Note: Descriptions are shown in the official language in which they were submitted.
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MELANOCORTIN RECEPTOR BINDING MIMETIBODIES,
COMPOSITIONS, METHODS AND USES
Cross-Reference to Related Applications
This application is a continuation-in-part of United States
Application Serial No. 11/257,851, filed 25 October 2005, which
claims priority to United States Provisional Application Serial No.
60/621,960, filed 25 October 2004. This application also claims the
benefit of United States Provisional Application Serial No.
60/972,018, filed 13 September 2007. The entire contents of each of
the aforementioned applications is incorporated herein by reference
in their entirety.
Field of the Invention
The present invention relates to melanocortin receptor binding
mimetibodies, polynucleotides encoding the mimetibodies, cells
comprising the polynucleotides or expressing the mimetibodies, and
methods of making and using the foregoing.
Background of the Invention
Obesity is a chronic disease manifested by an excess of fat
mass in proportion to body size. Today, every third American is
considered overweight (Body Mass Index (BMI) >25 kg/m2), thus
prompting the United States Centers for Disease Control and
Prevention (CDC) to declare that obesity is reaching epidemic
proportions (Cummings and Schwartz, Annu. Rev. Med. 54:453-
471((2003)). The importance of treating obesity is emphasized by
the fact that this disease is either the underlying cause, or a risk
factor, for developing diseases such as Type 2 Diabetes, congestive
heart failure, osteoarthritis and sleep apnea among others.
Additionally, obesity is linked to "Metabolic Syndrome" which
is a medical condition characterized by obesity, atherogenic
dyslipidemia, elevated blood pressure and insulin resistance.
Metabolic Syndrome affects an increasing number of people in the
United States. Importantly, it has been shown that even a modest
decrease in body weight (5-10% of initial body weight) may
significantly improve Metabolic Syndrome conditions and decrease the
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risk factors for developing obesity-associated disease (Wing et al.,
Arch. Intern. Med. 147:1749-1753 (1987); Tuomilehto et al., New
Engl. J. Med. 344:1343-1350 (2001); Knowler et al., New Engl. J Med.
346:393-403 (2002); Franz et al., Diabetes Care 25:148-198 (2002)).
Additionally, treatment of obesity may be important from a mental
health perspective due to the social stigma often attached to obese
individuals in some cultures.
Melanocortin receptors play a major role in the regulation of
overall energy balance and obesity in both humans and rodents.
Alpha-melanocyte stimulating hormone (alpha-MSH) is a 13 amino acid
peptide hormone that is an important component of the melanocortin
system. Alpha-MSH is produced by the proteolytic processing of
proopiomelanocortin (POMC) released by the pituitary gland. Alpha-
MSH binds with high affinity to the melanocortin 4 receptor (MC4R),
but also binds melanocortin receptor 3 (MC3R) and melanocortin
receptor 5 (MC5R) with lower affinity. MC4R is a G-coupled protein
receptor found in the brain which, when stimulated by alpha-MSH
binding, causes decreased food intake and increased fat oxidation.
Ultimately, stimulation of melanocortin receptors such as MC4R
results in weight loss.
In humans and rodents, loss of function mutations in the
different components of the melanocortin system are closely
correlated with obesity and related conditions. In mice, mutations
within POMC, or MC4R and MC3R produce obesity, insulin resistance
and hyperphagia (Goodfellow and Saunders, Curr. Topics Med. Chem. 3:
855-883 (2003); Huszar et al., Cell 88:131-141 (1997); Yaswen et
al., Nat. Med. 5: 1066-1070 (1999)). In man, mutations within POMC
or MC4R lead to the development of obesity associated with increased
food intake (Krude et al., Nat. Genet. 19:155-157 (1998); Yeo et
al., Nature Genetics 20:111-112 (1998); Branson et al., New Engl. J.
Med. 348: 1096-1103 (2003); Vaisse et al., J. Clin. Invest.
106):253-262 (2000); Ho and MacKenzie, J. Biol. Chem. 275: 35816-
35822 (1999)).
Weight loss can result from the pharmacological stimulation of
melanocortin system activity. In rodents pharmacological
stimulation of melanocortin receptors such as MC4R leads to
decreased food intake, increased energy expenditure and weight loss
(Pierroz et al., Diabetes 51: 1337-1345 (2002)). In man, the
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intranasal administration of alpha-MSH to stimulate MC4R in non-
obese men results in decreased body weight due to the loss of fat-
but not lean body mass (Fehm et al., J. Clin. Endo. Metabol. 86:
1144-1148 (2001)).
Obesity is currently treated, with only limited success, by
several different strategies. These strategies primarily involve
"life-style" changes (e.g., diet and exercise), small molecule-based
pharmaceutical therapies or surgical removal of a portion of the
stomach (gastric by-pass surgery). Additionally, weight loss
stimulating melanocortin receptor binding peptides such as alpha-MSH
are of limited use as pharmaceuticals due to the extremely short serum
half-life of such peptides.
Alpha-MSH also plays a role in enhancing male erectile activity.
Targeting the melanocortin receptor with the synthetic melanocortin
receptor activator molecule melanotan II (MTII) produced an unexpected
side effect of enhancing erectile dysfunction (20). MTII has also
been shown to initiate erections in rodents and humans without sexual
stimulation in contrast to selective MC4 receptor agonists. Thus,
both MC3 and MC4 receptors are likely necessary for complete
proerectile erections. Clinical data showed a statistically
significant erectile response in healthy male subjects following
intranasal or subcutaneous administration of the MTII derivative, PT-
141 (bremelanotide) (22, 23). Male erectile dysfunction (ED) is
currently treated primarily with PDE5 inhibitors such as VIAGRA ,
CIALIS and LEVITRA . However, these agents are required to be taken
orally approximately one hour before sexual activity.
Bremelanotide is also being tested for use in treating female
sexual dysfunction (FSD). The American Foundation for Urologic
Disease defines FSD as: "The persistent or recurrent inability to
attain or maintain sufficient sexual excitement, causing personal
distress. It may be expressed as a lack of subjective excitement or a
lack of genital or other somatic responses." FSD consists of four
components, hypoactive sexual desire disorder, female sexual arousal
disorder (FSAD), anorgasmia and dyspareunia. Some form of FSD appears
to be prevalent in approximately 43 percent of the female population.
Laumann et al., JAMA 281, 537-544 (1999).
Alpha-MSH also functions as a cytokine antagonist that inhibits
inflammation caused by some of the most prominent mediators of local
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inflammation (31). For example, alpha-MSH inhibits production and
action of proinflammatory cytokines and chemokines (32, 33). Alpha-
MSH also inhibits macrophage production of cytotoxic nitric oxide (NO)
and neopterin (34,35), prostaglandin E synthesis (37). Alpha-MSH also
activates descending anti-inflammatory neural pathways dependent on
peripheral beta 2-adrenergic receptors (38) and increases production
of interleukin-10 (39).
In allergic inflammation, the anti-inflammatory effects of
alpha-MSH peptides were confirmed in acute skin inflammation induced
by nonspecific irritants and cytokines (42-46). In addition to its
suppressive effect on induction and elicitation of contact
hypersensitivity, alpha-MSH induces hapten-specific tolerance in mice
through IL-10 release (47).
In gouty (acute) arthritis, ACTH had an anti-inflammatory effect
in a rat model of gouty arthritis. The same authors also showed that
targeting MC3R subtype could be useful for clinical management of
human gouty arthritis and possibly other acute arthritis (48). In
rheumatoid arthritis, treatment of rats, who had a preclinical
adjuvant-induced rheumatoid arthritis, with alpha-MSH significantly
reduced joint pathology. Effectiveness of alpha-MSH was reported to
be similar to that of prednisolone.
In inflammatory bowel disease, alpha-MSH administered to mice
with dextran sulfate-induced colitis had reduced fecal blood and less
weight loss compared to mice receiving placebo (51). Alpha-MSH
administration reduced colonic macroscopic lesions in both acute and
chronic colitis induced by trinitrobenzosulfonic acid in rats (52).
In a mouse model of bilateral renal ischemia, alpha-MSH
significantly reduced ischemia-induced renal damage (32).
In liver inflammation and fibrosis, alpha-MSH gene therapy
reversed established liver fibrosis in CC14-treated mice (60) . In
another study, alpha-MSH inhibited systemic NO production, hepatic
neutrophil infiltration and increased hepatic mRNA abundance for TNF-
alpha and neutrophil and monocyte chemokines (33).
In ischemic brain damage (stroke), alpha-MSH treatment abolishes
intracerebral proinflammatory cytokine gene expression after transient
cerebral ischemia and indicates that systemically administered
melanocortins may exert neuroprotective efects in cerebral ischemia.
This study showed that alpha-MSH reduced activation of intracerebral
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TNF-alpha and IL1 beta gene expression after arterial occlusion and
reperfusion (40). In another study, melanocortins provided strong
protection, with a broad therapeutic window, against inflammatory,
apoptotic (incl DNA damage), and histopathological and behavioral
consequences of brain ischemia by activating CNS melanocortin 4 (MC4)
receptors (41).
In peripheral neuropathies, alpha-MSH and ACTH demonstrated that
both peptides stimulated axonal outgrowth from fetal spinal cord
slices in vitro in a dose-dependent manner (53). Also, alpha-MSH
promoted sprouting and neurite elongation from dissociated rat spinal
and sensory neurons (55).
A need exists for additional treatments for the conditions
discussed above and in particular for melanocortin receptor binding
molecules with a potentially fast onset of action that overcome the
short serum half-life of melanocortin receptor binding peptides such
as alpha-MSH.
Brief Description of the Drawings
Fig. 1 shows elements of a melanocortin receptor binding
mimetibody polypeptide.
Fig 2 is a cartoon of a melanocortin receptor binding
mimetibody.
Fig. 3 shows the amino acid (SEQ ID NO: 62) and cDNA (SEQ ID
NO: 61) sequences of a melanocortin receptor binding alpha-MSH
mimetibody. The amino terminal portions of individual mimetibody
elements are underlined.
Fig. 4 shows alpha-MSH mimetibody binding to MC4R in a
competitive binding assay.
Fig. 5 shows alpha-MSH mimetibody activation of MC4R in cells
expressing a high level of MC4R.
Fig. 6 shows alpha-MSH mimetibody activation of MC4R in cells
expressing a low level of MC4R.
Fig. 7 shows alpha-MSH mimetibody-mediated decrease in animal
food intake.
Fig. 8 shows alpha-MSH mimetibody-mediated decrease in animal
body weight.
Summary of the Invention
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One aspect of the invention is a polypeptide according to
formula (I):
(Mp-Lk- (V2) y-Hg-CH2-CH3) (t)
(I)
where Mp is a biologically active melanocortin receptor binding
molecule fragment of SEQ ID NO: 87, 89, 91, 93, 95, 97, or 282, Lk
is a polypeptide or chemical linkage, V2 is a portion of a C-
terminus of an immunoglobulin variable region, Hg is at least a
portion of an immunoglobulin variable hinge region, CH2 is an
immunoglobulin heavy chain CH2 constant region and CH3 is an
immunoglobulin heavy chain CH3 constant region, y is 0 or 1, and t
is independently an integer from 1 to 10.
Another aspect of the invention is a polypeptide comprising a
polypeptide having the sequence shown in SEQ ID NO: 121, 123, 127,
129, 132, 134, 137, 139, 142, 144, 147, 149, 152, 154, 157, 159,
162, 164, 167, 169, 172, 174, 177, 179, 182, 184, 187, 189, 192,
194, 197, 199, 202, 204, 207, 209, 212, 214, 217, 219, 222, 224,
227, 229, 232, 234, 237, 239, 242, 244, 251, 253, 256, 258, 261,
263, 266, or 268.
Another aspect of the invention is a polypeptide having the
sequence shown in SEQ ID NO: 212.
Another aspect of the invention is a polynucleotide comprising
a polynucleotide having the sequence shown in SEQ ID NO: 120, 122,
126, 128, 131, 133, 136, 138, 141, 143, 146, 148, 151, 153, 156,
158, 161, 163, 166, 168, 171, 173, 176, 178, 181, 183, 186, 188,
191, 193, 196, 198, 201, 203, 206, 208, 211, 213, 216, 218, 221,
223, 226, 228, 231, 233, 236, 238, 241, 243, 250, 252, 255, 257,
260, 262, 265, or 267 or a polynucleotide having a sequence
complementary to the sequence shown in SEQ ID NO: 120, 122, 126,
128, 131, 133, 136, 138, 141, 143, 146, 148, 151, 153, 156, 158,
161, 163, 166, 168, 171, 173, 176, 178, 181, 183, 186, 188, 191,
193, 196, 198, 201, 203, 206, 208, 211, 213, 216, 218, 221, 223,
226, 228, 231, 233, 236, 238, 241, 243, 250, 252, 255, 257, 260,
262, 265, or 267.
Another aspect of the invention is a polynucleotide comprising
a polynucleotide encoding the polypeptide having the sequence shown
in SEQ ID NO: 121, 123, 127, 129, 132, 134, 137, 139, 142, 144, 147,
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149, 152, 154, 157, 159, 162, 164, 167, 169, 172, 174, 177, 179,
182, 184, 187, 189, 192, 194, 197, 199, 202, 204, 207, 209, 212,
214, 217, 219, 222, 224, 227, 229, 232, 234, 237, 239, 242, 244,
251, 253, 256, 258, 261, 263, 266, or 268.
Another aspect of the invention is a pharmaceutical
composition comprising a mimetibody composition of the invention.
Another aspect of the invention is a method of modifying the
biological activity of a melanocortin receptor in a cell, tissue or
organ, comprising contacting a mimetibody composition of the
invention with the cell, tissue or organ.
Another aspect of the invention is a method of modulating at
least one melanocortin receptor mediated condition comprising
administering a mimetibody composition of the invention to a patient
in need thereof.
Detailed Description of the Invention
All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as though fully set forth.
The present invention provides polypeptides having the
properties of binding a melanocortin receptor and mimicking
different isotypes of antibody immunoglobulin molecules such as IgA,
IgD, IgE, IgG, or IgM, and any subclass thereof, such as IgA1, IgA2,
IgG1, IgG2, IgG3 or IgG4, or combinations thereof, herein after
generally referred to as "mimetibodies." In some embodiments, the
mimetibody polypeptides of the invention contain an alpha melanocyte
stimulating hormone peptide (alpha-MSH) sequence and are designated
melanocortin receptor binding alpha-MSH mimetibody. Such alpha-MSH
mimetibody polypeptides can bind melanocortin receptor 4 (MC4R) and
MCR5 with equal affinity and MC5R with lower affinity. One result
of such melanocortin receptor binding can be the stimulation or
inhibition of melanocortin receptor activity. Stimulation or
inhibition of melanocortin receptor activity can be useful for
treatment of melanocortin receptor mediated conditions.
In one embodiment, the polypeptides of the invention have the
generic formula (I):
(Mp-Lk- (V2) y-Hg-CH2-CH3) (t)
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(I)
where Mp is a melanocortin receptor binding molecule, Lk is a
polypeptide or chemical linkage, V2 is a portion of a C-terminus of
an immunoglobulin variable region, Hg is at least a portion of an
immunoglobulin variable hinge region, CH2 is an immunoglobulin heavy
chain CH2 constant region and CH3 is an immunoglobulin heavy chain
CH3 constant region, y is 0 or 1, and t is independently an integer
of 1 to 10.
As used herein, "melanocortin receptor binding molecule" means
a molecule, which can bind at least one melanocortin receptor such
as Homo sapiens MC4R (SEQ ID NO: 77). Examples of other Homo
sapiens melanocortin receptors include MCR1 (SEQ ID NO: 71), MCR2
(SEQ ID NO: 73), MCR3 (SEQ ID NO: 75), and MCR5 (SEQ ID NO: 79). A
given peptide chain is a "melanocortin receptor" if it has at least
85% amino acid sequence identity to a known melanocortin receptor
sequence or the mature form of a known melanocortin receptor and can
function as a G-protein coupled receptor. Percent identity between
two peptide chains can be determined by pairwise alignment using the
default settings of the AlignX module of Vector NTI v.9Ø0
(Invitrogen Corp., Carslbad, CA). An exemplary melanocortin
receptor binding molecule is the 13 amino acid alpha-MSH peptide
having the amino acid sequence shown in SEQ ID NO: 2. Other
melanocortin receptor binding molecules include biologically active
fragments of SEQ ID NO: 2 and other amino acid sequences that can
bind a melanocortin receptor. The term "biologically active
fragment" as used herein, refers to a portion of an alpha-MSH
peptide that can bind to a melanocortin receptor such as MC4R. The
peptide sequence HFRW (SEQ. ID. NO. 81) is an exemplary
"biologically active fragment" of the alpha-MSH peptide sequence
SYSMEHFRWGKPV (SEQ ID NO: 2). The HFRW fragment has been
incorporated into the structure of the synthetic melanocortin
receptor activator molecule melanotan II (MTII) (Fan et al., Nature
385: 165-168 (1997)).
Incorporation of melanocortin receptor binding molecules in
the mimetibody polypeptides of the invention provides for binding to
melanocortin receptors with a wide range of affinities. The
mimetibody polypeptides of the invention may bind a melanocortin
receptor with a Kd less than or equal to about 10-7, 10-8, 10-9, 10-10,
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10-11 or 10-12 M. The range of obtained IC50 values for aMSH peptide,
MTII peptide and aMSH mimetibody were 260-400 nM, 5-30 nM and 200-
300 nM, respectively. The affinity of a mimetibody polypeptide for
a melanocortin receptor can be determined experimentally using any
suitable method. Such methods may utilize Biacore or KinExA
instrumentation, ELISA or competitive binding assays. Mimetibody
polypeptides binding specific melanocortin receptors with a desired
affinity can be selected from libraries of variants or fragments by
techniques known to those skilled in the art.
An alpha-MSH peptide having the amino acid sequence shown in
SEQ ID NO: 2 may be modified to obtain other melanocortin receptor
binding molecules. Such modifications may comprise the
incorporation of C-[X]õ-C motifs into the peptide to
conformationally constrain the peptide through the formation of
disulfide bonds. In a C-[X]õ-C motif, C is a cysteine residue, X is
a amino acid residues and n is an integer necessary to acheive the
required conformational constraint. In this instance n can be as
little as 1 residue and as high as 50. Exemplary C-[X]õ-C modified
peptide sequences are shown in SEQ ID NOs: 4, 6, 8, 10, 89, 91, 93,
95, and 97. The C-[X]õ-C modified peptide sequences can be further
modified, if necessary, to prevent N-terminal clipping of mature
mimetibodies. For example, SEQ ID NO: 4 or SEQ ID NO: 97 can be
modified to remove the N-terminal S-Y-S sequence and replace it with
G-G as shown in SEQ ID NO: 282 or SEQ ID NO: 271, respectively.
The modification may also comprise the incorporation of a Wa-
[X]õ-Wa motif into the peptide to conformationally constrain the
peptide through the formation of a tryptophan zipper. In a Wa-[X]õ-
Wa motif W is tryptophan residue, X is an amino acid, a is an
integer ususlly 2, but can be from 1 to 10, and n is an integer
necessary to acheive the required conformational constraint. In
this instance n can be as little a 1 residue and as high as 50.
Exemplary Wa-[X]õ-Wa peptides are shown in SEQ ID NOs: 12, 14, 16
and 18. Further, the sequence HFRW (SEQ ID NO: 81) present in the
alpha-MSH peptide may also be modified by substituting any residue
in this sequence with any one of F, H, W and M; for example, HFRW
(SEQ ID NO: 81) can be substituted to FHWM (SEQ ID NO: 83).
In the polypeptides of the invention, the linker portion (Lk)
provides structural flexibility by allowing the mimetibody to have
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alternative orientations and binding properties. Exemplary linkers
include non-peptide chemical linkages or one to 20 amino acids
linked by peptide bonds, wherein the amino acids are selected from
the 20 naturally occurring amino acids or other amino acids (e.g. D-
amino acids, non-naturally occurring amino acids, or rare naturally
occuring amino acids). The linker portion can include a majority of
amino acids that are sterically unhindered, such as glycine, alanine
and serine and can include GS, poly GS (e.g. GSGS (SEQ ID NO: 20)),
GGSG (SEQ ID NO: 22), GSGGGS (SEQ ID NO: 24), GSGGGSG (SEQ ID NO:
26), GSSG (SEQ ID NO: 28), GGGS (SEQ ID NO: 85), GGGGS (SEQ ID NO:
99), GGGGSGGGGS (SEQ ID NO: 101), GGGGSGGGGSGGGGS (SEQ ID NO: 103),
GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 105) or any combination or polymer
thereof. Other exemplary linkers within the scope of the invention
may be longer than 20 residues and may include residues other than
glycine, alanine and serine.
In the polypeptides of the invention, V2 is a portion of a
carboxy terminal domain of an immunoglobulin variable region such as
a heavy chain variable region. Exemplary V2 amino acid sequences
are GTLVTVSS (SEQ ID NO: 32), TLVAVSS (SEQ ID NO: 34), and TLVTVSS
(SEQ ID NO: 249).
The (Mp-Lk- (V2) y-Hg-CH2-CH3) (t) mimetibody polypeptides of the
invention may comprise "y" V2 polypeptides where y is 0 (zero) or 1
(one). The amino acid sequences shown in SEQ ID NOs: 127, 129, 132,
134, 137, 139, 142, 144, 202, 204, 207, 209, 212, 214, 217, 219,
222, 224, 227, 229, 256, 259, 261, 264, 266, or 269 are exemplary of
mimetibody polypeptides comprising one V2 polypeptide. Stated
differently, these mimetibody polypeptides are examples of the
formula (Mp-Lk-(V2)y-Hg-CH2-CH3) (t) where y is one. The amino acid
sequences in SEQ ID NOs: 60, 62, 121, 123, 147, 149, 152, 154, 157,
159, 162, 164, 172, 174, 177, 179, 182, 184, 187, 189, 192, 194,
197, 199, 232, 234, 237, 239, 242, 244, 251, or 253 are exemplary of
mimetibody polypeptides that lack a V2 polypeptide. In other words,
these are mimetibody polypeptides of the formula (Mp-Lk-(V2)y-Hg-
CH2-CH3) (t) .
In the polypeptides of the invention, Hg is a portion of the
hinge domain of an immunoglobulin variable region such as a heavy
chain variable region. Exemplary Hg amino acid sequences include
EPKSCDKTHTCPPCP (SEQ ID NO: 36), EPKSADKTHTCPPCP (SEQ ID NO: 38),
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ESKYGPPCPSCP (SEQ ID NO: 40), ESKYGPPCPPCP (SEQ ID NO: 42), CPPCP
(SEQ ID NO: 44) and CPSC (SEQ ID NO: 46).
Hg amino acid sequences can be modified. Such modifications
can remove potential sites of 0-linked glycosylation. Such
modifications can also remove cysteine residues that may cause
aggregates or multimers of the polypeptides of the invention to
form.
One way to minimize 0-linked glycosylation in the mimetibodies
of the invention is to substitute Ala residues for Thr residues in
the Hg portion of the polypeptides of the invention. The Hg amino
acid sequence EPKSCDKTHACPPCP (SEQ ID NO: 107) is exemplary of such
a Thr to Ala substitution; this particular Hg substitution can also
be obtained by a Thr to Ala substitution at position 59 of SEQ ID
NO: 62.
One way to minimize aggregation or multimerization of the
mimetibodies of the invention is to substitute Ala residues for Cys
residues in the Hg portion of the polypeptides of the invention.
The Hg amino acid sequence EPKSADKTHTCPPCP (SEQ ID NO: 109) is
exemplary of such a Cys to Ala substitution; this particular Hg
substitution can also be obtained by a Cys to Ala substitution at
position 54 of SEQ ID NO: 62.
Modifications to the Hg amino acid sequences of the mimetibody
polypeptides of the invention can be made singly or in combination.
The Hg amino acid sequence EPKSADKTHACPPCP (SEQ ID NO: 111)
combines both the aforementioned substitutions; and can be obtained
by a Cys to Ala substitution at position 54 and a Thr to Ala
substitution at position 59 of SEQ ID NO: 62. Those skilled in the
art will recognize other amino acid residues that can be used to
make substitutions that remove 0-glycosylation sites and aggregation
or multimerization associated sites in the mimetibodies of the
invention. Such sites can also be deleted by removing amino acid
residues.
In the polypeptides of the invention, CH2 is an immunoglobulin
heavy chain CH2 constant region. Exemplary CH2 amino acid sequences
include:
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 48),
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
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TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 50),
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 52),
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 54),
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLSSPIEKTISKAK (SEQ ID NO: 117), and
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 246).
In the polypeptides of the invention, CH3 is an immunoglobulin
heavy chain CH3 constant region. Exemplary CH3 amino acid sequences
include:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 56),
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 58), and
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 119). It will
be recognized by those skilled in the art that the CH3 region of the
polypeptides of the invention may have its C-terminal amino acid
cleaved off when expressed in certain recombinant systems.
In the mimetibody polypeptides of invention Hg, CH2 or CH3 may
be of the IgG1 or IgG4 subclass. A sequence is of the IgG1 or IgG4
subclass if it is formed or developed from a yl or y4 heavy chain
respectively. A given peptide chain is a yl or y4 heavy chain if it
is at least 80% identical to a known yl or y4 heavy chain sequence of
a given species. Percent identity between two peptide chains can be
determined by pairwise alignment using the default settings of the
AlignX module of Vector NTI v.9Ø0 (Invitrogen Corp., Carlsbad,
CA).
In the mimetibody polypeptides of the invention Hg, CH2 or CH3
may individually be of the IgG2 or IgG4 subclass. The mimetibodies
of the invention may also comprise combinations of Hg, CH2 or CH3
elements from each subclass For example, Hg may be of the IgG4
subclass while CH2 and CH3 are of the IgG1 subclass. Alternatively,
Hg, CH2 and CH3 may all of the IgG4 or IgG2 subclass. The polypeptide
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
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VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65) is exemplary of a
polypeptide in which Hg (residues 1-15 of SEQ ID NO: 65), CH2
(residues 16-125 of SEQ ID NO: 65), and CH3 (residues 126-232 of SEQ
ID NO: 65) are all of the IgG2 subclass.
The IgG1 and IgG4 subclasses differ in the number of cysteines in
the hinge region. Most IgG type antibodies, such as IgG1, are
homodimeric molecules made up of two identical heavy (H) chains and
two identical light (L) chains, typically abbreviated H2L2. Thus,
these molecules are generally bivalent with respect to antigen binding
due to the formation of inter-heavy chain disulfide bonds and both
antigen binding (Fab) arms of the IgG molecule have identical binding
specificity. IgG4 isotype heavy chains, in contrast, contain a CPSC
(SEQ ID NO: 46) motif in their hinge regions capable of forming either
inter- or intra-heavy chain disulfide bonds, i.e., the two Cys
residues in the CPSC motif may disulfide bond with the corresponding
Cys residues in the other H chain (inter) or the two Cys residues
within a given CPSC motif may disulfide bond with each other (intra).
Since the HL pairs in those IgG4 molecules with intra-heavy chain
bonds in the hinge region are not covalently associated with each
other, they may dissociate into HL monomers that then reassociate with
HL monomers derived from other IgG4 molecules forming bispecific,
heterodimeric IgG4 molecules. In vivo isomerase enzymes may
facilitate this process. In a bispecific IgG antibody the two Fab
"arms" of the antibody molecule differ in the epitopes that they bind.
Substituting Ser residues in the hinge region of IgG4 with Pro
results in "IgG1-like behavior," i.e., the molecules form stable
disulfide bonds between heavy chains and therefore, are not
susceptible to HL exchange with other IgG4 molecules.
The mimetibody polypeptides of the invention may be made more
IgG4-like, or IgG2-like by the modification of sites which are
involved in disulfide bond formation and are present in the Hg-CH2-
CH3 portion of the mimetibody polypeptides. Such sites may be
modified by removal, deletion, insertion or substitution with other
amino acids. Typically, the cysteine residues present in disulfide
bond associated motifs are removed or substituted. Removal of these
sites may avoid covalent disulfide bonding with other cysteine-
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containing proteins present in the mimetibody producing host cell or
intra-heavy chain disulfide bonding in IgG4-based constructs while
still allowing for noncovalent dimerization of mimetibody Hg-CH2-CH3
domains. Modification of such sites can permit the formation of
bispecific mimetibody polypeptides with two different Mp portions or
prevent the formation of such bispecific species.
The IgG1 and IgG4 subclasses also differ in their ability to
mediate complement dependent cytotoxicity (CDC) and antibody-
dependent cellular cytotoxicity (ADCC). CDC is the lysing of a
target cell in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (Clq) to a molecule complexed
with a cognate antigen. IgG1 is a strong inducer of the complement
cascade and subsequent CDC activity, while IgG4 has little
complement-inducing activity. ADCC is a cell-mediated process in
which nonspecific cytotoxic cells that express Fc receptors (FcRs)
involved in ADCC (e.g., natural killer (NK) cells, neutrophils, and
macrophages) recognize bound antibody on a target cell and
subsequently cause lysis of the target cell. The IgG1 subclass
binds with high affinity to Fc receptors involved in ADCC and
contributes to ADCC, while IgG4 binds only weakly to such receptors
and has little ADCC inducing activity. The relative inability of
IgG4 to activate effector functions such as ADCC is desirable since
delivery of the mimetibody polypeptide to cells without cell killing
is possible.
The CDC and ADCC activity of the mimetibody polypeptides of
the invention may be modified by altering sites involved in CDC and
ADCC present in the Hg-CH2-CH3 portion of the mimetibody polypeptide.
Such sites may be modified by removal, deletion, insertion or
substitution with other amino acids. In the mimetibodies of the
invention sites involved in CDC, such as the Clq binding site, are
typically removed or otherwise modified to minimize CDC activity.
Additionally, Fc receptor binding sites involved in ADCC can also be
similarly modified in the mimetibodies of the invention. In
general, such modification will remove Fc receptor binding sites
involved in ADCC activity from the mimetibodies of the invention.
The substitution of Leu residues with Ala residues in the CH2
portion of the polypeptides of the invention is one example of a
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modification which can minimize ADCC activity in the polypeptides of
the invention. The CH2 amino acid sequences
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 52) and
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLSSPIEKTISKAK (SEQ ID NO: 54) are
exemplary of such a Leu to Ala substitution at residues 4 and 5 (in
these sequences). Further, the V1 domain can be removed such that
the N-terminus of the peptide is free following cleavage of the
signal peptide, and is accessible to and could be modified by
enzymes such as acetylases.
Antibodies of both the IgG4 and IgG1 isotypes contain FcRn
salvage receptor binding sites. The FcRn salvage receptor helps
maintain IgG antibody levels in the body by recycling or
transporting IgG type antibodies across enodothelial cell layers
such as those lining the inside of body cavities and blood vessels.
The FcRn salavage receptor does this by binding IgGs that have
entered endothelial cells by nonspecific pinocytosis and preventing
these IgG antibody molecules from being degraded in the lysosome of
the cell. The result of such FcRn receptor activity is that the
serum half-life of a molecule with an FcRn binding site is extended
relative to an otherwise identical molecule lacking such a site.
Exemplary mature mimetibody polypeptides of the invention
including a Ser to Pro substitution in the hinge region and CH2
domain Leu to Ala substitutions on an IgG4 backbone have the nucleic
and amino acid sequences shown in SEQ ID NOs: 211-215 and 250-254.
In particular, SEQ ID NOs: 212 and 214 show the amino acid sequence
of a mimetibody polypeptide having an Mp sequence as shown in SEQ ID
NO: 4 and an Lk sequence as shown in SEQ ID NO: 101 without and with
a signal sequence, respectively. SEQ ID NOs: 211 and 213 show
exemplary nucleic acid sequences encoding these polypeptides.
Further, SEQ ID NOs: 251 and 253 show the amino acid sequence of a
mimetibody polypeptide having an Mp sequence as shown in SEQ ID NO:
282 and an Lk sequence as shown in SEQ ID NO: 101 without and with a
signal sequence, respectively. SEQ ID NOs: 250 and 252 show
exemplary nucleic acid sequences encoding these polypeptides.
It is desirable that the Hg-CH2-CH3 portion of the mimetibodies
of the invention contain a FcRn binding site at the junction of the
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CH2 and CH3 regions. It is expected that such FcRn sites will
increase the serum half-life of the mimetibodies of the invention as
well as improve other pharmacokinetic properties relative to a
melanocortin receptor binding molecule, such as alpha-MSH alone. In
the mimetibodies of the invention FcRn sites may be modified or
added by removal, deletion, insertion or substitution of amino
acids. Typically, such modifications are used to improve the
binding of a given site to the FcRn. One example of a human FcRn
binding sites is the sequence MISRTPTVLHQHNHY (SEQ. ID. NO.: 69)
found in both IgG1 and IgG4 antibodies. Other FcRn binding sites are
well known by those skilled in the art.
Antibodies with different isotypes, such as IgG4 and IgG1, may
contain glycosylation sites. Glycosylation of these sites can alter
the properties and activites of antibody molecules. Antibody
molecules may be N-glycosylated or 0-glycosylated. N-glycosylation
of antibody amino acid residue side chains containing nitrogen atoms
(e.g., Asn) can modulate antibody Fc effector functions such as ADCC
by conferring a cytolytic activity to N-glycosylated antibody
molecules. This ADCC associated cytolytic activity causes the lysis
of cells effected by such N-glycosylated antibodies. Alternatively,
an antibody molecule may be 0-glycosylated by modification of amino
acid residue side chains containing oxygen atoms (e.g., Ser or Thr).
0-glycosylation can decrease the serum half-life of an antibody
molecule through increased lectin mediated clearance of 0-
glycosylated antibody molecules from the serum. Additionally, 0-
glycosylation can cause undesirable increases in antibody
heterogeneity due to differing extents of 0-glycosylation between
various antibody molecules. Lastly, both 0-glycosylation and N-
glycosylation can alter the structure dependent properties of
antibody molecules such as binding affinity and immunogenicity.
Like the antibody molecules they mimic, the mimetibody
polypeptides of the invention may also be post-translationally
modified by N-glycosylation and 0-glycosylation. In most instances,
it is desirable to limit the N-glycosylation of the mimetibodies of
the invention to minimize cytolytic activity. N-glycosylation can
be limited by the removal or substitution of amino acid residues,
such as Asn, which are typically N-glycosylated. It is also
desirable to limit mimetibody 0-glycosylation to minimize lectin-
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mediated clearance, mimetibody heterogeneity and the alteration of
structure dependent mimetibody properties such as binding affinity
and immunogenicity. One way to minimize 0-linked glycosylation in
the mimetibodies of the invention is to substitute Ala residues for
Thr residues in the V2 portion of the polypeptides of the invention.
The V2 amino acid sequence TLVAVSS (SEQ ID NO: 34) is exemplary of
such a Thr to Ala substitution; this particular V2 substitution can
also be obtained by a Thr to Ala substitution at postion 47 of SEQ
ID NO: 62. Those skilled in the art also will recognize other ways
to control N-linked and 0-linked glycosylation including modulation
of glycosylase enzyme activity.
The monomeric structure Mp-Lk- (V2) y-Hg-CH2-CH3 of the mimetibody
polypeptides of the invention can be linked to "t" other monomers
where t is an integer from 1 to 10. Such linking can occur through
non-covalent interactions or covalent linkages such as a Cys-Cys
disulfide bond. In this way multimeric structures such as dimers
and higher order multimers of the polypeptides of the invention can
be formed. It is expected that dimerization of the polypeptides of
the invention will increase the affinity of these polypeptides to
melanocortin receptors such as MC4R. The term "multimers" as used
herein means molecules that have quaternary structure and are formed
by the association of two or more subunits.
The polypeptides of the invention can optionally comprise at
the amino terminus, an amino terminal portion of an immunoglobulin
variable region, designated V1 as shown in Formula II:
(V1-Mp-Lk- (V2) v-Hg-CH2-CH3) (t)
(II)
Exemplary V1 amino acid sequences include QIQ, QVQ, QIQGG (SEQ ID
NO: 113), and QIQGGGG (SEQ ID NO: 115).
The polypeptides of the invention may also comprise secretory
signals necessary to facilitate protein secretion or other signals
necessary for protein trafficking in the cell. An exemplary
secretory signal sequence is MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 69).
Those skilled in the art will recognize other secretory signals.
In one embodiment the polypeptides of the invention comprise
polypeptides having the sequences shown in SEQ ID NO: 60, 62, 121,
123, 127, 129, 132, 134, 137, 139, 142, 144, 147, 149, 152, 154,
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157, 159, 162, 164, 167, 169, 172, 174, 177, 179, 182, 184, 187,
189, 192, 194, 197, 199, 202, 204, 207, 209, 212, 214, 217, 219,
222, 224, 227, 229, 232, 234, 237, 239, 242, 244, 251, 253, 256,
258, 261, 263, 266, or 268. These sequences exemplify melanocortin
receptor binding alpha-MSH polypetides either having, or lacking, an
amino terminal secretory signal sequence. SEQ ID NO: 62 represents
a (V1-Mp-Lk-(V2)y-Hg-CH2-CH3) (t) melanocortin receptor binding alpha-
MSH polypetide of generic formula (II) which has the secretory
signal MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 69) fused to its amino
terminus. SEQ ID NO: 60 represents a (Mp-Lk- (V2) y-Hg-CH2-CH3) (t)
melanocortin receptor binding alpha-MSH polypetide of generic
formula (I). No secretory signal is present in SEQ ID NO: 60.
Another aspect of the present invention is a polynucleotide
comprising, complementary to or having significant identity with, a
polynucleotide encoding at least one melanocortin receptor binding
mimetibody. Other aspects of the present invention include vectors
comprising at least one polynucleotide molecule encoding a
melanocortin receptor binding mimetibody. In a different aspect the
invention provides a cell comprising a vector of the invention or a
cell expressing a mimetibody polypeptide of the invention. The
polynucleotides, vectors and cells may be used to produce the
mimetibody polypeptides of the invention.
In one embodiment, the polynucleotides of the invention
comprise a polynucleotide having the sequence shown in SEQ ID NO:
59, 61, 120, 122, 126, 128, 131, 133, 136, 138, 141, 143, 146, 148,
151, 153, 156, 158, 161, 163, 166, 168, 171, 173, 176, 178, 181,
183, 186, 188, 191, 193, 196, 198, 201, 203, 206, 208, 211, 213,
216, 218, 221, 223, 226, 228, 231, 233, 236, 238, 241, 243, 250,
252, 255, 257, 260, 262, 265, or 267 or a polynucleotide having a
sequence complementary to the sequence shown in SEQ ID NO: 59, 61,
120, 122, 126, 128, 131, 133, 136, 138, 141, 143, 146, 148, 151,
153, 156, 158, 161, 163, 166, 168, 171, 173, 176, 178, 181, 183,
186, 188, 191, 193, 196, 198, 201, 203, 206, 208, 211, 213, 216,
218, 221, 223, 226, 228, 231, 233, 236, 238, 241, 243, 250, 252,
255, 257, 260, 262, 265, or 267. SEQ ID NO: 59 is a cDNA encoding a
(Mp-Lk- (V2) y-Hg-CH2-CH3) (t) melanocortin receptor binding alpha-MSH
polypetide of generic formula (I) which lacks a signal sequence.
SEQ ID NO: 61 is a cDNA encoding a (V1-Mp-Lk- (V2) Y-Hg-CH2-CH3) (t)
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melanocortin receptor binding alpha-MSH polypetide of generic
formula (II) which has a secretory signal fused to its amino
terminus.
In one embodiment, the polynucleotides of the invention
comprise a polynucleotide encoding the polypeptide having the
sequence shown in SEQ ID NO: 60, 62, 121, 123, 127, 129, 132, 134,
137, 139, 142, 144, 147, 149, 152, 154, 157, 159, 162, 164, 167,
169, 172, 174, 177, 179, 182, 184, 187, 189, 192, 194, 197, 199,
202, 204, 207, 209, 212, 214, 217, 219, 222, 224, 227, 229, 232,
234, 237, 239, 242, 244, 251, 253, 256, 258, 261, 263, 266, or 268.
Exemplary nucleic acid sequences that encode the polypeptide
sequences shown in SEQ ID NO 60, 62, 121, 123, 127, 129, 132, 134,
137, 139, 142, 144, 147, 149, 152, 154, 157, 159, 162, 164, 167,
169, 172, 174, 177, 179, 182, 184, 187, 189, 192, 194, 197, 199,
202, 204, 207, 209, 212, 214, 217, 219, 222, 224, 227, 229, 232,
234, 237, 239, 242, 244, 251, 253, 256, 258, 261, 263, 266, or 268
are shown in SEQ ID NO: 59, 61, 120, 122, 126, 128, 131, 133, 136,
138, 141, 143, 146, 148, 151, 153, 156, 158, 161, 163, 166, 168,
171, 173, 176, 178, 181, 183, 186, 188, 191, 193, 196, 198, 201,
203, 206, 208, 211, 213, 216, 218, 221, 223, 226, 228, 231, 233,
236, 238, 241, 243, 250, 252, 255, 257, 260, 262, 265, or 267,
respectively.
Also provided are polynucleotides encoding polypeptides that
are substantially identical to the polypeptides of the invention.
The term "substantially identical" in the context of polypeptides
means that a given polypeptide sequence is identical to a
polypeptide sequence of the invention, in particular the V1-Mp-Lk-
V2-Hg region, in at least 50% or at least about 60% or at least
about 70% or at least about 80% or at least about 90% or at least
about 95-98% of the amino acid residues. Percent identity between
two polypeptide sequences can be determined by pairwise alignment
using the default settings of the AlignX module of Vector NTI
v.9Ø0 (Invitrogen Corp., Carlsbad, CA). Those skilled in the art
would recognize polynucleotide sequences which would encode the
above-described polypeptides.
Typically, the polynucleotides of the invention are used in
expression vectors for the preparation of the mimetibody
polypeptides of the invention. Vectors within the scope of the
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invention provide necessary elements for eukaryotic expression and
include viral promoter driven vectors, such as CMV promoter driven
vectors, e.g., pcDNA3.1, pCEP4, and their derivatives, Baculovirus
expression vectors, Drosophila expression vectors, and expression
vectors that are driven by mammalian gene promoters, such as human
Ig gene promoters. Other examples include prokaryotic expression
vectors, such as T7 promoter driven vectors, e.g. pET41, lactose
promoter driven vectors and arabinose gene promoter driven vectors.
The present invention also relates to a cell that expresses a
mimetibody of the invention or comprises a vector of the invention.
Open reading frames encoding the mimetibody polypeptides of the
invention can be identified by translation of the positive strand
reading frame beginning with nucleotide residue 1601 of SEQ ID NOs:
63, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,
190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 254,
259, 264, or 269. The various signal peptide, V1, Mp, V2, Hg, CH2,
and CH3 portions of the mimetibody polypeptides of the invention,
which have been exemplified herein, are also present in these open
reading frames and can be identified using standard sequence
analysis methods, such as multiple sequence alignment or other
methods well know in the art. Such a cell can be prokaryotic or
eukaryotic. Exemplary eukaryotic cells are mammalian cells, such as
but not limited to, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2,
653, SP2/0, NSO, 293, HeLa, myeloma, lymphoma cells or any
derivative thereof. Most preferably, the eukaryotic cell is a
HEK293, NSO, SP2/0, or CHO cell. E. coli is an exemplary
prokaryotic cell. A cell according to the invention may be
generated by transfection, cell fusion, immortalization, or other
procedures that are well known in the art. Polynucleotides
transfected into a cell may be extrachromasomal or stably integrated
into the chromosome of the cell.
The mimetibodies of the invention can be made more compatible
with a given host cell by modification of the Hg-CH2-CH3 portion of
the polypeptide. For example, when a mimetibody of the invention is
expressed recombinantly in a bacterial cell such as E. coli, the
Pro-Ala sequence in the Hg element may be removed to prevent
digestion by the E. coli enzyme proline iminopeptidase. Similarly,
a portion of the Hg element can be deleted or substituted with other
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amino acids in the mimetibodies of the invention to prevent
heterogeneity in the products expressed in a selected host cell.
The present invention further provides a method to produce a
mimetibody polypeptide comprising the steps of culturing a cell of
the invention and purifying an expressed mimetibody polypeptide of
the invention. Cell components, such as those necessary for in
vitro transcription and translation, may also be used to express the
polypeptides of the invention. The present invention encompasses
mimetibodies produced by both methods. Expressed mimetibody
polypeptides can be recovered and purified from cells or cell
component based systems by methods well known in the art including,
but not limited to, protein A purification, ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylatpatite chromatography and lectin chromatography. High
performance liquid chroatography (HPLC) can also be employed for
purification. Typically purfication will require a combination of
several different methods.
Another aspect of the present invention is a pharmaceutical
composition comprising an effective amount of at least one
mimetibody polypeptide and a pharmaceutically acceptable carrier or
diluent. The term "effective amount" generally refers to the
quantity of mimetibody necessary for effective therapy, i.e., the
partial or complete alleviation of the symptom or disorder for which
treatment was sought. The composition can optionally comprise at
least one further compound, protein or composition useful for
treating obesity and the other conditions described below. The
parmaceutically acceptable carrier or diluent in the compositions
can be a solution, suspension, emulsion, colloid or powder. Those
skilled in the art will recognize other parmaceutically acceptable
carriers and diluents.
Another aspect of the present invention is a method of
modifying the biological activity of a melanocortin receptor in a
cell, tissue or organ comprising contacting the pharmaceutical
compositions of the invention with the cell, tissue or organ. The
method may be used to modify melanocortin receptor activity in the
brain, brain tissue, or brain cells. Alternatively, the method of
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the invention may be used to modify melanocortin receptor activity
in other peripheral cells or tissues such as muscle, or other organs
such as the stomach. Those skilled in the art will recognize other
cells, tissues or organs, which may be used.
Another aspect of the invention is a method of modulating at
least one melanocortin receptor-mediated condition comprising
administering a pharmaceutical composition of the invention to a
patient in need thereof. The pharmaceutical compositions of the
invention can be administered by any suitable route. Such routes
may be intrathecal, intranasal, peripheral (e.g., subcutaneous,
intramuscular, intradermal, intravenous) or by any other means known
in the art. As described previously, abnormal melanocortin receptor
activity has been implicated in a number of pathological conditions,
such as obesity and Type 2 diabetes. Stimulation of MC4R can cause
weight loss while inhibition may cause weight gain. The mimetibody
polypeptides of the invention may be also be used to modulate other
melanocortin receptor mediated conditions such as male and female
erectile dysfunction, inflammation, congestive heart failure,
central nervous system disorders, nerve damage, infectious disease,
pulmonary disease, skin disease, fever and pain.
The present invention is further described with reference to
the following examples. These examples are merely to illustrate
aspects of the present invention and are not intended as limitations
of this invention.
Example 1
Alpha-MSH Mimetibody and Expression Vector Construction
An alpha-MSH mimetibody protein comprising a secretory signal
sequence, an alpha-MSH peptide sequence, a linker sequence, VH
sequence, a hinge sequence, a human IgG1 CH2 sequence and a human
IgG1 CH3 sequence was designed (Fig. 3 and SEQ ID NO. 62) Analytical
data, e.g., mass spectroscopy, has confirmed that a mature
polypeptide is generated (61,344.6 for G1/G1 form). Nucleic acid
sequences encoding this alpha-MSH mimetibody protein (Fig. 3; SEQ ID
NO: 61) were generated using standard molecular biology techniques.
Nucleic acid sequences encoding the alpha-MSH mimetibody sequence
were subcloned into the p2389 expression vector to generate an
alpha-MSH mimetibody expression vector (SEQ ID NO: 63).
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Example 2
Alpha-MSH Mimetibody Expression
The alpha-MSH mimetibody was transiently expressed in HEK293E
cells. Cells were cultured using standard conditions and
transiently transfected with the alpha-MSH mimetibody expression
vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as
directed by the manufacturer. 24 h after transfection cells were
transferred to a serum free media formulation and cultured for 5
days. The culture media was then removed and centrifuged to remove
debris. Clarified media was incubated with Protein A-SepharoseTM
(HiTrap rProtein A FF, Amersham Biosciencies, Piscataway, NJ) and
proteins were eluted from the Protein A-SepharoseTM conjugate as
directed by the manufacturer. The eluted protein solution was then
further purified via SuperoseTM 12 size exclusion chromatography
(Superose 12 10/300 GL, Amersham Biosciencies, Piscataway, NJ) using
standard methods. Column eluant was then subjected to SDS-PAGE and
visualized by silver and Coomassie blue staining. Western blots
were then prepared and the blots were probed with either an Fc
specific primary antibody or an alpha-MSH specific primary antibody.
Together, the Western Blot and SDS-PAGE staining results indicated
that a purified alpha-MSH mimetibody, composed of two polypeptide
chains, had been obtained from the transiently transfected HEK293
cells.
Example 3
Alpha-MSH Mimetibody Binds MC4R
The alpha-MSH mimetibody binds to MC4R and can compete with
radiolabeled [Nle(4), D-Phe(7)]-alpha-MSH (NDP-alpha-MSH) agonist
molecules for MC4R binding (Fig. 4). MC4R is a receptor for alpha-
MSH. alpha-MSH binding to recombinantly expressed MC4R in HEK293
cell membranes (Perkin Elmer Life and Analytical Sciences, Boston,
MA) was examined by competive binding assays in which increasing
amounts of unlabeled MC4R agonists (positive controls) and the Fc
domain of a human antibody (negative control) were added to assay
cocktails containing [125I]-NDP-alpha-MSH as indicated in Fig. 4.
The unlabeled MC4R agonists were melanotan II (MTII; an alpha MSH
analog), alpha-MSH, and NDP-alpha-MSH. Alpha-MSH mimetibody binding
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to MC4R was stable after two weeks of storage at 4 C, -20 C, and -
80 C in PBS (phosphate buffered saline) as assessed by competive
binding assays.
Competivive binding assays were performed using Scintillation
Proximity Assays (Amersham Biosciences Corp, Piscataway, NJ) as
directed by the assay manufacturer. Assay cocktails contained
[1251]-NDP-alpha-MSH at EC80, i.e., -0.5 nM, 0.1 pg of MC4R
membranes, 1 mM MgS04, 1.5 mM CaC12, 25 mM Hepes, 0.2% BSA, 1 mM
1,10-phenthroline, an assay manufacturer recommended quantity of
protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis,
IN) and SPA beads. Light emission from Scintillation Proximity
Assay beads was measured with a Packard Top Count NXT Instrument
(Perkin Elmer Life and Analytical Sciences, Boston, MA) for 5
minutes.
Example 4
Alpha-MSH Mimetibody Activates MC4R
The alpha-MSH mimetibody can activate MC4R signalling to
increase cAMP production in CHOK1 cells expressing MC4R (Fig. 5 and
Fig. 6). MC4R is a seven transmembrane (7TM) G-protein coupled
receptor. Activation of MC4R by ligand or agonist results in an
increase in cyclic AMP levels (cAMP).
MC4R receptor activation assays were performed using two
different clonal CHOK1 cell lines stably transfected with a MC4R
expression vector and expressing MC4R. Clone 1 (Fig. 5) expressed
MC4R at high levels relative to Clone 2 (Fig. 6). Clone 1 and Clone
2 cells were grown as a monolayer using standard culture conditions
to a density of approximately 100,000 cells/well and then incubated
with increasing amounts (0-100 pM) of alpha-MSH, MTII, or alpha-MSH
mimetibody for 15 minutes as indicated in Fig. 5 and Fig. 6. Cells
were then lysed and cAMP assays were performed using the cAMP-Screen
Direct'' Chemiluminescent Immunoassay System (Applied Biosystems,
Foster City, CA) as directed by the manufacturer. EC50 values from
cAMP assays using Clone 1 (Fig. 5) and Clone 2 (Fig. 6) are listed
in Table 1 below
Table 1
Clone 1 Clone 2
alpha-MSH peptide EC50 = 3.29 nM EC50 = 9.46 nM
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(Positive control)
MT II EC50 = 0.52 nM EC50 = 0.52 nM
(Positive control)
alpha-MSH mimetibody EC50 = 14.36 nM EC50 = 52.4 nM
Example 5
Alpha-MSH Mimetibody Administration Decreases
Animal Food Intake and Body Weight
Alpha-MSH mimetibody administration to Rattus norvegicus brain
ventricules decreases animal food intake (Fig. 7) and body weight
(Fig. 8). Alpha-MSH mimetibody was supplied to brain ventricules by
intracerebroventricular injections (ICV) via a cannula surgically
inserted into the left lateral brain ventricle.
Cannulae were surgically inserted into male Sprague-Dawley or
Wistar rats weighing 250 g to 350 g. Cannula placement coordinates
were as follows: -0.8 mm from bregma, -4.5 mm ventral and -1.5
posterior-anterior. Animals recovered for 7 to 10 days after
surgery. Animals were acclimatized to the experimental procedures
by both daily handling and mock injection, in order to minimize
stress. In addition animals were submitted to the reversal of dark-
light cycle.
Proper cannula placement was confirmed by an angiotensin II
test. The test confirmed proper cannula placement if the ICV
administration of 10 ng of angiotensin II via the cannula caused the
rats to drink 5-10 ml of water in 30 minutes. Only animals that
passed this angiotensin II test were used in food intake
experiments.
Animals were fasted for 18-24 hours and alpha-MSH mimetibody,
alpha-MSH (positive control), or PBS (negative control) were then
administered to the brain ventricles via the cannula at an injection
rate of 9 pl/min. Each treatment group had a minimum of 7 animals.
Treatments and dosages were as indicated in Fig. 7 and Fig. 8.
Food and water was given to the animals after injection. The
amount of food and water consumed was measured at 0 h, 4 h, 24 h, 48
h and 72 h (Fig. 7) after injection. Body weight at 72 hours post
injection was measured as ahown in Fig. 8.
CA 02704446 2010-04-30
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The present invention now being fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
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