U.S. patent application number 11/893281 was filed with the patent office on 2008-02-28 for delivery of pharmaceutical agents via the human insulin receptor.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Ruben J. Boado, William M. Pardridge.
Application Number | 20080051564 11/893281 |
Document ID | / |
Family ID | 32325847 |
Filed Date | 2008-02-28 |
United States Patent
Application |
20080051564 |
Kind Code |
A1 |
Pardridge; William M. ; et
al. |
February 28, 2008 |
Delivery of pharmaceutical agents via the human insulin
receptor
Abstract
A humanized murine antibody is provided that binds to the human
insulin receptor (HIR). The humanized murine antibody is suitable
for use as a Trojan horse to deliver pharmaceutical agents to human
organs and tissue that express the HIR. The humanized murine
antibody is especially well suited for delivering
neuropharmaceutical agents from the blood stream to the brain
across the blood brain barrier (BBB). The humanized murine antibody
may be genetically fused to the pharmaceutical agent or it may be
linked to the pharmaceutical agent using an avidin-biotin
conjugation system.
Inventors: |
Pardridge; William M.;
(Pacific Palisades, CA) ; Boado; Ruben J.; (Agoura
Hills, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
32325847 |
Appl. No.: |
11/893281 |
Filed: |
August 14, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10307276 |
Nov 27, 2002 |
|
|
|
11893281 |
Aug 14, 2007 |
|
|
|
Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/2869 20130101; A61P 25/00 20180101; A61K 47/6849 20170801;
C07K 2317/24 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
530/387.3 |
International
Class: |
C07K 16/26 20060101
C07K016/26 |
Claims
1-24. (canceled)
25. A humanized antibody that binds to the human insulin
receptor.
26. The humanized antibody of claim 1, wherein the humanized
antibody competes with murine antibody 83-14 for binding to the
human insulin receptor.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to the delivery of
pharmaceutical agents from the blood stream to the human brain and
other organs or tissues that express the human insulin receptor.
More particularly, the present invention involves the development
of "humanized" monoclonal antibodies (MAb) that may be attached to
pharmaceutical agents to form compounds that are able to readily
bind to the human insulin receptor (HIR). The compounds are able to
cross the human blood brain barrier (BBB) by way of insulin
receptors located on the brain capillary endothelium. Once across
the BBB, the humanized monoclonal antibody/pharmaceutical agent
compounds are also capable of undergoing receptor mediated
endocytosis into brain cells via insulin receptors located on the
brain cells.
[0003] 2. Description of Related Art
[0004] The publications and other reference materials referred to
herein to describe the background of the invention and to provide
additional detail regarding its practice are hereby incorporated by
reference. For convenience, the reference materials are identified
by author and date and grouped in the appended bibliography.
[0005] The BBB is a system-wide membrane barrier that prevents the
brain uptake of circulating drugs, protein therapeutics, antisense
drugs, and gene medicines. Drugs or genes can be delivered to the
human brain for the treatment of serious brain disease either (a)
by injecting the drug or gene directly into the brain, thus
bypassing the BBB, or (b) by injecting the drug or gene into the
bloodstream so that the drug or gene enters the brain via the
transvascular route across the BBB. With intra-cerebral
administration of the drug, it is necessary to drill a hole in the
head and perform a procedure called craniotomy. In addition to
being expensive and highly invasive, this craniotomy based drug
delivery to the brain approach is ineffective, because the drug or
gene is only delivered to a tiny volume of the brain at the tip of
the injection needle. The only way the drug or gene can be
distributed widely in the brain is the transvascular route
following injection into the bloodstream. However this latter
approach requires the ability to undergo transport across the BBB
The BBB has proven to be a very difficult and stubborn barrier to
traverse safely.
[0006] Prior work has shown that drugs or gene medicines can be
ferried across the BBB using molecular Trojan horses that bind to
BBB receptor/transport systems. These Trojan horses may be modified
proteins, endogenous peptides, or peptidomimetic monoclonal
antibodies (MAb's). For example, HIR MAb 83-14 is a murine MAb that
binds to the human insulin receptor (HIR). This binding triggers
transport across the BBB of MAb 83-14 (Pardridge et al., 1995), and
any drug or gene payload attached to the MAb (Wu et al., 1997).
[0007] The use of molecular Trojan horses to ferry drugs or genes
across the BBB is described in U.S. Pat. Nos. 4,801,575 and
6,372,250. The linking of drugs to MAb transport vectors is
facilitated with use of avidin-biotin technology. In this approach,
the drug or protein therapeutic is monobiotinylated and bound to a
conjugate of the antibody vector and avidin or streptavidin. The
use of avidin-biotin technology to facilitate linking of drugs to
antibody-based transport vectors is described in U.S. Pat. No.
6,287,792. Fusion proteins have also been used where a drug is
genetically fused to the MAb transport vector.
[0008] HIRMAb 83-14 has been shown to rapidly undergo transport
across the BBB of a living Rhesus monkey, and to bind avidly to
isolated human brain capillaries, which are the anatomical
substrate of the human BBB (see Pardridge et al., 1995). In either
case, the activity of the HIRMAb 83-14 with respect to binding and
transport at the primate or human BBB is more than 10-fold greater
than the binding or transport of other peptidomimetic MAb's that
may target other BBB receptors such as the transferrin receptor
(Pardridge, 1997). To date, HIRMAb 83-14 is the most active BBB
transport vector known (Pardridge, 1997). On this basis, the HIRMAb
83-14 has proven to be a very useful agent for the delivery of
drugs to the primate brain in vivo, and would also be highly active
for brain drug or gene delivery to the brain in humans.
[0009] HIRMAb 83-14 cannot be used in humans because this mouse
protein will be immunogenic. Genetically engineered forms of HIRMAb
83-14 might be used in humans in either the form of a chimeric
antibody or a genetically engineered "humanized" HIRMAb. However,
in order to perform the genetic engineering and production of
either a chimeric or a humanized antibody, it is necessary to first
clone the variable region of the antibody heavy chain (VH) and the
variable region of the antibody light chain (VL). Following cloning
of the VH and VL genes, the genes must be sequenced and the amino
acid sequence deduced from the nucleotide sequence. With this amino
acid sequence, using technologies known to those skilled in the art
(Foote et al., 1992), it may be possible to perform humanization of
the murine HIRMAb 83-14. However, HIRMAb 83-14 may lose biological
activity following the humanization (Pichla et al. 1997).
Therefore, it is uncertain as to whether the murine HIRMAb can be
humanized with retention of biological activity.
[0010] A chimeric form of the HIRMAb 83-14 has been genetically
engineered, and the chimeric antibody binds to the HIR and is
transported into the primate brain (Coloma et al., 2000). However,
a chimeric antibody retains the entire mouse FR for both the VH and
the VL, and because of this, chimeric antibodies are still
immunogenic in humans (Bruggemann et al., 1989). In contrast to the
chimeric antibody, a humanized antibody would use the human FR
amino acid sequences for both the VH and the VL and retain only the
murine amino acids for the 3 complementarity determining regions
(CDRs) of the VH and 3 CDRs of the VL. Not all murine MAb's can be
humanized, because there is a loss of biological activity when the
murine FR's are replaced by human FR sequences (Pichla et al.,
1997). The biological activity of the antibody can be restored by
substituting back certain mouse FR amino acids (see U.S. Pat. No.
5,585,089). Nevertheless, even with FR amino acid
back-substitution, certain antibodies cannot be humanized with
retention of biological activity (Pichla et al., 1997). Therefore,
there is no certainty that the murine HIRMAb 83-14 can be humanized
even once the key murine CDR and FR amino acid sequences are
known.
SUMMARY OF THE INVENTION
[0011] In accordance with the present invention, it was discovered
that the murine HIRMAb 83-14 antibody can be humanized to provide a
biologically active humanized insulin receptor (HIR) antibody that
may be used in combination with drugs and diagnostic agents to
treat human beings in vivo. The HIR antibody may be conjugated to
the drug or diagnostic agent using avidin-biotin conjugation or the
HIR antibody/drug combination may be prepared as a fusion protein
using genetic engineering techniques. The HIR antibody is
especially well suited for delivering neuropharmaceutical agents to
the human brain across the BBB. The humanized character of the HIR
antibody significantly reduces immunogenic reactions in humans.
[0012] The humanized murine antibody of the present invention is
capable of binding to the HIR and includes a heavy chain (HC) of
amino acids and a light chain (LC) of amino acids which both
include variable and constant regions. The variable regions of the
HC and LC include complementarity determining regions (CDRs) that
are interspersed between framework regions (FRs).
[0013] The HC includes a first CDR located at the amino end of the
variable region, a third CDR located at the carboxyl end of the HC
variable region and a second CDR located between said first and
third CDRs. The amino acid sequences for the first CDR, the second
CDR, and the third CDR are SEQ. ID. NOS. 31, 33 and 35,
respectively, and combined equivalents thereof. The HC framework
regions include a first FR located adjacent to the amino end of the
first CDR, a second FR located between said first and second CDRs,
a third FR located between said second and third CDRs and a fourth
FR located adjacent to the carboxyl end of said third CDR. In
accordance with the present invention, the four FRs of the HC are
humanized such that the overall antibody retains biological
activity with respect to the HIR and is not immunogenic in
humans.
[0014] The LC also includes a first CDR located at the amino end of
the variable region, a third CDR located at the carboxyl end of the
variable region and a second CDR located between said first and
third CDRs. The amino acid sequences for the first CDR, the second
CDR, and the third CDR are SEQ. ID. NOS. 38, 40, and 42,
respectively, and combined equivalents thereof. The LC framework
regions include a first FR located adjacent to the amino end of
said first CDR, a second FR located between said first and second
CDRs, a third FR located between said second and third CDRs and a
fourth FR located adjacent to the carboxyl end of said third CDR.
Pursuant to the present invention, the four FRs of the LC are
humanized such that the overall antibody retains biological
activity with respect to the HIR and has minimal immunogenicity in
humans.
[0015] The constant regions of the murine antibody are also
modified to minimize immunogenicity in humans. The murine HC
constant region is replaced with the HC constant region from a
human immunoglobulin such as IgG1. The murine LC constant region is
replaced with a constant region from the LC of a human
immunoglobulin such as a kappa (.kappa.) LC constant region.
Replacement of the murine HC and LC constant regions with human
constant regions was found to not adversely affect the biological
activity of the humanized antibody with respect to HIR binding.
[0016] The present invention not only covers the humanized murine
antibodies themselves, but also covers pharmaceutical compositions
that are composed of the humanized antibody linked to a drug or
diagnostic agent. The humanized antibody is effective in delivering
the drug or diagnostic agent to the HIR in vivo to provide
transport across the BBB and/or endocytosis into cells via the HIR.
The compositions are especially well suited for intra venous (iv)
injection into humans for delivery of neuropharmaceutical agents to
the brain.
[0017] The above discussed and many other features and attendant
advantages of the present invention will become better understood
by reference to the detailed description when taken in conjunction
with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 shows the nucleotide sequence for the murine VH (SEQ.
ID. NO. 1) and murine VL (SEQ. ID. NO. 2) and deduced amino acid
sequence of the murine VH (SEQ. ID. NO. 3) and the murine VL (SEQ.
ID. NO. 4), which shows the 3 framework (FR) regions and the 4
complementarity determining regions (CDRs) of both the heavy chain
(HC) and the light chain (LC) of the 83-14 murine HIRMAb. The amino
acids denoted by an asterisk (*) were confirmed by amino acid
sequencing of either the intact murine LC or tryptic peptides of
the intact murine HC; for amino acid sequencing, the intact murine
HC or LC were purified from gels following purification of the
intact murine IgG from the hybridoma conditioned medium.
[0019] FIGS. 2A and 2B graphically show the results of a
radio-receptor assay on isolated human brain capillaries that were
obtained with a mechanical homogenization procedure from human
autopsy brain. These capillaries were incubated with
[.sup.25I]-labeled chimeric HIRMAb (Coloma et al., 2000) (FIG. 2A)
or [.sup.125I]-version 5 humanized HIRMAb (FIG. 2B). The data show
that both antibodies bind equally well to human brain capillaries,
which form the anatomical basis of the BBB in humans.
[0020] FIG. 3 shows the brain scan of a Rhesus monkey treated with
a humanized monoclonal antibody in accordance with the present
invention. The [.sup.125I]-labeled version 5 HIRMAb was injected
intravenously in an anesthetized rhesus monkey, and the animal was
euthanized 120 minutes later. The brain was rapidly removed and cut
into coronal hemispheric slabs, which were immediately frozen.
Cryostat sections (20 .mu.m) were cut and exposed to x-ray film.
The film was scanned to yield the image shown in FIG. 3. This image
shows the clear demarcations between the gray matter and white
matter of the primate brain. Owing to the higher vascular density
in gray matter, there is a greater uptake of the humanized HIRMAb,
relative to white matter.
[0021] FIG. 4 shows a comparison of the amino acid sequence for the
3 FRs and 3 CDRs of both the heavy chain and the light chain for
the following: (a) the version 5 humanized HIRMAb, (v) the original
murine 83-14 HIRMAb, and (c) the VH of the B43 human IgG or the VL
of the REI human IgG.
[0022] FIG. 5 shows the amino acid sequence of a fusion protein of
human .alpha.-L-iduronidase (IDUA) (SEQ. ID. NO. 48), which is
fused to the carboxyl terminus of the heavy chain (HC) of the
humanized monoclonal antibody to the human insulin receptor
(HIRMAb). The HC is comprised of a variable region (VH) and a
constant region (CH); the CH is further comprised of 3 sub-regions,
CH1 (SEQ. ID. NO. 44), CH2 (SEQ. ID. No. 45), and CH3 (SEQ. ID NO.
46); the CH1 and (CH2 regions are connected by a 12 amino acid
hinge region (SEQ. ID. NO. 47). The VH is comprised of 4 framework
regions (FR1=SEQ. ID. NO. 30; FR2=SEQ. ID. NO. 32; FR3=SEQ. ID. NO.
34; and FR4=SEQ. ID. NO. 36) and 3 complementarity determining
regions (CDR) (CDR1=SEQ. ID. NO. 31; CDR2=SEQ. ID. NO. 33; and
CDR3=SEQ. ID. NO. 35). The amino acid sequence shown for the CH is
well known in existing databases and corresponds to the CH sequence
of human IgG1. There is a single N-linked glycosylation site on the
asparagine (N) residue within the CH2 region of the CH, and there
are 6 potential N-linked glycosylation sites within the IDUA
sequence, as indicated by the underline.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention involves the humanization of the
murine monoclonal antibody identified as MAb 83-14 so that it can
be used in vivo in humans. As previously mentioned, MAb 83-14 has a
high affinity for the human insulin receptor at the human or rhesus
monkey blood-brain barrier (Pardridge, et al. 1995) and is a
candidate for use as a Trojan horse to transport
neuropharmaceutical agents across the BBB. As used herein, the term
"pharmaceutical agents" is intended to include any drug, gene or
chemical that is used to treat or diagnose disease in humans. The
term "neuropharmaceutical agent" covers pharmaceutical agents that
are used to treat brain disease. The present humanized antibody
Trojan horses are especially well suited for transporting
neuropharmaceutical agents from the blood stream to the brain
across the BBB.
[0024] The complete amino acid sequence for the variable region of
the HC and LC of murine Mab 83-14 was determined as described in
Example 1. The nucleotide sequence for the gene that expresses the
murine VH (SEQ. ID. NO. 1) and the murine VL (SEQ. ID. NO. 2) is
set forth in FIG. 1. The amino acid sequence for the murine VH
(SEQ. ID. NO. 3) and murine VL (SEQ. ID. NO. 4) is also set forth
in FIG. 1. The amino acid sequences for the variable regions of the
murine MAb 83-14 VH and VL are also set forth in FIG. 4 (SEQ. ID.
NOS. 3 AND 4, respectively). The humanized murine antibodies of the
present invention are prepared by modifying the amino acid
sequences of the variable regions of the murine antibody to more
closely resemble human antibody without destroying the ability of
the antibody to strongly bind to the HIR. In addition, the
humanized antibody includes constant regions that also correspond
to human antibody.
[0025] The humanized murine antibodies include a heavy chain of
amino acids (HC) that is composed of a constant region (CH) and a
variable region (VH). The variable region of the HC has an amino
end and a carboxyl end and includes three CDRs interspersed between
four FRs. The first CDR (CDR1) is located towards the amino end of
the VH with the third CDR (CDR3) being located towards the carboxyl
end of the HC. The amino acid sequences for murine MAb 83-14 HC
CDR1, CDR2, and CDR3 are set forth in SEQ. ID. NOS. 31, 33 and 35,
respectively. Since the HC CDRs are essential for antibody binding
to the HIR, it is preferred that the humanized antibodies have HC
CDRs with amino acid sequences that are identical to SEQ. ID. NOS.
31, 33 and 35. However, the humanized antibodies may include CDRs
in the HC that have amino acid sequences which are "individually
equivalent" to SEQ. ID. NOS. 31, 33 and 35. "Individually
equivalent" amino acid sequences are those that have at least 75
percent sequence identity and which do not adversely affect the
binding of the antibody to the HIR. Preferably, individually
equivalent amino acid sequences will have at least 85 percent
sequence identity with SEQ. ID. NOS. 31, 33 or 35. Even more
preferred are individually equivalent amino acid sequences having
at least 95 percent sequence identity.
[0026] The three VH CDR amino acid sequences may also be viewed as
a combined group of amino acid sequences (VH CDR1, VH CDR2 and VH
CDR3). The present invention also covers equivalents of the
combined group of VH CDR sequences. Such "combined equivalents" are
those that have at least 75 percent sequence identity with the
combined amino acid sequences SEQ. ID. NOS. 31, 32 and 35 and which
do not adversely affect the binding of the antibody to the HIR.
Preferably, combined equivalent amino acid sequences will have at
least 85 percent sequence identity with the combined sequences
found in SEQ. ID. NOS. 31, 33 and 35. Even more preferred are
combined equivalent amino acid sequences that have at least 95
percent sequence identity with the combined amino acid sequences
(SEQ. ID. NOS. 31, 33 and 35).
[0027] It is preferred that the VH CDR amino acid sequences meet
both the individual equivalency and combined equivalency
requirements set forth above. However, there are certain
situations, especially for the shorter CDRs, where one or more of
the CDRs may not meet the criteria for individual equivalence even
though the criteria for combined equivalence is met. In such
situations, the individual equivalency requirements are waived
provided that the combined equivalency requirements are met. For
example, VH CDR3 (SEQ. ID. NO. 35) is only 4 amino acids long. If
two amino acids are changed, then the individual sequence identity
is only 50% which is below the 75% floor for individual equivalence
set forth above. However, this particular sequence is still
suitable for use as part of a combined equivalent VH CDR group
provided that the sequence identity of the combined CDR1, CDR2 and
CDR3 sequences meet the group equivalency requirements.
[0028] The humanized murine antibodies also include a light chain
(LC) of amino acids that is composed of a constant region (CL) and
a variable region (VL). The variable region of the LC has an amino
end and a carboxyl end and includes three CDRs interspersed between
four FRs. The first CDR (CDR1) is located towards the amino end of
the VL with the third CDR (CDR3) being located towards the carboxyl
end of the VL. The amino acid sequences for murine MAb 83-14 LC
CDR1, CDR2, and CDR3 are set forth in SEQ. ID. NOS. 38, 40 and 42,
respectively. Since the VL CDRs are also important for antibody
binding to the HIR, it is preferred that the humanized antibodies
have LC CDRs with amino acid sequences that are identical to SEQ.
ID. NOS. 38, 40 and 42. However, the humanized antibodies may
include CDRs in the VL that have amino acid sequences which are
"individually equivalent" to SEQ. ID. NOS. 38, 40 or 42.
"Individually equivalent" amino acid sequences are those that have
at least 75 percent sequence identity and which do not adversely
affect the binding of the antibody to the HIR. Preferably,
individually equivalent amino acid sequences will have at least 85
percent sequence identity with SEQ. ID. NOS. 38, 40 or 42. Even
more preferred are individually equivalent amino acid sequences
having at least 95 percent sequence identity.
[0029] The three VL CDR, amino acid sequences may also be viewed as
a combined group of amino acid sequences (VL CDR1, VL CDR2 and VL
CDR3). The present invention also covers equivalents of the
combined group of VL CDR sequences. Such "combined equivalents" are
those that have at least 75 percent sequence identity with the
combined amino acid sequences SEQ. ID. NOS. 38, 40 and 42 and which
do not adversely affect the binding of the antibody to the HIR.
Preferably, combined equivalent amino acid sequences will have at
least 85 percent sequence identity with the combined sequences
found in SEQ. ID. NOS. 38, 40 and 42. Even more preferred are
combined equivalent amino acid sequences that have at least 95
percent sequence identity with the combined amino acid sequences
(SEQ. ID. NOS. 38, 40 and 42).
[0030] It is preferred that the VL CDR amino acid sequences meet
both the individual equivalency and combined equivalency
requirements set forth above. However, there are certain
situations, especially for the shorter CDRs, where one or more of
the CDRs may not meet the criteria for individual equivalence even
though the criteria for combined equivalence is met. In such
situations, the individual equivalency requirements are waived
provided that the combined equivalency requirements are met. For
example, VH CDR3 (SEQ. ID. NO. 42) is only 9 amino acids long. If
three amino acids are changed, then the individual sequence
identity is only 66% which is below the 75% floor for individual
equivalence set forth above. However, this particular sequence is
still suitable for use as part of a combined equivalent VL CDR
group provided that the sequence identity of the combined CDR1,
CDR2 and CDR3 sequences meet the group equivalency
requirements.
[0031] The first framework region (FR1) of the VH is located at the
amino end of the humanized antibody. The fourth framework region
(FR4) is located towards the carboxyl end of the humanized
antibody. Exemplary preferred amino acid sequences for the
humanized VH FR1, FR2, FR3 and FR4 are set forth in SEQ. ID. NOS.
30, 32, 34 and 36, respectively, and these preferred sequences
correspond to version 5 humanized HIRMAb (Table 3). The amino acid
sequence for FR2 (SEQ. ID. NO. 32) is identical to the amino acid
sequence of murine MAb 83-14 VH FR2 or the human IgG, B43 (See FIG.
4). The amino acid sequences for VH FR1 and FR4 (SEQ. ID. NOS. 30
and 36) correspond to the B43 human antibody framework regions that
have amino acid sequences that differ from murine MAb 83-14 (FIG.
4). The amino acid sequences for the VH FR3 (SEQ. ID. No. 34) of
the version 5 humanized HIRMAb corresponds to the VH FR3 of the
murine 83-14 antibody (Table 3). It is possible to modify the
preferred VH FR sequences without destroying the biological
activity of the antibody. Suitable alternate or equivalent FRs
include those that have at least 70 percent individual sequence
identity with SEQ. ID. NOS. 30, 32, 34 or 36 and do not destroy the
resulting antibodies ability to bind the HIR. Preferably, the
alternate FRs will have at least 80 percent sequence identity with
the preferred VH FR that is being replaced. Even more preferred are
alternate FRs that have at least 90 percent sequence identity with
the preferred VH FR that is being replaced.
[0032] The four VH FR amino acid sequences may also be viewed as a
combined group of amino acid sequences (VH FR1, VH FR2, VH FR3 and
VH FR4). The present invention also covers alternates or
equivalents of the combined group of VH FR sequences. Such
"combined equivalents" are those that have at least 70 percent
sequence identity with the combined amino acid sequences SEQ. ID.
NOS. 30, 32, 34 and 36 and which do not adversely affect the
binding of the antibody to the HIR. Preferably, combined equivalent
amino acid sequences will have at least 80 percent sequence
identity with the combined sequences found in SEQ. ID. NOS. 30, 32,
34 and 36. Even more preferred are combined equivalent amino acid
sequences that have at least 90 percent sequence identity with the
combined amino acid sequences (SEQ. ID. NOS. 30, 32, 34 and
36).
[0033] It is preferred that the alternate VH FR amino acid
sequences meet both the individual equivalency and combined
equivalency requirements set forth above. However, there are
certain situations, especially for the shorter FRs, where one or
more of the FRs may not meet the criteria for individual
equivalence even though the criteria for combined equivalence is
met. In such situations, the individual equivalency requirements
are waived provided that the combined equivalency requirements are
met.
[0034] The first framework region (FR1) of the LC is located at the
amino end of the VL of the humanized antibody. The fourth framework
region (FR4) is located towards the carboxyl end of the VL of the
humanized antibody. Exemplary preferred amino acid sequences for
the humanized VL FR1, FR2, FR3 and FR4 are set forth in SEQ. ID.
NOS. 37, 39, 41 and 43, respectively. The amino acid sequences for
VL FR1, FR2, FR3 and FR4 (SEQ. ID. NOS. 37, 39, 41 and 43)
correspond to the REI human antibody framework regions that have
amino acid sequences that differ from murine MAb 83-14 (See FIG.
4). It is possible to modify the preferred VL FR sequences without
destroying the biological activity of the antibody. Suitable
alternate or equivalent FRs include those that have at least 70
percent sequence identity with SEQ. ID. NOS. 37, 39, 41 and 43 and
do not destroy the resulting antibodies ability to bind the HIR.
Preferably, the equivalent or alternate FRs will have at least 80
percent sequence identity with the preferred VL FR that is being
replaced. Even more preferred are alternate FRs that have at least
90 percent sequence identity with the preferred VL FR that is being
replaced.
[0035] The four VL FR amino acid sequences may also be viewed as a
combined group of amino acid sequences (VL FR1, VL FR2, VL FR3 and
VL FR4). The present invention also covers alternates or
equivalents of the combined group of VL FR sequences. Such
"combined equivalents" are those that have at least 70 percent
sequence identity with the combined amino acid sequences SEQ. ID.
NOS. 37, 39, 41 and 43 and which do not adversely affect the
binding of the antibody to the HIR. Preferably, combined equivalent
amino acid sequences will have at least 80 percent sequence
identity with the combined sequences found in SEQ. ID. NOS. 37, 39,
41 and 43. Even more preferred are combined equivalent amino acid
sequences that have at least 90 percent sequence identity with the
combined amino acid sequences (SEQ. ID. NOS. 37, 39, 41 and
43).
[0036] It is preferred that the alternate VL FR amino acid
sequences meet both the individual equivalency and combined
equivalency requirements set forth above. However, there are
certain situations, especially for the shorter FRs, where one or
more of the FRs may not meet the criteria for individual
equivalence even though the criteria for combined equivalence is
met. In such situations, the individual equivalency requirements
are waived provided that the combined equivalency requirements are
met.
[0037] Version 5 is a preferred humanized antibody in accordance
with the present invention. The amino acid sequences for the VH and
VL of Version 5 are set forth in SEQ. ID. NOS. 5 and 6,
respectively. The preparation and identification of Version 5 is
set forth in more detail in Example 2, Table 3 and FIG. 4. The
amino acid sequences for the VH FRs of Version 5 correspond to the
preferred VH FR sequences set forth above (SEQ. ID. NOS. 30, 32, 34
and 36). In addition, the amino acid sequences for the VL FRs of
Version 5 correspond to the preferred VL FR sequences set forth
above (SEQ. ID. NOS. 37, 39, 41, 43). The VH and VL FRs of Version
5 are a preferred example of VH and VL LC FRs that have been
"humanized". "Humanized" means that the four framework regions in
either the HC or LC have been matched as closely as possible with
the FRs from a human antibody (HAb) without destroying the ability
of the resulting antibody to bind the HIR. The model human antibody
used for the HC is the B43 antibody, and the model human antibody
used for the LC is the REI antibody, and both the B43 and REI
antibody sequences are well known and available in public
databases. When the HC or LC FRs are humanized, it is possible that
one or more of the FRs will not correspond identically with the
chosen HAb template and may retain identity or similarity to the
murine antibody. The degree to which murine amino acid sequences
are left in the humanized FRs should be kept as low as possible in
order to reduce the possibility of an immunogenic reaction in
humans.
[0038] Examples of FRs that have been humanized are set forth in
Example 2 and Table 3. Framework regions from human antibodies that
correspond closely to the FRs of murine MAb 84-13 are chosen. The
human FRs are then substituted into the MAb 84-13 in place of the
murine FRs. The resulting antibody is then tested. The FRs, as a
group, are only considered to be humanized if the modified antibody
still binds strongly to the HIR receptor and has reduced
immunogenicity in humans. If the first test is not successful, then
the human FRs are modified slightly and the resulting antibody
tested. Exemplary human antibodies that have HC FRs that may be
used to humanize the HC FRs of MAb 84-13 include B43 human IgG
(SEQ. ID. NO. 12), which is deposited in Genbank (accession number
S78322), and other human IgG molecules with a VH homologous to the
murine 83-14 VH may be found by searching public databases, such as
the Kabat Database of immunoglobulin sequences. Exemplary human
antibodies that have LC FRs that may be used to humanize the LC FRs
of MAb 84-13 include human REI antibody (SEQ. ID. NO. 13), which is
deposited in Genbank (accession number 1WTLB), and other human IgG
molecules with a VL homologous to the murine 83-14 VL may be found
by searching public databases, such as the Kabat Database of
immunoglobulin sequences.
[0039] In order for the humanized antibody to function properly,
the HC and LC should each include a constant region. Any number of
different human antibody constant regions may be incorporated into
the humanized antibody provided that they do not destroy the
ability of the antibody to bind the HIR. Suitable human antibody HC
constant regions include human IgG1, IgG2, IgG3, or IgG4. The
preferred HC constant region is human IgG1. Suitable human antibody
LC constant regions include kappa (.kappa.) or lambda. Human
.kappa. LC constant regions are preferred.
[0040] The humanized antibody may be used in the same manner as any
of the other antibody targeting agents (Trojan Horses) that have
previously been used to deliver genes, drugs and diagnostic agents
to cells by accessing the HIR. The humanized antibody is typically
linked to a drug or diagnostic compound (pharmaceutical agent) and
combined with a suitable pharmaceutical carrier and administered
intravenously (iv). With suitable carriers, the drug/humanized
antibody complex could also be administered subcutaneously,
intramuscularly, intra-nasally, intra-thecally, or orally. There
are a number of ways that the humanized antibody may be linked to
the pharmaceutical agent. The humanized antibody may be fused to
either avidin or streptavidin and conjugated to a pharmaceutical
agent that has been mono-biotinylated in accordance with known
procedures that use the avidin-biotin linkage to conjugate antibody
Trojan Horses and pharmaceutical agents together. Alternatively,
the humanized antibody and pharmaceutical agent may be expressed as
a single fusion protein using known genetic engineering
procedures.
[0041] Exemplary pharmaceutical agents to which the humanized
antibody may be linked include small molecules, recombinant
proteins, synthetic peptides, antisense agents or nanocontainers
for gene delivery. Exemplary recombinant proteins include basic
fibroblast growth factor (bFGF), human .alpha.-L-iduronidase
(IDUA), or other neurotrophins, such as brain derived neurotrophic
factor, or other lysosomal enzymes. The use of Trojan Horses, such
as the present humanized antibody, for transporting bFGF across the
BBB is described in a co-pending United States patent application
(UC Case 2002-094-1, Attorney Docket 0180-0027) that is owned by
the same assignee as the present application and which was filed on
the same day as the present application)
[0042] Once the humanized antibody is linked to a pharmaceutical
agent, it is administered to the patient in the same manner as
other known conjugates or fusion proteins. The particular dose or
treatment regimen will vary widely depending upon the
pharmaceutical agent being delivered and the condition being
treated. The preferred route of administration is intravenous (iv).
Suitable carriers include saline or water buffered with acetate,
phosphate, TRIS or a variety of other buffers, with or without low
concentrations of mild detergents, such as one from the Tween
series of detergents. The humanized antibody/pharmaceutical agent
Trojan horse compound is preferably used to deliver
neuropharmaceutical agents across the BBB. However, the humanized
Trojan horse may also be used to deliver pharmaceutical agents, in
general, to any organ or tissue that carries the HIR.
[0043] The following examples describe how the humanized monoclonal
antibodies in accordance with the present invention were discovered
and additional details regarding their fabrication and use.
EXAMPLE 1
Cloning of Murine 83-14 VH and VL Genes
[0044] Poly A+ RNA was isolated from the 83-14 hybridoma cell line
(Soos et al., 1986), and used to produce complementary DNA (cDNA)
with reverse transcriptase. The cDNA was used with polymerase chain
reaction (PCR) amplification of either the 83-14 VH or 83-14 VL
gene using oligodeoxynucleotide (ODN) primers that specifically
amplify the VH and VL of murine antibody genes, and similar methods
are well known (Li et al., 1999). The sequences of PCR ODNs
suitable for PCR amplification of these gene fragments are well
known (Li., 1999). The PCR products were isolated from 1% agarose
gels and the expected 0.4 Kb VH and VL gene products were isolated.
The VH and VL gene fragments were sequentially subcloned into a
bacterial expression plasmid so as to encode a single chain Fv
(ScFv) antibody. The ScFv expression plasmid was then used to
transform E. Coli. Individual colonies were identified on agar
plates and liquid cultures were produced in LB medium. This medium
was used in immunocytochemistry of Rhesus monkey brain to identify
clones producing antibody that bound avidly to the Rhesus monkey
brain microvasculature or BBB. This immunocytochemistry test
identified those colonies secreting the functional 83-14 ScFv.
Following identification of the 83-14 VH and VL genes, the
nucleotide sequence was determined in both directions using
automatic DNA sequencing methods. The nucleotide sequence of the
murine 83-14 VH (SEQ. ID. NO. 1) and the murine VL (SEQ. ID. NO. 2)
gives the deduced amino acid sequence for the murine VH (SEQ. ID.
NO. 3) and the murine VL (SEQ. ID. NO. 4). The amino acid sequence
is given for all 3 CDRs and all 4 FRs of both the HC and the LC of
the murine 83-14 HIRMAb. The variable region of the LC is
designated VL, and the variable region of the HC is designated VH
in FIG. 1.
EXAMPLE 2
Iterative Humanization of the 83-14 HIRMAb: Version 1 Through
Version 5
[0045] Humanization of the 83-14 MAb was performed by CDR/FR
grafting wherein the mouse FRs in the 83-14 MAb are replaced by
suitable human FR regions in the variable regions of both the LC
and HC. The Kabat database was screened using the Match program.
Either the murine 83-14 VH or the VL amino acid sequence was
compared with human IgG VH or human .kappa. light chain VL
databases. Using the minimal mismatch possible, several human IgG
molecules were identified that contained FR amino sequences highly
homologous to the amino acid sequences of the murine 83-14 VH and
VL. The framework regions of the B43 human IgG1 heavy chain and the
REI human .kappa. light chain were finally selected for CDR/FR
grafting of the murine 83-14 HIRMAb.
[0046] Sets of 6 ODN primers, of 69-94 nucleotides in length, were
designed to amplify the synthetic humanized 83-14 VL and VH genes
(Tables 1 and 2). The ODN primers overlapped 24 nucleotides in both
the 5'- and 3'-ends, and secondary structure is analyzed with
standard software. Stable secondary structure producing T.sub.m of
>46.degree. C. was corrected by replacement of first, second, or
third letter codons to reduce the melting point of these structures
to 32-46.degree. C. In addition, primers corresponding to both 5'
and 3' ends were also designed, and these allowed for PCR
amplification of the artificial genes. These new sequences lack any
consensus N-glycosylation sites at asparagine residues.
TABLE-US-00001 TABLE 1 Oligodeoxynucleotides for CDR/FR grafting of
VL Primer 1 FWD (SEQ. ID. NO. 14)
5'TAGGATATCCACCATGGAGACCCCCGCCCAGCTGCTGTTCCTGTTGCT
GCTTTGGCTTCCAGATACTACCGGTGACATCCAGATGACCCAG-3' Primer 2 reverse
(SEQ. ID. NO. 15)
5'GTCCTGACTAGCCCGACAAGTAATGGTCACTCTGTCACCCACGCTGGC
GCTCAGGCTGCTTGGGCTCTGGGTCATCTGGATGTCGCCGGT-3' Primer 3 FWD (SEQ.
ID. NO. 16) 5'ATTACTTGTCGGGCTAGTCAGGACATTGGAGGAAACTTATATTGGTAC
CAACAAAAGCCAGGTAAAGCTCCAAAGTTACTGATCTACGCC-3' Primer 4 reverse
(SEQ. ID. NO. 17)
5'GGTGTAGTCGGTACCGCTACCACTACCACTGAATCTGCTTGGCACACC
AGAATCTAAACTAGATGTGGCGTAGATCAGTAACTTTGGAGC-3' Primer 5 FWD (SEQ.
ID. NO. 18) 5'AGTGGTAGCGGTACCGACTACACCTTCACCATCAGCAGCTTACAGCCA
GAGGACATCGCCACCTACTATTGCCTACAGTATTCTAGTTCT-3' Primer 6 reverse
(SEQ. ID. NO. 19)
5'CCCGTCGACTTCAGCCTTTTGATTTCCACCTTGGTCCCTTGTCCGAAC
GTCCATGGAGAACTAGAATACTGTAGGCAATA-3' 5-PCR primer FWD (SEQ. ID. NO.
20) 5'TAGGATATCCACCATGGAGACCCC-3' 3-PCR primer reverse (SEQ. ID.
NO. 21) 5'CCCGTCGACTTCAGCCTTTTGATT-3'
[0047] TABLE-US-00002 TABLE 2 Oligodeoxynucleotides for CDR/FR
grafting of VH PRIMER 1 FWD (SEQ. ID. NO. 22)
5'TAGGATATCCACCATGGACTGGACCTGGAGGGTGTTATGCCTGCTTGC
AGTGGCCCCCGGAGCCCACAGCCAAGTGCAGCTGCTCGAGTCTGGG-3' PRIMER 2 REVERSE
(SEQ. ID. NO. 23)
5'GTTTGTGAAGGTGTAACCAGAAGCCTTGCAGGAAATCTTCACTGAGGA
CCCAGGCCTCACCAGCTCAGCCCCAGACTCGAGCAGCTGCACTTG-3' PRIMER 3FWD (SEQ.
ID. NO. 24) 5'GCTTCTGGTTACACCTTCACAAACTACGATATACACTGGGTGAAGCAG
AGGCCTGGACAGGGTCTTGAGTGGATTGGATGGATTTATCCTGGA-3' PRIMER 4 REVERSE
(SEQ. ID. NO. 25)
5'GCTGGAGGATTCGTCTGCAGTCAGAGTGGCTTTGCCCTTGAATTTCTC
ATTGTACTTAGTACTACCATCTCCAGGATAAATCCATCCAATCCA-3' PRIMER 5 FWD (SEQ.
ID. NO. 26) 5'CTGACTGCAGACGAATCCTCCAGCACAGCCTACATGCAACTAAGCAGC
CTACGATCTGAGGACTCTGCGGTCTATTCTTGTGCAAGAGAGTGG-3' PRIMER 6 REVERSE
(SEQ. ID. NO. 27)
5'CATGCTAGCAGAGACGGTGACTGTGGTCCCTTGTCCCCAGTAAGCCCA
CTCTCTTGCACAAGAATAGAC-3' 5'-PCR PRIMER FWD (SEQ. ID. NO. 28)
5'TAGGATATCCACCATGGACTGGACCTG-3' 3'-PRC PRIMER REV (SEQ. ID. NO.
29) 5'CATGCTAGCAGAGACGGTGACTGTG-3'
[0048] The PCR was performed in a total volume of 100 .mu.L
containing 5 pmole each of 6 overlapping ODNs, nucleotides, and Taq
and Taq extender DNA polymerases. Following PCR, the humanized VH
and VL genes were individually ligated in a bacterial expression
plasmid and E. coli was transformed. Several clones were isolated,
individually sequenced, and clones containing no PCR-introduced
sequence errors were subsequently produced.
[0049] The humanized VH insert was released from the bacterial
expression plasmid with restriction endonucleases and ligated into
eukaryotic expression vectors described previously (Coloma et al.,
1992; U.S. Pat. No. 5,624,659). A similar procedure was performed
for the humanized VL synthetic gene. Myeloma cells were transfected
with the humanized light chain gene, and this cell line was
subsequently transfected with version 1 of the humanized heavy
chain gene (Table 3). The transfected myeloma cells were screened
in a 96-well ELISA to identify clones secreting intact human IgG.
After multiple attempts, no cell lines producing human IgG could be
identified. Conversely, Northern blot analysis indicated the
transfected cell lines produced the expected humanized 83-14 mRNA,
which proved the transfection of the cell line was successful.
These results indicated that version 1 of the humanized HIRMAb,
which contains no FR amino acid substitutions, was not secreted
from the cell, and suggested the humanized HC did not properly
assemble with the humanized LC. Version 1 was derived from a
synthetic HC gene containing FR amino acids corresponding to the
25C1'C1 antibody (Bejcek et al., 1995). Therefore, a new HC
artificial gene was prepared, which contained HC FR amino acids
derived from a different human IgG sequence, that of the B43 human
IgG (Bejcek et al., 1995), and this yielded version 2 of the
humanized HIRMAb (Table 3). However, the version 2 humanized HIRMAb
was not secreted by the transfected myeloma cell. Both versions 1
and 2 contain the same HC signal peptide (Table 3), which is
derived from Rechavi et al. (1983). In order to evaluate the effect
of the signal peptide on IgG secretion, the signal peptide sequence
was changed to that used for production of the chimeric HIRMAb
(Coloma et al., 2000), and the sequence of this signal peptide is
given in Table 3. Versions 2 and 3 of the humanized HIRMAb differed
only with respect to the signal peptide (Table 3). However, version
3 was not secreted from the myeloma cell, indicating the signal
peptide was not responsible for the lack of secretion of the
humanized HIRMAb.
[0050] The above findings showed that simply grafting the murine
83-14 CDRs on to human FR regions produced a protein that could not
be properly assembled and secreted. Prior work had shown that the
chimeric form of the HIRMAb was appropriately processed and
secreted in transfected myeloma lines (Coloma et al, 2000). This
suggested that certain amino acid sequences within the FRs of the
humanized HC or LC prevented the proper assembly and secretion of
the humanized HIRMAb. Therefore, chimeric/humanized hybrid
molecules were engineered. Version 4a contained the murine FR1 and
the humanized FR2, FR3, and FR4; version 4b contained the murine FR
3, and FR4 and the humanized FR1 and FR2 (Table 3). Both versions
4a and 4b were secreted, although version 4b was more active than
version 4a. These findings indicated amino acids within either FR3
or FR4 were responsible for the lack of secretion of the humanized
HIRMAb. The human and murine FR4 differed by only 1 amino acid
(Table 3); therefore, the sequence of FR4 was altered by
site-directed mutagenesis to correspond to the human sequence, and
this version was designated version 5 (Table 3). The version 5
HIRMAb corresponded to the original CDR-grated antibody sequence
with substitution of the human sequence in FR3 of the VH with the
original murine sequence for the FR3 in the VH. The same
CDR-grafted LC, without any FR substitutions, was used in
production of all versions of the humanized HIRMAb. This
corresponds with other work showing no FR changes in the LC may be
required (Graziano et al., 1995). TABLE-US-00003 TABLE 3 Iterations
of Genetic Engineering of Humanized HIRMAb Heavy Chain FR1 CDR1 FR2
Version 5 QVQLLESGAELVRPGSSVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
Version 4b QVQLLESGAELVRPGSSVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
Version 4a QVQLQESGPELVKPGALVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
Version 3 QVQLLESGAELVRPGSSVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
Version 2 QVQLLESGAELVRPGSSVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
Version 1 QVQLLESGAELVRPGSSVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG
murine QVQLQESGPELVKPGALVKISCKAS GYTFTNYDIH WVKQRPGQGLEWIG human
B43 QVQLLESGAELVRPGSSVKISCKAS GYAFSSYWMN WVKQRPGQGLEWIG 1 26 36
CDR2 FR3 Version 5 WIYPGDGSTKYNEKFKG
KATLTADKSSSTAYMHLSSLTSEKSAVYFCAR Version 4b WIYPGDGSTKYNEKFKG
KATLTADKSSSTAYMHLSSLTSEKSAVYFCAR Version 4a WIYPGDGSTKYNEKFKG
KATLTADESSSTAYMQLSSLRSEDSAVYSCAR Version 3 WIYPGDGSTKYNEKFKG
KATLTADESSSTAYMQLSSLRSEDSAVYSCAR Version 2 WIYPGDGSTKYNEKFKG
KATLTADESSSTAYMQLSSLRSEDSAVYSCAR Version 1 WIYPGDGSTKYNEKFKG
QATLTADKSSSTAYMQLSSLTSEDSAVYSCAR murine WIYPGDGSTKYNEKFKG
KATLTADKSSSTAYMHLSSLTSEKSAVYFCAR human B43 QIWPGDGDTNYNGKFKG
KATLTADESSSTAYMQLSSLRSEDSAVYSCAR 50 67 CDR3 FR4 Version 5
-----------EWAY WGQGTTVTVSA (SEQ. ID. NO. 5) Version 4b
-----------EWAY WGQGTLVTVSA (SEQ. ID. NO. 11) Version 4a
-----------EWAY WGQGTTVTVSA (SEQ. ID. NO. 10) Version 3
-----------EWAY WGQGTTVTVSA (SEQ. ID. NO. 9) Version 2
-----------EWAY WGQGTTVTVSA (SEQ. ID. NO. 8) Version 1
-----------EWAY WGQGTTVTVSA (SEQ. ID. NO. 7) murine -----------EWAY
WGQGTLVTVSA (SEQ. ID. NO. 3) human B43 RETTTVGRYYYAMDY WGQGTTVT---
(SEQ. ID. NO. 12) 99 99 103 113
[0051] Version 1 was designed using the FRs of the human 25C1C1 IgG
heavy chain (HC) variable region (VH). Version 1 did not produce
secreted hIgG from the transfected myeloma cells despite high
abundance of the HC mRNA determined by Northern blot analysis.
[0052] Version 2 was re-designed using the FRs of the human B43 IgG
HC variable region. The peptide signal #1 (MDWTWRVLCLLAVAPGAHS)
(SEQ. ID. NO. 49) in versions 1 and 2 was replaced by signal
peptide #2 (MGWSWVMLFLLSVTAGKGL) (SEQ. ID. NO. 50) in version 3.
The FRs and CDRs in version 2 and 3 are identical. The signal
peptide #2 was used for versions 4a, 4b and 5.
[0053] Verson 4a has human FRs 2, 3 and 4 and murine FR1.
[0054] Version 4b has human FRs 1 and 2, and murine FRs 3 and 4
[0055] Version 5 vas produced using the human FRs 1, 2 and 4 and
the murine FR3.
[0056] Versions 4a, 4b and 5 produced secreted hIgG, whereas
version 1, 2, and 3 did not secrete IgG. Among versions 4a, 4b, and
5, version 5 contains fewer murine framework amino acid
substitutions and is preferred.
[0057] The version 5 form of the protein was secreted intact from
the transfected myeloma lines. The secreted version 5 humanized
HIRMAb was purified by protein A affinity chromatography and the
affinity of this antibody for the HIR was tested with an
immunoradiometric assay (IRMA), which used [.sup.125I]-labeled
murine 83-14 MAb as the ligand as described previously (Coloma et
al., 2000). These results showed that the affinity of the antibody
for the HIR was retained. In the IRMA, the antigen was the
extracellular domain of the HIR, which was produced from
transfected CHO cells and purified by lectin affinity
chromatography of CHO cell conditioned medium. The dissociation
constant (K.sub.D) of the murine and Version 5 humanized 83-14
HIRMAb was 2.7.+-.0.4 nM and 3.7.+-.0.4 nM, respectively. These
results show that the 83-14 HIRMAb has been successfully humanized
using methods that (a) obtain the FR regions of the HC and of the
LC from different human immunoglobulin molecules, and (b) do not
require the use of molecular modeling of the antibody structure, as
taught in U.S. Pat. No. 5,585,089. Similar to other applications
(Graziano et al., 1995), no FR amino acid changes in the LC of the
antibody were required.
EXAMPLE 3
Binding of the Humanized HIRMAb to the Human BBB
[0058] Prior work has reported that the radiolabelled murine HIRMAb
avidly binds to human brain capillaries with percent binding
approximately 400% per mg protein at 60-120 minutes of incubation
(Pardridge et al., 1995). Similar findings were recorded with
radiolabelled Version 5 humanized HIRMAb in this example. When
human brain capillaries were incubated in a radioreceptor assay
with [.sup.125I] Version 5 humanized HIRMAb, the percent binding
approximated 400% per mg protein by 60 minutes of incubation at
room temperature, and approximated the binding to the human brain
capillary of the [.sup.125I-chimeric HIRMAb (see FIGS. 2A and 2B).
In contrast, the binding of a nonspecific IgG to human brain
capillaries is less than 5% per mg protein during a comparable
incubation period Pardridge et al., 1995). This example shows that
the Version 5 humanized HIRMAb was avidly bound and endocytosed by
the human brain capillary, which forms the BBB in vivo.
EXAMPLE 4
Transport of Humanized HIRMAb Across the Primate BBB In Vivo
[0059] The humanized Version 5 HIRMAb was radiolabelled with
125-Iodine and injected intravenously into the adult Rhesus monkey.
The animal was sacrificed 2 hours later and the brain was removed
and frozen. Cryostat sections (20 micron) were cut and applied to
X-ray film. Scanning of the film yielded an image of the primate
brain uptake of the humanized HIRMAb (FIG. 3). The white matter and
gray matter tracts of the primate brain are clearly delineated,
with a greater uptake in the gray matter as compared with the white
matter. The higher uptake of the human HIRMAb in the gray matter,
as compared with the white matter, is consistent with the 3-fold
higher vascular density in gray matter, and 3-fold higher
nonspecific IgG is injected into Rhesus monkeys there is no brain
uptake of the antibody (Pardridge et al., 1995). These film
autoradiography studies show that the humanized HIRMAb is able to
carry a drug (iodine) across the primate BBB in vivo. Based on the
high binding of the humanized HIRMAb to the human BBB (FIG. 2),
similar findings of high brain uptake in vivo would be recorded in
humans.
EXAMPLE 5
Affinity Maturation of the Antibody by CDR or FR Amino Acid
Substitution
[0060] The amino acid sequences of the VH of the HC and of the VL
of the LC are given in FIG. 4 for the Version 5 humanized HIRMAb,
the murine 83-14 HIRMAb, and either the B43 HC or the REI LC
antibodies. Given the CDR amino sequences in FIG. 4, those skilled
in the art of antibody engineering (Schier et al., 1996) may make
certain amino acid substitutions in the 83-14 HC or LC CDR
sequences in a process called "affinity maturation" or molecular
evolution. This may be performed either randomly or guided by x-ray
diffraction models of immunoglobulin structure, similar to single
amino acid changes made in the FR regions of either the HC or the
LC of an antibody (U.S. Pat. No. 5,585,089). Similarly, given the
FR amino acid sequences in FIG. 4, those skilled in the art can
make certain amino acid substitutions in the HC or LC FR regions to
further optimize the affinity of the HIRMAb for the target HIR
antigen. The substitutions should be made keeping in mind the
sequence identity limitations set forth previously for both the FR
and CDR regions. These changes may lead to either increased binding
or increased endocytosis or both.
EXAMPLE 6
Humanized HIRMAb/.alpha.-L-iduronidase Fusion Protein
[0061] .alpha.-L-iduronidase (IDUA) is the enzyme missing in
patients with Hurler syndrome or type I mucopolysaccharidosis
(MPS), which adversely affects the brain. The brain pathology
ultimately results in early death for children carrying this
genetic disease. IDUA enzyme replacement therapy (ERT) for patients
with MPS type I is not effective for the brain disease, because the
enzyme does not cross the BBB. This is a serious problem and means
the children with this disease will die early even though they are
on ERT. The enzyme could be delivered across the human BBB
following peripheral administration providing the enzyme is
attached to a molecular Trojan horse such as the humanized HIRMAb.
The IDUA may be attached to the humanized HIRMAb with avidin-biotin
technology. In this approach, the IDUA enzyme is mono-biotinylated
in parallel with the production of a fusion protein of the
humanized HIRMAb and avidin. In addition, the IDUA could be
attached to the humanized HIRMAb not with avidin-biotin technology,
but with genetic engineering that avoids the need for biotinylation
or the use of foreign proteins such as avidin. In this approach,
the gene encoding for IDUA is fused to the region of the humanized
HIRMAb heavy chain or light chain gene corresponding to the amino
or carboxyl terminus of the HIRMAb heavy or light chain protein.
Following construction of the fusion gene and insertion into an
appropriate prokaryotic or eukaryotic expression vector, the
HIRMAb/IDUA fusion protein is mass produced for purification and
manufacturing. The amino acid sequence and general structure of a
typical MAb/IDUA fusion protein is shown in FIG. 5 (SEQ. ID. NO.
48). In this construct, the enzyme is fused to the carboxy terminus
of the heavy chain (HC) of the humanized HIRMAb. The amino acid
sequence for the IDUA shown in FIG. 5 is that of the mature,
processed enzyme. Alternatively, the enzyme could be fused to the
amino terminus of the HIRMAb HC or the amino or carboxyl termini of
the humanized HIRMAb light chain (LC). In addition, one or more
amino acids within the IDUA sequence could be modified with
retention of the biological activity of the enzyme. Fusion proteins
of lysosomal enzymes and antibodies have been prepared and these
fusion proteins retain biological activity (Haisma et al., 1998).
The fusion gene encoding the fusion protein can be inserted in one
of several commercially available permanent expression vectors,
such as pCEP4, and cell lines can be permanently transfected and
selected with hygromycin or other selection agents. The conditioned
medium may be concentrated for purification of the recombinant
humanized HIRMAb/IDUA fusion protein.
EXAMPLE 7
Role of Light Chain (LC) in Binding of HIRMAb to the Human Insulin
Receptor
[0062] Myeloma cells (NSO) were transfected with a plasmid encoding
the either the humanized HIRMAb light chain or "surrogate light
chain", which was an anti-dansyl MAb light chain (Shin and
Morrison, 1990). The anti-dansyl light chain is derived from the
anti-dansyl IgG, where dansyl is a common hapten used in antibody
generation. Both the myeloma line transfected with the humanized
HIRMAb light chain, and the myleoma line transfected with the
surrogate light chain were subsequently transfected with a plasmid
encoding the heavy chain of the chimeric HIRMAb. One cell line
secreted an IgG comprised of the anti-HIRMAb chimeric heavy chain
and the anti-HIRMAb humanized light chain, and this IgG is
designated chimeric HIRMAb heavy chain/humanized HIRMAb light chain
IgG. The other cell line secreted an IgG comprised of a chimeric
HIRMAb heavy chain and the anti-dansyl light chain, and this IgG is
designated chimeric HIRMAb HC/dansyl LC IgG. Both cells lines
secreted IgG processed with either the humanized HIRMAb light chain
or the anti-dansyl light chain, as determined with a human IgG
ELISA on the myeloma supernatants. These data indicated the
chimeric HIRMAb heavy chain could be processed and secreted by
myeloma cells producing a non-specific or surrogate light chain.
The reactivity of these chimeric antibodies with the soluble
extracellular domain (ECD) of the HIR was determined by ELISA. The
HIR ECD was purified by lectin affinity chromatography of the
conditioned medium of CHO cells transfected with the HIR ECD as
described previously (Coloma et al., 2000). In the HIR ECD ELISA,
the murine 83-14 HIRMAb was used as a positive control and mouse
IgG2a was used as a negative control. The negative control produced
negligible ELISA signals; the standard curve with the murine 83-14
MAb gave a linear increase in absorbance that reached saturation at
1 .mu.g/ml murine 83-14 MAb. The immune reaction in the ELISA was
quantified with a spectrophotometer and maximum absorbance at 405
nm (A405) in this assay was 0.9. All isolated myeloma clones
secreting the chimeric HIRMAb heavy chain/humanized HIRMAb light
chain IgG were positive in the HIR ECD ELISA with immuno-reactive
levels that maximized the standard curve. In addition, the myeloma
clones secreting the chimeric HIRMAb HC/dansyl LC IgG also produced
positive signals in the HIR ECD ELISA, and the A405 levels were
approximately 50% of the A405 levels obtained with the chimeric
HIRMAb heavy chain/humanized HIRMAb light chain IgG. These findings
indicate the light chain plays a minor role in binding of the
HIRMAb to its target antigen, which is the extracellular domain of
the human insulin receptor. This interpretation is supported by the
finding that no FR substitutions in the humanized LC were required
to enable active binding of the humanized HIRMAb to the HIR ECD
(see Example 2). These findings show that large variations in the
amino acid sequence of the HIRMAb light chain (50% and more) can be
made with minimal loss of binding of the intact humanized HIRMAb to
the target HIR antigen. Accordingly, a wide variety of LC's may be
used to prepare humanized antibodies in accordance with the present
invention provided that they are compatible with the HC. The LC is
considered to be "compatible" with the HC if the LC can be combined
with the HC and not destroy the ability of the resulting antibody
to bind to the HIR. In addition, the LC must be human or
sufficiently humanized so that any immunogenic reaction in humans
is minimized. Routine experimentation can be used to determine
whether a selected human or humanized LC sequence is compatible
with the HC.
[0063] Having thus described exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
within disclosures are exemplary only and that various other
alternatives, adaptations and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the above preferred embodiments and examples, but
is only limited by the following claims.
BIBLIOGRAPHY
[0064] Bruggemann, M. et al. (1989) "The immunogenicity of chimeric
antibodies," J. Exp. Med., 170:2153-2157. [0065] Coloma, M. J.,
Lee, H. J., Kurihara, A., Landaw, E. M., Boado, R. J., Morrison, S.
L., and Pardridge, W. M. (2000) "Transport across the primate
blood-brain barrier of a genetically engineered chimeric monoclonal
antibody to the human insulin receptor," Pharm. Res., 17:266-274.
[0066] Coloma, M. J., Hastings, A., Wims, L. A., and Morrison, S.
L. (1992) "Novel vectors for the expression of antibody molecules
using variable regions generated by polymerase chain reaction," J.
Immunol. Methods, 152:89-104. [0067] Foote, J. and Winter, G.
(1992) "Antibody framework residues affecting the conformation of
the hypervariable loops," J. Mol. Biol., 224:487-499. [0068]
Graziano, R. F. et al. (1995) "Construction and characterization of
a humanized anti-.gamma.-Ig receptor type I (Fc.gamma.RI)
monoclonal antibody," J. Immunol., 155:4996-5002. [0069] Haisma, J.
J. et al. (2000) Construction and characterization of a fusion
protein of single-chain anti-CD20 antibody and human
.beta.-glucuronidase for antibody-directed enzyme prodrug therapy.
Blood. 92: 184-190. [0070] Li, J. Y., Sugimura, K., Boado, R. J.,
Lee, H. J., Zhang, C., Dubel, S., and Pardridge, W. M. (1999)
"Genetically engineered brain drug delivery vectors--cloning,
expression, and in vivo application of an anti-transferrin receptor
single chain antibody-streptavidin fusion gene and protein,"
Protein Engineering, 12:787-796. [0071] Miller, G. (2002) "Breaking
down barriers," Science, 297:1116-1118. [0072] Pardridge, W. M.
(1997) "Drug delivery to the brain," J. Cereb. Blood Flow.
Metabol., 17:713-731. [0073] Pardridge, W. M., Buciak, J. L., and
Friden, P. M. (1991) "Selective transport of anti-transferrin
receptor antibody through the blood-brain barrier in vivo," J.
Pharmacol. Exp. Ther., 259:66-70. [0074] Pardridge, W. M., Kang,
Y.-S., Buciak, J. L., and Yang, J. (1995) "Human insulin receptor
monoclonal antibody undergoes high affinity binding to human brain
capillaries in vitro and rapid transcytosis through the blood-brain
barrier in vivo in the primate," Pharm., Res. 12:807-816. [0075]
Pichla, W. L., Murali, R., and Burnett, R. M. (1997) "The crystal
structure of a Fab fragment to the meelanoma-associated GD2
ganglioside," J. Struct. Biol., 119:6-16. [0076] Rechavi, G. et al.
(1983) "Evolutionary aspects of immunoglobulin heavy chain variable
region (VH) gene subgroups". Proc. Natl. Acad. Sci. (U.S.A) 80:
855-859. [0077] Schier, R. et al. (1996) "Isolation of picomolar
affinity anti-c-erbB-2 single-chain Fv by molecular evolution of
the complementarity determining regions in the center of the
antibody binding site," J. Mol. Biol., 263:551-557. [0078] Shin, S.
U. and Morrison, S. L. (1990) "Expression and characterization of
an antibody binding specificity joined to insulin-like growth
factor 1: potential applications for cellular targeting". Proc.
Natl. Acad. Sci. U.S.A. 87: 5322-5326. [0079] Soos, M. A., et al.
(1986) "Monoclonal antibodies reacting with multiple epitopes on
the human insulin receptor". Biochem. J. 235: 199-208. [0080] U.S.
Pat. No. 5,624,659 (issued Apr. 29, 1997) "Method of Treating Brain
Tumors Expressing Tenascin" (Inventors: Darell D. Bigner and
Michael R. Zalutsky; Assignee: Duke University). [0081] U.S. Pat.
No. 6,287,792 (issued Sep. 11, 2001) "Drug delivery of antisense
oligonucleotides and peptides to tissues in vivo and to cells using
avidin-biotin technology (Inventors: William M. Pardridge and Ruben
J. Boado; Assignee: University of California). [0082] Vogel, C. L.
et al. (2002) "Efficacy and safety of trastuzumab as a single agent
in first-line treatment of HER2--overexpressing metastatic breast
cancer," J. Clin. Oncol., 20:719-726. [0083] Wu, D., Yang, J., and
Pardridge, W. M. (1997): "Drug targeting of a peptide
radiopharmaceutical through the primate blood-brain barrier in vivo
with a monoclonal antibody to the human insulin receptor." J. Clin.
Invest., 100: 1804-1812. [0084] Zhang, Y., Lee, H. J., Boado, R.
J., and Pardridge, W. M. (2002) "Receptor-mediated delivery of an
antisense gene to human brain cancer cells," J. Gene Med.,
4:183-194.
Sequence CWU 1
1
* * * * *