U.S. patent application number 16/457267 was filed with the patent office on 2020-03-05 for monovalent blood brain barrier shuttle modules.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Adrian HUGENMATTER, Ekkehard MOESSNER, Jens NIEWOEHNER, Francesca ROS, Petra RUEGER, Cuiying SHAO, Georg TIEFENTHALER, Gang XU.
Application Number | 20200071413 16/457267 |
Document ID | / |
Family ID | 52396646 |
Filed Date | 2020-03-05 |
![](/patent/app/20200071413/US20200071413A1-20200305-D00001.png)
United States Patent
Application |
20200071413 |
Kind Code |
A1 |
RUEGER; Petra ; et
al. |
March 5, 2020 |
MONOVALENT BLOOD BRAIN BARRIER SHUTTLE MODULES
Abstract
Herein is reported a blood brain barrier shuttle module
comprising a brain effector entity, a linker and one monovalent
binding entity which binds to a blood brain barrier receptor,
wherein the linker couples the effector entity to the monovalent
binding entity which binds to the blood brain barrier receptor
wherein the monovalent binding entity does not comprise the
variable domains of the anti-transferrin receptor antibody 8D3 (SEQ
ID NO: 01 and SEQ ID NO: 02) or of the variant anti-transferrin
receptor antibody 8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
Inventors: |
RUEGER; Petra; (Penzberg,
DE) ; TIEFENTHALER; Georg; (Sindelsdorf, DE) ;
MOESSNER; Ekkehard; (Kreuzlingen, CH) ; NIEWOEHNER;
Jens; (Muenchen, DE) ; HUGENMATTER; Adrian;
(Zurich, CH) ; SHAO; Cuiying; (Shanghai, CN)
; ROS; Francesca; (Bernried, DE) ; XU; Gang;
(Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
52396646 |
Appl. No.: |
16/457267 |
Filed: |
June 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15203057 |
Jul 6, 2016 |
10364292 |
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16457267 |
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PCT/EP2014/079353 |
Dec 29, 2014 |
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15203057 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/56 20130101;
C07K 2317/35 20130101; A61P 43/00 20180101; A61K 2039/505 20130101;
C07K 2317/31 20130101; C07K 2317/622 20130101; C07K 2319/33
20130101; C07K 16/2881 20130101; C07K 2317/55 20130101; A61P 25/00
20180101; C07K 16/18 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/18 20060101 C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 6, 2014 |
CN |
PCT/CN2014/070183 |
Claims
1. A blood brain barrier shuttle module comprising a brain effector
entity, optionally a linker and exactly one (monovalent) binding
entity which binds to a blood brain barrier transferrin receptor
(TfR), wherein the linker if present couples the effector entity to
the monovalent binding entity, which binds to the blood brain
barrier receptor, wherein the (monovalent) binding entity comprises
an anti-TfR antibody or fragment thereof comprising (a) a HVR-H1
comprising the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2
comprising the amino acid sequence of SEQ ID NO: 117; (c) a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 119; (d) a HVR-L1
comprising the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2
comprising the amino acid sequence of SEQ ID NO: 121; and (f) a
HVR-L3 comprising the amino acid sequence of SEQ ID NO: 122.
2. A blood brain barrier shuttle module according to claim 1,
wherein the monovalent binding entity comprises an anti-TfR
antibody or fragment thereof comprising a heavy chain variable
domain having the amino acid sequence of SEQ ID NO: 91 and a light
chain variable domain having the amino acid sequence of SEQ ID NO:
92.
3. The blood brain barrier shuttle module according to claim 1,
wherein the monovalent binding entity, which binds to the blood
brain barrier receptor, comprises a molecule selected from the
group consisting of a blood brain barrier receptor ligand, a full
length antibody, a scFv, an Fv, a scFab, and a VHH.
4. The blood brain barrier shuttle module according to claim 1,
wherein the blood brain barrier receptor is selected from the group
consisting of transferrin receptor, insulin receptor, insulin-like
growth factor receptor, low density lipoprotein receptor-related
protein 8, low density lipoprotein receptor-related protein 1 and
heparin-binding epidermal growth factor-like growth factor.
5. The blood brain barrier shuttle module according to claim 1, to
wherein the monovalent binding entity specifically binds to human
transferrin receptor and to cynomolgus transferrin receptor.
6. The blood brain barrier shuttle module according to claim 1,
wherein the monovalent binding entity, which binds to the blood
brain barrier receptor, comprises one scFab directed to the
transferrin receptor.
7. The blood brain barrier shuttle module according to claim 1,
wherein the monovalent binding entity, which binds to the blood
brain barrier receptor, comprises one scFv directed to the
transferrin receptor.
8. (canceled)
9. The blood brain barrier shuttle module according to claim 1,
wherein the brain effector entity is selected from the group
consisting of neurological disorder drugs, neurotrophic factors,
growth factors, enzymes, cytotoxic agents, antibodies directed to a
brain target, monoclonal antibodies directed to a brain target,
peptides directed to a brain target.
10. The blood brain barrier shuttle module according to claim 9,
wherein the brain target is selected from the group consisting of
.beta.-secretase 1, A.beta. (Abeta), epidermal growth factor,
epidermal growth factor receptor 2, tau, phosphorylated tau,
phosphorylated tau(pS422), apolipoprotein E4, alpha synuclein,
oligomeric fragments of alpha synuclein, CD20, huntingtin, prion
protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma
secretase, death receptor 6, amyloid precursor protein, p75
neurotrophin receptor and caspase 6.
11. The blood brain barrier shuttle module according to claim 1,
wherein the monovalent binding entity, which binds to the blood
brain receptor, is a polypeptide and the monovalent binding entity
is conjugated to the C-terminal end of the brain effector entity
either directly or via a linker.
12. The blood brain barrier shuttle module according to claim 1,
wherein the brain effector entity comprises a full length antibody
directed to a brain target.
13. A pharmaceutical formulation comprising a blood brain barrier
shuttle module according to claim 1.
14. Use of a blood brain barrier shuttle module according to claim
1 as a medicament.
15. The use according to claim 14, wherein the medicament is for
the treatment of a neurological disorder.
16. The blood brain barrier shuttle module according to claim 1 for
use to transport the brain effector entity across the blood brain
barrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/203,057, filed Jul. 6, 2016, which is a
continuation of International Application No. PCT/EP2014/079353,
having an international filing date of Dec. 29, 2014, which claims
benefit to International Application No. PCT/CN2014/070183, filed
Jan. 6, 2014, the entire contents of which are incorporated herein
by reference.
SEQUENCE LISTING
[0002] This application hereby incorporates by reference the
material of the electronic Sequence Listing filed concurrently
herewith. The material in the electronic Sequence Listing is
submitted as a text (.txt) file entitled
"P31922-US-1_Sequence_Listing_ST25" created on Jun. 19, 2019, which
has a file size of 137,498 bytes, and is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a blood brain barrier
shuttle module that has one binding specificity that specifically
binds to a blood brain barrier receptor (BBBR) and which is
monovalent with respect to this binding specificity and methods of
using this construct as blood barrier shuttle and in the treatment
of neurological disorders.
BACKGROUND
[0004] Brain penetration of neurological disorder drugs such alls
e.g. large biotherapeutic drugs or small molecule drugs having a
low brain penetration, is strictly limited by the extensive and
impermeable blood brain barrier (BBB) together with the other cell
component in the neurovascular unit (NVU). Many strategies to
overcome this obstacle have been tested and one is to utilize
transcytosis pathways mediated by endogenous receptors expressed on
the brain capillary endothelium (blood brain barrier receptor).
Recombinant proteins such as monoclonal antibodies or peptides have
been designed against these receptors to enable receptor-mediated
delivery of biotherapeutics to the brain. However, strategies to
maximize brain uptake while minimizing miss-sorting within the
brain endothelial cells (BECs), and the extent of accumulation
within certain organelles (especially organelles that lead to
degradation of the biotherapeutic) in BECs, remain unexplored.
[0005] Monoclonal antibodies and other biotherapeutics have huge
therapeutic potential for treatment of pathology in the central
nervous system (CNS). However, their route into the brain is
prevented by the BBB. Previous studies have illustrated that a very
small percentage (approximately 0.1%) of an IgG injected in the
bloodstream is able to penetrate into the CNS compartment
(Felgenhauer, Klin. Wschr. 52 (1974) 1158-1164). This will
certainly limit any pharmacological effect due to the low
concentration within CNS of the antibody.
[0006] Therefore, there is a need for delivery systems of
neurological disorder drugs across the BBB to shuttle the drugs
into the brain efficiently.
[0007] In WO 2014/033074 a blood brain barrier shuttle is
reported.
[0008] Mouse 8D3 anti-transferrin antibody and variable light chain
domain (VL) variant (L596V and L5981) thereof is reported by Boado,
R. J., et al. (Biotechnol. Bioeng. 102 (2009) 1251-1258).
SUMMARY
[0009] One aspect of the current invention is a blood brain barrier
shuttle module comprising a brain effector entity, a linker and one
monovalent binding entity which binds to a blood brain barrier
receptor, wherein the linker couples the effector entity to the
monovalent binding entity, which binds to the blood brain barrier
receptor, wherein the monovalent binding entity does not comprise
the variable domains of the anti-transferrin receptor antibody 8D3
(SEQ ID NO: 01 and SEQ ID NO: 02) or of the variant
anti-transferrin receptor antibody 8D3v (SEQ ID NO: 01 and SEQ ID
NO: 03).
[0010] The anti-transferrin receptor antibody 8D3 has a heavy chain
variable domain with the following amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 01) EVQLVESGGG LVQPGNSLTL SCVASGFTFS
NYGMHWIRQA PKKGLEWIAM IYYDSSKMNY ADTVKGRFTI SRDNSKNTLY LEMNSLRSED
TAMYYCAVPT SHYVVDVWGQ GVSVTVSS.
[0011] The anti-transferrin receptor antibody 8D3 has a light chain
variable domain with the following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 02) DIQMTQSPAS LSASLEEIVT ITCQASQDIG
NWLAWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ FSLKISRVQV EDIGIYYCLQ
AYNTPWTFGG GTKLELK.
[0012] The variant anti-transferrin receptor antibody 8D3v has the
same heavy chain variable domain as antibody 8D3 and a light chain
variable domain with mutants L104V and L1061 that has the following
amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 03) DIQMTQSPAS LSASLEEIVT ITCQASQDIG
NWLAWYQQKP GKSPQLLIYG ATSLADGVPS RFSGSRSGTQ FSLKISRVQV EDIGIYYCLQ
AYNTPWTFGG GTKVEIK.
[0013] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
barrier receptor is a polypeptide.
[0014] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
barrier receptor comprises a molecule selected from the group
consisting of a blood brain barrier receptor ligand, a full length
antibody, a scFv, an Fv, a scFab, and a VHH.
[0015] In one embodiment of the blood brain barrier shuttle module
the blood brain receptor is selected from the group consisting of
transferrin receptor, insulin receptor, insulin-like growth factor
receptor, low density lipoprotein receptor-related protein 8, low
density lipoprotein receptor-related protein 1 and heparin-binding
epidermal growth factor-like growth factor. In one embodiment the
blood brain receptor is the transferrin receptor.
[0016] In one embodiment the monovalent binding entity specifically
binds to human transferrin receptor and to cynomolgus transferrin
receptor.
[0017] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
barrier receptor comprises one scFab directed to the transferrin
receptor, more particularly, a scFab that specifically binds to an
epitope in the transferrin receptor comprised within the amino acid
sequence of SEQ ID NOs: 04, 05, or 06.
[0018] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
barrier receptor comprises one scFv directed to the transferrin
receptor, more particularly, a scFv recognizing an epitope in the
transferrin receptor comprised within the amino acid sequence of
SEQ ID NOs: 04, 05, or 06.
[0019] In one embodiment of the blood brain barrier shuttle module
the brain effector entity is selected from the group consisting of
neurological disorder drugs, neurotrophic factors, growth factors,
enzymes, cytotoxic agents, antibodies directed to a brain target,
monoclonal antibodies directed to a brain target, peptides directed
to a brain target.
[0020] In one embodiment of the blood brain barrier shuttle module
the brain target is selected from the group consisting of
.beta.-secretase 1, A.beta. (Abeta), epidermal growth factor,
epidermal growth factor receptor 2, tau, phosphorylated tau,
phosphorylated tau(p S422), apolipoprotein E4, alpha synuclein,
oligomeric fragments of alpha synuclein, CD20, huntingtin, prion
protein, leucine rich repeat kinase 2, parkin, presenilin 2, gamma
secretase, death receptor 6, amyloid precursor protein, p75
neurotrophin receptor and caspase 6.
[0021] In a particular embodiment of the blood brain barrier
shuttle module the brain effector entity is a polypeptide.
[0022] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
receptor is a polypeptide and the monovalent binding entity is
conjugated to the C-terminal end of the brain effector entity
either directly or via a linker.
[0023] In one embodiment of the blood brain barrier shuttle module
the brain effector entity comprises a full length antibody directed
to a brain target. In one embodiment the full length antibody is an
IgG.
[0024] In one preferred embodiment of the blood brain barrier
shuttle module the blood brain barrier shuttle comprises a full
length IgG antibody as brain effector entity, a linker and one
scFab as the monovalent binding entity which binds the blood brain
barrier receptor, wherein the scFab is conjugated to the C-terminal
end of the Fc-region of one of the heavy chains of the IgG antibody
via the linker.
[0025] In one embodiment of the blood brain barrier shuttle module
the first heavy chain of the antibody of the blood brain barrier
shuttle directed to a brain target comprises a first dimerization
module and the second heavy chain of the antibody of the blood
brain barrier shuttle to a brain target comprises a second
dimerization module allowing heterodimerization of the two heavy
chains.
[0026] In one embodiment of the blood brain barrier shuttle module
the first dimerization module of the first heavy chain of the
antibody of the blood brain barrier shuttle directed to the brain
target comprises knobs and the dimerization module of the second
heavy chain of the antibody of the blood brain barrier shuttle
directed to the brain target comprises holes according to the
knobs-into-holes strategy.
[0027] In one embodiment of the blood brain barrier shuttle module
the linker is a peptidic linker. In one embodiment the peptidic
linker has an amino acid sequence with a length of at least 25
amino acids. In one embodiment the peptidic linker has an amino
acid sequence with a length of 30 to 50 amino acids. In one
embodiment the peptidic linker is (G4S)6G2 (SEQ ID NO: 07) or
(G4S)4 (SEQ ID NO: 08).
[0028] The following three embodiments are directed to a blood
brain barrier shuttle module wherein the brain effector entity is a
polypeptide with the proviso that the brain effector entity is not
a full length antibody, in particular not a full length IgG.
[0029] In one embodiment of the blood brain barrier shuttle module
the monovalent binding entity which binds to the blood brain
barrier receptor comprises a CH2-CH3 Ig entity and one scFab
(comprising a first linker), which binds to the blood brain barrier
receptor, wherein the scFab is coupled to a C-terminal end of the
CH2-CH3 Ig entity by a second linker.
[0030] In one embodiment of the blood brain barrier shuttle module
the blood brain barrier shuttle comprises a brain effector entity,
a linker, a CH2-CH3 Ig domain, a second linker and one scFab, which
binds to the blood brain barrier receptor, wherein the brain
effector entity is conjugated by a first linker to an N-terminal
end of the CH2-CH3 Ig domain and the scFab is conjugated to a
C-terminal end of the CH2-CH3 Ig domain by a second linker.
[0031] In one embodiment of the blood brain barrier shuttle module
the CH2-CH3 Ig entity is a CH2-CH3 IgG entity.
[0032] Further aspects of the current invention are an (isolated)
nucleic acid encoding the blood brain barrier shuttle module as
reported herein, a host cell comprising the (isolated) nucleic acid
encoding the blood brain barrier shuttle module, and a
pharmaceutical formulation comprising the blood brain barrier
shuttle module.
[0033] The blood brain barrier shuttle module as reported herein
can be used as a medicament, in particular it can be used for the
treatment of a neurological disorder such as e.g. Alzheimer's
disease.
[0034] The blood brain barrier shuttle module as reported herein
can be used to transport the brain effector entity across the blood
brain barrier.
[0035] In a particular embodiment, the heavy chain of the IgG
antibody of the blood brain barrier shuttle module as reported
herein conjugated at its C-terminal end of the Fc-region to the
scFab as monovalent binding entity, which binds to the blood brain
barrier receptor, has the following structure: [0036] IgG heavy
chain, [0037] linker conjugating the C-terminal end of the
Fc-region of the IgG heavy chain to the N-terminal end of the VL
domain of the scFab, [0038] variable light chain domain (VL) and
C-kappa light chain domain of the scFab, [0039] linker conjugating
the C-terminal end of the C-kappa light chain domain of the scFab
to the N-terminal end of the VH domain of the scFab, [0040]
variable heavy chain domain (VH) of the scFab antibody and IgG CH1
heavy chain domain.
[0041] One aspect as reported herein is a fusion polypeptide to
transport a brain effector entity across the blood brain barrier
comprising a CH2-CH3 Ig entity, a linker and one scFab that
specifically binds to a blood brain barrier receptor, wherein the
scFab is conjugated to a C-terminal end of the CH2-CH3 Ig entity by
the linker, wherein scFab does not comprise the variable domains of
the anti-transferrin receptor antibody 8D3 (SEQ ID NO: 01 and SEQ
ID NO: 02) or of the variant anti-transferrin receptor antibody
8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
[0042] One aspect as reported herein is a fusion polypeptide to
transport a brain effector entity across the blood brain barrier
comprising a CH2-CH3 Ig entity, a linker and one scFv that
specifically binds to a blood brain barrier receptor, wherein the
scFv is conjugated to a C-terminal end of the CH2-CH3 Ig entity by
the linker, wherein scFv does not comprise the variable domains of
the anti-transferrin receptor antibody 8D3 (SEQ ID NO: 01 and SEQ
ID NO: 02) or of the variant anti-transferrin receptor antibody
8D3v (SEQ ID NO: 01 and SEQ ID NO: 03).
[0043] In one embodiment the fusion polypeptide further comprises a
linker at the N-terminal end of the CH2-CH3 Ig entity to conjugate
the brain effector entity to the N-terminal end of the CH2-CH3 Ig
entity.
[0044] In one embodiment of the fusion polypeptide the brain
effector entity is selected from the group consisting of
neurological disorder drugs, neurotrophic factors, growth factors,
enzymes, cytotoxic agents, antibody fragments or peptides directed
to a brain target selected from the group consisting of scFv, Fv,
scFab, Fab, VHH, F(ab')2.
[0045] In one embodiment of the fusion polypeptide the scFab or the
scFv that specifically binds to the blood brain barrier receptor
specifically binds to the transferrin receptor. In one embodiment
the scFab or the scFv specifically binds to an epitope of the
transferrin receptor comprised within the amino acid sequence of
SEQ ID NO: 04, 05 or 06.
[0046] In embodiment of the fusion polypeptide the linker is a
peptidic linker. In one embodiment the peptidic linker has an amino
acid sequence with a length of at least 15 amino acids. In one
embodiment the peptidic linker has a length of 20 to 50 amino
acids. In one embodiment the peptidic linker has the amino acid
sequence (G4S)6G2, (SEQ ID NO: 07) or (G45)4 (SEQ ID NO: 08).
[0047] In one embodiment of the fusion polypeptide the CH2-CH3 Ig
entity is a CH2-CH3 IgG entity.
[0048] Further aspects of the current invention are an isolated
nucleic acid encoding the fusion polypeptide as reported herein and
a host cell comprising the nucleic acid encoding the fusion
polypeptide as reported herein.
[0049] One aspect as reported herein is a conjugate comprising a
fusion polypeptide as reported herein and a brain effector entity
conjugated to an N-terminal end of the CH2-CH3 Ig entity of the
fusion polypeptide as reported herein via a linker.
[0050] In one embodiment of the conjugate the brain effector entity
is a neurotrophic factor and the linker conjugating the
neurotrophic factor to the N-terminal end of the CH2-CH3 Ig entity
is a peptidic linker.
[0051] Further aspects as reported herein are a pharmaceutical
formulation comprising the conjugate as reported herein and a
pharmaceutical carrier, the use of the conjugate as reported
herein, in particular the use of the conjugate for the treatment of
a neurodegenerative disorder in particular Alzheimer's disease.
[0052] The monovalent binding entity that specifically binds to a
blood brain barrier receptor can be conjugated to any terminus of
the light or heavy chain of the antibody either directly or via a
peptidic linker. In one embodiment the monovalent binding entity is
conjugated to a C-terminus of the heavy chain.
[0053] The C-terminus of a heavy chain of an antibody can be a
complete C-terminus ending with the amino acid residues PGK. The
C-terminus of the heavy chain can be a shortened C-terminus in
which one or two of the C-terminal amino acid residues have been
removed. In one embodiment the C-terminus of the heavy chain is a
shortened C-terminus ending with the amino acid residues PG.
[0054] The monovalent binding entity can be conjugated to the
respective antibody chain either directly or via a peptidic linker.
In one embodiment the peptidic linker has the amino acid sequence
GGSGGGGSGGGGSGGGGS (SEQ ID NO: 09).
[0055] The monovalent binding entity can be an antibody scFv
fragment. In one embodiment the monovalent binding entity is a scFv
comprising in N- to C-terminal order a light chain variable
domain-a light chain constant domain-a peptidic linker-a heavy
chain variable domain-the heavy chain constant domain 1.
[0056] In one embodiment the monovalent binding entity is a scFv
fragment of an anti-transferrin receptor-antibody with a (G45)6
peptidic linker (SEQ ID NO: 10).
[0057] In one embodiment the blood-brain-barrier receptor is
selected from the group consisting of transferrin receptor, insulin
receptor, insulin-like growth factor receptor, low density
lipoprotein receptor-related protein 8, low density lipoprotein
receptor-related protein 1 and heparin-binding epidermal growth
factor-like growth factor. In one embodiment blood-brain-barrier
receptor is a human blood-brain-barrier receptor. In one embodiment
the blood-brain-barrier receptor is the transferrin receptor and
the antibody does not inhibit the binding of the transferrin
receptor to transferrin. In one embodiment the blood-brain-barrier
receptor is the human transferrin receptor.
[0058] In one embodiment the peptidic linker conjugating the
monovalent binding entity to the brain effector entity is an amino
acid sequence with a length of at least 15 amino acids. In one
embodiment the peptidic linker has a length of 18 to 25 amino
acids.
[0059] In one embodiment, the brain effector entity is a full
length antibody. In one embodiment the brain effector entity is a
full length antibody of the subclass IgG1 or IgG4.
[0060] In one embodiment the monovalent binding entity is an
anti-blood brain barrier receptor antibody or a blood brain barrier
receptor binding fragment thereof In one embodiment, the anti-blood
brain barrier receptor antibody or fragment thereof does not impair
the binding of the blood brain barrier receptor to one or more of
its native ligands. In another embodiment, the anti-blood brain
barrier receptor antibody specifically binds to human transferrin
receptor in such a manner that it does not inhibit binding of the
human transferrin receptor to human transferrin.
[0061] In one embodiment the blood brain barrier shuttle module is
effector silent.
[0062] In one embodiment the brain effector entity is a full length
antibody comprising an Fc-region, wherein in case the Fc-region is
of the human subclass IgG1 the Fc-region comprises the mutations
L234A, L235A and P329G (numbering according to the EU index of
Kabat), or in case the Fc-region is of the human subclass IgG4 the
Fc-region comprises the mutations S228P, L235E and P329G (numbering
according to the EU index of Kabat).
BRIEF DESCRIPTION OF DRAWING
[0063] FIG. 1 depicts the assay scheme for the assay used in the
transcytosis screening.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. DEFINITIONS
[0064] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0065] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0066] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more hypervariable regions
(HVRs), compared to a parent antibody which does not possess such
alterations, such alterations resulting in an improvement in the
affinity of the antibody for antigen.
[0067] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0068] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0069] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0070] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG3, IgG4, IgA.sub.1, and IgA.sub.2.
The heavy chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively.
[0071] "Effector functions" refer to those biological activities
attributable to the Fc-region of an antibody, which vary with the
antibody class. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0072] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0073] The term "Fc-region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc-regions and variant Fc-regions. In one embodiment, a human IgG
heavy chain Fc-region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc-region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc-region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat, 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.
[0074] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0075] The terms "full length antibody", "intact antibody", and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc-region
as defined herein.
[0076] The terms "host cell", "host cell line", and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0077] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat, E. A. et al.,
Sequences of Proteins of Immunological Interest, 5th ed., Bethesda
Md. (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment,
for the VL, the subgroup is subgroup kappa I as in Kabat et al.,
supra. In one embodiment, for the VH, the subgroup is subgroup III
as in Kabat et al., supra.
[0078] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0079] The term "hypervariable region" or "HVR", as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and form structurally defined loops
("hypervariable loops"), and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3).
[0080] HVRs herein include [0081] (a) hypervariable loops occurring
at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32
(H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J.
Mol. Biol. 196 (1987) 901-917); [0082] (b) CDRs occurring at amino
acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1),
50-65 (H2), and 95-102 (H3) (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); [0083] (c) antigen contacts occurring at
amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b
(H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol.
262: 732-745 (1996)); and [0084] (d) combinations of (a), (b),
and/or (c), including HVR amino acid residues 46-56 (L2), 47-56
(L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2),
93-102 (H3), and 94-102 (H3).
[0085] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0086] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g. cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0087] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman, S. et al., J. Chromatogr. B 848 (2007) 79-87.
[0088] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0089] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0090] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0091] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0092] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0093] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0094] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0095] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject., A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0096] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0097] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt, T. J. et al. Kuby
Immunology, 6th ed., W. H. Freeman and Co., N.Y. (2007), page 91) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano, S. et al., J. Immunol. 150
(1993) 880-887; Clackson, T. et al., Nature 352 (1991)
624-628).
[0098] The term "vector", as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors".
[0099] The term "blood-brain barrier" (BBB) denotes the
physiological barrier between the peripheral circulation and the
brain and spinal cord which is formed by tight junctions within the
brain capillary endothelial plasma membranes, creating a tight
barrier that restricts the transport of molecules into the brain,
even very small molecules such as urea (60 Daltons). The BBB within
the brain, the blood-spinal cord barrier within the spinal cord,
and the blood-retinal barrier within the retina are contiguous
capillary barriers within the CNS, and are herein collectively
referred to as the blood-brain barrier or BBB. The BBB also
encompasses the blood-CSF barrier (choroid plexus) where the
barrier is comprised of ependymal cells rather than capillary
endothelial cells.
[0100] The term "central nervous system" (CNS) denotes the complex
of nerve tissues that control bodily function, and includes the
brain and spinal cord.
[0101] The term "blood-brain barrier receptor" (BBBR) denotes an
extracellular membrane-linked receptor protein expressed on brain
endothelial cells which is capable of transporting molecules across
the BBB or be used to transport exogenous administrated molecules.
Examples of BBBR include but are not limited to transferrin
receptor (TfR), insulin receptor, insulin-like growth factor
receptor (IGF-R), low density lipoprotein receptors including
without limitation low density lipoprotein receptor-related protein
1 (LRP1) and low density lipoprotein receptor-related protein 8
(LRP8), and heparin-binding epidermal growth factor-like growth
factor (HB-EGF). An exemplary BBBR is the transferrin receptor
(TfR).
[0102] The term "brain effector entity" denotes a molecule that is
to be transported to the brain across the BBB. The effector entity
typically has a characteristic therapeutic activity that is desired
to be delivered to the brain. Effector entities include
neurological disorder drugs and cytotoxic agents such as e.g.
polypeptides and antibodies, in particular monoclonal antibodies or
fragments thereof directed to a brain target.
[0103] The term "monovalent binding entity" denotes a molecule able
to bind specifically and in a monovalent binding mode to a BBBR.
The blood brain shuttle module and/or conjugate as reported herein
are characterized by the presence of a single unit of a monovalent
binding entity i.e. the blood brain shuttle module and/or conjugate
of the present invention comprise exactly one unit of the
monovalent binding entity. The monovalent binding entity includes
but is not limited to polypeptides, full length antibodies,
antibody fragments including Fab, Fab', Fv fragments, single-chain
antibody molecules such as e.g. single chain Fab, scFv. The
monovalent binding entity can be, for example, a scaffold protein
engineered using state of the art technologies like phage display
or immunization. The monovalent binding entity can also be a
polypeptide. In certain embodiments, the monovalent binding entity
comprises a CH2-CH3 Ig domain and a single chain Fab (scFab)
directed to a blood brain barrier receptor. The scFab is coupled to
the C-terminal end of the CH2-CH3 Ig domain by a linker. In certain
embodiments, the scFab is directed to the transferrin receptor.
[0104] The term "monovalent binding mode" denotes a specific
binding to the BBBR where the interaction between the monovalent
binding entity and the BBBR takes place through one single epitope.
The monovalent binding mode prevents any
dimerization/multimerization of the BBBR due to a single epitope
interaction point. The monovalent binding mode prevents that the
intracellular sorting of the BBBR is altered.
[0105] The term "epitope" denotes any polypeptide determinant
capable of specific binding to an antibody. In certain embodiments,
epitope determinants include chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl, or
sulfonyl, and, in certain embodiments, may have specific three
dimensional structural characteristics, and or specific charge
characteristics. An epitope is a region of an antigen that is bound
by an antibody.
[0106] The "transferrin receptor" (TfR) is a transmembrane
glycoprotein (with a molecular weight of about 180,000 Da) which is
composed of two disulfide-bonded sub-units (each of apparent
molecular weight of about 90,000 Da) and is involved in iron uptake
in vertebrates. In one embodiment, the TfR herein is human TfR
comprising the amino acid sequence as reported in Schneider et al.
(Nature 311 (1984) 675-678).
[0107] The term "neurological disorder" denotes a disease or
disorder which affects the CNS and/or which has an etiology in the
CNS. Exemplary CNS diseases or disorders include, but are not
limited to, neuropathy, amyloidosis, cancer, an ocular disease or
disorder, viral or microbial infection, inflammation, ischemia,
neurodegenerative disease, seizure, behavioral disorders, and a
lysosomal storage disease. For the purposes of this application,
the CNS will be understood to include the eye, which is normally
sequestered from the rest of the body by the blood-retina barrier.
Specific examples of neurological disorders include, but are not
limited to, neurodegenerative diseases (including, but not limited
to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger
syndrome, olivopontocerebellar atrophy, Parkinson's disease,
multiple system atrophy, striatonigral degeneration, tauopathies
(including, but not limited to, Alzheimer disease and supranuclear
palsy), prion diseases (including, but not limited to, bovine
spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome,
kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting
disease, and fatal familial insomnia), bulbar palsy, motor neuron
disease, and nervous system heterodegenerative disorders
(including, but not limited to, Canavan disease, Huntington's
disease, neuronal ceroid-lipofuscinosis, Alexander's disease,
Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome,
Halervorden-Spatz syndrome, lafora disease, Rett syndrome,
hepatolenticular degeneration, Lesch-Nyhan syndrome, and
Unverricht-Lundborg syndrome), dementia (including, but not limited
to, Pick's disease, and spinocerebellar ataxia), cancer (e.g. of
the CNS and/or brain, including brain metastases resulting from
cancer elsewhere in the body).
[0108] The term "neurological disorder drug" denotes a drug or
therapeutic agent that treats one or more neurological disorder(s).
Neurological disorder drugs include, but are not limited to, small
molecule compounds, antibodies, peptides, proteins, natural ligands
of one or more CNS target(s), modified versions of natural ligands
of one or more CNS target(s), aptamers, inhibitory nucleic acids
(i.e., small inhibitory RNAs (siRNA) and short hairpin RNAs
(shRNA)), ribozymes, and small molecules, or active fragments of
any of the foregoing. Exemplary neurological disorder drugs are
described herein and include, but are not limited to: antibodies,
aptamers, proteins, peptides, inhibitory nucleic acids and small
molecules and active fragments of any of the foregoing that either
are themselves or specifically recognize and/or act upon (i.e.,
inhibit, activate, or detect) a CNS antigen or target molecule such
as, but not limited to, amyloid precursor protein or portions
thereof, amyloid beta, beta-secretase, gamma-secretase, tau,
alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma
or other CNS cancer markers, and neurotrophins. Non-limiting
examples of neurological disorder drugs and the corresponding
disorders they may be used to treat: Brain-derived neurotrophic
factor (BDNF), Chronic brain injury (Neurogenesis), Fibroblast
growth factor 2 (FGF-2), Anti-Epidermal Growth Factor Receptor
Brain cancer, (EGFR)-antibody, glial cell-line derived neural
factor Parkinson's disease, (GDNF), Brain-derived neurotrophic
factor (BDNF) Amyotrophic lateral sclerosis, depression, Lysosomal
enzyme Lysosomal storage disorders of the brain, Ciliary
neurotrophic factor (CNTF) Amyotrophic lateral sclerosis,
Neuregulin-1 Schizophrenia, Anti-HER2 antibody (e.g. trastuzumab)
Brain metastasis from HER2-positive cancer.
[0109] The term "imaging agent" denotes a compound that has one or
more properties that permit its presence and/or location to be
detected directly or indirectly. Examples of such imaging agents
include proteins and small molecule compounds incorporating a
labeled entity that permits detection.
[0110] The terms "CNS antigen" and "brain target" denote an antigen
and/or molecule expressed in the CNS, including the brain, which
can be targeted with an antibody or small molecule. Examples of
such antigen and/or molecule include, without limitation:
beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth
factor receptor (EGFR), human epidermal growth factor receptor 2
(HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20,
huntingtin, prion protein (PrP), leucine rich repeat kinase 2
(LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death
receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin
receptor (p75NTR), and caspase 6. In one embodiment, the antigen is
BACE1.
[0111] The term "that specifically binds" denotes an antibody
selectively or preferentially binding to an antigen. The binding
affinity is generally determined using a standard assay, such as
Scatchard analysis, or surface plasmon resonance technique (e.g.
using BIACORE.RTM.).
[0112] The term "CH2-CH3 Ig entity" as used herein refers to a
protein entity derived from immunoglobulin CH2 or CH3 domains. The
"CH2-CH3 Ig entity" comprises two "CH2-CH3" polypeptides forming a
dimer. The immunoglobulin can be IgG, IgA, IgD, IgE or IgM. In one
embodiment, the CH2-CH3 Ig entity derived from an IgG
immunoglobulin and is referred to herein as "CH2-CH3 IgG entity".
The term includes native sequence of CH2-CH3 domains and variant
CH2-CH3 domains. In one embodiment, the "CH2-CH3 Ig entity" derives
from human heavy chain CH2-CH3 IgG domain which extends from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, the C-terminal lysine (Lys447) of the Fc region may
or may not be present. Unless otherwise specified herein, numbering
of amino acid residues in the CH2-CH3 domain region or constant
region is according to the EU numbering system, also called the EU
index, as described in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991.
[0113] A "conjugate" is fusion protein of the present invention
conjugated to one or more heterologous molecule(s), including but
not limited to a label, neurological disorder drug or cytotoxic
agent.
[0114] The term "linker" denotes a chemical linker or a single
chain peptidic linker that covalently connects different entities
of the blood brain barrier shuttle module and/or the fusion
polypeptide and/or the conjugate as reported herein. The linker
connects for example the brain effector entity to the monovalent
binding entity. For example, if the monovalent binding entity
comprises a CH2-CH3 Ig entity and a scFab directed to the blood
brain barrier receptor, then the linker conjugates the scFab to the
C-terminal end of the CH3-CH2 Ig entity. The linker conjugating the
brain effector entity to the monovalent binding entity (first
linker) and the linker connecting the scFab to the C-terminal end
of the CH2-CH3 Ig domain (second linker) can be the same or
different.
[0115] Single chain peptidic linkers, comprising of from one to
twenty amino acid residues joined by peptide bonds, can be used. In
certain embodiments, the amino acids are selected from the twenty
naturally-occurring amino acids. In certain other embodiments, one
or more of the amino acids are selected from glycine, alanine,
proline, asparagine, glutamine and lysine. In other embodiments,
the linker is a chemical linker. In certain embodiments, the linker
is a single chain peptidic linker with an amino acid sequence with
a length of at least 25 amino acid residues, in one preferred
embodiment with a length of 32 to 50 amino acid residues. In one
embodiment the peptidic linker is a (G.times.S)n linker with
G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8),
in one embodiment with x=4, n=6 or 7, in one preferred embodiment
with x=4, n=7. In one embodiment the linker is (G45)4 (SED ID NO:
08). In one embodiment the linker is (G4S)6G2 (SEQ ID NO: 07).
[0116] Conjugation may be performed using a variety of chemical
linkers. For example, the monovalent binding entity or the fusion
polypeptide and the brain effector entity may be conjugated using a
variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). The linker may be a "cleavable
linker" facilitating release of the effector entity upon delivery
to the brain. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al, Cancer Res. 52 (1992)
127-131; U.S. Pat. No. 5,208,020) may be used.
[0117] Covalent conjugation can either be direct or via a linker.
In certain embodiments, direct conjugation is by construction of a
polypeptide fusion (i.e. by genetic fusion of the two genes
encoding the monovalent binding entity towards the BBBR and
effector entity and expressed as a single polypeptide (chain)). In
certain embodiments, direct conjugation is by formation of a
covalent bond between a reactive group on one of the two portions
of the monovalent binding entity against the BBBR and a
corresponding group or acceptor on the brain effector entity. In
certain embodiments, direct conjugation is by modification (i.e.
genetic modification) of one of the two molecules to be conjugated
to include a reactive group (as non-limiting examples, a sulfhydryl
group or a carboxyl group) that forms a covalent attachment to the
other molecule to be conjugated under appropriate conditions. As
one non-limiting example, a molecule (i.e. an amino acid) with a
desired reactive group (i.e. a cysteine residue) may be introduced
into, e.g., the monovalent binding entity towards the BBBR antibody
and a disulfide bond formed with the neurological drug. Methods for
covalent conjugation of nucleic acids to proteins are also known in
the art (i.e., photocrosslinking, see, e.g., Zatsepin et al. Russ.
Chem. Rev. 74 (2005) 77-95). Conjugation may also be performed
using a variety of linkers. For example, a monovalent binding
entity and a effector entity may be conjugated using a variety of
bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as bis-(p-diazoniumb
enzoyl)-ethylenediamine), diisocyanates (such as toluene
2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-2,4-dinitrobenzene). Peptidic linkers, comprised of
from one to twenty amino acid residues joined by peptide bonds, may
also be used. In certain such embodiments, the amino acid residues
are selected from the twenty naturally-occurring amino acids. In
certain other such embodiments, one or more of the amino acid
residues are selected from glycine, alanine, proline, asparagine,
glutamine and lysine. The linker may be a "cleavable linker"
facilitating release of the effector entity upon delivery to the
brain. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al, Cancer Res. 52 (1992) 127-131; U.S. Pat. No.
5,208,020) may be used.
II. COMPOSITIONS AND METHODS
[0118] In one aspect, the invention is based, in part, on the
finding that the blood brain barrier shuttle modules as reported
herein can be used to deliver a brain effector entity across the
blood brain barrier into the brain. In certain embodiments, the
blood brain barrier shuttle module comprises a monovalent binding
entity that specifically binds to a blood brain barrier receptor,
such as the transferrin receptor. The blood brain barrier shuttle
modules as reported herein are useful, e.g., for the diagnosis or
treatment of neurological disorders, such as Alzheimer's disease,
Parkinson's Disease and Alzheimer's Disease with Parkinson's
Disease co-morbidity.
A. Exemplary Anti-Blood Brain Barrier Receptor Antibodies
[0119] Monovalent binding entities that specifically bind to a
blood brain barrier receptor can be characterized with respect to
their binding and transcytosis properties: [0120] efficient cell
binding of BBBR expressing cells as monovalent binding entity,
[0121] efficient in vitro transcytosis as monovalent binding
entity, [0122] human--cynomolgus cross-reactivity (e.g. in BIAcore
and FACS experiments).
[0123] The transcytosis screening was performed in an hCMEC/D3
based assay. The assay was performed in a pulse-chase mode. The
hCMEC/D3 brain endothelial cells were incubated with the monovalent
binding entity for 1 hour, washed thereafter and the following
parameters were determined 0 hours and 4 hours post washing: [0124]
i) amount of monovalent binding entity taken up into the cells
during the loading phase, [0125] ii) basolateral amount of
monovalent binding entity 4 hours post loading and washing; [0126]
iii) apical amount of monovalent binding entity 4 hours post
loading and washing; [0127] iv) amount of monovalent binding entity
in the cells (by cell lysis) 0 hours and 4 hours after loading and
washing; [0128] v) total amount of monovalent binding entity 0
hours and 4 hours after loading and washing.
[0129] In order to be eligible as monovalent binding entity in a
blood brain barrier shuttle module as reported herein the
monovalent binding entity has to be i) taken up by the hCMEC/D3
cells (endocytosis), ii) transported outside the hCMEC/D3 cells
(exocytosis), and iii) stable inside the hCMEC/D3 cells (no or low
transport to the endosome for degradation).
[0130] Thus, in one embodiment the monovalent binding entity is
characterized in a hCMEC/D3 based assay by i) an (substantial)
uptake into the hCMEC/D3 cells during a one-hour loading period,
ii) a release into the apical and/or basolateral compartment after
the loading period and a washing step within 4 hours after the
washing, and iii) a low (intracellular) degradation rate.
[0131] In one embodiment the loading is at a concentration of about
2.67 .mu.g/mL monovalent binding entity for one hour.
[0132] It has been found that a monovalent binding entity in order
to be eligible as monovalent binding entity of a blood brain
barrier shuttle module as reported herein has to show in the above
described hCMEC/D3 based assay the following threshold values:
[0133] i) an amount of monovalent binding entity taken up into the
cells during the loading phase of 400 pg or more, [0134] ii)
basolateral amount of monovalent binding entity 4 hours post
loading and washing of 100 pg or more, and [0135] iii) apical
amount of monovalent binding entity 4 hours post loading and
washing of 150 pg or more.
[0136] The mouse anti-human transferrin-receptor antibody 128.1
(for variable region sequences see WO 93/10819 and SEQ ID NO: 11
and 12) can be taken as reference. In this case the monovalent
binding entity in order to be eligible as monovalent binding entity
of a blood brain barrier shuttle module as reported herein has to
show in the above described hCMEC/D3 based assay the following
threshold values: [0137] i) an amount of monovalent binding entity
taken up into the cells during the loading phase of 20% or more of
the loading of antibody 128.1, [0138] ii) basolateral amount of
monovalent binding entity 4 hours post loading and washing of 15%
or more of the basolateral amount of antibody 128.1; and [0139]
iii) apical amount of monovalent binding entity 4 hours post
loading and washing of 15% or more of the apical amount of antibody
128.1.
[0140] The hCMEC/D3 based assay was performed as follows (this is
one embodiment of all aspects as reported herein):
[0141] Medium and supplements for hCMEC/D3 (see WO 2006/056879 and
Weksler, B. B., et al., FASEB J. 19 (2005) 1872-1874) can be
obtained from Lonza. hCMEC/D3 cells (passages 26-29) are/can be
cultured to confluence on collagen-coated coverslips (microscopy)
or flasks in EBM2 medium containing 2.5% FBS, a quarter of the
supplied growth factors and fully complemented with supplied
hydrocortisone, gentamycin and ascorbic acid.
[0142] For all transcytosis assays, high density pore
(1.times.10.sup.8 pores/cm.sup.2) PET membrane filter inserts (0.4
.mu.m pore size, 12 mm diameter) are/can be used in 12-well cell
culture plates. Media volumes are calculated to be 400 .mu.L and
1600 .mu.L for apical and basolateral chambers, respectively.
Apical chambers of filter inserts are/can be coated with rat tail
collagen I (7.5 .mu.g/cm.sup.2) followed by fibronectin (5
.mu.g/mL), each incubation lasting for 1 h at RT. hCMEC/D3 cells
are/can be grown to confluent monolayers (.about.2.times.10.sup.5
cells/cm.sup.2) for 10-12 days in EBM2 medium. Empty filters
are/can be blocked in PBS containing 1% BSA for 1 hour or overnight
(o/n) before assay and then calibrated for at least 1 hour in EBM2
before the assay.
[0143] The assay (for assay scheme see FIG. 1) was performed in
serum-free EBM2 medium which was otherwise reconstituted as
described herein. On day of the assay, cells are serum-starved for
60 min. to deplete the natural ligand of the blood brain barrier
receptor in question. Filter inserts with or without (but blocked
overnight in complete medium) cells were incubated apically with
monoclonal antibodies in question (monovalent binding entity) for 1
hour at 37.degree. C. The monolayers were washed at room
temperature (RT) in serum-free medium apically (400 .mu.L) and
basolaterally (1600 .mu.L) three times for 3-5 min. each.
Pre-warmed medium was added to the apical chamber and the filters
transferred to a fresh 12 well plate (blocked overnight with PBS
containing 1% BSA) containing 1600 .mu.L pre-warmed medium. At this
point, filters with or without cells were lysed in 500 .mu.L RIPA
buffer in order to determine specific antibody (monovalent binding
entity) uptake. The remaining filters were incubated at 37.degree.
C. or at 4.degree. C. and samples were collected at various time
points to determine apical and/or basolateral release of the
antibody (monovalent binding entity). The content of antibody in
the samples can be quantified using a highly sensitive IgG ELISA
(see Example 10). For each time point, data should be generated
from two empty filters and three filter cell cultures.
[0144] The results for 69 anti-transferrin receptor antibodies are
shown in the Table below. Antibody 128.1 was used as reference.
TABLE-US-00004 values of reference transcytosis total total
antibody loading basolateral apical 128.1 for 0 h 4 h 4 h set [pg]
[pg] [pg] 1 2072 896 1547 2 2951 797 1690 3 3448 1034 1843 4 864
274 375 5 3027 957 1679 6 2133 638 1187 7 3490 1138 1966
transcytosis total total relative relative relative loading
basolateral apical loading to basolateral of apical of 0 h 4 h 4 h
reference reference reference clone [pg] [pg] [pg] set [%] [%] [%]
No. 0116 872 70 203 1 42 8 13 No. 0127 293 56 160 1 10 7 9 No. 0128
1155 231 746 3 33 22 40 No. 0134 151 18 154 3 4 2 8 No. 0143 651
156 475 2 22 20 28 No. 0146 1623 357 748 2 55 45 44 No. 0150 298 36
205 3 9 3 11 No. 0279 768 77 131 4 89 28 35 No. 0280 221 42 133 4
26 15 35 No. 0281 34 3 16 4 4 1 4 No. 0282 574 14 45 4 66 5 12 No.
0283 112 24 52 4 13 9 14 No. 0284 32 0 24 4 4 0 6 No. 0285 21 0 18
4 2 0 5 No. 0286 130 29 93 4 15 11 25 No. 0291 419 61 96 4 48 22 26
No. 0292 118 27 95 4 14 10 25 No. 0293 354 28 88 4 41 10 23 No.
0294 25 0 12 4 3 0 3 No. 0295 101 21 75 4 12 8 20 No. 0297 95 17 64
4 11 6 17 No. 0298 195 10 59 4 23 4 16 No. 0299 705 170 294 4 82 64
79 No. 0300 632 103 86 4 73 38 23 No. 0301 61 10 41 4 7 4 11 No.
0302 420 118 217 4 49 43 58 No. 0304 665 157 282 4 77 58 75 No.
0305 154 33 111 4 18 12 30 No. 0313 175 36 132 5 6 4 8 No. 0316 84
0 72 5 3 0 4 No. 0318 130 21 82 5 4 2 5 No. 0319 406 104 343 5 13
11 20 No. 0322 172 84 96 5 6 9 6 No. 0323 794 175 478 5 26 18 28
No. 0324 193 32 144 5 6 3 9 No. 0325 664 166 483 5 22 17 29 No.
0329 744 192 449 6 35 30 38 No. 0335 601 0 45 6 28 0 4 No. 0339 967
227 580 6 45 36 49 No. 0343 883 195 574 6 41 34 48 No. 0346 1684
424 832 6 79 67 70 No. 0348 838 205 626 6 39 32 53 No. 0349 1538
181 396 6 72 28 33 No. 0350 940 257 637 6 44 40 54 No. 0411 1655
512 955 6 78 80 80 No. 0431 1738 511 493 6 81 80 42 No. 0432 890
265 421 6 42 41 35 No. 0433 957 274 370 6 45 43 31 No. 0434 1033
264 403 6 48 41 34 No. 0435 1356 351 503 6 64 55 42 No. 0441 1256
158 383 6 59 25 32 No. 0445 758 133 196 6 36 21 17 No. 0447 451 133
320 6 21 21 18 No. 0449 728 214 377 6 34 34 32 No. 0451 797 174 442
6 37 27 37 No. 0452 822 291 490 6 39 46 41 No. 0453 720 220 500 6
34 34 42 No. 0486 1434 454 503 7 41 40 26 No. 0487 4583 685 1364 7
131 60 69 No. 0488 991 176 658 7 28 15 33 No. 0489 2124 370 1179 7
61 33 60 No. 0490 4011 581 1388 7 115 51 71 No. 0491 1310 244 762 7
38 21 39 No. 0492 2962 516 1052 7 85 45 54 No. 0493 1006 215 885 7
29 19 45 No. 0494 3510 748 1503 7 101 66 76 No. 0495 1236 184 617 7
35 16 31 No. 0496 2178 539 1402 7 64 47 71 No. 0497 2984 725 1809 7
85 64 92
[0145] Antibody 299 shows a transcytosis loading of 705 pg, whereof
after 4 hours 170 pg (=24% of loading) can be found in the
basolateral compartment and 294 pg (=42% of loading) can be found
in the apical compartment.
[0146] Antibody 494 shows a transcytosis loading of 3510 pg,
whereof after 4 hours 748 pg (=21% of loading) can be found in the
basolateral compartment and 1503 pg (=43% of loading) can be found
in the apical compartment.
[0147] The antibodies fulfilling the criteria as outlined above are
embodiments of the current invention.
[0148] Thus, one aspect as reported herein is an anti-transferrin
receptor antibody or transferrin receptor binding fragment thereof
comprising
[0149] (1) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 13 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 14, or
[0150] (2) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 15 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 16, or
[0151] (3) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 17 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 18, or
[0152] (4) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 19 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 20, or
[0153] (5) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 21 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 22, or
[0154] (6) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 23 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 24, or
[0155] (7) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 25 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 26, or
[0156] (8) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 27 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 28, or
[0157] (9) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 29 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 30, or
[0158] (10) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 31 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 32, or
[0159] (11) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 33 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 34, or
[0160] (12) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 35 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 36, or
[0161] (13) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 37 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 38, or
[0162] (14) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 39 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 40, or
[0163] (15) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 41 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 42, or
[0164] (16) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 43 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 44, or
[0165] (17) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 45 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 46, or
[0166] (18) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 47 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 48, or
[0167] (19) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 49 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 50, or
[0168] (20) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 51 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 52, or
[0169] (21) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 53 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 54, or
[0170] (22) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 55 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 56, or
[0171] (23) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 57 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 58, or
[0172] (24) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 59 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 60, or
[0173] (25) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 61 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 62, or
[0174] (26) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 63 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 64, or
[0175] (27) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 65 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 66, or
[0176] (28) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 67 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 68, or
[0177] (29) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 69 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 70, or
[0178] (30) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 71 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 72, or
[0179] (31) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 73 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 74, or
[0180] (32) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 75 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 76, or
[0181] (33) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 77 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 78, or
[0182] (34) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 79 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 80, or
[0183] (35) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 81 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 82, or
[0184] (36) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 83 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 84, or
[0185] (37) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 85 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 86, or
[0186] (38) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 87 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 88, or
[0187] (39) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 89 and a light chain variable domain that has the amino
acid sequence of SEQ ID NO: 90, or
[0188] (40) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 91 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 92.
[0189] (41) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 93 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 94, or
[0190] (42) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 95 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 96, or
[0191] (43) a heavy chain variable domain that has the amino acid
sequence of SEQ ID NO: 97 and a light chain variable domain that
has the amino acid sequence of SEQ ID NO: 98.
[0192] One preferred aspect as reported herein is an
anti-transferrin receptor antibody or transferrin receptor binding
fragment thereof comprising a heavy chain variable domain that has
the amino acid sequence of SEQ ID NO: 23 and a light chain variable
domain that has the amino acid sequence of SEQ ID NO: 24.
[0193] One preferred aspect as reported herein is an
anti-transferrin receptor antibody or transferrin receptor binding
fragment thereof comprising a heavy chain variable domain that has
the amino acid sequence of SEQ ID NO: 91 and a light chain variable
domain that has the amino acid sequence of SEQ ID NO: 92.
[0194] The respective amino acid sequences are depicted in the
following Table.
TABLE-US-00005 heavy chain variable domain amino light chain
variable domain amino clone acid sequence (SEQ ID NO:) acid
sequence (SEQ ID NO:) No. 0128 QSVEESGGRLVTPGTPLTLTCTVSGIDLS
DIQMTQSPSSLSASVGDRVTITCRASQSI SYYMSWVRQAPGKGLEWIGII
SNYLNWYQQKPGKAPKLLIYD YPSGYAYYTSWAKGRFTISKTSTTVDLK
ASSLESGVPSRFSGSGSGTDFTLTISSLQP MTSLTTEDTATYFCARDVGDGG
EDFATYYCQQSGSTPYTFGQ GTFDIWGQGTMVTVSS (13) GTKLEIKR (14) No. 0143
GVQCQSLEESGGRLVTPGGSLTLTCTVS DIQMTQSPSSLSVSVGDRVTITCRASQGI
GIDLSTYSMGWVRQSPGKGLEY SNYLAWYQQKPGKAPELLIYA
IGIISSSDNTYYTSWAKGRFTISKTSTTVD ASILQGGVPSRFSGSGSGTDFTLTISSLQ
LKITSPTTEDTATYFCARGY PEDVATYYCQQYNTYPPTFGQ NGGWKTPMDVWGQRTTVTVSS
(15) GTKLEIKR (16) No. 0146 GVQCEVQLVESGGGLVQPGGSLRLSCA
DIQMTQSPSSLSASVGDRVTITCRASQSI ASGFTFGDFAMNWVRQAPGKGLE
SSYLNWYQQKPGKAPKLLIYS WVSGISASGVSTYYADSVKGRVTISRDN
ASSLQSGVPSRFSGSRSGTEFTLTISSLQP SKNTLNLQMNSLRAEDTAVYYC
EDFATYYCQQSYNTPLTFGG ARDPCDASNDYGYCKLDVWGQGTTVT GTKVEIKR (18) VSS
(17) No. 0279 QSVEESGGRLVTPGTPLTLTCTVSGIDLS
AYDMTQTPASVEVAVGGTVTIKCQASQ SYAMGWVRQAPGKGLEWIGYI
SISNYLAWYQQKPGQPPKLLIYR WSGGSADYASWAKGRFTISKTSTTVDL
ASTLASGVSSRFKGSGSGTDFTLTISGV KIPSPTMEDTATYFCARGDAEYG
GCADAATYYCQQTLSTINIDNT DANGFAIWGPGTLVTVSL (19) FGGGTEVVVKR (20) No.
0291 QSVEESGGRLVTPGTPLTLTCTVSGFSLS AYDMTQTPASVEVAVGGTVTIKCQASQ
SGAMTWVRQAPGKGLEWIGYI SISSYLAWYQQKPGQPPKLLIYR
WSGGSTDYASWAKGRFTISKTSTTVDLK ASTLASGVSSRFKGSGSGTQFTLTISDLE
ITSPTTEDTATYFCARGYGADY CADAATYYCQQGLSTINVDNT PDFGDANGFDPWGPGTLVTVSS
(21) FGGGTEVVVKR (22) No. 0299 QSMEESGGRLVTPGTPLTLTCTVSGFSLS
AYDMTQTPASVEVAVGGTVTIKCQASQ SYAMSWVRQAPGKGLEWIGYI
SISSYLSWYQQKPGQRPKLLIYR WSGGSTDYASWAKGRFTISKTSTTVDLK
ASTLASGVSSRFKGSGSGTQFTLTISGVE ITSPTTEDTATYFCARRYGTSY
CADAATYYCQQCYSSSNVDNT PDYGDANGFDPWGPGTLVTVSS (23) FGGGTEVVVKR (24)
No. 0300 QSVEESGGRLVTPGTPLTLTCTASEFSLS ADVVMTQTPASVEAAVGGTVTIKCQAS
NYYMSWVRQAPGKGLEYIGFI QSISSYFAWYQQKPGQPPKLLIY
HTDGSTNYASWVNGRFTISRTSTTVDLK RASKLATGVPSRFKGSGSGTEFTLTISDL
TTSLTTEDTATYFCCRYLVSGG ECADAATYYCQTCGSISTYGG TIAFDLWGPGTLVTVSS (25)
AFGGGTEVVVKR (26) No. 0302 QSLEESGGRLVTPGGSLTLTCTVSGFSLS
AFEMTQTPSSVSEPVGGTVTIKCQASEN TYAMTWVRQAPGKGLEWIGII
IYSFLAWYQQKPGQPPKLLIYD YAGTDITWYASWAKGRFTISKTSTTVDL
ASDLASGVPSRFKGSGSGTQFTLTISDLE SITSPTTEDTATYFCAKVPYTG
CADAATYYCQQGYSGNVILNA VSDYLYYFDLWGPGTLVTVSS (27) FGGGTEVVVKR (28)
No. 0304 QSMEESGGRLVTPGTPLTLTCTVSGFSLS AYDMTQTPASVEVAVGGTVTIKCQASQ
SYAMSWVRQAPGKGLEWIGYI SISSYLSWYQQKPGQRPKLLIYR
WSGGSTDYASWAKGRFTISKTSTTVDLK ASTLASGVSSRFKGSGSGTQFTLTISGVE
ITSPTTEDTATYFCARRYGTSY CADAATYYCQQCYSSSNVDNT PDYGDANGFDPWGPGTLVTVSS
(29) FGGGTEVVVKR (30) No. 0323 QSVEESGGRLVTPGTPLPLTCTVSGFSLS
AYDMTQTPASVEVAVGGTVTIKCQASQ NYAMGWFRQAPGKGLEWIGYI
SISIYLAWYQQKPGQPPKLLIYK WSGGSTDYASWAKGRFTISKTSTTVDLK
ASTLASGVPSRFKGSGSGTEFTLTISDLE MTSLTTEDTATYFCARADTVYR
CADAATYYCQQAYSYSNVDNV DDYGWSLWGPGTLVTVSS (31) FGGGTEVVVKR (32) No.
0325 QSVEESGGRLVTPGTPLTLTCTVSGFSLS AYDMTQTPASVEVAVGGTVTIKCQASE
NYNMGWVRQAPGKGLEWIGII DIESYLAWYQQKPGQRPKLLIYA
SDAGSAWYASWVKGRFTISKTSTTVDL VSTLASGVSSRFKGSGSGTEYTLTISGV
KITSPTTEDAATYFCARGDAAAY QCADAATYYCQQGYSSSNLDNA TTGTEFFSLWGPGTLVTVSS
(33) FGGGTEVVVRR (34) No. 0329 GVQCQSLEESGGGLVKPGGTLTLTCTVS
AYDMTQTPASVEAAVGGTVTIKCQASQ GFSLNSYSMTWVRQAPGKGLEW
SITSYLAWYQQKPGQPPKLLIYR IGYIWSGGSADYANWAKGRFTISKTSTT
ASTLASGVSSRFKGSGSGTQFTLTISGVE VDLKMTSLTAEDTATYFCGRGY
CADAATYYCQQTYRYMNVDNV DTTDNGFNLWGPGTLVTVSS (35) FGGGTEVVVKR (36)
No. 0339 GVQCQSVEESGGRLVTPGTPLTLTCTVS AYDMTQTPSSVEAAVGGTVTIKCQASQ
GIDLSSHAMTWVRQAPGKGLEW SISNYLAWYQQKPGQPPELLIYR
IGYIWSGGSADYASWAKGRFTISKTSSTT ASTLASGVSSRFRGSGSGTDFTLTISGVG
VDLKIPSPTTEDTATYFCARG CADAATYYCQQGLSSSYVDNT ADEYGDANGFNIWGPGTLVTVSL
(37) FGGGTEVVVKR (38) No. 0343 GVQCQEQLKESGGGLVTPGTPLTLTCTA
ADVVMTQTPSPVSGAVGGTVTIKCQAS SGFSLSIYYMSWVRQAPGKGLD
QSIDSYLSWYQQKPGQPPKLLIY WIGFIYVDSSSAHYASWAKGRFTISKTST
SASTLASGVSSRFKGSGSGTQFTLTISDL TVDLKITSPTTEDTATYFCAR
ECADAATYYCQCTYYDSSSIG DVDSSYFWGFNLWGPGTLVTVSS (39) GFGGGTEVVVKR
(40) No. 0346 GVQCQSVEESGGRLVTPGTPLTLTCTVS
DVVMTQTPSSVSEPVGGTVTINCQASEN GFSLSSNAINWVRQAPGKGLEW
IYSSLDWYQQKPGQPPKLLIYS IGYIYTDGNTYYASWAKGRFTISKTSTT
ASNLASGVSSRFKGSRSGTEYTLTISDLE VDLKITSPTTEDTATYFCARGV
CADAATYYCQCIDGGNRGKPF GYSDLWGPGTLVTVSS (41) GGGTEVVVKR (42) No.
0348 GVQCQSVEESGGRLVTPGTPLTLTCTAS AQVLTQTASSVSAAVGGTVTISCQSSQS
GFSLNSVYMSWVRQAPGKGLEW VWNNNFLSWYQQKPGQPPKLLI
IGIIYASGSIYYASWAKGRFTISRTSSTTV YLASTLASGVPSRFKGSGSGTQFTLTISD
DLKMTSLTTEDTATYFCVRS LECDDAATYYCAGVYSDNIFA ADYDSGMPFDLWGPGTLVTVSS
(43) FGGGTEVVVKR (44) No. 0349 GVQCQSVEESGGRLVTPGTPLTLTCTVS
ADVVMTQTPASVEAAVGGTVTIKCQAS GFSLSSSYMAWVRQAPGKGLEY
QSISSYFAWYQQKPGQPPKLLIY IGFIHTDGSTYYATWVNGRFTISRTSTTV
RASNLATGVPSRFKGSGSGTEFTLTISDL TLKMTSLTTEDTATYFCARYL
ECADAATYYCQSCGSISTYGG VGSGAVAFDLWGPGTLVTVSS (45) AFGGGTEVVVKR (46)
No. 0350 GVQCQSVEESGGRLVTPGTPLTLTCTVS AYDMTQTPASVSEPVGGTVTIKCQASQ
GFSLSSYAMGWVRQAPGKGLEW SISSYLSWYQQKPGQPPKLLIYR
IGYIWSGGSTDYATWAKGRFTISKTSTT ASTLESGVPSRFKGSGSGTEFTLTISDLE
VDLSITSPTTEDTAAYFCVRSA CADAATYYCQQGLSVIHVDNT GSGDAMGFNLWGPGTLVTVSS
(47) FGGGTEVVVKR (48) No. 0411 QSVEESGGRLVTPGTPLTLTCTVSGFSLS
AYDMTQTPASVSAAVGGTVSINCQASQ NYAMTWVRQAPGKGLEWIGYI
SISGYLSWYQQKPGQRPKLLIYR WSGGSTDYATWAKGRFTISKTSTTVDL
ASTLASGVSSRFKGSGSGTQFTLTISGVE KITSPTTEDTATYFCARGYGAGY
CADAATYYCQQGYSIINVDNT PDYGDANGFDPWGPGTLVTVSS (49) FGGGTEVVVKR (50)
No. 0431 QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCAREE EDEADYYCNSRDDYGDGVFGG SSYISGFDYWGQGTLVTVSS
(51) GTKLTVL (52) No. 0432 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASLTITGAQA VYMELSSLRSEDTAVYYCAREE
EDEADYYCNSMTESMNYVFGG SWWLSGLDYWGQGTLVTVSS (53) GTKLTVL (54) No.
0433 QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCAREE EDEADYYCNSIDSSGNHDVFG STWFSGFDYWGQGTLVTVSS
(55) GGTKLTVL (56) No. 0434 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASLTITGAQA VYMELSSLRSEDTAVYYCAREY
EDEADYYCNSPDSIAYGVVFG PWYIWSYDYWGQGTLVTVSS (57) GGTKLTVL (58) No.
0435 QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCARES EDEADYYCNSEDSESNLVFGG YFLSGFDYWGQGTIVTVSS
(59) GTKLTVL (60) No. 0441 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASLTITGAQA VYMELSSLRSEDTAVYYCARSW
EDEADYYCNSIDSALDHVFGG FGEWMDYWGQGTLVTVSS (61) GTKLTVL (62) No. 0445
QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCARDS EDEADYYCNSRDMSLNVVFGG WYGWAFDYWGQGTLVTVSS
(63) GTKLTVL (64) No. 0447 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASLTITGAQA VYMELSSLRSEDTAVYYCARES
EDEADYYCNSRDSTLIVVVFG FYGWAFDYWGQGTLVTVSS (65) GGTKLTVL (66) No.
0449 QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCARET EDEADYYCNSPDSSVNYMVFG WGWVAFDYWGQGTLVTVSS
(67) GGTKLTVL (68) No. 0451 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASITITGAQA VYMELSSLRSEDTAVYYCAREE
EDEADYYCNSRDIIGDAVFGG YYYLSGFDYWGQGTLVTVSS (69) GTKLTVL (70) No.
0452 QVQLVQSGAEVKKPGASVKVSCKASGY SSELTQDPAVSVALGQTVRITCQGDSLR
TFTSYYMHWVRQAPGQGLEWMGI SYYASWYQQKPGQAPVLVIYGK
INPSGGSTSYAQKFQGRVTMTRDTSTST NNRPSGIPDRFSGSSSGNTASLTITGAQA
VYMELSSLRSEDTAVYYCAREA EDEADYYCNSIDSEGNDIVFG WDYLSSMDYWGQGTLVTVSS
(71) GGTKLTVL (72) No. 0453 QVQLVQSGAEVKKPGASVKVSCKASGY
SSELTQDPAVSVALGQTVRITCQGDSLR TFTSYYMHWVRQAPGQGLEWMGI
SYYASWYQQKPGQAPVLVIYGK INPSGGSTSYAQKFQGRVTMTRDTSTST
NNRPSGIPDRFSGSSSGNTASLTITGAQA VYMELSSLRSEDTAVYYCARDV
EDEADYYCNSIDLSMDIVFGG YGHLDYWGQGTLVTVSS (73) GTKLTVL (74) No. 0486
EVQLQQSGPELEKPGASVKISCKASGYS DVLMTQTPLSLPVSLGDQASISCRSTQS
FTGYNMNWVKQSNGERLEWIGS VVHSDGITHLEWYLQKPGQSPK
IDPFYGGTTYNQKFKDKATLTVDKPSST LLIYKVFNRFYGVPDRFSGSGSGTDFTL
AYMQLTSLTAEDSAVYYCAREG KISRVEAEDLGVYYCFQGSHVP GNYYGPWFAYWGQGTLVTVSA
(75) WTFGGGTKLEIKR (76) No. 0487 DVQLVESGGGLVQPGGSRKLSCAASGFT
DVVMTQTPLTLSVTIGQPASISCKSSQSL FSSFGMHWVRQAPEKGLEWVAY
LYTIGKTYLNWLLQRPGQSPK ISSGSGTIYYADTVKGRFTISRDNPKNTL
RLIYLVSKLDSGVPDRFSGSGSGTDFTL FLQMTTLRSEDTAMYYCARSY
KISRVEAEDLGVYYCFQSTHFP YSDFGHAMDYWGQGTSVTVSS (77) LTFGAGTKLELRR
(78) No. 0488 DVQLVESGGGLVQPGGSRKLSCAASGFT
DIQMTQSPASLSATVGETVTITCRTSGNI FSTFGMHWVRQTPERGLEWVAY
HNYLAWYQQKQGKSPQLLVYN ISSGGTNIYYADPVKGRFTISRDNPKKTL
GQTLAEGVPSKFSGSGSGTQYSLKINSL FLQMTGLRSEDTAIYYCARDG
QPEDFGSYFCHQYFYFPLTFGT FGNYWFAYWGQGTLVTVSA (79) GTKLEIKR (80) No.
0489 EVQLQQSGTELVRPGALVRLSCKASDFN DIVMTQSHKFMSTSVGDRVTFTCKASQ
IKDYYLHWVKQRPEQGLEWVGW DVRSAVAWYQQKAGQSPKLLIYS
IDPETLNTIYGPKFQGKASITADTSSNTA ASYRYTGVPDRFTGSGSGTDFTFTISSV
YLQLSGLTSEDIAVYYCTSST QAEDLSVYYCQQHYTTPLTFGA VISYWHFDVWGAGTSVTVSS
(81) GTKLELKR (82) No. 0490 DVQLVESGGGLVQPGGSRKLSCAASGFT
DVVMTQTPLTLSVTIGQPASISCKSSQSL FSSFGMHWVRQAPEKGLEWVAY
LYTIGKTYLNWLLQRPGQSPK ISSGSGTIYYADTMKGRFTISRDNPKNTL
RLIYLVSKLDSGVPDRFSGSGSGTDFTL FLQMTTLRSEDTAMYYCARSY
KISRVEAEDLGVYYCFQSTHFP YSDFGHAMDYWGQGTSVTVSS (83) LTFGAGTKLELRR
(84) No. 0491 DVQLVESGGGLVQPGGSRKLSCAASGFT
DIQMTQSPASLSATVGETVTITCRTSGNI FSTFGMHWVRQTPERGLEWVAY
HNYLAWYQQKQGKSPQLLVYN ISSGSTNIYYADPVKGRFTISRDNPKKTL
GQTLAEGVPSKFSGSGSGTQYSLKINSL FLQMTGLRSEDTAMYYCARDG
QPEDFGSYFCHQYFYFPLTFGT FGNYWFAYWGQGTLVTVSA (85) GTKLEIKR (86) No.
0492 DVQLVESGGGLVQPGGSRKLSCAASGFT DVVMTQTPLTLSVTIGQPASISCKSSQSL
FSSFGMHWVRQAPEKGLEWVAY LYTQGKTYLNWLLQRPGQSPK
ISSGSSTIYYADTVKGRFTISRDNPKNTLF RLIYLVSKLDSGVPDRFSGSGSGTDFTL
LQMASLRSEDTAMYYCARSY KISRVEAEDLGVYYCLQSTHFP YGNFGFAMDYWGQGTSVTVSS
(87) LTFGAGTKLELKR (88) No. 0493 DVQLVESGGGLVQPGGSRKLSCAASGFT
DIQMTQSPASLSATVGETVTITCRTSGNI FSTFGMHWVRQTPERGLEWVAY
HNYLAWYQQKQGKSPQLLVYN ISSDSSNIYYADPVKGRFTISRDNPKKTLF
GQTLAEGVPSKFSGSGSGTQYSLKINSL LQMTGLRSEDTAIYYCARDG
QPEDFGSYFCHQYFYFPLTFGT FGNYWFAYWGQGTLVTVSA (89) GTKLEIKR (90) No.
0494 EVQLQQSGAVLVKPGASVKLSCPASGFN KIVMTQSPKSMSMSVGERVTLNCRASE
IKDTYIHWVIQRPEQGLEWIGR SVDTYVSWYQQKPEQSPELLIYG
IDPANGDTKCDPKFQVKATITADTSSNT ASNRYTGVPDRFTGSGSATDFTLTISSV
AYLQLSSLTSEDTAVYFCVRDY QAEDLADYYCGQTYNYPLTFGA LYPYYFDFWGQGTTLTVSS
(91) GTKLELKR (92) No. 0495 EVQLQQSGPELVKPGASVKMSCRASGY
QIVLTQSPAIMSASPGEKVTLTCSANSSI SFPSYVVHWVKQKPGQGLEWIGY
RNMHWYQQKTGTSPKRWIYDT INPYTDGTEYNEKFKGKATLTSDKSSST
SNLASGVPSRFSGSGSGTSYSLTISSMEA AYMELSSLTSEDSAVYYCARGF
EDAATYYCHQRSSFPYTFGGG YYYSMDYWGQGTSVTVSS (93) TKLEIKR (94)
No. 0496 QVQLIQSGAELVRPGASVNLSCKASGYT DIQMTQSSSSFSVSLGDRVTVTCKTTEDI
FTDYYINWVKQRPGQGLEWIAR YNRLAWYQHKPGNAPRLLISG
IYPGSDITYYNEKFKGKATLTAEKSSSTA ATGLETGVPSRFSGSGSGKDYTLTITSL
YMQLSSLTSEDSAVYFCARSP QTEDVATYYCLQYWSTPYTFGG
PHYVGNRYYALDYWGQGTSVTVSS (95) GTKLEIKR (96) No. 0497
QVQLQQPGAEFVKPGASVKMSCKASGY DIKMTQSPSSMYASLGERVTITCKASQD
TFTSYWITWVKQRPGQGLEWIGD INSYLNWFQQKPGKSPKTLIYR
IYPGSGSNKYNEKFKSKATLTVDTSSSTA ANRLVDGVPSRFSGSGSGQDYSLTISSL
YMHLSSLTSEDSAVYYCARER DYEDMGIYYCLQYDEFPYTFGG
PTYYGSSAWFDYWGQGTLVTVSA (97) GTKLEIKR (98)
[0195] One aspect as reported herein is a humanized
anti-transferrin receptor antibody or transferrin receptor binding
fragment thereof comprising
[0196] (1) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 13 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 14, or
[0197] (2) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 15 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 16, or
[0198] (3) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 17 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 18, or
[0199] (4) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 19 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 20, or
[0200] (5) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 21 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 22, or
[0201] (6) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 23 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 24, or
[0202] (7) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 25 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 26, or
[0203] (8) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 27 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 28, or
[0204] (9) a humanized heavy chain variable domain derived from the
heavy chain variable domain that has the amino acid sequence of SEQ
ID NO: 29 and a humanized light chain variable domain derived from
the light chain variable domain that has the amino acid sequence of
SEQ ID NO: 30, or
[0205] (10) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 31 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 32, or
[0206] (11) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 33 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 34, or
[0207] (12) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 35 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 36, or
[0208] (13) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 37 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 38, or
[0209] (14) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 39 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 40, or
[0210] (15) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 41 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 42, or
[0211] (16) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 43 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 44, or
[0212] (17) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 45 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 46, or
[0213] (18) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 47 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 48, or
[0214] (19) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 49 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 50, or
[0215] (20) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 51 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 52, or
[0216] (21) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 53 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 54, or
[0217] (22) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 55 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 56, or
[0218] (23) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 57 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 58, or
[0219] (24) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 59 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 60, or
[0220] (25) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 61 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 62, or
[0221] (26) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 63 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 64, or
[0222] (27) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 65 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 66, or
[0223] (28) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 67 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 68, or
[0224] (29) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 69 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 70, or
[0225] (30) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 71 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 72, or
[0226] (31) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 73 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 74, or
[0227] (32) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 75 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 76, or
[0228] (33) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 77 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 78, or
[0229] (34) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 79 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 80, or
[0230] (35) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 81 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 82, or
[0231] (36) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 83 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 84, or
[0232] (37) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 85 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 86, or
[0233] (38) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 87 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 88, or
[0234] (39) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 89 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 90, or
[0235] (40) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 91 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 92.
[0236] (41) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 93 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 94, or
[0237] (42) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 95 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 96, or
[0238] (43) a humanized heavy chain variable domain derived from
the heavy chain variable domain that has the amino acid sequence of
SEQ ID NO: 97 and a humanized light chain variable domain derived
from the light chain variable domain that has the amino acid
sequence of SEQ ID NO: 98.
[0239] One preferred aspect as reported herein is a humanized
anti-transferrin receptor antibody or transferrin receptor binding
fragment thereof comprising a humanized heavy chain variable domain
derived from the heavy chain variable domain that has the amino
acid sequence of SEQ ID NO: 23 and a humanized light chain variable
domain derived from the light chain variable domain that has the
amino acid sequence of SEQ ID NO: 24.
[0240] One aspect as reported herein is a humanized anti-human
transferrin receptor antibody comprising (a) a HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 110; and (c) a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 112. In one
embodiment the antibody further comprises (d) a HVR-L1 comprising
the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising
the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3
comprising the amino acid sequence of SEQ ID NO: 115.
[0241] One aspect as reported herein is a humanized anti-human
transferrin receptor antibody comprising (a) a HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 111; and (c) a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 112. In one
embodiment the antibody further comprises (d) a HVR-L1 comprising
the amino acid sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising
the amino acid sequence of SEQ ID NO: 114; and (f) a HVR-L3
comprising the amino acid sequence of SEQ ID NO: 115.
[0242] In one aspect, the invention provides an anti-transferrin
receptor antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) a HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 109; (b) a HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 110; (c) a HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 112; (d) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 113; (e) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 114; and (f) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 115.
[0243] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO:
109; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO:
110; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 112. In one embodiment, the antibody comprises a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 112. In another
embodiment, the antibody comprises a HVR-H3 comprising the amino
acid sequence of SEQ ID NO: 112 and a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 115. In a further embodiment, the
antibody comprises a HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 112, a HVR-L3 comprising the amino acid sequence of SEQ
ID NO: 115, and a HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 111. In a further embodiment, the antibody comprises (a) a
HVR-Hl comprising the amino acid sequence of SEQ ID NO: 109; (b) a
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and
(c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO:
112.
[0244] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 113; (b) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 114; and (c) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 115. In one embodiment, the antibody
comprises (a) a HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 113; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID
NO: 114; and (c) a HVR-L3 comprising the amino acid sequence of SEQ
ID NO: 115.
[0245] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) a HVR-H1 comprising the amino
acid sequence of SEQ ID NO: 109, (ii) a HVR-H2 comprising the amino
acid sequence of SEQ ID NO: 110, and (iii) a HVR-H3 comprising an
amino acid sequence selected from SEQ ID NO: 112; and (b) a VL
domain comprising at least one, at least two, or all three VL HVR
sequences selected from (i) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 113, (ii) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 114, and (c) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 115.
[0246] In another aspect, the invention provides an antibody
comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 109; (b) a HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 110; (c) a HVR-H3 comprising the amino acid sequence of SEQ
ID NO: 112; (d) a HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 113; (e) a HVR-L2 comprising the amino acid sequence of SEQ
ID NO: 114; and (f) a HVR-L3 comprising an amino acid sequence
selected from SEQ ID NO: 115.
[0247] One preferred aspect as reported herein is a humanized
anti-transferrin receptor antibody or transferrin receptor binding
fragment thereof comprising a humanized heavy chain variable domain
derived from the heavy chain variable domain that has the amino
acid sequence of SEQ ID NO: 91 and a humanized light chain variable
domain derived from the light chain variable domain that has the
amino acid sequence of SEQ ID NO: 92.
[0248] One aspect as reported herein is a humanized anti-human
transferrin receptor antibody comprising (a) a HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 117; and (c) a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 119. In one
embodiment the antibody further comprises (d) a HVR-L1 comprising
the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising
the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3
comprising the amino acid sequence of SEQ ID NO: 122.
[0249] One aspect as reported herein is a humanized anti-human
transferrin receptor antibody comprising (a) a HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 118; and (c) a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 119. In one
embodiment the antibody further comprises (d) a HVR-L1 comprising
the amino acid sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising
the amino acid sequence of SEQ ID NO: 121; and (f) a HVR-L3
comprising the amino acid sequence of SEQ ID NO: 122.
[0250] In one aspect, the invention provides an anti-transferrin
receptor antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) a HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 116; (b) a HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 117; (c) a HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 119; (d) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 120; (e) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 121; and (f) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 122.
[0251] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) a HVR-H1 comprising the amino acid sequence of SEQ ID NO:
116; (b) a HVR-H2 comprising the amino acid sequence of SEQ ID NO:
117; and (c) a HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 119. In one embodiment, the antibody comprises a HVR-H3
comprising the amino acid sequence of SEQ ID NO: 119. In another
embodiment, the antibody comprises a HVR-H3 comprising the amino
acid sequence of SEQ ID NO: 119 and a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 122. In a further embodiment, the
antibody comprises a HVR-H3 comprising the amino acid sequence of
SEQ ID NO: 119, a HVR-L3 comprising the amino acid sequence of SEQ
ID NO: 122, and a HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 118. In a further embodiment, the antibody comprises (a) a
HVR-H1 comprising the amino acid sequence of SEQ ID NO: 116; (b) a
HVR-H2 comprising the amino acid sequence of SEQ ID NO: 118; and
(c) a HVR-H3 comprising the amino acid sequence of SEQ ID NO:
119.
[0252] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 120; (b) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 121; and (c) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 122. In one embodiment, the antibody
comprises (a) a HVR-L1 comprising the amino acid sequence of SEQ ID
NO: 120; (b) a HVR-L2 comprising the amino acid sequence of SEQ ID
NO: 121; and (c) a HVR-L3 comprising the amino acid sequence of SEQ
ID NO: 122.
[0253] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) a HVR-H1 comprising the amino
acid sequence of SEQ ID NO: 116, (ii) a HVR-H2 comprising the amino
acid sequence of SEQ ID NO: 117, and (iii) a HVR-H3 comprising an
amino acid sequence selected from SEQ ID NO: 119; and (b) a VL
domain comprising at least one, at least two, or all three VL HVR
sequences selected from (i) a HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 120, (ii) a HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 121, and (c) a HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 122.
[0254] In another aspect, the invention provides an antibody
comprising (a) a HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 116; (b) a HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 117; (c) a HVR-H3 comprising the amino acid sequence of SEQ
ID NO: 119; (d) a HVR-L1 comprising the amino acid sequence of SEQ
ID NO: 120; (e) a HVR-L2 comprising the amino acid sequence of SEQ
ID NO: 121; and (f) a HVR-L3 comprising an amino acid sequence
selected from SEQ ID NO: 122.
[0255] One aspect as reported herein is a transferrin shuttle
module comprising a full length IgG antibody as brain effector
entity, a linker and one scFab as the monovalent binding entity,
which binds the transferrin receptor, wherein the scFab is
conjugated to the C-terminal end of the Fc-region of one of the
heavy chains of the IgG antibody via the linker.
[0256] One aspect as reported herein is a transferrin shuttle
module comprising a full length IgG antibody as brain effector
entity, a linker and one scFv as the monovalent binding entity,
which binds the transferrin barrier receptor, wherein the scFv is
conjugated to the C-terminal end of the Fc-region of one of the
heavy chains of the IgG antibody via the linker.
[0257] In one preferred embodiment the monovalent binding entity
comprises the HVRs of SEQ ID NOs:109, 110, 112, 113, 114, and 115,
or of SEQ ID NOs:116, 117, 119, 120, 121, and 122.
[0258] In a further aspect, an anti-transferrin receptor antibody
according to any of the above embodiments may incorporate any of
the features, singly or in combination, as described in Sections
1-6 below:
[0259] 1. Antibody Affinity
[0260] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled antigen in the presence of a titration series
of unlabeled antigen, then capturing bound antigen with an anti-Fab
antibody-coated plate (see, e.g., Chen, Y. et al., J. Mol. Biol.
293 (1999) 865-881). To establish conditions for the assay,
MICROTITER.degree. multi-well plates (Thermo Scientific) are coated
overnight with 5 .mu.g/mL of a capturing anti-Fab antibody (Cappel
Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked
with 2% (w/v) bovine serum albumin in PBS for two to five hours at
room temperature (approximately 23.degree. C.). In a non-adsorbent
plate (Nunc #269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed
with serial dilutions of a Fab of interest (e.g., consistent with
assessment of the anti-VEGF antibody, Fab-12, in Presta, L. G. et
al., Cancer Res. 57 (1997) 4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for a
longer period (e.g., about 65 hours) to ensure that equilibrium is
reached. Thereafter, the mixtures are transferred to the capture
plate for incubation at room temperature (e.g., for one hour). The
solution is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.L/well of scintillant (MICROSCINT-20.TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0261] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CMS chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/mL (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.L/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block non-reacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.L/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. Evaluation Software version 3.2) by
simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (Kd) is
calculated as the ratio k.sub.off/k.sub.on (see, e.g., Chen, Y. et
al., J. Mol. Biol. 293 (1999) 865-881). If the on-rate exceeds
10.sup.6 M.sup.-1 s.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0262] 2. Antibody Fragments
[0263] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab').sub.2, Fv, and scFv fragments, and
other fragments described below. For a review of certain antibody
fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003) 129-134.
For a review of scFv fragments, see, e.g., Plueckthun, A., In; The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and
Moore (eds.), Springer-Verlag, New York (1994), pp. 269-315; see
also WO 93/16185; U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046.
[0264] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP 0
404 097; WO 1993/01161; Hudson, P. J. et al., Nat. Med. 9 (2003)
129-134; and Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90
(1993) 6444-6448. Triabodies and tetrabodies are also described in
Hudson, P. J. et al., Nat. Med. 9 (20039 129-134).
[0265] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516).
[0266] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0267] 3. Chimeric and Humanized Antibodies
[0268] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison, S. L. et al., Proc. Natl.
Acad. Sci. USA 81 (1984) 6851-6855). In one example, a chimeric
antibody comprises a non-human variable region (e.g., a variable
region derived from a mouse, rat, hamster, rabbit, or non-human
primate, such as a monkey) and a human constant region. In a
further example, a chimeric antibody is a "class switched" antibody
in which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0269] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0270] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro, J. C. and Fransson, J., Front. Biosci.
13 (2008) 1619-1633, and are further described, e.g., in Riechmann,
I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl.
Acad. Sci. USA 86 (1989) 10029-10033; U.S. Pat. Nos. 5, 821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri, S. V. et al.,
Methods 36 (2005) 25-34 (describing specificity determining region
(SDR) grafting); Padlan, E. A., Mol. Immunol. 28 (1991) 489-498
(describing "resurfacing"); Dall'Acqua, W. F. et al., Methods 36
(2005) 43-60 (describing "FR shuffling"); and Osbourn, J. et al.,
Methods 36 (2005) 61-68 and Klimka, A. et al., Br. J. Cancer 83
(2000) 252-260 (describing the "guided selection" approach to FR
shuffling).
[0271] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims, M. J. et al., J. Immunol.
151 (1993) 2296-2308; framework regions derived from the consensus
sequence of human antibodies of a particular subgroup of light or
heavy chain variable regions (see, e.g., Carter, P. et al., Proc.
Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L. G. et al.,
J. Immunol. 151 (1993) 2623-2632); human mature (somatically
mutated) framework regions or human germline framework regions
(see, e.g., Almagro, J. C. and Fransson, J., Front. Biosci. 13
(2008) 1619-1633); and framework regions derived from screening FR
libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 (1997)
10678-10684 and Rosok, M. J. et al., J. Biol. Chem. 271 (19969
22611-22618).
[0272] 4. Library-Derived Antibodies
[0273] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom, H. R. et al., Methods in
Molecular Biology 178 (2001) 1-37 and further described, e.g., in
the McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T.
et al., Nature 352 (1991) 624-628; Marks, J. D. et al., J. Mol.
Biol. 222 (1992) 581-597; Marks, J. D. and Bradbury, A., Methods in
Molecular Biology 248 (2003) 161-175; Sidhu, S. S. et al., J. Mol.
Biol. 338 (2004) 299-310; Lee, C. V. et al., J. Mol. Biol. 340
(2004) 1073-1093; Fellouse, F. A., Proc. Natl. Acad. Sci. USA 101
(2004) 12467-12472; and Lee, C. V. et al., J. Immunol. Methods 284
(2004) 119-132.
[0274] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter, G. et al., Ann.
Rev. Immunol. 12 (1994) 433-455. Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self-antigens without any
immunization as described by Griffiths, A. D. et al., EMBO J. 12
(1993) 725-734. Finally, naive libraries can also be made
synthetically by cloning non-rearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro, as described by Hoogenboom, H. R. and Winter, G., J. Mol.
Biol. 227 (1992) 381-388. Patent publications describing human
antibody phage libraries include, for example: U.S. Pat. No.
5,750,373, and US 2005/0079574, US 2005/0119455, US 2005/0266000,
US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936,
and US 2009/0002360.
[0275] 5. Multispecific Antibodies
[0276] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for the
transferrin receptor and the other is for any other antigen.
Bispecific antibodies may also be used to localize cytotoxic agents
to cells which express the transferrin receptor. Bispecific
antibodies can be prepared as full length antibodies or antibody
fragments.
[0277] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO
93/08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659),
and "knob-in-hole" engineering (see, e.g., U.S. Pat. No.
5,731,168). Multi-specific antibodies may also be made by
engineering electrostatic steering effects for making antibody
Fc-heterodimeric molecules (WO 2009/089004); cross-linking two or
more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980,
and Brennan, M. et al., Science 229 (1985) 81-83); using leucine
zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.
A. et al., J. Immunol. 148 (1992) 1547-1553; using "diabody"
technology for making bispecific antibody fragments (see, e.g.,
Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993)
6444-6448); and using single-chain Fv (scFv) dimers (see, e.g.
Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing
trispecific antibodies as described, e.g., in Tutt, A. et al., J.
Immunol. 147 (1991) 60-69).
[0278] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies", are also included
herein (see, e.g. US 2006/0025576).
[0279] The antibody or fragment herein also includes a "Dual Acting
Fab" or "DAF" comprising an antigen binding site that binds to the
transferrin receptor as well as another, different antigen (see, US
2008/0069820, for example).
[0280] The antibody or fragment herein also includes multispecific
antibodies described in WO 2009/080251, WO 2009/080252, WO
2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO
2010/136172, WO 2010/145792, and WO 2010/145793.
[0281] In one embodiment of all aspects as reported herein the
anti-transferrin receptor antibody is a bispecific antibody.
[0282] One aspect as reported herein is a bivalent, bispecific
antibody comprising [0283] a) a first light chain and a first heavy
chain of an antibody specifically binding to a first antigen, and
[0284] b) a second light chain and a second heavy chain of an
antibody specifically binding to a second antigen, wherein the
variable domains VL and VH of the second light chain and the second
heavy chain are replaced by each other, [0285] wherein the first
antigen or the second antigen is human transferrin receptor.
[0286] The antibody under a) does not contain a modification as
reported under b) and the heavy chain and the light chain under a)
are isolated chains.
[0287] In the antibody under b) within the light chain [0288] the
variable light chain domain VL is replaced by the variable heavy
chain domain VH of said antibody, and [0289] within the heavy
chain, the variable heavy chain domain VH is replaced by the
variable light chain domain VL of said antibody.
[0290] In one embodiment [0291] i) in the constant domain CL of the
first light chain under a) the amino acid at position 124
(numbering according to Kabat) is substituted by a positively
charged amino acid, and wherein in the constant domain CH1 of the
first heavy chain under a) the amino acid at position 147 or the
amino acid at position 213 (numbering according to Kabat EU index)
is substituted by a negatively charged amino acid, or [0292] ii) in
the constant domain CL of the second light chain under b) the amino
acid at position 124 (numbering according to Kabat) is substituted
by a positively charged amino acid, and wherein in the constant
domain CH1 of the second heavy chain under b) the amino acid at
position 147 or the amino acid at position 213 (numbering according
to Kabat EU index) is substituted by a negatively charged amino
acid.
[0293] In one preferred embodiment [0294] i) in the constant domain
CL of the first light chain under a) the amino acid at position 124
is substituted independently by lysine (K), arginine (R) or
histidine (H) (numbering according to Kabat) (in one preferred
embodiment independently by lysine (K) or arginine (R)), and
wherein in the constant domain CH1 of the first heavy chain under
a) the amino acid at position 147 or the amino acid at position 213
is substituted independently by glutamic acid (E) or aspartic acid
(D) (numbering according to Kabat EU index), or [0295] ii) in the
constant domain CL of the second light chain under b) the amino
acid at position 124 is substituted independently by lysine (K),
arginine (R) or histidine (H) (numbering according to Kabat) (in
one preferred embodiment independently by lysine (K) or arginine
(R)), and wherein in the constant domain CH1 of the second heavy
chain under b) the amino acid at position 147 or the amino acid at
position 213 is substituted independently by glutamic acid (E) or
aspartic acid (D) (numbering according to Kabat EU index).
[0296] In one embodiment in the constant domain CL of the second
heavy chain the amino acids at position 124 and 123 are substituted
by K (numbering according to Kabat EU index).
[0297] In one embodiment in the constant domain CH1 of the second
light chain the amino acids at position 147 and 213 are substituted
by E (numbering according to EU index of Kabat).
[0298] In one preferred embodiment in the constant domain CL of the
first light chain the amino acids at position 124 and 123 are
substituted by K, and in the constant domain CH1 of the first heavy
chain the amino acids at position 147 and 213 are substituted by E
(numbering according to Kabat EU index).
[0299] In one embodiment in the constant domain CL of the second
heavy chain the amino acids at position 124 and 123 are substituted
by K, and wherein in the constant domain CH1 of the second light
chain the amino acids at position 147 and 213 are substituted by E,
and in the variable domain VL of the first light chain the amino
acid at position 38 is substituted by K, in the variable domain VH
of the first heavy chain the amino acid at position 39 is
substituted by E, in the variable domain VL of the second heavy
chain the amino acid at position 38 is substituted by K, and in the
variable domain VH of the second light chain the amino acid at
position 39 is substituted by E (numbering according to Kabat EU
index).
[0300] One aspect as reported herein is a bivalent, bispecific
antibody comprising
[0301] 1a) a first light chain and a first heavy chain of an
antibody specifically binding to a first antigen, and [0302] b) a
second light chain and a second heavy chain of an antibody
specifically binding to a second antigen, wherein the variable
domains VL and VH of the second light chain and the second heavy
chain are replaced by each other, and wherein the constant domains
CL and CH1 of the second light chain and the second heavy chain are
replaced by each other, [0303] wherein the first antigen or the
second antigen is human transferrin receptor.
[0304] The antibody under a) does not contain a modification as
reported under b) and the heavy chain and the light chain and a)
are isolated chains.
[0305] In the antibody under b) within the light chain the variable
light chain domain VL is replaced by the variable heavy chain
domain VH of said antibody, and the constant light chain domain CL
is replaced by the constant heavy chain domain CH1 of said
antibody; and [0306] within the heavy chain, the variable heavy
chain domain VH is replaced by the variable light chain domain VL
of said antibody, and the constant heavy chain domain CH1 is
replaced by the constant light chain domain CL of said
antibody.
[0307] One aspect as reported herein is a bivalent, bispecific
antibody comprising [0308] a) a first light chain and a first heavy
chain of an antibody specifically binding to a first antigen, and
[0309] b) a second light chain and a second heavy chain of an
antibody specifically binding to a second antigen, wherein the
constant domains CL and CH1 of the second light chain and the
second heavy chain are replaced by each other,
[0310] wherein the first antigen or the second antigen is human
transferrin receptor.
[0311] The antibody under a) does not contain a modification as
reported under b) and the heavy chain and the light chain under a)
are isolated chains.
[0312] In the antibody under b) [0313] within the light chain the
constant light chain domain CL is replaced by the constant heavy
chain domain CH1 of said antibody; [0314] and within the heavy
chain, the constant heavy chain domain CH1 is replaced by the
constant light chain domain CL of said antibody.
[0315] One aspect as reported herein is a multispecific antibody
comprising [0316] a) a full length antibody specifically binding to
a first antigen and consisting of two antibody heavy chains and two
antibody light chains, and [0317] b) one, two, three or four single
chain Fab fragments specifically binding to one to four further
antigens (i.e. a second and/or third and/or fourth and/or fifth
antigen, preferably specifically binding to one further antigen,
i.e. a second antigen), [0318] wherein said single chain Fab
fragments under b) are fused to said full length antibody under a)
via a peptidic linker at the C- or N-terminus of the heavy or light
chain of said full length antibody, [0319] wherein the first
antigen or one of the further antigens is human transferrin
receptor.
[0320] In one embodiment one or two identical single chain Fab
fragments binding to a second antigen are fused to said full length
antibody via a peptidic linker at the C-terminus of the heavy or
light chains of said full length antibody.
[0321] In one embodiment one or two identical single chain Fab
fragments binding to a second antigen are fused to said full length
antibody via a peptidic linker at the C-terminus of the heavy
chains of said full length antibody.
[0322] In one embodiment one or two identical single chain Fab
fragments binding to a second antigen are fused to said full length
antibody via a peptidic linker at the C-terminus of the light
chains of said full length antibody.
[0323] In one embodiment two identical single chain Fab fragments
binding to a second antigen are fused to said full length antibody
via a peptidic linker at the C-terminus of each heavy or light
chain of said full length antibody.
[0324] In one embodiment two identical single chain Fab fragments
binding to a second antigen are fused to said full length antibody
via a peptidic linker at the C-terminus of each heavy chain of said
full length antibody.
[0325] In one embodiment two identical single chain Fab fragments
binding to a second antigen are fused to said full length antibody
via a peptidic linker at the C-terminus of each light chain of said
full length antibody.
[0326] One aspect as reported herein is a trivalent, bispecific
antibody comprising [0327] a) a full length antibody specifically
binding to a first antigen and consisting of two antibody heavy
chains and two antibody light chains, [0328] b) a first polypeptide
consisting of [0329] ba) an antibody heavy chain variable domain
(VH), or [0330] bb) an antibody heavy chain variable domain (VH)
and an antibody constant domain 1 (CH1), [0331] wherein said first
polypeptide is fused with the N-terminus of its VH domain via a
peptidic linker to the C-terminus of one of the two heavy chains of
said full length antibody, [0332] c) a second polypeptide
consisting of [0333] ca) an antibody light chain variable domain
(VL), [0334] or [0335] cb) an antibody light chain variable domain
(VL) and an antibody light chain constant domain (CL), [0336]
wherein said second polypeptide is fused with the N-terminus of the
VL domain via a peptidic linker to the C-terminus of the other of
the two heavy chains of said full length antibody, [0337] and
[0338] wherein the antibody heavy chain variable domain (VH) of the
first polypeptide and the antibody light chain variable domain (VL)
of the second polypeptide together form an antigen-binding site
specifically binding to a second antigen, [0339] and [0340] wherein
the first antigen or the second antigen is human transferrin
receptor.
[0341] In one embodiment the antibody heavy chain variable domain
(VH) of the polypeptide under b) and the antibody light chain
variable domain (VL) of the polypeptide under c) are linked and
stabilized via an interchain disulfide bridge by introduction of a
disulfide bond between the following positions: [0342] i) heavy
chain variable domain position 44 to light chain variable domain
position 100, or [0343] ii) heavy chain variable domain position
105 to light chain variable domain position 43, or [0344] iii)
heavy chain variable domain position 101 to light chain variable
domain position 100 (numbering always according to Kabat EU
index).
[0345] Techniques to introduce unnatural disulfide bridges for
stabilization are described e.g. in WO 94/029350, Rajagopal, V., et
al., Prot. Eng. (1997) 1453-59; Kobayashi, H., et al., Nuclear
Medicine & Biology, Vol. 25, (1998) 387-393; or Schmidt, M., et
al., Oncogene (1999) 18 1711-1721. In one embodiment the optional
disulfide bond between the variable domains of the polypeptides
under b) and c) is between heavy chain variable domain position 44
and light chain variable domain position 100. In one embodiment the
optional disulfide bond between the variable domains of the
polypeptides under b) and c) is between heavy chain variable domain
position 105 and light chain variable domain position 43 (numbering
always according to EU index of Kabat). In one embodiment a
trivalent, bispecific antibody without said optional disulfide
stabilization between the variable domains VH and VL of the single
chain Fab fragments is preferred.
[0346] One aspect as reported herein is a tri specific or
tetraspecific antibody, comprising [0347] a) a first light chain
and a first heavy chain of a full length antibody which
specifically binds to a first antigen, and [0348] b) a second
(modified) light chain and a second (modified) heavy chain of a
full length antibody which specifically binds to a second antigen,
wherein the variable domains VL and VH are replaced by each other,
and/or wherein the constant domains CL and CH1 are replaced by each
other, and [0349] c) wherein one to four antigen binding peptides
which specifically bind to one or two further antigens (i.e. to a
third and/or fourth antigen) are fused via a peptidic linker to the
C- or N-terminus of the light chains or heavy chains of a) and/or
b), [0350] wherein the first antigen or the second antigen or one
of the further antigens is human transferrin receptor.
[0351] The antibody under a) does not contain a modification as
reported under b) and the heavy chain and the light chain and a)
are isolated chains.
[0352] In one embodiment the trispecific or tetraspecific antibody
comprises under c) one or two antigen binding peptides which
specifically bind to one or two further antigens.
[0353] In one embodiment the antigen binding peptides are selected
from the group of a scFv fragment and a scFab fragment.
[0354] In one embodiment the antigen binding peptides are scFv
fragments.
[0355] In one embodiment the antigen binding peptides are scFab
fragments.
[0356] In one embodiment the antigen binding peptides are fused to
the C-terminus of the heavy chains of a) and/or b).
[0357] In one embodiment the trispecific or tetraspecific antibody
comprises under c) one or two antigen binding peptides which
specifically bind to one further antigen.
[0358] In one embodiment the trispecific or tetraspecific antibody
comprises under c) two identical antigen binding peptides which
specifically bind to a third antigen. In one preferred embodiment
such two identical antigen binding peptides are fused both via the
same peptidic linker to the C-terminus of the heavy chains of a)
and b). In one preferred embodiment the two identical antigen
binding peptides are either a scFv fragment or a scFab
fragment.
[0359] In one embodiment the trispecific or tetraspecific antibody
comprises under c) two antigen binding peptides which specifically
bind to a third and a fourth antigen. In one embodiment said two
antigen binding peptides are fused both via the same peptide
connector to the C-terminus of the heavy chains of a) and b). In
one preferred embodiment said two antigen binding peptides are
either a scFv fragment or a scFab fragment.
[0360] One aspect as reported herein is a bispecific, tetravalent
antibody comprising [0361] a) two light chains and two heavy chains
of an antibody, which specifically bind to a first antigen (and
comprise two Fab fragments), [0362] b) two additional Fab fragments
of an antibody, which specifically bind to a second antigen,
wherein said additional Fab fragments are fused both via a peptidic
linker either to the C- or N-termini of the heavy chains of a), and
[0363] wherein in the Fab fragments the following modifications
were performed [0364] i) in both Fab fragments of a), or in both
Fab fragments of b), the variable domains VL and VH are replaced by
each other, and/or the constant domains CL and CH1 are replaced by
each other, or [0365] ii) in both Fab fragments of a) the variable
domains VL and VH are replaced by each other, and the constant
domains CL and CH1 are replaced by each other, and [0366] in both
Fab fragments of b) the variable domains VL and VH are replaced by
each other, or the constant domains CL and CH1 are replaced by each
other, or [0367] iii) in both Fab fragments of a) the variable
domains VL and VH are replaced by each other, or the constant
domains CL and CH1 are replaced by each other, and [0368] in both
Fab fragments of b) the variable domains VL and VH are replaced by
each other, and the constant domains CL and CH1 are replaced by
each other, or [0369] v) in both Fab fragments of a) the variable
domains VL and VH are replaced by each other, and in both Fab
fragments of b) the constant domains CL and CH1 are replaced by
each other, or [0370] v) in both Fab fragments of a) the constant
domains CL and CH1 are replaced by each other, and in both Fab
fragments of b) the variable domains VL and VH are replaced by each
other, [0371] wherein the first antigen or the second antigen is
human transferrin receptor.
[0372] In one embodiment said additional Fab fragments are fused
both via a peptidic linker either to the C-termini of the heavy
chains of a), or to the N-termini of the heavy chains of a).
[0373] In one embodiment said additional Fab fragments are fused
both via a peptidic linker either to the C-termini of the heavy
chains of a).
[0374] In one embodiment said additional Fab fragments are fused
both via a peptide connector to the N-termini of the heavy chains
of a).
[0375] In one embodiment in the Fab fragments the following
modifications are performed: [0376] i) in both Fab fragments of a),
or in both Fab fragments of b), the variable domains VL and VH are
replaced by each other, and/or the constant domains CL and CH1 are
replaced by each other.
[0377] In one embodiment in the Fab fragments the following
modifications are performed: [0378] i) in both Fab fragments of a)
the variable domains VL and VH are replaced by each other, and/or
[0379] the constant domains CL and CH1 are replaced by each
other.
[0380] In one embodiment in the Fab fragments the following
modifications are performed: [0381] i) in both Fab fragments of a)
the constant domains CL and CH1 are replaced by each other.
[0382] In one embodiment in the Fab fragments the following
modifications are performed: [0383] i) in both Fab fragments of b)
the variable domains VL and VH are replaced by each other, and/or
[0384] the constant domains CL and CH1 are replaced by each
other.
[0385] In one embodiment in the Fab fragments the following
modifications are performed: [0386] i) in both Fab fragments of b)
the constant domains CL and CH1 are replaced by each other.
[0387] One aspect as reported herein is a bispecific, tetravalent
antibody comprising: [0388] a) a (modified) heavy chain of a first
antibody, which specifically binds to a first antigen and comprises
a first VH-CH1 domain pair, wherein to the C-terminus of said heavy
chain the N-terminus of a second VH-CH1 domain pair of said first
antibody is fused via a peptidic linker, [0389] b) two light chains
of said first antibody of a), [0390] c) a (modified) heavy chain of
a second antibody, which specifically binds to a second antigen and
comprises a first VH-CL domain pair, wherein to the C-terminus of
said heavy chain the N-terminus of a second VH-CL domain pair of
said second antibody is fused via a peptidic linker, and [0391] d)
two (modified) light chains of said second antibody of c), each
comprising a CL-CH1 domain pair, [0392] wherein the first antigen
or the second antigen is human transferrin receptor.
[0393] One aspect as reported herein is a bispecific antibody
comprising [0394] a) the heavy chain and the light chain of a first
full length antibody that specifically binds to a first antigen,
and [0395] b) the heavy chain and the light chain of a second full
length antibody that specifically binds to a second antigen,
wherein the N-terminus of the heavy chain is connected to the
C-terminus of the light chain via a peptidic linker, [0396] wherein
the first antigen or the second antigen is human transferrin
receptor.
[0397] The antibody under a) does not contain a modification as
reported under b) and the heavy chain and the light chain are
isolated chains.
[0398] One aspect as reported herein is a bispecific antibody
comprising [0399] a) a full length antibody specifically binding to
a first antigen and consisting of two antibody heavy chains and two
antibody light chains, and [0400] b) an Fv fragment specifically
binding to a second antigen comprising a VH.sup.2 domain and a
VL.sup.2 domain, wherein both domains are connected to each other
via a disulfide bridge, [0401] wherein only either the VH.sup.2
domain or the VL.sup.2 domain is fused via a peptidic linker to the
heavy or light chain of the full length antibody specifically
binding to a first antigen, [0402] wherein the first antigen or the
second antigen is human transferrin receptor.
[0403] In the bispecific the heavy chains and the light chains
under a) are isolated chains.
[0404] In one embodiment the other of the VH.sup.2 domain or the
VL.sup.2 domain is not fused via a peptide linker to the heavy or
light chain of the full length antibody specifically binding to a
first antigen.
[0405] In all aspects as reported herein the first light chain
comprises a VL domain and a CL domain and the first heavy chain
comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain
and a CH3 domain.
[0406] In one embodiment of all aspects the antibody as reported
herein is a multispecific antibody, which requires
heterodimerization of at least two heavy chain polypeptides, and
wherein the antibody specifically binds to human transferrin
receptor and a second non-human transferrin receptor antigen.
[0407] Several approaches for CH3-modifications in order to support
heterodimerization have been described, for example 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, WO 2013/096291, which are herein
included by reference. Typically, in the approaches known in the
art, the CH3 domain of the first heavy chain and the CH3 domain of
the second heavy chain are both engineered in a complementary
manner so that the heavy chain comprising one engineered CH3 domain
can no longer homodimerize with another heavy chain of the same
structure (e.g. a CH3-engineered first heavy chain can no longer
homodimerize with another CH3-engineered first heavy chain; and a
CH3-engineered second heavy chain can no longer homodimerize with
another CH3-engineered second heavy chain). Thereby the heavy chain
comprising one engineered CH3 domain is forced to heterodimerize
with another heavy chain comprising the CH3 domain, which is
engineered in a complementary manner. For this embodiment of the
invention, the CH3 domain of the first heavy chain and the CH3
domain of the second heavy chain are engineered in a complementary
manner by amino acid substitutions, such that the first heavy chain
and the second heavy chain are forced to heterodimerize, whereas
the first heavy chain and the second heavy chain can no longer
homodimerize (e.g. for steric reasons).
[0408] The different approaches for supporting heavy chain
heterodimerization known in the art, that were cited and included
above, are contemplated as different alternatives used in a
multispecific antibody according to the invention, which comprises
a "non-crossed Fab region" derived from a first antibody, which
specifically binds to a first antigen, and a "crossed Fab region"
derived from a second antibody, which specifically binds to a
second antigen, in combination with the particular amino acid
substitutions described above for the invention.
[0409] The CH3 domains of the multispecific antibody as reported
herein can be 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 heterodimerization 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 further 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.
[0410] In one preferred embodiment the multispecific antibody as
reported herein 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" (numbering according to Kabat EU index). 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 Y349C mutation into the CH3 domain
of the "knobs chain" and a E356C mutation or a S354C mutation into
the CH3 domain of the "hole chain". Thus in a another preferred
embodiment, the multispecific antibody as reported herein comprises
the Y349C and T366W mutations in one of the two CH3 domains and the
E356C, T366S, L368A and Y407V mutations in the other of the two CH3
domains or the multispecific antibody as reported herein comprises
the Y349C and T366W mutations in one of the two CH3 domains and the
S354C, T366S, L368A and Y407V mutations in the other of the two CH3
domains (the additional Y349C mutation in one CH3 domain and the
additional E356C or S354C mutation in the other CH3 domain forming
a interchain disulfide bridge) (numbering according to Kabat EU
index).
[0411] But also other knobs-in-holes technologies as described by
EP 1 870 459A1, can be used alternatively or additionally. In one
embodiment the multispecific antibody as reported herein comprises
the R409D and K370E mutations in the CH3 domain of the "knobs
chain" and the D399K and E357K mutations in the CH3 domain of the
"hole-chain" (numbering according to Kabat EU index).
[0412] In one embodiment the multispecific antibody as reported
herein comprises a T366W mutation in the CH3 domain of the "knobs
chain" and the T366S, L368A and Y407V mutations in the CH3 domain
of the "hole chain" and additionally the R409D and K370E mutations
in the CH3 domain of the "knobs chain" and the D399K and E357K
mutations in the CH3 domain of the "hole chain" (numbering
according to the Kabat EU index).
[0413] In one embodiment the multispecific antibody as reported
herein comprises the Y349C and T366W mutations in one of the two
CH3 domains and the S354C, T366S, L368A and Y407V mutations in the
other of the two CH3 domains, or the multispecific antibody as
reported herein comprises the Y349C and T366W mutations in one of
the two CH3 domains and the S354C, T366S, L368A and Y407V mutations
in the other of the two CH3 domains and additionally the R409D and
K370E mutations in the CH3 domain of the "knobs chain" and the
D399K and E357K mutations in the CH3 domain of the "hole chain"
(numbering according to the Kabat EU index).
[0414] Apart from the "knob-into-hole technology" other techniques
for modifying the CH3 domains of the heavy chains of a
multispecific antibody to enforce heterodimerization are known in
the art. These technologies, especially the ones described 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 are contemplated
herein as alternatives to the "knob-into-hole technology" in
combination with a multispecific antibody as reported herein.
[0415] In one embodiment of a multispecific antibody as reported
herein the approach described in EP 1870459 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. This approach is based on the
introduction of charged amino acids with opposite charges at
specific amino acid positions in the CH3/CH3-domain-interface
between both, the first and the second heavy chain.
[0416] Accordingly, this embodiment relates to a multispecific
antibody as reported herein, wherein in the tertiary structure of
the antibody the CH3 domain of the first heavy chain and the CH3
domain of the second heavy chain form an interface that is located
between the respective antibody CH3 domains, wherein the respective
amino acid sequences of the CH3 domain of the first heavy chain and
the CH3 domain of the second heavy chain each comprise a set of
amino acids that is located within said interface in the tertiary
structure of the antibody, wherein from the set of amino acids that
is located in the interface in the CH3 domain of one heavy chain a
first amino acid is substituted by a positively charged amino acid
and from the set of amino acids that is located in the interface in
the CH3 domain of the other heavy chain a second amino acid is
substituted by a negatively charged amino acid. The multispecific
antibody according to this embodiment is herein also referred to as
"CH3(+/-)-engineered multispecific antibody" (wherein the
abbreviation "+/-" stands for the oppositely charged amino acids
that were introduced in the respective CH3 domains).
[0417] In one embodiment of said CH3(+/-)-engineered multispecific
antibody as reported herein the positively charged amino acid is
selected from K, R and H, and the negatively charged amino acid is
selected from E or D.
[0418] In one embodiment of said CH3(+/-)-engineered multispecific
antibody as reported herein the positively charged amino acid is
selected from K and R, and the negatively charged amino acid is
selected from E or D.
[0419] In one embodiment of said CH3(+/-)-engineered multispecific
antibody as reported herein the positively charged amino acid is K,
and the negatively charged amino acid is E.
[0420] In one embodiment of said CH3(+/-)-engineered multispecific
antibody as reported herein in the CH3 domain of one heavy chain
the amino acid R at position 409 is substituted by D and the amino
acid K at position is substituted by E, and in the CH3 domain of
the other heavy chain the amino acid D at position 399 is
substituted by K and the amino acid E at position 357 is
substituted by K (numbering according to Kabat EU index).
[0421] In one embodiment of a multispecific antibody as reported
herein the approach described in WO2013/157953 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. In one embodiment of said
multispecific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid T at position 366 is substituted by K,
and in the CH3 domain of the other heavy chain the amino acid L at
position 351 is substituted by D (numbering according to Kabat EU
index). In another embodiment of said multispecific antibody as
reported herein, in the CH3 domain of one heavy chain the amino
acid T at position 366 is substituted by K and the amino acid L at
position 351 is substituted by K, and in the CH3 domain of the
other heavy chain the amino acid L at position 351 is substituted
by D (numbering according to Kabat EU index).
[0422] In another embodiment of said multispecific antibody as
reported herein, in the CH3 domain of one heavy chain the amino
acid T at position 366 is substituted by K and the amino acid L at
position 351 is substituted by K, and in the CH3 domain of the
other heavy chain the amino acid L at position 351 is substituted
by D (numbering according to Kabat EU index). Additionally at least
one of the following substitutions is comprised in the CH3 domain
of the other heavy chain: the amino acid Y at position 349 is
substituted by E, the amino acid Y at position 349 is substituted
by D and the amino acid L at position 368 is substituted by E
(numbering according to Kabat EU index). In one embodiment the
amino acid L at position 368 is substituted by E (numbering
according to Kabat EU index).
[0423] In one embodiment of a multispecific antibody as reported
herein the approach described in WO2012/058768 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. In one embodiment of said
multispecific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid L at position 351 is substituted by Y
and the amino acid Y at position 407 is substituted by A, and in
the CH3 domain of the other heavy chain the amino acid T at
position 366 is substituted by A and the amino acid K at position
409 is substituted by F (numbering according to Kabat EU index). In
another embodiment, in addition to the aforementioned
substitutions, in the CH3 domain of the other heavy chain at least
one of the amino acids at positions 411 (originally T), 399
(originally D), 400 (originally S), 405 (originally F), 390
(originally N) and 392 (originally K) is substituted (numbering
according to Kabat EU index). Preferred substitutions are: [0424]
substituting the amino acid T at position 411 by an amino acid
selected from N, R, Q, K, D, E and W (numbering according to Kabat
EU index), [0425] substituting the amino acid D at position 399 by
an amino acid selected from R, W, Y, and K (numbering according to
Kabat EU index), [0426] substituting the amino acid S at position
400 by an amino acid selected from E, D, R and K (numbering
according to Kabat EU index), [0427] substituting the amino acid F
at position 405 by an amino acid selected from I, M, T, S, V and W
(numbering according to Kabat EU index; [0428] substituting the
amino acid N at position 390 by an amino acid selected from R, K
and D (numbering according to Kabat EU index; and [0429]
substituting the amino acid K at position 392 by an amino acid
selected from V, M, R, L, F and E (numbering according to Kabat EU
index).
[0430] In another embodiment of said multispecific antibody as
reported herein (engineered according to WO2012/058768), in the CH3
domain of one heavy chain the amino acid L at position 351 is
substituted by Y and the amino acid Y at position 407 is
substituted by A, and in the CH3 domain of the other heavy chain
the amino acid T at position 366 is substituted by V and the amino
acid K at position 409 is substituted by F (numbering according to
Kabat EU index). In another embodiment of said multispecific
antibody as reported herein, in the CH3 domain of one heavy chain
the amino acid Y at position 407 is substituted by A, and in the
CH3 domain of the other heavy chain the amino acid T at position
366 is substituted by A and the amino acid K at position 409 is
substituted by F (numbering according to Kabat EU index). In said
last aforementioned embodiment, in the CH3 domain of said other
heavy chain the amino acid K at position 392 is substituted by E,
the amino acid T at position 411 is substituted by E, the amino
acid D at position 399 is substituted by R and the amino acid S at
position 400 is substituted by R (numbering according to Kabat EU
index).
[0431] In one embodiment of a multispecific antibody as reported
herein the approach described in WO 2011/143545 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. In one embodiment of said
multispecific antibody as reported herein, amino acid modifications
in the CH3 domains of both heavy chains are introduced at positions
368 and/or 409 (numbering according to Kabat EU index).
[0432] In one embodiment of a multispecific antibody as reported
herein the approach described in WO 2011/090762 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. WO 2011/090762 relates to
amino acid modifications according to the "knob-into-hole"
technology. In one embodiment of said CH3(KiH)-engineered
multispecific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid T at position 366 is substituted by W,
and in the CH3 domain of the other heavy chain the amino acid Y at
position 407 is substituted by A (numbering according to Kabat EU
index). In another embodiment of said CH3(KiH)-engineered multi
specific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid T at position 366 is substituted by Y,
and in the CH3 domain of the other heavy chain the amino acid Y at
position 407 is substituted by T (numbering according to Kabat EU
index).
[0433] In one embodiment of a multispecific antibody as reported
herein, which is of IgG2 isotype, the approach described in WO
2011/090762 is used to support heterodimerization of the first
heavy chain and the second heavy chain of the multispecific
antibody.
[0434] In one embodiment of a multispecific antibody as reported
herein, the approach described in WO 2009/089004 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. In one embodiment of said
multispecific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid K or N at position 392 is substituted by
a negatively charged amino acid (in one preferred embodiment by E
or D, in one preferred embodiment by D), and in the CH3 domain of
the other heavy chain the amino acid D at position 399 the amino
acid E or D at position 356 or the amino acid E at position 357 is
substituted by a positively charged amino acid (in one preferred
embodiment K or R, in one preferred embodiment by K, in one
preferred embodiment the amino acids at positions 399 or 356 are
substituted by K) (numbering according to Kabat EU index). In one
further embodiment, in addition to the aforementioned
substitutions, in the CH3 domain of the one heavy chain the amino
acid K or R at position 409 is substituted by a negatively charged
amino acid (in one preferred embodiment by E or D, in one preferred
embodiment by D) (numbering according to Kabat EU index). In one
even further embodiment, in addition to or alternatively to the
aforementioned substitutions, in the CH3 domain of the one heavy
chain the amino acid K at position 439 and/or the amino acid K at
position 370 is substituted independently from each other by a
negatively charged amino acid (in one preferred embodiment by E or
D, in one preferred embodiment by D) (numbering according to Kabat
EU index).
[0435] In one embodiment of a multispecific antibody as reported
herein, the approach described in WO 2007/147901 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody. In one embodiment of said
multispecific antibody as reported herein, in the CH3 domain of one
heavy chain the amino acid K at position 253 is substituted by E,
the amino acid D at position 282 is substituted by K and the amino
acid K at position 322 is substituted by D, and in the CH3 domain
of the other heavy chain the amino acid D at position 239 is
substituted by K, the amino acid E at position 240 is substituted
by K and the amino acid K at position 292 is substituted by D
(numbering according to Kabat EU index).
[0436] In one embodiment of a multispecific antibody as reported
herein, the approach described in WO 2007/110205 is used to support
heterodimerization of the first heavy chain and the second heavy
chain of the multispecific antibody
[0437] In one embodiment of all aspects and embodiments as reported
herein the multispecific antibody is a bispecific antibody or a
trispecific antibody. In one preferred embodiment of the invention
the multispecific antibody is a bispecific antibody.
[0438] In one embodiment of all aspects as reported herein, the
antibody is a bivalent or trivalent antibody. In one embodiment the
antibody is a bivalent antibody.
[0439] In one embodiment of all aspects as reported herein, the
multispecific antibody has a constant domain structure of an IgG
type antibody. In one further embodiment of all aspects as reported
herein, the multispecific antibody is characterized in that said
multispecific antibody is of human subclass IgG1, or of human
subclass IgG1 with the mutations L234A and L235A. In one further
embodiment of all aspects as reported herein, the multispecific
antibody is characterized in that said multispecific antibody is of
human subclass IgG2. In one further embodiment of all aspects as
reported herein, the multispecific antibody is characterized in
that said multispecific antibody is of human subclass IgG3. In one
further embodiment of all aspects as reported herein, the
multispecific antibody is characterized in that said multispecific
antibody is of human subclass IgG4 or, of human subclass IgG4 with
the additional mutation S228P. In one further embodiment of all
aspects as reported herein, the multispecific antibody is
characterized in that said multispecific antibody is of human
subclass IgG1 or human subclass IgG4. In one further embodiment of
all aspects as reported herein, the multispecific antibody is
characterized in that said multispecific antibody is of human
subclass IgG1 with the mutations L234A and L235A (numbering
according to Kabat EU index). In one further embodiment of all
aspects as reported herein, the multispecific antibody is
characterized in that said multispecific antibody is of human
subclass IgG1 with the mutations L234A, L235A and P329G (numbering
according to Kabat EU index). In one further embodiment of all
aspects as reported herein, the multispecific antibody is
characterized in that said multispecific antibody is of human
subclass IgG4 with the mutations S228P and L235E (numbering
according to Kabat EU index). In one further embodiment of all
aspects as reported herein, the multispecific antibody is
characterized in that said multispecific antibody is of human
subclass IgG4 with the mutations S228P, L235E and P329G (numbering
according to Kabat EU index).
[0440] In one embodiment of all aspects as reported herein, an
antibody comprising a heavy chain including a CH3 domain as
specified herein, comprises an additional C-terminal glycine-lysine
dipeptide (G446 and K447, numbering according to Kabat EU index).
In one embodiment of all aspects as reported herein, an antibody
comprising a heavy chain including a CH3 domain, as specified
herein, comprises an additional C-terminal glycine residue (G446,
numbering according to Kabat EU index).
[0441] 6. Antibody Variants
[0442] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0443] a) Substitution, Insertion and Deletion Variants
[0444] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs.
[0445] Conservative substitutions are shown in Table 1 under the
heading of "conservative substitutions". More substantial changes
are provided in Table 1 under the heading of "exemplary
substitutions," and as further described below in reference to
amino acid side chain classes. Amino acid substitutions may be
introduced into an antibody of interest and the products screened
for a desired activity, e.g., retained/improved antigen binding,
decreased immunogenicity, or improved ADCC or CDC.
TABLE-US-00006 TABLE 1 Original Exemplary Conservative Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine; Ile; Val; Met;
Ala; Ile Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine
[0446] Amino acids may be grouped according to common side-chain
properties: [0447] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu,
Ile; [0448] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0449] (3) acidic: Asp, Glu; [0450] (4) basic: His, Lys, Arg;
[0451] (5) residues that influence chain orientation: Gly, Pro;
[0452] (6) aromatic: Trp, Tyr, Phe.
[0453] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0454] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0455] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, P. S., Methods Mol. Biol. 207 (2008) 179-196), and/or
residues that contact antigen, with the resulting variant VH or VL
being tested for binding affinity. Affinity maturation by
constructing and reselecting from secondary libraries has been
described, e.g., in Hoogenboom, H. R. et al. in Methods in
Molecular Biology 178 (2002) 1-37. In some embodiments of affinity
maturation, diversity is introduced into the variable genes chosen
for maturation by any of a variety of methods (e.g., error-prone
PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A
secondary library is then created. The library is then screened to
identify any antibody variants with the desired affinity. Another
method to introduce diversity involves HVR-directed approaches, in
which several HVR residues (e.g., 4-6 residues at a time) are
randomized. HVR residues involved in antigen binding may be
specifically identified, e.g., using alanine scanning mutagenesis
or modeling. CDR-H3 and CDR-L3 in particular are often
targeted.
[0456] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0457] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham, B. C. and Wells,
J. A., Science 244 (1989) 1081-1085. In this method, a residue or
group of target residues (e.g., charged residues such as Arg, Asp,
His, Lys, and Glu) are identified and replaced by a neutral or
negatively charged amino acid (e.g., alanine or polyalanine) to
determine whether the interaction of the antibody with antigen is
affected. Further substitutions may be introduced at the amino acid
locations demonstrating functional sensitivity to the initial
substitutions. Alternatively, or additionally, a crystal structure
of an antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0458] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0459] b) Glycosylation Variants
[0460] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0461] Where the antibody comprises an Fc-region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc-region (see, e.g., Wright, A.
and Morrison, S. L., TIBTECH 15 (1997) 26-32). The oligosaccharide
may include various carbohydrates, e.g., mannose, N-acetyl
glucosamine (GlcNAc), galactose, and sialic acid, as well as a
fucose attached to a GlcNAc in the "stem" of the biantennary
oligosaccharide structure. In some embodiments, modifications of
the oligosaccharide in an antibody of the invention may be made in
order to create antibody variants with certain improved
properties.
[0462] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc-region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc-region (EU numbering of Fc-region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US 2003/0157108; US 2004/0093621. Examples of publications related
to "defucosylated" or "fucose-deficient" antibody variants include:
US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140;
Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249;
Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622.
Examples of cell lines capable of producing defucosylated
antibodies include Lec13 CHO cells deficient in protein
fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986)
533-545; US 2003/0157108; and WO 2004/056312, especially at Example
11), and knockout cell lines, such as alpha-1,6-fucosyltransferase
gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki, N. et
al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al.,
Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).
[0463] Antibody variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc-region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878; US 6,602,684; and US
2005/0123546. Antibody variants with at least one galactose residue
in the oligosaccharide attached to the Fc-region are also provided.
Such antibody variants may have improved CDC function. Such
antibody variants are described, e.g., in WO 1997/30087; WO
1998/58964; and WO 1999/22764.
[0464] c) Fc-region Variants
[0465] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc-region of an antibody provided
herein, thereby generating an Fc-region variant. The Fc-region
variant may comprise a human Fc-region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0466] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half-life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of
Ravetch, J. V. and Kinet, J. P., Annu. Rev. Immunol. 9 (1991)
457-492. Non-limiting examples of in vitro assays to assess ADCC
activity of a molecule of interest is described in U.S. Pat. No.
5,500,362 (see, e.g. Hellstrom, I. et al., Proc. Natl. Acad. Sci.
USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl.
Acad. Sci. USA 82 (1985) 1499-1502); U.S. Pat. No. 5,821,337 (see
Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95
(1998) 652-656. C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro, H. et al., J.
Immunol. Methods 202 (1996) 163-171; Cragg, M. S. et al., Blood 101
(2003) 1045-1052; and Cragg, M. S. and M. J. Glennie, Blood 103
(2004) 2738-2743). FcRn binding and in vivo clearance/half-life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int. Immunol. 18 (2006)
1759-1769).
[0467] Antibodies with reduced effector function include those with
substitution of one or more of Fc-region residues 238, 265, 269,
270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0468] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276
(2001) 6591-6604).
[0469] In certain embodiments, an antibody variant comprises an
Fc-region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc-region (EU numbering of residues).
[0470] In some embodiments, alterations are made in the Fc-region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie, E.
E. et al., J. Immunol. 164 (2000) 4178-4184.
[0471] Antibodies with increased half-lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer, R. L. et al., J.
Immunol. 117 (1976) 587-593, and Kim, J. K. et al., J. Immunol. 24
(1994) 2429-2434), are described in US 2005/0014934. Those
antibodies comprise an Fc-region with one or more substitutions
therein which improve binding of the Fc-region to FcRn. Such Fc
variants include those with substitutions at one or more of
Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,
312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,
e.g., substitution of Fc-region residue 434 (U.S. Pat. No.
7,371,826).
[0472] See also Duncan, A. R. and Winter, G., Nature 322 (1988)
738-740; U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351
concerning other examples of Fc-region variants.
[0473] d) Cysteine Engineered Antibody Variants
[0474] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc-region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0475] e) Antibody Derivatives
[0476] In certain embodiments, an antibody provided herein may be
further modified to contain additional non-proteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or non-branched. The
number of polymers attached to the antibody may vary, and if more
than one polymer is attached, they can be the same or different
molecules. In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0477] In another embodiment, conjugates of an antibody and
non-proteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the non-proteinaceous
moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad.
Sci. USA 102 (2005) 11600-11605). The radiation may be of any
wavelength, and includes, but is not limited to, wavelengths that
do not harm ordinary cells, but which heat the non-proteinaceous
moiety to a temperature at which cells proximal to the
antibody-non-proteinaceous moiety are killed.
[0478] B. Recombinant Methods and Compositions
[0479] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-transferrin
receptor antibody described herein is provided. Such nucleic acid
may encode an amino acid sequence comprising the VL and/or an amino
acid sequence comprising the VH of the antibody (e.g., the light
and/or heavy chains of the antibody). In a further embodiment, one
or more vectors (e.g., expression vectors) comprising such nucleic
acid are provided. In a further embodiment, a host cell comprising
such nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an
anti-transferrin receptor antibody is provided, wherein the method
comprises culturing a host cell comprising a nucleic acid encoding
the antibody, as provided above, under conditions suitable for
expression of the antibody, and optionally recovering the antibody
from the host cell (or host cell culture medium).
[0480] For recombinant production of an anti-transferrin receptor
antibody, nucleic acid encoding an antibody, e.g., as described
above, is isolated and inserted into one or more vectors for
further cloning and/or expression in a host cell. Such nucleic acid
may be readily isolated and sequenced using conventional procedures
(e.g., by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0481] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, K. A., In: Methods in
Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), Humana Press,
Totowa, N.J. (2003), pp. 245-254, describing expression of antibody
fragments in E. coli.) After expression, the antibody may be
isolated from the bacterial cell paste in a soluble fraction and
can be further purified.
[0482] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, T. U., Nat. Biotech. 22
(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006)
210-215.
[0483] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0484] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0485] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham, F. L.
et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells
(BHK); mouse sertoli cells (TM4 cells as described, e.g., in
Mather, J. P., Biol. Reprod. 23 (1980) 243-252); monkey kidney
cells (CV1); African green monkey kidney cells (VERO-76); human
cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo
rat liver cells (BRL 3A); human lung cells (W138); human liver
cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as
described, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci.
383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including DHFR.sup.- CHO cells (Urlaub, G. et al., Proc.
Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines
such as Y0, NS0 and Sp2/0. For a review of certain mammalian host
cell lines suitable for antibody production, see, e.g., Yazaki, P.
and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, B. K. C.
(ed.), Humana Press, Totowa, N.J. (2004), pp. 255-268.
[0486] C. Assays
[0487] Anti-transferrin receptor antibodies provided herein may be
identified, screened for, or characterized for their
physical/chemical properties and/or biological activities by
various assays known in the art.
[0488] 1. Binding assay
[0489] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
alphaLISA, Western blot, antibody or reverse phase array, etc.
[0490] In an exemplary ELISA or alphaLISA assay, transferrin
receptor in solution (cell supernatant, cell or tissue lysates,
body fluids etc.) is bound by a capture antibody, which
specifically binds to a first epitope on the transferrin receptor,
or transferrin receptor in a certain conformation and a detection
antibody coupled to a detection entity, which specifically binds to
a second epitope or conformation of the transferrin receptor. The
readout is based on the detection entity (chemiluminescence,
fluorescence, energy transfer induced luminescence etc.).
[0491] In the case of antibody array, antibodies are spotted onto
glass or nitrocellulose chips. The slides are blocked and incubated
with transferrin receptor containing solution, washed to remove
unbound antibodies and bound antibodies are detected with a
fluorescently labeled corresponding secondary antibody. The
fluorescence signal is measured by a fluorescence slide scanner.
Similarly, for a reverse phase array, recombinant transferrin
receptor, cell supernatant, cell or tissue lysates, body fluids
etc. are spotted onto glass or nitrocellulose chips. The slides are
blocked and individual arrays are incubated with an antibody
against a specific epitope on the transferrin receptor. Unbound
antibodies are washed off and bound antibodies are detected with a
fluorescently labeled corresponding secondary antibody. The
fluorescence signal is measured by a fluorescence slide scanner
(Dernick, G., et al., J. Lipid Res. 52 (2011) 2323-2331).
[0492] D. Methods and Compositions for Diagnostics and
Detection
[0493] In certain embodiments, any of the anti-transferrin receptor
antibodies provided herein is useful for detecting the presence of
human transferrin receptor in a biological sample. The term
"detecting" as used herein encompasses quantitative or qualitative
detection.
[0494] In certain embodiments, a biological sample comprises a cell
or tissue, such as brain tissue.
[0495] In one embodiment, an anti-transferrin receptor antibody for
use in a method of diagnosis or detection is provided. In a further
aspect, a method of detecting the presence of the transferrin
receptor in a biological sample is provided. In certain
embodiments, the method comprises contacting the biological sample
with an anti-transferrin receptor antibody as described herein
under conditions permissive for binding of the anti-transferrin
receptor antibody to the transferrin receptor, and detecting
whether a complex is formed between the anti-transferrin receptor
antibody and the transferrin receptor. Such method may be an in
vitro or in vivo method. In one embodiment, an anti-transferrin
receptor antibody is used to select subjects eligible for therapy
with an anti-transferrin receptor antibody, e.g. where the
transferrin receptor is a biomarker for selection of patients.
[0496] Exemplary disorders that may be diagnosed using an antibody
of the invention include neurodegeneration with brain iron
accumulation type 1 (NBIA1), pure autonomic failure, Down's
syndrome, complex of Guam, and several Lewy body disorders, such as
diffuse Lewy body disease (DLBD), the Lewy body variant of
Alzheimer's disease (LBVAD), certain forms of Gaucher' s disease,
and Parkinson's Disease dementia (PDD).
[0497] In certain embodiments, labeled anti-transferrin receptor
antibodies are provided. Labels include, but are not limited to,
labels or moieties that are detected directly (such as fluorescent,
chromophoric, electron-dense, chemiluminescent, and radioactive
labels), as well as moieties, such as enzymes or ligands, that are
detected indirectly, e.g., through an enzymatic reaction or
molecular interaction. Exemplary labels include, but are not
limited to, the radioisotopes .sup.32P, .sup.14C, .sup.125I,
.sup.3H, and .sup.131I, fluorophores such as rare earth chelates or
fluorescein and its derivatives, rhodamine and its derivatives,
dansyl, umbelliferone, luciferases, e.g., firefly luciferase and
bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),
alkaline phosphatase, .beta.-galactosidase, glucoamylase, lysozyme,
saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as
uricase and xanthine oxidase, coupled with an enzyme that employs
hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.
[0498] E. Pharmaceutical Formulations
[0499] Pharmaceutical formulations of an anti-transferrin receptor
antibody as described herein are prepared by mixing such antibody
having the desired degree of purity with one or more optional
pharmaceutically acceptable carriers (Remington's Pharmaceutical
Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of
lyophilized formulations or aqueous solutions. Pharmaceutically
acceptable carriers are generally nontoxic to recipients at the
dosages and concentrations employed, and include, but are not
limited to: buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyl dimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride; benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
poly(vinylpyrrolidone); amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as polyethylene glycol (PEG). Exemplary
pharmaceutically acceptable carriers herein further include
interstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rhuPH20, are described in US 2005/0260186 and US
2006/0104968. In one aspect, a sHASEGP is combined with one or more
additional glycosaminoglycanases such as chondroitinases.
[0500] Exemplary lyophilized antibody formulations are described in
U.S. Pat. No. 6,267,958. Aqueous antibody formulations include
those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the
latter formulations including a histidine-acetate buffer.
[0501] The formulation herein may also contain more than one active
ingredients as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such active ingredients are suitably
present in combination in amounts that are effective for the
purpose intended.
[0502] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methyl methacrylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nanoparticles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed.) (1980).
[0503] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules.
[0504] The formulations to be used for in vivo administration are
generally sterile. Sterility may be readily accomplished, e.g., by
filtration through sterile filtration membranes.
III. ARTICLES OF MANUFACTURE
[0505] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an antibody of the invention. The label
or package insert indicates that the composition is used for
treating the condition of choice. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic or otherwise therapeutic agent. The article of
manufacture in this embodiment of the invention may further
comprise a package insert indicating that the compositions can be
used to treat a particular condition. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline,
[0506] Ringer's solution and dextrose solution. It may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
and syringes.
[0507] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the invention in
place of or in addition to an anti-transferrin receptor
antibody.
VI. EXAMPLES
[0508] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
[0509] Materials and Methods
[0510] Recombinant DNA Techniques
[0511] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0512] Gene and Oligonucleotide Synthesis
[0513] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany). The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany).
[0514] Reagents
[0515] All commercial chemicals, antibodies and kits were used as
provided according to the manufacturer's protocol if not stated
otherwise.
Example 1
[0516] Immunization of Rabbits and Mice
[0517] Immunization of Mice
[0518] NMRI mice were immunized genetically, using a plasmid
expression vector coding for full-length human or cynomolgus TfR by
intradermal application of 100 .mu.g vector DNA, followed by
electroporation (2 square pulses of 1000 V/cm, duration 0.1 ms,
interval 0.125 s; followed by 4 square pulses of 287.5 V/cm,
duration 10 ms, interval 0.125 s. Mice received either 6 or 7
consecutive immunizations at days 0, 14, 28, 42, 56, 70, and 84.
The fourth and sixth immunizations were performed with vector
coding for cynomolgus TfR; vector coding for human TfR was used for
all other immunizations. Blood was taken at days 36, 78 and 92 and
serum prepared, which was used for titer determination by ELISA
(see below). Animals with highest titers were selected for boosting
at day 96, by intravenous injection of either 10.sup.6 human TF-1
cells or 50 .mu.g of recombinant human soluble TfR lacking the
helical domain (extracellular domain of the human TfR beginning at
Leu122, ending at Asn608, expressed in HEK293F cells as an
N-terminal fusion to human Fc-region and purified by protein A
affinity chromatography and size exclusion chromatography, and
monoclonal antibodies were isolated by hybridoma technology, based
on their ability to bind human and cynomolgus transferrin receptor
expressed on the surface of stably transfected CHO-K1 cells (see
Example 4).
[0519] Immunization of Rabbits
[0520] New Zealand White rabbits or transgenic rabbits expressing a
humanized antibody repertoire were immunized genetically, using a
plasmid expression vector coding for full-length human or
cynomolgus TfR, by intradermal application of 400 .mu.g vector DNA,
followed by electroporation (5 square pulses of 750 V/cm, duration
10 ms, interval 1 s.). Rabbits received 6 consecutive immunizations
at days 0, 14, 28, 56, 84 and 112. The fourth and sixth
immunizations were performed with vector coding for cynomolgus TfR;
vector coding for human TfR was used for all other immunizations.
Blood (10% of estimated total blood volume) was taken at days 35,
63, 91 and 119. Serum was prepared, which was used for titer
determination by ELISA (see below), and peripheral mononuclear
cells were isolated, which were used as a source of
antigen-specific B cells in the B cell cloning process (see Example
2).
[0521] Determination of Serum Titers (ELISA)
[0522] Human recombinant soluble TfR (R&D Systems Cat. No.
2474-TR) was immobilized on a 96-well NUNC Maxisorb plate at 3
.mu.g/mL, 100 .mu.L/well, in PBS, followed by: blocking of the
plate with 2% CroteinC in PBS, 200 .mu.L/well; application of
serial dilutions of antisera, in duplicates, in 0.5% CroteinC in
PBS, 100 .mu.L/well; detection with (1) HRP-conjugated goat
anti-mouse antibody (Jackson Immunoresearch/Dianova 115-036-071;
1/16000) for all mouse sera, (2) HRP-conjugated donkey anti-rabbit
IgG antibody (Jackson Immunoresearch/Dianova 711-036-152; 1/16000)
for all rabbit sera, (3) rabbit anti-human IgG antibody
(Pierce/Thermo Scientific 31423; 1/5000) for sera from transgenic
rabbits only, (4) biotinylated goat anti-human kappa antibody
(Southern Biotech/Biozol 2063-08, 1/5000) and streptavidin-HRP for
sera from transgenic rabbits only; diluted in 0.5% CroteinC in PBS,
100 .mu.L/well. For all steps, plates were incubated for 1 h at
37.degree. C. Between all steps, plates were washed 3 times with
0.05 Tween 20 in PBS. Signal was developed by addition of BM Blue
POD Substrate soluble (Roche), 100 .mu.L/well; and stopped by
addition of 1 M HCl, 100 .mu.L/well. Absorbance was read out at 450
nm, against 690 nm as reference. Titer was defined as dilution of
antisera resulting in half-maximal signal.
Example 2
B-Cell Cloning from Rabbits
[0523] Isolation of Rabbit Peripheral Blood Mononuclear Cells
(PBMC)
[0524] Blood samples were taken of in summary 6 animals (2
wild-type (wt) rabbits and 4 transgenic (tg) rabbits). These
rabbits derived from 2 different immunization campaigns: first
campaign with 2 wt and 2 tg rabbits and second campaign with 2 tg
rabbits (see also the example "Immunization of rabbits"). EDTA
containing whole blood was diluted twofold with 1.times. PBS (PAA,
Pasching, Austria) before density centrifugation using lympholyte
mammal (Cedarlane Laboratories, Burlington, Ontario, Canada)
according to the specifications of the manufacturer. The PBMCs were
washed twice with 1.times. PBS.
[0525] EL-4 B5 Medium
[0526] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented
with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM glutamine, 1%
penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM
sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and
0.05 mM .beta.-mercaptoethanol (Gibco, Paisley, Scotland)
[0527] Depletion of Cells
[0528] First immunization campaign: Sterile 6-well plates (cell
culture grade) covered with a confluent monolayer of CHO cells were
used to deplete macrophages/monocytes through unspecific adhesion
as well as non-specifically binding lymphocytes.
[0529] Second immunization campaign: The depletion step using wells
covered with CHO cells was omitted since we could not exclude those
B-cells producing antibodies that are cross-reactive to hamster
transferrin receptor antibodies would be depleted. Therefore, blank
sterile 6-well plates (cell culture grade) were used to deplete
macrophages and monocytes through unspecific adhesion enabling
potential B-lymphocytes producing hamster cross-reactive (and
possibly mouse cross-reactive) surface antibodies to reach the next
step in the workflow.
[0530] For each immunization campaign: each well was filled at
maximum with 4 mL medium and up to 6.times.10.sup.6 PBMCs from the
immunized rabbit and allowed to bind for 1 h at 37.degree. C. in
the incubator. The cells in the supernatant (peripheral blood
lymphocytes (PBLs)) were used for the antigen panning step.
[0531] Enrichment of B-cells on the Human Transferrin Receptor
[0532] 6-well tissue culture plates covered with a monolayer of
human transferrin receptor-positive CHO cells were seeded with up
to 6.times.10.sup.6 PBLs per 4 mL medium and allowed to bind for 1
h at 37.degree. C. in the incubator. Non-adherent cells were
removed by carefully washing the wells 1-2 times with 1.times. PBS.
The remaining sticky cells were detached by trypsin for 10 min. at
37.degree. C. in the incubator. Trypsination was stopped with EL-4
B5 medium. The cells were kept on ice until the immune fluorescence
staining.
[0533] Immune Fluorescence Staining and Flow Cytometry
[0534] The anti-IgG FITC (AbD Serotec, Dusseldorf, Germany) was
used for single cell sorting. For surface staining, cells from the
depletion and enrichment step were incubated with the anti-IgG FITC
antibody in PBS and incubated for 45 min. in the dark at 4.degree.
C. After staining the PBMCs were washed two fold with ice cold PBS.
Finally, the PBMCs were resuspended in ice cold PBS and immediately
subjected to the FACS analyses. Propidium iodide in a concentration
of 5 .mu.g/mL (BD Pharmingen, San Diego, Calif., USA) was added
prior to the FACS analyses to discriminate between dead and live
cells.
[0535] A Becton Dickinson FACSAria equipped with a computer and the
FACSDiva software (BD Biosciences, USA) were used for single cell
sort.
[0536] B-Cell Cultivation
[0537] The cultivation of the rabbit B-cells was prepared by a
method similar to that described by Zubler et al. (1985). Briefly,
single sorted rabbit B-cells were incubated in 96-well plates with
200 .mu.L/well EL-4 B5 medium containing Pansorbin Cells (1:100000)
(Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbit thymocyte
supernatant (charge TSN-M13 (10242), MicroCoat, Bernried, Germany)
and gamma-irradiated murine EL-4-B5 thymoma cells (2.5
.times.10.sup.4/well) for 7 days at 37.degree. C. in an atmosphere
of 5% CO.sub.2 in the incubator. The supernatants of the B-cell
cultivation were removed for screening and the remaining cells were
harvested immediately and were frozen at -80.degree. C. in 100
.mu.L RLT buffer (Qiagen, Hilden, Germany).
Example 3
[0538] Phage Display for Selection and Production of Monovalent
Anti-TfR IgGs
[0539] Selection by Phage Display
[0540] Generation of antibodies binding to human and cynomolgus TfR
was carried out by phage display using standard protocols (Silacci
et al, Proteomics, 5 (2005) 2340-2350). A synthesized gene for
hTfR-Fc(KiH)-Avi (KiH=knobs-into-holes, Avi=AviTag) antigen was
cloned by connecting the hTfR ECD to the N-terminal hinge of a
human hole Fc-region, which carried a C-terminal Avi-tag (SEQ ID
NO: 99), and ligation into a mammalian expression vector. All our
mammalian expression vectors carry a MPSV promoter for initiation
of transcription and translation, where transcription is terminated
by a synthetic polyA signal sequence located downstream of the ORF.
In addition, the vector contains an EBV oriP sequence for
autonomous replication in EBV-EBNA expressing cell lines. The
correct ORF was verified by sequencing. This vector was used for
expression in HEK293 EBNA suspension cells, by co-expression with
an empty knob-Fc-region and the BirA protein, and adding 1 mM of
biotin to the culture medium. The resulting biotinylated protein
was purified by protein A affinity chromatography, followed by size
exclusion chromatography. The cyTfR-Fc(KiH)-Avi antigen was
produced respectively (SEQ ID NO: 100).
[0541] The library used was a fully synthetic library using human
frameworks of the VH1, 3, 4, and 5 families, as well as V-kappa1,
-2, and -3, and V-lambda3. Randomized amino-acids were in CDR-H3
and CDR-L3. Library pools were generated, using each heavy chain VH
together with all different light chain libraries. Selections were
carried out in solution according to the following procedure: 1.
binding of approx. 10.sup.12 phageimid particles of each library to
100 nM biotinylated TfR-avi-his for 0.5 h in a total volume of 1
mL, 2. capture of biotinylated TfR-avi-his and specifically bound
phage particles by addition of 5.4.times.10.sup.7
streptavidin-coated magnetic beads for 10 min., 3. washing of beads
using 5-10.times.1 mL PBS/Tween 20 and 5-10.times.1 mL PBS, 4.
elution of phage particles by addition of 1 mL 100 mM TEA
(triethylamine) for 10 min. and neutralization by adding 500 .mu.L
1 M Tris/HCl pH 7.4 and 5. Re-infection of exponentially growing E.
coli TG1 bacteria, infection with helper phage VCSM13 and
subsequent PEG/NaCl precipitation of phageimid particles to be used
in subsequent selection rounds. Selections were carried out over
3-5 rounds using either constant or decreasing (from 10.sup.-7 M to
2.times.10.sup.-9M) antigen concentrations. In round 2, capture of
antigen/phage complexes was performed using neutravidin plates
instead of streptavidin beads. Since the binders generated against
the recombinant soluble antigen often showed only weak binding on
cells, we introduced two additional selection steps using cells
displaying the native antigen after the second or third round of
enrichment on the recombinant antigen. Here, the two cell-lines
TF-1, and NCI-H460 were used (both available from ATCC). In brief,
1*10.sup.6 cells were incubated with approx. 10.sup.12 phage
particles for 1 h on ice to avoid receptor mediated
internalization. Washing was performed by 5-10 centrifugation steps
using PBST buffer (PBS containing 1 Tween-20). Entire cells were
used to infect the TG1 bacteria for phage rescue.
[0542] Specific binders were identified by ELISA as follows: 100
.mu.L of 10 nM biotinylated TfR-avi-his per well were coated on
neutravidin plates. Fab-containing bacterial supernatants were
added and binding Fabs were detected via their FLAG-tags by using
an anti-FLAG/HRP secondary antibody. ELISA-positive clones were
bacterially expressed as soluble Fab fragments in 96-well format
and supernatants were subjected to a kinetic screening experiment
by SPR-analysis using ProteOn XPR36 (BioRad). Clones expressing
Fabs with the highest affinity constants were identified and the
corresponding phageimids were sequenced.
[0543] Purification of Fab Antibodies
[0544] Top10 cells were individually transfected with the phageimid
plasmid of all cell ELISA positive clones. The cells were grown in
media and production of Fab antibodies was induced. Fab antibodies
were isolated by periplasmic preparation and purified using
IMAC.
[0545] FACS Analysis
[0546] Purified Fab antibodies were applied to TF-1 cells in
various concentrations. Cell bound Fab antibodies were detected
using a fluorescent labeled anti-Fab antibody and measurements by
FACS. EC.sub.50 values were calculated.
[0547] Production and Characterization of the Selected Clones
[0548] Conversion into the IgG Format
[0549] Selected clones with a complex half live (t1/2) between 6 to
20 minutes or an EC.sub.50 on cells between 10 nM and 500 nM, with
a distinct cell binding signal either on FACS or cell ELISA, and
cross-reactive binding against both the hTfR-Fc(KiH)-Avi and the
cyTfR-Fc(KiH)-Avi antigen were chosen for conversion into the IgG
format.
[0550] Therefore, the VL domain was amplified by PCR and cloned
into a mammalian expression vector, as described above, directly
upstream of a CL domain. In addition, the VH domain was amplified
by PCR and cloned directly upstream of a CH-Fc domain. The
sequences of both expression vectors were determined.
[0551] Production of IgG Antibodies
[0552] HEK293 EBNA suspension cells were transfected with both, the
LC and HC encoding, plasmids and cultivated for 7 days. The
supernatant was cleared by sterile filtration. IgG concentration
was determined by protein A chromatography.
[0553] Determination of EC50 using the TagLite Technology
[0554] The gene of the ECD of hTfR or cyTfR, respectively,
including the transmembrane domain was ligated downstream to
SNAP-tag into a mammalian expression vector. This vector was used
to transfect HEK293 EBNA suspension cells, resulting in display of
the TfR with a C-terminal SNAP tag fusion ((SEQ ID NO: 101, SEQ ID
NO: 102). The SNAP tag was specifically labeled with SNAP-Lumi4Tb
(Cisbio). The labeling efficiency determined by measuring the
emission of Terbium at 615 nm. Labeled cells were stored at
-80.degree. C.
[0555] In presence of anti-humanFc-d2 IgG antibody, labeled cells
were incubated with IgG supernatant in various dilutions and the
FRET signals (emission of donor dye Lumi4Tb: 615 nM and acceptor
dye d2: 665 nM) were measured after 4 hours. The EC.sub.50 values
were calculated, resulting in 25 IgGs, which bound to both hTfR and
cyTfR.
[0556] Determination of Cell Binding by FACS
[0557] The gene of full-length hTfR or cyTfR, respectively, was
cloned into a mammalian expression vector. This vector was used for
transfection of CHO EBNA suspension cells. IgG supernatant was
directly applied to the TfR displaying cells. After washing with
PBST, the antigen-antibody complex was detected using an anti-huFc
IgG-HRP antibody conjugate, followed by development with
3,3'-5,5'-Tetramethylbenzidine. Functional display was verified
using commercially available anti-TfR antibody. All 25 TagLite
positive clones exhibited a strong binding signal.
[0558] Production and Purification of Monovalent IgG like
Molecules
[0559] The VH domain was cloned into a mammalian expression vector
encoding the human knob IgG1 HC, which carries the L234A, L235A,
and P329G mutations. This vector was used for expression in HEK293
EBNA suspension cells, co-expressing the LC (vector described
above) and an empty hole-Fc domain carrying the L234A, L235A,
P329G, H435R and Y436F mutations. The resulting protein was
purified by protein A affinity chromatography. In cases of
monomeric protein was >95%, determined by analytical size
exclusion chromatography, the protein was further purified by size
exclusion chromatography.
Example 4
[0560] Identification of Human and Cynomolgus TfR-Binding
Antibodies by Cell ELISA
[0561] To screen rabbit B-cell or mouse hybridoma supernatants for
antibodies recognizing human and cynomolgus TfR, a cell ELISA using
stably transfected CHO-K1 cells way employed. Stable transfectants
were obtained by transfecting CHO-K1 cells with expression plasmids
containing expression cassettes for the human or cynomolgus TfR as
well as for neomycin-phosphotransferase. After transfection, cells
were diluted in growth medium containing 500 .mu.g/mL G418 (Life
Technologies). After appearance of growing clones, cells were
detached, stained with MEM-75 (Abcam) or 13E4 (Life Technologies)
and PE-labeled secondary antibodies for human or cynomolgus TfR,
and highly fluorescent cells sorted as single cells into
96-well-plate wells (FACS Aria). After 7 days of growth, clones
were again checked for TfR expression and best expressing clones
selected for cell ELISA experiments.
[0562] Briefly, 15,000 cells were seeded per well of a 384-well
plate and incubated for 18 hat 37.degree. C., 5% CO.sub.2.
Supernatant was removed using an automated washer (BIOTEK), and 30
.mu.L of antibody-containing supernatant added to each well,
followed by 24 .mu.L of growth medium. After 2 hours of incubation,
wells were emptied and 30 .mu.L of 0.05% glutaraldehyde in PBS
added for 45 min. at RT. After 3 washes with PBS/0.025% Tween20
(PBST), 30 .mu.L of anti-rabbit-HRP or anti-mouse-HRP (Southern
Biotech) diluted 1:5000 in Blocking buffer was added and plates
incubated for 1 hour at RT. Wells were washed 6 times with PBST and
signal was generated using 30 .mu.L of TMB per well and absorbance
measured at 450 nm.
Example 5
Cloning and Expression of Anti-TfR Antibodies
[0563] Recombinant DNA Techniques
[0564] Standard methods were used to manipulate DNA as described in
Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
[0565] Gene and Oligonucleotide Synthesis
[0566] Desired gene segments were prepared by chemical synthesis at
Geneart GmbH (Regensburg, Germany). The synthesized gene fragments
were cloned into an E. coli plasmid for propagation/amplification.
The DNA sequences of subcloned gene fragments were verified by DNA
sequencing. Alternatively, short synthetic DNA fragments were
assembled by annealing chemically synthesized oligonucleotides or
via PCR. The respective oligonucleotides were prepared by metabion
GmbH (Planegg-Martinsried, Germany).
[0567] PCR Amplification of V-Domains
[0568] Total RNA was prepared from B-cells lysate (resuspended in
RLT buffer-Qiagen-Cat. N.degree. 79216) using the NucleoSpin 8/96
RNA kit (Macherey&Nagel; 740709.4, 740698) according to
manufacturer's protocol. RNA was eluted with 60 .mu.L RNAse free
water. 6 .mu.L of RNA was used to generate cDNA by reverse
transcriptase reaction using the Superscript III First-Strand
Synthesis SuperMix (Invitrogen 18080-400) and an oligo-dT-primer
according to the manufacturer's instructions. All steps were
performed on a Hamilton ML Star System. 4 .mu.L of cDNA were used
to amplify the immunoglobulin heavy and light chain variable
regions (VH and VL) with the AccuPrime SuperMix (Invitrogen
12344-040) in a final volume of 50 .mu.L using the primers rbHC.up
and rbHC.do for the heavy chain, rbLC.up and rbLC.do for the light
chain of Wild Type Rabbit B cells and BcPCR_FHLC_leader.fw and
BcPCR_huCkappa.rev for the light chain of transgenic rabbit B-cells
(see Table below). All forward primers were specific for the signal
peptide (of respectively VH and VL) whereas the reverse primers
were specific for the constant regions (of respectively VH and VL).
The PCR conditions for the RbVH+RbVL were as follows: Hot start at
94.degree. C. for 5 min.; 35 cycles of 20 sec. at 94.degree. C., 20
sec. at 70.degree. C., 45 sec. at 68.degree. C., and a final
extension at 68.degree. C. for 7 min. The PCR conditions for the
HuVL were as follows: Hot start at 94.degree. C. for 5 min.; 40
cycles of 20 sec. at 94.degree. C., 20 sec. at 52.degree. C., 45
sec. at 68.degree. C., and a final extension at 68.degree. C. for 7
min.
TABLE-US-00007 rbHC.up (SEQ ID NO: 103)
AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC rbHCf.do (SEQ ID NO: 104)
CCATTGGTGAGGGTGCCCGAG rbLC.up (SEQ ID NO: 105)
AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC rbLC.do (SEQ ID NO: 106)
CAGAGTRCTGCTGAGGTTGTAGGTAC BcPCR_FHLC_leader.fw (SEQ ID NO: 107)
ATGGACATGAGGGTCCCCGC BcPCR_huCkappa.rev (SEQ ID NO: 108)
GATTTCAACTGCTCATCAGATGGC
[0569] 8 .mu.L of 50 .mu.L PCR solution were loaded on a 48 E-Gel
2% (Invitrogen G8008-02). Positive PCR reactions were cleaned using
the NucleoSpin Extract II kit (Macherey&Nagel; 740609250)
according to manufacturer's protocol and eluted in 50 .mu.L elution
buffer. All cleaning steps were performed on a Hamilton ML Starlet
System.
[0570] Recombinant Expression of Rabbit Monoclonal Bivalent
Antibodies
[0571] For recombinant expression of rabbit monoclonal bivalent
antibodies, PCR-products coding for VH or VL were cloned as cDNA
into expression vectors by the overhang cloning method (R S Haun et
al., BioTechniques (1992) 13, 515-518; MZ Li et al., Nature Methods
(2007) 4, 251-256). The expression vectors contained an expression
cassette consisting of a 5' CMV promoter including intron A, and a
3' BGH poly adenylation sequence. In addition to the expression
cassette, the plasmids contained a pUC18-derived origin of
replication and a beta-lactamase gene conferring ampicillin
resistance for plasmid amplification in E. coli. Three variants of
the basic plasmid were used: one plasmid containing the rabbit IgG
constant region designed to accept the VH regions while two
additional plasmids containing rabbit or human kappa LC constant
region to accept the VL regions.
[0572] Linearized expression plasmids coding for the kappa or gamma
constant region and VL/VH inserts were amplified by PCR using
overlapping primers.
[0573] Purified PCR products were incubated with T4 DNA-polymerase
which generated single-strand overhangs. The reaction was stopped
by dCTP addition.
[0574] In the next step, plasmid and insert were combined and
incubated with recA which induced site specific recombination. The
recombined plasmids were transformed into E. coli. The next day the
grown colonies were picked and tested for correct recombined
plasmid by plasmid preparation, restriction analysis and
DNA-sequencing.
[0575] For antibody expression, the isolated HC and LC plasmids
were transiently co-transfected into HEK293 cells and the
supernatants were harvested after 1 week.
[0576] Generation of Vectors for the Expression of Rabbit
Monoclonal Monovalent Antibodies
[0577] For recombinant expression of selected candidates as
monoclonal monovalent antibodies rabbit constant regions of all VH
chains were converted into human constant regions enclosing the
knob-mutation in the CH3 segment. For VL chains derived from rabbit
wild-type B-cells, rabbit C kappa constant regions were converted
into human. 4 .mu.L of cDNA of the selected candidates were used to
amplify the immunoglobulin heavy and light chain variable regions
with the AccuPrime SuperMix (Invitrogen 12344-040) in a final
volume of 50 .mu.L with forward primers specific for the signal
peptide and reverse primers specific for the CDR3-J region with (at
the 3' end) overlap sequence (20 bp) homologous to the human
constant regions (respectively of VH and VL). The
[0578] PCR conditions for the VH and VL chain amplification were as
follows: Hot start at 94.degree. C. for 5 min.; 35 cycles of 20
sec. at 94.degree. C., 20 sec. at 68.degree. C., 45 sec. at
68.degree. C., and a final extension at 68.degree. C. for 7
min.
[0579] PCR-products coding for VH or VL were cloned as cDNA into
expression vectors by the overhang cloning method (RS Haun et al.,
BioTechniques (1992) 13, 515-518; MZ Li et al., Nature Methods
(2007) 4, 251-256). The expression vectors contained an expression
cassette consisting of a 5' CMV promoter including intron A, and a
3' BGH poly adenylation sequence. In addition to the expression
cassette, the plasmids contained a pUC18-derived origin of
replication and a beta-lactamase gene conferring ampicillin
resistance for plasmid amplification in E. coli. Two variants of
the basic plasmid were used: one plasmid containing the human IgG
constant region designed to accept the new amplified VH chain and a
second plasmid containing the human kappa LC constant region to
accept the VL chain.
[0580] Linearized Expression Plasmids Coding for the Kappa or Gamma
Constant Region and VL/VH Inserts were Amplified by PCR using
Overlapping Primers.
[0581] Purified PCR products were incubated with T4 DNA-polymerase
which generated single-strand overhangs. The reaction was stopped
by dCTP addition.
[0582] In the next step, plasmid and insert were combined and
incubated with recA which induced site specific recombination. The
recombined plasmids were transformed into E. coli. The next day the
grown colonies were picked and tested for correct recombined
plasmid by plasmid preparation, restriction analysis and
DNA-sequencing.
Example 6
Transient Expression of the Monovalent Anti-TfR Antibodies
[0583] The antibodies were generated in vivo in transiently
transfected HEK293 cells (human embryonic kidney cell line
293-derived) cultivated in F17 Medium (Invitrogen Corp.). For
transfection "293-Free" Transfection Reagent (Novagen) was used.
Antibodies and antibody-based modified molecules as described above
were expressed from individual expression plasmids. Transfections
were performed as specified in the manufacturer's instructions.
Recombinant protein-containing cell culture supernatants were
harvested three to seven days after transfection. Supernatants were
stored at reduced temperature (e.g. --80.degree. C.) until
purification.
[0584] General information regarding the recombinant expression of
human immunoglobulins in e.g. HEK293 cells is given in: Meissner,
P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
Example 7
Purification of One-Armed Transferrin Receptor Antibodies in High
Throughput
[0585] The 50 mL clarified supernatants containing one armed
antibodies in 96 deep-well plates were loaded on 200 .mu.L
MabSelectSuRe columns. After washing steps with PBS at pH 7.4,
proteins were eluted with 2.5 mM HCl using Tecan/Atoll-system
resulting in 0.5 mL eluate. Eluate was neutralized by 2 M Tris pH
8. Purified proteins were quantified using a Nanodrop
spectrophotometer and analyzed by CE-SDS under denaturing and
reducing conditions and analytical SEC. To obtain protein with high
purity (>95%) a large proportion of the antibodies have to be
purified further on size exclusion chromatography to separate from
half antibody, knob-knob antibodies and higher aggregates. In the
following 500 .mu.L of the samples were injected on Superdex200
10/300GL in 20 mM histidine containing 140 mM NaCl pH 6.0 using
Dionex UltiMate 3000. This method allows fractionating 25-30
samples/day and therefore allows polishing a large number of
screening hits in one-armed format. Fractions were pooled and
analyzed again as described above.
Example 8
hCMEC/D3 Cell Culture for Transcytosis Assays
[0586] Medium and supplements for hCMEC/D3 (Weksler, B. B. et al.,
FASEB J. 19 (2005), 1872-1874) were obtained from Lonza. hCMEC/D3
cells (passages 26-29) were cultured to confluence on
collagen-coated coverslips (microscopy) or flasks in EBM2 medium
containing 2.5% FBS, a quarter of the supplied growth factors and
fully complemented with supplied hydrocortisone, gentamycin and
ascorbic acid.
[0587] For all transcytosis assays, high density pore (1
.times.10.sup.8 pores/cm.sup.2) PET membrane filter inserts (0.4
.mu.m, 12 mm diameter) were used in 12-well cell culture plates.
Optimum media volumes were calculated to be 400 .mu.L and 1600
.mu.L for apical and basolateral chambers, respectively. Apical
chambers of filter inserts were coated with rat tail collagen I
(7.5 .mu.g/cm.sup.2) followed by fibronectin (5 .mu.g/mL), each
incubation lasting for 1 hour at RT. hCMEC/D3 cells were grown to
confluent monolayers (approx. 2.times.10.sup.5 cells/cm.sup.2) for
10-12 days in EMB2 medium.
Example 9
Transcytosis Assay of Monovalent Antibodies
[0588] The entire assay was performed in serum-free EBM2 medium
which was otherwise reconstituted as described in Example 1. Filter
inserts with cells were incubated apically with monovalent
antibodies (concentration: 2.67 .mu.g/mL) for 1 hour at 37.degree.
C. following which the entire apical and basolateral media were
collected. From these values, paracellular flux was calculated. The
monolayers were washed at RT in serum-free medium apically (400
.mu.L) and basolaterally (1600 .mu.L) 3 .times.3-5 min. each. All
the washes were collected to monitor efficiency of removal of the
unbound antibody. Pre-warmed medium was added to the apical chamber
and the filters transferred to a fresh 12 well plate (blocked
overnight with PBS containing 1% BSA) containing 1600 .mu.L
pre-warmed medium. At this point, cells on filters were lysed in
500 .mu.L RIPA buffer in order to determine specific antibody
uptake. The remaining filters were incubated at 37.degree. C. and
samples collected at various time points to determine apical and/or
basolateral release of antibody. The content of antibody in the
samples was quantified using a highly sensitive IgG ELISA (see
Example 3). For each time point, data were generated from three
filter cell cultures.
Example 10
Sensitive IgG ELISA after Transcytosis Assay
[0589] The entire procedure was performed at RT using an automated
washer for the wash steps. A 384-well plate was coated with 30
.mu.L/well of 1 pg/mL anti-human/mouse-IgG, Fc.gamma.-specific in
PBS for 2 hours followed by 1 hour incubation in blocking buffer
PBS containing 1% BSA or 1 CroteinC for human and mouse IgG assays,
respectively). Serially diluted samples from the transcytosis assay
and standard concentrations of the antibody used in the
transcytosis assay were added to the plate and incubated for 2
hours. After four washes, 30 .mu.L/well of 50 ng/mL
anti-human/mouse-F(ab)2-Biotin in blocking buffer was added and
incubated for a further 2 hours. Following 6 washes, 30 .mu.L/well
of 50 ng/mL (huIgG assay) or 100 ng/mL (mIgG assay)
Poly-HRP40-Streptavidin (Fitzgerald; in PBS containing 1% BSA and
0.05% Tween-20) was added and incubated for 30 min. After 4 washes,
immune complexes were detected by addition of 30 .mu.L/well of BM
Chemiluminescence Substrate (Roche). The luminescence signal was
measured using a luminescence plate reader and concentration
calculated using the fitted standard curve. The sensitivity of the
assay ranged from 10 .mu.g/mL to 10 ng/mL.
Example 11
Epitope Mapping by Cell ELISA of CHO Cells Transfected with hTfR
Mutants
[0590] In order to be able determine the epitope regions on human
transferrin receptor (hTfR), mutations were introduced into the
hTfR sequence at positions where a cluster of surface-exposed amino
acids had different amino acids in the aligned mouse TfR sequence
(see Table below), following the rationale that in spite of the
significant homology between human and mouse TfR (77% identity), no
antibodies directed to the extracellular part are known which show
good cross-reactivity between both orthologous. Cloning of plasmids
with the corresponding mutations is described above. To map binding
of human TfR binders to those epitopes, CHO-K1 cells were
transiently transfected with the described plasmids and antibody
binding measured in a cell ELISA. Briefly, 10.sup.4 cells were
plated per well of a 96-well plate the day before experiment in
normal growth medium (RPMI/10% FCS). The other day, medium was
changed to OPTI-MEM Serum-Reduced Medium (Gibco), and 10 .mu.L of a
mixture of 1200 OPTI-MEM, 12 .mu.g plasmid DNA and 12 .mu.L
XtremeGENE transfection reagent (Roche) were added to the wells
after 30 minutes of pre-incubation. Cells were incubated for 2 days
at 37.degree. C./7.5% CO2, then medium was removed and TfR
antibodies added at concentrations between 1 nM and 100 nM in
growth medium, followed by 2 h incubation at 4.degree. C.
Afterwards, antibody solutions were replaced by 0.05%
glutaraldehyde in PBS and cells fixed for 15 min. at RT, then
washed twice with PBS and incubated with HRP-conjugated
anti-human-Fc secondary antibody (BioRad; 1:2000 in ELISA Blocking
Reagent (Roche)) for 1.5 hours at RT. Signal was generated after 3
washes with PBS using 50 .mu.L of TMB per well and absorbance
measured at 450 nm.
TABLE-US-00008 Plasmid # mutations in hTfR 10188 -- 18909
Thr518Asp/Gln520Lys/Phe521Ser/Gln524Arg 18910 Arg325Gln 18911
Ser355Ala/Asp356Arg/Lys358Asn/Thr359Ile 18912
Asp204Gln/Lys205Ser/Arg208Asn 18913
Lys574Gly/Glu575Ala/Ile577Thr/Glu578Gln 18914
Ala196Ile/Gln197Gly/Asn198Gln/Ser199Asn/
Val200Met/Ile201Val/Ile202Thr/Val203Ile/
Asp204Val/Lys205Gln/Asn206Ser/Gly207Asn/
Arg208Gly/Leu209Asn/Val210Leu/Tyr211Asp/ Leu212Pro 18974
Asp245Glu/Tyr247Ser/Thr248Tyr/Pro249Ser
Example 12
Surface Plasmon Resonance-Based Binding Assay for Human
TfR--Antibody Interaction
[0591] The binding experiment were carried out on a BIAcore B 4000
(GE Healthcare) equipped with C1 sensorchip (GE Healthcare, cat.
no. BR1005-35) pre-treated with anti-human Fab antibody (GE
Healthcare, cat. no 28-9583-25) using a standard amine coupling
chemistry procedure accordingly to the vendor's manual.
[0592] For kinetic measurements the sample antibody was immobilized
applying a contact time of 60 seconds and a flow rate of 10
.mu.L/min in phosphate buffer saline pH 7.4, 0.05% Tween 20 at
25.degree. C. Recombinant His6-tagged human transferrin receptor
(R&D systems, cat.no 2474-TR-050) was applied in increasing
concentrations and the signal monitored over the time. An average
time span of 150 seconds of association time and 600 seconds of
dissociation time at 30 .mu.L/min flow rate was recorded. Data were
fit using a 1:1 binding model (Langmuir isotherm).
Sequence CWU 1
1
1221118PRTMus musculus 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Asn1 5 10 15Ser Leu Thr Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met His Trp Ile Arg Gln Ala Pro
Lys Lys Gly Leu Glu Trp Ile 35 40 45Ala Met Ile Tyr Tyr Asp Ser Ser
Lys Met Asn Tyr Ala Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Glu Met Asn Ser
Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Val Pro Thr
Ser His Tyr Val Val Asp Val Trp Gly Gln Gly Val 100 105 110Ser Val
Thr Val Ser Ser 1152107PRTMus musculus 2Asp Ile Gln Met Thr Gln Ser
Pro Ala Ser Leu Ser Ala Ser Leu Glu1 5 10 15Glu Ile Val Thr Ile Thr
Cys Gln Ala Ser Gln Asp Ile Gly Asn Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45Tyr Gly Ala Thr
Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser
Gly Thr Gln Phe Ser Leu Lys Ile Ser Arg Val Gln Val65 70 75 80Glu
Asp Ile Gly Ile Tyr Tyr Cys Leu Gln Ala Tyr Asn Thr Pro Trp 85 90
95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100
1053107PRTArtificial Sequencevariant anti-transferrin receptor
antibody 8D3v has a light chain variable domain with mutants L104V
and L106I 3Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Leu Glu1 5 10 15Glu Ile Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile
Gly Asn Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
Gln Leu Leu Ile 35 40 45Tyr Gly Ala Thr Ser Leu Ala Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Gln Phe Ser Leu Lys
Ile Ser Arg Val Gln Val65 70 75 80Glu Asp Ile Gly Ile Tyr Tyr Cys
Leu Gln Ala Tyr Asn Thr Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105415PRTArtificial SequenceEpitope mapping
peptide 4Ile Gly Gln Asn Met Val Thr Ile Val Gln Ser Asn Gly Asn
Leu1 5 10 15515PRTArtificial SequenceEpitope mapping peptide 5Asn
Met Val Thr Ile Val Gln Ser Asn Gly Asn Leu Asp Pro Val1 5 10
15615PRTArtificial SequenceEpitope mapping peptide 6Gln Ser Asn Gly
Asn Leu Asp Pro Val Glu Ser Pro Glu Gly Tyr1 5 10
15732PRTArtificial SequencePeptide linker 7Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30820PRTArtificial SequencePeptide linker 8Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
20918PRTArtificial Sequencepeptidic linker 9Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly1 5 10 15Gly
Ser1030PRTArtificial Sequencepeptidic linker 10Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 3011137PRTMus
musculus 11Met Glu Trp Ser Trp Val Met Leu Phe Leu Leu Ser Gly Thr
Ala Gly1 5 10 15Val Arg Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys 20 25 30Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe 35 40 45Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser
His Gly Glu Asn Leu 50 55 60Glu Trp Ile Gly Arg Ile Asn Pro His Asn
Gly Gly Thr Asp Tyr Asn65 70 75 80Gln Lys Phe Lys Asp Lys Ala Pro
Leu Thr Val Asp Lys Ser Ser Asn 85 90 95Thr Ala Tyr Met Glu Leu Leu
Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg
Gly Tyr Tyr Tyr Tyr Ser Leu Asp Tyr Trp Gly 115 120 125Gln Gly Thr
Ser Val Thr Val Ser Ser 130 13512128PRTMus musculus 12Met Asp Phe
Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser1 5 10 15Val Ile
Leu Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile 20 25 30Met
Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 35 40
45Ser Ser Ile Asp Tyr Ile His Trp Tyr Gln Gln Lys Ser Gly Thr Ser
50 55 60Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val
Pro65 70 75 80Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile 85 90 95Ser Ser Met Glu Pro Glu Asp Ala Ala Thr Tyr Tyr
Cys His Gln Arg 100 105 110Asn Ser Tyr Pro Trp Thr Phe Gly Gly Gly
Thr Arg Leu Glu Ile Arg 115 120 12513116PRTArtificial Sequenceclone
0128 VH 13Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Thr Pro1 5 10 15Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser
Ser Tyr Tyr 20 25 30Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Ile Ile Tyr Pro Ser Gly Tyr Ala Tyr Tyr Thr
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu Lys Met Thr65 70 75 80Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Asp Val 85 90 95Gly Asp Gly Gly Gly Thr Phe
Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110Thr Val Ser Ser
11514108PRTArtificial Sequenceclone 128 VL 14Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Ser Leu Glu 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 Ser Gly Ser Thr Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10515121PRTArtificial Sequenceclone 0143 VH 15Gly Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr1 5 10 15Pro Gly Gly Ser
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu 20 25 30Ser Thr Tyr
Ser Met Gly Trp Val Arg Gln Ser Pro Gly Lys Gly Leu 35 40 45Glu Tyr
Ile Gly Ile Ile Ser Ser Ser Asp Asn Thr Tyr Tyr Thr Ser 50 55 60Trp
Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp65 70 75
80Leu Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95Ala Arg Gly Tyr Asn Gly Gly Trp Lys Thr Pro Met Asp Val Trp
Gly 100 105 110Gln Arg Thr Thr Val Thr Val Ser Ser 115
12016108PRTArtificial Sequenceclone 0143 VL 16Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Val Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ile Leu Gln Gly 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 Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
10517129PRTArtificial Sequenceclone 0146 VH 17Gly Val Gln Cys Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val1 5 10 15Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 20 25 30Phe Gly Asp
Phe Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu
Trp Val Ser Gly Ile Ser Ala Ser Gly Val Ser Thr Tyr Tyr 50 55 60Ala
Asp Ser Val Lys Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys65 70 75
80Asn Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
85 90 95Val Tyr Tyr Cys Ala Arg Asp Pro Cys Asp Ala Ser Asn Asp Tyr
Gly 100 105 110Tyr Cys Lys Leu Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 125Ser18108PRTArtificial Sequenceclone 0146 VL
18Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Asn Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg 100 10519118PRTArtificial Sequenceclone 0279 VH 19Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10
15Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr Ala
20 25 30Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
Gly 35 40 45Tyr Ile Trp Ser Gly Gly Ser Ala Asp Tyr Ala Ser Trp Ala
Lys Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
Lys Ile Pro65 70 75 80Ser Pro Thr Met Glu Asp Thr Ala Thr Tyr Phe
Cys Ala Arg Gly Asp 85 90 95Ala Glu Tyr Gly Asp Ala Asn Gly Phe Ala
Ile Trp Gly Pro Gly Thr 100 105 110Leu Val Thr Val Ser Leu
11520111PRTArtificial Sequenceclone 0279 VL 20Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1 5 10 15Gly Thr Val Thr
Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr Arg
Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Val Gly Cys65 70 75
80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Leu Ser Thr Ile Asn
85 90 95Ile Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg
100 105 11021122PRTArtificial Sequenceclone 0291 VH 21Gln Ser Val
Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu Thr
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gly Ala 20 25 30Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40
45Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys Gly
50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile
Thr65 70 75 80Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
Arg Gly Tyr 85 90 95Gly Ala Asp Tyr Pro Asp Phe Gly Asp Ala Asn Gly
Phe Asp Pro Trp 100 105 110Gly Pro Gly Thr Leu Val Thr Val Ser Ser
115 12022111PRTArtificial Sequenceclone 0291 VL 22Ala Tyr Asp Met
Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1 5 10 15Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr
Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55
60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys65
70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Leu Ser Thr Ile
Asn 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val Val Lys
Arg 100 105 11023122PRTArtificial Sequenceclone 0299 VH 23Gln Ser
Met Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ala 20 25
30Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala Lys
Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys
Ile Thr65 70 75 80Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
Ala Arg Arg Tyr 85 90 95Gly Thr Ser Tyr Pro Asp Tyr Gly Asp Ala Asn
Gly Phe Asp Pro Trp 100 105 110Gly Pro Gly Thr Leu Val Thr Val Ser
Ser 115 12024111PRTArtificial Sequenceclone 0299 VL 24Ala Tyr Asp
Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1 5 10 15Gly Thr
Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40
45Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly
50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu
Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Cys Tyr Ser
Ser Ser Asn 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val
Val Lys Arg 100 105 11025117PRTArtificial Sequenceclone 0300 VH
25Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1
5 10 15Leu Thr Leu Thr Cys Thr Ala Ser Glu Phe Ser Leu Ser Asn Tyr
Tyr 20 25 30Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr
Ile Gly 35 40 45Phe Ile His Thr Asp Gly Ser Thr Asn Tyr Ala Ser Trp
Val Asn Gly 50 55 60Arg Phe Thr Ile Ser Arg Thr Ser Thr Thr Val Asp
Leu Lys Thr Thr65 70 75 80Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys Cys Arg Tyr Leu 85 90 95Val Ser Gly Gly Thr Ile Ala Phe Asp
Leu Trp Gly Pro Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11526112PRTArtificial Sequenceclone 0300 VL 26Ala Asp Val Val Met
Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val1 5 10 15Gly Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser 20 25 30Tyr Phe Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu 35 40 45Ile Tyr
Arg Ala Ser Lys Leu Ala Thr Gly Val Pro Ser Arg Phe Lys 50 55 60Gly
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Asp Leu Glu65 70 75 80Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Thr
Cys Gly Ser Ile Ser 85 90 95Thr Tyr Gly Gly Ala Phe Gly Gly Gly Thr
Glu Val Val Val Lys Arg 100 105 11027121PRTArtificial Sequenceclone
302 VH 27Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Gly Ser1 5 10 15Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
Thr Tyr Ala 20 25 30Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Ile Ile Tyr Ala Gly Thr Asp Ile Thr Trp Tyr
Ala Ser Trp Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr
Thr Val Asp Leu Ser Ile65 70 75 80Thr Ser Pro Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala Lys Val 85 90 95Pro Tyr Thr Gly Val Ser Asp
Tyr Leu Tyr Tyr Phe Asp Leu Trp Gly 100 105 110Pro Gly Thr Leu Val
Thr Val Ser Ser 115 12028111PRTArtificial Sequenceclone 0302 VL
28Ala Phe Glu Met Thr Gln Thr Pro Ser Ser Val Ser Glu Pro Val Gly1
5 10 15Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Asn Ile Tyr Ser
Phe 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
Leu Ile 35 40 45Tyr Asp Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg
Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Asp Leu Glu Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Gly Tyr Ser Gly Asn Val 85 90 95Ile Leu Asn Ala Phe Gly Gly Gly Thr
Glu Val Val Val Lys Arg 100 105 11029122PRTArtificial Sequenceclone
0304 VH 29Gln Ser Met Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Thr Pro1 5 10 15Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
Ser Tyr Ala 20 25 30Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu Lys Ile Thr65 70 75 80Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Arg Tyr 85 90 95Gly Thr Ser Tyr Pro Asp Tyr
Gly Asp Ala Asn Gly Phe Asp Pro Trp 100 105 110Gly Pro Gly Thr Leu
Val Thr Val Ser Ser 115 12030111PRTArtificial Sequenceclone 0304 VL
30Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1
5 10 15Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu
Leu Ile 35 40 45Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg
Phe Lys Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Gly Val Glu Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Cys Tyr Ser Ser Ser Asn 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr
Glu Val Val Val Lys Arg 100 105 11031118PRTArtificial Sequenceclone
0323 VH 31Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Thr Pro1 5 10 15Leu Pro Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser
Asn Tyr Ala 20 25 30Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile Gly 35 40 45Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala
Ser Trp Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu Lys Met Thr65 70 75 80Ser Leu Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala Arg Ala Asp 85 90 95Thr Val Tyr Arg Asp Asp Tyr
Gly Trp Ser Leu Trp Gly Pro Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 11532111PRTArtificial Sequenceclone 0323 VL 32Ala Tyr Asp Met
Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1 5 10 15Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ile Tyr 20 25 30Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr
Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys65
70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Tyr Ser
Asn 85 90 95Val Asp Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys
Arg 100 105 11033120PRTArtificial Sequenceclone 0325 VH 33Gln Ser
Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1 5 10 15Leu
Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr Asn 20 25
30Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45Ile Ile Ser Asp Ala Gly Ser Ala Trp Tyr Ala Ser Trp Val Lys
Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys
Ile Thr65 70 75 80Ser Pro Thr Thr Glu Asp Ala Ala Thr Tyr Phe Cys
Ala Arg Gly Asp 85 90 95Ala Ala Ala Tyr Thr Thr Gly Thr Glu Phe Phe
Ser Leu Trp Gly Pro 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12034111PRTArtificial Sequenceclone 0325 VL 34Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly1 5 10 15Gly Thr Val Thr
Ile Lys Cys Gln Ala Ser Glu Asp Ile Glu Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Val Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60Ser
Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Gly Val Gln Cys65 70 75
80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Ser Asn
85 90 95Leu Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val Arg Arg
100 105 11035120PRTArtificial Sequenceclone 0329 VH 35Gly Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Lys1 5 10 15Pro Gly
Gly Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 20 25 30Asn
Ser Tyr Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40
45Glu Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Ala Asp Tyr Ala Asn
50 55 60Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp65 70 75 80Leu Lys Met Thr Ser Leu Thr Ala Glu Asp Thr Ala Thr
Tyr Phe Cys 85 90 95Gly Arg Gly Tyr Asp Thr Thr Asp Asn Gly Phe Asn
Leu Trp Gly Pro 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12036111PRTArtificial Sequenceclone 0329 VL 36Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser Val Glu Ala Ala Val Gly1 5 10 15Gly Thr Val Thr
Ile Lys Cys Gln Ala Ser Gln Ser Ile Thr Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr Arg
Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50 55 60Ser
Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val Glu Cys65 70 75
80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Arg Tyr Met Asn
85 90 95Val Asp Asn Val Phe Gly Gly Gly Thr Glu Val Val Val Lys Arg
100 105 11037123PRTArtificial Sequenceclone 0339 VH 37Gly Val Gln
Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr1 5 10 15Pro Gly
Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu 20 25 30Ser
Ser His Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40
45Glu Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Ala Asp Tyr Ala Ser
50 55 60Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr
Val65 70 75 80Asp Leu Lys Ile Pro Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe 85 90 95Cys Ala Arg Gly Ala Asp Glu Tyr Gly Asp Ala Asn
Gly Phe Asn Ile 100 105 110Trp Gly Pro Gly Thr Leu Val Thr Val Ser
Leu 115 12038111PRTArtificial Sequenceclone 0339 VL 38Ala Tyr Asp
Met Thr Gln Thr Pro Ser Ser Val Glu Ala Ala Val Gly1 5 10 15Gly Thr
Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Glu Leu Leu Ile 35 40
45Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Arg Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Val Gly
Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Leu Ser
Ser Ser Tyr 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val Val
Val Lys Arg 100 105 11039123PRTArtificial Sequenceclone 0343 VH
39Gly Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Gly Leu Val1
5 10 15Thr Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe
Ser 20 25 30Leu Ser Ile Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly 35 40 45Leu Asp Trp Ile Gly Phe Ile Tyr Val Asp Ser Ser Ser
Ala His Tyr 50 55 60Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Thr Thr65 70 75 80Val Asp Leu Lys Ile Thr Ser Pro Thr Thr
Glu Asp Thr Ala Thr Tyr 85 90 95Phe Cys Ala Arg Asp Val Asp Ser Ser
Tyr Phe Trp Gly Phe Asn Leu 100 105 110Trp Gly Pro Gly Thr Leu Val
Thr Val Ser Ser 115 12040112PRTArtificial Sequenceclone 0343 VL
40Ala Asp Val Val Met Thr Gln Thr Pro Ser Pro Val Ser Gly Ala Val1
5 10 15Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Asp
Ser 20 25 30Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys
Leu Leu 35 40 45Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Ser Ser
Arg Phe Lys 50 55 60Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile
Ser Asp Leu Glu65 70 75 80Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln
Cys Thr Tyr Tyr Asp Ser 85 90 95Ser Ser Ile Gly Gly Phe Gly Gly Gly
Thr Glu Val Val Val Lys Arg 100 105 11041116PRTArtificial
Sequenceclone 0346 VH 41Gly Val Gln Cys Gln Ser Val Glu Glu Ser Gly
Gly Arg Leu Val Thr1 5 10 15Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu 20 25 30Ser Ser Asn Ala Ile Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Ile Gly Tyr Ile Tyr Thr
Asp Gly Asn Thr Tyr Tyr Ala Ser 50 55 60Trp Ala Lys Gly Arg Phe Thr
Ile Ser Lys Thr Ser Thr Thr Val Asp65 70 75 80Leu Lys Ile Thr Ser
Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Gly Val
Gly Tyr Ser Asp Leu Trp Gly Pro Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 11542110PRTArtificial Sequenceclone 0346 VL 42Asp Val Val
Met Thr Gln Thr Pro Ser Ser Val Ser Glu Pro Val Gly1 5 10 15Gly Thr
Val Thr Ile Asn Cys Gln Ala Ser Glu Asn Ile Tyr Ser Ser 20 25 30Leu
Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly
50 55 60Ser Arg Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu
Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Ile Asp Gly
Gly Asn Arg 85 90 95Gly Lys Pro Phe Gly Gly Gly Thr Glu Val Val Val
Lys Arg 100 105 11043122PRTArtificial Sequenceclone 0348 VH 43Gly
Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr1 5 10
15Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
20 25 30Asn Ser Val Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 35 40 45Glu Trp Ile Gly Ile Ile Tyr Ala Ser Gly Ser Ile Tyr Tyr
Ala Ser 50 55 60Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Thr Ser Ser
Thr Thr Val65 70 75 80Asp Leu Lys Met Thr Ser Leu Thr Thr Glu Asp
Thr Ala Thr Tyr Phe 85 90 95Cys Val Arg Ser Ala Asp Tyr Asp Ser Gly
Met Pro Phe Asp Leu Trp 100 105 110Gly Pro Gly Thr Leu Val Thr Val
Ser Ser 115 12044111PRTArtificial Sequenceclone 0348 VL 44Ala Gln
Val Leu Thr Gln Thr Ala Ser Ser Val Ser Ala Ala Val Gly1 5 10 15Gly
Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Trp Asn Asn 20 25
30Asn Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
35 40 45Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg
Phe 50 55 60Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser
Asp Leu65 70 75 80Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly
Val Tyr Ser Asp 85 90 95Asn Ile Phe Ala Phe Gly Gly Gly Thr Glu Val
Val Val Lys Arg 100 105 11045121PRTArtificial Sequenceclone 0349 VH
45Gly Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr1
5 10 15Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu 20 25 30Ser Ser Ser Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 35 40 45Glu Tyr Ile Gly Phe Ile His Thr Asp Gly Ser Thr Tyr
Tyr Ala Thr 50 55 60Trp Val Asn Gly Arg Phe Thr Ile Ser Arg Thr Ser
Thr Thr Val Thr65 70 75 80Leu Lys Met Thr Ser Leu Thr Thr Glu Asp
Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Tyr Leu Val Gly Ser Gly Ala
Val Ala Phe Asp Leu Trp Gly 100 105 110Pro Gly Thr Leu Val Thr Val
Ser Ser 115 12046112PRTArtificial Sequenceclone 0349 VL 46Ala Asp
Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val1 5 10 15Gly
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser 20 25
30Tyr Phe Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
35 40 45Ile Tyr Arg Ala Ser Asn Leu Ala Thr Gly Val Pro Ser Arg Phe
Lys 50 55 60Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp
Leu Glu65 70 75 80Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Cys
Gly Ser Ile Ser 85 90 95Thr Tyr Gly Gly Ala Phe Gly Gly Gly Thr Glu
Val
Val Val Lys Arg 100 105 11047121PRTArtificial Sequenceclone 0350 VH
47Gly Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr1
5 10 15Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu 20 25 30Ser Ser Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 35 40 45Glu Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Thr Asp
Tyr Ala Thr 50 55 60Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Thr Thr Val Asp65 70 75 80Leu Ser Ile Thr Ser Pro Thr Thr Glu Asp
Thr Ala Ala Tyr Phe Cys 85 90 95Val Arg Ser Ala Gly Ser Gly Asp Ala
Met Gly Phe Asn Leu Trp Gly 100 105 110Pro Gly Thr Leu Val Thr Val
Ser Ser 115 12048111PRTArtificial Sequenceclone 0350 VL 48Ala Tyr
Asp Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly1 5 10 15Gly
Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Ser Tyr 20 25
30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile
35 40 45Tyr Arg Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys
Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu
Glu Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Leu
Ser Val Ile His 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr Glu Val
Val Val Lys Arg 100 105 11049122PRTArtificial Sequenceclone 0411 VH
49Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro1
5 10 15Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr
Ala 20 25 30Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Ile Gly 35 40 45Tyr Ile Trp Ser Gly Gly Ser Thr Asp Tyr Ala Thr Trp
Ala Lys Gly 50 55 60Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp
Leu Lys Ile Thr65 70 75 80Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys Ala Arg Gly Tyr 85 90 95Gly Ala Gly Tyr Pro Asp Tyr Gly Asp
Ala Asn Gly Phe Asp Pro Trp 100 105 110Gly Pro Gly Thr Leu Val Thr
Val Ser Ser 115 12050111PRTArtificial Sequenceclone 0411 VL 50Ala
Tyr Asp Met Thr Gln Thr Pro Ala Ser Val Ser Ala Ala Val Gly1 5 10
15Gly Thr Val Ser Ile Asn Cys Gln Ala Ser Gln Ser Ile Ser Gly Tyr
20 25 30Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu
Ile 35 40 45Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe
Lys Gly 50 55 60Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly
Val Glu Cys65 70 75 80Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly
Tyr Ser Ile Ile Asn 85 90 95Val Asp Asn Thr Phe Gly Gly Gly Thr Glu
Val Val Val Lys Arg 100 105 11051120PRTArtificial Sequenceclone
0431 VH 51Gln 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 Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ser Ser Tyr
Ile Ser Gly Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser 115 12052107PRTArtificial Sequenceclone 0431 VL 52Ser
Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10
15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala
20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile
Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser
Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala
Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp
Asp Tyr Gly Asp Gly 85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 10553120PRTArtificial Sequenceclone 0432 VH 53Gln 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 Ser Tyr 20 25 30Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Glu Ser Trp Trp Leu Ser Gly Leu Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12054107PRTArtificial Sequenceclone 0432 VL 54Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Met Thr Glu Ser Met Asn Tyr
85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10555120PRTArtificial Sequenceclone 0433 VH 55Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Glu Ser Thr Trp Phe Ser Gly Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12056108PRTArtificial Sequenceclone 0433 VL 56Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ile Asp Ser Ser Gly Asn His
85 90 95Asp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10557120PRTArtificial Sequenceclone 0434 VH 57Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Tyr Pro Trp Tyr Ile Trp Ser Tyr Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12058108PRTArtificial Sequenceclone 0434 VL 58Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Pro Asp Ser Ile Ala Tyr Gly
85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10559119PRTArtificial Sequenceclone 0435 VH 59Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Tyr Phe Leu Ser Gly Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Ile Val Thr Val Ser Ser 11560107PRTArtificial
Sequenceclone 0435 VL 60Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser
Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser
Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser
Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Asn Ser Glu Asp Ser Glu Ser Asn Leu 85 90 95Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 10561118PRTArtificial Sequenceclone
0441 VH 61Gln 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 Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Trp Phe Gly Glu
Trp Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 11562107PRTArtificial Sequenceclone 0441 VL 62Ser Ser Glu Leu
Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg
Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly
Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55
60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65
70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ile Asp Ser Ala Leu Asp
His 85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10563119PRTArtificial Sequenceclone 0445 VH 63Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Ser Trp Tyr Gly Trp Ala Phe Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 11564107PRTArtificial
Sequenceclone 0445 VL 64Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser
Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser
Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser
Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Asn Ser Arg Asp Met Ser Leu Asn Val 85 90 95Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 10565119PRTArtificial Sequenceclone
0447 VH 65Gln 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 Ser Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr
Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Phe Tyr Gly
Trp Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 11566108PRTArtificial Sequenceclone 0447 VL 66Ser Ser Glu
Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val
Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40
45Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala
Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Thr
Leu Ile Val 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 10567119PRTArtificial Sequenceclone 0449 VH 67Gln 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 Ser Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Thr Trp Gly Trp Val Ala
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11568108PRTArtificial Sequenceclone 0449 VL 68Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Pro Asp Ser Ser Val Asn Tyr
85 90 95Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10569120PRTArtificial Sequenceclone 0451 VH 69Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Glu Tyr Tyr Tyr Leu Ser Gly Phe Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12070107PRTArtificial Sequenceclone 0451 VL 70Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Ile Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ile Ile Gly Asp Ala
85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10571120PRTArtificial Sequenceclone 0452 VH 71Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Trp Asp Tyr Leu Ser Ser Met Asp Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12072108PRTArtificial Sequenceclone 0452 VL 72Ser Ser Glu Leu Thr
Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile
Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys
Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ile Asp Ser Glu Gly Asn Asp
85 90 95Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10573117PRTArtificial Sequenceclone 0453 VH 73Gln 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 Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Asp Val Tyr Gly His Leu Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 11574107PRTArtificial
Sequenceclone 0453 VL 74Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser
Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp Ser
Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala Ser
Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp Tyr
Tyr Cys Asn Ser Ile Asp Leu Ser Met Asp Ile 85 90 95Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 10575121PRTArtificial Sequenceclone
0486 VH 75Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro
Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
Thr Gly Tyr 20 25 30Asn Met Asn Trp Val Lys Gln Ser Asn Gly Glu Arg
Leu Glu Trp Ile 35 40 45Gly Ser Ile Asp Pro Phe Tyr Gly Gly Thr Thr
Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys
Pro Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Thr Ser Leu Thr Ala
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Gly Gly Asn Tyr
Tyr Gly Pro Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ala 115 12076113PRTArtificial Sequenceclone 0486 VL
76Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly1
5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Thr Gln Ser Val Val His
Ser 20 25 30Asp Gly Ile Thr His Leu Glu Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Phe Asn Arg Phe Tyr
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His Val Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110Arg77121PRTArtificial
Sequenceclone 0487 VH 77Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr Ile Ser Ser Gly Ser Gly
Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Thr Thr
Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Tyr
Tyr Ser Asp Phe Gly His Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Ser Val Thr Val Ser Ser 115 12078113PRTArtificial Sequenceclone
0487 VL 78Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr
Ile Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu
Leu Tyr Thr 20 25 30Ile Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg
Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu
Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95Thr His Phe Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Arg 100 105
110Arg79119PRTArtificial Sequenceclone 0488 VH 79Asp Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30Gly Met
His Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val 35 40 45Ala
Tyr Ile Ser Ser Gly Gly Thr Asn Ile Tyr Tyr Ala Asp Pro Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Lys Thr Leu Phe65
70 75 80Leu Gln Met Thr Gly Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95Ala Arg Asp Gly Phe Gly Asn Tyr Trp Phe Ala Tyr Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala
11580108PRTArtificial Sequenceclone 0488 VL 80Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Thr Val Gly1 5 10 15Glu Thr Val Thr
Ile Thr Cys Arg Thr Ser Gly Asn Ile His Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn
Gly Gln Thr Leu Ala Glu Gly Val Pro Ser Lys Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75
80Glu Asp Phe Gly Ser Tyr Phe Cys His Gln Tyr Phe Tyr Phe Pro Leu
85 90 95Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys Arg 100
10581120PRTArtificial Sequenceclone 0489 VH 81Glu Val Gln Leu Gln
Gln Ser Gly Thr Glu Leu Val Arg Pro Gly Ala1 5 10 15Leu Val Arg Leu
Ser Cys Lys Ala Ser Asp Phe Asn Ile Lys Asp Tyr 20 25 30Tyr Leu His
Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Val 35 40 45Gly Trp
Ile Asp Pro Glu Thr Leu Asn Thr Ile Tyr Gly Pro Lys Phe 50 55 60Gln
Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75
80Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Ile Ala Val Tyr Tyr Cys
85 90 95Thr Ser Ser Thr Val Ile Ser Tyr Trp His Phe Asp Val Trp Gly
Ala 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115
12082108PRTArtificial Sequenceclone 0489 VL 82Asp Ile Val Met Thr
Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Thr
Phe Thr Cys Lys Ala Ser Gln Asp Val Arg Ser Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ser Val Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100
10583121PRTArtificial Sequenceclone 0490 VH 83Asp Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr
Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Met 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75
80Leu Gln Met Thr Thr Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Ser Tyr Tyr Ser Asp Phe Gly His Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser 115
12084113PRTArtificial Sequenceclone 0490 VL 84Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser
Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr 20 25 30Ile Gly Lys
Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Lys
Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Ser
85 90 95Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
Arg 100 105 110Arg85119PRTArtificial Sequenceclone 0491 VH 85Asp
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe
20 25 30Gly Met His Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp
Val 35 40 45Ala Tyr Ile Ser Ser Gly Ser Thr Asn Ile Tyr Tyr Ala Asp
Pro Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Lys
Thr Leu Phe65 70 75 80Leu Gln Met Thr Gly Leu Arg Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Phe Gly Asn Tyr Trp Phe
Ala Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala
11586108PRTArtificial Sequenceclone 0491 VL 86Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Thr Val Gly1 5 10 15Glu Thr Val Thr
Ile Thr Cys Arg Thr Ser Gly Asn Ile His Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn
Gly Gln Thr Leu Ala Glu Gly Val Pro Ser Lys Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75
80Glu Asp Phe Gly Ser Tyr Phe Cys His Gln Tyr Phe Tyr Phe Pro Leu
85 90 95Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys Arg 100
10587121PRTArtificial Sequenceclone 0492 VH 87Asp Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met His
Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val 35 40 45Ala Tyr
Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thr Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe65 70 75
80Leu Gln Met Ala Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Ser Tyr Tyr Gly Asn Phe Gly Phe Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Ser Val Thr Val Ser Ser 115
12088113PRTArtificial Sequenceclone 0492 VL 88Asp Val Val Met Thr
Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly1 5 10 15Gln Pro Ala Ser
Ile Ser Cys Lys Ser Ser Gln Ser Leu
Leu Tyr Thr 20 25 30Gln Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg
Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu
Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Leu Gln Ser 85 90 95Thr His Phe Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105
110Arg89119PRTArtificial Sequenceclone 0493 VH 89Asp Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Arg Lys
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30Gly Met
His Trp Val Arg Gln Thr Pro Glu Arg Gly Leu Glu Trp Val 35 40 45Ala
Tyr Ile Ser Ser Asp Ser Ser Asn Ile Tyr Tyr Ala Asp Pro Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Lys Thr Leu Phe65
70 75 80Leu Gln Met Thr Gly Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95Ala Arg Asp Gly Phe Gly Asn Tyr Trp Phe Ala Tyr Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ala
11590108PRTArtificial Sequenceclone 0493 VL 90Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Thr Val Gly1 5 10 15Glu Thr Val Thr
Ile Thr Cys Arg Thr Ser Gly Asn Ile His Asn Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45Tyr Asn
Gly Gln Thr Leu Ala Glu Gly Val Pro Ser Lys Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro65 70 75
80Glu Asp Phe Gly Ser Tyr Phe Cys His Gln Tyr Phe Tyr Phe Pro Leu
85 90 95Thr Phe Gly Thr Gly Thr Lys Leu Glu Ile Lys Arg 100
10591119PRTArtificial Sequenceclone 0494 VH 91Glu Val Gln Leu Gln
Gln Ser Gly Ala Val Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu
Ser Cys Pro Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His
Trp Val Ile Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45Gly Arg
Ile Asp Pro Ala Asn Gly Asp Thr Lys Cys Asp Pro Lys Phe 50 55 60Gln
Val Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75
80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95Val Arg Asp Tyr Leu Tyr Pro Tyr Tyr Phe Asp Phe Trp Gly Gln
Gly 100 105 110Thr Thr Leu Thr Val Ser Ser 11592108PRTArtificial
Sequenceclone 0494 VL 92Lys Ile Val Met Thr Gln Ser Pro Lys Ser Met
Ser Met Ser Val Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Arg Ala Ser
Glu Ser Val Asp Thr Tyr 20 25 30Val Ser Trp Tyr Gln Gln Lys Pro Glu
Gln Ser Pro Glu Leu Leu Ile 35 40 45Tyr Gly Ala Ser Asn Arg Tyr Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Ala Thr Asp Phe
Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Asp
Tyr Tyr Cys Gly Gln Thr Tyr Asn Tyr Pro Leu 85 90 95Thr Phe Gly Ala
Gly Thr Lys Leu Glu Leu Lys Arg 100 10593118PRTArtificial
Sequenceclone 0495 VH 93Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Arg Ala Ser Gly
Tyr Ser Phe Pro Ser Tyr 20 25 30Val Val His Trp Val Lys Gln Lys Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asn Pro Tyr Thr Asp
Gly Thr Glu Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr
Ser Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Phe
Tyr Tyr Tyr Ser Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val
Thr Val Ser Ser 11594107PRTArtificial Sequenceclone 0495 VL 94Gln
Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10
15Glu Lys Val Thr Leu Thr Cys Ser Ala Asn Ser Ser Ile Arg Asn Met
20 25 30His Trp Tyr Gln Gln Lys Thr Gly Thr Ser Pro Lys Arg Trp Ile
Tyr 35 40 45Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met
Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser
Ser Phe Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 100 10595124PRTArtificial Sequenceclone 0496 VH 95Gln Val Gln
Leu Ile Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10 15Ser Val
Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr
Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Ala Arg Ile Tyr Pro Gly Ser Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Glu Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95Ala Arg Ser Pro Pro His Tyr Val Gly Asn Arg Tyr
Tyr Ala Leu Asp 100 105 110Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 115 12096108PRTArtificial Sequenceclone 0496 VL 96Asp Ile
Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly1 5 10 15Asp
Arg Val Thr Val Thr Cys Lys Thr Thr Glu Asp Ile Tyr Asn Arg 20 25
30Leu Ala Trp Tyr Gln His Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45Ser Gly Ala Thr Gly Leu Glu Thr Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Leu
Gln Thr65 70 75 80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Trp
Ser Thr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 100 10597123PRTArtificial Sequenceclone 0497 VH 97Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Phe Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp
Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Asp Ile Tyr Pro Gly Ser Gly Ser Asn Lys Tyr Asn Glu Lys Phe
50 55 60Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr65 70 75 80Met His Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Arg Pro Thr Tyr Tyr Gly Ser Ser Ala
Trp Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ala 115 12098108PRTArtificial Sequenceclone 0497 VL 98Asp Ile Lys
Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly1 5 10 15Glu Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu
Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40
45Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp
Tyr65 70 75 80Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu
Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 10599916PRTArtificial SequencehuTfR-Fc(kih)-Avi 99Met Gly Trp
Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His
Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp 20 25 30His
Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35 40
45Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
50 55 60Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val65 70 75 80Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val 85 90 95Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser 100 105 110Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu 115 120 125Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala 130 135 140Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro145 150 155 160Gln Val Cys
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 165 170 175Val
Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 180 185
190Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
195 200 205Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser
Lys Leu 210 215 220Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser225 230 235 240Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 245 250 255Leu Ser Pro Gly Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Ser 260 265 270Gly Gly Gly Gly Ser
Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu Ser 275 280 285Glu Lys Leu
Asp Ser Thr Asp Phe Thr Gly Thr Ile Lys Leu Leu Asn 290 295 300Glu
Asn Ser Tyr Val Pro Arg Glu Ala Gly Ser Gln Lys Asp Glu Asn305 310
315 320Leu Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe Lys Leu Ser
Lys 325 330 335Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val Lys
Asp Ser Ala 340 345 350Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly
Arg Leu Val Tyr Leu 355 360 365Val Glu Asn Pro Gly Gly Tyr Val Ala
Tyr Ser Lys Ala Ala Thr Val 370 375 380Thr Gly Lys Leu Val His Ala
Asn Phe Gly Thr Lys Lys Asp Phe Glu385 390 395 400Asp Leu Tyr Thr
Pro Val Asn Gly Ser Ile Val Ile Val Arg Ala Gly 405 410 415Lys Ile
Thr Phe Ala Glu Lys Val Ala Asn Ala Glu Ser Leu Asn Ala 420 425
430Ile Gly Val Leu Ile Tyr Met Asp Gln Thr Lys Phe Pro Ile Val Asn
435 440 445Ala Glu Leu Ser Phe Phe Gly His Ala His Leu Gly Thr Gly
Asp Pro 450 455 460Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln
Phe Pro Pro Ser465 470 475 480Arg Ser Ser Gly Leu Pro Asn Ile Pro
Val Gln Thr Ile Ser Arg Ala 485 490 495Ala Ala Glu Lys Leu Phe Gly
Asn Met Glu Gly Asp Cys Pro Ser Asp 500 505 510Trp Lys Thr Asp Ser
Thr Cys Arg Met Val Thr Ser Glu Ser Lys Asn 515 520 525Val Lys Leu
Thr Val Ser Asn Val Leu Lys Glu Ile Lys Ile Leu Asn 530 535 540Ile
Phe Gly Val Ile Lys Gly Phe Val Glu Pro Asp His Tyr Val Val545 550
555 560Val Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser
Gly 565 570 575Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met Phe
Ser Asp Met 580 585 590Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser
Ile Ile Phe Ala Ser 595 600 605Trp Ser Ala Gly Asp Phe Gly Ser Val
Gly Ala Thr Glu Trp Leu Glu 610 615 620Gly Tyr Leu Ser Ser Leu His
Leu Lys Ala Phe Thr Tyr Ile Asn Leu625 630 635 640Asp Lys Ala Val
Leu Gly Thr Ser Asn Phe Lys Val Ser Ala Ser Pro 645 650 655Leu Leu
Tyr Thr Leu Ile Glu Lys Thr Met Gln Asn Val Lys His Pro 660 665
670Val Thr Gly Gln Phe Leu Tyr Gln Asp Ser Asn Trp Ala Ser Lys Val
675 680 685Glu Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe Leu Ala
Tyr Ser 690 695 700Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp
Thr Asp Tyr Pro705 710 715 720Tyr Leu Gly Thr Thr Met Asp Thr Tyr
Lys Glu Leu Ile Glu Arg Ile 725 730 735Pro Glu Leu Asn Lys Val Ala
Arg Ala Ala Ala Glu Val Ala Gly Gln 740 745 750Phe Val Ile Lys Leu
Thr His Asp Val Glu Leu Asn Leu Asp Tyr Glu 755 760 765Arg Tyr Asn
Ser Gln Leu Leu Ser Phe Val Arg Asp Leu Asn Gln Tyr 770 775 780Arg
Ala Asp Ile Lys Glu Met Gly Leu Ser Leu Gln Trp Leu Tyr Ser785 790
795 800Ala Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu Thr Thr Asp
Phe 805 810 815Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys Lys
Leu Asn Asp 820 825 830Arg Val Met Arg Val Glu Tyr His Phe Leu Ser
Pro Tyr Val Ser Pro 835 840 845Lys Glu Ser Pro Phe Arg His Val Phe
Trp Gly Ser Gly Ser His Thr 850 855 860Leu Pro Ala Leu Leu Glu Asn
Leu Lys Leu Arg Lys Gln Asn Asn Gly865 870 875 880Ala Phe Asn Glu
Thr Leu Phe Arg Asn Gln Leu Ala Leu Ala Thr Trp 885 890 895Thr Ile
Gln Gly Ala Ala Asn Ala Leu Ser Gly Asp Val Trp Asp Ile 900 905
910Asp Asn Glu Phe 915100916PRTArtificial SequencecyTfR-Fc(kih)-Avi
100Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1
5 10 15Val His Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu
Trp 20 25 30His Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu 35 40 45Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 50 55 60Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val65 70 75 80Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val 85 90 95Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 100 105 110Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 115 120 125Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 130 135 140Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro145 150 155
160Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
165 170 175Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala 180 185 190Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr 195 200 205Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Val Ser Lys Leu 210 215 220Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser225 230 235 240Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 245 250 255Leu Ser Pro
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser 260
265 270Gly Gly Gly Gly Ser Leu Tyr Trp Asp Asp Leu Lys Arg Lys Leu
Ser 275 280 285Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser Thr Ile Lys
Leu Leu Asn 290 295 300Glu Asn Leu Tyr Val Pro Arg Glu Ala Gly Ser
Gln Lys Asp Glu Asn305 310 315 320Leu Ala Leu Tyr Ile Glu Asn Gln
Phe Arg Glu Phe Lys Leu Ser Lys 325 330 335Val Trp Arg Asp Gln His
Phe Val Lys Ile Gln Val Lys Asp Ser Ala 340 345 350Gln Asn Ser Val
Ile Ile Val Asp Lys Asn Gly Gly Leu Val Tyr Leu 355 360 365Val Glu
Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys Ala Ala Thr Val 370 375
380Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys Lys Asp Phe
Glu385 390 395 400Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val Ile
Val Arg Ala Gly 405 410 415Lys Ile Thr Phe Ala Glu Lys Val Ala Asn
Ala Glu Ser Leu Asn Ala 420 425 430Ile Gly Val Leu Ile Tyr Met Asp
Gln Thr Lys Phe Pro Ile Val Lys 435 440 445Ala Asp Leu Ser Phe Phe
Gly His Ala His Leu Gly Thr Gly Asp Pro 450 455 460Tyr Thr Pro Gly
Phe Pro Ser Phe Asn His Thr Gln Phe Pro Pro Ser465 470 475 480Gln
Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr Ile Ser Arg Ala 485 490
495Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp Cys Pro Ser Asp
500 505 510Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser Glu Asn
Lys Ser 515 520 525Val Lys Leu Thr Val Thr Asn Val Leu Lys Glu Thr
Lys Ile Leu Asn 530 535 540Ile Phe Gly Val Ile Lys Gly Phe Val Glu
Pro Asp His Tyr Val Val545 550 555 560Val Gly Ala Gln Arg Asp Ala
Trp Gly Pro Gly Ala Ala Lys Ser Ser 565 570 575Val Gly Thr Ala Leu
Leu Leu Lys Leu Ala Gln Met Phe Ser Asp Met 580 585 590Val Leu Lys
Asp Gly Phe Gln Pro Ser Arg Ser Ile Ile Phe Ala Ser 595 600 605Trp
Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr Glu Trp Leu Glu 610 615
620Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr Tyr Ile Asn
Leu625 630 635 640Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
Ser Ala Ser Pro 645 650 655Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met
Gln Asp Val Lys His Pro 660 665 670Val Thr Gly Arg Ser Leu Tyr Gln
Asp Ser Asn Trp Ala Ser Lys Val 675 680 685Glu Lys Leu Thr Leu Asp
Asn Ala Ala Phe Pro Phe Leu Ala Tyr Ser 690 695 700Gly Ile Pro Ala
Val Ser Phe Cys Phe Cys Glu Asp Thr Asp Tyr Pro705 710 715 720Tyr
Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu Val Glu Arg Ile 725 730
735Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu Val Ala Gly Gln
740 745 750Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn Leu Asp
Tyr Glu 755 760 765Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu Arg Asp
Leu Asn Gln Tyr 770 775 780Arg Ala Asp Val Lys Glu Met Gly Leu Ser
Leu Gln Trp Leu Tyr Ser785 790 795 800Ala Arg Gly Asp Phe Phe Arg
Ala Thr Ser Arg Leu Thr Thr Asp Phe 805 810 815Arg Asn Ala Glu Lys
Arg Asp Lys Phe Val Met Lys Lys Leu Asn Asp 820 825 830Arg Val Met
Arg Val Glu Tyr Tyr Phe Leu Ser Pro Tyr Val Ser Pro 835 840 845Lys
Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser Gly Ser His Thr 850 855
860Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg Gln Asn Asn
Ser865 870 875 880Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
Leu Ala Thr Trp 885 890 895Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser
Gly Asp Val Trp Asp Ile 900 905 910Asp Asn Glu Phe
915101954PRTArtificial SequencehuTfR-SNAP tag 101Met Met Asp Gln
Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser
Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30Asn Ser
His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45Asp
Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55
60Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly65
70 75 80Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys
Thr 85 90 95Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Val Arg Glu
Glu Pro 100 105 110Gly Glu Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp
Asp Asp Leu Lys 115 120 125Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr
Asp Phe Thr Gly Thr Ile 130 135 140Lys Leu Leu Asn Glu Asn Ser Tyr
Val Pro Arg Glu Ala Gly Ser Gln145 150 155 160Lys Asp Glu Asn Leu
Ala Leu Tyr Val Glu Asn Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser
Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190Lys
Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Arg 195 200
205Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly
Thr Lys225 230 235 240Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn
Gly Ser Ile Val Ile 245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala
Glu Lys Val Ala Asn Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val
Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285Pro Ile Val Asn Ala
Glu Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300Thr Gly Asp
Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln305 310 315
320Phe Pro Pro Ser Arg Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu
Gly Asp 340 345 350Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg
Met Val Thr Ser 355 360 365Glu Ser Lys Asn Val Lys Leu Thr Val Ser
Asn Val Leu Lys Glu Ile 370 375 380Lys Ile Leu Asn Ile Phe Gly Val
Ile Lys Gly Phe Val Glu Pro Asp385 390 395 400His Tyr Val Val Val
Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser
Gly Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430Phe
Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440
445Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala
Phe Thr465 470 475 480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr
Ser Asn Phe Lys Val 485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu
Ile Glu Lys Thr Met Gln Asn 500 505 510Val Lys His Pro Val Thr Gly
Gln Phe Leu Tyr Gln Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu
Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr
Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp545 550 555
560Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575Ile Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala
Ala Glu 580 585 590Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp
Val Glu Leu Asn 595 600 605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu
Leu Ser Phe Val Arg Asp 610 615 620Leu Asn Gln Tyr Arg Ala Asp Ile
Lys Glu Met Gly Leu Ser Leu Gln625 630 635 640Trp Leu Tyr Ser Ala
Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp
Phe Gly Asn Ala Glu Lys Thr Asp Arg Phe Val Met Lys 660 665 670Lys
Leu Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Leu Ser Pro 675 680
685Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700Gly Ser His Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu
Arg Lys705 710 715 720Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe
Arg Asn Gln Leu Ala 725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala
Ala Asn Ala Leu Ser Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu
Phe Thr Arg Tyr Pro Tyr Asp Val Pro 755 760 765Asp Tyr Ala Ser Gly
Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu 770 775 780Asp Ser Pro
Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu785 790 795
800His Glu Ile Lys Leu Leu Gly Ser Gly Thr Ser Ala Ala Asp Ala Val
805 810 815Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro
Leu Met 820 825 830Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln
Pro Glu Ala Ile 835 840 845Glu Glu Phe Pro Val Pro Ala Leu His His
Pro Val Phe Gln Gln Glu 850 855 860Ser Phe Thr Arg Gln Val Leu Trp
Lys Leu Leu Lys Val Val Lys Phe865 870 875 880Gly Glu Val Ile Ser
Tyr Gln Gln Leu Ala Ala Leu Ala Gly Asn Pro 885 890 895Ala Ala Thr
Ala Ala Val Lys Thr Ala Leu Ser Gly Asn Pro Val Pro 900 905 910Ile
Leu Ile Pro Cys His Arg Val Val Ser Ser Ser Gly Ala Val Gly 915 920
925Gly Tyr Glu Gly Gly Leu Ala Val Lys Glu Trp Leu Leu Ala His Glu
930 935 940Gly His Arg Leu Gly Lys Pro Gly Leu Gly945
950102954PRTArtificial SequencecyTfR-SNAP tag 102Met Met Asp Gln
Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser
Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30Asn Ser
His Val Glu Met Lys Leu Gly Val Asp Glu Glu Glu Asn Thr 35 40 45Asp
Asn Asn Thr Lys Ala Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly 50 55
60Asn Ile Cys Tyr Gly Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly65
70 75 80Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys
Thr 85 90 95Glu Cys Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu
Glu Pro 100 105 110Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp
Asp Asp Leu Lys 115 120 125Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr
Asp Phe Thr Ser Thr Ile 130 135 140Lys Leu Leu Asn Glu Asn Leu Tyr
Val Pro Arg Glu Ala Gly Ser Gln145 150 155 160Lys Asp Glu Asn Leu
Ala Leu Tyr Ile Glu Asn Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser
Lys Val Trp Arg Asp Gln His Phe Val Lys Ile Gln Val 180 185 190Lys
Asp Ser Ala Gln Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly 195 200
205Leu Val Tyr Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys
210 215 220Ala Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly
Thr Lys225 230 235 240Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn
Gly Ser Ile Val Ile 245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala
Glu Lys Val Ala Asn Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val
Leu Ile Tyr Met Asp Gln Thr Lys Phe 275 280 285Pro Ile Val Lys Ala
Asp Leu Ser Phe Phe Gly His Ala His Leu Gly 290 295 300Thr Gly Asp
Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asn His Thr Gln305 310 315
320Phe Pro Pro Ser Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr
325 330 335Ile Ser Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu
Gly Asp 340 345 350Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys
Met Val Thr Ser 355 360 365Glu Asn Lys Ser Val Lys Leu Thr Val Thr
Asn Val Leu Lys Glu Thr 370 375 380Lys Ile Leu Asn Ile Phe Gly Val
Ile Lys Gly Phe Val Glu Pro Asp385 390 395 400His Tyr Val Val Val
Gly Ala Gln Arg Asp Ala Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser
Ser Val Gly Thr Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430Phe
Ser Asp Met Val Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440
445Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr
450 455 460Glu Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala
Phe Thr465 470 475 480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr
Ser Asn Phe Lys Val 485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu
Ile Glu Lys Thr Met Gln Asp 500 505 510Val Lys His Pro Val Thr Gly
Arg Ser Leu Tyr Gln Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu
Lys Leu Thr Leu Asp Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr
Ser Gly Ile Pro Ala Val Ser Phe Cys Phe Cys Glu Asp545 550 555
560Thr Asp Tyr Pro Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu
565 570 575Val Glu Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala
Ala Glu 580 585 590Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp
Thr Glu Leu Asn 595 600 605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu
Leu Leu Phe Leu Arg Asp 610 615 620Leu Asn Gln Tyr Arg Ala Asp Val
Lys Glu Met Gly Leu Ser Leu Gln625 630 635 640Trp Leu Tyr Ser Ala
Arg Gly Asp Phe Phe Arg Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp
Phe Arg Asn Ala Glu Lys Arg Asp Lys Phe Val Met Lys 660 665 670Lys
Leu Asn Asp Arg Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro 675 680
685Tyr Val Ser Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser
690 695 700Gly Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu
Arg Arg705 710 715 720Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe
Arg Asn Gln Leu Ala 725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala
Ala Asn Ala Leu Ser Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu
Phe Thr Arg Tyr Pro Tyr Asp Val Pro 755 760 765Asp Tyr Ala Ser Gly
Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu 770 775 780Asp Ser Pro
Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu785 790 795
800His Glu Ile Lys Leu Leu Gly Ser Gly Thr Ser Ala Ala Asp Ala Val
805 810 815Glu Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro
Leu Met 820 825 830Gln Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln
Pro Glu Ala Ile 835 840 845Glu Glu Phe Pro Val Pro Ala Leu His His
Pro Val Phe Gln Gln Glu 850
855 860Ser Phe Thr Arg Gln Val Leu Trp Lys Leu Leu Lys Val Val Lys
Phe865 870 875 880Gly Glu Val Ile Ser Tyr Gln Gln Leu Ala Ala Leu
Ala Gly Asn Pro 885 890 895Ala Ala Thr Ala Ala Val Lys Thr Ala Leu
Ser Gly Asn Pro Val Pro 900 905 910Ile Leu Ile Pro Cys His Arg Val
Val Ser Ser Ser Gly Ala Val Gly 915 920 925Gly Tyr Glu Gly Gly Leu
Ala Val Lys Glu Trp Leu Leu Ala His Glu 930 935 940Gly His Arg Leu
Gly Lys Pro Gly Leu Gly945 95010337DNAArtificial SequencerbHC.up
103aagcttgcca ccatggagac tgggctgcgc tggcttc 3710421DNAArtificial
SequencerbHCf.do 104ccattggtga gggtgcccga g 2110534DNAArtificial
SequencerbLC.up 105aagcttgcca ccatggacay gagggccccc actc
3410626DNAArtificial SequencerbLC.do 106cagagtrctg ctgaggttgt
aggtac 2610720DNAArtificial SequenceBcPCR_FHLC_leader.fw
107atggacatga gggtccccgc 2010824DNAArtificial
SequenceBcPCR_huCkappa.rev 108gatttcaact gctcatcaga tggc
241096PRTArtificial Sequenceclone 299 HVR-H1 109Phe Ser Leu Ser Ser
Tyr1 51102PRTArtificial Sequenceclone 299 HVR-H2s 110Trp
Ser111115PRTArtificial Sequenceclone 299 HVR-H2 111Tyr Ile Trp Trp
Ser Gly Gly Ser Thr Asp Tyr Ala Ser Trp Ala1 5 10
1511214PRTArtificial Sequenceclone 299 HVR-H3 112Gly Thr Ser Tyr
Pro Asp Tyr Gly Asp Ala Asn Gly Phe Asp1 5 101137PRTArtificial
Sequenceclone 299 HVR-L1 113Ser Gln Ser Ile Ser Ser Tyr1
51143PRTArtificial Sequenceclone 299 HVR-L2 114Arg Ala
Ser11159PRTArtificial Sequenceclone 299 HVR-L3 115Cys Tyr Ser Ser
Ser Asn Val Asp Asn1 51167PRTArtificial Sequenceclone 494 HVR-H1
116Gly Phe Asn Ile Lys Asp Thr1 51173PRTArtificial Sequenceclone
494 HVR-H2s 117Ala Asn Gly111817PRTArtificial Sequenceclone 494
HVR-H2 118Arg Ile Asp Pro Ala Asn Gly Asp Thr Lys Cys Asp Pro Lys
Phe Gln1 5 10 15Val1198PRTArtificial Sequenceclone 494 HVR-H3
119Tyr Leu Tyr Pro Tyr Tyr Phe Asp1 51207PRTArtificial
Sequenceclone 494 HVR-L1 120Ser Glu Ser Val Asp Thr Tyr1
51213PRTArtificial Sequenceclone 494 HVR-L2 121Gly Ala
Ser11226PRTArtificial Sequenceclone 494 HVR-L3 122Thr Tyr Asn Tyr
Pro Leu1 5
* * * * *