U.S. patent application number 17/505763 was filed with the patent office on 2022-04-21 for agonistic trkb binding molecules for the treatment of eye diseases.
The applicant listed for this patent is Boehringer Ingelheim International GmbH. Invention is credited to Remko Alexander BAKKER, Peter Michael BENZ, Holger FUCHS, Fei HAN, Sandeep KUMAR, Sarah LOW, Justin M. SCHEER, Leo THOMAS.
Application Number | 20220119536 17/505763 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-21 |
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United States Patent
Application |
20220119536 |
Kind Code |
A1 |
BENZ; Peter Michael ; et
al. |
April 21, 2022 |
AGONISTIC TRKB BINDING MOLECULES FOR THE TREATMENT OF EYE
DISEASES
Abstract
The invention relates to agonistic TrkB binding molecules,
methods for improving agonistic TrkB binders, their use in
medicine, pharmaceutical compositions comprising the same, and
methods of using the same as agents for treatment and/or prevention
of diseases of the eye.
Inventors: |
BENZ; Peter Michael;
(Veitshoechheim, DE) ; BAKKER; Remko Alexander;
(Biberach an der Riss, DE) ; FUCHS; Holger;
(Warthausen, DE) ; HAN; Fei; (Acton, MA) ;
KUMAR; Sandeep; (Ridgefield, CT) ; LOW; Sarah;
(Carmel, NY) ; SCHEER; Justin M.; (Ridgefield,
CT) ; THOMAS; Leo; (Biberach an der Riss,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Boehringer Ingelheim International GmbH |
Ingelheim am Rhein |
|
DE |
|
|
Appl. No.: |
17/505763 |
Filed: |
October 20, 2021 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 27/02 20060101 A61P027/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 2020 |
EP |
20203030.0 |
Claims
1. A TrkB binding molecule comprising or consisting of two scFs,
wherein each scFv binds specifically to TrkB.
2. The TrkB binding molecule according to claim 1, wherein the
scFvfs are connected to each other via a hinge region or a
linker.
3. The TrkB binding molecule according to claim 1, wherein the TrkB
binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv')2
or scFv-CH.sub.3.
4. The TrkB binding molecule according to claim 1 further
comprising an Ig molecule.
5. The TrkB binding molecule of claim 4, wherein the Ig molecule is
a monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, a fragment of an
antibody, such as a Fc, Fv, Fab, Fab', or F(ab')2 fragment, a
single chain antibody, such as a single chain variable fragment
(scFv), a Small Modular Immunopharmaceutical (SMIP), a domain
antibody, a nanobody, or a diabody.
6. The TrkB binding molecule of claim 4, wherein each scFv is fused
to the C-terminus of the heavy chain of the Ig molecule.
7. The TrkB binding molecule of claim 4 wherein each scFv is fused
to the N-terminus of the heavy chain of the Ig molecule.
8. The TrkB binding molecule according to claim 4, wherein the Ig
molecule is an IgG, F(ab), or F(ab')2.
9. The TrkB binding molecule according to claim 4, wherein the Ig
molecule comprises or consists of an Fc region.
10. The TrkB binding molecule according to claim 4, wherein each
scFv is fused to the Ig molecule by a peptide linker, preferably a
peptide linker having a length of about 4 to 20 amino acids.
11. The TrkB binding molecule according to claim 1, wherein the two
scFvs bind to the same epitope or bind to a different epitope.
12. The TrkB binding molecule according to claims 1, wherein the
two scFvfs are each TrkB agonists, preferably partial agonists.
13. The TrkB binding molecule according to claim 1, wherein the
TrkB binding molecule is a TrkB agonist.
14. The TrkB binding molecule according to claim 1, wherein the
TrkB binding molecule is bispecific and tetravalent.
15. A method for improving the efficacy of an agonistic TrkB
binder, wherein the agonistic TrkB binder contains a light chain
variable domain (VL) and a heavy chain variable domain (VH), (i)
generating a first single chain variable fragment (scFv) with the
VL and the VH of the agonistic TrkB binder, (ii) generating a
second scFv with the same or substantially the same VL and the VH
of the agonistic TrkB binder of step (i) or with the VL and the VH
of a different agonistic TrkB binder, (iii) including the first and
the second scFv into a TrkB binding molecule, wherein the TrkB
binding molecule comprises at least the two agonistic TrkB binding
scFv's from step (i) and (ii), wherein the TrkB binding molecule
has a higher efficacy compared to the efficacy of the agonistic
TrkB binder, and wherein the efficacy is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
16. The method according to claim 15, wherein the agonistic TrkB
binder is a partial agonist.
17. The method according to claim 15, wherein the efficacy of the
TrkB binding molecule compared to the efficacy of the agonistic
TrkB binder is higher by at least 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
100%.
18. The method according to claim 15, wherein the TrkB binding
molecule is about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 100% as efficacious as compared to BDNF.
19. The method according to claim 15, wherein the two agonistic
TrkB binding scFvs bind to the same epitope or bind to a different
epitope.
20. The method according to claim 15, wherein the two agonistic
TrkB binding scFvs are identical.
21. The method according to claim 15, wherein the scFvs are
connected to each other via a hinge region or a linker.
22. The method according to claim 21, wherein the TrkB binding
molecule is in the format of an Fc-scFv, scFv-Fc, (scFv')2 or
scFv-CH.sub.3.
23. The method according according to claim 15, wherein the TrkB
binding molecule further comprises an Ig molecule.
24. The method according to claim 23, wherein the Ig molecule is a
monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, a fragment of an
antibody, such as a Fv, Fab, Fab', or F(ab')2 fragment, a single
chain antibody, such as a single chain variable fragment (scFv), a
Small Modular Immunopharmaceutical (SMIP), a domain antibody, a
nanobody, or a diabody.
25. The method according to claim 23, wherein each scFv is fused to
the C-terminus of the heavy chain of the Ig molecule.
26. The method according to claim 23, wherein each scFv is fused to
the N-terminus of the heavy chain of the Ig molecule.
27. The method according to claim 23, wherein the Ig molecule is an
IgG, F(ab), or F(ab')2.
28. The method according to claim 23, wherein the Ig molecule
comprises or consists of an Fc region.
29. The method according to claim 23, wherein each scFv is fused to
the Ig molecule by a peptide linker, preferably a peptide linker
having a length of about 4 to 20 amino acids.
30. The method according to claim 15, wherein the TrkB binding
molecule is a TrkB agonist.
31. The method according to claim 15, wherein the agonistic TrkB
binder is bivalent.
32. The method according to claim 15, wherein the agonistic TrkB
binder is a bivalent partial agonist.
33. A method for improving the efficacy of a bivalent partial
agonistic TrkB binder, wherein the bivalent partial agonistic TrkB
binder contains a light chain variable domain (VL) and a heavy
chain variable domain (VH), (i) generating a first single chain
variable fragment (scFv) with the VL and the VH of the agonistic
TrkB binder, (ii) generating a second scFv with the same or
substantially the same VL and the VH of the agonistic TrkB binder
of step (i), (iii) including the first and the second scFv into a
TrkB binding molecule, wherein the TrkB binding molecule comprises
at least the two agonistic TrkB binding scFv's from step (i) and
(ii), wherein the TrkB binding molecule has a higher efficacy
compared to the efficacy of the bivalent partial agonistic TrkB
binder, and wherein the efficacy is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
34. The method according to claim 33, wherein the bivalent partial
agonistic TrkB binder is an IgG.
35. The method according to claim 33, wherein the TrkB binding
molecule is in the format of an an Fc-scFv, scFv-Fc, (scFv')2 or
scFv-CH.sub.3.
36. The method according to claim 15, wherein the TrkB binding
molecule is bispecific and tetravalent.
37. A method to produce or generate a TrkB binding molecule having
improved efficacy compared to an agonistic TrkB binder it is based
upon, wherein the agonistic TrkB binder contains a light chain
variable domain (VL) and a heavy chain variable domain (VH), (i)
generating a first single chain variable fragment (scFv) with the
VL and the VH of the agonistic TrkB binder, (ii) generating a
second scFv with the same or substantially the same VL and the VH
of the agonistic TrkB binder of step (i), (iii) including the first
and the second scFv into a TrkB binding molecule, wherein the TrkB
binding molecule comprises at least the two agonistic TrkB binding
scFv's from step (i) and (ii), wherein the TrkB binding molecule
has a higher efficacy compared to the efficacy of the agonistic
TrkB binder, and wherein the efficacy is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
38. An isolated nucleic acid molecule encoding (i) the heavy chain
or heavy chain variable domain, and/or (ii) the light chain or
light chain variable domain of the TrkB binding molecule according
to claim1.
39. A viral vector comprising the isolated nucleic acid molecule
according to claim 38.
40. An expression vector comprising a nucleic acid molecule
according to claim 38.
41. A host cell transfected with an expression vector of claim
40.
42. A method of manufacturing a TrkB binding molecule according to
claim 1 comprising (a) cultivating the host cell transfected with
an expression vector comprising a nucleic acid molecule encoding
(i) the heavy chain or heavy chain variable domain, and/or (ii) the
light chain or light chain variable domain of the TrkB binding
molecule according to claim 1 under conditions allowing expression
of the molecule; and, (b) recovering the molecule; and optionally
c) further purifying and/or modifying and/or formulating the
molecule.
43. A method for the treatment of eye or retinal or
neurodegenerative diseases comprising administration of a TrkB
binding molecule according to claim 1.
44. The method according to claim 43, wherein said eye or retinal
or neurodegenerative diseases are selected from the group
consisting of macular degeneration, age-related macular
degeneration, wet age-related macular degeneration (wAMD), retinal
vein occlusion (RVO), diabetic retinopathy, diabetic macular edema,
retinitis pigmentosa, inherited retinal dystrophy, inherited
macular dystrophy, myopic degeneration, geographic atrophy, retinal
artery occlusions, endophthalmitis, uveitis, cystoid macular edema,
choroidal neovascular membrane secondary to any retinal diseases,
optic neuropathies, glaucoma, retinal detachment, toxic
retinopathy, radiation retinopathy, and traumatic retinopathy,
prodromal and mild-to-moderate alzheimer's diseases, delaying
disease progression of patients with Alzheimer's disease,
Huntington's disease, Parkinson's disease, major depressive
disorder, schizophrenia, cognitive impairment associated with
schizophrenia, prevention of first-episode psychosis in individuals
with attenuated psychosis syndrome, prevention of relapse in
patients with schizophrenia, treatment-resistant depression,
hyperphagia, obesity or metabolic syndrome, hearing loss, in
particular for cis platin induced hearing loss as well as noise and
age-related hearing loss.
45. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the TrkB binding molecule according to claim
1.
Description
SEQUENCE LISTING
[0001] The instant 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 Oct. 14, 2021, is named 01-3516-sequencelisting.txt and is
848,690 bytes in size.
FIELD OF THE INVENTION
[0002] This invention relates to agonistic TrkB binding molecules,
methods for improving agonistic TrkB binders, methods for producing
or generating agonistic TrkB binding molecuels, their use in
medicine, pharmaceutical compositions comprising the same, and
methods of using the same as agents for treatment and/or prevention
of diseases e.g. of the eye.
BACKGROUND OF THE INVENTION
[0003] Tropomyosin receptor kinase B (TrkB), also known as tyrosine
receptor kinase B, or BDNF/NT-3 growth factors receptor or
neurotrophic tyrosine kinase, receptor, type 2, is a protein that
in humans is encoded by the NTRK2 gene (Genbank ID: 4915). TrkB is
a receptor for brain-derived neurotrophic factor (BDNF).
[0004] The neurotrophic tyrosine kinase receptor B (TrkB; gene
symbol: NTRK2) is expressed by retinal neurons and glial cells. In
the normal retina, TrkB signaling counteracts cell stress and
promotes cell survival. In the diseased eye, such as in diabetic
retinopathy or geographic atrophy, loss and functional impairments
of retinal neurons and glial cells occur which cause visual
impairments and vision loss. Activating TrkB signaling above the
basal level (which is reduced in diabetic retinopathy), can
counteract the loss and functional impairments of neurons and glial
cells, thus improving visual function. Furthermore, TrkB activation
has the potential to regenerate lost synaptic connections in the
diseased eye, thereby promoting the regain of visual function. Upon
ligand binding, TrkB undergoes homodimerization followed by
autophosphorylation. Dependent on the phosphorylation sites (Y516,
Y702, Y706, Y707 or Y817) different signal transduction pathways
are activated, including the activity of PLC.gamma.1 or different
subforms of AKT and ERK which regulate distinct overlapping
signalling cascades inducing axonal/neurite outgrowth, increasing
synaptic plasticity, or increasing cell survival.
[0005] Agonistic anti-TrkB antibodies have been described in the
US20100196390 and US20100150914 as well as their proposed use in
the treatment of e.g. Charcot-Marie-Tooth disease or diabetes.
[0006] However, there remains a significant need for new more
efficacious agonistic molecules that can be used to activate the
TrkB pathway and thereby allow their use in therapeutic
interventions of e.g. neurodegenerative and psychiatric
disorders.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the surprising finding
that the design of the TrkB binding sites (two scFv's) in the TrkB
binding molecules of the invention apparently supports an optimal
sterical formation of TrkB binding and activation. The invention is
directed to a TrkB binding molecule comprising or consisting of two
scFv's, wherein each scFv binds specifically to TrkB and both
together act as a TrkB agonist. Without wishing to be bound by
theory the inventors believe that by combining two scFv's into a
TrkB binding molecule, an optimal sterical formation of is achieved
which results in the observed full TrkB agonist activity.
[0008] In a first aspect, the invention relates to a TrkB binding
molecule comprising or consisting of two scFv's, wherein each scFv
binds specifically to TrkB.
[0009] In an embodiment relating to the first aspect, the scFv's
are connected to each other via a hinge region or a linker.
[0010] In an embodiment relating to the first aspect, the TrkB
binding molecule is in the format of an Fc-scFv, scFv-Fc, (scFv')2
or scFv-CH.sub.3.
[0011] In an embodiment relating to the first aspect or any of it's
embodiments, the TrkB binding molecule further comprises an Ig
molecule. Further relating to this embodiment, the Ig molecule is a
monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, a fragment of an
antibody, such as a Fc, Fv, Fab, Fab', or F(ab')2 fragment, a
single chain antibody, such as a single chain variable fragment
(scFv), a Small Modular Immunopharmaceutical (SMIP), a domain
antibody, a nanobody, or a diabody. Further relating to these
embodiments, each scFv is fused to the C- terminus of the heavy
chain of the Ig molecule. Further relating to these embodiments,
each scFv is fused to the N- terminus of the heavy chain of the Ig
molecule. Further relating to these embodiments, the Ig molecule is
an IgG, F(ab), or F(ab')2. Further relating to these embodiments,
the Ig molecule comprises or consists of an Fc region. In a further
related embodiment, each scFv is fused to the Ig molecule by a
peptide linker, preferably a peptide linker having a length of
about 4 to 20 amino acids.
[0012] In an embodiment relating to the first aspect or any of it's
embodiments, the two scFv's bind to the same epitope or bind to a
different epitope.
[0013] In an embodiment relating to the first aspect or any of it's
embodiments, the two scFv's are each TrkB agonists, preferably
partial agonists.
[0014] In an embodiment relating to the first aspect or any of it's
embodiments, the TrkB binding molecule is a TrkB agonist.
[0015] In an embodiment relating to the first aspect or any of it's
embodiments, the TrkB binding molecule is bispecific and
tetravalent.
[0016] In a second aspect, the invention relates to a method for
improving the efficacy of an agonistic TrkB binder, wherein the
agonistic TrkB binder contains a light chain variable domain (VL)
and a heavy chain variable domain (VH), [0017] (i) generating a
first single chain variable fragment (scFv) with the VL and the VH
of the agonistic TrkB binder, [0018] (ii) generating a second scFv
with the same or substantially the same VL and the VH of the
agonistic TrkB binder of step (i) or with the VL and the VH of a
different agonistic TrkB binder, [0019] (iii) including the first
and the second scFv into a TrkB binding molecule, wherein the TrkB
binding molecule comprises at least the two agonistic TrkB binding
scFv's from step (i) and (ii), wherein the TrkB binding molecule
has a higher efficacy compared to the efficacy of the agonistic
TrkB binder, and wherein the efficacy is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
[0020] In an embodiment relating to the second aspect or any of
it's embodiments, the agonistic TrkB binder is a partial
agonist.
[0021] In an embodiment relating to the second aspect or any of
it's embodiments, the efficacy of the TrkB binding molecule
compared to the efficacy of the agonistic TrkB binder is higher by
at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.
[0022] In an embodiment relating to the second aspect or any of
it's embodiments, the TrkB binding molecule is about at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as efficacious
as compared to BDNF.
[0023] In an embodiment relating to the second aspect or any of
it's embodiments, wherein the two agonistic TrkB binding scFv's
bind to the same epitope or bind to a different epitope.
[0024] In an embodiment relating to the second aspect or any of
it's embodiments, the two agonistic TrkB binding scFv's are
identical.
[0025] In an embodiment relating to the second aspect or any of
it's embodiments, the scFv's are connected to each other via a
hinge region or a linker.
[0026] In an embodiment relating to the second aspect or any of
it's embodiments, the TrkB binding molecule is in the format of an
Fc-scFv, scFv-Fc, (scFv')2 or scFv-CH.sub.3.
[0027] In an embodiment relating to the second aspect or any of
it's embodiments, the TrkB binding molecule further comprises an Ig
molecule.
[0028] In an embodiment relating to the second aspect or any of
it's embodiments, the Ig molecule is a monoclonal antibody, a human
monoclonal antibody, a humanized monoclonal antibody, a chimeric
antibody, a fragment of an antibody, such as a Fv, Fab, Fab', or
F(ab')2 fragment, a single chain antibody, such as a single chain
variable fragment (scFv), a Small Modular Immunopharmaceutical
(SMIP), a domain antibody, a nanobody, or a diabody.
[0029] In an embodiment relating to the second aspect or any of
it's embodiments, each scFv is fused to the C-terminus of the heavy
chain of the Ig molecule.
[0030] In an embodiment relating to the second aspect or any of
it's embodiments, each scFv is fused to the N-terminus of the heavy
chain of the Ig molecule.
[0031] In an embodiment relating to the second aspect or any of
it's embodiments, the Ig molecule is an IgG, F(ab), or F(ab')2.
[0032] In an embodiment relating to the second aspect or any of
it's embodiments, the Ig molecule comprises or consists of an Fc
region.
[0033] In an embodiment relating to the second aspect or any of
it's embodiments, each scFv is fused to the Ig molecule by a
peptide linker, preferably a peptide linker having a length of
about 4 to 20 amino acids.
[0034] In an embodiment relating to the second aspect or any of
it's embodiments, the TrkB binding molecule is a TrkB agonist.
[0035] In an embodiment relating to the second aspect or any of
it's embodiments, the agonistic TrkB binder is bivalent.
[0036] In an embodiment relating to the second aspect or any of
it's embodiments, the agonistic TrkB binder is a bivalent partial
agonist.
[0037] In a third aspect, the invention relates to a method for
improving the efficacy of a bivalent partial agonistic TrkB binder,
wherein the bivalent partial agonistic TrkB binder contains a light
chain variable domain (VL) and a heavy chain variable domain (VH),
[0038] (i) generating a first single chain variable fragment (scFv)
with the VL and the VH of the agonistic TrkB binder, [0039] (ii)
generating a second scFv with the same or substantially the same VL
and the VH of the agonistic TrkB binder of step (i), [0040] (iii)
including the first and the second scFv into a TrkB binding
molecule, wherein the TrkB binding molecule comprises at least the
two agonistic TrkB binding scFv's from step (i) and (ii), wherein
the TrkB binding molecule has a higher efficacy compared to the
efficacy of the bivalent partial agonistic TrkB binder, and wherein
the efficacy is the maximum response as determined by incubating
CHO cells stably expressing a TrkB receptor with the agonistic TrkB
binder or the TrkB binding molecule and measuring the TrkB
phosphorylation at Y706/707 in the cell lysate of the treated CHO
cells.
[0041] In an embodiment relating to the third aspect or any of it's
embodiments, the bivalent partial agonistic TrkB binder is an
IgG.
[0042] In an embodiment relating to the third aspect or any of it's
embodiments, the TrkB binding molecule is in the format of an an
Fc-scFv, scFv-Fc, (scFv')2 or scFv-CH.sub.3.
[0043] In an embodiment relating to the third aspect or any of it's
embodiments, the TrkB binding molecule is bispecific and
tetravalent.
[0044] In a related aspect the invention is directed to a method to
produce or generate a TrkB binding molecule having improved
efficacy compared to an agonistic TrkB binder it is based upon,
wherein the agonistic TrkB binder contains a light chain variable
domain (VL) and a heavy chain variable domain (VH), [0045] (i)
generating a first single chain variable fragment (scFv) with the
VL and the VH of the agonistic TrkB binder, [0046] (ii) generating
a second scFv with the same or substantially the same VL and the VH
of the agonistic TrkB binder of step (i), [0047] (iii) including
the first and the second scFv into a TrkB binding molecule, wherein
the
[0048] TrkB binding molecule comprises at least the two agonistic
TrkB binding scFv's from step (i) and (ii),
wherein the TrkB binding molecule has a higher efficacy compared to
the efficacy of the agonistic TrkB binder, and wherein the efficacy
is the maximum response as determined by incubating CHO cells
stably expressing a TrkB receptor with the agonistic TrkB binder or
the TrkB binding molecule and measuring the TrkB phosphorylation at
Y706/707 in the cell lysate of the treated CHO cells.
[0049] In a fourth aspect, the invention relates to an isolated
nucleic acid molecule encoding (i) the heavy chain or heavy chain
variable domain, and/or (ii) the light chain or light chain
variable domain of the TrkB binding molecule according to the first
aspect or any embodiments related to the first aspect.
[0050] In a fifth aspect, the invention relates to a viral vector
comprising the isolated nucleic acid molecule of the fourth
aspect.
[0051] In a sixth aspect, the invention relates to an expression
vector comprising a nucleic acid molecule according to the fifth
aspect.
[0052] In a seventh aspect, the invention relates to a host cell
transfected with an expression vector according to the sixth
aspect.
[0053] In an eight aspect, the invention relates to a method of
manufacturing a TrkB binding molecule according to the first aspect
or any embodiments related to the first aspect comprising [0054]
(a) cultivating the host cell of the seventh aspect under
conditions allowing expression of the molecule; and, [0055] (b)
recovering the molecule; and optionally [0056] (c) further
purifying and/or modifying and/or formulating the molecule.
[0057] In a ninth aspect, the invention relates to the TrkB binding
molecule according to the first aspect or any embodiments related
to the first aspect for use in medicine, wherein the use is the
treatment of eye or retinal or neurodegenerative diseases. In a
related embodiment, the TrkB binding molecule is for the use
according to the ninth aspect, wherein the use is for the treatment
and/or prevention of macular degeneration, age-related macular
degeneration, wet age-related macular degeneration (wAMD), retinal
vein occlusion (RVO), diabetic retinopathy, diabetic macular edema,
retinitis pigmentosa, inherited retinal dystrophy, inherited
macular dystrophy, myopic degeneration, geographic atrophy, retinal
artery occlusions, endophthalmitis, uveitis, cystoid macular edema,
choroidal neovascular membrane secondary to any retinal diseases,
optic neuropathies, glaucoma, retinal detachment, toxic
retinopathy, radiation retinopathy, and traumatic retinopathy,
prodromal and mild-to-moderate alzheimer's diseases, delaying
disease progression of patients with Alzheimer's disease,
Huntington's disease, Parkinson's disease, major depressive
disorder, schizophrenia, cognitive impairment associated with
schizophrenia, prevention of first-episode psychosis in individuals
with attenuated psychosis syndrome, prevention of relapse in
patients with schizophrenia, treatment-resistant depression,
hyperphagia, obesity or metabolic syndrome.
[0058] In a tenth aspect, the invention relates to a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and
the TrkB binding molecule according to the first aspect or any
embodiments related to the first aspect.
BRIEF DESCRIPTION OF THE FIGURES
[0059] FIG. 1: Illustration of the design of VEGF-TrkB single
binding molecules from series 1 to series 4.
[0060] FIG. 2 A-B: (A) TrkB phosphorylation (Y706/707) or (B)
ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was
measured in CHO cells stably expressing human TrkB after incubation
with growing concentrations of the indicated molecules. Data
represent mean+/-SEM.
[0061] FIG. 3 A-D: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing (A) cyno TrkB, (B) rabbit TrkB, (C) rat
TrkB or (D) mouse TrkB after incubation with growing concentrations
of the indicated molecules. Data represent mean+/-SEM.
[0062] FIG. 4 A-C: Trk phosphorylation (Y706/707) was measured in
CHO cells stably expressing (A) human TrkA, (B) human TrkB, or (C)
human TrkC after incubation with growing concentrations of the
indicated molecules. The natural ligands NGF for TrkA, BDNF for
TrkB, and NT-3 for TrkC were used as controls. Data represent
mean+/-SEM.
[0063] FIG. 5 A-B: (A) CHO cells stably expressing human TrkB were
incubated with the indicated concentrations of the natural TrkB
ligand BDNF (in duplicate), 1 nM BDNF with the indicated
concentrations of the agonistic TrkB antibody C2 (in triplicate) or
1 nM BDNF with the indicated concentrations of the Doppelmab
TPP-11736 (in triplicate). (B) TrkB internalization was assessed by
immunofluorescence staining of surface TrkB receptors followed by
confocal microscopy analysis. Dark and light fields of the heatmap
represent high and low percentage of cells above fluorescence
threshold, respectively.
[0064] FIG. 6: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of the indicated
molecules, with or without pre-incubation of 200 ng/mL human
VEGF-A. Incubation only with VEGF-A served as control. Data
represent the mean. For clarity error bars are omitted.
[0065] FIG. 7: Neuroprotective function of TrkB activation in a rat
model of diabetes-induced retinal neurodegeneration. Animals were
treated with STZ to induce hyperglycemia. The retinal function was
then assessed by electroretinography (ERG) and rod-driven B-wave
implicit time delays immediately before and two weeks after
intravitreal application of the agonistic TrkB antibody C2 or
Doppelmab TPP-11736; mean+/-SEM; .sup.n.s.p>0.05,
non-significant; **p<0.01; ***p<0.001; one-way Anova with
Tukey multi-comparison test. Anti-TNP served as isotype control
antibody.
[0066] FIG. 8 A-B: VEGF-A scavenging was assessed by measuring VEGF
receptor 2 phosphorylation (Y1175-VEGFR2). For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated
molecules. (A) Comparison of Doppelmabs TPP-11735, -736, -737 and
-738. (B) Comparison of TPP-11736 and TPP-11738 with EYLEA.RTM.
(aflibercept). Non-stimulated cells (Basal) and 50 ng/ml human VEGF
without antibody treatment served as control. Data represent
mean+/-SEM.
[0067] FIG. 9 A-B: VEGF-A scavenging was assessed by ERK1/2
phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2). For this
purpose, human retinal microvascular endothelial cells (HRMECs)
were starved and then incubated with 50 ng/mL human VEGF with or
without pre-incubation with growing concentrations of the indicated
molecules. (A) Comparison of Doppelmabs TPP-11735, -736, -737 and
-738. (B) Comparison of TPP-11736, -738 with EYLEA.RTM.
(aflibercept). Non-stimulated cells (Basal) and 50 ng/ml human VEGF
without antibody treatment served as control. Data represent
mean+/-SEM.
[0068] FIG. 10: VEGF-A scavenging was assessed by measuring p38
MAPK phosphorylation (T180/Y182). For this purpose, human retinal
microvascular endothelial cells (HRMECs) were starved and then
incubated with 50 ng/mL human VEGF with or without pre-incubation
with growing concentrations of the indicated molecules.
Non-stimulated cells (Basal) and 50 ng/ml human VEGF without
antibody treatment served as control. Data represent
mean+/-SEM.
[0069] FIG. 11 A-E: Human retinal microvascular endothelial cells
(HRMECs) were starved and then incubated with 10 ng/mL human VEGF
with or without pre-incubation with growing concentrations of the
indicated molecules: (A) TPP-11735, (B) TPP-11736, (C) EYLEA.RTM.
(aflibercept), (D) TPP-11737, (E) TPP-11738. Molecule
concentrations are given in mol/L. VEGF-A scavenging was assessed
by image-based quantification of HRMEC cell numbers. Relative cell
numbers are shown. Cell numbers at t=0 were set to one.
Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM.
[0070] FIG. 12: TrkB phosphorylation (Y706/707) was measured in CHO
cells stably expressing human TrkB after incubation with growing
concentrations of the indicated molecules. Data represent
mean+/-SEM.
[0071] FIG. 13: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of the indicated
molecules. Data represent mean+/-SEM.
[0072] FIG. 14 A-B: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175) or (B) ERK1/2
phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2). For this
purpose, human retinal microvascular endothelial cells (HRMECs)
were starved and then incubated with 50 ng/mL human VEGF with or
without pre-incubation with growing concentrations of the indicated
molecules. Non-stimulated cells (Basal) and 50 ng/ml human VEGF
without molecule treatment served as control. Data represent
mean+/-SEM.
[0073] FIG. 15: VEGF-A scavenging was assessed by image-based
quantification of HRMEC cell numbers. For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 10 ng/mL human VEGF with or without
pre-incubation with 0.5 nM Doppelmabs or 1 nM EYLEA.RTM.
(aflibercept). Images were recorded every four hours for a total
period of 96 hours. Relative cell numbers are shown. Cell numbers
at t=0 were set to one. Non-stimulated cells served as control
(Basal). Data represent mean+/-SEM.
[0074] FIG. 16: Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. For this purpose, spheroids of human
retinal microvascular endothelial cells (HRMECs) were embedded in a
collagen matrix. Endothelial sprouting was induced for 24 hours by
incubation with 50 ng/mL human VEGF with or without pre-incubation
with 2.5 nM of the indicated Doppelmabs or 5 nM EYLEA.RTM.
(aflibercept) for 24 hours. Non-stimulated cells served as control
(Basal). Data represent mean+/-SEM. n
[0075] FIG. 17 A-C: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation
(T202/Y204-ERK1) (T185/Y187-ERK2), or (C) p38 MAPK phosphorylation
(T180/Y182). For this purpose, human retinal microvascular
endothelial cells (HRMECs) were starved and then incubated with 50
ng/mL human VEGF with or without pre-incubation with growing
concentrations of Doppelmab TPP-11736 or TPP-13788 (B20 IgG). 50
ng/ml human VEGF without antibody treatment served as control. Data
represent mean+/-SEM.
[0076] FIG. 18: Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. For this purpose, spheroids of human
retinal microvascular endothelial cells (HRMECs) were embedded in a
collagen matrix. Endothelial sprouting was induced for 24 hours by
incubation with 50 ng/mL human VEGF with or without pre-incubation
with 2.5 nM of Doppelmab TPP-11736 or 2.5 nM TPP-13788 (B20 IgG).
Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM.
[0077] FIG. 19 A-C: (A) TrkB phosphorylation (Y706/707) or (B &
C) ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was
measured in CHO cells stably expressing human TrkB after incubation
with growing concentrations of the indicated molecules. Data
represent mean+/-SEM.
[0078] FIG. 20 A-B: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing (A) cyno TrkB or (B) rat TrkB after
incubation with growing concentrations of the indicated molecules.
Data represent mean+/-SEM.
[0079] FIG. 21 A-C: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation
(T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation
(Y419). For this purpose, human retinal microvascular endothelial
cells (HRMECs) were starved and then incubated with 50 ng/mL human
VEGF with or without pre-incubation with growing concentrations of
the indicated molecules. 50 ng/ml human VEGF without antibody
treatment served as control. Data represent mean+/-SEM.
[0080] FIG. 22: Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. For this purpose, spheroids of human
retinal microvascular endothelial cells (HRMECs) were embedded in a
collagen matrix. Endothelial sprouting was induced for 24 hours by
incubation with 50 ng/mL human VEGF with or without pre-incubation
with 2.5 nM of the indicated molecules. Non-stimulated cells served
as control (Basal). Data represent mean+/-SEM. n.s. p>0.05
non-significant, ****p<0.0001.
[0081] FIG. 23 A-D: VEGF-A scavenging was assessed by image-based
quantification of HRMEC cell numbers. For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations indicated molecules: (A)
TPP-11736, (B) TPP-14936, (C) TPP-14937, or (D) EYLEA.RTM.
(aflibercept). Binding molecule/EYLEA.RTM. (aflibercept)
concentrations are given in mol/L. Images were recorded every four
hours for a total period of 84 hours. Relative cell numbers are
shown. Cell numbers at t=0 were set to one. Non-stimulated cells
served as control (Basal). Data represent mean+/-SEM.
[0082] FIG. 24 A-B: (A) TrkB phosphorylation (Y706/707) or (B)
ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was
measured in CHO cells stably expressing human TrkB after incubation
with growing concentrations of the indicated molecules of the
second series. Data represent mean+/-SEM.
[0083] FIG. 25: Cartoon depicting the proposed underlying effect of
VEGF induced clustering with the single binding molecules resulting
in increased efficacy and potency of TrkB activation.
[0084] FIG. 26 A-B: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing (A) cyno TrkB or (B) rat TrkB after
incubation with growing concentrations of the indicated molecules
of the second series. Data represent mean+/-SEM.
[0085] FIG. 27 A-C: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB after incubation with
growing concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941
or (C) C2 antibody with or without pre-incubation with 200 ng/mL
human VEGF-A. Data represent the mean+/-SEM.
[0086] FIG. 28 A-C: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of Doppelmabs (A)
TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without
pre-incubation with 200 ng/mL human VEGF-A. Data represent the
mean+/-SEM.
[0087] FIG. 29 A-B: (A) TrkB phosphorylation (Y706/707) or (B)
ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was
measured in CHO cells stably expressing human TrkB after incubation
with growing concentrations of either human VEGF-A or with growing
concentrations of BDNF alone, or growing concentrations of BDNF
with a fixed concentration of 200 ng/mL hVEGF. Data represent the
mean+/-SEM.
[0088] FIG. 30 A-C: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing cyno TrkB after incubation with growing
concentrations of Doppelmabs (A) TPP-14940, (B) TPP-14941 or (C) C2
antibody with or without pre-incubation with 200 ng/mL human
VEGF-A. Data represent the mean+/-SEM.
[0089] FIG. 31 A-C: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing cyno
TrkB after incubation with growing concentrations of Doppelmabs (A)
TPP-14940, (B) TPP-14941 or (C) C2 antibody with or without
pre-incubation with 200 ng/mL human VEGF-A. Data represent the
mean+/-SEM.
[0090] FIG. 32 A-C: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation
(T202/Y204 -ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation
(Y419). For this purpose, human retinal microvascular endothelial
cells (HRMECs) were starved and then incubated with 50 ng/mL human
VEGF with or without pre-incubation with growing concentrations of
the indicated molecules. 50 ng/ml human VEGF without antibody
treatment served as control. Data represent mean+/-SEM.
[0091] FIG. 33 A-C: Human retinal microvascular endothelial cells
(HRMECs) were starved and then incubated with 10 ng/mL human VEGF
with or without pre-incubation with growing concentrations of the
indicated molecules: (A) TPP-11736, (B) TPP-14938 (C) TPP-14939.
Molecule concentrations are given in mol/L. VEGF-A scavenging was
assessed by image-based quantification of HRMEC cell numbers.
Relative cell numbers are shown. Cell numbers at t =0 were set to
one. Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM.
[0092] FIG. 34 A-C: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation
(T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation
(Y419). For this purpose, human retinal microvascular endothelial
cells (HRMECs) were starved and then incubated with 50 ng/mL human
VEGF with or without pre-incubation with growing concentrations of
the indicated molecules. 50 ng/ml human VEGF without antibody
treatment served as control. Data represent mean+/-SEM.
[0093] FIG. 35 A-D: VEGF-A scavenging was assessed by image-based
quantification of HRMEC cell numbers. For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of (A) TPP-14936, (B)
TPP-14937, (C) TPP-14940 or (D) TPP-14941. Molecule concentrations
are given in mol/L. Images were recorded every four hours for a
total period of 84 hours. Relative cell numbers are shown. Cell
numbers at t=0 were set to one. Non-stimulated cells served as
control (Basal). Data represent mean+/-SEM.
[0094] FIG. 36 A-C: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1175), (B) ERK1/2 phosphorylation
(T202/Y204-ERK1) (T185/Y187-ERK2) or (C) Src phosphorylation
(Y419). For this purpose, human retinal microvascular endothelial
cells (HRMECs) were starved and then incubated with 50 ng/mL human
VEGF with or without pre-incubation with growing concentrations of
the indicated molecules. 50 ng/ml human VEGF without antibody
treatment served as control. Data represent mean+/-SEM.
[0095] FIG. 37 A-D: VEGF-A scavenging was assessed by image-based
quantification of HRMEC cell numbers. For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of (A) TPP-14938, (B)
TPP-14939, (C) TPP-14940 or (D) TPP-14941. Molecule concentrations
are given in mol/L. Images were recorded every four hours for a
total period of 84 hours. Relative cell numbers are shown. Cell
numbers at t=0 were set to one. Non-stimulated cells served as
control (Basal). Data represent mean+/-SEM.
[0096] FIG. 38: Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. For this purpose, spheroids of human
retinal microvascular endothelial cells (HRMECs) were embedded in a
collagen matrix. Endothelial sprouting was induced for 24 hours by
incubation with 50 ng/mL human VEGF with or without pre-incubation
with 2.5 nM of the indicated molecules. Non-stimulated cells served
as control (Basal). Data represent mean+/-SEM. n.s. p>0.05 no
significant difference; .sctn.p<0.0001 compared to 50 ng/mL
hVEGF; #p>0.05 no significant difference compared to 50 ng/mL
hVEGF. ***p<0.001; one-way Anova with Tukey multi comparison
test.
[0097] FIG. 39 A-B: (A) Time protocol showing the experimental
procedure. Fifteen minutes after intravitreal (ivt) administration
of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA.RTM.
(aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13
pmol human VEGF-A per eye was administered by ivt injection. PBS
injection served as control. Twenty-four hours later 1 mL/kg of an
Evans Blue (EB) solution (45 mg/mL in 0.9% saline) were
administered by intravenous (iv) injection for 30 minutes before
the eyes were isolated and fixed. Plasma samples were collected at
the same point in time to confirm equal systemic EB exposure. (B)
Quantification of VEGF-A-induced hyperpermeability in the retinas
of Brown Norway rats was done by measuring EB extravasation in
retinal flatmounts by confocal microscopy. Eyes were cut along
along the Ora serrata, lens and vitreous were removed and the eye
cup was fixed in paraformaldehyde (4%) for 1 h at 4.degree. C. and
then transferred to PBS overnight at 4.degree. C. The retinae were
separated from the outer segments (sclera and choroidea) and
transferred to a glass slide and cut four times to achieve a flat
cloverleaf-like structure. The tissue was covered with mounting
medium (Vectashield.RTM. antifade mounting media H-1200 containing
the DNA stain DAPI) and a coverslip was put on top to obtain a
retinal flatmount. The samples were excited at a wavelength of 639
nm and emission of Evans Blue at 669 nm was recorded with a LSM 700
confocal laser scanning-microscope (Carl Zeiss, Jena; gain 800,
laser strength 2%, 5 stacks of 60 .mu.m) and images of the retinal
flatmounts with maximum intensity projection were obtained.
Analysis of fluorescence intensity sum was done after opening the
images in the program ImageJ with a threshold of 30. ***p<0.001;
*p<0.05; n.s. p>0.05; #p>0.05 non-significant vs.
TPP-11737+PBS. One-way Anova with Tukey multi comparison test,
n=9-17.
[0098] FIG. 40 A-D: (A & B) TrkB phosphorylation (Y706/707) or
(C & D) ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of BDNF or
growing concentrations of the indicated molecules.
[0099] FIG. 41 A-B: (A) CHO cells stably expressing human TrkB were
incubated with the indicated concentrations of the natural TrkB
ligand BDNF (in duplicate), or 1 nM BDNF with the indicated
concentrations of the Doppelmabs (each in triplicate). (B) TrkB
internalization was assessed by immunofluorescence staining of
surface TrkB receptors followed by confocal microscopy analysis.
Dark and light fields of the heatmap represent high and low
percentage of cells above fluorescence threshold, respectively.
[0100] FIG. 42: VEGF-A scavenging was assessed by measuring VEGF
receptor 2 phosphorylation (Y1175). For this purpose, human retinal
microvascular endothelial cells (HRMECs) were starved and then
incubated with 50 ng/mL human VEGF with or without pre-incubation
with growing concentrations of the indicated molecules. 50 ng/ml
human VEGF without molecule treatment served as control. Data
represent mean+/-SEM.
[0101] FIG. 43: Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. For this purpose, spheroids of human
retinal microvascular endothelial cells (HRMECs) were embedded in a
collagen matrix. Endothelial sprouting was induced for 24 hours by
incubation with 50 ng/mL human VEGF with or without pre-incubation
with 2.5 nM of the indicated molecules. Non-stimulated cells served
as control (Basal). Data represent mean+/-SEM. n.s. p>0.05 no
significant difference; .sctn.p<0.0001 compared to 50 ng/mL
hVEGF; #p>0.05 no significant difference compared to 50 ng/mL
hVEGF. ***p<0.001; one-way Anova with Tukey multi comparison
test.
[0102] FIG. 44 A-D: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB after incubation with
growing concentrations of Doppelmabs (A) TPP-22180 vs. TPP-22204;
(B) TPP-22192 vs. TPP-22216; (C) TPP-22190 vs. TPP-22214; (D)
TPP-22191 vs. TPP-22215. Data represent mean+/-SEM.
[0103] FIG. 45 A-B: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing rat TrkB after incubation with growing
concentrations of Doppelmabs (A) TPP-22180 vs. TPP-22204; (B)
TPP-22192 vs. TPP-22216. Data represent mean+/-SEM.
[0104] FIG. 46 A-C: Trk phosphorylation (Y706/707) was measured in
CHO cells stably expressing (A) human TrkA, (B) human TrkB or (C)
human TrkC after incubation with growing concentrations the C2
antibody or the Doppelmabs TPP-22204 or TPP-22214. The natural
ligands NGF for TrkA, BDNF for TrkB, and NT-3 for TrkC were used as
controls. Data represent mean+/-SEM.
[0105] FIG. 47 A-B: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB receptor after incubation
with (A) growing concentrations of the C2 antibody with or without
constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF or (B) growing
concentrations of the Doppelmab TPP-22214 with or without constant
concentrations of 0.3 nM, 1 nM or 3 nM BDNF. Data represent the
mean+/-SEM.
[0106] FIG. 48 A-D: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB receptor after incubation
with growing concentrations of Doppelmab TPP-22214 with or without
pre-incubation with (A) 200 ng/mL human VEGF-A (hVEGF), (B) 50
ng/mL hVEGF, (C) 10 ng/mL hVEGF or (D) 2 ng/mL hVEGF. Data
represent the mean+/-SEM.
[0107] FIG. 49 A-D: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of Doppelmab
TPP-22214 with or without pre-incubation with (A) 200 ng/mL human
VEGF-A (hVEGF), (B) 50 ng/mL hVEGF, (C) 10 ng/mL hVEGF or (D) 2
ng/mL hVEGF. Data represent the mean+/-SEM.
[0108] FIG. 50: Size of Dopplemab complex was assessed using size
exclusion chromatography combined with a multi-angle light
scattering detector. The differential refractive index (black, dark
grey, light grey and dashed lines) and light scattering were
monitored over the time it takes for the proteins to elute from the
size exclusion column. The light scattering data is used to
determine the molar mass at each point. The molar mass at the
midpoint of each peak is denoted by a star and measured using the
right axis. TPP-22214 alone (dashed line) or in complex with VEGF
in various ratios was studied (black 20:1, dark grey 4:1, light
grey 1:1). A thru D represent possible complex schematics based on
the measured molar masses.
[0109] FIG. 51 A-C: TrkB internalization was assessed by
immunofluorescence staining the surface TrkB receptors without
permeabilization of the cells followed by confocal microscopy
analysis. CHO cells with stable expression of human TrkB were
incubated with (A) growing concentrations of the natural TrkB
ligand BDNF, (B) growing concentrations of TPP-22214 or (C) 1 nM
BDNF with growing concentrations of TPP-22214. Data represent the
percent of cells with surface TrkB staining intensity above
threshold; mean+/-SEM.
[0110] FIG. 52: Neuroprotective function of TrkB activation in a
rat model of diabetes-induced retinal neurodegeneration. Animals
were treated with STZ to induce hyperglycemia. The retinal function
was then assessed by electroretinography (ERG) and rod-driven
B-wave implicit time delays immediately before and two weeks after
intravitreal application of the agonistic TrkB antibody C2 or
Doppelmab TPP-22214; mean+/-SEM; .sup.n.s.p>0.05;
non-significant (n.s.), ****p<0.0001; one-way Anova with Tukey
multi-comparison test. Anti-TNP served as isotype control
antibody.
[0111] FIG. 53 A-B: VEGF-A scavenging was assessed by measuring
VEGF receptor 2 phosphorylation (Y1175). For this purpose, human
retinal microvascular endothelial cells (HRMECs) were starved and
then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated
molecules. (A) Comparison of Doppelmabs TPP-14941, TPP-22216,
TPP-22192, TPP-22204, and TPP-22180. (B) Comparison of Doppelmabs
TPP-14940, TPP-14941, TPP-22190, TPP-22214, TPP-22191 and
TPP-22215. Non-stimulated cells (Basal) and 50 ng/ml human VEGF
without molecule treatment served as control. Data represent
mean+/-SEM.
[0112] FIG. 54 A-B: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 (VEGFR2) phosphorylation (Y1175) or (B) ERK1/2
phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2). For this
purpose, human retinal microvascular endothelial cells (HRMECs)
were starved and then incubated with 50 ng/mL human VEGF with or
without pre-incubation with growing concentrations of TPP-22214 or
EYLEA.RTM. (aflibercept). 50 ng/ml human VEGF without molecule
treatment served as control. Data represent mean+/-SEM.
[0113] FIG. 55 A-E: Human retinal microvascular endothelial cells
(HRMECs) were starved and then incubated with 10 ng/mL human VEGF
with or without pre-incubation with growing concentrations of the
indicated molecules: (A) TPPP-22204, (B) TPP-22214c (C) TPP-22216
or (D) EYLEA.RTM. (aflibercept). Molecule concentrations are given
in mol/L. VEGF-A scavenging was assessed by image-based
quantification of HRMEC cell numbers. Images were recorded every
four hours for a total period of 84 hours. Relative cell numbers
are shown. Cell numbers at t=0 were set to one. Non-stimulated
cells served as control (Basal). Data represent mean+/-SEM. (E)
shows a plot of the difference of the area under the growth curves
and the basal curves vs. the concentration of EYLEA.RTM.
(aflibercept) or TPP-22214.
[0114] FIG. 56 A-B: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y1214) or (B) p38-MAPK
phosphorylation (T180/Y182). For this purpose, human retinal
microvascular endothelial cells (HRMECs) were starved and then
incubated with 50 ng/mL human VEGF with or without pre-incubation
with growing concentrations of TPP-22214 or EYLEA.RTM.
(aflibercept). 50 ng/ml human VEGF without molecule treatment
served as control. Data represent mean+/-SEM.
[0115] FIG. 57 A-B: Spheroids of human retinal microvascular
endothelial cells (HRMECs) were embedded in a collagen matrix.
Endothelial sprouting was induced for 24 hours by incubation with
50 ng/mL human VEGF with or without pre-incubation with 2.5 nM of
TPP-22204, TPP-22214, TPP-22215, TPP-22216 or 5 nM EYLEA.RTM.
(aflibercept). (A) Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. Non-stimulated cells served as control
(Basal). Data represent mean+/-SEM. n.s. p>0.05 non-significant,
****p<0.0001. (B) shows representative maximum projection images
from spheroids after 24 hours of sprouting under basal conditions
or after stimulation with 50 ng/mL human VEGF without or with
pre-incubation with 2.5 nM TPP-22214 or 5 nM EYLEA.RTM.
(aflibercept). Bar=100 .mu.m.
[0116] FIG. 58 A-B: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 phosphorylation (Y951) or (B) Src phosphorylation
(Y419). For this purpose, human retinal microvascular endothelial
cells (HRMECs) were starved and then incubated with 50 ng/mL human
VEGF with or without pre-incubation with growing concentrations of
TPP-22214 or EYLEA.RTM. (aflibercept). 50 ng/ml human VEGF without
antibody treatment served as control. Data represent
mean+/-SEM.
[0117] FIG. 59 A-B: TPP-22214 prevented human VEGF-A-induced
hyperpermeability in the rat retina. (A) Time protocol showing the
experimental procedure. Fifteen minutes after intravitreal (ivt)
administration of the anti-VEGF compound (13 or 26 pmol per eye of
EYLEA.RTM. (aflibercept) or TPP-14940) or the control (26 pmol
TPP-11737), 13 pmol human VEGF-A per eye was administered by ivt
injection. PBS injection served as control. Twenty-four hours later
1 mL/kg of an Evans Blue (EB) solution (45 mg/mL in 0.9% saline)
were administered by intravenous (iv) injection for 30 minutes
before the eyes were isolated and fixed. Plasma samples were
collected at the same point in time to confirm equal systemic EB
exposure. (B) Quantification of VEGF-A-induced hyperpermeability in
the retinas of Brown Norway rats was done by measuring EB
extravasation in retinal flatmounts by confocal microscopy. Eyes
were cut along along the Ora serrata, lens and vitreous were
removed and the eye cup was fixed in paraformaldehyde (4%) for 1 h
at 4.degree. C. and then transferred to PBS overnight at 4.degree.
C. The retinae were separated from the outer segments (sclera and
choroidea) and transferred to a glass slide and cut four times to
achieve a flat cloverleaf-like structure. The tissue was covered
with mounting medium (Vectashield.RTM. antifade mounting media
H-1200 containing the DNA stain DAPI) and a coverslip was put on
top to obtain a retinal flatmount. The samples were excited at a
wavelength of 639 nm and emission of Evans Blue at 669 nm was
recorded with a LSM 700 confocal laser scanning-microscope (Carl
Zeiss, Jena; gain 800, laser strength 2%, 5 stacks of 60 .mu.m) and
images of the retinal flatmounts with maximum intensity projection
were obtained. Analysis of fluorescence intensity sum was done
after opening the images in the program ImageJ with a threshold of
30. ***p<0.001; *p<0.05; n.s. p>0.05. One-way Anova with
Tukey multi comparison test, n=9-17. Incubation with 67: 1 molar
ratio of EYLEA.RTM. (aflibercept) : VEGF is shown for
comparison.
[0118] FIG. 60 A-B: (A) Functional characterization of the TkrB
extracellular domain (TrkB-ECD). CHO cells with stable expression
of human TrkB were incubated with growing concentrations of the
natural ligand BDNF or 10 nM BDNF with growing concentrations of
TrkB-ECD. TrkB activation was assessed by measuring TrkB
phosphorylation (Y706/707). (B) Impact of TrkB-ECD binding of
TPP-22214 on inhibition of VEGF-induced VEGFR2 phosphorylation.
Human retinal microvascular endothelial cells (HRMECs) were starved
and then incubated with 50 ng/mL human VEGF, 50 ng/mL human VEGF
with growing concentrations of TPP-22214 or 50 ng/mL human VEGF
with growing concentrations of TPP-22214 and 100 nM TrkB-ECD. HRMEC
incubation with growing concentrations of TrkB-ECD with or without
50 ng/mL VEGF and unstimulated cells (Basal) served as control.
VEGF-A scavenging was assessed by measuring VEGF receptor 2
phosphorylation (Y1175). Data represent mean+/-SEM.
[0119] FIG. 61 A-B: (A) TrkB phosphorylation (Y706/707) or (B)
ERK1/2 phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2) was
measured in CHO cells stably expressing human TrkB after incubation
with growing concentrations of the indicated molecules. Data
represent mean+/-SEM.
[0120] FIG. 62 A-C: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB after incubation with
growing concentrations of Doppelmabs (A) TPP-23457, (B) TPP-23459
or (C) TPP-6830 antibody with or without pre-incubation with 200
ng/mL human VEGF-A. Data represent the mean+/-SEM.
[0121] FIG. 63 A-C: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with growing concentrations of Doppelmabs (A)
TPP-23457, (B) TPP-23459 or (C) TPP-6830 antibody with or without
pre-incubation with 200 ng/mL human VEGF-A. Data represent the
mean+/-SEM.
[0122] FIG. 64 A-B: TrkB phosphorylation (Y706/707) was measured in
CHO cells stably expressing human TrkB receptor after incubation
with (A) growing concentrations of the C2 antibody with or without
constant concentrations of 0.3 nM, 1 nM or 3 nM BDNF or (B) growing
concentrations of the Doppelmab TPP-23457 with or without constant
concentrations of 0.3 nM, 1 nM or 3 nM BDNF. Data represent the
mean+/-SEM.
[0123] FIG. 65 A-B: ERK1/2 phosphorylation (T202/Y204-ERK1)
(T185/Y187-ERK2) was measured in CHO cells stably expressing human
TrkB after incubation with (A) growing concentrations of the C2
antibody with or without constant concentrations of 0.3 nM, 1 nM or
3 nM BDNF or (B) growing concentrations of the Doppelmab TPP-23457
with or without constant concentrations of 0.3 nM, 1 nM or 3 nM
BDNF. Data represent the mean+/-SEM.
[0124] FIG. 66 A-B: VEGF-A scavenging was assessed by measuring (A)
VEGF receptor 2 (VEGFR2) phosphorylation (Y1175) or (B) ERK1/2
phosphorylation (T202/Y204-ERK1) (T185/Y187-ERK2). For this
purpose, human retinal microvascular endothelial cells (HRMECs)
were starved and then incubated with 50 ng/mL human VEGF with or
without pre-incubation with growing concentrations of TPP-22215,
TPP-23457, TPP-23459 or EYLEA.RTM. (aflibercept). 50 ng/ml human
VEGF without molecule treatment served as control. Data represent
mean+/-SEM.
[0125] FIG. 67 A-C: TrkB binders identified via naive Phage display
or via B-cell to Phage were formatted either as IgG, scFv-Fc or
Fc-scFv. The potency (EC.sub.50) for each hit and each of the three
molecule formats was determined by measuring the TrkB
phosphorylation (Y706/707) in CHO cells stably expressing human
TrkB receptor after incubation with growing concentrations of the
molecules. Figures A-C show the pairwise alignment (scatter plots
with line of equality) of the EC.sub.50 of the molecules with
Figure (A) comparing the EC.sub.50 of IgG (x-axis) against scFv-Fc
(y-axis) for each hit, Figure (B) comparing the EC.sub.50 of IgG
(x-axis) against Fc-scFv (y-axis) for each hit, and Figure (C)
comparing the EC.sub.50 of scFv-Fc (x-axis) against Fc-scFv
(y-axis) for each hit.
[0126] FIG. 68 A-C: TrkB binders identified via naive Phage display
or via B-cell to Phage were formatted either as IgG, scFv-Fc or
Fc-scFv. The efficacy for each hit and each of the three molecule
formats was determined by measuring the TrkB phosphorylation
(Y706/707) in CHO cells stably expressing human TrkB receptor after
incubation with growing concentrations of the molecules. BDNF
treated cells were measured as well and the efficacy of BDNF was
set to 100% as control. Figures A-C show the pairwise alignment
(scatter plots with line of equality) of the efficacy of the
molecules. The efficacy of each molecule is expressed as percentage
in comparison to the natural ligand BDNF. Figure (A) compares the
efficacy of IgG (x-axis) against scFv-Fc (y-axis) for each hit,
Figure (B) compares the efficacy of IgG (x-axis) against Fc-scFv
(y-axis) for each hit, and Figure (C) compares the efficacy of
scFv-Fc (x-axis) against Fc-scFv (y-axis) foreach hit.
[0127] FIG. 69 A-H: Selected hits identified via naive Phage
display or via B-cell to Phage were formatted either as IgG,
scFv-Fc or Fc-scFv. The efficacy for each hit and each of the three
molecule formats was determined by measuring the TrkB
phosphorylation (Y706/707) in CHO cells stably expressing human
TrkB receptor after incubation with growing concentrations of the
molecules. BDNF treated cells were measured as well and the
efficacy of BDNF was set to 1.0 as control. The molecule
concentration is plotted on the x-axis and the y-axis shows the
TrkB phosphorylation relative to BDNF. Figures A-H show in total 8
selected hits and for each hit the three different formats IgG,
scFv-Fc and Fc-scFv in direct comparison. The lowest compound
concentration was solvent alone. Data represent mean.+-.SEM.
[0128] FIG. 70 A-C: Schematic of the three different molecule
formats: (A) IgG, (B) scFv-Fc and (C) Fc-scFv.
[0129] FIG. 71: A selected TrkB binder was formatted either as IgG,
scFv-Fc or as two scFvs connected to each other via two different
peptide linkers (scFv-linker1-scFv and scFv-linker2-scFv). The
resulting molecules were tested for their ability to activate TrkB
by measuring the TrkB phosphorylation (Y706/707) in CHO cells
stably expressing human TrkB receptor after incubation with growing
concentrations of the molecules. BDNF treated cells were measured
as well and served as reference. The molecule concentration is
plotted on the x-axis and the y-axis shows the TrkB
phosphorylation. The lowest compound concentration was solvent
alone. Data represent mean.+-.SEM.
[0130] FIG. 72: A selected TrkB binder was formatted either as IgG
or scFv-Fc. In addition, the scFv-Fc was cleaved by a cysteine
protease below the hinge to remove the Fc portion, creating two
scFvs connected to each other via the hinge region
(scFv-hinge-scFv). All three molecules were tested for their
ability to activate TrkB by measuring the TrkB phosphorylation
(Y706/707) in CHO cells stably expressing human TrkB receptor after
incubation with growing concentrations of the molecules. The
molecule concentration is plotted on the x-axis and the y-axis
shows the TrkB phosphorylation. The lowest compound concentration
was solvent alone. Data represent mean.+-.SEM.
DETAILED DESCRIPTION OF THE INVENTION
[0131] The present invention is based on the concept of combining
an antigen binding site that binds specifically to Vascular
Endothelial Growth Factor (VEGF) with an antigen binding site that
binds specifically to Tropomyosin receptor kinase B (TrkB) within a
single binding molecule.
[0132] It is important to point out that until the present
invention it had not been disclosed or even remotely contemplated
to prepare binding molecules targeting these two antigens.
Accordingly, the inventors prepared binding molecules including at
least one antigen binding site that binds specifically to VEGF and
at least one antigen binding site that binds specifically to
TrkB.
[0133] The initial goal of the inventors was to design a single
binding molecule which had comparable efficacy and/or potency when
compared to their respective individual binders, i.e. the
individual VEGF or TrkB binder which only bind to their respective
target. This was already considered challenging in itself, as it
was not fully understood but expected that formatting of the
individual binders into a single binding molecule would negatively
impact the original efficacy and/or potency of the individual
binders within the single binding molecule.
[0134] It was surprisingly found that combining TrkB activation
with VEGF scavenging in a single binding molecule resulted in an
unexpected increase of both efficacy as wells as potency of TrkB
activation. The single binding molecules according to the invention
show full TrkB agonist activity--opposed to partial agonist
activity of the individual TrkB binder. Moreover, potency of TrkB
activation is further enhanced after binding of VEGF to the single
binding molecule.
[0135] Without wishing to be bound by theory it appears that VEGF
induced clustering with the single binding molecules of the
invention may be responsible for the observed increase in potency
of TrkB activation. In addition, independently of the proposed VEGF
induced clustering mechanism, apparently the design of the TrkB
binding sites in the binding molecules of the invention support an
optimal sterical formation of TrkB binding which resulted in the
observed full TrkB agonist activity. Advantageously, by combining
VEGF-scavenging (resulting in inhibition of vascular dysfunction
and leakage) with the activation of the neuroprotective
TrkB-receptor (resulting in reduction of neuronal death) the
patients now benefit from a single injection. This is important as
retinal specialists are not in favor to inject the same eye with
more than one treatment on the same day. Therefore, two separate
treatments addressing wAMD and GA would require two separate
treatment visits per eye. In summary, the binding molecules
according to the invention are useful for treatment and/or
prevention of loss in visual function and thereby improvement in
quality of life.
[0136] In one aspect, the present invention provides a binding
molecule, in particular a molecule having at least one antigen
binding site that binds specifically to vascular endothelial growth
factor VEGF, preferably VEGF-A and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB) having one or more of the properties described herein
below.
[0137] In another aspect, a binding molecule of the present
invention binds with high affinity to human TrkB. In an embodiment
relating to this aspect, a binding molecule of the present
invention binds to human TrkB at a K.sub.D<500 nM. In another
embodiment, a binding molecule of the present invention binds to
human TrkB at a KD<450 nM. In another embodiment, a binding
molecule of the present invention binds to human TrkB at a
K.sub.D<400 nM. In another embodiment, a binding molecule of the
present invention binds to human TrkB at a K.sub.D<300 nM. In
another embodiment, a binding molecule of the present invention
binds to human TrkB at a KD<250 nM. In an embodiment relating to
this aspect, a binding molecule of the present invention binds to
human TrkB at a K.sub.D<200 nM. In another embodiment, a binding
molecule of the present invention binds to human TrkB at a
K.sub.D<150 nM. In another embodiment, a binding molecule of the
present invention binds to human TrkB at a K.sub.D<100 nM. In
another embodiment, a binding molecule of the present invention
binds to human TrkB at a K.sub.D<50 nM.
[0138] In another aspect, a binding molecule of the present
invention--under conditions comprising pre-incubation with 200
ng/mL human VEGF-A--activates TrkB with high potency. In an
embodiment relating to this aspect, a binding molecule of the
present invention activates human TrkB with an EC.sub.50<100 nM.
In a further embodiment, a binding molecule of the present
invention activates human TrkB with an EC.sub.50<90 nm. In a
further embodiment, a binding molecule of the present invention
activates human TrkB with an EC.sub.50<80 nm. In a further
embodiment, a binding molecule of the present invention activates
human TrkB with an EC.sub.50<70 nm. In a further embodiment, a
binding molecule of the present invention activates human TrkB with
an EC.sub.50<60 nm. In a further embodiment, a binding molecule
of the present invention activates human TrkB with an
EC.sub.50<50 nm. In a further embodiment, a binding molecule of
the present invention activates human TrkB with an EC.sub.50<40
nm. In a further embodiment, a binding molecule of the present
invention activates human TrkB with an EC.sub.50<30 nm. In a
further embodiment, a binding molecule of the present invention
activates human TrkB with an EC.sub.50<20 nm. In a further
embodiment, a binding molecule of the present invention activates
human TrkB with an EC.sub.50<10 nm.
[0139] In another aspect, a binding molecule of the present
invention binds with high affinity to human VEGF, preferably
VEGF-A. In an embodiment relating to this aspect, a binding
molecule of the present invention binds to human VEGF-A at a
K.sub.D<1 nM. In another embodiment, a binding molecule of the
present invention binds to human VEGF-A at a K.sub.D<900 pM. In
another embodiment, a binding molecule of the present invention
binds to human VEGF-A at a K.sub.D<800 pM. In another
embodiment, a binding molecule of the present invention binds to
human VEGF-A at a K.sub.D<700 pM. In another embodiment, a
binding molecule of the present invention binds to human VEGF-A at
a K.sub.D<600 pM. In another embodiment, a binding molecule of
the present invention binds to human VEGF-A at a K.sub.D<500 pM.
In another embodiment, a binding molecule of the present invention
binds to human VEGF-A at a K.sub.D<400 pM.
[0140] In another aspect, a binding molecule of the present
invention inhibits VEGF-A phosphorylation (Tyr1175) with high
potency. In an embodiment relating to this aspect, a binding
molecule of the present invention inhibits human VEGF-A
phosphorylation (Tyr1175) with an IC.sub.50<1 nM. In an
embodiment relating to this aspect, a binding molecule of the
present invention inhibits human VEGF-A phosphorylation (Tyr1175)
with an IC.sub.50<900 pM. In an embodiment relating to this
aspect, a binding molecule of the present invention inhibits human
VEGF-A phosphorylation (Tyr1175) with an IC.sub.50<800 pM. In an
embodiment relating to this aspect, a binding molecule of the
present invention inhibits human VEGF-A phosphorylation (Tyr1175)
with an IC.sub.50<700 pM. In an embodiment relating to this
aspect, a binding molecule of the present invention inhibits human
VEGF-A phosphorylation (Tyr1175) with an IC.sub.50<600 pM. In an
embodiment relating to this aspect, a binding molecule of the
present invention inhibits human VEGF-A phosphorylation (Tyr1175)
with an IC.sub.50<500 pM. In an embodiment relating to this
aspect, a binding molecule of the present invention inhibits human
VEGF-A phosphorylation (Tyr1175) with an IC.sub.50<400 pM. In an
embodiment relating to this aspect, a binding molecule of the
present invention inhibits human VEGF-A phosphorylation (Tyr1175)
with an IC.sub.50<300 pM.
[0141] In another aspect, a binding molecule of the present
invention is more potent in inducing activation of TrkB downstream
signaling pathways than the natural TrkB ligand, BDNF. In a further
aspect, a binding molecule of the present invention regulates gene
expression through TrkB-mediated signaling pathways in a comparable
pattern to that of BDNF.
[0142] In yet another aspect, a binding molecule of the present
invention does not reduce BDNF induced ERK phosphorylation. In a
further aspect, a binding molecule of the present invention is
specific for TrkB phosphorylation and/or activation and does not
unspecifically phosphorylate/activate TrkA or TrkC.
[0143] In one aspect, the binding molecules according to the
invention are useful to prevent neurodegeneration and loss of
retinal function in a disease-related animal model. In yet a
further aspect a binding molecule of the present invention protects
neurons, glial cells and/or the neurovascular unit in the retina of
patients with e.g. macular degeneration, age-related macular
degeneration, geographic atrophy or diabetic retinopathy by
stimulating TrkB-dependent survival signaling pathways and thereby
providing neuroprotection.
[0144] In another aspect a binding molecule of the present
invention regenerates axons/dendrites and/or synapses in the retina
after disease onset in e.g. macular degeneration, age-related
macular degeneration, geographic atrophy or diabetic retinopathy
and thereby resulting in neuroregeneration. In one aspect, a
binding molecule of the present invention can be formulated to high
concentrations for intravitreal injections into the eye.
[0145] In another aspect, the binding molecules according to the
invention show superior VEGF-A scavenging compared to the current
anti-VEGF standard of care compound aflibercept (EYLEA.RTM.
(aflibercept)) in terms of inhibition of VEGF-induced signaling,
endothelial cell proliferation/sprouting and/or inhibition of
VEGF-induced hyperpermeability in the rat retina.
[0146] In another aspect, the binding molecules according to the
invention show a substantially longer vitreal half-life in rabbit
PK studies compared to e.g. EYLEA.RTM. (aflibercept) or other IgG
antibodies. Accordingly, the binding molecules according to the
invention are suitable for a quarterly injection or even less
frequent injections.
[0147] In another aspect, the binding molecules according to the
invention are useful to target the high unmet need of patients with
wAMD and at risk of developing geographic atrophy. In a related
embodiment the binding molecules according to the invention are
uniquely useful for treating patients with wAMD and preventing the
development of geographic atrophy. In a further related embodiment,
the development of geographic atrophy is delayed or the severity of
the disease is reduced, i.e. a reduction of the onset and/or the
rate of progression of geographic atrophy. In this respect
treatment of patients with the binding molecules according to the
invention may already be initiated before the definite diagnosis of
geographic atrophy, i.e. for patients being at risk of developing
geographic atrophy.
[0148] In another aspect, the binding molecules according to the
invention are useful to target the high unmet need of patients with
retinal vein occlusion (CRVO).
[0149] In another aspect, the binding molecules according to the
invention improve the ERG-deficit implicit time when compared to
the current standard of care (EYLEA.RTM. (aflibercept)).
[0150] In one aspect, binding of the binding molecules according to
the invention to TrkB does not induce receptor internalization. In
a related aspect, binding of the binding molecules according to the
invention to TrkB will result in less receptor internalization when
compared to the natural ligand BDNF.
[0151] In one aspect, VEGF-A binding to the binding molecules
according to the invention increases the potency of TrkB
activation.
[0152] Said increase in potency may be measured e.g. in vitro by
determining the EC.sub.50 for TrkB or Erk1/2 phosphorylation in an
appropriate cell model after treatment with the single binding
molecules, e.g such as in CHO cells overexpressing (human) TrkB
(cf. examples). The single binding molecules will be tested with or
without pre-incubating the cells with appropriate concentrations of
human VEGF-A (hVEGF), such as 200 ng/mL, 50 ng/mL, 10 ng/mL or 2
ng/mL. After pre-incubation of the cells with human VEGF-A the
single binding molecules of the invention will show an increase in
potency, as measured by EC.sub.50 for TrkB or Erk1/2
phosphorylation of at least approximately 2-fold, 3-fold, 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold or at least approximately
10-fold increase in potency when compared to cells not
pre-incubated with VEGF-A.
[0153] In another aspect, the binding molecules according to the
invention show full TrkB agonist activity. Hence, the binding
molecules according to the invention are as efficacious in
activating TrkB as the natural ligand BDNF.
[0154] In another aspect, the binding molecules according to the
invention can simultaneously activate the TrkB receptor and
scavenge VEGF, preferably VEGF-A.
[0155] Definitions
[0156] Terms not specifically defined herein should be given the
meanings that would be given to them by one of skill in the art in
light of the disclosure and the context. As used in the
specification, however, unless specified to the contrary, the
following terms have the meaning indicated and the following
conventions are adhered to.
[0157] As used herein the term "antigen binding site" comprises a
heavy chain variable domain (VH) and a light chain variable domain
(VL) derived from an antibody. In such case, each variable domain
comprises 3 CDRs. In one aspect, an antigen binding site according
to the present invention or certain portions of the protein is
generally derived from an antibody. The generalized structure of
antibodies or immunoglobulin molecules is well known to those of
skill in the art.
[0158] "Antibodies" or "immunoglobulin molecules" (also known as
immunoglobulins, abbreviated Ig) are gamma globulin proteins that
can be found in blood or other bodily fluids of vertebrates, and
are used by the immune system to identify and neutralize foreign
objects, such as bacteria and viruses. They are typically made of
basic structural units - each with two large heavy chains and two
small light chains - to form, for example, monomers with one unit,
dimers with two units or pentamers with five units. Antibodies can
bind, by non-covalent interaction, to other molecules or structures
known as antigens. This binding is specific in the sense that an
antibody will only bind to a specific structure with high affinity.
The unique part of the antigen recognized by an antibody is called
an epitope, or antigenic determinant. The part of the antibody
binding to the epitope is sometimes called paratope and resides in
the so-called variable domain, or variable region (Fv) of the
antibody. The variable domain comprises three so-called
complementary-determining region (CDR's) spaced apart by framework
regions (FR's).
[0159] Within the context of this invention, reference to CDR's is
based on different definitions, such as e.g. CCG, also referred to
as IMGT (Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E,
Truong L, Thouvenin-Contet V, Lefranc G. "IMGT unique numbering for
immunoglobulin and T cell receptor variable domains and Ig
superfamily V-like domains." Dev Comp Immunol. 2003 Jan.;
27(1):55-77; Giudicelli V,
[0160] Brochet X, Lefranc MP. "IMGT/V-QUEST: IMGT standardized
analysis of the immunoglobulin (IG) and T cell receptor (TR)
nucleotide sequences". Cold Spring Harb Protoc. 2011; 2011(6):
695-715. An alternative definition of CDRs is based on Chothia
(Chothia and Lesk, J. Mol. Biol. 1987, 196: 901-917), together with
Kabat (E. A. Kabat, T. T. Wu, H. Bilofsky, M. Reid-Miller and H.
Perry, Sequence of Proteins of Immunological Interest, National
Institutes of Health, Bethesda (1983)).
[0161] The expressions "variable domains" or "variable region" or
Fv as used herein denotes each of the pair of light and heavy
chains which is involved directly in binding the antibody to the
antigen. The variable domain of a light chain is abbreviated as
"VL" and the variable domain of a heavy chain is abbreviated as
"VH". The variable light and heavy chain domains have the same
general structure and each domain comprises four framework (FR)
regions whose sequences are widely conserved, connected by three
HVRs (or CDRs). The framework regions adopt a beta-sheet
conformation and the CDRs may form loops connecting the beta-sheet
structure. The CDRs in each chain are held in their
three-dimensional structure by the framework regions and form
together with the CDRs from the other chain the antigen binding
site. The antibody's heavy and light chain CDR3 regions play a
particularly important role in the binding specificity/affinity of
the antibodies according to the invention and therefore provide a
further object of the invention. The term "constant domains" or
"constant region" as used within the current application denotes
the sum of the domains of an antibody other than the variable
region. Such constant domains and regions are well known in the
state of the art and e.g. described by Kabat et al. ("Sequence of
proteins of immunological interest", US Public Health Services, NIH
Bethesda, Md., Publication No. 91).
[0162] The "Fc part" of an antibody is not involved directly in
binding of an antibody to an antigen,but exhibit various effector
functions. An "Fc part of an antibody" is a term well known to the
skilled artisan and defined on the basis of papain cleavage of
antibodies. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies or immunoglobulins are
divided in the classes: IgA, IgD, IgE, IgG and IgM. According to
the heavy chain constant regions the different classes of
immunoglobulins are called .gamma., .delta., , .gamma., and .mu.
respectively. Several of these may be further divided into
subclasses (isotypes), e.g. IgGI, IgG2, IgG3, and IgG4, IgAl, and
IgA2. The Fc part of an antibody is directly involved in ADCC
(antibody dependent cell-mediated cytotoxicity) and CDC
(complement-dependent cytotoxicity) based on complement activation,
Clq binding and Fc receptor binding. Complement activation (CDC) is
initiated by binding of complement factor Clq to the Fc part of
most IgG antibody subclasses. While the influence of an antibody on
the complement system is dependent on certain conditions, binding
to Clq is caused by defined binding sites in the Fc part. Such
binding sites are known in the state of the art and described e.g.
by Boakle et al., Nature 282 (1975) 742-743, Lukas et al., J.
Immunol. 127 (1981) 2555-2560, Brunhouse and Cebra, Mol. Immunol.
16 (1979) 907-917, Burton et al., Nature 288 (1980) 338-344,
Thommesen et al., Mol. Immunol. 37 (2000) 995-1004, Idusogie et
al., J. Immunol. 164 (2000) 4178-4184, Hezareh et al., J. Virology
75 (2001) 12161-12168, Morgan et al., Immunology 86 (1995) 319-324,
EP 0307434. Such binding sites are e.g. L234, L235, D270, N297,
E318, K320, K322, P331 and P329 (numbering according to EU index of
Kabat, see below). Most crucial among these residues in mediating
C1q and Fcgamma receptor binding in IgG1 are L234 and L235 (Hezareh
et al., J. Virology 75 (2001) 12161-12168). Antibodies of subclass
IgGI and IgG3 usually show complement activation and Clq and C3
binding, whereas IgG2 and IgG4 do not activate the complement
system and do not bind Clq and C3.
[0163] The art has further developed antibodies and made them
versatile tools in medicine and technology. Thus, in the context of
the present invention the terms "antibody molecule" or "antibody"
or "Ig molecule" (used synonymously herein) do not only include
antibodies as they may be found in nature, comprising e.g. two
light chains and two heavy chains, or just two heavy chains as in
camelid species, but furthermore encompasses all molecules
comprising at least one paratope with binding specificity to an
antigen and structural similarity to a variable domain of an
immunoglobulin.
[0164] Thus, an antibody or immunoglobulin or Ig molecule may
comprise a monoclonal antibody, a human antibody, a humanized
antibody, a chimeric antibody, a fragment of an antibody, in
particular a Fv, Fab, Fab', or F(ab')2 fragment, a single chain
antibody, in particular a single chain variable fragment (scFv), a
Small Modular Immunopharmaceutical (SMIP), a domain antibody, a
nanobody, a diabody. The antibody may have an effector function,
such as ADCC or CDC, that is usually mediated by the Fc part
(antibody constant region) of the antibody, or it may have no
effector function, e.g. by lacking a Fc part or having a blocked,
masked Fc part, in essence a Fc part that is not or insufficiently
recognized by immune cells or immune system components, like the
complement system. Monoclonal antibodies (mAb) are monospecific
antibodies that are identical in amino acid sequence. They may be
produced by hybridoma technology from a hybrid cell line (called
hybridoma) representing a clone of a fusion of a specific
antibody-producing B cell with a myeloma (B cell cancer) cell
(Kohler G, Milstein C. Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 1975; 256:495-7.).
Alternatively, monoclonal antibodies may be produced by recombinant
expression in host cells (Norderhaug L, Olafsen T, Michaelsen T E,
Sandlie I. (May 1997). "Versatile vectors for transient and stable
expression of recombinant antibody molecules in mammalian cells." J
Immunol Methods 204 (1): 77-87; see also below). A "recombinant
antibody" or "recombinant binding molecule" is an antibody or
binding molecule which has been produced by a recombinantly
engineered host cell. It is optionally isolated or purified.
[0165] For application in man, it is often desirable to reduce
immunogenicity of antibodies originally derived from other species,
like mouse. This can be done by construction of chimeric
antibodies, or by a process called "humanization". In this context,
a "chimeric antibody" is understood to be antibody comprising a
sequence part (e.g. a variable domain) derived from one species
(e.g. mouse) fused to a sequence part (e.g. the constant domains)
derived from a different species (e.g. human). A "humanized
antibody" is an antibody comprising a variable domain originally
derived from a non-human species, wherein certain amino acids have
been mutated to make the overall sequence of that variable domain
more closely resemble to a sequence of a human variable domain.
Methods of chimerisation and humanization of antibodies are
well-known in the art (Billetta R, Lobuglio A F. "Chimeric
antibodies". Int Rev Immunol. 1993; 10(2-3): 165-76; Riechmann L,
Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies
for therapy". Nature: 332:323).
[0166] A "humanized" antibody refers to an antibody comprising
amino acid residues from non-human hypervariable regions (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. complementary determining regions (CDRs))
correspond to those of a non-human antibody, and all or
substantially the entire framework regions (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.
[0167] Furthermore, technologies have been developed for creating
antibodies based on sequences derived from the human genome, for
example by phage display or use of transgenic animals (WO 90/05144;
D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D.
Griffiths and G. Winter (1991) "By-passing immunisation. Human
antibodies from V-gene libraries displayed on phage." J.Mol.Biol.,
222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S.
Carmen and L. Jermutus, "Concepts in antibody phage display".
Briefings in Functional Genomics and Proteomics 2002 1(2): 189-203;
Lonberg N, Huszar D. "Human antibodies from transgenic mice". Int
Rev Immunol. 1995; 13(1): 65-93.; Bruggemann M, Taussig M J.
"Production of human antibody repertoires in transgenic mice". Curr
Opin Biotechnol. 1997 Aug.; 8(4): 455-8.). Such antibodies are
"human antibodies" in the context of the present invention.
[0168] Antibody can also include fragments of immunoglobulins which
retain antigen binding properties, like Fab, Fab', or F(ab')2
fragments. Such fragments may be obtained by fragmentation of
immunoglobulins e.g. by proteolytic digestion, or by recombinant
expression of such fragments. For example, immunoglobulin digestion
can be accomplished by means of routine techniques, e.g. using
papain or pepsin (WO 94/29348). Papain digestion of antibodies
typically produces two identical antigen binding fragments,
so-called Fab fragments, each with a single antigen binding site,
and a residual Fc fragment. Pepsin treatment yields an F(ab')2. In
Fab molecules, the variable domains are each fused to an
immunoglobulin constant domain, preferably of human origin. Thus,
the heavy chain variable domain may be fused to a CH1 domain (a
so-called Fd fragment), and the light chain variable domain may be
fused to a CL domain. Fab molecules may be produced by recombinant
expression of respective nucleic acids in host cells, see
below.
[0169] A number of technologies have been developed for placing
variable domains of immunoglobulins, or molecules derived from such
variable domains, in a different molecular context. Those should be
also considered as "antibodies" in accordance with the present
invention. In general, these antibody molecules are smaller in size
compared to immunoglobulins,and may comprise a single amino acid
chain or several amino acid chains. For example, a single-chain
variable fragment (scFv) is a fusion of the variable regions of the
heavy and light chains of immunoglobulins, linked together with a
short linker, usually serine (S) or glycine (G) (WO 88/01649; WO
91/17271; Huston et al; International Reviews of Immunology, Volume
10, 1993, 195-217). "Single domain antibodies" or "nanobodies"
harbour an antigen-binding site in a single Ig-like domain (WO
94/04678; WO 03/050531, Ward et al., Nature. 1989 Oct. 12;
341(6242): 544-6; Revets et al., Expert Opin Biol Ther. 5(1):
111-24, 2005). One or more single domain antibodies with binding
specificity for the same or a different antigen may be linked
together. Diabodies are bivalent antibody molecules consisting of
two amino acid chains comprising two variable domains (WO 94/13804,
Holliger et al., Proc Natl Acad Sci U S A. 1993 Jul. 15; 90(14):
6444-8). Other examples of antibody-like molecules are
immunoglobulin super family antibodies (IgSF; Srinivasan and
Roeske, Current Protein Pept. Sci. 2005, 6(2): 185-96). A different
concept leads to the so-called Small Modular Immunopharmaceutical
(SMIP) which comprises a Fv domain linked to single-chain hinge and
effector domains devoid of the constant domain CH1 (WO
02/056910).
[0170] As used herein, the term "binding" or "specifically binding"
refers to the binding of an antibody or antigen binding site (e.g.,
in the binding molecule described herein) to an epitope of the
antigen in an in-vitro assay. Affinity is the interaction between a
single antigen-binding site on an antibody molecule and a single
epitope. It is expressed by the equilibrium association constant
Ka=kon/koff, or the equilibrium dissociation constant
Kd=koff/kon.
[0171] The binding affinity of an antibody molecule may be enhanced
by a process known as affinity maturation (Marks et al., 1992,
Biotechnology 10:779-783; Barbas, et al., 1994, Proc. Nat. Acad.
Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169: 147-155).
Affinity matured antibodies are therefore also embraced in the
present invention.
[0172] An epitope is a region of an antigen that is bound by an
antibody or antigen binding moiety. The term "epitope" includes any
polypeptide determinant capable of specific binding to an antibody
or antigen binding moiety. In certain embodiments, epitope
determinants include chemically active surface groupings of
molecules such as amino acids, glycan side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific
three-dimensional structural characteristics, and/or specific
charge characteristics. Conformational and non-conformational
epitopes are distinguished in that the binding to the former but
not the latter is lost in the presence of denaturing solvents. As
used herein, the terms "binding" and "specific binding" refer to
the binding of the antibody or antigen binding moiety to an epitope
of the antigen in an in vitro assay, preferably in a plasmon
resonance assay (BlAcore.RTM., GE-Healthcare Uppsala, Sweden) with
purified wild-type antigen.
[0173] A "single chain Fv fragment" (scFv) is a polypeptide
comprising an antibody heavy chain variable domain (VH), a linker,
and an antibody light chain variable domain (VL), wherein said
antibody domains and said linker have one of the following orders
in N-terminal to C-terminal direction: a) VH-linker-VL, b)
VL-linker-VH; and wherein said linker is a polypeptide of 15 to 25
amino acids, preferably 20 amino acids, in length.
[0174] As used herein, the terms "identical" or "percent identity,"
in the context of two or more nucleic acids or polypeptide
sequences, refer to two or more sequences or subsequences that are
the same or have a specified percentage of nucleotides or amino
acid residues that are the same, when compared and aligned for
maximum correspondence. To determine the percent identity, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of a first amino acid or nucleic
acid sequence for optimal alignment with a second amino or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions (e.g., overlapping positions).times.100). In some
embodiments, the two sequences that are compared are the same
length after gaps are introduced within the sequences, as
appropriate (e.g., excluding additional sequence extending beyond
the sequences being compared). For example, when variable region
sequences are compared, the leader and/or constant domain sequences
are not considered. For sequence comparisons between two sequences,
a "corresponding" CDR refers to a CDR in the same location in both
sequences (e.g., CDR-H1 of each sequence).
[0175] The determination of percent identity or percent similarity
between two sequences can be accomplished using a mathematical
algorithm. A preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of two sequences is the
algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA
87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl.
Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into
the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.
Biol. 215:403-410. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12, to obtain nucleotide
sequences homologous to a nucleic acid encoding a protein of
interest. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3, to obtain amino acid sequences
homologous to a protein of interest. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis
and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA
described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA
85:2444-8. Within FASTA, ktup is a control option that sets the
sensitivity and speed of the search. If ktup=2, similar regions in
the two sequences being compared are found by looking at pairs of
aligned residues; if ktup=1, single aligned amino acids are
examined. ktup can be set to 2 or 1 for protein sequences, or from
1 to 6 for DNA sequences. The default if ktup is not specified is 2
for proteins and 6 for DNA. Alternatively, protein sequence
alignment may be carried out using the CLUSTAL W algorithm, as
described by Higgins et al., 1996, Methods Enzymol.
266:383-402.
[0176] The "therapeutically effective amount" of the molecule to be
administered is the minimum amount necessary to prevent,
ameliorate, or treat clinical symptoms of e.g. eye or retinal or
neurodegenerative diseases, in particular the minimum amount which
is effective to these disorders.
[0177] As used herein, the terms "monovalent", "bivalent",
"tetravalent" refer to the number (one, two or four, respectively)
of antigen binding elements in a binding molecule.
[0178] As used herein, the terms "monospecific", "bispecific" refer
to the number (one, two) of different antigens or epitopes a
binding molecule specifically binds.
[0179] By way of example, a typical monoclonal antibody (MAb) is
bivalent and monospecific, with two antigen-binding arms that both
recognize the same epitope. Bispecific antibodies have two
antigen-binding sites, which are capable of recognizing and binding
two different antigens or epitopes.
[0180] TrkB Binding
[0181] Tropomyosin receptor kinase B (TrkB), also known as tyrosine
receptor kinase B, or BDNF/NT-3 growth factors receptor or
neurotrophic tyrosine kinase, receptor, type 2, is a protein that
in humans is encoded by the NTRK2 gene (Genbank ID: 4915). TrkB is
a receptor for brain-derived neurotrophic factor (BDNF).
[0182] The neurotrophic tyrosine kinase receptor B (TrkB; gene
symbol: NTRK2) is expressed by retinal neurons and glial cells. In
the normal retina, TrkB signaling counteracts cell stress and
promotes cell survival. In the diseased eye, such as in diabetic
retinopathy or geographic atrophy, loss and functional impairments
of retinal neurons and glial cells occur which cause visual
impairments and vision loss. Activating TrkB signaling above the
basal level (which is reduced in diabetic retinopathy), can
counteract the loss and functional impairments of neurons and glial
cells, thus improving visual function. Furthermore, TrkB activation
has the potential to regenerate lost synaptic connections in the
diseased eye, thereby promoting the regain of visual function. Upon
ligand binding, TrkB undergoes homodimerization followed by
autophosphorylation. Dependent on the phosphorylation sites (Y516,
Y702, Y706, Y707 or Y817) different signal transduction pathways
are activated, including the activity of PLC.gamma.1 or different
subforms of AKT and ERK which regulate distinct overlapping
signalling cascades inducing axonal/neurite outgrowth, increasing
synaptic plasticity, or increasing cell survival.
[0183] The TrkB-binding components of the binding molecules
according to the invention, e.g. scFv or scFv2 specifically bind to
native or recombinant human TrkB. By binding to TrkB the binding
molecules of the invention thereby activate TrkB signaling, hence
the binding molecules of the invention act as TrkB agonists.
[0184] The binding molecules of the present invention may recognize
specific "TrkB antigen epitope" and "TrkB epitope". In particular,
the binding molecules of the invention bind to an epitope in the
extracellular domain of human TrkB. Preferably, the TrkB-binding
components of the binding molecule is a TrkB agonist and more
preferably a full agonist.
[0185] The extracellular domain of human TrkB essentially comprises
the following sequence (SEQ ID NO.226):
TABLE-US-00001 CPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPEN
ITEIFIANQKRLEIINEDDVEAYVGLRNLTIVDSG
LKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRH
LDLSELILVGNPFTCSCDIMWIKTLQEAKSSPDTQ
DLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLT
VEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNE
TSHTQGSLRITNISSDDSGKQISCVAENLVGEDQD
SVNLTVHFAPTITFLESPTSDHHWCIPFTVKGNPK
PALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQ
LDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWP
GIDDGANPNYPDVIYEDYGTAANDIGDTTNRSNEI PSTDVTDKTGREH
[0186] As used herein, the terms "TrkB antigen epitope" and "TrkB
epitope" refer to a molecule (e.g., a peptide) or a fragment of a
molecule capable of binding to an antigen binding site of the
binding molecule or binding molecules fragments according to the
invention that binds specifically to Tropomyosin receptor kinase B.
These terms further include, for example, a TrkB antigenic
determinant recognized by any of the binding molecules or binding
molecules fragments of the present invention, which has a light and
heavy chain CDR combination selected from:
[0187] Light chain CDRs comprising the amino acid sequences of SEQ
ID NO: 201 (CDR1), SEQ ID NO: 202 (CDR2) and SEQ ID NO: 203 (CDR3),
and Heavy chain CDRs comprising the amino acid sequences of SEQ ID
NO: 204 (CDR1), SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or
Heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:
207 (CDR1), SEQ ID NO: 208 (CDR2) and SEQ ID NO: 209 (CDR3); or
Heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:
210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ ID NO: 212 (CDR3).
[0188] TrkB antigen epitopes can be included in proteins, protein
fragments, peptides or the like. The epitopes are most commonly
proteins, short oligopeptides, oligopeptide mimics (i.e., organic
compounds that mimic antibody binding properties of the TrkB
antigen), or combinations thereof.
[0189] VEGF Binding
[0190] Vascular endothelial growth factor (VEGF) is one of the most
important pro-angiogenic factors, also termed VEGF-A or vascular
permeability factor (VPF). VEGF belongs to a gene family that
includes placenta growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D,
VEGF-E and VEGF-F. Alternative splicing of mRNA of a single gene of
human VEGF results in at least six isoforms (VEGF121, VEGF145,
VEGF165, VEGF183, VEGF189, and VEGF206), VEGF165 being the most
abundant isoform.
[0191] Two VEGF tyrosine kinase receptors (VEGFR) have been
identified that interact with VEGF, i.e. VEGFR-1 (also known as
Flt-1) and VEGFR-2 (also known as KDR or FIK-1). VEGFR-1 has the
highest affinity for VEGF, while VEGFR- 2 has a somewhat lower
affinity for VEGF. Ferrara (Endocrine Rev. 2004, 25: 581-611)
provide a detailed description of VEGF, the interaction with its
receptors and its function in normal and pathological processes can
be found in Hoeben et al. Pharmacol. Rev. 2004, 56: 549-580.
[0192] VEGF has been reported to be a pivotal regulator of both
normal and abnormal angiogenesis (Ferrara and Davis-Smyth,
Endocrine Rev. 1997, 18: 4-25; Ferrara J. MoL Med. 1999, 77:
527-543). Compared to other growth factors that contribute to the
processes of vascular formation, VEGF is unique in its high
specificity for endothelial cells within the vascular system.
[0193] VEGF is in particular involved in eye diseases. The
concentration of VEGF in eye fluids is highly correlated with the
presence of active proliferation of blood vessels in patients with
diabetic and other ischemia-related retinopathies. Furthermore,
studies have demonstrated the localization of VEGF in choroidal
neovascular membranes in patients affected by age-related macular
degeneration (AMD).
[0194] The VEGF-binding components of the binding molecule
according to the invention, e.g. immunoglobulin (Ig) molecules,
have specificity for VEGF in that they bind specifically to one or
more epitopes within the VEGF molecule. By binding to VEGF the
binding molecules of the invention act as VEGF antagonists.
Preferably, the binding molecules of the invention bind
specifically to VEGF-A.
[0195] Specific binding of a VEGF-binding component to its antigen
VEGF can be determined in any suitable manner known per se,
including, for example, the assays described herein, Scatchard
analysis and/or competitive binding assays, such as
radioimmunoassays (RIA), enzyme immunoassays (EIA and ELISA) and
sandwich competition assays, and the different variants thereof
known per se in the art. The same is true for an TrkB-binding
component when binding to its antigen.
[0196] With regard to the antigen VEGF, a VEGF-binding component of
the invention, e.g. an immunoglobulin (Ig) molecule, is not limited
with regard to the species. Thus, the immunoglobulin (Ig) molecule
preferably binds to human VEGF, if intended for therapeutic
purposes in humans. However, immunoglobulin (Ig) molecules that
bind to VEGF from another mammalian species are also within the
scope of the invention. An immunoglobulin (Ig) molecule binding to
one species form of VEGF may cross-react with VEGF, which has a
different sequence than the human one, from one or more other
species. For example immunoglobulin (Ig) molecules binding to human
VEGF may exhibit cross reactivity with VEGF from one or more other
species of primates and/or with VEGF from one or more species of
animals that are used in animal models for diseases, for example
monkey, mouse, rat, rabbit, pig, dog, and in particular in animal
models for diseases and disorders associated with VEGF-mediated
effects on angiogenesis (such as the species and animal models
mentioned herein). Immunoglobulin (Ig) molecules that show such
cross-reactivity are advantageous in a research and/or drug
development, since it allows the immunoglobulin single variable
domains of the invention to be tested in acknowledged disease
models such as monkeys, in particular Cynomolgus or Rhesus, or mice
and rats.
[0197] Preferably, in view of cross-reactivity with one or more
VEGF molecules from species other than human that is/are intended
for use as an animal model during development of a therapeutic VEGF
antagonist, a VEGF-binding component recognizes an epitope in a
region of the VEGF of interest that has a high degree of identity
with human VEGF.
[0198] Hence, the VEGF-binding components of the binding molecule
according to the invention, e.g. an immunoglobulin (Ig) molecule
recognizes an epitope which is, totally or in part, located in a
region of VEGF that is relevant for binding to its receptor.
According to preferred aspects, the VEGF-binding components of the
binding molecule according to the invention block VEGF receptor
activation, preferably substantially and most preferably
totally.
[0199] As described above, the ability of a VEGF-binding component
to block the interaction between VEGF and its receptors can be
determined by an Amplified Luminescent Proximity Homogeneous Assay
(AlphaScreen.RTM.), a competition ELISA, or a plasmon resonance
(SPR) based assay (Biacore.RTM.), as described in the Examples.
[0200] TrkB & VEGF Binding Molecules
[0201] The present invention relates to binding molecules that have
binding specificities for at least two different targets. In
relation to the present invention, the binding molecules are
derived from antibodies. Techniques for making binding molecules
include, but are not limited to, recombinant co-expression of two
immunoglobulin heavy chain- light chain pairs having different
specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO
93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and
"knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731, 168).
Binding molecules of the invention may also be made by engineering
electrostatic steering effects for making antibody Fc-heterodimeric
molecules (WO 2009/089004A1); cross- linking two or more antibodies
or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et
al., Science, 229: 81 (1985)); using leucine zippers to produce
bi-specific antibodies (see, e.g., Kostelny et al., Immunol.,
148(5): 1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al.,/Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al./Immunol. 147: 60 (1991).
[0202] In one embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF),
preferably VEGF-A and at least one antigen binding site that binds
specifically to Tropomyosin receptor kinase B (TrkB). In a related
embodiment, the binding molecule is bispecific and tetravalent.
[0203] In another embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF) and at
least one antigen binding site that binds specifically to
Tropomyosin receptor kinase B (TrkB), wherein the at least one
antigen binding site that binds specifically to VEGF is an
immunoglobulin (Ig) molecule, preferably an IgG molecule. In a
related embodiment, the binding molecule is bispecific and
tetravalent.
[0204] In another embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF) and at
least one antigen binding site that binds specifically to
Tropomyosin receptor kinase B (TrkB), wherein the at least one
antigen binding site that binds specifically to TrkB comprises one
or more scFv(s), preferably in a VL-VH orientation from N-to
C-terminus. In a related embodiment, the binding molecule is
bispecific and tetravalent.
[0205] In another embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF) and at
least one antigen binding site that binds specifically to
Tropomyosin receptor kinase B (TrkB), wherein the at least one
antigen binding site that binds specifically to VEGF is an
immunoglobulin (Ig) molecule, preferably an IgG molecule, and
wherein the at least one antigen binding site that binds
specifically to TrkB is fused to the C- terminus of the heavy chain
of the Ig molecule. In a related embodiment, the binding molecule
is bispecific and tetravalent.
[0206] In another embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF) and at
least one antigen binding site that binds specifically to
Tropomyosin receptor kinase B (TrkB), wherein the at least one
antigen binding site that binds specifically to VEGF is an
immunoglobulin (Ig) molecule, preferably an IgG molecule, and
wherein the at least one antigen binding site that binds
specifically to TrkB comprises one or more scFv(s), preferably in a
VL-VH orientation from N-to C-terminus. In a related embodiment,
the binding molecule is bispecific and tetravalent.
[0207] In another embodiment, the binding molecule comprises or
consists of at least one antigen binding site that binds
specifically to Vascular Endothelial Growth Factor (VEGF) and at
least one antigen binding site that binds specifically to
Tropomyosin receptor kinase B (TrkB), wherein the at least one
antigen binding site that binds specifically to VEGF is an
immunoglobulin (Ig) molecule, preferably an IgG molecule, and
wherein the at least one antigen binding site that binds
specifically to TrkB comprises one or more scFv(s), preferably in a
VL-VH orientation from N-to C-terminus, and is fused to the C-
terminus of the heavy chain of the Ig molecule. In a related
embodiment, the binding molecule is bispecific and tetravalent.
[0208] In a preferred embodiment, the binding molecules of the
invention comprise or consists of (i) two heavy chains each
comprising or consisting of a heavy chain variable region specific
for VEGF, constant IgG domains and an scFv specific for TrkB and
(ii) two light chains each comprising or consisting of a light
chain variable region specific for VEGF.
[0209] In addition, these single chain Fv fragments might be
further stabilized by incorporation of disulfide bonds between the
VH and VL domains, within the VH domain, or within the VL domain,
via incorporation of cysteine residues. The term N-terminus denotes
the first amino acid of the polypeptide chain while the term
C-terminus denotes the last amino acid of the C-terminus of the
polypeptide chain. Hence an embodiment of the invention is wherein
the one or more scFv(s) comprises additional cysteine residues to
form disulfide bonds.
[0210] In one embodiment, the present invention provides a binding
molecule which is a multi-specific binding protein comprising (i)
an Ig molecule specifically binding to VEGF with two heavy and two
light chains, and (ii) two scFv molecules (scFv(s)) each
specifically binding to TrkB. Preferably, each heavy chain of the
Ig molecule has one scFv fused to its C-terminus, thereby forming a
bispecific tetravalent binding protein.
[0211] In one embodiment, the present invention provides a binding
molecule (also referred to herein multi-specific binding protein or
a modified Ig molecule) with: [0212] (i) two heavy chains, each
comprising from N to C terminus: [0213] a heavy chain variable
domain specific for VEGF (e.g., murine, humanized or human VH
domain) [0214] constant domains of an IgG (e.g. human IgG1 or IgG4)
[0215] a peptide linker (e.g.a GS mini linker) and [0216] an scFv
specific for TrkB (e.g. an scFv comprising from N to C terminus a
VH domain (e.g. murine, humanized or human VH domain) a linker and
a VL domain (e.g. murine, humanized or human VL domain), or vice
versa a VL domain a linker and a VH domain); and [0217] (ii) two
light chains, each comprising from N to C-terminus: a light chain
variable domain specific for VEGF (e.g. murine, humanized or human
VL domain), a light chain constant domain (e.g., a human kappa
chain).
[0218] The present invention provides a binding molecule having at
least one antigen binding site that binds specifically to vascular
endothelial growth factor (VEGF) and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB).
[0219] Methods of preparing binding sites that bind to specific
target antigens are well known in the art. The skilled person can
readily use these methods to devise an antigen binding site having
the necessary specificity for the VEGF or TrkB target antigens.
[0220] Methods of generating antibodies and antibody fragments are
well known in the art. For example, antibodies may be generated via
any one of several methods which employ induction of in vivo
production of antibody molecules, screening of immunoglobulin
libraries (Orlandi et al, 1989. Proc. Natl. Acad. Sci. U.S.A.
86:3833-3837; Winter et al 1991, Nature 349:293-299) or generation
of monoclonal antibody molecules by cell lines in culture. These
include, but are not limited to, the hybridoma technique, the human
B-cell hybridoma technique, and the Epstein-Barr virus
(EBV)-hybridoma technique (Kohler et al 1975. Nature 256:4950497;
Kozbor et al 1985. J. Immunol. Methods 81:31-42; Cote et al 1983.
Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al 1984. Mol.
Cell. Biol. 62:109-120).
[0221] Using methods known in the art and described herein it would
be routine for the person skilled in the art to prepare antibodies
having a binding site with the necessary specificity for the VEGF
and/or TrkB target antigens as well as binding molecules described
herein. Isolation of the binding domains from such antibodies is a
routine practice and indeed further information on methods that can
be used to generate antibodies and binding molecules as described
herein are provided in the accompanying examples.
[0222] The present inventors conceived binding molecules against
VEGF/TrkB of the invention as shown in Table 1 and a selection of
those molecules were prepared and are discussed in the accompanying
examples.
TABLE-US-00002 TABLE 1 Binding Molecule Sequence SEQ ID NO:
TPP-11735 DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 1 Light
Chain LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-11735
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 2 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCA
ASGFTINASWIHWVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRF
TISADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQ
GTLVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGD
RVTITCRASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVEIK TPP-11736
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 3 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-11736
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 4 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITC
RASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVEIKGGSEGK
SSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGFTINA
SWIHWVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSK
NTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS TPP-11737
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 5 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-11737
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 6 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCA
ASGFTISDYWIHWVRQAPGKGLEWVAGITPAGGYTYYADSVKGRF
TISADTSKNTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQG
TLVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDR
VTITCRASQDVSTAVAWYQQKPGKAPKLLTYSASFLYSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIK TPP-11738
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 7 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-11738
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 8 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITC
RASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKGGSEGK
SSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGFTISD
YWIHWVRQAPGKGLEWVAGITPAGGYTYYADSVKGRFTISADTSK
NTAYLQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS TPP-14936
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 9 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14936
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 10 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCA
ASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRF
TFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVW
GQGTLVTVSSGGSEGKSSGSGSESKSTGGSDIQLTQSPSSLSASV
GDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE IK TPP-14937
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 11 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14937
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 12 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSDIQLTQSPSSLSASVGDRVTITC
SASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGSEGK
SSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTH
YGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSK
STAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTV SS TPP-16061
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 13 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-16061
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 14 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RASQVIRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQSNTSPLTFGQGTKVEIKGGSEGK
SSGSGSESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGFTINA
SWIHWVRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSK
NTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS TPP-16062
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 15 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-16062
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 16 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQV
IRRSLAWYQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQSNTSPLTFGQGTKVEIKGGSEGKSSGSG
SESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHW
VRQAPGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYL
QMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS TPP-16063
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 17 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-16063
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 18 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQVIRRSL
AWYQQKPGKAPKLLIYAASNLASGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQSNTSPLTFGQGTKVEIKGGSEGKSSGSGSESKS
TGGSEVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAP
GKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSL
RAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVSS TPP-16064
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 19 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-16064
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 20 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSGGSDIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQ
QKPGKAPKLLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQSNTSPLTFGQGTKVEIKGGSEGKSSGSGSESKSTGGS
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKGL
EWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCARWGHSTSPWAMDYWGQGTLVTVSS TPP-19984
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 21 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19984
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 22 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYG
MNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKST
AYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS
GGSEGKSSGSGSESKSTGGSDIQLTQSPSSLSASVGDRVTITCSA
SQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK TPP-19985
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 23 Light Chain
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19985
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 24 Heavy Chain
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCA
ASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRF
TFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVW
GQGTLVTVSSGGSEGKSSGSGSESKSTGGSDIQLTQSPSSLSASV
GDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVE IK TPP-14938
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAPK 25 Light Chain
LLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SNTSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14938
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKGL 26 Heavy Chain
EWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCARWGHSTSPWAMDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
GGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKAS
GYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTM
TRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQ
GTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGD
RVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRF
SGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-14939
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAPK 27 Light Chain
LLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SNTSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14939
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKGL 28 Heavy Chain
EWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCARWGHSTSPWAMDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSS
GSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-14940
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 29 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14940
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 30 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-14941
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 31 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-14941
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 32 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-19986
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 33 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19986
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 34 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-19987
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 35 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19987
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 36 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-19988
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 37 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19988
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 38 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-19989
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 39 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-19989
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 40 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22171
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 41 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22171
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 42 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22172
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 43 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
TPP-22172 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 44 Heavy
Chain EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22173
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 45 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22173
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 46 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22174
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 47 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22174
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 48 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22175
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 49 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22175
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 50 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22176
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 51 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22176
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 52 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22177
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 53 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22177
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 54 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22178
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 55 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22178
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 56 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22179
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 57 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22179
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 58 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22180
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 59 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22180
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 60 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIK TPP-22181
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 61 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22181
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 62 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-22182
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 63 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22182
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 64 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK
TPP-22183 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 65 Light
Chain VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22183
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 66 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-22184
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 67 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22184
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 68 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-22185
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 69 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22185
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 70 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-22186
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 71 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22186
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 72 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLE IK TPP-22187
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 73 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22187
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 74 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22188
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 75 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22188
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 76 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22189
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 77 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22189
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 78 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22190
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 79 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22190
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 80 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22191
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 81 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22191
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 82 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22192
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 83 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22192
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 84 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22193
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 85 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22193
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 86 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22194
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 87 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22194
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 88 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22195
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 89 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22195
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 90 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22196
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 91 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22196
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 92 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22197
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 93 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22197
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 94 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22198
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 95 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22198
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 96 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGQGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22199
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 97 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22199
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 98 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22200
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 99 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22200
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 100 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22201
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 101 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22201
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 102 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22202
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 103 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22202
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 104 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22203
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 105 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22203
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 106 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22204
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 107 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22204
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 108 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD
IIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTST
VYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
GGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASVGDRVTITCRT
SENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTD
YTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIK TPP-22205
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 109 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22205
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 110 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22206
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 111 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22206
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 112 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22207
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 113 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22207
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 114 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22208
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 115 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22208
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 116 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22209
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 117 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22209
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 118 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22210
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 119 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22210
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 120 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCK
ASGYTFTNYDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRV
TMTRDTSTSTVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYW
GQGTTVTVSSGGSEGKSSGSGSESKSTGGSDIQMTQSPSSLSASV
GDRVTITCRTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPS
RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLE IK TPP-22211
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 121 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22211
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 122 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22212
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 123 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22212
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 124 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22213
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 125 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22213
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 126 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22214
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 127 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22214
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 128 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS
TPP-22215 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 129 Light
Chain VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22215
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 130 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22216
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 131 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22216
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 132 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNL
AWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSL
QPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKS
TGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAP
GQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSL
RSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS TPP-22217
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 133 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22217
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 134 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22218
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 135 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22218
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 136 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22219
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 137 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22219
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 138 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22220
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 139 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22220
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 140 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22221
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 141 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22221
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 142 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS TPP-22222
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 143 Light Chain
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TPP-22222
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 144 Heavy Chain
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC
RTSENVYSNLAWYQQKPGKAPKLLIYAASNLQSGVPSRFSGSGSG
TDYTFTISSLQPEDIATYYCQHFWGSPFTFGCGTKLEIKGGSEGK
SSGSGSESKSTGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTN
YDIIWVRQAPGQCLEWMGYINPYNDGTKYNEKFKGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARLLKYRRFRYYAIDYWGQGTTVTV SS VEGF binder 145
Ranibizumab L-CDR1 VEGF binder 146 Ranibizumab L-CDR2 VEGF binder
147 Ranibizumab L-CDR3 VEGF binder G Y D F T H Y G M N 148
Ranibizumab H-CDR1 (CCG) VEGF binder W I N T Y T G E P T Y A A D F
K R 149 Ranibizumab H-CDR2 (CCG) VEGF binder Y P Y Y Y G T S H W Y
F D V 150 Ranibizumab H-CDR3 (CCG) VEGF binder H Y G M N 151
Ranibizumab H-CDR1 (Kabat) VEGF binder W I N T Y T G E P T Y A A D
F K R 152 Ranibizumab H-CDR2 (Kabat) VEGF binder Y P Y Y Y G T S H
W Y F D V 153 Ranibizumab H-CDR3 (Kabat) VEGF binder G Y D F T H Y
154 Ranibizumab H-CDR1 (Chothia) VEGF binder N T Y T G E 155
Ranibizumab H-CDR2 (Chothia) VEGF binder Y P Y Y Y G T S H W Y F D
V 156 Ranibizumab H-CDR3 (Chothia) VEGF binder B20 157 L-CDR1 VEGF
binder B20 158 L-CDR2 VEGF binder B20 159 L-CDR3 VEGF binder B20 G
F T I N A S W I H 160 H-CDR1 (CCG) VEGF binder B20 A I Y P Y S G Y
T N Y A D S V K G 161
H-CDR2 (CCG) VEGF binder B20 W G H S T S P W A M D Y 162 H-CDR3
(CCG) VEGF binder B20 A S W I H 163 H-CDR1 (Kabat) VEGF binder B20
A I Y P Y S G Y T N Y A D S V K G 164 H-CDR2 (Kabat) VEGF binder
B20 W G H S T S P W A M D Y 165 H-CDR3 (Kabat) VEGF binder B20 G F
T I N A S 166 H-CDR1 (Chothia) VEGF binder B20 Y P Y S G Y 167
H-CDR2 (Chothia) VEGF binder B20 W G H S T S P W A M D Y 168 H-CDR3
(Chothia) VEGF binder G6 169 L-CDR1 VEGF binder G6 170 L-CDR2 VEGF
binder G6 171 L-CDR3 VEGF binder G6 G F T I S D Y W I H 172 H-CDR1
(CCG) VEGF binder G6 G I T P A G G Y T Y Y A D S V K G 173 H-CDR2
(CCG) VEGF binder G6 F V F F L P Y A M D Y 174 H-CDR3 (CCG) VEGF
binder G6 D Y W I H 175 H-CDR1 (Kabat) VEGF binder G6 G I T P A G G
Y T Y Y A D S V K G 176 H-CDR2 (Kabat) VEGF binder G6 F V F F L P Y
A M D Y 177 H-CDR3 (Kabat) VEGF binder G6 G F T I S D Y 178 H-CDR1
(Chothia) VEGF binder G6 T P A G G Y 179 H-CDR2 (Chothia) VEGF
binder G6 F V F F L P Y A M D Y 180 H-CDR3 (Chothia) VEGF binder
B20 DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAPK 181 Variable
Light LLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ Chain (VL)
SNTSPLTFGQGTKVEIK VEGF binder B20
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKGL 182 Variable Heavy
EWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAED Chain (VH)
TAVYYCARWGHSTSPWAMDYWGQGTLVTVSS VEGF binder G6
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK 183 Variable Light
LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ Chain (VL)
SYTTPPTFGQGTKVEIK VEGF binder G6
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGL 184 Variable Heavy
EWVAGITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAED Chain (VH)
TAVYYCARFVFFLPYAMDYWGQGTLVTVSS VEGF binder B20
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKPGKAPK 185 Light Chain
LLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SNTSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VEGF binder B20
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQAPGKGL 186 Heavy Chain
EWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCARWGHSTSPWAMDYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD
KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG VEGF binder G6
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPK 187 Light Chain
LLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VEGF binder G6
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQAPGKGL 188 Heavy Chain
EWVAGITPAGGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAED
TAVYYCARFVFFLPYAMDYWGQGTLVTVSSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG VEGF binder
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 189 Ranibizumab
VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ Variable Light
YSTVPWTFGQGTKVEIK Chain (VL) VEGF binder
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 190 Ranibizumab
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED Variable Heavy
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS Chain (VH) VEGF binder
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 191 Ranibizumab (1Q)
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED Variable Heavy
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS Chain (VH) VEGF binder
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 192 Ranibizumab (6Q)
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED Variable Heavy
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS Chain (VH) VEGF binder
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 193 Ranibizumab
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ (70G)
YSTVPWTFGQGTKVEIK Variable Light Chain (VL) VEGF binder
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 194 Ranibizumab
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED (1Q/6Q)
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS Variable Heavy Chain (VH) VEGF
binder DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 195
Ranibizumab VLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ Light
Chain YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VEGF binder
EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 196 Ranibizumab
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED Heavy Chain
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG VEGF binder
QVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 197 Ranibizumab (1Q)
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED Heavy Chain
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG VEGF binder
EVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL Ranibizumab (6Q)
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED 198 Heavy Chain
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG VEGF binder
DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPK 199 Ranibizumab
VLIYFTSSLHSGVPSRFSGSGSGTGFTLTISSLQPEDFATYYCQQ (70G)
YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL Light Chain
LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VEGF binder
QVQLVQSGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGL 200 Ranibizumab
EWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED (1Q/6Q)
TAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLA Heavy Chain
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG TrkB binder C2 201
L-CDR1 TrkB binder C2 202 L-CDR2 TrkB binder C2 203 L-CDR3 TrkB
binder C2 G Y T F T N Y D I I 204 H-CDR1 (CCG) TrkB binder C2 Y I N
PY N D G T K Y N E K F K G 205 H-CDR2 (CCG) TrkB binder C2 L L K Y
R R F R Y Y A I D Y 206 H-CDR3 (CCG) TrkB binder C2 N Y D I I 207
H-CDR1 (Kabat) TrkB binder C2 Y I N P Y N D G T K Y N E K F K G 208
H-CDR2 (Kabat) TrkB binder C2 L L K Y R R F R Y Y A I D Y 209
H-CDR3 (Kabat) TrkB binder C2 G Y T F T N Y 210 H-CDR1 (Chothia)
TrkB binder C2 N P Y N D G 211 H-CDR2 (Chothia) TrkB binder C2 L L
K Y R R F R Y Y A I D Y 212 H-CDR3 (Chothia) TrkB binder C2
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 213 (100C)
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH Variable Light
FWGSPFTFGCGTKLEIK Chain (VL) TrkB binder C2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQCL 214 (44C)
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED Variable Heavy
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS Chain (VH) TrkB binder C2
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 215 Variable Light
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH Chain (VL)
FWGSPFTFGQGTKLEIK
TrkB binder C2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 216
Variable Heavy EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED Chain
(VH) TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSS 6GS GGSGGS 217 10L1
GGGGSGGGGS 218 15L1 GGGGSGGGGSGGGGS 219 20L1 GGGGSGGGGSGGGGSGGGGS
220 20L3 GGSEGKSSGSGSESKSTGGS 221 TrkB binder
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 222 VL-VH
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH (44C/100C)
FWGSPFTFGCGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSG
AEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQCLEWMGYINP
YNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
LLKYRRFRYYAIDYWGQGTTVTVSS TrkB binder
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQCL 223 VH-VL
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED (44C/1000)
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSGGSEGKSSGSGS
ESKSTGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQ
QKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPED
IATYYCQHFWGSPFTFGCGTKLEIK TrkB binder
DIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQQKPGKAPK 224 VL-VH
LLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH
FWGSPFTFGQGTKLEIKGGSEGKSSGSGSESKSTGGSQVQLVQSG
AEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGLEWMGYINP
YNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
LLKYRRFRYYAIDYWGQGTTVTVSS TrkB binder
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIIWVRQAPGQGL 225 VH-VL
EWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSED
TAVYYCARLLKYRRFRYYAIDYWGQGTTVTVSSGGSEGKSSGSGS
ESKSTGGSDIQMTQSPSSLSASVGDRVTITCRTSENVYSNLAWYQ
QKPGKAPKLLIYAASNLQSGVPSRFSGSGSGTDYTFTISSLQPED
IATYYCQHFWGSPFTFGQGTKLEIK 4GS GGGS 227
[0223] In a further embodiment the binding molecule of the
invention or the TrkB binding molecule may be based on any of the
below disclosed TrkB binders which are also disclosed in
WO2018/224630.
TABLE-US-00003 TABLE 2 228 TrkB binder 277 L-CDR1 TrkB binder 229
277 L-CDR2 TrkB binder 230 277 L-CDR3 TrkB binder G Y T F T G Y W M
H 231 277 H-CDR1 (CCG) TrkB binder Y I N P S T D Y T E Y N Q K F K
D 232 111 H-CDR2 (CCG) TrkB binder S R T G N Y 233 277 H-CDR3 (CCG)
TrkB binder G Y W M H 234 277 H-CDR1 (Kabat) TrkB binder Y I N P S
T D Y T E Y N Q K F K D 235 277 H-CDR2 (Kabat) TrkB binder S R T G
N Y 236 277 H-CDR3 (Kabat) TrkB binder G Y T F T G Y 237 277 H-CDR1
(Chothia) TrkB binder N P S T D Y 238 277 H-CDR2 (Chothia) TrkB
binder S R T G N Y 239 277 H-CDR3 (Chothia) TrkB binder
DIVMTQSPDSLAVSLGERATIN 240 277-gr_VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
light chain VAVYYCQQYYSYPYTFGQGTKL EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 241 277-gr_VH, KASGYTFTGYWMHWVRQAPGQG
(humanized) LEWMGYINPSTDYTEYNQKFKD variable RVTMTRDTSTSTVYMELSSLRS
heavy EDTAVYYCARSRTGNYWGQGTL chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIS 242 277-33_VL: CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
light chain VAVYYCQQYYSYPYTFGGGTKL EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 243 277-33_VH, KASGYTFTGYWMHWVRQRPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTLTRDTSTSTVYMELSSLTS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATISC 244 277-35_VL: KSSQSLLYSSNQKKYLAWYQQKP
(humanized) GQPPKLLIYWASTRESGVPDRFS variable
GSGSGTDFTLTISSLQAEDVAVY Light YCQQYYSYPYTFGGGTKLEIK chain TrkB
binder QVQLVQSGAEVKKPGASVKVSC 245 277-35 VH, KASGYTFTGYWMHWVRQRPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTLTRDTSTSTVYMELSSLRS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIN 246 277-42_VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
Light VAVYYCQQYYSYPYTFGGGTKL chain EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 247 277-42_VH, KASGYTFTGYWMHWVRQRPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTLTRDTSTSTVYMELSSLRS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIN 248 277-44 VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQSPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
Light VAVYYCQQYYSYPYTFGQGTKL chain EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 249 277-44_VH, KASGYTFTGYWMHWVRQAPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTMTRDTSTSTVYMELSSLTS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIN 250 277-48_VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
Light VAVYYCQQYYSYPYTFGQGTKL chain EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 251 277-48_VH, KASGYTFTGYWMHWVRQRPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTMTRDTSTSTVYMELSSLRS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIS 252 277-51 _VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
light chain VAVYYCQQYYSYPYTFGGGTKL EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 253 277-51_VH, KASGYTFTGYWMHWVRQRPGQ
(humanized) GLEWIGYINPSTDYTEYNQK variable FKDRATLTRDTSTSTVYMELSS
heavy LRSEDTAVYYCARSRTGNYWGQ chain GTTVTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIS 254 277-64_VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQPPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
light chain VAVYYCQQYYSYPYTFGQGTKL EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 255 277-64 VH, KASGYTFTGYWMHWVRQAPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD variable RVTMTRDTSTSTVYMELSSLRS
heavy EDTAVYYCARSRTGNYWGQGTT chain VTVSS TrkB binder
DIVMTQSPDSLAVSLGERATIN 256 277-67_VL, CKSSQSLLYSSNQKNYLAWYQQ
(humanized) KPGQSPKLLIYWASTRESGVPD variable RFSGSGSGTDFTLTISSLQAED
light chain VAVYYCQQYYSYPYTFGQGTKL EIK TrkB binder
QVQLVQSGAEVKKPGASVKVSC 257 277-67 VH, KASGYTFTGYWMHWVRQAPGQG
(humanized) LEWIGYINPSTDYTEYNQKFKD Variable RVTMTRDTSTSTVYMELSSLRS
EDTAVYYCARSRTGNYWGQG heavy TTVTVSS chain
[0224] In a preferred embodiment, the binding molecule comprises at
least one antigen binding site that binds specifically to Vascular
Endothelial Growth Factor (VEGF) and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB), wherein the antigen binding site that binds specifically to
TrkB comprises light chain CDRs comprising or consisting of the
amino acid sequences of SEQ ID NO: 201 (CDR1), SEQ ID NO: 202
(CDR2) and SEQ ID NO: 203 (CDR3) and heavy chain CDRs comprising or
consisting of the amino acid sequences of SEQ ID NO: 204 (CDR1),
SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or heavy chain
CDRs comprising or consisting of the amino acid sequences of SEQ ID
NO: 207 (CDR1), SEQ ID NO: 208 (CDR2) and SEQ ID NO: 209 (CDR3); or
heavy chain CDRs comprising or consisting of the amino acid
sequences of SEQ ID NO: 210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ
ID NO: 212 (CDR3); and wherein the antigen binding site that binds
specifically to VEGF comprises light chain CDRs comprising or
consisting of the amino acid sequences of SEQ ID NO: 145 (CDR1),
SEQ ID NO: 146 (CDR2) and SEQ ID NO: 147 (CDR3) and heavy chain
CDRs comprising or consisting of the amino acid sequences of SEQ ID
NO: 148 (CDR1), SEQ ID NO: 149 (CDR2) and SEQ ID NO: 150 (CDR3); or
heavy chain CDRs comprising or consisting of the amino acid
sequences of SEQ ID NO: 151 (CDR1), SEQ ID NO: 152 (CDR2) and SEQ
ID NO: 153 (CDR3); or heavy chain CDRs comprising or consisting of
the amino acid sequences of SEQ ID NO: 154 (CDR1), SEQ ID NO: 155
(CDR2) and SEQ ID NO: 156 (CDR3).
[0225] In a further preferred embodiment, the binding molecule
comprises at least one antigen binding site that binds specifically
to Vascular Endothelial Growth Factor (VEGF) and at least one
antigen binding site that binds specifically to Tropomyosin
receptor kinase B (TrkB), wherein the antigen binding site that
binds specifically to TrkB comprises a light chain variable domain
comprising or consisting of an amino acid sequence at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 213 or 215 and a heavy chain variable
domain comprising or consisting of an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 214 or 216; and wherein the
antigen binding site that binds specifically to VEGF comprises a
light chain variable domain comprising or consisting of an amino
acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 189 or 193 and a
heavy chain variable domain comprising or consisting of an amino
acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 190, 191, 192 or
194.
[0226] In a further preferred embodiment, the binding molecule
comprises at least one antigen binding site that binds specifically
to Vascular Endothelial Growth Factor (VEGF) and at least one
antigen binding site that binds specifically to Tropomyosin
receptor kinase B (TrkB), wherein the antigen binding site that
binds specifically to TrkB comprises a light chain variable domain
comprising or consisting of an amino acid sequence of SEQ ID NO:
213 or 215 and a heavy chain variable domain comprising or
consisting of an amino acid sequence of SEQ ID NO: 214 or 216; and
wherein the antigen binding site that binds specifically to VEGF
comprises a light chain variable domain comprising or consisting of
an amino acid sequence of SEQ ID NO: 189 or 193 and a heavy chain
variable domain comprising or consisting of an amino acid sequence
of SEQ ID NO: 190, 191, 192 or 194.
[0227] In a further preferred embodiment, the binding molecule
comprises at least one antigen binding site that binds specifically
to Vascular Endothelial Growth Factor (VEGF) and at least one
antigen binding site that binds specifically to Tropomyosin
receptor kinase B (TrkB), wherein the antigen binding site that
binds specifically to TrkB comprises a light chain variable domain
comprising or consisting of an amino acid sequence of SEQ ID NO:
213 and a heavy chain variable domain comprising or consisting of
an amino acid sequence of SEQ ID NO: 214; and wherein the antigen
binding site that binds specifically to VEGF comprises a light
chain variable domain comprising or consisting of an amino acid
sequence of SEQ ID NO: 193 and a heavy chain variable domain
comprising or consisting of an amino acid sequence of SEQ ID NO:
191.
[0228] In a further preferred embodiment, the binding molecule
comprises at least one antigen binding site that binds specifically
to Vascular Endothelial Growth Factor (VEGF) and at least one
antigen binding site that binds specifically to Tropomyosin
receptor kinase B (TrkB), wherein the antigen binding site that
binds specifically to TrkB comprises a light chain variable domain
comprising or consisting of an amino acid sequence at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 213 or 215 and a heavy chain variable
domain comprising or consisting of an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 214 or 216, wherein the light
chain CDRs in the light chain variable domain consist of the amino
acid sequences of SEQ ID NO: 201 (CDR1), SEQ ID NO: 202 (CDR2) and
SEQ ID NO: 203 (CDR3) and the heavy chain CDRs in the heavy chain
variable domain consist of the amino acid sequences of SEQ ID NO:
204 (CDR1), SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or the
heavy chain CDRs in the heavy chain variable domain consist of the
amino acid sequences of SEQ ID NO: 207 (CDR1), SEQ ID NO: 208
(CDR2) and SEQ ID NO: 209 (CDR3); or the heavy chain in the heavy
chain variable domain CDRs consist of the amino acid sequences of
SEQ ID NO: 210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ ID NO: 212
(CDR3); and wherein the antigen binding site that binds
specifically to VEGF comprises a light chain variable domain
comprising or consisting of an amino acid sequence at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 189 or 193 and a heavy chain variable
domain comprising or consisting of an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 190, 191, 192 or 194, wherein the
light chain CDRs in the light chain variable domain consist of the
amino acid sequences of SEQ ID NO: 145 (CDR1), SEQ ID NO: 146
(CDR2) and SEQ ID NO: 147 (CDR3) and the heavy chain CDRs in the
heavy chain variable domain consist of the amino acid sequences of
SEQ ID NO: 148 (CDR1), SEQ ID NO: 149 (CDR2) and SEQ ID NO: 150
(CDR3); or the heavy chain CDRs in the heavy chain variable domain
consist of the amino acid sequences of SEQ ID NO: 151 (CDR1), SEQ
ID NO: 152 (CDR2) and SEQ ID NO: 153 (CDR3); or the heavy chain
CDRs in the heavy chain variable domain consist of the amino acid
sequences of SEQ ID NO: 154 (CDR1), SEQ ID NO: 155 (CDR2) and SEQ
ID NO: 156 (CDR3).
[0229] In a further preferred embodiment, the binding molecule
comprises at least one antigen binding site that binds specifically
to Vascular Endothelial Growth Factor (VEGF) and at least one
antigen binding site that binds specifically to Tropomyosin
receptor kinase B (TrkB), wherein the antigen binding site that
binds specifically to TrkB comprises a light chain variable domain
comprising or consisting of an amino acid sequence of SEQ ID NO:
213 and a heavy chain variable domain comprising or consisting of
an amino acid sequence of SEQ ID NO: 214, wherein the light chain
CDRs in the light chain variable domain consist of the amino acid
sequences of SEQ ID NO: 201 (CDR1), SEQ ID NO: 202 (CDR2) and SEQ
ID NO: 203 (CDR3) and the heavy chain CDRs in the heavy chain
variable domain consist of the amino acid sequences of SEQ ID NO:
204 (CDR1), SEQ ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or the
heavy chain CDRs in the heavy chain variable domain consist of the
amino acid sequences of SEQ ID NO: 207 (CDR1), SEQ ID NO: 208
(CDR2) and SEQ ID NO: 209 (CDR3); or the heavy chain in the heavy
chain variable domain CDRs consist of the amino acid sequences of
SEQ ID NO: 210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ ID NO: 212
(CDR3); and wherein the antigen binding site that binds
specifically to VEGF comprises a light chain variable domain
comprising or consisting of an amino acid sequence of SEQ ID NO:
193 and a heavy chain variable domain comprising or consisting of
an amino acid sequence of SEQ ID NO: 191, wherein the light chain
CDRs in the light chain variable domain consist of the amino acid
sequences of SEQ ID NO: 145 (CDR1), SEQ ID NO: 146 (CDR2) and SEQ
ID NO: 147 (CDR3) and the heavy chain CDRs in the heavy chain
variable domain consist of the amino acid sequences of SEQ ID NO:
148 (CDR1), SEQ ID NO: 149 (CDR2) and SEQ ID NO: 150 (CDR3); or the
heavy chain CDRs in the heavy chain variable domain consist of the
amino acid sequences of SEQ ID NO: 151 (CDR1), SEQ ID NO: 152
(CDR2) and SEQ ID NO: 153 (CDR3); or the heavy chain CDRs in the
heavy chain variable domain consist of the amino acid sequences of
SEQ ID NO: 154 (CDR1), SEQ ID NO: 155 (CDR2) and SEQ ID NO: 156
(CDR3).
[0230] In one embodiment, the binding molecule comprises at least
one antigen binding site that binds specifically to Vascular
Endothelial Growth Factor (VEGF) and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB), wherein the antigen binding site that binds specifically to
TrkB comprises light chain CDRs comprising or consisting of the
amino acid sequences of SEQ ID NO: 228 (CDR1), SEQ ID NO: 229
(CDR2) and SEQ ID NO: 230 (CDR3) and heavy chain CDRs comprising or
consisting of the amino acid sequences of SEQ ID NO: 231 (CDR1),
SEQ ID NO: 232 (CDR2) and SEQ ID NO: 23 (CDR3); or heavy chain CDRs
comprising or consisting of the amino acid sequences of SEQ ID NO:
234 (CDR1), SEQ ID NO: 235 (CDR2) and SEQ ID NO: 236 (CDR3); or
heavy chain CDRs comprising or consisting of the amino acid
sequences of SEQ ID NO: 237 (CDR1), SEQ ID NO: 238 (CDR2) and SEQ
ID NO: 239 (CDR3); and wherein the antigen binding site that binds
specifically to VEGF comprises light chain CDRs comprising or
consisting of the amino acid sequences of SEQ ID NO: 145 (CDR1),
SEQ ID NO: 146 (CDR2) and SEQ ID NO: 147 (CDR3) and heavy chain
CDRs comprising or consisting of the amino acid sequences of SEQ ID
NO: 148 (CDR1), SEQ ID NO: 149 (CDR2) and SEQ ID NO: 150 (CDR3); or
heavy chain CDRs comprising or consisting of the amino acid
sequences of SEQ ID NO: 151 (CDR1), SEQ ID NO: 152 (CDR2) and SEQ
ID NO: 153 (CDR3); or heavy chain CDRs comprising or consisting of
the amino acid sequences of SEQ ID NO: 154 (CDR1), SEQ ID NO: 155
(CDR2) and SEQ ID NO: 156 (CDR3).
[0231] In another embodiment, the binding molecule comprises at
least one antigen binding site that binds specifically to Vascular
Endothelial Growth Factor (VEGF) and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB), wherein the antigen binding site that binds specifically to
TrkB comprises a light chain variable domain and a heavy chain
variable domain, each comprising or consisting of an amino acid
sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequences of SEQ ID NO: 240 and 241,
242 and 243, 244 and 245, 246 and 247, 248 and 249, 250 and 251,
252 and 253, 254 and 255, or 256 and 257; and wherein the antigen
binding site that binds specifically to VEGF comprises a light
chain variable domain comprising or consisting of an amino acid
sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 189 or 193 and a
heavy chain variable domain comprising or consisting of an amino
acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 190, 191, 192 or
194.
[0232] In a further preferred embodiment relating to any of the
foregoing embodiments, the binding molecule is bispecific and
tetravalent. For the avoidance of doubt, by binding of the binding
molecules of the invention to their respective targets VEGF and
TrkB, the binding molecules of the inventions act as VEGF
antagonists or TrkB agonists, respectively.
[0233] In a further preferred embodiment relating to any of the
foregoing embodiments, the antigen binding site that binds
specifically to VEGF is an immunoglobulin (Ig) molecule and the at
least one antigen binding site that binds specifically to TrkB
comprises one or more scFv(s).
[0234] In a further preferred embodiment relating to any of the
foregoing embodiments, the one or more scFv(s) is fused to the C-
terminus of the heavy chain of the Ig molecule.
[0235] In a further preferred embodiment relating to any of the
foregoing embodiments the at least one antigen binding site that
binds specifically to VEGF is an immunoglobulin (Ig) molecule, more
preferably an IgG, and the at least one antigen binding site that
binds specifically to TrkB comprises one or more scFv(s), more
preferably two scFv.
[0236] The antibody molecule or binding molecule described herein
may be fused (as a fusion protein) or otherwise linked (by covalent
or non-covalent bonds) to other molecular entities having a desired
impact on the properties of the antibody molecule. For example, it
may be desirable to improve pharmacokinetic properties of antibody
or binding molecules described herein, stability e.g. in body
fluids such as blood, in particular in the case of single chain
antibodies or domain antibodies. A number of technologies have been
developed in this regard, in particular to prolong the half-life of
such antibody molecules in the circulation, such as pegylation (WO
98/25971; WO 98/48837; WO 2004081026), fusing or otherwise
covalently attaching the antibody molecule to another antibody
molecule having affinity to a serum protein like albumin (WO
2004041865; WO 2004003019), or expression of the antibody molecule
as fusion protein with all or part of a serum protein like albumin
or transferrin (WO 01/79258).
[0237] Since the Fc region of an antibody interacts with a number
of Fc receptors, which results in a number of important functional
capabilities (which are referred to as "effector functions"), the
antibody is, in certain embodiments, a full length antibody or an
antibody that contains a portion of the Fc region, the latter as
long as the antibody exhibits specific binding both to the relevant
portion of the antigen and to Fc receptors and complements. The
choice of the type and length of the constant region depends on
whether effector functions like complement fixation or
antibody-dependent cell-mediated cytotoxicity are desirable
features, and on the desired pharmacological properties of the
antibody protein.
[0238] In an embodiment of the invention, stability of the scFv
moiety can be increased by incorporation of two cysteine residues
in close 3-dimensional proximity to form a disulfide bond within
the scFv. To effect stabilization through engineered disulfide
bonds, residues at these positions are preferably substituted with
cysteine residues.
[0239] As demonstrated in the accompanying examples, the inventors
have shown that a TrkB scFv having a VL-VH orientation from N-to
C-terminus can function in the binding molecules of the invention
to activate TrkB signalling. While a TrkB scFv having a VH-VL
orientation from N-to C-terminus can also function, the activity
may be reduced in this orientation. Hence a preferred embodiment of
the invention is where the order is VL-VH from N-to C-terminus.
[0240] A further preferred embodiment of the invention is wherein
the one or more scFv(s) specifically binding to TrkB is fused to
the Ig molecule (e.g., human IgG1, IgG1(KO), IgG1FcRnmut, IgG4Pro)
specifically binding to VEGF by a peptide linker, preferably a
peptide linker having a length of about 4 to 20 amino acids (e.g.,
anyone of 6, 9, 10, 12, or 15). Preferably the scFv is fused to the
C- terminus of the heavy chain of the Ig molecule. Preferably the
Ig molecule is an IgG.
[0241] Methods of linking scFv molecules to the C- terminus of the
heavy chain of the IgG molecule or linking the variable domains
within scFv molecules are well known in the art. Typically a small
linker sequence of glycine and serine (termed a GS mini-linker)
amino acids is used. The number of amino acids in the linker can
vary, from 4 (GGGS) (SEQ ID NO: 227), 6 (GGSGGS) (SEQ ID NO: 217),
10 (GGGGSGGGGS) (SEQ ID NO: 218), 15 (GGGGSGGGGSGGGGS) (SEQ ID NO:
219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 220) or more. In
practice, normally the linker is formed by combining the nucleic
acid molecule encoding the IgG of interest (which in the present
case would include the nucleic acid encoding the variable domain of
the heavy chain for the VEGF binding site and constant domains of
the IgG type) with the nucleic acid encoding the desired scFv
(which in the present case would include the nucleic acid encoding
the variable domain of the heavy and light chain, either in VL-VH
or VH-VL orientation for the TrkB binding site) interspaced by the
nucleic acid molecule encoding the linker sequence (e.g. a GS mini
linker of any one of 5, 10, 15, or 20 amino acids, preferably a
linker of SEQ ID NO: 218). Then as further explained below this
complete HC-scFv encoding nucleic acid molecule is placed within an
expression vector and introduced to appropriate host cells such
that the complete IgG heavy chain-scFv single polypeptide is
formed.
[0242] Preferably the GS mini-linker between the scFV molecule and
the C-terminus of the heavy chain of the IgG molecule is 10L1 (SEQ
ID NO: 218).
[0243] In an embodiment of the invention, binding to complement
product C1q or Fc gamma receptor by the binding molecule in this
invention is ablated by utilization of the IgG4 constant region or
of the IgG1 constant region with directed L to A mutagenesis at
positions 234 and 235.
[0244] In an embodiment of the invention, the binding molecule of
the invention may have an Fc region, or the relevant section
thereof, that has been engineered to avoid unintended cross-linking
by soluble Fc gamma receptors or complement Cl q. In one
embodiment, such binding molecule or antibody variant has much
lower affinities to Fc gamma receptors and complement C1q than the
parent antibody. (In the following, if not otherwise stated, the
term "parent" in the context of an antibody molecule, or in the
context of IgG or the Fc region, refers to the non-engineered
antibody molecule, Fc region or IgG, respectively, from which the
mutated (engineered) molecule is derived.). Hence an embodiment of
the invention is wherein the Ig molecule comprises a Fc variant
having a reduced affinity to Fc gamma receptors or complement
receptors or both compared to a wildtype Fc region. Such Ig
molecule is referred to herein as IgG1(KO).
[0245] Also contemplated is a binding molecule comprising an Fc
region, or the relevant section thereof, that has been engineered
to modify serum levels (half-life) by optimizing its interaction
with the neonatal Fc receptor (FcRn), e.g. by a point mutation in
the CH2 domain at position H310A or a point mutation at position
H435A). Such Ig molecule is referred to herein as IgG1 FcRnmut.
[0246] Further contemplated is a binding molecule comprising an Ig
molecule which comprises a hinge region variant of IgG4 that
ablates swapping of the heavy chains with other IgG4 molecules.
Such Ig molecule is referred to herein as IgG4Pro.
[0247] A further aspect of the invention provides isolated nucleic
acid molecules that encode the binding molecule of the invention or
the antibody molecule of the invention, or an expression vector
comprising such a nucleic acid molecule(s).
[0248] In some embodiments the binding molecules of the invention
or antibody molecule of the invention comprise antibody heavy chain
and/or light chain polypeptides. As can be appreciated by the
skilled person, nucleic acid molecules can be readily prepared
which encode the heavy chain polypeptides, light chain
polypeptides, or heavy chain polypeptides and light chain
polypeptides.
[0249] Nucleic acid molecules coding for the light chain and the
heavy chain may be synthesized chemically and enzymatically by
Polymerase Chain Reaction (PCR) using standard methods. First,
suitable oligonucleotides can be synthesized with methods known in
the art (e.g. Gait, 1984), which can be used to produce a synthetic
gene. Methods to generate synthetic genes from oligonucleotides are
known in the art (e.g. Stemmer et al., 1995; Ye et al., 1992;
Hayden et Mandecki, 1988; Frank et al., 1987).
[0250] The nucleic acid molecules of the invention include, but are
not limited to, the DNA molecules encoding the polypeptide
sequences shown in the sequence listing. Also, the present
invention also relates to nucleic acid molecules that hybridize to
the DNA molecules encoding the polypeptide sequences shown in the
sequence listing under high stringency binding and washing
conditions, as defined in WO 2007/042309. Preferred molecules (from
an mRNA perspective) are those that have at least 75% or 80%
(preferably at least 85%, more preferably at least 90% and most
preferably at least 95%) homology or sequence identity with one of
the DNA molecules described herein. By way of example, in view of
expressing the antibodies in eukaryotic cells, the DNA sequences
shown in the sequence listing have been designed to match codon
usage in eukaryotic cells. If it is desired to express the
antibodies in E. coli, these sequences can be changed to match E.
coli codon usage. Variants of DNA molecules of the invention can be
constructed in several different ways, as described e.g. in WO
2007/042309.
[0251] A further aspect of the invention provides a method of
production of a binding or antibody molecule described herein,
comprising: [0252] (a) cultivating the host cell of the invention
under conditions allowing expression of the molecule; and, [0253]
(b) recovering the molecule; and optionally
[0254] An embodiment of this aspect of the invention is wherein the
method of production further comprises step (c) further purifying
and/or modifying and/or formulating the binding molecule of the
invention.
[0255] For producing the binding molecules or antibodies of the
invention, the DNA molecules encoding full-length light and/or
heavy chains or fragments thereof are inserted into an expression
vector such that the sequences are operatively linked to
transcriptional and translational control sequences.
[0256] For manufacturing the binding molecules or antibodies of the
invention, the skilled artisan may choose from a great variety of
expression systems well known in the art, e.g. those reviewed by
Kipriyanov and Le Gall, Curr Opin Drug Discov Devel. 2004 Mar.;
7(2):233-42.
[0257] Expression vectors include plasmids, retroviruses, cosmids,
EBV-derived episomes, and the like. The expression vector and
expression control sequences are selected to be compatible with the
host cell. The antibody light chain gene and the antibody heavy
chain gene or the gene of the heavy chain of the binding molecule
described herein (e.g. the gene comprising an immunoglobulin heavy
chain sequence attached with its C-terminus to a scFv sequence) can
be inserted into separate vectors. In certain embodiments, both DNA
sequences, light and heavy chain sequences, are inserted into the
same expression vector. Convenient vectors are those that encode a
functionally complete human CH or CL immunoglobulin sequence, with
appropriate restriction sites engineered so that any VH or VL
sequence can be easily inserted and expressed, as described above.
The constant chain is usually kappa or lambda for the antibody
light chain, for the antibody heavy chain, it can be, without
limitation, any IgG isotype (IgG 1 , IgG2, IgG3, IgG4) or other
immunoglobulins, including allelic variants.
[0258] The recombinant expression vector may also encode a signal
peptide that facilitates secretion of the antibody chain (e.g., the
heavy and light chains of the binding molecules or antibodies
described herein) from a host cell. The DNA encoding the antibody
chain may be cloned into the vector such that the signal peptide is
linked in-frame to the amino terminus of the mature antibody chain
DNA. The signal peptide may be an immunoglobulin signal peptide or
a heterologous peptide from a non-immunoglobulin protein.
Alternatively, the DNA sequence encoding the antibody chain (e.g.,
the heavy and light chains of the binding molecules or antibodies
described herein) may already contain a signal peptide
sequence.
[0259] In addition to the DNA sequences encoding the antibody
chains (e.g., the heavy and light chains of the binding molecules
or antibodies described herein), the recombinant expression vectors
carry regulatory sequences including promoters, enhancers,
termination and polyadenylation signals and other expression
control elements that control the expression of the antibody chains
in a host cell. Examples for promoter sequences (exemplified for
expression in mammalian cells) are promoters and/or enhancers
derived from (CMV) (such as the CMV Simian Virus 40 (SV40) (such as
the SV40 promoter/enhancer), adenovirus, (e. g., the adenovirus
major late promoter (AdMLP)), polyoma and strong mammalian
promoters such as native immunoglobulin and actin promoters.
Examples for polyadenylation signals are BGH polyA, SV40 late or
early polyA; alternatively, 3'UTRs of immunoglobulin genes etc. can
be used.
[0260] The recombinant expression vectors may also carry sequences
that regulate replication of the vector in host cells (e. g.
origins of replication) and selectable marker genes. Nucleic acid
molecules encoding the heavy chain or an antigen-binding portion
thereof and/or the light chain or an antigen-binding portion
thereof of a binding molecule or antibody described herein, and
vectors comprising these DNA molecules can be introduced into host
cells, e.g. bacterial cells or higher eukaryotic cells, e.g.
mammalian cells, according to transfection methods well known in
the art, including liposome-mediated transfection,
polycation-mediated transfection, protoplast fusion,
microinjections, calcium phosphate precipitation, electroporation
or transfer by viral vectors.
[0261] Preferably, the nucleic acid molecules encoding the heavy
chain and the light chain of the binding molecules or antibodies
described herein are present on two vectors which are
co-transfected into the host cell, preferably a mammalian cell.
[0262] Hence a further aspect provides a host cell comprising an
expression vector comprising a nucleic acid molecule encoding the
heavy chain and an expression vector comprising a nucleic acid
molecule encoding the light chain of the binding molecules or
antibodies described herein.
[0263] Mammalian cell lines available as hosts for expression are
well known in the art and include, inter alia, Chinese hamster
ovary (CHO, CHO-DG44) cells, NSO, SP2/0 cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
carcinoma cells (e. g., Hep G2), A549 cells, 3T3 cells or the
derivatives/progenies of any such cell line. Other mammalian cells,
including but not limited to human, mice, rat, monkey and rodent
cells lines, or other eukaryotic cells, including but not limited
to yeast, insect and plant cells, or prokaryotic cells such as
bacteria may be used. The binding molecules of the invention are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the binding molecule in the
host cells.
[0264] Binding molecules and antibody molecules as described herein
are preferably recovered from the culture medium as a secreted
polypeptide or it can be recovered from host cell lysates if for
example expressed without a secretory signal. It is necessary to
purify the binding molecules or antibody molecules described herein
using standard protein purification methods used for recombinant
proteins and host cell proteins in a way that substantially
homogenous preparations of the binding molecule or antibody as
described herein are obtained. By way of example, state-of-the art
purification methods useful for obtaining the binding molecules and
antibodies of the invention include, as a first step, removal of
cells and/or particulate cell debris from the culture medium or
lysate. The binding molecule or antibody is then purified from
contaminant soluble proteins, polypeptides and nucleic acids, for
example, by fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, Sephadex
chromatography, chromatography on silica or on a cation exchange
resin. As a final step in the process for obtaining a VEGF and TrkB
single binding molecule as described herein, the purified binding
molecule may be dried, e.g. lyophilized, as described below for
therapeutic applications.
[0265] A further aspect of the invention provides the binding
molecules of the invention for use in medicine. It will be
understood that this use in medicine and the uses for treatment of
diseases as described in the following also includes the TrkB
binding molecules, scFv and antibodies according to the
invention.
[0266] The binding molecules of the invention are indicated e.g.
for use in the therapy/treatment of eye or retinal or
neurodegenerative diseases, preferably for the treatment of
neural/neuronal eye or retinal diseases. In a further aspect, the
present invention relates to methods for the treatment and/or
prevention of eye or retinal or neurodegenerative diseases, which
method comprises the administration of an effective amount of the
binding molecule of the invention to a human being (e.g. an
individual suffering from wAMD or being at risk of developing
geographic atrophy), thereby ameliorating one or more symptoms of
the eye or retinal or neurodegenerative diseases.
[0267] The terms "treatment" and "therapy" and the like, as used
herein, are meant to include therapeutic as well as prophylactic,
or suppressive measures for a disease or disorder leading to any
clinically desirable or beneficial effect, including but not
limited to alleviation or relief of one or more symptoms,
regression, slowing or cessation of progression of the disease or
disorder. Thus, for example, the term treatment includes the
administration of a binding molecule prior to or following the
onset of a symptom of a disease or disorder thereby preventing or
removing one or more signs of the disease or disorder. As another
example, the term includes the administration of a binding molecule
after clinical manifestation of the disease to combat the symptoms
of the disease. Further, administration of a binding molecule after
onset and after clinical symptoms have developed where
administration affects clinical parameters of the disease or
disorder, such as the degree of tissue injury or the amount or
extent of metastasis, whether or not the treatment leads to
amelioration of the disease, comprises "treatment" or "therapy" as
used herein. Moreover, as long as the compositions of the invention
either alone or in combination with another therapeutic agent
alleviate or ameliorate at least one symptom of a disorder being
treated as compared to that symptom in the absence of use of the
binding molecule, the result should be considered an effective
treatment of the underlying disorder regardless of whether all the
symptoms of the disorder are alleviated or not.
[0268] A binding molecule of the invention can be administered to a
subject having or at risk of having an eye or retinal disease. The
invention further provides for the use of a binding molecule in the
manufacture of a medicament for prevention and/or treatment of an
eye or retinal disease. The term "subject" as used herein means any
mammalian patient to which a binding molecule can be administered,
including, e.g., humans and non-human mammals, such as primates,
rodents, and dogs. Subjects specifically intended for treatment
using the methods described herein include humans. The binding
molecule can be administered either alone or in combination with
other compositions.
[0269] In one embodiment, the binding molecules of the invention
are used for the treatment of macular degeneration, age-related
macular degeneration, diabetic retinopathy, diabetic macular edema,
retinitis pigmentosa, inherited retinal dystrophy, inherited
macular dystrophy, myopic degeneration, geographic atrophy,
geographic atrophy secondary to age-related macular degeneration,
retinal artery occlusions, endophthalmitis, uveitis, cystoid
macular edema, choroidal neovascular membrane secondary to any
retinal diseases, optic neuropathies, glaucoma, retinal detachment,
toxic retinopathy, radiation retinopathy, and traumatic
retinopathy, prodromal and mild-to-moderate alzheimer's diseases,
delaying disease progression of patients with Alzheimer's disease,
Huntington's disease, Parkinson's disease, major depressive
disorder, schizophrenia, cognitive impairment associated with
schizophrenia, prevention of first-episode psychosis in individuals
with attenuated psychosis syndrome, prevention of relapse in
patients with schizophrenia, treatment-resistant depression,
hyperphagia, obesity or metabolic syndrome.
[0270] In a preferred embodiment, the binding molecules of the
invention have utility in the treatment of wAMD. Further preferred,
the binding molecules of the invention have utility in the
treatment of wAMD and treatment of geographic atrophy. In a yet
further preferred embodiment the binding molecules of the invention
have utility in the treatment of geographic atrophy secondary to
age-related macular degeneration. In a further preferred embodiment
the binding molecules of the invention have utility in the
treatment of geographic atrophy or the treatment of patients at
risk for developing geographic atrophy. In a further preferred
embodiment the binding molecules of the invention have utility in
the prevention of geographic atrophy. In a most preferred
embodiment the binding molecules of the invention have utility in
the treatment of wAMD in patients at risk for developing geographic
atrophy.
[0271] In another embodiment the binding molecules of the invention
may be useful for treatment of hearing loss, in particular for cis
platin induced hearing loss as well as noise and age-related
hearing loss.
[0272] The binding molecule of the invention is administered by any
suitable means, including intravitreal, oral, parenteral,
subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
addition, the binding molecule of the invention is suitably
administered by pulse infusion, particularly with declining doses.
In one aspect, the dosing is given by injections, most preferably
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Preferably, the
binding molecule of the invention is given through an intravitreal
injection into the eye.
[0273] In a further aspect, a binding molecule of the invention is
used in combination with a device useful for the administration of
the binding molecule, such as a syringe, injector pen, or other
device. In a further aspect, a binding molecule of the invention is
comprised in a kit of parts, for example also including a package
insert with instructions for the use of the binding molecule.
[0274] The efficacy of the binding molecules of the invention, and
of compositions comprising the same, can be tested using any
suitable in vitro assay, cell-based assay, in vivo assay and/or
animal model known per se, or any combination thereof, depending on
the specific disease involved. Suitable assays and animal models
will be clear to the skilled person, and for example include the
assays and animal models used in the Examples below.
[0275] The actual pharmaceutically effective amount or therapeutic
dosage will of course depend on factors known by those skilled in
the art such as age and weight of the patient, route of
administration and severity of disease. In any case the binding
molecule of the invention will be administered at dosages and in a
manner which allows a pharmaceutically effective amount to be
delivered based upon patient's unique condition.
[0276] The binding molecules of the invention may be used on their
own or in combination with other pharmacologically active
ingredients, such as state-of-the-art or standard-of-care
compounds.
[0277] Hence a further aspect of the invention provides a
pharmaceutical composition comprising a binding molecule of the
invention, together with a pharmaceutically acceptable carrier and
optionally one or more further active ingredients.
[0278] For intravitreal injection of the binding molecules
generally longer intervals between treatments are preferred. In one
embodiment the binding molecule of the invention is administered
every 6 weeks, preferably every 7 weeks, also preferred every 8
weeks, further preferred every 9 weeks, more preferred every 10
weeks, further preferred every 11 weeks, and more preferred every
12 weeks. In a further preferred embodiment the TrkB-antibody is
administered once every 3 months.
[0279] Depending on the specific binding molecule of the invention
and its specific pharmacokinetic and other properties the frequency
of administration may be even longer, such as every 13 weeks, 14
weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks or 20
weeks.
[0280] Alternatively, other dosage regimens may be applied
including e.g. a loading dose, wherein the binding molecules of the
invention may be injected monthly for 3 loading doses and then
every 12 weeks. Likewise, the frequency of administration may also
be extended in such cases and may be even longer, such as every 13
weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks
or 20 weeks.
[0281] The predicted estimated human dose of approximately 2.5
mg/eye corresponds to a 50 mg/mL formulation in which 50 .mu.L will
be injected into the eye.
[0282] To be used in therapy, the binding molecule of the invention
is formulated into pharmaceutical compositions appropriate to
facilitate administration to animals or humans. Typical
formulations of the binding molecule or antibody molecule described
herein can be prepared by mixing the binding with physiologically
acceptable carriers, excipients or stabilizers, in the form of
lyophilized or otherwise dried formulations or aqueous solutions or
aqueous or non-aqueous suspensions. Carriers, excipients, modifiers
or stabilizers are nontoxic at the dosages and concentrations
employed. They include buffer systems such as phosphate, citrate,
acetate and other inorganic or organic acids and their salts;
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; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone
or polyethylene glycol (PEG); amino acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, oligosaccharides or polysaccharides
and other carbohydrates including glucose, mannose, sucrose,
trehalose, dextrins or dextrans; chelating agents such as EDTA;
sugar alcohols such as, mannitol or sorbitol; salt-forming
counter-ions such as sodium; metal complexes (e.g., Zn-protein
complexes); and/or ionic or non-ionic surfactants such as TWEEN.TM.
(polysorbates), PLURONICS.TM. or fatty acid esters, fatty acid
ethers or sugar esters. Also organic solvents can be contained in
the formulation such as ethanol or isopropanol. The excipients may
also have a release-modifying or absorption-modifying function.
[0283] Usually, aqueous solutions or suspensions will be preferred.
Generally, suitable formulations for therapeutic proteins such as
the binding molecules of the invention are buffered protein
solutions, such as solutions including the protein in a suitable
concentration (such as from 0.001 to 400 mg/ml, preferably from
0.005 to 200 mg/ml, more preferably 0.01 to 200 mg/ml, more
preferably 1.0 -100 mg/ml.
[0284] However, it will be clear to the person skilled in the art
that the ingredients and the amounts thereof as given above do only
represent one, preferred option. Alternatives and variations
thereof will be immediately apparent to the skilled personor can
easily be conceived starting from the above disclosure.
[0285] TrkB Binding Molecule
[0286] Based on the surprising finding that the design of the TrkB
binding sites (two scFv's) in the binding molecules of the
invention apparently supports an optimal sterical formation of TrkB
binding and activation--which may be independent of a further
antigen binding site, such as a VEGF binding site or the VEGF
induced clustering mechanism--the invention is further directed to
a TrkB binding molecule comprising or consisting of two scFv's,
wherein each scFv binds specifically to TrkB and both together act
as a TrkB agonist.
[0287] Without wishing to be bound by theory the inventors believe
that by combining two scFv's into a TrkB binding molecule, an
optimal sterical formation of is achieved which results in the
observed full TrkB agonist activity. Thus, in one aspect the TrkB
binding molecules are full TrkB agonists, i.e. the TrkB binding
molecules are as efficacious in activating TrkB as the natural
ligand BDNF.
[0288] Hence, in it's broadest form the invention relates to a TrkB
binding molecule comprising or consisting of two scFv's, wherein
each scFv binds specifically to TrkB.
[0289] Specific binding of a an scFv and in particular a TrkB
binding molecule to its antigen TrkB can be determined in any
suitable manner known per se, including, for example, the assays
described herein, Scatchard analysis and/or competitive binding
assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA
and ELISA) and sandwich competition assays, and the different
variants thereof known per se in the art.
[0290] A TrkB binding molecule is considered as efficacious as BDNF
if it shows at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% of the efficacy of BDNF. For the purpose of comparing the
efficacy both BDNF as well as the TrkB binding molecule will be
tested by measuring/determing the efficacy, which is the maximum
response as determined by incubating CHO cells stably expressing a
TrkB receptor with BDNF or the TrkB binding molecule and measuring
the TrkB phosphorylation at Y706/707 in the cell lysate of the
treated CHO cells. Further details are described in the
Examples.
[0291] On a related note it was also observed that some of the TrkB
binding molecules were not as efficacious as BDNF, i.e. were not
full TrkB agonists but nonetheless exhibited a very high efficacy.
Hence, in another aspect the TrkB binding molecules may be about at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as efficacious
as compared to BDNF as determined by incubating CHO cells stably
expressing a TrkB receptor with BDNF or the TrkB binding molecule
and measuring the TrkB phosphorylation at Y706/707 in the cell
lysate of the treated CHO cells. Further details are described in
the Examples.
[0292] It is understood that the two scFv's in the TrkB binding
molecule are connected to each other to form a TrkB binding
molecule, in particular a bivalent TrkB binding molecule. The two
scFv's in the TrkB binding molecule may be fused or otherwise
covalently attached to each other. Alternatively, the two scFv's
may be linked via a peptide linker, preferably a peptide linker
e.g. having a length of about 4 to 20 amino acids and more
preferably a flexible peptide linker. In any case also other
linkers may be used having lengths ranging from 4 to 100 amino
acids and being flexible, helical or rigid. In some instances a
longer linker and/or rigid linker may be preferable. The scFv's in
the TrkB binding molecule may also be connected to each other via a
hinge region. It is well understood by the skilled artisan that the
hinge region is a short sequence of the heavy chains of antibodies
linking the Fab (Fragment antigen binding) region to the Fc
(Fragment crystallisable) region.
[0293] In one alternative the TrkB binding molecule comprises or
consists of two scFv's that are linked via a peptide linker. In
another alternative the TrkB binding molecule comprises or consists
of two scFv's that are linked via a hinge region. In another
alternative the TrkB binding molecule consists of two scFv's linked
via a hinge region.
[0294] Each scFv in the TrkB binding molecule may bind to the same
or a different epitope within the TrkB protein. Preferably, both
scFv bind to the same TrkB epitope. In a related embodiment the two
scFv's are identical. In this embodiment both scFv will be based on
the same VL and VH sequence. In another embodiment the two scFv's
are substantially the same, i.e. both scFv's have the same CDR
regions but may have some variability in the framework region. In a
related embodiment the two scFv's have the same CDR regions and the
remaining framework regions are at least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% identical to each other. In another embodiment both
scFv's are different from each other, i.e. have a different VL
and/or VH sequence.
[0295] The scFv's in the TrkB binding molecule may comprise from N
to C terminus a VH domain (e.g. murine, humanized or human VH
domain) a linker and a VL domain (e.g. murine, humanized or human
VL domain), or vice versa a VL domain a linker and a VH domain).
Preferably the scFv's in the TrkB binding molecule have a VL-VH
orientation from N-to C-terminus.
[0296] In addition, the single chain Fv fragments might be further
stabilized by incorporation of disulfide bonds between the VH and
VL domains, within the VH domain, or within the VL domain, via
incorporation of cysteine residues. The term N-terminus denotes the
first amino acid of the polypeptide chain while the term C-terminus
denotes the last amino acid of the C-terminus of the polypeptide
chain. Hence in one embodiment the one or both scFv(s) comprise
additional cysteine residues to form disulfide bonds.
[0297] In one embodiment the TrkB binding molecule further
comprises an Ig molecule. Related to this embodiment, the Ig
molecule may be a monoclonal antibody, a human monoclonal antibody,
a humanized monoclonal antibody, a chimeric antibody, a fragment of
an antibody, such as a Fv, Fab, Fab', or F(ab')2 fragment, a single
chain antibody, such as a single chain variable fragment (scFv), a
Small Modular Immunopharmaceutical (SMIP), a domain antibody, a
nanobody, or a diabody. Further relating to this embodiment, each
scFv may be fused to the C-terminus of the heavy chain of the Ig
molecule. Alternatively, each scFv may be fused to the N-terminus
of the heavy chain of the Ig molecule. Preferably, the Ig molecule
is an IgG, F(ab), or F(ab')2. Preferably, the Ig molecule comprises
or consists of an Fc region. In a related embodiment, each scFv may
be fused to the Ig molecule by a peptide linker, preferably a
peptide linker having a length of about 4 to 20 amino acids. In any
case also other linkers may be used having lengths ranging from 4
to 100 amino acids and being flexible, helical or rigid. In some
instances a longer linker and/or rigid linker may be
preferable.
[0298] In a preferred embodiment the TrkB binding molecule is in
the format of an Fc-scFv, scFv-Fc, (scFv')2 or scFv-CH.sub.3. The
scFv-Fc format is depicted in FIG. 70B and comprises or consists of
two polypeptide chains, each polypeptide chain containing a scFv, a
CH2 and a CH3 domain of an IgG, wherein both polypeptide chains
form together a dimer through the disulfide bonds in the hinge
region and each scFv is fused to the hinge region. The Fc-scFv
format is depicted in FIG. 70C and comprises or consists of two
polypeptide chains, each polypeptide chain containing a CH2 and a
CH3 domain of an IgG and a scFv, wherein both polypeptide chains
form together a dimer through the disulfide bonds in the hinge
region and each scFv is fused to the C-temrinus of the CH3 domain
of one polypeptide chain. It will be understood that the Fc-scFv
format as depicted in FIG. 70C may further comprise one or more Fab
domains.
[0299] In an embodiment relating to any of the aforementioned
embodiments, the TrkB binding molecule is a TrkB agonist.
Preferably the TrkB binding molecule is about at least 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as efficacious as
compared to BDNF.
[0300] In an embodiment relating to any of the aforementioned
embodiments, the TrkB binding molecule is bispecific and
tetravalent, more preferably a Doppelmab.
[0301] By adding a further Ig molecule optimal sterical formation
of the two scFv's within the TrkB binding molecule for achievement
of the full TrkB agonist effect is supported. This effect is
believed to be independent of the specificity of the Ig molecule as
the effect is based on the sterical formation of the TrkB binding
molecule and not the specificity of the Ig molecule. Hence, the Ig
molecule may be designed to bind to any target. Since, it is
believed that the observed full agonist effect is mainly based on
the sterical formation of the scFv's within the TrkB binding
molecule, it follows that the Ig molecule may also not bind to any
target specifically or does not contain any target specific binding
domain at all.
[0302] In a further preferred embodiment the Ig molecule comprises
or consists of an Fc region. Preferably, in this embodiment each
scFv is fused to the C- terminus of the heavy chain of the Fc
region. In a further embodiment relating to this embodiment each
scFv is fused to the Fc region by a peptide linker, preferably a
peptide linker having a length of about 4 to 20 amino acids.
[0303] Also here, it is understood that the two scFv's and the Ig
molecule in the TrkB binding molecule are connected to each other
to form a TrkB binding molecule. The scFv's may be fused or
otherwise covalently attached directly to the Ig molecule or they
may be linked to the Ig molecule via a linker, preferably a peptide
linker e.g. having a length of about 4 to 20 amino acids and more
preferably a flexible peptide linker.
[0304] Preferably the scFv are fused to the C-terminus of the heavy
chain of the Ig molecule. Preferably, the Ig molecule is an IgG,
F(ab), or F(ab')2. Hence, in a preferred embodiment the TrkB
binding molecule comprises or consists of two scFv's and an IgG,
F(ab), or F(ab')2 wherein each scFv binds specifically to TrkB. In
a related preferred embodiment the TrkB binding molecule is
bispecific and tetravalent and comprises or consists of two scFv's
and an IgG or F(ab')2 wherein each scFv binds specifically to TrkB.
In a preferred embodiment the Ig molecule and more preferably the
IgG, F(ab), or F(ab')2 binds specifically to VEGF.
[0305] Methods of linking scFv molecules to the C-terminus of the
heavy chain of the Ig molecule e.g. such as an IgG molecule or
linking the variable domains within scFv molecules are well known
in the art. Typically, a small linker sequence of glycine and
serine (termed a GS mini-linker) amino acids is used. The number of
amino acids in the linker can vary, from 4 (GGGS) (SEQ ID NO: 227),
6 (GGSGGS) (SEQ ID NO: 217), 10 (GGGGSGGGGS) (SEQ ID NO: 218), 15
(GGGGSGGGGSGGGGS) (SEQ ID NO: 219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ
ID NO: 220) or more. In practice, normally the linker is formed by
combining the nucleic acid molecule encoding the Ig of interest
with the nucleic acid encoding the desired scFv (which in the
present case would include the nucleic acid encoding the variable
domain of the heavy and light chain, either in VL-VH or VH-VL
orientation for the TrkB binding site) interspaced by the nucleic
acid molecule encoding the linker sequence (e.g. a GS mini linker
of any one of 5, 10, 15, or 20 amino acids, preferably a linker of
SEQ ID NO: 218). Then as explained beforehand this complete HC-scFv
encoding nucleic acid molecule is placed within an expression
vector and introduced to appropriate host cells such that the
complete Ig heavy chain-scFv single polypeptide is formed.
[0306] Preferably the GS mini-linker between the scFv molecule and
the C-terminus of the heavy chain of the Ig molecule is 10L1 (SEQ
ID NO: 218).
[0307] In one aspect, a TrkB binding molecule is more potent in
inducing activation of TrkB downstream signaling pathways than the
natural TrkB ligand, BDNF. In a further aspect, a TrkB binding
molecule regulates gene expression through TrkB-mediated signaling
pathways in a comparable pattern to that of BDNF.
[0308] In a further aspect, a TrkB binding molecule is specific for
TrkB phosphorylation and/or activation and does not unspecifically
phosphorylate/activate TrkA or TrkC.
[0309] In one embodiment, the TrkB binding molecule comprises or
consists of: [0310] (i) two heavy chains, each comprising from N to
C terminus: [0311] (optionally) a heavy chain variable domain
(e.g., murine, humanized or human VH domain) [0312] constant
domains, preferably of an IgG (e.g. human IgG1 or IgG4) [0313]
(optionally) a peptide linker (e.g.a GS mini linker) and [0314] an
scFv specific for TrkB (e.g. an scFv comprising from N to C
terminus a VH domain (e.g. murine, humanized or human VH domain) a
linker and a VL domain (e.g. murine, humanized or human VL domain),
or vice versa a VL domain a linker and a VH domain); and [0315]
(ii) two light chains, each comprising from N to C-terminus: [0316]
(optionally) a light chain variable domain (e.g. murine, humanized
or human VL domain), [0317] a light chain constant domain,
preferably of an IgG (e.g., a human kappa chain).
[0318] In one embodiment, the TrkB binding molecule comprises or
consists of: [0319] (i) two heavy chains, each comprising from N to
C terminus: [0320] an scFv specific for TrkB (e.g. an scFv
comprising from N to C terminus a VH domain (e.g. murine, humanized
or human VH domain) a linker and a VL domain (e.g. murine,
humanized or human VL domain), or vice versa a VL domain a linker
and a VH domain); [0321] (optionally) a peptide linker (e.g.a GS
mini linker) and [0322] constant domains, preferably of an IgG
(e.g. human IgG1 or IgG4), more preferably a hinge-CH2-CH3.
[0323] In one embodiment, the TrkB binding molecule comprises or
consists of: [0324] (i) two heavy chains, each comprising from N to
C terminus: [0325] constant domains, preferably of an IgG (e.g.
human IgG1 or IgG4), more preferably a hinge-CH2-CH3, [0326]
(optionally) a peptide linker (e.g.a GS mini linker) and [0327] an
scFv specific for TrkB (e.g. an scFv comprising from N to C
terminus a VH domain (e.g. murine, humanized or human VH domain) a
linker and a VL domain (e.g. murine, humanized or human VL domain),
or vice versa a VL domain a linker and a VH domain);
[0328] In a further related aspect, the invention provides for a
method to improve the efficacy of an agonistic TrkB binder. The
inventors have surprisingly found that agonistic TrkB binders and
in particular partial agonistic TrkB binders, such as IgG could be
improved in their efficacy when applying the inventive method.
Related to this aspect the invention provides for a method to
produce or generate a TrkB binding molecule having improved
efficacy compared to the agonistic TrkB binder it is based
upon.
[0329] The method comprises generating a first scFv using the
variable light and heavy chain domains of an agonistic TrkB binder.
Subsequently generating a second scFv, which is either identical or
different to the first scFv and linking or attaching both scFv in
one molecule to yield a bivalent TrkB binding molecule comprising
both scFv's. In its broadest sense the method can be used to
improve the efficacy of any agonistic TrkB binder and to produce or
generate a TrkB binding molecule having improved efficacy compared
to the agonistic TrkB binder it is based upon. It will be
understood that the following teachings how to improve the efficacy
of agonistic TrkB binders apply mutatis mutandis to the method to
produce or generate a TrkB binding molecule having improved
efficacy compared to the agonistic TrkB binder it is based
upon.
[0330] "Agonistic TrkB binder(s)" as used herein refers to binding
molecules characterized by comprising a light chain variable domain
(VL) and a heavy chain variable domain (VH) that form together the
antigen binding part of the binding molecule and specifically bind
to the TrkB receptor and act as agonists. Many different formats
containing a light chain variable domain (VL) and a heavy chain
variable domain (VH) are known to the skilled artisan and a
non-exhaustive list includes, monoclonal antibody, human monoclonal
antibody, humanized monoclonal antibody, chimeric antibody,
fragment of an antibody, such as Fv, Fab, Fab', or F(ab')2
fragment, single chain antibody, such as a single chain variable
fragment (scFv), Small Modular Immunopharmaceutical (SMIP), domain
antibody, nanobody, or diabody.
[0331] In particular, the method can be used to improve the
efficacy of agonistic TrkB binders that were identified and
characterized e.g. via screenings and appropriate assays. The
method is especially useful to improve the efficacy of agonistic
TrkB binding antibodies, such as IgG. It will be understood that
the agonistic TrkB binder may also be a scFv.
[0332] In the art numerous agonistic TrkB binders are described and
the skilled artisan will be able to choose freely among those to
improve their efficacy according to the method as described
herein.
[0333] Hence, in one aspect a TrkB binding molecule generated
according to the method of the invention will be more efficacious,
i.e. will show a higher efficacy to activate TrkB compared to the
agonistic TrkB binder that served as the basis to generate the TrkB
binding molecule.
[0334] The efficacy as used herein describes the maximum response
that can be achieved with a drug. The effect of the drug is plotted
against the concentraion in a graph, to give the
concentration-response curve. The increasing concentrations used
are displayed by the X axis and the half maximal and maximal
responses are displayed by the Y axis. The highest point on the
curve shows the maximum response (efficacy) and is referred to as
the Emax. Efficacy (Emax) is the maximum effect which can be
expected from this drug (i.e. when this magnitude of effect is
reached, increasing the dose will not produce a greater magnitude
of effect). For the purpose of the invention it is not the absolute
increase in efficacy that is important but the relative increase
that is achieved by generating the TrkB binding molecule from the
VL and VH of the agonistic TrkB binder.
[0335] In one embodiment the invention provides for a method for
improving the efficacy of an agonistic TrkB binder, wherein the
agonistic TrkB binder contains a light chain variable domain (VL)
and a heavy chain variable domain (VH), comprising [0336] (i)
generating a first single chain variable fragment (scFv) with the
VL and the VH of the agonistic TrkB binder, [0337] (ii) generating
a second scFv with the same or substantially the same VL and the VH
of the agonistic TrkB binder of step (i) or with the VL and the VH
of a different agonistic TrkB binder, [0338] (iii) including the
first and the second scFv into a TrkB binding molecule, wherein the
TrkB binding molecule comprises at least the two agonistic TrkB
binding scFv's from step (i) and (ii), wherein the TrkB binding
molecule has a higher efficacy compared to the efficacy of the
agonistic TrkB binder, and wherein the efficacy is the maximum
response as determined by incubating CHO cells stably expressing a
TrkB receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
[0339] In a related embodiment the agonistic TrkB binder is a
partial agonist.
[0340] In a related embodiment the efficacy of the TrkB binding
molecule compared to the efficacy of the agonistic TrkB binder is
higher by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. For the
purpose of comparing the efficacy both the agonistic TrkB binder as
well as the TrkB binding molecule will be tested by
measuring/determing the efficacy, which is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells. Further details are described
in the Examples.
[0341] In a related embodiment the TrkB binding molecule is about
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%
as efficacious as compared to BDNF. Here as well, for the purpose
of comparing the efficacy both BDNF as well as the TrkB binding
molecule will be tested by measuring/determing the efficacy, which
is the maximum response as determined by incubating CHO cells
stably expressing a TrkB receptor with BDNF or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells. Further details are described
in the Examples.
[0342] In a related embodiment the two scFv's are identical. In
this embodiment both scFv will be based on the same VL and VH of
the same agonistic TrkB binder. In another embodiment the two
scFv's are substantially the same, i.e. both scFv's have the same
CDR regions but may have some variability in the framework region.
In a related embodiment the two scFv's have the same CDR regions
and the remaining framework regions are at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% identical to each other. In another embodiment
both scFv's are different from each other, i.e. are based on two
different agonistic TrkB binders. In said embodiment the efficacy
of the TrkB binding molecule will be both improved compared to both
agonistic TrkB binders it is based upon.
[0343] In another embodiment the two agonistic TrkB binding scFv's
bind to the same epitope or bind to a different epitope.
[0344] In the broadest sense the TrkB binding molecule is formed by
the first and the second scFv, i.e. both are included to form the
TrkB binding. Within the TrkB binding molecule both scFv's may be
fused or otherwise covalently attached directly to each other to
form the TrkB binding molecule (scFv-scFv). Alternatively, the
scFv's may also be linked to each other via a linker
(scFv-linker-scFv). In a further alternative, the scFv's may be
linked to each other via a hinge region (scFv-hinge-scFv). In yet a
further alternative, the scFv's are not directly linked to each
other but are part of an Ig molecule, i.e. both scFv are attached
to the Ig molecule.
[0345] Hence, in a related embodiment the TrkB binding molecule
further comprises an Ig molecule. The Ig molecule may be a
monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, a fragment of an
antibody, such as a Fc, Fv, Fab, Fab', or F(ab')2 fragment, a
single chain antibody, such as a single chain variable fragment
(scFv), a Small Modular Immunopharmaceutical (SMIP), a domain
antibody, a nanobody, or a diabody.
[0346] In a related embodiment each scFv is fused to the C-terminus
of the heavy chain of the Ig molecule. Alternatively, each scFv is
fused to the N-terminus of the heavy chain of the Ig molecule or
replaces an antigen binding domain at the N-terminus.
[0347] In a preferred embodiment the Ig molecule is an IgG, F(ab),
or F(ab')2. In a further preferred embodiment the Ig molecule
comprises or consists of an Fc region. In a more preferred
embodiment the TrkB binding molecule is in the format of an
Fc-scFv, scFv-Fc, (scFv').sub.2 or scFv-CH.sub.3.
[0348] In a related embodiment each scFv is fused to the Ig
molecule by a peptide linker, preferably a peptide linker having a
length of about 4 to 20 amino acids.
[0349] In a further embodiment the scFv's may be connected to each
other via a hinge region or a linker only. Here as well, it will be
understood that the two scFv's in the TrkB binding molecule are
connected to each other to form the TrkB binding molecule. The
scFv's may be fused or otherwise covalently attached directly to
each other or they may be linked via a linker, preferably a peptide
linker e.g. having a length of about 4 to 20 amino acids and more
preferably a flexible peptide linker.
[0350] In a preferred embodiment the TrkB binding molecule
comprises or consists of two scFv's that are connected via a
linker. Methods of linking scFv molecules are well known in the
art. Typically, a small linker sequence of glycine and serine
(termed a GS mini-linker) amino acids is used. The number of amino
acids in the linker can vary, from 4 (GGGS) (SEQ ID NO: 227), 6
(GGSGGS) (SEQ ID NO: 217), 10 (GGGGSGGGGS) (SEQ ID NO: 218), 15
(GGGGSGGGGSGGGGS) (SEQ ID NO: 219), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ
ID NO: 220) or more.
[0351] In another preferred embodiment the TrkB binding molecule
comprises or consists of two scFv's that are connected via a hinge
region only. Such molecule can be generated for example by
subjecting a scFv-Fc molecule to pepsin treatment. Other enzymes
that may be used are known to the skilled artisan, such as
Fabricator, which is a cysteine protease that cleaves IgGs and
Fc-fusions at one specific single site just below the hinge region.
It will be understood that other methods, substances and/or enzymes
can be used equally to generate two scFv's that are connected via a
hinge region only. Such methods, substances and/or enzymes may
result in hinge regions with different lengths depending on the
specific cleavage site. In another embodiment the TrkB binding
molecule consists of two scFv's and a hinge region connecting the
two scFv's.
[0352] In an embodiment relating to all other embodiments the TrkB
binding molecule is a TrkB agonist. In a further preferred
embodiment the agonistic TrkB binder is bivalent and the resulting
TrkB binding molecule is either bivalent or tetravalent. In a
related embodiment the agonistic TrkB binder is a bivalent partial
agonist.
[0353] In a preferred embodiment, the invention provides for a
method for improving the efficacy of a bivalent partial agonistic
TrkB binder, wherein the bivalent partial agonistic TrkB binder
contains a light chain variable domain (VL) and a heavy chain
variable domain (VH), [0354] (i) generating a first single chain
variable fragment (scFv) with the VL and the VH of the agonistic
TrkB binder, [0355] (ii) generating a second scFv with the same or
substantially the same VL and the VH of the agonistic TrkB binder
of step (i), [0356] (iii) including the first and the second scFv
into a TrkB binding molecule, wherein the TrkB binding molecule
comprises at least the two agonistic TrkB binding scFv's from step
(i) and (ii), wherein the TrkB binding molecule has a higher
efficacy compared to the efficacy of the bivalent partial agonistic
TrkB binder, and wherein the efficacy is the maximum response as
determined by incubating CHO cells stably expressing a TrkB
receptor with the agonistic TrkB binder or the TrkB binding
molecule and measuring the TrkB phosphorylation at Y706/707 in the
cell lysate of the treated CHO cells.
[0357] In a related embodiment the bivalent partial agonistic TrkB
binder is an IgG. In a preferred embodiment the TrkB binding
molecule is in the format of an an Fc-scFv, scFv-Fc, (scFv')2 or
scFv-CH.sub.3. In a further preferred embodiment the TrkB binding
molecule is bispecific and tetravalent.
[0358] The general observation was made that compared to an IgG
both the scFv-Fc and the Fc-scFv format were more efficacious than
the IgG they were based upon, i.e. efficacy: IgG
scFv-Fc.about.Fc-scFv. With respect to potency the IgG showed a
similar potency as the scFv-Fc whereas the Fc-scFv was slightly
less potent, i.e. potency: IgG.about.scFv-Fc Fc-scFv.
[0359] In one aspect the TrkB binding molecule is as potent as the
IgG molecule it is based upon. In another aspect the TrkB binding
molecule is as potent and more efficacious than the IgG molecule it
is based upon. In another aspect the TrkB binding molecule is more
efficacious than the IgG molecule it is based upon. In another
aspect the TrkB binding molecule is more efficacious and slightly
less potent than the IgG molecule it is based upon.
[0360] The invention also relates to an isolated nucleic acid
molecule encoding (i) the heavy chain or heavy chain variable
domain, and/or (ii) the light chain or light chain variable domain
of the TrkB binding molecule according any of the aforementioned
embodiments.
[0361] The invention also relates to a viral vector comprising the
isolated nucleic acid molecule of the TrkB binding molecules. The
invention further relates to an expression vector comprising a
nucleic acid molecule of the TrkB binding molecules. The invention
relates further to a host cell transfected with an expression
vector comprising a nucleic acid molecule of the TrkB binding
molecules.
[0362] In a further aspect the invention provides for a method of
manufacturing a TrkB binding molecule according to any of the
aforementioned embodiments comprising [0363] (a) cultivating the
host cell under conditions allowing expression of the molecule;
and, [0364] (b) recovering the molecule; and optionally [0365] (c)
further purifying and/or modifying and/or formulating the
molecule.
[0366] In a further aspect, the invention relates to the TrkB
binding molecule according to any of the aforementioned embodiments
for use in medicine, wherein the use is the treatment of eye or
retinal or neurodegenerative diseases. In a related embodiment, the
TrkB binding molecule is used for the treatment and/or prevention
of macular degeneration, age-related macular degeneration, wet
age-related macular degeneration (wAMD), retinal vein occlusion
(RVO), diabetic retinopathy, diabetic macular edema, retinitis
pigmentosa, inherited retinal dystrophy, inherited macular
dystrophy, myopic degeneration, geographic atrophy, retinal artery
occlusions, endophthalmitis, uveitis, cystoid macular edema,
choroidal neovascular membrane secondary to any retinal diseases,
optic neuropathies, glaucoma, retinal detachment, toxic
retinopathy, radiation retinopathy, and traumatic retinopathy,
prodromal and mild-to-moderate alzheimer's diseases, delaying
disease progression of patients with Alzheimer's disease,
Huntington's disease, Parkinson's disease, major depressive
disorder, schizophrenia, cognitive impairment associated with
schizophrenia, prevention of first-episode psychosis in individuals
with attenuated psychosis syndrome, prevention of relapse in
patients with schizophrenia, treatment-resistant depression,
hyperphagia, obesity or metabolic syndrome.
[0367] In another embodiment the TrkB binding molecules may be
useful for treatment of hearing loss, in particular for cis platin
induced hearing loss as well as noise and age-related hearing
loss.
[0368] In a further aspect, the invention relates to a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and the TrkB binding molecule according to any of the
aforementioned embodiment.
[0369] Items:
[0370] A first item relates to a binding molecule comprising at
least one antigen binding site that binds specifically to Vascular
Endothelial Growth Factor (VEGF) and at least one antigen binding
site that binds specifically to Tropomyosin receptor kinase B
(TrkB). Related to this first item, the binding molecule is
bispecific and tetravalent. Further relating to the binding
molecule according to the first item the at least one antigen
binding site that binds specifically to VEGF is an immunoglobulin
(Ig) molecule. Further relating to the binding molecule according
to the first item, the at least one antigen binding site that binds
specifically to TrkB is fused to the C- terminus of the heavy chain
of the Ig molecule. Further relating to the binding molecule
according to the first item, the at least one antigen binding site
that binds specifically to TrkB comprises one or more scFv(s).
Further relating to the binding molecule according to the first
item the at least one antigen binding site that binds specifically
to VEGF is an immunoglobulin (Ig) molecule and the at least one
antigen binding site that binds specifically to TrkB comprises one
or more scFv(s). Further relating to this, the one or more scFv(s)
have a VL-VH orientation from N-to C-terminus. Yet further relating
to this, the one or more scFv(s) is fused to the C- terminus of the
heavy chain of the Ig molecule. Further relating to the binding
molecule according to the first item, the Ig molecule is a
monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, an (scFv).sub.2 or a
fragment of an antibody such as a F(ab')2 fragment. Further
relating to the binding molecule according to the first item, the
Ig molecule is an IgG or F(ab')2. Further relating to the binding
molecule according to the first item, the one or more scFv(s) is
fused to the Ig molecule by a peptide linker, preferably a peptide
linker having a length of about 4 to 20 amino acids.
[0371] Further relating to the binding molecule according to the
first item, the antigen binding site that binds specifically to
TrkB is selected from: [0372] a) an antigen binding site comprising
light chain CDRs comprising the amino acid sequences of SEQ ID NO:
201 (CDR1), SEQ ID NO: 202 (CDR2) and SEQ ID NO: 203 (CDR3), and
[0373] an antigen binding site comprising heavy chain CDRs
comprising the amino acid sequences of SEQ ID NO: 204 (CDR1), SEQ
ID NO: 205 (CDR2) and SEQ ID NO: 206 (CDR3); or [0374] an antigen
binding site comprising heavy chain CDRs comprising the amino acid
sequences of SEQ ID NO: 207 (CDR1), SEQ ID NO: 208 (CDR2) and SEQ
ID NO: 209 (CDR3); or [0375] an antigen binding site comprising
heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:
210 (CDR1), SEQ ID NO: 211 (CDR2) and SEQ ID NO: 212 (CDR3).
[0376] Further relating to the binding molecule according to the
first item, the antigen binding site that binds specifically to
TrkB is selected from: [0377] a) an antigen binding site comprising
a light chain variable domain comprising an amino acid sequence at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 213 or 215, and a heavy chain
variable domain comprising an amino acid sequence at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 214 or 216.
[0378] Further relating to the binding molecule according to the
first item, the binding molecule comprises the amino acid sequence
of SEQ ID NO: 222, or the amino acid sequence of SEQ ID NO: 223, or
the amino acid sequence of SEQ ID NO: 224, or the amino acid
sequence of SEQ ID NO: 225.
[0379] Further relating to the binding molecule according to the
first item, the antigen binding site that binds specifically to
VEGF is selected from the group consisting of antigen binding sites
a) to c): [0380] a) an antigen binding site comprising light chain
CDRs comprising the amino acid sequences of SEQ ID NO: 145 (CDR1),
SEQ ID NO: 146 (CDR2) and SEQ ID NO: 147 (CDR3), and [0381] an
antigen binding site comprising heavy chain CDRs comprising the
amino acid sequences of SEQ ID NO: 148 (CDR1), SEQ ID NO: 149
(CDR2) and SEQ ID NO: 150 (CDR3); or [0382] an antigen binding site
comprising heavy chain CDRs comprising the amino acid sequences of
SEQ ID NO: 151 (CDR1), SEQ ID NO: 152 (CDR2) and SEQ ID NO: 153
(CDR3); or [0383] an antigen binding site comprising heavy chain
CDRs comprising the amino acid sequences of SEQ ID NO: 154 (CDR1),
SEQ ID NO: 155 (CDR2) and SEQ ID NO: 156 (CDR3); [0384] b) an
antigen binding site comprising light chain CDRs comprising the
amino acid sequences of SEQ ID NO: 157 (CDR1), SEQ ID NO: 158
(CDR2) and SEQ ID NO: 159 (CDR3), and [0385] an antigen binding
site comprising heavy chain CDRs comprising the amino acid
sequences of SEQ ID NO: 160 (CDR1), SEQ ID NO: 161 (CDR2) and SEQ
ID NO: 162 (CDR3); or [0386] an antigen binding site comprising
heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:
163 (CDR1), SEQ ID NO: 164 (CDR2) and SEQ ID NO: 165 (CDR3); or
[0387] an antigen binding site comprising heavy chain CDRs
comprising the amino acid sequences of SEQ ID NO: 166 (CDR1), SEQ
ID NO: 167 (CDR2) and SEQ ID NO: 168 (CDR3); [0388] c) an antigen
binding site comprising light chain CDRs comprising the amino acid
sequences of SEQ ID NO: 169 (CDR1), SEQ ID NO: 170 (CDR2) and SEQ
ID NO: 171 (CDR3), and [0389] an antigen binding site comprising
heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:
172 (CDR1), SEQ ID NO: 173 (CDR2) and SEQ ID NO: 174 (CDR3); or
[0390] an antigen binding site comprising heavy chain CDRs
comprising the amino acid sequences of SEQ ID NO: 175 (CDR1), SEQ
ID NO: 176 (CDR2) and SEQ ID NO: 177 (CDR3); or [0391] an antigen
binding site comprising heavy chain CDRs comprising the amino acid
sequences of SEQ ID NO: 178 (CDR1), SEQ ID NO: 179 (CDR2) and SEQ
ID NO: 180 (CDR3).
[0392] Further relating to the binding molecule according to the
first item, the antigen binding site that binds specifically to
VEGF is selected from the group consisting of antigen binding sites
a) to d): [0393] a) an antigen binding site comprising a light
chain variable domain comprising an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 181 and a heavy chain variable
domain comprising an amino acid sequence at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or at least 99% identical to the amino acid sequence
of SEQ ID NO: 182; [0394] b) an antigen binding site comprising a
light chain variable domain comprising an amino acid sequence at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 183 and a heavy chain variable
domain comprising an amino acid sequence at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or at least 99% identical to the amino acid sequence
of SEQ ID NO: 184; [0395] c) an antigen binding site comprising a
light chain variable domain comprising an amino acid sequence at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 189 and a heavy chain variable
domain comprising an amino acid sequence at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or at least 99% identical to the amino acid sequence
of SEQ ID NO: 190, 191, 192 or 194; [0396] d) an antigen binding
site comprising a light chain variable domain comprising an amino
acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 193 and a heavy
chain variable domain comprising an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 190, 191, 192 or 194.
[0397] Further relating to the binding molecule according to the
first item, the antigen binding site that binds specifically to
VEGF is selected from the group consisting of antigen binding sites
a) to d): [0398] a) an antigen binding site comprising a light
chain comprising an amino acid sequence at least 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or at least 99% identical to the amino acid sequence
of SEQ ID NO: 185 and a heavy chain comprising an amino acid
sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 186; [0399] b)
an antigen binding site comprising a light chain comprising an
amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
least 99% identical to the amino acid sequence of SEQ ID NO: 187
and a heavy chain comprising an amino acid sequence at least 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or at least 99% identical to the amino
acid sequence of SEQ ID NO: 188; [0400] c) an antigen binding site
comprising a light chain comprising an amino acid sequence at least
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to the
amino acid sequence of SEQ ID NO: 195 and a heavy chain comprising
an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at
least 99% identical to the amino acid sequence of SEQ ID NO: 196,
197, 198 or 200; [0401] d) an antigen binding site comprising a
light chain comprising an amino acid sequence at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or at least 99% identical to the amino acid
sequence of SEQ ID NO: 199 and a heavy chain comprising an amino
acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%
identical to the amino acid sequence of SEQ ID NO: 196, 197, 198 or
200.
[0402] A second item relates to a binding molecule comprising a
light chain comprising an amino acid sequence at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or at least 99% identical to the amino acid
sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91,
93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,
121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 or 143, and a
heavy chain comprising an amino acid sequence at least 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or at least 99% identical to the amino acid
sequence of SEQ ID NO: 2, 4, 6 ,8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120,
122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, or 144.
[0403] A third item relates to a binding molecule comprising: (i) a
light chain comprising the amino acid sequence of SEQ ID NO: 41 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 42,
or (ii) a light chain comprising the amino acid sequence of SEQ ID
NO: 43 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 44, or (iii) a light chain comprising the amino acid
sequence of SEQ ID NO: 45 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 46, or (iv) a light chain comprising
the amino acid sequence of SEQ ID NO: 47 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 48, or (v) a light
chain comprising the amino acid sequence of SEQ ID NO: 49 and a
heavy chain comprising the amino acid sequence of SEQ ID NO: 50, or
(vi) a light chain comprising the amino acid sequence of SEQ ID NO:
51 and a heavy chain comprising the amino acid sequence of SEQ ID
NO: 52, or (vii) a light chain comprising the amino acid sequence
of SEQ ID NO: 53 and a heavy chain comprising the amino acid
sequence of SEQ ID NO: 54, or (viii) a light chain comprising the
amino acid sequence of SEQ ID NO: 55 and a heavy chain comprising
the amino acid sequence of SEQ ID NO: 56, or (ix) a light chain
comprising the amino acid sequence of SEQ ID NO: 57 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 58, or (x) a
light chain comprising the amino acid sequence of SEQ ID NO: 59 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 60,
or (xi) a light chain comprising the amino acid sequence of SEQ ID
NO: 61 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 62, or (xii) a light chain comprising the amino acid
sequence of SEQ ID NO: 63 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 64, or (xiii) a light chain comprising
the amino acid sequence of SEQ ID NO: 65 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 66, or (xiv) a
light chain comprising the amino acid sequence of SEQ ID NO: 67 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 68,
or (xv) a light chain comprising the amino acid sequence of SEQ ID
NO: 69 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 70, or (xvi) a light chain comprising the amino acid
sequence of SEQ ID NO: 71 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 72, or (xvii) a light chain comprising
the amino acid sequence of SEQ ID NO: 73 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 74, or (xviii) a
light chain comprising the amino acid sequence of SEQ ID NO: 75 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 76,
or (xix) a light chain comprising the amino acid sequence of SEQ ID
NO: 77 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 78, or (xx) a light chain comprising the amino acid sequence
of SEQ ID NO: 79 and a heavy chain comprising the amino acid
sequence of SEQ ID NO: 80, or (xxi) a light chain comprising the
amino acid sequence of SEQ ID NO: 81 and a heavy chain comprising
the amino acid sequence of SEQ ID NO: 82, or (xxii) a light chain
comprising the amino acid sequence of SEQ ID NO: 83 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 84, or
(xxiii) a light chain comprising the amino acid sequence of SEQ ID
NO: 85 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 86, or (xxiv) a light chain comprising the amino acid
sequence of SEQ ID NO: 87 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 88, or (xxv) a light chain comprising
the amino acid sequence of SEQ ID NO: 89 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 90, or (xxvi) a
light chain comprising the amino acid sequence of SEQ ID NO: 91 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 92,
or (xxvii) a light chain comprising the amino acid sequence of SEQ
ID NO: 93 and a heavy chain comprising the amino acid sequence of
SEQ ID NO: 94, or (xxviii) a light chain comprising the amino acid
sequence of SEQ ID NO: 95 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 96, or (xxix) a light chain comprising
the amino acid sequence of SEQ ID NO: 97 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 98, or (xxx) a
light chain comprising the amino acid sequence of SEQ ID NO: 99 and
a heavy chain comprising the amino acid sequence of SEQ ID NO: 100,
or (xxxi) a light chain comprising the amino acid sequence of SEQ
ID NO: 101 and a heavy chain comprising the amino acid sequence of
SEQ ID NO: 102, or (xxxii) a light chain comprising the amino acid
sequence of SEQ ID NO: 103 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 104, or (xxxiii) a light chain
comprising the amino acid sequence of SEQ ID NO: 105 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 106, or
(xxxiv) a light chain comprising the amino acid sequence of SEQ ID
NO: 107 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 108, or (xxxv) a light chain comprising the amino acid
sequence of SEQ ID NO: 109 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 110, or (xxxvi) a light chain
comprising the amino acid sequence of SEQ ID NO: 111 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 112, or
(xxxvii) a light chain comprising the amino acid sequence of SEQ ID
NO: 113 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 114, or (xxxviii) a light chain comprising the amino acid
sequence of SEQ ID NO: 115 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 116, or (xxxix) a light chain
comprising the amino acid sequence of SEQ ID NO: 117 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 118, or (xl)
a light chain comprising the amino acid sequence of SEQ ID NO: 119
and a heavy chain comprising the amino acid sequence of SEQ ID NO:
120, or (xli) a light chain comprising the amino acid sequence of
SEQ ID NO: 121 and a heavy chain comprising the amino acid sequence
of SEQ ID NO: 122, or (xlii) a light chain comprising the amino
acid sequence of SEQ ID NO: 123 and a heavy chain comprising the
amino acid sequence of SEQ ID NO: 124, or (xliii) a light chain
comprising the amino acid sequence of SEQ ID NO: 125 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 126, or
(xliv) a light chain comprising the amino acid sequence of SEQ ID
NO: 127 and a heavy chain comprising the amino acid sequence of SEQ
ID NO: 128, or (xlv) a light chain comprising the amino acid
sequence of SEQ ID NO: 129 and a heavy chain comprising the amino
acid sequence of SEQ ID NO: 130, or (xlvi) a light chain comprising
the amino acid sequence of SEQ ID NO: 131 and a heavy chain
comprising the amino acid sequence of SEQ ID NO: 132, or (xlvii) a
light chain comprising the amino acid sequence of SEQ ID NO: 133
and a heavy chain comprising the amino acid sequence of SEQ ID NO:
134, or (xlviii) a light chain comprising the amino acid sequence
of SEQ ID NO: 135 and a heavy chain comprising the amino acid
sequence of SEQ ID NO: 136, or (xlix) a light chain comprising the
amino acid sequence of SEQ ID NO: 137 and a heavy chain comprising
the amino acid sequence of SEQ ID NO: 138, or (I) a light chain
comprising the amino acid sequence of SEQ ID NO: 139 and a heavy
chain comprising the amino acid sequence of SEQ ID NO: 140, or (Ii)
a light chain comprising the amino acid sequence of SEQ ID NO: 141
and a heavy chain comprising the amino acid sequence of SEQ ID NO:
142, or (lii) a light chain comprising the amino acid sequence of
SEQ ID NO: 143 and a heavy chain comprising the amino acid sequence
of SEQ ID NO: 144.
[0404] A fourth item relates to a TrkB binding molecule comprising
or consisting of two scFv's, wherein each scFv binds specifically
to TrkB.
[0405] Further relating to the fourth item the TrkB binding
molecule further comprises an Ig molecule.
[0406] Further relating to the fourth item, the Ig molecule is a
monoclonal antibody, a human monoclonal antibody, a humanized
monoclonal antibody, a chimeric antibody, a fragment of an
antibody, such as a Fv, Fab, Fab', or F(ab')2 fragment, a single
chain antibody, such as a single chain variable fragment (scFv), a
Small Modular Immunopharmaceutical (SMIP), a domain antibody, a
nanobody, or a diabody.
[0407] Further relating to the fourth item, each scFv is fused to
the C-terminus of the heavy chain of the Ig molecule. Further
related to the fourth item, the Ig molecule is an IgG, F(ab),
F(ab')2. Further related to the fourth item, the Ig molecule
comprises or consists of an Fc region. Further related to the
fourth item, the TrkB binding molecule is bispecific and
tetravalent.
[0408] A fifth item relates to an scFv binding specifically to TrkB
comprising (i) a light chain variable domain comprising the amino
acid sequence of SEQ ID NO: 213 and a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO: 214 or (ii) a
light chain variable domain comprising the amino acid sequence of
SEQ ID NO: 215 and a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO: 216.
[0409] Further related to the fifth item, the scFv comprises the
amino acid sequence of SEQ ID NO: 222, or the amino acid sequence
of SEQ ID NO: 223, or the amino acid sequence of SEQ ID NO: 224, or
the amino acid sequence of SEQ ID NO: 225.
[0410] A sixth item relates to an antibody molecule binding
specifically to VEGF comprising: [0411] (i) a light chain variable
domain comprising the amino acid sequence of SEQ ID NO: 189, and a
heavy chain variable domain comprising the amino acid sequence of
SEQ ID NO: 191; or a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO: 192; or a heavy chain variable
domain comprising the amino acid sequence of SEQ ID NO: 194; [0412]
(ii) a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO: 190, and a light chain variable domain
comprising the amino acid sequence of SEQ ID NO: 192; or a light
chain variable domain comprising the amino acid sequence of SEQ ID
NO: 193.
[0413] Further related to the sixth item, the antibody molecule
comprises: a light chain comprising the amino acid sequence of SEQ
ID NO: 195 or 199, and a heavy chain comprising the amino acid
sequence of SEQ ID NO: 196, 197, 198 or 200.
[0414] A seventh item relates to an isolated nucleic acid molecule
encoding (i) the heavy chain or heavy chain variable domain, and/or
(ii) the light chain or light chain variable domain of a binding
molecule of any one of the aforementioned items.
[0415] An eigth item relates to an isolated nucleic acid molecule
encoding (i) the heavy chain or heavy chain variable domain, and/or
(ii) the light chain or light chain variable domain of a TrkB
binding molecule according of any one of the aforementioned
items.
[0416] A ninth item relates to an isolated nucleic acid molecule
encoding (i) the heavy chain variable domain, and/or (ii) the light
chain variable domain of an scFv according to any one of the
aforementioned items.
[0417] A tenth item relates to an isolated nucleic acid molecule
encoding (i) the heavy chain or heavy chain variable domain, and/or
(ii) the light chain or light chain variable domain of an antibody
molecule according to any one of the aforementioned items.
[0418] An eleventh item relates to a viral vector comprising the
isolated nucleic acid molecule according to seventh, eighth, ninth
or tenth item.
[0419] A twelfth item relates to an expression vector comprising a
nucleic acid molecule according to the eleventh item.
[0420] A thirteenth item relates to a host cell transfected with an
expression vector according to the twelfth item.
[0421] A fourteenth item relates to a method of manufacturing a
binding molecule, a TrkB binding molecule, a scFv, or an antibody
molecule according to any of the aforementioned items, comprising
[0422] (a) cultivating the host cell of the thirteenth aspect under
conditions allowing expression of the molecule; and, [0423] (b)
recovering the molecule; and optionally [0424] (c) further
purifying and/or modifying and/or formulating the molecule.
[0425] A fifteenth item relates to the binding molecule, a TrkB
binding molecule, the scFv, or the antibody molecule according to
any of the aforementioned items for use in medicine.
[0426] Related to the fiftteenth item, the binding molecule, the
TrkB binding molecule, the scFv, or the antibody molecule for the
use according to the fifteenth item, wherein the use is the
treatment of eye or retinal or neurodegenerative diseases.
[0427] Further relating to the fifteenth item, the binding
molecule, the TrkB binding molecule, the scFv, or the antibody
molecule for the use according to the fifteenth item, wherein the
use is for the treatment of neural/neuronal eye or retinal
diseases.
[0428] Further relating to the fifteenth item, the binding
molecule, the TrkB binding molecule, the scFv, or the antibody
molecule for the use according to the fifteenth aspect, wherein the
use is for the treatment of macular degeneration, age-related
macular degeneration, diabetic retinopathy, diabetic macular edema,
retinitis pigmentosa, inherited retinal dystrophy, inherited
macular dystrophy, myopic degeneration, geographic atrophy, retinal
artery occlusions, endophthalmitis, uveitis, cystoid macular edema,
choroidal neovascular membrane secondary to any retinal diseases,
optic neuropathies, glaucoma, retinal detachment, toxic
retinopathy, radiation retinopathy, and traumatic retinopathy,
prodromal and mild-to-moderate alzheimer's diseases, delaying
disease progression of patients with Alzheimer's disease,
Huntington's disease, Parkinson's disease, major depressive
disorder, schizophrenia, cognitive impairment associated with
schizophrenia, prevention of first-episode psychosis in individuals
with attenuated psychosis syndrome, prevention of relapse in
patients with schizophrenia, treatment-resistant depression,
hyperphagia, obesity or metabolic syndrome.
[0429] Further relating to the fifteenth item, the binding
molecule, the TrkB binding molecule, the scFv, or the antibody
molecule for the use according to the fifteenth item, wherein the
use is for the treatment of macular degeneration and in particular
wet age-related macular degeneration (wAMD). Further relating to
the fifteenth item, the binding molecule, the TrkB binding
molecule, the scFv, or the antibody molecule for the use according
to the fifteenth item, wherein the use is for the treatment of
retinal vein occlusion (RVO). Further relating to the fifteenth
item, the binding molecule, the TrkB binding molecule, the scFv, or
the antibody molecule for the use according to the fifteenth item,
wherein the use is for the treatment and/or prevention of
geographic atrophy.
[0430] A sixteenth item relates to a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and the binding
molecule, the TrkB binding molecule, the scFv, or the antibody
molecule according to any of the aforementioned items. A
seventeemth item relates to a method of treating or preventing an
eye or retinal or neurodegenerative disease comprising
administering to a patient in need thereof a therapeutically
effective amount of the binding molecule, the TrkB binding
molecule, the scFv, or the antibody molecule according to any of
the aforementioned items. An eightteenth item relates to the use of
the binding molecule, the TrkB binding molecule, the scFv or the
antibody molecule according to any of the aforementioned items for
preparing a pharmaceutical composition for treating or preventing
an eye or retinal or neurodegenerative diseases.
[0431] The invention is now described by way of the following
non-limiting examples.
EXAMPLES
Methods
[0432] Cultivation of CHO Cells Expressing Trk Receptors
[0433] CHO cells expressing the human TrkB receptor (ThermoFisher
Scientific, #K1491), and customised CHO cells expressing the human
TrkA or TrkC receptor were cultured in DMEM (Lonza, #BE12-604F)
supplemented with 10% fetal bovine serum, glutamax, non-essential
amino acids, 20 mM HEPES, 5 .mu.g/mL blasticidin and 200 .mu.g/mL
zeocin. Customized CHO cells expressing cyno, rabbit or rat TrkB
receptor were cultured in a 1:1 mixture of Hams F12 (Lonza
#BE12-615F) and DMEM (Lonza #BE12-604F) with 5% fetal bovine serum,
8 mM Glutamine, 0.5 mg/mL G418. Customised CHO cells expressing
mouse TrkB receptor were cultured DMEM (Lonza #BE12-604F)
supplemented with 10% fetal bovine serum, glutamax, 10 mM HEPES and
0.8 mg/mL G418.
[0434] Analysis of TrkA/B/C and ERK1/2 Phosphorylation in CHO Cells
Expressing Human, Cyno, Rabbit, Rat, or MouseTrk Receptors
[0435] Five thousand CHO cells expressing the respective Trk
receptor were seeded in each cavity of a 384 well clear tissue
culture plate (BD Falcon, # 353963) and incubated in a humidified
incubator at 37.degree. C. and 5% CO.sub.2. Twenty-four hours after
seeding, the supernatant of the cells was replaced with
room-temperature starvation medium (DMEM with 0.1% BSA (Sigma,
#A-3059) but without other supplements). After 15 minutes,
starvation medium with increasing concentrations of human BDNF
(R&D #248-BD or Bachem #H-5594), human NGF (Biovision
#4303R-20), human NT-3 (Sigma #N1905), agonistic antibodies, or
isotype controls was added in triplicate for 45 minutes at room
temperature to stimulate TrkB and ERK1/2 phosphorylation.
Starvation medium alone served as control.
[0436] In some experiments, BDNF or the agonistic antibodies were
pre-incubated for one hour without or with 2, 10, 50, or 200 ng/mL
human VEGF (R&D Systems #293-VE-050), prior to stimulation of
the cells. In these experiments, incubation with human VEGF alone
served as control.
[0437] To analyze whether or not an agonistic TrkB antibody limits
the BDNF-induced phosphorylation of TrkB and/or downstream ERK1/2
phosphorylation, CHO cells expressing the human TrkB receptor were
incubated with growing concentrations of the antibody without or
with a constant concentration of 0.3 nM, 1 nM or 3 nM BDNF.
[0438] After stimulation, cell supernatants were removed and cells
were lysed for 20 minutes on wet ice in lysis buffer (1x Triton
lysis buffer (Cell Signaling Technology #9803-S), supplemented with
complete mini protease inhibitor tablets (Roche #04693124001) and
phosphatase inhibitor cocktail 2 (Sigma #P5726) and 3 (Sigma
#P0044), and 1 mM PMSF (Sigma #93482)). The resulting lysate was
used for quantification of TrkA phosphorylation at Y680/681, TrkB
phosphorylation at Y706/707, or TrkC phosphorylation at Y709/710
using a commercially available assay (Perkin Elmer
#ALSU-PTRKAB-A10K), according to the manufacturer's instructions.
Quantification of ERK1/2 phosphorylation at T202/Y204 (ERK1) and
T185/Y187 (ERK2) was done similarly using another commercially
available assay (Perkin Elmer #TGRES10K or ALSU-PERK-A10K),
according to the manufacturer's instructions. Light emission of the
acceptor beads, reflecting the phosphorylation events, was recorded
at 570 nm on a Perking Elmer Envision.RTM. microplate reader. Date
were prepared for presentation with GraphPad Prism (version 8),
including the raw data (mean.+-.SEM) and a non-linear regression
(log(agonist) vs. response (three parameters)).
[0439] Functional Characterization of the TkrB Extracellular Domain
(TrkB-ECD)
[0440] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural ligand BDNF or
10 nM BDNF with growing concentrations of TrkB-ECD (R&D Systems
#1494-TB). TrkB activation was assessed by measuring TrkB
phosphorylation on Y706/707 as outlined above. Data represent
mean+/-SEM.
[0441] TrkB Receptor Internalization Assay
[0442] Twenty-four hours before stimulation, 25000 CHO cells
expressing human TrkB receptor were seeded in each cavity of a 96
well black clear bottom tissue culture plate (PerkinElmer,
#6055300) and incubated in a humidified incubator at 37.degree. C.
and 5% CO.sub.2. The supernatant of the CHO/hTrkB cells was
replaced with starvation medium (DMEM with 0.1% BSA (Sigma,
#A-3059) and 20 mM Hepes (Lonza, # BE17-737F)) and the cells were
incubate for 30 minutes at 37.degree. C. Cells were then stimulated
with increasing concentrations of BDNF (R&D #248-BD or Bachem
#H-5594) or agonistic TrkB antibodies alone, or a combination of 1
nM BDNF with increasing concentrations of the agonistic antibodies
in starvation medium for 50 minutes at 37.degree. C. Cells were
fixed for 20 minutes in 4% paraformaldehyde, washed, blocked in 5%
normal donkey serum in PBS for one hour, and incubated over night
at room temperature with 1 pg/mL goat anti-TrkB antibody (R&D
Systems, #AF-397), followed by extensive washing and incubation in
2 .mu.g/mL AlexaFluor.RTM. 647 donkey anti-goat antibody
(ThermoFisher Scientific, #A21447) and 1 .mu.g/mL Hoechst #H3570
for 2 hours at room temperature. After extensive washing, cells
were stained for one hour with 2 .mu.g/mL HCS-Cellmask.RTM. green
stain (ThermoFisher Scientific, #H32714) in PBS/0.05% Tween-20.
Cell surface receptors were imaged on a PerkinElmer Opera
Phenix.TM. High Content Screening System equipped with a 20.times.
water objective and analyzed with PerkinElmer Harmony High-Content
Imaging and Analysis Software. Data are either presented as
heatmap, with dark and light fields of the heatmap representing
high and low percentage of cells above fluorescence threshold,
respectively, or as diagram representing the percent of cells with
surface TrkB staining intensity above threshold. In the latter
case, data were prepared for presentation with GraphPad Prism
(version 8), including the raw data (mean.+-.SEM) and a non-linear
regression (log(agonist) vs.
[0443] response (three parameters))
[0444] Inhibition of VEGF-Induced VEGF Receptor 2 Phosphorylation
and Downstream Signalling
[0445] Human retinal microvascular endothelial cells (HRMEC; Cell
Systems #ACBR1181) were cultured on gelatine- (Millipore #ES-006B)
coated plates in endothelial cell basal medium (Promocell #C-22210)
with supplements (Promocell #C-39210) and 10 U/mL
penicillin/streptomycin, each. For analysis of VEGF-scavenging,
12000 cells were seeded in each cavity of a 96 well clear tissue
culture plate using normal growth medium and incubated in a
humidified incubator at 37.degree. C. and 5% CO.sub.2. Twenty-four
hours later, the medium was replaced with starvation medium
(Promocell #C-22210 supplemented with 0.1% BSA (Sigma, #A-3059) and
10 U/mL penicillin/streptomycin) and cells were starved for 20
hours in a humidified incubator at 37.degree. C. and 5% CO.sub.2.
Before stimulation of VEGFR2 signalling, 50 ng/mL human VEGF
(R&D Systems #293-VE-050) was pre-incubated without or with
antagonistic antibodies or EYLEA.RTM. (aflibercept) for 30-60
minutes at room temperature in starvation medium and then
pre-warmed to 37.degree. C. for 15 minutes. Cell stimulation was
accomplished for 5 minutes on a 37.degree. C. warming plate by
adding the pre-formed VEGF-antibody/EYLEA.RTM. (aflibercept)
complexes or VEGF alone to the cells. Starvation medium alone
served as control. Cells were lysed in lysis buffer (Perkin Elmer
#ALSU-PVGFR-A500) for 10 minutes at room temperature and then
incubated for additional 10 minutes on ice. According to the
manufacturer's instructions, commercially available assays were
used to quantify VEGFR2 phosphorylation at Y1175 (Perkin Elmer
#ALSU-PVGFR-A500 or #ALSU-PVGFR-A10K), VEGFR2 phosphorylation at
Y1214 (Perkin Elmer #ALSU-PVGFR-C500), VEGFR2 phosphorylation at
Y951 (Perkin Elmer #ALSU-PVGFR-B500), ERK1/2 phosphorylation at
T202/Y204 (ERK1) and T185/Y187 (ERK2) (Perkin Elmer #TGRES10K or
ALSU-PERK-A10K), Src phosphorylation at Y419 (Perkin Elmer
#ALSU-PSRC-A10K), p38-MAPK phosphorylation at Thr180/Tyr182 (Perkin
Elmer #ALSU-PP38-B500). Light emission of the acceptor beads,
reflecting above mentioned phosphorylation events, was recorded at
570 nm on a Perking Elmer EnVision.RTM. microplate reader and
prepared for presentation with GraphPad Prism (version 8),
including the raw data (mean.+-.SEM) and a non-linear regression
(log(agonist) vs. response (three parameters)).
[0446] To investigate the impact of TrkB-binding on VEGF-scavenging
by the bispecific and tetravalent Doppelmabs, inhibition of
VEGF-induced VEGFR2 phosphorylation was assessed in starved HRMEC
in the absence or presence of the TrkB-ECD. Prior to stimulation,
growing concentrations of the respective Doppelmab were incubated
with 100 nM TrkB-ECD (R&D Systems #1494-TB) in starvation
medium at room temperature for one hour, followed by incubation
with 50 ng/mL human VEGF for another hour. As controls, HRMEC were
incubated with (i) starvation medium alone, (ii) 50 ng/mL human
VEGF, (iii) pre-formed complexes (one hour at room temperature) of
50 ng/mL human VEGF with growing concentrations of TrkB-ECD, (iv)
pre-formed complexes (one hour at room temperature) of 50 ng/mL
human VEGF with growing concentrations of the respective Doppelmab,
and (v) growing concentrations of the TrkB-ECD alone. VEGF-A
scavenging was assessed by measuring VEGF receptor 2 (VEGFR2)
phosphorylation at Y1175 as outlined above.
[0447] Inhibition of VEGF-Induced HRMEC Proliferation
[0448] Three thousand HRMEC were seeded in each cavity of a clear
flat bottom 96 well plate using endothelial cell basal medium
(Promocell #C-22210) with supplements (Promocell #C-39210) and 10
U/mL penicillin/streptomycin, each, and incubated in a humidified
incubator at 37.degree. C. and 5% CO.sub.2. Sixteen hours later,
the growth medium was replaced by starvation medium (endothelial
cell basal medium (Promocell #C-22210) supplemented with 2% fetal
bovine serum) and cells were starved for eight hours in a
humidified incubator at 37.degree. C. and 5% CO.sub.2. Before
stimulation, human VEGF (R&D Systems #293-VE-050) was
pre-incubated without or with antagonistic antibodies or EYLEA.RTM.
(aflibercept) for 60 minutes at room temperature in starvation
medium. Cell stimulation was done by adding the pre-formed
VEGF-antibody/EYLEA.RTM. (aflibercept) complexes or VEGF alone to
the cells. Starvation medium alone served as control (basal
proliferation). Cell proliferation was assessed by automated, phase
contrast image-based quantification of the total HRMEC nuclear
areas (Essen Bioscience, IncuCyte S3), which was considered to be
proportional to the HRMEC numbers. Four images per well were
recorded with a 10.times. objective every four hours for a total
period of 96 hours. Data represent relative cell numbers (cell
number at time point t/cell number at t=0) as a function of time;
cell numbers at t=0 were set to one. For some experiments, the area
between each growth curve and the basal growth curve (proliferation
in starvation medium) was plotted against the decadic logarithm of
the respective compound concentration. Data were prepared for
presentation with GraphPad Prism (version 8), including the raw
data (mean.+-.SEM) and a non-linear regression (log(agonist) vs.
response (three parameters)) if applicable.
[0449] Inhibition of VEGF-Induced HRMEC Sprouting
[0450] Inhibition of VEGF-induced sprouting was assessed in a
HRMEC-based spheroid assay. HRMEC were resuspended in normal growth
medium containing 20% Methocel.TM. modified cellulose media (1.2%
methylcellulose and 10% fetal bovine serum in endothelial basal
medium) and 25 .mu.l drops containing 500 HRMEC were applied on
square petri dishes. The plates were turned upside down to
cultivate the cells in hanging drops, which allows the spontaneous
formation of spheroids. After 24 hours, the spheroids were
harvested and embedded in a Methocel.TM. modified cellulose
media--collagen mixture (80% Methocoel and 20% FCS 1:1 mixed with 3
mg/mL rat tail collagen I (Corning #354236) in M199 medium) in 48
well plates and incubated in a humidified incubator at 37.degree.
C. and 5% CO.sub.2 for 30 minutes to accomplish collagen
polymerization. Before stimulation, human VEGF (R&D Systems
#293-VE-050) was pre-incubated without or with antagonistic
antibodies or EYLEA.RTM. (aflibercept) for 60 minutes at room
temperature in basal endothelial growth medium supplemented with 2%
fetal bovine serum. Stimulation of sprouting was done by adding the
pre-formed VEGF-antibody/EYLEA.RTM. (aflibercept) complexes or VEGF
alone to the spheroids for 24 hours. Basal endothelial growth
medium supplemented with 2% fetal bovine serum without VEGF served
as control. After fixation in 4% paraformaldehyde, cells were
extensively washed and stained over night with 2 pg/mL
HCS-Cellmask.TM. green stain (ThermoFisher Scientific, #H32714) in
PBS containing 0.2% Triton X-100. Spheroids were analyzed on a
ZEISS LSM 780 confocal microscope. The three-dimensional sprouting
of HRMEC was quantified from maximum projection images of Z-stacks
either manually and expressed as accumulated sprout length per
spheroid (mm), or semi-automated with the Zeiss ZEN-imaging
software and expressed as spheroid perimeter (pixel). Data were
prepared for presentation with GraphPad Prism (version 8) and
represent mean.+-.SEM.
Electroretinography
[0451] General Procedure
[0452] Electroretinography (ERG) is a non-invasive
electrophysiological technique to assess light- induced electrical
activity of different retinal neurons,and allows for quantifying
different aspects of retinal function such as dim light or color
vision. ERGs were measured as the potential change between a
corneal and a reference electrode using the Espion E3 ERG recording
system (Diagnosys LLC). Prior to ERG recordings, animals were dark
adapted for at least 2 h, and anesthetized by i.p. injection of
ketamine (Ketanest.RTM., ca. 100 mg/kg) and xylazin (Rompun.TM.,
ca. 7 mg/kg). The animals were placed on a heated stage to maintain
the body temperature constant at 37.degree. C. Pupils were dilated
with topical Tropicamid and 10% phenylephrine. A drop of
Methocel.TM. 2% solution (Omni Vision) was placed on the cornea to
prevent corneas eyes from drying during recordings. Recordings were
performed simultaneously from both eyes with gold loop electrodes.
The reference electrode was a toothless alligator clip wetted with
Methocel.TM. media and attached to the cheek of the animal. For
electrical grounding, a clip was attached to the tail of the
animal. ERG signals were sampled at 1 kHz and recorded with 0.15 Hz
low-frequency and 500 Hz high-frequency cutoffs. The light stimuli
consisted of full-field flashes (duration.about.4 ms) delivered by
a set of light-emitting diodes or a Xenon light bulb (for flashes
.gtoreq.1 cds/m.sup.2). All flashes were produced by a Ganzfeld
stimulator (ColorDome; Diagnosys), either in darkness or on
background light.
[0453] ERG Protocols
[0454] ERG responses were first recorded from dark-adapted animals
(for isolating rod-driven responses), followed by recordings from
animals adapted to red background light (50 cd/m.sup.2, for
isolating UV cone-driven ERG responses) and finally adapted to
green-blue background light (25.5 cd/m.sup.2, for isolating M
cone-driven ERG responses).
[0455] In case of dark-adapted ERGs, responses were evoked by a
series of flashes ranging from 110.sup.-5 to 100 cds/m.sup.2. For
flashes with the luminance of 110.sup.-5 and 310.sup.-5
cds/m.sup.2, responses of 20 trials were averaged. For the flashes
between 110.sup.-4 up to 0.05 cds/m.sup.2, responses of 10 trials
were averaged, for the flash of 0.1 cds/m.sup.2, responses of 8
trials and for the flash of 1 cds/m.sup.2, 5 trials were averaged.
The last flashes of 10 cds/m.sup.2, we recorded responses of 3
trials and for the final flash 100 cds/m.sup.2, a single flash was
recorded.
[0456] Intervals between individual flashes were chosen to ensure
that the retina recovered completely from each flash (no
indications of flash-induced reduction of response amplitudes or
shortening of implicit times). Based on these criteria, the
inter-flash intervals were 2 s for the 110.sup.-5 and 310.sup.-5
cds/m.sup.2 flashes, 5 s for flashes between 110.sup.-4 up to 0.05
cds/m.sup.2, 10 s for the flash of 0.1 cds/m.sup.2, and 20 s for
the flash of 1 cds/m.sup.2. After the single flashes of 10
cds/m.sup.2 and 100 cds/m.sup.2, there was a recovery time of 30 s
and 120 s, respectively.
[0457] For recordings of UV cone-driven and M cone-driven
responses, animals were light-adapted for 2 min first to a red
background light and afterwards to a green background light. Light
responses were evoked by UV flashes of 0.02, 0.04, 0.08, 0.17,
0.35, 0.83, 1.66, 2.90, and 4.15 .mu.W/m.sup.2 and respectively by
M cone flashes from 0.1 up to 110 cds/m.sup.2 flashes. All
responses of 10 trials were averaged with inter-flash intervals of
3 s.
[0458] Animals and STZ Treatment
[0459] Male Brown Norway rats (BN rats) were obtained from Charles
River (Germany). Hyperglycemia was induced by i.p. injections of
STZ (65 mg/kg body weight). Non- or poorly responding animals were
not included into the study, i.e. animals with blood glucose
concentrations <20 mM at day 7 post STZ application. Body weight
and blood glucose levels were monitored regularly. STZ was
administered ca. 3 weeks before intravitreal dosing.
Dosing and Intravitreal Injections
[0460] For ivt injections, rats were anesthetized with 2.5-3%
isoflurane (Forene; Abbvie). A drop of 4 mg/ml
oxybuprocainhydrochlorid (Novesine.RTM.; Omnivision) was
administered for topical local anesthesia. 5 .mu.L were injected
via a 34-gauge needle (fitted on a 10 .mu.l Hamilton glass syringe)
into the vitreous just behind the limbus in each eye.
[0461] Data Analysis
[0462] ERGs were measured as the potential change between a corneal
and a reference electrode using the Espion E3 ERG recording system
(Diagnosys LLC).
[0463] Before calculating with MATLAB.TM. software the different
sets of each ERG flash were proved to be consistent before
calculating a mean curve from each flash.
[0464] To determine ERG a-/b-wave amplitudes ERG data were
processed and analyzed using the MATLAB.TM. software (version
R2014a; MathWorks). In our case we sorted the data with an
individual macro and prepared a file for our Matlab routines.
[0465] The b-wave amplitude was calculated from the bottom of the
a-wave response to the peak of the b-wave peak. The b-wave implicit
time was measured as the time after the flash stimulus needed to
reach the peak of the b-wave.
[0466] The amplitudes of b-waves as a function of the stimulus
intensity were fitted by using a least-square fitting procedure
(GraphPad Prism, Version 6.01 and later on an upper Version of
GraphPad Prism). The a-wave amplitude was calculated from baseline
(zero line) to the negative a-wave response.
[0467] Statistical analysis was performed by one-way ANOVA.
Example 1
Overall Design of Binding Molecules Recognizing Human VEGF and
Human TrkB
[0468] FIG. 1
[0469] The present inventors have developed binding molecules that
bind VEGF and TrkB and act as VEGF antagonists and TrkB agonists
respectively. The molecular design used has an IgG antibody (termed
the "master antibody") which has specificity for one target
antigen, with scFvs of a different specificity coupled to the C
terminus of the heavy chain. A schematic of the design is shown in
FIG. 1. Preferably the binding molecule is bispecific and
tetravalent.
[0470] The bispecific molecule contains flexible peptide sequences
between the variable heavy (VH) and variable light (VL) domains of
the scFv, and the scFv domains are linked to the master IgG
antibody via further series of linkers. In one configuration, the
scFv is oriented such that the VL domain forms the "N-terminal" end
of the scFv and is thus fused to the C-terminus of the heavy chain
of the master antibody while the VH forms the C-terminus of the
scFv and indeed the whole heavy chain polypeptide. However, it can
be appreciated that this "N-VL-VH-C" structure can be reversed,
i.e. "N-VH-VL-C".
[0471] To test feasibility of this concept, a number of different
bispecific molecules based on the format depicted in FIG. 1 were
prepared and further optimized over several cycles of molecule
design starting from Series 1 through to Series 4.
[0472] The following Examples explain the methods used to generate
the bispecific molecule of the different Series that binds VEGF and
TrkB as well as variations in the format and the biological
activity of these molecules.
Example 2
Preparation of Binding Domains that Recognize VEGF and TrkB
[0473] FIG. 1
[0474] As can be appreciated, to prepare bispecific molecules
binding to human VEGF and TrkB, it is necessary to obtain variable
domains that bind to their individual target antigens.
[0475] For this purpose, Immunoglobulin (Ig) VH and VL genes were
obtained from different VEGF and TrkB binders and formatted into
the bispecific molecules of the invention. In total, three
different VEGF binding domains were obtained from the individual
VEGF binders termed B20, G6, and Ranibizumab. For binding to TrkB
the binding domains were obtained from the individual TrkB binder
termed C2 (WO2010086828).
[0476] Formatting of the individual binders into the bispecific
molecules of the invention is performed by routine methods known to
the skilled artisan. Briefly, to construct the gene segment
encoding the scFv, pairs of VL and VH genes encoding the variable
domains were joined by a gene segment encoding a flexible linker of
peptide sequence GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 221). The
resulting scFv-encoding gene segments were in turn cloned in-frame
to the 3' end of a gene encoding the heavy chain of a human IgG
antibody. These coding segments were synthesized by overlapping PCR
methods and cloned into the expression vector pTT5.
[0477] The pairs of VL and VH genes encoding the Fab.sub.(2) part
were then formatted into the bispecific format outlined in Example
1. The VH genes were cloned into pTT5 expression vector as an
in-frame fusion at the 5' end of a gene encoding human Ig.gamma.. A
gene encoding the scFv binder was cloned in frame at the 3' end of
the same Ig.gamma. encoding segment. Similarly, the VL genes were
cloned into pTT5 expression vector as an in-frame fusion with a
gene encoding human IgG kappa light chain.
[0478] In-Fusion.RTM. HD Cloning Kit (Clonetech, U.S.A.) was used
in the above procedure for directional cloning of VH and VL genes.
PCR primers for VL/VH with 15 bp extensions complementary to the
ends of the linearized vector were synthesized. PCR was performed
using the manufacturer's standard protocol and the amplicons were
purified or treated with Cloning Enhancer, then cloned into the
appropriate vector. E. coli were then transformed according to
manufacturer's instructions (Clonetech, U.S.A.). DNA mini-preps
were sequenced.
[0479] Each expression vector contains eukaryotic promoter elements
for the chain-encoding gene, the gene encoding the signal sequence
and the heavy or light chain, an expression cassette for a
prokaryotic selection marker gene such as ampicillin, and an origin
of replication. These DNA plasmids were propagated in ampicillin
resistant E. coli colonies and purified.
[0480] The expression vectors were transfected into CHO-E cells.
Transfected CHO-E cells growing in suspension in serum-free media
were cultivated in shake flasks under agitation at 140 rpm,
37.degree. C. and 5% CO.sub.2 and kept at conditions of exponential
growth. On the day of transfection, cells were chemically
transfected with 1 mg of light chain plasmid and 0.5 mg of heavy
chain plasmid. They were then seeded at 1 to 2.times.10.sup.6
cells/ml in 1 L of Gibco.RTM. FreeStyle.TM. CHO expression medium
(LifeTechnologies, NY, US). Cells were then incubated under orbital
shaking for 10 to 12 days with one-time feeding of 150 ml
commercial feed solution to allow expression of the proteins.
Binding molecule titers in the cell culture supernatants were
determined using an Octet.RTM. instrument (Pall ForteBio, CA, US)
and protA biosensor tips according to manufacturer's
instructions.
[0481] Recombinant binding molecules were purified from culture
supernatant by Protein A affinity chromatography using
MabSelect.TM. SuRe.TM. (Cytiva) followed by either size exclusion
chromatography (Superdex.RTM. 200 column, Cytiva) or cation
exchange chromatography (POROS.TM.50 HS, ThermoFisher) and stored
in 60 mM NaOAc buffer, 100 mM NaCI (pH 5.0). Purity and degree of
heterogeneity of the samples were assessed by mass spectrometry and
analytical size exclusion chromatography. All samples were
confirmed to have a monomer content of .gtoreq.90% and contain
<10% impurities prior to functional testing.
[0482] This resulted in the generation of the bispecific,
tetravalent binding molecules through the different Series 1 to
Series 4 as shown below in Table 3 and in the following are also
referred to as Doppelmabs (DMabs) (cf. also Vekataramani et al.,
Biochemical and Biophysical Research Communications, Volume 504,
Issue 1, 26 Sep. 2018, Pages 19-24).
TABLE-US-00004 TABLE 3 Fab ScFv/Orientation Linker Series
Modifications TPP-11735 C2 B20 VH-VL 20L3 1 TPP-11736 C2 B20 VL-VH
20L3 1 TPP-11737 C2 G6 VH-VL 20L3 1 TPP-11738 C2 G6 VL-VH 20L3 1
TPP-14936 C2 Ranibizumab VH-VL 20L3 1 TPP-14937 C2 Ranibizumab
VL-VH 20L3 1 TPP-16061 C2 B20 VL-VH 20L1 1 TPP-16062 C2 B20 VL-VH
15L1 1 TPP-16063 C2 B20 VL-VH 10L1 1 TPP-16064 C2 B20 VL-VH 6GS 1
TPP-19984 C2 Ranibizumab VH-VL 10L1 1 TPP-19985 C2 Ranibizumab
VH-VL 20L1 1 TPP-14938 B20 C2 VH-VL 20L3 2 TPP-14939 B20 C2 VL-VH
20L3 2 TPP-14940 Ranibizumab C2 VH-VL 20L3 2 TPP-14941 Ranibizumab
C2 VL-VH 20L3 2 TPP-19986 Ranibizumab C2 VH-VL 10L1 3 TPP-19987
Ranibizumab C2 VH-VL 20L1 3 TPP-19988 Ranibizumab C2 VL-VH 10L1 3
TPP-19989 Ranibizumab C2 VL-VH 20L1 3 TPP-22171 Ranibizumab C2
VH-VL 10L1 4 CC TPP-22173 Ranibizumab C2 VL-VH 10L1 4 CC TPP-22180
Ranibizumab C2 VH-VL 10L1 4 1Q6Q70G TPP-22187 Ranibizumab C2 VL-VH
10L1 4 1Q TPP-22188 Ranibizumab C2 VL-VH 10L1 4 6Q TPP-22189
Ranibizumab C2 VL-VH 10L1 4 70G TPP-22190 Ranibizumab C2 VL-VH 10L1
4 1Q70G TPP-22191 Ranibizumab C2 VL-VH 10L1 4 6Q70G TPP-22192
Ranibizumab C2 VL-VH 10L1 4 1Q6Q70G TPP-22204 Ranibizumab C2 VH-VL
10L1 4 CC1Q6Q70G TPP-22211 Ranibizumab C2 VL-VH 10L1 4 CC1Q
TPP-22212 Ranibizumab C2 VL-VH 10L1 4 CC6Q TPP-22213 Ranibizumab C2
VL-VH 10L1 4 CC70G TPP-22214 Ranibizumab C2 VL-VH 10L1 4 CC1Q70G
TPP-22215 Ranibizumab C2 VL-VH 10L1 4 CC6Q70G TPP-22216 Ranibizumab
C2 VL-VH 10L1 4 CC1Q6Q70G TPP-23457 Ranibizumab TPP-6830 VH-VL 10L1
4 TPP-23459 Ranibizumab TPP-6830 VL-VH 10L1 4
[0483] Briefly, the following design activities were performed:
[0484] Series 1
[0485] In Series 1 binding molecules were generated having an
antigen-binding fragment (Fab) as TrkB binding part in the
bispecific binding molecule. The VEGF binding part was formatted as
scFv(2) and different VEGF binders were evaluated, e.g.
[0486] B20, G6 or Ranibizumab. Furthermore, different VL-VH or
VH-VL orientation were tested and several linkers, such as 20L3,
20L1, 15L1, 10L1, 6GS were used in the design of the binding
molecules.
[0487] Series 2
[0488] In Series 2 binding molecules were generated having an
antigen-binding fragment (Fab) as VEGF binding part in the
bispecific binding molecule and again different VEGF binders were
evaluated. This time the TrkB binding part was formatted as
scFv.sub.(2) and different VL-VH or VH-VL orientation were
tested.
[0489] Series 3
[0490] In Series 3 binding molecules were generated based on
Ranibizumab as antigen-binding fragment (Fab) and VEGF binding part
in the bispecific binding molecule. The TrkB binding part was again
formatted as scFv.sub.(2) and based on the C2 TrkB binder.
Permutations in VL-VH or VH-VL orientation and different linkers
were tested.
[0491] Series 4
[0492] In the final Series 4, different mutations were introduced
into the Ranibizumab binding part, such as VHE1Q, VHE6Q, and/or
VLD70G and the stabilizing effect of CC bridge was analyzed in the
TrkB binding scFv parts.
[0493] The results of these design activities and their impact on
the properties and biological activity are now described in more
detail in the following examples.
Example 3
Comparison of Human TrkB Activation by C2, BDNF and the Four
Doppelmabs TPP-11735, 736, 737 and 738 of the First Series
[0494] FIG. 2 A-B
[0495] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, the C2 antibody, an IgG1 isotype control or the four
Doppelmabs TPP-11735, TPP-11736, TPP-11737, TPP-11738. TrkB
activation was assessed by measuring (A) TrkB phosphorylation on
Y706/707 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and
T185/Y187 (ERK2), respectively, downstream of TrkB. The lowest
compound concentration is solvent alone.
[0496] Results:
[0497] TrkB activation by DMabs was virtually identical to the
parental C2 molecule. Also, the DMabs showed the same properties as
the parental C2 molecule and acted as partial TrkB agonists having
an efficacy of TrkB activation at around -40-50% of BDNF (pTrkB).
Although, the DMab did incorporate the entire (Fab).sub.2 portion
of the parental C2 antibody without changing the sequence or the
orientation/layout this was a very encouraging result, as it showed
that formatting into this specific format did not negatively affect
the ability of the TrkB binder to activate TrkB and TrkB downstream
signalling. As expected the isotype control did not activate
TrkB.
Example 4
Comparison of Cyno/Rabbit/Rat/Mouse TrkB Activation by C2, BDNF and
the Doppelmabs TPP-11735/736 of the First Series
[0498] FIG. 3A-D
[0499] CHO cells with stable expression of (A) cyno TrkB, (B)
rabbit TrkB, (C) rat TrkB or (D) mouse TrkB were incubated with
growing concentrations of the natural TrkB ligand BDNF, the C2
antibody, or the Doppelmabs TPP-11735 or TPP-11736. TrkB activation
was assessed by measuring TrkB phosphorylation on Y706/707. The
lowest compound concentration is solvent alone. Data represent
mean+/-SEM.
[0500] Results:
[0501] Again, TrkB activation by DMabs was virtually identical to
the parental C2 molecule and activation was comparable to
activation of human TrkB receptor. Also in this assay the DMabs
showed the same properties as the parental C2 molecule and acted as
partial TrkB agonists having an efficacy of TrkB activation at
around .about.40-50% of BDNF (pTrkB).
Example 5
Selectivity of TPP-11735/736/737/738 Mediated TrkB Activation
[0502] FIG. 4A-C
[0503] CHO cells with stable expression of (A) human TrkA, (B)
human TrkB, or (C) human TrkC were incubated with growing
concentrations of the C2 antibody or the four Doppelmabs TPP-11735,
TPP-11736, TPP-11737, TPP-11738. Activation of the Trk receptors
was assessed by measuring receptor phosphorylation on Y706/707.
Incubation with growing concentrations of the natural ligands for
TrkA (NGF), TrkB (BDNF) and TrkC (NT-3) were used as controls. The
lowest compound concentration is solvent alone. Data represent the
mean+/-SEM.
[0504] Results:
[0505] None of the tested Doppelmabs activated either TrkA or TrkC.
All Doppelmabs were very specific/selective for TrkB.
Example 6
Comparison of C2-, TPP-11736- and/or BDNF-Induced Internalization
of Human TrkB Receptor
[0506] FIG. 5A-B
[0507] (A) CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, or 1 nM BDNF with growing concentrations of the C2 antibody
or the Doppelmab TPP-11736. (B) TrkB internalization was assessed
by immunofluorescence staining of surface TrkB receptors without
permeabilization of the cells, followed by confocal microscopy
analysis. Dark and light fields of the heatmap represent high and
low percentage of cells above fluorescence threshold,
respectively.
[0508] Results:
[0509] BDNF induced TrkB receptor internalization (lanes 1+2;
heatmap fields are getting darker from top to bottom).
[0510] The antibody C2 and TPP-11736 decreased the BDNF-induced
internalization of the TrkB receptor (lanes 3-5 and 6-8; note that
heatmap fields are getting darker from bottom to top).
Example 7
Comparison of Human TrkB Activation by C2 and TPP-11736 (First
Series) in the Presence or Absence of Human VEGF
[0511] FIG. 6
[0512] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the C2 tool antibody or
the Doppelmab TPP-11736, without or with pre-incubation with 200
ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by
measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187
(ERK2), downstream of TrkB. Incubation with growing concentrations
of human VEGF-A without antibody served as control. The lowest
compound concentration is solvent alone. Data represent the mean.
For clarity, error bars are omitted.
[0513] Results:
[0514] Incubation with human VEGF alone did not change ERK1/2
phosphorylation.
[0515] Also, pre-incubation with human VEGF did neither change the
potency nor the efficacy of ERK1/2 phosphorylation by C2 or
TPP-11736 substantially.
Example 8
Neuroprotective Efficacy of DMab 11736 and C2 in STZ-Induced
Diabetic Rats
[0516] FIG. 7
[0517] Neuroprotective function of TrkB activation in a rat model
of diabetes-induced retinal neurodegeneration using IVT injection
of an agonistic TrkB tool antibody (C2) as well as Doppelmab
TPP-11736. Animals were treated with STZ to induce hyperglycemia.
The retinal function was assessed by electroretinography (ERG)
before and after treatment. Diabetes induction led to delayed
implicit times within 3 weeks after STZ treatment. At this point in
time, animals were intravitreally dosed with an isotype control
antibody (anti-TNP) or C2 (19 .mu.g/5 .mu.l, each), or an equimolar
amount of TPP-11736 (25 .mu.g/5 .mu.l). After two weeks of
treatment, ERG recordings were repeated and analyzed. Rod-driven
B-wave implicit time delays immediately before and two weeks after
intravitreal application of the antibodies are shown in FIG. 7;
mean+/-SEM; .sup.n.s.p>0.05, non-significant; **p<0.01;
***p<0.001; one-way Anova with Tukey multi-comparison test.
[0518] Results:
[0519] Two weeks after administration, anti-TNP antibody treatment
did not reduce diabetes induced rod driven b-wave implicit time as
compared to the point in time before anti-TNP treatment (15.4 ms at
t=2 weeks vs. 13.3 ms at t=0; p>0.05, non-significant).
[0520] Two weeks after administration, TPP-11736 and C2 treated
animals showed a significant reduction in diabetes induced rod
driven b-wave implicit time delay as compared to the point in time
before antibody treatment (C2: 8.62 ms at t=2 weeks vs. 14.4 ms at
t=0; ***p<0.01; TPP-11736 10.3 ms at t=2 weeks vs. 16.1 ms at
t=0; **p<0.01).
[0521] In agreement with the earlier measurements in in vitro
assays of TrkB activation, C2 and TPP-11736 showed a similar
neuroprotective efficacy in vivo.
Example 9
Comparison of the Human VEGF-A Scavenging by EYLEA.RTM.
(Aflibercept) and the Four Doppelmabs TPP-11735, 736, 737 and 738
of the First Series-Inhibition of VEGF-Induced VEGFR2
Phosphorylation
[0522] FIG. 8A-B
[0523] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules or EYLEA.RTM. (aflibercept).
[0524] VEGF-A scavenging was assessed by measuring VEGF receptor 2
(VEGFR2) phosphorylation on Y1175. (A) Comparison of Doppelmabs
TPP-11735, -736, -737 and -738. (B) Comparison of TPP-11736 and
TPP-11738 with EYLEA.RTM. (aflibercept). Non-stimulated cells
(Basal) and 50 ng/ml human VEGF without antibody treatment served
as control . Data represent mean+/-SEM.
[0525] Results:
[0526] TPP-11737 vs. TPP-11738: These molecules had both the G6
anti-VEGF molecule as scFv. 737 is connected in a VH-VL
orientation, whereas 738 has the VL-VH orientation. It turned out
that the VL-VH orientation worked a lot better based on the G6
anti-VEGF antibody.
[0527] TPP-11735 vs. TPP-11736: These molecules had both the B20
anti-VEGF molecule as scFv. For the B20 VEGF antibody in this
assay, there was not much of a performance difference between the
two orientations.
[0528] TPP-11736 vs. TPP-11738: These molecules had either the B20
(VL-VH) or the G6 (VL-VH) anti-VEGF molecule as scFv. Both binding
molecules showed similar potency (1050 .about.4 nM), but TPP-11736
was more efficacious.
[0529] EYLEA.RTM. (aflibercept) (IC50=0.7 nM) is more potent than
compared to either TPP-11736 or TPP-11738 (.about.4 nM).
Example 10
Comparison of Human VEGF-A Scavenging by EYLEA.RTM. (Aflibercept)
and the Four Doppelmabs TPP-11735, 736, 737 and 738 of the First
Series-Inhibition of VEGF-Induced ERK1/2 Phosphorylation
FIG. 9 A-B
[0530] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated
antibodies or EYLEA.RTM. (aflibercept).
[0531] VEGF-A scavenging was assessed by TrkB activation was
assessed by measuring ERK1/2 phosphorylation on T202/Y204 (ERK1)
and T185/Y187 (ERK2). (A) Comparison of Doppelmabs TPP-11735, -736,
-737 and -738. (B) Comparison of TPP-11736, -738 with EYLEA.RTM.
(aflibercept). Non-stimulated cells (Basal) and 50 ng/ml human VEGF
without antibody treatment served as control. Data represent
mean+/-SEM.
[0532] Results:
[0533] TPP-11737 vs. TPP-11738: These molecules had both the G6
anti-VEGF molecule as scFv. 737 is connected in a VH-VL
orientation, whereas 738 has the VL-VH orientation. It turned out
that the VL-VH orientation worked a lot better based on the G6
anti-VEGF antibody.
[0534] TPP-11735 vs. TPP-11736: These molecules had both the B20
anti-VEGF molecule as scFv. For the B20 VEGF antibody in this
assay, there was not much of a performance difference between the
two orientations.
[0535] TPP-11736 vs. TPP-11738: These molecules had either the B20
(VL-VH) or the G6 (VL-VH) anti-VEGF molecule as scFv. Both binding
molecules showed similar potency (IC.sub.50 .about.4 nM), but
TPP-11736 was more efficacious.
[0536] EYLEA.RTM. (aflibercept) (IC50=2 nM) is more potent than
compared to either TPP-11736 or TPP-11738 (.about.10 nM).
Example 11
Comparison of the Human VEGF-A Scavenging by EYLEA.RTM.
(Aflibercept) and the Doppelmab TPP-11736 of the First
Series-Inhibition of VEGF-Induced p38 MAPK Phosphorylation
[0537] FIG. 10
[0538] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of TPP-11736 or
EYLEA.RTM. (aflibercept). VEGF-A scavenging was assessed by
measuring p38 MAPK phosphorylation on T180/Y182. Non-stimulated
cells (Basal) and 50 ng/ml human VEGF without antibody treatment
served as control. Data represent mean+/-SEM.
[0539] Results:
[0540] EYLEA.RTM. (aflibercept) (IC.sub.50=.about.0.74 nM) was
around 10-times more potent than TPP-11736 (.about.7.4 nM)
Example 12
Comparison of the Human VEGF-A Scavenging by the Four Doppelmabs
TPP-11735, 736, 737 and 738 of the First Series and EYLEA.RTM.
(Aflibercept)-Inhibition of VEGF-Induced HRMEC Proliferation
[0541] FIG. 11 A-E
[0542] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules or EYLEA.RTM. (aflibercept). Molecule concentrations are
given in mol/L. VEGF-A scavenging was assessed by automated,
image-based quantification of HRMEC cell numbers (IncuCyte). Images
were recorded every four hours for a total period of 96 hours.
Relative cell numbers are shown. Cell numbers at t=0 were set to
one. Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM.
[0543] Results:
[0544] TPP-11737 vs. TPP-11738: These molecules have both the G6
anti-VEGF molecule as scFv. TPP-11737 is connected in a VH-VL
orientation, whereas TPP-11738 has a VL-VH orientation. Obviously,
the VL-VH orientation worked a lot better in the case of G6. There
is virtually no effect of TPP-11737.
[0545] TPP-11735 (VH-VL) vs. TPP-11736 (VL-VH): These molecules
have both the B20 anti-VEGF molecule as scFv. Again, the VL-VH
orientation performed better.
[0546] 736 vs. 738: B20 (VL-VH) vs. G6 (VL-VH): In this assay the
TPP-11736 (B20) was clearly more efficacious
[0547] Finally, EYLEA.RTM. (aflibercept) was more efficacious than
TPP-11736.
[0548] Summary of findings Series 1-TPP-11735, TPP-11736,
TPP-11737, TPP-11738
[0549] TrkB Activation:
[0550] TrkB activation by DMabs was virtually identical to the
parental C2 molecule. This was not unexpected since the entire
(Fab).sub.2 portion of the C2 TrkB binder was incorporated into the
DMabs without changing the sequence or the orientation/layout.
Also, all Doppelmabs (and C2) were only partial TrkB agonists.
[0551] VEGF-Scavenging:
[0552] Apparently, the VL-VH orientation worked better for G6 and
B20 as VEGF binders and B20 (VL-VH) was more efficacious than G6
(VL-VH). Also, both Doppelmabs (B20 and G6) were inferior to
EYLEA.RTM. (aflibercept) in vitro.
[0553] Based on these initial findings the inventors set out to
generate and test in the following examples different linkers for
B20 in VL-VH orientation to improve VEGF-scavenging of Doppelmabs
(TPP-16061 to TPP-16064).
Example 13
Comparison of Human TrkB Activation by C2, BDNF and the Four
Doppelmabs TPP-16061, 16062, 16063 and 16064 of the First Series;
TrkB Phosphorylation
[0554] FIG. 12
[0555] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, the C2 antibody, TPP-11736 (B20, scFv, 20L3, VL-VH), or four
Doppelmabs with different linkers TPP-16061 (B20, scFv, 20L1,
VL-VH), TPP-16062 (B20, scFv, 15L1, VL-VH), TPP-16063 (B20, scFv,
10L1, VL-VH), TPP-16064 (B20, scFv, 6GS, VL-VH). TrkB activation
was assessed by measuring TrkB phosphorylation on Y706/707. The
lowest compound concentration was solvent alone. Data represent
mean. For clarity, error bars were omitted.
[0556] Results:
[0557] TrkB activation by DMabs TPP-16061 to 16064 was virtually
identical to the parental C2 molecule and TPP-11736. TrkB
activation appeared to be largely independent of the linker
variations between the Fc and the scFv (anti-VEGF) portion of the
protein. Although this was expected - because the linkers were
located distant to the TrkB (Fab).sub.2 fragment between the Fc and
the scFv (anti-VEGF) portion of the protein--this was nonetheless a
good confirmation that different linkers can be utilized without
impacting the activity of the binding molecule. Finally, all
displayed binding molecules only showed partial TrkB agonist
activity.
Example 14
Comparison of Human TrkB Activation by BDNF, TPP-11736 and the Two
Doppelmabs TPP-16061 & 16062 of First Series; ERK1/2
Phosphorylation
[0558] FIG. 13
[0559] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, TPP-11736 (B20, scFv, 20L3, VL-VH), or two Doppelmabs with
different linkers TPP-16061 (B20, scFv, 20L1, VL-VH) and TPP-16062
(B20, scFv, 15L1, VL-VH). TrkB activation was assessed by measuring
ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187
(ERK2).
[0560] The lowest compound concentration was solvent alone. Data
represent mean+/-SEM.
[0561] Results:
[0562] TrkB activation by DMabs TPP-16061 and 16062 was virtually
identical to TPP-11736. Also here it showed that TrkB activation
was largely independent of the linker variations and the displayed
binding molecules showed only partial TrkB agonist activity.
Example 15
Comparison of the human VEGF-A Scavenging by the Four Doppelmabs
TPP-16061, 062, 063 and 064 of the First Series with TPP-11736-(A)
Inhibition of VEGF-Induced VEGFR2 Phosphorylation, (B) ERK1/2
Phosphorylation
[0563] FIG. 14A-B
[0564] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated
antibodies; TPP-11736 (B20, scFv, 20L3, VL-VH), or four Doppelmabs
with different linkers TPP-16061 (B20, scFv, 20L1, VL-VH),
TPP-16062 (B20, scFv, 15L1, VL-VH), TPP-16063 (B20, scFv, 10L1,
VL-VH), TPP-16064 (B20, scFv, 6GS, VL-VH). VEGF-A scavenging was
assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation
on Y1175 or (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and
T185/Y187 (ERK2), respectively. Non-stimulated cells (Basal) and 50
ng/ml human VEGF without antibody treatment served as control. Data
represent mean+/-SEM.
[0565] Results:
[0566] The variation of the linkers had virtually no impact on the
potency or efficacy of VEGF-scavenging. This was somewhat
unexpected--given that the linkers were located in proximity to the
VEGF binding sites--but further confirmed that different linkers
can be utilized without impacting the activity of the binding
molecule.
Example 16
Comparison of the Human VEGF-A Scavenging by the Four Doppelmabs
TPP-16061, 062, 063 and 064 of the First Series-Inhibition of
VEGF-Induced HRMEC Proliferation
[0567] FIG. 15
[0568] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with 0.5 nM Doppelmabs or 1 nM EYLEA.RTM.
(aflibercept). VEGF-A scavenging was assessed by automated,
image-based quantification of HRMEC cell numbers (IncuCyte). Images
were recorded every four hours for a total period of 96 hours.
Relative cell numbers are shown. Cell numbers at t=0 were set to
one. Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM.
[0569] Results:
[0570] Similar to the phosphorylation assays shown in example 15,
the linker variants did not perform better (or worse) compared to
the parental molecule TPP-11736. EYLEA.RTM. (aflibercept) was used
at 1 nM because EYLEA.RTM. (Aflibercept) Was supposed to be a
mono-valent molecule, whereas the Doppelmabs are bi-valent. Under
these conditions, EYLEA.RTM. (aflibercept) performed better in
inhibition of VEGF-induced proliferation of HRMEC.
Example 17
Comparison of the Human VEGF-A Scavenging by EYLEA.RTM.
(Aflibercept), TPP-11736 and the Four Doppelmabs TPP-16061, 062,
063 and 064 of the First Series-Inhibition of VEGF-Induced HRMEC
Sprouting
[0571] FIG. 16
[0572] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hours by incubation with 50 ng/mL human VEGF
with or without pre-incubation with 2.5 nM of the indicated
Doppelmabs or 5 nM EYLEA.RTM. (aflibercept) for 24 hours.
Endothelial sprouting was assessed by confocal microscopy and
displayed spheroid perimeter obtained from maximum projections of
Z-stacks. Non-stimulated cells served as control (Basal). Data
represent mean+/-SEM. n.s. p>0.05 non-significant vs. 50 ng/mL
hVEGF+2.5 nM TPP-11736.
[0573] Results:
[0574] Similar to the phosphorylation assays shown in example 15,
the linker variants did not perform better (or worse) compared to
the parental molecule TPP-11736. EYLEA.RTM. (aflibercept) was used
at 1 nM because EYLEA.RTM. (aflibercept) is supposed to be a
mono-valent molecule, whereas the Doppelmabs are bi-valent. Under
these conditions, EYLEA.RTM. (aflibercept) performed better in
inhibition of VEGF-induced proliferation of HRMEC.
[0575] Summary of findings Series 1-TPP-16061, TPP-16062,
TPP-16063, TPP-16064
[0576] TrkB activation: No difference/improvement compared to the
parental molecule TPP-11736 was observed.
[0577] VEGF-scavenging: No difference/improvement compared to the
parental molecule TPP-11736.
[0578] Based on these findings the inventors set out to compare the
VEGF-scavenging of TPP-11736 (B20 as scFv) with TPP-13788 (B20 IgG)
to test if re-formatting of the B20 as a Fab would improve
VEGF-scavenging.
Example 18
Comparison of the Human VEGF-A Scavenging by TPP-11736 and
TPP-13788 (B20 IgG)-Inhibition of VEGF-Induced Phosphorylation of
VEGFR2, ERK1/2 and p38 MAPK
[0579] FIG. 17 A-C
[0580] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF without or with
pre-incubation with growing concentrations of Doppelmab TPP-11736
(B20 anti-VEGF as scFv) or TPP-13788 (B20 IgG). VEGF-A scavenging
was assessed by measuring (A) VEGF receptor 2 (VEGFR2)
phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204
(ERK1) and T185/Y187 (ERK2), or (C) p38 MAPK phosphorylation on
T180/Y182. 50 ng/ml human VEGF without molecule treatment served as
control. Data represent mean+/-SEM. The Table 4 below reports the
corresponding best-fit IC.sub.50 values (non-linear regression;
log(agonist) vs. response (three parameters).
TABLE-US-00005 TABLE 4 TPP-11736 TPP-13788 IC.sub.50 (VEGFR2
phosphorylation) 2.6 nM 1.0 nM IC.sub.50 (ERK1/2 phosphorylation)
9.1 nM 3.0 nM IC.sub.50 (p38 MAPK phosphorylation) 7.7 nM 2.4
nM
[0581] Results:
[0582] The tested B20 IgG TPP-13788 displays an approximately
3-fold increased VEGF-A scavenging potency vs. TPP-11736
Example 19
[0583] Comparison of the human VEGF-A scavenging by TPP-11736 and
TPP-13788 (B20 IgG)-Inhibition of VEGF-Induced HRMEC Sprouting
[0584] FIG. 18
[0585] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hours by incubation with 50 ng/mL human VEGF
without or with pre-incubation with 2.5 nM of Doppelmab TPP-11736
or 2.5 nM TPP-13788 (B20 IgG). Endothelial sprouting was assessed
by confocal microscopy and displayed spheroid perimeter obtained
from maximum projections of Z-stacks. Non-stimulated cells served
as control (Basal). Data represent mean+/-SEM. n.s. p>0.05
non-significant vs. 50 ng/mL hVEGF+2.5 nM TPP-11736.
[0586] Results:
[0587] Despite the improvements of VEGF-scavenging in the VEGFR2,
ERK1/2 and p38 MAPK phosphorylation assay, TPP-13788 (B20 IgG)
mediated inhibition of VEGF-induced sprouting was not better than
that of TPP-11736.
Example 20
Comparison of Human TrkB Activation by BDNF, TPP-11736 and the Two
Doppelmabs TPP-14936 and TPP-14937 of the First Series; TrkB and
ERK1/2 Phosphorylation
[0588] FIG. 19 A-C
[0589] CHO Cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, TPP-11736 (B20, scFv, 20L3, VL-VH), or Two the Doppelmabs
TPP-13936 (Ranibizumab, scFv, 20L3, VH-VL) or TPP-14937
(Ranibizumab, scFv, 20L3, VL-VH). TrkB activation was assessed by
measuring (A) TrkB phosphorylation on Y706/707, (B & C) ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
respectively. The lowest compound concentration was solvent alone.
Data represent mean+/-SEM.
[0590] Results:
[0591] TrkB activation by DMabs TPP-14936 and TPP-14937 was
virtually identical to TPP-11736. This was expected since the
TrkB-binding component of the binding molecules was always the C2
as Fab fragment.
Example 21
Comparison of Cyno and Rat TrkB Activation by BDNF, TPP-11736 and
the Doppelmab TPP-14936; TrkB Phosphorylation
[0592] FIG. 20 A-B
[0593] CHO cells with stable expression of (A) cyno TrkB or (B) rat
TrkB were incubated with growing concentrations of the natural TrkB
ligand BDNF, TPP-11736 (B20, scFv, 20L3, VL-VH) or TPP-14936
(Ranibizumab, scFv, 20L3, VH-VL). TrkB activation was assessed by
measuring TrkB phosphorylation on Y706/707. The lowest compound
concentration was solvent alone. Data represent mean+/-SEM.
[0594] Results:
[0595] TrkB activation by DMab TPP-14936 was virtually identical to
TPP-11736. Again, this is was expected since the TrkB-binding
component of the two molecules is C2 as Fab fragment in both
cases.
Example 22
Comparison of the Human VEGF-A Scavenging by TPP-11736, TPP-14936
or TPP-14937-Inhibition of VEGF-Induced Phosphorylation of VEGFR2,
ERK1/2 and Src
[0596] FIG. 21 A-C
[0597] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the Doppelmabs
TPP-11736 (B20, scFv, 20L3, VL-VH), TPP-14936 (Ranibizumab, scFv,
20L3, VH-VL) or TPP-14937 (Ranibizumab, scFv, 20L3, VL-VH). VEGF-A
scavenging was assessed by measuring (A) VEGF receptor 2 (VEGFR2)
phosphorylation on Y1175, (B) ERK1/2 phosphorylation on T202/Y204
and T185/Y187, or (C) Src phosphorylation on Y419. 50 ng/ml human
VEGF without antibody treatment served as control. Data represent
mean+/-SEM. The Table 5 below reports the corresponding best-fit
IC.sub.50 values and the absolute efficacy values (bottom plateau),
respectively (non-linear regression; log(agonist) vs. response
(three parameters).
TABLE-US-00006 TABLE 5 TPP-11736 TPP-14936 TPP-14937 IC.sub.50
(VEGFR2 phosphorylation) 2.4 nM 1.9 nM 4.3 nM Bottom plateau
(VEGFR2 phosphorylation; counts) 482 -1097 -1407 IC.sub.50 (ERK1/2
phosphorylation) 5.6 nM 3.5 nM 5.6 nM Bottom plateau (ERK1/2
phosphorylation, counts) 3688 -340.8 -926.9 IC.sub.50 (Src
phosphorylation) 4.0 nM 3.4 nM 5.8 nM Bottom plateau (Src
phosphorylation, counts) 6300 4654 4571
[0598] Results:
[0599] Potency of VEGF-scavenging (IC.sub.50) is similar between
TPP-11736 and TPP-14936/TPP-14937.
[0600] Potency of VEGF-scavenging (IC.sub.50) by TPP-14936 is
somewhat better than TPP-14937.fwdarw.VH-VL better than VL-VH
orientation of Ranibizumab scFv.
[0601] VEGF scavenging by TPP-14936 and TPP-14937 was more
efficacious than TPP-11736 (bottom values were smaller).
Example 23
Comparison of the Human VEGF-A Scavenging by TPP-11736 and the
Doppelmabs TPP-14936 and TPP-14937-Inhibition of VEGF-Induced HRMEC
Sprouting
[0602] FIG. 22
[0603] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hours by incubation with 50 ng/mL human VEGF
with or without pre-incubation with 2.5 nM of Doppelmab TPP-11736
(B20, scFv, 20L3, VL-VH), TPP-14936 (Ranibizumab, scFv, 20L3,
VH-VL) or TPP-14937 (Ranibizumab, scFv, 20L3, VL-VH). Endothelial
sprouting was assessed by confocal microscopy and displayed
spheroid perimeter obtained from maximum projections of Z-stacks.
Non-stimulated cells served as control (Basal). Data represent
mean+/-SEM. n.s. p>0.05 non-significant, ****p<0.0001.
[0604] Results:
[0605] Although VEGF-scavenging by TPP-14936 and TPP-14937 was
similar in the VEGFR2/ERK1_2/Src phosphorylation assay, there were
dramatic differences in the inhibition of VEGF-induced sprouting.
No significant difference between TPP-11736 and TPP-14937, however,
inhibition of VEGF-induced HRMEC sprouting by TPP-14936 was a lot
more efficacious than TPP-14937 or TPP-11736.
[0606] This unexpected finding indicated that the VH-VL orientation
of the anti-VEGF scFv is critical for the performance in this
assay: VH-VL is much better than VL-VH. Interestingly, the VL-VH
orientation was better than VH-VL in case of TPP-11735 vs. 736
(B20) and also in case of TPP-11737 vs. 738 (G6).
Example 24
Comparison of the Human VEGF-A Scavenging by the Doppelmabs
TPP-11736, TPP-14936 and TPP-14937, and EYLEA.RTM.
(Aflibercept)-Inhibition of VEGF-Induced HRMEC Proliferation
[0607] FIG. 23 A-D
[0608] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules or EYLEA.RTM. (aflibercept). Binding molecule/EYLEA.RTM.
(aflibercept) concentrations are given in mol/L. VEGF-A scavenging
was assessed by automated, image-based quantification of HRMEC cell
numbers (IncuCyte). Images were recorded every four hours for a
total period of 84 hours. Relative cell numbers are shown. Cell
numbers at t=0 were set to one. Non-stimulated cells served as
control (Basal). Data represent mean+/-SEM.
[0609] Results:
[0610] TPP-14936 and TPP-14937 displayed a better potency and
especially efficacy of inhibition of VEGF-induced HRMEC
proliferation compared to TPP-11736. TPP-14936 was more potent than
TPP-14937.
[0611] Very surprisingly TPP-14936 and TPP-14937 were more
efficacious than EYLEA.RTM. (aflibercept). Both Doppelmabs reduced
the proliferation even below baseline. In contrast to EYLEA.RTM.
(aflibercept), they showed full inhibition of VEGF-induced
proliferation. However, EYLEA.RTM. (aflibercept) was still more
potent than TPP-14936 and 14937.
[0612] Summary of findings Series 1-TPP-14936/14937
[0613] TrkB activation: No difference/improvement compared to the
TPP-11736 were observed. Since both binding molecules had the C2
binder as Fab fragment this was kind of expected.
[0614] VEGF-Scavenging:
[0615] Phosphorylation assay: Potency of VEGF-scavenging
(IC.sub.50) was similar between TPP-11736 and TPP-14936/TPP-14937.
VEGF scavenging by TPP-14936 and TPP-14937 was more efficacious
than TPP-11736 (bottom values).
[0616] Sprouting assay: No significant difference between TPP-11736
and TPP-14937. Inhibition of VEGF-induced HRMEC sprouting by
TPP-14936 was a lot more efficacious than TPP-14937 or TPP-11736.
Unexpectedly, VH-VL orientation of anti-VEGF Ranibizumab was much
better than VL-VH.
[0617] Proliferation assay: TPP-14936 and TPP-14937 displayed a
better potency and especially efficacy of inhibition of
VEGF-induced HRMEC proliferation compared to TPP-11736. TPP-14936
was more potent than TPP-14937 and TPP-14936 and TPP-14937 were
both more efficacious than EYLEA.RTM. (aflibercept).
[0618] Based on these findings the inventors set out to better
understand the reverse layout of Doppelmabs and tried
Ranibizumab/B20 as Fab to improve potency (efficacy) of
VEGF-scavenging and the C2 TrkB binder as scFv. The following
binding molecules were now generated and tested in the following
examples (series 2): TPP-14938 (B20 Fab), TPP-14939 (B20 Fab),
TPP-14940 (Ranibizumab Fab), TPP-14941 (Ranibizumab Fab).
Example 25
Comparison of Human TrkB Activation by BDNF, TPP-11736 and the Four
Doppelmabs TPP-14938, TPP-14939, TPP-14940 and TPP-14941 of the
Second Series; TrkB and ERK1/2 Phosphorylation
[0619] FIG. 24 A-B & FIG. 25
[0620] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, TPP-11736 (C2 as Fab), or four Doppelmabs TPP-14938 (C2 as
scFv, 20L3, VH-VL), TPP-14939 (C2 as scFv, 20L3, VL-VH), TPP-14940
(C2 as scFv, 20L3, VH-VL), and TPP-14941 (C2 as scFv, 20L3, VL-VH)
of the second series. TrkB activation was assessed by (A) measuring
TrkB phosphorylation on Y706/707 or by (B) measuring ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
respectively. The lowest compound concentration was solvent alone.
Data represent mean+/-SEM.
[0621] Results:
[0622] TPP-11736 contained the C2 CDRs as Fabs. The four Doppelmabs
TPP-14938, TPP-14939, TPP-14940 and TPP-14941 contained C2 as
scFv.
[0623] In strong contrast to TPP-11736 or the original C2
antibody--both being partial TrkB receptor agonists--all four new
Doppelmabs now showed full TrkB receptor agonists activity. TrkB
activation by those Doppelmabs was now as efficacious as the
natural ligand BDNF. This was totally unexpected since the original
C2 antibody only showed partial TrkB receptor agonist. Without
wishing to be bound by theory it appears that VEGF induced
clustering with the single binding molecules, as well as the
sterical formation of the binding molecules, may be responsible for
the observed increase in efficacy and potency of TrkB activation
(see also FIG. 25).
[0624] Potency of TrkB activation was also somewhat better with the
Doppelmabs TPP-14938 & TPP-14940 as compared to TPP-14939 &
TPP-14941. Thus the "VH-VL" orientation of the C2 scFv showed
better potency of TrkB activation.
Example 26
Comparison of Cyno/Rat TrkB Activation by BDNF, TPP-11736 and the
Four Doppelmabs TPP-14938, TPP-14939, TPP-14940 and TPP-14941 of
the Second Series; TrkB and ERK1/2 Phosphorylation
[0625] FIG. 26 A-B
[0626] CHO cells with stable expression of (A) cyno TrkB or (B) rat
TrkB were incubated with growing concentrations of the natural TrkB
ligand BDNF, TPP-11736 (C2 as Fab), or two Doppelmabs TPP-14940 (C2
as scFv, 20L3, VH-VL) and TPP-14941 (C2 as scFv, 20L3, VL-VH) of
the second series. TrkB activation was assessed by measuring TrkB
phosphorylation on Y706/707. The lowest compound concentration wa
solvent alone. Data represent mean+/-SEM.
[0627] Results:
[0628] The results of example 25 with human TrkB could be
reproduced in the cyno and rat modell. Also here, in strong
contrast to TPP-11736, the new Doppelmabs were full TrkB receptor
agonists. TrkB activation by those Doppelmabs was as efficacious as
the natural ligand BDNF.
[0629] Likewise, potency of TrkB activation was somewhat better
with the Doppelmab TPP-14940 as compared to TPP-14941. Thus the
VH-VL orientation of the C2 scFv showed better potency of TrkB
activation.
Example 27
Comparison of Human TrkB Activation (TrkB Phosphorylation) by C2,
TPP-14940 and TPP-14941 (Second Series) in the Presence or Absence
of Human VEGF-Synergistic Effect
[0630] FIG. 27 A-C
[0631] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmabs (A) TPP-14940,
(B) TPP-14941, or (C) C2 with or without pre-incubation with 200
ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by
measuring TrkB phosphorylation on Y706/707. The lowest compound
concentration was solvent alone. Data represent the mean+/-SEM.
[0632] Results:
[0633] Surprisingly, pre-incubation with human VEGF dramatically
improved the potency of TrkB phosphorylation (TrkB activation) by
TPP-14941 (C2 as scFv, 20L3, VL-VH).
[0634] The impact of VEGF on the potency of TrkB phosphorylation
(TrkB activation) was greater for TPP-14941 compared to TPP-14940.
Thus, although both molecules showed synergistic effects on potency
of TrkB phosphorylation, it appears that the orientation/geometry
of the TrkB activating part of the Doppelmabs (scFv VL-VH vs. scFv
VH-VL vs. Fab) seems to have an impact on the synergistic effect,
with scFv having a VL-VH orientation being preferred.
[0635] VEGF pre-incubation did not impact on the potency of TrkB
phosphorylation (TrkB activation) by C2 (control).
Example 28
Comparison of Human TrkB Activation (ERK1/2 Phosphorylation) by C2,
TPP-14940 and TPP-14941 (Second Series) in the Presence or Absence
of Human VEGF-Synergistic Effect
[0636] FIG. 28 A-C
[0637] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmabs (A) TPP-14940,
(B) TPP-14941, or (C) C2 with or without pre-incubation with 200
ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by
measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187
(ERK2), downstream of TrkB. The lowest compound concentration is
solvent alone. Data represent the mean+/-SEM.
[0638] Results:
[0639] Here as well, surprisingly, pre-incubation with human VEGF
dramatically improved the potency of ERK1/2 phosphorylation (TrkB
activation) by TPP-14941 (C2 as scFv, 20L3, VL-VH).
[0640] Also here, the impact of VEGF on the potency of ERK1/2
phosphorylation (TrkB activation) was greater for TPP-14941
compared to TPP-14940. Again, both molecules showed synergistic
effects on potency of TrkB phosphorylation, but it appears that the
orientation/geometry of the TrkB activating part of the Doppelmabs
(scFv VL-VH vs. scFv VH-VL vs. Fab) seems to have an impact on the
synergistic effect with scFv having a VL-VH orientation being
preferred.
[0641] As shown previously, VEGF pre-incubation did also not impact
on the potency of ERK1/2 phosphorylation (TrkB activation) by
TPP-11736 (series 1, C2 as Fab). Finally, VEGF pre-incubation did
not impact on the potency of ERK1/2 phosphorylation (TrkB
activation) by C2 (control).
Example 29
Comparison of Human TrkB Activation (TrkB/ERK Phosphorylation) by
VEGF Alone and in Combination with BDNF-Control Experiments for the
Synergistic Effect
[0642] FIG. 29 A-B
[0643] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of human VEGF-A (hVEGF) alone
or growing concentrations of BDNF, with or without a fixed
concentration of 200 ng/mL hVEGF. TrkB activation was assessed by
measuring (A) TrkB phosphorylation on Y706/707 or (B) ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
downstream of TrkB, respectively. The lowest compound concentration
was solvent alone. Data represent the mean+/-SEM.
[0644] Results:
[0645] hVEGF alone did not induce TrkB or ERK1/2
phosphorylation.
[0646] The BDNF dose-response-curve is largely independent of
hVEGF.
Example 30
Comparison of Cyno TrkB Activation (TrkB Phosphorylation) by C2,
TPP-14940 and TPP-14941 (Second Series) in the Presence or Absence
of Human VEGF-Synergistic Effect
[0647] FIG. 30 A-C
[0648] CHO cells with stable expression of cyno TrkB were incubated
with growing concentrations of Doppelmabs (A) TPP-14940, (B)
TPP-14941, or (C) C2 with or witout pre-incubation with 200 ng/mL
human VEGF-A (hVEGF). TrkB activation was assessed by measuring
TrkB phosphorylation on Y706/707. The lowest compound concentration
was solvent alone. Data represent the mean+/-SEM.
[0649] Results:
[0650] Also in this model, pre-incubation with human VEGF
dramatically improved the potency of TrkB phosphorylation (TrkB
activation) by TPP-14941 (C2 as scFv, 20L3, VL-VH). The impact of
VEGF on the potency of TrkB phosphorylation (TrkB activation) by
TPP-14940 (C2 as scFv, 20L3, HL) was smaller.
Example 31
Comparison of cyno TrkB Activation (ERK1/2 Phosphorylation) by C2,
TPP-14940 and TPP-14941 (Second Series) in the Presence or Absence
of Human VEGF-Synergistic Effect
[0651] FIG. 31 A-C
[0652] CHO cells with stable expression of cyno TrkB were incubated
with growing concentrations of Doppelmabs (A) TPP-14940, (B)
TPP-14941, or (C) C2 with or without pre-incubation with 200 ng/mL
human VEGF-A (hVEGF). TrkB activation was assessed by measuring
ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
downstream of TrkB. The lowest compound concentration was solvent
alone. Data represent the mean+/-SEM.
[0653] Results:
[0654] Also in this model, pre-incubation with human VEGF
dramatically improved the potency of ERK1/2 phosphorylation (TrkB
activation) by TPP-14941 (C2 as scFv, 20L3, VL-VH). The impact of
VEGF on the potency of ERK1/2 phosphorylation (TrkB activation) by
TPP-14940 (C2 as scFv, 20L3, HL) was smaller.
Example 32
Comparison of the Human VEGF-A Scavenging by TPP-11736 (B20 as
scFv), TPP-14938 (B20 as Fab) or TPP-14939 (B20 as Fab)-Inhibition
of VEGF-Induced Phosphorylation of VEGFR2, ERK1/2 and Src
[0655] FIG. 32 A-C
[0656] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the Doppelmabs
TPP-11736 (B20 as scFv), TPP-14938 (B20 as Fab) or TPP-14939 (B20
as Fab). VEGF-A scavenging was assessed by measuring (A) VEGF
receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C)
Src phosphorylation on Y419. 50 ng/ml human VEGF without antibody
treatment served as control. Data represent mean+/-SEM. The Table 6
below reports the corresponding best-fit IC.sub.50 values and the
absolute efficacy values (bottom plateaus), respectively
(non-linear regression; log(agonist) vs. response (three
parameters).
TABLE-US-00007 TABLE 6 TPP-11736 TPP-14938 TPP-14939 IC.sub.50
(VEGFR2 phosphorylation) 2.36 nM 1.02 nM 0.742 nM Bottom plateau
(VEGFR2 phosphorylation, counts) 482 129 242 IC.sub.50 (ERK1/2
phosphorylation) 5.59 nM 2.71 nM 2.51 nM Bottom plateau (ERK1/2
phosphorylation, counts) 3688 1331 1144 IC.sub.50 (Src
phosphorylation) 3.97 nM 1.80 nM 1.16 nM Bottom plateau (Src
phosphorylation, counts) 6300 5485 5538
[0657] Results:
[0658] Potency of VEGF-scavenging (1050) by TPP-14938/39 was
somewhat better than TPP-11736.
[0659] VEGF scavenging by TPP-14938 and TPP-14939 was more
efficacious than TPP-11736 (bottom values are smaller).
[0660] Overall the differences between B20 scFv and B20 Fab were
rather small and correspond well to the previous results shown in
FIG. 17/18: TPP-11736 vs. TPP-13788 (B20 IgG). In conclusion, B20
tolerates reformatting from Fab to scFv apparently relatively
well.
Example 33
Comparison of the Human VEGF-A Scavenging by TPP-11736 (B20 scFv),
TPP-14938 (B20 Fab) or TPP-14939 (B20 Fab)-Inhibition of
VEGF-Induced Proliferation of HRMEC
[0661] FIG. 33 A-C
[0662] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules. Molecule concentrations are given in mol/L. VEGF-A
scavenging was assessed by automated, image-based quantification of
HRMEC cell numbers (IncuCyte). Images were recorded every four
hours for a total period of 84 hours. Relative cell numbers are
shown. Cell numbers at t=0 were set to one. Non-stimulated cells
served as control (Basal). Data represent mean+/-SEM.
[0663] Results:
[0664] Reformatting B20 scFv (TPP-11736) into B20 Fab (TPP-14938
& TPP-14939) did neither improve the potency nor the efficacy
of VEGF-A-scavenging in the proliferation assay. Indeed, the B20
Fab based antibodies seemed to be somewhat less potent/efficacious
than TPP-11736.
Example 34
Comparison of Human VEGF-A Scavenging by TPP-14936 (Ranibizumab,
scFv, HL) or TPP-14937 (Ranibizumab, scFv, LH) with TPP-14940 &
TPP-14941 (Ranibizumab Fab)-Inhibition of VEGF-Induced
Phosphorylation of VEGFR2, ERK1/2 and Src
[0665] FIG. 34 A-C
[0666] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the Doppelmabs
TPP-11736 (B20 as scFv), TPP-14936 (Ranibizumab as scFv, VH-VL),
TPP-14937 (Ranibizumab as scFv, VL-VH), TPP-14940 (Ranibizumab as
Fab), or TPP-14941 (Ranibizumab as Fab). VEGF-A scavenging was
assessed by measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation
on Y1175, (B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and
T185/Y187 (ERK2), or (C) Src phosphorylation on Y419. 50 ng/ml
human VEGF without antibody treatment served as control. Data
represent mean+/-SEM. Below Table 7 reports the corresponding
best-fit 1050 values and the absolute efficacy values (bottom
plateaus), respectively (non-linear regression; log(agonist) vs.
response (three parameters).
TABLE-US-00008 TABLE 7 TPP-11736 TPP-14936 TPP-14937 TPP-14940
TPP-14941 IC.sub.50 (VEGFR2 phosphorylation) 2.36 nM 1.90 nM 4.29
nM 0.373 nM 0.292 nM Bottom plateau (VEGFR2 phosphorylation,
counts) 482 -1097 -1407 -567 -763 IC.sub.50 (ERK1/2
phosphorylation) 5.59 nM 3.49 nM 5.58 nM 0.802 nM 0.430 nM Bottom
plateau (ERK1/2 phosphorylation, counts) 3688 -340 -926 438 1147
IC.sub.50 (Src phosphorylation) 3.97 nM 3.40 nM 5.83 nM 0.276 nM
0.393 nM Bottom plateau (Src phosphorylation, counts) 6300 4654
4571 4771 4367
[0667] Results:
[0668] Reformatting Ranibizumab from scFv to Fab dramatically
improved the potency of VEGF-scavenging in all three assays
(10-fold better IC.sub.50than Ranibizumab scFv or reference
molecule TPP-11736).
[0669] Efficacy of VEGF scavenging by the Ranibizumab-based
molecules was a lot better than TPP-11736 (bottom values).
[0670] However, Ranibizumab did not tolerate reformatting from Fab
to scFv very well (in contrast to B20, see earlier examples).
Example 35
Comparison of Human VEGF-A Scavenging by TPP-14936 (Ranibizumab as
scFv, VH-VL) or TPP-14937 (Ranibizumab as scFv, VL-VH) with
TPP-14940 & TPP-14941 (Ranibizumab Fab)-Inhibition of
VEGF-Induced Proliferation of HRMEC
[0671] FIG. 35 A-D
[0672] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules. Molecule concentrations are given in mol/L. VEGF-A
scavenging was assessed by automated, image-based quantification of
HRMEC cell numbers (IncuCyte). Images were recorded every four
hours for a total period of 84 hours. Relative cell numbers are
shown. Cell numbers at t=0 were set to one. Non-stimulated cells
served as control (Basal). Data represent mean+/-SEM.
[0673] Results:
[0674] Reformatting Ranibizumab as scFv (TPP-11736/TPP-14937) into
Ranibizumab Fab (TPP-14940 & TPP-14941) significantly improved
the potency of VEGF-A-scavenging in the proliferation assay.
[0675] With TPP-14940 & TPP-14941, full VEGF-inhibition was
already seen at a concentration of 0.25 nM, whereas 1 nM TPP-14936
and 4 nM TPP-14937 were needed for full inhibition,
respectively.
[0676] The efficacy of VEGF-A-inhibition was similar among all four
molecules (all full inhibitors).
Example 36
Comparison of Human VEGF-A Scavenging by TPP-14938 or TPP-14939
(B20 Fab) with TPP-14940 & TPP-14941 (Ranibizumab
Fab)-Inhibition of VEGF-Induced Phosphorylation of VEGFR2, ERK1/2
and Src
[0677] FIG. 36 A-C
[0678] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the Doppelmabs
TPP-11736 (B20 as scFv), TPP-13938 (B20 as Fab) or TPP-13939 (B20
as Fab), TPP-14940 (Ranibizumab as Fab), or TPP-14941 (Ranibizumab
as Fab). VEGF-A scavenging was assessed by measuring (A) VEGF
receptor 2 (VEGFR2) phosphorylation on Y1175, (B) ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2), or (C)
Src phosphorylation on Y419. 50 ng/ml human VEGF without molecule
treatment served as control. Data represent mean+/-SEM. Below Table
8 reports the corresponding best-fit IC.sub.50 values and the
absolute efficacy values (bottom plateaus), respectively
(non-linear regression; log(agonist) vs. response (three
parameters).
TABLE-US-00009 TABLE 8 TPP-11736 TPP-14938 TPP-14939 TPP-14940
TPP-14941 IC.sub.50 (VEGFR2 phosphorylation) 2.36 nM 1.02 nM 0.742
nM 0.373 nM 2.92 nM Bottom plateau (VEGFR2 phosphorylation, counts)
482 129 242 -567 -763 IC.sub.50 (ERK1/2 phosphorylation) 5.59 nM
2.71 nM 2.51 nM 0.802 nM 0.430 nM Bottom plateau (ERK1/2
phosphorylation, counts) 3688 1331 1144 438 1147 IC.sub.50 (Src
phosphorylation) 3.97 nM 1.80 nM 1.16 nM 0.276 nM 0.393 nM Bottom
plateau (Src phosphorylation, counts) 6300 5485 5538 4771 4367
[0679] Results:
[0680] Doppelmabs with Ranibizumab Fab showed clearly better
potency of VEGF-scavenging than Doppelmabs based on B20 Fab in all
three assays.
[0681] Doppelmabs with Ranobizumab Fab showed clearly better
efficacy of VEGF-scavenging than Doppelmabs based on B20 Fab in all
three assays (bottom values).
Example 37
Comparison of Human VEGF-A Scavenging by TPP-14938 or TPP-14939
(B20 as Fab) with TPP-14940 & TPP-14941 (Ranibizumab as
Fab)-Inhibition of VEGF-Induced Proliferation of HRMEC
[0682] FIG. 37 A-D
[0683] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules. Molecule concentrations are given in mol/L. VEGF-A
scavenging was assessed by automated, image-based quantification of
HRMEC cell numbers (IncuCyte). Images were recorded every four
hours for a total period of 84 hours. Relative cell numbers are
shown. Cell numbers at t=0 were set to one. Non-stimulated cells
served as control (Basal). Data represent mean+/-SEM.
[0684] Results:
[0685] Potency and efficacy of inhibition of VEGF-induced
proliferation by the Ranibizumab Fab-based molecules TPP-14940
& TPP-14941 was clearly superior compared to the B20 Fab-based
molecules TPP-14938 & TPP-14939.
[0686] Even at the highest concentration (16 nM), TPP-14938 &
TPP-14939 failed to fully inhibit VEGF-induced sprouting.
[0687] In contrast, TPP-14940 & TPP-14941 fully inhibited
VEGF-induced proliferation already at a 64-fold lower concentration
of 0.25 nM.
Example 38
Comparison of Human VEGF-A Scavenging by Doppelmabs TPP-11736 (B20
as scFv), TPP-14936 (Ranibizumab as scFv, VH-VL), TPP-14937
(Ranibizumab as scFv, VL-VH), TPP-14938 (B20 as Fab) or TPP-14939
(B20 Fab), TPP-14940 (Ranibizumab as Fab), TPP-14941 (Ranibizumab
as Fab) or EYLEA.RTM. (Aflibercept)-Inhibition of VEGF-Induced
HRMEC Sprouting
[0688] FIG. 38
[0689] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hours by incubation with 50 ng/mL human VEGF
with or without pre-incubation with 2.5 nM of Doppelmab TPP-11736
(B20 scFv), TPP-14936 (Ranibizumab scFv, VH-VL), TPP-14937
(Ranibizumab scFv, VL-VH), TPP-14938 (B20 Fab), TPP-14939 (B20
Fab), TPP-14940 (Ranibizumab Fab), TPP-14941 (Ranibizumab Fab) or
5nM EYLEA.RTM. (aflibercept). Endothelial sprouting was assessed by
confocal microscopy and displayed spheroid perimeter obtained from
maximum projections of Z-stacks. Non-stimulated cells served as
control (Basal). Data represent mean+/-SEM. n.s. p>0.05 no
significant difference; .sctn.p<0.0001 compared to 50 ng/mL
hVEGF; #p>0.05 no significant difference compared to 50 ng/mL
hVEGF. ***p<0.001; one-way Anova with Tukey multi comparison
test.
[0690] Results:
[0691] The Ranibizumab Fab based Doppelmabs TPP-14940 &
TPP-14941 were clearly the best VEGF-scavengers in this assay. In
contrast to EYLEA.RTM. (aflibercept), TPP-14940 & TPP-14941
fully inhibited VEGF-induced sprouting; no statistically
significant difference was seen between the endothelial sprouting
in the absence of VEGF (Basal) and 50 ng/mL hVEGF pre-incubated
with 2.5 nM TPP-14940 or TPP-14941.
[0692] Inhibition of VEGF-induced EC sprouting by TPP-14940 &
TPP-14941 was significantly better than inhibition by any other
Doppelmab.
[0693] Inhibition of VEGF-induced EC sprouting by TPP-14940 &
TPP-14941 was significantly better than inhibition by EYLEA.RTM.
(aflibercept).
[0694] Furthermore, there was no statistically significant
difference between the endothelial sprouting stimulated with 50
ng/mL hVEGF and 50 ng/mL hVEGF pre-incubated with EYLEA.RTM.
(aflibercept).
Example 39
Comparison of Human VEGF-A Scavenging by Doppelmab TPP-11940 and
EYLEA.RTM. (Aflibercept)-Inhibition of Human VEGF-A-Induced Retinal
Hyperpermeability in Brown Norway Rats
[0695] FIG. 39 A-B
[0696] TPP-14940 (but not EYLEA.RTM. (aflibercept)) prevented human
VEGF-A-induced hyperpermeability in the rat retina. (A) Time
protocol showing the experimental procedure. Fifteen minutes after
intravitreal (ivt) administration of the anti-VEGF compound (13 or
26 pmol per eye of EYLEA.RTM. (aflibercept) or TPP-14940) or the
control (26 pmol TPP-11737), 13 pmol human VEGF-A per eye was
administered by ivt injection. PBS injection served as control.
Twenty-four hours later 1 mL/kg of an Evans Blue (EB) solution (45
mg/mL in 0.9% saline) were administered by intravenous (iv)
injection for 30 minutes before the eyes were isolated and fixed.
Plasma samples were collected at the same point in time to confirm
equal systemic EB exposure. (B) Quantification of VEGF-A-induced
hyperpermeability in the retinas of Brown Norway rats was done by
measuring EB extravasation in retinal flatmounts by confocal
microscopy. Eyes were cut along along the Ora serrata, lens and
vitreous were removed and the eye cup was fixed in paraformaldehyde
(4%) for 1 h at 4.degree. C. and then transferred to PBS overnight
at 4.degree. C. The retinae were separated from the outer segments
(sclera and choroidea) and transferred to a glass slide and cut
four times to achieve a flat cloverleaf-like structure. The tissue
was covered with mounting medium (Vectashield.RTM. antifade
mounting media H-1200 containing the DNA stain DAPI) and a
coverslip was put on top to obtain a retinal flatmount. The samples
were excited at a wavelength of 639 nm and emission of Evans Blue
at 669 nm was recorded with a
[0697] LSM 700 confocal laser scanning-microscope (Carl Zeiss,
Jena; gain 800, laser strength 2%, 5 stacks of 60 .mu.m) and images
of the retinal flatmounts with maximum intensity projection were
obtained. Analysis of fluorescence intensity sum was done after
opening the images in the program ImageJ with a threshold of 30.
***p<0.001; *p<0.05; n.s. p>0.05; #p>0.05
non-significant vs. TPP-11737+PBS. One-way Anova with Tukey multi
comparison test, n=9 -17.
[0698] Results:
[0699] Under control conditions, intravitreal VEGF-A injection
increased the vascular permeability by about 60%. TPP-11737 was
used as control antibody because it has the same molecular format
(Doppelmab) as TPP-14940 but in earlier in vitro assays the
compound did not scavange VEGF.
[0700] At a 1:1 and a 2:1 molecular ratio of binding molecule vs
VEGF, TPP-14940 completely blocked vascular hyperpermeability. In
sharp contrast, however, EYLEA.RTM. (aflibercept) failed to
significantly reduce vascular leakage under the same conditions and
even at the doubled concentration.
[0701] Summary of findings Series 2--TPP-14938, TPP-14939,
TPP-14940, TPP-14941
[0702] TrkB Activation:
[0703] In strong contrast to TPP-11736, all four new Doppelmabs
were full TrkB receptor agonists. TrkB activation by those
Doppelmabs was as efficacious as the natural ligand BDNF.
[0704] Potency of TrkB activation was somewhat better in Doppelmabs
with "VH-VL" orientation TPP-14938/40 vs. TPP-14939 &
TPP-14941.
[0705] Surprisingly, pre-incubation with human VEGF dramatically
improved the potency of TrkB phosphorylation (TrkB activation) by
TPP-14941 [C2, scFv, 20L3, VL-VH] pointing to a synergistic
effect.
[0706] In contrast to TPP-14941, the impact of VEGF on the potency
of TrkB phosphorylation (TrkB activation) by TPP-14940 [C2, scFv,
20L3, VH-VL] was comparatively smaller.
[0707] VEGF-Scavenging:
[0708] Reformatting B20 scFv (TPP-11736) into B20 Fab (TPP-14938
& TPP-14939) did neither improve the potency nor the efficacy
of VEGF-A-scavenging in the proliferation assay.
[0709] Reformatting Ranibizumab scFv (TPP-11736/TPP-14937) into
Ranibizumab Fab (TPP-14940 & TPP-14941) significantly improved
the potency of VEGF-A-scavenging in the proliferation assay.
[0710] Full VEGF-inhibition was already seen at a concentration as
low as 0.25 nM with TPP-14940 & TPP-14941.
[0711] Potency and efficacy of inhibition of VEGF-induced
proliferation by the Ranibizumab Fab-based molecules TPP-14940
& TPP-14941 was superior compared to the B20 Fab-based
molecules TPP-14938 & TPP-14939.
[0712] Since the inventors observed linker clipping in the binding
molecules (data not shown) in the following examples further
molecules were derived on the basis of TPP-14940: TPP-19986 (VH-VL,
10L1), TPP-19987 (VH-VL, 20L1); and on the basis of TPP-14941:
TPP-19988 (VL-VH, 10L1), TPP-19989 (VL-VH, 20L1), which were all
based on Ranibizumab as Fab and C2 as scFv.
Example 40
Comparison of Human TrkB Activation by BDNF, TPP-14940 [C2, scFv,
20L3, VH-VL] or its Linker Variants TPP-19986 [C2, scFv, 10L1,
VH-VL] or TPP-19987 [C2, scFv, 20L1, VH-VL], or TPP-14941 [C2,
scFv, 20L3, VL-VH] and its Linker Variants TPP-19988 [C2, scFv,
10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH]; TrkB and ERK1/2
Phosphorylation--Series 3
[0713] FIG. 40 A-D
[0714] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, TPP-14940 [C2, scFv, 20L3, VH-VL] or its linker variants
TPP-19986 [C2, scFv, 10L1, VH-VL] or TPP-19987 [C2, scFv, 20L1,
VH-VL], or TPP-14941 [C2, scFv, 20L3, V-VH] and its linker variants
TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1,
VL-VH]. TrkB activation was assessed by (A & B) measuring TrkB
phosphorylation on Y706/707 or by (C & D) measuring ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
respectively. The lowest compound concentration was solvent alone.
Data represent mean+/-SEM. The Tables 9 and 10 below report the
corresponding best-fit EC.sub.50 values of TrkB and ERK1/2
phosphorylation, respectively (non-linear regression; log(agonist)
vs. response (three parameters).
TABLE-US-00010 TABLE 9 BDNF TPP-14940 TPP-19986 TPP-19987 EC.sub.50
(pTrkB) 1.22 nM 3.21 nM 2.00 nM 2.16 nM EC.sub.50 (pERK) 0.507 nM
0.475 nM 0.406 nM 0.381 nM
TABLE-US-00011 TABLE 10 BDNF TPP-14941 TPP-19988 TPP-19989
EC.sub.50 (pTrkB) 1.28 nM 6.28 nM 7.83 nM 7.37 nM EC.sub.50 (pERK)
0.282 nM 1.29 nM 2.05 nM 1.77 nM
[0715] Results:
[0716] There was no significant difference between the parental
molecules and the respective linker variants. Potency and efficacy
of TrkB and ERK1/2 phosphorylation were virtually identical. This
was noteworthy because the linkers were located close to the TrkB
binding site but yet did not impact the biological activity of the
binding molecule.
[0717] All tested molecules were full TrkB agonists.
[0718] Again, as shown earlier, potency of TrkB/ERK phosphorylation
was better for TPP-14940 molecule and its linker variants (VH-VL
orientation) compared to the TPP-14941 molecule and its linker
variants (VL-VH orientation).
Example 41
Comparison of Human TrkB Internalization by BDNF, TPP-14940 [C2,
scFv, 20L3, VH-VL] or TPP-14941 [C2, scFv, 20L3, VL-VH] and the
linker variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2,
scFv, 20L1, VL-VH]-Series 3
[0719] FIG. 41 A-B
[0720] CHO cells with stable expression of cyno TrkB were incubated
with growing concentrations of the natural TrkB ligand BDNF, or 1
nM BDNF with growing concentrations of the Doppelmabs of the first
series TPP-14940 [C2, scFv, 20L3, VH-VL] or TPP-14941 [C2, scFv,
20L3, VL-VH] and the linker variants TPP-19988 [C2, scFv, 10L1,
VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH]. TrkB internalization
was assessed by immunofluorescence staining the surface TrkB
receptors without permeabilization of the cells followed by
confocal microscopy analysis. Dark and light fields of the heatmap
represent high and low percentage of cells above florescence
threshold, respectively.
[0721] Results:
[0722] BDNF induced TrkB receptor internalization. Although
TPP-14940, TPP-14941, TPP-19988 and TPP-19989 were full TrkB
receptor agonists, none of the Doppelmabs increased the
BDNF-induced receptor internalization but rather decreased the
BDNF-induced internalization of the TrkB receptor (note: heatmap
fields are getting darker from bottom to top).
Example 42
Comparison of Human VEGF-A Scavenging by TPP-14940 and its Linker
Variant TPP-19986, and TPP-14941 and its Linker Variant
TPP-19988-Inhibition of VEGF-Induced Phosphorylation of
VEGFR2-Series 3
[0723] FIG. 42
[0724] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the Doppelmabs
TPP-14940 and its linker variant TPP-19986, and TPP-14941 and its
linker variant TPP-19988. VEGF-A scavenging was assessed by
measuring VEGF receptor 2 (VEGFR2) phosphorylation on Y1175. 50
ng/ml human VEGF without molecule treatment served as control. Data
represent mean+/-SEM. The Table 11 below reports the corresponding
best-fit IC.sub.50 values of the inhibition of VEGFR2
phosphorylation (non-linear regression; log(agonist) vs. response
(three parameters).
TABLE-US-00012 TABLE 11 TPP- TPP- TPP- TPP- TPP- TPP- 14940 19986
19987 14941 19988 19989 IC.sub.50 (VEGFR2 0.280 nM 0.142 nM 0.188
nM 0.225 nM 0.281 nM 0.268 nM phosphorylation)
[0725] Results:
[0726] There was no significant difference between the parental
molecules TPP-14940/TPP-14941 and the respective linker variants.
Potency and efficacy of inhibition of VEGF-induced VEGFR2
phosphorylation was virtually identical.
Example 43
Comparison of Human VEGF-A Scavenging by Doppelmabs TPP-14940 [C2,
scFv, 20L3, VH-VL] or its linker variants TPP-19986 [C2, scFv,
10L1, VH-VL] or TPP-19987 [C2, scFv, 20L1, VH-VL], or TPP-14941
[C2, scFv, 20L3, VL-VH] and its linker variants TPP-19988 [C2,
scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv, 20L1, VL-VH]--Inhibition
of VEGF-Induced HRMEC Sprouting-Series 3
[0727] FIG. 43
[0728] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hours by incubation with 50 ng/mL human VEGF
with or without pre-incubation with 2.5 nM of Doppelmabs TPP-11736
(B20 scFv), TPP-14940 [C2, scFv, 20L3, VH-VL] or its linker
variants TPP-19986 [C2, scFv, 10L1, VH-VL] or TPP-19987 [C2, scFv,
20L1, VH-VL], or TPP-14941 [C2, scFv, 20L3, VL-VH] and its linker
variants TPP-19988 [C2, scFv, 10L1, VL-VH] or TPP-19989 [C2, scFv,
20L1, VL-VH]. Endothelial sprouting was assessed by confocal
microscopy and displayed spheroid perimeter obtained from maximum
projections of Z-stacks. Non-stimulated cells served as control
(Basal). Data represent mean+/-SEM. n.s. p>0.05 no significant
difference; .sctn.p<0.0001 compared to 50 ng/mL hVEGF;
#p>0.05 no significant difference compared to 50 ng/mL hVEGF.
***p<0.001; one-way Anova with Tukey multi comparison test.
[0729] Results:
[0730] No significant difference was observed between the parental
molecules TPP-14940/TPP-14941 and the respective linker variants.
All molecules fully inhibited VEGF-induced sprouting of the
HRMEC.
[0731] Summary of Findings Series 3-TPP-19986, 19987, 19988,
19989
[0732] TrkB Activation:
[0733] No significant differences were observed between the
parental molecules (TPP-14940/14941) and the respective linker
variants.
[0734] All tested binding molecules were full TrkB receptor
agonists. VH-VL orientation and its linker variants were still more
potent.
[0735] No change in receptor internalization was observed. This was
very surprising considering that the new Doppelmabs were, similar
to BDNF, full TrkB receptor agonists.
[0736] VEGF-Scavenging
[0737] No significant differences were observed between the
parental molecules (TPP-14940/14941) and the respective linker
variants.
[0738] CMC Properties:
[0739] Linker clipping was no longer observed with the use of the
linkers 10L1 & 20L1 linker (data not shown).
[0740] As next steps, a final series of binding molecules were
designed (Series 4) to improve CMC as well as Biophysics properties
of the binding molecules but always trying to preserve the superior
biological function of the binding molecules of the invention.
Example 44
Comparison of Human TrkB Activation by Doppelmabs without or with
Disulfide Bridge (CC) in the TrkB scFv Portion; TrkB
Phosphorylation-Series 4
[0741] FIG. 44 A-D
[0742] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the indicated Doppelmabs
with/without CC bridge in the anti-TrkB scFv portion. (A) TPP-22180
vs. TPP-22204; (B) TPP-22192 vs. TPP-22216; (C) TPP-22190 vs.
TPP-22214; (D) TPP-22191 vs. TPP-22215. TrkB activation was
assessed by measuring TrkB phosphorylation on Y706/707. The lowest
compound concentration was solvent alone. Data represent
mean+/-SEM. The Table 12 below reports the corresponding best-fit
EC.sub.50 values, and the absolute or relative efficacy values (top
plateaus) of TrkB activation, respectively (non-linear regression;
log(agonist) vs. response (three parameters).
TABLE-US-00013 TABLE 12 TrkB-phosphorylation efficacy relative to
TrkB-phosphorylation TrkB-phosphorylation parental clone potency
efficacy without CC bridge TPP Figure Description (EC.sub.50, nM)
(AlphaLISA counts) (%) TPP-22180 A (1Q6Q70G_TrkB-C2-VH-VL_10L1)
8.09 6642 100 TPP-22204 A (1Q6Q70G_TrkB-C2-VH-VL_10L1, CC) 3.88
7613 115 TPP-22192 B (1Q6Q70G_TrkB-C2-VL-VH_10L1) 12.2 5642 100
TPP-22216 B (1Q6Q70G_TrkB-C2-VL-VH_10L1, CC) 4.33 8407 149
TPP-22190 C (1Q70G_TrkB-C2-VL-VH_10L1) 9.95 4225 100 TPP-22214 C
(1Q70G_TrkB-C2-VL-VH_10L1, CC) 2.18 5102 121 TPP-22191 D
(6Q70G_TrkB-C2-VL-VH_10L1) 5.49 4848 100 TPP-22215 D
(6Q70G_TrkB-C2-VL-VH_10L1, CC) 1.12 5793 119
[0743] Results:
[0744] The disulfide bridge was introduced mainly to improve the
CMC properties of the binding molecules. Surprisingly, by including
the disulfide bridge in the scFv anti-TrkB part further improved
both potency and efficacy of TrkB activation. A greater impact was
observed in the binding molecule having the VL-VH orientation.
Example 45
Comparison of Rat TrkB Activation by Doppelmabs with or without
Disulfide Bridge (CC) in the TrkB scFv Portion; TrkB
Phosphorylation-Series 4
[0745] FIG. 45 A-B
[0746] CHO cells with stable expression of rat TrkB were incubated
with growing concentrations of the indicated Doppelmabs
with/witouth CC bridge in the anti-TrkB scFv portion. (A) TPP-22180
vs. TPP-22204; (B) TPP-22192 vs. TPP-22216. TrkB activation was
assessed by measuring TrkB phosphorylation on Y706/707. The lowest
compound concentration was solvent alone. Data represent
mean+/-SEM. The Table 13 below reports the corresponding best-fit
EC.sub.50 values and the absolute or relative efficacy values (top
plateaus) of TrkB activation, respectively (non-linear regression;
log(agonist) vs. response (three parameters).
TABLE-US-00014 TABLE 13 TrkB-phosphorylation efficacy relative
TrkB-phosphorylation TrkB-phosphorylation to parental clone potency
efficacy without CC bridge TPP Figure Description (EC.sub.50, nM)
(AlphaLISA counts) (%) TPP-22180 A (1Q6Q70G_TrkB-C2-VH-VL_10L1)
4.21 19676 100 TPP-22204 A (1Q6Q70G_TrkB-C2-VH-VL_10L1, CC) 2.01
21787 111 TPP-22192 B (1Q6Q70G_TrkB-C2-VL-VH_10L1) 18.9 21213 100
TPP-22216 B (1Q6Q70G_TrkB-C2-VL-VH_10L1, CC) 3.31 25808 122
[0747] Results:
[0748] Including the disulfide bridge in the scFv anti-TrkB part
improved both potency and efficacy of TrkB activation. Again, the
impact on VL-VH orientated binding molecules appeared to be larger.
Overall, the data was in good agreement with the data obtained for
human TrkB.
Example 46
Selectivity of TPP-22204/22214-Mediated TrkB Activation
[0749] FIG. 46
[0750] CHO cells with stable expression of (A) human TrkA, (B)
human TrkB, or (C) human TrkC were incubated with growing
concentrations of the C2 antibody or the Doppelmabs TPP-22204 or
TPP-22214. Activation of the Trk receptors was assessed by
measuring receptor phosphorylation on Y706/707. Incubation with
growing concentrations of the natural ligands for TrkA (NGF), TrkB
(BDNF) and TrkC (NT-3) were used as controls. The lowest compound
concentration was solvent alone. Data represent the mean+/-SEM.
[0751] Results:
[0752] Both Doppelmabs were very specific/selective for TrkB--None
of them activated either TrkA or TrkC .
Example 47
Impact of TPP-22214 on BDNF-Induced TrkB Activation
[0753] FIG. 47 A-B
[0754] CHO cells with stable expression of human TrkB receptor were
incubated with growing concentrations of (A) C2 antibody or (B)
Doppelmab TPP-22214 with or without a constant concentration of 0.3
nM, 1 nM or 3 nM BDNF. Activation of TrkB was assessed by measuring
receptor phosphorylation on Y706/707. Data represent the
mean+/-SEM.
[0755] Results:
[0756] In strong contrast to C2, TPP-22214 did not limit
BDNF-induced TrkB activation.
Example 48
Comparison of Human TrkB Activation (TrkB Phosphorylation) by
TPP-22214 in the Presence or Absence of Human VEGF
[0757] FIG. 48 A-D
[0758] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmab TPP-22214 with
or without pre-incubation with (A) 200 ng/mL human VEGF-A (hVEGF),
(B) 50 ng/mL hVEGF, (C) 10 ng/mL hVEGF, or (D) 2 ng/mL hVEGF. TrkB
activation was assessed by measuring TrkB phosphorylation on
Y706/707. The lowest compound concentration was solvent alone. Data
represent the mean+/-SEM.
[0759] Results:
[0760] As shown before, pre-incubation with 200 ng/mL human VEGF
improved potency of TrkB phosphorylation (TrkB activation) by
TPP-22214. More importantly, this synergistic effect was largely
retained at 50, 10 and even 2 ng/mL hVEGF. Similar effects were
also obtained in other species such as cyno (data not shown).
Example 49
Comparison of Human TrkB Activation (ERK Phosphorylation) by
TPP-22214 in the Presence or Absence of Human VEGF
[0761] FIG. 49 A-D
[0762] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmab TPP-22214 with
or without pre-incubation with 200 ng/mL (A) human VEGF-A (hVEGF),
(B) 50 ng/mL hVEGF, (C) 10 ng/mL hVEGF, or (D) 2 ng/mL hVEGF. TrkB
activation was assessed by measuring ERKI /2 phosphorylation on
T202/Y204 (ERK1) and T185/Y187 (ERK2), downstream of TrkB. The
lowest compound concentration was solvent alone. Data represent the
mean+/-SEM.
[0763] Results:
[0764] Likewise, pre-incubation with 200 ng/mL human VEGF improved
also the potency of ERK phosphorylation downstream of TrkB
activation by TPP-22214. Also in this assay, the synergistic effect
was largely retained at concentrations as low as 2 ng/mL hVEGF.
Similar effects were also obtained in other species such as cyno
(data not shown).
Example 50
VEGF Binding Potentiates TrkB Activation-Studying Proposed
Mechanism
[0765] FIG. 50
[0766] Complex of TPP-22214 and hVEGF was made by mixing together
the samples at the described molar ratio (1:1, 4:1 or 20:1) in
1.times. Dulbecco's PBS. Samples were incubated for 1 hour at room
temperature prior to analysis. An Agilent 1200 was used in series
with mini-DAWN.RTM. TREOS.RTM. compact SEC-MALs detector and
Optilab.RTM. T-REX detector (Wyatt Technologies Corp). The samples
were run in 1.times. Dulbecco's PBS as the mobile phase at a flow
rate of 0.6 ml/min. 100 .mu.l of the complex or TPP-22214 alone was
injected on a Superose.RTM. 6 Increase column (30 cm.times.10 mm,
Cytiva), where molecules were subjected to separation by
hydrodynamic volume. The data were then analyzed using Astra.RTM.
software version 6.1.1.17 (Wyatt Technologies Corp.).
[0767] Results:
[0768] TPP-22214 forms complexes in the presence of VEGF-A with
largest complexes formed at 1:1 molar ratio. These complexes
suggest complexing of the Doppelmab and VEGF beyond 1:1 ratio.
These larger complexes can lead to clustering of the TrkB receptors
on the cell surface. Experimental data support the proposed
mechanism.
Example 51
Comparison of Human TrkB Internalization by Growing Concentrations
of BDNF or TPP-22214, and Growing Concentrations of TPP-22214 in
the Presence of 1 nM BDNF
[0769] FIG. 51 A-C
[0770] CHO cells with stable expression of human TrkB were
incubated with (A) growing concentrations of the natural TrkB
ligand BDNF, (B) growing concentrations of TPP-22214, or (C) 1 nM
BDNF with growing concentrations of TPP-22214. TrkB internalization
was assessed by immunofluorescence staining the surface TrkB
receptors without permeabilization of the cells followed by
confocal microscopy analysis. Data represent the percent of cells
with surface TrkB staining intensity above threshold;
mean+/-SEM.
[0771] Results:
[0772] (A) Incubation with BDNF induced TrkB receptor
internalization.
[0773] (B) TPP-22214 did not induce TrkB receptor
internalization.
[0774] (C) By increasing concentrations of TPP-22214 BDNF-induced
receptor internalization was decrease/abolished.
Example 52
Neuroprotective Efficacy of TPP-22214 and C2 in STZ-Induced
Diabetic Rats
[0775] FIG. 52
[0776] Neuroprotective function of TrkB activation in a rat model
of diabetes-induced retinal neurodegeneration using IVT injection
of an agonistic TrkB antibody (C2) as well as Doppelmab TPP-22214.
Animals were treated with STZ to induce hyperglycemia. The retinal
function was assessed by electroretinography (ERG) before and after
treatment. Diabetes induction led to delayed implicit times within
3 weeks after STZ treatment. At this point in time, animals were
intravitreally dosed with an isotype control antibody (anti-TNP) or
C2 (19 .mu.g/5 .mu.l, each), or an equimolar amount of TPP-22214
(25 .mu.g/5.mu.l). Rod-driven B-wave implicit time delays
immediately before and two weeks after intravitreal application of
the molecules are depicted; mean+/-SEM; .sup.n.s.p>0.05;
non-significant (n.s.), ****p<0.0001; one-way Anova with Tukey
multi-comparison test.
[0777] Results:
[0778] Two weeks after administration, anti-TNP antibody treatment
did not reduce the diabetes induced rod driven b-wave implicit time
as compared to the point in time before anti-TNP treatment. Indeed,
the implicit time delay was even significantly increased as
compared to the point in time before anti-TNP antibody application
(17.1 ms at t=2 weeks vs. 11.2 ms at t=0 weeks; ****p<0.0001).
This indicates that the STZ-induced retinal damage was not fully
established at t=0 weeks and that anti-TNP treatment could not stop
the further increase in the implicit time delay.
[0779] Two weeks after administration, C2 antibody treatment
blocked a further increase of the implicit time delay. However, C2
antibody treatment did not significantly reduce the diabetes
induced rod driven b-wave implicit time as compared to the point in
time before C2 treatment (10.3 ms at t=2 weeks vs. 12.8 ms at t=0
weeks, .sup.n.s.p>0.05; non-significant).
[0780] Two weeks after administration, TPP-22214 antibody treatment
blocked a further increase of the implicit time delay and even
significantly reduced the diabetes induced rod driven b-wave
implicit time by more than 40% as compared to the point in time
before TPP-22214 treatment (8.3 ms at t=2 weeks vs. 14.2 ms at t=0
weeks, ****p<0 . 0001).
Example 53
Comparison of Human VEGF-A Scavenging by Doppelmabs without or with
Disulfide Bridge (CC) and the Parental Molecules
TPP-14940/TPP-14941-Inhibition of VEGF-Induced Phosphorylation of
VEGFR2
[0781] FIG. 53 A-B
[0782] VEGF-A scavenging was assessed by measuring VEGF receptor 2
(VEGFR2) phosphorylation on Y1175. 50 ng/ml human VEGF without
molecule treatment served as control. Data represent mean+/-SEM.
Below Table 14 reports the corresponding best-fit IC.sub.50 values
(non-linear regression; log(agonist) vs. response (three
parameters).
TABLE-US-00015 TABLE 14 Inhibition of VEGF-induced TPP Figure
Description VEGFR2 phosphorylation (IC50, nM) TPP-14941 A
(TrkB-C2-VL-VH,_20L3) 0.225 TPP-22180 A (1Q6Q70G
TrkB-C2-VH-VL_10L1) 0.210 TPP-22204 A (1Q6Q70G TrkB-C2-VH-VL_10L1,
CC) 0.199 TPP-22192 A (1Q6Q70G TrkB-C2-VL-VH_10L1) 0.264 TPP-22216
A (1Q6Q70G TrkB-C2-VL-VH_10L1, CC) 0.331 TPP-14940 B
TrkB-C2-VH-VL,_20L3) 0.165 TPP-14941 B (TrkB-C2-VL-VH,_20L3) 0.173
TPP-22190 B (1Q70G TrkB-C2-VL-VH_10L1) 0.235 TPP-22214 B (1Q70G
TrkB-C2-VL-VH_10L1, CC) 0.242 TPP-22191 B (6Q70G
TrkB-C2-VL-VH_10L1) 0.233 TPP-22215 B (6Q70G TrkB-C2-VL-VH_10L1,
CC) 0.195
[0783] Results:
[0784] There was no significant difference between the parental
molecules TPP-14940/TPP-14941 and the respective Doppelmab variants
with or without disulfide bridge. Potency and efficacy of
inhibition of VEGF-induced VEGFR2 phosphorylation was virtually
identical.
Example 54
Comparison of Human VEGF-A Scavenging by TPP-22214 or EYLEA.RTM.
(Aflibercept)-Inhibition of VEGF-Induced Phosphorylation of VEGFR2
on Y1175 and ERK1/2 (Both Related to EC Proliferation)
[0785] FIG. 54 A-B
[0786] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of TPP-22214 or
EYLEA.RTM. (aflibercept). VEGF-A scavenging was assessed by
measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1175 or
(B) ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187
(ERK2). 50 ng/ml human VEGF without molecule treatment served as
control. Data represent mean+/-SEM. Table 15 below reports the
corresponding best-fit values form the non-linear regression
(log(agonist) vs. response (three parameters). Best-fit values that
significantly differ between EYLEA.RTM. (aflibercept) and TPP-22214
(P<0.05) are shown in bold face. P-values are also reportet in
Table 15.
TABLE-US-00016 TABLE 15 Figure Eyela TPP-22214 P-value Inhibition
of VEGF-induced VEGFR2 phosphorylation (Y1175) Top plateau
(AlphaLISA counts) A 17523 18104 0.4629 IC.sub.50 (nM) A 0.339
0.186 -- LogIC.sub.50 A -9.470 -9.731 0.0245 Bottom plateau
(AlphaLISA counts) A 4031 -761.9 <0.0001 Inhibition of
VEGF-induced ERK1/2 phosphorylation (T202/Y204 (ERK1) and T185/Y187
(ERK2) Top plateau (AlphaLISA counts) B 108769 111446 0.7349
IC.sub.50 (nM) B 1.95 0.415 - LogIC.sub.50 B -8.709 -9.382 0.0087
Bottom plateau (AlphaLISA counts) B 21745 -7737 0.0024
[0787] Results:
[0788] Compared to EYLEA.RTM. (aflibercept), TPP-22214 fully
inhibited VEGF-induced phosphorylation of VEGFR2 on Y1175 and of
ERK1/2. The improved efficacy in VEGF-scavenging of TPP-22214 can
also be seen on the highly significant differences in the values of
the bottom plateaus (see Table 15). Finally, TPP-22214 was
significantly more potent in VEGF-scavenging than EYLEA.RTM.
(aflibercept).
Example 55
Comparison of Human VEGF-A Scavenging by TPP-22204, TPP-22214 or
TPP-22216 with EYLEA.RTM. (Aflibercept)-Inhibition of VEGF-Induced
Proliferation of HRMEC
[0789] FIG. 55 A-E
[0790] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 10 ng/mL human VEGF with or without
pre-incubation with growing concentrations of the indicated binding
molecules: (A) TPPP-22204, (B) TPP-22214; (C) TPP-22216 or (D)
EYLEA.RTM. (aflibercept). VEGF-A scavenging was assessed by
automated, image-based quantification of HRMEC cell numbers
(IncuCyte). Images were recorded every four hours for a total
period of 84 hours. Relative cell numbers are shown. Cell numbers
at t=0 were set to one. Non-stimulated cells served as control
(Basal). Data represent mean+/-SEM. (E) Plot of the difference of
the area under the growth curves and the basal curves vs. the
concentration of EYLEA.RTM. (aflibercept) or TPP-22214. Below Table
16 reports the corresponding best-fit values form the non-linear
regression (log(agonist) vs. response (three parameters). Best-fit
values that significantly differ between EYLEA.RTM. (aflibercept)
and TPP-22214 (P<0.05) are shown in bold face. P-values are also
reportet in Table 16.
TABLE-US-00017 TABLE 16 Inhibition of VEGF-induced HRMEC
proliferation Delta (area under the curve vs. basal) Figure Eyela
TPP-22214 P-value Top plateau (arbitrary units) E 128.9 128.3
0.9406 IC.sub.50 (pM) E 171 88.7 -- LogIC.sub.50 E -9.767 -10.05
0.0093 Bottom plateau (arbitrary units) E 13.3 -14.2 <0.0001
[0791] Results:
[0792] Potency and efficacy of inhibition of VEGF-induced
proliferation by the Doppelmabs was clearly superior to EYLEA.RTM.
(aflibercept) (highly significant difference in the values of
LogIC.sub.50 and bottom plateau between TPP-22214 and EYLEA.RTM.
(aflibercept)).
[0793] Even at the highest concentration (16 nM), EYLEA.RTM.
(aflibercept) failed to fully inhibit VEGF-induced sprouting. In
contrast all Doppelmabs fully inhibit VEGF-induced proliferation
already at a 64-fold lower concentration of 0.25 nM.
Example 56
Comparison of Human VEGF-A Scavenging by TPP-22214 or EYLEA.RTM.
(Aflibercept)-Inhibition of VEGF-Induced Phosphorylation of VEGFR2
on Y1214 and p38-MAPK
[0794] FIG. 56 A-B
[0795] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of TPP-22214 or
EYLEA.RTM. (aflibercept). VEGF-A scavenging was assessed by
measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y1214 or
(B) p38-MAPK phosphorylation on T180/Y182. 50 ng/ml human VEGF
without molecule treatment served as control. Data represent
mean+/-SEM. Below Table 17 reports the corresponding best-fit
values form the non-linear regression (log(agonist) vs. response
(three parameters). Best-fit values that significantly differ
between EYLEA.RTM. (aflibercept) and TPP-22214 (P<0.05) are
shown in bold face. P-values are also reportet in Table 17.
TABLE-US-00018 TABLE 17 Figure Eyela TPP-22214 P-value Inhibition
of VEGF-induced VEGFR2 phosphorylation (Y1214) Top plateau
(AlphaLISA counts) A 7150 7275 0.7759 IC.sub.50 (nM) A 0.437 0.278
-- LogIC.sub.50 A -9.360 -9.555 0.1991 Bottom plateau (AlphaLISA
counts) A 1429 -245.0 <0.0001 Inhibition of VEGF-induced p38-
MAPK phosphorylation (T180/Y182) Top plateau (AlphaLISA counts) B
23071 25363 0.1608 IC.sub.50 (nM) B 0.748 0.472 -- LogIC.sub.50 B
-9.126 -9.326 0.4202 Bottom plateau (AlphaLISA counts) B 8894 -1489
<0.0001
[0796] Results:
[0797] In strong contrast to EYLEA.RTM. (aflibercept), TPP-22214
fully inhibited VEGF-induced phosphorylation of VEGFR2 on Y1214 and
p38-MAPK. The more efficacious VEGF-scavenging of TPP-22214 can be
seen from the highly significant difference in the values of the
bottom plateaus in Table 17. Moreover, a trend was observed that
the potency of VEGF-scavenging by TPP-22214 is better than that of
EYLEA.RTM. (aflibercept).
Example 57
Comparison of the Human VEGF-A Scavenging by TPP-22204, TPP-22214,
TPP-22215 or TPP-22216 with EYLEA.RTM. (Aflibercept)--Inhibition of
VEGF-Induced HRMEC Sprouting
[0798] FIG. 57 A-B
[0799] Spheroids of human retinal microvascular endothelial cells
(HRMECs) were embedded in a collagen matrix. Endothelial sprouting
was induced for 24 hoursvby incubation with 50 ng/mL human VEGF
with or without pre-incubation with 2.5 nM of TPP-22204, TPP-22214,
TPP-22215 or TPP-22216, or 5 nM EYLEA.RTM. (aflibercept). (A)
Endothelial sprouting was assessed by confocal microscopy and
displayed spheroid perimeter obtained from maximum projections of
Z-stacks. Non-stimulated cells served as control (Basal). Data
represent mean+/-SEM. n.s. p>0.05 non-significant,
****p<0.0001. (B) shows representative maximum projection images
from spheroids after 24 hours of sprouting under basal conditions
or after stimulation with 50 ng/mL human VEGF with or without
pre-incubation with 2.5 nM TPP-22214 or 5 nM EYLEA.RTM.
(aflibercept). Bar=100 .mu.m.
[0800] Results:
[0801] Similar to the Y1214 VEGFR2 phosphorylation assays, the
Doppelmabs were a lot more efficacious in inhibiting VEGF-induced
HRMEC sprouting. Moreover, the Doppelmabs were able to fully
inhibit VEGF-induced endothelial cell sprouting. Noteworthy,
EYLEA.RTM. (aflibercept) was used at the double molecular
concentration as compared to the Doppelmabs and still was not able
to fully inhibit cell sprouting.
Example 58
Comparison of Human VEGF-A Scavenging by TPP-22214 or EYLEA.RTM.
(Aflibercept)-Inhibition of VEGF-Induced Phosphorylation of VEGFR2
on Y951 and Src
[0802] FIG. 58 A-B
[0803] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of TPP-22214 or
EYLEA.RTM. (aflibercept). VEGF-A scavenging was assessed by
measuring (A) VEGF receptor 2 (VEGFR2) phosphorylation on Y951 or
(B) Src phosphorylation on Y419. 50 ng/ml human VEGF without
antibody treatment served as control. Data represent mean+/-SEM.
Below Table 18 reports the corresponding best-fit values form the
non-linear regression (log(agonist) vs. response (three
parameters). Best-fit values that significantly differ between
EYLEA.RTM. (aflibercept) and TPP-22214 (P<0.05) are shown in
bold face. P-values are also reported in Table 18.
TABLE-US-00019 TABLE 18 Figure Eyela TPP-22214 P-value Inhibition
of VEGF-induced VEGFR2 phosphorylation (Y951) Top plateau
(AlphaLISA counts) A 20491 20757 0.8567 IC.sub.50 (nM) A 0.540
0.340 -- LogIC.sub.50 A -9.268 -9.469 0.2856 Bottom plateau
(AlphaLISA counts) A 5133 -743.9 <0.0001 Inhibition of
VEGF-induced Src phosphorylation (Y419) Top plateau (AlphaLISA
counts) B 3425 3405 0.8832 IC.sub.50 (nM) B 0.933 0.426 -
LogIC.sub.50 B -9.370 -9.030 0.2219 Bottom plateau (AlphaLISA
counts) B 2207 1629 <0.0001
[0804] Results:
[0805] In strong contrast to EYLEA.RTM. (aflibercept), TPP-22214
fully inhibited VEGF-induced phosphorylation of VEGFR2 on Y951 and
of Src on Y419. The more efficacious VEGF-scavenging of TPP-22214
can be seen from the highly significant difference in the values of
the bottom plateaus (Table 18).
Example 59
Comparison of the Human VEGF-A Scavenging by Doppelmab TPP-22214
and EYLEA.RTM. (Aflibercept)-Inhibition of Human VEGF-A-Induced
Retinal Hyperpermeability in Brown Norway Rats
[0806] FIG. 59 A-B
[0807] TPP-22214 prevented human VEGF-A-induced hyperpermeability
in the rat retina. (A) Time protocol showing the experimental
procedure. Fifteen minutes after intravitreal (ivt) administration
of the anti-VEGF compound (13 or 26 pmol per eye of EYLEA.RTM.
(aflibercept) or TPP-14940) or the control (26 pmol TPP-11737), 13
pmol human VEGF-A per eye was administered by ivt injection. PBS
injection served as control. Twenty-four hours later 1 mL/kg of an
Evans Blue (EB) solution (45 mg/mL in 0.9% saline) were
administered by intravenous (iv) injection for 30 minutes before
the eyes were isolated and fixed. Plasma samples were collected at
the same point in time to confirm equal systemic EB exposure. (B)
Quantification of VEGF-A-induced hyperpermeability in the retinas
of Brown Norway rats was done by measuring EB extravasation in
retinal flatmounts by confocal microscopy. Eyes were cut along
along the Ora serrata, lens and vitreous were removed and the eye
cup was fixed in paraformaldehyde (4%) for 1 h at 4.degree. C. and
then transferred to PBS overnight at 4.degree. C. The retinae were
separated from the outer segments (sclera and choroidea) and
transferred to a glass slide and cut four times to achieve a flat
cloverleaf-like structure. The tissue was covered with mounting
medium (Vectashield.RTM. H-1200 antifade mounting media containing
the DNA stain DAPI) and a coverslip was put on top to obtain a
retinal flatmount. The samples were excited at a wavelength of 639
nm and emission of Evans Blue at 669 nm was recorded with a LSM 700
confocal laser scanning-microscope (Carl Zeiss, Jena; gain 800,
laser strength 2%, 5 stacks of 60 .mu.m) and images of the retinal
flatmounts with maximum intensity projection were obtained.
Analysis of fluorescence intensity sum was done after opening the
images in the program ImageJ with a threshold of 30. ***p<0.001;
*p<0.05; n.s. p>0.05. One-way Anova with Tukey multi
comparison test, n=9-17. Incubation with 67: 1 molar ratio of
EYLEA.RTM. (aflibercept): VEGF is shown for comparison.
[0808] Results:
[0809] Under control conditions, intravitreal VEGF-A injection
increased the vascular permeability by about 60%. Note: TPP-11737
was used as control molecule because it had the same molecular
format (Doppelmab) as TPP-22214 but in earlier in vitro assays did
not scavange VEGF (see above).
[0810] At a 1:1 molecular ratio of binding molecule vs VEGF,
TPP-22214 completely blocked vascular hyperpermeability. In sharp
contrast, however, EYLEA.RTM. (aflibercept) failed to significantly
reduce vascular leakage under the same conditions and even at the
doubled concentration. Only at a very high molecular ratio of 67:1,
EYLEA.RTM. (aflibercept) was able to inhibit human VEGF-induced
vascular leakage.
Example 60
Impact of TrkB-Binding on VEGF-Scavenging by TPP-22214-Comparison
of Human VEGF-A Scavenging by TPP-22214 in the Presence or Absence
of the TrkB Extracellular Domain
[0811] FIG. 60 A-B
[0812] (A) Functional characterization of the TkrB extracellular
domain (TrkB-ECD). CHO cells with stable expression of human TrkB
were incubated with growing concentrations of the natural ligand
BDNF or 10 nM BDNF with growing concentrations of TrkB-ECD. TrkB
activation was assessed by measuring TrkB phosphorylation on
Y706/707. The lowest compound concentration was solvent alone. Data
represent mean+/-SEM.
[0813] (B) Impact of TrkB-ECD binding of TPP-22214 on inhibition of
VEGF-induced VEGFR2 phosphorylation. Human retinal microvascular
endothelial cells (HRMECs) were starved and then incubated with 50
ng/mL human VEGF, 50 ng/mL human VEGF with growing concentrations
of TPP-22214, or 50 ng/mL human VEGF with growing concentrations of
TPP-22214 and 100 nM TrkB-ECD. HRMEC incubation with growing
concentrations of TrkB-ECD with or without 50 ng/mL VEGF and
unstimulated cells (Basal) served as control. VEGF-A scavenging was
assessed by measuring VEGF receptor 2 (VEGFR2) phosphorylation on
Y1175. Data represent mean+/-SEM.
[0814] Results:
[0815] Figure (A): The pre-incubation of BDNF with growing
concentrations of TrkB-ECD reduced activation of the TrkB receptor
dramatically. This showed that the TrkB-ECD is able to bind and
scavenge the BDNF, which is expected from a functional
TrkB-ECD.
[0816] Figure (B): VEGF-scavenging by TPP-22214 was independent of
the presence or absence of the TrkB-ECD. Both curves were nearly
identical. This indicated that TrkB-binding of TPP-22214 did per se
not limit VEGF-scavenging of the Doppelmab suggesting that the
Doppelmab can simultaneously activate the TrkB receptor and
scavenge VEGF.
Example 61
Engineering Efforts to Improve CMC Properties Series 3 to series
4
[0817] Biophysical properties of molecules from series 3 and series
4 were assessed. During production of the molecules the quality
after protein A measured by the percentage of monomer was tested
using analytical size exclusion chromatography. Additionally, the
thermal stability and aggregation onset of each molecule was
assessed in 10 mM Histidine pH 6.0. The thermal stability was
measured using a thermal shift assay which measures the unfolding
of the protein with temperature using Sypro-orange dye. Each Tm was
calculated as the peak maxima of the first derivative of
fluorescence signal across temperature. Aggregation onset was
measured using dynamic light scattering to measure the hydrodynamic
radius as a function of temperature. Finally, the storage stability
of various Doppelmabs was tested by measuring aggregation after 2
weeks at either 40.degree. C. or 5.degree. C. Aggregation was
measured using analytical size exclusion chromatography.
[0818] Molecular modeling was used to identify single point
mutations that could potentially raise the pl of the Fv portion of
Ranibizumab and therefore potentially improve its solubility,
without affecting its conformational stability. This computational
design exercise suggested three framework mutations, namely, VH
E1.fwdarw.Q, VH E6.fwdarw.Q and VL D70G, where VH and VL refer to
the variable portions of the heavy and light chains in Ranibizumab
Fv, respectively. The mutations in heavy chain, VH E1.fwdarw.Q and
VH E6.fwdarw.Q, correspond to the human germline residues at these
positions. The mutation in the light chain VL D70.fwdarw.G is
expected to disrupt a solvent exposed negatively charged patch of
the Ranibizumab Fv.
[0819] The engineering efforts have increased the overall
biophysical properties of the Dopplemabs and translated into
improved CMC properties for the whole molecule. An increase in
percent monomer after protein A was seen for most molecules tested.
This improves the overall manufacturability of the molecules. The
improvements seen in T.sub.m1 and T.sub.agg indicate that the
engineering improved the conformational and colloidal stability of
the molecules. This further translates into a better stability
profile both at 40.degree. C. and 5.degree. C.
TABLE-US-00020 TABLE 19 TPP- TPP- TPP- TPP- TPP- TPP- TPP- 19988
22173 22204 22192 22216 22190 22214 TrkB- TrkB- 1Q6Q70G_TrkB-
1Q6Q70G_TrkB- 1Q6Q70G_TrkB- 1Q70G_TrkB- 1Q70G_TrkB- C2-VL- C2-VL-
C2-VH- C2-VL- C2-VL- C2-VL- C2-VL- VH VH-CC VL_10L1_CC VH_10L1
VH_10L1_CC VH_10L1 VH_10L1_CC Quality % Monomer 70.9 91.49 87.5
74.6 87.2 78.7 87.3 after Protein A Thermal T.sub.m1 (.degree. C.)
60 62.7 60.2 60.1 63.0 59.8 61.9 Shift T.sub.m2 (.degree. C.) 66.7
69.7 70.8 71.4 70.1 68.3 70.8 Assay Aggregation T.sub.agg (.degree.
C.) 54 61 58.0 53.0 62.0 53.0 60.0 Onset Temperature 2 week .DELTA.
% 9.46 0.32 0.1 6.2 0.1 1.6 0.1 stability Monomer at 50 at
40.degree. C. mg/mL .DELTA. % 5.08 0.12 0.0 0.6 0.1 0.8 0.1 Monomer
at 5.degree. C.
Example 62
Determination of Vitreous Half-Life of TPP-22214
[0820] New Zealand white female rabbits received bilateral
intravitreal administration of TPP-22214 or Bevacizumab. Vitreous
samples were collected at various time points and concentrations
for TPP-22214 or Bevacizumab were determined by ELISA. Animals
underwent ocular examination prior and at regular intervals
following intravitreal administration.
TABLE-US-00021 TABLE 20 Targeting TrkB & VEGF VEGF Molecule
TPP-22214 Bevacizumab Bevacizumab Ranibizumab Aflibercept Format
Doppelmab IgG IgG Fab Fusion protein Molecular weight (kDa) 202 149
149 48 115 Vitreous half-life (days) 6.7 4.3 4.3 2.9 3.9
Publication Experimentally Experimentally Bakri, S., Bakri, S.,
Park, S., determined determined Ophthalmology Ophthalmology IOVS,
2007, 114 (5): 2007, 114 (12): 2016, 57 (6), 855-859 2179-82
2613
[0821] Table 20 shows the results of the experimentally determined
vitreous half-life of TPP-22214 and Bevacizumab and compares the
inhouse experimentally determined values with the known literature
values of Bevacizumab, Ranibizumab and Aflibercept. The reported
half-life of Bevacizumab is identical to the
in-house/experimentally derived value confirming the accuracy of
the applied method and the comparability of the data sets.
TPP-22214 was shown to have an in vivo half-life in rabbits which
was approximately 50% higher compared to e.g Bevacizumab (6.7 days
vs 4.3 days).
TABLE-US-00022 TABLE 21 Ocular VEGF (aqueous humor, wet AMD
patients) Hata M, IOVS Cabral T, Int J Hsu, M., Sci Rep Sato T, Sci
Rep 2017, 58 (1): Retin Vitr 2017, Publication 2016, 6:34631 2018;
8:1098 292-298 3:6 VEGF pg/mL pM pg/mL pM pg/mL pM pg/mL pM
concentration 546 28 228 12 90.0 4.7 180 9.4
[0822] Table 21 reports on the literature values of ocular VEGF
concentrations in wet age-related macular degeneration (wAMD)
patients.
TABLE-US-00023 TABLE 22 Dosing interval ofTPP-22214 (calculation
based on the human t.sub.1/2 of Bevacizumab) Species Human Eye
Volume (mL) 4.5 Target Dose in 0.05 mL (mg) 2 Trough concentration
(pM) 400 Number t.sub.1/2 above trough concentration 12.4
Bevacizumab human t.sub.1/2 from literature (days) 9.7 Days above
trough concentration 121 Dosing interval (month) 3.95
[0823] Based on the values shown in Table 21 and just by using the
human intravitreal half-life (t.sub.1/2) of Bevacizumab (9.7 days;
Hutton-Smith, L., Mol. Pharmaceutics, 2016, 13, 2941-2950) a
conservative estimation of the human dosing frequency of TPP-22214
was calculated (Table 21). At a dose of 2 mg/eye a dosing interval
of 4 months is plausible.
Example 63
Comparison of Human TrkB Activation by BDNF, TPP-6830 and the Two
Doppelmabs TPP-23457 and TPP-23459
[0824] FIG. 61 A-B
[0825] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of the natural TrkB ligand
BDNF, TPP-6830 (a further monoclonal TrkB antibody), or two
Doppelmabs TPP-23457 (TPP-6380 as scFv, 10L1, VH-VL; Ranibizumab as
Fab) and TPP-23459 (TPP6380 as scFv, 10L1, VL-VH; Ranibizumab as
Fab).
[0826] The new Doppelmab molecules were now based on a different
TrkB binder (TPP-6830) to evaluate whether the observed effects
with the Doppelmabs based on the C2 TrkB binder could be also
reproduced with a different TrkB binder.
[0827] Results:
[0828] Again, in strong contrast to TPP-6830 (the original TrkB
antibody being a partial TrkB receptor agonists), the two new
Doppelmabs TPP-23457 and TPP-23459 Doppelmabs now showed full TrkB
receptor agonists activity. TrkB activation by those Doppelmabs was
at least as efficacious as the natural ligand BDNF.
[0829] This further supports the theory that the sterical formation
of the binding molecules may be responsible for the observed
increase in efficacy independent of a specific TrkB binder.
Example 64
Comparison of Human TrkB Activation (TrkB Phosphorylation) by
TPP-6830 (TrkB Monoclonal Antibody) and the Two Doppelmabs
TPP-23457 and TPP-23459 in the Presence or Absence of Human
VEGF
[0830] FIG. 62 A-C
[0831] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmabs (A) TPP-23457,
(B) TPP-23459, or (C)TPP-6830 with or without pre-incubation with
200 ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by
measuring TrkB phosphorylation on Y706/707. The lowest compound
concentration was solvent alone. Data represent the mean+/-SEM.
[0832] Results:
[0833] Again, pre-incubation with human VEGF dramatically improved
the potency of TrkB phosphorylation (TrkB activation) by TPP-23457
(TPP-6830 as scFv, 10L1, VH-VL; Ranibizumab as Fab) and TPP-23459
(TPP-6380 as scFv, 10L1, VL-VH; Ranibizumab as Fab).
[0834] VEGF pre-incubation did not impact on the potency of TrkB
phosphorylation (TrkB activation) by TPP-6830 (control).
[0835] This further supports the theory that the observed increase
in potency is independent of a specific TrkB binder.
Example 65
Comparison of Human TrkB Activation (ERK Phosphorylation) by
TPP-6830 (TrkB Monoclonal Antibody) and the Two Doppelmabs
TPP-23457 and TPP-23459 in the Presence or Absence of Human
VEGF
[0836] FIG. 63 A-C
[0837] CHO cells with stable expression of human TrkB were
incubated with growing concentrations of Doppelmabs (A) TPP-23457,
(B) TPP-23459, or (C)TPP-6830 with or without pre-incubation with
200 ng/mL human VEGF-A (hVEGF). TrkB activation was assessed by
measuring ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187
(ERK2), downstream of TrkB. The lowest compound concentration is
solvent alone. Data represent the mean+/-SEM.
[0838] Results:
[0839] Here as well, pre-incubation with human VEGF dramatically
improved the potency of ERK1/2 phosphorylation (TrkB activation) by
TPP-23457 (TPP-6830 as scFv, 10L1, VH-VL; Ranibizumab as Fab) and
TPP-23459 (TPP-6380 as scFv, 10L1, VL-VH; Ranibizumab as Fab).
[0840] As shown previously, VEGF pre-incubation did not impact on
the potency of ERK1/2 phosphorylation (TrkB activation) by TPP-6830
(control) further supporting the theory that the observed increase
in potency is independent of a specific TrkB binder.
Example 66
Impact of TPP-23457 on BDNF-Induced TrkB Activation (TrkB
Phosphorylation
[0841] FIG. 64 A-B
[0842] CHO cells with stable expression of human TrkB receptor were
incubated with growing concentrations of (A) C2 antibody or (B)
Doppelmab TPP-23457 with or without a constant concentration of 0.3
nM, 1 nM or 3 nM BDNF. Activation of TrkB was assessed by measuring
receptor phosphorylation on Y706/707. Data represent the
mean+/-SEM.
[0843] Results:
[0844] In strong contrast to C2, TPP-23457 did not limit
BDNF-induced TrkB activation.
Example 67
Impact of TPP-23457 on BDNF-Induced TrkB Activation (Erk
Phosphorylation
[0845] FIG. 65 A-B
[0846] CHO cells with stable expression of human TrkB receptor were
incubated with growing concentrations of (A) C2 antibody or (B)
Doppelmab TPP-23457 with or without a constant concentration of 0.3
nM, 1 nM or 3 nM BDNF. Activation of TrkB was assessed by measuring
ERK1/2 phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2),
downstream of TrkB. Data represent the mean+/-SEM.
[0847] Results:
[0848] In strong contrast to C2, TPP-23457 did not limit
BDNF-induced Erk phosphorylation downstream of TrkB.
Example 68
Comparison of Human VEGF-A Scavenging by TPP-22214, TPP-23457, or
TPP-23459 Inhibition of VEGF-Induced Phosphorylation of VEGFR2 on
Y1175 and ERK1/2 (Both Related to EC Proliferation)
[0849] FIG. 66 A-B
[0850] Human retinal microvascular endothelial cells (HRMECs) were
starved and then incubated with 50 ng/mL human VEGF with or without
pre-incubation with growing concentrations of TPP-22214, TPP-23457,
or TPP23459. VEGF-A scavenging was assessed by measuring (A) VEGF
receptor 2 (VEGFR2) phosphorylation on Y1175 or (B) ERK1/2
phosphorylation on T202/Y204 (ERK1) and T185/Y187 (ERK2). 50 ng/ml
human VEGF without molecule treatment served as control. Data
represent mean+/-SEM.
[0851] Results:
[0852] VEGF-scavenging of the Doppelmabs TPP-23457 and TPP-23459
was virtually identical to TPP-22215.
[0853] As expected, also Doppelmbab(s) based on a different TrkB
scFvs binder (TPP-6830 and not C2) did not have any impact the
VEGF-scavenging function of the Fabs.
Example 69
Comparison of Different TrkB Binders Identified Via Naive Phage
Display or Via B-Cell to Phage and Formatted Either as IgG, scFv-Fc
or Fc-scFv
[0854] FIG. 67 A-C & 68 A-C
[0855] In total 39 TrkB binders were identified via naive Phage
display (27 hits) or B-cell to Phage (12 hits). Of those, selected
molecules were expressed either as IgG, scFv-Fc or Fc-scFv in order
to analyse the effects of the molecule format on the potency and
efficacy of the TrkB binders.
[0856] For this purpose, the potency (EC.sub.50) and efficacy for
each hit and each of the three molecule formats was determined by
measuring the TrkB phosphorylation (Y706/707) in CHO cells stably
expressing human TrkB receptor after incubation with growing
concentrations of the molecules. BDNF treated cells were measured
as well and the efficacy of BDNF was set to 100% as control.
[0857] FIGS. 67/68 A-C show the pairwise alignment (scatter plots
with line of equality) of the potency or efficacy of the molecules.
The potency is expressed as EC.sub.50 and the efficacy of each
molecule is expressed as percentage in comparison to the natural
ligand BDNF (whereas BDNF is set to 100%). Figure (A) compares the
EC50/efficacy of IgG (x-axis) against scFv-Fc (y-axis) for each
hit, Figure (B) compares the EC50/efficacy of IgG (x-axis) against
Fc-scFv (y-axis) for each hit, and Figure (C) compares the
EC50/efficacy of scFv-Fc (x-axis) against Fc-scFv (y-axis) foreach
hit.
[0858] Result Interpretation:
[0859] If a hit in the pairwise alignments is directly on the line
of equality this is to be interpreted that there is no difference
in EC50 or efficacy between those molecule formats. Hits that shift
either above the line or under the line respectively are indicative
for a change in EC50 or efficacy based on the molecule format. A
clear trend can be observed when comparing the different molecule
formats.
[0860] EC50: IgG.about.scFv-Fc Fc-scFv
[0861] Efficacy: IgG scFv-Fc.about.Fc-scFv
Example 70
Comparison of Different TrkB Binders Identified Via Naive Phage
Display or Via B-Cell to Phage and Formatted Either as IgG, scFv-Fc
or Fc-scFv
[0862] FIG. 69 A-H
[0863] Selected hits identified via naive Phage display or via
B-cell to Phage were formatted either as IgG, scFv-Fc or Fc-scFv.
The efficacy for each hit and each of the three molecule formats
was determined by measuring the TrkB phosphorylation (Y706/707) in
CHO cells stably expressing human TrkB receptor after incubation
with growing concentrations of the molecules. BDNF treated cells
were measured as well and the efficacy of BDNF was set to 1.0 as
control. The molecule concentration is plotted on the x-axis and
the y-axis shows the TrkB phosphorylation relative to BDNF. Figures
A-H show in total 8 selected hits and for each hit the three
different formats IgG, scFv-Fc and Fc-scFv in direct
comparison.
[0864] Result Interpretation:
[0865] Consistent with FIG. 67/68 A-C, the data show a clear trend
that TrkB binders formated either as scFv-Fc or Fc-scFv show a
comparable efficacy of TrkB phosphorylation, which is substantially
higher as compared to the same binders formatted as IgG. Efficacy:
IgG scFv-Fc.about.Fc-scFv. Furthermore, the data show a clear trend
that TrkB binders formated as IgG or scFv-Fc induce TrkB
phosphorylation at compareable EC50 values, which are substantially
lower than EC50 values of the same binders formatted as Fc-scFv.
EC50: IgG.about.scFv-Fc Fc-scFv.
TABLE-US-00024 TABLE_23A FIG. 69 A Best-fit TPP-30881 TPP-31078
TPP-33556 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.0275 0.01428
0.03602 Top 0.7002 1.159 0.9322 LogEC50 -8.109 -8.27 -7.916 EC50
7.779E-09 5.365E-09 1.212E-08 Span 0.6727 1.144 0.8962
TABLE-US-00025 TABLE_23 B FIG. 69 B Best-fit TPP-30883 TPP-31080
TPP-33558 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.02601 0.01949
0.04562 Top 0.3928 0.9257 0.8759 LogEC50 -8.23 -8.173 -7.32 EC50
5.894E-09 6.72E-09 4.781E-03 Span 0.3668 0.9062 0.8303
TABLE-US-00026 TABLE_23C FIG. 69 C Best-fit TPP-29343 TPP-31153
TPP-33633 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.01054 -0.005552
0.03392 Top 0.2948 0.5956 0.6472 LogEC50 -9.695 -9.794 -9.051 EC50
2.018E-10 1.606E-10 8.889E-10 Span 0.2843 0.6012 0.6132
TABLE-US-00027 TABLE_23D FIG. 69 D Best-fit TPP-30882 TPP-31079
TPP-33557 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.02037 0.005932
0.04287 Top 0.2325 0.8548 0.7599 LogEC50 -9.094 -9.115 -8.029 EC50
8.06E-10 7.676E-10 9.358E-09 Span 0.2122 0.8489 0.717
TABLE-US-00028 TABLE_23E FIG. 69 E Best-fit TPP-11734 TPP-30013
TPP-33632 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.01078 0.001248
0.02738 Top 0.4667 0.9764 0.7525 LogEC50 -9.507 -8.394 -8.197 EC50
3.109E-10 4.039E-09 6.35E-09 Span 0.4559 0.9752 0.7251
TABLE-US-00029 TABLE_23F FIG. 69 F Best-fit TPP-30905 TPP-31102
TPP-33580 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.02457 0.01821
0.03792 Top 0.7594 1.163 0.9601 LogEC50 -8.345 -8.562 -7.291 EC50
4.517E-09 2.74E-09 5.123E-08 Span 0.7348 1.145 0.9222
TABLE-US-00030 TABLE_23G FIG. 69 G Best-fit TPP-30870 TPP-31067
TPP-33545 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.02424 0.01671
0.05056 Top 0.2698 0.671 0.536 LogEC50 -8.679 -8.594 -7.418 EC50
2.096E-09 2.547E-09 3.816E-08 Span 0.2456 0.6543 0.4854
TABLE-US-00031 TABLE_23H FIG. 69 H Best-fit TPP-30866 TPP-31063
TPP-33540 values (IgG) (scFv-Fc) (Fc-scFv) Bottom 0.02577 0.01643
0.03682 Top 0.1649 0.8018 1.227 LogEC50 -8.023 -8.08 -6.978 EC50
9.49E-09 8.322E-09 1.053E-07 Span 0.1391 0.7854 1.191
Example 71
Comparison of TrkB Binders Formatted Either as IgG, scFv-Fc or as
Two scFvs Connected to Each Other Via a Peptide Linker
(scFv-Linker-scFv)
[0866] FIG. 71
[0867] A selected TrkB binder was formatted either as IgG, scFv-Fc
or as two scFvs connected to each other via two different peptide
linkers (scFv-linkerl -scFv and scFv-linker2-scFv). The resulting
molecules were tested for their ability to activate TrkB by
measuring the TrkB phosphorylation (Y706/707) in CHO cells stably
expressing human TrkB receptor after incubation with growing
concentrations of the molecules. BDNF treated cells were measured
as well and served as reference. The molecule concentration is
plotted on the x-axis and the y-axis shows the TrkB
phosphorylation. The lowest compound concentration was solvent
alone. Data represent mean.+-.SEM.
[0868] Result Interpretation
[0869] Consistent with previous results, the efficacy of TrkB
phosphorylation by TrkB binders formatted into a scFv-Fc is
substantially higher than that of the corresponding IgG.
Importantly, efficacy of TrkB phosphorylation by TrkB binders
formatted as two scFvs connected to each other via two different
peptide linkers (scFv-linker1-scFv and scFv-linker2-scFv) was
comparable to the efficacy of the scFv-Fc format and thus
substantially higher than the IgG. This demonstrates that the
increased TrkB phosphorylation efficacy by scFv's is not limited to
scFv-Fc formats but can also be achieved by connecting two scFv's
with different peptide linkers.
Example 72
Comparison of Different TrkB Binder Formats IgG vs. scFv-Fv and
scFv-hinge-scFv
[0870] FIG. 72
[0871] A selected TrkB binder was formatted either as IgG or
scFv-Fc. In addition, the scFv-Fc was cleaved with the cysteine
protease FabRICATOR.RTM. enzyme (Genovis) below the hinge to remove
the Fc portion, creating two scFv's connected to each other via the
hinge region (scFv-hinge-scFv). All three molecules were tested for
their ability to induce TrkB phosphorylation on Y706/707.
[0872] Result Interpretation
[0873] Consistent with previous results, the efficacy of TrkB
phosphorylation by TrkB binders formatted into a scFv-Fc is
substantially higher than that of the corresponding IgG.
Importantly, efficacy of TrkB phosphorylation induced by the
cleaved scFv-Fc (scFv-hinge-scFv) was comparable to the efficacy of
the scFv-Fc format and thus substantially higher than the
corresponding IgG. This demonstrates that the increased TrkB
phosphorylation efficacy by scFv's is not limited to scFv-Fc
formats but can also be achieved by connecting two scFv's with the
hinge in the absence of the Fc fragment.
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=US20220119536A1).
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=US20220119536A1).
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