U.S. patent application number 16/423718 was filed with the patent office on 2019-09-19 for conditionally active biological proteins.
This patent application is currently assigned to BioAtla, LLC. The applicant listed for this patent is BioAtla, LLC. Invention is credited to Hwai Wen Chang, Gerhard Frey, Jay M. Short.
Application Number | 20190284551 16/423718 |
Document ID | / |
Family ID | 54480489 |
Filed Date | 2019-09-19 |
United States Patent
Application |
20190284551 |
Kind Code |
A1 |
Short; Jay M. ; et
al. |
September 19, 2019 |
Conditionally Active Biological Proteins
Abstract
This disclosure relates to a method of generating conditionally
active biologic proteins from wild type proteins, in particular
therapeutic proteins, which are reversibly or irreversibly
inactivated at some physiological conditions. For example,
conditionally active biologic proteins are active in tumors, but
virtually inactive at other body parts, or conditionally active
antibodies capable of crossing blood-brain-barrier.
Inventors: |
Short; Jay M.; (Del Mar,
CA) ; Chang; Hwai Wen; (San Marcos, CA) ;
Frey; Gerhard; (San Diego, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
BioAtla, LLC |
San Diego |
CA |
US |
|
|
Assignee: |
BioAtla, LLC
San Diego
CA
|
Family ID: |
54480489 |
Appl. No.: |
16/423718 |
Filed: |
May 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15308659 |
Nov 3, 2016 |
10329556 |
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PCT/US2015/030086 |
May 11, 2015 |
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16423718 |
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62153001 |
Apr 27, 2015 |
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62043080 |
Aug 28, 2014 |
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61992415 |
May 13, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/475 20130101; G01N 2333/705 20130101; C07K 16/28 20130101;
C07K 14/54 20130101; C12N 15/1058 20130101; C07K 2319/30 20130101;
C07K 16/00 20130101; C12Q 1/68 20130101; C07K 2319/00 20130101;
C07K 2317/31 20130101; C07K 2317/52 20130101; G01N 33/6854
20130101; C12N 15/09 20130101; C07K 14/52 20130101; C07K 14/575
20130101; C12N 9/00 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; G01N 33/68 20060101 G01N033/68; C07K 16/28 20060101
C07K016/28; C12N 15/09 20060101 C12N015/09; C12Q 1/68 20060101
C12Q001/68; C07K 14/52 20060101 C07K014/52; C07K 16/00 20060101
C07K016/00; C12N 9/00 20060101 C12N009/00; C07K 14/475 20060101
C07K014/475; C07K 14/575 20060101 C07K014/575; C07K 14/54 20060101
C07K014/54 |
Claims
1. A method of preparing a conditionally active antibody for
crossing the blood-brain barrier, the method comprising the steps
of: i. evolving DNA which encodes a parent antibody against a
blood-brain barrier receptor using one or more evolutionary
techniques to create mutant DNAs; ii. expressing the mutant DNAs to
obtain mutant antibodies; iii. subjecting the mutant antibodies to
an assay under a first physiological condition in blood plasma and
to an assay under a second physiological condition in brain
extracellular fluid; and iv. selecting the conditionally active
antibody from the mutant antibodies which exhibit both (a) an
affinity to the blood-brain barrier receptor in the assay under the
first physiological condition, and (b) an affinity selected from
the group consisting of a decreased affinity to the blood-brain
barrier receptor in the assay under the second physiological
condition in comparison with the affinity in the assay under the
first physiological condition and no affinity to the blood-brain
barrier receptor in the assay under the second physiological
condition.
2. The method of claim 1, further comprising the step of
conjugating the conditionally active antibody to a molecule.
3. The method of claim 2, wherein the conjugating step comprises
forming a covalent bond between the conditionally active antibody
and the molecule.
4. The method of claim 2, wherein the conjugating step comprises
forming a non-covalent bond between the conditionally active
antibody and the molecule.
5. The method of claim 2, wherein the molecule is selected from the
group consisting of cytokines, interleukins, enzymes, hormones,
growth factors, cytotoxic agents, chemotherapy drugs, radioactive
particles, antibodies and diagnostic agents.
6. The method of claim 2, wherein the molecule is conjugated to the
Fc region of the conditionally active antibody.
7. The method of claim 1, further comprising the step of
introducing at least one amino acid substitution in the Fc region
of the conditionally active antibody.
8. The method of claim 7, wherein the at least one amino acid
substitution is two or more amino acid substitutions.
9. The method of claim 1, further comprising the step of
engineering the conditionally active antibody to be
multispecific.
10. The method of claim 9, wherein the selecting step selects the
conditionally active antibody which also exhibits an affinity to an
antigen in addition to an affinity for the blood-brain barrier
receptor.
11. The method of claim 1, wherein the blood-brain barrier receptor
is selected from the group consisting of a transferrin receptor, an
insulin receptor, an insulin-like growth factor receptor, low
density lipoprotein receptor-related protein 1, low density
lipoprotein receptor-related protein 8, and a heparin-binding
epidermal growth factor-like growth factor.
12. The method of claim 1, wherein in the affinity for the
blood-brain barrier receptor in the assay under the first
physiological condition is measured by the conditionally active
antibody's IC50 for inhibiting binding of the blood-brain barrier
receptor's natural ligand, and the IC50 of the conditionally active
antibody is from about 1 nM to about 100 .mu.M.
13. The method of claim 1, wherein the evolving step comprises
mutating a Fc region of the antibody.
14. The method of claim 1, wherein the evolving step comprises a
technique selected from PCR, error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, gene site saturated
mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase
chain reaction, oligonucleotide synthesis, and combinations
thereof.
15. The method of claim 1, wherein the expression step comprises
expressing the mutant DNA in a host cell selected from a bacterial
cell, a fungal cell, an insect cell, a mammalian cell,
adenoviruses, and a plant cell.
16. The method of claim 15, wherein the host cell is a mammalian
cell selected from a Bowes melanoma cell, a COS-7 cell, a C127
cell, a 3T3 cell, a CHO cell, a HeLa cell and a BHK cell.
17. A conditionally active antibody prepared by the method of claim
1, wherein the conditionally active antibody is reversibly
inactivated under the second physiological condition.
18. The conditionally active antibody of claim 17, wherein the
conditionally active antibody is conjugated to a molecule that is
released under the second physiological condition.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/308,659, filed on Nov. 3, 2016, which, in
turn is a 371 continuation of International application no.
PCT/US2015/030086, filed May 11, 2015, which, in turn, claims the
benefit of U.S. Provisional application No. 62/153,001, filed Apr.
27, 2015, U.S. Provisional application No. 62/043,080, filed Aug.
28, 2014, and U.S. Provisional application No. 61/992,415, filed
May 13, 2014.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to the field of protein evolution
and activity. Specifically, this disclosure relates to a method of
generating conditionally active biologic proteins from wild type
proteins, in particular therapeutic proteins, which are reversibly
or irreversibly inactivated at the wild type normal physiological
conditions as well as to such conditionally active biologic
proteins and uses of such conditional active biologic proteins.
BACKGROUND OF THE DISCLOSURE
[0003] There is a considerable body of literature describing the
potential for evolving proteins for a variety of characteristics,
especially enzymes for example, to be stabilized for operation at
different conditions. For example, enzymes have been evolved to be
stabilized at higher temperatures, with varying activity. In
situations where there is an activity improvement at the high
temperature, a substantial portion of the improvement can be
attributed to the higher kinetic activity commonly described by the
Q10 rule where it is estimated that in the case of an enzyme the
turnover doubles for every increase of 10 degrees Celsius. In
addition, there exist examples of natural mutations that
destabilize proteins at their normal operating conditions, such as
wild-type temperature activity of the molecule. For temperature
mutants, these mutants can be active at the lower temperature, but
typically are active at a reduced level compared to the wild type
molecules (also typically described by a reduction in activity
guided by the Q10 or similar rules).
[0004] It is desirable to generate useful molecules that are
conditionally activated, for example virtually inactive at
wild-type conditions but are active at other than wild-type
conditions at a level that is equal or better than at wild-type
conditions, or that are activated or inactivated in certain
microenvironments, or that are activated or inactivated over time.
Besides temperature, other conditions for which the proteins can be
evolved or optimized include at least pH, osmotic pressure,
osmolality, oxidation and electrolyte concentration. Other
desirable properties that can be optimized during evolution include
chemical resistance, and proteolytic resistance.
[0005] Many strategies for evolving or engineering molecules have
been published. US 2010/0189651 discloses an engineered antibody
containing an antibody or antibody fragment linked with a masking
moiety. Such an engineered antibody can be further coupled to a
cleavable moiety, resulting in an antibody that can be
conditionally activated. The cleavable moiety is capable of being
cleaved, reduced, or photolysed. The antibody can exhibit a
conformation such that the antibody is more accessible to a target
after removal of the masking moiety by cleavage, reduction, or
photolysis of the cleavable moiety.
[0006] US 2013/0101555 discloses engineered activatable proprotein
compositions. An activatable proprotein contains a functional
protein coupled to a peptide mask, and further coupled to an
activatable linker. In a non-activated state, the peptide mask
inhibits binding of the functional protein to its target or binding
partner. In an activated state, the peptide mask does not inhibit
binding of the functional protein to its target or binding partner.
Proproteins can provide for reduced toxicity and adverse side
effects that could otherwise result from binding of a functional
protein at non-treatment sites if it were not inhibited from
binding to its binding partner at such non-treatment sites.
Proproteins containing the peptide mask can also have a longer in
vivo or serum half-life than the corresponding functional protein
not containing the peptide mask.
[0007] US 2011/0229489 discloses antibodies with pH dependent
binding to antigens such that the affinity for antigen binding at
physiological pH (i.e., pH 7.4) is greater than at endosomal pH
(i.e., pH 6.0 or 5.5). Such pH-dependent antibodies preferentially
dissociate from the antigen in the endosome. This can increase
antibody half-life, as compared to antibodies with equivalent
K.sub.DS at pH 7.4 but with no pH dependent binding, when the
antigen is one that undergoes antigen-mediated clearance (e.g.,
PCSK9). Antibodies with pH-dependent binding can decrease total
antigen half-life when the antigen undergoes reduced clearance
after being bound to an antibody.
[0008] US 2013/0266579 discloses a conditionally active anti-EGFR
antibody. The anti-EGFR antibody exhibits a ratio of binding
activity to human epidermal growth factor receptor (EGFR) for
conditions in a tumor environment to conditions in a non-tumor
environment of at least 3.0. The conditions in a tumor environment
comprise one or both of a pH of from 5.6 to 6.8 or a lactate
concentration of from 5 mM to 20 mM, and a protein concentration
from 10 mg/mL to 50 mg/mL. The conditions in a non-tumor
environment comprise one or both of a pH of from 7.0 to 7.8 or a
lactate concentration of from 0.5 mM to 5 mM, and a protein
concentration of from 10 mg/mL to 50 mg/mL. The anti-EGFR antibody
is said to be conditionally active under conditions that may be
found in a tumor microenvironment.
[0009] Pardoll et al, "The blockade of immune checkpoints in cancer
immunotherapy," Nature Review Cancer, vol. 12, pages 252-264, 2012
describes a cancer therapy that involves activating host
anti-tumour immunity by blockading host immune system checkpoints.
Such a blockade may be achieved by inhibiting immune checkpoint
proteins such as receptors on T-cells, including cytotoxic
T-lymphocyte-associated antigen 4 (CTLA4) and death protein 1
(PD1). Antibodies against these immune checkpoint proteins have
been developed for cancer therapy.
[0010] Engineering or evolving a protein to be inactive or
virtually inactive (less than 10% activity and especially 1%
activity) at its wild type operating condition, while maintaining
activity equivalent or better than its wild type condition at new
conditions, requires that the destabilizing mutation(s) co-exist
with activity increasing mutations that do not counter the
destabilizing effect. It is expected that destabilization would
reduce the protein's activity greater than the effects predicted by
standard rules such as Q10, therefore the ability to evolve
proteins that work efficiently at lower temperature, for example,
while being inactivated under their normal operating condition,
creates an unexpected new class of conditionally active
proteins.
[0011] Throughout this application, various publications are
referenced by author and date. The disclosures of these
publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art as known to those skilled therein as of the date
of the disclosure described and claimed herein.
SUMMARY OF THE DISCLOSURE
[0012] In one aspect, the present invention provides a method of
preparing a conditionally active biological protein, the method
comprising the steps of: i. selecting wild-type biological protein;
ii. evolving the DNA which encodes the wild-type biological protein
using one or more evolutionary techniques to create mutant DNAs;
iii. expressing the mutant DNAs to obtain mutant biological
proteins; iv. subjecting the mutant biological proteins and the
wild-type biological protein to an assay under a first
physiological condition selected from physiological conditions of a
first location selected from the group consisting of synovial
fluid, a tumor microenvironment and a stem cell niche, and to an
assay under a second physiological condition selected from
physiological conditions of a second location in a body that is
different from the first location; and v. selecting the
conditionally active biologic protein from the mutant biologic
proteins which exhibit both (a) an increased activity in the assay
under the first physiological condition compared to the wild-type
biologic protein, and (b) a decreased activity in the assay under
the second physiological condition compared to the wild-type
biologic protein.
[0013] In another aspect, the present invention provides a method
of preparing a conditionally active antibody for crossing the
blood-brain barrier, the method comprising the steps of: i.
selecting a wild-type antibody against a blood-brain barrier
receptor; ii. evolving the DNA which encodes the wild-type antibody
using one or more evolutionary techniques to create mutant DNAs;
iii. expressing the mutant DNAs to obtain mutant antibodies; iv.
subjecting the mutant antibodies and the wild-type antibody to an
assay under a first physiological condition in blood plasma and to
an assay under a second physiological condition in brain
extracellular fluid; and v. selecting the conditionally active
antibody from the mutant antibodies which exhibit both: (a) a
decrease in affinity to the blood-brain barrier receptor in the
assay under the first physiological condition compared to the
wild-type antibody, and (b) an affinity selected from the group
consisting of an increased affinity to the blood-brain barrier
receptor in the assay under the second physiological condition and
no affinity to the blood-brain barrier receptor in the assay under
the second physiological condition.
[0014] In yet another aspect, the method of the present invention
further comprises the step of conjugating the conditionally active
biological protein to a molecule.
[0015] In yet another aspect, the method of the present invention
further comprises the step of introducing at least one amino acid
substitution in the Fc region of a conditionally active
antibody.
[0016] In yet another aspect, the method of the present invention
further comprises the step of engineering the conditionally active
antibody to be multispecific.
[0017] In yet another aspect, the present invention provides a
conditionally active biological protein. In yet another aspect, the
conditionally active biological protein is a conditionally active
antibody.
Definitions
[0018] In order to facilitate understanding of the examples
provided herein, certain frequently occurring methods and/or terms
will be defined herein.
[0019] As used herein in connection with a measured quantity, the
term "about" refers to the normal variation in that measured
quantity that would be expected by the skilled artisan making the
measurement and exercising a level of care commensurate with the
objective of the measurement and the precision of the measuring
equipment used. Unless otherwise indicated, "about" refers to a
variation of +/-10% of the value provided.
[0020] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, an array of spatially
localized compounds (e.g., a VLSIPS peptide array, polynucleotide
array, and/or combinatorial small molecule array), biological
macromolecule, a bacteriophage peptide display library, a
bacteriophage antibody (e.g., scFv) display library, a polysome
peptide display library, or an extract made from biological
materials such as bacteria, plants, fungi, or animal (particular
mammalian) cells or tissues. Agents are evaluated for potential
enzyme activity by inclusion in screening assays described herein
below. Agents are evaluated for potential activity as conditionally
active biologic therapeutic enzymes by inclusion in screening
assays described herein below.
[0021] The term "amino acid" as used herein refers to any organic
compound that contains an amino group (--NH.sub.2) and a carboxyl
group (--COOH); preferably either as free groups or alternatively
after condensation as part of peptide bonds. The "twenty naturally
encoded polypeptide-forming alpha-amino acids" are understood in
the art and refer to: alanine (ala or A), arginine (arg or R),
asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or
C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly
or G), histidine (his or H), isoleucine (ile or I), leucine (leu or
L), lysine (lys or K), methionine (met or M), phenylalanine (phe or
F), proline (pro or P), serine (ser or S), threonine (thr or T),
tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or
V).
[0022] The term "amplification" as used herein means that the
number of copies of a polynucleotide is increased.
[0023] The term "antibody" as used herein refers to intact
immunoglobulin molecules, as well as fragments of immunoglobulin
molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that
are capable of binding to an epitope of an antigen. These antibody
fragments, which retain some ability to selectively bind to an
antigen (e.g., a polypeptide antigen) of the antibody from which
they are derived, can be made using well known methods in the art
(see, e.g., Harlow and Lane, supra), and are described further, as
follows. Antibodies can be used to isolate preparative quantities
of the antigen by immunoaffinity chromatography. Various other uses
of such antibodies are to diagnose and/or stage disease (e.g.,
neoplasia) and for therapeutic application to treat disease, such
as for example: neoplasia, autoimmune disease, AIDS, cardiovascular
disease, infections, and the like. Chimeric, human-like, humanized
or fully human antibodies are particularly useful for
administration to human patients.
[0024] An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0025] An Fab' fragment of an antibody molecule can be obtained by
treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0026] An (Fab')2 fragment of an antibody can be obtained by
treating a whole antibody molecule with the enzyme pepsin, without
subsequent reduction. A (Fab')2 fragment is a dimer of two Fab'
fragments, held together by two disulfide bonds.
[0027] An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0028] The term "antibody-dependent cell-mediated cytotoxicity" or
"ADCC" refers to a form of cytotoxicity in which secreted
immunoglobulin binds to Fc receptors (FcRs) present on certain
cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and
macrophages) that enables these cytotoxic effector cells to bind
specifically to an antigen-bearing target cell and subsequently
kill the target cell with cytotoxins. Ligand specific high-affinity
IgG antibodies directed to the surface of target cells stimulate
the cytotoxic cells via affinity to the ADCC domain on the IgG to
attack the cell bound to the IgG antibody via the Fab region. Lysis
of the target cell is extracellular, which requires direct
cell-to-cell contact, and does not involve complement.
[0029] The ability of any particular antibody to mediate lysis of
the target cell by ADCC can be assayed. To assess ADCC activity, an
antibody of interest is mixed with the target cells displaying the
target ligand in combination with immune effector cells, which may
be activated by the antigen antibody complexes resulting in
cytolysis of the target cell. Cytolysis is often detected by the
release of a label (e.g., radioactive substrates, fluorescent dyes
or natural intracellular proteins) from the lysed cells. Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Specific examples of in
vitro ADCC assays are described in Bruggemann et al, 1987, J. Exp.
Med., vol. 166, page 1351 ; Wilkinson et al, 2001, J. Immunol.
Methods, vol. 258, page 183; Patel et al, 1995 J. Immunol. Methods,
vol. 184, page 29 (each of which is incorporated by reference).
Alternatively, or additionally, ADCC activity of the antibody of
interest may be assessed in vivo, e.g., in an animal model, such as
that disclosed in Clynes et al, 1998, PNAS USA, vol. 95, page 652,
the contents of which are incorporated by reference in its
entirety.
[0030] The term "Antibody-dependent cellular phagocytosis" or
"ADCP" refers to a process by which antibody-coated cells are
internalized, either in whole or in part, by phagocytic immune
cells (e.g., macrophages, neutrophils and dendritic cells) that
bind to an immunoglobulin Fc region.
[0031] The term "blood-brain barrier" or "BBB" refers to the
physiological barrier between the peripheral circulation and the
brain and spinal cord which is formed by tight junctions within the
brain capillary endothelial plasma membranes, creating a tight
barrier that restricts the transport of molecules into the brain,
even very small molecules such as urea (60 Daltons). The
blood-brain barrier within the brain, the blood-spinal cord barrier
within the spinal cord, and the blood-retinal barrier within the
retina are contiguous capillary barriers within the central nerve
system (CNS), and are herein collectively referred to as the
"blood-brain barrier" or "BBB." The BBB also encompasses the
blood-cerebral spinal fluid barrier (choroid plexus) where the
barrier is comprised of ependymal cells rather than capillary
endothelial cells.
[0032] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. A "tumor" comprises one
or more cancerous cells. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
[0033] A "comparison window," as used herein, refers to a
conceptual segment of at least 20 contiguous nucleotide positions
wherein a polynucleotide sequence may be compared to a reference
sequence of at least 20 contiguous nucleotides and wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 percent or less
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith (Smith and
Waterman, 1981 "Comparison of biosequences", Adv Appl Math,
2:482-489; Smith and Waterman, 1981, "Overlapping genes and
information theory", J Theor Biol, 91:379-380; Smith and Waterman,
J Mol Biol, "Identification of common molecular subsequences",
1981, 147:195-197; Smith et al., 1981, ""Comparative biosequence
metrics", J Mol Evol, 18:38-46), by the homology alignment
algorithm of Needleman (Needleman and Wunsch, 1970, "A general
method applicable to the search for similarities in the amino acid
sequence of two proteins" J Mol Biol, 48(3):443-453), by the search
of similarity method of Pearson (Pearson and Lipman, 1988,
"Improved tools for biological sequence comparison", Proc Nat Acad
Sci USA, 85:2444-2448), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected.
[0034] The term "complement-dependent cytotoxicity (CDC)" refers to
a process initiated by binding of complement factor C1q to the Fc
part of most IgG antibody subclasses. Binding of C1q to an antibody
is caused by defined protein-protein interactions at the so called
binding site. Such Fc part binding sites are known in the state of
the art (see above). Such Fc part binding sites are, e.g.,
characterized by the amino acids L234, L235, D270, N297, E318,
K320, K322, P331, and P329 (numbering according to EU index of
Kabat). Antibodies of subclass IgG1, IgG2, and IgG3 usually show
complement activation including C1q and C3 binding, whereas IgG4
does not activate the complement system and does not bind C1q
and/or C3. C1q is a polypeptide that includes a binding site for
the Fc region of an immunoglobulin. C1q together with two serine
proteases, C1r and C1s, forms the complex CI, the first component
of the complement dependent cytotoxicity (CDC) pathway.
[0035] The term "conditionally active biologic protein" refers to a
variant, or mutant, of a wild-type or a parent protein which is
more or less active than the parent or wild-type protein under one
or more normal physiological conditions. This conditionally active
protein also exhibits activity in selected regions of the body
and/or exhibits increased or decreased activity under aberrant, or
permissive, physiological conditions. Normal physiological
conditions are those of temperature, pH, osmotic pressure,
osmolality, oxidation and electrolyte concentration which would be
considered within a normal range at the site of administration, or
at the tissue or organ at the site of action, to a subject. An
aberrant condition is that which deviates from the normally
acceptable range for that condition. In one aspect, the
conditionally active biologic protein is virtually inactive at
wild-type conditions but is active at other than wild-type
conditions at a level that is equal or better than at wild-type
conditions. For example, in one aspect, an evolved conditionally
active biologic protein is virtually inactive at body temperature,
but is active at lower temperatures. In another aspect, the
conditionally active biologic protein is reversibly or irreversibly
inactivated at the wild type conditions. In a further aspect, the
wild-type protein is a therapeutic protein. In another aspect, the
conditionally active biologic protein is used as a drug, or
therapeutic agent. In yet another aspect, the protein is more or
less active in highly oxygenated blood, such as, for example, after
passage through the lung or in the lower pH environments found in
the kidney.
[0036] The term "conditionally active antibody" refers to a
variant, or mutant, of a wild-type or parent antibody which is more
or less active compared to the parent or wild-type antibody under
one or more normal physiological conditions. This conditionally
active antibody also exhibits activity in selected regions of the
body and/or exhibits increased or decreased activity under
aberrant, or permissive, physiological conditions. In one aspect,
the conditionally active antibody is virtually inactive under
normal physiological conditions but is active under non-normal
physiological conditions at a level that is equal or better than
under normal physiological conditions. For example, an evolved
conditionally active antibody is virtually inactive at normal body
temperature, but is active at lower body temperatures. In another
aspect, the conditionally active antibody is reversibly or
irreversibly inactivated under normal physiological conditions. In
a further aspect, the wild-type antibody is a therapeutic antibody.
In another aspect, the conditionally active antibody is used as a
drug, or therapeutic agent. In yet another aspect, the antibody is
more or less active in highly oxygenated blood, for example, after
passage through the lung or in the lower pH environments found in
the kidney.
[0037] "Conservative amino acid substitutions" refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0038] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormones such as human growth hormones, N-methionyl
human growth hormones, and bovine growth hormones; parathyroid
hormones; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and luteinizing hormone (LH);
hepatic growth factor; fibroblast growth factor; prolactin;
placental lactogen; tumor necrosis factor-a and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-a and TGF-.beta.; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons
such as interferon-a, -.beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis
factor such as TNF-a or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0039] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof. "Digestion" of DNA refers to catalytic
cleavage of the DNA with a restriction enzyme that acts only at
certain sequences in the DNA. The various restriction enzymes used
herein are commercially available and their reaction conditions,
cofactors and other requirements were used as would be known to the
ordinarily skilled artisan. For analytical purposes, typically 1
microgram of plasmid or DNA fragment is used with about 2 units of
enzyme in about 20 microliters of buffer solution. For the purpose
of isolating DNA fragments for plasmid construction, typically 5 to
50 micrograms of DNA are digested with 20 to 250 units of enzyme in
a larger volume. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the manufacturer.
Incubation times of about 1 hour at 37 degrees C. are ordinarily
used, but may vary in accordance with the supplier's instructions.
After digestion the reaction is electrophoresed directly on a gel
to isolate the desired fragment.
[0040] The term "DNA shuffling" is used herein to indicate
recombination between substantially homologous but non-identical
sequences, in some embodiments DNA shuffling may involve crossover
via non-homologous recombination, such as via cer/lox and/or
flp/frt systems and the like. DNA shuffling can be random or
non-random.
[0041] The term "drug" or "drug molecule" refers to a therapeutic
agent including a substance having a beneficial effect on a human
or animal body when it is administered to the human or animal body.
Preferably, the therapeutic agent includes a substance that can
treat, cure or relieve one or more symptoms, illnesses, or abnormal
conditions in a human or animal body or enhance the wellness of a
human or animal body.
[0042] An "effective amount" is an amount of a conditionally active
biologic protein or fragment which is effective to treat or prevent
a condition in a living organism to whom it is administered over
some period of time, e.g., provides a therapeutic effect during a
desired dosing interval.
[0043] As used herein, the term "electrolyte" is used to define a
mineral in the blood or other body fluids that carries a charge.
For example, in one aspect, the normal physiological condition and
aberrant condition can be conditions of "electrolyte
concentration". In one aspect, the electrolyte concentration to be
tested is selected from one or more of ionized calcium, sodium,
potassium, magnesium, chloride, bicarbonate, and phosphate
concentration. For example, in one aspect, normal range of serum
calcium is 8.5 to 10.2 mg/dL. In this aspect, aberrant serum
calcium concentration may be selected from either above or below
the normal range, m another example, in one aspect, normal range of
serum chloride is 96-106 milliequivalents per liter (mEq/L). In
this aspect, aberrant serum chloride concentration may be selected
from either above or below the normal range, in another example, in
one aspect, a normal range of serum magnesium is from 1.7-2.2
mg/dL. In this aspect, an aberrant serum magnesium concentration
may be selected from either above or below the normal range, in
another example, in one aspect, a normal range of serum phosphorus
is from 2.4 to 4.1 mg/dL. In this aspect, aberrant serum phosphorus
concentration may be selected from either above or below the normal
range. In another example, in one aspect, a normal range of serum,
or blood, sodium is from 135 to 145 mEq/L. In this aspect, aberrant
serum, or blood, sodium concentration may be selected from either
above or below the normal range. In another example, in one aspect,
a normal range of serum, or blood, potassium is from 3.7 to 5.2
mEq/L. In this aspect, aberrant serum, or blood, potassium
concentration maybe selected from either above or below the normal
range. In a further aspect, a normal range of serum bicarbonate is
from 20 to 29 mEq/L. In this aspect, aberrant serum, or blood,
bicarbonate concentration may be selected from either above or
below the normal range. In a different aspect, bicarbonate levels
can be used to indicate normal levels of acidity (pH), in the
blood. The term "electrolyte concentration" may also be used to
define the condition of a particular electrolyte in a tissue or
body fluid other than blood or plasma. In this case, the normal
physiological condition is considered to be the clinically normal
range for that tissue or fluid. In this aspect, aberrant tissue or
fluid electrolyte concentration may be selected from either above
or below the normal range.
[0044] As used in this disclosure, the term "epitope" refers to an
antigenic determinant on an antigen, such as an enzyme polypeptide,
to which the paratope of an antibody, such as an enzyme-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics. As used herein "epitope" refers to that portion of
an antigen or other macromolecule capable of forming a binding
interaction that interacts with the variable region binding body of
an antibody. Typically, such binding interaction is manifested as
an intermolecular contact with one or more amino acid residues of a
CDR.
[0045] As used herein, an "enzyme" is a protein with specific
catalytic properties. Factors such as, for example, substrate
concentration, pH, temperature and presence or absence of
inhibitors can affect the rate of catalysis. Typically, for a wild
type enzyme, Q10 (the temperature coefficient) describes the
increase in reaction rate with a 10 degree C. rise in temperature.
For wild type enzymes, the Q10=2 to 3; in other words, the rate of
reaction doubles or triples with every 10 degree increase in
temperature. At high temperatures, proteins denature. At pH values
slightly different from an enzymes optimum value, small changes
occur in the charges of the enzyme and perhaps the substrate
molecule. The change in ionization can affect the binding of the
substrate molecule. At extreme pH levels, the enzyme will produce
denaturation, where the active site is distorted, and the substrate
molecule will no longer fit.
[0046] As used herein, the term "evolution", or "evolving", refers
to using one or more methods of mutagenesis to generate a novel
polynucleotide encoding a novel polypeptide, which novel
polypeptide is itself an improved biological molecule &/or
contributes to the generation of another improved biological
molecule. In a particular non-limiting aspect, the present
disclosure relates to evolution of conditionally active biologic
proteins from a parent wild type protein. In one aspect, for
example, evolution relates to a method of performing both
non-stochastic polynucleotide chimerization and non-stochastic
site-directed point mutagenesis disclosed in U.S. patent
application publication 2009/0130718, which is incorporated herein
by reference. More particularly, the present disclosure provides
methods for evolution of conditionally active biologic enzymes
which exhibit reduced activity at normal physiological conditions
compared to a wild-type enzyme parent molecule, but enhanced
activity under one or more aberrant conditions compared to the
wild-type enzyme.
[0047] The terms "fragment", "derivative" and "analog" when
referring to a reference polypeptide comprise a polypeptide which
retains at least one biological function or activity that is at
least essentially same as that of the reference polypeptide.
Furthermore, the terms "fragment", "derivative" or "analog" are
exemplified by a "pro-form" molecule, such as a low activity
proprotein that can be modified by cleavage to produce a mature
enzyme with significantly higher activity.
[0048] The term "full length antibody" refers to an antibody which
comprises an antigen-binding variable region (VH or VL) as well as
a light chain constant domain (CL) and heavy chain constant
domains, CH1, CH2 and CH3. The constant domains may be native
sequence constant domains (e.g. human native sequence constant
domains) or amino acid sequence variants thereof.
[0049] Depending on the amino acid sequence of the constant domain
of their heavy chains, full length antibodies can be assigned to
different "classes". There are five major classes of full length
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called alpha,
delta, epsilon, gamma, and mu, respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0050] A method is provided herein for producing from a template
polypeptide a set of progeny polypeptides in which a "full range of
single amino acid substitutions" is represented at each amino acid
position. As used herein, "full range of single amino acid
substitutions" is in reference to the 20 naturally encoded
polypeptide-forming alpha-amino acids, as described herein.
[0051] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (nitrons) between individual coding segments
(exons).
[0052] "Genetic instability", as used herein, refers to the natural
tendency of highly repetitive sequences to be lost through a
process of reductive events generally involving sequence
simplification through the loss of repeated sequences. Deletions
tend to involve the loss of one copy of a repeat and everything
between the repeats.
[0053] The term "growth factor" refers to proteins that promote
growth, and include, for example, hepatic growth factors;
fibroblast growth factors; vascular endothelial growth factors;
nerve growth factors such as NGF-.beta.; platelet-derived growth
factors; transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma., and colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF).
As used herein, the term growth factor includes proteins from
natural sources or from recombinant cell culture and biologically
active equivalents of the native-sequence growth factor, including
synthetically produced small-molecule entities and pharmaceutically
acceptable derivatives and salts thereof.
[0054] The term "heterologous" means that one single-stranded
nucleic acid sequence is unable to hybridize to another
single-stranded nucleic acid sequence or its complement. Thus areas
of heterology means that areas of polynucleotides or
polynucleotides have areas or regions within their sequence which
are unable to hybridize to another nucleic acid or polynucleotide.
Such regions or areas are for example areas of mutations.
[0055] The term "hormone" refers to polypeptide hormones, which are
generally secreted by glandular organs with ducts. Included among
the hormones are, for example, growth hormones such as human growth
hormones, N-methionyl human growth hormones, and bovine growth
hormones; parathyroid hormones; thyroxine; insulin; proinsulin;
relaxin; estradiol; hormone-replacement therapy; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
or testolactone; prorelaxin; glycoprotein hormones such as follicle
stimulating hormones (FSH), thyroid stimulating hormones (TSH), and
luteinizing hormones (LH); prolactin, placental lactogen, mouse
gonadotropin-associated peptide, gonadotropin-releasing hormones;
inhibin; activin; mullerian-inhibiting substance; and
thrombopoietm. As used herein, the term hormone includes proteins
from natural sources or from recombinant cell culture and
biologically active equivalents of the native-sequence hormone,
including synthetically produced small-molecule entities and
pharmaceutically acceptable derivatives and salts thereof.
[0056] The term "identical" or "identity" means that two nucleic
acid sequences have the same sequence or a complementary sequence.
Thus, "areas of identity" means that regions or areas of a
polynucleotide or the overall polynucleotide are identical or
complementary to areas of another polynucleotide.
[0057] The term "immune system checkpoint," or "immune checkpoint",
refers to one or more inhibitory pathways in the immune system that
contribute to the maintenance of self-tolerance or modulation of
the duration and amplitude of physiological immune responses to
minimize collateral tissue damages. The immune checkpoint functions
as a safeguard for preventing the immune system from attacking host
molecules or cells (self-tolerance). When the immune checkpoints
are inhibited, the immune system, especially the T-cells, becomes
super activated, which may lead to a loss of self-tolerance. The
loss of self-tolerance may result in host molecules, cells, and/or
tissues being attacked by the immune system thereby causing
collateral tissue damage, in addition to attacking foreign
molecules or cells. When these immune checkpoints are not
inhibited, the immune system can achieve a balance between
self-tolerance and attacking foreign molecules and cells in the
body.
[0058] It has been found that tumor tissue and possibly certain
pathogens have the ability to cope with the immune checkpoints to
reduce the effectiveness of host immune response, resulting in
tumor growth and/or chronic infection (see, e.g., Pardoll, Nature
Reviews Cancer, vol. 12, pages 252-264, 2012; Nirschl & Drake,
Clin Cancer Res, vol. 19, pages 4917-4924, 2013). However, a
super-activated immune system initiated by inhibition of the immune
checkpoints is much more sensitive and thus can detect and attack
tumors. Thus, for cancer therapy, immune checkpoint inhibition is a
desirable goal in order to allow the immune system to participate
in the fight against tumors. The problem that must be addressed is
how to super-activate the immune system to fight tumors, while
minimizing the potential for collateral damage to other parts of
the body.
[0059] The term "immune checkpoint inhibitor" as used herein refers
to molecules that totally or partially reduce, inhibit, interfere
with or modulate one or more immune checkpoint proteins. Immune
checkpoint proteins regulate the immune system, especially T-cells,
activation or function. Numerous immune checkpoint proteins are
known, such as cytotoxic T-lymphocyte antigen 4 (CTLA4) and its
ligands CD 80 and CD86, and programmed cell death 1 protein (PD1)
and its ligands PDL1 and PDL2 (Pardoll, Nature Reviews Cancer, vol.
12, pages 252-264, 2012). These proteins are responsible for
interactions that inhibit T-cell responses. Immune checkpoint
proteins regulate and maintain self-tolerance, as well as the
duration and amplitude of physiological immune responses. Immune
checkpoint inhibitors may include antibodies or may be derived from
antibodies. For example, antibodies that bind to CTLA4, PD-1, or
PD-L1 function as immune checkpoint inhibitors.
[0060] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or enzyme present in a living animal is not
isolated, but the same polynucleotide or enzyme, separated from
some or all of the coexisting materials in the natural system, is
isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or enzymes could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0061] The term "isolated nucleic acid" is used to define a nucleic
acid, e.g., a DNA or RNA molecule, that is not immediately
contiguous with the 5' and 3' flanking sequences with which it
normally is immediately contiguous when present in the naturally
occurring genome of the organism from which it is derived. The term
thus describes, for example, a nucleic acid that is incorporated
into a vector, such as a plasmid or viral vector; a nucleic acid
that is incorporated into the genome of a heterologous cell (or the
genome of a homologous cell, but at a site different from that at
which it naturally occurs); and a nucleic acid that exists as a
separate molecule, e.g., a DNA fragment produced by PCR
amplification or restriction enzyme digestion, or an RNA molecule
produced by in vitro transcription. The term also describes a
recombinant nucleic acid that forms part of a hybrid gene encoding
additional polypeptide sequences that can be used, for example, in
the production of a fusion protein.
[0062] The term "joint damage" is used in the broadest sense and
refers to any damage or partial or complete destruction to any part
of one or more joints, including the connective tissue and
cartilage, where damage includes structural and/or functional
damage of any cause, and may or may not cause joint pain/arthalgia.
It includes, without limitation, joint damage associated with or
resulting from inflammatory joint disease as well as
non-inflammatory joint disease. This damage may be caused by any
condition, such as an autoimmune disease such as lupus (e.g.,
systemic lupus erythematosus), arthritis (e.g., acute and chronic
arthritis, rheumatoid arthritis (RA) including juvenile-onset
rheumatoid arthritis, juvenile idiopathic arthritis (JIA), or
juvenile RA (JRA)). Other conditions and diseases include
rheumatoid synovitis, gout or gouty arthritis, acute immunological
arthritis, chronic inflammatory arthritis, degenerative arthritis,
type II collagen-induced arthritis, infectious arthritis, septic
arthritis, Lyme arthritis, proliferative arthritis, psoriatic
arthritis, Still's disease, vertebral arthritis, osteoarthritis,
arthritis chronica progrediente, arthritis deformans, polyarthritis
chronica primaria, reactive arthritis, menopausal arthritis,
estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid
spondylitis), rheumatic autoimmune disease other than RA,
significant systemic involvement secondary to RA (including but not
limited to vasculitis, pulmonary fibrosis or Felty's syndrome),
Sjogren's syndrome, particular secondary such syndrome. Further
conditions include secondary limited cutaneous vasculitis with RA,
seronegative spondyloarthropathy, Lyme disease, inflammatory bowel
disease, scleroderma, inflammatory myopathy, mixed connective
tissue disease, any overlap syndrome, bursitis, tendonitis,
osteomyelitis, infectious diseases, including influenza, measles
(rubeola), rheumatic fever, Epstein-Barr viral syndrome, hepatitis,
mumps, rebella (German measles), and varicella (chickenpox),
Chondromalacia patellae, collagenous colitis, autoimmune disorders
associated with collagen disease, joint inflammation, unusual
exertion or overuse such as sprains or strains, injury including
fracture, gout, especially found in the big toe, as well as caused
by neurological disorders, hemophilic disorders (for example,
hemophilic arthropathy), muscular disorders, progressive disorders,
bone disorders, cartilage disorders, and vascular disorders. For
purposes herein, joints are points of contact between elements of a
skeleton (of a vertebrate such as an animal) with the parts that
surround and support it include, but are not limited to, hips,
joints between the vertebrae of the spine, joints between the spine
and pelvis (sacroiliac joints), joints where the tendons and
ligaments attach to bones, joints between the ribs and spine,
shoulders, knees, feet, elbows, hands, fingers, ankles and toes,
but especially joints in the hands and feet.
[0063] As used herein "ligand" refers to a molecule, such as a
random peptide or variable segment sequence, that is recognized by
a particular receptor. As one of skill in the art will recognize, a
molecule (or macromolecular complex) can be both a receptor and a
ligand. In general, the binding partner having a smaller molecular
weight is referred to as the ligand and the binding partner having
a greater molecular weight is referred to as a receptor.
[0064] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Sambrook
et al., (1982). Molecular Cloning: A Laboratory Manual. Cold Spring
Harbour Laboratory, Cold Spring Harbor, N.Y., p. 146; Sambrook et
al., Molecular Cloning: a laboratory manual, 2.sup.nd Ed., Cold
Spring Harbor Laboratory Press, 1989). Unless otherwise provided,
ligation may be accomplished using known buffers and conditions
with 10 units of T4 DNA ligase ("ligase") per 0.5 micrograms of
approximately equimolar amounts of the DNA fragments to be
ligated.
[0065] As used herein, "linker" or "spacer" refers to a molecule or
group of molecules that connects two molecules, such as a DNA
binding protein and a random peptide, and serves to place the two
molecules in a preferred configuration, e.g., so that the random
peptide can bind to a receptor with minimal steric hindrance from
the DNA binding protein.
[0066] The term "mammalian cell surface display" refers to a
technique whereby a protein or antibody, or a portion of an
antibody, is expressed and displayed on a mammalian host cell
surface for screening purposes; for example, by screening for
specific antigen binding by a combination of magnetic beads and
fluorescence-activated cell sorting. In one aspect, mammalian
expression vectors are used for simultaneous expression of
immunoglobulins as both a secreted and cell surface bound form as
in DuBridge et al., US 2009/0136950, which is incorporated herein
by reference for the disclosure of this aspect. In another aspect,
the techniques are employed for a viral vector encoding for a
library of antibodies or antibody fragments are displayed on the
cell membranes when expressed in a cell as in Gao et al., US
2007/0111260, incorporated herein by reference for the disclosure
of this aspect.
[0067] Whole IgG surface display on mammalian cells is known. For
example, Akamatsuu et al. developed a mammalian cell surface
display vector, suitable for directly isolating IgG molecules based
on their antigen-binding affinity and biological activity. Using an
Epstein-Barr virus-derived episomal vector, antibody libraries were
displayed as whole IgG molecules on the cell surface and screened
for specific antigen binding by a combination of magnetic beads and
fluorescence-activated cell sorting. Plasmids encoding antibodies
with desired binding characteristics were recovered from sorted
cells and converted to the form for production of soluble IgG. See
Akamatsuu et al. J. Immunol. Methods, vol. 327, pages 40-52, 2007,
incorporated herein by reference. Ho et al. used human embryonic
kidney 293T cells that are widely used for transient protein
expression for cell surface display of single-chain Fv antibodies
for affinity maturation. Cells expressing a rare mutant antibody
with higher affinity were enriched 240-fold by a single-pass cell
sorting from a large excess of cells expressing WT antibody with a
slightly lower affinity. Furthermore, a highly enriched mutant was
obtained with increased binding affinity for CD22 after a single
selection of a combinatory library randomizing an intrinsic
antibody hotspot. See Ho et al., "Isolation of anti-CD22 Fv with
high affinity by Fv display on human cells," Proc Natl Acad Sci
USA, vol. 103, pages 9637-9642, 2006, incorporated herein by
reference.
[0068] B cells specific for an antigen can also be used. Such B
cells were directly isolated from peripheral blood mononuclear
cells (PBMC) of human donors. Recombinant, antigen-specific
single-chain Fv (scFv) libraries are generated from this pool of B
cells and screened by mammalian cell surface display by using a
Sindbis virus expression system. This method allows isolating
antigen-specific antibodies by a single round of FACS. The variable
regions (VRs) of the heavy chains (HCs) and light chains (LCs) were
isolated from positive clones and recombinant fully human
antibodies produced as whole IgG or Fab fragments. In this manner,
several hypermutated high-affinity antibodies binding the Q.beta.
virus like particle (VLP), a model viral antigen, as well as
antibodies specific for nicotine were isolated. All antibodies
showed high expression levels in cell culture. The human
nicotine-specific mAbs were validated preclinically in a mouse
model. See Beerli et al., "Isolation of human monoclonal antibodies
by mammalian cell display," Proc Natl Acad Sci USA, vol. 105, pages
14336-14341, 2008, incorporated herein by reference.
[0069] Yeast cell surface display may also be used in the present
invention, for example, see Kondo and Ueda, "Yeast cell-surface
display-applications of molecular display," Appl. Microbiol.
Biotechnol., vol. 64, pages 28-40, 2004, which describes for
example, a cell-surface engineering system using the yeast
Saccharomyces cerevisiae. Several representative display systems
for the expression in yeast S. cerevisiae are described in Lee et
al, "Microbial cell-surface display," TRENDS in Bitechnol., vol.
21, pages 45-52, 2003. Also Boder and Wittrup, "Yeast surface
display for screening combinatorial polypeptide libraries," Nature
Biotechnol., vol. 15, pages 553, 1997.
[0070] As used herein "microenvironment" means any portion or
region of a tissue or body that has constant or temporal, physical
or chemical differences from other regions of the tissue or regions
of the body.
[0071] As used herein, a "molecular property to be evolved"
includes reference to molecules comprised of a polynucleotide
sequence, molecules comprised of a polypeptide sequence, and
molecules comprised in part of a polynucleotide sequence and in
part of a polypeptide sequence. Particularly relevant--but by no
means limiting--examples of molecular properties to be evolved
include protein activities at specified conditions, such as related
to temperature; salinity; osmotic pressure; pH; oxidation, and
concentration of glycerol, DMSO, detergent, &/or any other
molecular species with which contact is made in a reaction
environment. Additional particularly relevant--but by no means
limiting--examples of molecular properties to be evolved include
stabilities--e.g. the amount of a residual molecular property that
is present after a specified exposure time to a specified
environment, such as may be encountered during storage.
[0072] The term "multispecific antibody" as used herein is an
antibody having binding specificities for at least two different
epitopes. Exemplary multispecific antibodies may bind both a BBB-R
and a brain antigen. Multispecific antibodies can be prepared as
full-length antibodies or antibody fragments (e.g. F(ab')2
bispecific antibodies). Engineered antibodies binding two, three or
more (e.g. four) antigens are contemplated (see, e.g., US
2002/0004587 A1). One or more wild-type antibody(s) may be
engineered to be multispecific, or two antibodies may be engineered
to comprise a multispecific antibody. Multispecific antibodies can
be multifunctional.
[0073] The term "mutations" means changes in the sequence of a
wild-type nucleic acid sequence or changes in the sequence of a
peptide. Such mutations may be point mutations such as transitions
or transversions. The mutations may be deletions, insertions or
duplications.
[0074] As used herein, the degenerate "N,N,G/T" nucleotide sequence
represents 32 possible triplets, where "N" can be A, C, G or T.
[0075] The term "naturally-occurring" as used herein as applied to
the object refers to the fact that an object can be found in
nature. For example, a polypeptide or polynucleotide sequence that
is present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally occurring.
Generally, the term naturally occurring refers to an object as
present in a non-pathological (un-diseased) individual, such as
would be typical for the species.
[0076] As used herein, "normal physiological conditions", or "wild
type operating conditions", are those conditions of temperature,
pH, osmotic pressure, osmolality, oxidation and electrolyte
concentration which would be considered within a normal range at
the site of administration, or the site of action, in a
subject.
[0077] As used herein, a "nucleic acid molecule" is comprised of at
least one base or one base pair, depending on whether it is
single-stranded or double-stranded, respectively. Furthermore, a
nucleic acid molecule may belong exclusively or chimerically to any
group of nucleotide-containing molecules, as exemplified by, but
not limited to, the following groups of nucleic acid molecules:
RNA, DNA, genomic nucleic acids, non-genomic nucleic acids,
naturally occurring and not naturally occurring nucleic acids, and
synthetic nucleic acids. This includes, by way of non-limiting
example, nucleic acids associated with any organelle, such as the
mitochondria, ribosomal RNA, and nucleic acid molecules comprised
chimerically of one or more components that are not naturally
occurring along with naturally occurring components.
[0078] Additionally, a "nucleic acid molecule" may contain in part
one or more non-nucleotide-based components as exemplified by, but
not limited to, amino acids and sugars. Thus, by way of example,
but not limitation, a ribozyme that is in part nucleotide-based and
in part protein-based is considered a "nucleic acid molecule".
[0079] In addition, by way of example, but not limitation, a
nucleic acid molecule that is labeled with a detectable moiety,
such as a radioactive or alternatively a nonradioactive label, is
likewise considered a "nucleic acid molecule".
[0080] he terms "nucleic acid sequence coding for" or a "DNA coding
sequence of or a "nucleotide sequence encoding" a particular
enzyme--as well as other synonymous terms--refer to a DNA sequence
which is transcribed and translated into an enzyme when placed
under the control of appropriate regulatory sequences. A "promotor
sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The promoter is part of the DNA
sequence. This sequence region has a start codon at its 3'
terminus. The promoter sequence does include the minimum number of
bases where elements necessary to initiate transcription at levels
detectable above background. However, after the RNA polymerase
binds the sequence and transcription is initiated at the start
codon (3' terminus with a promoter), transcription proceeds
downstream in the 3' direction. Within the promoter sequence will
be found a transcription initiation site (conveniently defined by
mapping with nuclease S1) as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0081] The terms "nucleic acid encoding an enzyme (protein)" or
"DNA encoding an enzyme (protein)" or "polynucleotide encoding an
enzyme (protein)" and other synonymous terms encompasses a
polynucleotide which includes only coding sequence for the enzyme
as well as a polynucleotide which includes additional coding and/or
non-coding sequence.
[0082] In one preferred embodiment, a "specific nucleic acid
molecule species" is defined by its chemical structure, as
exemplified by, but not limited to, its primary sequence. In
another preferred embodiment, a specific "nucleic acid molecule
species" is defined by a function of the nucleic acid species or by
a function of a product derived from the nucleic acid species.
Thus, by way of non-limiting example, a "specific nucleic acid
molecule species" may be defined by one or more activities or
properties attributable to it, including activities or properties
attributable to its expressed product.
[0083] The instant definition of "assembling a working nucleic acid
sample into a nucleic acid library" includes the process of
incorporating a nucleic acid sample into a vector-based collection,
such as by ligation into a vector and transformation of a host. A
description of relevant vectors, hosts, and other reagents as well
as specific non-limiting examples thereof are provided hereinafter.
The instant definition of "assembling a working nucleic acid sample
into a nucleic acid library" also includes the process of
incorporating a nucleic acid sample into a non-vector-based
collection, such as by ligation to adaptors. Preferably the
adaptors can anneal to PCR primers to facilitate amplification by
PCR.
[0084] Accordingly, in a non-limiting embodiment, a "nucleic acid
library" is comprised of a vector-based collection of one or more
nucleic acid molecules. In another preferred embodiment a "nucleic
acid library" is comprised of a non-vector-based collection of
nucleic acid molecules. In yet another preferred embodiment a
"nucleic acid library" is comprised of a combined collection of
nucleic acid molecules that is in part vector-based and in part
non-vector-based. Preferably, the collection of molecules
comprising a library is searchable and separable according to
individual nucleic acid molecule species.
[0085] The present disclosure provides a "nucleic acid construct"
or alternatively a "nucleotide construct" or alternatively a "DNA
construct". The term "construct" is used herein to describe a
molecule, such as a polynucleotide (e.g., an enzyme polynucleotide)
which may optionally be chemically bonded to one or more additional
molecular moieties, such as a vector, or parts of a vector. In a
specific--but by no means limiting--aspect, a nucleotide construct
is exemplified by DNA expression constructs suitable for the
transformation of a host cell.
[0086] An "oligonucleotide" (or synonymously an "oligo") refers to
either a single stranded polydeoxynucleotide or two complementary
polydeoxynucleotide strands which may be chemically synthesized.
Such synthetic oligonucleotides may or may not have a 5' phosphate.
Those that do not will not ligate to another oligonucleotide
without adding a phosphate with an ATP in the presence of a kinase.
A synthetic oligonucleotide will ligate to a fragment that has not
been dephosphorylated. To achieve polymerase-based amplification
(such as with PCR), a "32-fold degenerate oligonucleotide that is
comprised of, in series, at least a first homologous sequence, a
degenerate N,N,G/T sequence, and a second homologous sequence" is
mentioned. As used in this context, "homologous" is in reference to
homology between the oligo and the parental polynucleotide that is
subjected to the polymerase-based amplification.
[0087] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the coding sequence.
Operably linked means that the DNA sequences being linked are
typically contiguous and, where necessary to join two protein
coding regions, contiguous and in reading frame.
[0088] A coding sequence is "operably linked to" another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences are ultimately processed
to produce the desired protein.
[0089] As used herein the term "parental polynucleotide set" is a
set comprised of one or more distinct polynucleotide species.
Usually this term is used in reference to a progeny polynucleotide
set which is preferably obtained by mutagenization of the parental
set, in which case the terms "parental", "starting" and "template"
are used interchangeably.
[0090] The term "patient", "individual" or "subject", refers to an
animal, for example a mammal, such as a human, who is the object of
treatment. The subject, or patient, may be either male or female.
Mammals include, but are not limited to, domesticated animals
(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans
and non-human primates such as monkeys), rabbits, and rodents
(e.g., mice and rats). In certain embodiments, the individual or
subject is a human.
[0091] As used herein the term "physiological conditions" refers to
temperature, pH, osmotic pressure, ionic strength, viscosity, and
like biochemical parameters which are compatible with a viable
organism, and/or which typically exist intracellularly in a viable
cultured yeast cell or mammalian cell. For example, the
intracellular conditions in a yeast cell grown under typical
laboratory culture conditions are physiological conditions.
Suitable in vitro reaction conditions for in vitro transcription
cocktails are generally physiological conditions. In general, in
vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH
6.5-8.5, 20-45 degrees C. and 0.001-10 mM divalent cation (e.g.,
Mg.sup.++'', Ca.sup.++); preferably about 150 mM NaCl or KCl, pH
7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent
nonspecific protein (e.g., BSA). A non-ionic detergent (Tween,
NP-40, Triton X-100) can often be present, usually at about 0.001
to 2%, typically 0.05-0.2% (v/v). Particular aqueous conditions may
be selected by the practitioner according to conventional methods.
For general guidance, the following buffered aqueous conditions may
be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with
optional addition of divalent cation(s) and/or metal chelators
and/or non-ionic detergents and/or membrane fractions and/or
anti-foam agents and/or scintillants. Normal physiological
conditions refer to conditions of temperature, pH, osmotic
pressure, osmolality, oxidation and electrolyte concentration in
vivo in a patient or subject at the site of administration, or the
site of action, which would be considered within the normal range
in a patient.
[0092] Standard convention (5' to 3') is used herein to describe
the sequence of double stranded polynucleotides.
[0093] The term "polyepitopic specificity" refers to the ability of
a multispecific or multifunctional antibody to specifically bind to
two or more different epitopes on the same target or on different
targets.
[0094] The term "epitope" refers to a specific amino acid sequence,
modified amino acid sequence, or protein secondary or tertiary
structure which is recognized by an antibody.
[0095] The term "population" as used herein means a collection of
components such as polynucleotides, portions or polynucleotides or
proteins. A "mixed population" means a collection of components
which belong to the same family of nucleic acids or proteins (i.e.,
are related) but which differ in their sequence (i.e., are not
identical) and hence in their biological activity.
[0096] A molecule having a "pro-form" refers to a molecule that
undergoes any combination of one or more covalent and noncovalent
chemical modifications (e.g. glycosylation, proteolytic cleavage,
dimerization or oligomerization, temperature-induced or pH-induced
conformational change, association with a co-factor, etc.) en route
to attain a more mature molecular form having a property difference
(e.g. an increase in activity) in comparison with the reference
pro-form molecule. When two or more chemical modifications (e.g.
two proteolytic cleavages, or a proteolytic cleavage and a
deglycosylation) can be distinguished en route to the production of
a mature molecule, the reference precursor molecule may be termed a
"pre-pro-form" molecule.
[0097] As used herein, the term "pseudorandom" refers to a set of
sequences that have limited variability, such that, for example,
the degree of residue variability at another position, but any
pseudorandom position is allowed some degree of residue variation,
however circumscribed.
[0098] "Quasi-repeated units", as used herein, refers to the
repeats to be re-assorted and are by definition not identical.
Indeed the method is proposed not only for practically identical
encoding units produced by mutagenesis of the identical starting
sequence, but also the reassortment of similar or related sequences
which may diverge significantly in some regions. Nevertheless, if
the sequences contain sufficient homologies to be reasserted by
this approach, they can be referred to as "quasi-repeated"
units.
[0099] As used herein, "receptor" refers to a molecule that has an
affinity for a given ligand. Receptors can be naturally occurring
or synthetic molecules. Receptors can be employed in an unaltered
state or as aggregates with other species. Receptors can be
attached, covalently or non-covalently, to a binding member, either
directly or via a specific binding substance. Examples of receptors
include, but are not limited to, antibodies, including monoclonal
antibodies and antisera reactive with specific antigenic
determinants (such as on viruses, cells, or other materials), cell
membrane receptors, complex carbohydrates and glycoproteins,
enzymes, and hormone receptors.
[0100] The term "recombinant antibody", as used herein, refers to
an antibody (e.g. a chimeric, humanized, or human antibody or
antigen-binding fragment thereof) that is expressed by a
recombinant host cell comprising nucleic acid encoding the
antibody. Examples of "host cells" for producing recombinant
antibodies include: (1) mammalian cells, for example, Chinese
Hamster Ovary (CHO), COS, myeloma cells (including Y0 and NS0
cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insect
cells, for example, sf9, sf21 and Tn5; (3) plant cells, for example
plants belonging to the genus Nicotiana (e.g. Nicotiana tabacum);
(4) yeast cells, for example, those belonging to the genus
Saccharomyces (e.g. Saccharomyces cerevisiae) or the genus
Aspergillus (e.g. Aspergillus niger); (5) bacterial cells, for
example Escherichia. coli cells or Bacillus subtilis cells,
etc.
[0101] "Recombinant" enzymes refer to enzymes produced by
recombinant DNA techniques, i.e., produced from cells transformed
by an exogenous DNA construct encoding the desired enzyme.
"Synthetic" enzymes are those prepared by chemical synthesis.
[0102] "Reductive reassortment", as used herein, refers to the
increase in molecular diversity that is accrued through deletion
(and/or insertion) events that are mediated by repeated
sequences.
[0103] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence," "comparison window," "sequence identity," "percentage of
sequence identity," and "substantial identity."
[0104] A "reference sequence" is a defined sequence used as a basis
for a sequence comparison; a reference sequence may be a subset of
a larger sequence, for example, as a segment of a full-length cDNA
or gene sequence given in a sequence listing, or may comprise a
complete cDNA or gene sequence. Generally, a reference sequence is
at least 20 nucleotides in length, frequently at least 25
nucleotides in length, and often at least 50 nucleotides in length.
Since two polynucleotides may each (1) comprise a sequence (i.e., a
portion of the complete polynucleotide sequence) that is similar
between the two polynucleotides and (2) may further comprise a
sequence that is divergent between the two polynucleotides,
sequence comparisons between two (or more) polynucleotides are
typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare
local regions of sequence similarity.
[0105] "Repetitive Index (RI)", as used herein, is the average
number of copies of the quasi-repeated units contained in the
cloning vector.
[0106] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over the window of comparison. The term "percentage of sequence
identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, U, or I) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. This "substantial identity", as used herein,
denotes a characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence having at least 80 percent
sequence identity, preferably at least 85 percent identity, often
90 to 95 percent sequence identity, and most commonly at least 99
percent sequence identity as compared to a reference sequence of a
comparison window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison.
[0107] As known in the art "similarity" between two enzymes is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one enzyme to the sequence of a second
enzyme. Similarity may be determined by procedures which are
well-known in the art, for example, a BLAST program (Basic Local
Alignment Search Tool at the National Center for Biological
Information).
[0108] As used herein, the term "single-chain antibody" refers to a
polypeptide comprising a VH domain and a VL domain in polypeptide
linkage, generally liked via a spacer peptide, and which may
comprise additional amino acid sequences at the amino- and/or
carboxy-termini. For example, a single-chain antibody may comprise
a tether segment for linking to the encoding polynucleotide. As an
example a scFv is a single-chain antibody. Single-chain antibodies
are generally proteins consisting of one or more polypeptide
segments of at least 10 contiguous amino substantially encoded by
genes of the immunoglobulin superfamily (e.g, see The
Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay,
in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbits,
eds., (1989) Academic press: San Diego, Calif., pp. 361-368, which
is incorporated herein by reference), most frequently encoded by a
rodent, non-human primate, avian, porcine bovine, ovine, goat, or
human heavy chain or light chain gene sequence. A functional
single-chain antibody generally contains a sufficient portion of an
immunoglobulin superfamily gene product so as to retain the
property of binding to a specific target molecule, typically a
receptor or antigen (epitope).
[0109] The members of a pair of molecules (e.g., an
antibody-antigen pair or a nucleic acid pair) are said to
"specifically bind" to each other if they bind to each other with
greater affinity than to other, non-specific molecules. For
example, an antibody raised against an antigen to which it binds
more efficiently than to a non-specific protein can be described as
specifically binding to the antigen. (Similarly, a nucleic acid
probe can be described as specifically binding to a nucleic acid
target if it forms a specific duplex with the target by base
pairing interactions (see above).)
[0110] "Specific hybridization" is defined herein as the formation
of hybrids between a first polynucleotide and a second
polynucleotide (e.g., a polynucleotide having a distinct but
substantially identical sequence to the first polynucleotide),
wherein substantially unrelated polynucleotide sequences do not
form hybrids in the mixture.
[0111] The term "treating" includes: (1) preventing or delaying the
appearance of clinical symptoms of the state, disorder or condition
developing in an animal that may be afflicted with or predisposed
to the state, disorder or condition but does not yet experience or
display clinical or subclinical symptoms of the state, disorder or
condition; (2) inhibiting the state, disorder or condition (i.e.,
arresting, reducing or delaying the development of the disease, or
a relapse thereof in case of maintenance treatment, of at least one
clinical or subclinical symptom thereof); and/or (3) relieving the
condition (i.e., causing regression of the state, disorder or
condition or at least one of its clinical or subclinical symptoms).
The benefit to a patient to be treated is either statistically
significant or at least perceptible to the patient or to the
physician.
[0112] The term "variant" refers to polynucleotides or polypeptides
of the disclosure modified at one or more base pairs, codons,
introns, exons, or amino acid residues (respectively) of a
wild-type protein parent molecule. Variants can be produced by any
number of means including methods such as, for example, error-prone
PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,
sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,
recursive ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, saturation mutagenesis
and any combination thereof. Techniques for producing variant
proteins having reduced activity compared to the wild-type protein
at a normal physiological condition of e.g., one or more conditions
of temperature, pH, osmotic pressure, osmolality, oxidation and
electrolyte concentration; and enhanced activity at an aberrant
condition, are disclosed herein. Variants may additionally be
selected for the properties of enhanced chemical resistance, and
proteolytic resistance, compared to the wild-type protein.
[0113] As used herein, the term "wild-type" means that the
polynucleotide does not comprise any mutations, and includes a
template protein used as a parent molecule for evolution or other
engineering. The "wild-type protein" preferably has some desired
properties, such as higher binding affinity, or enzymatic activity,
which may be obtained by screening of a library of proteins for a
desired properties, including better stability in different
temperature or pH environments, or improved selectivity and/or
solubility. A "wild type protein", "wild-type protein", "wild-type
biologic protein", or "wild type biologic protein", refers to a
protein which can be isolated from nature that will be active at a
level of activity found in nature and will comprise the amino acid
sequence found in nature. The terms "parent molecule", "target
protein" and "template" can also refer to the wild-type
protein.
DETAILED DESCRIPTION
[0114] The present disclosure is directed to methods of engineering
or evolving proteins to generate new molecules that are reversibly
or irreversibly inactivated at the wild type condition, but active
at non-normal conditions at the same or equivalent level as the
activity at the wild-type condition. These new proteins are
referred to as conditionally active proteins herein. Methods of
producing these proteins have been described in US 2012/0164127,
which is incorporated herein by reference in its entirety.
Conditionally active proteins are particularly valuable for
development of novel therapeutics that are active for short or
limited periods of time within the host. This is particularly
valuable where extended operation of the protein at the given dose
would be harmful to the host, but where limited activity is
required to perform the desired therapy. Examples of beneficial
applications include topical or systemic treatments at high dose,
as well as localized treatments in high concentration. Inactivation
under the physiological condition can be determined by a
combination of the dosing and the rate of inactivation of the
protein. This condition based inactivation is especially important
for enzyme therapeutics where catalytic activity cause substantial
negative effects in a relatively short period of time.
[0115] The present disclosure is also directed to methods of
engineering or evolving proteins to generate new molecules that are
different from wild type molecules in that they are reversibly or
irreversibly activated or inactivated over time, or activated or
inactivated only when they are in certain microenvironments in the
body, including in specific organs in the body. In some
embodiments, the conditionally active proteins are antibodies
against a suitable antigen.
Target Wild-Type Proteins
[0116] Any therapeutic protein can serve as a target protein, or
wild-type protein, for production of a conditionally active
biologic protein. In one aspect, the target protein is a wild-type
enzyme. Currently used therapeutic enzymes include urokinase and
streptokinase, used in the treatment of blood clots; and
hyaluronidase, used as an adjuvant to improve the absorption and
dispersion of other drugs, in one aspect, the wild-type protein
selected for generation of a conditionally active biologic protein
can be a currently used therapeutic enzyme, in order to avoid or
minimize deleterious side effects associated with the wild-type
protein or enzyme. Alternatively, an enzyme not in current usage as
a therapeutic can be selected for generation of a conditionally
active biologic protein. Certain non-limiting examples will be
discussed in further detail below.
[0117] Therapeutic proteins are those which can be used in medicine
either alone or in conjunction with other therapies to treat
various diseases or medical conditions, such as antibodies,
enzymes, immune regulators, growth factors, hormones and cytokines.
The conditionally active biologic proteins of the disclosure could
be appropriate for use in one or more indications including the
treatment of circulatory disorders, arthritis, multiple sclerosis,
autoimmune disorders, cancer, dermatologic conditions and use in
various diagnostic formats. Depending on the protein and
indication, the conditionally active biologic protein could be
administered in parenteral, topical or oral formulations as
discussed below.
[0118] Some representative target wild-type proteins include
enzymes, antibodies, cytokines, receptors, DNA binding proteins,
chelating agents, and hormones. More examples include industrial
and pharmaceutical proteins, such as ligands, cell surface
receptors, antigens, transcription factors, signaling modules, and
cytoskeletal proteins. Some suitable classes of enzymes are
hydrolases such as proteases, carbohydrases, lipases; isomerases
such as racemases, epimerases, tautomerases, or mutases;
transferases, kinases, oxidoreductases, and phophatases.
[0119] The target wild-type proteins can be discovered by
generating and screening a library for a protein with a desired
properties, such as enzymatic activity, binding
affinity/selectivity, thermostability, tolerance of high or low pH,
expression efficiency, or other biological activities.
[0120] The target wild-type proteins may be discovered by screening
a cDNA library. A cDNA library is a combination of cloned cDNA
(complementary DNA) fragments inserted into a collection of host
cells, which together constitute some portion of the transcriptome
of the organism. cDNA is produced from fully transcribed mRNA and
therefore contains the coding sequence for expressed proteins of an
organism. The information in cDNA libraries is a powerful and
useful tool for discovery of proteins with desired properties by
screening the libraries for proteins with the desire property.
[0121] In embodiments where the target wild-type proteins are
antibodies, the wild-type antibodies can be discovered by
generating and screening antibody libraries. The antibody libraries
can be either polyclonal antibody libraries or monoclonal antibody
libraries. A polyclonal antibody library against an antigen can be
generated by direct injection of the antigen into an animal or by
administering the antigen to a non-human animal. The antibodies so
obtained represent a library of polyclonal antibodies that bind to
the antigen. For preparation of monoclonal antibody libraries, any
technique which provides antibodies produced by continuous cell
line cultures can be used. Examples include the hybridoma
technique, the trioma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (see, e.g., Cole (1985)
in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
pp. 77-96). Techniques described for the generating single chain
antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to
produce a single chain antibody library.
[0122] There are other methods for generation and screening of
antibody libraries for discovery of the wild-type antibody. For
example, fully human antibody display libraries can be utilized.
Such a library is a population of antibodies displayed on the
surface of host cell(s). Preferably, the antibody library is
representative of the human repertoire of antibodies in that they
have the capability of binding to a wide range of antigens. Because
the antibodies are displayed on the surface of cells, the effective
affinity (due to avidity) of each antibody in the library is
increased. Unlike other popular library types, such as phage
display libraries, where avidity of the antibodies for screening
and identification purposes is less desirable, the super avidity
provided by cell surface display in the present invention, is
desirable. Cell surface display libraries enable the identification
of low, medium and high binding affinity antibodies, as well as the
identification of non-immunogenic and weak epitopes in the
screening or selection step.
Circulatory Disorders-Thrombosis and Thrombolytic Therapy.
[0123] A thrombus (blood clot) is defined as a solid mass derived
from blood constituents that forms in the circulatory system. The
thrombus is formed by a series of events involving blood
coagulation factors, platelets, red blood cells, and interactions
with the vessel wall. A platelet is an intravascular aggregation of
platelets, fibrin and entrapped blood cells which can cause
vascular obstruction. By obstructing or blocking blood flow, the
thrombus deprives downstream tissue of oxygen supply. Fragments
(emboli) of the thrombus may break away and obstruct smaller
vessels. Arterial thrombus formation is precipitated by any of a
variety of factors including an underlying
stenosis-atherosclerosis, a low flow state-cardiac function,
hypercoagubility as in cancer or a coagulation factor deficiency,
or a foreign body such as a stent or catheter. A thrombus leading
to arterial ischemia can result in limb or tissue injury, acute
myocardial infarction (AMI), stroke, amputation, or bowel
infarction. Major causes of morbidity and mortality are the
formation of arterial thrombi (coronary arterial thrombi and
cerebral arterial thrombi) and pulmonary thrombi. Venous thrombus
formation can occur due to endothelial injury such as trauma,
stasis due to e.g. immobility, or hypercoagulability, but
atherosclerosos is not a factor. Treatment strategies include
mechanical thrombectomy, pharmacomechanical thrombectomy and
thrombolysis. Thrombotic therapy is used to minimize formation and
aid in removal of thrombi.
[0124] Thrombotic therapy includes the use of antiplatelet agents
which inhibit platelet activation, anticoagulant therapies, and/or
thrombolytic therapy to degrade blood clots. Examples of
antiplatelets include aspirin, dipyridamole, and ticlopidine.
Examples of anticoagulants include heparin, warfarin, hirudin, and
activated human protein C. Examples of thrombolytics include tissue
plasminogen activator (tPA)/tPA variants, urokinase and
streptokinase. The thrombolytics display a catalytic mode of
action.
[0125] Thrombolytic therapy in acute myocardial infarction is well
established. Use of thrombolytic agents has become standard
emergency treatment. Although effective, these products achieve
complete reperfusion in only about 50% of patients and side effects
include risk of hemorrhage (in particular intracranial bleeding) as
well as hypertension. The degradation of blood clots from a damaged
or diseased vessel is termed "fibrinolysis" or the "fibrinolytic
process". Fibrinolysis is a proteolytic process, by a plasminogen
activator which activates the protein plasminogen, thereby forming
plasmin. Plasmin proteolytically degrades the fibrin strands of the
blood clot to dissolve the clot. Fibrin specific plasminogen
activators include tissue plasminogen activators or variants.
Non-specific plasminogen activators can include streptokinase and
urokinase.
[0126] Certain commonly used thrombolytic therapies utilize one of
several available tissue plasminogen activator (tPA) variants. For
example, tPA based product variants which have been previously
approved for use are Alteplase (rt-PA), Reteplase (r-PA) and
Tenecteplase (TNK). Approved uses for tPA variants include, for
example, acute myocardial infarction for the improvement of
ventricular function following AMI, the reduction of incidence of
congestive heart failure, and reduction of mortality associated
with AMI, management of ischemic stroke in adults for improving
neurological recovery and reducing incidence of disability,
management of acute massive pulmonary embolism in adults for the
lysis of acute pulmonary emboli, and for the lysis of pulmonary
emboli accompanied by unstable hemodynamics.
[0127] Another commonly used thrombolytic therapy utilizes
urokinase. Urokinase is a standard lytic agent used in the
management of peripheral vascular disease.
[0128] Streptokinase is a protein secreted by several species of
streptococci that can bind and activate human plasminogen.
Complexes of streptokinase with human plasminogen can
hydrolytically activate other unbound plasminogen by activating
through bond cleavage to produce plasmin. The usual activation of
plasminogen is through the proteolysis of the Arg561-Val562 bond.
The amino group of Val562 then forms a salt-bridge with Asp740,
which causes a conformational change to produce the active protease
plasmin. Plasmin is produced in the blood to break down fibrin, the
major constituent of blood clots.
[0129] Streptokinase is used as an effective clot-dissolving
medication in some cases of myocardial infarction (heart attack),
pulmonary embolism (lung blood clots), and deep venous thrombosis
(leg blood clots). Streptokinase belongs to a group of medications
called fibrinolytics. Streptokinase is given as soon as possible
after the onset of a heart attack to dissolve clots in the arteries
of the heart wall and reduce damage to the heart muscle.
Streptokinase is a bacterial product, so the body has the ability
to build up immunity against the protein. Therefore, it is
recommended that this product should not be given again after four
days from the first administration, as it may not be as effective
and cause an allergic reaction. For this reason it is usually given
only after a first heart attack, and further thrombotic events are
typically treated with tissue plasminogen activator (TPA).
Streptokinase is also sometimes used to prevent post-operative
adhesions.
[0130] Side effects of streptokinase include bleeding (major and
minor), hypotension, and respiratory depression as well as possible
allergic reaction. In addition, anticoagulants, agents that alter
platelet function (e.g. aspirin, other NSAIDs, dipyridamole) may
increase risk of bleeding.
[0131] Administration of the thrombolytics is generally by infusion
or by bolus intravenous dose; or by a mechanical infusion system.
Adverse effects can include serious intracranial, gastrointestinal,
retroperitoneal, or pericardial bleeding. If bleeding occurs the
administration must be discontinued immediately.
[0132] In certain embodiments of the disclosure, tPA, streptokinase
or urokinase is selected as the target, or wild-type protein.
[0133] In one embodiment, the methods of the disclosure are used to
select for a conditionally active recombinant or synthetic
streptokinase variant with high activity at aberrant temperature
conditions below normal physiological conditions; and substantial
deactivation or inactivation at normal physiological conditions
(e.g. 37 degrees C.). In one aspect, the aberrant temperature
condition is room temperature, e.g. 20-25 degrees C. In another
aspect, the disclosure provides a method of treating a stroke or
heart attack, the method comprising administering a high dose of
the conditionally active streptokinase variant to stroke or heart
attack victims in order to clear clots, yet allow for rapid
inactivation of the streptokinase variant to avoid excessive
bleeding.
Circulatory Disorders-Renin/Angiotensin
[0134] The renin-angiotensin system is a hormone system that
regulates blood pressure and water (fluid) balance. The kidneys
secrete renin when the blood volume is low. Renin is an enzyme
which hydrolyzes angiotensinogen secreted from the liver into the
peptide angiotensin I. Angiotensin I is further cleaved in the
lungs by endothelial-bound angiotensin converting enzyme (ACE) into
angiotensin II, the most vasoactive peptide. Angiotensin II causes
the blood vessels to constrict, resulting in increased blood
pressure. However, angiotensin .pi. also stimulates the secretion
of the hormone aldosterone from the adrenal cortex. Aldosterone
causes the tubules of the kidneys to increase the resorption of
sodium and water. This increases the volume of fluid in the body,
which also increases blood pressure. An over-active
renin-angiotensin system leads to vasoconstriction and retention of
sodium and water. These effects lead to hypertension. There are
many drugs which interrupt different steps in this system to lower
blood pressure. These drugs are one of the main ways to control
high blood pressure (hypertension), heart failure, kidney failure,
and harmful effects of diabetes.
[0135] Hypovolemic shock is an emergency condition in which severe
blood and/or fluid loss makes the heart unable to adequately
perfuse the body's cells with oxygenated blood. Blood loss can be
from trauma, injuries and internal bleeding. The amount of
circulating blood may drop due to excessive fluid loss from burns,
diarrhea, excessive perspiration or vomiting. Symptoms of
hypovolemic shock include anxiety, cool clammy skin, confusion,
rapid breathing, or unconsciousness. Examination shows signs of
shock including low blood pressure, low body temperature, and rapid
pulse, which may be weak or thready. Treatment includes intravenous
fluids; blood or blood products; treatment for shock; and
medication such as dopamine, dobutamine, epinephrine and
norepinephrine to increase blood pressure and cardiac output.
[0136] In one embodiment, the disclosure provides a method of
selecting for a conditionally active recombinant renin variant to
be reversibly deactivated at normal physiological temperature, but
reactivated at the aberrant lower temperatures in a patient with
hypovolemic shock. The conditionally active protein can be used to
treat hypovolemic shock to help increase the volume of fluid in the
body, and increase blood pressure. Circulatory Disorders-Reynaud's
phenomenon
[0137] Reynaud's phenomenon (RP) is a vasospastic disorder causing
discoloration of the fingers, toes and occasionally other
extremities. Emotional stress and cold are classic triggers of the
phenomenon. When exposed to cold temperatures, the extremities lose
heat. The blood supply to fingers and toes is normally slowed to
preserve the body's core temperature. Blood flow is reduced by the
narrowing of small arteries under the skin of the extremities.
Stress causes similar reaction to cold in the body. Li Reynaud's,
the normal response is exaggerated. The condition can cause pain,
discoloration, and sensations of cold and numbness. The phenomenon
is the result of vasospasms that decrease the blood supply to the
respective regions, in Reynaud's disease (Primary Raynaud's
phenomenon), the disease is idiopathic. Li Raynaud's syndrome
(Secondary Reynaud's), the phenomenon is caused by some other
instigating factor. Measurement of hand-temperature gradients is
one tool to distinguish between the primary and secondary forms.
The primary form can progress to the secondary form, and in extreme
cases, the secondary form can progress to necrosis or gangrene of
the fingertips.
[0138] Raynaud's phenomenon is an exaggeration of responses to cold
or emotional stress. Primary RP is essentially mediated by
microvascular vasospasm. Hyperactivation of the sympathetic system
causes extreme vasoconstriction of the peripheral blood vessels,
leading to hypoxia. Chronic, recurrent cases can result in atrophy
of the skin, subcutaneous tissue, and muscle. It can also rarely
result in ulceration and ischemic gangrene.
[0139] Traditional treatment options for Reynaud's phenomenon
include prescription medication that dilates blood vessels and
promotes circulation. These include calcium channel blockers, such
as nifedipine or diltiazem; alpha blockers, which counteract the
actions of norepinephrine, a hormone that constricts blood vessels,
such as prazosin or doxazosin; and vasodilators, to relax blood
vessels, such as nitroglycerin cream, or the angiotensin II
inhibitor losartan, sildenafil, or prostaglandins. Fluoxetine, a
selective serotonin reuptake inhibitor and other antidepressant
medications may reduce the frequency and severity of episodes due
to psychological stressors. These drugs may cause side effects such
as headache, flushing and ankle edema. A drug may also lose
effectiveness over time.
[0140] The regulation of cutaneous vasoconstriction and
vasodilation involves altered sympathetic nerve activity and a
number of neuronal regulators, including adrenergic and
non-adrenergic, as well as REDOX signaling and other signaling such
as the RhoA/ROCK pathway. Vasoconstriction of vascular smooth
muscle cells (vSMC) in the skin is thought to be activated by
norepinephrine mediated by alpha1 and alpha2 adrenoreceptors.
Alpha2C-ARs translocate from the trans Golgi to the cell surface of
the vSMC where they respond to stimulation and signaling of these
responses involves the RhoA/Rhokinase (ROCK) signaling pathway.
Cold stimulation in cutaneous arteries results in the immediate
generation of reactive oxygen species (ROS) in the vSMC
mitochondria. ROS are involved in the REDOX signaling through the
RhoA/ROCK pathway. RhoA is a GTP-binding protein whose role is the
regulation of actin-myosin dependent processes such as migration
and cell contraction in vSMC. Non-adrenergic neuropeptides with
known function in vasculature with possible involvement in RP
include calcitonin gene-related peptide (CGRP), Substance P (SP),
Neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP).
Fonseca et al., 2009, "Neuronal regulators and vascular dysfunction
in Raynaud's phenomenon and systemic sclerosis", Curr. Vascul.
Pharmacol. 7:34-39.
[0141] New therapies for RP include alpha-2c adrenergic receptor
blockers, protein tyrosine kinase inhibitors, Rho-kinase inhibitors
and calcitonin gene related peptide.
[0142] Calcitonin gene related peptide (CGRP) is a member of the
calcitonin family of peptides and exists in two forms; alpha-CGRP
and beta-CGRP. Alpha-CGRP is a 37-amino acid peptide formed from
alternative splicing of the calcitonin/CGRP gene. CGRP is one of
the most abundant peptides produced in peripheral and central
neurons. It is a potent peptide vasodilator and can function in the
transmission of pain. Migraine is a common neurological disorder
that is associated with an increase in CGRP levels. CGRP dilates
intracranial blood vessels and transmits vascular nociception. CGRP
receptor antagonists have been tested as treatments for migraines.
Arulmani et al., 2004, "Calcitonin gene-related peptide and it role
in migraine pathophysiology", Eur. J. Pharmacol. 500(1-3): 315-330.
At least three receptor subtypes have been identified and CGRP acts
through G protein-coupled receptors whose presence and changes in
function modulate the peptide's effect in various tissues. CGRP's
signal transduction through the receptors is dependent on two
accessory proteins: receptor activity modifying protein 1 (RAMP1)
and receptor component protein (RCP). Ghatta 2004, Calcitonin
gene-related peptide: understanding its role. Indian J. Pharmacol.
36(5): 277-283. One study of the effects of intravenous infusion of
three vasodilators: endothelium-dependent vasodilator adenosine
triphosphate (ATP), endothelium-independent vasodilator
prostacyclin (epoprostenol; PGI2), and CGRP, to patients with
Reynaud's phenomenon, and a similar number of age and sex matched
controls, using laser Doppler flowmetry (LDF) showed CGRP induced
flushing of the face and hands by a rise in skin blood flow in the
Reynaud's patients, whereas in controls CGRP caused flushing only
in the face. PGI2 caused similar rises in blood flow in hands and
face of both groups. ATP did not cause any significant changes in
blood flow in hands or face of the patients, but increased blood
flow to the face of controls. Shawket et al., 1989, "Selective
suprasensitivity to calcitonin-gene-related peptide in the hands in
Reynaud's phenomenon". The Lancet, 334(8676):1354-1357. In one
aspect, the wild-type protein target molecule is CGRP.
[0143] In one embodiment, the disclosure provides methods of
selecting for conditionally active recombinant protein variants of
proteins associated with Reynaud's syndrome to be reversibly
deactivated at normal physiological temperature, but reactivated at
the aberrant lower temperatures in digits. The conditionally active
proteins can be used to treat Reynaud's phenomenon, to prevent or
reduce loss of digit function due to low circulation. Circulatory
disorders-Vasopressin
[0144] Arginine vasopressin (AVP, vasopressin, antidiuretic hormone
(ADH)) is a peptide hormone found in most mammals that controls
reabsorption of molecules in the tubules of the kidney by affecting
tissue permeability. One of the most important roles of vasopressin
is to regulate water retention in the body. In high concentrations
it raises blood pressure by introducing moderate vasoconstriction.
Vasopressin has three effects which result in increased urine
osmolality (increased concentration) and decreased water excretion.
First, vasopressin causes an increase in the permeability of water
of the collecting duct cells in the kidney allowing water
resorption and excretion of a smaller volume of concentrated urine
(antidiuresis). This occurs through insertion of aquaporin-2 water
channels into the apical membrane of the collecting duct cells.
Secondly, vasopressin causes an increase in the permeability of the
inner medullary portion of the collecting duct to urea, allowing
increased reabsorption urea into the medullary interstitium.
Thirdly, vasopressin causes stimulation of sodium and chloride
reabsorption in the thick ascending limb of the loop of Heme by
increasing the activity of the
Na.sup.+-K.sup.+-2Cl.sup.''-cotransporter. NaCl reabsorption drives
the process of countercurrent multiplication, which furnishes the
osmotic gradient for aquaporin mediated water reabsorption in the
medullary collecting ducts.
[0145] The hypertonic interstitial fluid surrounding the collecting
ducts of the kidney provides a high osmotic pressure for the
removal of water. Transmembrane channels made of proteins called
aquaporins are inserted in the plasma membrane greatly increasing
its permeability to water. When open, an aquaporin channel allows 3
billion molecules of water to pass through each second. Insertion
of aquaporin-2 channels requires signaling by vasopressin.
Vasopressin binds to receptors (called V2 receptors) on the
basolateral surface of the cells of the collecting ducts. Binding
of the hormone triggers a rising level of cAMP within the cell.
This "second messenger" initiates a chain of events culminating in
the insertion of aquaporin-2 channels in the apical surface of the
collecting duct cells. The aquaporins allow water to move out of
the nephron, increasing the amount of water re-absorbed from the
forming urine back into the bloodstream.
[0146] The main stimulus for the release of vasopressin from the
pituitary gland is increased osmolality of the blood plasma.
Anything that dehydrates the body, such as perspiring heavily
increases the osmotic pressure of the blood and turns on the
vasopressin to V2 receptor to aquaporin-2 pathway. As a result, as
little as 0.5 liters/day of urine may remain of the original 180
liters/day of nephric filtrate. The concentration of salts in urine
can be as high as four times that of the blood. If the blood should
become too dilute, as would occur from drinking a large amount of
water, vasopressin secretion is inhibited and the aquaporin-2
channels are taken back into the cell by endocytosis. The result is
that a large volume of watery urine is formed with a salt
concentration as little as one-fourth of that of the blood.
[0147] Decreased vasopressin release or decreased renal sensitivity
to AVP leads to diabetes insipidus, a condition featuring
hypernatremia (increased blood sodium concentration), polyuria
(excess urine production), and polydipsia (thirst).
[0148] High levels of AVP secretion (syndrome of inappropriate
antidiuretic hormone, SIADH) and resultant hyponatremia (low blood
sodium levels) occurs in brain diseases and conditions of the lungs
(Small cell lung carcinoma). In the perioperative period, the
effects of surgical stress and some commonly used medications
(e.g., opiates, syntocinon, anti-emetics) lead to a similar state
of excess vasopressin secretion. This may cause mild hyponatremia
for several days.
[0149] Vasopressin agonists are used therapeutically in various
conditions, and its long-acting synthetic analogue desmopressin is
used in conditions featuring low vasopressin secretion, as well as
for control of bleeding (in some forms of von Willebrand disease)
and in extreme cases of bedwetting by children. Terlipressin and
related analogues are used as vasoconstrictors in certain
conditions. Vasopressin infusion has been used as a second line of
management in septic shock patients not responding to high dose of
inotropes (e.g., dopamine or norepinephrine). A vasopressin
receptor antagonist is an agent that interferes with action at the
vasopressin receptors. They can be used in the treatment of
hyponatremia.
[0150] In one embodiment, the disclosure provides methods to select
for conditionally active biologic recombinant or synthetic protein
variants of proteins involved in the vasopressin response to be
reversibly deactivated at normal physiological osmotic pressure,
but reactivated at aberrant osmotic pressure in the blood. In
another embodiment, variants of proteins involved in the
vasopressin response are activated under hyponatremic conditions,
but inactivated at normal serum sodium concentrations. In one
aspect, hyponatremic conditions are those where serum sodium<135
mEq/L.
Cancer-Angiostatin
[0151] Angiostatin is a naturally occurring protein in several
animal species. It acts as an endogenous angiogenesis inhibitor
(i.e., it blocks the growth of new blood vessels). Angiostatin is
able to suppress tumor cell growth and metastasis through
inhibition of endothelial cell proliferation and migration.
Angiostatin is a 38 kD fragment of plasmin (which is itself a
fragment of plasminogen). Angiostatin comprises the kringles 1 to 3
of plasminogen. Angiostatin is produced, for example, by autolytic
cleavage of plasminogen, involving extracellular disulfide bond
reduction by phosphoglycerate kinase. Angiostatin can also be
cleaved from plasminogen by different matrix metalloproteinases
(MMPs) including MMP2, MMP 12 and MMP9, and serine proteases
(neutrophil elastase, prostate-specific antigen (PSA)). In vivo
angiostatin inhibits tumor growth and keeps experimental metastasis
in a dormant state. Angiostatin is elevated in animals with primary
tumors and other inflammatory and degenerative diseases.
[0152] Angiostatin is known to bind many proteins including
angiomotin and endothelial cell surface ATO synthase, but also
integrins, annexin II, C-met receptor, NG2-proteoglycans,
tissue-plasminogen activator, chondroitin sulfate glycoproteins,
and CD26. One study shows that IL-12, a TH1 cytokine with potent
antiangiogenic activity, is a mediator of angiostatin's activity.
Albin", J. Translational Medicine. Jan. 4, 2009, 7:5. Angiostatin
binds and inhibits ATP synthase on the endothelial cell surface.
ATP synthase also occurs on the surface of a variety of cancer
cells. Tumor cell surface ATP synthase was found to be more active
at low extracellular pH; a hallmark of tumor microenvironment.
Angiostatin was found to affect tumor cell surface ATP synthase
activity at acidic extracellular pH (pHe). At low extracellular pH,
angiostatin was directly anti-tumorigenic. At low pH, angiostatin
and anti-beta-subunit antibody induce intracellular acidification
of A549 cancer cells, as well as a direct toxicity that is absent
in tumor cells with low levels of extracellular ATP synthase. It
was hypothesized that the mechanism of tumor cytotoxicity is
dependent on intracellular pH deregulation due to inhibition of
cell surface ATP synthase. Chi and Pizzo, "Angiostatin is directly
cytotoxic to tumor cells at low extracellular pH: a mechanism
dependent on cell surface-associated ATP synthase", Cancer Res.,
2006, 66(2): 875-82.
[0153] In one embodiment, the disclosure provides a method for
identification of conditionally active angiostatin variant which is
less active than wild-type angiostatin at normal physiological
blood pH, but exhibits enhanced activity at low pH. Low pH is
defined as being less than normal physiological pH. In one aspect,
low pH is less than about pH 7.2. In a particular aspect, low pH is
about pH 6.7.
[0154] In one aspect, the conditionally active angiostatin variant
can be formulated and utilized as an anticancer agent.
Autoimmune Disease-Conditionally Active Biological Response
Modifiers
[0155] Rheumatoid arthritis is an autoimmune disease characterized
by aberrant immune mechanisms that lead to joint inflammation and
swelling with progressive destruction of the joints. RA can also
affect the skin, connective tissue and organs in the body.
Traditional treatment includes non-steroidal anti-inflammatory
drugs (NSAIDS), COX-2 inhibitors, and disease-modifying
anti-rheumatic drugs (DMARDS) such as methotrexate. None of the
traditional treatment regimes is ideal, especially for long term
use.
[0156] Biological response modifiers, which target inflammatory
mediators, offer a relatively new approach to the treatment of
rheumatoid arthritis and other autoimmune diseases. Such biological
response modifiers include antibodies, or active portions thereof,
against various inflammatory mediators such as IL-6, IL-6 receptor,
TNF-alpha, IL-23 and IL-12.
[0157] Some of the first biological response modifiers were
medications targeting tumor necrosis factor alpha (TNF-a), a
pro-inflammatory cytokine involved in the pathogenesis of RA.
Several anti-TNF-alpha medications are currently marketed for the
treatment of RA. For example, Enbrel.RTM. (etanercept, Amgen) is a
TNF-alpha blocker. Etanercept is a dimeric fusion protein
consisting of the extracellular ligand-binding portion of the human
75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to
the Fc portion of human IgG1. The Fc component of etanercept
contains the CH2 domain, the CH3 domain and hinge region, but not
the CH1 domain of IgG1. Etanercept is produced in a Chinese hamster
ovary (CHO) mammalian cell expression system. It consists of 934
amino acids and an apparent molecular weight of about 150
kilodaltons. Enbrel.RTM. is used to treat rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis and plaque psoriasis.
Serious side effects of Enbrel.RTM. include infections including
tuberculosis, fungal infection, bacterial or viral infection due to
opportunistic pathogens. Sepsis can also occur. Lymphoma, or other
malignancies have also been reported.
[0158] Remicade.RTM. (infliximab) is a chimeric anti-TNF-alpha
IgGkI monoclonal antibody composed of human constant and murine
variable regions. Remicade is administered by intravenous injection
and is used to treat rheumatoid arthritis, psoriasis, Crohn's
disease, ulcerative colitis, and ankylosing spondylitis. Side
effects of Remicade include serious infection or sepsis, and rarely
certain T-cell lymphomas. Other side effects include
hepatotoxicity, certain severe hematologic events, hypersensitivity
reactions and certain severe neurological events.
[0159] Other biological response modifiers include humanized
anti-interleukin-6 (IL-6) receptor antibodies. IL-6 is a cytokine
that contributes to inflammation, swelling and joint damage in RA.
One humanized anti-IL-6 receptor antibody, Actemra (tocilizumab,
Roche), is approved by the FDA and European Commission to treat
adult patients with rheumatoid arthritis. Actemra is also approved
in Japan for treatment of RA and juvenile idiopathic arthritis
(sJIA). Phase III studies showed that treatment with Actemra as a
monotherapy, or a combination with MTX or other DMARDs, reduced
signs and symptoms of RA compared with other therapies. Actemra is
a humanized anti-human IL-6 receptor monoclonal antibody that
competitively blocks the binding of IL-6 to its receptor. Thus, it
inhibits the proliferative effects of IL-6, which lead to synovial
thickening and pannus formation in RA. Serious side effects of
Actemra, include serious infections and hypersensitivity reactions
including a few cases of anaphylaxis. Other side effects include
upper respiratory tract infection, headache, nasopharyngitis,
hypertension and increased ALT.
[0160] Another common autoimmune disease is psoriasis. An
overactive immune system can lead to high levels of IL-12 and
IL-23, two cytokine proteins that have been found in psoriatic skin
plaques. IL-12 and IL-23 are involved in inflammatory and immune
responses such as natural killer cell activation and CD4+ T-cell
differentiation and activation.
[0161] One treatment for moderate or severe psoriasis involves
subcutaneous injection of Stelara.TM. (ustekinumab, Centocor Ortho
Biotech, Inc.) a humanized IgGIk monoclonal antibody against the
p40 subunit of the IL-12 and IL-23 cytokines. Stelara has been
shown to provide relief from certain symptoms associated with
psoriatic plaques, such as plaque thickness, scaling and redness.
The formulation for Stelara includes L-histidine and L-histidine
monohydrochloride monohydrate, polysorbate 80, and sucrose in
aqueous solution. Use of Stelara.TM. affects the immune system, and
may increase chances of infection, including tuberculosis, and
infections caused by bacteria, fungi or viruses; as well as
increase the risk of certain types of cancer.
[0162] Side effects of the biological response modifiers are
significant and are caused in part by high levels following
injection into patients renders patients susceptible to serious
infection or death. This is a major side effect associated with
this important class of drugs. One challenge is avoiding the high
initial level of activity from the dose of antibody required to
provide a long treatment effect following injection.
Conditionally Active Biological Antibodies for Brains
[0163] It has long been a challenge to deliver drugs, especially
large molecules such as antibodies, to the brain because brain
penetration by drugs is severely limited by the largely impermeable
BBB. Fortunately, the BBB has endogenous transport systems that are
mediated by a BBB receptor (BBB-R), which is a specific receptor
that allows transport of macromolecules across the BBB. For
example, an antibody that can bind to a BBB-R may be transported
across BBB using the endogenous transport systems. Such an antibody
may serve as a vehicle for transport of drugs or other agents
across BBB by using the endogenous BBB receptor mediated transport
system that traverses the BBB. Such antibodies need not have high
affinity to a BBB-R. Antibodies that are not conditionally active
antibodies with low affinities for BBB-R have been described as
crossing the BBB more efficiently than a high affinity antibody, as
described in US 2012/0171120 (incorporated herein by reference).
Unlike traditional antibodies, conditionally active antibodies are
not required to have low affinity for BBB-R to cross the BBB and
remain inside the brain. Conditionally active antibodies can have
high affinity for the BBB-R on the blood side of the BBB, and
little or no affinity on the brain side of the BBB. Drugs, such as
drug conjugates, may be coupled to a conditionally active antibody
to be transported with the antibody across the BBB into the
brain.
[0164] A BBB-R is a transmembrane receptor protein expressed on
brain endothelial cells which is capable of transporting molecules
across the blood-brain barrier. Examples of BBB-R include
transferrin receptor (TfR), insulin receptor, insulin-like growth
factor receptor (IGF-R), low density lipoprotein receptors
including without limitation low density lipoprotein
receptor-related protein 1 (LRP1) and low density lipoprotein
receptor-related protein 8 (LRP8), and heparin-binding epidermal
growth factor-like growth factor (HB-EGF). An exemplary BBB-R
herein is a transferrin receptor (TfR). The TfR is a transmembrane
glycoprotein (with a molecular weight of about 180,000) composed of
two disulphide-bonded sub-units (each of apparent molecular weight
of about 90,000) involved in iron uptake in vertebrates.
[0165] In some embodiments, the present invention provides a
conditionally active antibody generated from a parent or wild-type
antibody against a BBB-R. The conditionally active antibody binds
the BBB-R on the blood side of the BBB, and has a lower affinity to
the BBB-R than the parent or wild-type antibody on the brain side
of the BBB. In some other embodiments, the conditionally active
antibody has affinity to the BBB-R than the wild type or parent
antibody on the blood side of the BBB, and has no affinity to the
BBB-R on the brain side of the BBB.
[0166] Blood plasma is a body fluid that is very different from
brain extracellular fluid (ECF). As discussed by Somjen ("Ions in
the Brain: Normal Function, Seizures, and Stroke," Oxford
University Press, 2004, pages 16 and 33) and Redzic ("Molecular
biology of the blood-brain and the blood-cerebrospinal fluid
barriers: similarities and differences," Fluids and Barriers of the
CNS, vol. 8:3, 2011), the brain extracellular fluid has
significantly less K.sup.+, more Mg.sup.2+ and H.sup.+ than blood
plasma. The differences in ion concentrations between blood plasma
and brain ECF lead to significant differences in osmotic pressure
and osmolality between the two fluids. Table 1 shows the
concentrations of common ions in millimoles for both blood plasma
and brain ECF.
TABLE-US-00001 TABLE 1 Common ions in plasma (arterial plasma) and
brain extracellular fluid (CSF) ARTERIAL PLASMA CSF HUMAN RAT HUMAN
RAT Na.sup.+ 150 148 147 152 K.sup.+ 4.6 5.3 2.9 3.4 Ca, total 2.4
3.1 1.14 1.1 Ca.sup.2+, free 1.4 1.5 1.0 1.0 pCa Mg, total 0.86 0.8
1.15 1.3 Mg.sup.2+, free 0.47 0.44 0.7 0.88 H.sup.+ 0.000039
0.000032 0.000047 0.00005 pH 7.41 7.5 7.3 7.3 Cl.sup.- 99 119
HCO.sub.3.sup.- 26.8 31 23.3 28
[0167] Brain ECF also contains significantly more lactate than
blood plasma and significantly less glucose than blood plasma
(Abi-Saab et al., "Striking Differences in Glucose and Lactate
Levels Between Brain Extracellular Fluid and Plasma in Conscious
Human Subjects: Effects of Hyperglycemia and Hypoglycemia," Journal
of Cerebral Blood Flow & Metabolism, vol. 22, pages 271-279,
2002).
[0168] Thus, there are several physiological conditions that are
different between the two sides of the BBB, such as pH,
concentrations of various substances (such as lactose, glucose, K+,
Mg2+), osmotic pressure and osmolality. For the physiological
condition of pH, human blood plasma has a higher pH than human
brain ECF. For the physiological condition of K+concentration ,
brain ECF has a lower K+ concentration than human blood plasma. For
the physiological condition of Mg2+ concentration, the human brain
ECF has significantly more Mg2+ than human blood plasma. For the
physiological condition of osmotic pressure, the human brain ECF
has an osmotic pressure that is different from that of human blood
plasma. In some embodiments, the physiological conditions of brain
ECF may be the composition, pH, osmotic pressure and osmolality of
brain ECF of patients with a particular neurological disorder,
which may be different from the physiological condition of the
brain ECF of the general population.
[0169] The present invention thus provides a method for evolving a
DNA that encodes a template antibody against a BBB-R to create a
mutant DNA library. The mutant DNA library is then expressed to
obtain mutant antibodies. The mutant antibodies are screened for a
conditionally active antibody that has binds to the BBB-R under at
least one blood plasma physiological condition and has a low or no
affinity to the BBB-R under at least one brain physiological
condition in the brain ECF compared to the template antibody. Thus,
the selected mutant antibody has a low or high affinity to the
BBB-R at the blood plasma side and a low or no affinity to the
BBB-R at the brain ECF side. This selected mutant antibody is
useful as a conditionally active antibody for transport across the
BBB.
[0170] Such a conditionally active antibody is advantageous for
crossing the BBB and remaining in the brain ECF. The low affinity
to the BBB-R at the brain side lowers the rate (or removes) the
conditionally active antibody is transported back across the BBB
out of the brain and back into the blood relative to the template
antibody.
[0171] In some other embodiments, the present invention provides a
method for evolving a DNA that encodes a template antibody against
a BBB-R to create a mutant DNA library. The mutant DNA library is
then expressed to obtain mutant antibodies. The mutant antibodies
are screened for a conditionally active antibody that binds to the
BBB-R under at least one blood plasma physiological condition and
little or no affinity to the BBB-R under at least one brain
physiological condition. Thus, the selected mutant antibody has
affinity to the BBB-R at the plasma side and little or no affinity
to the BBB-R at the brain ECF side. This selected mutant antibody
is a conditionally active antibody.
[0172] Such a conditionally active antibody is advantageous in
crossing the BBB and remaining in the brain ECF. After binding to
the BBB-R at the blood plasma side, the conditionally active
antibody is transported across the BBB, and the little to no
affinity to the BBB-R at the brain ECF side means that the
conditionally active antibody is unlikely to be transported out of
the brain.
[0173] The affinity of the conditionally active antibody to a BBB-R
may be measured by its half maximal inhibitory concentration
(IC50), which is a measure of how much of the antibody is needed to
inhibit the binding of a known BBB-R ligand to the BBB-R by 50%. A
common approach is to perform a competitive binding assay, such as
competitive ELISA assay. An exemplary competitive ELISA assay to
measure IC50 on TfR (a BBB-R) is one in which increasing
concentrations of anti-TfR antibody compete against biotinylated
TfR.sup.A for binding to TfR. The anti-TfR antibody competitive
ELISA may be performed in Maxisorp plates (Neptune, N.J.) coated
with 2.5 .mu.g/ml of purified murine TfR extracellular domain in
PBS at 4.degree. C. overnight. Plates are washed with PBS/0.05%
Tween 20 and blocked using Superblock blocking buffer in PBS
(Thermo Scientific, Hudson, N.H.). A titration of each individual
anti-TfR antibody (1:3 serial dilution) is combined with
biotinylated anti-TfR.sup.A (0.5 nM final concentration) and added
to the plate for 1 hour at room temperature. Plates are washed with
PBS/0.05% Tween 20, and HRP-streptavidin (Southern Biotech,
Birmingham) is added to the plate and incubated for 1 hour at room
temperature. Plates are washed with PBS/0.05% Tween 20, and
biotinylated anti-TfR.sup.A bound to the plate is detected using
TMB substrate (BioFX Laboratories, Owings Mills).
[0174] A high IC50 indicates that more of the conditionally active
antibody is required to inhibit binding of the known ligand of a
BBB-R, and thus that the antibody's affinity for that BBB-R is
relatively low. Conversely, a low IC50 indicates that less of the
conditionally active antibody is required to inhibit binding of the
known ligand, and thus that the antibody's affinity for that BBB-R
is relatively high.
[0175] In some embodiments, the IC50 of the conditionally active
antibodies from a BBB-R in the blood plasma may be from about 1 nM
to about 100 .mu.M, or from about 5 nM to about 100 .mu.M, or from
about 50 nM to about 100 .mu.M, or from about 100 nM to about 100
.mu.M, or from about 5 nM to about 10 .mu.M, or from about 30 nM to
about 1 .mu.M, or from about 50 nM to about 1 .mu.M.
Conditionally Active Biological Proteins for Synovial Fluid
[0176] Joint diseases are a major cause of disability and early
retirement in the industrialized countries. Joint diseases often
lead to damage at a joint which is difficult to repair. Synovial
fluid is a body fluid that is found in the synovial cavity of the
joints (e.g., knee, hip, shoulder) of a human or animal body
between the cartilage and synovium of facing articulating surfaces.
Synovial fluid provides nourishment to the cartilage and also
serves as a lubricant for the joints. The cells of the cartilage
and synovium secrete fluid that serve as a lubricant between the
articulating surfaces. Human synovial fluid comprises approximately
85% water. It is derived from the dialysate of blood plasma, which
itself is made up of water, dissolved proteins, glucose, clotting
factors, mineral ions, hormones, etc. Proteins such as albumin and
globulins are present in synovial fluid and are believed to play an
important role in the lubricating the joint area. Some other
proteins are also found in human synovial fluid, including the
glycoproteins such as alpha-1-acid glycoprotein (AGP),
alpha-1-antitrypsin (A1AT) and lubricin.
[0177] Synovial fluid has a composition that is very different from
other parts of the body. Thus, synovial fluid has physiological
conditions that are different from other parts of the body, such as
the blood plasma. For example, synovial fluid has less than about
10 mg/dL of glucose whereas the mean normal glucose level in human
blood plasma is about 100 mg/dL, fluctuating within a range between
70 and 100 mg/dL throughout the day. In addition, the total protein
level in the synovial fluid is about one third of the blood plasma
protein level since large molecules such as proteins do not easily
pass through the synovial membrane into the synovial fluid. It has
also been found that the pH of human synovial fluid is higher than
the pH in human plasma (Jebens et al., "On the viscosity and pH of
synovial fluid and the pH of blood," The Journal of Bone and Joint
Surgery, vol. 41 B, pages 388-400, 1959; Farr et al., "Significance
of the hydrogen ion concentration in synovial fluid in Rheumatoid
Arthritis," Clinical and Experimental Rheumatology, vol. 3, pages
99-104, 1985).
[0178] Thus, the synovial fluid has several physiological
conditions that are different from those of the other parts of
body, such as the physiological conditions in the blood plasma. The
synovial fluid has a pH that is higher than other parts of the
body, especially the blood plasma. The synovial fluid has a lower
concentration of glucose than other parts of the body, such as
blood plasma. The synovial fluid also has a lower concentration of
protein than other parts of the body, such as blood plasma.
[0179] Several antibodies have been used to treat joint disease by
introducing the antibodies into the synovial fluid. For example,
the synovial fluid in an injured joint is known to contain many
factors which have an influence on the progression of
osteoarthritis (see, for example, Fernandes, et al., "The Role of
Cytokines in Osteoarthritis Pathophysiology", Biorheology, vol. 39,
pages 237-246, 2002). Cytokines, such as Interleukin-1 (IL-I) and
Tumor Necrosis Factor-.alpha. (TNF-.alpha.), which are produced by
activated synoviocytes, are known to upregulate matrix
metalloproteinase (MMP) gene expression. Upregulation of MMP leads
to degradation of the matrix and non-matrix proteins in the joints.
Antibodies that neutralize cytokines may stop the progression of
osteoarthritis.
[0180] Using antibodies as drug is a promising strategy for the
treatment of joint diseases. For example, antibodies (such as
antibody against aggrecan or aggrecanase) have been developed to
treat osteoarthritis, which has by far the greatest prevalence
among joint diseases (WO1993/022429A1). An antibody against
acetylated high-mobility group box 1 (HMGB1) has been developed for
diagnosis or treatment of joint diseases that are inflammatory,
autoimmune, neurodegenerative or malignant diseases/disorders, such
as arthritis. This antibody may be used to detect the acetylated
form of HMGB1 in synovial fluid (WO 2011/157905A1). Another
antibody (CD20 antibody) has also been developed to treat damage to
connective tissue and cartilage of the joints.
[0181] However, the antigens of these antibodies are often
expressed in other parts of the body carrying important
physiological functions. Antibodies against these antigens, though
efficacious in treating joint diseases, may also significantly
interfere with the normal physiological functions of these antigens
in other parts of the body. Therefore, severe side effects may be
experienced by patients. It is thus desirable to develop
therapeutics, such as antibodies against cytokines or other
antigens that can preferentially bind to their antigens (proteins
or other macromolecules) at higher affinity in the synovial fluid,
while not binding or only weakly binding to the same antigens in
other parts of the body in order to reduce side effects.
[0182] Such conditionally active biological proteins may be
conditionally active antibodies. In some embodiments, the present
invention also provides conditionally active biological proteins
that are proteins other than antibodies. For example, a
conditionally active immune regulator may be developed by the
present invention for preferentially regulating the immune response
in the synovial fluid, which may less or no effect on the immune
response at other parts of the body.
[0183] The conditionally active biological proteins may be
conditionally active suppressors of cytokine signaling (SOCS). Many
of these SOCS are involved in inhibiting the JAK-STAT signaling
pathway. The conditionally active suppressors of cytokine signaling
can preferentially suppress the cytokine signaling in the synovial
fluid, while not or to a lesser extent suppressing the cytokine
signaling in other parts of the body.
[0184] In some embodiments, the present invention provides a
conditionally active biological protein derived from a wild-type
biological protein. The conditionally active biological protein has
a lower activity under at least one physiological condition in
certain parts of the body such as in blood plasma than the
wild-type biological protein, and has a higher activity than the
wild-type biological protein under at least one physiological
condition in the synovial fluid. Such conditionally active
biological proteins can preferentially function in the synovial
fluid, but not or to a lesser extent act upon other parts of the
body. Consequently, such conditionally active biological proteins
may have reduced side effects.
[0185] In some embodiments, the conditionally active biological
proteins are antibodies against an antigen in or exposed to
synovial fluid. Such antigens may be any proteins involved in
immune response/inflammation in a joint disease, though the antigen
is often a cytokine. The conditionally active antibody has a lower
affinity to the antigen than the wild-type antibody for the same
antigen under at least one physiological condition in other parts
of the body (such as blood plasma), while has higher affinity for
the antigen than the wild-type antibody under at least one
physiological condition of synovial fluid. Such conditionally
active antibodies can bind weakly or not at all to the antigen in
other parts of the body, but bind, for example bind strongly and
tightly or bind stronger to the antigen in synovial fluid.
Conditionally Active Biological Proteins for Tumors
[0186] Cancer cells in a solid tumor are able to form a tumor
microenvironment in their surroundings to support the growth and
metastasis of the cancer cells. A tumor microenvironment is the
cellular environment in which the tumor exists, including
surrounding blood vessels, immune cells, fibroblasts, other cells,
soluble factors, signaling molecules, an extracellular matrix, and
mechanical cues that can promote neoplastic transformation, support
tumor growth and invasion, protect the tumor from host immunity,
foster therapeutic resistance, and provide niches for dormant
metastases to thrive. The tumor and its surrounding
microenvironment are closely related and interact constantly.
Tumors can influence their microenvironment by releasing
extracellular signals, promoting tumor angiogenesis and inducing
peripheral immune tolerance, while the immune cells in the
microenvironment can affect the growth and evolution of cancerous
cells. See Swarts et al. "Tumor Microenvironment Complexity:
Emerging Roles in Cancer Therapy," Cancer Res, vol., 72, pages
2473-2480, 2012.
[0187] The tumor microenvironment is often hypoxic. As the tumor
mass increases, the interior of the tumor grows farther away from
existing blood supply, which leads to difficulties in fully
supplying oxygen to the tumor microenvironment. The partial oxygen
pressure in the tumor environment is below 5 mm Hg in more than 50%
of locally advanced solid tumors, in comparison with a partial
oxygen pressure at about 40 mm Hg in blood plasma. In contrast,
other parts of the body are not hypoxic. The hypoxic environment
leads to genetic instability, which is associated with cancer
progression, via downregulating nucleotide excision repair and
mismatch repair pathways. Hypoxia also causes the upregulation of
hypoxia-inducible factor 1 alpha (HIF1-.alpha.), which induces
angiogenesis, and is associated with poorer prognosis and the
activation of genes associated with metastasis. See Weber et al.,
"The tumor microenvironment," Surgical Oncology, vol. 21, pages
172-177, 2012 and Blagosklonny, "Antiangiogenic therapy and tumor
progression," Cancer Cell, vol. 5, pages 13-17, 2004.
[0188] In addition, tumor cells tend to rely on energy generated
from lactic acid fermentation, which does not require oxygen. So
tumor cells are less likely to use normal aerobic respiration that
does require oxygen. A consequence of using lactic acid
fermentation is that the tumor microenvironment is acidic (pH
6.5-6.9), in contrast to other parts of the body which are
typically either neutral or slightly basic. For example, human
blood plasma has a pH of about 7.4. See Estrella et al., "Acidity
Generated by the Tumor Microenvironment Drives Local Invasion,"
Cancer Research, vol. 73, pages 1524-1535, 2013. The nutrient
availability in the tumor microenvironment is also low due to the
relatively high nutrient demand of the proliferating cancer cells,
in comparison with cells located in other parts of the body.
[0189] Further, the tumor microenvironment also contains many
distinct cell types not commonly found in other parts of the body.
These cell types include endothelial cells and their precursors,
pericytes, smooth muscle cells, Wbroblasts, carcinoma-associated
Wbroblasts, myoWbroblasts, neutrophils, eosinophils, basophils,
mast cells, T and B lymphocytes, natural killer cells and antigen
presenting cells (APC) such as macrophages and dendritic cells
(Lorusso et al., "The tumor microenvironment and its contribution
to tumor evolution toward metastasis," Histochem Cell Biol, vol.
130, pages 1091-1103, 2008).
[0190] Accordingly, the tumor microenvironment has at least several
physiological conditions that are different from those of other
parts of body, such as the physiological conditions in blood
plasma. The tumor microenvironment has a pH (acidic) that is lower
than other parts of the body, especially the blood plasma (pH 7.4).
The tumor microenvironment has a lower concentration of oxygen than
other parts of the body, such as blood plasma. Also, the tumor
microenvironment has a lower nutrient availability than other parts
of the body, especially the blood plasma. The tumor
microenvironment also has some distinct cell types that are not
commonly found in other parts of the body, especially the blood
plasma.
[0191] Some cancer drugs include antibodies that can penetrate into
the tumor microenvironment and act upon the cancer cells therein.
Antibody-based therapy for cancer is well established and has
become one of the most successful and important strategies for
treating patients with haematological malignancies and solid
tumors. There is a broad array of cell surface antigens that are
expressed by human cancer cells that are overexpressed, mutated or
selectively expressed in cancer cells compared with normal tissues.
These cell surface antigens are excellent targets for antibody
cancer therapy.
[0192] Cancer cell surface antigens that may be targeted by
antibodies fall into several different categories. Haematopoietic
differentiation antigens are glycoproteins that are usually
associated with clusters of differentiation (CD) groupings and
include CD20, CD30, CD33 and CD52. Cell surface differentiation
antigens are a diverse group of glycoproteins and carbohydrates
that are found on the surface of both normal and tumor cells.
Antigens that are involved in growth and differentiation signaling
are often growth factors and growth factor receptors. Growth
factors that are targets for antibodies in cancer patients include
CEA2, epidermal growth factor receptor (EGFR; also known as
ERBB1)12, ERBB2 (also known as HER2)13, ERBB3 (REF. 18), MET (also
known as HGFR)19, insulin-like growth factor 1 receptor (IGF1R)20,
ephrin receptor A3 (EPHA3)21, tumor necrosis factor (TNF)-related
apoptosis-inducing ligand receptor 1 (TRAILR1; also known as
TNFRSF10A), TRAILR2 (also known as TNFRSF10B) and receptor
activator of nuclear factor-.kappa.B ligand (RANKL; also known as
TNFSF11)22. Antigens involved in angiogenesis are usually proteins
or growth factors that support the formation of new
microvasculature, including vascular endothelial growth factor
(VEGF), VEGF receptor (VEGFR), integrin .alpha.V.beta.3 and
integrin .alpha.5.beta.1 (REF. 10). Tumor stroma and the
extracellular matrix are indispensable support structures for a
tumor. Stromal and extracellular matrix antigens that are
therapeutic targets include fibroblast activation protein (FAP) and
tenascin. See Scott et al., "Antibody therapy of cancer," Nature
Reviews Cancer, vol. 12, pages 278-287, 2012.
[0193] In addition to antibodies, other biological proteins have
also shown promise in treating cancers. Examples include tumor
suppressors such as Retinoblastoma protein (pRb), p53, pVHL, APC,
CD95, ST5, YPEL3, ST7, and ST14. Some proteins that induce
apoptosis in cancer cells may also be introduced into tumors for
shrinking the size of tumors. There are at least two mechanisms
that can induce apoptosis in tumors: the tumor necrosis
factor-induced mechanism and the Fas-Fas ligand-mediated mechanism.
At least some of the proteins involved in either of the two
apoptotic mechanisms may be introduced to tumors for treatment.
[0194] Cancer stem cells are cancer cells that have the ability to
give rise to all cell types found in a particular cancer sample,
and are therefore tumor-forming. They may generate tumors through
the stem cell processes of self-renewal and differentiation into
multiple cell types. It is believed that cancer stem cells persist
in tumors as a distinct population and cause relapse and metastasis
by giving rise to new tumors. Development of specific therapies
targeted at cancer stem cells may improve the survival and quality
of life of cancer patients, especially for sufferers of metastatic
disease.
[0195] These drugs for treating tumors often interfere with normal
physiological functions in other parts of the body besides tumors.
For example, proteins inducing apoptosis in tumors may also induce
apoptosis in some other parts of the body thus causing side
effects. In embodiments where an antibody is used to treat tumors,
the antigen of the antibody may also be expressed in other parts of
the body where they perform normal physiological functions. For
example, monoclonal antibody bevacizumab (targeting vascular
endothelial growth factor) to stop tumor blood vessel growth. This
antibody can also prevent blood vessel growth or repair in other
parts of the body, thus causing bleeding, poor wound healing, blood
clots, and kidney damage. Development of a conditionally active
biological protein that concentrates on targeting mainly or solely
tumors is highly desirable for more effective tumor therapies.
[0196] In some embodiments, the present invention provides a
conditionally active biological protein generated from a wild-type
biological protein that may be a candidate for tumor treatment. The
conditionally active biological protein has lower activity under at
least one physiological condition in parts of the body other than
the tumor microenvironment such as blood plasma than the wild-type
biological protein, while it has higher activity under at least one
physiological condition in the tumor microenvironment than the
wild-type biological protein. Such conditionally active biological
proteins can preferentially act upon cancer cells in the tumor
microenvironment for treating tumors, and thus will be less likely
to cause side effects. In the embodiment where the biological
protein is an antibody against an antigen on the surface of the
tumor cells where the antigen is exposed to the tumor
microenvironment, the conditionally active antibody has lower
affinity to the antigen than the wild-type antibody in other parts
of the body, e.g. a non-tumor microenvironment, while it has higher
affinity to the antigen than the wild-type antibody in the tumor
microenvironment. Such conditionally active antibodies can bind
weakly or not at all to the antigen in other parts of the body, but
have greater binding, or bind strongly and tightly, to the antigen
in the tumor microenvironment.
[0197] In some embodiments, the conditionally active antibody is an
antibody against an immune checkpoint protein, resulting in
inhibition of the immune checkpoints. Such conditionally active
antibodies have an increased binding affinity to the immune
checkpoint protein in a tumor microenvironment in comparison to the
wild-type antibody from which the conditionally active antibody is
derived, and a decreased binding affinity to the immune checkpoint
protein in a non-tumor microenvironment in comparison to the
wild-type antibody from which the conditionally active antibody is
derived.
[0198] The immune checkpoints function as endogenous inhibitory
pathways for the immune system to maintain self-tolerance and
modulate the duration and extent of immune response to antigenic
stimulation, i.e., foreign molecules, cells and tissues See
Pardoll, Nature Reviews Cancer, vol. 12, pages 252-264, 2012.
Inhibition of immune checkpoints by suppressing one or more
checkpoint proteins can cause super-activation of the immune
system, especially T-cells, thus inducing the immune system to
attack tumors. Checkpoint proteins suitable for the present
invention include CTLA4 and its ligands CD80 and CD86, PD1 and its
ligands PDL1 and PDL2, T cell immunoglobulin and mucin protein-3
(TIM3) and its ligand GALS, B and T lymphocyte attenuator (BTLA)
and its ligand HVEM (herpesvirus entry mediator), receptors such as
killer cell immunoglobulin-like receptor (KIR), lymphocyte
activation gene-3 (LAG3) and adenosine A2a receptor (A2aR), as well
as ligands B7-H3 and B7-H4. Additional suitable immune checkpoint
proteins are described in Pardoll, Nature Reviews Cancer, vol. 12,
pages 252-264, 2012 and Nirschl & Drake, Clin Cancer Res, vol.
19, pages 4917-4924, 2013, the disclosures of which are hereby
incorporated herein by reference.
[0199] CTLA-4 and PD1 are two of the best known immune checkpoint
proteins. CTLA-4 can down-regulate pathways of T-cell activation
(Fong et al., Cancer Res. 69(2):609-615, 2009; and Weber, Cancer
Immunol. Immunother, 58:823-830, 2009). Blockading CTLA-4 has been
shown to augment T-cell activation and proliferation. Inhibitors of
CTLA-4 include anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind
to CTLA-4 and block the interaction of CTLA-4 with its ligands CD80
or CD86 thereby blocking the down-regulation of the immune
responses elicited by the interaction of CTLA-4 with its
ligand.
[0200] The checkpoint protein PD1 is known to suppress the activity
of T cells in peripheral tissues at the time of an inflammatory
response to infection and to limit autoimmunity. An in vitro PD1
blockade can enhance T-cell proliferation and cytokine production
in response to stimulation by specific antigen targets or by
allogeneic cells in mixed lymphocyte reactions. A strong
correlation between PD1 expression and reduced immune response was
shown to be caused by the inhibitory function of PD1, i.e., by
inducing immune checkpoints (Pardoll, Nature Reviews Cancer, 12:
252-264, 2012). A PD1 blockade can be accomplished by a variety of
mechanisms including antibodies that bind PD1 or its ligands, PDL1
or PDL2.
[0201] Past research has discovered antibodies against several
checkpoint proteins (CTLA4, PD1, PD-L1). These antibodies are
effective in treating tumors by inhibiting the immune checkpoints
thereby super-activating the immune system, especially the T-cells,
for attacking tumors (Pardoll, Nature Reviews Cancer, vol. 12,
pages 252-264, 2012). However, the super-activated T-cells may also
attack host cells and/or tissues, resulting in collateral damage to
a patient's body. Thus, therapy based on use of these known
antibodies for inhibition of immune checkpoints is difficult to
manage and the risk to the patient is a serious concern. For
example, an FDA approved antibody against CTLA-4 carries a black
box warning due to its high toxicity.
[0202] The present invention addresses the problem of collateral
damage by super-activated T-cells by providing conditionally active
antibodies against immune checkpoint proteins. These conditionally
active antibodies preferentially activate the immune checkpoints in
a tumor-microenvironment. At the same time, the immune checkpoints
in the non-tumor-microenvironment(s), e.g. normal body tissue, are
not inhibited or are less inhibited by the conditionally active
antibodies such that in the non-tumor microenvironment the
potential for collateral damage to the body is reduced. This goal
is achieved by engineering the conditionally active antibody to be
more active in the tumor microenvironment than in the non-tumor
microenvironment.
[0203] In some embodiments, the conditionally active antibody
against an immune checkpoint protein may have a ratio of binding
activity to an immune checkpoint protein in the
tumor-microenvironment to the binding activity to the same immune
checkpoint protein in a non-tumor microenvironment of at least
about 1.1, or at least about 1.2, or at least about 1.4, or at
least about 1.6, or at least about 1.8, or at least about 2, or at
least about 2.5, or at least about 3, or at least about 5, or at
least about 7, or at least about 8, or at least about 9, or at
least about 10, or at least about 15, or at least about 20. A
typical assay for measuring the binding activity of an antibody is
an ELISA assay.
[0204] Highly immunogenic tumors, such as malignant melanoma, are
most vulnerable to a super-activated immune system achieved by
immune system manipulation. Thus the conditionally active
antibodies against immune checkpoint proteins may be especially
effective for treating such highly immunogenic tumors. However,
other types of tumors are also vulnerable to a super-activated
immune system.
[0205] In some embodiments, the conditionally active antibodies
against the immune checkpoint proteins may be used in combination
therapy. For example, combination therapy may include a
conditionally active antibody against a tumor cell surface molecule
(tumor specific antigen) and a conditionally active antibody
against an immune checkpoint protein. In one embodiment, both the
binding activity of the conditionally active antibody to the tumor
cell surface molecule and the binding activity of the conditionally
active antibody to the immune checkpoint protein may reside in a
single protein, i.e., a bispecific conditionally active antibody as
disclosed herein. In some further embodiments, combination therapy
may include a conditionally active antibody against a tumor cell
surface molecule (tumor specific antigen) and two or more
conditionally active antibodies against two or more different
immune checkpoint proteins. In one embodiment, all of these binding
activities may reside in a single protein, i.e., a multispecific
antibody as disclosed herein.
[0206] Since the conditionally active antibodies are more active in
a tumor microenvironment in comparison with the activity of the
wild-type antibody against the same tumor cell surface molecule or
checkpoint protein from which the conditionally active antibody is
derived, these combination therapies can provide both an enhanced
efficacy and a significant reduction in toxicity. The reduced
toxicity of these conditionally active antibodies, especially the
antibodies against the immune checkpoint proteins, can allow safe
use of potent antibodies, such as ADC antibodies as described
herein, as well as a higher dose of the antibodies.
[0207] In some embodiments, the conditionally active antibodies
against the checkpoint proteins may be in a prodrug form. For
example, the conditionally active antibodies may be prodrugs that
have no desired drug activity before being cleaved and turned into
a drug form. The prodrugs may be cleaved preferentially in a
tumor-microenvironment, either because the enzyme that catalyzes
such cleavage exists preferentially in the tumor-microenvironment
or because the conditionally active antibodies make the cleavage
site more accessible in a tumor microenvironment, in comparison
with the accessibility of the cleavage site in a non-tumor
microenvironment. Conditionally active biological proteins for stem
cell niches, including tumor stem cells
[0208] Stem cells exist in an environment called stem cell niche in
the body, which constitutes a basic unit of tissue physiology,
integrating signals that mediate the response of stem cells to the
needs of organisms. Yet the niche may also induce pathologies by
imposing aberrant functions on stem cells or other targets. The
interplay between stem cells and their niches creates the dynamic
system necessary for sustaining tissues, and for the ultimate
design of stem-cell therapeutics (Scadden, "The stem-cell niche as
an entity of action," Nature, vol. 441, pages 1075-1079, 2006).
Common stem cell niches in vertebrates include the germline stem
cell niche, the hematopoietic stem cell niche, the hair follicle
stem cell niche, the intestinal stem cell niche, and the
cardiovascular stem cell niche.
[0209] The stem cell niche is a specialized environment that is
different from other parts of the body (e.g. blood plasma)
(Drummond-Barbosa, "Stem Cells, Their Niches and the Systemic
Environment: An Aging Network," Genetics, vol. 180, pages
1787-1797, 2008; Fuchs, "Socializing with the Neighbors: Stem Cells
and Their Niche," Cell, vol. 116, pages 769-778, 2004). The stem
cell niche is hypoxic where oxidative DNA damage is reduced. Direct
measurements of oxygen levels have revealed that bone marrow is, in
general, quite hypoxic (.about.1%-2% O2), in comparison to blood
plasma (Keith et al., "Hypoxia-Inducible Factors, Stem Cells, and
Cancer," Cell, vol. 129, pages 465-472, 2007; Mohyeldin et al.,
"Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell
Niche," Cell Stem Cell, vol. 7, pages 150-161, 2010). In addition,
the stem cell niches need to have several other factors to regulate
stem cell characteristics within the niches: extracellular matrix
components, growth factors, cytokines, and factors of the
physiochemical nature of the environment including the pH, ionic
strength (e.g. Ca.sup.2+ concentration) and metabolites.
[0210] Accordingly, the stem cell niche has at least several
physiological conditions that are different from those of the other
parts of body, such as the physiological conditions in the blood
plasma. The stem cell niche has a lower oxygen concentration (1-2%)
than other parts of the body, especially the blood plasma. Other
physiological conditions for the stem cell niche including pH and
ionic strength, may also be different from other parts of the
body.
[0211] Stem cell therapy is an interventional strategy that
introduces new adult stem cells into damaged tissue in order to
treat disease or injury. This strategy depends on the ability of
stem cells to self-renew and give rise to subsequent offspring with
variable degrees of differentiation capacities. Stem cell therapy
offers significant potential for regeneration of tissues that can
potentially replace diseased and damaged areas in the body, with
minimal risk of rejection and side effects. Therefore, delivering a
drug (biological protein (e.g. antibody) or chemical compound) to
the stem cell niche for influencing the renewal and differentiation
of stem cells is an important part of stem cell therapy.
[0212] There are several examples on how the stem cell niches
influence the renewal and/or differentiation of the stem cells in
mammals. The first is in the skin, where the .beta.-1 integrin is
known to be differentially expressed on primitive cells and to
participate in constrained localization of a stem-cell population
through interaction with matrix glycoprotein ligands. Second, in
the nervous system, the absence of tenascin C alters neural
stem-cell number and function in the subventricular zone. Tenascin
C seems to modulate stem-cell sensitivity to fibroblast growth
factor 2 (FGF2) and bone morphogenetic protein 4 (BMP4), resulting
in increased stem-cell propensity. Third, another matrix protein,
the Arg-Gly-Asp-containing sialoprotein, osteopontin (OPN), has now
been demonstrated to contribute to haematopoietic stem cell
regulation. OPN interacts with several receptors known to be on
haematopoietic stem cells, CD44, and .alpha.4 and .alpha.5.beta.1
integrins. OPN production can vary markedly, particularly with
osteoblast activation. Animals deficient in OPN have an increased
HS-cell number, because a lack of OPN leads to superphysiologic
stem-cell expansion under stimulatory conditions. Therefore, OPN
seems to serve as a constraint on haematopoietic stem cell numbers,
limiting the number of stem cells under homeostatic conditions or
with stimulation. See Scadden, "The stem-cell niche as an entity of
action," Nature, vol. 441, pages 1075-1079, 2006.
[0213] Xie et al. "Autocrine signaling based selection of
combinatorial antibodies that transdifferentiate human stem cells,"
Proc Natl Acad Sci USA, vol. 110, pages 8099-8104, 2013) discloses
a method of using antibodies to influence stem cell
differentiation. The antibodies are agonists for a granulocyte
colony stimulating factor receptor. Unlike the natural
granulocyte-colony stimulating factor that activates cells to
differentiate along a predetermined pathway, the isolated agonist
antibodies transdifferentiated human myeloid lineage CD34+ bone
marrow cells into neural progenitors. Melidoni et al. ("Selecting
antagonistic antibodies that control differentiation through
inducible expression in embryonic stem cells," Proc Natl Acad Sci
USA, vol. 110, pages 17802-17807, 2013) also discloses a method of
using an antibody to interfere the interaction between FGF4 and its
receptor FGFR1.beta., therefore block the autocrine FGF4-mediated
embryonic stem cell differentiation.
[0214] Knowledge of the functions of ligands/receptors in stem cell
differentiation has enabled the strategy of applying biological
proteins to interfere with these ligands/receptors for the purpose
of regulating or even directing stem cell differentiation. The
ability to control differentiation of genetically unmodified human
stem cells through the administration of antibodies into the stem
cell niche can provide new ex vivo or in vivo approaches to stem
cell-based therapeutics. In some embodiments, the present invention
provides a conditionally active biological protein generated from a
wild-type biological protein that is capable of entering the stem
cell niches, including cancer stem cells, to regulate stem cell or
tumor development. The conditionally active biological protein has
lower activity than the wild-type biological protein under at least
one physiological condition in other parts of the body, while it
has higher activity than the wild-type biological protein under at
least one physiological condition in the stem cell niche, for
example the cancer stem cell environment. Such conditionally active
biological proteins will be less likely to cause side effects and
preferentially act in the stem cell niche to regulate renewal and
differentiation of stem cells. In some embodiments, the
conditionally active biological proteins are antibodies. Such
conditionally active antibodies can bind weakly or not at all to
their antigens in other parts of the body, but bind strongly and
tightly to the antigens in the stem cell niche.
[0215] The conditionally active proteins for the synovial fluid,
tumor microenvironment and stem cell niches of the present
invention are generated by a method for evolving a DNA that encodes
a wild-type biological protein to create a mutant DNA library. The
mutant DNA library is then expressed to obtain mutant proteins. The
mutant proteins are screened for a conditionally active biological
protein that has a higher activity than the wild-type biological
protein under at least one physiological condition of a first part
of the body selected from the group consisting of synovial fluid,
tumor microenvironment, and stem cell niches, and has lower
activity than the wild-type biological protein under at least one
physiological condition at a second part of the body that is
different from the first part of the body. The second part of the
body may be the blood plasma. Such selected mutant biological
proteins are conditionally active biological proteins that have
high activity in the first part of the body but low activity in the
second parts of the body.
[0216] Such conditionally active biological proteins are
advantageous in lowering side effects of the wild-type protein,
since the conditionally active biological protein has lower
activity in the other parts of the body where the conditionally
active biological protein is not intended to act. For instance, if
the conditionally active biological protein is intended to be
introduced into the tumor microenvironment, the fact that the
conditionally active biological protein has low activity in parts
of the body other than the tumor microenvironment means such
conditionally active biological protein will be less likely to
interfere with normal physiological functions in parts of the body
other than the tumor microenvironment. At the same time, the
conditionally active biological protein has high activity in the
tumor microenvironment, which gives the conditionally active
biological protein a higher efficacy in treating tumors.
[0217] Because of the reduced side effects, the conditionally
active biological protein will allow a significantly higher dose of
the protein to be safely used, in comparison with the wild-type
biological protein. This is especially beneficial for an antibody
against a cytokine or a growth factor, because antibodies against
the cytokine or growth factor may interfere with normal
physiological functions of the cytokine or growth factor in other
parts of the body. By using a conditionally active biological
protein, with reduced side effects, higher doses may be used to
achieve higher efficacy.
[0218] The conditionally active biological proteins for acting in
one of a synovial fluid, tumor microenvironment, or stem cell niche
can also enable new drug targets to be used. Using traditional
biological proteins as therapeutics may cause unacceptable side
effects. For example, inhibition of an epidermal growth factor
receptor (EGFR) can very effectively suppress tumor growth.
However, a drug inhibiting EGFR will also suppress growth at the
skin and gastrointestinal (GI) tract. The side effects render EGFR
unsuitable as a tumor drug target. Using a conditionally active
antibody that binds to EGFR at high affinity in only the tumor
microenvironment, but not or at very low affinity at any other
parts of the body, will significantly reduce the side effects and
at the same time suppress tumor growth. In this case, EGFR may
become an effective new tumor drug target by using conditionally
active antibodies.
[0219] In another example, suppressing cytokines is often
beneficial in repairing joint damage. However, suppressing
cytokines in other parts of the body also may suppress the immune
response of the body, causing an immune deficiency. Thus, cytokines
in synovial fluid are not ideal targets for developing traditional
antibody drugs for treatment of joint damage. However, by using
conditionally active antibodies that preferentially bind to
cytokines in the synovial fluid, while not or only weakly to the
same cytokines in other parts of the body, the side effect of
immune deficiency can be dramatically reduced. Therefore, cytokines
in synovial fluid may become suitable targets for repairing joint
damage by using conditionally active antibodies.
Conditionally Active Viral Particles
[0220] Viral particles have long been used as delivery vehicles for
transporting proteins, nucleic acid molecules, chemical compounds
or radioactive isotopes to a target cell or tissue. Viral panicles
that are commonly used as delivery vehicles include retoviruses,
adenoviruses, lentivirus, herpes virus, and adeno-associated
viruses. The viral particles recognize their target cells through a
surface protein that serves as a recognition protein for specific
binding to a cellular protein that serves as target protein of the
target cells, often in a ligand-receptor binding system (Lentz,
"The recognition event between virus and host cell receptor: a
target for antiviral agents," J. of Gen. Virol., vol. 71, pages
751-765, 1990, incorporated herein by reference). For example, the
viral recognition protein may be a ligand for a receptor on the
target cells. The specificity between a ligand and a receptor
allows the viral particles to specifically recognize and deliver
their content to a target cell.
[0221] Techniques for developing artificial viral particles from
wild-type viruses are well known to a person skilled in the art.
Known artificial viral particles as delivery vehicles include these
based on retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO
93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO
93/10218; U.S. Pat. No. 4,777,127; GB Patent No, 2,200,651; EP 0
345 242; and WO 91/02805), alphavirus Sindbis virus vectors,
Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus
(ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated viruses (see, e.g., WO 94/12649, WO 93/03769; WO
93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).
[0222] Generally, the artificial viral particles are constructed by
inserting a foreign recognition protein into a virus particle,
often replacing the native recognition protein by recombinant
technology. The foreign recognition protein may be, for example, an
antibody, a receptor, a ligand or a collagen binding domain. The
present invention provides a conditionally active recognition
protein that is inactive or less active for binding to a cell at a
normal physiological condition, and that is active or more active
for binding to a cell at an aberrant condition. The conditionally
active recognition protein can thereby preferentially bind to
target cells of diseased tissue and/or at a disease site based on
the presence of an abnormal condition at that site and avoid or
only minimally bind to the cells of normal tissue where a normal
physiological condition exists. The conditionally active
recognition protein may be expressed and displayed on the surface
of a viral particle.
[0223] In some embodiments, the present invention provides a method
of evolving a wild-type recognition protein and screening for a
conditionally active recognition protein. The conditionally active
recognition protein is less active in binding to a cell than the
wild-type recognition protein under a normal physiological
condition, and more active in binding to a cell than the wild-type
recognition protein under an aberrant condition. Such a
conditionally active recognition protein may be inserted into a
viral particle by well-known recombinant technology to generate a
conditionally active viral particle.
[0224] In another embodiment, the present invention provides a
conditionally active viral particle comprising a conditionally
active recognition protein, which allows the conditionally active
viral particle to recognize and bind with the target cells of
diseased tissue or at a disease site, but not the cells of normal
tissue. Such a conditionally active viral particle can
preferentially deliver therapeutics within the viral particle to
the disease tissue or disease site, while the conditionally active
viral particle delivers less or does not deliver the therapeutics
to the cells of normal tissue.
[0225] In some embodiments, the target cells at a disease site are
inside a zone or microenvironment with an abnormal pH (e.g., pH
6.5) or an abnormal temperature, in comparison with the pH or
temperature in other parts of the body that are healthy or not
suffering from the particular disease or disease state. In this
embodiment, the conditionally active recognition protein is less
active than a wild-type recognition protein in binding with a
target protein of a target cell at a normal physiological pH or
temperature, and more active than a wild-type recognition protein
in binding with the target protein of a target cell at an abnormal
pH or temperature. In this manner, the recognition protein will
preferentially bind at a site where an abnormal pH or temperature
is encountered thereby delivering a treatment to the site of a
disease.
[0226] In one embodiment, the viral particle may comprise a
conditionally active antibody of the present invention, and
especially the variable region of an antibody (e.g., Fab, Fab',
Fv). Such a conditionally active antibody can bind to the target
protein (as antigen) of a target cell with lower affinity than a
wild-type antibody under a normal physiological condition which may
be encountered at a location with normal tissue, and a higher
affinity than the wild-type antibody under aberrant condition which
may be encountered at a disease site or diseased tissue. The
conditionally active antibody may be derived from the wild-type
antibody according to the method of the present invention.
[0227] In an embodiment, the target protein on the target cell
includes tyrosine kinase growth factor receptors which are
overexpressed on the cell surfaces in, for example, many tumors.
Exemplary tyrosine kinase growth factors are VEGF receptors, FGF
receptors, PDGF receptors, IGF receptors, EGF receptors, TGF-alpha
receptors, TGF-beta receptors, HB-EGF receptors, ErbB2 receptors,
ErbB3 receptors, and ErbB4 receptors. Conditionally active DNA/RNA
modifying proteins
[0228] DNA/RNA modifying proteins have been discovered as a form of
new genome-engineering tools, particularly one called CRISPR, which
can allow researchers to perform microsurgery on genes, precisely
and easily changing a DNA sequence at exact locations on a
chromosome (genome editing, Mali et al., "Cas9 as a versatile tool
for engineering biology," Nature Methods, vol. 10, pages 957-963,
2013). For example, sickle-cell anemia is caused by a single base
mutation, which can potentially be corrected using DNA/RNA
modifying proteins. The technology may precisely delete or edit
bits of a chromosome, even by changing a single base pair (Makarova
et al., "Evolution and classification of the CRISPR-Cas systems,"
Nature Reviews Microbiology, vol. 9, pages 467-477, 2011).
[0229] Genome editing with CRISPR has the ability to quickly and
simultaneously make multiple genetic changes to a cell. Many human
illnesses, including heart disease, diabetes, and neurological
diseases, are affected by mutations in multiple genes. This
CRISPR-based technology has the potential to reverse the disease
causing mutations and cure these diseases or at least reduce the
severity of these diseases. Genome editing relies on CRISPR
associated (Cas) proteins (a family of enzymes) for cutting the
genomic DNA. Typically, the Cas protein is guided by a small guide
RNA to a targeted region in the genome, where the guide RNA matches
the target region. Because the Cas protein has little or no
sequence specificity, the guide RNA serves as a pointer for the Cas
protein to achieve precise genome editing. In one embodiment, one
Cas protein may be used with multiple guide RNAs to simultaneously
correct multiple gene mutations.
[0230] There are many Cas proteins. Examples include Cas1, Cas2,
Cas3', Cas3'', Cas4, Cas5, Cas6, Cas6e, Cas6f, Cas7, Cas8a1,
Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Csy1, Csy2, Csy3, Cse1,
Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,
Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10,
Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4
((Makarova et al., "Evolution and classification of the CRISPR-Cas
systems," Nature Reviews Microbiology, vol. 9, pages 467-477,
2011).
[0231] To conduct genome editing, the Cas protein has to enter the
target cell. Cells in a subject may have a different intracellular
pH inside of the cells. Some cells in diseased tissue have an
abnormal intracellular pH. For example, some tumor cells tend to
have an alkaline intracellular pH of about 7.12-7.65, while cells
in normal tissue have a neutral intracellular pH ranging from
6.99-7.20. See Cardone et al., "The role of disturbed pH dynamics
and the Na(+)/H(+) exchanger in metatasis," Nat. Rev. Cancer, vol.
5, pages 786-795, 2005. In chronic hypoxia, the cells in diseased
tissue have an intracellular pH of about 7.2-7.5, also higher than
the intracellular pH of normal tissue (Rios et al., "Chronic
hypoxia elevates intracellular pH and activates Na+/H+ exchange in
pulmonary arterial smooth muscle cells," American Journal of
Physiology--Lung Cellular and Molecular Physiology, vol. 289, pages
L867-L874, 2005). Further, in ischemia cells, the intracellular pH
is typical! in a range of 6.55-6.65, which is lower than the
intracellular pH of normal tissue (Haqberg, "Intracellular pH
during ischemic in skeletal muscle: relationship to membrane
potential, extracellular pH, tissue lactic acid and ATP," Pflugers
Arch., vol. 404, pages 342-347, 1985). More examples of abnormal
intracellular pH in diseased tissue are discussed in Han et al.,
"Fluorescent Indicators for Intracellular pH," Chem Rev., vol. 110,
pages 2709-2728, 2010.
[0232] The present invention provides a method for producing a
conditionally active Cas protein from a wild-type Cas protein,
where the conditionally active Cas protein has a decreased
enzymatic activity relative to the activity of the wild-type Cas
protein under a normal physiological condition inside a normal
cell, and an increased enzymatic activity relative to the activity
of the wild-type Cas protein under an aberrant condition inside a
target cell such as one of the diseased cells discussed above. In
some embodiments, the normal physiological condition is an
intracellular pH about neutral, and the aberrant condition is a
different intracellular pH that is above or below neutral. In an
embodiment, the aberrant condition is an intracellular pH of from
7.2 to 7.65 or an intracellular pH of from 6.5-6.8.
[0233] In some embodiments, the conditionally active Cas protein
may be delivered to a target cell using the conditionally active
viral particle of the present invention. The conditionally active
viral particle comprises the conditionally active Cas protein and
at least one guide RNA for directing the Cas protein to the
location at which Cas protein will edit the genomic DNA.
Method of Generating Conditionally Active Biological Proteins
[0234] One or more mutagenesis techniques are employed to evolve
the DNA which encodes the wild-type protein to create a library of
mutant DNA; the mutant DNA is expressed to create a library of
mutant proteins; and the library is subjected to a screening assay
under a normal physiological condition and under one or more
aberrant conditions. Conditionally active biologic proteins are
selected from those proteins which exhibit both (a) a decrease in
activity in the assay at the normal physiological condition
compared to the wild-type protein, and (b) an increase in activity
in the assay under the aberrant condition compared to the wild-type
protein. Alternatively, conditionally active biologic proteins are
selected from those proteins which exhibit changes in activity,
reversibly or irreversibly, in two or more different physiological
conditions. In some embodiments, the wild-type protein is an
antibody.
Generation of Evolved Molecules from a Parent Molecule
[0235] Conditionally active proteins can be generated through a
process of mutagenesis and screening for individual mutations for a
reduction in activity at the wild-type condition with activity at
non wild-type conditions remaining the same or better than the
activity at the wild-type condition.
[0236] The disclosure provides for a method for generating a
nucleic acid variant encoding a polypeptide having enzyme activity,
wherein the variant has an altered biological activity from that
which naturally occurs, the method comprising (a) modifying the
nucleic acid by (i) substituting one or more nucleotides for a
different nucleotide, wherein the nucleotide comprises a natural or
non-natural nucleotide; (ii) deleting one or more nucleotides,
(iii) adding one or more nucleotides, or (iv) any combination
thereof. In one aspect, the non-natural nucleotide comprises
inosine. In another aspect, the method further comprises assaying
the polypeptides encoded by the modified nucleic acids for altered
enzyme activity, thereby identifying the modified nucleic acid(s)
encoding a polypeptide having altered enzyme activity. In one
aspect, the modifications of step (a) are made by PCR, error-prone
PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,
sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,
recursive ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, gene site saturated
mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase
chain reaction, oligonucleotide synthesis, any DNA-generating
technique and any combination thereof. In another aspect, the
method further comprises at least one repetition of the
modification step (a).
[0237] The disclosure further provides a method for making a
polynucleotide from two or more nucleic acids, the method
comprising: (a) identifying regions of identity and regions of
diversity between two or more nucleic acids, wherein at least one
of the nucleic acids comprises a nucleic acid of the disclosure;
(b) providing a set of oligonucleotides which correspond in
sequence to at least two of the two or more nucleic acids; and, (c)
extending the oligonucleotides with a polymerase, thereby making
the polynucleotide.
[0238] Any technique of mutagenesis can be employed in various
embodiments of the disclosure. Stochastic or random mutagenesis is
exemplified by a situation in which a parent molecule is mutated
(modified or changed) to yield a set of progeny molecules having
mutation(s) that are not predetermined. Thus, in an in vitro
stochastic mutagenesis reaction, for example, there is not a
particular predetermined product whose production is intended;
rather there is an uncertainty--hence randomness--regarding the
exact nature of the mutations achieved, and thus also regarding the
products generated. Stochastic mutagenesis is manifested in
processes such as error-prone PCR and stochastic shuffling, where
the mutation(s) achieved are random or not predetermined. The
variant forms can be generated by error-prone transcription, such
as an error-prone PCR or use of a polymerase which lacks
proof-reading activity (see, Liao (1990) Gene 88: 107-111), of the
first variant form, or, by replication of the first form in a
mutator strain (mutator host cells are discussed in further detail
below, and are generally well known). A mutator strain can include
any mutants in any organism impaired in the functions of mismatch
repair. These include mutant gene products of mutS, mutT, mutH,
mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment
is achieved by genetic mutation, allelic replacement, selective
inhibition by an added reagent such as a small compound or an
expressed antisense RNA, or other techniques. Impairment can be of
the genes noted, or of homologous genes in any organism.
[0239] Current mutagenesis methods in widespread use for creating
alternative proteins from a starting molecule are
oligonucleotide-directed mutagenesis technologies, error-prone
polymerase chain reactions (error-prone PCR) and cassette
mutagenesis, in which the specific region to be optimized is
replaced with a synthetically mutagenized oligonucleotide. In these
cases, a number of mutant sites are generated around certain sites
in the original sequence.
[0240] In oligonucleotide-directed mutagenesis, a short sequence is
replaced with a synthetically mutagenized oligonucleotide. In
oligonucleotide-directed mutagenesis, a short sequence of the
polynucleotide is removed from the polynucleotide using restriction
enzyme digestion and is replaced with a synthetic polynucleotide in
which various bases have been altered from the original sequence.
The polynucleotide sequence can also be altered by chemical
mutagenesis. Chemical mutagens include, for example, sodium
bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
Other agents which are analogues of nucleotide precursors include
nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.
Generally, these agents are added to the PCR reaction in place of
the nucleotide precursor thereby mutating the sequence.
Intercalating agents such as proflavine, acriflavine, quinacrine
and the like can also be used. Random mutagenesis of the
polynucleotide sequence can also be achieved by irradiation with
X-rays or ultraviolet light. Generally, plasmid polynucleotides so
mutagenized are introduced into E. coli and propagated as a pool or
library of hybrid plasmids.
[0241] Error-prone PCR uses low-fidelity polymerization conditions
to introduce a low level of point mutations randomly over a long
sequence. In a mixture of fragments of unknown sequence,
error-prone PCR can be used to mutagenize the mixture.
[0242] In cassette mutagenesis, a sequence block of a single
template is typically replaced by a (partially) randomized
sequence. Reidhaar-Olson J F and Sauer R T: Combinatorial cassette
mutagenesis as a probe of the informational content of protein
sequences. Science 241(4861):53-57, 1988.
[0243] Alternatively, any technique of non-stochastic or non-random
mutagenesis can be employed in various embodiments of the
disclosure. Non-stochastic mutagenesis is exemplified by a
situation in which a parent molecule is mutated (modified or
changed) to yield a progeny molecule having one or more
predetermined mutations. It is appreciated that the presence of
background products in some quantity is a reality in many reactions
where molecular processing occurs, and the presence of these
background products does not detract from the non-stochastic nature
of a mutagenesis process having a predetermined product.
Site-saturation mutagenesis and synthetic ligation reassembly are
examples of mutagenesis techniques where the exact chemical
structure(s) of the intended product(s) are predetermined.
[0244] One method of site-saturation mutagenesis is disclosed in
U.S. patent application publication 2009/0130718, which is
incorporated herein by reference. This method provides a set of
degenerate primers corresponding to codons of a template
polynucleotide, and performs polymerase elongation to produce
progeny polynucleotides, which contain sequences corresponding to
the degenerate primers. The progeny polynucleotides can be
expressed and screened for directed evolution. Specifically, this
is a method for producing a set of progeny polynucleotides,
comprising the steps of (a) providing copies of a template
polynucleotide, each comprising a plurality of codons that encode a
template polypeptide sequence; and (b) for each codon of the
template polynucleotide, performing the steps of (1) providing a
set of degenerate primers, where each primer comprises a degenerate
codon corresponding to the codon of the template polynucleotide and
at least one adjacent sequence that is homologous to a sequence
adjacent to the codon of the template polynucleotide; (2) providing
conditions allowing the primers to anneal to the copies of the
template polynucleotides; and (3) performing a polymerase
elongation reaction from the primers along the template; thereby
producing progeny polynucleotides, each of which contains a
sequence corresponding to the degenerate codon of the annealed
primer; thereby producing a set of progeny polynucleotides.
[0245] Site-saturation mutagenesis relates to the directed
evolution of nucleic acids and screening of clones containing the
evolved nucleic acids for resultant activity(ies) of interest, such
nucleic acid activity(ies) &/or specified protein, particularly
enzyme, activity(ies) of interest.
[0246] Mutagenized molecules provided by this technique may have
chimeric molecules and molecules with point mutations, including
biological molecules that contain a carbohydrate, a lipid, a
nucleic acid, &/or a protein component, and specific but
non-limiting examples of these include antibiotics, antibodies,
enzymes, and steroidal and non-steroidal hormones.
[0247] Site saturation mutagenesis relates generally to a method
of: 1) preparing a progeny generation of molecule(s) (including a
molecule that is comprised of a polynucleotide sequence, a molecule
that is comprised of a polypeptide sequence, and a molecule that is
comprised in part of a polynucleotide sequence and in part of a
polypeptide sequence), that is mutagenized to achieve at least one
point mutation, addition, deletion, &/or chimerization, from
one or more ancestral or parental generation template(s); 2)
screening the progeny generation molecule(s)--preferably using a
high throughput method--for at least one property of interest (such
as an improvement in an enzyme activity or an increase in stability
or a novel chemotherapeutic effect); 3) optionally obtaining
&/or cataloguing structural &/or and functional information
regarding the parental &/or progeny generation molecules; and
4) optionally repeating any of steps 1) to 3).
[0248] In site saturation mutagenesis, there is generated (e.g.
from a parent polynucleotide template)--in what is termed "codon
site-saturation mutagenesis"--a progeny generation of
polynucleotides, each having at least one set of up to three
contiguous point mutations (i.e. different bases comprising a new
codon), such that every codon (or every family of degenerate codons
encoding the same amino acid) is represented at each codon
position. Corresponding to--and encoded by--this progeny generation
of polynucleotides, there is also generated a set of progeny
polypeptides, each having at least one single amino acid point
mutation. In a preferred aspect, there is generated--in what is
termed "amino acid site-saturation mutagenesis"--one such mutant
polypeptide for each of the 19 naturally encoded
polypeptide-forming alpha-amino acid substitutions at each and
every amino acid position along the polypeptide. This yields--for
each and every amino acid position along the parental
polypeptide--a total of 20 distinct progeny polypeptides including
the original amino acid, or potentially more than 21 distinct
progeny polypeptides if additional amino acids are used either
instead of or in addition to the 20 naturally encoded amino
acids.
[0249] Other mutagenesis techniques can also be employed which
involve recombination and more specifically a method for preparing
polynucleotides encoding a polypeptide by a method of in vivo
re-assortment of polynucleotide sequences containing regions of
partial homology, assembling the polynucleotides to form at least
one polynucleotide and screening the polynucleotides for the
production of polypeptide(s) having a useful property.
[0250] In another aspect, mutagenesis techniques exploit the
natural property of cells to recombine molecules and/or to mediate
reductive processes that reduce the complexity of sequences and
extent of repeated or consecutive sequences possessing regions of
homology.
[0251] Various mutagenesis techniques can be used alone or in
combination to provide a method for generating hybrid
polynucleotides encoding biologically active hybrid polypeptides
with enhanced activities. In accomplishing these and other objects,
there has been provided, in accordance with one aspect of the
disclosure, a method for introducing polynucleotides into a
suitable host cell and growing the host cell under conditions that
produce a hybrid polynucleotide.
[0252] Chimeric genes have been made by joining 2 polynucleotide
fragments using compatible sticky ends generated by restriction
enzyme(s), where each fragment is derived from a separate
progenitor (or parental) molecule. Another example is the
mutagenesis of a single codon position (i.e. to achieve a codon
substitution, addition, or deletion) in a parental polynucleotide
to generate a single progeny polynucleotide encoding for a single
site-mutagenized polypeptide.
[0253] Further, in vivo site specific recombination systems have
been utilized to generate hybrids of genes, as well as random
methods of in vivo recombination, and recombination between
homologous but truncated genes on a plasmid. Mutagenesis has also
been reported by overlapping extension and PCR.
[0254] Non-random methods have been used to achieve larger numbers
of point mutations and/or chimerizations, for example comprehensive
or exhaustive approaches have been used to generate all the
molecular species within a particular grouping of mutations, for
attributing functionality to specific structural groups in a
template molecule (e.g. a specific single amino acid position or a
sequence comprised of two or more amino acids positions), and for
categorizing and comparing specific grouping of mutations.
[0255] Any of these or other methods of evolving can be employed in
the present disclosure to generate a new population of molecules
(library) from one or more parent molecules.
[0256] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector, and transfected into an appropriate
host cell.
[0257] In some embodiments, the evolving step is directed at the Fc
region of a wild-type antibody. In these embodiments, the Fc region
of the wild-type antibody is modified in the resultant
conditionally active antibody. The Fc regions that may be modified
include the Fc region of an antibody (e.g., in a full-length IgG
antibody including full-length IgG1, IgG2, IgG3 or IgG4 antibodies,
a chimeric antibody, or a humanized antibody), or in a fusion
protein that contains a Fc region, or a part of a Fc region
(referred to as an "immunoglobulin (Ig) fusion protein", "Fc fusion
protein", or "Fc fusion polypeptide").
[0258] Modified Fc regions of antibodies have been described in the
art, including in US2006/0104989. The modified Fc regions can have
a single amino acid substitution (also referred to as a Fc variant
herein) relative to the sequence of a corresponding unmodified
(wild-type or parent) Fc region, and may have one or more
properties that differ from a corresponding wild-type or parent
having an unmodified Fc region as well as from other antibodies
having modified Fc regions that have been described in the art.
Such properties may include, for example, increased binding to one
or more Fc receptors and/or modified binding under different pH
conditions.
[0259] The modified Fc regions can be incorporated into any
antibody or Fc fusion polypeptide using standard molecular biology
techniques, and all such modified antibodies and Fc fusion
polypeptides are intended to be encompassed by the invention. Fc
refers to the last two constant region Ig domains of IgA, IgD, and
IgG, and the last three constant region Ig domains of IgE and IgM,
and the flexible hinge N-terminal to these domains. For IgA and
IgM, Fc may include the J chain. Fc is bound by receptors, FcRs,
which are present on certain cells. As the affinity of the
interaction between Fc and certain FcRs present on particular cells
correlates with targeted cytotoxicity, and clinical efficacy in
humans correlates with the allotype of high or low affinity
polymorphic forms of certain FcRs, an antibody or fusion
polypeptide with a Fc region optimized for binding to one or more
FcRs may result in more effective destruction of cancer cells.
[0260] In certain embodiments, modified Fc regions impart improved
properties to a polypeptide or a complex which includes a
polypeptide into which the Fc region is incorporated, e.g., a
complex such as a full-length antibody, chimeric antibody or
humanized antibody which includes an Ig heavy chain having an
modified Fc region, such as increased or modified binding to one or
more FcRs, and/or increased or modified antibody dependent cellular
cytotoxicity (ADCC), as compared to a corresponding polypeptide or
complex, such as an antibody, incorporating a corresponding
unmodified (a wild-type or parent) Fc region, or a different
modified Fc region. In some embodiments of the invention, modified
Fc regions impart increased or decreased half life to a
molecule.
[0261] In one embodiment of the invention, a modified Fc region of
the invention contains one substitution. In other embodiments, a
modified Fc region of the invention contains two, three, four, five
or more substitutions in combination, which may additively or
synergistically enhance the properties of the modified Fc regions.
In another embodiment, the invention includes a polypeptide having
a modified Fc region, i.e., it is an Fc fusion polypeptide that
contains one of the substitutions. In one embodiment, the non-Fc
region of the fusion polypeptide includes a target binding
molecule. In other embodiments, the invention includes a
polypeptide having a modified Fc region of the invention that
contains two, three, four, five, six, ten, twelve, or more
substitutions in combination.
[0262] In addition to the polypeptide, protein or other complex,
e.g., a conjugate, incorporating an modified Fc region, the
invention also encompasses polynucleotides and expression vectors
encoding a modified Fc region or polypeptides having a modified Fc
region, including libraries of those polynucleotides and expression
vectors, host cells into which such polynucleotides or expression
vectors have been introduced, for instance, so that the host cell
produces a polypeptide having the modified Fc region, libraries of
host cells, and methods of making, culturing or manipulating the
host cells or libraries of host cells. For instance, the invention
includes culturing such host cells so that a polypeptide with a
modified Fc region is produced, e.g., secreted or otherwise
released from the host cell. Pharmaceutical compositions and kits
which include a polypeptide, protein or other complex with an
modified Fc region, and/or polynucleotides, expression vectors or
host cells encoding polypeptides having such a modified Fc region,
are also encompassed. Moreover, use of a polypeptide, protein or
conjugate with an modified Fc region, such as in Fc receptor
binding assays or to induce ADCC activity in vitro or in vivo, is
also encompassed by the invention. The invention also provides a
polypeptide, protein, conjugate, polynucleotide, expression vector,
and/or host cell of the invention for use in medical therapy, as
well as the use of a polypeptide, protein or other complex,
polynucleotide, expression vector, and/or host cell of the
invention for the manufacture of a medicament, e.g., useful to
induce ADCC activity in vitro or in vivo.
[0263] A "parent Fc", as used herein, can be a naturally occurring
Fc region of an IgA, IgD, IgE, IgG or IgM class of antibody.
Alternatively, the source of a parent Fc is a Fc region from a
naturally occurring antibody, including IgG1, IgG1, IgG3, IgG4,
IgA1, or IgA2. A parent Fc region to be modified may be selected
for its FcR binding affinity and/or FcR binding pattern, and an
modified Fc region has at least an enhanced affinity for at least
one FcR, but may otherwise have the same pattern of FcR binding, as
the parent Fc region.
[0264] A parent Fc region is preferably one that interacts with one
or more FcRs, including but not limited to Fc.gamma.Rs,
Fc.alpha.Rs, Fc.mu.Rs, Fc.delta.Rs, FcRn, and viral Fc.gamma.R. A
modified Fc region derived from such a parent Fc region is one that
has an enhanced interaction with one or more FcRs and enhanced
ADCC, relative to the parent Fc region. ADCC generally requires the
Fc region to be combined with a binding domain (e.g., an antibody
variable domain). Methods to detect FcR binding and ADCC are known
to the art.
[0265] FcRs are defined by their specificity for immunoglobulin
isotypes and are well known in the art.
[0266] An Fc containing fusion includes a polypeptide where a Fc
region with favorable FcR binding, and optionally favorable
pharmacokinetics, is linked to one or more molecules. The linkage
may be synthetic in nature, e.g., via chemical conjugation, or via
recombinant expression, i.e., a fusion polypeptide is formed. Thus,
the molecule linked to a Fc region may be a molecule useful to
isolate or purify the Fc region, e.g., a tag such as a Flag-tag,
Strep-tag, glutathione S transferase, maltose binding protein (MBP)
or a His-tag, or other heterologous polypeptide, e.g., a ligand for
a receptor, an extracellular domain of a receptor, or a variable
region of a heavy Ig chain, and/or another molecule.
[0267] A vector encoding a modified Fc region or a Fc region
containing polypeptide such as an Ig heavy chain with a modified Fc
region or other Fc fusion polypeptide may be introduced into a host
cell, optionally along with other vectors, e.g., a vector encoding
an Ig light chain, or into a host cell modified to express another
polypeptide such as an Ig light chain, or into an in vitro
transcription/transcription reaction, so as to express the encoded
polypeptide. The modified Fc region, Ig heavy chain and Ig light
chain may also be expressed in the same vector and introduced into
a host cell. For some expression systems, host cells may be
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying desired
sequences. A resulting polypeptide with a modified Fc region is
optionally isolated, e.g., from host cell supernatants, and
screened for one or more activities.
[0268] In one embodiment, the Fc region may be one that is anchored
to the surface of a cell, such as a host cell, e.g., via fusion
with a transmembrane domain.
[0269] Suitable host cells for expressing the polynucleotide in the
vectors are the prokaryotic, yeast, or higher eukaryotic cells.
Suitable prokaryotes for this purpose include eubacteria, such as
Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Kiebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis, Pseudomonas such
as P. aeruginosa, and Streptomyces. Eukaryotic microbes such as
filamentous fungi or yeast are also suitable cloning or expression
hosts for polypeptide variant-encoding vectors. Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces hosts such as,
e.g., K. lactis, K. fragilis, K. bulgaricus, K. wickeramii, K.
waltii, K. drosophilarum, K. thermotolerans, and K. marxianus;
Pichia pastoris, Candida, Trichoderma reesia, Schwanniomyces such
as Schwanniomyces occidentalis; and filamentous fungi such as
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts may
be employed.
[0270] Suitable host cells for the expression of glycosylated
polypeptides are derived from multicellular organisms. Examples of
invertebrate cells for expression of glycosylated polypeptide
include plant and insect cells. Examples of eukaryotic cell
generation, screening and production hosts include 3T3 mouse
fibroblast cells, BHK21 Syrian hamster fibroblast cells, MDCK, dog
epithelial cells, Hela human epithelial cells, PtK1 rat kangaroo
epithelial cells, SP2/0 mouse plasma cells, and NS0 mouse mouse
plasma cells, HEK 293 human embryonic kidney cells, COS monkey
kidney cells, CHO, CHO-S Chinese hamster ovary cells, R1 mouse
embryonic cells, E14.1 mouse embryonic cells, H1 human embryonic
cells, H9 human embryonic cells, PER C.6, and human embryonic
cells. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda, Aedes aegypti, Aedes albopictus, Drosophila
melanogaster, and Bombyx mori may be used. For instance, viral
vectors maybe used to introduce a polynucleotide, particularly for
transfection of Spodoptera frugiperda cells. Plant cell cultures of
cotton, corn, potato, soybean, petunia, tomato, and tobacco can
also be utilized as hosts. Examples of useful vertebrate cells
include mammalian cells, e.g., human, simian, canine, feline,
bovine, equine, caprine, ovine, swine, or rodent, e.g., rabbit,
rat, mink or mouse cells, such as CHO cells. Transgenic plants and
animals may be employed as expression systems, although
glycosylation patterns in those cells may be different from human
glycoproteins. In one embodiment, transgenic rodents are employed
as expression systems. Bacterial expression may also be employed.
Although bacterially expressed proteins lack glycosylation, other
alterations may compensate for any reduced activity such as poor
stability and solubility, which may result from prokaryotic
expression.
[0271] Optionally, an Fc region or Fc containing polypeptide is
isolated from host cells, e.g., from host cell supernatants, or an
in vitro transcription/translation mixture, yielding a composition.
An isolated polypeptide in the composition is one which has been
isolated from at least one other molecule found in host cells, host
cell supernatants or the transcription/translation mixture, e.g.,
by fractionation on immunoaffinity or ion-exchange columns; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on
an anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; or ligand affinity chromatography. For some
applications, the isolated polypeptide in the composition is the
predominant species present (i.e., on a molar basis it is more
abundant than any other individual species in the composition), and
preferably comprises at least about 50 percent (on a molar basis),
more preferably more than about 85%, about 90%, about 95%, and
about 99, of all macromolecular species present. The isolated Fc
region or Fc containing polypeptide may be subjected to further in
vitro alterations, e.g., treated with enzymes or chemicals such as
proteases, molecules such as those which alter glycosylation or
ones that are useful to conjugate (couple) the isolated Fc region
or Fc region containing polypeptide to another molecule such as a
label including but not limited to fluorescent labels (e.g., FITC,
rhodamine, lanthanide, phosphors), enzymatic labels (e.g.,
horseradish peroxidase, /3-galactosidase, luciferase, alkaline
phosphatase), chemiluminescent labels, biotinyl groups, avidin
groups, or polypeptide epitopes recognized by a secondary reporter
(e.g., leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags), sugars, lipids,
fats, paramagnetic molecules or sound wave emitters, metals, or
synthetic polymers.
[0272] Methods to screen for activities associated with
polypeptides or complexes that incorporate a Fc region, including
but not limited to FcR binding (see, for example, U.S. Pat. Nos.
6,737,056, 7,217,797, and 8,088,376, all incorporated herein by
reference), are well known to the art. For instance, to assess ADCC
activity of a Fc containing polypeptide, an in vitro and/or in vivo
ADCC assay, may be performed using varying effector:target ratios,
e.g., PBMC and NK cells or in a animal model, respectively. In one
embodiment, Fc containing polypeptides expressed by host cells are
screened for enhanced FcR receptor binding affinity or activity in
vitro and/or in vivo and/or ADCC activity in vitro and/or in vivo.
In one embodiment, the binding of a FcR by a Fc containing
polypeptide with an modified Fc region is greater than the binding
of that receptor by a corresponding polypeptide with an unmodified
Fc region.
[0273] Thus, by introducing amino acid sequence modifications
described herein in a wild-type or parent Fc region or a Fc region
containing polypeptide, which wild-type or parent Fc region
preferably elicits ADCC and optionally is a human Fc region, e.g.,
a native sequence human Fc region human IgG sequence, a modified Fc
region is obtained which binds FcR with better affinity and
mediates ADCC in the presence of human effector cells more
effectively than the wild-type or parent Fc region or Fc region
containing polypeptide. Soluble FcRs such as recombinant soluble
human CD16 and recombinant soluble human CD32 can be contacted with
one or more different modified Fc regions in parallel, and modified
Fc regions having one or more substitutions that enhance binding to
human CD16 but not to human CD32, relative to an unmodified Fc
region, are identified. Those substitutions may be combined with
other substitutions that enhance binding. A combination of
substitutions in an Fc region or Fc region containing polypeptide
may yield a combinatorially modified Fc region, or a
combinatorially modified Fc region containing polypeptide with
synergistically enhanced properties.
[0274] Other methods to identify polypeptides with modified Fc
regions, including antibodies with an modified Fc region, with
desirable properties, and thus a corresponding polynucleotide
sequence, may be employed alone or in combination with methods
described above, include using modeling, e.g., 3D-modeling, of
modified Fc regions, preferably in the context of the molecule to
be screened for activity, e.g., an antibody with the Fc region, to
select for Fc regions with particular characteristics.
Characteristics that may be screened for by modeling include, but
are not limited to, a particular angle near FcR binding sites,
hinge architecture, and intra- and inter-molecular chain
interactions, e.g., substitutions that promote or disrupt
hydrophobic interactions or stabilize conformation in a particular
region. Thus, a 3D model of an Fc region containing polypeptide
having at least one or more substituted positions may be employed
to identify combinations of substitutions to be introduced into a
polynucleotide for expression in host cells.
[0275] The Fc variants, whether or not incorporated into a
heterologous polypeptide, e.g., incorporated into a Fc fusion with
a ligand for a cell surface receptor, e.g., CTLR-4 ligand or heavy
chain of an antibody, or conjugated to a molecule of interest, as
well as polynucleotides and host cells encoding those variants,
optionally in combination with one or more other agents, e.g.,
therapeutic or research reagents, are useful in a variety of
methods, e.g., in screening methods, prophylactic methods,
therapeutic methods, veterinary methods and agricultural methods.
The one or more other agents include other Fc region or Fc region
containing polypeptides, including those with unmodified Fc
regions. In one embodiment, an Fc variant is incorporated into an
antibody or other Fc fusion polypeptide and that antibody or Fc
fusion polypeptide, optionally in conjunction with one or more
other useful compositions, is employed to target particular
cells.
[0276] In one embodiment, an Fc variant containing antibody or an
antigen-binding fragment thereof targets and optionally kills
target cells that bear the target antigen. In another embodiment, a
Fc variant containing antibody or an antigen-binding fragment
thereof targets and activates cells that bear the target antigen,
e.g., thereby increasing expression of another antigen, such as a
viral or cellular antigen. In one embodiment, the Fc variants or
polypeptides incorporating an Fc variant may be used to prevent,
inhibit or treat various conditions or diseases, in humans and
non-humans, including non-human mammals. For example, an antibody
containing a modified Fc region may be administered to a human or
non-human animal which is at risk of, e.g., prone to having a
disease, prior to the onset of the disease and so prevent or
inhibit one or more symptoms of that disease. A Fc region or Fc
region containing polypeptide, or a conjugate thereof, may be
administered after clinical manifestation of a disease in a human
or non-human animal to inhibit or treat the disease. In one
embodiment, a pharmaceutical composition comprising an antibody or
Fc fusion polypeptide is administered to a human or non-human
animal with an autoimmune, immunological, infectious, inflammatory,
neurological, or neoplastic disease, e.g., cancer.
[0277] Fc regions or a Fc region containing polypeptides may be
administered alone or in combination with one or more other
therapeutic agents, including but not limited to cytotoxic agents,
e.g., chemotherapeutic agents, cytokines, growth inhibitory agents,
anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,
cardioprotectants, or other therapeutic agents, in amounts that are
effective for the purpose intended. The skilled medical
practitioner can determine empirically the appropriate dose or
doses of therapeutic agents including Fc regions or Fc region
containing polypeptides may thus be administered concomitantly with
one or more other therapeutic regimens. For example, an antibody or
Fc fusion polypeptide may be administered to a patient along with
chemotherapy or other therapy, e.g., other agents such as an
anti-angiogenic agent, a cytokine, radioisotope therapy, or both
chemotherapy and other therapies. In one embodiment, the antibody
or Fc fusion may be administered in conjunction with one or more
other antibodies or Fc fusions, which may or may not comprise a Fc
variant. In one embodiment, a Fc containing polypeptide is
administered with a chemotherapeutic agent, i.e., a chemical
compound useful in the treatment of cancer. A chemotherapeutic or
other cytotoxic agent may be administered as a prodrug, i.e., it is
in a form of a pharmaceutically active substance that is less
cytotoxic to cells compared to the drug and is capable of being
converted into the drug.
[0278] Pharmaceutical compositions are also contemplated having an
Fc region, an Fc fusion polypeptide, antibodies having an Fc
region, or conjugates thereof, optionally formulated with one or
more other agents. Formulations of antibodies, Fc regions, or Fc
region-containing polypeptides, or conjugates are prepared for
storage by mixing the antibodies, Fc regions, or Fc
region-containing polypeptides, or conjugates, having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed., 1980), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
antioxidants; alkyl parabens; low molecular weight (less than about
10 residues) polypeptides; hydrophilic polymers; amino acids;
monosaccharides; and other carbohydrates; chelating agents;
fillers; binding agents; additives; coloring agents; salt-forming
counter-ions; metal complexes; and/or non-ionic surfactants. Other
formulations include lipid or surfactant based formulations, and
microparticle or nanoparticle based formulations, including
sustained release dosage formulations, which are prepared by
methods known in the art.
[0279] The concentration of the Fc region, antibody or other Fc
region containing polypeptide in the formulation may vary from
about 0.1 to 100 weight %. In a preferred embodiment, the
concentration of the Fc region, antibody or Fc fusion polypeptide
is in the range of 0.001 to 2.0 M. In order to treat a patient, an
effective dose of the Fc region, or antibody or other Fc
region-containing polypeptide, and conjugates thereof, may be
administered.
[0280] By "therapeutically effective dose" herein is meant a dose
that produces the effects for which it is administered. Dosages may
range from 0.01 to 100 mg/kg of body weight or greater, for example
0.1, 1, 10, or 50 mg/kg of body weight, with 1 to 30 mg/kg being
preferred, although other dosages may provide beneficial results.
The amount administered is selected to prevent treat a particular
condition or disease. Administration of the Fc region, or antibody
or other Fc region-containing polypeptide, and conjugates thereof,
may be continuous or intermittent, depending, for example, upon the
recipient's physiological condition, whether the purpose of the
administration is therapeutic or prophylactic, and other factors
known to skilled practitioners. The administration of the Fc
region, or antibody or other Fc region-containing polypeptide, and
conjugates thereof, may be essentially continuous over a
preselected period of time or may be in a series of spaced doses.
Both local and systemic administration is contemplated.
[0281] Administration of the pharmaceutical composition comprising
a Fc region, an antibody or other Fc containing polypeptide and
conjugates, preferably in the form of a sterile aqueous solution,
may be done in a variety of ways, including, but not limited to,
orally, subcutaneously, intravenously, intranasally, intraotically,
transdermally, topically, intraperitoneally, intramuscularly,
intrapulmonary, inhalable technology, vaginally, parenterally,
rectally, and intraocularly. In some instances, for example for the
treatment of wounds, inflammation, etc., the antibody or Fc fusion
may be directly applied as a solution or spray.
[0282] Some references describing techniques that may be used in
the evolving step of the present invention include Molecular
Cloning: A Laboratory Manual (Sambrook et al, 3rd Ed., Cold Spring
Harbor Laboratory Press, (2001); Harlow and Lane, Antibodies: A
Laboratory Manual Cold Spring Harbor Laboratory Press, New York,
1988; Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Service, National
Institutes of Health, Bethesda (1991); Carter et al., Nucleic Acids
Res., 13:4431 (1985) Kunkel et al., Proc. Natl. Acad. Sci. USA,
82:488 (1987); Higuchi, in PCR Protocols, .rho..rho.. 177-183
(Academic Press, 1990); Vallette et al., Nuc. Acids Res., 17:723
(1989) Wells et al., Gene, 34:315 (1985); Gazzano-Santoro et al.,
J. Immunol. Methods, 202:163 (1996); Green et al., Nature Genet.,
7:13 (1994); Lonberg et al., Nature, 368:856 (1994); Taylor et al.,
Int. Immun., 6:579 (1994); McCafferty et al., Nature, 348:552
(1990); Johnson and Chiswell, Current Opinion in Structural
Biology, 3 :5564 (1993); Dall'Acqua, et al., The Journal of
Immunology, 169: 5171-5180 (2002); Yeung, et al., The Journal of
Immunology, 182: 7663-7671 (2009); Zalevsky, et al., Nature
Biotechnology; doi: 10.1038/nbt.1601 (published online 17 Jan.
2010); and Dall'Acqua, et al., The Journal of Biological Chemistry,
Vol 281, Num 33, 23515-23524 (2006), the disclosures of which are
hereby incorporated by reference in their entirety.
Expression of Evolved Molecules
[0283] Once a library of mutant molecules is generated, DNA can be
expressed using routine molecular biology techniques. Thus, protein
expression can be directed using various known methods.
[0284] For example, briefly, a wild type gene can be evolved using
any variety of random or non-random methods such as those indicated
herein. Mutant DNA molecules are then digested and ligated into
vector DNA, such as plasmid DNA using standard molecular biology
techniques. Vector DNA containing individual mutants is transformed
into bacteria or other cells using standard protocols. This can be
done in an individual well of a multi-well tray, such as a 96-well
tray for high throughput expression and screening. The process is
repeated for each mutant molecule.
[0285] Polynucleotides selected and isolated as described are
introduced into a suitable host cell. A suitable host cell is any
cell which is capable of promoting recombination and/or reductive
reassortment. The selected polynucleotides are preferably already
in a vector which includes appropriate control sequences. The host
cell can be a higher eukaryotic cell, such as a mammalian cell, or
a lower eukaryotic cell, such as a yeast cell, or preferably, the
host cell can be a prokaryotic cell, such as a bacterial cell.
Introduction of the construct into the host cell can be effected by
calcium phosphate transfection, DEAE-Dextran mediated transfection,
or electroporation (e.g. Ecker and Davis, 1986, Inhibition of gene
expression in plant cells by expression of antisense RNA, Proc Natl
Acad Sci USA, 83:5372-5376).
[0286] As representative examples of expression vectors which may
be used, there may be mentioned viral particles, baculovirus,
phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus and yeast). Thus, for example, the DNA may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a,
.rho.KK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5
(Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any
other plasmid or other vector may be used so long as they are
replicable and viable in the host. Low copy number or high copy
number vectors may be employed with the present disclosure.
[0287] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic
promoters include CMV immediate early, HSV thymidine kinase, early
and late SV40, LTRs from retrovirus, and mouse metallothionein-1.
Selection of the appropriate vector and promoter is well within the
level of ordinary skill in the art. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression. Promoter regions can be
selected from any desired gene using chloramphenicol transferase
(CAT) vectors or other vectors with selectable markers. In
addition, the expression vectors preferably contain one or more
selectable marker genes to provide a phenotypic trait for selection
of transformed host cells such as dihydrofolate reductase or
neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0288] Therefore, in another aspect of the disclosure, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs
containing consecutive sequences (original encoding sequences),
their insertion into an appropriate vector, and their subsequent
introduction into an appropriate host cell. The reassortment of the
individual molecular identities occurs by combinatorial processes
between the consecutive sequences in the construct possessing
regions of homology, or between quasi-repeated units. The
reassortment process recombines and/or reduces the complexity and
extent of the repeated sequences, and results in the production of
novel molecular species. Various treatments may be applied to
enhance the rate of reassortment. These could include treatment
with ultra-violet light, or DNA damaging chemicals, and/or the use
of host cell lines displaying enhanced levels of "genetic
instability". Thus the reassortment process may involve homologous
recombination or the natural property of quasi-repeated sequences
to direct their own evolution.
[0289] In one aspect, the host organism or cell comprises a gram
negative bacterium, a gram positive bacterium or a eukaryotic
organism. In another aspect of the disclosure, the gram negative
bacterium comprises Escherichia coli, or Pseudomonas fluorescens.
In another aspect of the disclosure, the gram positive bacterium
comprise Streptomyces diversa, Lactobacillus gasseri, Lactococcus
lactis, Lactococcus cremoris, or Bacillus subtilis. In another
aspect of the disclosure, the eukaryotic organism comprises
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia
pastoris, Kluyveromyces lactis, Hansenula plymorpha, or Aspergillus
niger. As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; and plant cells. The selection
of an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0290] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described in "SV40-transformed simian
cells support the replication of early SV40 mutants" (Gluzman,
1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0291] The cells are then propagated and "reductive reassortment"
is effected. The rate of the reductive reassortment process may be
stimulated by the introduction of DNA damage if desired, in vivo
reassortment is focused on "inter-molecular" processes collectively
referred to as "recombination" which in bacteria, is generally
viewed as a "RecA-dependent" phenomenon. The disclosure can rely on
recombination processes of a host cell to recombine and re-assort
sequences, or the cells' ability to mediate reductive processes to
decrease the complexity of quasi-repeated sequences in the cell by
deletion. This process of "reductive reassortment" occurs by an
"intra-molecular", RecA-independent process. The end result is a
reassortment of the molecules into all possible combinations.
[0292] Host cells containing the polynucleotides of interest can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying genes.
The culture conditions, such as temperature, pH and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0293] Protein expression can be induced by a variety of known
methods, and many genetic systems have been published for induction
of protein expression. For example, with appropriate systems, the
addition of an inducing agent will induce protein expression. Cells
are then pelleted by centrifugation and the supernatant removed.
Periplasmic protein can be enriched by incubating the cells with
DNAse, RNAse, and lysozyme. After centrifugation, the supernatant,
containing the new protein, is transferred to a new multi-well tray
and stored prior to assay.
[0294] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0295] The clones which are identified as having the desired
activity may then be sequenced to identify the polynucleotide
sequence encoding an enzyme having the enhanced activity.
[0296] The polypeptides that are identified from such libraries can
be used for therapeutic, diagnostic, research and related purposes,
and/or can be subjected to one or more additional cycles of
shuffling and/or selection. The disclosure provides for a fragment
of the conditionally active biologic protein which is at least 10
amino acids in length, and wherein the fragment has activity.
[0297] The disclosure provides for a codon-optimized polypeptide or
a fragment thereof, having enzyme activity, wherein the codon usage
is optimized for a particular organism or cell. Narum et al.,
"Codon optimization of gene fragments encoding Plasmodium
falciparum merzoite proteins enhances DNA vaccine protein
expression and immunogenicity in mice". Infect. Immun. 2001
December, 69(12):7250-3 describes codon-optimization in the mouse
system. Outchkourov et al., "Optimization of the expression of
Equistatin in Pichia pastoris, protein expression and
purification", Protein Expr. Purif 2002 February; 24(1): 18-24
describes codon-optimization in the yeast system. Feng et al.,
"High level expression and mutagenesis of recombinant human
phosphatidylcholine transfer protein using a synthetic gene:
evidence for a C-terminal membrane binding domain" Biochemistry
2000 Dec. 19, 39(50): 15399-409 describes codon-optimization in E.
coli. Humphreys et al., "High-level periplasmic expression in
Escherichia coli using a eukaryotic signal peptide: importance of
codon usage at the 5' end of the coding sequence", Protein Expr.
Purif. 2000 Nov. 20(2):252-64 describes how codon usage affects
secretion in E. coli.
[0298] The evolution of a conditionally active biologic protein can
be aided by the availability of a convenient high throughput
screening or selection process.
[0299] Once identified, polypeptides and peptides of the disclosure
can be synthetic, or be recombinantly generated polypeptides.
Peptides and proteins can be recombinantly expressed in vitro or in
vivo. The peptides and polypeptides of the disclosure can be made
and isolated using any method known in the art. Polypeptide and
peptides of the disclosure can also be synthesized, whole or in
part, using chemical methods well known in the art. See e.g.,
Caruthers (1980) "New chemical methods for synthesizing
polynucleotides", Nucleic Acids Res. Symp. Ser. 215-223; Horn
(1980), "Synthesis of oligonucleotides on cellulose. Part II:
design and synthetic strategy to the synthesis of 22
oligodeoxynucleotides coding for Gastric Inhibitory Polypeptide
(GIP)", Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,
Therapeutic Peptides and Proteins, Formulation, Processing and
Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.
For example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) "A strategy for a
convergent synthesis of N-linked glycopeptides on a solid support",
Science 269:202; Merrifield (1997) "Concept and early development
of solid-phase peptide synthesis", Methods Enzymol. 289:3-13) and
automated synthesis may be achieved, e.g., using the ABI 43 IA
Peptide Synthesizer (Perkin Elmer) in accordance with the
instructions provided by the manufacturer.
[0300] The peptides and polypeptides of the disclosure can also be
glycosylated. The glycosylation can be added post-translationally
either chemically or by cellular biosynthetic mechanisms, wherein
the latter incorporates the use of known glycosylation motifs,
which can be native to the sequence or can be added as a peptide or
added in the nucleic acid coding sequence. The glycosylation can be
O-linked or N-linked.
[0301] The peptides and polypeptides of the disclosure, as defined
above, include all "mimetic" and "peptidomimetic" forms. The terms
"mimetic" and "peptidomimetic" refer to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of the polypeptides of the disclosure.
The mimetic can be either entirely composed of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of
partly natural peptide amino acids and partly non-natural analogs
of amino acids. The mimetic can also incorporate any amount of
natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's
structure and/or activity. As with polypeptides of the disclosure
which are conservative variants, routine experimentation will
determine whether a mimetic is within the scope of the disclosure,
i.e., that its structure and/or function is not substantially
altered.
[0302] Polypeptide mimetic compositions of the disclosure can
contain any combination of non-natural structural components. In
alternative aspect, mimetic compositions of the disclosure include
one or all of the following three structural groups: a) residue
linkage groups other than the natural amide bond ("peptide bond")
linkages; b) non-natural residues in place of naturally occurring
amino acid residues; or c) residues which induce secondary
structural mimicry, i.e., to induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like. For example, a polypeptide of the
disclosure can be characterized as a mimetic when all or some of
its residues are joined by chemical means other than natural
peptide bonds. Individual peptidomimetic residues can be joined by
peptide bonds, other chemical bonds or coupling means, such as,
e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g.,
.about.C(.dbd.O).about.CH.sub.2.about. for --C(.dbd.O).about.NH--),
aminomethylene (CH.sub.2-NH), ethylene, olefin (CH.dbd.CH), ether
(CH.sub.2.about.O), thioether (CH.sub.2.about.S), tetrazole
(CN.sub.4-), thiazole, retroamide, thioamide, or ester (see, e.g.,
Spatola (1983) in Chemistry and Biochemistry of Amino Acids,
Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, N. Y.).
[0303] A polypeptide of the disclosure can also be characterized as
a mimetic by containing all or some non-natural residues in place
of naturally occurring amino acid residues. Non-natural residues
are well described in the scientific and patent literature; a few
exemplary non-natural compositions useful as mimetics of natural
amino acid residues and guidelines are described below. Mimetics of
aromatic amino acids can be generated by replacing by, e.g., D- or
L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;
D- or L-1,-2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine;
D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine;
D- or L-2-indole(alkyl)alanines; and, D- or L-alkylanines, where
alkyl can be substituted or unsubstituted methyl, ethyl, propyl,
hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl,
or a non-acidic amino acids. Aromatic rings of a non-natural amino
acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic
rings.
[0304] Mimetics of acidic amino acids can be generated by
substitution by, e.g., non-carboxylate amino acids while
maintaining a negative charge; (phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can
also be selectively modified by reaction with carbodiimides
(R'.about.N--C--N--R') such as, e.g.,
1-cyclohexyl-3(2-mo.phi.holinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions. Mimetics of basic amino
acids can be generated by substitution with, e.g., (in addition to
lysine and arginine) the amino acids ornithine, citrulline, or
(guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where
alkyl is defined above. Nitrile derivative (e.g., containing the
CN-moiety in place of COOH) can be substituted for asparagine or
glutamine. Asparaginyl and glutaminyl residues can be deaminated to
the corresponding aspartyl or glutamyl residues. Arginine residue
mimetics can be generated by reacting arginyl with, e.g., one or
more conventional reagents, including, e.g., phenylglyoxal,
2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, preferably
under alkaline conditions. Tyrosine residue mimetics can be
generated by reacting tyrosyl with, e.g., aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane can be used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Cysteine residue mimetics can be
generated by reacting cysteinyl residues with, e.g.,
alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
and corresponding amines; to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteine residue mimetics can also
be generated by reacting cysteinyl residues with, e.g.,
bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic
acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl
disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate;
2-chloromercuri-4 nitrophenol; or,
chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be
generated (and amino terminal residues can be altered) by reacting
lysinyl with, e.g., succinic or other carboxylic acid anhydrides.
Lysine and other alpha-amino-containing residue mimetics can also
be generated by reaction with imidoesters, such as methyl
picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione,
and transamidase-catalyzed reactions with glyoxylate. Mimetics of
methionine can be generated by reaction with, e.g., methionine
sulfoxide. Mimetics of proline include, e.g., pipecolic acid,
thiazolidine carboxylic acid, 3- or 4-hydroxy proline,
dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
Histidine residue mimetics can be generated by reacting histidyl
with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of
proline and lysine; phosphorylation of the hydroxyl groups of seryl
or threonyl residues; methylation of the alpha-amino groups of
lysine, arginine and histidine; acetylation of the N-terminal
amine; methylation of main chain amide residues or substitution
with N-methyl amino acids; or amidation of C-terminal carboxyl
groups.
[0305] A residue, e.g., an amino acid, of a polypeptide of the
disclosure can also be replaced by an amino acid (or peptidomimetic
residue) of the opposite chirality. Thus, any amino acid naturally
occurring in the L-configuration (which can also be referred to as
the R or S, depending upon the structure of the chemical entity)
can be replaced with the amino acid of the same chemical structural
type or a peptidomimetic, but of the opposite chirality, referred
to as the D-amino acid, but also can be referred to as the R- or
S-form.
[0306] The mimetic polypeptides of the present invention may be
synthesized using any protein chemical synthesis techniques. In a
typical in vitro protein synthesis process, a peptide is extended
in length by one amino acid through forming a peptide bond between
the peptide and an amino acid. The formation of the peptide bond is
carried out using a ligation reaction, which can use a natural
amino acid or a non-natural amino acid. Thus, in this manner
non-natural amino acids can be introduced into the polypeptides of
the present invention to make mimetics.
[0307] The conditionally active biologic proteins can also be
synthesized, as a whole or in part, using chemical protein
synthesis methods well known in the art. See e.g., Caruthers (1980)
"New chemical methods for synthesizing polynucleotides", Nucleic
Acids Res. Symp. Ser. 215-223; Horn (1980), "Synthesis of
oligonucleotides on cellulose. Part II: design and synthetic
strategy to the synthesis of 22 oligodeoxynucleotides coding for
Gastric Inhibitory Polypeptide (GIP).sup.1", Nucleic Acids Res.
Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and
Proteins, Formulation, Processing and Delivery Systems (1995)
Technomic Publishing Co., Lancaster, Pa. For example, peptide
synthesis can be performed using various solid-phase techniques
(see e.g., Roberge (1995) "A strategy for a convergent synthesis of
N-linked glycopeptides on a solid support", Science 269:202;
Merrifield (1997) "Concept and early development of solid-phase
peptide synthesis", Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 43 IA Peptide
Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer.
[0308] Solid-phase chemical peptide synthesis methods can also be
used to synthesize the polypeptide or fragments thereof. Such
methods have been known in the art since the early 1960's
(Merrifield, R. B., "Solid-phase synthesis.I. The synthesis of a
tetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (See also
Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd
Ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have
recently been employed in commercially available laboratory peptide
design and synthesis kits (Cambridge Research Biochemicals). Such
commercially available laboratory kits have generally utilized the
teachings of H. M. Geysen et al., "Use of peptide synthesis to
probe viral antigens for epitopes to a resolution of a single amino
acid," Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for
synthesizing peptides upon the tips of a multitude of "rods" or
"pins" all of which are connected to a single plate.
[0309] The mimetic polypeptides of the present invention may also
be produced by recombinant techniques, which produce a polypeptide
by inserting a coding sequence of the polypeptide into an
expression vector and utilizing the protein translation machinery
of a eukaryotic cell production host. The protein translation
machinery reads the codons of the coding sequence and uses tRNA to
bring in the encoded amino acid to produce the polypeptide. There
are several techniques can be used to alter the protein translation
machinery to allow it to incorporate a non-natural amino acid into
a recombinant polypeptide. A proven approach depends on the
recognition of the non-natural amino acid by aminoacyl-tRNA
synthetases, which, in general, require high selectivity to insure
the fidelity of protein translation. These synthetases may be
engineered to relax the substrate specificity such that a
non-natural amino acid may be linked to a tRNA, which then brings
the non-natural amino acid to the protein translation machinery to
be incorporated into a polypeptide. For example, it was found that
replacement of Ala.sup.294by Gly in Escherichia coli
phenylalanyl-tRNA synthetase (PheRS) increases the size of
substrate binding pocket, and results in the acylation of
tRNA.sup.Phe by p-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P.
Kast and H. Hennecke, Biochemistry, 33:7107 (1994). An Escherichia
coli strain harboring this mutant PheRS allows the incorporation of
p-Cl-phenylalanine or p-Br-phenylalanine in place of phenylalanine,
See, e.g., M. Ibba and H. Hennecke, FEBS Lett., 364:272 (1995);
and, N. Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBS Lett.,
467:37 (2000). Similarly, a point mutation Phe130Ser near the amino
acid binding site of Escherichia coli tyrosyl-tRNA synthetase was
shown to allow azatyrosine to be incorporated more efficiently than
tyrosine. See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K.
Takaku, Y. Monden, M. Kitabatake, D. Soll and S. Nishimura, J.
Biol. Chem., 275:40324 (2000).
[0310] The first method involves reassigning sense codon, which
engineers at least one aminoacyl-tRNA synthetase. The enzyme
normally adds a natural amino acid to a tRNA to be transported to
the protein translation machinery for protein synthesis. However,
an aminoacyl-tRNA synthetase for a particular tRNA may be altered
such that it can have certain level of promiscuity to charge a
non-natural amino acid non-specifically to the tRNA to activate the
tRNA. The activated tRNA can carry the non-natural amino acid to
the protein translation machinery (e.g. ribosomes) and add the
non-natural amino acid to a peptide where a codon of the coding
sequence calls for that particular tRNA. In other words, the codon
for that particular tRNA has been reassigned to non-natural amino
acids. The recombinant polypeptide comprises 19 natural amino acids
and at least one non-natural amino acid. The one natural amino acid
in the polypeptide has been replaced by at least one non-natural
amino acid. The successful substitution of a natural amino acid
with non-natural amino acid relies on the use of auxotrophic
expression hosts deficient in the biosynthesis of that natural
amino acid. Employment of such hosts limits competition from the
natural amino acid for the reassigned sense codon, and improves the
incorporation efficiency and yield of target proteins. Codons for
many amino acids (including Met, Pro, Tyr, Phe, Leu, Val etc.) have
been reassigned, and more than 60 non-natural amino acids have been
incorporated into proteins via this method. See Hendrickson et al.,
"Incorporation of nonnatural amino acids into proteins," Annu. Rev.
Biochem., vol. 73, pages 147-176, 2004; Voloshchuk et al.,
"Incorporation of unnatural amino acids for synthetic biology,"
Mol. Biosyst., vol. 6, pages 65-80, 2010, both incorporated herein
by reference.
[0311] The main limitation of this method is that the non-natural
amino acid will replace the natural amino acid throughout the
polypeptide sequence, which may restrict its application if such
global substitution is undesirable. One solution is to mutate sites
where substitution is undesirable to other natural amino acids so
that only the desired site(s) are reserved for the non-natural
amino acid. With this modification, the method can introduce a
non-natural amino acid site-specifically at any desired site to
produce mimetic polypeptides.
[0312] Another method for producing mimetic recombinant
polypeptides is by using wobble codons. Wobble codons refer to
codons that are decoded by tRNAs via non-classical Watson-Crick
base-pairing. The non-classical (or wobble) pairing is enabled
through modification at the tRNA's 1.sup.st anticodon base (which
pairs with the 3rd base to the codon triplet), as proposed in the
"Wobble Hypothesis". For example, many organisms have only one tRNA
to decode two codons for Phe: UUU and UUC. As a result, the GAA
anticodon on the tRNA binds to the UUC codon via Watson-Crick
base-pairing, and to the UUU codon via "wobble" base-pairing.
[0313] Because of the wobble pairing between codon and anticodon,
one tRNA may pair with several codons, and a given codon may pair
with more than one tRNA. Taking advantage of this property, a
wobble codon may be assigned to a non-natural amino acid to
generate a recombinant protein that contains natural amino acids
and at least one non-natural amino acid. For example, Phe is
normally encoded by two codons UUC and UUU, with both codons
recognized by a single tRNA. By expressing an orthogonal pair of
aminoacyl-tRNA synthetase and tRNA, with specificity for a
non-natural amino acid and containing the "AAA" anticodon,
efficient introduction of the non-natural amino acid at UUU codons
can be achieved (Kwon et al., "Breaking the degeneracy of the
genetic code," J. Am. Chem. Soc., vol. 125, pages 7512-7513, 2003,
incorporated herein by reference). With this method, Phe can be
essentially quantitatively assigned to the UUC codon, and a
non-natural amino acid to the UUU wobble codon. Furthermore,
multiple copies of a non-natural amino acid can be introduced site
specifically into a polypeptide.
[0314] The third method for generating recombinant mimetic
polypeptide is by using biased codons. The preferred codons differ
between organisms, and even between different tissues or cell types
of the same organism. The cellular content of tRNA species is a
determining factor on the rates and amounts of protein synthesized.
As a consequence, recombinant protein production in heterologous
host cells is often codon-optimized to match the preferred host
cell codon bias (The codon usage database for different organisms
and codon analysis of a given gene can be found at:
http://www.kazusa.or.jp/codon/).
[0315] The biased codon usage provides another method to introduce
non-natural amino acids into recombinant polypeptides. For example,
out of the six degenerate codons for Arg, AGG and AGA are rarely
used in E. coli. Introduction of an orthogonal pair of
aminoacyl-tRNA synthetase and tRNA that pairs with the AGG codon
into an E. coli expression host may enable linking a non-natural
amino acid to the tRNA. Therefore, the tRNA with a non-natural
amino acid linked thereto can bring the non-natural amino acid to
the codon AGG, where normally Arg may be encoded. This method has
been proven feasible with an in vitro cell-free biased system,
where chemically synthesized non-natural amino acid linked tRNA
that pairs with the AGG codon was incorporated at AGG codons
(Hohsaka et al., FEBS Letters, vol. 344, pages 171-174, 1994). The
method could be adapted to an E. coli cell-based expression system
if an aminoacyl-tRNA synthetase can be engineered to link a
non-natural orthogonal to a tRNA.
[0316] Similarly, a bias codon may be assigned to a non-natural
amino acid in mammalian cells that exhibit codon bias. For example,
through study of human papillomavirus gene expression in different
mammalian cells, Frazer and his colleagues have found that
papillomavirus protein expression is determined by the codon usage
and tRNA availability. Substantial differences in the tRNA pools
were discovered between differentiated and undifferentiated
keratinocytes (Zhao et al., "Gene codon composition determines
differentiation-dependent expression of a viral capsid gene in
keratinocytes in vitro and in vivo," Mol. Cell Biol., vol. 25,
pages 8643-8655, 2005), and the observed bias in their tRNA may be
the reason that papillomavirus replicates exclusively in epithelial
cells. For example, in CHO and Cos1 cells, it seems that TCG is a
bias and thus might be assigned to a non-natural amino acid.
[0317] As the codon bias phenomenon is wide-spread in different
eukaryotic organisms, utilization of such codons for site-specific
incorporation of non-natural amino acids could be applied in many
eukaryotic cell production hosts. The limitation would be the
engineering of the aminoacyl-tRNA synthetase to link a non-natural
orthogonal to a tRNA that can pair with a biased codon in the
production hosts.
[0318] A fourth method for producing a mimetic polypeptide is by
suppressing a stop codon. Generally, protein translation terminates
at one of the three stop codons (encoded by UAG (amber), UAA
(ochre) and UGA (opal)) by the action of protein release factors
(RF). However, occasional read-through of a stop codon with an
amino acid has been observed to happen naturally in a variety of
species. The suppression is caused by either mutations in the tRNA
anticodon or mismatches of the codon-anticodon (Beier & Grimm,
"Misreading of termination codons in eukaryotes by natural nonsense
suppressor tRNAs," Nucleic Acids Res., vol. 29, pages 4767-4782,
2001). Utilization of stop codon suppression represents another way
to producing proteins containing non-natural amino acids, and
generally involves the introduction of an aminoacyl-tRNA synthetase
that can link a non-natural amino acid to a tRNA that can pair with
a stop codon. For example, an aminoacyl-tRNA synthetase and tRNA
that pairs with the amber stop codon has been developed to
introduce a non-natural amino acid site-specifically at amber
codons, as it is the least frequently used stop codon in both
eukaryotic (23% in humans) and prokaryotic genomes (7% in E. coli)
(Liu et al., "Genetic incorporation of unnatural amino acids into
proteins in mammalian cells," Nat. Methods, vol. 4, pages 239-244,
2007). Ochre and opal stop codons have been used for the
introduction of non-natural amino acids as well (Kohrer et al.,
"Complete set of orthogonal 21.sup.St aminoacyl-tRNA
synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant
suppression of three different termination codons in an mRNA in
mammalian cells," Nucleic Acids Res., vol. 32, pages 6200-6211,
2004). So far, over 70 non-natural amino acids have been
site-specifically incorporated into recombinant proteins by this
method (Liu & Schultz, "Adding new chemistries to the genetic
code," Annu. Rev. Biochem., vol. 79, pages 413-444, 2010).
Typically, over 95% non-natural amino acid incorporation efficiency
(defined as occupancy rate of non-natural amino acid in the
full-length product) at the desired site can be obtained, making it
one of the most frequently used methods for non-natural amino acid
incorporation.
[0319] The present invention also encompasses any other techniques
known to a person skilled in the art for introducing non-natural
amino acids into a recombinant polypeptide. Some of the techniques
involve using four-base-pair codons (Anderson et al., "An expanded
genetic code with a functional quadruplet codon. Proc. Natl. Acad.
Sci. U.S.A., vol. 101, pages 7566-7571, 2004). More discussion
about producing mimetic recombinant polypeptides may be found in
U.S. Pat. No. 7,045,337 and WO2010132341A2, both of which are
hereby incorporated herein by reference.
[0320] The disclosure also provides methods for modifying the
polypeptides of the disclosure by either natural processes, such as
post-translational processing (e.g., phosphorylation, acylation,
etc), or by chemical modification techniques. Modifications can
occur anywhere in the polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
PEGylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment
of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of a
phosphatidylinositol, cross-linking cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristolyation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, and transfer-RNA mediated
addition of amino acids to protein such as arginylation. See, e.g.,
Creighton, T. E., Proteins--Structure and Molecular Properties 2nd
Ed., W. H. Freeman and Company, New York (1993); Posttranslational
Covalent Modification of Proteins, B. C. Johnson, Ed., Academic
Press, New York, pp. 1-12 (1983).
[0321] Solid-phase chemical peptide synthesis methods can also be
used to synthesize the polypeptide or fragments of the disclosure.
Such methods have been known in the art since the early 1960's
(Merrifield, R. B., "Solid-phase synthesis.I. The synthesis of a
tetrapeptide", J. Am. Chem. Soc, 85:2149-2154, 1963) (See also
Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd
Ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have
recently been employed in commercially available laboratory peptide
design and synthesis kits (Cambridge Research Biochemicals). Such
commercially available laboratory kits have generally utilized the
teachings of H. M. Geysen et al., "Use of peptide synthesis to
probe viral antigens for epitopes to a resolution of a single amino
acid," Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for
synthesizing peptides upon the tips of a multitude of "rods" or
"pins" all of which are connected to a single plate. When such a
system is utilized, a plate of rods or pins is inverted and
inserted into a second plate of corresponding wells or reservoirs,
which contain solutions for attaching or anchoring an appropriate
amino acid to the pin's or rod's tips. By repeating such a process
step, i.e., inverting and inserting the rod's and pin's tips into
appropriate solutions, amino acids are built into desired peptides.
In addition, a number of available FMOC peptide synthesis systems
are available. For example, assembly of a polypeptide or fragment
can be carried out on a solid support using an Applied Biosystems,
Inc. Model 431 A.TM. automated peptide synthesizer. Such equipment
provides ready access to the peptides of the disclosure, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques.
[0322] The synthetic polypeptide or fragment thereof can be
recovered and purified by known methods including ammonium sulfate
or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the polypeptide. If desired, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0323] The disclosure provides for a conditionally active protein
variant preparation or formulation which comprises at least one of
the protein variants, wherein the preparation is liquid or dry. The
protein formulation optionally includes a buffer, cofactor, second
or additional protein, or one or more excipients. In one aspect the
formulation is utilized as a therapeutic conditionally active
biologic protein which is active under aberrant or
non-physiological conditions, but less active or inactive under
normal physiological conditions of, e.g., temperature, pH, or
osmotic pressure, oxidation or osmolality.
[0324] Standard purification techniques can be employed for either
recombinant or synthetic conditionally active biologic
proteins.
Screening of Mutants to Identify Reversible or Nonreversible
Mutants
[0325] Identifying desirable molecules is most directly
accomplished by measuring protein activity at the permissive
condition and the wild type condition. The mutants with the largest
ratio of activity (permissive/wild type) can then be selected and
permutations of the point mutations are generated by combining the
individual mutations using standard methods. The combined
permutation protein library is then screened for those proteins
displaying the largest differential activity between the permissive
and wild type condition.
[0326] Activity of supernatants can be screened using a variety of
methods, for example using high throughput activity assays, such as
fluorescence assays, to identify protein mutants that are sensitive
at whatever characteristic one desires (temperature, pH, etc). For
example, to screen for temporally sensitive mutants, the enzymatic
or antibody activity of each individual mutant is determined at
lower temperatures (such as 25 degrees Celsius), and at
temperatures which the original protein functions (such as 37
degrees Celsius), using commercially available substrates.
Reactions can initially be performed in a multi well assay format,
such as a 96-well assay, and confirmed using a different format,
such as a 14 ml tube format.
[0327] The disclosure further provides a screening assay for
identifying a enzyme, the assay comprising: (a) providing a
plurality of nucleic acids or polypeptides; (b) obtaining
polypeptide candidates to be tested for enzyme activity from the
plurality; (c) testing the candidates for enzyme activity; and (d)
identifying those polypeptide candidates which exhibit elevated
enzyme activity under aberrant or non-physiological conditions, and
decreased enzyme activity compared to the wild-type enzyme protein
under normal physiological conditions of, e.g., temperature, pH,
oxidation, osmolality, electrolyte concentration or osmotic
pressure.
[0328] In one aspect, the method further comprises modifying at
least one of the nucleic acids or polypeptides prior to testing the
candidates for conditional biologic activity, in another aspect,
the testing of step (c) further comprises testing for improved
expression of the polypeptide in a host cell or host organism, in a
further aspect, the testing of step (c) further comprises testing
for enzyme activity within a pH range from about pH 3 to about pH
12. In a further aspect, the testing of step (c) further comprises
testing for enzyme activity within a pH range from about pH 5 to
about pH 10. In a further aspect, the testing of step (c) further
comprises testing for enzyme activity within a pH range from about
pH 6 to about pH 8. In a further aspect, the testing of step (c)
further comprises testing for enzyme activity at pH 6.7 and pH 7.5.
In another aspect, the testing of step (c) further comprises
testing for enzyme activity within a temperature range from about 4
degrees C. to about 55 degrees C. In another aspect, the testing of
step (c) further comprises testing for enzyme activity within a
temperature range from about 15 degrees C. to about 47 degrees C.
In another aspect, the testing of step (c) further comprises
testing for enzyme activity within a temperature range from about
20 degrees C. to about 40 degrees C. In another aspect, the testing
of step (c) further comprises testing for enzyme activity at the
temperatures of 25 degrees C. and 37 degrees C. In another aspect,
the testing of step (c) further comprises testing for enzyme
activity under normal osmotic pressure, and aberrant (positive or
negative) osmotic pressure, In another aspect, the testing of step
(c) further comprises testing for enzyme activity under normal
electrolyte concentration, and aberrant (positive or negative)
electrolyte concentration. The electrolyte concentration to be
tested is selected from one of calcium, sodium, potassium,
magnesium, chloride, bicarbonate and phosphate concentration, in
another aspect, the testing of step (c) further comprises testing
for enzyme activity which results in a stabilized reaction
product.
[0329] In another aspect, the disclosure provides for a purified
antibody that specifically binds to the polypeptide of the
disclosure or a fragment thereof, having enzyme activity, In one
aspect, the disclosure provides for a fragment of the antibody that
specifically binds to a polypeptide having enzyme activity.
Antibodies and Antibody-Based Screening Methods
[0330] The disclosure provides isolated or recombinant antibodies
that specifically bind to an antigen of the disclosure. These
antibodies can be used to isolate, identify or quantify the
antigens of the disclosure or related polypeptides. These
antibodies can be used to isolate other polypeptides within the
scope the disclosure or other related proteins. The antibodies can
be designed to bind to an active site of an enzyme. Thus, the
disclosure provides methods of inhibiting enzymes using the
antibodies of the disclosure.
[0331] The antibodies can be used in immunoprecipitation, staining,
immunoaffinity columns, and the like. If desired, nucleic acid
sequences encoding for specific antigens can be generated by
immunization followed by isolation of polypeptide or nucleic acid,
amplification or cloning and immobilization of polypeptide onto an
array of the disclosure. Alternatively, the methods of the
disclosure can be used to modify the structure of an antibody
produced by a cell to be modified, e.g., an antibody's affinity can
be increased or decreased. Furthermore, the ability to make or
modify antibodies can be a phenotype engineered into a cell by the
methods of the disclosure.
[0332] Methods of immunization, producing and isolating antibodies
(polyclonal and monoclonal) are known to those of skill in the art
and described in the scientific and patent literature, see, e.g.,
Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991);
Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical
Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New
York, N.Y. (1986); Kohler (1975) "Continuous cultures of fused
cells secreting antibody of predefined specificity", Nature
256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring
Harbor Publications, New York. Antibodies also can be generated in
vitro, e.g., using recombinant antibody binding site expressing
phage display libraries, in addition to the traditional in vivo
methods using animals. See, e.g., Hoogenboom (1997) "Designing and
optimizing library selection strategies for generating
high-affinity antibodies", Trends Biotechnol. 15:62-70; and Katz
(1997) "Structural and mechanistic determinants of affinity and
specificity of ligands discovered or engineered by phage display",
Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
[0333] Polypeptides or peptides can be used to generate antibodies
which bind specifically to the polypeptides, e.g., the enzymes, of
the disclosure. The resulting antibodies may be used in
immunoaffinity chromatography procedures to isolate or purify the
polypeptide or to determine whether the polypeptide is present in a
biological sample. In such procedures, a protein preparation, such
as an extract, or a biological sample is contacted with an antibody
capable of specifically binding to one of the polypeptides of the
disclosure.
[0334] In immunoaffinity procedures, the antibody is attached to a
solid support, such as a bead or other column matrix. The protein
preparation is placed in contact with the antibody under conditions
in which the antibody specifically binds to one of the polypeptides
of the disclosure. After a wash to remove non-specifically bound
proteins, the specifically bound polypeptides are eluted.
[0335] The ability of proteins in a biological sample to bind to
the antibody may be determined using any of a variety of procedures
familiar to those skilled in the art. For example, binding may be
determined by labeling the antibody with a detectable label such as
a fluorescent agent, an enzymatic label, or a radioisotope.
Alternatively, binding of the antibody to the sample may be
detected using a secondary antibody having such a detectable label
thereon. Particular assays include ELISA assays, sandwich assays,
radioimmunoassays, and Western Blots.
[0336] Polyclonal antibodies generated against the polypeptides of
the disclosure can be obtained by direct injection of the
polypeptides into an animal or by administering the polypeptides to
a non-human animal. The antibody so obtained will then bind the
polypeptide itself. In this manner, even a sequence encoding only a
fragment of the polypeptide can be used to generate antibodies
which may bind to the whole native polypeptide. Such antibodies can
then be used to isolate the polypeptide from cells expressing that
polypeptide.
[0337] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique, the trioma
technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0338] Techniques described for the production of single chain
antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to
produce single chain antibodies to the polypeptides of the
disclosure. Alternatively, transgenic mice may be used to express
humanized antibodies to these polypeptides or fragments thereof.
Antibodies generated against the polypeptides of the disclosure may
be used in screening for similar polypeptides (e.g., enzymes) from
other organisms and samples. In such techniques, polypeptides from
the organism are contacted with the antibody and those polypeptides
which specifically bind the antibody are detected. Any of the
procedures described above may be used to detect antibody
binding.
Screening Methodologies and "On-line" Monitoring Devices
[0339] In practicing the methods of the disclosure, a variety of
apparatus and methodologies can be used to in conjunction with the
polypeptides and nucleic acids of the disclosure, e.g., to screen
polypeptides for enzyme activity, to screen compounds as potential
modulators, e.g., activators or inhibitors, of an enzyme activity,
for antibodies that bind to a polypeptide of the disclosure, for
nucleic acids that hybridize to a nucleic acid of the disclosure,
to screen for cells expressing a polypeptide of the disclosure and
the like.
Arrays or "Biochips"
[0340] Nucleic acids or polypeptides of the disclosure can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
disclosure. For example, in one aspect of the disclosure, a
monitored parameter is transcript expression of an enzyme gene. One
or more, or, all the transcripts of a cell can be measured by
hybridization of a sample comprising transcripts of the cell, or,
nucleic acids representative of or complementary to transcripts of
a cell, by hybridization to immobilized nucleic acids on an array,
or "biochip." By using an "array" of nucleic acids on a microchip,
some or all of the transcripts of a cell can be simultaneously
quantified. Alternatively, arrays comprising genomic nucleic acid
can also be used to determine the genotype of a newly engineered
strain made by the methods of the disclosure. Polypeptide arrays"
can also be used to simultaneously quantify a plurality of
proteins. The present disclosure can be practiced with any known
"array," also referred to as a "microarray" or "nucleic acid array"
or "polypeptide array" or "antibody array" or "biochip," or
variation thereof. Arrays are generically a plurality of "spots" or
"target elements," each target element comprising a defined amount
of one or more biological molecules, e.g., oligonucleotides,
immobilized onto a defined area of a substrate surface for specific
binding to a sample molecule, e.g., mRNA transcripts.
[0341] In practicing the methods of the disclosure, any known array
and/or method of making and using arrays can be incorporated in
whole or in part, or variations thereof, as described, for example,
in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606;
6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452;
5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752;
5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752;
5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313;
WO 96/17958; see also, e.g., Johnston (1998) "Gene chips: Array of
hope for understanding gene regulation", Curr. Biol. 8:R171-R174;
Schummer (1997) "Inexpensive Handheld Device for the Construction
of High-Density Nucleic Acid Arrays", Biotechniques 23:1087-1092;
Kern (1997) "Direct hybridization of large-insert genomic clones on
high-density gridded cDNA filter arrays", Biotechniques 23:120-124;
Solinas-Toldo (1997) "Matrix-Based Comparative Genomic
Hybridization: Biochips to Screen for Genomic Imbalances", Genes,
Chromosomes & Cancer 20:399-407; Bowtell (1999) "Options
Available--From Start to Finish--for Obtaining Expression Data by
Microarray", Nature Genetics Supp. 21:25-32. See also published
U.S. patent applications Nos. 20010018642; 20010019827;
20010016322; 20010014449; 20010014448; 20010012537;
20010008765.
Capillary Arrays
[0342] Capillary arrays, such as the GIGAMATRIX.TM. Diversa
Corporation, San Diego, Calif., can be used in the methods of the
disclosure. Nucleic acids or polypeptides of the disclosure can be
immobilized to or applied to an array, including capillary arrays.
Arrays can be used to screen for or monitor libraries of
compositions (e.g., small molecules, antibodies, nucleic acids,
etc.) for their ability to bind to or modulate the activity of a
nucleic acid or a polypeptide of the disclosure. Capillary arrays
provide another system for holding and screening samples. For
example, a sample screening apparatus can include a plurality of
capillaries formed into an array of adjacent capillaries, wherein
each capillary comprises at least one wall defining a lumen for
retaining a sample. The apparatus can further include interstitial
material disposed between adjacent capillaries in the array, and
one or more reference indicia formed within of the interstitial
material. A capillary for screening a sample, wherein the capillary
is adapted for being bound in an array of capillaries, can include
a first wall defining a lumen for retaining the sample, and a
second wall formed of a filtering material, for filtering
excitation energy provided to the lumen to excite the sample. A
polypeptide or nucleic acid, e.g., a ligand, can be introduced into
a first component into at least a portion of a capillary of a
capillary array. Each capillary of the capillary array can comprise
at least one wall defining a lumen for retaining the first
component. An air bubble can be introduced into the capillary
behind the first component. A second component can be introduced
into the capillary, wherein the second component is separated from
the first component by the air bubble. A sample of interest can be
introduced as a first liquid labeled with a detectable particle
into a capillary of a capillary array, wherein each capillary of
the capillary array comprises at least one wall defining a lumen
for retaining the first liquid and the detectable particle, and
wherein the at least one wall is coated with a binding material for
binding the detectable particle to the at least one wall. The
method can further include removing the first liquid from the
capillary tube, wherein the bound detectable particle is maintained
within the capillary, and introducing a second liquid into the
capillary tube. The capillary array can include a plurality of
individual capillaries comprising at least one outer wall defining
a lumen. The outer wall of the capillary can be one or more walls
fused together. Similarly, the wall can define a lumen that is
cylindrical, square, hexagonal or any other geometric shape so long
as the walls form a lumen for retention of a liquid or sample. The
capillaries of the capillary array can be held together in close
proximity to form a planar structure. The capillaries can be bound
together, by being fused (e.g., where the capillaries are made of
glass), glued, bonded, or clamped side-by-side. The capillary array
can be formed of any number of individual capillaries, for example,
a range from 100 to 4,000,000 capillaries. A capillary array can
form a micro titer plate having about 100,000 or more individual
capillaries bound together.
Engineering Conditionally Active Biological Proteins
[0343] The conditionally active biological proteins of the present
invention, including the conditionally active antibodies against
BBB-R and conditionally active antibodies for synovial fluid, tumor
microenvironments and stem cell niches, including cancer stem
cells, may be engineered by one or more protein engineering
techniques described herein. Non-limiting examples of protein
engineering techniques also include antibody conjugation,
engineering multispecific antibodies, engineering Fc region of the
antibodies.
Conjugating Conditionally Active Biological Proteins
[0344] The conditionally active biological proteins provided by the
present invention may be conjugated to a molecule. Because the
conditionally active biological protein preferentially acts in the
brain, synovial fluid, a tumor microenvironment, or a stem cell
niche, the conditionally active biological protein may be
conjugated to a molecule (therapeutic or diagnostic agent), which
will be transported to the brain, synovial fluid, tumor
microenvironment or stem cell niche with the conditionally active
biological proteins. In some embodiments, the molecule has
non-specific toxicity, which may be reduced by being conjugated to
the conditionally active biological proteins, to thus
preferentially act on the disease site.
[0345] In some embodiments, the conjugated molecule on the
conditionally active biological protein may be optionally released
from the conditionally active biological protein once the
conditionally active biological protein has reached its intended
location such as a brain, synovial fluid, a tumor microenvironment,
or a stem cell niche. In these embodiments, the conditionally
active biological proteins may act as a delivery vehicle for
transporting the conjugated molecules (such as therapeutics or
diagnostics) into a brain, synovial fluid, a tumor
microenvironment, or stem cell niches. Once inside the brain,
synovial fluid, a tumor microenvironment, or stem cell niches, the
conjugated molecule can be released for treatment of disease.
[0346] The conjugation of the conditionally active biological
protein with a molecule (therapeutics or diagnostics) can be
covalent conjugation or non-covalent. Covalent conjugation can
either be direct or via a linker. In certain embodiments, direct
conjugation is by construction of a fusion protein (i.e., by
genetic fusion of the two genes encoding the conditionally active
antibody and neurological disorder drug and expression as a single
protein). In certain embodiments, direct conjugation is by
formation of a covalent bond between a reactive group on one of the
two portions of the conditionally active antibody and a
corresponding group or acceptor on the neurological drug/imaging
agent. In certain embodiments, direct conjugation is by
modification (i.e., genetic modification) of one of the two
molecules to be conjugated to include a reactive group (as
non-limiting examples, a sulfhydryl group or a carboxyl group) that
forms a covalent attachment to the other molecule to be conjugated
under appropriate conditions. As one non-limiting example, a
molecule (i.e., an amino acid) with a desired reactive group (i.e.,
a cysteine residue) may be introduced into, e.g., the conditionally
active antibody and a disulfide bond formed with the neurological
drug. Methods for covalent conjugation of nucleic acids to proteins
are also known in the art (i.e., photocrosslinking, see, e.g.,
Zatsepin et al. Russ. Chem. Rev., 74: 77-95 (2005)) Non-covalent
conjugation can be by any non-covalent attachment means, including
hydrophobic bonds, ionic bonds, electrostatic interactions, and the
like, as will be readily understood by one of ordinary skill in the
art.
[0347] Conjugation may also be performed using a variety of
linkers. For example, a conditionally active antibody and a
neurological drug may be conjugated using a variety of bifunctional
protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Peptide linkers, comprised of from one to twenty amino acids joined
by peptide bonds, may also be used. In certain such embodiments,
the amino acids are selected from the twenty naturally-occurring
amino acids. In certain other such embodiments, one or more of the
amino acids are selected from glycine, alanine, proline,
asparagine, glutamine and lysine. The linker may be a "cleavable
linker" facilitating release of the neurological drug upon delivery
to the brain. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Res., 52:127-131
(1992); U.S. Pat. No. 5,208,020) may be used. Some examples of
cross-linker reagents for antibody conjugation include BMPS, EMCS,
GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH,
sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate).
[0348] The conjugated therapeutic agent may be toxic to the body,
such as a radioactive particle, chemotherapy drug, or a cell toxin
(i.e., cytotoxin). These therapeutic agents are highly toxic to the
body. Using the conditionally active antibodies of the present
invention to deliver the conjugated therapeutic agent to the
disease site can significantly reduce the toxic effects of these
therapeutic agents. The technology for conjugating radioactive
particles to antibodies is known in the art. Ibritumomab tiuxetan
(Zevalin.RTM.) and tositumomab (Bexxar.RTM.) are examples of
radioactive particle conjugated monoclonal antibodies. Both are
antibodies against the CD20 antigen conjugated with a different
radioactive particle. Similarly, the technology for conjugating
chemotherapy drugs to antibodies is also known in the art. There
are two marketed antibodies that are conjugated with a chemotherapy
drug: brentuximab vedotin (Adcetris.RTM.) and ado-trastuzumab
emtansine (Kadcyla.TM.). Brentuximab vedotin is made up of an
antibody that targets the CD30 antigen (found on B cells and T
cells), attached to a chemo drug called MMAE. Ado-trastuzumab
emtansine is made of an antibody that targets the HER2 protein
attached to a chemotherapy drug called DM1. The technology for
conjugating a cell toxin to an antibody is also known in the art.
For example, denileukin diftitox (Ontak.RTM., a cancer drug)
consists of an immune system protein known as interleukin-2 (IL-2)
attached to a toxin from the germ that causes diphtheria.
[0349] It is contemplated that any radioactive particles,
chemotherapy drugs, and cell toxins may be conjugated to the
conditionally active biological proteins of the present invention
in order to reduce the side effects of these agents.
[0350] In some embodiments, the radioactive particles conjugated to
the conditionally active biological proteins for treatment of an
abnormal tissue comprise particles impregnated with one or more
radioactive isotopes, and have sufficient radioactivity for
locoregional ablation of cells in the abnormal tissue. The
particles may comprise glass, metal, resin, albumin, or polymer.
Metal in the radioactive particles may be selected from iron,
gadolinium, and calcium. Examples of the one or more radioactive
isotopes in the radioactive particles are selected from the group
consisting of Gallium-67 (.sup.67Ga), Yttrium-90 (.sup.90Y),
Gallium-68 (.sup.68Ga), Thallium-201 (.sup.201Tl), Strontium-89
(.sup.89Sr), Indium-III (.sup.IIIIn), Iodine-131 (.sup.131I),
Samarium-153 (.sup.153Sm), Technetium-99m (.sup.99mTc), Rhenium-186
(.sup.186Re), Rhenium-188 (.sup.188Re), Copper-62 (.sup.62Cu), and
Copper-64 (.sup.64Cu). Preferably the radioactive isotope(s) in the
composition emit beta radiations, gamma radiations, and/or
positrons.
[0351] In some embodiments, the chemotherapy drugs conjugated to
the conditionally active biological proteins are selected from the
group consisting of anthracyclines, topoisomerase I and/or II
inhibitors, spindle poison plant alkaloids, alkylating agents,
anti-metabolites, ellipticine and harmine.
[0352] Anthracyclines (or anthracycline antibiotics) are derived
from Streptomyces bacteria. These compounds are used to treat a
wide range of cancers, including for example hepatocellular
carcinoma, leukemias, lymphomas, and breast, uterine, ovarian, and
lung cancers. Anthracyclines include, but are not limited to
doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin,
pirarubicin, zorubicin, aclarubicin, detorubicin, carminomycin,
morpholinodoxorubicin, morpholinodaunorubicin,
methoxymorpholinyldoxorubicin, and their pharmaceutically
acceptable salts thereof.
[0353] Topoisomerases are essential enzymes that maintain the
topology of DNA. Inhibition of type I or type II topoisomerases
interferes with both transcription and replication of DNA by
upsetting proper DNA supercoiling. Some type I topoisomerase
inhibitors include camptothecins derivatives Camptothecin
derivatives refer to camptothecin analogs such as irinotecan,
topotecan, hexatecan, silatecan, lutortecan, karenitecin (BNP1350),
gimatecan (ST1481), belotecan (CKD602), or their pharmaceutically
acceptable salts. Examples of type II topoisomerase inhibitors
include, but are not limited to, amsacrine, etoposide, etoposide
phosphate and teniposide. These are semisynthetic derivatives of
epipodophyllotoxins, alkaloids naturally occurring in the root of
American Mayapple (Podophyllum peltatum).
[0354] Spindle poison plant alkaloids are derived from plants and
block cell division by preventing microtubule function, essential
for cell division. These alkaloids include, but are not limited to,
vinca alkaloids (like vinblastine, vincristine, vindesine,
vinorelbine and vinpocetine) and taxanes. Taxanes include, but are
not limited to, paclitaxel, docetaxel, larotaxel, cabazitaxel,
ortataxel, tesetaxel, and their pharmaceutically acceptable
salts.
[0355] Alkylating agents are so named because of their ability to
add alkyl groups to many electronegative groups under conditions
present in cells. They impair cell function by forming covalent
bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in
biologically important molecules. Noteworthy, their cytotoxicity is
thought to result from inhibition of DNA synthesis. Alkylating
agents include, but are not limited to, mechlorethamine,
cyclophosphamide, chlorambucil, ifosfamide and platinum compounds
such as oxaliplatin, cisplatin or carboplatin.
[0356] An anti-metabolite is a chemical that inhibits the use of a
metabolite, which is part of normal metabolism. Such substances are
often similar in structure to the metabolite that they interfere
with. The presence of anti-metabolites halts cell growth and cell
division.
[0357] Purine or pyrimidine analogues prevent the incorporation of
nucleotides into DNA, stopping DNA synthesis and thus cell
divisions. They also affect RNA synthesis. Examples of purine
analogues include azathioprine, mercaptopurine, thioguanine,
fludarabine, pentostatin and cladribine. Examples of pyrimidine
analogues include 5-fluorouracil (5FU), which inhibits thymidylate
synthase, floxuridine (FUDR) and cytosine arabinoside
(Cytarabine).
[0358] Antifolates are chemotherapy drugs which impair the function
of folic acids. A well-known example is methotrexate, which is a
folic acid analogue that inhibits the enzyme dihydrofolate
reductase (DHFR), and thus prevents the formation of
tetrahydrofolate. Tetrahydrofolate is essential for purine and
pyrimidine synthesis. This leads to inhibited production of DNA,
RNA and proteins (as tetrahydrofolate is also involved in the
synthesis of amino acids serine and methionine). Other antifolates
include, but are not limited to, trimethoprim, raltitrexed,
pyrimethamine and pemetrexed.
[0359] Other chemotherapy drugs may also be conjugated to the
conditionally active biological proteins, such as ellipticine and
harmine. Ellipticine is a natural plant alkaloid product which is
isolated from the evergreen tree of the Apocynaceae family.
Ellipticine and its derivatives such as 9-hydroxyellipticinium,
N2-methyl-9-hydroxyellipticinium,
2-(diethyiamino-2-ethyl)9-hydroxyellipticinium acetate,
2-(diisopropylamino-ethyl)9-hydroxy-ellipticinium acetate and
2-(beta piperidino-2-ethyl)9-hydroxyellipticinium are all effective
chemotherapy drugs.
[0360] Harmine is a natural plant alkaloid product which was
isolated from the Peganum harmala seeds. Harmine-based chemotherapy
drugs include harmine, harmaline, harmol, harmalol and harman, and
quinazoline derivatives: vasicine and vasicinone.
[0361] In some embodiments, the cell toxins conjugated to the
conditionally active biological proteins include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Other toxins include, for example,
ricin, CC-1065 and analogues, the duocarmycins. Still other toxins
include diptheria toxin, and snake venom (e.g., cobra venom).
[0362] In one embodiment, a pyrrolobenzodiazepine may be conjugated
to a conditionally active biological protein. Pyrrolobenzodiazepine
(PBD) dimers are a class of rationally designed DNA minor groove,
sequence selective, cross-linking agents, which cross-link the two
DNA strands thus preventing DNA replication and cell division. The
PBDs may be used as chemotherapy agents. This class of chemotherapy
agents exhibits picomolar or subpicomolar activity in inhibiting
tumor cell growth. The synthetic PBDs, when conjugated to a
conditionally active antibody, can be guided towards a tumor site
for inhibition of tumor cell growth. PBDs may use different
conjugation sites for linking to a conditionally active antibody.
For example, two suitable PBDs are show below.
##STR00001##
[0363] In some embodiments, the conditionally active biological
proteins of the present invention may be conjugated to a diagnostic
agent. A diagnostic agent used in the present invention can include
any diagnostic agent known in the art, as provided, for example, in
the following references: Armstrong et al, Diagnostic Imaging,
5.sup.th Ed., Blackwell Publishing (2004); Torchilin, V. P., Ed.,
Targeted Delivery of Imaging Agents, CRC Press (1995);
Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET
and SPECT, Springer (2009). A diagnostic agent can be detected by a
variety of methods, including using the agent to provide and/or
enhance a detectable signal that includes, but is not limited to,
gamma-emitting, radioactive, echogenic, optical, fluorescent,
absorptive, magnetic or tomography signals. Techniques for imaging
the diagnostic agent can include, but are not limited to, single
photon emission computed tomography (SPECT), magnetic resonance
imaging (MRI), optical imaging, fluorescence imaging, positron
emission tomography (PET), computed tomography (CT), x-ray imaging,
gamma ray imaging, and the like.
[0364] In some embodiments, a diagnostic agent can include
chelators that bind, e.g., to metal ions to be used for a variety
of diagnostic imaging techniques. Exemplary chelators include but
are not limited to ethylenediaminetetraacetic acid (EDTA),
[4-(1,4,8,11-tetraazacyclotetradec-1-yl) methyljbenzoic acid
(CPTA), Cyclohexanediaminetetraacetic acid (CDTA),
ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA),
diethylenetriaminepentaacetic acid (DTPA), citric acid,
hydroxyethyl ethylenediamine triacetic acid (HEDTA), iminodiacetic
acid (IDA), triethylene tetraamine hexaacetic acid (TTHA),
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic
acid) (DOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic
acid (TETA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA), and derivatives thereof.
[0365] A radioisotope can be incorporated into some of the
diagnostic agents described herein and can include radionuclides
that emit gamma rays, positrons, beta and alpha particles, and
X-rays. Suitable radionuclides include but are not limited to Ac,
As, At, .sup.nB, .sup.128Ba, .sup.212Bi, .sup.75Br, .sup.77Br,
.sup.14C, .sup.109Cd, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.18F,
.sup.67Ga, .sup.68Ga, .sup.3H, .sup.123I, .sup.125I, .sup.130I,
.sup.131I, .sup.111In, .sup.177Lu, .sup.13N, .sup.150, .sup.32P,
.sup.33P, .sup.212Pb, .sup.103Pd, .sup.186Re, .sup.188Re,
.sup.47Sc, .sup.153Sm, .sup.89Sr, .sup.111Tc, .sup.88Y and
.sup.90Y. In certain embodiments, radioactive agents can include
.sup.mIn-DTPA, .sup.99mTc(CO)3-DTPA, .sup.99mTc(CO).sub.3-ENPy2,
.sup.62/64/67Cu-TETA, .sup.99mTc(CO).sub.3-IDA, and
.sup.99mTc(CO).sub.3triamines (cyclic or linear). In other
embodiments, the agents can include DOTA and its various analogs
with .sup.111In, .sup.177Lu, .sup.153Sm, .sup.88/90Y,
.sup.62/64/67Cu, or .sup.67/68Ga. In some embodiments, the
liposomes can be radiolabeled, for example, by incorporation of
lipids attached to chelates, such as DTPA-lipid, as provided in the
following references: Phillips et al, Wiley Interdisciplinary
Reviews: Nanomedicine and Nanobiotechnology, vol. 1, pages 69-83
(2008); Torchilin, V. P. & Weissig, V., Eds. Liposomes 2nd Ed.
: Oxford Univ. Press (2003); Elbayoumi, T. A. & Torchilin, V.
P., Eur. J. Nucl. Med. Mol. Imaging, 33: 1196-1205 (2006);
Mougin-Degraef, M. et al, Int'l J. Pharmaceutics, 344: 110-1 17
(2007).
[0366] In other embodiments, the diagnostic agents may include
optical agents such as fluorescent agents, phosphorescent agents,
chemiluminescent agents, and the like. Numerous agents (e.g., dyes,
probes, labels, or indicators) are known in the art and can be used
in the present invention. (See, e.g., Invitrogen, The Handbook--A
Guide to Fluorescent Probes and Labeling Technologies, Tenth
Edition (2005)). Fluorescent agents can include a variety of
organic and/or inorganic small molecules or a variety of
fluorescent proteins and derivatives thereof. For example,
fluorescent agents can include but are not limited to cyanines,
phthalocyanines, porphyrins, indocyanines, rhodamines,
phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines,
fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones,
tetracenes, quinolines, pyrazines, corrins, croconiums, acridones,
phenanthridines, rhodamines, acridines, anthraquinones,
chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine
dyes, indolenium dyes, azo compounds, azulenes, azaazulenes,
triphenyl methane dyes, indoles, benzoindoles, indocarbocyanines,
benzoindocarbocyanines, and BODIPY.TM. derivatives having the
general structure of 4,4-difiuoro-4-bora-3a,4a-diaza-s-indacene,
and/or conjugates and/or derivatives of any of these. Other agents
that can be used include, but are not limited to, fluorescein,
fluorescein-polyaspartic acid conjugates, fluorescein-polyglutamic
acid conjugates, fluorescein-polyarginine conjugates, indocyanine
green, indocyanine-dodecaaspartic acid conjugates, indocyanine
(NIRD)-polyaspartic acid conjugates, isosulfan blue, indole
disulfonates, benzoindole disulfonate,
bis(ethylcarboxymethyl)indocyanine,
bis(pentylcarboxymethyl)indocyanine, polyhydroxyindole sulfonates,
polyhydroxybenzoindole sulfonate, rigid heteroatomic indole
sulfonate, indocyaninebispropanoic acid, indocyaninebishexanoic
acid,
3,6-dicyano-2,5-[(N,N,N',N'-tetrakis(carboxymethyl)amino]pyrazine,
3,6-[(N,N,N',N'-tetrakis(2-hydroxyethyl)amino]pyrazine-2,5-dicarboxylic
acid, 3,6-bis(N-azatedino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-morpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-piperazino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid,
3,6-bis(N-thiomorpholino)pyrazine-2,5-dicarboxylic acid S-oxide,
2,5-dicyano-3,6-bis(N-thiomorpholino)pyrazine S,S-dioxide,
indocarbocyaninetetrasulfonate, chloroindocarbocyanine, and
3,6-diaminopyrazine-2,5-dicarboxylic acid.
[0367] In yet other embodiments, the diagnostic agents may include
contrast agents that are generally well known in the art,
including, for example, superparamagnetic iron oxide (SPIO),
complexes of gadolinium or manganese, and the like. (See, e.g.,
Armstrong et al, Diagnostic Imaging, 5.sup.th Ed., Blackwell
Publishing (2004)). In some embodiments, a diagnostic agent can
include a magnetic resonance (MR) imaging agent. Exemplary magnetic
resonance agents include but are not limited to paramagnetic
agents, superparamagnetic agents, and the like. Exemplary
paramagnetic agents can include but are not limited to Gadopentetic
acid, Gadoteric acid, Gadodiamide, Gadolinium, Gadoteridol ,
Mangafodipir, Gadoversetamide, Ferric ammonium citrate, Gadobenic
acid, Gadobutrol, or Gadoxetic acid. Superparamagnetic agents can
include but are not limited to superparamagnetic iron oxide and
Ferristene. In certain embodiments, the diagnostic agents can
include x-ray contrast agents as provided, for example, in the
following references: H. S Thomsen, R. N. Muller and R. F. Mattrey,
Eds., Trends in Contrast Media, (Berlin: Springer-Verlag, 1999); P.
Dawson, D. Cosgrove and R. Grainger, Eds., Textbook of Contrast
Media (ISIS Medical Media 1999); Torchilin, V. P., Curr. Pharm.
Biotech., vol. 1, pages 183-215 (2000); Bogdanov, A. A. et al, Adv.
Drug Del. Rev., Vol. 37, pages 279-293 (1999); Sachse, A. et al.,
Investigative Radiology, vol. 32, pages 44-50 (1997). Examples of
x-ray contrast agents include, without limitation, iopamidol,
iomeprol, iohexol, iopentol, iopromide, iosimide, ioversol,
iotrolan, iotasul, iodixanol, iodecimol, ioglucamide, ioglunide,
iogulamide, iosarcol, ioxilan, iopamiron, metrizamide, iobitridol
and iosimenol. In certain embodiments, the x-ray contrast agents
can include iopamidol, iomeprol, iopromide, iohexol, iopentol,
ioversol, iobitridol, iodixanol, iotrolan and iosimenol.
[0368] In some embodiments, the conditionally active biological
proteins may be conjugated to another protein, such as
interleukins, cytokines, enzymes, growth factors, or other
antibodies. Some examples of such proteins include, for example,
tumor necrosis factor, .alpha.-interferon (EFN-.alpha.),
.beta.-interferon (IFN-.beta.), nerve growth factor (NGF), platelet
derived growth factor (PDGF), tissue plasminogen activator (TPA),
an apoptotic agent (e.g., TNF-.alpha., TNF-.beta., AIM I as
disclosed in WO 97/33899), AIM II (see WO 97/34911), Fas Ligand
(Takahashi et al., J. Immunol., vol. 6, pages 1567-1574, 1994), and
VEGI (WO 99/23105), a thrombotic agent or an anti-angiogenic agent
(e.g., angiostatin or endostatin); or a biological response
modifier such as, for example, a lymphokine (e.g., interleukin-1
("IL-I"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"), and
granulocyte colony stimulating factor ("G-CSF")), or a growth
factor (e.g., growth hormone ("GH")).
[0369] In some embodiments, the conditionally active antibodies for
crossing the BBB as provided by the present invention may be
conjugated to a drug for treating a neurological disorder. The drug
will be transported across the BBB with the antibodies and remain
in the brain for treating the neurological disorder. The
neurological disorder refers to a disease or disorder which affects
the CNS and/or which has an etiology in the CNS. Exemplary CNS
diseases or disorders include, but are not limited to, neuropathy,
amyloidosis, cancer, an ocular disease or disorder, viral or
microbial infection, inflammation, ischemia, neurodegenerative
disease, seizure, behavioral disorders, and a lysosomal storage
disease. For the purposes of this application, the CNS will be
understood to include the eye, which is normally sequestered from
the rest of the body by the blood-retina barrier. Specific examples
of neurological disorders include, but are not limited to,
neurodegenerative diseases (including, but not limited to, Lewy
body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,
olivopontocerebellar atrophy, Parkinson's disease, multiple system
atrophy, striatonigral degeneration, tauopathies (including, but
not limited to, Alzheimer disease and supranuclear palsy), prion
diseases (including, but not limited to, bovine spongiform
encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru,
Gerstmann-Straussler-Scheinker disease, chronic wasting disease,
and fatal familial insomnia), bulbar palsy, motor neuron disease,
and nervous system heterodegenerative disorders (including, but not
limited to, Canavan disease, Huntington's disease, neuronal
ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome,
Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz
syndrome, lafora disease, Rett syndrome, hepatolenticular
degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg
syndrome), dementia (including, but not limited to, Pick's disease,
and spinocerebellar ataxia), cancer (e.g. of the CNS and/or brain,
including brain metastases resulting from cancer elsewhere in the
body).
[0370] The drugs for treating the neurological disorder include,
but are not limited to, antibodies, peptides, proteins, natural
ligands of one or more CNS target(s), modified versions of natural
ligands of one or more CNS target(s), aptamers, inhibitory nucleic
acids (i.e., small inhibitory RNAs (siRNA) and short hairpin RNAs
(shRNA)), ribozymes, and small molecules, or active fragments of
any of the foregoing. Exemplary neurological disorder drugs
include, but are not limited to: antibodies, aptamers, proteins,
peptides, inhibitory nucleic acids and small molecules and active
fragments of any of the foregoing that either are themselves or
specifically recognize and/or act upon (i.e., inhibit, activate, or
detect) a CNS antigen or target molecule such as, but not limited
to, amyloid precursor protein or portions thereof, amyloid beta,
beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin,
huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer
markers, and neurotrophins. Non-limiting examples of neurological
disorder drugs and disorders they may be used to treat include
anti-BACE1 antibody for treating Alzheimer's, acute and chronic
brain injury, stroke; anti-Abeta antibody for treating Alzheimer's
disease; neurotrophin for treating stroke, acute brain injury,
spinal cord injury; brain-derived neurotrophic factor (BDNF) and
fibroblast growth factor 2 (FGF-2) for treating chronic brain
injury (neurogenesis); anti-Epidermal Growth Factor Receptor
(EGFR)-antibodies for treating brain cancer; Glial cell-line
derived neural factor (GDNF) for treating Parkinson's disease;
brain-derived neurotrophic factor (BDNF) for treating Amyotrophic
lateral sclerosis and depression; lysosomal enzyme for treating
lysosomal storage disorders of the brain; Ciliary neurotrophic
factor (CNTF) for treating Amyotrophic lateral sclerosis;
Neuregulin-1 for treating Schizophrenia; and anti-HER2 antibody
(e.g. trastuzumab) for treating brain metastasis from HER2-positive
cancer.
[0371] In some embodiments, the conjugation of the conditionally
active biological proteins may be on the Fc region of the
antibodies. The conjugating molecules, compound or drugs described
above may be conjugated to the Fc region, as described in U.S. Pat.
No. 8,362,210 (incorporated herein by reference). For example, Fc
region may be conjugated to a cytokine or a toxin to be delivered
to the site where the conditionally active antibody displays
preferentially activity. Methods for conjugating polypeptides to
the Fc region of antibodies are known in the art. See, e.g., U.S.
Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851,
5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP
307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO
96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et
al., Proc. Natl. Acad. Sci. USA, vol. 88, pages 10535-10539, 1991;
Traunecker et al., Nature, vol. 331, pages 84-86, 1988; Zheng et
al., J. Immunol., vol. 154, pages 5590-5600, 1995; and ViI et al.,
Proc. Natl. Acad. Sci. USA, vol. 89, pages 11337-11341, 1992, which
are incorporated herein by reference in their entireties.
Engineering Multispecific Conditionally Active Antibodies
[0372] When the conditionally active biological proteins are
conditionally active antibodies, the conditionally active
antibodies may be engineered to generated multispecific
conditionally active antibodies. The multispecific antibody is an
antibody with polyepitopic specificity, as described in WO
2013/170168, incorporated herein by reference in its entirety.
Multispecific antibodies include, but are not limited to, an
antibody comprising a heavy chain variable domain (V.sub.H) and a
light chain variable domain (VL), where the V.sub.HV.sub.L unit has
polyepitopic specificity, antibodies having two or more V.sub.L and
V.sub.H domains where each V.sub.HV.sub.L unit binds to a different
epitope, antibodies having two or more single variable domains with
each single variable domain binding to a different epitope, and
antibodies comprising one or more antibody fragments as well as
antibodies comprising antibody fragments that have been linked
covalently or non-covalently.
[0373] To construct multispecific antibodies, including bispecific
antibodies, antibody fragments having at least one free sulfhydryl
group are obtained. The antibody fragments may be obtained from
full-length conditionally active antibodies. The conditionally
active antibodies may be digested enzymatically to produce antibody
fragments. Exemplary enzymatic digestion methods include, but are
not limited to, pepsin, papain and Lys-C. Exemplary antibody
fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv,
diabodies (Db); tandem diabodies (taDb), linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.,
vol. 8, pages 1057-1062 (1995)); one-armed antibodies, single
variable domain antibodies, minibodies (Olafsen et al (2004)
Protein Eng. Design & Sel., vol. 17, pages 315-323),
single-chain antibody molecules, fragments produced by a Fab
expression library, anti-idiotypic (anti-Id) antibodies, CDR
(complementary determining region), and epitope-binding fragments.
Antibody fragments may also be produced using DNA recombinant
technology. The DNA encoding the antibody fragments may be cloned
into plasmid expression vectors or phagemid vectors and expressed
directly in E. Coli. Antibody enzymatic digestion methods, DNA
cloning and recombinant protein expression methods are well known
to those skilled in the art.
[0374] Antibody fragments may be purified using conventional
techniques and are subjected to reduction to generate a free thiol
group. Antibody fragments having a free thiol group are reacted
with a crosslinker, for example, bis-maleimide. Such crosslinked
antibody fragments are purified and then reacted with a second
antibody fragment having a free thiol group. The final product in
which two antibody fragments are crosslinked is purified. In
certain embodiments, each antibody fragment is a Fab and the final
product, in which the two Fabs are linked through bis-maleimide, is
referred to herein as bismaleimido-(thio-Fab)2, or bis-Fab. Such
multispecific antibodies and antibody analogs, including bis-Fabs,
can be exploited to quickly synthesize a large number of antibody
fragment combinations, or structural variants of native antibodies
or particular antibody fragment combinations.
[0375] Multispecific antibodies can be synthesized with modified
crosslinkers such that additional functional moieties may be
attached to the multispecific antibody. Modified crosslinkers allow
for attachment of any sulfhydryl-reactive moiety. In one
embodiment, N-succinimidyl-S-acetylthioacetate (SAT A) is attached
to bis-maleimide to form bis-maleimido-acetylthioacetate (BMata).
After deprotection of the masked thiol group, any functional group
having a sulfhydryl-reactive (or thiol-reactive) moiety may be
attached to the multispecific antibodies.
[0376] Exemplary thiol-reactive reagents include a multifunctional
linker reagent, a capture, i.e. affinity, label reagent (e.g. a
biotin-linker reagent), a detection label (e.g. a fluorophore
reagent), a solid phase immobilization reagent (e.g. SEPHAROSE.TM.,
polystyrene, or glass), or a drug-linker intermediate. One example
of a thiol-reactive reagent is N-ethyl maleimide (NEM). Such
multispecific antibodies or antibody analogs having modified
crosslinkers may be further reacted with a drug moiety reagent or
other label. Reaction of a multispecific antibody or antibody
analog with a drug-linker intermediate provides a multispecific
antibody drug conjugate or antibody analog drug conjugate,
respectively.
[0377] Many other techniques for making multispecific antibodies
may also be used in the present invention. References (incorporated
herein by references) describing these techniques include: (1)
Milstein and Cuello, Nature, vol. 305, page 537 (1983)), WO
93/08829, and Traunecker et al., EMBO J., vol. 10, page 3655 (1991)
on recombinant co-expression of two immunoglobulin heavy
chain-light chain pairs having different specificities; (2) U.S.
Pat. No. 5,731,168 on "knob-in-hole" engineering; (3) WO
2009/089004A1 on engineering electrostatic steering effects for
making antibody Fc-heterodimeric molecules; (4) U.S. Pat. No.
4,676,980, and Brennan et al., Science, vol. 229, page 81 (1985) on
cross-linking two or more antibodies or fragments; (5) Kostelny et
al., J. Immunol., vol. 148, pages 1547-1553 (1992) on using leucine
zippers to produce bi-specific antibodies; (6) Hollinger et al.,
Proc. Natl. Acad. Sci. USA, vol. 90, pages 6444-6448 (1993) on
using "diabody" technology for making bispecific antibody
fragments; (7) Gruber et al., J. Immunol., vol. 152, page 5368
(1994) on using single-chain Fv (sFv) dimers; (8) Tutt et al. J.
Immunol. 147: 60 (1991) on preparing trispecific antibodies; and
(9) US 2006/0025576A1 and Wu et al. Nature Biotechnology, vol. 25,
pages 1290-1297 (2007) on engineered antibodies with three or more
functional antigen binding sites, including "Octopus antibodies" or
"dual-variable domain immunoglobulins" (DVDs).
[0378] Multispecific antibodies of the present invention might also
be generated as described in WO/2011/109726, incorporated herein by
reference in its entirety.
[0379] In one embodiment, the conditionally active antibody for
crossing the BBB is engineered to make a multispecific antibody
(e.g. a bispecific antibody). This multispecific antibody comprises
a first antigen binding site which binds a BBB-R and a second
antigen binding site which binds a brain antigen. At least the
first antigen binding site for BBB-R is conditionally active. A
brain antigen is an antigen expressed in the brain, which can be
targeted with an antibody or small molecule. Examples of such
antigens include, without limitation: beta-secretase 1 (BACE1),
amyloid beta (Abeta), epidermal growth factor receptor (EGFR),
human epidermal growth factor receptor 2 (HER2), Tau,
apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion
protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin,
presenilin 1, presenilin 2, gamma secretase, death receptor 6
(DR6), amyloid precursor protein (APP), p75 neurotrophin receptor
(p75NTR), and caspase 6. In one embodiment, the antigen is
BACE1.
[0380] Multispecific antibodies have high selectivity at
preferentially targeting tissues containing all or most of the
targets (antigens) that a multispecific antibody can bind to. For
example, a bispecific antibody provides selectivity for target
cells by displaying greater preference to target cells that express
both of the antigens recognized by the bispecific antibody, in
comparison with non-target cells that may express only one of the
antigens. Therefore, due to the dynamism of the system, there are
more bispecific antibodies being bound to the target cells than
non-target cells at equilibrium.
Engineering the Fc Region of Conditionally Active Antibodies
[0381] When the conditionally active biological proteins are
conditionally active antibodies, the conditionally active
antibodies may be engineered at their fragment crystallizable
region (Fc region). The Fc region is the tail region of an antibody
that interacts with cell surface receptors called Fc receptors and
some proteins of the complement system. Unlike the Fab region that
is specific for each antigen, the Fc region of all antibodies in a
class is the same for each species regardless which antigen the
antibody binds.
[0382] The Fc receptors are members of the immunoglobulin gene
superfamily of proteins. Fc receptors are found on a number of
cells in the immune system including phagocytes like macrophages
and monocytes, granulocytes like neutrophils and eosinophils, and
lymphocytes of the innate immune system (natural killer cells) or
adaptive immune system (e.g., B cells). After binding with an
antibody, the Fc receptor activates these cells and allows these
cells to identify and eliminate antigens (such as microbial
pathogens) that are bound on the Fab region of the antibody. The Fc
receptor mediated killing mechanisms include complement-dependent
cytotoxicity (CDC), antibody-dependent cellular cytotoxicity
(ADCC), and antibody-dependent cellular phagocytosis (ADCP).
[0383] In some embodiments, the Fc region is engineered to
introduce mutations such as amino acid substitutions in the Fc
region. Such substitution in the Fc region may increase the
half-life of the mutated antibody in serum. For example, the
half-life of an IgG antibody is correlated with its pH-dependent
binding to neonatal receptor FcRn, which is expressed on the
surface of endothelial cells and protects the IgG in a pH-dependent
manner from degradation. Several amino acid substitutions at the Fc
region, such as T250Q/M428L and M252Y/S254T/T256E+H433K/N434F, have
shown increased binding affinity of the antibody to FcRn and extend
the half-life of the antibody.
[0384] Amino acid substitutions may also be introduced to the Fc
region to alter effector functions. For example, human antibodies
in the IgG class bind to Fcy receptors (Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIIa), the inhibitory Fc.gamma.RIIb receptor, and the
first component of complement (C1q) with different affinities,
yielding very different effector functions among different
antibodies. Binding of IgG antibody to Fc.gamma.Rs or C1q depends
on residues located in the hinge domain and the CH2 domain of the
antibody. Amino acid substitutions in human antibodies IgG1 or IgG2
residues at positions 233-236 and antibody IgG4 residues at
positions 327, 330 and 331 can greatly reduce ADCC and CDC.
Furthermore, alanine substitution at different positions in the Fc
region, including K322, significantly reduced complement
activation. Many more examples of engineering the Fc region are
described in U.S. Pat. No. 8,362,210, which is incorporated by
reference in its entirety.
[0385] In some embodiments, the Fc region of an antibody may be
engineered to be capable of recognizing an antigen (US
2010/0256340, incorporated herein by reference). At least one,
preferably two, extra Fab fragments may be linked onto the Fc
region of an antibody. In some embodiments, the extra Fab fragments
are conditionally active. For example, the antibody of the present
invention for crossing the BBB may contain such an extra Fab
fragment with affinity for a BBB-R on the plasma side and little or
no affinity to the BBB-R on the brain side. The antibody can also
bind to multiple brain antigens, thus may have a higher selectivity
for preferentially acting on sites where these antigens are
present.
Pharmaceutical Compositions
[0386] The present disclosure provides at least one composition
comprising (a) a conditionally active biologic protein; and (b) a
suitable carrier or diluent. The present disclosure also provides
at least one composition comprising (a) a conditionally active
biologic protein encoding nucleic acid as described herein; and (b)
a suitable carrier or diluent. The carrier or diluent can
optionally be pharmaceutically acceptable, according to known
carriers or diluents. The composition can optionally further
comprise at least one further compound, protein or composition. In
some embodiment, the conditionally active biologic protein is a
conditionally active antibody.
[0387] The conditionally active biologic protein may be in the form
of a pharmaceutically acceptable salt. Pharmaceutically acceptable
salts means which can be generally used as salts of an therapeutic
protein in pharmaceutical industry, including for example, salts of
sodium, potassium, calcium and the like, and amine salts of
procaine, dibenzylamine, ethylenediamine, ethanolamine,
methylglucamine, taurine, and the like, as well as acid addition
salts such as hydrochlorides, and basic amino acids and the
like.
[0388] The present disclosure further provides at least one
conditionally active biologic protein method or composition, for
administering a therapeutically effective amount to modulate or
treat at least one parent molecule related condition in a cell,
tissue, organ, animal or patient and/or, prior to, subsequent to,
or during a related condition, as known in the art and/or as
described herein. Thus, the disclosure provides a method for
diagnosing or treating a condition associated with the wild-type
protein in a cell, tissue, organ or animal, comprising contacting
or administering a composition comprising an effective amount of at
least one conditionally active biologic protein of the disclosure
with, or to, the cell, tissue, organ or animal. The method can
optionally further comprise using an effective amount of 0.001-50
mg/kilogram of a conditionally active biologic protein of the
disclosure to the cells, tissue, organ or animal. The method can
optionally further comprise using the contacting or the
administrating by at least one mode selected from parenteral,
subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
The method can optionally further comprise administering, prior,
concurrently, or after the conditionally active biologic protein
contacting or administering at least one composition comprising an
effective amount of at least one compound or protein selected from
at least one of a detectable label or reporter, a TNF antagonist,
an antirheumatic, a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAK)), an analgesic, an anesthetic, a
sedative, a local anesthetic, a neuromuscular blocker, an
antimicrobial, an antipsoriatic, a corticosteriod, an anabolic
steroid, an erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog thereof, a cytotoxic or other
anti-cancer agent, an anti-metabolite such as methotrexate, or an
antiproliferative agent.
[0389] The present disclosure further provides at least one
conditionally active biologic protein method for diagnosing at
least one wild-type protein related condition in a cell, tissue,
organ, animal or patient and/or, prior to, subsequent to, or during
a related condition, as known in the art and/or as described
herein.
[0390] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention. A variety of aqueous
carriers can be used, e.g., buffered saline and the like. These
solutions are sterile and generally free of undesirable matter.
These compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
conditionally active biologic protein in these formulations can
vary widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the patient's
needs.
[0391] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Pharmaceutical compositions and
formulations of the invention for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in appropriate and suitable dosages. Such carriers enable
the pharmaceuticals to be formulated in unit dosage forms as
tablets, pills, powder, dragees, capsules, liquids, lozenges, gels,
syrups, slurries, suspensions, etc., suitable for ingestion by the
patient. Pharmaceutical preparations for oral use can be formulated
as a solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
additional compounds, if desired, to obtain tablets or dragee
cores. Suitable solid excipients are carbohydrate or protein
fillers include, e.g., sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose such as methyl cellulose,
hydroxypropyhnethyl cellulose, or sodium carboxy-methylcellulose;
and gums including arabic and tragacanth; and proteins, e.g.,
gelatin and collagen. Disintegrating or solubilizing agents may be
added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate. Tablet
forms can include one or more of lactose, sucrose, mannitol,
sorbitol, calcium phosphates, corn starch, potato starch,
tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide, croscannellose sodium, talc, magnesium stearate,
stearic acid, and other excipients, colorants, fillers, binders,
diluents, buffering agents, moistening agents, preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically
acceptable carriers.
[0392] The invention provides aqueous suspensions comprising a
conditionally active biologic protein, in admixture with excipients
suitable for the manufacture of aqueous suspensions. Such
excipients include a suspending agent, such as sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethylene oxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
(e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty
acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan
mono-oleate). The aqueous suspension can also contain one or more
preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or
more coloring agents, one or more flavoring agents and one or more
sweetening agents, such as sucrose, aspartame or saccharin.
Formulations can be adjusted for osmolality.
[0393] Lozenge forms can comprise the active ingredient in a
flavor, usually sucrose and acacia or tragacanth, as well as
pastilles comprising the active ingredient in an inert base, such
as gelatin and glycerin or sucrose and acacia emulsions, gels, and
the like containing, in addition to the active ingredient, carriers
known in the art. It is recognized that the conditionally active
biologic protein, when administered orally, must be protected from
digestion. This is typically accomplished either by complexing the
conditionally active biologic protein with a composition to render
it resistant to acidic and enzymatic hydrolysis or by packaging the
conditionally active biologic protein in an appropriately resistant
carrier such as a liposome. Means of protecting proteins from
digestion are well known in the art. The pharmaceutical
compositions can be encapsulated, e.g., in liposomes, or in a
formulation that provides for slow release of the active
ingredient.
[0394] The packaged conditionally active biologic protein, alone or
in combination with other suitable components, can be made into
aerosol formulations (e.g., they can be "nebulized") to be
administered via inhalation. Aerosol formulations can be placed
into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. Suitable
formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a
suppository base. Suitable suppository bases include natural or
synthetic triglycerides or paraffin hydrocarbons, in addition, it
is also possible to use gelatin rectal capsules which consist of a
combination of the packaged nucleic acid with a base, including,
for example, liquid triglycerides, polyethylene glycols, and
paraffin hydrocarbons.
[0395] Dermal or topical delivery compositions of the invention may
include in addition to a conditionally active biologic protein, a
pharmaceutically acceptable carrier in a cream, ointment, solution
or hydrogel formulation, and other compounds so long as the added
component does not deleteriously affect delivery of the therapeutic
protein. Conventional pharmaceutically acceptable emulsifiers,
surfactants, suspending agents, antioxidants, osmotic enhancers,
extenders, diluents and preservatives may also be added. Water
soluble polymers can also be used as carriers.
[0396] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and nonaqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives, in the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. In one aspect, parenteral modes of
administration are preferred methods of administration for
compositions comprising a conditionally active biologic protein.
The compositions may conveniently be administered in unit dosage
form and may be prepared by any of the methods well-known in the
pharmaceutical art, for example as described in Remington's
Pharmaceutical Sciences, Mack Publishing Co. Easton Pa., 18.sup.th
Ed., 1990. Formulations for intravenous administration may contain
a pharmaceutically acceptable carrier such as sterile water or
saline, polyalkylene glycols such as polyethylene glycol, oils of
vegetable origin, hydrogenated naphthalenes and the like. Also see
and adapt the description in U.S. Pat. No. 4,318,905.
[0397] The formulations of packaged compositions comprising a
conditionally active biologic protein can be presented in unit-dose
or multi-dose sealed containers, such as ampoules and vials.
Injection solutions and suspensions can be prepared from sterile
powders, granules, and tablets of the kind previously
described.
[0398] The present disclosure also provides at least one
conditionally active biologic protein composition, device and/or
method of delivery for diagnosing of at least one wild-type protein
related condition, according to the present disclosure.
[0399] Also provided is a composition comprising at least one
conditionally active biologic protein and at least one
pharmaceutically acceptable carrier or diluent. The composition can
optionally further comprise an effective amount of at least one
compound or protein selected from at least one of a detectable
label or reporter, a cytotoxic or other anti-cancer agent, an
anti-metabolite such as methotrexate, an antiproliferative agent, a
cytokine, or a cytokine antagonist, a TNF antagonist, an
antirheumatic, a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local anesthetic, a neuromuscular blocker, an
antimicrobial, an antipsoriatic, a corticosteriod, an anabolic
steroid, an erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a
stimulant, an asthma medication, a beta agonist, an inhaled
steroid, an epinephrine or analog.
[0400] Also provided is a medical device, comprising at least one
conditionally active biologic protein of the disclosure, wherein
the device is suitable to contacting or administering the at least
one conditionally active biologic protein by at least one mode
selected from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or
transdermal.
[0401] In a further aspect, the disclosure provides a kit
comprising at least one conditionally active biologic protein or
fragment of the disclosure in lyophilized form in a first
container, and an optional second container comprising sterile
water, sterile buffered water, or at least one preservative
selected from the group consisting of phenol, m-cresol, p-cresol,
o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride,
alkylparaben, benzalkonium chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous
diluent. In one aspect, in the kit, the concentration of
conditionally active biologic protein or specified portion or
variant in the first container is reconstituted to a concentration
of about 0.1 mg/ml to about 500 mg/ml with the contents of the
second container, in another aspect, the second container further
comprises an isotonicity agent. In another aspect, the second
container further comprises a physiologically acceptable buffer. In
one aspect, the disclosure provides a method of treating at least
one wild-type protein mediated condition, comprising administering
to a patient in need thereof a formulation provided in a kit and
reconstituted prior to administration.
[0402] Also provided is an article of manufacture for human
pharmaceutical or diagnostic use, comprising packaging material and
a container comprising a solution or a lyophilized form of at least
one conditionally active biologic protein of the present
disclosure. The article of manufacture can optionally comprise
having the container as a component of a parenteral, subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracelebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, or transdermal delivery device or
system.
[0403] The present disclosure further provides any disclosure
described herein.
EXAMPLE 1
General Description of a Multiwall Assay (for Example, 96-Well
Assay) for Temperature Mutants
[0404] Fluorescent substrate is added to each well of a multiwall
plate, at both wild-type and new, lower reaction temperatures (for
example, either 37.degree. C. or 25.degree. C. as mentioned above)
for an appropriate time period. Fluorescence is detected by
measuring fluorescence in a fluorescent plate reader at appropriate
excitation and emission spectra (for example, 320 nm exitation/405
nm emission). Relative fluorescence units (RFU) are determined.
Supernatant from wild type molecule and plasmid/vector transformed
cells are used as positive and negative controls. Duplicate
reactions are performed for each sample, reaction temperature, and
positive and negative control.
[0405] Mutants that are active at the lower temperature (for
example, the mutants active at 25.degree. C.) and that have a
decrease in activity at the wild type temperature (for example, a
10%, 20%, 30%, 40% or more decrease in activity at 37.degree. C.),
thus having a ratio of activities greater than or equal to about
1.1 or more (e.g., the ratio of the activities at 25.degree. C. or
37.degree. C. (25.degree. C./37.degree. C.) is greater than or
equal to 1.1 or more), can be deemed to be putative primary
temperature sensitive hits. These putative primary temperature
sensitive hits can then be rescreened, using the same assay, to
confirm any primary hits.
EXAMPLE 2
General Description of a Different Assay Format for Confirmation of
Activity (for Example, a 14-mL Assay) for Temperature Mutants
[0406] Mutants that are identified as temperature sensitive primary
hits are expressed in 14 ml culture tubes and their enzymatic
activity is measured at wild type (for example, 37.degree. C.) and
the lower temperature (for example, 25.degree. C.). Protein is
expressed and purified as described above for the multiwall format,
with the exception that the expression is performed in different
format (14 ml tubes) rather than the multiwall (96-well plate)
format.
[0407] Each mutant supernatant is transferred to a multiwall plate,
for example a 96-well microplate. Fluorescent substrate is added to
each tube at the indicated reaction temperatures (wild-type, lower
temperature) for a required period of time. Wild-type molecules are
used as a positive control and supernatant from cells transformed
with only vector is used as a negative control. Fluorescence is
detected by measuring fluorescence in a fluorescent plate reader at
the appropriate emission spectra (for example, 320 nm exitation/405
ran emission). Relative fluorescence units (RFU) are determined.
Duplicate reactions can are performed for each sample, reaction
temperature, and positive and negative control.
[0408] Mutants that are active at the lower temperatures (for
example, 25.degree. C.) but that demonstrate at least a 30% or more
decreased activity at wild type (for example, 37.degree. C.), thus
have a ratio of activity at lower temperature (for example,
25.degree. C.) to wild type temperature (for example, 37.degree.
C.) equal to or greater than 1.5, are identified as temperature
sensitive hits.
[0409] The activities of mutants at the lower temperature (for
example 25.degree. C.) are compared to the activity of the
wild-type molecule at the wild-type temperature (for example
37.degree. C.). If molecules are more active than the wild-type
molecules at the lower temperature (for example 25.degree. C.), as
indicated by a residual activity>1, preferably 2 or greater than
2, and if the mutants demonstrate an overall decrease in activity
when compared to the wild-type molecule at the wild-type
temperature (37.degree. C.), the phenotype of the mutants as
temperature sensitive mutants can be confirmed.
EXAMPLE 3
General Description of Further Evolution of Hits Discovered
[0410] If desired, a new, combinatorial variant library is
generated from all or selected mutant hits previously identified.
The new library can be designed to contain every possible
combination of amino acid variants for each of the selected
mutants, and rescreened as described for new hits.
[0411] It is to be understood, however, that even though numerous
characteristics and advantages of the present invention have been
set forth in the foregoing description, together with details of
the structure and function of the invention, the disclosure is
illustrative only, and changes may be made in detail, especially in
matters of shape, size and arrangement of parts within the
principles of the invention to the full extent indicated by the
broad general meanings of the terms in which the appended claims
are expressed.
* * * * *
References