and anti-Digoxygenin detection antibody (POD)
were added stepwise to a 96-well streptavidin-coated microtiter
plate and incubated after every step for 1h at room temperature.
The plate is washed three times after each step to remove unbound
substances. Finally, the peroxidase-bound complex is visualized by
adding ABTS substrate solution to form a colored reaction product.
The reaction product intensity, which is photometrically determined
at 405 nm (with reference wavelength at 490 nm), is proportional to
the analyte concentration in the serum sample. The calibration
range of the standard curve for the constructs was 0.156 to 10
ng/ml, where 3 ng/ml is the lower limit of quantification (LLOQ).
FIG. 28C shows the observed decrease in concentration over the
time.
[0681] The tested constructs 2.1 and 2.3 are stable enough in the
body and possess PK parameters in a suitable range for
pharmaceutical development. It can also be concluded from the
results that construct 2.1 is slightly more stable.
[0682] 6.7 FAP Prevalence in Human Tumors
[0683] The prevalence of FAP in human tumors was evaluated as
described in WO 2014/161845 to get an understanding on possible
clinical use of FAP-targeted constructs.
[0684] Rat anti-human Seprase antibody (IgG2a, clone D8) from
Vitatex (MABS1001) was used to immunostain 2,5 .mu.m FFPET sections
from various tumour indications on the Ventana Benchmark XT.
Sections were subjected to standard CC1 treatment followed by
antibody incubation for 60' at 37.degree. C. at a concentration of
5 .mu.g/mL in Dako antibody diluent (S3022) and positive staining
was detected using the Ultraview DAB detection system (Ventana
#760-4456). Matched isotype antibody from Abcam (ab18450) was used
as the negative control. FAP+ stromal infiltrate was present in
human tumors of different indications including head and neck
squamous cell carcinoma (HNSCC), breast cancer, colorectal cancer
(CRC), pancreatic cancer (PAC), gastric cancer, non-small-cell lung
carcinoma (NSCLC) and Mesothelioma marking potentially interesting
clinical indications for a FAP-targeted constructs (Table 42).
TABLE-US-00045 TABLE 42 FAP prevalence in human tumors % cases with
moderate to high grade of FAP.sup.+ No. of samples Tumor Type
infiltrate investigated HNSCC 90 10 Breast Cancer 77 105 triple
negative BC 80 7 CRC 77 90 PAC 74 19 Gastric Cancer 68 28 NSCLC 66
90 Mesothelioma 60 10
Example 7
[0685] 7.1 Preparation of CD19 (8B8-018) Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0686] 7.1.1 Preparation, Purification and Characterization of CD19
Antigen Fc Fusion for Phage Display Campaign
[0687] In order to express and purify the human and cynomolgus CD19
ectodomain in a monomeric state (human CD19 see SEQ ID NO:31), the
respective DNA fragment was fused to a human IgG1 Fc gene segment
containing the "knob" mutations (human: SEQ ID NO: 186; cynomolgus:
SEQ ID NO: 188) and was transfected with an "Fc-hole" (SEQ ID NO:
86) counterpart (Merchant et al., 1998). An IgA cleavage site
(PTPPTP; SEQ ID NO: 378) was introduced between an antigen
ectodomain and the Fc knob chain. An Avi tag for directed
biotinylation was introduced at the C-terminus of the antigen-Fc
knob chain and mutations H435R and Y436F were introduced in the Fc
hole for purification purposes (Jendeberg L. et al, J.
Immunological methods, 1997). Combination of the antigen-Fc knob
chain containing the S354C/T366W mutations (human: SEQ ID NO: 187;
cynomolgus: SEQ ID NO: 189), with a Fc hole chain containing the
Y349C/T366S/L368A/Y407V mutations (SEQ ID NO: 90) allows generation
of a heterodimeric Fc fusion fragment which includes a single copy
of the CD19 ectodomain (in analogy to the 4-1BB construct in FIG.
5C). Table 43 lists the cDNA and amino acid sequences of the
antigen Fc-fusion construct.
TABLE-US-00046 TABLE 43 cDNA and Amino acid sequences of monomeric
human and cynomolgus CD19 antigen Fc(kih) fusion molecule SEQ ID
NO: Antigen Sequence 86 Nucleotide see Table 32 sequence Fc hole
chain 186 Nucleotide CCCGAGGAACCCCTGGTCGTGAAGGTGGAAGAGGGCGACAAT
sequence GCCGTGCTGCAGTGCCTGAAGGGCACCTCCGATGGCCCTACC human CD19
CAGCAGCTGACCTGGTCCAGAGAGAGCCCCCTGAAGCCCTTC antigen Fc
CTGAAGCTGTCTCTGGGCCTGCCTGGCCTGGGCATCCATATG knob chain
AGGCCTCTGGCCATCTGGCTGTTCATCTTCAACGTGTCCCAG avi tag
CAGATGGGCGGCTTCTACCTGTGTCAGCCTGGCCCCCCATCT
GAGAAGGCTTGGCAGCCTGGCTGGACCGTGAACGTGGAAGGA
TCCGGCGAGCTGTTCCGGTGGAACGTGTCCGATCTGGGCGGC
CTGGGATGCGGCCTGAAGAACAGATCTAGCGAGGGCCCCAGC
AGCCCCAGCGGCAAACTGATGAGCCCCAAGCTGTACGTGTGG
GCCAAGGACAGACCCGAGATCTGGGAGGGCGAGCCTCCTTGC
CTGCCCCCTAGAGACAGCCTGAACCAGAGCCTGAGCCAGGAC
CTGACAATGGCCCCTGGCAGCACACTGTGGCTGAGCTGTGGC
GTGCCACCCGACTCTGTGTCTAGAGGCCCTCTGAGCTGGACC
CACGTGCACCCTAAGGGCCCTAAGAGCCTGCTGAGCCTGGAA
CTGAAGGACGACAGGCCCGCCAGAGATATGTGGGTCATGGAA
ACCGGCCTGCTGCTGCCTAGAGCCACAGCCCAGGATGCCGGC
AAGTACTACTGCCACAGAGGCAACCTGACCATGAGCTTCCAC
CTGGAAATCACCGCCAGACCCGTGCTGTGGCACTGGCTGCTG
AGAACAGGCGGCTGGAAGGTCGACGCTAGCGGTGGTAGTCCG
ACACCTCCGACACCCGGGGGTGGTTCTGCAGACAAAACTCAC
ACATGCCCACCGTGCCCAGCACCTGAAGCCGCAGGGGGACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG
CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG
GTCTCCAACAAAGCCCTCGGAGCCCCCATCGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACC
CTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGC
CTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG
ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCC
GGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGAATGG CACGAG 90 Polypeptide
see Table 32 sequence Fc hole chain 187 Polypeptide
PEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPF sequence
LKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPS human CD19
EKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPS antigen Fc
SPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQD knob chain
LTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLE avi tag
LKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFH
LEITARPVLWHWLLRTGGWKVDASGGSPTPPTPGGGSADKTH
TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYT
LPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 188 Nucleotide
CCCCAGGAACCCCTGGTCGTGAAGGTGGAAGAGGGCGACAAT sequence
GCCGTGCTCCAGTGCCTGGAAGGCACCTCCGATGGCCCTACA cynomolgus
CAGCAGCTCGTGTGGTGCAGAGACAGCCCCTTCGAGCCCTTC CD19 antigen
CTGAACCTGTCTCTGGGCCTGCCTGGCATGGGCATCAGAATG Fc knob
GGCCCTCTGGGCATCTGGCTGCTGATCTTCAACGTGTCCAAC chain avi tag
CAGACCGGCGGCTTCTACCTGTGTCAGCCTGGCCTGCCAAGC
GAGAAGGCTTGGCAGCCTGGATGGACCGTGTCCGTGGAAGGA
TCTGGCGAGCTGTTCCGGTGGAACGTGTCCGATCTGGGCGGC
CTGGGATGCGGCCTGAAGAACAGAAGCAGCGAGGGCCCTAGC
AGCCCCAGCGGCAAGCTGAATAGCAGCCAGCTGTACGTGTGG
GCCAAGGACAGACCCGAGATGTGGGAGGGCGAGCCTGTGTGT
GGCCCCCCTAGAGATAGCCTGAACCAGAGCCTGAGCCAGGAC
CTGACAATGGCCCCTGGCAGCACACTGTGGCTGAGCTGTGGC
GTGCCACCCGACTCTGTGTCCAGAGGCCCTCTGAGCTGGACA
CACGTGCGGCCAAAGGGCCCTAAGAGCAGCCTGCTGAGCCTG
GAACTGAAGGACGACCGGCCCGACCGGGATATGTGGGTGGTG
GATACAGGCCTGCTGCTGACCAGAGCCACAGCCCAGGATGCC
GGCAAGTACTACTGCCACAGAGGCAACTGGACCAAGAGCTTT
TACCTGGAAATCACCGCCAGACCCGCCCTGTGGCACTGGCTG
CTGAGAATCGGAGGCTGGAAGGTCGACGCTAGCGGTGGTAGT
CCGACACCTCCGACACCCGGGGGTGGTTCTGCAGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAAGCCGCAGGGGGA
CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC
ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC
GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCGGAGCCCCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC
CTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
TCCGGAGGCCTGAACGACATCTTCGAGGCCCAGAAGATTGA TGGCACGAG 189 Polypeptide
PQEPLVVKVEEGDNAVLQCLEGTSDGPTQQLVWCRDSPFEPF sequence
LNLSLGLPGMGIRMGPLGIWLLIFNVSNQTGGFYLCQPGLPS cynomolgus
EKAWQPGWTVSVEGSGELFRWNVSDLGGLGCGLKNRSSEGPS CD19 antigen
SPSGKLNSSQLYVWAKDRPEMWEGEPVCGPPRDSLNQSLSQD Fc knob
LTMAPGSTLWLSCGVPPDSVSRGPLSWTHVRPKGPKSSLLSL chain avi tag
ELKDDRPDRDMWVVDTGLLLTRATAQDAGKYYCHRGNWTKSF
YLEITARPALWHWLLRIGGWKVDASGGSPTPPTPGGGSADKT
HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVY
TLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE
[0688] For the production of the monomeric antigen/Fc fusion
molecules, exponentially growing suspension CHO cells were
co-transfected with two plasmids encoding the two components of
fusion protein (knob and hole chains) using standard methods.
[0689] Secreted protein was purified from cell culture supernatant
by affinity chromatography using Protein A, followed by size
exclusion chromatography. For affinity chromatography, the
supernatant was loaded on a MABSELECT SURE.RTM. column volume
(CV)=5-15 mL, resin from GE Healthcare) equilibrated with Sodium
Phosphate (20 mM), Sodium Citrate (20 mM), 0.5M sodium chloride
buffer (pH 7.5). Unbound protein was removed by washing with at
least 6 column volumes of the same buffer. The bound protein was
eluted using a linear gradient; step 1, 10 CV from 0 to 60% elution
buffer (20 mM sodium citrate, 500 mM Sodium chloride buffer (pH
2.5)); step 2, 2 CV from 60 to 100% elution buffer. For the linear
gradient an additional 2 column volumes step elution with 100%
elution buffer was applied.
[0690] The pH of collected fractions was adjusted by adding 1/40
(v/v) of 2M Tris, pH8.0. The protein was concentrated and filtered
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 2 mM MOPS, 150 mM sodium chloride,
0.02% (w/v) sodium azide solution of pH 7.4.
[0691] Table 44 summarizes the yield and final monomer content of
monomeric human and cynomolgus CD19 antigen Fc(kih) fusion
protein.
TABLE-US-00047 TABLE 44 Biochemical analysis of monomeric human and
cynomolgus CD19 antigen Fc(kih) fusion protein Monomer [%] Yield
Construct (SEC) [mg/l] monomeric human CD19 Fc(kih) fusion protein
91 0.2 monomeric cynomolgus CD19 Fc(kih) fusion 95 3.56 protein
[0692] Part of the purified antigen was in vitro biotinylated using
the BirA biotin-protein ligase standard reaction kit (Avidity, Cat.
#BirA500) according to the manufacturer's instructions. The
biotinylation degree for the human CD19-containing fusion was 94%,
for the respective cynomolgus CD19 construct 100%. The biotinylated
protein was then used for selection, screening and characterization
of affinity-matured 8B8-derived clones devoid of the de-amidation
hotspots N27d and N28.
[0693] 7.1.2 Generation of Anti-CD19 Clone 8B8-018
[0694] 7.1.2.1 Immunization and Generation of Mouse Anti-Human CD19
Antibodies (Hybridomas)
[0695] Balb/c mice were immunized six times and boosted with
CD19-transfected HEK293 cells (mean receptor density 35,000 per
cell). The immune response was monitored by testing serum samples
with a CD19-cell-ELISA on human CD19-transfected NIH-3T3 cells.
Spleen cells from mice with sufficient titers of anti-human CD19
antibody were used for immortalization by fusion with mouse myeloma
cell line P3X63 Ag8.653. Three fusions were carried out and
hybridoma supernatants screened by cell-ELISA on human
CD19-transfected NIH-3T3 cells and FACS binding assay using Daudi
(CD19+) and CD19- cells for anti-human CD19 specific antibodies
(see Example 1 of WO 2011/147834).
[0696] 7.1.2.2 Hybridoma Screening and Cell Biological Functional
Evaluation of Anti-CD19 Antibody
Cell-ELISA for Screening Antibodies Against Human CD19
[0697] A cell ELISA was applied for screening of hybridomas, and to
identify those hybridomas that secrete antibodies against
human-CD19. NIH3T3 cells transfected with human-CD19 were used as
positive cells; non-transfected NIH3T3 cells were used as negative
control cells. For the assessment of the positive hybridomas the OD
ratio between transfected and non-transfected NIH3T3 cells was
quantified. [0698] Culture Medium: DMEM high glucose (4.5 mg/ml),
10% FCS, Na-Pyruvate, NEAA, Glutamine [0699] Antibodies positive
control: anti CD19 monoclonal antibody (IgG1) Pharmingen Cat
#555409 c=1 mg/ml [0700] Detection antibody: Goat anti-Mouse IgG
(H+L) HRP Conjugate Bio-Rad Cat #170-06516 [0701] Dilution 1:2000
in 1.times.ELISA Blocking Reagent [0702] Other reagents:
Fibronectin Roche Cat #838039 c=1 mg/ml [0703] Glutardialdehyde:
25% stock solution//Grade Agar Scientific #R102 final
concentration: 0.05% in PBS [0704] ELISA Blocking Reagent:
10.times. stock solution//Roche Cat #1112589 [0705] TMB substrate:
Roche Cat #11432559 [0706] Stop Solution: 1 M H2SO4 [0707] BioRad
Cat #170-6516 Dilution 1:2000 in 1.times.ELISA Blocking Reagent
Day 1:
[0707] [0708] Fibronectin coating: 5 .mu.g/cm.sup.2 in PBS; 96 well
plate=32 cm.sup.2; 160 .mu.g/plate in 6 ml [0709] PBS, 50
.mu.l/well [0710] incubate 45 min at RT, aspirate coating solution
[0711] Seed 1.25.times.104 cells/well in 50 .mu.l culture medium in
a 96 well plate [0712] incubate 40 hours at 37.degree. C. [0713]
add to upper half of the plate: NIH3T3 cells expressing CD19 [0714]
add to lower half of the plate: non-transfected NIH3T3 cells
Day 3:
[0714] [0715] Addition of positive control antibody or samples
(supernatant or mouse serum) in 50 .mu.l culture medium [0716]
incubate for 2 h at 4.degree. C. [0717] Remove medium, fix cells
with 100 .mu.l Glutardialdehyde (0.05% in PBS) [0718] Wash two
times with 200 .mu.l PBS [0719] Addition of detection antibody
1:2000, 50 .mu.l/well [0720] incubate 2 h at RT [0721] wash three
times with 200 .mu.l PBS [0722] add 50 .mu.l TMB, incubate for 30
min. at RT, [0723] stop by addition of 25 .mu.l 1 M H2SO4; read
extinction at 450 nm/620 nm [0724] Calculation of results: ratio OD
NIH3T3 CD19: OD NIH3T3 non-transfected The selected antibody
demonstrated specific binding to CD19 transfected NIH3T3 cells as
compared to untransfected NIH3T3 cells (see Example 2 of WO
2011/147834).
[0725] 7.1.2.3 Humanization of Anti-CD19 Antibody
[0726] The CD19 binding specificity of the murine antibody was
transferred onto a human acceptor framework to eliminate potential
immunogenicity issues arising from sequence stretches that the
human body will recognize as foreign. This was done by engrafting
the entire complementary determining regions (CDR) of the murine
(donor) antibody onto a human (acceptor) antibody framework, and is
called CDR-grafting or antibody humanization.
[0727] The murine amino acid sequence was aligned with a collection
of human germ-line antibody V genes, and sorted according to
sequence identity and homology. Before selecting one particular
acceptor sequence, the so-called canonical loop structures of the
donor antibody have to be determined (Morea, V., et al., Methods,
Vol 20, Issue 3 (2000) 267-279). These canonical loop structures
are determined by the type of residues present at the so-called
canonical positions. These positions lie (partially) outside of the
CDR regions, and have to be kept functionally equivalent in the
final construct in order to retain the CDR conformation of the
parental (donor) antibody. The human germ-line sequence VBASE_VH1_1
was chosen as the acceptor for the heavy chain and sequence
VBASE_VK2_5 was chosen for the light chain.
[0728] 7.1.2.4 Removal of Deamidation Hotspots
[0729] It has been found that the wild-type humanized anti-human
CD19 antibody has three deamidation hotspots in the HVR-L1: NSNGNT
(SEQ ID NO: 190). Additionally it has been found that in the HVR-H2
a further deamidation hotspot is present: KFNG (SEQ ID NO: 191). To
address the deamidation hotspot in the HVR-H2 an N (Asn) to Q (Gln)
point mutation at position 64 (numbering according to Kabat) has
been introduced. Thus, the antibody as reported herein has a HVR-H2
comprising the amino acid sequence TEKFQGRVGTM (SEQ ID NO:
192).
[0730] To address the deamidation hotspots in the light chain and
to obtain a humanized anti-human CD19 antibody with improved
deamidation stability individual mutations at Kabat position 27d,
27e, 28 and 29 and a double mutation at positions 27e and 28
(numbering according to Kabat) were introduced. In total 9 variants
(var.1 to var.9) of the wild-type humanized antibody (var.0) have
been generated (see Table 45A and Table 45B).
TABLE-US-00048 TABLE 45A Variants of humanized wild-type CD19
antibody Kabat Kabat Variant position LC: position: 6, 4 HC: var.0:
wt SEQ ID QSLENSNGNTYLN wt SEQ ID TEKFNGKATM NO: 379 W NO: 389
var.1: N27dH SEQ ID QSLEHSNGNTYLN SEQ ID TEKFQGRVTM NO: 380 W NO:
192 var.2: N27dQ SEQ ID QSLEQSNGNTYLN SEQ ID TEKFQGRVTM NO: 381 W
NO: 192 var.3: S27eA SEQ ID QSLENANGNTYLN SEQ ID TEKFQGRVTM NO: 382
W NO: 192 var.4: 527eV SEQ ID QSLENVNGNTYLN SEQ ID TEKFQGRVTM NO:
383 W NO: 192 var.5: S27eP SEQ ID QSLENPNGNTYLN SEQ ID TEKFQGRVTM
NO: 384 W NO: 192 var.6: N28Q SEQ ID QSLENSQGNTYLN SEQ ID
TEKFQGRVTM NO: 385 W NO: 192 var.7: G29A SEQ ID QSLENSNANTYLN SEQ
ID TEKFQGRVTM NO: 386 W NO: 192 var.8: G29V SEQ ID QSLENSNVNTYLN
SEQ ID TEKFQGRVTM NO: 387 W NO: 192 var.9: S27eP/N28S SEQ ID
QSLENPSGNTYLN SEQ ID TEKFQGRVTM NO: 388 W NO: 192
TABLE-US-00049 TABLE 45B variant parameter 0 1 2 3 4 5 6 7 8 9
K.sub.D 5 250 136 2 1 6 54 4 16 45 (BIACORE .RTM.) [nM] t.sub.1/2
-- 0.1 1.1 105.2 191.5 43.6 4.4 51.5 17.6 4 [min] human CD19 46 0
75 84 85 95 91 72 83 83 binding after pH 7.4 incubation [%] human
CD19 90 0 95 95 97 99 97 86 91 87 binding after pH 6.0 incubation
[%] SEC main peak >95 >95 >95 >95 >95 >95 >95
>95 >95 -- after incubation [%]
[0731] It has been found that with a single mutation at position
27e according to Kabat from S (serine) to P (proline) all
deamidation hotspots in the HVR-L1 can be addressed. This is a
mutation not of the deamidation prone N (asparagine) residue but of
a neighboring residue.
[0732] Thus, the antibody as reported herein has a HVR-L1
comprising the amino acid sequence LENPNGNT (SEQ ID NO: 193). In
one embodiment the humanized anti-human CD19 antibody comprises a
HVR-L1 that has the amino acid sequence LENPSGNT (SEQ ID NO:
194).
[0733] Additionally these antibodies maintain the cross-reactivity
to cynomolgus CD19 as shown in the following Table 46.
TABLE-US-00050 EC50 [.mu.g/ml] var.0 var.5 var.9 huCD19 ECD 0.087
0.084 0.089 cyCD19 ECD 0.313 0.255 0.435
[0734] The wild-type humanized anti-human CD19 antibody (var.0)
shows after purification approx. 7.5% deamidation. After storage
for two weeks at pH 7.4 the amount of deamidated antibody is
increased to approx. 18.5%. The variant antibody with an S27eP
mutation (var.5) shows approx. 2% deamidation and 2% succinimide
formation after purification. During storage at pH 7.4 for two
weeks only approx. 7.5% deamidated antibody is present. Var. 5 is
named clone 8B8-018 and was elected for the preparation of
CD19-targeted TNF family ligand trimer-containing antigen binding
molecules.
[0735] 7.1.3 Preparation of Monovalent CD19(8B8-018) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 3.1)
[0736] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
29A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL. A polypeptide containing one ectodomain of 4-1BB ligand
(71-254) and fused to the human IgG1-CH domain, was cloned as
described in FIG. 29B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0737] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998).
To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0738] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1. The Pro329Gly, Leu234Ala and
Leu235Ala mutations have been introduced in the constant region of
the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
[0739] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CD19-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CD19 binding Fab (FIG. 30A, Construct 3.1).
[0740] Table 47 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 3.1).
TABLE-US-00051 TABLE 47 cDNA and amino acid sequences of monovalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 3.1). SEQ ID NO: Description Sequence 129 Nucleotide see
Table 3 sequence Dimeric hu 4-1BBL (71- 254)-CL* Fc knob chain 130
Nucleotide see Table 3 sequence Monomeric hu 4-1BBL (71-254)- CH1*
203 Nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence
anti- ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)
GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc hole chain
AGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA
ACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC
AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC
ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGAC
ACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTAC
GGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACA
GTAACCGTGAGCTCCGCTAGCACCAAGGGCCCCTCCGTG
TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGG
CACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA
CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTT
CTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTT
CTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT
GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCAC
CGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGG
GATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGC
AGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
GTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 204 Nucleotide
GACATCGTCATGACCCAGACACCCCTGTCCCTCTCTGTG sequence anti-
ACCCCTGGCCAGCCAGCCTCAATTAGCTGCAAGTCCTCT CD19(8B8-018)
CAAAGTCTGGAGAACCCCAATGGGAACACTTACCTTAAT light chain
TGGTATCTGCAGAAACCCGGACAATCCCCTCAACTCCTG
ATCTACAGGGTCTCTAAGAGATTCTCAGGCGTGCCAGAT
CGCTTTAGCGGTTCCGGGTCTGGCACAGACTTCACCTTG
AAGATTAGTCGGGTTGAAGCTGAGGATGTGGGAGTCTA
TTACTGTCTGCAGCTCACTCATGTGCCCTACACCTTTGGT
CAGGGCACAAAACTGGAGATCAAGCGGACCGTGGCCGC
TCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCT
GAAGTCCGGCACCGCCAGCGTGGTGTGCCTGCTGAACA
ACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTG
GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGT
GACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGT
CCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGC
ACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTG
TCCAGCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC 115 Dimeric hu 4- see Table
3 1BBL (71-254)- CL* Fc knob chain 116 Monomeric hu see Table 3
4-1BBL (71-254)- CH1* 205 anti-CD19(8B8-
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc hole
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chain
YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRD
ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 206
anti-CD19(8B8- DIVMTQTPLSLSVTPGQPASISCKSSQSLENPNGNTYLNWY 018) light
chain LQKPGQSPQLLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRV
EAEDVGVYYCLQLTHVPYTFGQGTKLEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC *for
charged residues
[0741] 7.1.4 Preparation of Monovalent CD19(8B8-018) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains without Charged Residues
(Construct 3.2)
[0742] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy as
depicted in FIG. 29A, but without amino acid mutations in the CL
domain: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human CL. A polypeptide containing one ectodomain of
4-1BB ligand (71-254) and fused to the human IgG1-CH1 domain, was
cloned in analogy as depicted in FIG. 29B, but without amino acid
mutations in the CH1 domain: human 4-1BB ligand, (G.sub.4S).sub.2
(SEQ ID NO:13) connector, human CH1.
[0743] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1.
[0744] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831. Combination of the dimeric ligand-Fc
knob chain containing the S354C/T366W mutations, the monomeric CH1
fusion, the targeted anti-CD19-Fc hole chain containing the
Y349C/T366S/L368A/Y407V mutations and the anti-CD19 light chain
allows generation of a heterodimer, which includes an assembled
trimeric 4-1BB ligand and a CD19-binding Fab (FIG. 30B, Construct
3.2).
[0745] Table 48 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 3.2).
TABLE-US-00052 TABLE 48 cDNA and amino acid sequences of monovalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 3.2). SEQ ID NO: Description Sequence 165 Nucleotide
sequence dimeric ligand see Table 22 (71-254)-CL Fc knob chain 166
Nucleotide sequence monomeric hu see Table 22 4-1BBL (71-254)-CH1
203 Nucleotide sequence anti-CD19(8B8-018) see Table 47 Fc hole
chain 204 Nucleotide sequence anti-CD19(8B8-018) see Table 47 light
chain 117 Dimeric ligand (71-254)-CL Fc knob see Table 22 chain 118
Monomeric ligand (71-254)-CH1 see Table 22 205 anti-CD19(8B8-018)
Fc hole chain see Table 47 206 anti-CD19(8B8-018) light chain see
Table 47
[0746] 7.1.5 Preparation of Bivalent CD19(8B8-018) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
(Construct 3.3)
[0747] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 29C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 29D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0748] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole, the knob
or the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the anti-CD19 huIgG1 hole dimeric ligand chain
containing the Y349C/T366S/L368A/Y407V mutations, the anti-CD19
huIgG1 knob monomeric ligand chain containing the S354C/T366W
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
two CD19 binding Fabs (FIG. 30C, construct 3.3).
[0749] Table 49 shows the cDNA and amino acid sequences of the
bivalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule (construct
3.3).
TABLE-US-00053 TABLE 49 Base pair sequences of bivalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand Fc (kih) PGLALA
fusion (construct 3.3) SEQ ID NO: Description Sequence 207
Nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence anti-
ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B 8-018)
GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc hole dimeric
AGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA ligand chain
ACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC
AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC
ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGAC
ACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTAC
GGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACA
GTAACCGTGAGCTCCGCTAGCACCAAGGGCCCCTCCGTG
TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGG
CACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA
CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTT
CTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTT
CTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT
GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCAC
CGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGG
GATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGC
AGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
GTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA
GAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACT
GCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGC
CCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTA
CAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTG
GCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAA
CTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGT
GTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGC
TGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGG
GAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCC
TGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTC
AGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCA
AGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATC
TAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCG
GGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTG
GTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGC
TGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACT
GGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGT
GGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCT
GGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGAT
CTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTG
CTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGC
CACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGG
TTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCG
CCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGC
ATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGA
CTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTGCCA AGCCCTAGATCAGAA 208
Nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence anti-
ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)
GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc knob
AGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA monomeric ligand
ACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC
AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC
ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGAC
ACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTAC
GGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACA
GTAACCGTGAGCTCCGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA
CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCT
TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGA
GATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTG
GTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGG
GAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTA
CTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGG
GCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCC
CGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA
GAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACT
GCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGC
CCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTA
CAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTG
GCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAA
CTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGT
GTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGC
TGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCC
TGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGG
GAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCC
TGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTC
AGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCA AGAAGCGAA 204 Nucleotide
see Table 47 sequence anti- CD19(8B8-018) light chain 209
anti-CD19(8B8- QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc hole
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA dimeric ligand
YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT chain
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRD
ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQ
GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKED
TKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP
LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG
QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP
SPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQL
VAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVV
AKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAG
AAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH
LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 210 anti-CD19(8B8-
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc knob
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA monomeric ligand
YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRD
ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQ
GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKED
TKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP
LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG
QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLP SPRSE 206 anti-CD19(8B8-
see Table 47 018) light chain
[0750] 7.1.6 Preparation of Monovalent CD19 (8B8-018) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 3.4)
[0751] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy to the one
depicted in FIG. 29A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CL. A polypeptide containing one ectodomain
of 4-1BB ligand (71-248) and fused to the human IgG1-CH domain, was
cloned in nalogy to the one described in FIG. 29B: human 4-1BB
ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human CH.
[0752] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998).
To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0753] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1. The Pro329Gly, Leu234Ala and
Leu235Ala mutations have been introduced in the constant region of
the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the dimeric ligand-Fc knob chain containing the
S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CD19-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CD19 binding Fab (FIG. 30D, construct 3.4).
[0754] Table 50 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 3.4).
TABLE-US-00054 TABLE 50 cDNA and amino acid sequences of monovalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 3.4). SEQ ID NO: Description Sequence 169 Nucleotide see
Table 24 sequence dimeric ligand (71-248)-- CL* Fc knob chain 170
Nucleotide see Table 24 sequence monomeric hu 4-1BBL (71-248)--
CH1* 203 Nucleotide see Table 47 sequence anti- CD19(8B8-018) Fc
hole chain 204 Nucleotide see Table 47 sequence anti- CD19(8B8-018)
light chain 119 Dimeric ligand see Table 24 (71-248)--CL* Fc knob
chain 120 Monomeric see Table 24 ligand (71-248)- CH1* 205
anti-CD19(8B8- see Table 47 018) Fc hole chain 206 anti-CD19(8B8-
see Table 47 018) light chain *charged residues
[0755] 7.1.7 Preparation of Monovalent CD19(8B8-018) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains without Charged Residues
(Construct 3.5)
[0756] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy as
depicted in FIG. 29A, but without amino acid mutations in the CL
domain: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human CL. A polypeptide containing one ectodomain of
4-1BB ligand (71-248) and fused to the human IgG1-CH1 domain, was
cloned in analogy as depicted in FIG. 29B, but without amino acid
mutations in the CH1 domain: human 4-1BB ligand, (G.sub.4S).sub.2
(SEQ ID NO:13) connector, human CH1.
[0757] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1. The Pro329Gly, Leu234Ala and
Leu235Ala mutations have been introduced in the constant region of
the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the dimeric ligand-Fc knob chain containing the
S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CD19-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CD19-binding Fab (FIG. 30E, Construct 3.5).
[0758] Table 51 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 3.5).
TABLE-US-00055 TABLE 51 cDNA and amino acid sequences of monovalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 3.5). SEQ ID NO: Description Sequence 171 Nucleotide see
Table 25 sequence dimeric ligand (71-248)-- CL Fc knob chain 172
Nucleotide see Table 25 sequence monomeric ligand (71-248)-CH1 203
Nucleotide see Table 47 sequence anti- CD19(8B8-018) Fc hole chain
204 Nucleotide see Table 47 sequence anti- CD19(8B8-018) light
chain 173 Dimeric ligand see Table 25 (71-248)--CL Fc knob chain
174 Monomeric see Table 25 ligand (71-248)- CH1 205 anti-CD19(8B8-
see Table 47 018) Fc hole chain 206 anti-CD19(8B8- see Table 47
018) light chain
[0759] 7.1.8 Preparation of Bivalent CD19(8B8-018) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
(Construct 3.6)
[0760] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 29C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 29D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0761] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CD19, clone 8B8-018, were subcloned
in frame with either the constant heavy chain of the hole, the knob
or the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the anti-CD19 huIgG1 hole dimeric ligand chain
containing the Y349C/T366S/L368A/Y407V mutations, the anti-CD19
huIgG1 knob monomeric ligand chain containing the S354C/T366W
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
two CD19 binding Fabs (FIG. 30F, construct 3.6).
[0762] Table 52 shows the cDNA and amino acid sequences of the
bivalent CD19(8B8-018) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule (construct
3.6).
TABLE-US-00056 TABLE 52 cDNA and amino acid sequences of bivalent
CD19(8B8-018) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion (construct 3.6) SEQ ID NO: Description Sequence 211
Nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence anti-
ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)
GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc hole dimeric
AGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA ligand (71-248)
ACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC chain
AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC
ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGAC
ACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTAC
GGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACA
GTAACCGTGAGCTCCGCTAGCACCAAGGGCCCCTCCGTG
TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGG
CACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA
CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTT
CTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTT
CTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT
GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCAC
CGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGG
GATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGC
AGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
GTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA
GAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACT
GCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGC
CCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTA
CAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTG
GCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAA
CTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGT
GTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGC
TGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCC
TGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGG
GAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCC
TGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTC
AGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCGG
CGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGC
TGTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGAC
AGGGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGC
TGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGC
TGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAA
GAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGT
GTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGT
GGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCA
TCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACT
GGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGC
CAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGC
ATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCAT
ACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCA
GGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGA GATCCCAGCCGGGCTC 212
Nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGTCAAGAA sequence anti-
ACCCGGGGCTTCTGTGAAGGTTTCATGCAAGGCAAGCG CD19(8B8-018)
GATACACCTTCACCGACTATATCATGCATTGGGTCAGGC Fc knob
AGGCCCCTGGCCAAGGTCTCGAATGGATGGGCTACATTA monomeric (71-
ACCCATATAATGATGGCTCCAAATACACCGAGAAGTTTC 248) ligand
AGGGAAGAGTCACTATGACATCTGACACCAGTATCAGC
ACTGCTTACATGGAGCTGTCCCGCCTTCGGTCTGATGAC
ACCGCAGTGTATTACTGTGCCAGGGGCACATATTACTAC
GGCTCAGCTCTGTTCGACTATTGGGGGCAGGGAACCACA
GTAACCGTGAGCTCCGCTAGCACCAAGGGCCCATCGGT
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA
CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACC
GTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCT
TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT
CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGA
GATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTG
GTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGG
GAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCAC
CCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTA
CTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGG
GCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCC
CGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGA
GAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACT
GCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGC
CCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTA
CAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTG
GCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAA
CTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGT
GTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGC
TGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCC
TGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGG
GAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCC
TGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTC
AGAGTGACCCCCGAGATTCCTGCCGGGCTC 204 Nucleotide see Table 47 sequence
anti- CD19(8B8-018) light chain 213 anti-CD19(8B8-
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fc hole
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA dimeric ligand
YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT (71-248) chain
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRD
ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQ
GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKED
TKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP
LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG
QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLG
GGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNV
LLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV
YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA
RARHAWQLTQGATVLGLFRVTPEIPAGL 214 anti-CD19(8B8-
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ 018) Fe knob
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA monomeric (71-
YMELSRLRSDDTAVYYCARGTYYYGSALFDYWGQGTTVT 248) ligand
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRD
ELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQ
GMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKED
TKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP
LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG
QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 206 anti-CD19(8B8- see
Table 47 018) light chain
[0763] 7.2 Preparation of CD19 (8B8-Derived Affinity Matured)
Targeted 4-1BB Ligand Trimer-Containing Fc Fusion Antigen Binding
Molecules and Corresponding Control Molecules
[0764] 7.2.1 Generation of 8B8-Derived Affinity-Matured Anti-CD19
Binders Devoid of Hotspots
[0765] 7.2.1.1 Selection of Affinity Matured CD19-Specific
Antibodies
[0766] De-amidation of the asparagine residues at positions 27d and
28, located in CDR1 of the light chain of the humanized clone 8B8,
leads to a significant reduction in the biological activity.
Therefore, 2 phage display libraries were generated in which a)
both asparagine residues at positions 27d and 28 were eliminated
and b) additional CDRs of heavy and light chain were randomized in
order to select for 8B8 variants with an improved affinity.
[0767] 7.2.1.2 Generation of 8B8 Affinity Maturation Libraries
Devoid of LCDR1 Hotspots
[0768] Generation of affinity-matured 8B8-derived antibodies
without the de-amidation sites N27d and N28, located in LCDR1, was
carried out by phage display using standard protocols (Silacci et
al, 2005). In a first step, the V.sub.L and V.sub.H DNA sequences
of the humanized parental clone 8B8 (SEQ ID NO: 215 and SEQ ID NO:
216) were cloned into our phagemid which was then used as a
template for randomization. In a next step, two libraries were
generated for the selection of favourable clones by phage display.
In order to eliminate the above-mentioned hotspot positions, a
LCDR1 randomization primer (SEQ ID NO: 217) that only allowed amino
acids S T Q E at positions 27d and 28 was used for both libraries.
Maturation library 1 was randomized in CDR1 and 2 of both the light
and the heavy chain, while maturation library 2 was randomized in
CDR1 and 3 of the light chain and in CDR3 of the heavy chain. The
randomized positions in the respective CDR regions are shown in
FIGS. 31A-1 to 31A-2. For the generation of the maturation library
1, randomized in CDR1 and 2 of both the light and the heavy chain,
three fragments were assembled by "splicing by overlapping
extension" (SOE) PCR and cloned into the phage vector (FIGS. 31B-1
to 31B-2). The following primer combinations were used to generate
the library fragments: fragment 1 (LMB3 (SEQ ID NO: 222) and CD19
L1 reverse random (SEQ ID NO: 217), fragment 2 (CD19 L2 forward
random (SEQ ID NO: 218) and CD19 HI reverse random (SEQ ID NO:
219), and fragment 3 (CD19 H2 forward random (SEQ ID NO: 220) and
CD19 H3 reverse constant (SEQ ID NO: 221) (Table 53). After
assembly of sufficient amounts of full length randomized fragment,
it was digested with NcoI/NheI alongside with identically treated
acceptor phagemid vector. A 3-fold molar excess of library insert
was ligated with 10 .mu.g of phagemid vector. Purified ligations
were used for 20 transformations resulting in 2.times.10 exp9
transformants. Phagemid particles displaying the 8B8 affinity
maturation library were rescued and purified by PEG/NaCl
purification to be used for selections.
[0769] The generation of the second library, randomized in CDR1 and
3 of the light chain and in CDR3 of the heavy chain, was done
similarly. The following primer combinations were used to generate
the library fragments: fragment 1 (LMB3 (SEQ ID NO: 222) and CD19
L1 reverse random (SEQ ID NO: 217), fragment 2 (CD19 L1 forward
constant (SEQ ID NO 223) and CD19 L3 reverse random (SEQ ID NO
224), and fragment 3 (CD19 L3 forward constant (SEQ ID NO: 225) and
CD19 H3 reverse random (SEQ ID NO: 226) (Table 54). After assembly
of sufficient amounts of full length randomized fragment, it was
digested with NcoI/KpnI alongside with identically treated acceptor
phagemid vector. A 3-fold molar excess of library insert was
ligated with 20 ug of phagemid vector. Purified ligations were used
for 40 transformations resulting in 2.times.10 exp9 transformants.
Phagemid particles displaying the 8B8 affinity maturation library
were rescued and purified by PEG/NaCl purification to be used for
selections.
TABLE-US-00057 TABLE 53 Primers for 8B8 affinity maturation and
hotspot removal library L1_L2/H1_H2 SEQ ID Name Sequence 217 CD19
L1 CAG CTG CGG GCT CTG ACC CGG TTT CTG GAG ATA reverse CCA GTT CAG
1 CGT 2 GCC 3 GGA 4 TTC CAG AGA TTG random GCT GGA TTT GCA AGA AAT
G 1: 40% Y, 6% A/S/T/G/P/D/N/E/Q/V, 2: 40% N, 6%
A/S/T/Y/G/P/D/E/Q/V, 3: 25% S/T/Q/E, 4: 25% S/T/Q/E 218 CD19 L2 CTC
CAG AAA CCG GGT CAGAGC CCG CAG CTG CTG forward ATC TAC 5 GTA TCT 6
CGC 7 8 GGC GTT 9 GAT CGT TTC random AGC GGT TCT GGA TCC GGC ACC 5:
30% R, 20% E, 5% A/S/T/Y/G/P/D/N/Q/V. 6: 30% K, 20% S, 5%
A/N/T/Y/G/P/D/E/Q/V, 7: 40% F, 5% A/S/T/Y/G/P/D/E/Q/V/I/L, 8: 40%
S, 6.6% A/T/Y/G/P/D/E/Q/V, 9: 50% P, 50% L 219 CD19 H1 CAT CCA CTC
CAG ACC CTG GCC CGG GGC CTGACG reverse AAC CCA 10 CAT 11 12 13 14
GAA 15 GTA ACC AGA TGC random TTT GCA GCT CAC TTT AAC GGAAGC 10:
52% H, 4% G/A/S/P/T/N/Y/D/E/Q/V/I, 11: 30% I, 15% Y, 5%
G/A/S/T/P/N/H/D/E/Q/V, 12: 52% Y, 4% G/A/S/P/T/N/H/D/E/Q/V/I, 13:
30% D, 15% G, 5% A/S/P/Y/N/H/D/E/Q/V/I, 14: 52% T, 4%
G/A/S/P/Y/N/H/D/E/Q/V/I, 15: 52% T, 4% G/A/S/P/Y/N/H/D/E/Q/V/I 220
CD19 H2 CAG GCC CCG GGC CAG GGT CTG GAG TGG ATG GGC forward 16 ATT
17 CCA 18 19 20 21 TCC 22 TAT ACC 23 AAA TTC random CAG GGC CGC GTC
ACG ATG ACC 16: 45% Y, 5% A/S/P/TN/H/D/E/Q/V/I, 17: 52% N, 4%
G/A/S/P/Y/T/H/D/E/Q/V/I, 18: 40% Y, 5% G/A/S/P/T/N/H/D/E/Q/V/I, 19:
30% N, 15% S, 5% G/A/T/P/Y/H/D/E/Q/V/I, 20: 30% D, 15% G, 5%
A/S/T/P/Y/N/H/E/Q/V/I, 21: 52% G, 4% N/A/S/P/Y/T/H/D/E/Q/V/I, 22:
30% K, 15% N, 4% G/A/S/P/Y/T/H/D/E/Q/V/I, 23: 30% E, 15% Q, 5%
G/A/S/T/P/Y/N/H/D/V/I 221 CD19 H3 CGTCACCGGTTCGGGGAAGTAGTCCTTGACCAG
reverse constant 222 LMB3 CAGGAAACAGCTATGACCATGATTAC
TABLE-US-00058 TABLE 54 Primers for 8B8 affinity maturation and
hotspot removal library L1_L3/H3 SEQ ID Name Sequence 223 CD19 L1
TGGTATCTCCAGAAACCGGGTCAGAGCCCGCAG forward constant 217 CD19 L1 See
Table 53 reverse random 224 CD19 L3 TTT AAT TTC CAG TTT AGT TCC TTG
ACC GAA GGT 24 reverse 25 26 27 28 29 CTG CAG ACA ATA GTA GAC GCC
AAC random GTC TTC AGC 24: 52% Y, 4% G/A/S/T/N/P/D/E/Q/V/L/I, 25:
52% P, 4% G/A/S/T/Y/N/H/D/E/QN/I, 26: 42% V, 10% L, 4%
G/A/S/T/Y/N/P/D/E/Q/V/I, 27: 52% H, 4% G/A/S/T/Y/N/P/D/E/Q/V/I, 28:
42% T, 10% I, 4% G/A/S/T/Y/N/P/D/E/Q/V/L, 29: 45% L, 11% G, 4%
A/S/T/Y/N/P/D/E/Q/V/I 225 CD19 L3
ACCTTCGGTCAAGGAACTAAACTGGAAATTAAACG forward constant 226 CD19 H3 TT
GGT GCT AGC AGA GCT TAC GGT CAC CGT GGT reverse ACC TTG GCC CCA GTA
ATC AAA 30 31 32 33 34 35 36 37 random 38 GCG TGC ACA ATA GTA AAC
AGC GGT GTC 30: 50% L, 3.8% G/A/S/T/P/H/Y/N/D/E/Q/V/I, 31: 50% A,
4.2% G/S/T/P/H/Y/N/D/E/Q/V/I, 32: 50% S, 4.2%
G/A/T/P/H/Y/N/D/E/QN/I, 33: 50% G, 4.2% S/A/T/P/H/Y/N/D/E/Q/V/I,
34: 50% Y, 4.2% G/A/T/P/H/S/N/D/E/Q/V/I, 35: 50% Y, 4.2%
G/A/T/P/H/S/N/D/E/Q/V/I, 36: 50% Y, 4.2% G/A/T/P/H/S/N/D/E/Q/V/I,
37: 50% T, 4.2% G/A/Y/P/H/S/N/D/E/Q/V/I, 38: 50% G, 4.2%
Y/A/T/P/H/S/N/D/E/Q/V/I 222 LMB3 See Table 53
[0770] 7.2.1.3 Selection of Affinity Matured 8B8-Derived Clones
Devoid of LCDR1 Hotspots N27d and N28
[0771] For the selection of affinity-matured clones devoid of the
LCDR1 hotspots N27d and N28, two selection approaches by phage
display were performed:
[0772] In the first approach, the selection was executed on human
CD19-Fc fusion protein using both phage display libraries. Panning
rounds were performed in solution according to the following
pattern: 1. binding of .about.10.sup.12 phagemid particles to 30 nM
biotinylated CD19-Fc protein for 0.5 h in a total volume of 1 ml,
2. capture of biotinylated CD19-Fc protein and specifically bound
phage particles by addition of 5.4.times.10.sup.7
streptavidin-coated magnetic beads for 10 min, 3. washing of beads
using 5.times.1 ml PBS/TWEEN.RTM. 20 (polysorbate 20) and 5.times.1
ml PBS, 4. elution of phage particles by addition of 1 ml 100 mM
TEA for 10 min and neutralization by adding 500 ul 1M Tris/HCl pH
7.4, 5. re-infection of exponentially growing E. coli TG1 bacteria,
and 6. infection with helperphage VCSM13 and subsequent PEG/NaCl
precipitation of phagemid particles to be used in subsequent
selection rounds. Selections were carried out over 3 rounds using
decreasing antigen concentrations (30.times.10.sup.-9M,
10.times.10.sup.-9M, and 3.times.10.sup.-9M). In round 2 and 3,
capture of antigen:phage complexes was performed using neutravidin
plates instead of streptavidin beads. Neutravidin plates were
washed with 5.times.PBS/TWEEN.RTM. 20 (polysorbate 20) and
5.times.PBS. In round 3, the neutravidin plate was incubated
overnight in 2 liters PBS for an "off-rate" selection before phage
was eluted from the plate. Furthermore, cynomolgus CD19-Fc protein
was used in round 2 in order to enrich cross-reactive binders.
[0773] In the second selection approach, the phage panning was
executed on cells transiently expressing either the human or
cynomolgus CD19 ECD on the cell surface. For the transient
transfection of HEK cells, expression plasmids were generated that
harbor the DNA sequences (from 5' to 3') for the following protein
segments: A Flag tag, a SNAP tag, the CD19 ECD of either human or
cynomolgus origin, and the transmembrane region of the
Platelet-derived growth factor receptor (PDGFR) (SEQ ID NOs: 227
and 228). The expression of the respective proteins (SEQ ID NOs:
229 and 230) on the cell surface was confirmed by flow cytometry
using an anti-Flag antibody for detection. Both libraries were
exposed in the first selection round to cells either expressing the
human or cynomolgus CD19 ECD-containing protein fusion. For the
subsequent panning rounds, the species of the CD19 ECD was
alternated accordingly. Cells transiently transfected with an
irrelevant membrane protein were used for pre-clearing.
[0774] Panning rounds were performed according to the following
pattern:
1. Transfection of HEK cells with constructs expressing either CD19
ECD or an irrelevant transmembrane protein according to the
standard procedure described before, 2. Incubation of the cells for
total 48h at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere, 3. Isolation of cells by centrifugation (3 min at
250.times.g) and re-suspension of 1.times.10E7 CD19 ECD-positive
cells and 1.times.10E7 negative cells in PBS/5% BSA, respectively,
3. Pre-clearing of unspecific phage by incubating the phage library
with 1.times.107 CD19-negative cells for 60 min at 4.degree. C.
using a gently rotating tube rotator, 4. Centrifugation of cells at
250.times.g for 3 min and transfer of supernatant into a fresh tube
and addition of 1.times.10E7 CD19-positive cells and incubation for
60 min at 4.degree. C. by gentle rotation on a tube rotator, 5.
Washing of cells by centrifugation for 1 min at 250.times.g,
aspiration of the supernatant, and re-suspension in 1 ml PBS (8
times), 6. Phage elution with 1 ml 100 mM TEA, incubation for 5 min
at RT, and neutralization of the eluate with 500 ul 1M Tris-HCl,
pH7.6, 7. re-infection of exponentially growing E. coli TG1
bacteria, and 8. infection with helperphage VCSM13 and subsequent
PEG/NaCl precipitation of phagemid particles to be used in
subsequent selection rounds. Selections were carried out over 3
rounds.
[0775] For both selection approaches, specific binders were
identified by ELISA as follows: 100 ul of 30 nM biotinylated
CD19-Fc protein per well were coated on neutravidin plates.
Fab-containing bacterial supernatants were added and binding Fabs
were detected via their Flag-tags using an anti-Flag/HRP secondary
antibody.
[0776] Clones that were ELISA-positive on recombinant human CD19
were further tested in a cell-based ELISA using cells that were
transiently transfected with the human CD19 ECD-containing
expression plasmid (SEQ ID NO: 227). This analysis was performed as
follows: 48 h after transfection, HEK cells were harvested and
centrifuged at 250.times.g for 5 min. Cells were then re suspended
in ice-cold PBS BSA 2% to 4.times.10.sup.6 cells/ml and incubated
for 20 min on ice to block unspecific binding sites.
4.times.10.sup.5 cells in 100 ul were distributed to each well of a
96 well plate and centrifuged at 250.times.g and 4.degree. C. for 3
min. Supernatant was aspirated off and 50 ul bacterial supernatant
containing soluble Fab fragments was diluted with 50 ul ice-cold
PBS/BSA 2%, added to the plate, mixed with the cells and incubated
for 1 h at 4.degree. C. Afterwards, cells were washed 3 times with
ice cold PBS before 100 ul PBS BSA 2% per well containing a 1:2000
dilution of anti-Fab-HRP antibody were added. After an incubation
time of 1 h, cells were washed again 3 times with ice-cold PBS. For
the development, 100 ul "1-step ultra TMB-ELISA" substrate was
added per well. After an incubation time of 10 minutes, supernatant
was transferred to a new 96-well plate containing 40 ul H2504 1M
per well and absorbance was measured 450 nM. Clones exhibiting
significant signals over background were subjected to a kinetic
screening experiment by SPR-analysis using ProteOn XPR36.
[0777] 7.2.1.4 Identification of Affinity-Matured 8B8-Derived
Variants by SPR
[0778] In order to further characterize the ELISA-positive clones,
the off-rate was measured by surface plasmon resonance and compared
with the parental humanized clone 8B8.
[0779] For this experiment, 7000 RU of polyclonal anti-human Fab
antibody were immobilized on all 6 channels of a GLM chip by Amine
coupling (NaAcetate pH4.5, 25 .mu.l/min, 240s) (vertical
orientation). Each antibody-containing bacterial supernatant was
filtered and 2-fold diluted with PBS, and then injected for 360s at
25 .mu.l/minute to achieve immobilization levels of between 100 and
400 response units (RU) in vertical orientation. Injection of
monomeric CD19-Fc: For one-shot kinetics measurements, injection
direction was changed to horizontal orientation, three-fold
dilution series of purified monomeric CD19-Fc (varying
concentration ranges between 150 and 6 nM) were injected
simultaneously at 50 .mu.l/min along separate channels 1-4, with
association times of 180 s, and dissociation times of 300 s. A
human IgG Fc fragment (150 nM) was injected in channel 5 as a
negative control for specific binding to monomeric CD19-Fc Buffer
(PBST) was injected along the sixth channel to provide an "in-line"
blank for referencing. Regeneration was performed by two pulses of
10 mM glycine pH 1.5 and 50 mM NaOH for 30s at 90 ul/min
(horizontal orientation). Dissociation rate constants (k.sub.off)
were calculated using a simple one-to-one Langmuir binding model in
ProteOn Manager v3.1 software by simultaneously fitting the
sensorgrams. Clones expressing Fabs with the slowest dissociation
rate constants were identified (Table 55). Of note, the
dissociation rate constants of clones 5A07 and 5B08 could not be
determined due to inadequate fitting. Nevertheless, both clones
were selected because results obtained suggested a very slow
dissociation. The variable domains of the corresponding phagemids
were sequenced. Importantly, both asparagine residue in LCDR1
(position 27d and 28) were replaced by a serine or a threonine,
demonstrating that both de-amidation sites were removed. An
alignment is shown in FIGS. 32A and 32B. The CDRs of the best
clones are listed in Table 56 (variable regions of the light chain)
and Table 57 (variable regions of the heavy chain) (clone 5H09:
(SEQ ID NO:231-236); clone 7H07: (SEQ ID NO:237-242); clone 2B03:
(SEQ ID NO: 243-248); clone 2B11: (SEQ ID NO:249-254); clone 5A07:
(SEQ ID NO:255-260); clone 5B08: (SEQ ID NO:261-266); clone 5D08:
(SEQ ID NO:267-272).
TABLE-US-00059 TABLE 55 Dissociation constants of selected clones
obtained in screening analysis with bacterial supernatant clone
Dissociation constant kd (1/s) Parental 8B8 3.01E-4 5H09 2.58E-4
7H07 5.75E-5 2B03 3.24E-5 2B11 4.37E-6 5A07 n.d. 5B08 n.d. 5D08
1.95E-4
TABLE-US-00060 TABLE 56 CDR sequences of the selected 8B8 light
chains SEQ SEQ SEQ ID ID ID clone NO CDR-L1 NO CDR-L2 NO CDR-L3
5H09 231 KSSQSLESSTGNTYLN 232 RVSKRFS 233 LQLIDYPVT 7H07 237
KSSQSLETSTGNTYLN 238 RVSKRFS 239 LQATHIPYT 2B03 243
KSSQSLETSTGNTYLN 244 RVSKRFS 245 LQLTHVPYT 2B11 249
KSSQSLETSTGTTYLN 250 RVSKRFS 251 LQLLEDPYT 5A07 255
KSSQSLETSTGNTYLN 256 RVSKRFS 257 LQPGHYPGT 5B08 261
KSSQSLETSTGNTYLN 262 RVSKRFS 263 LQLDSYPNT 5D08 267
KSSQSLETSTGNTYLN 268 RVSKRFS 269 LQLTHEPYT
TABLE-US-00061 TABLE 57 CDR sequences of the selected 8B8 heavy
chains SEQ SEQ SEQ ID CDR- ID ID clone NO H1 NO CDR-H2 NO CDR-H3
5H09 234 DYIMH 235 YINPYNDGSKYTEKFQG 236 GTYYYG SALFDY 7H07 240
DYIMH 241 YINPYNDGSKYTEKFQG 242 GTYYYG SELFDY 2B03 246 DYITH 247
YINPYNDGSKYTEKFQG 248 GTYYYG PDLFDY 2B11 252 DYIMH 253
YINPYNDGSKYTEKFQG 254 GTYYYG PQLFDY 5A07 258 DYIMH 259
YINPYNDGSKYTEKFQG 260 GTYYYG SALFDY 5B08 264 DYIMH 265
YINPYNDGSKYTEKFQG 266 GTYYYG PQLFDY 5D08 270 DYIMH 271
YINPYNDGSKYTEKFQG 272 GTYYYG SELFDY
[0780] 7.2.2 Characterization of Affinity-Matured 8B8-Derived
Antibodies
[0781] 7.2.2.1 Cloning of Variable Antibody Domains into Expression
Vectors
[0782] The variable regions of heavy and light chain DNA sequences
of the selected anti-CD19 binders were subcloned in frame with
either the constant heavy chain or the constant light chain of
human IgG1. In the heavy chain, Pro329Gly, Leu234Ala and Leu235Ala
mutations have been introduced in order to abrogate binding to Fc
gamma receptors according to the method described in International
Patent Appl. Publ. No. WO 2012/130831 A1.
[0783] The cDNA and amino acid sequences of the anti-CD19 IgGs are
shown in Table 58 and Table 59, respectively. All antibody-encoding
sequences were cloned into an expression vector, which drives
transcription of the insert with a chimeric MPSV promoter and
contains a synthetic polyA signal sequence located at the 3' end of
the CDS. In addition, the vector contains an EBV OriP sequence for
episomal maintenance of the plasmid.
TABLE-US-00062 TABLE 58 cDNA and amino acid sequences of anti-CD19
clone 8B8 in P329GLALA human IgG1 format SEQ Clone ID and NO: Chain
Sequence 273 8B8 GATGCTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGA
Parental GATCAAGCCTCCATCTCTTGCAGGTCTAGTCAGAGCCTTGAAAACAGT light
AATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTC chain
TCCACAACTCCTGATCTACAGGGTTTCCAAACGATTTTCTGGGGTCCT
AGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTCACACTGAAAA
TCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTCTGCCTACAA
CTTACACATGTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAT
AAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG
AGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCT
ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG
GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT
ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCAC
AAAGAGCTTCAACAGGGGAGAGTGT 274 8B8
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGC parental
TTCAGTGAAGATGGCCTGCAAGGCTTCTGGATACACATTCACTGACTA heavy
TATTATGCACTGGGTGAAGCAGAAGACTGGGCAGGGCCTTGAGTGGA chain
TTGGATATATTAATCCTTACAATGATGGTTCTAAGTACACTGAGAAGT
TCAACGGCAAGGCCACACTGACTTCAGACAAATCTTCCATCACAGCC
TACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTAC
TGTGCAAGAGGGACCTATTATTATGGTAGCGCCCTCTTTGACTACTGG
GGCCAAGGCACCACTCTCACAGTCTCCTCGGCTAGCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGC
GGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG
TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTG
TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCC
CAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAA
CTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC
CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAG
CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT
TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGA
GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAA
CGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA
GAAGAGCCTCTCCCTGTCTCCGGGTAAA 275 8B8
DAVMTQTPLSLPVSLGDQASISCRSSQSLENSNGNTYLNWYLQKPGQSP Parental
QLLIYRVSKRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQLTHVP light
YTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLIVNFYPREAKVQW chain
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
276 8B8 EVQLQQSGPELVKPGASVKMACKASGYTFTDYIMHWVKQKTGQGLEWI parental
GYINPYNDGSKYTEKFNGKATLTSDKSSITAYMELSSLTSEDSAVYYCAR heavy
GTYYYGSALFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL chain
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
TABLE-US-00063 TABLE 59 cDNA and amino acid sequences of affinity
matured anti-CD19 clones in P329GLALA human IgG1 format SEQ Clone
ID and NO: Chain Sequence 277 2B11
GATATTGTCATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTCC chain
ACCGGCACCACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGAG
CCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTCC
TGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAAT
CAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAGCT
GCTGGAAGATCCATACACCTTCGGTCAAGGAACGAAACTGGAAATTA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG
AGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT
TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTC
CAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG
ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGAC
TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 278 2B11
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGCG
TACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTAT
TGTGCACGCGGTACCTACTACTACGGTCCACAGCTGTTTGATTACTGG
GGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
AGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG
ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG
GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGC
TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC
ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 279 2B11
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGTTYLNWYLQKPGQSPQL light
LIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLLEDPY chain
TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC 280 2B11
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGPQLFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 281
7H07 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTCC chain
ACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGAG
CCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTCC
TGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAAT
CAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAGG
CAACCCATATCCCATACACCTTCGGTCAAGGAACTAAACTGGAAATT
AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG
GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC
TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 282 7H07
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGCG
TACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTAT
TGTGCACGCGGTACCTACTACTACGGTTCTGAACTGTTTGATTACTGG
GGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCCC
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC
AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
AGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG
ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG
GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA
GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGC
TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA
ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAC
ACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 283 7H07
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYLNWYLQKPGQSPQL light
LIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQATHIPYT chain
FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC 284 7H07
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGSELFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 285
2B03 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTC chain
CACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGA
GCCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTC
CTGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAA
TCAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAG
TTGACCCACGTTCCGTACACCTTCGGTCAAGGAANNAAACTGGAAAT
TAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 286 2B03
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCACGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGC
GTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTA
TTGTGCACGCGGTACCTACTACTACGGTCCAGATCTGTTTGATTACTG
GGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 287 2B03
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYLNWYLQKPGQSPQ light
LLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHVP chain
YTFGQGXKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 288 2B03
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYITHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGPDLFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 289
5A07 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTC chain
CACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGA
GCCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTC
CTGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAA
TCAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAG
CCAGGTCATTACCCAGGTACCTTCGGTCAAGGAACTAAACTGGAAAT
TAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 290 5A07
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGC
GTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTA
TTGTGCACGCGGTACTTACTACTACGGTTCCGCCCTCTTTGATTACTG
GGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 291 5A07
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYLNWYLQKPGQSPQ light
LLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQPGHYP chain
GTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 292 5A07
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGSALFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 293
5D08 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTC chain
CACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGA
GCCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTC
CTGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAA
TCAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAG
CTGACCCATGAACCATACACCTTCGGTCAAGGAACTAAACTGGAAAT
TAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 294 5D08
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGC
GTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTA
TTGTGCACGCGGTACCTACTACTACGGTTCTGAACTGTTTGATTACTG
GGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 295 5D08
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYLNWYLQKPGQSPQ light
LLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLTHEP chain
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 296 5D08
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGSELFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 297
5B08 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAAACCTC chain
CACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGA
GCCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTC
CTGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAA
TCAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAG
CTGGATTCTTACCCAAACACCTTCGGTCAAGGAACTAAACTGGAAAT
TAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 298 5B08
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGC
GTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTA
TTGTGCACGCGGTACCTACTACTACGGTCCACAGCTGTTTGATTACTG
GGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 299 5B08
DIVMTQTPLSLSVTPGQPASISCKSSQSLETSTGNTYLNWYLQKPGQSPQ light
LLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLDSYP chain
NTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 300 5B08
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGPQLFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 301
5H09 GATATTGTTATGACTCAAACTCCACTGTCTCTGTCCGTGACCCCGGGT light
CAGCCAGCGAGCATTTCTTGCAAATCCAGCCAATCTCTGGAATCTTCC chain
ACCGGCAACACGTACCTGAACTGGTATCTCCAGAAACCGGGTCAGAG
CCCGCAGCTGCTGATCTACCGTGTATCTAAGCGCTTCTCCGGCGTTCC
TGATCGTTTCAGCGGTTCTGGATCCGGCACCGACTTTACTCTGAAAAT
CAGCCGTGTGGAAGCTGAAGACGTTGGCGTCTACTATTGTCTGCAGC
TGATCGATTACCCAGTTACCTTCGGTCAAGGAACTAAACTGGAAATT
AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA
GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGG
CCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 302 5H09
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGC heavy
TTCCGTTAAAGTGAGCTGCAAAGCATCTGGTTACACCTTCACTGACTA chain
TATCATGCACTGGGTTCGTCAGGCCCCGGGCCAGGGTCTGGAGTGGA
TGGGCTACATTAACCCATACAACGACGGTTCCAAATATACCGAGAAA
TTCCAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCCACCGC
GTACATGGAACTGTCTAGACTGCGTTCTGACGACACCGCTGTTTACTA
TTGTGCACGCGGTACCTACTACTACGGTTCTGCACTGTTTGATTACTG
GGGCCAAGGTACCACGGTGACCGTAAGCTCTGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTT
CCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCC
CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG
AAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT
GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA
GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTT
CTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 303 5H09
DIVMTQTPLSLSVTPGQPASISCKSSQSLESSTGNTYLNWYLQKPGQSPQ light
LLIYRVSKRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQLIDYP chain
VTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA
CEVTHQGLSSPVTKSFNRGEC 304 5H09
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQAPGQGLEW heavy
MGYINPYNDGSKYTEKFQGRVTMTSDTSISTAYMELSRLRSDDTAVYYC chain
ARGTYYYGSALFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK
[0784] 7.2.2.2 Affinity Determination of Selected Antibodies by
SPR
[0785] For the exact determination of the affinities by SPR, the
selected anti-CD19 antibodies were produced by co-transfecting
HEK293-EBNA cells with the mammalian expression vectors using
polyethylenimine. The cells were transfected with the corresponding
expression vectors in a 1:1 ratio ("vector heavy chain": "vector
light chain") according to the standard procedure. 7 days after
transfection, the antibody titer in the supernatant was measured
and all titers were equilibrated to 10 .mu.g/ml.
[0786] The Affinity (K.sub.D) of the parental antibody 8B8 as well
as it derivatives was measured by SPR using a ProteOn XPR36
instrument (Biorad) at 25.degree. C. 7000 RU of polyclonal
anti-human Fab antibody were immobilized on all 6 channels of a GLM
chip by Amine coupling (NaAcetate pH4.5, 25 ul/min, 240s) (vertical
orientation). Each antibody-containing HEK supernatant was
filtered, diluted with PBST (10 mM phosphate, 150 mM sodium
chloride pH 7.4, 0.005% Tween 20) to a concentration of 10 ug/ml,
and then injected at a for 360s at 25 .mu.l/minute to achieve
immobilization levels between 500 and 800 response units (RU) in
vertical orientation. Injection of monomeric CD19-Fc: For one-shot
kinetics measurements, injection direction was changed to
horizontal orientation, three-fold dilution series of purified
monomeric CD19-Fc (varying concentration ranges between 150 and 6
nM) were injected simultaneously at 50 .mu.l/min along separate
channels 1-4, with association times of 180s, and dissociation
times of 300s. A human IgG Fc fragment (150 nM) was injected in
channel 5 as a negative control for specific binding to monomeric
CD19-Fc. Buffer (PBST) was injected along the sixth channel to
provide an "in-line" blank for referencing. An overview of the
respective sensorgrams is shown in FIGS. 33A-33H. Regeneration was
performed by two pulses of 10 mM glycine pH 1.5 and 50 mM NaOH for
30s at 90 ul/min (vertical orientation). Association rate constants
(k.sub.on) and dissociation rate constants (k.sub.off) were
calculated using a simple one-to-one Langmuir binding model in
ProteOn Manager v3.1 software by simultaneously fitting the
association and dissociation sensorgrams. The equilibrium
dissociation constant (K.sub.D) was calculated as the ratio
k.sub.off/k.sub.on. A summary of the kinetic and thermodynamic data
is shown in Table 60. The dissociation constant of all
affinity-matured clones was improved compared to their parental
clone 8B8.
TABLE-US-00064 TABLE 60 Summary of the kinetic and thermodynamic
data for the interaction between anti-CD19 huIgG1 and human CD19
clone ka (1/Ms) kd (1/s) KD (M) Parental 8B8 5.66E+4 1.34E-4
2.36E-9 5H09 7.91E+4 1.50E-5 1.89E-10 7H07 7.45E+4 5.57E-5 7.47E-10
2B03 6.02E+4 5.00E-5 8.31E-10 2B11 6.34E+4 3.14E-5 4.95E-10 5A07
6.98E+4 3.07E-5 4.40E-10 5B08 6.81E+4 5.26E-5 7.72E-10 5D08 8.88E+4
8.44E-5 9.51E-10
[0787] 7.2.2.3 Preparation and Purification of Anti-CD19 IgG1 P329G
LALA
[0788] The selected anti-CD19 antibodies were produced by
co-transfecting HEK293-EBNA cells with the mammalian expression
vectors using polyethylenimine. The cells were transfected with the
corresponding expression vectors in a 1:1 ratio ("vector heavy
chain": "vector light chain").
[0789] For the production in 500 mL shake flasks, 400 million
HEK293 EBNA cells were seeded 24 hours before transfection. Before
the transfection, cells were centrifuged for 5 minutes at
210.times.g, and the supernatant was replaced by pre-warmed CD CHO
medium. Expression vectors (200 .mu.g of total DNA) were mixed in
20 mL CD CHO medium. After addition of 540 .mu.L PEI, the solution
was vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO2 atmosphere.
After the incubation, 160 mL of F17 medium was added and cells were
cultured for 24 hours. One day after transfection 1 mM valproic
acid and 7% Feed with supplements were added. After culturing for 7
days, the supernatant was collected by centrifugation for 15
minutes at 210.times.g. The solution was sterile filtered (0.22
.mu.m filter), supplemented with sodium azide to a final
concentration of 0.01% (w/v), and kept at 4.degree. C.
[0790] Purification of antibody molecules from cell culture
supernatants was carried out by affinity chromatography using
Protein A as described above for purification of antigen Fc
fusions. The protein was concentrated and filtered prior to loading
on a HILOAD.RTM. Superdex 200 column (GE Healthcare) equilibrated
with 20 mM Histidine, 140 mM NaCl solution of pH 6.0.
[0791] The protein concentration of purified antibodies was
determined by measuring the OD at 280 nm, using the molar
extinction coefficient calculated on the basis of the amino acid
sequence. Purity and molecular weight of the antibodies were
analyzed by CE-SDS in the presence and absence of a reducing agent
(Invitrogen, USA) using a LabChipGXII (Caliper). The aggregate
content of antibody samples was analyzed using a TSKGEL.RTM. G3000
SW XL analytical size-exclusion column (Tosoh) equilibrated in a 25
mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-Arginine
Monohydrocloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C. (Table 61).
TABLE-US-00065 TABLE 61 Biochemical analysis of anti-CD19 P329G
LALA IgG1 clones Yield Monomer CE-SDS Clone [mg/l] [%] (non red)
Parental 8B8 25.3 100 99.1 2B11 35.4 100 98.4 7H07 89.8 100 99.4
2B03 182 100 100 5A07 90.2 100 99.4 5D08 90.2 100 99.3 5B08 24.1
99.6 100 5H09 29.9 100 98.1
[0792] For the preparation of bispecific constructs clone 2B11 was
chosen because it lacks the three deamidation hotspots.
[0793] The DNA sequence encoding part of the ectodomain (amino acid
71-254 and 71-248) of human 4-1BB ligand was synthetized according
to the P41273 sequence of Uniprot database.
[0794] 7.2.3 Preparation of Monovalent CD19 (8B8-2B11) Targeted
4-1BB Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 4.1)
[0795] The construct 4.1 was prepared as described for construct
3.1 (FIG. 30A), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0796] Table 62 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-2B11) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 4.1).
TABLE-US-00066 TABLE 62 cDNA and amino acid sequences of monovalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 4.1). SEQ ID NO: Description Sequence 129 Nucleotide see
Table 3 sequence Dimeric hu 4-1BBL (71-254)- CL* Fc knob chain 130
Nucleotide see Table 3 sequence Monomeric hu 4-1BBL (71-254)- CH1*
305 Nucleotide CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence
anti- ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B 11)
TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc hole chain
GGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA
ACCCATACAACGACGGTTCCAAATATACCGAGAAATTC
CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC
ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGAC
ACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTAC
GGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACG
GTGACCGTAAGCTCTGCTAGCACCAAGGGCCCCTCCGTG
TTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGG
CACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGA
CCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTT
CTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTT
CTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGT
GGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCAC
CGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC
GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGC
CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC
CCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGG
GATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGC
AGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACC
ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
GTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA
GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 277 Nucleotide see
Table 59 sequence anti- CD19(8B8-2B11) light chain 115 Dimeric hu
4- see Table 3 1BBL (71-254)- CL* Fc knob chain 116 Monomeric hu
see Table 3 4-1BBL (71-254)- CH1* 306 anti-CD19(8B8-2B11)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ Fc hole
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA chain
YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRD
ELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 206
anti-CD19(8B8-2b11) see Table 59 light chain *for charged
residues
[0797] 7.2.4 Preparation of Monovalent CD19(8B8-2B11) Targeted
4-1BB Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains without Charged
Residues (Construct 4.2)
[0798] The construct 4.2 was prepared as described for construct
3.2 (FIG. 30B), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0799] Table 63 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-2B11) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 4.2).
TABLE-US-00067 TABLE 63 cDNA and amino acid sequences of monovalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 4.2). SEQ ID NO: Description Sequence 165 Nucleotide see
Table 22 sequence dimeric ligand (71-254)-- CL Fc knob chain 166
Nucleotide see Table 22 sequence monomeric hu 4-1BBL (71-254)-- CH1
305 Nucleotide see Table 62 sequence anti- CD19(8B8-2B11) Fc hole
chain 277 Nucleotide see Table 59 sequence anti- CD19(8B8-2B11)
light chain 117 Dimeric ligand see Table 22 (71-254)--CL Fc knob
chain 118 Monomeric see Table 22 ligand (71-254)-- CH1 306
anti-CD19(8B8- see Table 62 2B11) Fc hole chain 279 anti-CD19(8B8-
see Table 59 018) light chain
[0800] 7.2.5 Preparation of Bivalent CD19(8B8-2B11) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
(Construct 4.3)
[0801] The construct 4.3 was prepared as described for construct
3.3 (FIG. 30C), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0802] Table 64 shows the cDNA and amino acid sequences of the
bivalent CD19 (8B8-2B11) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule (construct
4.3).
TABLE-US-00068 TABLE 64 cDNA and amino acid sequences of bivalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand Fc (kih) PGLALA
fusion (construct 4.3) SEQ ID NO: Description Sequence 307
Nucleotide CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence anti-
ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)
TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc hole dimeric
GGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA ligand chain
ACCCATACAACGACGGTTCCAAATATACCGAGAAATTC
CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC
ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGAC
ACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTAC
GGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACG
GTGACCGTAAGCTCTGCTAGCACCAAGGGCCCCTCCGTGT
TCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC
AGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAG
CCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCG
GCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCT
GTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCC
TGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT
GCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
GCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA
GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCG
GGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCA
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGC
GGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTG
AGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGG
GCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATC
GATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTG
GCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACAC
CAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTG
TTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAG
GATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTG
AGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGG
ATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTT
GGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGA
GGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACA
CGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCC
AAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATC
TAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGG
CTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGG
CTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTAC
TCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGAC
TGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAA
GCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCG
GGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCT
CTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTG
CACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAG
GCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGC
ATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATAC
TGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGG
GCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCC
TGCCGGACTGCCAAGCCCTAGATCAGAA 308 Nucleotide
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence anti-
ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)
TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc knob
GGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA monomeric ligand
ACCCATACAACGACGGTTCCAAATATACCGAGAAATTC
CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC
ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGAC
ACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTAC
GGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACG
GTGACCGTAAGCTCTGCTAGCACCAAGGGCCCATCGGTCT
TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAG
AGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGG
TCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGA
GAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCC
CCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAA
ACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTG
TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTA
CACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCT
GAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAG
GGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGAT
CGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCT
GGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACA
CCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT
GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAA
GGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCT
GAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTG
GATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATT
TGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAG
AGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGAC
ACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCT
GTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTC CAAGAAGCGAA 277
Nucleotide see Table 59 sequence anti- CD19(8B8-018) light chain
309 anti-CD19(8B8-2B11) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ Fc
hole APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA dimeric ligand
YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT chain
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG
APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR
WQQGNVFSCS1MHEALH1VHYTQKSLSLSPGGGGGSGGGGSR
EGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP
GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE
GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQ
GRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP
EIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQG
MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKEL
VVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA
AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTE
ARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 310 anti-CD19(8B8-2B11)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ Fc knob
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA monomeric ligand
YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG
APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPEN1VYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALH1VHYTQKSLSLSPGGGGGSGGGGS
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD
PGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSE 279
anti-CD19(8B8-018) see Table 59 light chain
[0803] 7.2.6 Preparation of Monovalent CD19(8B8-2B11) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 4.4)
[0804] The construct 4.4 was prepared as described for construct
3.4 (FIG. 30D), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0805] Table 65 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-2B11) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 4.4).
TABLE-US-00069 TABLE 65 cDNA and amino acid sequences of monovalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 4.4). SEQ ID NO: Description Sequence 169 Nucleotide see
Table 24 sequence dimeric ligand (71-248)-- CL* Fc knob chain 170
Nucleotide see Table 24 sequence monomeric hu 4-1BBL (71-248)--
CH1* 305 Nucleotide see Table 62 sequence anti- CD19(8B8-2B11) Fc
hole chain 277 Nucleotide see Table 59 sequence anti-
CD19(8B8-2B11) light chain 119 Dimeric ligand see Table 24
(71-248)--CL* Fc knob chain 120 Monomeric see Table 24 ligand
(71-248)- CH1* 306 anti-CD19(8B8- see Table 62 2B11) Fc hole chain
279 anti-CD19(8B8- see Table 59 2B11) light chain *charged
residues
[0806] 7.2.7 Preparation of Monovalent CD19(8B8-2B11) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains without Charged
Residues (Construct 4.5)
[0807] The construct 4.5 was prepared as described for construct
3.5 (FIG. 30E), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0808] Table 66 shows the cDNA and amino acid sequences of the
monovalent CD19(8B8-2B11) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 4.5).
TABLE-US-00070 TABLE 66 cDNA and amino acid sequences of monovalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 4.5). SEQ ID NO: Description Sequence 171 Nucleotide see
Table 25 sequence dimeric ligand (71-248)-- CL Fc knob chain 172
Nucleotide see Table 25 sequence monomeric ligand (71-248)-CH1 305
Nucleotide see Table 62 sequence anti- CD19(8B8-2B11) Fc hole chain
277 Nucleotide see Table 59 sequence anti- CD19(8B8-2B11) light
chain 173 Dimeric ligand see Table 25 (71-248)--CL Fc knob chain
174 Monomeric see Table 25 ligand (71-248)- CH1 306 anti-CD19(8B8-
see Table 62 2B11) Fc hole chain 279 anti-CD19(8B8- see Table 59
2B11) light chain
[0809] 7.2.8 Preparation of Bivalent CD19(8B8-2B11) Targeted 4-1BB
Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen Binding
(Construct 4.6)
[0810] The construct 4.6 was prepared as described for construct
3.6 (FIG. 30F), but using the variable region of heavy and light
chain DNA sequences encoding a binder specific for CD19, clone
8B8-2B11.
[0811] Table 67 shows the cDNA and amino acid sequences of the
bivalent CD19(8B8-2B11) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule (construct
3.6).
TABLE-US-00071 TABLE 67 cDNA and amino acid sequences of bivalent
CD19(8B8-2B11) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion (construct 4.6) SEQ ID NO: Description Sequence 311
Nucleotide CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence anti-
ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)
TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc hole dimeric
GGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA ligand (71-248)
ACCCATACAACGACGGTTCCAAATATACCGAGAAATTC chain
CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC
ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGAC
ACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTAC
GGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACG
GTGACCGTAAGCTCTGCTAGCACCAAGGGCCCCTCCGTGT
TCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCAC
AGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAG
CCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCG
GCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCT
GTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCC
TGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCT
GCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA
GCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA
GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCG
GGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCA
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA
GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGC
GGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTG
AGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGG
GAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATC
GATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTG
GCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACAC
CAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTG
TTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAG
GATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTG
AGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGG
ATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTT
GGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGA
GGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACA
CGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTG
TTCAGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCG
GCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCT
GTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGACAG
GGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGCTGAT
TGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCA
GGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATAC
AAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGT
TTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGG
CTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGC
GCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGA
CCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCG
GGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCG
CCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCAT
GCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGT
TTCGCGTGACACCTGAGATCCCAGCCGGGCTC 312 Nucleotide
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAA sequence anti-
ACCGGGCGCTTCCGTTAAAGTGAGCTGCAAAGCATCTGG CD19(8B8-2B11)
TTACACCTTCACTGACTATATCATGCACTGGGTTCGTCA Fc knob monomeric
GGCCCCGGGCCAGGGTCTGGAGTGGATGGGCTACATTA (71-248) ligand
ACCCATACAACGACGGTTCCAAATATACCGAGAAATTC
CAGGGCCGCGTCACGATGACCAGCGACACTTCTATCTCC
ACCGCGTACATGGAACTGTCTAGACTGCGTTCTGACGAC
ACCGCTGTTTACTATTGTGCACGCGGTACCTACTACTAC
GGTCCACAGCTGTTTGATTACTGGGGCCAAGGTACCACG
GTGACCGTAAGCTCTGCTAGCACCAAGGGCCCATCGGTCT
TCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG
GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGC
TGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC
GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA
GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA
ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAG
AGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGG
TCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGA
GAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCC
CCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAA
ACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTG
TTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTA
CACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGG
CGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCT
GAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAG
GGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGAT
CGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCT
GGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACA
CCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT
GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAA
GGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCT
GAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTG
GATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCAT
TTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCA
GAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGA
CACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCC
TGTTCAGAGTGACCCCCGAGATTCCTGCCGGGCTC 277 Nucleotide see Table 59
sequence anti- CD19(8B8-2B11) light chain 313 anti-CD19(8B8-2B11)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ Fc hole
APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA dimeric ligand
YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT (71-248) chain
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG
APIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR
WQQGNVFSCS1MHEALH1VHYTQKSLSLSPGGGGGSGGGGSR
EGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP
GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE
GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQ
GRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP
EIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQL
VAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKA
GVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALT
VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHA WQLTQGATVLGLFRVTPEIPAGL
314 anti-CD19(8B8-2B11) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIMHWVRQ Fc
knob monomeric APGQGLEWMGYINPYNDGSKYTEKFQGRVTMTSDTSISTA (71-248)
ligand YMELSRLRSDDTAVYYCARGTYYYGPQLFDYWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG
APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPEN1VYKTTPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALH1VHYTQKSLSLSPGGGGGSGGGGS
REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSD
PGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAG
EGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGF
QGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGL 279
anti-CD19(8B8-018) see Table 59 light chain
[0812] 7.3 Preparation of Untargeted Split Trimeric 4-1BB Ligand Fc
Fusion and Human IgG as Control Molecules
[0813] 7.3.1 Preparation of Untargeted Human 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules (Control
Molecules)
[0814] These control molecules were prepared as described above for
the CD19 targeted construct 3.1 (termed control B), 3.3 (termed
control C), 3.4 (termed control D) and 3.5 (termed control E) with
the only difference that the anti-CD19 binder (VH-VL) was replaced
by a germline control, termed DP47, not binding to the antigen (see
FIGS. 30A-F).
[0815] Table 68 shows, respectively, the cDNA and amino acid
sequences of the monovalent DP47-untargeted split trimeric 4-1BB
ligand (71-254) Fc (kih) fusion containing crossed CH-CL with
charged residues, control B.
[0816] Table 69 shows, respectively, the cDNA and amino acid
sequences of the bivalent DP47-untargeted split trimeric 4-1BB
ligand (71-254) Fc (kih) fusion, control C.
[0817] Table 70 shows, respectively, the cDNA and amino acid
sequences of the monovalent DP47-untargeted split trimeric 4-1BB
ligand (71-248) Fc (kih) fusion containing CH-CL cross with charged
residues, control D.
[0818] Table 71 shows, respectively, the cDNA and amino acid
sequences of the monovalent DP47-untargeted split trimeric 4-1BB
ligand (71-248) Fc (kih) fusion without charged residues in the
CH-CL cross, control E.
TABLE-US-00072 TABLE 68 cDNA and amino acid sequences of monovalent
DP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih)
fusion with CH-CL cross and with charged residues (control B). SEQ
ID NO: Description Sequence 96 nucleotide sequence dimeric hu
4-1BBL see Table 3 (71-254)--CL* Fc knob chain 97 nucleotide
sequence monomeric hu see Table 3 4-1BBL (71-254)--CH1* 79
nucleotide sequence DP47 Fc hole chain see Table 18 80 nucleotide
sequence DP47 light chain see Table 18 98 Dimeric hu 4-1BBL
(71-254)--CL* Fc see Table 3 knob chain 99 Monomeric hu see Table 3
4-1BBL (71-254)--CH1* 81 DP47 Fc hole chain see Table 18 82 DP47
light chain see Table 18 *charges residues
TABLE-US-00073 TABLE 69 cDNA and amino acid sequences of bivalent
DP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih)
fusion (control C). SEQ ID NO: Description Sequence 177 nucleotide
sequence DP47 Fc hole chain see Table 27 fused to dimeric hu 4-1BBL
(71-254) 178 nucleotide sequence DP47 Fc knob chain see Table 27
fused to monomeric hu 4-1BBL (71-254) 80 nucleotide sequence DP47
light chain see Table 18 179 DP47 Fc hole chain fused to dimeric hu
4- see Table 27 1BBL (71-254) 180 DP47 Fc knob chain fused to
monomeric see Table 27 hu 4-1BBL (71-254) 82 DP47 light chain see
Table 18
TABLE-US-00074 TABLE 70 cDNA and amino acid sequences of monovalent
DP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih)
fusion with CH-CL cross and with charged residues (control D). SEQ
ID NO: Description Sequence 169 nucleotide sequence dimeric hu
4-1BBL see Table 24 (71-248)--CL* Fc knob chain 170 nucleotide
sequence monomeric hu see Table 24 4-1BBL (71-248)--CH1* 79
nucleotide sequence DP47 Fc hole chain see Table 18 80 nucleotide
sequence DP47 light chain see Table 18 119 Dimeric hu 4-1BBL
(71-254)--CL* Fc see Table 24 knob chain 120 Monomeric hu see Table
24 4-1BBL (71-254)--CH1* 81 DP47 Fc hole chain see Table 18 82 DP47
light chain see Table 18 *charged residues
TABLE-US-00075 TABLE 71 cDNA and amino acid sequences of monovalent
DP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih)
fusion with CH-CL cross and without charged residues (control E).
SEQ ID NO: Description Sequence 171 nucleotide sequence dimeric hu
4- see Table 25 1BBL (71-248)--CL Fc knob chain 172 nucleotide
sequence monomeric hu see Table 25 4-1BBL (71-248)--CH1 79
nucleotide sequence DP47 Fc hole see Table 18 chain 80 nucleotide
sequence DP47 light chain see Table 18 173 Dimeric hu 4-1BBL
(71-248)--CL see Table 25 Fc knob chain 174 Monomeric hu see Table
25 4-1BBL (71-248)--CH1 81 DP47 Fc hole chain see Table 18 82 DP47
light chain see Table 18
[0819] 7.3.2 Antibodies as Control Molecules
[0820] Two control human IgG1 containing PGLALA were prepared.
[0821] Table 72 shows the cDNA and amino acid sequences of the
anti-CD19 huIgG1 PGLALA (clone 8B8-018), i.e. control G.
[0822] Table 73 shows the cDNA and amino acid sequences of germline
control DP47 huIgG1 PGLALA (control F).
TABLE-US-00076 TABLE 72 cDNA and amino acid sequences of anti-
CD19(8B8-018) huIgG1 PGLALA (control G) SEQ ID NO: Description
Sequence 315 nucleotide CAGGTCCAGCTGGTGCAGTCCGGCGCCGAGGT sequence
CAAGAAACCCGGGGCTTCTGTGAAGGTTTCAT CD19(8B8-018)
GCAAGGCAAGCGGATACACCTTCACCGACTAT heavy chain
ATCATGCATTGGGTCAGGCAGGCCCCTGGCCA (huIgG1
AGGTCTCGAATGGATGGGCTACATTAACCCAT PGLALA)
ATAATGATGGCTCCAAATACACCGAGAAGTTT CAGGGAAGAGTCACTATGACATCTGACACCAG
TATCAGCACTGCTTACATGGAGCTGTCCCGCC TTCGGTCTGATGACACCGCAGTGTATTACTGT
GCCAGGGGCACATATTACTACGGCTCAGCTCT GTTCGACTATTGGGGGCAGGGAACCACAGTAA
CCGTGAGCTCCGCAAGTACTAAGGGCCCATCG GTCTTCCCCCTGGCACCCTCCTCCAAGAGCAC
CTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTG
TCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAG
GACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCCAGCAGCTTGGGCACCCAGACCTACAT
CTGCAACGTGAATCACAAGCCCAGCAACACCA AGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAAGCAGCTGGGGGACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCGTGGT
GGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAATG GCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCGGAGCCCCCATCGAGAAAACCATCT CCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGATGAGC TGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGT GGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTACAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC GTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCC TGTCCCCGGGCAAA 204 nucleotide see
Table 47 sequence CD19(8B8-018) light chain 316 CD19(8B8-018)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYI heavy chain
MHWVRQAPGQGLEWMGYINPYNDGSKYTEKF (huIgG1
QGRVTMTSDTSISTAYMELSRLRSDDTAVYYCA PGLALA)
RGTYYYGSALFDYWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALGAPIEKTISKAKGQPREPQVYTLPPSRDE
LTKNQVSLTCLVKGFYPIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK 206 CD19(8B8-018) see Table 47 light
chain
TABLE-US-00077 TABLE 73 cDNA and amino acid sequences of germline
control DP47 huIgG1 PGLALA (control F) SEQ ID NO: Description
Sequence 181 nucleotide sequence DP47 see Table 29 heavy chain (hu
IgG1 PGLALA) 80 DP47 light chain see Table 18 182 DP47 heavy chain
(hu see Table 29 IgG1 PGLALA) 82 DP47 light chain see Table 18
[0823] 7.4 Production of CD19-Targeted Split Trimeric 4-1BB Ligand
Fc Fusion Antigen Binding Molecules and their Control Molecules
[0824] The targeted and untargeted split trimeric 4-1BB ligand Fc
(kih) fusion antigen binding molecule encoding sequences were
cloned into a plasmid vector, which drives expression of the insert
from an MPSV promoter and contains a synthetic polyA sequence
located at the 3' end of the CDS. In addition, the vector contains
an EBV OriP sequence for episomal maintenance of the plasmid.
[0825] The split trimeric 4-1BB ligand Fc (kih) fusion antigen
binding molecule was produced by co-transfecting HEK293-EBNA cells
with the mammalian expression vectors using polyethylenimine. The
cells were transfected with the corresponding expression vectors.
For variants 1,2,4,5 and it's control B, D and E, at a 1:1:1:1
ratio ("vector dimeric ligand-CL- knob chain": "vector monomeric
ligand fusion-CH1": "vector anti-CD19 Fab-hole chain": "vector
anti-CD19 light chain"). For variant 3, 6 and it's control C, at a
1:1:1 ratio ("vector huIgG1 Fc hole dimeric ligand chain": "vector
huIgG1 Fc knob monomeric ligand chain": "vector anti-CD19 light
chain"). Human IgGs, used as control in the assay, were produced as
for the bispecific construct (for transfection only a vector for
light and a vector for heavy chain were used at a 1:1 ratio).
[0826] For production in 500 mL shake flasks, 300 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 10 minutes at 210.times.g,
and the supernatant was replaced by 20 mL pre-warmed CD CHO medium.
Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CD
CHO medium. After addition of 540 .mu.L PEI, the solution was
vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere. After the incubation, 160 mL of Excell medium
supplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM
valproic acid was added and cells were cultured for 24 hours. One
day after transfection 12% Feed (amino acid and glucose) were
added. After culturing for 7 days, the supernatant was collected by
centrifugation for 30-40 minutes at least 400.times.g. The solution
was sterile filtered (0.22 .mu.m filter), supplemented with sodium
azide to a final concentration of 0.01% (w/v), and kept at
4.degree. C.
[0827] The split trimeric 4-1BB ligand Fc (kih) fusion antigen
binding molecule, as well as the IgG, was purified from cell
culture supernatants by affinity chromatography using Protein A,
followed by size exclusion chromatography. For affinity
chromatography, the supernatant was loaded on a MABSELECT SURE.RTM.
column (CV=5-15 mL, resin from GE Healthcare) equilibrated with
sodium phosphate (20 mM), sodium sitrate (20 mM) buffer (pH 7.5).
Unbound protein was removed by washing with at least 6 column
volumes of the same buffer. The bound protein was eluted using
either a linear gradient (20 CV) or a step elution (8 CV) with 20
mM sodium citrate, 100 mM sodium chloride, 100 mM glycine buffer
(pH 3.0). For the linear gradient an additional 4 column volumes
step elution was applied.
[0828] The pH of collected fractions was adjusted by adding 1/10
(v/v) of 0.5M sodium phosphate, pH8.0. The protein was concentrated
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 20 mM histidine, 140 mM sodium
chloride, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20) solution of pH
6.0.
[0829] The protein concentration was determined by measuring the
optical density (OD) at 280 nm, using a molar extinction
coefficient calculated on the basis of the amino acid sequence.
Purity and molecular weight of the targeted trimeric 4-1BB ligand
Fc (kih) fusion was analyzed by SDS-PAGE in the presence and
absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining
with Coomassie SIMPLYBLUE.TM. SafeStain (Invitrogen USA). The
aggregate content of samples was analyzed using a TSKGEL.RTM. G3000
SW XL analytical size-exclusion column (Tosoh) equilibrated in 25
mM K.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-arginine
monohydrochloride, 0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at
25.degree. C.
[0830] Table 74 summarizes the yield and final monomer content of
the CD19 targeted split trimeric 4-1BB ligand Fc (kih) fusion
antigen molecules.
TABLE-US-00078 TABLE 74 Biochemical analysis of CD19 targeted split
trimeric 4-1BB ligand Fc (kih) fusion antigen binding molecules
Monomer [%] Yield Construct (SEC) [mg/l] monovalent CD19(8B8-018)
targeted split trimeric 98 8.6 4-1BB ligand (71-254) Fc fusion
anitgcontaining CH-CL cross with charged residues (construct 3.1)
bivalent CD19(8B8-018) targeted split 100 11.3 trimeric 4-1BB
ligand (71-254) Fc fusion (construct 3.3) monovalent CD19(8B8-018)
targeted split trimeric 99 11.5 4-1BB ligand (71-248) Fc fusion
containing CH-CL cross with charged residues (construct 3.4)
monovalent CD19(8B8-018) targeted split trimeric 97 13.3 4-1BB
ligand (71-248) Fc fusion containing CH-CL cross without charged
residues (construct 3.5) bivalent CD19(8B8-018) targeted split
trimeric 96 19.9 4-1BB ligand (71-248) Fc fusion (construct 3.6)
monovalent CD19(8B8-2B11) targeted split 99.2 21.2 trimeric 4-1BB
ligand (71-248) Fc fusion containing CH-CL cross with charged
residues (construct 4.4)
[0831] Table 75 summarizes the yield and final monomer content of
the DP47 untargeted split trimeric 4-1BB ligand Fc (kih) fusion,
both monovalent (control B, D and E) and bivalent (control C).
TABLE-US-00079 TABLE 75 Biochemical analysis of DP47 untargeted
split trimeric 4-1BB ligand Fc (kih) fusion Monomer [%] Yield
Construct (SEC) [mg/l] monovalent DP47-untargeted split trimeric 99
15.4 human 4-1BB ligand (71-254) Fc (kih) fusion (control B)
bivalent DP47 untargeted split trimeric human 98 12.6 4-1BB ligand
(71-254) Fc (kih) fusion (control C) monovalent DP47-untargeted
split trimeric 99.5 25.9 human 4-1BB ligand (71-254) Fc (kih)
fusion (control D) monovalent DP47-untargeted split trimeric 93.3
4.1 human 4-1BB ligand (71-254) Fc (kih) fusion (control E)
[0832] Table 76 summarizes the yield and final monomer content of
anti-CD19 (8B8-018) and germline DP47 human IgG1 PGLALA (control
F).
TABLE-US-00080 TABLE 76 Biochemical analysis of control human IgG1
PGLALA Monomer [%] Yield Construct (SEC) [mg/l] anti-CD19(8B8-018)
huIgG1 PGLALA 100 36.6 germline DP47 human IgG1 PGLALA 100 50
Example 8
Functional Characterization of the CD19 Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0833] 8.1. Surface Plasmon Resonance (Affinity)
[0834] Binding of CD19 targeted split trimeric 4-1BB ligand Fc
fusion antigen binding molecules (constructs 3.4 and 3.6) to the
recombinant 4-1BB Fc(kih) and CD19 was assessed by surface plasmon
resonance (SPR). All SPR experiments were performed on a
BIACORE.RTM. T200 instrument at 25.degree. C. with HBS-EP as
running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005%
Surfactant P20, Biacore, Freiburg/Germany).
[0835] Interaction with Human and Cynomolgus 4-1BB
[0836] Anti-human Fab antibody (Biacore, Freiburg/Germany) was
directly coupled on a CMS chip at pH 5.0 using the standard amine
coupling kit (Biacore, Freiburg/Germany). The immobilization level
was approximately 8000 RU. The CD19 targeted split trimeric 4-1BB
ligand Fc fusions were captured for 60 seconds at 2 and 5 nM
(control D was also injected). Recombinant human or cynomolgus
4-1BB avi His was passed at a concentration range from 2.7 to 2000
nM (3-fold dilution) with a flow of 30 .mu.L/minutes through the
flow cells over 120 seconds. The dissociation was monitored for 180
seconds. Bulk refractive index differences were corrected for by
subtracting the response obtained on reference flow cell. Here, the
antigens were flown over a surface with immobilized anti-human Fab
antibody but on which HBS-EP has been injected rather than the
antibodies.
[0837] Interaction with Human CD19
[0838] Anti-human Fab antibody (Biacore, Freiburg/Germany) was
directly coupled on a CMS chip at pH 5.0 using the standard amine
coupling kit (Biacore, Freiburg/Germany). The immobilization level
was approximately 8000 RU. The CD19 targeted split trimeric 4-1BB
ligand Fc fusions, or the control antibody (anti-CD19(8B8-018)
huIgG1 PGLALA) were captured for 60 seconds at 20 nM. Recombinant
human CD19-Fc(kih) was passed at a concentration range from 7.8 to
500 nM (2-fold dilution) with a flow of 30 .mu.L/minutes through
the flow cells over 120 seconds. The dissociation was monitored for
120/1800 seconds. Bulk refractive index differences were corrected
for by subtracting the response obtained on reference flow cell.
Here, the antigens were flown over a surface with immobilized
anti-human Fab antibody but on which HBS-EP has been injected
rather than the antibodies.
[0839] Kinetic constants were derived using the Biacore T200
Evaluation Software (vAA, Biacore AB, Uppsala/Sweden), to fit rate
equations for 1:1 Langmuir binding by numerical integration.
[0840] The bispecific constructs 3.4, 3.6 and control D bind
similarly to 4-1BB. Table 77 shows the average with standard
deviation (in parenthesis) from the two experiments (using the
construct capture solution either at 2 nM or 5 nM). The bispecific
constructs 3.4 and 3.6 bind human CD19 with a similar affinity as
the IgG. Affinity constants for the interaction were determined by
fitting to a 1:1 Langmuir binding. For measurements with hu4-1BB
and cy4-1BB, average and standard deviation (in parenthesis) are
shown (two experiments with 2 or 5 nM capture solution).
TABLE-US-00081 TABLE 77 Binding of CD19 targeted split trimeric
4-1BB ligand Fc fusion to recombinant human (hu) 4-1BB, cynomolgus
(cy) 4-1BB and human (hu) CD19. Antigen ka (1/Ms) kd (1/s) KD (M)
monovalent CD19(8B8- hu 7.2E+04 2.5E-02 3.4E-07 018) targeted split
trimeric 4-1BB (5.9E+03) (1.0E-05) (2.8E-08) 4-1BB ligand (71-248)
Fc cy 1.2E+05 1.3E-02 1.1E-07 (kih) fusion containing 4-1BB
(8.6E+03) (1.8E-04) (9.9E-09) CH-CL cross with charged hu 2.77E+04
2.67E-04 9.64E-09 residues CD19 (construct 3.4) bivalent
CD19(8B8-018) hu 6.9E+04 2.4E-02 3.5E-07 targeted split trimeric 4-
4-1BB (1.7E+03) (1.5E-04) (1.1E-08) 1BB ligand (71-248) Fc cy
1.1E+05 1.4E-02 1.3E-07 (kih) fusion 4-1BB (7.7E+03) (3.1E-04)
(1.3E-08) (construct 3.6) hu 2.55E+04 2.69E-04 1.06E-08 CD19
monovalent DP47 hu 7.3E+04 2.6E-02 3.5E-07 untargeted split
trimeric 4-1BB (3.9E+03) (6.3E-04) (1.0E-08) human 4-1BB ligand
(71- cy 1.2E+05 1.4E-02 1.2E-07 248) Fc (kih) fusion with 4-1BB
(1.9E+03) (1.0E-04) (2.9E-09) CH-CL cross and with charged residues
(control D) anti-CD19(8B8-018) hu 2.12E+04 2.61E-04 1.23E-08 huIgG1
PGLALA CD19
[0841] 8.2. Surface Plasmon Resonance (Simultaneous Binding)
[0842] The capacity of binding simultaneously human 4-1BB Fc(kih)
and human CD19 was assessed by surface plasmon resonance (SPR). All
SPR experiments were performed on a BIACORE.RTM. T200 instrument at
25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4,
0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,
Freiburg/Germany). Biotinylated human 4-1BB Fc(kih) was directly
coupled to a flow cell of a streptavidin (SA) sensor chip.
Immobilization levels up to 250 resonance units (RU) were used.
[0843] The CD19 targeted trimeric split 4-1BBL constructs
(constructs 3.1, 3.3, 3.4, 3.5, 3.6, 4.4) were passed at a
concentration range of 200 nM with a flow of 30 .mu.L/minute
through the flow cells over 90 seconds and dissociation was set to
zero sec. Human CD19 was injected as second analyte with a flow of
30 .mu.L/minute through the flow cells over 90 seconds at a
concentration of 500 nM (FIG. 34). The dissociation was monitored
for 120 sec. Bulk refractive index differences were corrected for
by subtracting the response obtained in a reference flow cell,
where no protein was immobilized.
[0844] As can be seen in the graphs of FIGS. 35A to 35F, all
bispecific constructs could bind simultaneously human 4-1BB and
human CD19.
Example 9
[0845] Functional characterization of the CD-19 targeted 4-1BB
ligand trimer-containing Fc fusion antigen binding molecules
[0846] 9.1. Binding on Activated Human PMBCs of the CD19-Targeted
4-1BB Ligand Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecules
[0847] To determine binding of 4-1BBL trimer-containing Fc fusion
antigen binding molecules to human PBMCs, different titrated
concentrations of the CD19-targeted 4-1BBL trimer-containing Fc
fusion antigen binding molecules were used in the assay as
described in Example 5.2.
[0848] FIGS. 36A-1 to 36A-3 and 36B-1 to 36B-3 show the binding of
Constructs 3.1, 3.3, 3.4, 3.5 and 3.6 as prepared in Example 7 on
activated 4-1BB-expressing CD4+ T cells and CD8+ T cells,
respectively. Gates were set on living CD45+CD3+CD4+ or
CD45+CD3+CD8+ T cells and MFI of PE-conjugated AffiniPure
anti-human IgG IgG Fc.gamma.-fragment-specific goat F(ab')2
fragment were blotted against the titrated concentration of
targeted split trimeric 4-1BB ligand Fc fusion variants. Table 78
shows the EC.sub.50 values as measured for Constructs 3.1, 3.3.
3.4, 3.5 and 3.6 and control molecules.
TABLE-US-00082 TABLE 78 Binding on activated 4-1BB-expressing CD4 +
T cells and CD8 + T cells EC.sub.50 [nM] EC.sub.50 [nM] Construct
4-1BB.sup.+CD8.sup.+ 4-1BB.sup.+CD4.sup.+ Control B 0.05 0.26
Control C 0.02 0.30 Control D 0.04 0.28 Control E 0.13 1.22 3.1
0.03 0.28 3.3 0.01 0.29 3.4 0.15 2.04 3.5 0.04 1.03 3.6 0.05
0.21
[0849] 9.2 Binding to CD19-Expressing Tumor Cells
[0850] For binding assays on CD19-expressing tumor cells, the
following human CD19-expressing lymphoma cell lines were used:
diffuse large non-Hodgkin B cell lymphoma (B-NHL) cell line
SU-DHL-8 (DSMZ ACC573), acute B cell precursor lymphoid leukemia
cell line Nalm6 (DSMZ ACC-128), diffuse large cell lymphoblast
lymphoma cell line Toledo (ATCC CRL-2631) and diffuse large B cell
lymphoma cell line OCI-Ly18 (DSMZ ACC-699). The assays were
preformed as described for the FAP-expressing MV-3 and WM-266-4
tumor cell lines in Example 5.3.
[0851] Gates were set on living tumor cells and MFI of
PE-conjugated AffiniPure anti-human IgG IgG
Fc.gamma.-fragment-specific goat F(ab')2 fragment were blotted
against the titrated concentration of targeted split trimeric 4-1BB
ligand Fc fusion constructs.
[0852] FIGS. 37A-1 to 37A-3 show the binding of Constructs 3.1,
3.3, 3.4, 3.5 and 3.6 as prepared in Example 7.1 to diffuse large
non-Hodgkin B cell lymphoma (B-NHL) cell line SU-DHL-8 and in FIGS.
37B-1 to 37B-3 the binding of Constructs 3.1, 3.3, 3.4, 3.5 and 3.6
to acute B cell precursor lymphoid leukemia cell line Nalm6 is
presented. FIGS. 37C-1 to 37C-3 show the binding of Constructs 3.1,
3.3, 3.4, 3.5 and 3.6 to diffuse large cell lymphoblast lymphoma
cell line Toledo and FIGS. 37D-1 to 37D-3 show the binding of
Constructs 3.1, 3.3, 3.4, 3.5 and 3.6 to diffuse large B cell
lymphoma cell line OCI-Ly18. Table 79 shows the EC50 values as
measured for Constructs 3.1, 3.3, 3.4, 3.5 and 3.6 and control
molecules.
TABLE-US-00083 TABLE 79 Binding to CD19-expressing tumor cells
EC.sub.50 [nM] EC.sub.50 [nM] EC.sub.50 [nM] EC.sub.50 [nM]
Construct SU-DHL-8 Nalm6 Toledo OCI-Ly18 3.1 0.64 0.43 0.29 0.29
3.3 0.15 0.14 0.10 0.09 3.4 0.31 0.39 0.29 0.26 3.5 0.54 0.43 0.27
0.31 3.6 0.14 0.12 0.09 0.10 control G 0.09 0.10 0.06 0.07
Example 10
Biological Activity of the CD19-Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0853] 10.1. NF-.kappa.B Activation in HeLa Cells Expressing Human
4-1BB
[0854] HeLa cells expressing human 4-1BB and NF-.kappa.B-luciferase
were generated as described in Example 6.1.
[0855] NF-.kappa.B Activation in Hela Cells Expressing Human 4-1BB
Co-Cultured with CD19-Expressing Tumor Cells
[0856] NF-.kappa.B-luciferase human-4-1BB HeLa cells were harvested
and resuspended in DMEM medium supplied with 10% (v/v) FBS and 1%
(v/v) GlutaMAX-I to a concentration of 0.2.times.10.sup.6 cells/ml.
100 .mu.l (2.times.10.sup.4 cells) of this cell suspension were
transferred to each well of a sterile white 96-well flat bottom
tissue culture plate with lid (greiner bio-one, Cat. No. 655083)
and the plate were incubated at 37.degree. C. and 5% CO.sub.2
overnight. The next day 50 .mu.L of medium containing titrated
concentrations of CD19-targeted 4-1BB ligand trimer-containing Fc
fusion antigen binding molecules (CD19 split 4-1BBL trimer) or
DP47-untargeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules (DP47 split 4-1BBL trimer) were added.
CD19-expressing B cell lymphoma cell lines (diffuse large
non-Hodgkin B cell lymphoma (B-NHL) cell line SU-DHL-8 (DSMZ
ACC573) and human non-Hodgkin's B cell lymphoma cell line Pfeiffer
(ATCC CRL-2632)) were resuspended in DMEM medium supplied with 10%
(v/v) FBS and 1% (v/v) GlutaMAX-I to a concentration of
2.times.10.sup.6 cells/ml.
[0857] Suspension of CD19-expressing B cell lymphoma cell (50 final
ratio 1:5) or only medium were added to each well and plates were
incubated for 6 hours at 37.degree. C. and 5% CO.sub.2. Cells were
washed two times with 200 .mu.L/well DPBS. 40 .mu.l freshly
prepared Reporter Lysis Buffer (Promega, Cat-No: E3971) were added
to each well and the plate were stored over night at -20.degree. C.
The next day frozen cell plate and Detection Buffer (Luciferase
1000 Assay System, Promega, Cat. No. E4550) were thawed at room
temperature. 100 .mu.L of detection buffer were added to each well
and luciferase activity was measured as fast as possible using a
SpectraMax M5/M5e microplate reader and a SoftMax Pro Software
(Molecular Devices) counting light emission in URL (units of
released light for 0.5s/well) or Victor3 1420 multilabel counter
plate reader (Perkin Elmer) and the Perkin Elmer 2030 Manager
Software counting light emission as counts per seconds (CPS) and
blotted against the concentration of tested constructs.
[0858] CD19-targeted 4-1BB ligand trimer-containing Fc fusion
antigen binding molecules Constructs 3.1 and 3.3 triggered
activation of the NF-kB signaling pathway in the reporter cell line
in the presence of CD19-expressing B cell lymphoma cells. In
contrast, the untargeted control molecules failed to trigger such
an effect at any of the tested concentrations (FIGS. 38A to
38C).
Example 11
[0859] 11.1 Preparation of CEA (T84.66-LCHA) Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0860] 11.1.1 Humanization of Anti-CEA Clone T84.66
[0861] Novel humanized variants of the murine antibody T84.66
(Wagener et al., J Immunol 130, 2308 (1983), Neumaier et al., J
Immunol 135, 3604 (1985)) were developed by grafting of the CDRs
onto human germline framework acceptor sequences.
[0862] Humanization of an antibody from non-human origin consists
essentially of transplanting the CDR residues from the non-human
antibody (donor) onto the framework of a human (acceptor) antibody.
Normally the acceptor framework is selected by aligning the
sequence of the donor to a collection of potential acceptor
sequences and choosing one that has either reasonable homology to
the donor, or shows similar amino acids at some positions critical
for structure and activity. In the present case, the search for the
antibody acceptor framework was performed by aligning the mouse
T84.66 protein (NCBI Acc No: CAA36980 for the heavy chain (SEQ ID
NO:317), and CAA36979 (SEQ ID NO:318) for the light chain) sequence
to a collection of human germ-line sequences and picking that human
sequence that showed high sequence identity. Here, the sequence
IGHV1-69*08 from the IMGT database was chosen as the heavy chain
framework acceptor sequence (IMGT Acc No. Z14309, SEQ ID NO:319),
and the IGKV3-11*01 sequence (IMGT Acc No. X01668, SEQ ID NO:320)
was chosen to be the framework acceptor for the light chain. Onto
these two acceptor frameworks, the three complementary determining
regions (CDRs) of the mouse heavy and light variable domains were
grafted. Since the framework 4 (FR4) region is not part of the
variable region of the germ line V gene, the alignment for that
position was done individually. The JH4 sequence was chosen for the
heavy chain, and the JK2 sequence was chosen for the light
chain.
[0863] 11.1.2 Binding of Different Humanized Variants of T84.66 IgG
to Cells
[0864] The binding of different humanized variants of T84.66 IgG
was tested on CEA-expressing human gastric adenocarcinoma cells
(MKN45, DSMZ ACC 409).
[0865] Cells were harvested, counted, checked for viability and
re-suspended at 2.times.10.sup.6 cells/ml in FACS buffer (100 .mu.l
PBS 0.1% BSA). 100 .mu.l of cell suspension (containing
0.2.times.10.sup.6 cells) were incubated in round-bottom 96-well
plate for 30 min at 4.degree. C. with increasing concentrations of
the CEA IgG (4 ng/ml-60 .mu.g/ml), washed twice with cold PBS 0.1%
BSA, re-incubated for further 30 min at 4.degree. C. with the
PE-conjugated AffiniPure F(ab')2 Fragment goat anti-human IgG Fcg
Fragment Specific secondary antibody (Jackson Immuno Research Lab
PE #109-116-170), washed twice with cold PBS 0.1% BSA and
immediately analyzed by FACS using a FACS CantoII (Software FACS
Diva). Binding curves and EC50 values were obtained and calculated
using GraphPadPrism5.
[0866] FIG. 39 shows the different binding pattern of selected
humanized variants of the T84.66 IgG to human CEA, expressed on
MKN45 cells. Based on the calculated EC50 binding values (Table
80), the humanized variant 1 was selected for further
evaluation.
TABLE-US-00084 TABLE 80 Binding of different humanized variants of
T84.66 IgGs to cells (EC50 values, based on binding curves shown in
FIG. 39, calculated by Graph Pad Prism). EC50 (.mu.g/ml) Parental
chimeric T84.66 0.99 Humanized variant 1 1.5 Humanized variant 2
8.6 Humanized variant 3 1.4 Humanized variant 4 3.1 Humanized
variant 5 -- Humanized variant 6 --
[0867] Humanized variant 1 is termed in the following T84.66-LCHA.
The amino acid sequences of its CDRs and of the VH and VL as well
as the aminoacid sequences of the VH and VL domain of the parental
chimeric T84.66 clone are shown in Table 81.
TABLE-US-00085 TABLE 81 Amino acid sequences of the variable
domains of CEA clone T84.66-LCHA and its parental antibody T84.66
SEQ ID Description Sequence NO: CEA CDR-H1 DTYMH 321 CEA CDR-H2
RIDPANGNSKYVPKFQG 322 CEA CDR-H3 FGYYVSDYAMAY 323 CEA CDR-L1
RAGESVDIFGVGFLH 324 CEA CDR-L2 RASNRAT 325 CEA CDR-L3 QQTNEDPYT 326
Parental CEA EVQLQQSGAELVEPGASVK 327 binder VH LSCTASGFNIKDTYMHWVK
QRPEQGLEWIGRIDPANGN SKYVPKFQGKATITADTSS NTAYLQLTSLTSEDTAVYY
CAPFGYYVSDYAMAYWGQG TSVTVSS Parental CEA DIVLTQSPASLAVSLGQRA 328
binder VL TMSCRAGESVDIFGVGFLH WYQQKPGQPPKLLIYRASN
LESGIPVRFSGTGSRTDFT LIIDPVEADDVATYYCQQT NEDPYTFGGGTKLEIK Humanized
QVQLVQSGAEVKKPGSSVK 329 CEA binder VSCKASGFNIKDTYMHWVR CEA (T84.66-
QAPGQGLEWMGRIDPANGN LCHA) VH SKYVPKFQGRVTITADTST
STAYMELSSLRSEDTAVYY CAPFGYYVSDYAMAYWGQG TLVTVSS Humanized
EIVLTQSPATLSLSPGERA 330 CEA binder TLSCRAGESVDIFGVGFLH CEA (T84.66-
WYQQKPGQAPRLLIYRASN LCHA) VL RATGIPARFSGSGSGTDFT
LTISSLEPEDFAVYYCQQT NEDPYTFGQGTKLEIK
[0868] 11.2 Preparation of CEA (T84.66-LCHA) Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0869] Different fragments of the DNA sequence encoding part of the
ectodomain (amino acid 71-254 and 71-248) of human 4-1BB ligand
were synthetized according to the P41273 sequence of Uniprot
database (SEQ ID NO:42).
[0870] 11.2.1 Preparation of Monovalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 5.1)
[0871] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
29A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL. A polypeptide containing one ectodomain of 4-1BB ligand
(71-254) and fused to the human IgG1-CH domain, was cloned as
described in FIG. 29B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0872] To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0873] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the hole
or the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
[0874] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CEA-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CEA light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CEA binding Fab (FIG. 40A, Construct 5.1).
[0875] Table 82 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 5.1).
TABLE-US-00086 TABLE 82 cDNA and amino acid sequences of monovalent
CEA(T84.66-LCHA) targeted split trimeric 4- 1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 5.1).* for charged residues SEQ ID NO: Description
Sequence 129 Nucleotide see Table 3 sequence Dimeric hu 4-1BBL (71-
254)-CL* Fc knob chain 130 Nucleotide see Table 3 sequence
Monomeric hu 4-1BBL (71- 254)-CH1* 331 Nucleotide
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAA sequence anti-
GTGAAGAAACCCGGCAGCAGCGTGAAGGTG CEA(T84.66-
TCCTGCAAGGCCAGCGGCTTCAACATCAAG LCHA) Fc hole
GACACCTACATGCACTGGGTGCGCCAGGCC chain CCTGGACAGGGACTGGAATGGATGGGCAGA
ATCGACCCCGCCAACGGCAACAGCAAATAC GTGCCCAAGTTCCAGGGCAGAGTGACCATC
ACCGCCGACACCAGCACCTCCACCGCCTAC ATGGAACTGAGCAGCCTGCGGAGCGAGGAC
ACCGCCGTGTACTACTGTGCCCCCTTCGGC TACTACGTGTCCGACTACGCCATGGCCTAT
TGGGGCCAGGGCACACTCGTGACCGTGTCC TCTGCTAGCACCAAGGGCCCCTCCGTGTTC
CCCCTGGCCCCCAGCAGCAAGAGCACCAGC GGCGGCACAGCCGCTCTGGGCTGCCTGGTC
AAGGACTACTTCCCCGAGCCCGTGACCGTG TCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGT TCTGGCCTGTATAGCCTGAGCAGCGTGGTC
ACCGTGCCTTCTAGCAGCCTGGGCACCCAG ACCTACATCTGCAACGTGAACCACAAGCCC
AGCAACACCAAGGTGGACAAGAAGGTGGAG CCCAAGAGCTGCGACAAAACTCACACATGC
CCACCGTGCCCAGCACCTGAAGCTGCAGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAA
CCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTG
AGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAATGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAA
GCCCTCGGCGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTGCACCCTGCCCCCATCCCGGGAT GAGCTGACCAAGAACCAGGTCAGCCTCTCG
TGCGCAGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACTCCGACGGCTCCTTCTTCCTC
GTGAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTAC ACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA
332 Nucleotide GAGATCGTGCTGACCCAGAGCCCTGCCACC sequence anti-
CTGTCACTGTCTCCAGGCGAGAGAGCCACC CEA(T84.66-
CTGAGCTGTAGAGCCGGCGAGAGCGTGGAC LCHA) light
ATCTTCGGCGTGGGATTTCTGCACTGGTAT chain CAGCAGAAGCCCGGCCAGGCCCCCAGACTG
CTGATCTACAGAGCCAGCAACCGGGCCACA GGCATCCCCGCCAGATTTTCTGGCTCTGGC
AGCGGCACCGACTTCACCCTGACAATCAGC AGCCTGGAACCCGAGGACTTCGCCGTGTAC
TACTGCCAGCAGACCAACGAGGACCCCTAC ACCTTTGGCCAGGGCACCAAGCTGGAAATC
AAGCGTACGGTGGCTGCACCATCTGTCTTC ATCTTCCCGCCATCTGATGAGCAGTTGAAA
TCTGGAACTGCCTCTGTTGTGTGCCTGCTG AATAACTTCTATCCCAGAGAGGCCAAAGTA
CAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAG
GACAGCAAGGACAGCACCTACAGCCTCAGC AGCACCCTGACGCTGAGCAAAGCAGACTAC
GAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGT 115 Dimeric hu 4- see Table 3 1BBL
(71-254)- CL* Fc knob chain 116 Monomeric hu see Table 3 4-1BBL
(71- 254)-CH1* 333 anti-CEA QVQLVQSGAEVKKPGSSVKVSCKASGFNIK
(T84.66-LCHA) DTYMHWVRQAPGQGLEWMGRIDPANGNSKY Fc hole chain
VPKFQGRVTITADTSTSTAYMELSSLRSED TAVYYCAPFGYYVSDYAMAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPEAAG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALGAPIEKTIS
KAKGQPREPQVCTLPPSRDELTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 334 anti-CEA
EIVLTQSPATLSLSPGERATLSCRAGESVD (T84.66-LCHA)
IFGVGFLHWYQQKPGQAPRLLIYRASNRAT light chain
GIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQTNEDPYTFGQGTKLEIKRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC
[0876] 11.2.2 Preparation of Monovalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains without Charged
Residues (Construct 5.2)
[0877] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy as
depicted in FIG. 29A, but without amino acid mutations in the CL
domain: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human CL. A polypeptide containing one ectodomain of
4-1BB ligand (71-254) and fused to the human IgG1-CH1 domain, was
cloned in analogy as depicted in FIG. 29B, but without amino acid
mutations in the CH1 domain: human 4-1BB ligand, (G.sub.4S).sub.2
(SEQ ID NO:13) connector, human CH1.
[0878] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the hole
or the constant light chain of human IgG1.
[0879] The Pro329Gly, Leu234Ala and Leu235Ala mutations have been
introduced in the constant region of the knob and hole heavy chains
to abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831. Combination of the dimeric ligand-Fc
knob chain containing the S354C/T366W mutations, the monomeric CH1
fusion, the targeted anti-CEA-Fc hole chain containing the
Y349C/T366S/L368A/Y407V mutations and the anti-CEA light chain
allows generation of a heterodimer, which includes an assembled
trimeric 4-1BB ligand and a CEA-binding Fab (FIG. 40B, Construct
5.2).
[0880] Table 83 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 5.2).
TABLE-US-00087 TABLE 83 cDNA and amino acid sequences of monovalent
CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 5.2) SEQ ID NO: Description Sequence 165 Nucleotide
sequence dimeric see Table 22 ligand (71-254)--CL Fc knob chain 166
Nucleotide sequence monomeric see Table 22 hu 4-1BBL (71-254)--CH1
331 Nucleotide sequence anti-CEA see Table 82 (T84.66-LCHA) Fc hole
chain 332 Nucleotide sequence anti-CEA see Table 82 (T84.66-LCHA)
light chain 117 Dimeric ligand (71-254)--CL Fc see Table 22 knob
chain 118 Monomeric ligand (71-254)--CH1 see Table 22 333 anti-CEA
(T84.66-LCHA) Fc see Table 82 hole chain 334 anti-CEA (T84.66-LCHA)
light see Table 82 chain
[0881] 11.2.3 Preparation of Bivalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen
Binding (Construct 5.3)
[0882] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 29C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 29D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0883] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the
hole, the knob or the constant light chain of human IgG1. The
Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced
in the constant region of the knob and hole heavy chains to
abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831. Combination of the anti-CEA huIgG1
hole dimeric ligand chain containing the Y349C/T366S/L368A/Y407V
mutations, the anti-CEA huIgG1 knob monomeric ligand chain
containing the S354C/T366W mutations and the anti-CEA light chain
allows generation of a heterodimer, which includes an assembled
trimeric 4-1BB ligand and two CEA binding Fabs (FIG. 40C, construct
5.3).
[0884] Table 84 shows the cDNA and amino acid sequences of the
bivalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule (construct
5.3).
TABLE-US-00088 TABLE 84 cDNA and amino acid sequences of bivalent
CEA(T84.66-LCHA) targeted split trimeric 4-1BB ligand (71-254) Fc
(kih) PGLALA fusion (construct 5.3) SEQ ID NO: Description Sequence
335 Nucleotide CAGGTGCAGCTGGTGCAGTCTGGCGCCGA sequence anti-
AGTGAAGAAACCCGGCAGCAGCGTGAAGG CEA(T84.66-
TGTCCTGCAAGGCCAGCGGCTTCAACATC LCHA) Fc hole
AAGGACACCTACATGCACTGGGTGCGCCA dimeric 4-1BBL
GGCCCCTGGACAGGGACTGGAATGGATGG (71-254) chain
GCAGAATCGACCCCGCCAACGGCAACAGC AAATACGTGCCCAAGTTCCAGGGCAGAGT
GACCATCACCGCCGACACCAGCACCTCCA CCGCCTACATGGAACTGAGCAGCCTGCGG
AGCGAGGACACCGCCGTGTACTACTGTGC CCCCTTCGGCTACTACGTGTCCGACTACG
CCATGGCCTATTGGGGCCAGGGCACACTC GTGACCGTGTCCTCTGCTAGCACCAAGGG
CCCCTCCGTGTTCCCCCTGGCCCCCAGCA GCAAGAGCACCAGCGGCGGCACAGCCGCT
CTGGGCTGCCTGGTCAAGGACTACTTCCC CGAGCCCGTGACCGTGTCCTGGAACAGCG
GAGCCCTGACCTCCGGCGTGCACACCTTC CCCGCCGTGCTGCAGAGTTCTGGCCTGTA
TAGCCTGAGCAGCGTGGTCACCGTGCCTT CTAGCAGCCTGGGCACCCAGACCTACATC
TGCAACGTGAACCACAAGCCCAGCAACAC CAAGGTGGACAAGAAGGTGGAGCCCAAGA
GCTGCGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTA
ACAACAGCACGTCCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGCCCTCGGCGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTGCACCCTGCCCCCATC
CCGGGATGAGCTGACCAAGAACCAGGTCA GCCTCTCGTGCGCAGTCAAAGGCTTCTAT
CCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGC TCCTTCTTCCTCGTGAGCAAGCTCACCGT
GGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTGGAGGCGGCGGAA
GCGGAGGAGGAGGATCCAGAGAGGGCCCT GAGCTGAGCCCCGATGATCCTGCTGGACT
GCTGGACCTGCGGCAGGGCATGTTTGCTC AGCTGGTGGCCCAGAACGTGCTGCTGATC
GATGGCCCCCTGTCCTGGTACAGCGATCC TGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAA CTGGTGGTGGCCAAGGCCGGCGTGTACTA
CGTGTTCTTTCAGCTGGAACTGCGGAGAG TGGTGGCCGGCGAAGGATCTGGCTCTGTG
TCTCTGGCCCTGCATCTGCAGCCTCTGAG AAGCGCTGCTGGCGCTGCAGCTCTGGCAC
TGACAGTGGATCTGCCTCCTGCCAGCTCC GAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCC AGAGGCTGGGAGTGCATCTGCACACAGAG
GCCAGGGCTAGACACGCCTGGCAGCTGAC ACAGGGCGCTACAGTGCTGGGCCTGTTCA
GAGTGACCCCCGAGATTCCAGCCGGCCTG CCTTCTCCAAGAAGCGAAGGCGGAGGCGG
ATCTGGCGGCGGAGGATCTAGAGAGGGAC CCGAACTGTCCCCTGACGATCCAGCCGGG
CTGCTGGATCTGAGACAGGGAATGTTCGC CCAGCTGGTGGCTCAGAATGTGCTGCTGA
TTGACGGACCTCTGAGCTGGTACTCCGAC CCAGGGCTGGCAGGGGTGTCCCTGACTGG
GGGACTGTCCTACAAAGAAGATACAAAAG AACTGGTGGTGGCTAAAGCTGGGGTGTAC
TATGTGTTTTTTCAGCTGGAACTGAGGCG GGTGGTGGCTGGGGAGGGCTCAGGATCTG
TGTCCCTGGCTCTGCATCTGCAGCCACTG CGCTCTGCTGCTGGCGCAGCTGCACTGGC
TCTGACTGTGGACCTGCCACCAGCCTCTA GCGAGGCCAGAAACAGCGCCTTCGGGTTC
CAAGGACGCCTGCTGCATCTGAGCGCCGG ACAGCGCCTGGGAGTGCATCTGCATACTG
AAGCCAGAGCCCGGCATGCTTGGCAGCTG ACTCAGGGGGCAACTGTGCTGGGACTGTT
TCGCGTGACACCTGAGATCCCTGCCGGAC TGCCAAGCCCTAGATCAGAA 336 Nucleotide
CAGGTGCAGCTGGTGCAGTCTGGCGCCGA sequence anti-
AGTGAAGAAACCCGGCAGCAGCGTGAAGG CEA(T84.66-
TGTCCTGCAAGGCCAGCGGCTTCAACATC LCHA) Fc knob
AAGGACACCTACATGCACTGGGTGCGCCA monomeric 41-
GGCCCCTGGACAGGGACTGGAATGGATGG BBL (71-254)
GCAGAATCGACCCCGCCAACGGCAACAGC AAATACGTGCCCAAGTTCCAGGGCAGAGT
GACCATCACCGCCGACACCAGCACCTCCA CCGCCTACATGGAACTGAGCAGCCTGCGG
AGCGAGGACACCGCCGTGTACTACTGTGC CCCCTTCGGCTACTACGTGTCCGACTACG
CCATGGCCTATTGGGGCCAGGGCACACTC GTGACCGTGTCCTCTGCTAGCACCAAGGG
CCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCC CGAACCGGTGACGGTGTCGTGGAACTCAG
GCGCCCTGACCAGCGGCGTGCACACCTTC CCGGCTGTCCTACAGTCCTCAGGACTCTA
CTCCCTCAGCAGCGTGGTGACCGTGCCCT CCAGCAGCTTGGGCACCCAGACCTACATC
TGCAACGTGAATCACAAGCCCAGCAACAC CAAGGTGGACAAGAAAGTTGAGCCCAAAT
CTTGTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAG CCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCC TCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGCCCTCGGCGCCCCCATCGAGAAAA
CCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCCTG
CAGAGATGAGCTGACCAAGAACCAGGTGT CCCTGTGGTGTCTGGTCAAGGGCTTCTAC
CCCAGCGATATCGCCGTGGAGTGGGAGAG CAACGGCCAGCCTGAGAACAACTACAAGA
CCACCCCCCCTGTGCTGGACAGCGACGGC AGCTTCTTCCTGTACTCCAAACTGACCGT
GGACAAGAGCCGGTGGCAGCAGGGCAACG TGTTCAGCTGCAGCGTGATGCACGAGGCC
CTGCACAACCACTACACCCAGAAGTCCCT GAGCCTGAGCCCCGGCGGAGGCGGCGGAA
GCGGAGGAGGAGGATCCAGAGAGGGCCCT GAGCTGAGCCCCGATGATCCTGCTGGACT
GCTGGACCTGCGGCAGGGCATGTTTGCTC AGCTGGTGGCCCAGAACGTGCTGCTGATC
GATGGCCCCCTGTCCTGGTACAGCGATCC TGGACTGGCTGGCGTGTCACTGACAGGCG
GCCTGAGCTACAAAGAGGACACCAAAGAA CTGGTGGTGGCCAAGGCCGGCGTGTACTA
CGTGTTCTTTCAGCTGGAACTGCGGAGAG TGGTGGCCGGCGAAGGATCTGGCTCTGTG
TCTCTGGCCCTGCATCTGCAGCCTCTGAG AAGCGCTGCTGGCGCTGCAGCTCTGGCAC
TGACAGTGGATCTGCCTCCTGCCAGCTCC GAGGCCCGGAATAGCGCATTTGGGTTTCA
AGGCAGGCTGCTGCACCTGTCTGCCGGCC AGAGGCTGGGAGTGCATCTGCACACAGAG
GCCAGGGCTAGACACGCCTGGCAGCTGAC ACAGGGCGCTACAGTGCTGGGCCTGTTCA
GAGTGACCCCCGAGATTCCAGCCGGCCTG CCTTCTCCAAGAAGCGAA 332 Nucleotide see
Table 82 sequence anti- CEA(T84.66- LCHA) light chain 337 anti-
QVQLVQSGAEVKKPGSSVKVSCKASGFNI CEA(T84.66-
KDTYMHWVRQAPGQGLEWMGRIDPANGNS LCHA) Fc hole
KYVPKFQGRVTITADTSTSTAYMELSSLR dimeric 41-BBL
SEDTAVYYCAPFGYYVSDYAMAYWGQGTL (71-254) chain
VTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVCTLPPSRDELTKNQVSLSCAVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLVSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGGGGGSGGGGSREGP
ELSPDDPAGLLDLRQGMFAQLVAQNVLLI DGPLSWYSDPGLAGVSLTGGLSYKEDTKE
LVVAKAGVYYVFFQLELRRVVAGEGSGSV SLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGL
PSPRSEGGGGSGGGGSREGPELSPDDPAG LLDLRQGMFAQLVAQNVLLIDGPLSWYSD
PGLAGVSLTGGLSYKEDTKELVVAKAGVY YVFFQLELRRVVAGEGSGSVSLALHLQPL
RSAAGAAALALTVDLPPASSEARNSAFGF QGRLLHLSAGQRLGVHLHTEARARHAWQL
TQGATVLGLFRVTPEIPAGLPSPRSE 338 anti- QVQLVQSGAEVKKPGSSVKVSCKASGFNI
CEA(T84.66- KDTYMHWVRQAPGQGLEWMGRIDPANGNS LCHA) Fc knob
KYVPKFQGRVTITADTSTSTAYMELSSLR monomeric 4-
SEDTAVYYCAPFGYYVSDYAMAYWGQGTL 1BBL (71-254)
VTVSSASTKGPSVFPLAPSSKSTSGGTAA chain LGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPP
CPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPR
EPQVYTLPPCRDELTKNQVSLWCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGGGGGSGGGGSREGP
ELSPDDPAGLLDLRQGMFAQLVAQNVLLI DGPLSWYSDPGLAGVSLTGGLSYKEDTKE
LVVAKAGVYYVFFQLELRRVVAGEGSGSV SLALHLQPLRSAAGAAALALTVDLPPASS
EARNSAFGFQGRLLHLSAGQRLGVHLHTE ARARHAWQLTQGATVLGLFRVTPEIPAGL PSPRSE
334 anti- see Table 82 CEA(T84.66- LCHA) light chain
[0885] 11.2.4 Preparation of Monovalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 5.4)
[0886] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy to the one
depicted in FIG. 29A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CL. A polypeptide containing one ectodomain
of 4-1BB ligand (71-248) and fused to the human IgG1-CH domain, was
cloned in analogy to the one described in FIG. 29B: human 4-1BB
ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human CH.
[0887] The polypeptide encoding the dimeric 4-1BB ligand fused to
human CL domain was subcloned in frame with the human IgG1 heavy
chain CH2 and CH3 domains on the knob (Merchant, Zhu et al. 1998).
To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0888] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the hole
or the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the dimeric ligand-Fc knob chain containing the
S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CD19-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CEA binding Fab (FIG. 40D, construct 5.4).
[0889] Table 85 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 5.4).
TABLE-US-00089 TABLE 85 cDNA and amino acid sequences of monovalent
CEA(T84.66-LCHA) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross with charged residues
(construct 5.4). SEQ ID NO: Description Sequence 169 Nucleotide
sequence dimeric ligand see Table 24 (71-248)--CL* Fc knob chain
170 Nucleotide sequence monomeric hu see Table 24 4-1BBL
(71-248)--CH1* 331 Nucleotide sequence anti- see Table 82
CEA(T84.66-LCHA) Fc hole chain 332 Nucleotide sequence anti- see
Table 82 CEA(T84.66-LCHA) light chain 119 Dimeric ligand
(71-248)--CL* Fc see Table 24 knob chain 120 Monomeric ligand
(71-248)--CH1* see Table 24 333 anti-CEA(T84.66-LCHA) Fc hole see
Table 62 chain 334 anti-CEA(T84.66-LCHA) light chain see Table 59
*charged residues
[0890] 11.2.5 Preparation of Monovalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding Molecule with Crossed CH1-CL Domains without Charged
Residues (Construct 5.5)
[0891] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned in analogy as
depicted in FIG. 29A, but without amino acid mutations in the CL
domain: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human CL. A polypeptide containing one ectodomain of
4-1BB ligand (71-248) and fused to the human IgG1-CH1 domain, was
cloned in analogy as depicted in FIG. 29B, but without amino acid
mutations in the CH1 domain: human 4-1BB ligand, (G.sub.4S).sub.2
(SEQ ID NO:13) connector, human CH1.
[0892] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the hole
or the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the dimeric ligand-Fc knob chain containing the
S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CEA-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CEA light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CD19-binding Fab (FIG. 40E, Construct 5.5).
[0893] Table 86 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule containing
crossed CH-CL cross without charged residues (construct 5.5).
TABLE-US-00090 TABLE 86 cDNA and amino acid sequences of monovalent
CEA(T84.66-LCHA) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion containing CH-CL cross without charged residues
(construct 5.5). SEQ ID NO: Description Sequence 171 Nucleotide
sequence dimeric ligand see Table 25 (71-248)--CL Fc knob chain 172
Nucleotide sequence monomeric see Table 25 ligand (71-248)--CH1 331
Nucleotide sequence anti- see Table 82 CEA(T84.66-LCHA) Fc hole
chain 332 Nucleotide sequence anti- see Table 82 CEA(T84.66-LCHA)
light chain 173 Dimeric ligand (71-248)--CL Fc see Table 25 knob
chain 174 Monomeric ligand (71-248)--CH1 see Table 25 333
anti-CEA(T84.66-LCHA) Fc see Table 82 hole chain 334
anti-CEA(T84.66-LCHA) light see Table 82 chain
[0894] 11.2.6 Preparation of Bivalent CEA (T84.66-LCHA) Targeted
4-1BB Ligand (71-248) Trimer-Containing Fc (Kih) Fusion Antigen
Binding (Construct 5.6)
[0895] A polypeptide containing two ectodomains of 4-1BB ligand
(71-248), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 29C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 29D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0896] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66-LCHA, were
subcloned in frame with either the constant heavy chain of the
hole, the knob or the constant light chain of human IgG1. The
Pro329Gly, Leu234Ala and Leu235Ala mutations have been introduced
in the constant region of the knob and hole heavy chains to
abrogate binding to Fc gamma receptors according to the method
described in WO 2012/130831. Combination of the anti-CEA huIgG1
hole dimeric ligand chain containing the Y349C/T366S/L368A/Y407V
mutations, the anti-CEA huIgG1 knob monomeric ligand chain
containing the S354C/T366W mutations and the anti-CEA light chain
allows generation of a heterodimer, which includes an assembled
trimeric 4-1BB ligand and two CEA binding Fabs (FIG. 40F, construct
5.6).
[0897] Table 87 shows the cDNA and amino acid sequences of the
bivalent CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion antigen binding molecule (construct
5.6).
TABLE-US-00091 TABLE 87 cDNA and amino acid sequences of bivalent
CEA (T84.66-LCHA) targeted split trimeric 4-1BB ligand (71-248) Fc
(kih) fusion (construct 5.6) SEQ ID Descrip- NO: tion Sequence 339
Nucleo- CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA tide
ACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCG sequence
GCTTCAACATCAAGGACACCTACATGCACTGGGTGCGC anti-CEA
CAGGCCCCTGGACAGGGACTGGAATGGATGGGCAGAAT (T84.66-
CGACCCCGCCAACGGCAACAGCAAATACGTGCCCAAGT LCHA) Fc
TCCAGGGCAGAGTGACCATCACCGCCGACACCAGCACC hole
TCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGA dimeric
GGACACCGCCGTGTACTACTGTGCCCCCTTCGGCTACT 4-1BBL
ACGTGTCCGACTACGCCATGGCCTATTGGGGCCAGGGC (71-248)
ACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCC chain
CTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCA
GCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGG
AGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGC
TGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTC
ACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACAT
CTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCAC
ACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA
CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCT
GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCA
GCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG
GCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA
GCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGA
GGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGA
TGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGT
TTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGAT
GGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGG
CGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACA
CCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTAC
GTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGG
CGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGC
AGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCA
CTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCG
GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACC
TGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACA
GAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGG
CGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGA
TTCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGGA
GGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGATCC
CGCTGGGCTGCTGGATCTGAGACAGGGCATGTTCGCTC
AGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCT
CTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTC
CCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAG
AACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTT
TTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGG
CTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCAC
TGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACT
GTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAG
CGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCG
CCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCC
AGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAAC
TGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCAG CCGGGCTC 340 Nucleo-
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAA tide
ACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCG sequence
GCTTCAACATCAAGGACACCTACATGCACTGGGTGCGC anti-CEA
CAGGCCCCTGGACAGGGACTGGAATGGATGGGCAGAAT (T84.66-
CGACCCCGCCAACGGCAACAGCAAATACGTGCCCAAGT LCHA) Fc
TCCAGGGCAGAGTGACCATCACCGCCGACACCAGCACC knob
TCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGA monomeric
GGACACCGCCGTGTACTACTGTGCCCCCTTCGGCTACT (71-248)
ACGTGTCCGACTACGCCATGGCCTATTGGGGCCAGGGC 4-1BBL
ACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCC chain
ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCT
CTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC
TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCAC
ACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA
CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG
GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT
CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGT
CCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGAT
ATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAA
CAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACG
GCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAG
AGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT
GATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT
CCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGA
GGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGA
TGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGT
TTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGAT
GGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGG
CGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACA
CCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTAC
GTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGG
CGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGC
AGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCA
CTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCG
GAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACC
TGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACA
GAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGG
CGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGA TTCCTGCCGGGCTC 332 Nucleo-
see Table 82 tide sequence anti-CEA (T84.66- LCHA) light chain 341
anti-CEA QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVR (T84.66-
QAPGQGLEWMGRIDPANGNSKYVPKFQGRVTITADTST LCHA)
STAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQG Fc hole
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD dimeric
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV 4-1BBL
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH (71-248)
TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV chain
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSG
GGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID
GPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYY
VFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHT
EARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGG
GSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP
LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVF
FQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALT
VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEA RARHAWQLTQGATVLGLFRVTPEIPAGL
342 anti-CEA QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDTYMHWVR (T84.66-
QAPGQGLEWMGRIDPANGNSKYVPKFQGRVTITADTST LCHA)
STAYMELSSLRSEDTAVYYCAPFGYYVSDYAMAYWGQG Fc knob
TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD monomeric
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV (71-248)
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH 4-1BBL
TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV chain
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK
AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSG
GGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID
GPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYY
VFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA
LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHT
EARARHAWQLTQGATVLGLFRVTPEIPAGL 334 anti- see Table 82 CD19(8B8-
018) light chain
[0898] 11.2.7 Preparation of Monovalent CEA (T84.66) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule with Crossed CH1-CL Domains with Charged Residues
(Construct 5.7)
[0899] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and
fused to the human IgG1-CL domain, was cloned as depicted in FIG.
29A: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CL. A polypeptide containing one ectodomain of 4-1BB ligand
(71-254) and fused to the human IgG1-CH domain, was cloned as
described in FIG. 29B: human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID
NO:13) connector, human CH.
[0900] To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0901] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66, were subcloned in
frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1. The Pro329Gly, Leu234Ala and
Leu235Ala mutations have been introduced in the constant region of
the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
[0902] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-CD19-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-CD19 light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
a CEA binding Fab. Construct 5.7 corresponds to Construct 5.1 as
shown in FIG. 40A.
[0903] Table 88 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66) targeted split trimeric 4-1BB ligand
(71-254) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 5.7).
TABLE-US-00092 TABLE 88 cDNA and amino acid sequences of monovalent
CEA(T84.66) targeted split trimeric 4-1BB ligand (71-254) Fc (kih)
fusion containing crossed CH-CL with charged residues (construct
5.7).* for charged residues SEQ ID Descrip- NO: tion Sequence 129
Nucleo- see Table 3 tide sequence Dimeric hu 4-1BBL (71-254)- CL*
Fc knob chain 130 Nucleo- see Table 3 tide sequence Monomeric hu
4-1BBL (71-254)- CH1* 343 Nucleo-
GAGGTGCAGCTGCAGCAGTCTGGCGCCGAACTGG tide
TGGAACCTGGCGCCTCTGTGAAGCTGAGCTGTAC sequence
CGCCAGCGGCTTCAACATCAAGGACACCTACATG anti-
CACTGGGTCAAGCAGCGGCCTGAGCAGGGCCTGG CEA(T84.66)
AATGGATCGGCAGAATCGACCCCGCCAACGGCAA Fc hole
CAGCAAATACGTGCCCAAGTTCCAGGGCAAGGCC chain
ACCATCACCGCCGACACCAGCAGCAACACAGCCT
ACCTGCAGCTGACCAGCCTGACCTCCGAGGACAC
CGCCGTGTACTACTGCGCCCCCTTCGGCTACTGA
CTGTCCGACTACGCCATGGCCTATTGGGGCCAGG
GCACAAGCGTGACCGTGTCCTCTGCTAGCACCAA
GGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGC
AAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAGCCCGTGAC
CGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTG
GCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCC
TTCTAGCAGCCTGGGCACCCAGACCTACATCTGC
AACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAAGCT
GCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA
CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTGCACCCTGCCCCCATCCCGGGATGAGCTGAC
CAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA 344
Nucleo- GACATCGTGCTGACCCAGAGCCCTGCCTCTCTGG tide
CCGTGTCTCTGGGACAGAGGGCCACCATGTCTTG sequence
CAGAGCCGGCGAGAGCGTGGACATCTTCGGCGTG anti-
GGATTTCTGCACTGGTATCAGCAGAAGCCCGGCC CEA(T84.66)
AGCCCCCCAAGCTGCTGATCTACAGAGCCAGCAA light chain
CCTGGAAAGCGGCATCCCCGTGCGGTTTAGCGGC
ACCGGCAGCAGAACCGACTTCACCCTGATCATCG
ACCCCGTGGAAGCCGACGACGTGGCCACCTACTA
CTGCCAGCAGACCAACGAGGACCCCTACACCTTT
GGCGGAGGCACCAAGCTGGAAATCAAGCGTACGG
TGGCTGCACCATCTGTCTTCATCTTCCCGCCATC
TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA
ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG
GACAGCAAGGACAGCACCTACAGCCTCAGCAGCA
CCCTGACGCTGAGCAAAGCAGACTACGAGAAACA
CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC
CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT 115 Dimeric hu see
Table 3 4-1BBL (71-254)- CL* Fc knob chain 116 Monomeric see Table
3 hu 4-1BBL (71-254)- CH1* 345 anti-CEA
EVQLQQSGAELVEPGASVKLSCTASGFNIKDTYM (T84.66)
HWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKA Fc hole
TITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYY chain
VSDYAMAYWGQGTSVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI
SKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 346 anti-CEA
DIVLTQSPASLAVSLGQRATMSCRAGESVDIFGV (T84.66)
GFLHWYQQKPGQPPKLLIYRASNLESGIPVRFSG light chain
TGSRTDFTLIIDPVEADDVATYYCQQTNEDPYTF
GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
[0904] 11.2.8 Preparation of Bivalent CEA (T84.66) Targeted 4-1BB
Ligand (71-254) Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecule (Construct 5.8)
[0905] A polypeptide containing two ectodomains of 4-1BB ligand
(71-254), separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers was
fused to the C-terminus of human IgG1 Fc hole chain, as depicted in
FIG. 29C: human IgG1 Fc hole, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand, (G.sub.4S).sub.2 (SEQ ID NO:13)
connector, human 4-1BB ligand. A polypeptide containing one
ectodomain of 4-1BB ligand (71-254) and fused to the C-terminus of
human IgG1 Fc knob chain as described in FIG. 29D: human IgG1 Fc
knob, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human 4-1BB
ligand.
[0906] The variable region of heavy and light chain DNA sequences
encoding a binder specific for CEA, clone T84.66, were subcloned in
frame with either the constant heavy chain of the hole, the knob or
the constant light chain of human IgG1. The Pro329Gly, Leu234Ala
and Leu235Ala mutations have been introduced in the constant region
of the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
Combination of the anti-CEA huIgG1 hole dimeric ligand chain
containing the Y349C/T366S/L368A/Y407V mutations, the anti-CEA
huIgG1 knob monomeric ligand chain containing the S354C/T366W
mutations and the anti-CEA light chain allows generation of a
heterodimer, which includes an assembled trimeric 4-1BB ligand and
two CEA binding Fabs. Construct 5.8 corresponds to Construct 5.3 as
shown in FIG. 40C.
[0907] Table 89 shows the cDNA and amino acid sequences of the
bivalent CEA (T84.66) targeted split trimeric 4-1BB ligand (71-254)
Fc (kih) fusion antigen binding molecule (construct 5.8).
TABLE-US-00093 TABLE 89 cDNA and amino acid sequences of bivalent
CEA(T84.66) targeted split trimeric 4-1BB ligand (71-254) Fc (kih)
PGLALA fusion (construct 5.8) SEQ ID Descrip- NO: tion Sequence 347
Nucleo- GAGGTGCAGCTGCAGCAGTCTGGCGCCGAACTGG tide
TGGAACCTGGCGCCTCTGTGAAGCTGAGCTGTAC sequence
CGCCAGCGGCTTCAACATCAAGGACACCTACATG anti-
CACTGGGTCAAGCAGCGGCCTGAGCAGGGCCTGG CEA(T84.66)
AATGGATCGGCAGAATCGACCCCGCCAACGGCAA Fc hole
CAGCAAATACGTGCCCAAGTTCCAGGGCAAGGCC dimeric 4-
ACCATCACCGCCGACACCAGCAGCAACACAGCCT 1BBL
ACCTGCAGCTGACCAGCCTGACCTCCGAGGACAC (71-254)
CGCCGTGTACTACTGCGCCCCCTTCGGCTACTAC chain
GTGTCCGACTACGCCATGGCCTATTGGGGCCAGG
GCACAAGCGTGACCGTGTCCTCTGCTAGCACCAA
GGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGC
AAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAGCCCGTGAC
CGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGC
GTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTG
GCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCC
TTCTAGCAGCCTGGGCACCCAGACCTACATCTGC
AACGTGAACCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAAGCT
GCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA
CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTGCACCCTGCCCCCATCCCGGGATGAGCTGAC
CAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAA
GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCA
GGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAG
AAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCG
GAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGA
GCTGAGCCCCGATGATCCTGCTGGACTGCTGGAC
CTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCC
AGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGGACACCAAAG
AACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT
GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCC
GGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGC
ATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGC
AGCTCTGGCACTGACAGTGGATCTGCCTCCTGCC
AGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTC
AAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAG
GCTGGGAGTGCATCTGCACACAGAGGCCAGGGCT
AGACACGCCTGGCAGCTGACACAGGGCGCTACAG
TGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCC
AGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGA
GGCGGATCTGGCGGCGGAGGATCTAGAGAGGGAC
CCGAACTGTCCCCTGACGATCCAGCCGGGCTGCT
GGATCTGAGACAGGGAATGTTCGCCCAGCTGGTG
GCTCAGAATGTGCTGCTGATTGACGGACCTCTGA
GCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTC
CCTGACTGGGGGACTGTCCTACAAAGAAGATACA
AAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACT
ATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGT
GGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCT
CTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCG
CAGCTGCACTGGCTCTGACTGTGGACCTGCCACC
AGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGG
TTCCAAGGACGCCTGCTGCATCTGAGCGCCGGAC
AGCGCCTGGGAGTGCATCTGCATACTGAAGCCAG
AGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCA
ACTGTGCTGGGACTGTTTCGCGTGACACCTGAGA TCCCTGCCGGACTGCCAAGCCCTAGATCAGAA
348 Nucleotide GAGGTGCAGCTGCAGCAGTCTGGCGCCGAACTGG sequence
TGGAACCTGGCGCCTCTGTGAAGCTGAGCTGTAC anti-
CGCCAGCGGCTTCAACATCAAGGACACCTACATG CEA(T84.66)
CACTGGGTCAAGCAGCGGCCTGAGCAGGGCCTGG Fc knob
AATGGATCGGCAGAATCGACCCCGCCAACGGCAA monomeric
CAGCAAATACGTGCCCAAGTTCCAGGGCAAGGCC 4-1BBL
ACCATCACCGCCGACACCAGCAGCAACACAGCCT (72-254)
ACCTGCAGCTGACCAGCCTGACCTCCGAGGACAC chain
CGCCGTGTACTACTGCGCCCCCTTCGGCTACTAC
GTGTCCGACTACGCCATGGCCTATTGGGGCCAGG
GCACAAGCGTGACCGTGTCCTCTGCTAGCACCAA
GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCC
AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC
GGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG
GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACCCAGACCTACATCTGC
AACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAAGCT
GCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCC
TGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GGTCACGTCCTCACCGTCCTGCACCAGGACTGGC
TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAA
CAAAGCCCTCGGCGCCCCCATCGAGAAAACCATC
TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTACACCCTGCCCCCCTGCAGAGATGAGCTGAC
CAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAG
GGCTTCTACCCCAGCGATATCGCCGTGGAGTGGG
AGAGCAACGGCCAGCCTGAGAACAACTACAAGAC
CACCCCCCCTGTGCTGGACAGCGACGGCAGCTTC
TTCCTGTACTCCAAACTGACCGTGGACAAGAGCC
GGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGT
GATGCACGAGGCCCTGCACAACCACTACACCCAG
AAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCG
GAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGA
GCTGAGCCCCGATGATCCTGCTGGACTGCTGGAC
CTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCC
AGAACGTGCTGCTGATCGATGGCCCCCTGTCCTG
GTACAGCGATCCTGGACTGGCTGGCGTGTCACTG
ACAGGCGGCCTGAGCTACAAAGAGGACACCAAAG
AACTGGTGGTGGCCAAGGCCGGCGTGTACTACGT
GTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCC
GGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGC
ATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGC
AGCTCTGGCACTGACAGTGGATCTGCCTCCTGCC
AGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTC
AAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAG
GCTGGGAGTGCATCTGCACACAGAGGCCAGGGCT
AGACACGCCTGGCAGCTGACACAGGGCGCTACAG
TGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCC AGCCGGCCTGCCTTCTCCAAGAAGCGAA 344
Nucleotide see Table 88 sequence anti- CEA(T84.66) light chain 349
anti- EVQLQQSGAELVEPGASVKLSCTASGFNIKDTYM CEA(T84.66)
HWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKA Fc hole
TITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYY dimeric 4-
VSDYAMAYWGQGTSVTVSSASTKGPSVFPLAPSS 1BBL(71-
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG 254) chain
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI
SKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLD
LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL
TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVA
GEGSGSVSLALHLQPLRSAAGAAALALTVDLPPA
SSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA
RHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGG
GGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLV
AQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDT
KELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLA
LHLQPLRSAAGAAALALTVDLPPASSEARNSAFG
FQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGA TVLGLFRVTPEIPAGLPSPRSE 350 anti-
EVQLQQSGAELVEPGASVKLSCTASGFNIKDTYM CEA(T84.66)
HWVKQRPEQGLEWIGRIDPANGNSKYVPKFQGKA Fc knob
TITADTSSNTAYLQLTSLTSEDTAVYYCAPFGYY monomeric
VSDYAMAYWGQGTSVTVSSASTKGPSVFPLAPSS 4-1BBL
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG (71-254)
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC chain
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTI
SKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLD
LRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSL
TGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVA
GEGSGSVSLALHLQPLRSAAGAAALALTVDLPPA
SSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARA RHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE
346 anti- see Table 88 CEA(T84.66) light chain
[0908] 11.3 Preparation of Untargeted Split Trimeric 4-1BB Ligand
Fc Fusion Molecules and Human IgG as Control Molecules
[0909] 11.3.1 Preparation of Untargeted Human 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules (Control
Molecules)
[0910] These control molecules were prepared as described above for
the CEA targeted construct 3.1 (termed control B), 3.3 (termed
control C), 3.4 (termed control D) and 3.5 (termed control E) with
the only difference that the anti-CD19 binder (V.sub.H-V.sub.L) was
replaced by a germline control, termed DP47, not binding to the
antigen (see FIGS. 40A to 40F). The cDNA and amino acid sequences
of control B, the monovalent DP47-untargeted split trimeric 4-1BB
ligand (71-254) Fc (kih) fusion containing crossed CH-CL with
charged residues, are shown in Table 68 above (see Example 7.3.1).
Table 69 shows the cDNA and amino acid sequences of the bivalent
DP47-untargeted split trimeric 4-1BB ligand (71-254) Fc (kih)
fusion, control C. Table 70 shows the cDNA and amino acid sequences
of the monovalent DP47-untargeted split trimeric 4-1BB ligand
(71-248) Fc (kih) fusion containing CH-CL cross with charged
residues, control D. Table 71 shows the cDNA and amino acid
sequences of the monovalent DP47-untargeted split trimeric 4-1BB
ligand (71-248) Fc (kih) fusion without charged residues in the
CH-CL cross, control E.
[0911] 11.3.2 Antibodies as Control Molecules
[0912] An additional control used in the assays, termed control F,
was an untargeted DP47, germline control, human IgG1, containing
the Pro329Gly, Leu234Ala and Leu235Ala mutations, to abrogate
binding to Fc gamma receptors. The cDNA and amino acid sequences of
control F can be found in Table 73 above.
[0913] 11.4 Production of CEA-Targeted Split Trimeric 4-1BB Ligand
Fc Fusion Antigen Binding Molecules and their Control Molecules
[0914] The targeted and untargeted split trimeric 4-1BB ligand Fc
(kih) fusion antigen binding molecule encoding sequences were
cloned into a plasmid vector, which drives expression of the insert
from an MPSV promoter and contains a synthetic polyA sequence
located at the 3' end of the CDS. In addition, the vector contains
an EBV OriP sequence for episomal maintenance of the plasmid.
[0915] The split trimeric 4-1BB ligand Fc (kih) fusion was produced
by co-transfecting HEK293-EBNA cells with the mammalian expression
vectors using polyethylenimine. The cells were transfected with the
corresponding expression vectors. For variants 1,2,4,5 and it's
control B, D and E, at a 1:1:1:1 ratio ("vector dimeric ligand-CL-
knob chain": "vector monomeric ligand fusion-CH1": "vector anti-CEA
Fab-hole chain": "vector anti-CEA light chain"). For variant 3, 6
and it's control C, at a 1:1:1 ratio ("vector huIgG1 Fc hole
dimeric ligand chain": "vector huIgG1 Fc knob monomeric ligand
chain": "vector anti-CEA light chain"). Human IgGs, used as control
in the assay, were produced as for the bispecific construct (for
transfection only a vector for light and a vector for heavy chain
were used at a 1:1 ratio).
[0916] For production in 500 mL shake flasks, 300 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 10 minutes at 210.times.g,
and the supernatant was replaced by 20 mL pre-warmed CD CHO medium.
Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CD
CHO medium. After addition of 540 .mu.L PEI, the solution was
vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere. After the incubation, 160 mL of Excell medium
supplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM
valproic acid was added and cells were cultured for 24 hours. One
day after transfection 12% Feed (amino acid and glucose) were
added. After culturing for 7 days, the supernatant was collected by
centrifugation for 30-40 minutes at least 400.times.g. The solution
was sterile filtered (0.22 .mu.m filter), supplemented with sodium
azide to a final concentration of 0.01% (w/v), and kept at
4.degree. C.
[0917] The split trimeric 4-1BB ligand Fc (kih) fusion, as well as
the IgG, was purified from cell culture supernatants by affinity
chromatography using Protein A, followed by size exclusion
chromatography. For affinity chromatography, the supernatant was
loaded on a MABSELECT SURE.RTM. column (CV=5-15 mL, resin from GE
Healthcare) equilibrated with Sodium Phosphate (20 mM), Sodium
Citrate (20 mM) buffer (pH 7.5). Unbound protein was removed by
washing with at least 6 column volumes of the same buffer. The
bound protein was eluted using either a linear gradient (20 CV) or
a step elution (8 CV) with 20 mM sodium citrate, 100 mM Sodium
chloride, 100 mM Glycine buffer (pH 3.0). For the linear gradient
an additional 4 column volumes step elution was applied.
[0918] The pH of collected fractions was adjusted by adding 1/10
(v/v) of 0.5M sodium phosphate, pH8.0. The protein was concentrated
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium
chloride, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20) solution of pH
6.0.
[0919] The protein concentration was determined by measuring the
optical density (OD) at 280 nm, using a molar extinction
coefficient calculated on the basis of the amino acid sequence.
Purity and molecular weight of the targeted trimeric 4-1BB ligand
Fc (kih) fusion antigen binding molecule was analyzed by SDS-PAGE
in the presence and absence of a reducing agent (5 mM
1,4-dithiotreitol) and staining with Coomassie SIMPLYBLUE.TM.
SafeStain (Invitrogen USA) or CE-SDS using Caliper LabChip GXII
(Perkin Elmer). The aggregate content of samples was analyzed using
a TSKGEL.RTM. G3000 SW XL analytical size-exclusion column (Tosoh)
equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-Arginine
Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 running buffer at
25.degree. C.
[0920] Table 90 summarizes the yield and final monomer content of
the CEA targeted split trimeric 4-1BB ligand Fc (kih) fusion
antigen binding molecules.
TABLE-US-00094 TABLE 90 Biochemical analysis of CEA targeted split
trimeric 4-1BB ligand Fc (kih) fusion. Monomer [%] Yield Construct
(SEC) [mg/l] monovalent CEA(T84.66-LCHA) targeted split 98 1.4
trimeric 4-1BB ligand (71-248) Fc fusion containing CH-CL cross
with charged residues (construct 5.4) bivalent CEA(T84.66-LCHA)
targeted split trimeric 98 0.4 4-1BB ligand (71-248) Fc fusion
(construct 5.6) monovalent CEA(T84.66) targeted split trimeric 97
15 4-1BB ligand (71-254) Fc fusion containing CH-CL cross with
charged residues (construct 5.7) bivalent CEA(T84.66) targeted
split trimeric 4-1BB 96 2 ligand (71-254) Fc fusion (construct
5.8)
[0921] Table 91 summarizes the yield and final monomer content of
the DP47 untargeted split trimeric 4-1BB ligand Fc (kih) fusion
molecules, both monovalent (control B, D and E) and bivalent
(control C), and of the germline DP47 human IgG1 PGLALA (control
F).
TABLE-US-00095 TABLE 91 Biochemical analysis of DP47 untargeted
split trimeric 4-1BB ligand Fc (kih) fusion Monomer [%] Yield
Construct (SEC) [mg/l] monovalent DP47-untargeted split trimeric
human 99 15.4 4-1BB ligand (71-254) Fc (kih) fusion (control B)
bivalent DP47 untargeted split trimeric human 4-1BB 98 12.6 ligand
(71-254) Fc (kih) fusion (control C) monovalent DP47-untargeted
split trimeric human 99.5 25.9 4-1BB ligand (71-254) Fc (kih)
fusion (control D) monovalent DP47-untargeted split trimeric human
93.3 4.1 4-1BB ligand (71-254) Fc (kih) fusion (control E) germline
DP47 human IgG1 PGLALA 100 50
Example 12
Functional Characterization of the CEA Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0922] 12.1 Surface Plasmon Resonance (Simultaneous Binding)
Production of Hu NA3B3A2 as Antigen for CEA Targeted Trimeric Split
4-1BBL Constructs
[0923] The antigen used to assess binding by SPR to CEA was a
hybrid molecule composed of A3 and B3 domains from human CEACAM5
(CEA) and N and A2 domains from human CEACAM1 (BGP1) similarly to
what has been described for NABA (Durbin H. et al, Proc Natl Acad
Sci USA. 1994 May 10; 91(10):4313-7). The antigen is termed here
NA3B3A2 and a schematic description can be found in FIG. 41A.
[0924] Table 92 shows the nucleotide and amino acid sequences of hu
NA3B3A2-avi-His.
TABLE-US-00096 TABLE 92 Nucleotide and amino acid sequences of hu
NA3B3A2-avi-His SEQ ID NO: Antigen Sequence 351 nucleo-
CAGCTGACCACCGAGTCCATGCCCTTCAACGTGGCCG tide
AGGGCAAAGAGGTGCTGCTGCTGGTCCACAACCTGCC sequence
CCAGCAGCTGTTCGGCTACAGCTGGTACAAGGGCGAG hu
CGGGTGGACGGCAACCGGCAGATCGTGGGCTACGCCA NA3B3A2-
TCGGCACCCAGCAGGCCACACCCGGCCCTGCCAATAG avi His
CGGCAGAGAGACAATCTACCCCAACGCCAGCCTGCTG
ATCCAGAACGTGACCCAGAACGACACCGGCTTCTACA
CACTCCAAGTCATCAAGAGCGACCTGGTCAACGAGGA
AGCCACCGGCCAGTTCCACGTGTACCCCGAGCTGCCC
AAGCCCAGCATCAGCAGCAACAACAGCAAGCCCGTGG
AAGATAAGGACGCCGTGGCCTTTACCTGCGAGCCCGA
GGCCCAGAACACCACCTACCTGTGGTGGGTCAACGGC
CAGAGCCTGCCCGTGTCCCCCAGACTCCAGCTGAGCA
ACGGCAACAGAACCCTGACCCTGTTCAACGTGACCCG
GAATGACGCCAGAGCCTACGTGTGCGGCATCCAGAAC
AGCGTGTCCGCCAACCGCAGCGACCCCGTGACCCTGG
ATGTGCTGTACGGCCCCGACACCCCCATCATCAGCCCC
CCTGACAGCAGCTACCTGAGCGGCGCCAACCTGAACC
TGAGCTGCCACAGCGCCAGCAACCCCAGCCCTCAGTA
CAGCTGGCGGATCAACGGCATCCCCCAGCAGCACACC
CAGGTGCTGTTTATCGCCAAGATCACCCCCAACAACA
ACGGCACCTACGCCTGCTTCGTGTCCAACCTGGCCACC
GGCCGGAACAACAGCATCGTGAAGTCCATCACCGTGT
CCGCCTCCCTGAGCCCCGTGGTGGCCAAGCCTCAGAT
CAAGGCCAGCAAGACCACCGTGACCGGCGACAAGGA
CAGCGTGAACCTGACCTGCTCCACCAACGATACCGGC
ATCAGCATCCGGTGGTTCTTCAAGAATCAGTCCCTGCC
CAGCAGCGAGCGGATGAAGCTGAGCCAGGGCAACAT
CACCCTGTCCATCAACCCCGTGAAAAGAGAGGACGCC
GGCACCTATTGGTGCGAGGTGTTCAACCCCATCAGCA
AGAACCAGAGCGACCCCATCATGCTGAACGTGAACTA
CAACGCCCTGCCCCAAGAAAACCTGATCAATGTTGAT
CTGGAAGTGCTGTTCCAGGGCCCAGGCAGCGGCCTGA
ACGACATCTTCGAAGCCCAGAAAATCGAGTGGCACGA GGCCAGAGCCCACCACCACCATCACCAC
352 human QLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGER NA3B3A2-
VDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQ avi-His
NVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPS
ISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLP
VSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSAN
RSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSA
SNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACF
VSNLATGRNNSIVKSITVSASLSPVVAKPQIKASKTTV
TGDKDSVNLTCSTNDTGISIRWFFKNQSLPSSERMKLS
QGNITLSINPVKREDAGTYWCEVFNPISKNQSDPIMLN
VNYNALPQENLINVDLEVLFQGPGSGLNDIFEAQKIEW HEARAHHHHHH
[0925] Protein production was performed as described above for the
Fc-fusion protein (Example 7.1.1). Secreted proteins were purified
from cell culture supernatants by chelating chromatography,
followed by size exclusion chromatography. The first
chromatographic step was performed on a Ni-NTA SUPERFLOW' Cartridge
(5 ml, Qiagen) equilibrated in 20 mM sodium phosphate, 500 nM
sodium chloride, pH7.4. Elution was performed by applying a
gradient over 12 column volume from 5% to 45% of elution buffer (20
mM sodium phosphate, 500 nM sodium chloride, 500 mM Imidazole,
pH7.4).
[0926] The protein was concentrated and filtered prior to loading
on a HILOAD.RTM. Superdex 75 column (GE Healthcare) equilibrated
with 20 mM Histidine, 140 mM NaCl, 0.01% TWEEN.RTM. 20 (polysorbate
20) pH 6.0. Table 93 summarizes the yield and final monomer content
of human NA3B3A2-avi-His.
TABLE-US-00097 TABLE 93 Biochemical analysis of human
NA3B3A2-avi-His Monomer [%] Yield Construct (SEC) [mg/l] human
NA3B3A2- 88 14.1 avi-His
[0927] The capacity of binding simultaneously human 4-1BB Fc (kih)
and human NA3B3A2 was assessed by surface plasmon resonance (SPR).
All SPR experiments were performed on a BIACORE.RTM. T200
instrument at 25.degree. C. with HBS-EP as running buffer (0.01 M
HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20,
Biacore, Freiburg/Germany). Biotinylated human 4-1BB Fc (kih) was
directly coupled to a flow cell of a streptavidin (SA) sensor chip.
Immobilization levels up to 250 resonance units (RU) were used.
[0928] The CEA targeted trimeric split 4-1BBL constructs
(constructs 5.4, 5.6, 5.7 and 5.8) were passed at a concentration
range of 200 nM with a flow of 30 .mu.L/minute through the flow
cells over 90 seconds and dissociation was set to zero seconds.
Human NA3B3A2 was injected as second analyte with a flow of 30
.mu.L/minute through the flow cells over 90 seconds at a
concentration of 500 nM (FIG. 41B). The dissociation was monitored
for 120 seconds. Bulk refractive index differences were corrected
for by subtracting the response obtained in a reference flow cell,
where no protein was immobilized.
[0929] As can be seen in the graphs of FIGS. 42A to 42D, all
bispecific constructs could bind simultaneously human 4-1BB and
human NA3B3A2.
[0930] 12.2. Binding on Activated Human PMBCs of the CEA-Targeted
4-1BB Ligand Trimer-Containing Fc (Kih) Fusion Antigen Binding
Molecules
[0931] To determine binding of 4-1BBL trimer-containing Fc fusion
antigen binding molecules to human PBMCs, different titrated
concentrations of the CEA-targeted 4-1BBL trimer-containing Fc
fusion antigen binding molecules were used in the assay as
described in Example 5.2.
[0932] FIGS. 43A to 43D show the binding of Constructs 5.4, 5.6,
5.7 and 5.8 as prepared in Example 11 on activated 4-1BB-expressing
CD4+ T cells and CD8+ T cells, respectively. Gates were set on
living CD45+CD3+CD4+ or CD45+CD3+CD8+ T cells and MFI of
PE-conjugated AffiniPure anti-human IgG Fc.gamma.-fragment-specific
goat F(ab')2 fragment were blotted against the titrated
concentration of targeted split trimeric 4-1BB ligand Fc fusion
variants. Table 94 shows the EC50 values as measured for Constructs
5.4, 5.6, 5.7 and 5.8 and control molecules.
TABLE-US-00098 TABLE 94 Binding on activated 4-1BB-expressing CD4 +
T cells and CD8 + T cells EC.sub.50 [nM] EC.sub.50 [nM] Construct
4-1BB.sup.+CD8.sup.+ 4-1BB.sup.+CD4.sup.+ Control B 0.05 0.26
Control C 0.02 0.30 Control D 0.04 0.28 Control E 0.13 1.22 5.4
0.13 0.35 5.6 0.06 0.34 5.7 0.0004 0.36 5.8 0.17 0.38
[0933] 12.2 Binding to CEA-Expressing Tumor Cells
[0934] For binding assays on CEA-expressing tumor cells, the
following human CEA-expressing lymphoma cell lines were used:
CEA-expressing tumor cell lines human gastric cancer cell line
MKN-45 (ATCC TCP-1008) and human colorectal adenocarcinoma cell
line LS180 (ATCC CL-187). The assays were preformed as described
for the FAP-expressing MV-3 and WM-266-4 tumor cell lines in
Example 5.3.
[0935] Gates were set on living tumor cells and MFI of
PE-conjugated AffiniPure anti-human IgG IgG
Fc.gamma.-fragment-specific goat F(ab')2 fragment were blotted
against the titrated concentration of targeted split trimeric 4-1BB
ligand Fc fusion constructs.
[0936] FIGS. 44A and 44B shows the binding of Constructs 5.7 as
prepared in Example 11.2.7 to human-CEA expressing human gastric
cell line MKN-45 (44A) and human colorectal adenocarcinoma cells
line LS180 (44B). Table 95 shows the EC.sub.50 values as measured
for human-CEA expressing human gastric cell line MKN-45.
TABLE-US-00099 TABLE 95 Binding to CEA-expressing tumor cells
EC.sub.50 [nM] EC.sub.50 [nM] Construct MKN45-8 LS180 5.7 11.6
14.4
Example 13
Biological Activity of the CEA-Targeted 4-1BB Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0937] 13.1. NF-.kappa.B Activation in HeLa Cells Expressing Human
4-1BB
[0938] HeLa cells expressing human 4-1BB and NF-.kappa.B-luciferase
were generated as described in Example 6.1.
[0939] NF-.kappa.B Activation in Hela Cells Expressing Human 4-1BB
Co-Cultured with Human CEA-Expressing Tumor Cells
[0940] NF-.kappa.B-luciferase human-4-1BB HeLa cells were harvested
and resuspended in DMEM medium supplied with 10% (v/v) FBS and 1%
(v/v) GlutaMAX-I to a concentration of 0.2.times.10.sup.6 cells/ml.
100 .mu.l (2.times.10.sup.4 cells) of this cell suspension were
transferred to each well of a sterile white 96-well flat bottom
tissue culture plate with lid (greiner bio-one, Cat. No. 655083)
and the plate were incubated at 37.degree. C. and 5% CO.sub.2
overnight. The next day 50 .mu.L of medium containing titrated
concentrations of CEA-targeted 4-1BB ligand trimer-containing Fc
fusion antigen binding molecules (CEA split 4-1BBL trimer) or
DP47-untargeted 4-1BB ligand trimer-containing Fc fusion antigen
binding molecules (DP47 split 4-1BBL trimer) were added.
CEA-expressing tumor cell lines human gastric cancer cell line
MKN-45 (ATCC TCP-1008) was resuspended in DMEM medium supplied with
10% (v/v) FBS and 1% (v/v) GlutaMAX-I to a concentration of
2.times.10.sup.6 cells/ml.
[0941] Suspension of CEA-expressing B cell lymphoma cell (50 final
ratio 1:5) or only medium were added to each well and plates were
incubated for 6 hours at 37.degree. C. and 5% CO.sub.2. Cells were
washed two times with 200 .mu.L/well DPBS. 40 .mu.l freshly
prepared Reporter Lysis Buffer (Promega, Cat-No: E3971) were added
to each well and the plate were stored over night at -20.degree. C.
The next day frozen cell plate and Detection Buffer (Luciferase
1000 Assay System, Promega, Cat. No. E4550) were thawed at room
temperature. 100 .mu.L of detection buffer were added to each well
and luciferase activity was measured as fast as possible using a
SpectraMax M5/M5e microplate reader and a SoftMax Pro Software
(Molecular Devices) counting light emission in URL (units of
released light for 0.5s/well) or Victor3 1420 multilabel counter
plate reader (Perkin Elmer) and the Perkin Elmer 2030 Manager
Software counting light emission as counts per seconds (CPS) and
blotted against the concentration of tested constructs.
[0942] CEA-targeted 4-1BB ligand trimer-containing Fc fusion
antigen binding molecules Constructs 5.7 and 5.8 triggered
activation of the NF-kB signaling pathway in the reporter cell line
in the presence of human gastric cancer cell line MKN-45 cells. In
contrast, the untargeted control molecules failed to trigger such
an effect at any of the tested concentrations (FIGS. 45A to 45D).
Table 96 shows the corresponding EC50 values.
TABLE-US-00100 Table 96 Binding to CEA-expressing tumor cells
EC.sub.50 [nM] MKN-45 EC.sub.50 [nM] Construct no tumor cells MKN4
5 5.4 3.1 0.34 5.6 2.05 0.21 5.7 0.85 0.05 5.8 1.52 0.45
Example 14
[0943] 14.1 Preparation of FAP Targeted OX40 Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecules
[0944] The DNA sequence encoding part of the ectodomain (amino
acids 51-183) of human OX40 ligand was synthetized according to the
P23510 sequence of Uniprot database. To decrease heterogeneity of
human OX40 ligand due to glycosylation asparagine residues at
position 90 and 114 were mutated to aspartic acid by site-directed
mutagenesis (according to Compaan D. M., Hymowitz S. G., Structure
(2006) 14(8), 1321-30).
[0945] A polypeptide containing two ectodomains of OX40 ligand,
separated by (G.sub.4S).sub.2 (SEQ ID NO:13) linkers, and fused to
the human IgG1-CL domain, was cloned as depicted in FIG. 46A: human
OX40 ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human OX40
ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector, human CL.
[0946] A polypeptide containing one ectodomain of OX40 ligand and
fused to the human IgG1-CH domain, was cloned as described in FIG.
46B: human OX40 ligand, (G.sub.4S).sub.2 (SEQ ID NO:13) connector,
human CH.
[0947] To improve correct pairing the following mutations have been
introduced in the crossed CH-CL. In the dimeric 4-1BB ligand fused
to human CL, E123R and Q124K. In the monomeric 4-1BB ligand fused
to human CH1, K147E and K213E.
[0948] The generation and preparation of the FAP binders is
described in WO 2012/020006 A2, which is incorporated herein by
reference.
[0949] The variable region of heavy and light chain DNA sequences
encoding a binder specific for FAP, clone 28H11, were subcloned in
frame with either the constant heavy chain of the hole or the
constant light chain of human IgG1. The Pro329Gly, Leu234Ala and
Leu235Ala mutations have been introduced in the constant region of
the knob and hole heavy chains to abrogate binding to Fc gamma
receptors according to the method described in WO 2012/130831.
[0950] Combination of the dimeric ligand-Fc knob chain containing
the S354C/T366W mutations, the monomeric CH1 fusion, the targeted
anti-FAP-Fc hole chain containing the Y349C/T366S/L368A/Y407V
mutations and the anti-FAP light chain allows generation of a
heterodimer, which includes an assembled trimeric OX40 ligand and a
FAP binding Fab (FIG. 46C, Construct 6.1).
[0951] Table 97 shows the cDNA and amino acid sequences of the
monovalent CEA (T84.66-LCHA) targeted split trimeric OX40 ligand
(51-183) Fc (kih) fusion antigen binding molecule with crossed
CH-CL and charged residues (construct 6.1).
TABLE-US-00101 TABLE 97 cDNA and amino acid sequences of monovalent
FAP(28H1) targeted split trimeric OX40 ligand Fc (kih) fusion
containing CH-CL cross with charged residues (construct 6.1).* for
charged residues SEQ ID Descrip- NO: tion Sequence 353 Nucleo-
CAGGTGTCCCACAGATACCCCAGAATCCAGAGCATCAA tide
GGTGCAGTTCACCGAGTACAAGAAAGAGAAGGGCTTCA sequence
TCCTGACCAGCCAGAAAGAGGACGAGATCATGAAGGTG Dimeric
CAGGACAACAGCGTGATCATCAACTGCGACGGCTTCTA hu OX40L
CCTGATCAGCCTGAAGGGCTACTTCAGCCAGGAAGTGG (51-183)-
ACATCAGCCTGCACTACCAGAAGGACGAGGAACCCCTG CL* Fc
TTCCAGCTGAAGAAAGTGCGGAGCGTGAACAGCCTGAT knob
GGTGGCCAGCCTGACCTACAAGGACAAGGTGTACCTGA chain
ACGTGACCACCGACAACACCAGCCTGGACGACTTCCAC
GTGAACGGCGGCGAGCTGATCCTGATTCACCAGAACCC
CGGCGAGTTCTGCGTGCTGGGAGGCGGAGGATCTGGCG
GAGGCGGATCTCAGGTGTCACACCGCTACCCCCGGATT
CAGTCCATTAAGGTGCAGTTTACAGAGTATAAGAAAGA
AAAAGGCTTTATTCTGACTTCCCAGAAAGAAGATGAGA
TTATGAAGGTGCAGGATAATTCTGTGATCATCAATTGT
GACGGCTTCTACCTGATCAGCCTGAAGGGCTACTTCAG
CCAGGAAGTGGACATCAGCCTGCACTACCAGAAGGACG
AGGAACCCCTGTTCCAGCTGAAGAAAGTGCGGAGCGTG
AACAGCCTGATGGTGGCCAGCCTGACCTACAAGGACAA
GGTGTACCTGAACGTGACCACCGACAACACCAGCCTGG
ACGACTTCCACGTGAACGGCGGCGAGCTGATCCTGATC
CACCAGAACCCTGGCGAGTTCTGCGTGCTGGGAGGCGG
AGGCTCCGGAGGGGGAGGATCTCGTACGGTGGCTGCAC
CATCTGTCTTTATCTTCCCACCCAGCGACCGGAAGCTG
AAGTCTGGCACAGCCAGCGTCGTGTGCCTGCTGAATAA
CTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGG
ACAATGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTG
ACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAG
CAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGC
ACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTG
TCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTG
CGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCTG
AAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCA
AAGCCCAAGGACACCCTGATGATCAGCCGGACCCCTGA
AGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACC
CTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAA
GTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC
TGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC
AGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG
GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT
TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 354 Nucleo-
CAGGTGTCCCACAGATACCCCAGAATCCAGAGCATCAA tide
GGTGCAGTTCACCGAGTACAAGAAAGAGAAGGGCTTCA sequence
TCCTGACCAGCCAGAAAGAGGACGAGATCATGAAGGTG Mono-
CAGGACAACAGCGTGATCATCAACTGCGACGGCTTCTA meric hu
CCTGATCAGCCTGAAGGGCTACTTCAGCCAGGAAGTGG OX40L
ACATCAGCCTGCACTACCAGAAGGACGAGGAACCCCTG (51-183)-
TTCCAGCTGAAGAAAGTGCGGAGCGTGAACAGCCTGAT CH1*
GGTGGCCAGCCTGACCTACAAGGACAAGGTGTACCTGA
ACGTGACCACCGACAACACCAGCCTGGACGACTTCCAC
GTGAACGGCGGCGAGCTGATCCTGATTCACCAGAACCC
CGGCGAGTTCTGCGTGCTGGGAGGCGGAGGTTCCGGAG
GCGGAGGATCTGCTAGCACAAAGGGCCCCAGCGTGTTC
CCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAAC
AGCCGCCCTGGGCTGCCTGGTGGAAGATTACTTCCCCG
AGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACA
AGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAG
CGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCA
GCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGT GGAACCCAAGTCCTGC 68 Nucleo-
see Table 2 tide sequence anti- FAP(28H1) Fc hole chain 69 Nucleo-
see Table 2 tide sequence anti- FAP(28H1) light chain 355 Dimeric
QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMK hu
VQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEE OX40L
PLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLD (51-183)-
DFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHR CL* Fc
YPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQDNS knob
VIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQL chain
KKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVN
GGELILIHQNPGEFCVLGGGGSGGGGSRTVAAPSVFI
FPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
YACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEA
AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPRE
PQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK
356 Mono- QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMK meric hu
VQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEE OX40L
PLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLD (51-183)-
DFHVNGGELILIHQNPGEFCVLGGGGSGGGGSASTKG CH1*
PSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDEKVEPKSC 18
anti- see Table 2 FAP(28H1) Fc hole chain 19 anti- see Table 2
FAP(28H1) light chain
[0952] 14.2 Preparation of Untargeted Human IgG1 as Control F
[0953] A control molecule used in the assays, termed control F
(FIG. 46D), was an untargeted DP47, germline control, human IgG1,
containing the Pro329Gly, Leu234Ala and Leu235Ala mutations, to
abrogate binding to Fc gamma receptors according to the method
described in International Patent Appl. Publ. No. WO 2012/130831).
Its preparation is described in Example 2.3, Table 29 shows the
cDNA and amino acid sequences of the cDNA and amino acid sequences
of the untargeted DP47 huIgG1 PGLALA (Control F).
[0954] 14.3 Production of FAP-Targeted Split Trimeric OX40 Ligand
Fc Fusion Antigen Binding Molecules and their Control Molecules
[0955] The targeted and untargeted split trimeric OX40 ligand Fc
(kih) fusion encoding sequences were cloned into a plasmid vector,
which drives expression of the insert from an MPSV promoter and
contains a synthetic polyA sequence located at the 3' end of the
CDS. In addition, the vector contains an EBV OriP sequence for
episomal maintenance of the plasmid.
[0956] The split trimeric OX40 ligand Fc (kih) fusion was produced
by co-transfecting HEK293-EBNA cells with the mammalian expression
vectors using polyethylenimine. The cells were transfected with the
corresponding expression vectors. For variants 1,2,4,5 and it's
control B, D and E, at a 1:1:1:1 ratio ("vector dimeric ligand-CL-
knob chain": "vector monomeric ligand fusion-CH1": "vector anti-FAP
Fab-hole chain": "vector anti-FAP light chain"). For variant 3, 6
and it's control C, at a 1:1:1 ratio ("vector huIgG1 Fc hole
dimeric ligand chain": "vector huIgG1 Fc knob monomeric ligand
chain": "vector anti-FAP light chain"). Human IgGs, used as control
in the assay, were produced as for the bispecific construct (for
transfection only a vector for light and a vector for heavy chain
were used at a 1:1 ratio).
[0957] For production in 500 mL shake flasks, 300 million HEK293
EBNA cells were seeded 24 hours before transfection. For
transfection cells were centrifuged for 10 minutes at 210.times.g,
and the supernatant was replaced by 20 mL pre-warmed CD CHO medium.
Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CD
CHO medium. After addition of 540 .mu.L PEI, the solution was
vortexed for 15 seconds and incubated for 10 minutes at room
temperature. Afterwards, cells were mixed with the DNA/PEI
solution, transferred to a 500 mL shake flask and incubated for 3
hours at 37.degree. C. in an incubator with a 5% CO2 atmosphere.
After the incubation, 160 mL of Excell medium supplemented with 6
mM L-Glutamine, 5 g/L PEPSOY and 1.2 mM valproic acid was added and
cells were cultured for 24 hours. One day after transfection 12%
Feed (amino acid and glucose) were added. After culturing for 7
days, the supernatant was collected by centrifugation for 30-40
minutes at least 400.times.g. The solution was sterile filtered
(0.22 .mu.m filter), supplemented with sodium azide to a final
concentration of 0.01% (w/v), and kept at 4.degree. C.
[0958] The split trimeric OX40 ligand Fc (kih) fusion antigen
binding molecule, as well as the IgG, was purified from cell
culture supernatants by affinity chromatography using Protein A,
followed by size exclusion chromatography. For affinity
chromatography, the supernatant was loaded on a MABSELECT SURE.RTM.
column (CV=5-15 mL, resin from GE Healthcare) equilibrated with
Sodium Phosphate (20 mM), Sodium Citrate (20 mM) buffer (pH 7.5).
Unbound protein was removed by washing with at least 6 column
volumes of the same buffer. The bound protein was eluted using
either a linear gradient (20 CV) or a step elution (8 CV) with 20
mM sodium citrate, 100 mM Sodium chloride, 100 mM Glycine buffer
(pH 3.0). For the linear gradient an additional 4 column volumes
step elution was applied.
[0959] The pH of collected fractions was adjusted by adding 1/10
(v/v) of 0.5M sodium phosphate, pH8.0. The protein was concentrated
prior to loading on a HILOAD.RTM. Superdex 200 column (GE
Healthcare) equilibrated with 20 mM Histidine, 140 mM sodium
chloride, 0.01% (v/v) TWEEN.RTM. 20 (polysorbate 20) solution of pH
6.0.
[0960] The protein concentration was determined by measuring the
optical density (OD) at 280 nm, using a molar extinction
coefficient calculated on the basis of the amino acid sequence.
Purity and molecular weight of the targeted trimeric 4-1BB ligand
Fc (kih) fusion was analyzed by SDS-PAGE in the presence and
absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining
with Coomassie SIMPLYBLUE.TM. SafeStain (Invitrogen USA) or CE-SDS
using Caliper LabChip GXII (Perkin Elmer). The aggregate content of
samples was analyzed using a TSKGEL.RTM. G3000 SW XL analytical
size-exclusion column (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM
NaCl, 200 mM L-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7
running buffer at 25.degree. C.
[0961] Table 98 summarizes the yield and final monomer content of
the FAP targeted split trimeric OX40 ligand Fc (kih) fusion antigen
binding molecule, and of the germline DP47 human IgG1 PGLALA
(control F).
TABLE-US-00102 TABLE 98 Biochemical analysis of OX40 targeted split
trimeric 4-1BB ligand Fc (kih) fusion. Monomer [%] Yield Construct
(SEC) [mg/l] monovalent FAP(28H1) targeted split trimeric OX40 93.8
19.7 ligand Fc fusion containing CH-CL cross with charged residues
(construct 6.1) germline DP47 human IgG1 PGLALA 100 50
Example 15
Functional Characterization of the Targeted OX40 Ligand
Trimer-Containing Fc Fusion Antigen Binding Molecule
[0962] 15.1 Binding to Human FAP-Expressing Tumor Cells
[0963] The binding to cell surface FAP was tested using WM-266-4
cells (ATCC CRL-1676). 0.5.times.105 WM-266-4 cells were added to
each well of a round-bottom suspension cell 96-well plates (greiner
bio-one, cellstar, Cat. No. 650185). Cells were stained for 120
minutes at 4.degree. C. in the dark in 50 .mu.L/well 4.degree. C.
cold FACS buffer (DPBS (Gibco by Life Technologies, Cat. No. 14190
326) w/BSA (0.1% v/w, Sigma-Aldrich, Cat. No. A9418) containing
titrated anti-OX40 antibody construct. After three times washing
with excess FACS buffer, cells were stained for 45 minutes at
4.degree. C. in the dark in 25 .mu.L/well 4.degree. C. cold FACS
buffer containing Fluorescein isothiocyanate (FITC)-conjugated
AffiniPure anti-human IgG Fc.gamma.-fragment-specific goat IgG
F(ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109 096
098).
[0964] Plates were finally resuspended in 90 .mu.L/well FACS-buffer
containing 0.2 .mu.g/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598)
and acquired the same day using 5-laser LSR-FORTESSA.RTM. (BD
Bioscience with DIVA software).
[0965] As shown in FIG. 47A, the monovalent FAP (28H1) targeted
split trimeric OX40 ligand Fc (kih) fusion antigen binding molecule
(FAP-OX40L) but not the negative control F efficiently bound to
human FAP-expressing target cells. EC50 values of binding to FAP
positive WM-266-4 was [6.9 nM].
[0966] 15.2 Binding to OX40 and FAP Negative Tumor Cells
[0967] The lack of binding to OX40 negative FAP negative tumor
cells was tested using A549 NucLight.TM. Red Cells
(Essenbioscience, Cat. No. 4491) expressing the NucLight.TM. Red
fluorescent protein restricted to the nucleus to allow separation
from unlabeled human FAP positive WM266-4 cells. Parental A549
(ATCC CCL-185) were transduced with the Essen CellPlayer
NucLight.TM. Red Lentivirus (Essenbioscience, Cat. No. 4476;
EF1.alpha., puromycin) at an MOI of 3 (TU/cell) in the presence of
8 .mu.g/ml polybrene following the standard Essen protocol.
[0968] A mixture of 5.times.104 unlabeled WM266-4 cells and
unlabeled A549 NucLight.TM. Red Cells in FACS buffer were added to
each well of a round-bottom suspension cell 96-well plates and
binding assay was performed as described in section 15.1.
[0969] As shown in FIG. 47B, FAP-OX40L did not bind to OX40
negative FAP negative human tumor cells.
[0970] 15.3 Binding to Human OX40 Expressing Cells: Naive and
Activated Human Peripheral Mononuclear Blood Leukocytes (PBMCs)
[0971] Buffy coats were obtained from the Zurich blood donation
center. To isolate fresh peripheral blood mononuclear cells (PBMCs)
the buffy coat was diluted with the same volume of DPBS (Gibco by
Life Technologies, Cat. No. 14190 326). 50 mL polypropylene
centrifuge tubes (TPP, Cat.-No. 91050) were supplied with 15 mL
HISTOPAQUE.RTM. reagent 1077 (SIGMA Life Science, Cat.-No. 10771,
polysucrose and sodium diatrizoate, adjusted to a density of 1.077
g/mL) and the buffy coat solution was layered above the
HISTOPAQUE.RTM. reagent 1077. The tubes were centrifuged for 30 min
at 400.times.g, room temperature and with low acceleration and no
break. Afterwards the PBMCs were collected from the interface,
washed three times with DPBS and resuspended in T cell medium
consisting of RPMI 1640 medium (Gibco by Life Technology, Cat. No.
42401-042) supplied with 10% Fetal Bovine Serum (FBS, Gibco by Life
Technology, Cat. No. 16000-044, Lot 941273, gamma-irradiated,
mycoplasma-free and heat inactivated at 56.degree. C. for 35 min),
1% (v/v) GlutaMAX I (GIBCO by Life Technologies, Cat. No. 35050
038), 1 mM Sodium-Pyruvat (SIGMA, Cat. No. S8636), 1% (v/v) MEM
non-essential amino acids (SIGMA, Cat.-No. M7145) and 50 .mu.M
.beta.-Mercaptoethanol (SIGMA, M3148).
[0972] PBMCs were used directly after isolation (binding on resting
human PBMCs) or they were stimulated to receive a strong human OX40
expression on the cell surface of T cells (binding on activated
human PBMCs). Therefore naive PBMCs were cultured for four days in
T cell medium supplied with 200 U/mL Proleukin (Novartis) and 2
ug/mL PHA-L (Sigma-Aldrich, L2769-10) in 6-well tissue culture
plate and then over night on pre-coated 6-well tissue culture
plates [4 ug/mL] anti-human CD3 (clone OKT3, eBioscience, Ca. No.
16-0037-85) and [2 ug/mL] anti-human CD28 (clone CD28.2,
eBioscience, Cat No. 16-0289-85] in T cell medium supplied with 200
U/mL Proleukin at 37.degree. C. and 5% CO.sub.2.
[0973] For detection of OX40 naive human PBMC and activated human
PBMC were mixed. To enable distinction of naive from activated
human PBMC naive cells were labeled prior to the binding assay
using the EFLUOR.RTM. 670 cell proliferation dye (eBioscience,
Cat.-No. 65-0840-85).
[0974] For labeling cells were harvested, washed with pre-warmed
(37.degree. C.) DPBS and adjusted to a cell density of
1.times.10.sup.7 cells/mL in DPBS. EFLUOR.RTM. 670 cell
proliferation dye (eBioscience, Cat.-No. 65-0840-85) was added to
the suspension of naive human PBMC at a final concentration of 2.5
mM and a final cell density of 0.5.times.10.sup.7 cells/mL in DPBS.
Cells were then incubated for 10 min at room temperature in the
dark. To stop labeling reaction 4 mL heat inactivated FBS were
added and cells were washed three times with T cell medium. A two
to one mixture of 1.times.10.sup.5 resting EFLUOR.RTM.670 labeled
human PBMC and 0.5.times.10.sup.5 unlabeled activated human PBMC
were then added to each well of a round-bottom suspension cell
96-well plates (greiner bio-one, cellstar, Cat. No. 650185).
[0975] Cells were stained for 120 minutes at 4.degree. C. in the
dark in 50 .mu.L/well 4.degree. C. cold FACS buffer containing
titrated anti-OX40 constructs. After three times washing with
excess FACS buffer, cells were stained for 45 minutes at 4.degree.
C. in the dark in 25 .mu.L/well 4.degree. C. cold FACS buffer
containing a mixture of fluorescently labeled anti-human CD4 (clone
RPA-T4, mouse IgG1 k, BioLegend, Cat.-No. 300532), anti-human CD8
(clone RPa-T8, mouse IgG1k, BioLegend, Cat.-No. 3010441) and
Fluorescein isothiocyanate (FITC)-conjugated AffiniPure anti-human
IgG Fc.gamma.-fragment-specific goat IgG F(ab') 2 fragment (Jackson
ImmunoResearch, Cat.-No. 109-096-098).
[0976] Plates were finally resuspended in 90 .mu.L/well FACS-buffer
containing 0.2 .mu.g/mL DAPI (Santa Cruz Biotec, Cat. No. Sc-3598)
and acquired the same day using 5-laser LSR-FORTESSA.RTM. (BD
Bioscience with DIVA software).
[0977] As shown in FIGS. 48A-1 and 48A-2 and 48B-1 to 48B-2,
FAP-OX40L did not bind to resting human CD4+ T-cells or CD8+
T-cells, which are negative for OX40. In contrast, FAP-OX40L bound
to activated CD8+ or CD4+ T-cells, which do express OX40. Binding
to CD4+ T-cells was much stronger than that to CD8+ T cells.
Activated human CD8+ T cells do express only a fraction of the OX40
levels detected on activated CD4+ T cells. Expression levels for
OX40 are depending on kinetic and strength of stimulation and
conditions were here optimized for OX40 expression on CD4+ T cells
but not for CD8+ T cells. Thus, only little OX40 expression was
induced on CD8 T cells. The EC 50 value of binding to OX40 positive
CD4+ or CD8+ T cells was [0.15 nM].
[0978] 15.4 NF.kappa.B Activation in HeLa Cells Expressing Human
OX40 and Reporter Gene NF.kappa.B-Luciferase
[0979] Agonistic binding of OX40 to its ligand induces downstream
signaling via activation of nuclear factor kappa B (NF.kappa.B) (A.
D. Weinberg et al., J. Leukoc. Biol. 2004, 75(6), 962-972). The
recombinant reporter cell line HeLa_hOx40_NFkB_Luc1 was generated
to express human OX40 on its surface. Additionally, it harbors a
reporter plasmid containing the luciferase gene under the control
of an NF.kappa.B-sensitive enhancer segment. OX40 triggering
induces dose-dependent activation of NF.kappa.B, which translocates
in the nucleus, where it binds on the NF.kappa.B sensitive enhancer
of the reporter plasmid to increase expression of the luciferase
protein. Luciferase catalyzes luciferin-oxidation resulting in
oxyluciferin which emits light. This can be quantified by a
luminometer. Thus, the capacity of the various anti-OX40 molecules
to induce NF.kappa.B activation in HeLa_hOx40_NFkB_Luc1 reporter
cells was analyzed as a measure for bioactivity.
[0980] Adherent HeLa_hOx40_NFkB_Luc1 cells were harvested using
cell dissociation buffer (Invitrogen, Cat.-No. 13151-014) for 10
minutes at 37.degree. C. Cells were washed once with DPBS and were
adjusted to a cell density of 1.33.times.10.sup.5 in assay media
comprising of MEM (Invitrogen, Cat.-No. 22561-021), 10% (v/v)
heat-inactivated FBS, 1 mM Sodium-Pyruvat and 1% (v/v)
non-essential amino acids. Cells were seeded in a density of
0.2*10.sup.5 cells per well in a sterile white 96-well flat bottom
tissue culture plate with lid (greiner bio-one, Cat. No. 655083)
and kept over night at 37.degree. C. and 5% CO.sub.2 in an
incubator (Hera Cell 150).
[0981] The next day, HeLa_hOx40_NFkB_Luc1 were stimulated for 5
hours by adding assay medium containing titrated FAP-OX40L or
negative control F. For testing the effect of hyper-crosslinking on
anti-OX40 antibodies, 25 .mu.L/well of medium containing secondary
antibody anti-human IgG Fc.gamma.-fragment-specific goat IgG F(ab')
2 fragment (Jackson ImmunoResearch, 109-006-098) were added in a
1:2 ratio (2 times more secondary antibody than the primary
antibody). After incubation, supernatant was aspirated and plates
washed two times with DPBS. Quantification of light emission was
done using the luciferase 100 assay system and the reporter lysis
buffer (both Promega, Cat.-No. E4550 and Cat-No: E3971) according
to manufacturer instructions. Briefly, cells were lysed for 10
minutes at -20.degree. C. by addition of 30 uL per well 1.times.
lysis buffer. Cells were thawed for 20 minutes at 37.degree. C.
before 90 uL per well provided luciferase assay reagent was added.
Light emission was quantified immediately with a SpectraMax M5/M5e
microplate reader (Molecular Devices, USA) using 500 ms integration
time, without any filter to collect all wavelengths. Emitted
relative light units (URL) were corrected by basal luminescence of
HeLa_hOx40_NFkB_Luc1 cells and were blotted against the logarithmic
primary antibody concentration using Prism4 (GraphPad Software,
USA). Curves were fitted using the inbuilt sigmoidal dose
response.
[0982] As shown in FIGS. 49A and 49B, a limited, dose dependent
NFkB activation was induced already by addition of FAP-OX40L (49A)
to the reporter cell line. Hyper-crosslinking of FAP-OX40L by
anti-human IgG specific secondary antibodies increased the
induction of NF.kappa.B-mediated luciferase-activation in a
concentration-dependent manner (49B).
[0983] Consequently, we tested the NFkB activating capacity of
FAP-OX40L with hyper-crosslinking of the constructs by FAP+ tumor
cell lines.
[0984] Tested tumor cell line was NIH/3T3-huFAP clone 39.
NIH/3T3-huFAP clone 39 was generated by the transfection of the
mouse embryonic fibroblast NIH/3T3 cell line (ATCC CRL-1658) with
the expression vector pETR4921 to express huFAP under 1.5 .mu.g/mL
Puromycin selection. The surface expression of FAP was quantified
using the Quifikit (Dako Cat. No. K0078) according to manufactures
instructions. The primary antibody used to detect cell surface FAP
expression was the human/mouse crossreactive clone F11-24 (mouse
IgG1, Calbiochem, Ca. No. OP188). The surface expression on
NIH/3T3-huFAP clone 39 was app. 90000 huFAP per cell.
[0985] As described herein before, adherent HeLa_hOx40_NFkB_Luc1
cells were cultured over night at a cell density of 0.2*10.sup.5
cells per well and were stimulated for 5 hours with assay medium
containing titrated FAP-OX40L. To test the effect of
hyper-crosslinking by cell surface FAP binding 25 .mu.L/well of
medium containing FAP+ tumor cells NIH/3T3-huFAP clone 39 were
co-cultured in a 3 to 1 ratio (three times as much FAP+ tumor cells
than reporter cells per well). Activated NF.kappa.B was quantified
by measuring light emission using luciferase 100 assay system and
the reporter lysis buffer (both Promega, Cat.-No. E4550 and Cat-No:
E3971.
[0986] As shown in FIG. 50A, the presence of FAP-expressing tumor
cells strongly increased induction of NF.kappa.B-mediated
luciferase-activation when FAP-OX40L was added. Area under the
curve of the respective blotted dose-response curves was quantified
as a marker for the agonistic capacity of each construct. As shown
in FIG. 50B, the presence of cell surface presented FAP ensured
higher cross-linking and thus a better agonistic effect of
FAP-OX40L then addition of an Fc specific secondary antibody.
[0987] 15.5 OX40 Mediated Costimulation of Suboptimally TCR
Triggered Resting Human PBMC and Hypercrosslinking by Cell Surface
FAP
[0988] It was shown in Example 15.4 that addition of FAP+ tumor
cells can strongly increase the NFkB activity induced by FAP
targeted OX40L in a human OX40 positive reporter cell lines by
providing strong oligomerization of OX40 receptors. Likewise, we
tested FAP-OX40L constructs in the presence of NIH/3T3-huFAP clone
39 cells for their ability to rescue suboptimal TCR stimulation of
resting human PBMC cells.
[0989] Human PBMC preparations contain (1) resting OX40 negative
CD4+ and CD8+ T cells and (2) antigen presenting cells with various
Fc-.gamma. receptor molecules on their cell surface e.g. B cells
and monocytes. Anti-human CD3 antibody of human IgG1 isotype can
bind with its Fc part to the present Fc-.gamma. receptor molecules
and mediate a prolonged TCR activation on resting OX40 negative
CD4+ and CD8+ T cells. These cells then start to express OX40
within several hours. Functional agonistic compounds against OX40
can signal via the OX40 receptor present on activated CD8+ and CD4+
T cells and support TCR-mediated stimulation.
[0990] Resting CFSE-labeled human PBMC were stimulated for five
days with a suboptimal concentration of anti-CD3 antibody in the
presence of irradiated FAP+ NIH/3T3-huFAP clone 39 cells and
titrated FAP-OX40L. Effects on T-cell survival and proliferation
were analyzed through monitoring of total cell counts and CFSE
dilution in living cells by flow cytometry.
[0991] Mouse embryonic fibroblast NIH/3T3-huFAP clone 39 cells (see
Example 15.4) were harvested using cell dissociation buffer
(Invitrogen, Cat.-No. 13151-014) for 10 minutes at 37.degree. C.
Cells were washed once with DPBS. NIH/3T3-huFAP clone 39 cells were
cultured at a density of 0.2*10.sup.5 cells per well in T cell
media in a sterile 96-well round bottom adhesion tissue culture
plate (TPP, Cat. No. 92097) over night at 37.degree. C. and 5%
CO.sub.2 in an incubator (Hera Cell 150). The next day they were
irradiated in an xRay irradiator using a dose of 4500 RAD to
prevent later overgrowth of human PBMC by the tumor cell line.
[0992] Human PBMCs were isolated by ficoll density centrifugation
as described in Example 15.3. Cells were then labeled with CFSE at
a cell density of 1.times.10.sup.6 cells/mL with CFDA-SE
(Sigma-Aldrich, Cat.-No. 2188) at a final concentration of [50 nM]
for 10 minutes at 37.degree. C. Thereafter, cells were washed twice
with excess DPBS containing FBS (10% v/v). Labeled cells were
rested in T-cell media at 37.degree. C. for 30 minutes. Thereafter,
non-converted CFDA-SE was removed by two additional washing steps
with DPBS. CFSE labeled resting human PBMC were added to each well
at a density of 0.5*10.sup.5 cells per well. Anti-human CD3
antibody (clone V9, human IgG1, described in Rodrigues et al., Int
0.1 Cancer Suppl 7, 45-50 (1992) and U.S. Pat. No. 6,054,297) at a
final concentration of [20 nM] and FAP-OX40L were added at the
indicated concentrations. Cells were activated for five days at
37.degree. C. and 5% CO.sub.2 in an incubator (Hera Cell 150).
Then, Cells were surface-stained with fluorescent dye-conjugated
antibodies anti-human CD4 (clone RPA-T4, BioLegend, Cat.-No.
300532) and CD8 (clone RPa-T8, BioLegend, Cat.-No. 3010441) for 20
min at 4.degree. C. After a washing step with FACS buffer, cells
were resuspended in 85 .mu.L/well FACS buffer and acquired using a
5-laser FORTESSA.RTM. flow cytometer (BD Bioscience with DIVA
software).
[0993] As shown in FIGS. 51A to 51D, hyper-crosslinking of
FAP-OX40L constructs by the present NIH/3T3-huFAP clone 39 cells
strongly promoted proliferation (see "Events" graphs, 51A and 51B)
and survival (see "proliferation" graphs, 51C and 51D) in TCR
stimulated human CD4 and CD8 T cells. In line with a lower
expression of OX40 on human CD8+ T cells the agonistic effect of
FAP-OX40L was less strong on CD8+ T cells than on CD4+ T cells.
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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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220259326A1).
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
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220259326A1).
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