U.S. patent application number 16/985477 was filed with the patent office on 2021-05-13 for treatment of ophthalmologic diseases.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Genentech, Inc., Hoffmann-La Roche Inc.. Invention is credited to Aaron Osborne, Jayashree Sahni, Robert James Weikert.
Application Number | 20210139576 16/985477 |
Document ID | / |
Family ID | 1000005384510 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210139576 |
Kind Code |
A1 |
Osborne; Aaron ; et
al. |
May 13, 2021 |
TREATMENT OF OPHTHALMOLOGIC DISEASES
Abstract
The current invention relates to the use of antibodies which
bind to VEGF and ANG2 for the treatment of ophthalmologic
diseases.
Inventors: |
Osborne; Aaron; (South San
Francisco, CA) ; Sahni; Jayashree; (Basel, CH)
; Weikert; Robert James; (Basel, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc.
Genentech, Inc. |
Little Falls
South San Francisco |
NJ
CA |
US
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000005384510 |
Appl. No.: |
16/985477 |
Filed: |
August 5, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2019/052704 |
Feb 5, 2019 |
|
|
|
16985477 |
|
|
|
|
62627103 |
Feb 6, 2018 |
|
|
|
62729333 |
Sep 10, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/31 20130101;
C07K 2317/565 20130101; C07K 16/22 20130101; A61K 2039/545
20130101; A61K 2039/505 20130101; A61P 27/02 20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; A61P 27/02 20060101 A61P027/02 |
Claims
1. A method of treating a patient suffering from an ocular vascular
disease the method comprising administering to the patient an
effective amount of a bispecific antibody which binds to human
vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2), wherein the bispecific antibody is
administered intravitreally every 8 weeks or less frequently.
2. A method of treating a patient suffering from an ocular vascular
disease the method comprising administering to the patient an
effective amount of a bispecific antibody which binds to human
vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2), wherein the patient gains 12 or more
letters of Best Corrected Visual Acuity (BCVA) measured using Early
Treatment Diabetic Retinopathy Study (ETDRS) like charts, compared
to the patient's BCVA letter score prior to the dosing of the
bispecific VEGF/ANG2 antibody.
3. The method according to claim 2, wherein the bispecific antibody
is administered intravitreally every 8 weeks or less
frequently.
4. The method according to any one of claims 1 to 3, wherein the
gain of letters in the BCVA/ETDRS letter score is measured at 4
weeks, and/or at 8 weeks, and/or at 12 weeks, and/or at 16 weeks,
and/or at 20 weeks, and/or at 24 weeks after treatment start,
respectively.
5. The method according to any one of claims 1 to 3, wherein the
gain of letters in the BCVA/ETDRS letter score is measured at 45
weeks, and/or at 46 weeks, and/or at 47 weeks, and/or at 48 weeks,
and/or at 49 weeks, and/or at 50 weeks, and/or at 51 weeks, and/or
at 52 weeks, and/or at 53 weeks, and/or at 54 weeks, and/or at 55
weeks, and/or at 56 weeks, and/or at 57 weeks, and/or at 58 weeks,
and/or at 59 weeks, and/or at 60 weeks after treatment start,
respectively.
6. The method according to any one of claims 1 to 5, wherein the
bispecific antibody is used to prolong the time to retreatment
and/or to prolong the time to loss of visual acuity and, wherein
the retreatment with the bispecific antibody is administered in
case of a disease activity which is determined as Central Subfield
Thickness (CST) increase by .gtoreq.50 .mu.m; and/or Best Corrected
Visual Acuity (BCVA/ETDRS) decrease by .gtoreq.5 letters.
7. The method according to any one of claims 1 to 6, wherein the
bispecific antibody is administered following a treatment
initiation of 3 to 7 monthly administrations.
8. The method according to any one of claims 1 to 7, wherein the
ocular vascular disease is selected from the group of: wet
age-related macular degeneration (wet AMD), neovascular AMD,
diabetic macular edema (DME), cystoid macular edema (CME),
non-proliferative diabetic retinopathy (NPDR), proliferative
diabetic retinopathy (PDR), macular edema secondary to central
retinal vein occlusion, secondary to hemiretinal vein occlusion or
secondary to branch vein occlusion, retinitis, conjunctivitis,
uveitis, choroiditis, choroidal neovascularization (CNV) secondary
to ocular inflammation including secondary to ocular histoplasmosis
or presumed histoplasmosis or choroiditis; myopic choroidal
neovascularization (mCNV), choroidal neovascularization secondary
to trauma, retinopathy of prematurity and rubeosis iridis/rubeotic
glaucoma.
9. The method according to of claim 8, wherein the ocular vascular
disease is DME.
10. The method according to claim 8, wherein the ocular vascular
disease is wet AMD or neovascular AMD.
11. The method according to any one of claims 1 to 10, wherein the
a bispecific antibody which binds to VEGF and to human ANG-2 is a
VEGF antagonist/inhibitor and an ANG2 antagonist/inhibitor or
inhibits binding of VEGF to its receptor VEGFR and inhibits binding
of ANG2 to its receptor TIE2.
12. The method according to any one of claims 1 to 11, wherein the
bispecific antibody is administered every 10 to 12 weeks.
13. The method according to any one of claims 1 to 11, wherein the
bispecific antibody is administered every 11 to 13 weeks
14. The method according to any one of claims 1 to 11, wherein the
bispecific antibody is administered every 12 to 14 weeks.
15. The method according to any one of claims 1 to 11, wherein the
bispecific antibody is administered every 13 to 15 weeks.
16. The method according to any one of claims 1 to 11, wherein the
bispecific antibody is administered every 14 to 16 weeks.
17. The method according to any one of claims 1 to 16, wherein the
bispecific antibody which binds to human VEGF and to human ANG2 is
a bispecific, bivalent anti-VEGF/ANG2 antibody comprising a first
antigen-binding site that specifically binds to human VEGF and a
second antigen-binding site that specifically binds to human ANG-2,
wherein i) said first antigen-binding site specifically binding to
VEGF comprises in the heavy chain variable domain a CDR3H region of
SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a CDR1H region of
SEQ ID NO:3, and in the light chain variable domain a CDR3L region
of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and a CDR1L region
of SEQ ID NO:6; and ii) said second antigen-binding site
specifically binding to ANG-2 comprises in the heavy chain variable
domain a CDR3H region of SEQ ID NO: 9, a CDR2H region of, SEQ ID
NO: 10, and a CDR1H region of SEQ ID NO: 11, and in the light chain
variable domain a CDR3L region of SEQ ID NO: 12, a CDR2L region of
SEQ ID NO: 13, and a CDR1L region of SEQ ID NO: 14, and wherein
iii) the bispecific antibody comprises a constant heavy chain
region of human IgG1 subclass comprising the mutations I253A,
H310A, and H435A and the mutations L234A, L235A and P329G
(numberings according to EU Index of Kabat).
18. The method according to claim 17, wherein i) said first
antigen-binding site specifically binding to VEGF comprises as
heavy chain variable domain VH an amino acid sequence of SEQ ID NO:
7, and as light chain variable domain VL an amino acid sequence of
SEQ ID NO: 8, and ii) said second antigen-binding site specifically
binding to ANG-2 comprises as heavy chain variable domain VH an
amino acid sequence of SEQ ID NO: 15, and as light chain variable
domain VL an amino acid sequence of SEQ ID NO: 16.
19. The method according to claim 18, wherein the bispecific
antibody which binds to human VEGF and human ANG2 comprises the
amino acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID
NO: 19, and of SEQ ID NO: 20.
20. The method according to any one of claims 17 to 19, wherein the
bispecific antibody is administered in a dose of about 5 to 7
mg.
21. The method according to any one of claims 17 to 19, wherein the
bispecific antibody is administered in a dose of about 6 mg.
22. The method according to claim 20 or 21, wherein the bispecific
antibody is administered at a concentration of about 30 mg/ml.
23. The method according to claim 20 or 21, wherein the bispecific
antibody is administered at a concentration of about 120 mg/ml.
24. The method according to any one of the claims 1 to 23 wherein
patients suffering from an ocular vascular disease have not been
previously treated with anti-VEGF treatment (e.g monotherapy).
25. The method according to any one of claims 1 to 23 wherein
patients suffering from an ocular vascular disease have been
previously treated with anti-VEGF treatment (e.g monotherapy).
26. The method according to any one of the preceding claims wherein
the ocular vascular disease is DME and the treatment of patients
suffering from DME includes a fixed every 8th week (Q8W) dosing
schedule following treatment initiation.
27. The method according to any one of the preceding claims wherein
the ocular vascular disease is DME and the treatment of patients
suffering from DME includes a fixed Q12W dosing schedule following
treatment initiation.
28. The method according to claim 27 wherein, following the
treatment initiation, first one dose cycle of Q8W follows before
the fixed Q12W dosing schedule.
29. The method according to any one of the preceding claims wherein
the ocular vascular disease is DME and the treatment of patients
suffering from DME includes following treatment initiation a dosing
schedule that extends the administration interval in stable absence
of disease, or shortens the interval if there is disease
activity.
30. The method according to claim 29 wherein such dosing schedule
includes that the patient receives Q8W or Q12W or Q16W dosing,
dependent on their disease state.
31. The method according to claim 28 or 29, wherein the stable
absence of disease is determined as Central Subfield Thickness
(CST) increased by <50 .mu.m; and/or Best Corrected Visual
Acuity (BCVA/ETDRS) decreased by <5 letters and the disease
activity is determined as Central Subfield Thickness (CST)
increased by .gtoreq.50 .mu.m; and/or Best Corrected Visual Acuity
(BCVA/ETDRS) decreased by .gtoreq.5 letters.
32. The method according to claim 28 or 29, wherein the stable
absence of disease is determined as Central Subfield Thickness
(CST) is below about 300 .mu.m (In one embodiment below 325 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Spectralis.TM. device; in one embodiment below 315 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Cirrus' device; in one embodiment below 315 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Topcon.TM. device; in one embodiment below 295 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Optovue.TM. device), and the disease activity is determined as
Central Subfield Thickness (CST) is above about 300 .mu.m (In one
embodiment above 325 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Spectralis.TM. device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Cirrus.TM. device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
33. The method according to any one of the preceding claims wherein
the treatment of patients suffering from AMD includes following
treatment initiation a dosing schedule that extends the
administration interval in stable absence of disease, or shortens
the interval if there is disease activity.
34. The method according to claim 33 wherein such dosing schedule
includes that the patient receives Q8W or Q12W or Q16W dosing,
dependent on their disease state.
35. The method according to claim 33 or 34, wherein the stable
absence of disease is determined as Central Subfield Thickness
(CST) increased by <50 .mu.m; and/or Best Corrected Visual
Acuity (BCVA/ETDRS) decreased by <5 letters and the disease
activity is determined as Central Subfield Thickness (CST)
increased by .gtoreq.50 .mu.m; and/or Best Corrected Visual Acuity
(BCVA/ETDRS) decreased by .gtoreq.5 letters.
36. The method according to claim 33 or 34, wherein the stable
absence of disease is determined as Central Subfield Thickness
(CST) is below about 300 .mu.m (In one embodiment below 325 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Spectralis.TM. device; in one embodiment below 315 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Cirrus.TM. device; in one embodiment below 315 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Topcon.TM. device; in one embodiment below 295 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Optovue.TM. device), and the disease activity is determined
as Central Subfield Thickness (CST) is above about 300 .mu.m (In
one embodiment above 325 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Spectralis.TM. device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Cirrus.TM. device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/EP2019/052704, filed Feb. 5, 2019, claiming
priority to provisional Application No. 62/627,103 filed Feb. 6,
2018 and provisional Application No. 62/729,333, filed Sep. 10,
2018, which are incorporated herein by reference in their
entireties.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Jul. 15,
2020, is named Sequence_Listing.txt and is 44,608 bytes in
size.
[0003] The current invention relates to the use of antibodies which
bind to VEGF and ANG2 for the treatment of ophthalmologic
diseases.
BACKGROUND OF THE INVENTION
[0004] Angiogenesis is implicated in the pathogenesis of a variety
of disorders which include solid tumors, intraocular neovascular
syndromes such as proliferative retinopathies or age-related
macular degeneration (AMD), rheumatoid arthritis, and psoriasis
(Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934;
Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217-239; and
Gamer, A., Vascular diseases, in: Pathobiology of ocular disease, A
dynamic approach, Garner, A., and Klintworth, G. K. (eds.), 2nd
edition, Marcel Dekker, New York (1994), pp. 1625-1710).
[0005] Ranibizumab (trade name Lucentis.RTM.) is a monoclonal
antibody fragment derived from the same parent murine antibody as
bevacizumab (Avastin.RTM.). However, it has been affinity matured
to provide stronger binding to VEGF-A (WO 98/45331). It is known
that systemic blockade of VEGF-A is associated with an increased
risk of certain adverse events, therefore ranibizumab is missing an
Fc part in order to reduce systemic exposure and the risk of
systemic toxicities. It is an anti-angiogenic agent that has been
approved to treat the "wet" type of age-related macular
degeneration (neovascular AMD), a common form of age-related vision
loss.
[0006] Corneal angiogenesis assays have shown that both ANG-1 and
ANG-2 had similar effects, acting synergistically with VEGF to
promote growth of new blood vessels. Asahara, T., et al., Circ.
Res. 83 (1998) 233-40. The possibility that there was a
dose-dependent endothelial response was raised by the observation
that in vitro at high concentration, ANG-2 can also be
pro-angiogenic (Kim, I., et al., Oncogene 19 (2000) 4549-52). At
high concentration, ANG-2 acts as an apoptosis survival factor for
endothelial cells during serum deprivation apoptosis through
activation of Tie2 via PI-3 Kinase and Akt pathway (Kim, I., et
al., Oncogene 19 (2000) 4549-52).
[0007] Ocular vascular diseases such as "wet" age related macular
degeneration (AMD) and proliferative diabetic retinopathy (PDR),
are due to abnormal choroidal or retinal neovascularization
respectively. Bleeding and leakage from these vessels can cause
retinal dysfunction and loss of cision Other retinal vascular
disease, such as diabetic macular edema (DME) and macular edema
secondary to retinal vein occlusion (RVO) are due to abnormal
retinal leakage leading to retinal swelling and impairing visual
function. These conditions are leading causes of visual loss in
industrialized nations. Since the retina consists of well-defined
layers of neuronal, glial, and vascular elements, relatively small
disturbances such as those seen in vascular proliferation or edema
can lead to significant loss of visual function. Inherited retinal
degenerations, such as Retinitis Pigmentosa (RP), are also
associated with vascular abnormalities, such as arteriolar
narrowing and vascular atrophy. They affect as many as 1 in 3500
individuals and are characterized by progressive night blindness,
visual field loss, optic nerve atrophy, arteriolar attenuation, and
central loss of vision often progressing to complete blindness.
[0008] Ischemic retinopathies are characterized by loss or
dysfunction of the retinal vasculature which results in a reduction
of blood flow and hypoxia. The retina responds to hypoxia by
generating signals to grow new blood vessels, but these new vessels
are usually fragile and disorganized. It is the growth of these
abnormal new vessels that creates most of the threat to vision
since they can leak, hemorrhage or lead to scarring that may end in
retinal detachment. Current treatments for ischemic retinopathies
seek to halt the growth of the pathological vessels but do not
address the underlying ischemia that drives their growth.
Furthermore, standard treatment for diabetic retinopathy, an
ischemic retinopathy that affects millions, involves destruction of
a portion of the retina with a laser in an attempt destroy ischemic
tissue in order to to stop new vessel growth and preserve central
vision. Strategies have been employed to block the function of
vascular endothelial growth factor (VEGF), a major promoter of
abnormal vessel growth and leakage. In the short term, anti-VEGF
therapy can improve vision, but it does not address the underlying
ischemia and in fact may exacerbate this condition as it inhibits
all vessel growth, including beneficial collaterals. There is also
the serious concern of systemic exposure of these drugs in elderly
and/or diabetic patients where new vessel growth may be required in
ischemic brains, hearts or limbs.
SUMMARY OF THE INVENTION
[0009] According to one aspect of the present invention, methods,
uses, bispecific antibodies (for use), medicaments or
pharmaceutical formulations are provided for the treatment of
patients suffering from an ocular vascular disease the method
comprising administering to the patient an effective amount of a
bispecific antibody which binds to human vascular endothelial
growth factor (VEGF) and to human angiopoietin-2 (ANG-2), [0010]
wherein the bispecific antibody is administered (is to be
administered) intravitreally every 8 weeks or less frequently (in
one embodiment every 9 weeks or less frequently; in one embodiment
every 10 weeks or less frequently; in one embodiment every 11 weeks
or less frequently; in one embodiment every 12 weeks or less
frequently; in one embodiment every 13 weeks or less frequently; in
one embodiment every 14 weeks or less frequently; in one embodiment
every 15 weeks or less frequently in one embodiment every 16 weeks
or less frequently).
[0011] One aspect of the invention is such method, use, bispecific
antibody (for use), medicament or pharmaceutical formulation (for
use) of/for treating a patient suffering from an ocular vascular
disease the method, use, bispecific antibody (for use), medicament
or pharmaceutical formulation (for use) comprising administering
(intravitreally) to the patient an effective amount of a bispecific
antibody which binds to human vascular endothelial growth factor
(VEGF) and to human angiopoietin-2 (ANG-2), wherein the patient
gains 12 or more letters (in one embodiment 13 or more letters, in
one embodiment 14 or more letters, in one embodiment 15 or more
letters) of Best Corrected Visual Acuity (BCVA) measured using
Early Treatment Diabetic Retinopathy Study (ETDRS) like charts,
compared to the patient's BCVA letter score prior to the dosing of
the bispecific VEGF/ANG2 antibody. In one embodiment the bispecific
antibody is administered intravitreally every 8 weeks or less
frequently. One embodiment of the invention is a method of treating
a patient suffering from an ocular vascular disease the method
comprising administering (intravitreally) to the patient an
effective amount of a bispecific antibody which binds to human
vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2), wherein the patient experiences an
improvement in vision subsequent to the administration of the
bispecific VEGF/ANG2 antibody as measured by gaining 12 or more
letters (in one embodiment 13 or more letters, in one embodiment 14
or more letters, in one embodiment 15 or more letters) of Best
Corrected Visual Acuity (BCVA) measured using Early Treatment
Diabetic Retinopathy Study (ETDRS) like charts, compared to the
patient's BCVA letter score prior to the dosing of the bispecific
VEGF/ANG2 antibody. In one embodiment the bispecific antibody is
administered intravitreally every 8 weeks or less frequently.
[0012] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively.
[0013] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 24 weeks, and/or at 25
weeks, and/or at 26 weeks, and/or at 27 weeks, and/or at 28 weeks,
and/or at 29 weeks, and/or at 30 weeks, and/or at 31 weeks, and/or
at 32 weeks, and/or at 33 weeks, and/or at 34 weeks, and/or at 35
weeks, and/or at 36 weeks, and/or at 37 weeks, and/or at 38 weeks,
and/or at 39 weeks, and/or at 40 weeks, and/or at 41 weeks, and/or
at 42 weeks, and/or at 43 weeks, and/or at 44 weeks, and/or at 45
weeks, and/or at 46 weeks, and/or at 47 weeks, and/or at 48 weeks,
and/or at 49 weeks, and/or at 50 weeks, and/or at 51 weeks, and/or
at 52 weeks, and/or at 53 weeks, and/or at 54 weeks, and/or at 55
weeks, and/or at 56 weeks, and/or at 57 weeks, and/or at 58 weeks,
and/or at 59 weeks, and/or at 60 weeks after treatment start,
respectively. In one embodiment of the invention the ocular
vascular disease is selected from the group of: wet age-related
macular degeneration (wet AMD), neovascular AMD, diabetic macular
edema (DME), cystoid macular edema (CME), non-proliferative
diabetic retinopathy (NPDR), proliferative diabetic retinopathy
(PDR), macular edema secondary to central retinal vein occlusion,
secondary to hemiretinal vein occlusion or secondary to branch vein
occlusion, retinitis, conjunctivitis, uveitis, choroiditis,
choroidal neovascularization (CNV) secondary to ocular inflammation
including secondary to ocular histoplasmosis or presumed
histoplasmosis or choroiditis; myopic choroidal neovascularization
(mCNV). And choroidal neovascularization secondary to trauma,
retinopathy of prematurity and rubeosis iridis/rubeotic
glaucoma.
[0014] In one embodiment of the invention the ocular vascular
disease is diabetic macular edema (DME).
[0015] In one embodiment of the invention the ocular vascular
disease is diabetic macular edema (DME) and the gain of letters in
the BCVA/ETDRS letter score is measured at about 9 to 15 month (in
one embodiment at 9 to 14 month, in one embodiment at 9 to 12
month) after treatment start.
[0016] In one embodiment of the invention the ocular vascular
disease is diabetic macular edema (DME) and the gain of letters in
the BCVA/ETDRS letter score is measured at 36 weeks, and/or at 37
weeks, and/or at 38 weeks, and/or at 39 weeks, and/or at 40 weeks,
and/or at 41 weeks, and/or at 42 weeks, and/or at 43 weeks, and/or
at 44 weeks, and/or at 45 weeks, and/or at 46 weeks, and/or at 47
weeks, and/or at 48 weeks, and/or at 49 weeks, and/or at 50 weeks,
and/or at 51 weeks, and/or at 52 weeks, and/or at 53 weeks, and/or
at 54 weeks, and/or at 55 weeks, and/or at 56 weeks, and/or at 57
weeks, and/or at 58 weeks, and/or at 59 weeks, and/or at 60 weeks
after treatment start, respectively.
[0017] These time points are quite early, typically maximum gains
are not reached until about month 6-9 in nAMD and m 9-12 in DME
[0018] In one embodiment of the invention the ocular vascular
disease is wet age-related macular degeneration (wet AMD) (, or
neovascular age-related macular degeneration (nAMD).
[0019] In one embodiment of the invention the ocular vascular
disease is wet age-related macular degeneration (wet AMD) (, or
neovascular age-related macular degeneration (nAMD) and the gain of
letters in the BCVA/ETDRS letter score is measured at about 9 to 15
month (in one embodiment at 6 to 9 month, in one embodiment at 6 to
12 month) after treatment start.
[0020] In one embodiment of the invention the ocular vascular
disease is wet age-related macular degeneration (wet AMD) (, or
neovascular age-related macular degeneration (nAMD) and the gain of
letters in the BCVA/ETDRS letter score is measured at 24 weeks,
and/or at 25 weeks, and/or at 26 weeks, and/or at 27 weeks, and/or
at 28 weeks, and/or at 29 weeks, and/or at 30 weeks, and/or at 31
weeks, and/or at 32 weeks, and/or at 33 weeks, and/or at 34 weeks,
and/or at 35 weeks, and/or at 36 weeks, and/or at 37 weeks, and/or
at 38 weeks, and/or at 39 weeks, and/or at 40 weeks, and/or at 41
weeks, and/or at 42 weeks, and/or at 43 weeks, and/or at 44 weeks,
and/or at 45 weeks, and/or at 46 weeks, and/or at 47 weeks, and/or
at 48 weeks, and/or at 49 weeks, and/or at 50 weeks, and/or at 51
weeks, and/or at 52 weeks, and/or at 53 weeks, after treatment
start, respectively.
[0021] In one embodiment of the invention the bispecific antibody
which binds to human VEGF and to human ANG2 is a bispecific,
bivalent anti-VEGF/ANG2 antibody comprising a first antigen-binding
site that specifically binds to human VEGF and a second
antigen-binding site that specifically binds to human ANG-2,
wherein [0022] i) said first antigen-binding site specifically
binding to VEGF comprises in the heavy chain variable domain a
CDR3H region of SEQ ID NO: 1, a CDR2H region of SEQ ID NO: 2, and a
CDR1H region of SEQ ID NO:3, and in the light chain variable domain
a CDR3L region of SEQ ID NO: 4, a CDR2L region of SEQ ID NO:5, and
a CDR1L region of SEQ ID NO:6; and [0023] ii) said second
antigen-binding site specifically binding to ANG-2 comprises in the
heavy chain variable domain a CDR3H region of SEQ ID NO: 9, a CDR2H
region of, SEQ ID NO: 10, and a CDR1H region of SEQ ID NO: 11, and
in the light chain variable domain a CDR3L region of SEQ ID NO: 12,
a CDR2L region of SEQ ID NO: 13, and a CDR1L region of SEQ ID NO:
14, and wherein [0024] iii) the bispecific antibody comprises a
constant heavy chain region of human IgG1 subclass comprising the
mutations I253A, H310A, and H435A and the mutations L234A, L235A
and P329G (numberings according to EU Index of Kabat).
[0025] In one embodiment of the invention the patients suffering
from an ocular vascular disease have not been previously treated
with anti-VEGF treatment (e.g monotherapy)(are treatment
naive).
[0026] In one embodiment of the invention the patients suffering
from an ocular vascular disease have been previously treated with
anti-VEGF treatment (e.g monotherapy).
[0027] In one embodiment of the invention the ocular vascular
disease is DME and the treatment of patients suffering from DME
includes a fixed every 8.sup.th week (Q8W) dosing schedule
following treatment initiation.
[0028] In one embodiment of the invention the ocular vascular
disease is DME and the treatment of patients suffering from DME
includes a fixed Q12W dosing schedule following treatment
initiation. In one embodiment of the invention following the
treatment initiation, first one dose cycle of Q8W follows before
the fixed Q12W dosing schedule.
[0029] In one embodiment of the invention the ocular vascular
disease is DME and the treatment of patients suffering from DME
includes following treatment initiation a dosing schedule that
extends the administration interval in stable absence of disease,
or shortens the interval if there is disease activity. In one
embodiment of the invention such dosing schedule includes that the
patient receives Q4W or Q8W or Q12W or Q16W dosing, dependent on
their disease state. In one embodiment of the invention the stable
absence of disease is determined as [0030] Central Subfield
Thickness (CST) increased by <50 .mu.m; and/or [0031] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by <5 letters and
the disease activity is determined as [0032] Central Subfield
Thickness (CST) increased by .gtoreq.50 .mu.m; and/or [0033] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by .gtoreq.5
letters.
[0034] In one embodiment of the invention the ocular vascular
disease is AMD and the treatment of patients suffering from AMD
includes following treatment initiation a dosing schedule that
extends the administration interval in stable absence of disease,
or shortens the interval if there is disease activity. In one
embodiment of the invention such dosing schedule includes that the
patient receives Q4W or Q8W or Q12W or Q16W dosing, dependent on
their disease state. In one embodiment of the invention the stable
absence of disease is determined as [0035] Central Subfield
Thickness (CST) increased by <50 .mu.m; and/or [0036] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by <5 letters and
the disease activity is determined as [0037] Central Subfield
Thickness (CST) increased by .gtoreq.50 .mu.m; and/or [0038] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by .gtoreq.5
letters.
DESCRIPTION OF THE FIGURES
[0039] FIG. 1: BCVA change of DME patients treated from Baseline
over Time to Week 24 (treatment naive patients). VA2 refers to the
bispecific anti-VEGF/ANG2 antibody RO6867461 comprising the amino
acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO:
19, and of SEQ ID NO: 20 (administered intravitreally with a 6.0 mg
or 1.5 mg dose), RBZ refers to ranibizumab (Lucentis.RTM.)
((administered intravitreally with a 0.3 mg dose))
[0040] FIG. 2: CST, central subfield thickness measured by SD OCT.
CST change of DME patients treated from Baseline over Time to Week
24 (treatment naive patients). The bispecific anti-VEGF/ANG2
antibody RO6867461 comprising the amino acid sequences of SEQ ID
NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20
(administered intravitreally with a 6.0 mg or 1.5 mg dose), was
compared to ranibizumab (Lucentis.RTM.) ((administered
intravitreally with a 0.3 mg dose)).
[0041] FIG. 3: Time to necessary retreatment based on disease
activity assessed by both: BCVA decreased by .gtoreq.5 letters and
CST increased by .gtoreq.50 .mu.m (after dosing has discontinued
(after 20 weeks or 6 monthly doses=Time post last intravitreal
(IVT) administration). The bispecific anti-VEGF/ANG2 antibody
RO6867461 comprising the amino acid sequences of SEQ ID NO: 17, of
SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20 (administered
intravitreally with a 6.0 mg or 1.5 mg dose), was compared to
ranibizumab (Lucentis.RTM.) ((administered intravitreally with a
0.3 mg dose)).
[0042] FIG. 4: Schematic comparison to other treatment options of
DME based on published results (Compared agents Lucentis.RTM.
(ranibizumab), Eylea.RTM. (aflibercept), brolucizumab and VA2
(RO6867461/RG7716).
[0043] FIG. 5: Overview of the study design for the evaluation of
the bispecific antibody RO6867461 administered at 12- and 16-week
intervals in patients with neovascular age-related macular
degeneration (nAMD).
[0044] FIG. 6: BCVA gains from baseline of patients with
neovascular age-related macular degeneration (nAMD) comparing the
bispecific antibody RO6867461 (comprising the amino acid sequences
of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID
NO: 20 (administered intravitreally with a 6.0 mg) at 12- and
16-week intervals and ranibizumab (Lucentis.RTM.) ((administered
intravitreally with a 0.3 mg dose)) at 4-week intervals.
[0045] FIG. 7: Change from baseline CST (measured via OCT) of
patients with neovascular age-related macular degeneration (nAMD)
comparing the bispecific antibody RO6867461 (comprising the amino
acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO:
19, and of SEQ ID NO: 20 (administered intravitreally with a 6.0
mg) at 12- and 16-week intervals and ranibizumab (Lucentis.RTM.)
((administered intravitreally with a 0.3 mg dose)) at 4-week
intervals.
DETAILED DESCRIPTION OF THE INVENTION
[0046] According to one aspect of the present invention, methods
are provided for the treatment of patients suffering from an ocular
vascular disease the method comprising administering to the patient
an effective amount of a bispecific antibody which binds to human
vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2), [0047] wherein the bispecific antibody is
administered (is to be administered) intravitreally every 8 weeks
or less frequently (in one embodiment every 9 weeks or less
frequently; in one embodiment every 10 weeks or less frequently; in
one embodiment every 11 weeks or less frequently; in one embodiment
every 12 weeks or less frequently; in one embodiment every 13 weeks
or less frequently; in one embodiment every 14 weeks or less
frequently; in one embodiment every 15 weeks or less
frequently).
[0048] One embodiment of the invention is a method of treating a
patient suffering from a ocular vascular disease the method
comprising administering to the patient an effective amount of a
bispecific antibody which binds to human vascular endothelial
growth factor (VEGF) and to human angiopoietin-2 (ANG-2), wherein
the patient gains 12 or more letters (in one embodiment 13 or more
letters, in one embodiment 14 or more letters, in one embodiment 15
or more letters) of Best Corrected Visual Acuity (BCVA) measured
using Early Treatment Diabetic Retinopathy Study (ETDRS) like
charts, compared to the patient's BCVA letter score prior to the
dosing of the bispecific VEGF/ANG2 antibody. In one embodiment the
bispecific antibody is administered (is to be administered)
intravitreally every 8 weeks or less frequently (in one embodiment
every 9 weeks or less frequently; in one embodiment every 10 weeks
or less frequently; in one embodiment every 11 weeks or less
frequently; in one embodiment every 12 weeks or less frequently; in
one embodiment every 13 weeks or less frequently; in one embodiment
every 14 weeks or less frequently; in one embodiment every 15 weeks
or less frequently).
[0049] One embodiment of the invention is a method of treating a
patient suffering from a ocular vascular disease the method
comprising administering to the patient an effective amount of a
bispecific antibody which binds to human vascular endothelial
growth factor (VEGF) and to human angiopoietin-2 (ANG-2), wherein
the patient experiences an improvement in vision subsequent to the
administration of the bispecific VEGF/ANG2 antibody as measured by
gaining 12 or more letters (in one embodiment 13 or more letters,
in one embodiment 14 or more letters, in one embodiment 15 or more
letters) of Best Corrected Visual Acuity (BCVA) measured using
Early Treatment Diabetic Retinopathy Study (ETDRS) like charts,
compared to the patient's BCVA letter score prior to the dosing of
the bispecific VEGF/ANG2 antibody. In one embodiment the bispecific
antibody is administered (is to be administered) intravitreally
every 8 weeks or less frequently (in one embodiment every 9 weeks
or less frequently; in one embodiment every 10 weeks or less
frequently; in one embodiment every 11 weeks or less frequently; in
one embodiment every 12 weeks or less frequently; in one embodiment
every 13 weeks or less frequently; in one embodiment every 14 weeks
or less frequently; in one embodiment every 15 weeks or less
frequently).
[0050] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively.
[0051] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 24 weeks, and/or at 25
weeks, and/or at 26 weeks, and/or at 27 weeks, and/or at 28 weeks,
and/or at 29 weeks, and/or at 30 weeks, and/or at 31 weeks, and/or
at 32 weeks, and/or at 33 weeks, and/or at 34 weeks, and/or at 35
weeks, and/or at 36 weeks, and/or at 37 weeks, and/or at 38 weeks,
and/or at 39 weeks, and/or at 40 weeks, and/or at 41 weeks, and/or
at 42 weeks, and/or at 43 weeks, and/or at 44 weeks, and/or at 45
weeks, and/or at 46 weeks, and/or at 47 weeks, and/or at 48 weeks,
and/or at 49 weeks, and/or at 50 weeks, and/or at 51 weeks, and/or
at 52 weeks, and/or at 53 weeks, and/or at 54 weeks, and/or at 55
weeks, and/or at 56 weeks, and/or at 57 weeks, and/or at 58 weeks,
and/or at 59 weeks, and/or at 60 weeks after treatment start,
respectively. In one embodiment of the invention the gain of
letters in the BCVA/ETDRS letter score is measured at 45 weeks,
and/or at 46 weeks, and/or at 47 weeks, and/or at 48 weeks, and/or
at 49 weeks, and/or at 50 weeks, and/or at 51 weeks, and/or at 52
weeks, and/or at 53 weeks, and/or at 54 weeks, and/or at 55 weeks,
and/or at 56 weeks, and/or at 57 weeks, and/or at 58 weeks, and/or
at 59 weeks, and/or at 60 weeks after treatment start,
respectively.
[0052] In one embodiment of the invention the method is used to
prolong the time to retreatment and/or to prolong the time to loss
of visual acuity and, wherein the retreatment with the bispecific
antibody is administered in case of a disease activity which is
determined as [0053] Central Subfield Thickness (CST) increase by
.gtoreq.50 .mu.m (in one embodiment using spectral domain optical
coherence tomography (SD-OCT)); and/or [0054] Best Corrected Visual
Acuity (BCVA/ETDRS) decrease by .gtoreq.5 letters.
[0055] One embodiment of the invention is a bispecific antibody
which binds to human vascular endothelial growth factor (VEGF) and
to human angiopoietin-2 (ANG-2), for use in the treatment of an
ocular vascular disease, [0056] wherein the bispecific antibody is
administered (is to be administered) intravitreally every 8 weeks
or less frequently (in one embodiment every 9 weeks or less
frequently; in one embodiment every 10 weeks or less frequently; in
one embodiment every 11 weeks or less frequently; in one embodiment
every 12 weeks or less frequently; in one embodiment every 13 weeks
or less frequently; in one embodiment every 14 weeks or less
frequently; in one embodiment every 15 weeks or less
frequently).
[0057] One embodiment of the invention is a bispecific antibody
which binds to human vascular endothelial growth factor (VEGF) and
to human angiopoietin-2 (ANG-2), for use in the treatment of a
patient suffering from an ocular vascular disease, wherein the
patient gains 12 or more letters (in one embodiment 13 or more
letters, in one embodiment 14 or more letters, in one embodiment 15
or more letters) of Best Corrected Visual Acuity (BCVA) measured
using Early Treatment Diabetic Retinopathy Study (ETDRS) like
charts, compared to the patient's BCVA letter score prior to the
dosing of the bispecific VEGF/ANG2 antibody. In one embodiment the
bispecific antibody is administered (is to be administered)
intravitreally every 8 weeks or less frequently (in one embodiment
every 9 weeks or less frequently; in one embodiment every 10 weeks
or less frequently; in one embodiment every 11 weeks or less
frequently; in one embodiment every 12 weeks or less frequently; in
one embodiment every 13 weeks or less frequently; in one embodiment
every 14 weeks or less frequently; in one embodiment every 15 weeks
or less frequently).
[0058] One embodiment of the invention is a bispecific antibody
which binds to human vascular endothelial growth factor (VEGF) and
to human angiopoietin-2 (ANG-2), for use in the treatment of a
patient suffering from an ocular vascular disease, wherein the
patient experiences an improvement in vision subsequent to the
(intravitreal) administration of the bispecific VEGF/ANG2 antibody
as measured by gaining 12 or more letters (in one embodiment 13 or
more letters, in one embodiment 14 or more letters, in one
embodiment 15 or more letters) of Best Corrected Visual Acuity
(BCVA) measured using Early Treatment Diabetic Retinopathy Study
(ETDRS) like charts, compared to the patient's BCVA letter score
prior to the dosing of the bispecific VEGF/ANG2 antibody. In one
embodiment the bispecific antibody is administered (is to be
administered) intravitreally every 8 weeks or less frequently (in
one embodiment every 9 weeks or less frequently; in one embodiment
every 10 weeks or less frequently; in one embodiment every 11 weeks
or less frequently; in one embodiment every 12 weeks or less
frequently; in one embodiment every 13 weeks or less frequently; in
one embodiment every 14 weeks or less frequently; in one embodiment
every 15 weeks or less frequently).
[0059] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively.
[0060] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 45 weeks, and/or at 46
weeks, and/or at 47 weeks, and/or at 48 weeks, and/or at 49 weeks,
and/or at 50 weeks, and/or at 51 weeks, and/or at 52 weeks, and/or
at 53 weeks, and/or at 54 weeks, and/or at 55 weeks, and/or at 56
weeks, and/or at 57 weeks, and/or at 58 weeks, and/or at 59 weeks,
and/or at 60 weeks after treatment start, respectively.
[0061] In one embodiment of the invention such bispecific antibody
(for use) is used to prolong the time to retreatment and/or to
prolong the time to loss of visual acuity and, wherein the
retreatment with the bispecific antibody is administered in case of
a disease activity which is determined as [0062] Central Subfield
Thickness (CST) increase by .gtoreq.50 .mu.m (in one embodiment
using spectral domain optical coherence tomography (SD-OCT));
and/or [0063] Best Corrected Visual Acuity (BCVA/ETDRS) decrease by
.gtoreq.5 letters.
[0064] One embodiment of the invention is a medicament or
pharmaceutical formulation comprising a bispecific antibody which
binds to human vascular endothelial growth factor (VEGF) and to
human angiopoietin-2 (ANG-2), for use in the treatment of an ocular
vascular disease, [0065] wherein the bispecific antibody is
administered (is to be administered) intravitreally every 8 weeks
or less frequently (in one embodiment every 9 weeks or less
frequently; in one embodiment every 10 weeks or less frequently; in
one embodiment every 11 weeks or less frequently; in one embodiment
every 12 weeks or less frequently; in one embodiment every 13 weeks
or less frequently; in one embodiment every 14 weeks or less
frequently; in one embodiment every 15 weeks or less
frequently).
[0066] One embodiment of the invention is a medicament or
pharmaceutical formulation comprising a bispecific antibody which
binds to human vascular endothelial growth factor (VEGF) and to
human angiopoietin-2 (ANG-2), for use in the treatment of a patient
suffering from an ocular vascular disease, wherein the patient
gains 12 or more letters (in one embodiment 13 or more letters, in
one embodiment 14 or more letters, in one embodiment 15 or more
letters) of Best Corrected Visual Acuity (BCVA) measured using
Early Treatment Diabetic Retinopathy Study (ETDRS) like charts,
compared to the patient's BCVA letter score prior to the dosing of
the bispecific VEGF/ANG2 antibody. In one embodiment the bispecific
antibody is administered (is to be administered) intravitreally
every 8 weeks or less frequently (in one embodiment every 9 weeks
or less frequently; in one embodiment every 10 weeks or less
frequently; in one embodiment every 11 weeks or less frequently; in
one embodiment every 12 weeks or less frequently; in one embodiment
every 13 weeks or less frequently; in one embodiment every 14 weeks
or less frequently; in one embodiment every 15 weeks or less
frequently).
[0067] One embodiment of the invention is a medicament or
pharmaceutical formulation comprising a bispecific antibody which
binds to human vascular endothelial growth factor (VEGF) and to
human angiopoietin-2 (ANG-2), for use in the treatment of a patient
suffering from an ocular vascular disease, wherein the patient
experiences an improvement in vision subsequent to the
(intravitreal) administration of the bispecific VEGF/ANG2 antibody
as measured by gaining 12 or more letters (in one embodiment 13 or
more letters, in one embodiment 14 or more letters, in one
embodiment 15 or more letters) of Best Corrected Visual Acuity
(BCVA) measured using Early Treatment Diabetic Retinopathy Study
(ETDRS) like charts, compared to the patient's BCVA letter score
prior to the dosing of the bispecific VEGF/ANG2 antibody. In one
embodiment the bispecific antibody is administered (is to be
administered) intravitreally every 8 weeks or less frequently (in
one embodiment every 9 weeks or less frequently; in one embodiment
every 10 weeks or less frequently; in one embodiment every 11 weeks
or less frequently; in one embodiment every 12 weeks or less
frequently; in one embodiment every 13 weeks or less frequently; in
one embodiment every 14 weeks or less frequently; in one embodiment
every 15 weeks or less frequently).
[0068] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively.
[0069] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 45 weeks, and/or at 46
weeks, and/or at 47 weeks, and/or at 48 weeks, and/or at 49 weeks,
and/or at 50 weeks, and/or at 51 weeks, and/or at 52 weeks, and/or
at 53 weeks, and/or at 54 weeks, and/or at 55 weeks, and/or at 56
weeks, and/or at 57 weeks, and/or at 58 weeks, and/or at 59 weeks,
and/or at 60 weeks after treatment start, respectively.
[0070] In one embodiment of the invention such medicament or
pharmaceutical formulation is used to prolong the time to
retreatment and/or to prolong the time to loss of visual acuity
and, wherein the retreatment with the bispecific antibody is
administered in case of a disease activity which is determined as
[0071] Central Subfield Thickness (CST) increase by .gtoreq.50
.mu.m (in one embodiment using spectral domain optical coherence
tomography (SD-OCT)); and/or [0072] Best Corrected Visual Acuity
(BCVA/ETDRS) decrease by .gtoreq.5 letters.
[0073] One embodiment of the invention is the use of a bispecific
antibody which binds to human vascular endothelial growth factor
(VEGF) and to human angiopoietin-2 (ANG-2), for the manufacture of
a medicament for use in the treatment of an ocular vascular
disease, [0074] wherein the bispecific antibody is administered (is
to be administered) intravitreally every 8 weeks or less frequently
(in one embodiment every 9 weeks or less frequently; in one
embodiment every 10 weeks or less frequently; in one embodiment
every 11 weeks or less frequently; in one embodiment every 12 weeks
or less frequently; in one embodiment every 13 weeks or less
frequently; in one embodiment every 14 weeks or less frequently; in
one embodiment every 15 weeks or less frequently).
[0075] One embodiment of the invention is the use of a bispecific
antibody which binds to human vascular endothelial growth factor
(VEGF) and to human angiopoietin-2 (ANG-2), for the manufacture of
a medicament for use in the treatment of an ocular vascular
disease, wherein the patient gains 12 or more letters (in one
embodiment 13 or more letters, in one embodiment 14 or more
letters, in one embodiment 15 or more letters) of Best Corrected
Visual Acuity (BCVA) measured using Early Treatment Diabetic
Retinopathy Study (ETDRS) like charts, compared to the patient's
BCVA letter score prior to the dosing of the bispecific VEGF/ANG2
antibody. In one embodiment the bispecific antibody is administered
(is to be administered) intravitreally every 8 weeks or less
frequently (in one embodiment every 9 weeks or less frequently; in
one embodiment every 10 weeks or less frequently; in one embodiment
every 11 weeks or less frequently; in one embodiment every 12 weeks
or less frequently; in one embodiment every 13 weeks or less
frequently; in one embodiment every 14 weeks or less frequently; in
one embodiment every 15 weeks or less frequently).
[0076] One embodiment of the invention is the use of a bispecific
antibody which binds to human vascular endothelial growth factor
(VEGF) and to human angiopoietin-2 (ANG-2), for the manufacture of
a medicament for use in the treatment of an ocular vascular
disease, wherein the patient experiences an improvement in vision
subsequent to the (intravitreal) administration of the bispecific
VEGF/ANG2 antibody as measured by gaining 12 or more letters (in
one embodiment 13 or more letters, in one embodiment 14 or more
letters, in one embodiment 15 or more letters) of Best Corrected
Visual Acuity (BCVA) measured using Early Treatment Diabetic
Retinopathy Study (ETDRS) like charts, compared to the patient's
BCVA letter score prior to the dosing of the bispecific VEGF/ANG2
antibody. In one embodiment the bispecific antibody is administered
(is to be administered) intravitreally every 8 weeks or less
frequently (in one embodiment every 9 weeks or less frequently; in
one embodiment every 10 weeks or less frequently; in one embodiment
every 11 weeks or less frequently; in one embodiment every 12 weeks
or less frequently; in one embodiment every 13 weeks or less
frequently; in one embodiment every 14 weeks or less frequently; in
one embodiment every 15 weeks or less frequently).
[0077] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively.
[0078] In one embodiment of the invention the gain of letters in
the BCVA/ETDRS letter score is measured at 45 weeks, and/or at 46
weeks, and/or at 47 weeks, and/or at 48 weeks, and/or at 49 weeks,
and/or at 50 weeks, and/or at 51 weeks, and/or at 52 weeks, and/or
at 53 weeks, and/or at 54 weeks, and/or at 55 weeks, and/or at 56
weeks, and/or at 57 weeks, and/or at 58 weeks, and/or at 59 weeks,
and/or at 60 weeks after treatment start, respectively.
[0079] In one embodiment of the invention medicament is used to
prolong the time to retreatment and/or to prolong the time to loss
of visual acuity and, wherein the retreatment with the bispecific
antibody is administered in case of a disease activity which is
determined as [0080] Central Subfield Thickness (CST) increase by
.gtoreq.50 .mu.m (in one embodiment using spectral domain optical
coherence tomography (SD-OCT)); and/or [0081] Best Corrected Visual
Acuity (BCVA/ETDRS) decrease by .gtoreq.5 letters.
[0082] In one embodiment BCVA determination in such method, use,
bispecific antibody (for use), medicament or pharmaceutical
formulation is based on the Early Treatment of Diabetic Retinopathy
Study (ETDRS) Protocol adapted visual acuity charts and is assessed
at a starting distance of 4 meters.
[0083] Such method, use, bispecific antibody (for use), medicament
or pharmaceutical formulation may comprise sequentially
administering initial doses ("treatment initiation") (e.g. 3 to 7
monthly administrations; in one embodiment the treatment initiation
includes 3 to 4 monthly administrations, in one embodiment the
treatment initiation includes 4 to 5 monthly administrations; in
one embodiment the treatment initiation includes 4 to 6 monthly
administrations; in one embodiment the treatment initiation
includes at least 4 monthly administrations; in one embodiment the
treatment initiation includes 5 to 7 monthly administrations, in
one embodiment the treatment initiation includes 6 monthly
administrations) followed by one or more secondary doses of a
therapeutically effective amount of the bispecific antibody,
medicament or pharmaceutical formulation.
[0084] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 10
to 12 weeks (following treatment initiation).
[0085] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 11
to 13 weeks (following treatment initiation).
[0086] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 12
to 14 weeks (following treatment initiation).
[0087] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is is administered every
13 to 15 weeks (following treatment initiation).
[0088] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 14
to 16 weeks (following treatment initiation).
[0089] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 10
to 11 weeks, or every 11 to 12 weeks, or every 12 to 13 weeks, or
every 13 to 14 weeks, or every 14 to 15 weeks, or every 15 to 16
weeks (following treatment initiation, respectively).
[0090] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered every 10
weeks, or every 11 weeks, or every 12 weeks, or every 13 weeks, or
every 14 weeks, or every 16 weeks (following treatment initiation,
respectively).
[0091] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered in a dose
of about 5 to 7 mg (at each treatment). In one embodiment the
bispecific antibody is is administered in a dose of 6 mg+/-10% (at
each treatment). In one embodiment the bispecific antibody is is
administered in a dose of about 6 mg (at each treatment). (in one
embodiment in a dose of 6 mg (at each treatment))
[0092] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation is administered in a
concentration of about 30 mg/ml of the bispecific antibody. In one
embodiment of the invention the bispecific antibody, medicament or
pharmaceutical formulation is administered in a concentration of
about 120 mg/ml of the bispecific antibody.
[0093] The terms "ocular vascular disease" and "vascular eye
disease" are used interchangeable herein and include, but are not
limited to intraocular neovascular syndromes such as diabetic
retinopathy, diabetic macular edema, retinopathy of prematurity,
neovascular glaucoma, (branch) retinal vein occlusions, central
retinal vein occlusions, macular degeneration, age-related macular
degeneration, retinitis pigmentosa, retinal angiomatous
proliferation, macular telangectasia, ischemic retinopathy, iris
neovascularization, intraocular neovascularization, comeal
neovascularization, retinal neovascularization, choroidal
neovascularization, and retinal degeneration. (Gamer, A., Vascular
diseases, In: Pathobiology of ocular disease, A dynamic approach,
Garner, A., and Klintworth, G. K., (eds.), 2nd edition, Marcel
Dekker, New York (1994), pp. 1625-1710). As used herein, ocular
vascular disorder refers to any pathological conditions
characterized by altered or unregulated proliferation and invasion
of new blood vessels into the structures of ocular tissues such as
the retina or cornea. In one embodiment the ocular vascular disease
is selected from the group consisting of: wet age-related macular
degeneration (wet AMD), neovascular AMD (nAMD), diabetic macular
edema (DME), cystoid macular edema (CME), non-proliferative
diabetic retinopathy (NPDR), proliferative diabetic retinopathy
(PDR), macular edema secondary to central retinal vein occlusion,
secondary to hemiretinal vein occlusion or secondary to branch vein
occlusion, retinitis, conjunctivitis, uveitis, choroiditis,
choroidal neovascularization (CNV) secondary to ocular inflammation
including secondary to ocular histoplasmosis or presumed
histoplasmosis or choroiditis; myopic choroidal neovascularization
(mCNV). And choroidal neovascularization secondary to trauma,
retinopathy of prematurity and rubeosis iridis/rubeotic glaucoma,
and other ophthalmic diseases wherein the eye disease or disorder
is associated with ocular neovascularization, vascular leakage,
and/or retinal edema. So the anti-VEGF/ANG2 bispecific antibodies
for use and the methods described herein are useful in the
prevention and treatment of wet AMD, nAMD CME, DME, NPDR, PDR, and
uveitis, also preferably wet AMD, nAMD, also preferably DME, CME,
NPDR and PDR, and also particularly wet AMD. In some embodiments,
the ocular vascular disease is selected from the group consisting
of wet age-related macular degeneration (wet AMD), neovascular
age-related macular degeneration (nAMD), (diabetic) macular edema,
retinal vein occlusions, retinopathy of prematurity, and diabetic
retinopathy.
[0094] Other diseases/conditions associated with comeal
neovascularization (or which may be the cause of corneal
neovascularization) include, but are not limited to, epidemic
keratoconjunctivitis, Vitamin A deficiency, contact lens overwear,
atopic keratitis, superior limbic keratitis, pterygium keratitis
sicca, sjogrens syndrome, acne rosacea, phylectenulosis, syphilis,
Mycobacteria infections, lipid degeneration, chemical burns,
bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes
zoster infections, protozoan infections, Kaposi sarcoma, Mooren
ulcer, Terrien's marginal degeneration, marginal keratolysis,
rheumatoid arthritis, systemic lupus, polyarteritis, trauma,
Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,
periphigoid radial keratotomy, and comeal graph rejection.
[0095] Diseases/conditions associated with retinal/choroidal
neovascularization (or which may be the cause of retinal/choroidal
neovascularization) include, but are not limited to, diabetic
retinopathy, macular degeneration, sickle cell anemia, sarcoid,
syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion,
artery occlusion, carotid obstructive disease, chronic
uveitis/vitritis, mycobacterial infections, Lyme's disease,
systemic lupus erythematosis, retinopathy of prematurity, retinitis
pigmentosa, retina edema (including macular edema), Eales disease,
Bechets disease, infections causing a retinitis or choroiditis,
presumed ocular histoplasmosis, Bests disease, myopia, optic (disc)
pits, Stargardts disease, pars planitis, chronic retinal
detachment, hyperviscosity syndromes, toxoplasmosis, trauma and
post-laser complications. Other diseases include, but are not
limited to, diseases associated with rubeosis (neovascularization
of the angle) and diseases caused by the abnormal proliferation of
fibrovascular or fibrous tissue including all forms of
proliferative vitreoretinopathy.
[0096] Retinopathy of prematurity (ROP) is a disease of the eye
that affects prematurely born babies. It is thought to be caused by
disorganized growth of retinal blood vessels which may result in
scarring and retinal detachment. ROP can be mild and may resolve
spontaneously, but may lead to (total) blindness in serious cases.
As such, all preterm babies are at risk for ROP, and very low birth
weight is an additional risk factor. Both oxygen toxicity and
relative hypoxia can contribute to the development of ROP.
[0097] Macular degeneration is a medical condition predominantly
found in elderly adults in which the center of the inner lining of
the eye, known as the macula area of the retina, suffers thinning,
atrophy, and in some cases, bleeding. This can result in loss of
central vision, which entails inability to see fine details, to
read, or to recognize faces. According to the American Academy of
Ophthalmology, it is the leading cause of central vision loss
(blindness) in the United States today for those over the age of
fifty years. Although some macular dystrophies that affect younger
individuals are sometimes referred to as macular degeneration, the
term generally refers to age-related macular degeneration (AMD or
ARMD).
[0098] "Age-related macular degeneration (AMD)", as used herein,
refers to a serious eye condition when the small central portion of
the retina, known as the macula, deteriorates. AMD includes wet AMD
and neovascular AMD. The wet form of AMD (wet AMD, wAMD or also
called neovascular AMD, nAMD) is characterized by the growth of
abnormal blood vessels from the choroid underneath the macula. This
is called choroidal neovascularization. These blood vessels leak
blood and fluid (below and) into the retina, causing (elevation of
the retina and) distortion of vision that makes straight lines look
wavy, as well as blind spots and loss of central vision. These
abnormal blood vessels eventually scar, leading to permanent loss
of central vision. The symptoms of AMD include dark, blurry areas
in the center of vision; and diminished or changed color
perception. AMD can be detected in a routine eye exam. One of the
most common early signs of macular degeneration is the presence of
drusen which are tiny yellow deposits under the retina and pigment
clumping.
[0099] Advanced AMD, which is responsible for profound vision loss,
has two forms: dry and wet. Central geographic atrophy, the dry
form of advanced AMD, results from atrophy to the retinal pigment
epithelial layer below the retina, which causes vision loss through
loss of photoreceptors (rods and cones) in the central part of the
eye. While no treatment is available for this condition, vitamin
supplements with high doses of antioxidants, lutein and zeaxanthin,
have been demonstrated by the National Eye Institute and others to
slow the progression of dry macular degeneration and in some
patients, improve visual acuity.
[0100] Retinitis pigmentosa (RP) is a group of genetic eye
conditions. In the progression of symptoms for RP, night blindness
generally precedes tunnel vision by years or even decades. Many
people with RP do not become legally blind until their 40s or 50s
and retain some sight all their life. Others go completely blind
from RP, in some cases as early as childhood. Progression of RP is
different in each case. RP is a type of hereditary retinal
dystrophy, a group of inherited disorders in which abnormalities of
the photoreceptors (rods and cones) or the retinal pigment
epithelium (RPE) of the retina lead to progressive visual loss.
Affected individuals first experience defective dark adaptation or
nyctalopia (night blindness), followed by reduction of the
peripheral visual field (known as tunnel vision) and, sometimes,
loss of central vision late in the course of the disease.
[0101] Macular edema occurs when fluid and protein deposits collect
on or under the macula of the eye, the central area of the retina
responsible for fine vision, causing it to thicken and swell. The
swelling may distort a person's central vision, as the macula is
near the center of the retina at the back of the eyeball. This area
holds tightly packed cones that provide sharp, clear central vision
to enable a person to see form, color, and detail that is directly
in the line of sight. Cystoid macular edema is a type of macular
edema that includes cyst formation.
[0102] "Diabetic Macular Edema" (DME), as used herein, refers to a
serious eye condition that affects people with diabetes (type 1 or
2). Macular edema occurs when blood vessels in the retina leak into
the macula and fluid and protein deposits collect on or under the
macula of the eye and causes it to thicken and swell (edema). The
swelling may distort a person's central vision, as the macula is
near the center of the retina at the back of the eyeball. The
primary symptoms of DME include, but are not limited to, blurry
vision, floaters, loss of contrast, double vision, and eventual
loss of vision. The pathology of DME is characterized by breakdown
of inner the blood-retinal barrier, normally preventing fluid
movement in the retina, thus allowing fluid to accumulate in the
retinal tissue, and presence of retinal thickening. DME is
presently diagnosed during an eye examination consisting of a
visual acuity test, which determines the smallest letters a person
can read on a standardized chart, a dilated eye exam to check for
signs of the disease, imaging tests such as optical coherence
tomography (OCT) or fluorescein angiography (FA) and tonometry, an
instrument that measures pressure inside the eye. The following
studies are also performed to determine treatment: optical
coherence tomography (OCT), fluorescein angiography, and color
stereo fundus photography. DME can be broadly characterized into
two main categories--Focal and Diffuse. Focal DME is characterized
by specific areas of separate and distinct leakage in the macula
with sufficient macular blood flow. Diffuse DME results from
leakage of the entire capillary bed surrounding the macula,
resulting from a breakdown of the inner blood-retina barrier of the
eye. In addition to Focal and Diffuse, DME is also categorized
based on clinical exam findings into clinically significant macular
edema (CSME), non-CSME and CSME with central involvement (CSME-CI),
which involves the fovea. The present invention includes methods to
treat the above-mentioned categories of DME.
[0103] Best Corrected Visual Acuity (BCVA) is determined using
methodology adapted from the 4-meter Early Treatment Diabetic
Retinopathy Study [ETDRS] protocol (using Early Treatment Diabetic
Retinopathy Study (ETDRS) like charts) and resulting in the
respective letter score.
[0104] Disease activity is determined e.g. via reduction of the
BCVA/ETDRs letter score and/or e.g. via the macular thickening by
spectral domain optical coherence tomography (SD-OCT) involving the
center of the macula as central subfield thickness (CST) (also
known as center subfoveal thickness). In one preferred embodiment
Central Subfield Thickness (CST) is determined using spectral
domain optical coherence tomography (SD-OCT): In one preferred
embodiment CST is measured by spectral domain optical coherence
tomography (SD-OCT) with a Spectralis.TM. device; in one preferred
embodiment CST is measured by spectral domain optical coherence
tomography (SD-OCT) with a Cirrus.TM. device; in one embodiment CST
is measured by spectral domain optical coherence tomography
(SD-OCT) with a Topcon.TM. device; in one embodiment CST is
measured by spectral domain optical coherence tomography (SD-OCT)
with a Optovue.TM. device). As used herein, the term "a patient
suffering from" refers to a human that exhibits one or more
symptoms or indications of, and/or who has been diagnosed with an
ocular vascular disease as described herein. The term "a patient
suffering from" may also include, e.g., subjects who, prior to
treatment, exhibit (or have exhibited) one or more indications of a
vascular eye disease such as, e.g., retinal angiogenesis,
neovascularization, vascular leak, retinal thickening of the center
of the fovea, hard, yellow exudates of the center of the fovea with
adjacent retinal thickening, and at least 1 disc area of retinal
thickening, any part of which is within 1 disc diameter of the
center of the fovea, blurry vision, floaters, loss of contrast,
double vision, and eventual loss of vision.
[0105] As used herein, the term "a patient suffering from" may
include a subset of population which is more susceptible to DME or
AMD or may show an elevated level of a DME-associated or an
AMD-associated biomarker. For example, "a subject in need thereof"
may include a subject suffering from diabetes for more than 10
years, have frequent high blood sugar levels or high fasting blood
glucose levels. In certain embodiments, the term "a patient
suffering from" includes a subject who, prior to or at the time of
administration of the bispecific anti-VEGF/ANG2 antibody, has or is
diagnosed with diabetes. In certain embodiments, the term "a
patient suffering from" includes a subject who, prior to or at the
time of administration of the anti-VEGF/ANG2 antibody, is more than
50 years old. In some embodiments, the term "a patient suffering
from" includes subjects who are smokers, or subjects with high
blood pressure or high cholesterol.
[0106] The present invention includes methods or bispecific
antibodies (for use), medicaments or pharmaceutical formulations
for treating, preventing or reducing the severity of an ocular
vascular disease comprising administering a therapeutically
effective amount of a bispecific anti-VEGF/ANG2 antibody (or a
medicament or pharmaceutical formulation comprising the bispecific
anti-VEGF/ANG2 antibody) to a subject in need thereof, wherein the
bispecific antibody, medicament or pharmaceutical formulation
comprising such bispecific anti-VEGF/ANG2 antibody is administered
(intravitreally) to the subject in multiple doses, e.g., as part of
a specific therapeutic dosing regimen.
[0107] One embodiment of the invention is the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein patients suffering from an
ocular vascular disease have not been previously treated with
anti-VEGF treatment (e.g monotherapy) (are treatment naive).
[0108] One embodiment of the invention is the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein patients suffering from an
ocular vascular disease have been previously treated with anti-VEGF
treatment (e.g monotherapy).
[0109] One embodiment of the invention is the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein the ocular vascular disease
is DME and the treatment of patients suffering from DME includes a
fixed every 8th week (Q8W) dosing schedule following treatment
initiation (In one embodiment the treatment initiation includes 5
to 7 monthly administrations; in one embodiment the treatment
initiation includes 6 monthly administrations).
[0110] One embodiment of the invention is the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein the ocular vascular disease
is DME and the treatment of patients suffering from DME includes a
fixed Q12W dosing schedule following treatment initiation (In one
embodiment the treatment initiation includes 5 to 7 monthly
administrations; in one embodiment the treatment initiation
includes 6 monthly administrations). In one embodiment following
the treatment initiation, first one dose cycle of Q8W follows
before the fixed Q12W dosing schedule.
[0111] One embodiment of the invention is the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein the ocular vascular disease
is DME and the treatment of patients suffering from DME includes
following treatment initiation a dosing schedule that extends the
administration interval in stable absence of disease, or shortens
the interval if there is disease activity (In one embodiment the
treatment initiation includes 3 to 7 monthly administrations; in
one embodiment the treatment initiation includes 3 to 5 monthly
administrations; in one embodiment the treatment initiation
includes at least 4 monthly administrations; in one embodiment the
treatment initiation includes 4 to 6 monthly administrations). In
one embodiment such dosing schedule includes that the patient
receives Q4W or Q8W or Q12W or Q16W dosing, dependent on their
disease state. In one embodiment the stable absence of disease is
determined as [0112] Central Subfield Thickness (CST) increased by
<50 .mu.m [0113] Best Corrected Visual Acuity (BCVA/ETDRS)
decreased by <5 letters and the disease activity is determined
as [0114] Central Subfield Thickness (CST) increased by .gtoreq.50
.mu.m [0115] Best Corrected Visual Acuity (BCVA/ETDRS) decreased by
.gtoreq.5 letters.
[0116] In one embodiment the stable absence of disease is
determined as [0117] Central Subfield Thickness (CST) is below
about 300 .mu.m (In one embodiment below 325 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Spectralis.TM. device; in one embodiment below 315 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Cirrus.TM. device; in one embodiment below 315 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Topcon.TM. device; in one embodiment below 295 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Optovue.TM. device), [0118] and the disease activity is determined
as [0119] Central Subfield Thickness (CST) is above about 300 .mu.m
(In one embodiment above 325 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Spectralis.TM. device;
in one embodiment above 315 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Cirrus.TM. device; in
one embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
[0120] One embodiment of the invention the method of treatment,
use, bispecific antibody (for use), medicament or pharmaceutical
formulation as described herein wherein the ocular vascular disease
is AMD (in one embodiment wet AMD) and the treatment of patients
suffering from AMD (in one embodiment wet AMD) includes following
treatment initiation a dosing schedule that extends the
administration interval in stable absence of disease, or shortens
the interval if there is disease activity (In one embodiment the
treatment initiation includes 3 to 7 monthly administrations; in
one embodiment the treatment initiation includes 3 to 5 monthly
administrations; in one embodiment the treatment initiation
includes at least 4 monthly administrations; in one embodiment the
treatment initiation includes 4 to 6 monthly administrations). In
one embodiment such dosing schedule includes that the patient
receives Q4W or Q8W or Q12W or Q16W dosing, dependent on their
disease state. In one embodiment the stable absence of disease is
determined as [0121] Central Subfield Thickness (CST) increased by
<50 .mu.m; and/or [0122] Best Corrected Visual Acuity
(BCVA/ETDRS) decreased by <5 letters and the disease activity is
determined as [0123] Central Subfield Thickness (CST) increased by
.gtoreq.50 .mu.m; and/or [0124] Best Corrected Visual Acuity
(BCVA/ETDRS) decreased by .gtoreq.5 letters.
[0125] In one embodiment the stable absence of disease is
determined as [0126] Central Subfield Thickness (CST) is below
about 300 .mu.m (In one embodiment below 325 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Spectralis.TM. device; in one embodiment below 315 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Cirrus.TM. device; in one embodiment below 315 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Topcon.TM. device; in one embodiment below 295 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Optovue.TM. device), [0127] and the disease activity is determined
as [0128] Central Subfield Thickness (CST) is above about 300 .mu.m
(In one embodiment above 325 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Spectralis.TM. device;
in one embodiment above 315 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Cirrus.TM. device; in
one embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
[0129] In one embodiment the vascular ocular disease in such
method, use, bispecific antibody (for use), medicament or
pharmaceutical formulation is wetAMD (nAMD).
[0130] As used herein, "antibody" refers to a binding protein that
comprises antigen-binding sites. The terms "binding site" or
"antigen-binding site" as used herein denotes the region(s) of an
antibody molecule to which a ligand actually binds. The term
"antigen-binding site" comprises an antibody heavy chain variable
domains (VH) and an antibody light chain variable domains (VL)
(pair of VH/VL).).
[0131] Antibody specificity refers to selective recognition of the
antibody for a particular epitope of an antigen. Natural
antibodies, for example, are monospecific.
[0132] "Bispecific antibodies" according to the invention are
antibodies which have two different antigen-binding specificities.
Antibodies of the present invention are specific for two different
antigens, VEGF as first antigen and ANG-2 as second antigen.
[0133] The term "monospecific" antibody as used herein denotes an
antibody that has one or more binding sites each of which bind to
the same epitope of the same antigen.
[0134] The term "valent" as used within the current application
denotes the presence of a specified number of binding sites in an
antibody molecule. As such, the terms "bivalent", "tetravalent",
and "hexavalent" denote the presence of two binding site, four
binding sites, and six binding sites, respectively, in an antibody
molecule. The bispecific antibodies according to the invention are
preferably "bivalent".
[0135] The terms "bispecific antibody which binds to human vascular
endothelial growth factor (VEGF) and to human angiopoietin-2
(ANG-2)", "bispecific anti-VEGF/ANG2 antibody" and bispecific
<VEGF/ANG2> antibody" as used herein are interchangeable and
refer to an antibody which has at least two different
antigen-binding sites, a first one which binds to VEGF and a second
one which binds to ANG2.
[0136] Bispecific anti-VEGF/ANG2 antibodies are e.g. described in
WO2010040508, WO2011/117329, WO2012/131078, WO2015/083978,
WO2017/197199, and WO2014/009465. WO2014/009465 describes
bispecific anti-VEGF/ANG2 antibodies especially designed for
treatment of ocular vascular diseases. The bispecific
anti-VEGF/ANG2 antibodies of WO2014/009465 (which is incorporated
herein in its entirety) are especially useful in the treatment and
treatment schedules of ocular vascular diseases as described
herein.
[0137] In one embodiment the bispecific antibody which binds to
human vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2) is a bispecific anti-VEGF/ANG2 antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0138] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0139] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, and wherein [0140] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 subclass comprising the mutations I253A, H310A, and
H435A and the mutations L234A, L235A and P329G (numberings
according to EU Index of Kabat).
[0141] In one embodiment such bispecific anti-VEGF/ANG2 antibody is
bivalent.
[0142] In one embodiment such bispecific anti-VEGF/ANG2 antibody is
characterized in that [0143] i) said first antigen-binding site
specifically binding to VEGF comprises as heavy chain variable
domain VH an amino acid sequence of SEQ ID NO: 7, and as light
chain variable domain VL an amino acid sequence of SEQ ID NO: 8,
and [0144] ii) said second antigen-binding site specifically
binding to ANG-2 comprises as heavy chain variable domain VH an
amino acid sequence of SEQ ID NO: 15, and as light chain variable
domain VL an amino acid sequence of SEQ ID NO: 16.
[0145] In one aspect of the invention such bispecific, bivalent
antibody according to the invention is characterized in comprising
[0146] a) the heavy chain and the light chain of a first full
length antibody that specifically binds to VEGF; [0147] b) the
modified heavy chain and modified light chain of a second full
length antibody that specifically binds to ANG-2, wherein the
constant domains CL and CH1 are replaced by each other.
[0148] This bispecific, bivalent antibody format for the bispecific
antibody specifically binding to human vascular endothelial growth
factor (VEGF) and human angiopoietin-2 (ANG-2) is described in WO
2009/080253 (including Knobs-into-Holes modified CH3 domains). The
antibodies based on this bispecific, bivalent antibody format are
named CrossMAbs.
[0149] In one embodiment such bispecific, bivalent anti-VEGF/ANG2
antibody is characterized in comprising [0150] a) as heavy chain of
the first full length antibody the amino acid sequence of SEQ ID
NO: 17, and as light chain of the first full length antibody the
amino acid sequence of SEQ ID NO: 18, and [0151] b) as modified
heavy chain of the second full length antibody the amino acid
sequence of SEQ ID NO: 19, and as modified light chain of the
second full length antibody the amino acid sequence of SEQ ID NO:
20.
[0152] In one embodiment such bispecific, bivalent anti-VEGF/ANG2
antibody is characterized in comprising the amino acid sequences of
SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID
NO: 20. In one preferred embodiment the bispecific, bivalent
anti-VEGF/ANG2 antibody is faricimab.
[0153] Accordingly, one embodiment of the invention is a
bispecific, bivalent antibody comprising a first antigen-binding
site that specifically binds to human VEGF and a second
antigen-binding site that specifically binds to human ANG-2,
characterized in comprising the amino acid sequences of SEQ ID NO:
17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20. In
one preferred embodiment the bispecific, bivalent anti-VEGF/ANG2
antibody is faricimab.
[0154] In on embodiment the CH3 domains of the bispecific, bivalent
antibody according to the invention is altered by the
"knob-into-holes" technology which is described in detail with
several examples in e.g. WO 96/027011, Ridgway J. B., et al.,
Protein Eng 9 (1996) 617-621; and Merchant, A. M., et al., Nat
Biotechnol 16 (1998) 677-681. In this method the interaction
surfaces of the two CH3 domains are altered to increase the
heterodimerisation of both heavy chains containing these two CH3
domains. Each of the two CH3 domains (of the two heavy chains) can
be the "knob", while the other is the "hole". The introduction of a
disulfide bridge stabilizes the heterodimers (Merchant, A. M, et
al., Nature Biotech 16 (1998) 677-681; Atwell, S., et al. J. Mol.
Biol. 270 (1997) 26-35) and increases the yield.
[0155] In a preferred aspect of the invention the bispecific
anti-VEGF/ANG2 antibodies according to the invention are
characterized in that the CH3 domain of one heavy chain and the CH3
domain of the other heavy chain each meet at an interface which
comprises an original interface between the antibody CH3
domains;
wherein said interface is altered to promote the formation of the
bispecific antibody, wherein the alteration is characterized in
that: a) the CH3 domain of one heavy chain is altered, so that
within the original interface the CH3 domain of one heavy chain
that meets the original interface of the CH3 domain of the other
heavy chain within the bispecific antibody, an amino acid residue
is replaced with an amino acid residue having a larger side chain
volume, thereby generating a protuberance within the interface of
the CH3 domain of one heavy chain which is positionable in a cavity
within the interface of the CH3 domain of the other heavy chain and
b) the CH3 domain of the other heavy chain is altered, so that
within the original interface of the second CH3 domain that meets
the original interface of the first CH3 domain within the
bispecific antibody an amino acid residue is replaced with an amino
acid residue having a smaller side chain volume, thereby generating
a cavity within the interface of the second CH3 domain within which
a protuberance within the interface of the first CH3 domain is
positionable.
[0156] Thus the bispecific anti-VEGF/ANG2 antibodies for use
described herein are preferably characterized in that [0157] the
CH3 domain of the heavy chain of the full length antibody of a) and
the CH3 domain of the heavy chain of the full length antibody of b)
each meet at an interface which comprises an alteration in the
original interface between the antibody CH3 domains; [0158] wherein
i) in the CH3 domain of one heavy chain [0159] an amino acid
residue is replaced with an amino acid residue having a larger side
chain volume, thereby generating a protuberance within the
interface of the CH3 domain of one heavy chain which is
positionable in a cavity within the interface of the CH3 domain of
the other heavy chain [0160] and wherein [0161] ii) in the CH3
domain of the other heavy chain [0162] an amino acid residue is
replaced with an amino acid residue having a smaller side chain
volume, thereby generating a cavity within the interface of the
second CH3 domain within which a protuberance within the interface
of the first CH3 domain is positionable.
[0163] Preferably said amino acid residue having a larger side
chain volume is selected from the group consisting of arginine (R),
phenylalanine (F), tyrosine (Y), tryptophan (W).
[0164] Preferably said amino acid residue having a smaller side
chain volume is selected from the group consisting of alanine (A),
serine (S), threonine (T), valine (V).
[0165] In one aspect of the invention both CH3 domains are further
altered by the introduction of cysteine (C) as amino acid in the
corresponding positions of each CH3 domain such that a disulfide
bridge between both CH3 domains can be formed.
[0166] In one embodiment, the bispecific antibody comprises a T366W
mutation in the CH3 domain of the "knobs chain" and T366S, L368A,
Y407V mutations in the CH3 domain of the "hole chain". An
additional interchain disulfide bridge between the CH3 domains can
also be used (Merchant, A. M, et al., Nature Biotech 16 (1998)
677-681) e.g. by introducing a S354C mutation into one CH3 domain
and a Y349C mutation into the other CH3 domain.
[0167] In a another preferred embodiment the bispecific antibody
comprises S354C and T366W mutations in one of the two CH3 domains
and Y349C, T366S, L368A, Y407V mutations in the other of the two
CH3 domains In a another preferred embodiment the bispecific
antibody comprises Y349C, T366W mutations in one of the two CH3
domains and S354C, T366S, L368A, Y407V mutations in the other of
the two CH3 domains (the additional Y349C or S354C mutation in one
CH3 domain and the additional S354C or Y349C mutation in the other
CH3 domain forming a interchain disulfide bridge) (numbering always
according to EU index of Kabat (Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)).
[0168] Other techniques for CH3-modifications to enforce the
heterodimerization are contemplated as alternatives of the
invention and described e.g. in WO 96/27011, WO 98/050431, EP
1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO
2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO
2013/157954 and WO 2013/096291.
[0169] In one embodiment the heterodimerization approach described
in EP 1 870 459A1 is used alternatively. This approach is based on
the introduction of substitutions/mutations of charged amino acids
with the opposite charge at specific amino acid positions of the in
the CH3/CH3 domain interface between both heavy chains. One
preferred embodiment for said multispecific antibodies are amino
acid R409D and K370E mutations in the CH3 domain of one heavy chain
and amino acid D399K and E357K mutations in the CH3 domain of the
other heavy chain of the multispecific antibody (numberings
according to Kabat EU index).
[0170] In another embodiment said multispecific antibody comprises
an amino acid T366W mutation in the CH3 domain of the "knobs chain"
and amino acid T366S, L368A and Y407V mutations in the CH3 domain
of the "hole chain"; and additionally comprises amino acid R409D
and K370E mutations in the CH3 domain of the "knobs chain" and
amino acid D399K and E357K mutations in the CH3 domain of the "hole
chain".
[0171] In one embodiment the heterodimerization approach described
in WO2013/157953 is used alternatively. In one embodiment the CH3
domain of one heavy chain comprises an amino acid T366K mutation
and the CH3 domain of the other heavy chain comprises an amino acid
L351D mutation. In a further embodiment the CH3 domain of the one
heavy chain further comprises an amino acid L351K mutation. In a
further embodiment the CH3 domain of the other heavy chain further
comprises an amino acid mutation selected from Y349E, Y349D and
L368E (in one embodiment L368E).
[0172] In one embodiment the heterodimerization approach described
in WO2012/058768 is used alternatively. In one embodiment the CH3
domain of one heavy chain comprises amino acid L351Y and Y407A
mutations and the CH3 domain of the other heavy chain comprises
amino acid T366A and K409F mutations. In a further embodiment the
CH3 domain of the other heavy chain further comprises an amino acid
mutation at position T411, D399, 5400, F405, N390 or K392. In one
embodiment said amino acid mutation is selected from the group
consisting of
a) T411N, T411R, T411Q, T411K, T411D, T411E and T411W,
b) D399R, D399W, D399Y and D399K,
c) S400E, S400D, S400R and S400K,
d) F4051, F405M, F405T, F405S, F405V and F405W,
e) N390R, N390K and N390D,
f) K392V, K392M, K392R, K392L, K392F and K392E.
[0173] In a further embodiment the CH3 domain of one heavy chain
comprises amino acid L351Y and Y407A mutations and the CH3 domain
of the other heavy chain comprises amino acid T366V and K409F
mutations. In a further embodiment the CH3 domain of one heavy
chain comprises an amino acid Y407A mutation and the CH3 domain of
the other heavy chain comprises amino acid T366A and K409F
mutations. In a further embodiment the CH3 domain of the other
heavy chain further comprises amino acid K392E, T411E, D399R and
S400R mutations.
[0174] In one embodiment the heterodimerization approach described
in WO2011/143545 is used alternatively. In one embodiment the amino
acid modification according to WO2011/143545 is introduced in the
CH3 domain of the heavy chain at a position selected from the group
consisting of 368 and 409.
[0175] In one embodiment the heterodimerization approach described
in WO2011/090762 which also uses the knob-into-hole technology
described above is used alternatively. In one embodiment the CH3
domain of one heavy chain comprises an amino acid T366W mutation
and the CH3 domain of the other heavy chain comprises an amino acid
Y407A mutation. In one embodiment the CH3 domain of one heavy chain
comprises an amino acid T366Y mutation and the CH3 domain of the
other heavy chain comprises an amino acid Y407T mutation.
[0176] In one embodiment the multispecific antibody is of IgG2
isotype and the heterodimerization approach described in
WO2010/129304 is used alternatively.
[0177] In one embodiment the heterodimerization approach described
in WO2009/089004 is used alternatively. In one embodiment the CH3
domain of one heavy chain comprises an amino acid substitution of
K392 or N392 with a negatively-charged amino acid (in one
embodiment glutamic acid (E) or aspartic acid (D); in a further
embodiment a K392D or N392D mutation) and the CH3 domain of the
other heavy chain comprises an amino acid substitution of D399,
E356, D356, or E357 with a positively-charged amino acid (in one
embodiment Lysine (K) or arginine (R), in a further embodiment a
D399K, E356K, D356K or E357K substitution; and in an even further
embodiment a D399K or E356K mutation). In a further embodiment the
CH3 domain of the one heavy chain further comprises an amino acid
substitution of K409 or R409 with a negatively-charged amino acid
(in one embodiment glutamic acid (E) or aspartic acid (D); in a
further embodiment a K409D or R409D mutation). In a further
embodiment the CH3 domain of the one heavy chain further or
alternatively comprises an amino acid substitution of K439 and/or
K370 with a negatively-charged amino acid (in one embodiment
glutamic acid (E) or aspartic acid (D)).
[0178] In one embodiment the heterodimerization approach described
in WO2007/147901 is used alternatively. In one embodiment the CH3
domain of one heavy chain comprises amino acid K253E, D282K and
K322D mutations and the CH3 domain of the other heavy chain
comprises amino acid D239K, E240K and K292D mutations.
[0179] In one embodiment the heterodimerization approach described
in WO2007/110205 is used alternatively.
[0180] In one embodiment the bispecific antibody which binds to
human vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2) is a bispecific anti-VEGF/ANG2 antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0181] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0182] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, and wherein [0183] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 subclass comprising the mutations I253A, H310A, and
H435A and the mutations L234A, L235A and P329G (numberings
according to EU Index of Kabat; and wherein [0184] iv) in the
constant heavy chain region a T366W mutation is comprised in one
CH3 domain and T366S, L368A, Y407V mutations are comprised the
other CH3 domain (numberings according to EU Index of Kabat).
[0185] In one embodiment the bispecific antibody which binds to
human vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2) is a bispecific anti-VEGF/ANG2 antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0186] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0187] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, and wherein [0188] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 subclass comprising the mutations I253A, H310A, and
H435A and the mutations L234A, L235A and P329G (numberings
according to EU Index of Kabat; and wherein [0189] iv) in the
constant heavy chain region a S354C and T366W mutations are
comprised in one CH3 domain and Y349C, T366S, L368A and Y407V
mutations are comprised the other CH3 domain (numberings according
to EU Index of Kabat).
[0190] In one embodiment such bispecific anti-VEGF/ANG2 antibody is
bivalent.
[0191] In one embodiment such bispecific anti-VEGF/ANG2 antibody is
characterized in that [0192] i) said first antigen-binding site
specifically binding to VEGF comprises as heavy chain variable
domain VH an amino acid sequence of SEQ ID NO: 7, and as light
chain variable domain VL an amino acid sequence of SEQ ID NO: 8,
and [0193] ii) said second antigen-binding site specifically
binding to ANG-2 comprises as heavy chain variable domain VH an
amino acid sequence of SEQ ID NO: 15, and as light chain variable
domain VL an amino acid sequence of SEQ ID NO: 16.
[0194] In one aspect of the invention such bispecific, bivalent
antibody according to the invention is characterized in comprising
[0195] a) the heavy chain and the light chain of a first full
length antibody that specifically binds to VEGF; [0196] b) the
modified heavy chain and modified light chain of a second full
length antibody that specifically binds to ANG-2, wherein the
constant domains CL and CH1 are replaced by each other.
[0197] The term "VEGF" as used herein refers to human vascular
endothelial growth factor (VEGF/VEGF-A,) the 165-amino acid human
vascular endothelial cell growth factor (amino acid 27-191 of
precursor sequence of human VEGF165: SEQ ID NO: 24; amino acids
1-26 represent the signal peptide), and related 121, 189, and 206
vascular endothelial cell growth factor isoforms, as described by
Leung, D. W., et al., Science 246 (1989) 1306-9; Houck et al., Mol.
Endocrin. 5 (1991) 1806-1814; Keck, P. J., et al., Science 246
(1989) 1309-12 and Connolly, D. T., et al., J. Biol. Chem. 264
(1989) 20017-24; together with the naturally occurring allelic and
processed forms of those growth factors. VEGF is involved in the
regulation of normal and abnormal angiogenesis and
neovascularization associated with tumors and intraocular disorders
(Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, R. A.,
et al., J. Clin. Invest. 91 (1993) 153-159; Brown, L. F., et al.,
Human Pathol. 26 (1995) 86-91; Brown, L. F., et al., Cancer Res. 53
(1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996)
931-934; and Dvorak, H. F., et al., Am. J. Pathol. 146 (1995)
1029-1039). VEGF is a homodimeric glycoprotein that has been
isolated from several sources and includes several isoforms. VEGF
shows highly specific mitogenic activity for endothelial cells. A
VEGF antagonist/inhibitor inhibits binding of VEGF to its receptor
VEGFR. Known VEGF antagonist/inhibitors include bispecific
anti-VEGF/ANG2 antibodies as described in WO2014/009465.
[0198] The term "ANG-2" as used herein refers to human
angiopoietin-2 (ANG-2) (alternatively abbreviated with ANGPT2 or
ANG2) (SEQ ID NO: 25) which is described e.g. in Maisonpierre, P.
C., et al, Science 277 (1997) 55-60 and Cheung, A. H., et al.,
Genomics 48 (1998) 389-91. The angiopoietins-1 (SEQ ID NO: 26) and
-2 were discovered as ligands for the Ties, a family of tyrosine
kinases that is selectively expressed within the vascular
endothelium (Yancopoulos, G. D., et al., Nature 407 (2000) 242-48).
There are now four definitive members of the angiopoietin family.
Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely
diverged counterparts of the same gene locus in mouse and man (Kim,
I., et al., FEBS Let, 443 (1999) 353-56; Kim, I., et al., J Biol
Chem 274 (1999) 26523-28). ANG-1 and ANG-2 were originally
identified in tissue culture experiments as agonist and antagonist,
respectively (see for ANG-1: Davis, S., et al., Cell 87 (1996)
1161-69; and for ANG-2: Maisonpierre, P. C., et al., Science 277
(1997) 55-60). All of the known angiopoietins bind primarily to its
receptor TIE2 (SEQ ID NO: 27), and both Ang-1 and -2 bind to TIE2
with an affinity of 3 nM (Kd) (Maisonpierre, P. C., et al., Science
277 (1997) 55-60). An ANG2 antagonist/inhibitor inhibits binding of
ANG2 to its receptor TIE2. Known ANG2 antagonist/inhibitors include
bispecific anti-VEGF/ANG2 antibodies as described in
WO2014/009465.
[0199] An antigen-binding sites of the bispecific antibody of the
invention contain six complementarity determining regions (CDRs)
which contribute in varying degrees to the affinity of the binding
site for antigen. There are three heavy chain variable domain CDRs
(CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs
(CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions
(FRs) is determined by comparison to a compiled database of amino
acid sequences in which those regions have been defined according
to variability among the sequences.
[0200] The antibodies of the invention comprise immunoglobulin
constant regions derived from human origin of one or more
immunoglobulin classes, wherein such immunoglobulin classes include
IgG, IgM, IgA, IgD, and IgE classes and, in the case of IgG and
IgA, their subclasses, especially IgG1 and IgG4.
[0201] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of a single amino acid composition.
[0202] The term "chimeric antibody" refers to an antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are preferred. Other preferred forms of
"chimeric antibodies" encompassed by the present invention are
those in which the constant region has been modified or changed
from that of the original antibody to generate the properties
according to the invention, especially in regard to C1q binding
and/or Fc receptor (FcR) binding. Such chimeric antibodies are also
referred to as "class-switched antibodies.". Chimeric antibodies
are the product of expressed immunoglobulin genes comprising DNA
segments encoding immunoglobulin variable regions and DNA segments
encoding immunoglobulin constant regions. Methods for producing
chimeric antibodies involve conventional recombinant DNA and gene
transfection techniques are well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
[0203] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and
Neuberger, M. S., et al., Nature 314 (1985) 268-270. Particularly
preferred CDRs correspond to those representing sequences
recognizing the antigens noted above for chimeric antibodies. Other
forms of "humanized antibodies" encompassed by the present
invention are those in which the constant region has been
additionally modified or changed from that of the original antibody
to generate the properties according to the invention, especially
in regard to C1q binding and/or Fc receptor (FcR) binding.
[0204] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germ line immunoglobulin sequences. Human antibodies are
well-known in the state of the art (van Dijk, M. A., and van de
Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human
antibodies can also be produced in transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
or a selection of human antibodies in the absence of endogenous
immunoglobulin production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge (see,
e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993)
2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258;
Brueggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human
antibodies can also be produced in phage display libraries
(Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992)
381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597).
The techniques of Cole, A., et al. and Boerner, P., et al. are also
available for the preparation of human monoclonal antibodies (Cole,
A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. L.,
p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991)
86-95). As already mentioned for chimeric and humanized antibodies
according to the invention the term "human antibody" as used herein
also comprises such antibodies which are modified in the constant
region to generate the properties according to the invention,
especially in regard to C1q binding and/or FcR binding, e.g. by
"class switching" i.e. change or mutation of Fc parts (e.g. from
IgG1 to IgG4 and/or IgG1/IgG4 mutation).
[0205] The term "recombinant antibody", as used herein, is intended
to include all human antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
isolated from a host cell such as a NSO or CHO cell or from an
animal (e.g. a mouse) that is transgenic for human immunoglobulin
genes or antibodies expressed using a recombinant expression vector
transfected into a host cell. Such recombinant antibodies have
variable and constant regions in a rearranged form. The recombinant
antibodies according to the invention have been subjected to in
vivo somatic hypermutation. Thus, the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germ line VH and VL
sequences, may not naturally exist within the human antibody germ
line repertoire in vivo.
[0206] The "variable domain" (variable domain of a light chain
(VL), variable domain of a heavy chain (VH) 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 domains of
variable human light and heavy chains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a n-sheet conformation and the
CDRs may form loops connecting the n-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 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.
[0207] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
CDRs on each chain are separated by such framework amino acids.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. CDR and FR regions are determined
according to the standard definition of Kabat, E. A., et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991).
[0208] The term "full length antibody" denotes an antibody
consisting of two "full length antibody heavy chains" and two "full
length antibody light chains". A "full length antibody heavy chain"
is a polypeptide consisting in N-terminal to C-terminal direction
of an antibody heavy chain variable domain (VH), an antibody
constant heavy chain domain 1 (CH1), an antibody hinge region (HR),
an antibody heavy chain constant domain 2 (CH2), and an antibody
heavy chain constant domain 3 (CH3), abbreviated as
VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant
domain 4 (CH4) in case of an antibody of the subclass IgE.
Preferably the "full length antibody heavy chain" is a polypeptide
consisting in N-terminal to C-terminal direction of VH, CH1, HR,
CH2 and CH3. A "full length antibody light chain" is a polypeptide
consisting in N-terminal to C-terminal direction of an antibody
light chain variable domain (VL), and an antibody light chain
constant domain (CL), abbreviated as VL-CL. The antibody light
chain constant domain (CL) can be .kappa. (kappa) or .lamda.
(lambda). The two full length antibody chains are linked together
via inter-polypeptide disulfide bonds between the CL domain and the
CH1 domain and between the hinge regions of the full length
antibody heavy chains. Examples of typical full length antibodies
are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA,
IgD, and IgE. The full length antibodies according to the invention
can be from a single species e.g. human, or they can be chimerized
or humanized antibodies. The full length antibodies according to
the invention comprise two antigen binding sites each formed by a
pair of VH and VL, which both specifically bind to the same
antigen. The C-terminus of the heavy or light chain of said full
length antibody denotes the last amino acid at the C-terminus of
said heavy or light chain. The N-terminus of the heavy or light
chain of said full length antibody denotes the last amino acid at
the N-terminus of said heavy or light chain.
[0209] The term "constant region" as used within the current
applications denotes the sum of the domains of an antibody other
than the variable region. The constant region is not involved
directly in binding of an antigen, but exhibits various effector
functions. Depending on the amino acid sequence of the constant
region of their heavy chains, antibodies are divided in the
classes: IgA, IgD, IgE, IgG and IgM, and several of these may be
further divided into subclasses, such as IgG1, IgG2, IgG3, and
IgG4, IgA1 and IgA2. The heavy chain constant regions that
correspond to the different classes of antibodies are called
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
light chain constant regions which can be found in all five
antibody classes are called .kappa. (kappa) and .lamda.
(lambda).
[0210] The terms "constant region derived from human origin" or
"human constant region" as used in the current application denotes
a constant heavy chain region of a human antibody of the subclass
IgG1, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or
lambda region. Such constant regions are well known in the state of
the art and e.g. described by Kabat, E. A., et al., Sequences of
Proteins of Immunological Interest, 5th ed., Public Health Service,
National Institutes of Health, Bethesda, Md. (1991) (see also e.g.
Johnson, G., and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218;
Kabat, E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975)
2785-2788). Within the application for the numbering of positions
and mutations the EU numbering system (EU Index) according to
Kabat, E. A., et al., Sequences of Proteins of Immunological
Interest, 5th ed., Public Health Service, National Institutes of
Health, Bethesda, Md. (1991) is used and referred to as "numbering
according to EU Index of Kabat".
[0211] In one embodiment the bispecific antibodies according to the
invention have a constant region of human IgG1 subclass (derived
from human IgG1 subclass). However, the C-terminal lysine (Lys447),
or the C-terminal glycine (Gly446) and the C-terminal lysine
(Lys447), of the Fc region may or may not be present.
[0212] In one embodiment the bispecific antibody as described
herein is of IgG1 isotype/subclass and comprises a constant heavy
chain domain of SEQ ID NO: 23 or the constant parts of the heavy
chain amino acid sequence of SEQ ID NO: 17 and of the heavy chain
amino acid sequence of SEQ ID NO: 18. In one embodiment
additionally the C-terminal glycine (Gly446) is present. In one
embodiment additionally the C-terminal glycine (Gly446) and the
C-terminal lysine (Lys447) is present.
[0213] Unless otherwise specified herein, numbering of amino acid
residues in the constant region is according to the EU numbering
system, also called the EU index of Kabat, as described in Kabat,
E. A. et al., Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, National Institutes of Health,
Bethesda, Md. (1991), NIH Publication 91-3242.
[0214] In one embodiment the bispecific antibody according to the
invention is of human IgG1 subclass with mutations L234A
(Leu235Ala), L235A (Leu234Ala) and P329G (Pro329Gly). Such antibody
has a reduced FcR binding (especially they show no more binding to
FcRgammaI, FcRgammaII and FcRgammaIII). This especially useful to
reduce potential side effects like e.g. thrombosis (Meyer, T., et
al., J. Thromb. Haemost. 7 (2009) 171-81).
[0215] While Pro329Ala mutation which was described already removes
only two third of the FcgammaRIIIa sandwich interaction, the
Pro329Gly in the antibodies according to the invention fully
imparts binding of the Fc part to FcgammaRIII. This is especially
useful as the binding to FcgammaRIII is involved in ADCC
(antibody-dependent cellular toxicity) which leads to cell death,
which may be helpful in the treatment of cancer diseases, but which
can cause serious side effect in the antibody based treatment of
other vascular or immunological diseases. So the antibodies
according to the invention of IgG1 subclass with mutations L234A,
L235A and P329G and IgG4 subclass with mutations S228P, L235E and
P329G are especially useful, as they both show no more binding to
FcRgammaI, FcRgammaII and FcRgammaIII.
[0216] An "effective amount" of an agent, e.g., a pharmaceutical
formulation or bispecific anti-VEGF/ANG2 antibody, refers to an
amount effective, at dosages and for periods of time necessary, to
achieve the desired therapeutic or prophylactic result.
[0217] In one embodiment of the invention the bispecific antibody,
medicament or pharmaceutical formulation as described herein is
administered via intravitreal application, e.g. via intravitreal
injection (is administered "intravitreally"). This can be performed
in accordance with standard procedures known in the art. See, e.g.,
Ritter et al., J. Clin. Invest. 116 (2006) 3266-76;
Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999)
196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-5.
[0218] In some embodiments, therapeutic kits of the invention can
contain one or more doses of the bispecific antibody described
present in a medicament or pharmaceutical formulation, a suitable
device for intravitreal injection of the medicament or
pharmaceutical formulation, and an instruction detailing suitable
subjects and protocols for carrying out the injection. In these
embodiments, the medicament or pharmaceutical formulation are
typically administered to the subject in need of treatment via
intravitreal injection. This can be performed in accordance with
standard procedures known in the art. See, e.g., Ritter et al., J.
Clin. Invest. 116 (2006) 3266-76; Russelakis-Cameiro et al.,
Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al.,
Arch. Neurol. 33 (1976) 183-5.
[0219] Regardless of the route of administration selected, the
bispecific antibody as described herein is formulated into
pharmaceutically acceptable dosage forms by conventional methods
known to those of skill in the art.
Description of the Amino Acid Sequences
TABLE-US-00001 [0220] SEQ ID NO: 1 heavy chain CDR3H,
<VEGF>ranibizumab SEQ ID NO: 2 heavy chain CDR2H,
<VEGF>ranibizumab SEQ ID NO: 3 heavy chain CDR1H,
<VEGF>ranibizumab SEQ ID NO: 4 light chain CDR3L,
<VEGF>ranibizumab SEQ ID NO: 5 light chain CDR2L,
<VEGF>ranibizumab SEQ ID NO: 6 light chain CDR1L,
<VEGF>ranibizumab SEQ ID NO: 7 heavy chain variable domain
VH, <VEGF>ranibizumab SEQ ID NO: 8 light chain variable
domain VL, <VEGF>ranibizumab SEQ ID NO: 9 heavy chain CDR3H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 10 heavy chain CDR2H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 11 heavy chain CDR1H,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 12 light chain CDR3L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 13 light chain CDR2L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 14 light chain CDR1L,
<ANG-2> Ang2i_LC10 variant SEQ ID NO: 15 heavy chain variable
domain VH, <ANG-2> Ang2i_LC10 variant SEQ ID NO: 16 light
chain variable domain VL, <ANG-2> Ang2i_LC10 variant SEQ ID
NO: 17 Heavy chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with AAA
mutations and P329G LALA mutations (VEGFang2-0016) SEQ ID NO: 18
Heavy chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA
mutations and P329G LALA mutations (VEGFang2-0016) SEQ ID NO: 19
Light chain 1 of <VEGF-ANG-2> CrossMAb IgG1 with AAA
mutations and P329G LALA mutations (VEGFang2-0016) SEQ ID NO: 20
Light chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA
mutations and P329G LALA mutations (VEGFang2-0016) SEQ ID NO: 21
kappa light chain constant region SEQ ID NO: 22 lambda light chain
constant region SEQ ID NO: 23 heavy chain constant region derived
from human IgG1 SEQ ID NO: 24 Human vascular endothelial growth
factor (VEGF); precursor sequence of human VEGF165 SEQ ID NO: 25
Human angiopoietin-2 (ANG-2) SEQ ID NO: 26 Human angiopoietin-1
(ANG-1) SEQ ID NO: 27 Human Tie-2 receptor
[0221] In the following, embodiments of the invention are listed:
[0222] 1. A bispecific antibody which binds to human vascular
endothelial growth factor (VEGF) and to human angiopoietin-2
(ANG-2), for use in the treatment of an ocular vascular disease,
[0223] wherein the bispecific antibody is administered (is to be
administered) intravitreally every 8 weeks or less frequently (in
one embodiment every 9 weeks or less frequently; in one embodiment
every 10 weeks or less frequently; in one embodiment every 11 weeks
or less frequently; in one embodiment every 12 weeks or less
frequently; in one embodiment every 13 weeks or less frequently; in
one embodiment every 14 weeks or less frequently; in one embodiment
every 15 weeks or less frequently). [0224] 2A. A bispecific
antibody which binds to human vascular endothelial growth factor
(VEGF) and to human angiopoietin-2 (ANG-2), for use in the
treatment of a patient suffering from an ocular vascular disease,
wherein the patient gains 12 or more letters (in one embodiment 13
or more letters, in one embodiment 14 or more letters, 15 or more
letters) of Best Corrected Visual Acuity (BCVA) measured using
Early Treatment Diabetic Retinopathy Study (ETDRS) like charts,
compared to the patient's BCVA letter score prior to the dosing of
the bispecific VEGF/ANG2 antibody. [0225] 2B. A bispecific antibody
which binds to human vascular endothelial growth factor (VEGF) and
to human angiopoietin-2 (ANG-2), for use in the treatment of a
patient suffering from an ocular vascular disease, wherein the
patient experiences an improvement in vision subsequent to the
administration of the bispecific VEGF/ANG2 antibody as measured by
gaining 12 or more letters (in one embodiment 13 or more letters,
in one embodiment 14 or more letters, 15 or more letters) of Best
Corrected Visual Acuity (BCVA) measured using Early Treatment
Diabetic Retinopathy Study (ETDRS) like charts, compared to the
patient's BCVA letter score prior to the dosing of the bispecific
VEGF/ANG2 antibody. [0226] 3. The bispecific antibody (for use)
according to any one of embodiments 2A to 2B, [0227] wherein the
bispecific antibody is administered (is to be administered)
intravitreally every 8 weeks or less frequently (in one embodiment
every 9 weeks or less frequently; in one embodiment every 10 weeks
or less frequently; in one embodiment every 11 weeks or less
frequently; in one embodiment every 12 weeks or less frequently; in
one embodiment every 13 weeks or less frequently; in one embodiment
every 14 weeks or less frequently; in one embodiment every 15 weeks
or less frequently). [0228] 4. The bispecific antibody (for use)
according to any one of embodiments 1 to 3, wherein the gain of
letters in the BCVA BCVA/ETDRS is measured at 4 weeks, and/or at 8
weeks, and/or at 12 weeks, and/or at 16 weeks, and/or at 20 weeks,
and/or at 24 weeks after treatment start, respectively. [0229] 5
The bispecific antibody (for use) according to any one of
embodiments 1 to 3, wherein the gain of letters in the BCVA
BCVA/ETDRS is measured at 45 weeks, and/or at 46 weeks, and/or at
47 weeks, and/or at 48 weeks, and/or at 49 weeks, and/or at 50
weeks, and/or at 51 weeks, and/or at 52 weeks, and/or at 53 weeks,
and/or at 54 weeks, and/or at 55 weeks, and/or at 56 weeks, and/or
at 57 weeks, and/or at 58 weeks, and/or at 59 weeks, and/or at 60
weeks after treatment start, respectively. [0230] 6. The bispecific
antibody (for use) according to any one of embodiments 1 to 5,
wherein the bispecific antibody is used to prolong the time to
retreatment and/or to prolong the time to loss of visual acuity
(e.g. Best Corrected Visual Acuity (BCVA) BCVA/ETDRS) and, wherein
the retreatment is deemed necessary in case of disease activity
which is determined as [0231] Central Subfield Thickness (CST)
increased by .gtoreq.50 .mu.m (in one embodiment using spectral
domain optical coherence tomography (SD-OCT)); and/or [0232] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by .gtoreq.5
letters. [0233] 7. The bispecific antibody (for use) according to
any one of embodiments 1 to 6 wherein the bispecific antibody is
administered following a treatment initiation of 3 to 7 monthly
administrations (in one embodiment the treatment initiation
includes 3 to 5 monthly administrations, in one embodiment the
treatment initiation includes 4 monthly administrations; in one
embodiment the treatment initiation includes 5 to 7 monthly
administrations, in one embodiment the treatment initiation
includes 6 monthly administrations). [0234] 8. The bispecific
antibody (for use) according to any one of embodiments 1 to 7,
wherein the ocular vascular disease is selected from the group of:
wet age-related macular degeneration (wet AMD), neovascular AMD,
diabetic macular edema (DME), cystoid macular edema (CME),
non-proliferative diabetic retinopathy (NPDR), proliferative
diabetic retinopathy (PDR), macular edema secondary to central
retinal vein occlusion, secondary to hemiretinal vein occlusion or
secondary to branch vein occlusion, retinitis, conjunctivitis,
uveitis, choroiditis, choroidal neovascularization (CNV) secondary
to ocular inflammation including secondary to ocular histoplasmosis
or presumed histoplasmosis or choroiditis; myopic choroidal
neovascularization (mCNV). And choroidal neovascularization
secondary to trauma, retinopathy of prematurity and rubeosis
iridis/rubeotic glaucoma. [0235] 9. The bispecific antibody (for
use) according to any one of embodiments 1 to 7 wherein the ocular
vascular disease is diabetic macular edema (DME). [0236] 10. The
bispecific antibody (for use) according to any one of embodiments 1
to 7, wherein the ocular vascular disease is wet age-related
macular degeneration (wet AMD), or neovascular age-related macular
degeneration (nAMD). [0237] 11. The bispecific antibody (for use)
according to any one of embodiments 1 to 10, wherein the bispecific
antibody which binds to VEGF and to human ANG-2 is a VEGF
antagonist/inhibitor and an ANG2 antagonist/inhibitor or inhibits
binding of VEGF to its receptor VEGFR and inhibits binding of ANG2
to its receptor TIE2. [0238] 12. The bispecific antibody (for use)
according to any one of embodiments 1 to 11, wherein the bispecific
antibody is administered every 10 to 12 weeks. [0239] 13. The
bispecific antibody (for use) according to any one of embodiments 1
to 11, wherein the bispecific antibody is administered every 11 to
13 weeks. [0240] 14. The bispecific antibody (for use) according to
any one of embodiments 1 to 11 wherein the bispecific antibody is
administered every 12 to 14 weeks. [0241] 15. The bispecific
antibody (for use) according to any one of embodiments 1 to 11
wherein the bispecific antibody is administered every 13 to 15
weeks. [0242] 16. The bispecific antibody (for use) according to
any one of embodiments 1 to 11 wherein the bispecific antibody is
administered every 14 to 16 weeks. [0243] 17. The bispecific
antibody (for use) according to any one of embodiments 1 to 16,
wherein the bispecific antibody which binds to human VEGF and to
human ANG2 is a bispecific, bivalent anti-VEGF/ANG2 antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0244] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0245] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, [0246] and wherein [0247] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 subclass comprising the mutations I253A, H310A, and
H435A and the mutations L234A, L235A and P329G (numberings
according to EU Index of Kabat). [0248] 18. The bispecific antibody
(for use) according to embodiment 17, wherein [0249] i) said first
antigen-binding site specifically binding to VEGF comprises as
heavy chain variable domain VH an amino acid sequence of SEQ ID NO:
7, and as light chain variable domain VL an amino acid sequence of
SEQ ID NO: 8, and [0250] ii) said second antigen-binding site
specifically binding to ANG-2 comprises as heavy chain variable
domain VH an amino acid sequence of SEQ ID NO: 15, and as light
chain variable domain VL an amino acid sequence of SEQ ID NO: 16.
[0251] 19. The bispecific antibody (for use) according to
embodiment 18, wherein the bispecific antibody which binds to human
VEGF and human ANG2 comprises the amino acid sequences of SEQ ID
NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20.
[0252] 20. The bispecific antibody (for use) according to any one
of embodiments 17 to 19, wherein the bispecific antibody is
administered in a dose of about 5 to 7 mg (at each treatment).
[0253] 21. The bispecific antibody (for use) according to any one
of embodiments 17 to 19, wherein the bispecific antibody is
administered in a dose of about 6 mg (at each treatment) (in one
embodiment in a dose of 6 mg+/-10% (at each treatment); (in one
embodiment in a dose of 6 mg (at each treatment))) [0254] 22. The
bispecific antibody (for use) according to any one of embodiments
20 to 21, wherein the bispecific antibody is administered at a
concentration of about 30 mg/ml. [0255] 23. The bispecific antibody
(for use) according to any one of embodiments 20 to 21, wherein the
bispecific antibody is administered at a concentration of about 120
mg/ml. [0256] 24. The bispecific antibody (for use) according to
any one of the preceding embodiments wherein patients suffering
from an ocular vascular disease have not been previously treated
with anti-VEGF treatment (e.g monotherapy) (are treatment naive).
[0257] 25. The bispecific antibody (for use) according to any one
of the preceding embodiments wherein patients suffering from an
ocular vascular disease have been previously treated with anti-VEGF
treatment (e.g monotherapy). [0258] 26. The bispecific antibody
(for use) according to the preceding embodiments wherein the ocular
vascular disease is DME and the treatment of patients suffering
from DME includes a fixed every 8.sup.th week (Q8W) dosing schedule
following treatment initiation (In one embodiment the treatment
initiation includes 5 to 7 monthly administrations; in one
embodiment the treatment initiation includes 6 monthly
administrations). [0259] 27. The bispecific antibody (for use)
according to the preceding embodiments wherein the ocular vascular
disease is DME and the treatment of patients suffering from DME
includes a fixed Q12W dosing schedule following treatment
initiation (In one embodiment the treatment initiation includes 5
to 7 monthly administrations; in one embodiment the treatment
initiation includes 6 monthly administrations). [0260] 28. The
bispecific antibody (for use) according to embodiment 27 wherein,
following the treatment initiation, first one dose cycle of Q8W
follows before the fixed Q12W dosing schedule. [0261] 29. The
bispecific antibody (for use) according to the preceding
embodiments wherein the ocular vascular disease is DME and the
treatment of patients suffering from DME includes following
treatment initiation a dosing schedule that extends the
administration interval in stable absence of disease, or shortens
the interval if there is disease activity (In one embodiment the
treatment initiation includes 3 to 7 monthly administrations; in
one embodiment the treatment initiation includes 4 to 6 monthly
administrations). [0262] 30. The bispecific antibody (for use)
according to embodiment 29 wherein such dosing schedule includes
that the patient receives Q8W or Q12W or Q16W dosing, dependent on
their disease state (in one embodiment Q4W or Q8W or Q12W or Q16W
dosing, dependent on their disease state) [0263] 31. The bispecific
antibody (for use) according to embodiment 29 or 30, wherein the
stable absence of disease is determined as [0264] Central Subfield
Thickness (CST) increased by <50 .mu.m; and/or [0265] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by <5 letters and
the disease activity is determined as [0266] Central Subfield
Thickness (CST) increased by .gtoreq.50 .mu.m; and/or [0267] Best
Corrected Visual Acuity (BCVA/ETDRS) decreased by .gtoreq.5
letters. [0268] 32. The method according to embodiment 29 or 30,
wherein the stable absence of disease is determined as [0269]
Central Subfield Thickness (CST) is below about 300 .mu.m (In one
embodiment below 325 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Spectralis.TM. device; in one
embodiment below 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Cirrus' device; in one
embodiment below 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment below 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device), [0270]
and the disease activity is determined as [0271] Central Subfield
Thickness (CST) is above about 300 .mu.m (In one embodiment above
325 .mu.m measured by spectral domain optical coherence tomography
(SD-OCT) with a Spectralis.TM. device; in one embodiment above 315
.mu.m measured by spectral domain optical coherence tomography
(SD-OCT) with a Cirrus.TM. device; in one embodiment above 315
.mu.m measured by spectral domain optical coherence tomography
(SD-OCT) with a Topcon.TM. device; in one embodiment above 295
.mu.m measured by spectral domain optical coherence tomography
(SD-OCT) with a Optovue.TM. device). [0272] 33. The bispecific
antibody (for use) according to the preceding embodiments wherein
the ocular vascular disease is AMD (in one embodiment wet AMD) and
the treatment of patients suffering from AMD (in one embodiment wet
AMD) includes following treatment initiation (In one embodiment the
treatment initiation includes 3 to 7 monthly administrations; in
one embodiment the treatment initiation includes 4 to 6 monthly
administrations) a dosing schedule that extends the administration
interval in stable absence of disease, or shortens the interval if
there is disease activity. [0273] 34. The bispecific antibody (for
use) according to embodiment 33 wherein such dosing schedule
includes that the patient receives Q8W or Q12W or Q16W dosing,
dependent on their disease state (in one embodiment Q4W or Q8W or
Q12W or Q16W dosing, dependent on their disease state). [0274] 35.
The bispecific antibody (for use) according to embodiment 33 or 34,
wherein the stable absence of disease is determined as
[0275] Central Subfield Thickness (CST) increased by <50 .mu.m;
and/or [0276] Best Corrected Visual Acuity (BCVA/ETDRS) decreased
by <5 letters and the disease activity is determined as [0277]
Central Subfield Thickness (CST) increased by .gtoreq.50 .mu.m;
and/or [0278] Best Corrected Visual Acuity (BCVA/ETDRS) decreased
by .gtoreq.5 letters. [0279] 36. The bispecific antibody (for use)
according to embodiment 33 or 34, wherein the stable absence of
disease is determined as [0280] Central Subfield Thickness (CST) is
below about 300 .mu.m (In one embodiment below 325 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Spectralis.TM. device; in one embodiment below 315 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Cirrus.TM. device; in one embodiment below 315 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Topcon.TM. device; in one embodiment below 295 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Optovue.TM. device), [0281] and the disease activity is determined
as [0282] Central Subfield Thickness (CST) is above about 300 .mu.m
(In one embodiment above 325 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Spectralis.TM. device;
in one embodiment above 315 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Cirrus.TM. device; in
one embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
[0283] In the following, embodiments of the invention are listed:
[0284] 1. A method of treating a patient suffering from an ocular
vascular disease the method comprising administering to the patient
an effective amount of a bispecific antibody which binds to human
vascular endothelial growth factor (VEGF) and to human
angiopoietin-2 (ANG-2), [0285] wherein the bispecific antibody is
administered intravitreally every 8 weeks or less frequently (in
one embodiment every 9 weeks or less frequently; in one embodiment
every 10 weeks or less frequently; in one embodiment every 11 weeks
or less frequently; in one embodiment every 12 weeks or less
frequently; in one embodiment every 13 weeks or less frequently; in
one embodiment every 14 weeks or less frequently; in one embodiment
every 15 weeks or less frequently). [0286] 2A. A method of treating
a patient suffering from an ocular vascular disease the method
comprising administering to the patient an effective amount of a
bispecific antibody which binds to human vascular endothelial
growth factor (VEGF) and to human angiopoietin-2 (ANG-2), wherein
the patient gains 12 or more letters (in one embodiment 13 or more
letters, in one embodiment 14 or more letters, in one embodiment 15
or more letters) of Best Corrected Visual Acuity (BCVA) measured
using Early Treatment Diabetic Retinopathy Study (ETDRS) like
charts, compared to the patient's BCVA letter score prior to the
dosing of the bispecific VEGF/ANG2 antibody. [0287] 2B. A method of
treating a patient suffering from a ocular vascular disease the
method comprising administering to the patient an effective amount
of a bispecific antibody which binds to human vascular endothelial
growth factor (VEGF) and to human angiopoietin-2 (ANG-2), wherein
the patient experiences an improvement in vision subsequent to the
administration of the bispecific VEGF/ANG2 antibody as measured by
gaining 12 or more letters (in one embodiment 13 or more letters,
in one embodiment 14 or more letters, in one embodiment 15 or more
letters) of Best Corrected Visual Acuity (BCVA) measured using
Early Treatment Diabetic Retinopathy Study (ETDRS) like charts,
compared to the patient's BCVA letter score prior to the dosing of
the bispecific VEGF/ANG2 antibody. [0288] 3. The method according
to any one of embodiments 2A to 2B, [0289] wherein the bispecific
antibody is administered (is to be administered) intravitreally
every 8 weeks or less frequently (in one embodiment every 9 weeks
or less frequently; in one embodiment every 10 weeks or less
frequently; in one embodiment every 11 weeks or less frequently; in
one embodiment every 12 weeks or less frequently; in one embodiment
every 13 weeks or less frequently; in one embodiment every 14 weeks
or less frequently; in one embodiment every 15 weeks or less
frequently). [0290] 4. The method according to any one of
embodiments 1 to 3, wherein the gain of letters in the BCVA/ETDRS
letter score is measured at 4 weeks, and/or at 8 weeks, and/or at
12 weeks, and/or at 16 weeks, and/or at 20 weeks, and/or at 24
weeks after treatment start, respectively. [0291] 5. The method
according to any one of embodiments 1 to 3, wherein the gain of
letters in the BCVA/ETDRS letter score is measured at 45 weeks,
and/or at 46 weeks, and/or at 47 weeks, and/or at 48 weeks, and/or
at 49 weeks, and/or at 50 weeks, and/or at 51 weeks, and/or at 52
weeks, and/or at 53 weeks, and/or at 54 weeks, and/or at 55 weeks,
and/or at 56 weeks, and/or at 57 weeks, and/or at 58 weeks, and/or
at 59 weeks, and/or at 60 weeks after treatment start,
respectively. [0292] 6. The method according to any one of
embodiments 1 to 5, wherein the bispecific antibody is used to
prolong the time to retreatment and/or to prolong the time to loss
of visual acuity and, wherein the retreatment with the bispecific
antibody is administered in case of a disease activity which is
determined as [0293] Central Subfield Thickness (CST) increase by
.gtoreq.50 .mu.m (in one embodiment using spectral domain optical
coherence tomography (SD-OCT)); and/or [0294] Best Corrected Visual
Acuity (BCVA/ETDRS) decrease by .gtoreq.5 letters. [0295] 7. The
method according to any one of embodiments 1 to 6, wherein the
bispecific antibody is administered following a treatment
initiation of 3 to 7 monthly administrations (in one embodiment the
treatment initiation includes 3 to 5 monthly administrations, in
one embodiment the treatment initiation includes 4 monthly
administrations in one embodiment the treatment initiation includes
5 to 7 monthly administrations, in one embodiment the treatment
initiation includes 6 monthly administrations). [0296] 8. The
method according to any one of embodiments 1 to 7, wherein the
ocular vascular disease is selected from the group of: wet
age-related macular degeneration (wet AMD), neovascular AMD,
diabetic macular edema (DME), cystoid macular edema (CME),
non-proliferative diabetic retinopathy (NPDR), proliferative
diabetic retinopathy (PDR), macular edema secondary to central
retinal vein occlusion, secondary to hemiretinal vein occlusion or
secondary to branch vein occlusion, retinitis, conjunctivitis,
uveitis, choroiditis, choroidal neovascularization (CNV) secondary
to ocular inflammation including secondary to ocular histoplasmosis
or presumed histoplasmosis or choroiditis; myopic choroidal
neovascularization (mCNV). And choroidal neovascularization
secondary to trauma, retinopathy of prematurity and rubeosis
iridis/rubeotic glaucoma. [0297] 9. The method according to any one
of embodiments 1 to 7, wherein the ocular vascular disease is
diabetic macular edema (DME). [0298] 10. The method according to
any one of embodiments 1 to 7, wherein the ocular vascular disease
is wet age-related macular degeneration (wet AMD), or neovascular
age-related macular degeneration (nAMD). [0299] 11. The method
according to any one of embodiments 1 to 10, wherein the a
bispecific antibody which binds to VEGF and to human ANG-2 is a
VEGF antagonist/inhibitor and an ANG2 antagonist/inhibitor or
inhibits binding of VEGF to its receptor VEGFR and inhibits binding
of ANG2 to its receptor TIE2. [0300] 12. The method according to
any one of embodiments 1 to 11, wherein the bispecific antibody is
administered every 10 to 12 weeks. [0301] 13. The method according
to any one of embodiments 1 to 11, wherein the bispecific antibody
is administered every 11 to 13 weeks [0302] 14. The method
according to any one of embodiments 1 to 11, wherein the bispecific
antibody is administered every 12 to 14 weeks. [0303] 15. The
method according to any one of embodiments 1 to 11, wherein the
bispecific antibody is administered every 13 to 15 weeks. [0304]
16. The method according to any one of embodiments 1 to 11, wherein
the bispecific antibody is administered every 14 to 16 weeks.
[0305] 17. The method according to any one of embodiments 1 to 16,
wherein the bispecific antibody which binds to human VEGF and to
human ANG2 is a bispecific, bivalent anti-VEGF/ANG2 antibody
comprising a first antigen-binding site that specifically binds to
human VEGF and a second antigen-binding site that specifically
binds to human ANG-2, wherein [0306] i) said first antigen-binding
site specifically binding to VEGF comprises in the heavy chain
variable domain a CDR3H region of SEQ ID NO: 1, a CDR2H region of
SEQ ID NO: 2, and a CDR1H region of SEQ ID NO:3, and in the light
chain variable domain a CDR3L region of SEQ ID NO: 4, a CDR2L
region of SEQ ID NO:5, and a CDR1L region of SEQ ID NO:6; and
[0307] ii) said second antigen-binding site specifically binding to
ANG-2 comprises in the heavy chain variable domain a CDR3H region
of SEQ ID NO: 9, a CDR2H region of, SEQ ID NO: 10, and a CDR1H
region of SEQ ID NO: 11, and in the light chain variable domain a
CDR3L region of SEQ ID NO: 12, a CDR2L region of SEQ ID NO: 13, and
a CDR1L region of SEQ ID NO: 14, and wherein [0308] iii) the
bispecific antibody comprises a constant heavy chain region of
human IgG1 subclass comprising the mutations I253A, H310A, and
H435A and the mutations L234A, L235A and P329G (numberings
according to EU Index of Kabat). [0309] 18. The method according to
embodiment 17, wherein [0310] i) said first antigen-binding site
specifically binding to VEGF comprises as heavy chain variable
domain VH an amino acid sequence of SEQ ID NO: 7, and as light
chain variable domain VL an amino acid sequence of SEQ ID NO: 8,
and [0311] ii) said second antigen-binding site specifically
binding to ANG-2 comprises as heavy chain variable domain VH an
amino acid sequence of SEQ ID NO: 15, and as light chain variable
domain VL an amino acid sequence of SEQ ID NO: 16. [0312] 19. The
method according to embodiment 18, wherein the bispecific antibody
which binds to human VEGF and human ANG2 comprises the amino acid
sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and
of SEQ ID NO: 20. [0313] 20. The method according to any one of
embodiments 17 to 19, wherein the bispecific antibody is
administered in a dose of about 5 to 7 mg (at each treatment).
[0314] 21. The method according to any one of embodiments 17 to 19,
wherein the bispecific antibody is administered in a dose of about
6 mg (at each treatment) (in one embodiment in a dose of 6 mg+/-10%
(at each treatment) (in one embodiment in a dose of 6 mg (at each
treatment))). [0315] 22. The method according to any one of
embodiments 20 to 21, wherein the bispecific antibody is
administered at a concentration of about 30 mg/ml. [0316] 23. The
method according to any one of embodiments 20 to 21, wherein the
bispecific antibody is administered at a concentration of about 120
mg/ml. [0317] 24. The method according to any one of the preceding
embodiments wherein patients suffering from an ocular vascular
disease have not been previously treated with anti-VEGF treatment
(e.g monotherapy) (are treatment naive). [0318] 25. The method
according to any one of the preceding embodiments wherein patients
suffering from an ocular vascular disease have been previously
treated with anti-VEGF treatment (e.g monotherapy). [0319] 26. The
method according to the preceding embodiments wherein the ocular
vascular disease is DME and the treatment of patients suffering
from DME includes a fixed every 8.sup.th week (Q8W) dosing schedule
following treatment initiation (In one embodiment the treatment
initiation includes 5 to 7 monthly administrations; in one
embodiment the treatment initiation includes 6 monthly
administrations). [0320] 27. The method according to the preceding
embodiments wherein the ocular vascular disease is DME and the
treatment of patients suffering from DME includes a fixed Q12W
dosing schedule following treatment initiation (In one embodiment
the treatment initiation includes 5 to 7 monthly administrations;
in one embodiment the treatment initiation includes 6 monthly
administrations). [0321] 28. The method according to embodiment 27
wherein, following the treatment initiation, first one dose cycle
of Q8W follows before the fixed Q12W dosing schedule. [0322] 29.
The method according to the preceding embodiments wherein the
ocular vascular disease is DME and the treatment of patients
suffering from DME includes following treatment initiation a dosing
schedule that extends the administration interval in stable absence
of disease, or shortens the interval if there is disease activity
(In one embodiment the treatment initiation includes 3 to 7 monthly
administrations; in one embodiment the treatment initiation
includes 4 to 6 monthly administrations). [0323] 30. The method
according to embodiment 29 wherein such dosing schedule includes
that the patient receives Q8W or Q12W or Q16W dosing, dependent on
their disease state (in one embodiment Q4W or Q8W or Q12W or Q16W
dosing, dependent on their disease state). [0324] 31. The method
according to embodiment 28 or 29, wherein the stable absence of
disease is determined as [0325] Central Subfield Thickness (CST)
increased by <50 .mu.m; and/or [0326] Best Corrected Visual
Acuity (BCVA/ETDRS) decreased by <5 letters and the disease
activity is determined as [0327] Central Subfield Thickness (CST)
increased by >50 .mu.m; and/or [0328] Best Corrected Visual
Acuity (BCVA/ETDRS) decreased by .gtoreq.5 letters. [0329] 32. The
method according to embodiment 28 or 29, wherein the stable absence
of disease is determined as [0330] Central Subfield Thickness (CST)
is below about 300 .mu.m (In one embodiment below 325 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Spectralis.TM. device; in one embodiment below 315 .mu.m
measured by spectral domain optical coherence tomography (SD-OCT)
with a Cirrus' device; in one embodiment below 315 .mu.m measured
by spectral domain optical coherence tomography (SD-OCT) with a
Topcon.TM. device; in one embodiment below 295 .mu.m measured by
spectral domain optical coherence tomography (SD-OCT) with a
Optovue.TM. device), [0331] and the disease activity is determined
as [0332] Central Subfield Thickness (CST) is above about 300 .mu.m
(In one embodiment above 325 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Spectralis.TM. device;
in one embodiment above 315 .mu.m measured by spectral domain
optical coherence tomography (SD-OCT) with a Cirrus' device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device). [0333]
33. The method according to the preceding embodiments wherein the
treatment of patients suffering from AMD (in one embodiment wet
AMD) includes following treatment initiation (In one embodiment the
treatment initiation includes 3 to 7 monthly administrations; in
one embodiment the treatment initiation includes 4 to 6 monthly
administrations) a dosing schedule that extends the administration
interval in stable absence of disease, or shortens the interval if
there is disease activity [0334] 34. The method according to
embodiment 33 wherein such dosing schedule includes that the
patient receives Q8W or Q12W or Q16W dosing, dependent on their
disease state (in one embodiment Q4W or Q8W or Q12W or Q16W dosing,
dependent on their disease state). [0335] 35. The method according
to embodiment 33 or 34, wherein the stable absence of disease is
determined as [0336] Central Subfield Thickness (CST) increased by
<50 .mu.m; and/or [0337] Best Corrected Visual Acuity
(BCVA/ETDRS) decreased by <5 letters [0338] and the disease
activity is determined as [0339] Central Subfield Thickness (CST)
increased by .gtoreq.50 .mu.m; and/or [0340] Best Corrected Visual
Acuity (BCVA/ETDRS) decreased by .gtoreq.5 letters. [0341] 36. The
method according to embodiment 33 or 34, wherein the stable absence
of disease is determined as [0342] Central Subfield Thickness (CST)
is below about 300
.mu.m (In one embodiment below 325 .mu.m measured by spectral
domain optical coherence tomography (SD-OCT) with a Spectralis.TM.
device; in one embodiment below 315 .mu.m measured by spectral
domain optical coherence tomography (SD-OCT) with a Cirrus.TM.
device; in one embodiment below 315 .mu.m measured by spectral
domain optical coherence tomography (SD-OCT) with a Topcon.TM.
device; in one embodiment below 295 .mu.m measured by spectral
domain optical coherence tomography (SD-OCT) with a Optovue.TM.
device), [0343] and the disease activity is determined as [0344]
Central Subfield Thickness (CST) is above about 300 .mu.m (In one
embodiment above 325 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Spectralis.TM. device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Cirrus' device; in one
embodiment above 315 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Topcon.TM. device; in one
embodiment above 295 .mu.m measured by spectral domain optical
coherence tomography (SD-OCT) with a Optovue.TM. device).
EXAMPLES
Treatment of Patient Suffering from Vascular Eye Diseases with a
Bispecific Antibody that Binds to Human VEGF and Human ANG2
Example 1A: Efficacy and Durability of Treatment of Patients
Suffering from Diabetic Macular Edema (DME)
Objectives
Primary Objective
[0345] The primary objective of this study were:
[0346] To evaluate the efficacy of the bispecific antibody that
binds to human VEGF and human ANG2 comprising the amino acid
sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and
of SEQ ID NO: 20 (this antibody VEGFang2-0016 and its production is
also described in detail in WO2014/009465 which is incorporated by
reference) compared with an active comparator in treatment naive
patients with center-involving diabetic macular edema (CI-DME).
Designations of this bispecific anti-VEGF/ANG2 antibody herein are
RO6867461 or RG7716 or VEGFang2-0016, or faricimab. Vials of
sterile, colorless to brownish, preservative-free solution of
RO6867461 for IVT administration of either 1.5 mg or 6 mg dose
every 4 weeks were used. The concentration of the bispecific
antibody was about 120 mg/ml.
Secondary Objectives
[0347] The secondary objectives for this study were as follows:
[0348] To investigate pharmacodynamics and anatomical outcomes
informing on the mechanism of action of RO6867461
[0349] To investigate the formation of plasma anti RO6867461
antibodies
[0350] To explore the duration of effect of RO6867461
Exploratory Objectives
[0351] The exploratory objectives for this study were as
follows:
[0352] To explore the predictive effect of previous IVT anti-VEGF
treatment on efficacy of RO6867461
[0353] To evaluate the efficacy and safety of RO6867461 compared
with the active comparator in patients with CI DME with previous
IVT anti-VEGF treatment.
[0354] To evaluate RO6867461 effects on plasma levels of markers of
angiogenesis and inflammation
[0355] To investigate RO6867461 concentration and, if sample volume
allows, biomarkers of angiogenesis and inflammation in aqueous
humor samples (optional) and vitreous (optional)
[0356] To evaluate improvement in diabetic retinopathy (DR)
severity score
Study Design
[0357] This was a multiple-center, multiple-dose, randomized,
active comparator-controlled, double masked, three parallel group,
36-week study in patients with CI-DME.
[0358] The three groups of this study were as follows:
Arm A: 0.3 mg ranibizumab IVT
Arm B: 1.5 mg RO6867461 IVT
Arm C: 6 mg RO6867461 IVT
[0359] Only one eye was selected as the study eye. Where both eyes
met all eligibility criteria, the eye with the worse BCVA was
defined as the study eye. Where both eyes met all eligibility
criteria and have the same BCVA letter score at Day 1, study eye
selection was at the investigator's discretion.
Number of Patients
[0360] Up to 210 patients were randomized.
[0361] Approximately 150 treatment-naive patients and approximately
60 patients who have been previously treated with IVT anti-VEGF
were enrolled in the study.
[0362] Approximately 50 treatment-naive patients were randomized on
each arm (1:1:1 randomization scheme) and approximately 30 patients
previously treated with IVT anti-VEGF were randomized into arms A
and C.
Target Population
[0363] Male and female patients of .gtoreq.18 years of age with
CI-DME.
Inclusion/Exclusion Criteria
Inclusion Criteria
[0364] Patients must have met the following criteria for study
entry:
Ocular Criteria for Study Eye:
[0365] Macular edema associated with DR defined as macular
thickening by spectral domain optical coherence tomography (SD-OCT)
involving the center of the macula: central subfield thickness
(CST) of .gtoreq.325 .mu.m with Spectralis.TM. (Heidelberg) at
screening (where Spectralis.TM. is not available, the following
devices and CST thresholds were acceptable: CST .gtoreq.315 .mu.m
for Cirrus.TM., CST .gtoreq.315 .mu.m for Topcon, CST .gtoreq.295
.mu.m for Optovue.TM.)
[0366] Decreased visual acuity attributable primarily to DME, with
best corrected visual acuity (BCVA) letter score of 73-24 letters
(inclusive) on Early Treatment Diabetic Retinopathy Study
(ETDRS)-like charts (20/40-20/320 Snellen equivalent) on Day 1.
[0367] Clear ocular media and adequate pupillary dilatation to
allow acquisition of good quality retinal images to confirm
diagnosis
General Criteria:
[0368] Diagnosis of diabetes mellitus (DM; Type 1 or Type 2), as
defined by the World Health Organization and/or American Diabetes
Association
[0369] Able and willing to provide written informed consent and to
comply with the study protocol according to International
Conference on Harmonisation (ICH) and local regulations.
Alternatively, a legally authorized representative must be able to
consent for the patient according to ICH and local regulations.
Age .gtoreq.18 Years
[0370] For women who were not postmenopausal (i.e. .gtoreq.12
months of non-therapy-induced amenorrhea, confirmed by FSH, if not
on hormone replacement) or surgically sterile (absence of ovaries
and/or uterus) agreement to remain abstinent or use combined
contraceptive methods that result in a failure rate of <1% per
year during the treatment period and at least through 4 weeks after
last dose.
[0371] Abstinence is only acceptable if it is in line with the
preferred and usual lifestyle of the patient. Periodic abstinence
(e.g., calendar, ovulation, symptothermal, or postovulation
methods) and withdrawal were not acceptable methods of
contraception;
[0372] Examples of contraceptive methods with an expected failure
rate of <1% per year include male sterilization, hormonal
implants, proper use of combined oral or injected hormonal
contraceptives, and certain intrauterine devices. Alternatively,
two methods (e.g., two barrier methods such as a condom and a
cervical cap) may be combined to achieve a failure rate of <1%
per year, barrier methods must always be supplemented with the use
of a spermicide.
[0373] For men: agreement to use a barrier method of contraception
during the treatment period for at least 4 weeks after the last
dose of study drug
[0374] Patients must be willing not to participate in any other
clinical trial including an investigational medical product (IMP)
or device up to completion of the current study.
Exclusion Criteria
[0375] Patients who meet any of the following criteria were
excluded from study entry:
Ocular Criteria for Study Eye:
[0376] Any signs of high-risk PDR defined as:
[0377] any vitreous or pre-retinal hemorrhage
[0378] NVE .gtoreq.1/2 disc area within an area equivalent to the
standard mydriatic ETDRS 7-field on clinical examination
[0379] NVD .gtoreq.1/3 disc area on clinical examination
[0380] Any IVT anti-VEGF treatment within 3 months prior to Day
1
[0381] Any panretinal photocoagulation (PRP) treatment prior to Day
1
[0382] Any macular laser photocoagulation within 3 months prior to
Day 1
[0383] History of Vitreoretinal Surgery
[0384] Any IVT or periocular corticosteroid treatment within 3
months prior to Day 1. Any history of Iluvien.RTM. or Ozurdex.RTM.
implants prior to Day 1 will not be permitted
[0385] Any cataract surgery or treatment for complications of
cataract surgery with steroids within 3 months prior to Day 1
History of Incisional Glaucoma Surgery
[0386] Uncontrolled glaucoma (e.g., progressive loss of visual
fields or defined as intraocular pressure [IOP].gtoreq.25 mmHg
despite treatment with anti-glaucoma medication)
Concurrent Ocular Conditions in the Study Eye:
History of Rubeosis
[0387] Any current or history of ocular disease other than DME that
may confound assessment of the macula or affect central vision
(e.g., age-related macular degeneration, retinal vein occlusion,
uveitis, angioid streaks, histoplasmosis, active or inactive
cytomegalovirus, pathological myopia, retinal detachment, macular
traction, macular hole, significant cataract)
[0388] Any current ocular condition for which, in the opinion of
the investigator, visual acuity loss would not improve from
resolution of macular edema (e.g., foveal atrophy, pigment
abnormalities, dense sub-foveal hard exudates, non-retinal
condition)
[0389] Any active ocular infection on Day 1
[0390] Any active intraocular inflammation (grade trace or above)
on Day 1
Characteristics for Fellow Eye:
[0391] Any anti-VEGF treatment within 7 days prior to Day 1
[0392] Any retinal condition that, in the opinion of the
investigator, might require anti-VEGF treatment within 7 days from
Day 1
General Criteria:
[0393] Any systemic anti-VEGF within 6 months prior to Day 1
[0394] Any major illness or major surgical procedure within 1 month
prior to Day 1
[0395] Any febrile illness within 1 week prior to Day 1
[0396] Any stroke or myocardial infarction within 12 months prior
to Day 1
[0397] Uncontrolled blood pressure (BP; defined as systolic >180
mmHg and/or diastolic >100 mmHg while patient at rest). If a
patient's initial reading exceeds these values, a second reading
may be taken either 30 or more minutes later on the same day or on
another day during the screening period. If the patient's BP needs
to be controlled by antihypertensive medication, the patient should
be taking the same medication continuously for at least 1 month
prior to Day 1.
[0398] Patients with glycosylated hemoglobin HbA1c >12% at
screening
[0399] Untreated diabetes mellitus or initiation of oral
anti-diabetic medication or insulin within 4 months prior to Day 1
or anticipated change of anti-diabetic medications within the
duration of the study
[0400] Renal failure requiring renal transplant, hemodialysis, or
peritoneal dialysis within 6 months prior to Day 1 or anticipated
to require hemodialysis or peritoneal dialysis at any time during
the study
[0401] History of other disease, metabolic dysfunction, physical
examination finding, or clinical laboratory finding giving
reasonable suspicion of a condition that contraindicated the use of
the IMP or that might affect interpretation of the results of the
study or renders the patient at high risk for treatment
complications in the opinion of the investigator
[0402] For females of childbearing potential, a positive blood
pregnancy test
[0403] Lactating female
[0404] Use of systemic corticosteroids within 1 month prior to Day
1
[0405] Any known hypersensitivity to active comparator,
fluorescein, any ingredient of the formulation used, dilating eye
drops, or any anesthetics and microbial drops used
[0406] Any other restriction accorded to the use of the active
comparator
[0407] Any treatment with an IMP in the 3 months prior to Day 1
Length of Study
[0408] The total duration of the study was up to 40 weeks (from
screening through study completion) for each enrolled patient as
follows:
[0409] Screening: up to 4 weeks
[0410] Baseline: Day 1
[0411] Study treatment administration period: from Day 1 to Week
20
[0412] Observational period: From Week 20 up to Week 36
[0413] Safety follow up call: During the observational period and 7
days after ranibizumab administration
End of Study
[0414] The end of the study was defined as the date when the last
patient last observation (LPLO) occurs. LPLO was expected to occur
36 weeks after the last patient is enrolled.
Efficacy and Pharmacodynamic Outcome Measures
[0415] The primary analysis population was treatment naive
patients. Additional analyses may be performed in the overall
population and in patients previously treated with IVT
anti-VEGF.
[0416] The primary efficacy outcome measure for this study was the
mean change in BCVA (ETDRS letters) from baseline at Week 24 in
treatment-naive patients.
[0417] Anatomic outcome measures by SD-OCT:
[0418] Mean change from baseline in foveal center point thickness
at Week 24
[0419] Mean change from baseline in mean CST (1 mm diameter) at
Week 24
[0420] Proportion of patients with resolution of subretinal and
intraretinal fluid at Week 24
[0421] Anatomic outcome measures by fundus fluorescein angiography
(FFA)
[0422] Proportion of patients with resolution of leakage at the
macula at Week 24
[0423] Change from baseline in the size of the foveal avascular
zone at Week 24
Exploratory Outcome Measures
[0424] The exploratory outcome measures for this study included but
were not limited to the following:
[0425] BCVA:
[0426] Difference in mean BCVA change from baseline between the
treatment-naive patients and patients with previous IVT anti-VEGF
(differential effect of RO6867461)
Durability-Related Exploratory Outcome Measures:
[0427] Time to increase of CST by .gtoreq.50 .mu.m and/or loss of
.gtoreq.5 letters of BCVA due to DME compared to values at Week
20
[0428] Time to retreatment with 0.3 mg ranibizumab after Week
20
Results
[0429] The primary efficacy analyses included all randomized
patients, with patients grouped according to the treatment assigned
at randomization.
[0430] The primary efficacy variable was the BCVA change from
baseline to Week 24. The primary efficacy analysis was performed
using a Mixed Model for Repeated Measurement (MMRM) model.
Best Corrected Visual Acuity
[0431] BCVA at a starting test distance of 4 meters was measured
prior to dilating eyes by a trained and certified VA examiner
masked to study drug arm assignment.
[0432] BCVA was measured by using the set of three Precision
Vision.TM. or Lighthouse distance acuity charts (modified ETDRS
Charts 1, 2, and R). A VA Manual was provided to the investigators.
VA examiner and VA examination room certifications were obtained
before any VA examinations were performed.
[0433] The BCVA examiner was masked to study eye and treatment
assignment and will only perform the refraction and BCVA assessment
(e.g. Visual Acuity Specification Manual). The BCVA examiner has
also been masked to the BCVA letter scores of a patient's previous
visits and only knew the patient's refraction data from previous
visits. The BCVA examiner was not allowed to perform any other
tasks involving direct patient care.
TABLE-US-00002 TABLE Baseline Ocular Characteristics in the Study
Eye Summary of Baseline Ocular Characteristics of Interest in the
Study Eye, All Patients, Treatment Naive Patients Protocol: BP30099
0.3 mg 1.5 mg 6 mg All Ranibizumab RO6867461 RO6867461 Patients (N
= 59) (N = 54) (N = 55) (N = 168) Best Corrected Visual Acuity
Result n 58 54 53 165 Mean (SD) 61.24 (9.87) .sup. 60.94 (11.11)
.sup. 60.15 (10.80) .sup. 60.79 (10.53) .sup. Median 64.00 63.50
63.00 63.00 Min-Max 33.0-73.0 35.0-85.0 25.0-73.0 25.0-85.0 Best
Corrected Visual Acuity Category n 58 54 53 165 20/40 or better 13
(22.4%) 15 (27.8%) 11 (20.8%) 39 (23.6%) 20/200 or worse 4 (6.9%) 3
(5.6%) 3 (5.7%) 10 (6.1%) Better than 20/200 but worse than 20/40
41 (70.7%) 36 (66.7%) 39 (73.6%) 116(70.3%) Baseline BCVA 20/40 or
better/worse than 20/40 n 58 54 53 165 Worse than 20/40 45 (77.6%)
39 (72.2%) 42 (79.2%) 126 (76.4%) 20/40 or better 13 (22.4%) 15
(27.8%) 11 (20.8%) 39 (23.6%) Baseline BCVA 20/200 or worse/better
than 20/200 n 58 54 53 165 Better than 20/200 54 (93.1%) 51 (94.4%)
50 (94.3%) 155 (93.9%) 20/200 or worse 4 (6.9%) 3 (5.6%) 3 (5.7%)
10 (6.1%) Central Subfield Thickness n 58 54 53 165 Mean (SD)
490.88 (139.01) .sup. 535.44 (163.13) .sup. 495.57 (132.70) .sup.
506.97 (145.95) .sup. Median 476.00 489.00 466.00 478.00 Min-Max
316.0-999.0 302.0-1000.0 234.0-825.0 234.0-1000.0
[0434] Primary Efficacy Outcome Measure is shown in FIG. 1. The
FIG. 1 displays the primary efficacy endpoint: BCVA change from
Baseline over Time to Week 24 for so far treatment naive patients.
VA2 refers to the bispecific anti-VEGF/ANG2 antibody RO6867461
comprising the amino acid sequences of SEQ ID NO: 17, of SEQ ID NO:
18, of SEQ ID NO: 19, and of SEQ ID NO: 20 (administered
intravitreally with a 6.0 mg or 1.5 mg dose), RBZ refers to
ranibizumab (Lucentis.RTM.) (administered intravitreally with a 0.3
mg dose).
Central Subfield Thickness (CST) Change from Baseline (Study
Eye)
[0435] A key secondary endpoint was the change from baseline in
CST, central subfield thickness. Results are shown in FIG. 2. The
bispecific anti-VEGF/ANG2 antibody RO6867461 comprising the amino
acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO:
19, and of SEQ ID NO: 20 (administered intravitreally with a 6.0 mg
or 1.5 mg dose), was compared to ranibizumab (Lucentis.RTM.)
(administered intravitreally with a 0.3 mg dose). This secondary
anatomical endpoint directionally supports BCVA primary outcome
Durability/Time to Retreament
[0436] Criteria for Treatment with Ranibizumab During Observational
Period
[0437] At each visit following the last dose of study treatment
(week 20 visit), BCVA was assessed and SD-OCT imaging was performed
(except for week 26 visit).
[0438] BCVA and CST values obtained at week 24 were compared to
those obtained at visit week 20. BCVA and CST values obtained at
weeks 28, 32 and 36 were compared to those of week 24.
[0439] If the patient met both of the following criteria the
patient received a single dose of 0.3 mg ranibizumab and exited the
study: [0440] CST increased by .gtoreq.50 .mu.m, [0441] BCVA
decreased by .gtoreq.5 letters due to DME
[0442] Results are shown in FIG. 3: FIG. 3 shows the time to
retreatment after dosing has discontinued (after 20 weeks or 6
monthly doses=Time post last intravitreal (IVT) administration)
based on disease activity assessed by both: BCVA decreased by
.gtoreq.5 letters and CST increased by .gtoreq.50 .mu.m. The
bispecific anti-VEGF/ANG2 antibody RO6867461 comprising the amino
acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO:
19, and of SEQ ID NO: 20 (administered intravitreally with a 6.0 mg
or 1.5 mg dose), was compared to ranibizumab (Lucentis.RTM.)
(administered intravitreally with a 0.3 mg dose).
[0443] For overview FIG. 4 represents a schematic comparison to
other treatment options of DME based on published results (The
following agents are compared Lucentis.RTM. (ranibizumab),
Eylea.RTM. (aflibercept), brolucizumab and VA2
(RO6867461/RG7716).
Example 1B: Efficacy and Durability of Treatment of Patients
Suffering from Diabetic Macular Edema (DME)
[0444] In a further study analogous to the above described study
under Example 1A, patients suffering from DME (e.g center-involving
diabetic macular edema (CI-DME)). are treated with the bispecific
antibody that binds to human VEGF and human ANG2 comprising the
amino acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID
NO: 19, and of SEQ ID NO: 20. As active comparator in treatment
e.g. aflibercept and/or ranibizumab and/or brolicuzimab will be
used. Patients include anti-VEGF treatment-naive patients (have not
been previously treated with anti-VEGF monotherapy with e.g. e.g.
aflibercept and/or ranibizumab and/or brolicuzimab)) and also a
group of patients which have been previously treated with anti-VEGF
monotherapy. Designations of the respective bispecific antibody
that binds to human VEGF and human ANG2 are RO6867461 or RG7716.
Vials of sterile, colorless to brownish, preservative-free solution
of RO6867461 for IVT administration of either 1.5 mg or 6 mg dose
are used.
[0445] One or more of the following dosing schedules are used:
[0446] a) patients suffering from DME will be treated with a fixed
Q8W dosing schedule following treatment initiation (e.g. 6 initial
monthly injections) [0447] b) patients suffering from DME will be
treated with a fixed Q12W dosing (in one schedule with one cycle of
Q8W dosing first), following treatment initiation (e.g. 6 initial
monthly injections) [0448] c) patients suffering from DME will be
treated following treatment initiation (e.g. with 3-7 initial
monthly injections) with a dosing regimen that extends the
injection interval in stable absence of disease, or shortens the
interval if there is disease activity. Such regimen includes e.g.
that patient receive Q4W/Q8W/Q12W/Q16W dosing, dependent on their
disease state
[0449] The disease stability assessment would be based on
best-corrected visual acuity (BCVA) and on CST as well as retinal
thickness based on Optical coherence tomography (OCT). Outcome
measure and results will be evaluated as described e.g. in Example
1A. Primary endpoints will be between 45 and 60 weeks.
[0450] In one embodiment patients suffering from DME are treatment
naive (have not been previously treated with anti-VEGF monotherapy
with e.g. aflibercept and/or ranibizumab and/or brolicuzimab)
[0451] In one embodiment patients suffering from DME have been
previously treated with anti-VEGF monotherapy with e.g. aflibercept
and/or ranibizumab and/or brolicuzimab.
[0452] In one embodiment patients suffering from DME will be
treated with a fixed Q8W dosing schedule following treatment
initiation (e.g. 6 initial monthly injections).
[0453] In one embodiment patients suffering from DME will be
treated with a fixed Q12W dosing (in one embodiment with one cycle
of Q8W dosing first), following treatment initiation (e.g. 6
initial monthly injections).
[0454] In one embodiment patients suffering from DME will be
treated following treatment initiation (e.g. with 3-7 initial
monthly injections) with a dosing regimen that extends the
injection interval in stable absence of disease, or shortens the
interval if there is disease activity. In one embodiment such
regimen includes that patient receive Q4w/Q8w/Q12w/Q16w dosing,
dependent on their disease state.
[0455] In one embodiment patients suffering from AMD will be
treated following treatment initiation (e.g. with 3-4 initial
monthly injections) with a dosing regimen that extends the
injection interval in stable absence of disease, or shortens the
interval if there is disease activity. In one embodiment such
regimen includes that patient receive Q4W/Q8W/Q12W/Q16W dosing,
dependent on their disease state.
Example 2A: Efficacy and Durability of Treatment of Patients
Suffering from Age-Related Macular Degeneration (AMD)
Objectives and Endpoints
[0456] This study has evaluated the efficacy, safety, and
pharmacokinetics of RO6867461 administered at 12- and 16-week
intervals in patients with neovascular age-related macular
degeneration (nAMD). RO6867461 is a bispecific antibody that binds
to human VEGF and human ANG2 comprising the amino acid sequences of
SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID
NO: 20 (this antibody VEGFang2-0016 and its production is also
described in detail in WO2014/009465 which is incorporated by
reference). Designations of this bispecific anti-VEGF/ANG2 antibody
herein are RO6867461 or RG7716 or VEGFang2-0016 or faricimab.
[0457] Specific objectives and corresponding endpoints for the
study are outlined below.
Objectives and Corresponding Endpoints
Primary Efficacy Objective
[0458] To evaluate the efficacy of RO6867461 on visual acuity when
administered at 12- and 16-week intervals
Corresponding Endpoint
[0458] [0459] Mean change from baseline BCVA at Week 40 using the
ETDRS-like charts Secondary Efficacy Objectives: 1) To evaluate the
efficacy of RO6867461 on additional visual acuity outcomes
Corresponding Endpoints
[0459] [0460] Mean change from baseline BCVA over time using the
ETDRS-like charts [0461] Proportion of patients gaining .gtoreq.15,
.gtoreq.10, .gtoreq.5, or .gtoreq.0 letters from baseline BCVA over
time [0462] Proportion of patients avoiding loss of .gtoreq.15,
.gtoreq.10, .gtoreq.5, or .gtoreq.0 letters from baseline BCVA over
time [0463] Proportion of patients with BCVA of 20/40 or better
over time [0464] Proportion of patients with BCVA of 20/200 or
worse over time 2) To evaluate the efficacy of RO6867461 on
anatomic outcome measures using SD-OCT
Corresponding Endpoints
[0464] [0465] Mean change from baseline in CFT over time [0466]
Mean change from baseline in mean CST (1 mm diameter) over time
[0467] Proportion of patients with intraretinal fluid, subretinal
fluid, cysts, or pigment epithelial detachment over time [0468] 3)
To evaluate the efficacy of RO6867461 on anatomic outcome measures
using FFA Corresponding Endpoints [0469] Mean change from baseline
in total area of CNV at Week 40 and Week 52 [0470] Mean change from
baseline in total area of CNV component at Week 40 and Week 52
[0471] Mean change from baseline in total area of leakage at Week
40 and Week 52 Exploratory Efficacy Objective [0472] To investigate
the incidence of disease activity at Week 24
Corresponding Endpoints
[0472] [0473] Proportion of patients with disease activity at Week
24
Safety Objective
[0473] [0474] To evaluate the safety of multiple IVT doses of
RO6867461 at 12- and 16-week intervals
Corresponding Endpoints
[0474] [0475] Incidence and severity of ocular adverse events
[0476] Incidence and severity of non-ocular adverse events [0477]
Other safety data, including but not limited to, reasons for
withdrawal from study, laboratory data, concomitant medications,
vital signs, and physical examination results will be listed and
summarized descriptively
Exploratory Pharmacokinetic/Pharmacodynamic Objectives
[0478] 1) To assess the systemic PK profile of RO6867461
Corresponding Endpoints
[0479] Plasma concentration of RO6867461 at specified timepoints 2)
To evaluate the RO6867461, ranibizumab, free VEGF-A, and Ang-2
profile in aqueous humor [0480] Relationship between aqueous humor
RO6867461 concentrations or PK parameters and free VEGF-A and Ang-2
concentrations
Corresponding Endpoints
[0480] [0481] Relationship between aqueous humor ranibizumab
concentrations or PK parameters and free VEGF-A and Ang-2
concentrations [0482] Time course of free VEGF-A and Ang-2
concentrations in aqueous humor
Immunogenicity Objective
[0482] [0483] To investigate the formation of plasma anti-RO6867461
antibodies
Corresponding Endpoints
[0483] [0484] Incidence of ADAs during the study
Exploratory Biomarker Objective
[0484] [0485] To explore levels of potential biomarkers of
angiogenesis and inflammation in aqueous humor at baseline and at
additional timepoints to assess their response to RO6867461
Corresponding Endpoints
[0485] [0486] Relationship between aqueous humor concentration of
potential biomarkers with primary and secondary endpoints
[0487] Abbreviations used above:
ADA=anti-drug antibody; Ang-2=angiopoietin-2; BCVA=best corrected
visual acuity; CFT=central foveal thickness; CNV=choroidal
neovascularization; CST=central subfield thickness; ETDRS=Early
Treatment Diabetic Retinopathy Study; FFA=fundus fluorescein
angiography; IVT=intravitreal; PK=pharmacokinetic; SD-OCT=spectral
domain optical coherence tomography; VEGF-A=vascular endothelial
growth factor A.
Study Design (FIG. 5 Presents an Overview of the Study Design)
Description of Study
[0488] This was a Phase II, multicenter, randomized, active
comparator-controlled, subject and outcome assessor masked,
parallel group, 52-week study to investigate the efficacy, safety,
and pharmacokinetics of RO6867461 administered at 12- and 16-week
intervals in treatment-naive patients with nAMD.
[0489] Approximately 75 patients were enrolled and randomized in a
2:2:1 ratio to one of three treatment arms: [0490] Arm A (Q12W): 6
mg RO6867461 intravitreally (IVT) every 4 weeks up to Week 12 (4
injections), followed by 6 mg RO6867461 IVT every 12 weeks up to
Week 48 (injections at Weeks 24, 36, and 48; 3 injections) [0491]
Arm B (Q16W): 6 mg RO6867461 IVT every 4 weeks up to Week 12 (4
injections), followed by 6 mg RO6867461 IVT every 16 weeks up to
Week 48 (injections at Weeks 28 and 44; 2 injections) A
protocol-defined assessment of disease activity at Week 24 requires
Arm B patients with active disease (see criteria below) to switch
to a 12-weekly dosing regimen of 6 mg RO6867461 for the remainder
of the study, with injections commencing at Week 24 and repeated at
Weeks 36 and 48. [0492] Arm C (comparator arm): 0.5 mg ranibizumab
IVT every 4 weeks for 48 weeks (13 injections) Only one eye will be
chosen as the study eye. The total duration of the study for each
patient will be up to 56 weeks, divided as follows: [0493]
Screening: up to 4 weeks prior to or on the same day as
randomization [0494] Randomization: Day 1 [0495] Study Treatment
Administration: from Day 1 to Week 48 [0496] Final Visit: Week
52
[0497] Patients have undergone a screening examination within 4
weeks of study treatment administration. The screening and Week
1/Day 1 (randomization) visit may have occurred as a combined visit
if all assessments (with the exception of informed consent) were
completed within 48 hours. During screening (or the combined
screening/Day 1 visit), the patient's eligibility was assessed,
including a central review of fundus photography (FP), spectral
domain optical coherence tomography (SD-OCT), and fundus
fluorescein angiography (FFA) to ensure that CNV secondary to AMD
meets the predefined ocular criteria in the study. Patients who
were deemed ineligible based on screening results for any of the
following reasons were allowed to be re-screened: [0498]
Uncontrolled blood pressure [0499] Administrative reason (e.g.,
unable to schedule Day 1 within 28 days from the screening visit)
[0500] Not meeting eligibility criteria for the study eye (in the
event the patient might be eligible to participate for the second
eye after the initial screening period)
[0501] At re-screening, all screening visit assessments were
performed (except for FFA imaging collection), provided the Central
Reading Center-eligible FFA images were taken within 4 weeks before
the new Day 1 visit (randomization).
[0502] On Day 1, eligible patients received their first IVT
administration of either RO6867461 or ranibizumab according to the
randomization schedule described above and following established
standard administration procedures. Patients returned to the eye
clinic 7 days after their first IVT administration and then every 4
weeks for study treatment administration and assessments as
outlined in the schedule of activities in the protocol. Sham IVT
administration was delivered to patients randomized to Arms A and B
to maintain masking throughout the study period.
[0503] All patients were assessed for disease activity at Week 24.
Patients randomized to Arm B who had active disease at Week 24 (see
criteria below) switched to the Q12W dosing regimen of 6 mg
RO6867461 for the remainder of the study, with injections
commencing at Week 24 and repeated at Weeks 36 and 48.
[0504] Determination of active disease was made if any of the
following criteria were met: [0505] Increase in central subfield
thickness (CST of .gtoreq.50 .mu.m on Spectralis.RTM. OCT compared
to average CST over last 2 visits (Weeks 16 and 20)
Or
[0505] [0506] Increase in CST of .gtoreq.75 .mu.m compared to
lowest CST recorded at either Week 16 or Week 20
Or
[0506] [0507] Decrease of at least 5 letters of best corrected
visual acuity (BCVA) compared with average BCVA over last 2 visits
(Weeks 16 and 20) due to nAMD disease activity
Or
[0507] [0508] Decrease of .gtoreq.10 letters of BCVA compared to
highest BCVA recorded at either Week 16 or Week 20 due to nAMD
disease activity Or [0509] Presence of new macular hemorrhage due
to nAMD activity
[0510] Patients will return for a final visit at Week 52. After the
final visit, adverse events should be followed up as outlined in
the protocol. Assessments performed in case of an unscheduled
visit(s) are at the discretion of the investigator
[0511] Number of Patients: Approximately 75 treatment-naive
patients with nAMD were expected to be enrolled and randomized in
this study in the United States.
Target Population
Inclusion Criteria
[0512] Patients met the following criteria for study entry: Ocular
Criteria for Study Eye [0513] Treatment-naive CNV secondary to AMD
(nAMD) [0514] Subfoveal CNV or juxtafoveal CNV with a subfoveal
component related to the CNV activity by FFA or SD-OCT (as
evidenced by subretinal fluid, subretinal hyper-reflective
material, evidence of leakage, or hemorrhage) [0515] CNV lesion of
all types (predominantly classic, minimally classic, or occult)
with: Total lesion size (including blood, atrophy, fibrosis, and
neovascularization) of .ltoreq.6 disc areas by FFA And CNV
component area of .gtoreq.50% of total lesion size by FFA And
Active CNV confirmed by FFA (evidence of leakage) And CNV exudation
confirmed by SD-OCT (presence of fluid) [0516] Clear ocular media
and adequate pupillary dilatation to allow acquisition of good
quality retinal images to confirm diagnosis General Criteria [0517]
Signed Informed Consent Form [0518] Age .gtoreq.50 years on Day 1
[0519] Ability to comply with the study protocol, in the
investigator's judgment [0520] For women of childbearing potential:
agreement to remain abstinent (refrain from heterosexual
intercourse) or use a contraceptive method with a failure rate of
<1% per year during the treatment period and for at least 28
days after the last dose of study treatment [0521] Patients must be
willing not to participate in any other clinical trial including an
investigational medicinal product (IMP) or device up to completion
of the current study
Exclusion Criteria
[0522] Patients who met any of the following criteria were excluded
from study entry:
Ocular Criteria for Study Eye
[0523] CNV due to causes other than AMD, such as ocular
histoplasmosis, trauma, pathological myopia, angioid streaks,
choroidal rupture, or uveitis [0524] Central serous
chorioretinopathy at screening [0525] Retinal pigment epithelial
tear involving the macula [0526] On FFA Subretinal hemorrhage of
>50% of the total lesion area and/or that involves the fovea
Fibrosis or atrophy of >50% of the total lesion area and/or that
involves the fovea [0527] Any prior or concomitant treatment for
CNV including (but not restricted to) IVT treatment (steroids,
anti-vascular endothelial growth factor [VEGF], tissue plasminogen
activator, ocriplasmin, C3F8 gas, air), periocular pharmacological
intervention, argon LASER photocoagulation, verteporfin
photodynamic therapy, diode laser, transpupillary thermotherapy, or
surgical intervention Cataract surgery within 3 months of baseline
assessments (Day 1) [0528] Any other intraocular surgery (pars
plana vitrectomy, glaucoma surgery, corneal transplant,
radiotherapy) [0529] Prior IVT treatment (including anti-VEGF
medication) except for management of cataract complication with
steroid IVT treatment Prior periocular pharmacological intervention
for other retinal diseases Concurrent Ocular Conditions [0530] Any
concurrent intraocular condition in the study eye (e.g., amblyopia,
aphakia, retinal detachment, cataract, diabetic retinopathy or
maculopathy, or epiretinal membrane with traction) that, in the
opinion of the investigator, could either reduce the potential for
visual improvement or require medical or surgical intervention
during the course of the study [0531] Active intraocular
inflammation (grade trace or above) in the study eye on Day 1
(prior to randomization) BCVA letter score of 73 to 24 letters
(inclusive) on Early Treatment Diabetic Retinopathy Study
(ETDRS)-like charts (20/40 to 20/320 Snellen equivalent) on Day 1
[0532] Current vitreous hemorrhage in the study eye [0533]
Uncontrolled glaucoma (e.g., progressive loss of visual fields or
defined as intraocular pressure [IOP].gtoreq.25 mmHg despite
treatment with anti-glaucoma medication) in the study eye [0534]
Spherical equivalent of refractive error demonstrating more than 8
diopters of myopia in the study eye [0535] History of idiopathic or
autoimmune-associated uveitis in either eye [0536] Active
infectious conjunctivitis, keratitis, scleritis, or endophthalmitis
in either eye on Day 1 (prior to randomization) General Criteria
[0537] Any major illness or major surgical procedure within 1 month
before screening [0538] Uncontrolled blood pressure ([BP] defined
as systolic >180 mmHg and/or diastolic >100 mmHg while
patient at rest). If a patient's initial reading exceeds these
values, a second reading may be taken later on the same day, or on
another day during the screening period. If the patient's BP is
controlled by antihypertensive medication, the patient should be
taking the same medication continuously for at least 30 days prior
to Day 1. [0539] Stroke or myocardial infarction within 3 months
prior to Day 1 [0540] History of other disease, metabolic
dysfunction, physical examination finding, or clinical laboratory
findings giving reasonable suspicion of a condition that
contraindicated the use of the investigational drug or that might
affect interpretation of the results of the study or renders the
patient at high risk for treatment complications in the opinion of
the investigator [0541] Pregnant or breastfeeding, or intending to
become pregnant during the study Women of childbearing potential
must have a negative urine pregnancy test result within 28 days
prior to initiation of study treatment. If the urine pregnancy test
is positive, it must be confirmed by a serum pregnancy test. [0542]
Known hypersensitivity to ranibizumab, fluorescein, any ingredients
of the formulation used, dilating eye drops, or any of the
anesthetic and antimicrobial drops used [0543] Treatment with
investigational therapy within 3 months prior to initiation of
study treatment
End of Study
[0544] The end of the study was defined as the date when the last
patient last visit (LPLV) occurs. LPLV was expected to occur 52
weeks after the last patient is enrolled.
Length of Study
[0545] The total length of the study, from screening of the first
patient to the end of the study, was expected to be approximately
18-19 months.
Investigational Medicinal Products Test Product
[0546] RO6867461 Drug Product (120 mg/mL) was provided as a
sterile, colorless to brownish liquid and contains no
preservatives. Vials of sterile, colorless to brownish,
preservative-free solution of RO6867461 for IVT administration of 6
mg dose every were used. The concentration of the bispecific
antibody was about 120 mg/ml.
[0547] Dosage and Administration,
[0548] RO6867461, Ranibizumab, and Sham
[0549] Patients were given a 50-.mu.L IVT injection of RO6867461 or
ranibizumab into the study eye, or a sham administration, according
to the randomization schedule as described below [0550] Arm A
(Q12W): 6 mg RO6867461 IVT every 4 weeks up to Week 12 (4
injections), followed by 6 mg RO6867461 IVT every 12 weeks up to
Week 48 (injections at Weeks 24, 36, and 48; 3 injections) [0551]
Arm B (Q16W): 6 mg RO6867461 IVT every 4 weeks up to Week 12 (4
injections), followed by 6 mg RO6867461 IVT every 16 weeks up to
Week 48 (injections at Weeks 28 and 44; 2 injections) [0552] Arm C
(comparator arm): 0.5 mg ranibizumab IVT every 4 weeks for 48 weeks
(13 injections)
[0553] Only one eye was chosen as the study eye.
Study Assessments
[0554] At timepoints when several assessments coincide, the
following sequence was suggested, at the discretion of the
investigator. The order could be adjusted to optimize site
personnel and patient's time management, except where explicitly
stated as mandatory (i.e., text in italics): [0555] Vital signs
[0556] Blood sampling: At visits where FFA is performed, blood
sampling and angiography can be performed from the same venous
cannula. Blood samples must be collected before angiography. [0557]
Ocular assessments and imaging
[0558] BCVA: BCVA must have been conducted before pupil dilation.
At screening and Day 1 visits, BCVA could be performed before vital
signs and blood sampling to avoid unnecessary investigations in
those patients who may be a screen failure as a result of BCVA
letter score.
[0559] Slitlamp examination
[0560] Pupil dilation
[0561] SD-OCT
[0562] FP (+infrared reflectance)
[0563] FFA
[0564] Dilated binocular indirect high-magnification
ophthalmoscopy
[0565] IOP: mandatory to be performed after all imaging
assessments, and the same method should be used throughout the
study period [0566] Aqueous humor sampling (optional)
Disease-Specific Assessments
[0567] Unless otherwise noted in schedule of activities (Appendix
1), all ocular assessments were performed for both eyes.
Best Corrected Visual Acuity
[0568] BCVA at a starting test distance of 4 meters was measured
prior to dilating eyes by a trained and certified visual acuity
(VA) examiner masked to study eye treatment assignment.
[0569] BCVA was measured using the set of three Precision
Vision.TM. or Lighthouse distance acuity charts (modified ETDRS
Charts 1, 2, and R). A VA Procedure Manual was provided to the
investigators. VA examiner and VA examination room certifications
were obtained before any VA examinations were performed.
[0570] The BCVA examiner was masked to the study eye and treatment
assignment and will perform the refraction and BCVA assessments
(e.g., VA Specification Manual). The BCVA examiner was also masked
to the BCVA letter scores of a patient's previous visits and may
only know patient refraction data from previous visits.
Additional Ocular Assessments
[0571] Additional ocular assessments which were performed during
the study include the following: [0572] Slitlamp examination
(scales for grading flare/cells and vitreous hemorrhage density are
detailed in Appendix 2) [0573] Dilated binocular indirect
high-magnification ophthalmoscopy [0574] IOP
[0575] The method of IOP measurement used for a patient remained
consistent throughout the study. IOP measurement of both eyes were
performed after all imaging.
[0576] At study treatment visits, IOP pressure was conducted prior
to study treatment administration and 30 (.+-.15) minutes
post-treatment administration in the study eye, and if IOP
.gtoreq.30 mmHg, IOP should be re-assessed 30 (.+-.15) minutes
later. If IOP continued to be elevated, treatment was undertaken at
the discretion of the investigator. [0577] Finger count vision
assessment
[0578] In the study eye, a post-treatment optic nerve head
perfusion was assessed for each patient immediately after study
treatment administration (maximum within 15 minutes after treatment
administration) by testing finger count vision, hand motion, or
light perception as appropriate.
Ocular Imaging
[0579] The Central Reading Center provided sites with the Central
Reading Center Manual and training materials for study-mandated
ocular imaging. Before study images were obtained, site personnel
and imaging systems (where applicable) was certified by the reading
center as specified in the Central Reading Center Manual. All study
subject ocular images were obtained only by trained and Central
Reading Center certified personnel on certified/registered
equipment at the study sites. A copy of all study subject ocular
images were transferred to the central reading center for storage
and for independent analysis, including for confirmation of
eligibility of defined image-related criteria.
Week 24 Assessment of Disease Activity
[0580] All patients were assessed for disease activity at Week 24.
Patients randomized to Arm B who had active disease at Week 24 (see
criteria below) switched to the Q12W dosing regimen of 6 mg
RO6867461 for the remainder of the study, with injections
commencing at Week 24 and repeated at Weeks 36 and 48.
[0581] Determination of active disease was made if any of the
following criteria are met: [0582] Increase in CST of >50 .mu.m
on Spectralis OCT compared to average CST over last 2 visits (Weeks
16 and 20)
Or
[0582] [0583] Increase in CST of .gtoreq.75 .mu.m compared to
lowest CST recorded at either Week 16 or Week 20
Or
[0583] [0584] Decrease of at least 5 letters of BCVA compared with
average BCVA over last 2 visits (Weeks 16 and 20), due to nAMD
disease activity
Or
[0584] [0585] Decrease of .gtoreq.10 letters of BCVA compared to
highest BCVA recorded at either Week 16 or Week 20 due to nAMD
disease activity
Or
[0585] [0586] Presence of new macular hemorrhage due to nAMD
activity
Results
Best Corrected Visual Acuity (BCVA) and Durability of BCVA Gains
(Time to Retreatment to Maintain BCVA Gain)
[0587] Primary Efficacy Outcome Measure is shown in FIG. 6. The
FIG. 6 displays the primary efficacy endpoint: BCVA change from
Baseline over Time to Week 40. RO6867461 refers to the bispecific
anti-VEGF/ANG2 antibody RO6867461 comprising the amino acid
sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO: 19, and
of SEQ ID NO: 20 (administered intravitreally with a 6.0 mg dose
either Q12W or Q16W), ranibizumab (Lucentis.RTM.) was administered
intravitreally with a 0.3 mg dose Q4W. The initial BCVA gains were
fully maintained for the RO6867461 Q12W or Q16W groups and in a
similar range as the ranibizumab (Lucentis.RTM.) Q4W group.
Central Subfield Thickness (CST) Change from Baseline (Study
Eye)
[0588] A key secondary endpoint was the change from baseline in
CST, central subfield thickness. Results are shown in FIG. 7. The
bispecific anti-VEGF/ANG2 antibody RO6867461 comprising the amino
acid sequences of SEQ ID NO: 17, of SEQ ID NO: 18, of SEQ ID NO:
19, and of SEQ ID NO: 20 (administered intravitreally with a 6.0 mg
dose either Q12W or Q16W), was compared to ranibizumab
(Lucentis.RTM.) (administered intravitreally with a 0.3 mg dose
Q4W). This secondary anatomical endpoint directionally supports
BCVA primary outcome There were grater reductions in CST with
bispecific anti-VEGF/ANG2 antibody RO6867461 during treatment
initiation than with ranibizumab.
Example 2B: Efficacy and Durability of Treatment of Patients
Suffering from Age-Related Macular Degeneration (AMD)
[0589] In a further study analogous to the above described study
under Example 2A, patients suffering from AMD (e.g. wet age-related
macular degeneration (wAMD), especially neovascular AMD) are
treated with the bispecific antibody that binds to human VEGF and
human ANG2 comprising the amino acid sequences of SEQ ID NO: 17, of
SEQ ID NO: 18, of SEQ ID NO: 19, and of SEQ ID NO: 20. As active
comparator in treatment e.g aflibercept and/or ranibizumab and/or
brolicuzimab will be used. Patients include anti-VEGF
treatment-naive patients (have not been previously treated with
anti-VEGF monotherapy with e.g. aflibercept and/or ranibizumab
and/or brolicuzimab) and also a group of patients which have been
previously treated with anti-VEGF monotherapy with e.g. aflibercept
and/or ranibizumab and/or brolicuzimab. Designations of the
respective bispecific antibody that binds to human VEGF and human
ANG2 are RO6867461 or RG7716. Vials of sterile, colorless to
brownish, preservative-free solution of RO6867461 for IVT
administration of either 1.5 mg or 6 mg dose are used.
[0590] E.g. the following dosing schedules is used: [0591] Patients
suffering from AMD will be treated following treatment initiation
(e.g. with 3-7 initial monthly injections) with a dosing regimen
that extends the injection interval in stable absence of disease,
or shortens the interval if there is disease activity. Such regimen
includes e.g. that patient receive Q4W/Q8W/Q12W/Q16W dosing,
dependent on their disease state
[0592] The disease stability assessment would be based on
best-corrected visual acuity (BCVA) and on CST as well as retinal
thickness based on Optical coherence tomography (OCT). Outcome
measure and results will be evaluated as described e.g. in Example
1A. Primary endpoints will be between 45 and 60 weeks.
Example 3
Binding to of the Anti-VEGF/ANG2 Antibody to VEGF, Ang2, FcgammaR
and FcRn
VEGF Isoforms Kinetic Affinity Including Assessment of
Species-Crossreactivity
[0593] Around 12000 resonance units (RU) of the capturing system
(10 .mu.g/ml goat anti human F(ab)'.sub.2; Order Code: 28958325; GE
Healthcare Bio-Sciences AB, Sweden) were coupled on a CMS chip (GE
Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit
supplied by the GE Healthcare. The sample and system buffer was
PBS-T (10 mM phosphate buffered saline including 0.05% Tween.RTM.
20) pH 7.4. The flow cell was set to 25.degree. C.--and the sample
block set to 12.degree. C.--and primed with running buffer twice.
The bispecific antibody was captured by injecting a 50 nM solution
for 30 sec at a flow of 5 .mu.l/min. Association was measured by
injection of human hVEGF121, mouse mVEGF120 or rat rVEGF164 in
various concentrations in solution for 300 sec at a flow of 30
.mu.l/min starting with 300 nM in 1:3 dilutions. The dissociation
phase was monitored for up to 1200 sec and triggered by switching
from the sample solution to running buffer. The surface was
regenerated by 60 sec washing with a Glycine pH 2.1 solution at a
flow rate of 30 .mu.l/min. Bulk refractive index differences were
corrected by subtracting the response obtained from a goat anti
human F(ab')2 surface. Blank injections are also subtracted
(=double referencing). For calculation of apparent K.sub.D and
other kinetic parameters the Langmuir 1:1 model was used. Results
are shown in Table 5.
Ang2 Solution Affinity Including Assessment of
Species-Crossreactivity
[0594] Solution affinity measures the affinity of an interaction by
determining the concentration of free interaction partners in an
equilibrium mixture. The solution affinity assay involves the
mixing of an <VEGF-ANG-2> bispecific antibody, kept at a
constant concentration, with a ligand (=Ang2) at varying
concentrations. Maximum possible resonance units (e.g. 17000
resonance units (RU)) of an antibody was immobilized on the CMS
chip (GE Healthcare BR-1005-30) surface at pH 5.0 using an amine
coupling kit supplied by the GE Healthcare. The sample and system
buffer was HBS-P pH 7.4. Flow cell was set to 25.degree. C. and
sample block to 12.degree. C. and primed with running buffer twice.
To generate a calibration curve increasing concentrations of Ang2
were injected into a BIAcore.TM. flowcell containing the
immobilized VEGF-ANG-2> bispecific antibody. The amount of bound
Ang2 was determined as resonance units (RU) and plotted against the
concentration. Solutions of each ligand (11 concentrations from 0
to 200 nM for the VEGF-ANG-2> bispecific antibody) were
incubated with 10 nM Ang2 and allowed to reach equilibrium at room
temperature. Free Ang2 concentrations were determined from
calibration curve generated before and after measuring the response
of solutions with known amounts of Ang2. A 4-parameter fit was set
with XLfit4 (IDBS Software) using Model 201 using free Ang2
concentration as y-axis and used concentration of antibody for
inhibition as x-axis. The affinity was calculated by determining
the inflection point of this curve. The surface was regenerated by
one time 30 sec washing with a 0.85% H3PO4 solution at a flow rate
of 30 .mu.l/min. Bulk refractive index differences were corrected
by subtracting the response obtained from a blank-coupled surface.
Results are shown in Table 6.
FcRn Steady State Affinity
[0595] For FcRn measurement a steady state affinity was used to
compare bispecific antibodies against each other. Human FcRn was
diluted into coupling buffer (10 .mu.g/ml, Na-Acetate pH5.0) and
immobilized on a C1-Chip (GE Healthcare BR-1005-35) by targeted
immobilization procedure using a BIAcore.TM. wizard to a final
response of 200 RU. Flow cell was set to 25.degree. C. and sample
block to 12.degree. C. and primed with running buffer twice. The
sample and system buffer was PBS-T (10 mM phosphate buffered saline
including 0.05% Tween.RTM. 20) pH 6.0. To assess different IgG
concentrations for each antibody, a concentration of 62.5 nM, 125
nM and 250 nM, 500 nM was prepared. Flow rate was set to 30
.mu.l/min and the different samples were injected consecutively
onto the chip surface choosing 180 sec association time. The
surface was regenerated by injected PBS-T pH 8 for 60 sec at a flow
rate of 30 .mu.l/min. Bulk refractive index differences were
corrected by subtracting the response obtained from a blank
surface. Buffer injections are also subtracted (=double
referencing). For calculation of steady state affinity the method
from the Bia-Evaluation software was used. Briefly, the RU values
(RU max) were plotted against the analysed concentrations, yielding
a dose-response curve. Based on a 2-parametric fit, the upper
asymptote is calculated, allowing the determination of the
half-maximal RU value and hence the affinity. Results are shown in
FIG. 5 and Table 7. Analogously the affinity to cyno, mouse and
rabbit FcRn can be determined.
FcgammaRIIIa measurement
[0596] For FcgammaRIIIa measurement a direct binding assay was
used. Around 3000 resonance units (RU) of the capturing system (1
.mu.g/ml Penta-His; Qiagen) were coupled on a CMS chip (GE
Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit
supplied by the GE Healthcare. The sample and system buffer was
HBS-P+ pH 7.4. The flow cell was set to 25.degree. C.--and sample
block to 12.degree. C.--and primed with running buffer twice. The
FcgammaRIIIa-His-receptor was captured by injecting a 100 nM
solution for 60 sec at a flow of 5 .mu.l/min. Binding was measured
by injection of 100 nM of bispecific antibody or monospecific
control antibodies (anti-Dig for IgG1 subclass and an IgG4 subclass
antibody) for 180 sec at a flow of 30 .mu.l/. The surface was
regenerated by 120 sec washing with Glycine pH 2.5 solution at a
flow rate of 30 .mu.l/min. Because FcgammaRIIIa binding differs
from the Langmuir 1:1 model, only binding/no binding was determined
with this assay. In a similar manner FcgammaRIa, and FcgammaRIIa
binding can be determined. Results are shown in FIG. 6, where it
follows that by introduction of the mutations P329G LALA no more
binding to FcgammaRIIIa could be detected.
Assessment of Independent VEGF- and Ang2-Binding to the
<VEGF-ANG-2> Bispecific Antibodies
[0597] Around 3500 resonance units (RU) of the capturing system (10
.mu.g/ml goat anti human IgG; GE Healthcare Bio-Sciences AB,
Sweden) were coupled on a CM4 chip (GE Healthcare BR-1005-34) at pH
5.0 by using an amine coupling kit supplied by the GE Healthcare.
The sample and system buffer was PBS-T (10 mM phosphate buffered
saline including 0.05% Tween.RTM. 20) pH 7.4. The temperature of
the flow cell was set to 25.degree. C. and of the sample block to
12.degree. C. Before capturing, the flow cell was primed with
running buffer twice.
[0598] The bispecific antibody was captured by injecting a 10 nM
solution for 60 sec at a flow of 5 .mu.l/min. Independent binding
of each ligand to the bispecific antibody was analysed by
determining the active binding capacity for each ligand, either
added sequentially or simultaneously (flow of 30 .mu.l/min): [0599]
1. Injection of human VEGF with a concentration of 200 nM for 180
sec (identifies the single binding of the antigen). [0600] 2.
Injection of human Ang2 with a concentration of 100 nM for 180 sec
(identifies single binding of the antigen). [0601] 3. Injection of
human VEGF with a concentration of 200 nM for 180 sec followed by
an additional injection of human Ang2 with a concentration of 100
nM for 180 sec (identifies binding of Ang2 in the presence of
VEGF). [0602] 4. Injection of human Ang2 with a concentration of
100 nM for 180 sec followed by an additional injection of human
VEGF with a concentration of 200 nM (identifies binding of VEGF in
the presence of Ang2). [0603] 5. Co-Injection of human VEGF with a
concentration of 200 nM and of human Ang2 with a concentration of
100 nM for 180 sec (identifies the binding of VEGF and of Ang2 at
the same time).
[0604] The surface was regenerated by 60 sec washing with a 3 mM
MgCl2 solution at a flow rate of 30 .mu.l/min. Bulk refractive
index differences were corrected by subtracting the response
obtained from a goat anti human IgG surface.
[0605] The bispecific antibody is able to bind both antigens mutual
independently if the resulting final signal of the approaches 3, 4
& 5 equals or is similar to the sum of the individual final
signals of the approaches 1 and 2. Results are shown in the Table
below, where VEGFang2-0016 (=RO6867461), is shown to be able to
bind mutual independently to VEGF and ANG2
Assessment of Simultaneous VEGF- and Ang2-Binding to the
<VEGF-ANG-2> Bispecific Antibodies
[0606] First, around 1600 resonance units (RU) of VEGF (20
.mu.g/ml) were coupled on a CM4 chip (GE Healthcare BR-1005-34) at
pH 5.0 by using an amine coupling kit supplied by the GE
Healthcare. The sample and system buffer was PBS-T (10 mM phosphate
buffered saline including 0.05% Tween.RTM. 20) pH 7.4. Flow cell
was set to 25.degree. C. and sample block to 12.degree. C. and
primed with running buffer twice. Second, 50 nM solution of the
bispecific antibody was injected for 180 sec at a flow of 30
.mu.l/min. Third, hAng-2 was injected for 180 sec at a flow of 30
.mu.l/min. The binding response of hAng-2 depends from the amount
of the bispecific antibody bound to VEGF and shows simultaneous
binding. The surface was regenerated by 60 sec washing with a 0.85%
H3PO4 solution at a flow rate of 30 .mu.l/min. Simultaneous binding
is shown by an additional specific binding signal of hAng2 to the
previous VEGF bound <VEGF-ANG-2> bispecific antibodies.
TABLE-US-00003 TABLE Results: Kinetic affinities to VEGF isoforms
from different species VEGFang2-0016 -apparent affinity Human VEGF
121 <1 pM (out of Biacore specification) mouseVEGF 120 no
binding Rat VEGF 164 14 nM
TABLE-US-00004 TABLE Results: Solution affinities to Ang2
VEGFang2-0016 KD [nM] humanAng2 20 cynoAng2 13 mouseAng2 13
rabbitAng2 11
TABLE-US-00005 TABLE Results: Affinity to FcRn of
<VEGF-ANG-2> bispecific antibodies VEGFang2-0016 [affinity]
Human FcRn no binding Cyno FcRn no binding Mouse FcRn no
binding
TABLE-US-00006 TABLE Results Binding to FcgammaRI - III
VEGFang2-0016 Fc.gamma.RIa No binding Fc.gamma.RIIa No binding
Fc.gamma.RIIIa No binding
TABLE-US-00007 TABLE Results: Independent binding of VEGF- and Ang2
to <VEGF-ANG-2> bispecific antibodies 3) first 4) first 5)
Co- VEGF Ang2 injection then then Ang2 + 1) Ang2 2) VEGF Ang2 VEGF
VEGF [RUmax] [RUmax] [RUmax] [RUmax] [RUmax] VEGFang2- 174 50 211
211 211 0016
Sequence CWU 1
1
27114PRTArtificialheavy chain CDR3H, <VEGF>ranibizumab 1Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val1 5
10217PRTArtificialheavy chain CDR2H, <VEGF>ranibizumab 2Trp
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys1 5 10
15Arg35PRTArtificialheavy chain CDR1H, <VEGF>ranibizumab 3His
Tyr Gly Met Asn1 549PRTArtificiallight chain CDR3L,
<VEGF>ranibizumab 4Gln Gln Tyr Ser Thr Val Pro Trp Thr1
557PRTArtificiallight chain CDR2L, <VEGF>ranibizumab 5Phe Thr
Ser Ser Leu His Ser1 5611PRTArtificiallight chain CDR1L,
<VEGF>ranibizumab 6Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu
Asn1 5 107123PRTArtificialheavy chain variable domain VH,
<VEGF>ranibizumab 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe
Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
1208107PRTArtificiallight chain variable domain VL,
<VEGF>ranibizumab 8Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Val Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105920PRTArtificialheavy chain
CDR3H, <ANG-2> Ang2i_LC10 variant 9Ser Pro Asn Pro Tyr Tyr
Tyr Asp Ser Ser Gly Tyr Tyr Tyr Pro Gly1 5 10 15Ala Phe Asp Ile
201017PRTArtificialheavy chain CDR2H, <ANG-2> Ang2i_LC10
variant 10Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys
Phe Gln1 5 10 15Gly115PRTArtificialheavy chain CDR1H, <ANG-2>
Ang2i_LC10 variant 11Gly Tyr Tyr Met His1 51211PRTArtificiallight
chain CDR3L, <ANG-2> Ang2i_LC10 variant 12Gln Val Trp Asp Ser
Ser Ser Asp His Trp Val1 5 10137PRTArtificiallight chain CDR2L,
<ANG-2> Ang2i_LC10 variant 13Asp Asp Ser Asp Arg Pro Ser1
51411PRTArtificiallight chain CDR1L, <ANG-2> Ang2i_LC10
variant 14Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His1 5
1015129PRTArtificialheavy chain variable domain VH, <ANG-2>
Ang2i_LC10 variant 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr
Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg
Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Pro
Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110Pro Gly
Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120
125Ser16110PRTArtificiallight chain variable domain VL,
<ANG-2> Ang2i_LC10 variant 16Ser Tyr Val Leu Thr Gln Pro Pro
Ser Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Gly
Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45Asp Asp Ser Asp Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser Gly Asn
Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly65 70 75 80Asp Glu
Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95Trp
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser 100 105
11017451PRTArtificialHeavy chain 1 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016)
17Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His
Tyr 20 25 30Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys
Ser Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr
Ser His Trp Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val145 150 155
160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala225 230 235 240Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255Leu Met Ala
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280
285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp
Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Gly Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu Pro Pro
Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val Ser Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390 395
400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln
Lys Ser Leu Ser 435 440 445Leu Ser Pro 45018461PRTArtificialHeavy
chain 2 of <VEGF-ANG-2> CrossMAb IgG1 with AAA mutations and
P329G LALA mutations (VEGFang2-0016) 18Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30Tyr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn
Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr
100 105 110Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr
Val Ser 115 120 125Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215
220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr
His225 230 235 240Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val 245 250 255Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ala Ser Arg Thr 260 265 270Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu 275 280 285Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser305 310 315 320Val
Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330
335Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile
340 345 350Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
Leu Pro 355 360 365Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Ser Cys Ala 370 375 380Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn385 390 395 400Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415Asp Gly Ser Phe Phe
Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445His
Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 450 455
46019214PRTArtificialLight chain 1 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016)
19Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val
Leu Ile 35 40 45Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ser Thr Val Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21020213PRTArtificialLight chain 2 of <VEGF-ANG-2> CrossMAb
IgG1 with AAA mutations and P329G LALA mutations (VEGFang2-0016)
20Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1
5 10 15Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser
Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg
Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Asp Ser Ser Ser Asp His 85 90 95Trp Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Ser Ser Ala Ser 100 105 110Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val145 150 155
160His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
165 170 175Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile 180 185 190Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val 195 200 205Glu Pro Lys Ser Cys 21021107PRTHomo
sapiens 21Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 10522105PRThomo sapiens 22Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly
Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45Lys Ala
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser65 70 75
80His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
85 90 95Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10523328PRTHomo
sapiens 23Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro 32524191PRTHomo
sapiens 24Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu
Leu Leu1 5 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met
Ala Glu Gly 20 25 30Gly Gly Gln Asn His His Glu Val Val Lys Phe Met
Asp Val Tyr Gln 35 40 45Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val
Asp Ile Phe Gln Glu 50 55 60Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys
Pro Ser Cys Val Pro Leu65 70 75 80Met Arg Cys Gly Gly Cys Cys Asn
Asp Glu Gly Leu Glu Cys Val Pro 85 90 95Thr Glu Glu Ser Asn Ile Thr
Met Gln Ile Met Arg Ile Lys Pro His 100 105 110Gln Gly Gln His Ile
Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125Glu Cys Arg
Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140Pro
Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr145 150
155 160Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg
Gln 165 170 175Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro
Arg Arg 180 185 19025496PRTHomo sapiens 25Met Trp Gln Ile Val Phe
Phe Thr Leu Ser Cys Asp Leu Val Leu Ala1 5 10 15Ala Ala Tyr Asn Asn
Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys 20 25 30Gln Tyr Gln Val
Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45Glu Met Asp
Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60Val Gln
Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu65 70 75
80Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys
85 90 95Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu
Ile 100 105 110Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile
Glu Ile Gly 115 120 125Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr
Arg Lys Leu Thr Asp 130 135 140Val Glu Ala Gln Val Leu Asn Gln Thr
Thr Arg Leu Glu Leu Gln Leu145 150 155 160Leu Glu His Ser Leu Ser
Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175Gln Thr Ser Glu
Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190Lys Lys
Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200
205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn
210 215 220Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr
Val Asn225 230 235 240Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu
Met Glu Thr Val Asn 245 250 255Asn Leu Leu Thr Met Met Ser Thr Ser
Asn Ser Ala Lys Asp Pro Thr 260 265 270Val Ala Lys Glu Glu Gln Ile
Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280 285Lys Ser Gly His Thr
Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300Ser Thr Glu
Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly305 310 315
320Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln
325 330 335Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser
Gly Glu 340 345 350Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr
Asn Gln Gln Arg 355 360 365Tyr Val Leu Lys Ile His Leu Lys Asp Trp
Glu Gly Asn Glu Ala Tyr 370 375 380Ser Leu Tyr Glu His Phe Tyr Leu
Ser Ser Glu Glu Leu Asn Tyr Arg385 390 395 400Ile His Leu Lys Gly
Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile 405 410 415Ser Gln Pro
Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430Cys
Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440
445Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln
450 455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys
Gly Ser465 470 475 480Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile
Arg Pro Ala Asp Phe 485 490 49526498PRTHomo sapiens 26Met Thr Val
Phe Leu Ser Phe Ala Phe Leu Ala Ala Ile Leu Thr His1 5 10 15Ile Gly
Cys Ser Asn Gln Arg Arg Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30Tyr
Asn Arg Ile Gln His Gly Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40
45Glu His Asp Gly Asn Cys Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr
50 55 60Asn Ala Leu Gln Arg Asp Ala Pro His Val Glu Pro Asp Phe Ser
Ser65 70 75 80Gln Lys Leu Gln His Leu Glu His Val Met Glu Asn Tyr
Thr Gln Trp 85 90 95Leu Gln Lys Leu Glu Asn Tyr Ile Val Glu Asn Met
Lys Ser Glu Met 100 105 110Ala Gln Ile Gln Gln Asn Ala Val Gln Asn
His Thr Ala Thr Met Leu 115 120 125Glu Ile Gly Thr Ser Leu Leu Ser
Gln Thr Ala Glu Gln Thr Arg Lys 130 135 140Leu Thr Asp Val Glu Thr
Gln Val Leu Asn Gln Thr Ser Arg Leu Glu145 150 155 160Ile Gln Leu
Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys Gln 165 170 175Leu
Leu Gln Gln Thr Asn Glu Ile Leu Lys Ile His Glu Lys Asn Ser 180 185
190Leu Leu Glu His Lys Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu
195 200 205Leu Asp Thr Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu
Val Thr 210 215 220Arg Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln
Leu Asn Arg Ala225 230 235 240Thr Thr Asn Asn Ser Val Leu Gln Lys
Gln Gln Leu Glu Leu Met Asp 245 250 255Thr Val His Asn Leu Val Asn
Leu Cys Thr Lys Glu Gly Val Leu Leu 260 265 270Lys Gly Gly Lys Arg
Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp 275 280 285Val Tyr Gln
Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr Ile 290 295 300Asn
Asn Met Pro Glu Pro Lys Lys Val Phe Cys Asn Met Asp Val Asn305 310
315 320Gly Gly Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu
Asp 325 330 335Phe Gln Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly
Asn Pro Ser 340 345 350Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe
Ala Ile Thr Ser Gln 355 360 365Arg Gln Tyr Met Leu Arg Ile Glu Leu
Met Asp Trp Glu Gly Asn Arg 370 375 380Ala Tyr Ser Gln Tyr Asp Arg
Phe His Ile Gly Asn Glu Lys Gln Asn385 390 395 400Tyr Arg Leu Tyr
Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln Ser 405 410 415Ser Leu
Ile Leu His Gly Ala Asp Phe Ser Thr Lys Asp Ala Asp Asn 420 425
430Asp Asn Cys Met Cys Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp
435 440 445Phe Asp Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr
Thr Ala 450 455 460Gly Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp
His Tyr Phe Lys465 470 475 480Gly Pro Ser Tyr Ser Leu Arg Ser Thr
Thr Met Met Ile Arg Pro Leu 485 490 495Asp Phe271124PRTHomo sapiens
27Met Asp Ser Leu Ala Ser Leu Val Leu Cys Gly Val Ser Leu Leu Leu1
5 10 15Ser Gly Thr Val Glu Gly Ala Met Asp Leu Ile Leu Ile Asn Ser
Leu 20 25 30Pro Leu Val Ser Asp Ala Glu Thr Ser Leu Thr Cys Ile Ala
Ser Gly 35 40 45Trp Arg Pro His Glu Pro Ile Thr Ile Gly Arg Asp Phe
Glu Ala Leu 50 55 60Met Asn Gln His Gln Asp Pro Leu Glu Val Thr Gln
Asp Val Thr Arg65 70 75 80Glu Trp Ala Lys Lys Val Val Trp Lys Arg
Glu Lys Ala Ser Lys Ile 85 90 95Asn Gly Ala Tyr Phe Cys Glu Gly Arg
Val Arg Gly Glu Ala Ile Arg 100 105 110Ile Arg Thr Met Lys Met Arg
Gln Gln Ala Ser Phe Leu Pro Ala Thr 115 120 125Leu Thr Met Thr Val
Asp Lys Gly Asp Asn Val Asn Ile Ser Phe Lys 130 135 140Lys Val Leu
Ile Lys Glu Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser145 150 155
160Phe Ile His Ser Val Pro Arg His Glu Val Pro Asp Ile Leu Glu Val
165 170 175His Leu Pro His Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser
Ala Arg 180 185 190Tyr Ile Gly Gly Asn Leu Phe Thr Ser Ala Phe Thr
Arg Leu Ile Val 195 200 205Arg Arg Cys Glu Ala Gln Lys Trp Gly Pro
Glu Cys Asn His Leu Cys 210 215 220Thr Ala Cys Met Asn Asn Gly Val
Cys His Glu Asp Thr Gly Glu Cys225 230 235 240Ile Cys Pro Pro Gly
Phe Met Gly Arg Thr Cys Glu Lys Ala Cys Glu 245 250 255Leu His Thr
Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly Gln Glu 260 265 270Gly
Cys Lys Ser Tyr Val Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser 275 280
285Cys Ala Thr Gly Trp Lys Gly Leu Gln Cys Asn Glu Ala Cys His Pro
290 295 300Gly Phe Tyr Gly Pro Asp Cys Lys Leu Arg Cys Ser Cys Asn
Asn Gly305 310 315 320Glu Met Cys Asp Arg Phe Gln Gly Cys Leu Cys
Ser Pro Gly Trp Gln 325 330 335Gly Leu Gln Cys Glu Arg Glu Gly Ile
Pro Arg Met Thr Pro Lys Ile 340 345 350Val Asp Leu Pro Asp His Ile
Glu Val Asn Ser Gly Lys Phe Asn Pro 355 360 365Ile Cys Lys Ala Ser
Gly Trp Pro Leu Pro Thr Asn Glu Glu Met Thr 370 375 380Leu Val Lys
Pro Asp Gly Thr Val Leu His Pro Lys Asp Phe Asn His385 390 395
400Thr Asp His Phe Ser Val Ala Ile Phe Thr Ile His Arg Ile Leu Pro
405 410 415Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr Val Ala
Gly Met 420 425 430Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu
Pro Lys Pro Leu 435 440 445Asn Ala Pro Asn Val Ile Asp Thr Gly His
Asn Phe Ala Val Ile Asn 450 455 460Ile Ser Ser Glu Pro Tyr Phe Gly
Asp Gly Pro Ile Lys Ser Lys Lys465 470 475 480Leu Leu Tyr Lys Pro
Val Asn His Tyr Glu Ala Trp Gln His Ile Gln 485 490 495Val Thr Asn
Glu Ile Val Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu 500 505 510Tyr
Glu Leu Cys Val Gln Leu Val Arg Arg Gly Glu Gly Gly Glu Gly 515 520
525His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser Ile Gly Leu Pro
530 535 540Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln Thr Thr
Leu Asn545 550 555 560Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu
Asp Asp Phe Tyr Val 565 570 575Glu Val Glu Arg Arg Ser Val Gln Lys
Ser Asp Gln Gln Asn Ile Lys 580 585 590Val Pro Gly Asn Leu Thr Ser
Val Leu Leu Asn Asn Leu His Pro Arg 595 600 605Glu Gln Tyr Val Val
Arg Ala Arg Val Asn Thr Lys Ala Gln Gly Glu 610 615 620Trp Ser Glu
Asp Leu Thr Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro625 630 635
640Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His Ser Ser Ala Val
645 650 655Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser Ser Ile
Thr Ile 660 665 670Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His
Val Asp Val Lys 675 680 685Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln
Leu Lys Gly Leu Glu Pro 690 695 700Glu Thr Ala Tyr Gln Val Asp Ile
Phe Ala Glu Asn Asn Ile Gly Ser705 710 715 720Ser Asn Pro Ala Phe
Ser His Glu Leu Val Thr Leu Pro Glu Ser Gln 725 730 735Ala Pro Ala
Asp Leu Gly Gly Gly Lys Met Leu Leu Ile Ala Ile Leu 740 745 750Gly
Ser Ala Gly Met Thr Cys Leu Thr Val Leu Leu Ala Phe Leu Ile 755 760
765Ile Leu Gln Leu Lys Arg Ala Asn Val Gln Arg Arg Met Ala Gln Ala
770 775 780Phe Gln Asn Val Arg Glu Glu Pro Ala Val Gln Phe Asn Ser
Gly Thr785 790 795 800Leu Ala Leu Asn Arg Lys Val Lys Asn Asn Pro
Asp Pro Thr Ile Tyr 805 810 815Pro Val Leu Asp Trp Asn Asp Ile Lys
Phe Gln Asp Val Ile Gly Glu 820 825 830Gly Asn Phe Gly Gln Val Leu
Lys Ala Arg Ile Lys Lys Asp Gly Leu 835 840 845Arg Met Asp Ala Ala
Ile Lys Arg Met Lys Glu Tyr Ala Ser Lys Asp 850 855 860Asp His Arg
Asp Phe Ala Gly Glu Leu Glu Val Leu Cys Lys Leu Gly865 870 875
880His His Pro Asn Ile Ile Asn Leu Leu Gly Ala Cys Glu His Arg Gly
885 890 895Tyr Leu Tyr Leu Ala Ile Glu Tyr Ala Pro His Gly Asn Leu
Leu Asp 900 905 910Phe Leu Arg Lys Ser Arg Val Leu Glu Thr Asp Pro
Ala Phe Ala Ile 915 920 925Ala Asn Ser Thr Ala Ser Thr Leu Ser Ser
Gln Gln Leu Leu His Phe 930 935 940Ala Ala Asp Val Ala Arg Gly Met
Asp Tyr Leu Ser Gln Lys Gln Phe945 950 955 960Ile His Arg Asp Leu
Ala Ala Arg Asn Ile Leu Val Gly Glu Asn Tyr 965 970 975Val Ala Lys
Ile Ala Asp Phe Gly Leu Ser Arg Gly Gln Glu Val Tyr 980 985 990Val
Lys Lys Thr Met Gly Arg Leu Pro Val Arg Trp Met Ala Ile Glu 995
1000 1005Ser Leu Asn Tyr Ser Val Tyr Thr Thr Asn Ser Asp Val Trp
Ser 1010 1015 1020Tyr Gly Val Leu Leu Trp Glu Ile Val Ser Leu Gly
Gly Thr Pro 1025
1030 1035Tyr Cys Gly Met Thr Cys Ala Glu Leu Tyr Glu Lys Leu Pro
Gln 1040 1045 1050Gly Tyr Arg Leu Glu Lys Pro Leu Asn Cys Asp Asp
Glu Val Tyr 1055 1060 1065Asp Leu Met Arg Gln Cys Trp Arg Glu Lys
Pro Tyr Glu Arg Pro 1070 1075 1080Ser Phe Ala Gln Ile Leu Val Ser
Leu Asn Arg Met Leu Glu Glu 1085 1090 1095Arg Lys Thr Tyr Val Asn
Thr Thr Leu Tyr Glu Lys Phe Thr Tyr 1100 1105 1110Ala Gly Ile Asp
Cys Ser Ala Glu Glu Ala Ala 1115 1120
* * * * *