U.S. patent application number 17/198029 was filed with the patent office on 2021-09-02 for enhanced delivery of drugs to the brain.
The applicant listed for this patent is Nascent Biotech, Inc.. Invention is credited to Mark C. Glassy, Rishab K Gupta.
Application Number | 20210269513 17/198029 |
Document ID | / |
Family ID | 1000005582959 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210269513 |
Kind Code |
A1 |
Glassy; Mark C. ; et
al. |
September 2, 2021 |
ENHANCED DELIVERY OF DRUGS TO THE BRAIN
Abstract
Compositions and methods related to antibodies or antibody
fragments which are capable of crossing the blood brain barrier are
provided.
Inventors: |
Glassy; Mark C.; (San Diego,
CA) ; Gupta; Rishab K; (Van Nuys, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nascent Biotech, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000005582959 |
Appl. No.: |
17/198029 |
Filed: |
March 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15261725 |
Sep 9, 2016 |
11028155 |
|
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17198029 |
|
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62217608 |
Sep 11, 2015 |
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62247490 |
Oct 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/2887 20130101; A61K 2039/505 20130101; C07K 2317/21
20130101; C07K 16/30 20130101; A61K 47/6865 20170801 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 16/30 20060101 C07K016/30; A61K 47/68 20060101
A61K047/68; C07K 16/28 20060101 C07K016/28 |
Claims
1.-46. (canceled)
47. An antibody comprising a heavy chain and a light chain, wherein
the heavy chain comprises a heavy chain acceptor framework of SEQ
ID NO: 1; and wherein the light chain comprises a light chain
acceptor framework of SEQ ID NO: 2, wherein the light chain CDR1,
CDR2 and CDR3 at amino acids 28-34, 50-56, and 91-97 of SEQ ID
NO:2, respectively, are replaced with the CDR1, CDR2 and CDR3 of a
desired antibody respectively; and wherein the heavy chain CDR1,
CDR2 and CDR3 at amino acids 31-35, 50-59, and 101-109 of SEQ ID
NO:1, respectively, are replaced with the CDR1, CDR2 and CDR3 of
the desired antibody, respectively.
48. The antibody of claim 47, wherein the desired antibody is
selected from the group consisting of ibritumomab, tositumomab,
ofatumumab, catumaxomab, gemtuzumab, alemtuzumab, eculizumab,
cetuximab, pantiumumab, trastuzumab, certoluzimab, bevacizumab,
ranibizumab, ipilimumab
49. The antibody of claim 47, wherein the heavy chain CDRs from the
desired antibody are from SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID
NO:11
50. The antibody of claim 47, wherein the light chain CDRs from the
desired antibody are from SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID
NO:11
51. The antibody of claim 47, wherein the recombinant antigen
binding protein has an isoelectric point of 8.0-9.0.
52. The antibody of claim 47, wherein the recombinant antigen
binding protein has an isoelectric point of about 8.7.
53. The antibody of claim 47, wherein the recombinant antigen
binding protein is capable of crossing the blood brain barrier.
54. A composition comprising the antibody of claim 47 and a
pharmaceutically acceptable carrier.
55. The composition of claim 54, wherein the composition is
formulated for delivery to the brain.
56. The composition of claim 54, wherein the composition is capable
of crossing the blood brain barrier.
57. The composition of claim 54, wherein the composition further
comprises one or more agents.
58. The composition of claim 57, wherein the agent is an imaging
agent.
59. The composition of claim 57, wherein the agent is a therapeutic
agent.
60. The composition of claim 59, wherein the therapeutic agent is a
chemotherapeutic agent.
61. The composition of claim 57, wherein the one or more agents are
conjugated to the antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/261,725, filed Sep. 9, 2016, which claims priority to U.S.
Provisional Application No. 62/217,608, filed Sep. 11, 2015 and
U.S. Provisional Application No. 62/247,490, filed Oct. 28, 2015,
all of which are incorporated herein by reference in their
entirety.
REFERENCE TO A "SEQUENCE LISTING," SUBMITTED AS ASCII TEXT FILED
VIA EFS-WEB
[0002] The Sequence Listing written in file
SeqListing-097408-1238323.txt created on Mar. 8, 2021, 32,925
bytes, machine format IBM-PC, MS-Windows operating system, in
accordance with 37 C.F.R. .sctn..sctn. 1.821 to 1.825, is hereby
incorporated by reference in its entirety for all purposes.
BACKGROUND
[0003] The blood brain barrier (BBB) poses a significant barrier
for the delivery of therapeutics to the brain. The BBB is a
protective endothelial tissue surrounding the CNS and poses a major
obstacle to the systemic delivery of therapeutic and diagnostic
agents for the treatment of neurological diseases. For example,
treatment of brain cancer or metastases of other solid tumors to
the brain is a highly unmet need. The lack of good treatments is
due to the invasive and infiltrating character of tumors in the
brain, and the inability of most effective biologic agents to cross
the BBB. If the BBB were leaky or can readily be overcome, then new
and useful drugs could be delivered to brain tissues. Previous
products designed to overcome or bypass the BBB have been difficult
to control thereby limiting their usefulness.
[0004] The present disclosure satisfies these and other needs by
providing antibodies or antibody fragments that are able to cross
the BBB.
SUMMARY
[0005] Described herein are compositions and methods related to
antibodies or antibody fragments which are able to cross the blood
brain barrier.
[0006] In a first aspect, disclosed herein is a recombinant antigen
binding protein comprising: (a) a heavy chain acceptor framework of
SEQ ID NO: 1 and at least one heterologous variable heavy chain CDR
specific for a desired antigen; and (b) a light chain acceptor
framework of SEQ ID NO: 2 and at least one heterologous variable
light chain CDR specific for a desired antigen.
[0007] In some embodiments, the recombinant antigen binding protein
comprises three heterologous variable heavy chain CDRs and three
heterologous variable light chain CDRs specific for a desired
antigen. In some embodiments, the antigen is selected from an
antigen listed in Table 1. In some embodiments, the variable heavy
chain CDR sequence is specific for an antigen listed in Table 1. In
some embodiments, the variable light chain CDR sequence is specific
for an antigen listed in Table 1.
[0008] In some embodiments, the recombinant antigen binding protein
has an isoelectric point of 8.0-9.0. In some embodiments, the
recombinant antigen binding protein has an isoelectric point of
about 8.7.
[0009] In some embodiments, the recombinant antigen binding protein
is capable of crossing the blood brain barrier.
[0010] In some embodiments, the heavy chain acceptor framework is
at least 90% identical to SEQ ID NO: 1. In some embodiments, the
light chain acceptor framework is at least 90% identical to SEQ ID
NO: 2.
[0011] In some embodiments, the antigen binding protein is a whole
immunoglobulin, scFv, Fab fragment, F(ab')2, Fab fragments linked
by a disulfide bridge at the hinge region, Fab' fragment, Fv,
single domain antibody (Dab), nanobody, or bispecific antibody.
[0012] In another aspect, disclosed herein is a nucleic acid
encoding the recombinant antigen binding protein of any of the
aspects and embodiments above.
[0013] In another aspect, disclosed herein is an expression vector
comprising the nucleic acid of any of the aspects and embodiments
above. In another aspect, disclosed herein is a host cell
comprising the expression vector of any of the aspects and
embodiments above. In some embodiments, the host cell is a
bacterial cell or eukaryotic cell, which can be a mammalian
cell.
[0014] In another aspect, disclosed herein is a method of
delivering a recombinant antigen binding protein across the blood
brain barrier comprising administering a recombinant antigen
binding protein in a therapeutically effective amount, wherein said
recombinant antigen binding protein comprises (a) a heavy chain
acceptor framework of SEQ ID NO: 1 and at least one heterologous
variable heavy chain CDR specific for a desired antigen; and (b) a
light chain acceptor framework of SEQ ID NO: 2 and at least one
heterologous variable light chain CDR specific for a desired
antigen.
[0015] In some embodiments, the recombinant antigen binding protein
comprises three heterologous variable heavy chain CDRs and three
heterologous variable light chain CDRs specific for a desired
antigen. In some embodiments, the antigen is selected from an
antigen listed in Table 1. In some embodiments, the variable heavy
chain CDR sequence is specific for an antigen listed in Table 1. In
some embodiments, the variable light chain CDR sequence is specific
for an antigen listed in Table 1.
[0016] In some embodiments, the recombinant antigen binding protein
has an isoelectric point of 8.0-9.0. In some embodiments, the
recombinant antigen binding protein has an isoelectric point of
about 8.7.
[0017] In some embodiments, the heavy chain acceptor framework is
at least 90% identical to SEQ ID NO: 1. In some embodiments, the
light chain acceptor framework is at least 90% identical to SEQ ID
NO: 2.
[0018] In some embodiments, the antigen binding protein is a whole
immunoglobulin, scFv, Fab fragment, F(ab')2, Fab fragments linked
by a disulfide bridge at the hinge region, Fab' fragment, Fv,
single domain antibody (Dab), nanobody, or bispecific antibody.
[0019] In a further aspect, disclosed herein is the use of the
recombinant antigen binding proteins of the above aspects and
embodiments for use in the treatment of cancer, infectious disease,
autoimmune disorders, or transplantation rejection.
[0020] Provided are methods of delivering an agent to the brain of
a subject, the method comprising administering to the subject a
composition comprising a conjugate comprising pritumumab and one or
more agents. Also provided are compositions and kits comprises a
composition comprising a conjugate comprising pritumumab and one or
more agents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic diagram illustrating the use of CDR
grafting to engineer new antigen binding specificities into the
pritumumab framework to generate new antigen binding proteins that
can cross the blood brain barrier. The CDRs of native pritumumab
allow binding to ectodomain vimentin (EDV). The native pritumumab
CDRs can be replaced with heterologous CDRs to generate binding
specificities to antigens, Ag1, Ag2, Ag3, and the like.
[0022] FIG. 2 is an image showing pritumumab crosses the blood
brain barrier and is detected in tumor tissue in the brain.
[0023] FIG. 3 is an image showing pritumumab distribution in normal
brain tissue.
[0024] FIG. 4 is an image showing pritumumab distribution in brain
tumor tissue.
[0025] FIG. 5 are images showing pritumumab specifically binds
tumor cells but not normal cells in a variety of tumor types.
DETAILED DESCRIPTION
[0026] Monoclonal antibodies (mAbs) are antibodies of a single
antigen specificity produced by identical immune cells, i.e.,
clones of a common germ cell. They offer unprecedented
opportunities for drug development because of their ability to
target almost any cell surface or secreted molecule with remarkable
specificity, efficacy, and safety.
[0027] As is well known, complementarity determining region (CDR)
segments are responsible for the ability of antibodies to bind to
their target antigens. Differences between the variable domains are
located on three loops known as hypervariable regions (HV-1, HV-2
and HV-3) or (CDR1, CDR2 and CDR3). CDRs are supported within the
variable domains by conserved framework regions. The present
disclosure relates to the use of the framework of a human natural
monoclonal antibody (PRITUMUMAB) to graft hypervariable regions
from other antibodies, such as, murine, chimerized, humanized, or
human monoclonal antibodies (mAbs) to generate new antigen binding
proteins that are able to cross the blood brain barrier.
[0028] The CDRs will be exchanged (as a group or individually)
between Abs of differing specificity and affinity. Swapping CDRs
(also called CDR grafting) is a technique that has been utilized
for the humanization of murine antibodies, and also for the
construction of more stable conventional antibody fragments. As
discussed herein, the framework of pritumumab is of human origin
and provides a unique characteristic of being able to cross the
BBB, and since the CDRs are both highly variable and selected for
binding affinity rather than stability it is proposed that
variation in CDRs on the framework of human IgG1 (pritumumab) is an
innovative approach to enhance clinical utility of monoclonal
antibodies that are highly target specific to treat various human
disorders but are not used due to their inability to cross the BBB.
As disclosed herein, we will construct a series of CDR-swap mutants
with the goal of understanding the contribution of the pritumumab
framework to deliver target specific monoclonal antibodies
containing CDRs of medical and therapeutic importance across the
BBB.
[0029] Specifically, the present inventors have shown that
pritumumab is a human antibody that readily crosses the BBB. By
placing different specificities and/or payloads on the antibody
then these specificities and/or payloads should be readily
delivered to the brain. The pritumumab heavy chain serves as a
carrier to cross the BBB and delivers the new specificity and/or
payload. Thus, the present disclosure exploits a natural human
antibody that readily crosses the BBB and uses this unique feature
to deliver additional drugs.
Definitions
[0030] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions or biological
systems, which can, of course, vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular aspects only, and is not intended to be limiting. As
used in this specification and the appended claims, the singular
forms "a", "an" and "the" include plural references unless the
content clearly dictates otherwise.
[0031] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of 20% or .+-.10%, more
preferably +5%, even more preferably +1%, and still more preferably
+0.1% from the specified value, as such variations are appropriate
to perform the disclosed methods.
[0032] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice for testing of the present
invention, the preferred materials and methods are described
herein.
[0033] "Vertebrate," "mammal," "subject," "mammalian subject," or
"patient" are used interchangeably and refer to mammals such as
human patients and non-human primates, as well as experimental
animals such as rabbits, rats, and mice, cows, horses, goats, and
other animals. Animals include all vertebrates, e.g., mammals and
non-mammals, such as mice, sheep, dogs, cows, avian species, ducks,
geese, pigs, chickens, amphibians, and reptiles.
[0034] "Treating" or "treatment" refers generally to either (i) the
prevention of disease, e.g., prophylaxis, or (ii) the reduction or
elimination of symptoms of a disease of interest, e.g., therapy.
Thus, treatment can be prophylactic (to prevent or delay the onset
of the disease, or to prevent the manifestation of clinical or
subclinical symptoms thereof) or therapeutic suppression or
alleviation of symptoms after the manifestation of the disease.
[0035] "Preventing" or "prevention" refers to prophylactic
administration with compositions of the invention.
[0036] "Therapeutically-effective amount" or "an effective amount"
refers to an amount of an antibody composition that is sufficient
to prevent a disease or to alleviate (e.g., mitigate, decrease,
reduce) at least one of the symptoms associated with a disease. It
is not necessary that the administration of the composition totally
eliminate the symptoms of the disease, as long as the benefits of
administration of the composition outweigh the detriments.
Likewise, the terms "treat" and "treating" in reference to a
disease, as used herein, are not intended to mean that the subject
is necessarily cured of the disease or that all clinical signs
thereof are eliminated, only that some alleviation or improvement
in the condition of the subject is effected by administration of
the composition.
Antibodies and Fragments
[0037] As used herein, the term "antibody" refers to any
immunoglobulin or intact molecule as well as to fragments thereof
that bind to a specific epitope. Such antibodies include, but are
not limited to polyclonal, monoclonal, chimeric, humanized, single
chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the
whole antibody and variants thereof. All isotypes are encompassed
by this term, including IgA, IgD, IgE, IgG, and IgM.
[0038] As used herein, the term "antibody fragment" refers
specifically to an incomplete or isolated portion of the full
sequence of the antibody which retains the antigen binding function
of the parent antibody. Examples of antibody fragments include Fab,
Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules; and multispecific antibodies
formed from antibody fragments.
[0039] An intact "antibody" comprises at least two heavy (H) chains
and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain is comprised of a heavy chain variable region
(abbreviated herein as HCVR or V.sub.H) and a heavy chain constant
region. The heavy chain constant region is comprised of three
domains, CH.sub.1, CH.sub.2 and CH.sub.3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
LCVR or V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxyl-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies can mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system. The term antibody includes
antigen-binding portions of an intact antibody that retain capacity
to bind. Examples of binding include (i) a Fab fragment, a
monovalent fragment consisting of the V.sub.L, V.sub.H, C.sub.L and
CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the V.sub.L and V.sub.H
domains of a single arm of an antibody, (v) a dAb fragment (Ward et
al., Nature, 341:544-546 (1989)), which consists of a VH domain;
and (vi) an isolated complementarity determining region (CDR).
[0040] The term "antigen binding protein" refers to a molecule that
contains all or a part of the antigen binding site of an antibody,
e.g. all or part of the heavy and/or light chain variable domain,
such that the antigen binding protein specifically recognizes a
target antigen. Non-limiting examples of antigen binding proteins
include full-length immunoglobulin molecules and scFvs, as well as
antibody fragments, including but not limited to (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains; (ii) a F(ab')2 fragment, a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fab' fragment, which is essentially a
Fab with part of the hinge region (see, Fundamental Immunology
(Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody, (vi) a
single domain antibody such as a Dab fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a V.sub.H or V.sub.L domain,
a Camelid (see Hamers-Casterman, et al., Nature 363:446-448 (1993),
and Dumoulin, et al., Protein Science 11:500-515 (2002)) or a Shark
antibody (e.g., shark Ig-NARs Nanobodies.TM.; and (vii) a nanobody,
a heavy chain variable region containing a single variable domain
and two constant domains.
[0041] The term "CDR" refers to one of the six hypervariable
regions within the variable domains of an antibody that mainly
contribute to antigen binding. One of the most commonly used
definitions for the six CDRs was provided by Kabat E. A. et al.,
(1991) Sequences of proteins of immunological interest. NIH
Publication 91-3242).
[0042] The term "antibody framework" as used herein refers to the
part of the variable domain, either VL or VH, which serves as a
scaffold for the antigen binding loops (CDRs) of this variable
domain. In essence it is the variable domain without the CDRs.
[0043] As used herein, the term "single chain antibodies" or
"single chain Fv (scFv)" refers to an antibody fusion molecule of
the two domains of the Fv fragment, V.sub.L and V.sub.H. Although
the two domains of the Fv fragment, V.sub.L and V.sub.H, are coded
for by separate genes, they can be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the V.sub.L and V.sub.H regions pair
to form monovalent molecules (known as single chain Fv (scFv); see,
e.g., Bird et al., Science, 242:423-426 (1988); and Huston et al.,
Proc Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chain
antibodies are included by reference to the term "antibody"
fragments and can be prepared by recombinant techniques or
enzymatic or chemical cleavage of intact antibodies.
[0044] As used herein, the term "human sequence antibody" includes
antibodies having variable and constant regions (if present)
derived from human germline immunoglobulin sequences. The human
sequence antibodies of the invention can include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo). Such antibodies can be
generated in non-human transgenic animals, e.g., as described in
PCT App. Pub. Nos. WO 01/14424 and WO 00/37504. However, the term
"human sequence antibody", as used herein, is not intended to
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences (e.g., humanized antibodies).
[0045] Also, recombinant immunoglobulins can be produced. See,
Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference
in its entirety and for all purposes; and Queen et al., Proc Natl
Acad Sci USA, 86:10029-10033 (1989).
[0046] As used herein, the term "monoclonal antibody" refers to a
preparation of antibody molecules of single molecular composition.
A monoclonal antibody composition displays a single binding
specificity and affinity for a particular epitope. Accordingly, the
term "human monoclonal antibody" refers to antibodies displaying a
single binding specificity which have variable and constant regions
(if present) derived from human germline immunoglobulin sequences.
In one aspect, the human monoclonal antibodies are produced by a
hybridoma which includes a B cell obtained from a transgenic
non-human animal, e.g., a transgenic mouse, having a genome
comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0047] As used herein, the term "antigen" refers to a substance
that prompts the generation of antibodies and can cause an immune
response. It can be used interchangeably in the present disclosure
with the term "immunogen". In the strict sense, immunogens are
those substances that elicit a response from the immune system,
whereas antigens are defined as substances that bind to specific
antibodies. An antigen or fragment thereof can be a molecule (i.e.,
an epitope) that makes contact with a particular antibody. When a
protein or a fragment of a protein is used to immunize a host
animal, numerous regions of the protein can induce the production
of antibodies (i.e., elicit the immune response), which bind
specifically to the antigen (given regions or three-dimensional
structures on the protein).
[0048] As used herein, the term "humanized antibody," refers to at
least one antibody molecule in which the amino acid sequence in the
non-antigen binding regions and/or the antigen-binding regions has
been altered so that the antibody more closely resembles a human
antibody, and still retains its original binding ability.
[0049] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison, et al., Proc Natl Acad Sci,
81:6851-6855 (1984), incorporated herein by reference in their
entirety) by splicing the genes from a mouse antibody molecule of
appropriate antigen specificity together with genes from a human
antibody molecule of appropriate biological activity can be used.
For example, the genes from a mouse antibody molecule can be
spliced together with genes from a human antibody molecule of
appropriate biological activity. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region.
[0050] In addition, techniques have been developed for the
production of humanized antibodies (see, e.g., U.S. Pat. Nos.
5,585,089 and 5,225,539, which are incorporated herein by reference
in their entirety). An immunoglobulin light or heavy chain variable
region consists of a "framework" region interrupted by three
hypervariable regions, referred to as complementarity determining
regions (CDRs). Briefly, humanized antibodies are antibody
molecules from non-human species having one or more CDRs from the
non-human species and a framework region from a human
immunoglobulin molecule.
[0051] Alternatively, techniques described for the production of
single chain antibodies can be adapted to produce single chain
antibodies against an immunogenic conjugate of the present
disclosure. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide. Fab and F(ab')2
portions of antibody molecules can be prepared by the proteolytic
reaction of papain and pepsin, respectively, on substantially
intact antibody molecules by methods that are well-known. See e.g.,
U.S. Pat. No. 4,342,566. Fab' antibody molecule portions are also
well-known and are produced from F(ab')2 portions followed by
reduction of the disulfide bonds linking the two heavy chain
portions as with mercaptoethanol, and followed by alkylation of the
resulting protein mercaptan with a reagent such as
iodoacetamide.
[0052] The amino acid positions can be indicated according to the
AHo numbering scheme. The AHo numbering system is described further
in Honegger, A. and Pluckthun, A. (2001) J. Mol. Biol.
309:657-670). Alternatively, the Kabat numbering system as
described further in Kabat et al. (Kabat, E. A., et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242) may be used. Conversion tables for the two different
numbering systems used to identify amino acid residue positions in
antibody heavy and light chain variable regions are provided in A.
Honegger, J. Mol. Biol. 309 (2001) 657-670.
[0053] In one aspect, the present disclosure provides a human
acceptor framework sequence for the grafting of CDRs from a
heterologous source. The human pritumumab framework was found to be
an especially useful framework.
[0054] Accordingly, the present invention provides an antigen
binding protein acceptor framework comprising i) a variable heavy
chain framework having at least 70% identity, preferably at least
75%, 80%, 85%, 90%, more preferably at least 95% identity, to SEQ
ID No. 1; and/or (ii) a variable light chain framework having at
least 70% identity, preferably at least 75%, 80%, 85%, 90%, more
preferably at least 95% identity, to SEQ ID No. 2. These sequences
are shown below:
TABLE-US-00001 SEQ ID NO. 1:
EVQLLESGGDLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSA
ITPSGGSTNYADSVKGRFTISRDNSQNTLYLQMNSLRVEDTAVYICGRVP
YRSTWYPLYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2:
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYA
ASSLHSKVPTQFSGSGSGTDFTLTISSLQPEDFATYYCLQYSTYPITEGG
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0055] A general method for grafting CDRs into human acceptor
frameworks has been disclosed by Winter in U.S. Pat. No. 5,225,539
and by Queen et al. in WO9007861A1, which are hereby incorporated
by reference in their entirety.
[0056] In exemplary embodiments of the methods of the invention,
the amino acid sequence of the CDR donor antibody is first
identified and the sequences aligned using conventional sequence
alignment tools (e.g., Needleman-Wunsch algorithm and Blossum
matrices). The introduction of gaps and nomenclature of residue
positions may be done using a conventional antibody numbering
system. For example, the AHo numbering system for immunoglobulin
variable domains may be used. The Kabat numbering scheme may also
be applied since it is the most widely adopted standard for
numbering the residues in an antibody. Kabat numbering may, e.g.,
be assigned using the SUBIM program. This program analyses variable
regions of an antibody sequence and numbers the sequence according
to the system established by Kabat and co-workers (Deret et al.
1995). The definition of framework and CDR regions is generally
done following the Kabat definition which is based on sequence
variability and is the most commonly used. Conversion tables for
the two different numbering systems used to identify amino acid
residue positions in antibody heavy and light chain variable
regions are provided in A. Honegger, J. Mol. Biol. 309 (2001)
657-670. The Kabat numbering system is described further in Kabat
et al. (Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242). The AHo numbering
system is described further in Honegger, A. and Pluckthun, A.
(2001) J. Mol. Biol. 309:657-670).
[0057] For example, the acceptor frameworks disclosed herein can be
used to generate a human or humanized antibody which retains the
binding properties of the antibody from which the CDRs are derived.
Accordingly, in a preferred embodiment the invention encompasses an
antigen binding protein acceptor framework as disclosed herein,
further comprising heavy chain CDR1, CDR2, and CDR3 and/or light
chain CDR1, CDR2, and CDR3 from a donor antigen binding protein.
Thus, in one embodiment, the invention provides an antigen binding
protein specific to a desired antigen comprising (i) variable heavy
and light chain CDRs; (ii) a human variable heavy chain framework
having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%,
or 100% identity to SEQ ID NO. 1; (iii) a human variable light
chain framework having at least 70%, preferably at least 75%, 80%,
85%, 90%, 95%, or 100% identity to SEQ ID NO. 2.
[0058] Examples of antibodies with CDRs useful in the practice of
the present disclosure include:
TABLE-US-00002 TABLE 1 Target/ First mAb Manufacturer Antigen
Indication Rituximab Roche/BiogenIDEC CD20 B-cell lymphoma
Ibritumomab Celldex Tositumomab GSK Ofatumumab GSK/Genmab CD3 CLL
Catumaxomab Trion CD3 and Ovarian cancer EPCAM ascites Gemtuzumab
Pfizer CD33 Acute myeloid Leukemi Alemtuzumab Genzyme CD52 B-cell
leukemia Eculizumab Alexion Complement Paroxysmal hemoglobinuria
Ca5 nocturnal Cetuximab Eli Lilly EGFR Colorectal cancer
Panitumumab Amgen Trastuzumab Roche HER2 Breast cancer Certoluzimab
UCB VEGF Crohn's disease Bevacizumab Roche Colorectal cancer
Ranibizumab Roche Macular degeneration Ipilimumab CD152 CTLA-4
Melanoma
[0059] Shown below is an alignment of the variable domains from a
number of monoclonal antibodies approved for cancer treatment, as
taken from Magdelaine-Beuzelin C, Kaas Q, Wehbi V, Ohresser M,
Jefferis R, Lefranc M-P, Watier H. Structure-function relationships
of the variable domains of monoclonal antibodies approved for
cancer treatment. Critical Reviews in Oncology/Hematology.
64:210-225, 2007. Cetuximab (SEQ ID NO:3) VH domain at amino acids
1-119 and V-kappa domain at amino acids 120 to 226, Rituximab (SEQ
ID NO:4) VH domain at amino acids 1 to 121 and V-kappa domain at
amino acids 122-226, Alemtuzmab (SEQ ID NO:5) VH domain at amino
acids 1 to 121 and V-kappa domain at amino acids 122-228,
Bevacizumab (SEQ ID NO:6) VH domain at amino acids 1 to 123 and
V-kappa domain at amino acids 124-230, Trastuzumab (SEQ ID NO:7) VH
domain at amino acids 1 to 119 and V-kappa domain at amino acids
120-225, Pertuzumab (SEQ ID NO:8) VH domain at amino acids 1 to 119
and V-kappa domain at amino acids 120-226, Panitumumab (SEQ ID
NO:9) VH domain at amino acids 1 to 96 and V-kappa domain at amino
acids 97-184.
TABLE-US-00003 VH domain FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT
certuximab SEQ ID NO: 3 (1-119) QVQLKQSGP.GLVQPSQSLSITCTVS
GFSL....TNYG VHWVRQSPGKGLEWLGV ##STR00001## rituximab.sup.1
QVQLQQPGA.ELVKPGASVKMSCKAS GYTF....TSYN MHWVKQTPGRGLEWIGA
IYPG..NGDT SEQ ID NO: 4 (1-121) alemtuzumab.sup.2 SEQ ID NO: 5
(1-121) QVQLQESGP.GLVRPSQTLSLTCTVS ##STR00002## MNWVRQPPGRGLEWIGF
##STR00003## bevacizumab SEQ ID NO: 6 (1-123)
EVQLVESGG.GLVQPGGSLRLSCAAS ##STR00004## ##STR00005## ##STR00006##
trastuzumab.sup.3 SEQ ID NO: 7 (1-119) EVQLVESGG.GLVQPGGSLRLSCAAS
GFNI....KDTY ##STR00007## IYPT..NGYT pertuzumab SEQ ID NO: 8
(1-119) EVQLVESGG.GLVQPGGSLRLSCAAS ##STR00008## MDWVRQAPGKGLEWVAD
##STR00009## panitumumab ........................VS GGSVS..SGDYY.
WTWIRQSPGKGLEWIGH IYYS...GNT SEQ ID NO: 9 (1-96) FR3-IMGT CDR3-IMGT
FR4IMGT certuximab SEQ ID NO: 3 (1-119)
DYNTPFT.SRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ##STR00010## WGQGTLVTVSA
rituximab.sup.1 SYNQKFX.GKATLTADKSSSTAYMQLSSLTSEDSAVYYC
ARSTYYG..GDWYFNV WGAGTTVTVSA SEQ ID NO: 4 (1-121) alemtuzumab.sup.2
SEQ ID NO: 5 (1-121) EYNPSVK.GRVTMLVDTSKNQFSLRLSSVTAADTAVYYC
##STR00011## WGQGSLVTVSS bevacizumab SEQ ID NO: 6 (1-123)
TYAADFK.RRFTFSLDTSKSTAYLQMNSLRAEDTAVYYC ##STR00012## WGQGTLVTVSS
trastuzumab.sup.3 SEQ ID NO: 7 (1-119)
RYADSVK.GRTISADTSKNTAYLQMNSLRAEDTAVYYC ##STR00013## WCQGTLVTVSS
pertuzumab SEQ ID NO: 8 (1-119)
IYNQRFK.GRFTLSVDRSKNTLYLQMNSLRAEDTAVYYC ##STR00014## WGQGTLVTVSS
panitumumab NYNPSLK.SRLTISIDTSKTQFSLKLSSVTAADTAIYYC
VRDRVT.....GAFDI WGQGTMVTVSS SEQ ID NO: 9 (1-96) V-kappa domain
FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT certuximab
DILLTQSPVILSVSPGERVSFSCRAS QSL......GTN IHWYQQRTNGSPRLLIK YA......S
SEQ ID NO: 3 (120-226) rituximab QIVLSQSPAILSASPGEKVTMTCRAS
SSV......SY IHWFQQKPGSSPKPWIY AT......S SEQ ID NO: 4 (122-226)
alemtuzumab DIQMTQSPSSLSASVGDRVTITCKAS QNI......DKY
LNWYQQKPGKAPKLLIY NT......N SEQ ID NO: 5 (122-228) bevacizumab
DIQMTQSPSSLSASVGDRVTITCSAS QDI......SNY LNWYQQKPGKAPKVLIY FT......S
SEQ ID NO: 6 (124-230) trastuzumab SEQ ID NO: 7 (120-225)
DIQMTQSPSSLSASVGDRVTITCRAS ##STR00015## VAWYQQKPGKAPKLLIY SA......S
pertuzumab DIQMTQSPSSLSASVGDRVTITCKAS QDV......SIG
VAWYQQKPGKAPKLLIY SA......S SEQ ID NO: 8 (120-226) panitumumab
...................TITCQAS QDI......SNY LNWYQQKPGKAPKLLIY DA......S
SEQ ID NO: 9 (97-184) V-kappa domain FR3-IMGT CDR3-IMGT FR4IMGT
certuximab SEQ ID NO: 3 (120-226)
ESISGIP.SRFSGSG..SGTDFTLSINSVESEDIASYYC ##STR00016## FGAGTKLELK.
rituximab NLASGVP.VRFSGSG..SGTSYSLTISRVEAEDAATYYC QQWTS....NPPT
FGGGTKLEIK SEQ ID NO: 4 (122-226) alemtuzumab SEQ ID NO: 5
(122-228) NLQTGVP.SRFSGSG..SGTDFTFTISSLQPEDIATYYC ##STR00017##
FGQGTKVEIK. bevacizumab SLHSGVP.SRFSGSG..SGTDFTLTISSLQPEDFATYYC
QQYST....VPWT FGQGTKVEIK. SEQ ID NO: 6 (124-230) trastuzumab SEQ ID
NO: 7 (120-225) FLYSGVP.SRFSGSR..SGTDFTLTISSLQPEDFATYYC
##STR00018## FGQGTKVEIK. pertuzumab
YRYTGVP.SRFSGSG..SGTDFTLTISSLQPEDFATYYC QQYYI....YPYT FGQGTKVEIK.
SEQ ID NO: 8 (120-226) panitumumab
LNETGCP.SRFSGSG..SGTDFTFTISSLQPEDIATYFC QHFDH....LPLA FGGGTKVEIK.
SEQ ID NO: 9 (97-184) .sup.1In WO9411026 PATENT, P15 > A15.
.sup.2In U.S. Pat. No. 5,846,534, F28 > S28 and T35 > 835.
.sup.3In U.S. Pat. No, 5,821,337, Y117 > V117. .sup.4In U.S.
Pat. No. 5,821,337, Y68 > E68.
[0060] The CDRs of ipilimumab are given in the US patent
2009/0074787 A1.
[0061] Shown below is an alignment of the VH and V-kappa domains of
bevacizumab (SEQ ID NO:10) VH domain at amino acids 1 to 123 and
V-kappa domain at amino acids 124-230 and ranibizumab (SEQ ID
NO:11) VH domain at amino acids 1 to 122 and V-kappa domain at
amino acids 123-229.
TABLE-US-00004 VH domain FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT
FR3-IMGT CDR3-IMGT FR4IMGT bevacizumab EVQLVESGG.GLV GYTF....TNYG
MNWVRQAPG INTY..TGEP TYAADFK.RRFTF AKYPHYYGSSHW WGQGTLVTVSS SEQ ID
QPGGSLRLSCAAS KGLEWVGW SLDTSKSTAYLQM YFDV NO: 10 NSLRAEDTAVYYC
(1-123) ranibizumab EVQLVESGG.GLV GYDF....THYG MNWVRQAPG INTY..TGEP
TYAADFK.RRFTF AKYPYYYGTSHW WGQGTLVTVSS SEQ ID QPGGSLRLSCAAS
KGLEWVGW SLDTSKSTAYLQM YFDV NO: 11 NSLRAEDTAVYYC (1-122) V-KAPPA
domain FR1-IMGT CDR1-IMGT FR2-IMGT CDR2-IMGT FR3-IMGT CDR3-IMGT
FR4IMGT bevacizumab DIQMTQSPSSLSA QDI......SNY LNWYQQKPG FT.......S
SLHSGVP.SRFSG QQYST...VPWT FGQGTKVEIK. SEQ ID SVGDRVTITCSAS
KAPKVLIY SG..SGTDFTLTI NO: 10 SSLQPEDFATYYC (124-230) ranibizumab
DIQLTQSPSSLSA QDI......SNY LNWYQQKPG FT.......S SLHSGVP.SRFSG
QQYST...VPWT FGQGTKVEIK. SEQ ID SVGDRVTITCSAS KAPKVLIY
SG..SGTDFTLTI NO: 11 SSLQPEDFATYYC (123-229)
[0062] In another aspect, the present invention features the
antibodies, or fragments thereof, disclosed herein conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates".
[0063] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0064] Since pritumumab can cross the blood brain barrier it can be
used as a delivery vehicle for delivering other agents, e.g.,
imaging or therapeutic agents to the brain or other tumor tissues.
Thus, provided are composition comprising an antibody comprising a
heavy chain and a light chain, the heavy chain comprising a
sequence that is at least 90% identical to SEQ ID NO:1 and the
light chain comprising a sequence that is at least 90% identical to
SEQ ID NO:2 and one or more agents, e.g., imaging or therapeutic
agents. Optionally, the agents are conjugated to the antibody.
Optionally, the therapeutic agent is a chemotherapeutic agent.
Optionally, the conjugate comprises a recombinant antigen binding
protein as described herein conjugated to one or more agents.
Optionally, the composition is formulated for delivery to the
brain. Optionally, the composition is capable of crossing the blood
brain barrier. Optionally, the heavy chain of the antibody
comprises SEQ ID NO:1 and the light chain comprises SEQ ID NO:2.
Optionally, the antibody is pritumumab.
[0065] Techniques for conjugating such therapeutic moieties to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0066] Also provided are methods of delivering an agent to the
brain of a subject. The method includes administering to the
subject a composition comprising an antibody comprising a heavy
chain and a light chain, the heavy chain comprising a sequence that
is at least 90% identical to SEQ ID NO:1 and the light chain
comprising a sequence that is at least 90% identical to SEQ ID NO:2
and one or more agents, e.g., imaging or therapeutic agents.
Optionally, the therapeutic agent is a chemotherapeutic agent.
Optionally, the heavy chain comprises SEQ ID NO:1 and the light
chain comprises SEQ ID NO:2. Optionally, the antibody is
pritumumab. Optionally, the antibody specifically binds tumor cells
but not normal cells.
[0067] Suitable therapeutic agents for use in the provided
compositions and methods, e.g., for conjugation to the provided
antibodies include, but are not limited to, therapeutic agent is
selected from the group consisting of analgesics, anesthetics,
analeptics, corticosteroids, anticholinergic agents,
anticholinesterases, anticonvulsants, antineoplastic agents,
allosteric inhibitors, anabolic steroids, antirheumatic agents,
psychotherapeutic agents, neural blocking agents, anti-inflammatory
agents, antihelmintics, antibiotics, anticoagulants, antifungals,
antihistamines, antimuscarinic agents, antimycobacterial agents,
antiprotozoal agents, antiviral agents, dopaminergics,
hematological agents, immunological agents, muscarinics, protease
inhibitors, vitamins, growth factors, and hormones. The choice of
agent and dosage can be determined readily by one of skill in the
art based on the given disease being treated.
[0068] As described herein, the antibodies can be linked or
conjugated to an imaging agent. Imaging agents and their use are
known. Optionally, the imaging agent is a "detectable moiety,"
which is a composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, chemical, or other physical means. For
example, useful labels include 32P, fluorescent dyes,
electron-dense reagents, enzymes (e.g., as commonly used in an
ELISA), biotin, digoxigenin, or haptens and proteins or other
entities which can be made detectable, e.g., by incorporating a
radiolabel into a peptide or antibody specifically reactive with a
target peptide. Any method known in the art for conjugating an
antibody to the label may be employed, e.g., using methods
described in Hermanson, Bioconjugate Techniques 1996, Academic
Press, Inc., San Diego. The detectable moiety can be selected from
the group consisting of gamma-emitters, beta-emitters, and
alpha-emitters, gamma-emitters, positron-emitters, X-ray-emitters
and fluorescence-emitters. Suitable fluorescent compounds include
fluorescein sodium, fluorescein isothiocyanate, phycoerythrin, and
Texas Red sulfonyl chloride, Allophycocyanin (APC), Cy5-PE,
CY7-APC, and Cascade yellow.
[0069] Optionally the detectable moiety can be visualized using
histochemical techniques, ELISA-like assays, confocal microscopy,
fluorescent detection, cell sorting methods, nuclear magnetic
resonance, radioimmunoscintigraphy, X-radiography, positron
emission tomography, computerized axial tomography, magnetic
resonance imaging, and ultrasonography.
Antibody Assays
[0070] A number of screening assays are known in the art for
assaying antibodies of interest to confirm their specificity and
affinity and to determine whether those antibodies cross-react with
other proteins.
[0071] The terms "specific binding" or "specifically binding" refer
to the interaction between the antigen and their corresponding
antibodies. The interaction is dependent upon the presence of a
particular structure of the protein recognized by the binding
molecule (i.e., the antigen or epitope). In order for binding to be
specific, it should involve antibody binding of the epitope(s) of
interest and not background antigens.
[0072] Once antibodies are produced, they are assayed to confirm
that they are specific for the antigen of interest and to determine
whether they exhibit any cross reactivity with other antigens. One
method of conducting such assays is a sera screen assay as
described in U.S. App. Pub. No. 2004/0126829, the contents of which
are hereby expressly incorporated herein by reference. However,
other methods of assaying for quality control are within the skill
of a person of ordinary skill in the art and therefore are also
within the scope of the present disclosure.
[0073] Antibodies, or antigen-binding fragments, variants or
derivatives thereof of the present disclosure can also be described
or specified in terms of their binding affinity to an antigen. The
affinity of an antibody for an antigen can be determined
experimentally using any suitable method. (See, e.g., Berzofsky et
al., "Antibody-Antigen Interactions," In Fundamental Immunology,
Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis
Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and
methods described herein). The measured affinity of a particular
antibody-antigen interaction can vary if measured under different
conditions (e.g., salt concentration, pH). Thus, measurements of
affinity and other antigen-binding parameters (e.g., K.sub.D,
K.sub.a, K.sub.d) are preferably made with standardized solutions
of antibody and antigen, and a standardized buffer.
[0074] The affinity binding constant (K.sub.aff) can be determined
using the following formula:
K a .times. f .times. f = ( n - 1 ) 2 .times. ( n .function. [ m
.times. A .times. b ' ] t - [ mAb ] t ) ##EQU00001##
in which:
n = [ m .times. A .times. g ] t [ mAg ' ] t ##EQU00002##
[0075] [mAb] is the concentration of free antigen sites, and [mAg]
is the concentration of free monoclonal binding sites as determined
at two different antigen concentrations (i.e., [mAg]t and
[mAg'].sub.t) (Beatty et al., J Imm Meth, 100:173-179 (1987)).
[0076] Surface plasmon resonance (SPR) can be used for detection
and measurement of antibody-antigen affinity and kinetics. (See,
e.g., Hearty, S., et al., Methods Mol. Biol., 907:411-42 (2012);
Malmqvist, M., Current Opinion in Immunology, 5: 282-286 (1993);
Chatellier, J, et al., J. Molecular Recognition, 9: 39-51 (1996);
Margulies, D. H., et al., Current Opinion in Immunology, 8: 262-270
(1996); Forbes, B. E., et al., Eur. J. Biochem., 269:961-968
(2002).)
[0077] The term "high affinity" for an antibody refers to an
equilibrium association constant (K.sub.aff) of at least about
1.times.10.sup.7 liters/mole, or at least about 1.times.10.sup.8
liters/mole, or at least about 1.times.10.sup.9 liters/mole, or at
least about 1.times.10.sup.10 liters/mole, or at least about
1.times.10.sup.11 liters/mole, or at least about 1.times.10.sup.12
liters/mole, or at least about 1.times.10.sup.13 liters/mole, or at
least about 1.times.10.sup.14 liters/mole or greater. "High
affinity" binding can vary for antibody isotypes. K.sub.D, the
equilibrium dissociation constant, is a term that is also used to
describe antibody affinity and is the inverse of K.sub.aff.
[0078] K.sub.D, the equilibrium dissociation constant, is a term
that is also used to describe antibody affinity and is the inverse
of K.sub.aff. If K.sub.D is used, the term "high affinity" for an
antibody refers to an equilibrium dissociation constant (K.sub.D)
of less than about 1.times.10.sup.-7 mole/liters, or less than
about 1.times.10.sup.-8 mole/liters, or less than about
1.times.10.sup.-9 mole/liters, or less than about
1.times.10.sup.-10 mole/liters, or less than about
1.times.10.sup.-11 mole/liters, or less than about
1.times.10.sup.-12 mole/liters, or less than about
1.times.10.sup.-13 mole/liters, or less than about
1.times.10.sup.-14 mole/liters or lower.
[0079] The production of antibodies according to the present
disclosure provides for antibodies with the characteristics of
those produced in the course of a physiological human immune
response, i.e. antibody specificities that can only be selected by
the human immune system. These antibodies can be used as
prophylactic or therapeutic agents upon appropriate
formulation.
[0080] In relation to a particular agent, a "neutralizing
antibody", "broadly neutralizing antibody", or "neutralizing
monoclonal antibody", all of which are used interchangeably herein,
is one that can neutralize the ability of that agent to function in
a host. In some embodiments, monoclonal antibodies produced in
accordance with the present disclosure have neutralizing activity,
where the antibody can neutralize at a concentration of 10.sup.-9M
or lower (e.g., 10.sup.-10M, 10.sup.-11M, 10.sup.-12M or
lower).
[0081] The immunoglobulin molecules of the present invention can be
of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass of
immunoglobulin molecule. In some embodiments, the antibodies are
antigen-binding antibody fragments (e.g., human) and include, but
are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs
(scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments comprising either a V.sub.L or V.sub.H domain.
Antigen-binding antibody fragments, including single-chain
antibodies, can comprise the variable region(s) alone or in
combination with the entirety or a portion of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the present
disclosure are antigen-binding fragments comprising any combination
of variable region(s) with a hinge region, CH1, CH2, and CH3
domains.
Recombinant Expression
[0082] The methods of the present disclosure also provide utilizing
a nucleic acid to generate a host cell that can express an antibody
of interest.
[0083] In some embodiments, the nucleotide sequence encoding a
desired antibody can be constructed and thereafter employed in a
heterologous expression system, e.g., 293 cells or CHO cells. In
some embodiments, an antibody can be recombinantly expressed by
obtaining one or more nucleic acids (e.g. heavy and/or light chain
genes) that encodes the antibody of interest and inserting the
nucleic acid into a host cell in order to permit expression of the
antibody of interest in that host.
[0084] Production of antibodies using recombinant DNA methods is
described, for example, in U.S. Pat. No. 4,816,567. For recombinant
production of the antibody, the nucleic acid encoding it is
isolated and inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression. DNA encoding a
monoclonal antibody is 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). Vectors that can be used generally
include, but are not limited to, one or more of the following: a
signal sequence, an origin of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription
termination sequence. Examples of such expression system components
are disclosed in, for example, U.S. Pat. No. 5,739,277. Suitable
host cells for cloning or expressing the DNA in the vectors herein
are the prokaryotic, yeast, or higher eukaryotic cells (see, e.g.,
U.S. Pat. No. 5,739,277).
Pharmaceutical Compositions
[0085] The presently disclosed subject matter provides
pharmaceutical compositions comprising the antibodies and antigen
binding proteins produced in accordance with the present
disclosure. In some embodiments, a pharmaceutical composition can
comprise one or more antibodies or antigen binding proteins
produced in using the methods disclosed herein. In some
embodiments, a panel of antibodies or antigen binding proteins
produced according to the present disclosure can be included in a
pharmaceutical composition. In some embodiments, the antibodies or
antigen binding proteins produced according to the present
disclosure can be included with one or more additional agents, for
example, antiviral or anticancer drugs or analgesics.
[0086] In some embodiments a pharmaceutical composition can also
contain a pharmaceutically acceptable carrier or adjuvant for
administration of the antibody or antigen binding protein. In some
embodiments, the carrier is pharmaceutically acceptable for use in
humans. The carrier or adjuvant should not itself induce the
production of antibodies harmful to the individual receiving the
composition and should not be toxic. Suitable carriers can be
large, slowly metabolized macromolecules such as proteins,
polypeptides, liposomes, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, ammo acid copolymers and
inactive virus particles.
[0087] Pharmaceutically acceptable salts can be used, for example
mineral acid salts, such as hydrochlorides, hydrobromides,
phosphates and sulphates, or salts of organic acids, such as
acetates, propionates, malonate and benzoates.
[0088] Pharmaceutically acceptable carriers in therapeutic
compositions can additionally contain liquids such as water,
saline, glycerol and ethanol. Additionally, auxiliary substances,
such as wetting or emulsifying agents or pH buffering substances,
can be present in such compositions. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries and suspensions,
for ingestion by the patient.
[0089] The compositions of the presently disclosed subject matter
can further comprise a carrier to facilitate composition
preparation and administration. Any suitable delivery vehicle or
carrier can be used, including but not limited to a microcapsule,
for example a microsphere or a nanosphere (Manome et al. (1994)
Cancer Res 54:5408-5413; Saltzman & Fung (1997) Adv Drug Deliv
Rev 26:209-230), a glycosaminoglycan (U.S. Pat. No. 6,106,866), a
fatty acid (U.S. Pat. No. 5,994,392), a fatty emulsion (U.S. Pat.
No. 5,651,991), a lipid or lipid derivative (U.S. Pat. No.
5,786,387), collagen (U.S. Pat. No. 5,922,356), a polysaccharide or
derivative thereof (U.S. Pat. No. 5,688,931), a nanosuspension
(U.S. Pat. No. 5,858,410), a polymeric micelle or conjugate
(Goldman et al. (1997) Cancer Res 57:1447-1451 and U.S. Pat. Nos.
4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and a
polysome (U.S. Pat. No. 5,922,545).
[0090] Antibody sequences can be coupled to active agents or
carriers using methods known in the art, including but not limited
to carbodiimide conjugation, esterification, sodium periodate
oxidation followed by reductive alkylation, and glutaraldehyde
crosslinking (Goldman et al. (1997) Cancer Res. 57:1447-1451; Cheng
(1996) Hum. Gene Ther. 7:275-282; Neri et al. (1997) Nat.
Biotechnol. 15:1271-1275; Nabel (1997) Vectors for Gene Therapy. In
Current Protocols in Human Genetics, John Wiley & Sons, New
York; Park et al. (1997) Adv. Pharmacol. 40:399-435; Pasqualini et
al. (1997) Nat. Biotechnol. 15:542-546; Bauminger & Wilchek
(1980) Meth. Enzymol. 70:151-159; U.S. Pat. No. 6,071,890; and
European Patent No. 0 439 095).
[0091] A therapeutic composition of the present invention comprises
in some embodiments a pharmaceutical composition that includes a
pharmaceutically acceptable carrier. Suitable formulations include
aqueous and non-aqueous sterile injection solutions which can
contain anti-oxidants, buffers, bacteriostats, bactericidal
antibiotics and solutes which render the formulation isotonic with
the bodily fluids of the intended recipient; and aqueous and
non-aqueous sterile suspensions which can include suspending agents
and thickening agents. The formulations can be presented in
unit-dose or multi-dose containers, for example sealed ampoules and
vials, and can be stored in a frozen or freeze-dried (lyophilized)
condition requiring only the addition of sterile liquid carrier,
for example water for injections, immediately prior to use. Some
exemplary ingredients are SDS in the range of in some embodiments
0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/or
mannitol or another sugar in the range of in some embodiments 10 to
100 mg/ml, in some embodiments about 30 mg/ml; and/or
phosphate-buffered saline (PBS). Any other agents conventional in
the art having regard to the type of formulation in question can be
used. In some embodiments, the carrier is pharmaceutically
acceptable. In some embodiments the carrier is pharmaceutically
acceptable for use in humans.
[0092] Pharmaceutical compositions of the present disclosure can
have a pH between 5.5 and 8.5, preferably between 6 and 8, and more
preferably about 7. The pH can be maintained by the use of a
buffer. The composition can be sterile and/or pyrogen free. The
composition can be isotonic with respect to humans. Pharmaceutical
compositions of the presently disclosed subject matter can be
supplied in hermetically-sealed containers.
[0093] Pharmaceutical compositions can include an effective amount
of one or more antibodies as described herein. In some embodiments,
a pharmaceutical composition can comprise an amount that is
sufficient to treat, ameliorate, or prevent a desired disease or
condition, or to exhibit a detectable therapeutic effect.
Therapeutic effects also include reduction in physical symptoms.
The precise effective amount for any particular subject will depend
upon their size and health, the nature and extent of the condition,
and therapeutics or combination of therapeutics selected for
administration. The effective amount for a given situation is
determined by routine experimentation as practiced by one of
ordinary skill in the art.
Treatment Regimens: Pharmacokinetics
[0094] The pharmaceutical compositions of the invention can be
administered in a variety of unit dosage forms depending upon the
method of administration. Dosages for typical antibody
pharmaceutical compositions are well known to those of skill in the
art. Such dosages are typically advisory in nature and are adjusted
depending on the particular therapeutic context or patient
tolerance. The amount antibody adequate to accomplish this is
defined as a "therapeutically effective dose." The dosage schedule
and amounts effective for this use, i.e., the "dosing regimen,"
will depend upon a variety of factors, including the stage of the
disease or condition, the severity of the disease or condition, the
general state of the patient's health, the patient's physical
status, age, pharmaceutical formulation and concentration of active
agent, and the like. In calculating the dosage regimen for a
patient, the mode of administration also is taken into
consideration. The dosage regimen must also take into consideration
the pharmacokinetics, i.e., the pharmaceutical composition's rate
of absorption, bioavailability, metabolism, clearance, and the
like. See, e.g., the latest Remington's; Egleton, Peptides 18:
1431-1439, 1997; Langer, Science 249: 1527-1533, 1990.
[0095] For purposes of the present invention, a therapeutically
effective amount of a composition comprising an antibody, contains
about 0.05 to 1500 .mu.g protein, preferably about 10 to 1000 .mu.g
protein, more preferably about 30 to 500 .mu.g and most preferably
about 40 to 300 .mu.g, or any integer between these values. For
example, antibodies of the invention can be administered to a
subject at a dose of about 0.1 .mu.g to about 200 mg, e.g., from
about 0.1 .mu.g to about 5 .mu.g, from about 5 .mu.g to about 10
.mu.g, from about 10 .mu.g to about 25 .mu.g, from about 25 .mu.g
to about 50 .mu.g, from about 50 .mu.g to about 100 .mu.g, from
about 100 .mu.g to about 500 .mu.g, from about 500 .mu.g to about 1
mg, from about 1 mg to about 2 mg, with optional boosters given at,
for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three
months, 6 months and/or a year later. It is understood that the
specific dose level for any particular patient depends upon a
variety of factors including the activity of the specific antibody
employed, the age, body weight, general health, sex, diet, time of
administration, route of administration, and rate of excretion,
drug combination and the severity of the particular disease
undergoing therapy.
[0096] Routes of administration include, but are not limited to,
oral, topical, subcutaneous, intramuscular, intravenous,
subcutaneous, intradermal, transdermal and subdermal. Depending on
the route of administration, the volume per dose is preferably
about 0.001 to 10 ml, more preferably about 0.01 to 5 ml, and most
preferably about 0.1 to 3 ml. Compositions can be administered in a
single dose treatment or in multiple dose treatments on a schedule
and over a time period appropriate to the age, weight and condition
of the subject, the particular antibody formulation used, and the
route of administration.
Kits
[0097] The invention provides kits comprising antibodies produced
in accordance with the present disclosure which can be used, for
instance, for therapeutic applications described above. The article
of manufacture comprises a container with a label. Suitable
containers include, for example, bottles, vials, and test tubes.
The containers can be formed from a variety of materials such as
glass or plastic. The container holds a composition which includes
an active agent that is effective for therapeutic applications,
such as described above. The active agent in the composition can
comprise the antibody. The label on the container indicates that
the composition is used for a particular therapy or non-therapeutic
application, and can also indicate directions for either in vivo or
in vitro use, such as those described above.
[0098] The following examples of specific aspects for carrying out
the present invention are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way.
EXAMPLES
Example 1. Pritumumab Penetrates the Blood Brain Barrier
[0099] To demonstrate pritumumab crosses the blood brain barrier,
human primary glioblastoma stem cells (GBM8 cells; 200K cells) were
injected intracranially into NSG mouse brain. After 35 days, mouse
was injected intraveneously (i.v.) via tail vein with 50 .mu.g of
Pritumumab-Alexa647 antibody. After 3.5 hours 20 .mu.g
fluorescein-labeled G. simplicifolia lectin that binds selectively
to mouse endothelial cells (GSL I-BSL I; Vector Laboratories, Inc.,
Burlingame, Calif.) was injected i.v. The mouse was then
euthanized, brain was removed, sliced, and various areas were
imaged by confocal microscopy (Nikon eclipse Ti). The results are
shown in FIG. 2. Pritumumab in present in brain tumor tissue but
not normal tissue. To further investigate these results, the
distribution of pritumumab in the brain was studied. In Normal
brain areas, the Pritumumab antibody is mostly in the normal,
intact blood vessels with very little leakage around vessels at 4
hours after injection (FIG. 3). In Tumor areas, the Pritumumab
antibody is mostly outside of tortuous, large, leaky tumor blood
vessels within the tumors at 4 hours after injection (FIG. 4).
[0100] The distribution of pritumumab was further studied in brain
and other tissues. SCID mice of either sex and typically weighing
between 20-25 g were housed in micro-isolator cages with autoclaved
bedding and autoclaved food and water. A limitation of this model
is that it involves the implantation of human glioma cells into
athymic nu/nu or SCID mice.
[0101] For intracranial implantation, aseptic surgical methods were
followed in accordance with UCSD animal research guidelines. Target
cells (both the U87 cell line and patient derived GBM8 glioma
cells) were concentrated into a compact suspension (1-2 ul). Mice
were anesthetized with ketamine/xylazine and the head was swabbed
with Betadine. Body temperature was maintained with a circulating
water pad. The tumor cell suspensions were injected stereotaxically
into the right frontal cortex at 1 ul/min using a Sul Hamilton
syringe. The syringe was left in place for 5 min before retraction
to prevent reflux of the injected material.
[0102] For imaging, MRI imaging of implanted tumors (both U87 and
patient derived GBM8 glioma cells) was performed every week as
tumors grew. After the appropriate scans the mice were sacrificed.
The MRI protocol was as follows. Mice were anesthetized with a
mixture of 1.5% isofluorane and 95% oxygen throughout imaging. MRI
was performed on a 4.7T horizontal bore system interfaced with a
commercial scanner console (Bruker BioSpin Corporation, MA) using a
homogeneous quadrature birdcage head coil. Coronal T2-weighted
images (T2WI) (TR/TEeff=2,000/72 ms) were acquired with a 2D fast
spin echo sequence at 1 mm slice thickness with 80 um.times.130 um
in-plane resolution. Magnevist Gd-DTPA was injected
intraeritoneally at 0.7 mmol/kg and coronal T1-weighted images
(T1WI) (TR/TEeff=417/24.5 ms) were acquired once before and twice
after contrast injection. Post-contrast imaging was performed at 12
min after injection.
[0103] Brain enhancement represents a measure of tumor
microvascular permerability and relates to how therapeutic agents
might be distributed within a tumor and whether contrast agents can
highlight a small tumor. The usual normalization method was used in
which a ratio of contrast enhancement is determined by comparing
tumor tissue to contralateral normal brain. A ratio of enhancement
comparing the tumor with cervical skeletal muscle
(sternocleidomastoid) was also calculated. This was performed to
provide a check for the tumor versus normal brain ratio since it is
possible that inaccuracies might arise from altered permeability in
the reference white matter due to tumor infiltration and edema.
[0104] The tumor to contralateral white matter ratio (TWR) and the
tumor-muscle enhancement ratio (TMR) were the mean signal intensity
of the enhanced tumor divided by mean signal intensity of white
matter and cervical muscle, respectively, at the 12 min
post-contrast T1WI. Maximum enhanced regions of interest (ROI) were
selected manually on the pre- and post-contrast images. The T1 and
T2 acquisitions for each animal were cross-referenced to ensure
proper ROI location.
[0105] For tumor morphology, all the GBM8 tumors (n=6) has a
well-demarcated margin and no central necrosis was observed. The
tumors had relatively uniform hyperintensity compared to
surrounding brain parenchyma on T2WI and all displayed clearly
visible contrast enhancement that was uniform.
[0106] The Results are shown in FIG. 5 and Table 2. FIG. 5 are
images of tissue slices showing pritumumab specifically binds to a
variety of tumor cells but not normal cells. Table 2 below shows
pritumumab specifically binds tumor cells but not normal cells.
Pritumumab was obtained from its hybridoma or manufactured in CHO
cells as described in, for example, Gupta, et al., the Journal of
Bioprocess Technology 98:318-326 (2013) ("Gupta").
TABLE-US-00005 TABLE 2 Distribution in normal and tumor cells of
pritumumab obtained from its hybridoma or manufactured in CHO
cells. Pritumumab - hybridoma Pritumumab - CHO malignant benign
normal malignant normal Brain 15/27 2/19 0/8 10/10 Normal brain 0/3
melanoma 10/10 Colon carcinoma 2/2 Tongue 1/1 ND 0/1 Salivary gland
0/1 ND ND Thyroid 2/2 ND ND Esophagus 1/1 ND 0/1 Lung 2/4 ND 0/1
9/9 Stomach 2/5 ND 0/2 Heart ND ND 0/1 Renal 1/1 ND 0/2 11/11
Adrenal 0/2 ND 0/1 Spleen 0/1 ND 0/3 Liver 0/2 ND 0/2 Pancreas 1/1
ND 0/1 9/9 Gall bladder 2/2 ND 0/1 Lymph node 0/17 ND 0/3 Breast
5/15 1/5 0/1 12/12 Ovary 6/8 ND 0/1 Uterus 2/3 ND 0/1 Cervix 7/10
0/2 Squamous 0/2 epithelium Endometrium 0/3 Blood vessels 0/4
Smooth muscle 0/5 Normal fat 0/2
Example 2. Generation of Antibodies with Enhanced Delivery to the
Brain Materials
[0107] Cell Lines: E. coli CJ236 (New England Biolabs, Beverly,
Mass.); E. coli SS320 (Lucigen, Middleton, Wis.); E. coli One
Shot.RTM. OmniMAX.TM. 2 T1R (Invitrogen, Grand Island, N.Y.);
Chinese hamster ovary (CHO) cells; HEK cells.
[0108] Tumor tissues and tumor cell lines: All tumor tissues will
be discards after their clinical and diagnostic application and the
cell lines will be procured from ATCC (USA). The tumor tissues will
include brain, breast, cervical, colon, liver, melanoma, ovarian,
and pancreatic cancers. Uterine smooth muscle will be used as
normal tissue control. Inclusion of these controls will determine
specificity of the recombinantly produced CDR swapped antibodies
which will be equivalent to the original human-human hybridoma
produced pritumumab particularly with respect to the
frame-work.
[0109] Media: One example of synthetic media is synthetic
low-density lipoprotein (sLDL), as a lipid supplement in serum-free
media (Hayavi and Halbert, 2005). The sLDL can be manufactured by
microfluidization of the lipid dissolve in solvent with aqueous
solutions, generating a non-toxic product with physico-chemical
characteristic of native LDL. The inclusion of 0.1-0.5 mM iron
citrate in chemically defined (animal-free) media has been shown to
increase mAb expression in CHO cells by around a third, involving
upregulation of genes associated with ribosome formation and
protein folding (Bai et al., 2011).
[0110] Antibodies/reagents: Specific antibodies and other
immunochemicals and routine reagents will be procured from
Sigma-Aldrich and Pierce Thermo Fisher.
[0111] Pritumumab: Pritumumab has been produced by Catalent using
the patented GPEx technology.
TABLE-US-00006 Primer design using Integrated DNA Technologies
Program "PRIME QUEST" for Pritumumab CDRs and Frameworks: Start
Stop Length Tm GC % HC-CDR1 primer sequence of pritumumab Sequence
Name; HC-CDR1 primer sequence of pritumumab Amplicon Length: 242
Forward: TCACCTTCAGCAACTATGCC (SEQ ID NO: 12) 2 22 20 67 50
Reverse: AAGGGTACCAAGTGCTTCTATATG (SEQ ID NO: 13) 220 244 24 62
41.7 HC-CDR2 primer sequence of pritumumab Sequence Name: HC-CDR2
primer sequence of pritumumab Amplicon Length: 281 Forward:
GAGTGGGTCTCAGCGATTAC (SEQ ID NO: 14) 58 78 20 62 55 Reverse:
AGAGGTGCTCTTGGAGGA (SEQ ID NO: 15) 321 339 18 62 55.6 HC-CDR3
primer of pritumumab Sequence Name: HC-CDR3 primer sequence of
pritumumab Amplicon Length: 213 Forward: CTATGCCATGAGCTGGGT (SEQ ID
NO: 16) 15 33 18 61 55.6 Reverse: TCTATATGGACTCTCCCACAGATA (SEQ ID
NO. 17) 204 228 24 62 41.7 Framework primer sequence of HC of
Pritumumab Set 1 Sequence Name: Framework primer sequence of HC of
Pritumumab - 1 Amplicon Length: 219 Forward: GCCTGGTCAAAGGCTTCTAT
(SEQ ID NO: 18) 641 661 20 62 50 Reverse: TCTTCTGCGTGTAGTGGTTG (SEQ
ID NO: 19) 840 860 20 62 50 Framework primer sequence of HC of
Pritumumab Set 2 Sequence Name: Framework primer sequence of HC of
Pritumumab - 2 Amplicon Length: 292 Forward: GCTGAATGGCAAGGAGTACA
(SEQ ID NO: 20) 480 500 20 62 50 Reverse: GAGCTTGCTGTAGAGGAAGAAG
(SEQ ID NO: 21) 750 772 22 62 50 Framework primer sequence of HC of
Pritumumab Set 3 Sequence Name: Framework primer sequence of HC of
Pritumumab -3 Amplicon Length: 658 Forward: CAAGGTGGACAAGAAAGTTGAG
(SEQ ID NO: 22) 168 190 22 61 45.5 Reverse: CATCACGGAGCATGAGAAGA
(SEQ ID NO: 23) 805 626 20 62 50 Framework primer sequence of HC of
Pritumumab Set 4 Sequence Name: Framework Primer sequence of HC of
Pritumumab Amplicon Length: 362 Forward: ATCACAAGCCCAGCAACA (SEQ ID
NO: 24) 149 167 31 62 50 Reverse: GACCTTGCACTTGTACTCCTT (SEQ ID NO:
25) 149 167 18 62 50 Framework primer sequence of HC of Pritumumab
Set 5 Sequence Name: Framework Primer sequence of HC of Pritumumab
Amplicon Length: 225 Forward: GTGCAAGGTCTCCAACAAAG (SEQ ID NO: 26)
500 521 21 63 47.5 Reverse: GCGTGGTCTTGTAGTTGTTCTC (SEQ ID NO: 27)
703 725 22 63 50 LC-CDR1 primer sequence of Pritumumab Sequence
Name: LC-CDR1 primers of Pritumumab Amplicon Length: 261 Forward:
GCATCTGTAGGAGACAGAGTCA (SEQ ID NO: 28) 37 59 22 63 50 Reverse:
GCCGAAGGTGATAGGGTAAGTA (SEQ ID NO: 29) 276 298 22 63 50 LC-CDR2
primer sequence of Pritumumab Sequence Name: LC-CDR2 primers of
Pritumumab Amplicon Length: 201 Forward: AGTCTCCATCCTCACTGTCT (SEQ
ID NO: 30) 17 37 20 62 50 Reverse: GAGTGAAATCTGTCCCAGATCC (SEQ ID
NO: 31) 196 218 22 62 50 LC-CDR3 primer sequence of Pritumumab
Sequence Name: LC-CDR primers of Priturnumab Amplicon Length: 239
Forward: GAGACAGAGTCACCATCACTTG (SEQ ID NO: 32) 47 69 22 62 50
Reverse: AGGGTAAGTACTATACTGTAGGCA (SEQ ID NO: 33) 262 286 24 62 411
LC Framework Primers sequence of Pritumumab Set 1 Sequence Name: LC
Framework Primers for Pritumumab - 1 Amplicon Length: 249 Forward:
CTGCACCATCTGTCTTCATCT (SEQ ID NO: 34) 32 53 21 62 47.6 Reverse:
AGGCGTAGACTTTGTGTTTCT (SEQ ID NO: 35) 260 281 21 62 32.9 LC
Framework Primers sequence of Pritumumab Set 2 Sequence Name: LC
Framework Primers for Pritumumab - 2 Amplicon Length: 223 Forward:
CTTCATCTTCCCGCCATCT (SEQ ID NO: 36) 45 64 19 61 52.5 Reverse:
GTGTTTCTCGTAGTCTGCTTTG (SEQ ID NO: 37) 248 268 22 61 45.5 LC
Framework Primers sequence of Pritumumab Set 3 Sequence Name: LC
Framework Primers for Pritumumab - 3 Amplicon Length: 209 Forward:
ATCTGGAACTGCCTCTGTTG (SEQ ID NO: 38) 78 98 20 62 50 Reverse:
CTTCGCAGGCGTAGACTTT (SEQ ID NO: 39) 268 237 19 62 52.6 LC Framework
Primers sequence of Pritumumab Set 4 Sequence Name: LC Framework
Primers for Pritumumab - 4 Amplicon Length: 241 Forward:
GTTGTGTGCCTGCTGAATAAC (SEQ ID NO: 40) 94 115 21 62 47.6 Reverse:
CCCTGTTGAAGCTCTTTGTGA (SEQ ID NO: 41) 314 335 21 63 47.6 LC
Framework Primers sequence of Pritumumab Set 5 Sequence Name: LC
Framework Primers for Pritumumab Amplicon Length: 215 Forward:
AGGTGGAGATCAAACGAACTG (SEQ ID NO: 42) 8 29 21 62 47.6 Reverse:
GCTGTAGGTGCTGTCCTTG (SEQ ID NO: 43) 204 223 19 62 57.9
Methods:
[0112] PCR Primers and Framework Cassette: Extended Packaging
Region. The PCR primers will be designed to amplify a portion of
the Moloney Murine Leukemia Virus extended packaging region that is
found in all gene constructs used in the GPEx.RTM. process. The
primers shown below amplify an 85 bp fragment of the EPR.
[0113] EPR PCR primers will include:
TABLE-US-00007 EPR1 (SEQ ID NO :44) 5'-GTTATGCGCCTGCGTCTGTAC-3'
EPR2 (SEQ ID NO: 45) 5'-CCGGGTGTTCAGAACTCGTC-3'
[0114] Heavy Chain. These PCR primers are designed to amplify a
portion of the human IgG1, IgG2, IgG3 and IgG4 constant regions.
The primers shown below will be amplified in a 92 bp fragment of
the constant region. These primers will be:
TABLE-US-00008 Human Ab heavy chain F (SEQ ID NO: 46)
5'-ACGGTGTCGTGGAACTCAG-3' Human Ab heavy chain R (SEQ ID NO: 47)
5'-CACGCTGCTGAGGGAGTAGAGTCC-3'
[0115] Light Chain. These PCR primers are designed to amplify a
portion of the human kappa constant region. The primers shown below
will be amplified in a 83 bp fragment of the constant region.
[0116] LC PCR Primers:
TABLE-US-00009 Human Ab light chain (kappa) F (SEQ ID NO: 48)
5'-CAAAGTACAGTGGAAGGTGGAT-3' Human Ab light chain (kappa) R (SEQ ID
NO: 49) 5'-GTGCTGTCCTTGCTGTCCTGCTCT-3'
[0117] Control. These PCR primers will be designed to amplify a
portion of the CHO .beta. 1, 4-galactosyltransferase gene. The
primers shown below amplify an 82 bp fragment of the CHO .beta. 1,
4-galactosyltransferase-1 gene.
[0118] Control Single Copy Gene PCR Primers:
TABLE-US-00010 CHO Internal 1 (SEQ ID NO: 50)
5'-AAAGATGGGCGGTCGTTATTC-3' CHO Internal 2 (SEQ ID NO: 51)
5'-CCTGCCGGTTGCGAAATGGGATAA-3'
[0119] DNA will be isolated from the cell lines using the DNeasy
genomic DNA purification kit (Catalog #69504, Qiagen, Valencia,
Calif.). PCR reactions will be set up on the SYBR.RTM. Green PCR
Master Mix (Catalog #4311034, Applied Biosystem, Foster City,
Calif.) under the conditions described below. The samples will be
run using the following cycling program on an iQ cycler from
Bio-Rad (Hercules, Calif.).
[0120] PCR Reaction Setup with Respect to PCR Components Final
Concentration:
SYBR.RTM. Green PCR Master Mix 12.5 L per reaction, Primer final
concentration: 125 nM for each primer Separate reactions will be
done for each of the different primer sets using 20 ng genomic DNA
for each 25 L reaction (Diluted in nuclease free water); Nuclease
free water will be added to bring final volume to 25 L. Each sample
(EPR and internal control) will be run in triplicate.
[0121] The Samples Will be Run Using the Following Cycling Program
on an iQ Cycler from Bio-Rad for PCR Program:
Step 1: 95.degree. C. 9 minutes (denature and activate polymerase)
Step 2: 94.degree. C. 15 seconds (denature) 60.degree. C. 1 minute
(combined anneal and synthesis steps) 40 cycles as in Step 2 The
gene copy index will be calculated by subtracting the Ct of the
transgene assay (EPR, HC or LC) from the Ct for the control assay
(.beta. 1, 4-galactosyltransferase-1).
[0122] Gene Cloning into Expression Retrovector:
[0123] Pritumumab (P-mAb) Heavy Chain grafted with CDR of interest
will be cloned into Expression Retrovector. In the first PCR
reaction P-mAb heavy chain variable region CDS with CPS-M's
(Catalent) proprietary bovine .alpha.-lactalbumin signal peptide
sequence will be amplified from the synthesized DNA fragment,
plasmid GDD2120.0001, using primers
SP75'(5'-TTTTAAGCTTGCCGCCACCATGATGTCCTTTGTCT-3' (SEQ ID NO:52)) and
P-mAbHC2 (5'-GCCAGGGGGAAGACCGATGGGCCCTTGGTGGAGGCAGAGGACACGGTCACGAG
GGTG CCCTGGCCCCAATA-3' (SEQ ID NO:53)). Primer SP75' will be added
to a Hind III site at the 5' end and Kozak translation initiation
sequence just before the translation start codon of the signal
peptide. Primer P-mAbHC2 will be amplified the variable region
sequences for in-frame fusion to the P-mAb heavy chain constant
region by addition of an overlap between the two sequences. In the
second PCR reaction the P-mAb heavy constant region will be
amplified to allow for fusion with the variable region using
primers P-mAbHC1
(5'TATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCCTCCACCAAGGGCCC
ATCGGTCTTCCCCCTGGC-3' (SEQ ID NO:54)) and INHC2
(5'-TTTCTCGAGATCTCATCATTTCCCGGGAGACAGGGAGAGGCTCTTCTGCGTGTAG TGGT-3'
(SEQ ID NO:55)) and GDD2110.0004 plasmid as a DNA template. The
GDD2110.0004 plasmid will be constructed by CPS-M previously and
will serve as the reaction source of the heavy chain constant
region sequence. Primer P-mAbHC1 will be a reverse compliment to
primer P-mAbHC2 and hence will serve the purpose of configuring the
amplified constant region sequence for in-frame fusion to the
variable region by addition of an overlap between the two
sequences. Primer INHC2 will encode the 3' end of the heavy
constant region and contributed an Xho I site for easy cloning. The
amplified products from PCR reactions 1 and 2 will be used as DNA
template with the outermost primers SP75' and INHC2 to join the
variable and the constant regions together and to amplify
full-length P-mAb heavy chain CDS. The resultant PCR product will
be digested with Hind III and Xho I restriction endonucleases and
ligated into the retrovector plasmid pFCSnewMCS-WPRE-SIN (new ori)
(GDD1008.0146) which will also be digested with the same
enzymes.
[0124] The resultant clones will be sequenced through the assembled
heavy chain gene and the flanking regions and a clone will be
confirmed to encode the desired full-length P-mAb heavy chain
CDS.
[0125] P-mAb Light Chain Gene Cloning into Expression Retrovector:
In the first PCR reaction P-mAb light chain variable region CDS
with CPS-M's proprietary bovine .alpha.-lactalbumin signal peptide
sequence will be amplified from the synthesized DNA fragment,
plasmid GDD2120.0001, using primers SP75'
(5'-TTTTAAGCTTGCCGCCACCATGATGTCCTTTGTCT-3' (SEQ ID NO:56)) and
PmabLC2 (5'-GCGGGAAGATGAAGACAGATGGTGCAGCCACAGTTCGCTTGATTTCCACCTTGGT
GCCTCCGCCGAAGGTGATAGG-3' (SEQ ID NO:57)). Primer SP75' will be
added to a Hind III site at the 5' end and Kozak translation
initiation sequence just before the translation start codon of the
signal peptide. Primer PmabLC2 amplified the light chain variable
region sequence for in-frame fusion to the P-mAb light chain
constant region by addition of an overlap between the two
sequences. In the second PCR reaction the P-mAb light chain
constant region will be amplified to allow for fusion with the
light chain variable region using primers PmabLC1
(5'-CCTATCACCTTCGGCGGAGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCA
CCATCTGTCTTCATCTTCCCGC-3' (SEQ ID NO:58)) and INLC2
(5'-TTTCTCGAGATCTCACTAACACTCTCCCCTGTTGAAGCTCT-3' (SEQ ID NO:59))
and GDD2103.0003 plasmid as the DNA template. The GDD2103.0003
plasmid will be constructed by CPS-M previously and in this
reaction will a source of the light chain constant region sequence.
Primer PmabLC1 will be a reverse compliment to primer PmabLC2 and
hence will serve the purpose of configuring the amplified constant
region sequence for in-frame fusion to the variable region. Primer
INLC2 encoded the 3' end of the light chain constant region and
contributed an Xho I site for easy cloning.
[0126] The amplified products from PCR reactions 1 and 2 will be
used as the DNA templates with the outermost primers SP75' and
INLC2 to join the variable and the constant regions and to amplify
full-length P-mAb light chain CDS. The resultant PCR product will
be digested with Hind III and Xho I restriction endonucleases and
ligated into the retrovector plasmid pFCS-newMCSWPRE-SIN (new ori)
(GDD1008.0146) which would have also been digested with the same
enzymes. Plasmid DNA isolated from the resultant clones will be
sequenced through the assembled light chain gene and the flanking
regions and a clone was confirmed to encode the desired full-length
P-mAb light chain CDS.
[0127] Development of Catalent Pharma Solutions-Middleton's
Expression Retrovector Construct pFCS-newMCS-WPRE-SIN (new ori)
(GDD1008.0146): The latest generation GPExo expression plasmid
pFCS-newMCS-WPRE-SIN (new ori) (GDD1008.0146) will be created by
adding WPRE, a post-transcriptional regulatory element whose
function is described below, to pFCS-newMCS-SIN (new ori)
(GDD1008.0136), which is also described below. Briefly,
pCNS-newMCS-WPRE (new ori), (GDD1008.0068), also described below,
was digested with ClaI, and the fragment containing WPRE will be
isolated and purified. The WPRE fragment will be ligated into the
major vector fragment purified from the digestion of
pFCS-newMCS-SIN (new ori) (GDD1008.0136) with ClaI. The recombinant
molecules will be screened using the restriction endonucleases
HindIII and NaeI to confirm the correct orientation of the WPRE
element. The sequence of the resultant vector across the ClaI
insertion sites will be confirmed.
[0128] The GPExoexpression plasmid pFCS-newMCS-SIN (new ori)
(GDD1008.0136) is a self-inactivating (SIN) vector featuring a
full-length human cytomegalovirus (CMV) immediate early
enhancer/promoter in the 5' LTR. The full-length human CMV promoter
and the mutated (SIN) version of 3' LTR will improve expression by
increasing viral titers and reducing 5' LTR promoter interference.
This vector is a legacy of the previous highly successful
expression vector pCS-newMCS-WPRE (new ori) (GDD1008.0074). Plasmid
pFCS-newMCS-SIN (new ori) (GDD1008.0136) will be constructed by
removal of an EcoRI/HindIII fragment encompassing a portion of the
3' part of the Extended Packaging Region (EPR) and Neo gene
(neomycin phosphotransferase, selectable marker) from plasmid
pFCNS-newMCS-SIN (new ori) (GDD1008.0140) and complementing the EPR
with the small EcoRI/HindIII fragment of pCSnewMCS-WPRE (new ori)
(GDD1008.0074) comprising its missing part. Plasmid pCSnewMCS-WPRE
(new ori) (GDD1008.0074) is GPExo previous generation expression
vector and history of its development is described below. Construct
pFCNS-newMCS-SIN (new ori) (GDD1008.0140) will be derived by
removing the ClaI restriction fragment containing WPRE sequence
from plasmid pFCNS-newMCS-WPRE-SIN (new ori) GDD1008.0141. Plasmid
pFCNS-newMCS-WPRE-SIN (new ori) (GDD1008.0141) will be created from
vector pCNS-newMCS-WPRE (new ori), (GDD1008.0068) by cloning the
full-length human CMV promoter amplified by PCR from plasmid
pLNC-MCS (GDD1008.0001) into the 5' LTR region upstream of the
Neomycin selectable marker gene. Finally, construct pCNS-newMCSWPRE
(new ori); (GDD1008.0068) will be developed by the addition of the
high-copy origin of replication from the plasmid pUC19 into plasmid
pCNS-newMCS-WPRE (GDD1008.0033). The origins and evolution of
plasmid pCNS-newMCS-WPRE (GDD1008.0033) are detailed below in the
description of development of GPExo previous basic expression
vector pCSnewMCS-WPRE (new ori) (GDD1008.0074).
[0129] The plasmid pCS-newMCS-WPRE (new ori) (GDD1008.0074) will be
originally derived from plasmid pLNCX II (GD0004). The pLNCX II
plasmid will be recreated at Catalent Pharma Solutions-Middleton by
removing the cc49 gene from the plasmid pLNC-cc49 (GDD1008.0049)
which has been received from the laboratory of Dr. Paul Sondel at
the University of Wisconsin-Madison. The pLNCX II plasmid is a
slight modification of the pLNCX plasmid (Genbank ACCESSION M28247)
created by A. D. Miller (removal of Eco RI site; Kashmiri et al.
Hybridoma 14: 461-473 1995 performed in the laboratory of Dr.
Jeffery Schlom at the National Institutes of Health). The pLNCX II
plasmid will be modified with oligonucleotides to create a multiple
cloning site following the hCMV promoter (plasmid pLNC-MCS
GDD1008.0001). In order to enhance production of retrovector
particles, the human CMV promoter will be used to replace the 5'
LTR U5 region of pLNC-MCS to create plasmid pCNC-MCS
(GDD1008.0085). When used in this way, the human CMV promoter on
the 5' end of the LTR does not get incorporated into retrovector
particles or inserted into production cell lines.
[0130] A segment from the Pol gene of the woodchuck Hepatitis B
virus will be obtained from Dr. Tom Hope then at the Salk Institute
(plasmid pBluescript II SK+ WPRE-B11). This fragment
(WPRE--Woodchuck Post-transcriptional Regulatory Element) enhances
export of mRNA lacking introns from the nucleus to the cytoplasm as
well as enhances expression of genes that include this sequence in
their mRNA. The plasmid pBluescript II SK+ WPRE-B11 sequence will
be mutated by its developers to eliminate promoter function and the
initiation codon for a fragment of the putative oncogene encoding
protein pX. The WPRE fragment will be inserted 3' from the multiple
cloning site to create plasmid pLNC-MCS-WPRE (GDD1008.0005). Later,
the WPRE fragment was transferred from pLNC-MCS-WPRE into pCNC-MCS
to create pCNCMCS-WPRE (GDD1008.0030).
[0131] The simian cytomegalovirus (sCMV) promoter will be procured
from Dr. Tom Hope at the University of Illinois as part of the
plasmid IEX, an expression plasmid for the HTLV Tax gene. Research
into the literature and intellectual property background on the
simian CMV promoter denotes that it is similar to the hCMV promoter
in having high constitutive activity, but will be available in the
public domain. The sCMV promoter fragment will be PCR amplified
from the IEX plasmid and used to replace the hCMV promoter in
pLNC-MCS, creating plasmid pSCMV-MCS (GDD1008.0018). The sCMV
promoter fragment will be later modified by PCR to remove a Sal I
site in the 5' end and cloned into pCNC-MCS-WPRE in place of the
hCMV promoter to create plasmid pCNS-MCS-WPRE (GDD1008.0031).
[0132] A second set of oligonucleotides will be used to add
additional restriction enzyme sites to the multiple cloning site to
create plasmid pCNSnewMCS-WPRE (GDD1008.0033). To reduce the burden
of excess production of neomycin phosphotransferase protein from
retrovector inserts in production cell lines, the NEO gene will be
removed from pCNS-newMCS-WPRE to create pCS-newMCS-WPRE
(GDD1008.0054). To improve yield from plasmid preps, the E. coli
origin of replication in pCS-newMCSWPRE will be replaced with the
origin of replication from the plasmid pUC19. This will create a
plasmid pCS-newMCS-WPRE (new ori), GDD1008.0074.
[0133] Cos-7 Expression: COS7 cells were obtained from the Health
Science Research Resources Bank (Osaka) is the haploid strain of
Saccharomyces. This unit describes the use of COS cells to
efficiently produce a desired protein in a short period of time.
These cells express high levels of the SV40 large tumor (T)
antigen, which is necessary to initiate viral DNA replication at
the SV40 origin. Three factors contribute to make COS cell
expression systems appropriate for the high-level, short-term
expression of proteins: (1) the high copy number achieved by SV40
origin-containing plasmids in COS cells 48 hr post-transfection,
(2) the availability of good COS cell expression/shuttle vectors,
and (3) the availability of simple methods for the efficient
transfection of COS cells. Each COS cell transfected with DNA
encoding a cell-surface antigen (in the appropriate vector) or
cytoplasmic protein will express several thousand to several
hundred thousand copies of the protein 72 hr post-transfection. If
the transfected DNA encodes a secreted protein, up to 10 .mu.g of
protein can be recovered from the supernatant of the transfected
COS cells 1 week post-transfection. COS cell transient expression
systems have also been used to screen cDNA libraries, to isolate
cDNAs encoding cell-surface proteins, secreted proteins, and DNA
binding proteins, and to test protein expression vectors rapidly
prior to the preparation of stable cell lines.
[0134] ELISA: A sandwich ELISA was developed using pritumumab
antibody as the capture reagent. Biotin-labeled recombinant
antibody will be used as the detection antibody. This homologous
antibody format would be possible assuming that the target antigen
would have multiple epitopes. Microtiter plates (96-well Nunc
Maxisorp) will be coated with purified unlabeled pritumumab
antibody at 10 .mu.g/ml concentration in 0.5M sodium carbonate pH
9.5 overnight at 25.degree. C. Plates will then be blocked with 1%
skim milk made in Tris-Buffered Saline (TBS) containing 5 mM EDTA
and 1% sucrose for 4 hours at 25.degree. C. Plates prepared in this
manner could be stored dried and sealed for at least 12 months. All
dilutions will be made in ImmunoBooster buffers (Bioworld
Consulting Laboratories, LLC) supplemented with 20 mM EDTA. Wash
buffer will be TBS containing 0.05% Tween-20 nonionic detergent. A
detergent extract of cultured human tumor cells will be used as a
source of antigen to derive a standard curve. Units will be in
cells/well. Extracts derived from human brain tumor cells will be
generated in a similar manner. As indicated above, all tumor cell
lines will be purchased from American Type Culture Collection
(Manassas, Va.) and grown in RPMI medium containing 10% FBS
(heat-inactivated) with 8 mM glutamine. To measure direct binding
of the antibody to the target antigen, cells will be grown in
serum-free medium for 5 days and the conditioned medium will be
filtered and stored in one large lot at 4.degree. C. The sandwich
ELISAs will be performed by diluting the cell extract standard on
each plate. All incubations were performed at 25.degree. C. and all
volumes will be 100 ul per well. The plates were incubated for 15
minutes and washed three times with wash buffer. The biotin-labeled
pritumumab antibody will then be added to the wells at 1 .mu.g/ml,
incubated for 15 minutes, and plates will be washed three times.
Peroxidase-conjugated streptavidin (1 5,000 dilution) will be added
to the plates for 15 minutes, and plates will be washed three times
with wash buffer and two times with TBS. The signal will be
developed by the addition of TMB substrate (BioFX Laboratories
Inc.) to the plates, incubation for 15 minutes, then the color
reaction will be stopped with the addition of 0.5M sulfuric acid.
The data was acquired by measuring absorbance at 450 nm, and
analyzed using GraphPad Prism or Microsoft Excel software
programs.
[0135] Immunohistochemistry (IHC): For IHC analyses, the antibody
will be purified, and will be covalently conjugated to HRP
(courtesy of American Qualex) and used to analyze various tissue
sections such as brain, breast, cervical, colon, liver, melanoma,
and pancreatic tissue sections. Tumor tissues left after needed
diagnostic and clinical evaluation of the donor will be processed
or left over paraffin blocks will be used to determine reactivity
in IHC of pritumumab or its hybrid CDR version. For this purpose,
Sum sections will be cut from the paraffin blocks, placed on slides
and dried and de-paraffinized overnight at 60.degree. C. The slides
will be heat treated for epitope retrieval using the Dako Target
Retrieval Solution at pH 9 (Dako cat #S2367) in conjunction with
pressure cooker for 30 minutes. The slides will then be stained on
Dako Autostainer by using 3% hydrogen peroxide for 5 minutes,
primary antibody (HRP-pritumumab or control) at 1:25 dilution for 1
hour, polymer-based Power Vision Plus detection solution (Leica cat
#PV6104) for 30 minute, and DAB for 10 minutes. The stained slides
will be counterstained in hematoxylin for 1 minute, dehydrated, and
cover-slip applied for pathological examination.
[0136] IEF (Isoelectric Focusing): IEF will be performed using
IsoGel Agarose of Lonza according their protocol using the pH range
from 3.5 to 9.5. The IEF plates will be stained first with
Coomassie Blue and then after de-staining with Silver stain.
[0137] Immunofluorescence Assay (FACS) Analysis with Soluble
Antibodies:
[0138] Freshly cultured tumor cells from established cell lines
(melanoma, lung, breast) will be used for FACS analysis as
described elsewhere. Cells from log-phase growing cultures with
greater than 95% viability will be gently scraped off, after
incubation with EDTA 0.02% (Sigma), and 3.times.105 cells per test
tube will be used. Soluble pritumumab or test antibody of the
selected GPEx clone will be prepared by methods described above.
Tumor cells will be incubated with the antibody (diluted 1/1 in FCS
RPMI 1640) at 37.degree. C. for 1 h in pretreated (1% BSA) plastic
tubes. After three washes with FCS RPMI 1640, cells will be
incubated with biotin labelled anti-human IgG antibody
(1:50)(Vector Lab) for 1 h at 4.degree. C. After three wash steps
(1% BSA PBS and PBS), Phycoerithrin Streptavidin (1:20) will be
added for 20 minutes at 20.degree. C. In repeat experiments FITC
labeled anti human IgG (Fab')2(1:25) (Sigma-Aldrich) will be used.
Immunofluorescence labelled cells will be finally fixed in 1%
Formalin-PBS. Ten thousand cells will be counted in a FACS Calibur
(BD Biosciences) and analyzed by Cell Quest.
[0139] Identification of CDRs and CDR Removal/Insertion--Polymerase
Chain Reaction (PCR):
[0140] PCR: Two novel approaches of recombinant PCR technology were
employed to graft the CDRs from murine monoclonal antibodies (mAb)
onto human antibody frameworks (Daugherty B L, DeMartino J A, Law M
F, Kawka D W, Singer I I, Mark G E (1991) Polymerase chain reaction
facilitates the cloning, CDR grafting, and rapid expression of a
murine monoclonal antibody directed against the CD18 component of
leukocyte integrins. Nucleic Acids Research, 19:2471-2476). One
approach relied on the availability of cloned human variable region
templates, whereas the other strategy is dependent only on human
variable region protein sequence data. The transient expression of
recombinant humanized antibody is generally driven by the
adenovirus major late promoter and can be detected within 48 hrs
post-transfection into non-lymphoid mammalian cells. Application of
these approaches enables the expression of a recombinant humanized
antibody just within 6 weeks after initiating the cDNA cloning of
the murine mAb.
[0141] Identification of CDRs and CDR Removal/Insertion: CDRs of
interest will be identified, removed and inserted from hybridoma
cells as detailed by Fields et al (Fields C, O'Connell D, Xiao S,
Lee G U, Billiald P, Muzard J: (2013) Creation of recombinant
antigen-binding molecules derived from hybridomas secreting
specific antibodies. Nature Protocols 8:1125-1148). This protocol
describes the design and development of recombinant monovalent
antigen-binding molecules derived from monoclonal antibodies
through rapid identification and cloning of the functional variable
heavy (VH) and variable light (VL) genes and the design and cloning
of a synthetic DNA sequence optimized for expression in recombinant
bacteria. Typically, monoclonal antibodies are obtained from mouse
hybridomas, which most often result from the fusion of B
lymphocytes from immunized mice with murine myeloma cells. The
protocol described here has previously been exploited for the
successful development of multiple antibody-based molecules
targeting a wide range of biomolecular targets. The protocol is
accessible for research groups who may not be specialized in this
area, and should permit the straightforward reverse engineering of
functional, recombinant antigen-binding molecules from hybridoma
cells secreting functional IgGs within 50 working days.
Furthermore, convenient strategies for purification of antibody
fragments are also described in this protocol.
[0142] Protein a Purification Method:
[0143] Purification of antibody molecules using protein A
chromatography: Purification of antibody proteins will be
essentially the same as the method used for purification of regular
mAbs. The protocol to be used is outlined below:
1. Prepare protein A column as instructed (GE Healthcare). 2.
Gently apply cell culture medium (diluted 1:1 with binding buffer)
to the column by layering onto the top of the resin. 3. Wash column
with 10 volumes of the wash/binding buffer, or until the absorbance
of eluate at 280 nm approaches the background level. 4. To each
collection tube add 100 ml 1 M Tris buffer (pH 8.0) so the eluate
could be immediately neutralized. 5. To elute the antibody, gently
add elution buffer to the top of the resin, collecting the eluate
in a prepared collection tube (0.9 ml/tube). 6. Repeat until the
entire volume has been collected, up to eight tubes. 7. Identify
positive fractions by adding 10-20 l of eluted fractions to 300 ml
of Coomassie Plus Protein Assay Reagent (Pierce) (in a microtiter
plate). Positive fractions show a blue reaction. 8. Combine
positive fractions and dialyze against 1000-fold of sample volume
of PBS overnight. 9. Measure OD280 of dialyzed sample. 10. Antibody
protein concentration can be determined UV at 280 nm. 11. Check
purity of the sample by SDS-PAGE. Single bands of about 200 kDa
should be observed for antibody molecules under nonreducing
condition, and two bands of 37.5 kDa (LC) and 62.5 kDa (HC) should
be seen under reducing conditions. 12. Store purified protein at
-20.degree. C.
[0144] SDS Page:
[0145] For analysis of monoclonal antibodies using polyacrylamide
gel electrophoresis, two hydrolytic fragments derived from the
heavy chain of mouse IgG1 will be produced during incubation of the
antibodies in Laemmli reducing sample buffer at 10.degree. C. for 5
min as described previously (Davagnino J, Wong C, Shelton L,
Mankarious S (1995) Acid hydrolysis of monoclonal antibodies. J
Immunol Method. 185:177-180). The cleavage sites will be identified
by amino terminal sequencing.
[0146] Western Blotting:
[0147] Expression levels of antibody in individual expression
systems will be evaluated by Western blotting according to the
procedure described previously (T. Matsuo, A. Yamamoto, T.
Yamamoto, K. Otsuki, N. Yamazaki, M. Kataoka, H. Terada, Y.
Shinohara, Replacement of C305 in heart/muscle-type isozyme of
human carnitine palmitoyltransferase I with aspartic acid and other
amino acids, Biochem. Genet. 48 (2010) 193-201). Specific antibody
will be prepared as stated above.
[0148] Measurements of Protein Concentration:
[0149] The protein concentration of mitochondrial fractions was
measured by use of a BCA protein assay kit with bovine serum
albumin as the standard.
[0150] Antibody Binding Affinity:
[0151] Robust generation of IgG bispecific antibodies has been a
long-standing challenge. Existing methods require extensive
engineering of each individual antibody, discovery of common light
chains, or complex and laborious biochemical processing. Here we
combine computational and rational design approaches with
experimental structural validation to generate antibody heavy and
light chains with orthogonal Fab interfaces. Parental monoclonal
antibodies incorporating these interfaces, when simultaneously
co-expressed, assemble into bispecific IgG with improved heavy
chain-light chain pairing. Bispecific IgGs generated with this
approach exhibit pharmacokinetic and other desirable properties of
native IgG, but bind target antigens monovalently. As such, these
CDR grafted reagents may be useful in many biotechnological and
therapeutic applications.
[0152] Affinity Measurement of the mAbs and CDR Grafted
Antibodies:
[0153] Affinities of the mAbs containing heavy chain-light chain
redesigns will be determined using surface plasmon resonance
(Biacore 3000, GE Lifesciences). Fabs will be generated from the WT
pritumumab IgG1 and CRD2 containing IgG1s. Goat anti-human IgG-Fc
(Jackson Immunolabs, cat #109-005-098) will be diluted to 40 g/ml
in 10 mM acetate, pH 5, and immobilized to a CM5 chip surface to
.about.10,000 RU using standard
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC)/N-hydroxysuccinimide (NHS) amine coupling protocols. Fc of
human antibodies that will be procured from different sources will
be captured on the sensorship surface by injection for 4 min at 5
.mu.l/min. The flow will be increased to 30 .mu.l/min and a
secondary injection of each Fab (at 50, 35, 20, 10, 5, 2 or 1 nM)
will be performed. The running buffer (and dilution buffer) will be
HBS-EP 10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% polysorbate 20.
The chip surface will be regenerated by two injections of 0.1 M
glycine, pH 2.0. The concentration series will be fitted to a 1:1
binding model to determine the binding (ka) and dissociation (kd)
rate constants and the equilibrium dissociation constant (KD).
[0154] Mass Spectrometric Determination of Light Chain Specificity
and CDR Grafted IgG Assembly:
[0155] Proteins will be purified on an Agilent 1100 HPLC using a
protein G (PG) ID sensor cartridge (Life Technologies). Purified
samples will be analyzed on an Agilent 6210 time-of-flight liquid
chromatography/mass spectrometry (LC/MS) system molecular weight
analyzer. Theoretical mass-averaged molecular weights of the light
chain and heavy chain components will be determined using the GPMaw
program (v. 8.20). For the light chain competition experiments, the
relative counts of the ionized light chains hitting the detector
will be used to quantify the ratio of designed versus WT light
chain bound to a designed or WT heavy chain.
[0156] Fab Protein Crystallization:
[0157] Fab proteins will be produced by proteolytic cleavage of the
full-length IgGs using papain. Pritumumab Fab (with CX) crystal
screening will be performed at 16 mg/ml protein. The crystals (thin
microcrystalline plates) should appear after 4 days in 100 mM
sodium acetate pH 4.6/30% PEG MME 2K/200 mM ammonium sulfate.
Crystals will be cryo-protected in reservoir solution with PEG MME
2K. Concentration will be increased by 10% and supplemented with
20% glycerol. Improved order within the variable domains will be
achieved by generating crystal in the same conditions, plus 10% MPD
at 12.4 mg/ml protein. The pritumumab Fab containing the CDR will
be crystallized using 15 mg/ml protein.
[0158] Structure Determination:
[0159] X-ray diffraction data will be collected under standard
cryogenic conditions at the Advanced Photon Source (Argonne
National Laboratory) using the LRL-CAT beamline and reduced to
structure factor amplitudes using MOSFLM, SCALA and TRUNCATE. All
structures will be solved using PHASER, refined using REFMAC, and
visualized and rebuilt using XTALVIEW/XFIT. Structures of
subsequent design mutants (CRD1 and an intermediate of CRD2) will
be solved by Phaser using this parent structure as a search model.
The parent Fab of pritumumab will be solved using the known
structure of pritumumab and the variable domains of the antibody.
The stereochemical quality of the atomic model will be monitored
using an automated quality control procedure.
[0160] Phage Display Library:
[0161] By using optimized procedures that are based on the
classical oligonucleotide-directed mutagenesis method of Kunkel et
al., very large phage-displayed antibody repertoires (>10.sup.10
members) can be constructed quite rapidly. Importantly, the method
is scalable and can be used to mutate up to four independent
regions concurrently with very high efficiency. First, a
dut-/ung-E. coli host will be used to propagate phage encapsulating
uracil-containing ssDNA (dU-ssDNA) template to which mutagenic
oligonucleotides are annealed. "Stop templates" contain stop codons
in the CDRs and ensure that only mutated antibodies are displayed,
as the parental stop template will fail to express a full-length
Fab-Pritumumab fusion protein. Residual template clones are
therefore eliminated from the phage pools during selections.
Diversity within a Fab library can be designed by using mutagenic
oligonucleotides that contain mixed bases at particular positions
to produce sets of degenerate codons. Alternatively, finer control
of codon usage can be achieved by using oligonucleotides
synthesized from sets of trinucleotides. By choosing particular
codons for specific amino acids, we biased the CDR amino acids to
those that are commonly found in natural antibodies or are
particularly well suited for antigen recognition. Annealed
mutagenic oligonucleotides to the ssDNA template will serve to
prime synthesis of a complementary DNA strand forming a synthetic
daughter strand lacking uracil. A ligase then fuses the synthesized
DNA fragments to from covalently closed circular double-stranded
heteroduplex DNA (CCC-dsDNA). The heteroduplex DNA will then be
electroporated into a highly competent strain of dut+/ung+E. coli,
SS320, where the synthesized strand is preferentially amplified
compared to the template strand.
[0162] Transformation into an E. coli host results in phagemid
replication as a double-stranded plasmid. Upon coinfection with
helper phage, single-stranded DNA (ssDNA) replication is initiated
and phagemid ssDNA is packaged into phage particles containing
phagemid-encoded protein, thereby providing physical linkage
between the phenotype of the Fab and the encoding phagemid
genotype. Helper phage, such as M13KO7, provides all proteins
necessary for assembly of phage particles with some phagemid
encoded fusion protein incorporated. These phage particles produced
by infected cells make up both the genetic barcode and interaction
readout of the Fab library as the host E. coli cells are never
introduced to antigens during selections.
[0163] While specific aspects have been described and illustrated,
such aspects should be considered illustrative only and not as
limiting in accordance with the accompanying claims.
[0164] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entirety for all purposes as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference for all purposes.
[0165] Although the foregoing compositions and methods has been
described in some detail by way of illustration and example for
purposes of clarity of understanding, it will be readily apparent
to one of ordinary skill in the art in light of the teachings
herein that certain changes and modifications can be made thereto
without departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
551450PRTArtificial SequenceSynthetic Construct 1Glu Val Gln Leu
Leu Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ala Ile Thr Pro Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Ile
Cys 85 90 95Gly Arg Val Pro Tyr Arg Ser Thr Trp Tyr Pro Leu Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 4502214PRTArtificial SequenceSynthetic Construct 2Asp
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 Asp Ile Ser Asn Tyr
20 25 30Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu
Ile 35 40 45Tyr Ala Ala Ser Ser Leu His Ser Lys Val Pro Thr Gln 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 Leu Gln Tyr
Ser Thr Tyr Pro Ile 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2103226PRTARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 3Gln Val Gln Leu Lys Gln Ser Gly Pro
Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30Gly Val His Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Ser Gly
Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60Ser Arg Leu Ser Ile
Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75 80Lys Met Asn
Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg Ala
Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ala Asp Ile Leu Leu Thr Gln Ser Pro Val
115 120 125Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys
Arg Ala 130 135 140Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln
Gln Arg Thr Asn145 150 155 160Gly Ser Pro Arg Leu Leu Ile Lys Tyr
Ala Ser Glu Ser Ile Ser Gly 165 170 175Ile Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu 180 185 190Ser Ile Asn Ser Val
Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln 195 200 205Gln Asn Asn
Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu 210 215 220Leu
Lys2254226PRTARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 4Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Leu 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 30Asn
Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40
45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe
Asn Val Trp Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ala Gln
Ile Val Leu Ser Gln Ser 115 120 125Pro Ala Ile Leu Ser Ala Ser Pro
Gly Glu Lys Val Thr Met Thr Cys 130 135 140Arg Ala Ser Ser Ser Val
Ser Tyr Ile His Trp Phe Gln Gln Lys Pro145 150 155 160Gly Ser Ser
Pro Lys Pro Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser 165 170 175Gly
Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 180 185
190Leu Thr Ile Ser Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
195 200 205Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys
Leu Glu 210 215 220Ile Lys2255228PRTARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 5Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro
Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe
Thr Asp Phe 20 25 30Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Arg Gly
Leu Glu Trp Ile 35 40 45Gly Phe Ile Arg Asp Lys Ala Lys Gly Tyr Thr
Thr Glu Tyr Asn Pro 50 55 60Ser Val Lys Gly Arg Val Thr Met Leu Val
Asp Thr Ser Lys Asn Gln65 70 75 80Phe Ser Leu Arg Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Glu Gly His
Thr Ala Ala Pro Phe Asp Tyr Trp Gly 100 105 110Gln Gly Ser Leu Val
Thr Val Ser Ser Asp Ile Gln Met Thr Gln Ser 115 120 125Pro Ser Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 130 135 140Lys
Ala Ser Gln Asn Ile Asp Lys Tyr Leu Asn Trp Tyr Gln Gln Lys145 150
155 160Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asn Thr Asn Asn Leu
Gln 165 170 175Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe 180 185 190Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp
Ile Ala Thr Tyr Tyr 195 200 205Cys Leu Gln His Ile Ser Arg Pro Arg
Thr Phe Gly Gln Gly Thr Lys 210 215 220Val Glu Ile
Lys2256230PRTARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 6Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp
Tyr Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Asp Ile Gln Met Thr 115 120 125Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val Thr Ile 130 135 140Thr Cys Ser Ala Ser Gln
Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln145 150 155 160Gln Lys Pro
Gly Lys Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser 165 170 175Leu
His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 180 185
190Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
195 200 205Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly
Gln Gly 210 215 220Thr Lys Val Glu Ile Lys225 2307225PRTARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 7Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln Met Thr Gln Ser Pro
115 120 125Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg 130 135 140Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr
Gln Gln Lys Pro145 150 155 160Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Ser Ala Ser Phe Leu Tyr Gly 165 170 175Val Pro Ser Arg Phe Gly Ser
Arg Ser Gly Thr Asp Phe Thr Leu Thr 180 185 190Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 195 200 205His Tyr Thr
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 210 215
220Lys2258226PRTARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 8Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Asp Ile Gln
Met Thr Gln Ser Pro Ser 115 120 125Ser Leu Ser Ala Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Lys Ala 130 135 140Ser Gln Asp Val Ser Ile
Gly Val Ala Trp Tyr Gln Gln Lys Pro Gly145 150 155 160Lys Ala Pro
Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly 165 170 175Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 180 185
190Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
195 200 205Gln Tyr Tyr Ile Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Val Glu 210 215 220Ile Lys2259184PRTARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 9Val Ser Gly Gly Ser Val Ser Ser Gly Asp Tyr Tyr Trp Arg
Trp Ile1 5 10 15Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile Gly His
Ile Tyr Tyr 20 25 30Ser Gly Asn Thr Asn Tyr Asn Pro Ser Lys Leu Ser
Arg Leu Thr Ile 35 40 45Ser Ile Asp Thr Ser Lys Thr Gln Phe Ser Leu
Lys Leu Ser Ser Val 50 55 60Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys
Val Arg Asp Arg Val Tyr65 70 75 80Gly Ala Phe Asp Ile Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser 85 90 95Thr Ile Thr Asp Gln Ala Ser
Gln Asp Ile Ser Asn Tyr Leu Asn Trp 100 105 110Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala 115 120 125Ser Asn Leu
Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 130 135 140Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile145 150
155 160Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu Ala Phe
Gly 165 170 175Gly Gly Thr Lys Val Glu Ile Lys
18010229PRTARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 10Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55
60Lys Arg Arg Phe Thr Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Ile
Gln Met Thr Gln 115 120 125Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr 130 135 140Cys Ser Ala Ser Gln Asp Ile Ser
Asn Tyr Leu Asn Trp Tyr Gln Gln145 150 155 160Lys Pro Gly Lys Ala
Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu 165 170 175His Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 180 185 190Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 195 200
205Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe
Gly Gln Gly Thr 210 215 220Lys Val Glu Ile Lys22511229PRTARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 11Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Trp Ile Asn Thr Tyr
Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60Lys Arg Arg Phe Thr
Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Lys Tyr
Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Ile Gln Leu Thr Gln
115 120 125Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr 130 135 140Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
Trp Tyr Gln Gln145 150 155 160Lys Pro Gly Lys Ala Pro Lys Val Leu
Ile Tyr Phe Thr Ser Ser Leu 165 170 175His Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp 180 185 190Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 195 200 205Tyr Cys Gln
Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr 210 215 220Lys
Val Glu Ile Lys2251220DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
12tcaccttcag caactatgcc 201324DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 13aagggtacca agtgcttcta tatg 241420DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 14gagtgggtct cagcgattac
201518DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 15agaggtgctc
ttggagga 181618DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
16ctatgccatg agctgggt 181724DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 17tctatatgga ctctcccaca gata 241820DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 18gcctggtcaa aggcttctat
201920DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 19tcttctgcgt
gtagtggttg 202020DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
20gctgaatggc aaggagtaca 202122DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 21gagcttgctg tagaggaaga ag 222222DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 22caaggtggac aagaaagttg ag
222320DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 23catcacggag
catgagaaga 202418DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
24atcacaagcc cagcaaca 182521DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 25gaccttgcac ttgtactcct t 212620DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 26gtgcaaggtc tccaacaaag
202722DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 27gcgtggtctt
gtagttgttc tc 222822DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
28gcatctgtag gagacagagt ca 222922DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 29gccgaaggtg atagggtaag ta 223020DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 30agtctccatc ctcactgtct
203122DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 31gagtgaaatc
tgtcccagat cc 223222DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
32gagacagagt caccatcact tg 223324DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 33agggtaagta ctatactgta ggca 243421DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 34ctgcaccatc tgtcttcatc t
213521DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 35aggcgtagac
tttgtgtttc t 213619DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
36cttcatcttc ccgccatct 193722DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 37gtgtttctcg tagtctgctt tg 223820DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 38atctggaact gcctctgttg
203919DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 39cttcgcaggc
gtagacttt 194021DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
40gttgtgtgcc tgctgaataa c 214121DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 41ccctgttgaa gctctttgtg a 214221DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 42aggtggagat caaacgaact g
214319DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 43gctgtaggtg
ctgtccttg 194421DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
44gttatgcgcc tgcgtctgta c 214520DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 45ccgggtgttc agaactcgtc 204619DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 46acggtgtcgt ggaactcag
194724DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 47cacgctgctg
agggagtaga gtcc 244822DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
48caaagtacag tggaaggtgg at 224924DNAARTIFICIAL SEQUENCESYNTHETIC
CONSTRUCT 49gtgctgtcct tgctgtcctg ctct 245021DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 50aaagatgggc ggtcgttatt c
215124DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT 51cctgccggtt
gcgaaatggg ataa 245235DNAARTIFICIAL SEQUENCESYNTHETIC CONSTRUCT
52ttttaagctt gccgccacca tgatgtcctt tgtct 355371DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 53gccaggggga agaccgatgg gcccttggtg
gaggcagagg acacggtcac gagggtgccc 60tggccccaat a 715471DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 54tattggggcc agggcaccct cgtgaccgtg
tcctctgcct ccaccaaggg cccatcggtc 60ttccccctgg c 715559DNAARTIFICIAL
SEQUENCESYNTHETIC CONSTRUCT 55tttctcgaga tctcatcatt tcccgggaga
cagggagagg ctcttctgcg tgtagtggt 59
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