NZ717700B2 - Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers - Google Patents
Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers Download PDFInfo
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- NZ717700B2 NZ717700B2 NZ717700A NZ71770012A NZ717700B2 NZ 717700 B2 NZ717700 B2 NZ 717700B2 NZ 717700 A NZ717700 A NZ 717700A NZ 71770012 A NZ71770012 A NZ 71770012A NZ 717700 B2 NZ717700 B2 NZ 717700B2
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Disclosed is a method of assessing the potential in vivo efficacy of a candidate anti-CGRP antibody or anti-CGRP antibody fragment, anti-CGRP receptor antibody or anti-CGRP receptor antibody fragment, or a polypeptide comprising a fragment of CGRP or CGRP receptor for treating photophobia or light aversion, comprising determining whether the candidate inhibits light aversive behaviour in a nestin/Ramp1 rodent administered CGRP compared to a rodent administered CGRP in the absence of the candidate. version, comprising determining whether the candidate inhibits light aversive behaviour in a nestin/Ramp1 rodent administered CGRP compared to a rodent administered CGRP in the absence of the candidate.
Description
USE OF ANTI-CGRP ANTIBODIES AND ANTIBODY FRAGMENTS
TO PREVENT OR INHIBIT PHOTOPHOBIA OR LIGHT AVERSION
IN SUBJECTS IN NEED THEREOF, ESPECIALLY MIGRAINE
SUFFERERS
RELATED APPLICATIONS
This is a divisional application of New Zealand patent application 618638, dated 21
May 2012, which is the national phase entry in New Zealand of PCT International
application (published ). This application claims
the benefit of U.S. Provisional Application No. 61/496,860 (Atty. Docket No.
67858.760000) filed June 14, 2011, entitled "USE OF ANTI-CGRP ANTIBODIES AND
ANTIBODY FRAGMENTS TO PREVENT OR INHIBIT PHOTOPHOBIA IN
SUBJECTS IN NEED THEREOF, ESPECIALLY MIGRAINE" and U.S. Provisional
Application No. 61/488,660 (Atty. Docket No. 67858.730300) filed May 20, 2011, entitled
"ANTI-CGRP COMPOSITIONS AND USE THEREOF" each of which is hereby
incorporated by reference in its entirety.
The instant application contains a Sequence Listing which has been submitted in
ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on May 21, 2012, is named 67858o730305.txt and is 203,941 bytes in
size.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention pertains to the discovery that polypeptides that inhibit the
CGRP/CGRP receptor interaction and/or antibodies and antibody fragments that
specifically bind CGRP or to a CGRP receptor may be used to inhibit CGRP-induced
photophobia when administered to a subject in need thereof. Polypeptides that inhibit the
CGRP/CGRP receptor interaction for use in the invention include by way of example
antibodies and antibody fragments specific to CGRP or the CGRP receptor and fragments
or variants of CGRP or the CGRP receptor that inhibit CGRP from interacting with CGRP
receptors. As photophobia is an adverse side-effect often associated with many disorders
including by way of example migraine with and without aura and other headache
conditions (as well as other indications disclosed infra) t these CGRP-receptor inhibitors,
e.g., antibodies and antibody fragments specific to CGRP or the CGRP receptor should be
well suited for inhibiting the photophobia often associated with migraine and other
headache conditions as well as for treating other conditions associated with photophobia.
The results also suggest that these antibodies and antibody fragments may be used to
prevent the onset of photophobia in subjects in need thereof such as individuals with a
chronic history of photophobia, e.g., as a result of migraine (with or without aura), other
headache condition, depression, agoraphobia or other conditions prone to photophobia if
the antibodies are administered prophylactically. The invention contemplates the use of
these anti-CGRP antibodies and antibody fragments as a monotherapy or in therapeutic
regimens with other active agents, e.g., analgesics, opioids, antidepressants or other actives
dependent on the condition and the individual treated.
The invention further contemplates methods of screening CGRP-receptor
inhibitors, e.g., anti-CGRP or anti-CGRP receptor antibodies and fragments thereof
(including Fab fragments) having binding specificity to human Calcitonin Gene Related
Peptide (hereinafter “CGRP”) or the CGRP receptor in specific animal models to determine
the in vivo effects thereof, most especially their ability to antagonize the photophobic side
effects of CGRP and to treat conditions involving photophobia including e.g., migraine.
Description of Related Art
Calcitonin Gene Related Peptide (CGRP) is produced as a multifunctional
neuropeptide of 37 amino acids in length. Two forms of CGRP, the CGRP-alpha and
CGRP-beta forms, exist in humans and have similar activities. CGRP-alpha and CGRP-
beta differ by three amino acids in humans, and are derived from different genes. The
CGRP family of peptides includes amylin, adrenomedullin, and calcitonin, although each
has distinct receptors and biological activities. Doods, H., Curr. Op. Invest. Drugs,
2(9):1261-78 (2001).
CGRP is released from numerous tissues such as trigeminal nerves, which when
activated release neuropeptides within the meninges, mediating neurogenic inflammation
that is characterized by vasodilation, vessel leakage, and mast-cell degradation. Durham,
P.L., New Eng. J. Med., 350 (11):1073-75 (2004). The biological effects of CGRP are
mediated via the CGRP receptor (CGRP-R), which consists of a seven-transmembrane
component, in conjunction with receptor-associated membrane protein (RAMP). CGRP-R
further requires the activity of the receptor component protein (RCP), which is essential for
an efficient coupling to adenylate cyclase through G proteins and the production of cAMP.
Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-78 (2001).
Migraines constitute a neurovascular disorder affecting approximately 10% of
the adult population in the U.S., and are typically accompanied by intense headaches.
Approximately 20-30% of migraine sufferers experience aura, comprising focal
neurological phenomena that precede and/or accompany the event. CGRP is believe to
play a prominent role in the development of migraines. For example, plasma
concentrations of CGRP were identified elevated in jugular venous blood during the
headache phase of migraines, to the exclusion of other neuropeptides. Moreover,
according to Arulmozhi et al, the following has been identified in migraine sufferers: (1) a
strong correlation between plasma CGRP concentrations and migraines; (2) the infusion of
CGRP produced a migraine-like headache; (3) baseline CGRP levels were elevated; and (4)
changes in plasma CGRP levels during migraine attacks significantly correlated with
headache intensity. Arulmozhi, D.K., et al., Vas. Pharma., 43: 176-187 (2005). In
addition, in the Journal of the International Association for the Study of Pain PII:S0304-
3959(11)00313-7; doi:10.1016/j.pain.2011.04.033, published online 06 June 2011, Hou et
al., reported that keratinocyte expression of calcitonin gene-related peptide β has
implications for neuropathic and inflammatory pain mechanisms.
One effective treatment for migraines is the administration of triptans, which are
a family of tryptamine-based drugs, including sumatriptan and rizatriptan. Members of this
family have an affinity for multiple serotonin receptors, including 5-HT , 5-HT , and 5-
1B 1D
HT . Members of this family of drugs selectively constrict cerebral vessels, but also cause
vasoconstrictive effects on coronary vessels. Durham, P.L., New Eng. J. Med., 350
(11):1073-75 (2004). There is a theoretical risk of coronary spasm in patients with
established heart disease following administration, and cardiac events after taking triptans
may rarely occur. Noted to be contraindicated for patients with coronary vascular disease.
Similarly, pain may often be addressed through the administration of certain
narcotics or non-steroidal anti-inflammatory drugs (NSAIDs). However, the
administration of these treatments may occur at the cost of certain negative consequences.
NSAIDs have the potential to cause kidney failure, intestinal bleeding, and liver
dysfunction. Narcotics have the potential to cause nausea, vomiting, imparied mental
functioning, and addiction. Therefore, it is desirable to identify alternative treatments for
pain in order to avoid certain of these negative consequences.
Aside from migraine, CGRP is believed to play a role in a multitude of diseases
and disorders, including but not limited to other headache conditions, and pain. Due to the
perceived involvement of CGRP in these diseases and disorders, there remains a need in
the art for compositions and methods useful for preventing or treating diseases and
disorders associated with CGRP, while avoiding adverse side effects. There in particular
remains a need in the art for compositions or methods that reduce or inhibit photophobia in
diseases or disorders associated with CGRP, such as migraines, headaches, and pain. It is
an object of the present invention to go someway towards meeting this need and/or to
provide the public with a useful choice.
Migraineurs typically develop worsening pain and migraine symptoms when
exposed to light, a phenomenon known as photophobia. Photophobia is also common in
ocular disorders, such as iritis and uveitis, and intracranial disorders, such as meningitis. In
the classic visual pathway, light activates rods and cones in the retina, which activate
retinal ganglion cells that project via the optic nerve, to the lateral geniculate nucleus,
superior colliculus, and then the visual cortex. This pathway includes image-forming and
non-image-forming data. A new pathway (non-image-forming information) allows
maintenance of normal circadian rhythms via the suprachiasmatic nucleus and is regulated
by intrinsically photosensitive retinal ganglion cells (ipRGCs). These ipRGCs are
independent of the rods and cones and contain melanopsin, a photopigment.
Noseda et al.(Noseda, R. et al. A neural mechanism for exacerbation of headache
by light. Nat. Neurosci. 13, 239–245 (2010)) studied blind individuals who had migraine
and correlated these findings with rat models involving tracing of ipRGC projections to
areas in perception of pain from the dura. Of the blind patients with migraine, 6 had no
light perception due to severe optic nerve damage or bilateral enucleation. These subjects
experienced abnormal sleep patterns and poor pupillary light responses. Their migraines
did not worsen with light exposure. In contrast, 14 blind subjects who were able to detect
light despite minimal perception of images had normal sleep patterns and a normal
pupillary light reflex. Despite widespread rod and cone degeneration, these patients had
worsening migraine symptoms with light exposure during migraine attacks, suggesting that
ipRGCs, and not rods and cones, are important in photophobia.
These retinal projections of non-image-forming brain areas project to the
contralateral dorsocaudal region of the posterior thalamus, as demonstrated by anterograde
tracing in the rat. ipRGC input to this area modulates dura-sensitive pain neurons, which
also project to this region. Thalamic neurons, dually sensitive to dural pain and light input,
project widely to multiple cortical regions, including the primary somatosensory cortex, the
primary and secondary motor cortices, the parietal association cortex, and the primary and
secondary visual cortices. These cortical projections may help explain other common
migraine symptoms, in addition to photophobia, such as motor weakness or incoordination,
visual disturbances, and poor concentration.
Photophobia also accompanies other less frequent but likewise disabling
conditions, such as cluster headache and other trigeminal autonomic cephalalgias and
blepharospasm. The mechanisms underlying photophobia involve the trigeminal system.
Photophobia in blind patients suggests contributions from a nonvisual pathway. In addition,
trigeminal autonomic cephalalgias, a less common group of primary headache disorders,
are characterized by unilateral trigeminal-mediated pain frequently associated with
ipsilateral photophobia .
Stimulation of trigeminal sensory neurons results in the release of neuropeptides
(including substance P and calcitonin gene-related peptide, producing blood vessel dilation
and mast cell, endothelial, and platelet activation (neurogenic inflammation), which leads
to migraine. (Buzzi MG, Dimitriadou V, Theoharides TC, Moskowitz MA . 5-
Hydroxytryptamine receptor agonists for the abortive treatment of vascular headaches
block mast cell, endothelial and platelet activation within the rat dura mater after trigeminal
stimulation. Brain Res 1992;583:137–149). CGRP is elevated in external jugular venous
blood during acute migraine pain, (Goadsby PJ, Edvinsson L, Ekman R . Vasoactive
peptide release in the extracerebral circulation of humans during migraine headache. Ann
Neurol 1990;28:183–187) and triptans reduce elevated CGRP levels. In animal models,
mice sensitized to CGRP demonstrate more light-aversive behavior when exposed to
exogenous CGRP. The administration of olcegepant, a CGRP receptor antagonist,
prevented photophobia in these mice. (See Recober A, Kaiser EA, Kuburas A, Russo AF.
Induction of multiple photophobic behaviors in a transgenic mouse sensitized to CGRP.
Neuropharmacology 2010;58:156–165).
However, while the use of anti-CGRP or anti-CGRP receptor antibodies and
fragments to treat migraine has been suggested, to the best of Applicant’s knowledge there
has been no report of any polypeptide CGRP antagonist or in particular an anti-CGRP or
anti-CGRP receptor antibody or antibody fragment able to alleviate or prevent the
photophobic side effects of CGRP in vivo. The development of novel polypeptides that act
as inhibitors of the CGRP/CGRP receptor interaction such as anti-CGRP or anti-CGRP
receptor antibodies or anti-CGRP or anti-CGRP receptor antibody fragments would be
beneficial for patients who either do not respond to current migraine therapeutics such as
triptans or who cannot take or tolerate them because of their potential vasoconstrictive
effects.
BRIEF SUMMARY OF THE INVENTION
This invention relates to the discovery that polypeptides which inhibit the
CGRP/CGRP receptor interaction such as anti-CGRP or anti-CGRP receptor antibodies
and anti-CGRP or anti-CGRP receptor antibody fragments (including Fab fragments)
having binding specificity to human Calcitonin Gene Related Peptide (hereinafter
“CGRP”) as well as fragments of CGRP and the CGRP receptor that inhibit the
CGRP/CGRP receptor interaction may be used to prevent or inhibit photophobia,
especially CGR associated photophobia. Herein we particularly exemplify an anti-CGRP
antibody identified as Ab3 infra, that very effectively alleviates or prevents photophobia,
especially the photophobic effects of CGRP. Other prefered examples for use in the
claimed therapies are Ab6 and Ab10 among others.
[00017a] In a first embodiment the invention provides a method of assessing the
potential in vivo efficacy of a candidate anti-CGRP antibody or anti-CGRP antibody
fragment, anti-CGRP receptor antibody or anti-CGRP receptor antibody fragment, or
polypeptide comprising a fragment of CGRP or CGRP receptor for treating photophobia or
light aversion, comprising determining whether the candidate anti-CGRP antibody or
antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide
comprising a fragment of CGRP or CGRP receptor inhibits light aversive behavior in a
nestin/Ramp1 rodent administered CGRP compared to a rodent administered CGRP in the
absence of the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor
antibody or antibody fragment, or polypeptide comprising a fragment of CGRP or CGRP
receptor.
Also described is the use of polypeptides which inhibit the CGRP/CGRP
receptor interaction such as anti-CGRP or anti-CGRP receptor antibodies and anti-CGRP
or anti-CGRP receptor antibody fragments (including Fab fragments) having binding
specificity to human Calcitonin Gene Related Peptide (hereinafter “CGRP”) as well as
fragments of CGRP and the CGRP receptor that inhibit the CGRP/CGRP receptor
interaction, preferably anti-CGRP antibodies and anti-CGRP antibody fragments for
treating or preventing photophobia. The present disclosure embraces the treatment or
prevention of any photophobia, and in particular includes treatment or prevention of
photophobia associated with migraine, and other disorders associated with photophobia
such as cluster headache and other trigeminal autonomic cephalalgias and blepharospasm,
depression, bipolar disorders, agoraphobia, meningitis, and photophobias associated with
eye related conditions, autism, chronic fatigue syndrome, menstrual migraines, and other
photopbia-associated conditions.
Also described are methods of screening polypeptides which inhibit the
CGRP/CGRP receptor interaction such as anti-CGRP or anti-CGRP receptor antibodies
and anti-CGRP or anti-CGRP receptor binding antibody fragments (including Fab
fragments) having binding specificity to human CGRP as well as fragments of CGRP and
the CGRP receptor that inhibit the CGRP/CGRP receptor interaction, in specific
photophobia animal models, e.g., the nestin/hRAMP1 rodent model disclosed infra, to
determine the in vivo effects thereof, especially the ability of these polypeptides to inhibit
the CGRP/CGRP receptor interaction in vivo and thereby antagonize the adverse in vivo
side effects of CGRP including photophobia and to treat CGRP conditions involving CGRP
associated photophobia including migraine and other disorders associated with
photophobia such as cluster headache and other trigeminal autonomic cephalalgias and
blepharospasm, depression, bipolar disorders, and other photophobia-associated conditions
identified herein.
Also described is a method of assessing the potential in vivo efficacy of a
candidate polypeptide which inhibit the CGRP/CGRP receptor interaction such as anti-
CGRP or anti-CGRP receptor antibodies and anti-CGRP or anti-CGRP receptor antibody
fragments (including Fab fragments) having binding specificity to CGRP as well as
fragments of CGRP and the CGRP receptor that inhibit the CGRP/CGRP receptor
interaction, preferably an anti-CGRP or anti-CGRP receptor antibody or antibody fragment
comprising determining whether the polypeptide, e.g., an antibody, inhibits light aversive
behavior in a transgenic rodent which exhibits photoaversion when administered CGRP
compared to the photoaversive behavior of the rodent administered CGRP in the absence of
the candidateCGRP/CGRP receptor inhibitor polypeptide.
Also, described is a method of assessing the potential in vivo efficacy of a
candidate polypeptide which inhibit the CGRP/CGRP receptor interaction such as anti-
CGRP or anti-CGRP receptor antibodies and anti-CGRP or anti-CGRP receptor antibody
fragments (including Fab fragments) as well as fragments of CGRP and the CGRP receptor
that inhibit the CGRP/CGRP receptor interaction, preferably an anti-CGRP antibody or
anti-CGRP receptor antibody or antibody fragment to treat a neurological condition or
other condition characterized by increased CGRP levels that result in photophobia.
Further, described is a method of assessing the potential in vivo efficacy of a
candidate polypeptide which inhibits the CGRP/CGRP receptor interaction such as anti-
CGRP or anti-CGRP receptor antibodies and anti-CGRP or anti-CGRP receptor antibody
fragments as well as fragments or variants of CGRP species and CGRP receptors that
inhibit the CGRP/CGRP receptor interaction, preferably anti-CGRP or anti-CGRP receptor
antibodies or antibody fragments to treat or prevent photophobia in migraine or chronic
migraine, menstrual or menopausal or other hormonal associated migraines, cluster
headaches or pain disorder associated with headache.
Still further, described is a method of determining a suitable therapeutic dosage
or dosage regimen of the candidate polypeptide CGRP/CGRP receptor inhibitor, e.g., anti-
CGRP or anti-CGRP receptor antibody or antibody fragment in humans based on the
effects of said polypeptide, e.g., an antibody or antibody fragment in a light aversive
behavioral Nestin/hRAMP1 rodent animal model described in detail infra.
Also described are methods of assessing based on results in a rodent CGRP
(Nestin/hRAMP1 animal model) a suitable therapeutic dosage or dosage regimen of the
candidate polypeptide, e.g., an anti-CGRP or anti-CGRP receptor antibody or antibody
fragment in humans.
In preferred embodiments the present disclosure is directed to therapeutic usage
of specific antibodies and fragments thereof having binding specificity for CGRP, in
particular antibodies having desired epitopic specificity, high affinity or avidity and/or
functional properties. In preferred embodiments this disclosure relates to assays and usage
of the antibodies described herein, comprising the sequences of the V , V and CDR
polypeptides described herein, and the polynucleotides encoding them. A preferred
embodiment of the disclosure is directed to chimeric or humanized antibodies and
fragments thereof (including Fab fragments) capable of binding to CGRP or the CGRP
receptor and/or inhibiting the biological activities mediated by the binding of CGRP to the
CGRP receptor (“CGRP-R”).
In another preferred embodiment of the disclosure, the assays and therapies use
full length antibodies and Fab fragments thereof that inhibit the CGRP-alpha-, CGRP-beta,
and rat CGRP-driven production of cAMP. In a further preferred embodiment of the
disclosure, full length and Fab fragments thereof are contemplated that reduce vasodilation
and inhibit or prevent photophobia in a recipient following administration.
In another embodiment of the disclosure, chimeric or humanized antibodies and
fragments thereof (including Fab fragments) capable of binding to CGRP or the CGRP
receptor are useful in methods directed to reducing, treating, or preventing photophobia
associated with one or more of the following conditions: migraines (with or without aura),
cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis
associated with cancer or tumor survival, weight loss, pain, hemiplagic migraines, cluster
headaches, menstrual migranes, migrainous neuralgia, chronic headaches, tension
headaches, general headaches, hot flashes, chronic paroxysomal hemicrania, secondary
headaches due to an underlying structural problem in the head or neck, cranial neuralgia,
sinus headaches (such as for example associated with sinusitis), headache-free migraine,
abdominal migraine, and allergy-induced headaches or migraines.
Common causes of photophobia include migraine headaches, cataracts, or severe
ophthalmologic diseases such as uveitis or corneal abrasion. A more extensive list of
disorders associated with photophobia includes eye related causes such as Achromatopsia,
Aniridia, Anticholinergic drugs may cause photophobia by paralyzing the iris sphincter
muscle, Aphakia (absence of the lens of the eye), Buphthalmos (abnormally narrow angle
between the cornea and iris), Cataracts, Cone dystrophy, Congenital abnormalities of the
eye, Viral conjunctivitis ("pink eye") Corneal abrasion, Corneal dystrophy, Corneal ulcer,
disruption of the corneal epithelium, such as that caused by a corneal foreign body or
keratitis, Ectopia lentis, Endophthalmitis, Eye trauma caused by disease, injury, or
infection such as chalazion, episcleritis, glaucoma, keratoconus, or optic nerve hypoplasia,
Hydrophthalmos, or congenital glaucoma Iritis, Optic neuritis, Pigment dispersion
syndrom, Pupillary dilation (naturally or chemically induced), Retinal detachment, Scarring
of the cornea or sclera and Uveitis.
In addition photophobia has nervous-system-related or urological causes
including: Autism spectrum disorders, Chiari malformation, Dyslexia, Encephalitis
including Myalgic encephalomyelitis aka Chronic fatigue syndrome, Meningitis,
Subarachnoid haemorrhage, Tumor of the posterior cranial fossa, as well as other causes
such as Ankylosing spondylitis, Albinism, Ariboflavinosis, Benzodiazepines (long term use
of or withdrawal from benzodiazepines), Chemotherapy, Chikungunya, Cystinosis, Ehlers-
Danlos syndrome, Hangover, Influenza, Infectious Mononucleosis, Magnesium deficiency,
Mercury poisoning, Migraine, Rabies, and Tyrosinemia type II, also known as "Richner-
Hanhart syndrome". Additionally it is known that photophobia is elevated in depression,
bipolar disorder and agoraphobia.
In another embodiment of the disclosure these antibodies and humanized
versions for treatment or prevention of photophobia may be derived from rabbit immune
cells (B lymphocytes) and may be selected based on their homology (sequence identity) to
human germ line sequences. These antibodies may require minimal or no sequence
modifications, thereby facilitating retention of functional properties after humanization. A
further embodiment of the disclosure is directed to fragments from anti-CGRP or anti-
CGRP receptor antibodies encompassing V , V and CDR polypeptides, e.g., derived from
rabbit immune cells and the polynucleotides encoding the same, as well as the use of these
antibody fragments and the polynucleotides encoding them in the creation of novel
antibodies and polypeptide compositions capable of binding to CGRP and/or
CGRP/CGRP-R complexes.
Also described are conjugates of anti-CGRP or anti-CGRP receptor antibodies
and binding fragments thereof for treatment or prevention of photophobia conjugated to
one or more functional or detectable moieties. The disclosure also contemplates methods
of making said chimeric or humanized anti-CGRP or anti-CGRP-R antibodies or anti-
CGRP/CGRP-R complex antibodies and binding fragments thereof for treatment or
prevention of photophobia. In one embodiment, binding fragments include, but are not
limited to, Fab, Fab', F(ab'), Fv, scFv fragments, SMIPs (small molecule
immunopharmaceuticals), camelbodies, nanobodies, and IgNAR.
Embodiments of the disclosure pertain to the use of polypeptide CGRP/CGRP
receptor inhibitors, e.g., anti-CGRP or anti-CGRP-R antibodies or antibody fragments and
CGRP or CGRP-R fragments, preferably anti-CGRP or anti-CGRP-R antibodies and
binding fragments thereof for the diagnosis, assessment and treatment of diseases and
disorders associated with CGRP or aberrant expression thereof especially for the treatment
or prevention of photophobia. Also described is the use of polypeptide CGRP/CGRP
receptor inhibitors, e.g., anti-CGRP or anti-CGRP receptor antibodies or CGRP or CGRP
receptor fragments, especially fragments of anti-CGRP antibodies for the diagnosis,
assessment and treatment of diseases and disorders associated with CGRP or aberrant
expression thereof especially for treatment or prevention of photophobia. Other
embodiments of the disclosure relate to the production of anti-CGRP or anti-CGRP
receptor antibodies or fragments thereof in recombinant host cells, for example mammalian
cells such as CHO, NSO or HEK 293 cells, or yeast cells (for example diploid yeast such
as diploid Pichia) and other yeast strains.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Figure 1 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab1.
Figure 2 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab2.
Figure 3 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab3.
Figure 4 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab4.
Figure 5 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab5.
Figure 6 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab6.
Figure 7 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab7.
Figure 8 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab8.
Figure 9 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab9.
Figure 10 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab10.
Figure 11 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab11.
Figure 12 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab12.
Figure 13 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab13.
Figure 14 provides polynucleotide and polypeptide sequences corresponding to
the full-length Antibody Ab14.
Figure 15 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab1, Ab2, Ab3, and Ab4.
Figure 16 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab5, Ab6, Ab7, and Ab8.
Figure 17 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab9, Ab10, and Ab14.
Figure 18 provides the CGRP-alpha ELISA binding data obtained following the
protocol in Example 1 infra for antibodies Ab11, Ab12, and Ab13.
Figure 19 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab1, Ab2, and Ab4, obtained following the protocol in Example 1 infra.
Figure 20 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibody Ab3, obtained following the protocol in Example 1 infra.
Figure 21 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab5 and Ab6, obtained following the protocol in Example 1 infra.
Figure 22 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab7, Ab8, Ab9, and Ab10, obtained following the protocol in Example 1
infra.
Figure 23 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibodies Ab11, Ab12, and Ab13, obtained following the protocol in Example 1 infra.
Figure 24 demonstrates the inhibition of CGRP-alpha-driven cAMP production
by antibody Ab14, obtained following the protocol in Example 1 infra.
Figure 25 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab1, Ab2, and Ab3, obtained following the protocol in Example 1 infra.
Figure 26 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab4, Ab5, and Ab6, obtained following the protocol in Example 1 infra.
Figure 27 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab7 and Ab8, obtained following the protocol in Example 1 infra.
Figure 28 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab9, Ab10, and Ab14, obtained following the protocol in Example 1 infra.
Figure 29 demonstrates the inhibition of CGRP-beta-driven cAMP production
by antibodies Ab11, Ab12, and Ab13, obtained following the protocol in Example 1 infra.
Figure 30 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab1, Ab2, Ab4, and Ab5, obtained following the protocol in Example 1 infra.
Figure 31 demonstrates the inhibition of rat CGRP -driven cAMP production by
antibodies Ab3 and Ab6, obtained following the protocol in Example 1 infra.
Figure 32 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab7 and Ab8, obtained following the protocol in Example 1 infra.
Figure 33 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab9, obtained following the protocol in Example 1 infra.
Figure 34 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab10, obtained following the protocol in Example 1 infra.
Figure 35 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibodies Ab11 and Ab12, obtained following the protocol in Example 1 infra.
Figure 36 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab13, obtained following the protocol in Example 1 infra.
Figure 37 demonstrates the inhibition of rat CGRP-driven cAMP production by
antibody Ab14, obtained following the protocol in Example 1 infra.
Figure 38 demonstrates the inhibition of binding of radiolabeled CGRP to
CGRP-R by antibodies Ab1-Ab13, obtained following the protocol in Example 6 infra.
Figure 39 demonstrates a reduction in vasodilation obtained by administering
antibodies Ab3 and Ab6 following capsaicin administration in a rat model, relative to a
control antibody, obtained following the protocol in Example 7 infra.
Figure 40 demonstrates a reduction in vasodilation obtained by administering
antibody Ab6 at differing concentrations following capsaicin administration in a rat model,
relative to a control antibody, obtained following the protocol in Example 7 infra.
Figure 41 shows the effect of ICV injection of CGRP in hRAMP1 tg mice and
control littermate mice and in particular contains data that shows that CGRP administration
decreases time in light behavior in the hRAMP1 tg mice relative to their control littermates.
Mice were injected hCGRP (2 ug) via ICV under anesthesia and allowed to recover for 30
minutes. Mice were placed individually in the two chamber light/dark boxes and
movement was recorded for 30 minutes. Six mice were run in parallel at a time in six
different boxes. Each group consisted of seven to nine mice.
Figure 42 contains data that compares the effect of systemic (IP) injection of
anti-CGRP antibody (Ab3) on CGRP driven light aversion. Ab3 in in vehicle, vehicle, and
control antibody in vehicle were administered at a dosage of 30 mg/kg in Nestin/RAMP1
mice and thereafter mice were administered CGRP via ICV administration. The data in the
left side of the graph is the total time in light (seconds) for the first 10 minutes post-CGRP
administration, and the data on the right side of the graph is the total time in light (seconds)
for the first 20 minutes measured post-CGRP injection. The data reveal that the mice who
received the anti-CGRP antibody Ab3 (disclosed infra) had a statistically significant
increase in the amount of time spent in the light relative to the mice who received the
controls.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
It is to be understood that this invention is not limited to the particular
methodology, protocols, cell lines, animal species or genera, and reagents described, as
such may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended claims. As used herein
the singular forms "a", "and", and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such
cells and reference to "the protein" includes reference to one or more proteins and
equivalents thereof known to those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
Calcitonin Gene Related Peptide (CGRP): As used herein, CGRP encompasses
not only the following Homo sapiens CGRP-alpha and Homo sapiens CGRP-beta amino
acid sequences available from American Peptides (Sunnyvale CA) and Bachem (Torrance,
CA):
CGRP-alpha: ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH (SEQ ID
NO: 281), wherein the N-terminal phenylalanine is amidated;
CGRP-beta: ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH (SEQ ID
NO: 282), wherein the N-terminal phenylalanine is amidated; but also any membrane-
bound forms of these CGRP amino acid sequences, as well as mutants (mutiens), splice
variants, isoforms, orthologues, homologues and variants of this sequence. In particular
CGRP heein encompases rodent (rat or mouse) CGRP as well as CGRP from other
mammals.
“CGRP receptor” or “CGRP-R” refers to the receptor binding partner of CGRP,
preferably the human CGRP receptor, but encompassing other species CGRP-R’s,
especially rodent (rat or mouse), non-human primate and other mammalian CGRP-R’s.
“CGRP/CGRP receptor inhibitor” herein refers to any polypeptide that inhibits
the interaction of CGRP and CGRP receptors, e.g., anti-CGRP or anti-CGRP-R antibodies
or antibody fragments and fragments of CGRP or CGRP-R polypeptides. Preferably these
inhibitors will inhibit this interaction in vitro and in vivo and will inhibit the adverse side
effects of CGRP including photoaversion or photophobia.
“Photophobia” herein refers to a symptom of abnormal intolerance to visual
perception of light, sometimes additionally defined by abnormal or irrational fear of light,
or by presence of actual physical photosensitivity of the eyes. In the present disclosure
photophobia includes in particular light aversion associated with migraine, cluster
headaches and other neurological causes of light aversive behavior that may trigger a
migraine or cluster headache. Patients may develop photophobia as a result of several
different medical conditions, related to the eye or the nervous system. Photophobia can be
caused by an increased response to light starting at any step in the visual system such as: (i)
too much light entering the eye, (ii) too much light can enter the eye if it is damaged, such
as with corneal abrasion and retinal damage, or if a pupil(s) is unable to normally constrict
(seen with damage to the oculomotor nerve, (iii) overstimulation of the photoreceptors in
the retina, (iv) excessive electric impulses to the optic nerve, and (v) excessive response in
the central nervous system.
Common causes of photophobia include migraine headaches, cataracts, or severe
ophthalmologic diseases such as uveitis or corneal abrasion. A more extensive list of
disorders associated with photophobia includes eye related causes such as Achromatopsia,
Aniridia, Anticholinergic drugs may cause photophobia by paralyzing the iris sphincter
muscle, Aphakia (absence of the lens of the eye), Buphthalmos (abnormally narrow angle
between the cornea and iris), Cataracts, Cone dystrophy, Congenital abnormalities of the
eye, Viral conjunctivitis ("pink eye") Corneal abrasion, Corneal dystrophy, Corneal ulcer,
disruption of the corneal epithelium, such as that caused by a corneal foreign body or
keratitis, Ectopia lentis, Endophthalmitis, Eye trauma caused by disease, injury, or
infection such as chalazion, episcleritis, glaucoma, keratoconus, or optic nerve hypoplasia,
Hydrophthalmos, or congenital glaucoma Iritis, Optic neuritis, Pigment dispersion
syndrom, Pupillary dilation (naturally or chemically induced), Retinal detachment, Scarring
of the cornea or sclera and Uveitis.
In addition photophobia has nervous-system-related or urological causes
including: Autism spectrum disorders, Chiari malformation, Dyslexia, Encephalitis
including Myalgic encephalomyelitis aka Chronic fatigue syndrome, Meningitis,
Subarachnoid haemorrhage, Tumor of the posterior cranial fossa, as well as other causes
such as Ankylosing spondylitis, Albinism, Ariboflavinosis, Benzodiazepines (long term use
of or withdrawal from benzodiazepines), Chemotherapy, Chikungunya, Cystinosis, Ehlers-
Danlos syndrome, Hangover, Influenza, Infectious Mononucleosis, Magnesium deficiency,
Mercury poisoning, Migraine, Rabies, and Tyrosinemia type II, also known as "Richner-
Hanhart syndrome". Additionally it is known that photophobia is elevated in depression,
bipolar disorder and agoraphobia.
“Migraine” from the Greek words hemi, meaning half, and kranion, meaning
skull) is a debilitating condition characterized by moderate to severe headaches, and
nausea. It is about three times more common in women than in men. The typical migraine
headache is unilateral (affecting one half of the head) and pulsating in nature and lasting
from 4 to 72 hours; symptoms include nausea, vomiting, photophobia (increased sensitivity
to light), phonophobia (increased sensitivity to sound); the symptoms are generally
aggravated by routine activity. Approximately one-third of people who suffer from
migraine headaches perceive an aura—unusual visual, olfactory, or other sensory
experiences that are a sign that the migraine will soon occur. Initial treatment of migraine
headaches typically is with analgesics for the headache, an antiemetic for the nausea, and
the avoidance of triggering conditions. Studies of twins indicate a 60- to 65-percent
genetic influence upon their propensity to develop migraine headaches. Moreover,
fluctuating hormone levels indicate a migraine relation: 75 percent of adult patients are
women, although migraine affects approximately equal numbers of prepubescent boys and
girls; propensity to migraine headache is known to disappear during pregnancy, although in
some women migraines may become more frequent during pregnancy.
“Effective treatment or prevention of photophobia” herein refers to inhibiting
light aversive behavior or photophobia or inhibiting the onset of light aversive behavior or
photophobia in a subject in need thereof, e.g., a subject having an active migraine attack or
cluster headache or a subject prone to migraine or cluster headaches, or one of the other
photophobia-asociated disorders identified herein after administration of an effective
amount of an CGRP/CGRP receptor inhibitor polypeptide according to the disclosure, e.g.,
an anti-CGRP antibody or antibody fragment according to the disclosure. The treatment
may be effected as a monotherapy or in association with another active agent such as
Topirimate or dihydroergotamine by way of example.
Mating competent yeast species: In the present disclosure this is intended to
broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such
species of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a
given ploidy may, under appropriate conditions, proliferate for an indefinite number of
generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential
mating can result in tetraploid strains through further mating or fusion of diploid strains.
The present disclosure contemplates the use of haploid yeast, as well as diploid or other
polyploid yeast cells produced, for example, by mating or spheroplast fusion.
In one embodiment of the disclosure, the mating competent yeast is a member of
the Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma;
Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania;
Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces;
Saturnispora; Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Other
types of yeast potentially useful in the invention include Yarrowia; Rhodosporidium;
Candida; Hansenula; Filobasium; Sporidiobolus; Bullera; Leucosporidium and
Filobasidella.
In a preferred embodiment of the disclosure, the mating competent yeast is a
member of the genus Pichia. In a further preferred embodiment of the disclosure, the
mating competent yeast of the genus Pichia is one of the following species: Pichia
pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia angusta). In a
particularly preferred embodiment of the disclosure, the mating competent yeast of the
genus Pichia is the species Pichia pastoris.
Haploid Yeast Cell: A cell having a single copy of each gene of its normal
genomic (chromosomal) complement.
Polyploid Yeast Cell: A cell having more than one copy of its normal genomic
(chromosomal) complement.
Diploid Yeast Cell: A cell having two copies (alleles) of essentially every gene
of its normal genomic complement, typically formed by the process of fusion (mating) of
two haploid cells.
Tetraploid Yeast Cell: A cell having four copies (alleles) of essentially every
gene of its normal genomic complement, typically formed by the process of fusion
(mating) of two haploid cells. Tetraploids may carry two, three, four or more different
expression cassettes. Such tetraploids might be obtained in S. cerevisiae by selective
mating homozygotic heterothallic a/a and alpha/alpha diploids and in Pichia by sequential
mating of haploids to obtain auxotrophic diploids. For example, a [met his] haploid can be
mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated
with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] x diploid [arg] to obtain
a tetraploid prototroph. It will be understood by those of skill in the art that reference to
the benefits and uses of diploid cells may also apply to tetraploid cells.
Yeast Mating: The process by which two haploid yeast cells naturally fuse to
form one diploid yeast cell.
Meiosis: The process by which a diploid yeast cell undergoes reductive division
to form four haploid spore products. Each spore may then germinate and form a haploid
vegetatively growing cell line.
Selectable Marker: A selectable marker is a gene or gene fragment that confers
a growth phenotype (physical growth characteristic) on a cell receiving that gene as, for
example through a transformation event. The selectable marker allows that cell to survive
and grow in a selective growth medium under conditions in which cells that do not receive
that selectable marker gene cannot grow. Selectable marker genes generally fall into
several types, including positive selectable marker genes such as a gene that confers on a
cell resistance to an antibiotic or other drug, temperature when two temperature sensitive
(“ts”) mutants are crossed or a ts mutant is transformed; negative selectable marker genes
such as a biosynthetic gene that confers on a cell the ability to grow in a medium without a
specific nutrient needed by all cells that do not have that biosynthetic gene, or a
mutagenized biosynthetic gene that confers on a cell inability to grow by cells that do not
have the wild type gene; and the like. Suitable markers include but are not limited to:
ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.
Expression Vector: These DNA vectors contain elements that facilitate
manipulation for the expression of a foreign protein within the target host cell.
Conveniently, manipulation of sequences and production of DNA for transformation is first
performed in a bacterial host, e.g. E. coli, and usually vectors will include sequences to
facilitate such manipulations, including a bacterial origin of replication and appropriate
bacterial selection marker. Selection markers encode proteins necessary for the survival or
growth of transformed host cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not survive in the culture
medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or
other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media. Exemplary vectors and methods for transformation of
yeast are described, for example, in Burke, D., Dawson, D., & Stearns, T. (2000). Methods
in yeast genetics: a Cold Spring Harbor Laboratory course manual. Plainview, N.Y.: Cold
Spring Harbor Laboratory Press.
Expression vectors for use in the methods of the disclosure will further include
yeast specific sequences, including a selectable auxotrophic or drug marker for identifying
transformed yeast strains. A drug marker may further be used to amplify copy number of
the vector in a yeast host cell.
The polypeptide coding sequence of interest is operably linked to transcriptional
and translational regulatory sequences that provide for expression of the polypeptide in
yeast cells. These vector components may include, but are not limited to, one or more of
the following: an enhancer element, a promoter, and a transcription termination sequence.
Sequences for the secretion of the polypeptide may also be included, e.g. a signal sequence,
and the like. A yeast origin of replication is optional, as expression vectors are often
integrated into the yeast genome. In one embodiment of the disclosure, the polypeptide of
interest is operably linked, or fused, to sequences providing for optimized secretion of the
polypeptide from yeast diploid cells.
Nucleic acids are "operably linked" when placed into a functional relationship
with another nucleic acid sequence. For example, DNA for a signal sequence is operably
linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the case of a secretory leader,
contiguous and in reading frame. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction sites or alternatively via a
PCR/recombination method familiar to those skilled in the art (Gateway Technology;
Invitrogen, Carlsbad California). If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accordance with conventional practice.
Promoters are untranslated sequences located upstream (5') to the start codon of
a structural gene (generally within about 100 to 1000 bp) that control the transcription and
translation of particular nucleic acid sequences to which they are operably linked. Such
promoters fall into several classes: inducible, constitutive, and repressible promoters (that
increase levels of transcription in response to absence of a repressor). Inducible promoters
may initiate increased levels of transcription from DNA under their control in response to
some change in culture conditions, e.g., the presence or absence of a nutrient or a change in
temperature.
The yeast promoter fragment may also serve as the site for homologous
recombination and integration of the expression vector into the same site in the yeast
genome; alternatively a selectable marker is used as the site for homologous
recombination. Pichia transformation is described in Cregg et al. (1985) Mol. Cell. Biol.
:3376-3385.
Examples of suitable promoters from Pichia include the AOX1 and promoter
(Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al. (2003)
Yeast 20(13):1097-108); glyceraldehydephosphate dehydrogenase promoter (GAP)
(Waterham et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998) Gene
216(1):93-102). The GAP promoter is a strong constitutive promoter and the AOX and
FLD1 promoters are inducible.
Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3,
PHO5, Pyk, and chimeric promoters derived therefrom. Additionally, non-yeast promoters
may be used herein such as mammalian, insect, plant, reptile, amphibian, viral, and avian
promoters. Most typically the promoter will comprise a mammalian promoter (potentially
endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides
for efficient transcription in yeast systems.
The polypeptides of interest may be produced recombinantly not only directly,
but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of the vector, or it may
be a part of the polypeptide coding sequence that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is recognized and processed
through one of the standard pathways available within the host cell. The S. cerevisiae
alpha factor pre-pro signal has proven effective in the secretion of a variety of recombinant
proteins from P. pastoris. Other yeast signal sequences include the alpha mating factor
signal sequence, the invertase signal sequence, and signal sequences derived from other
secreted yeast polypeptides. Additionally, these signal peptide sequences may be
engineered to provide for enhanced secretion in diploid yeast expression systems. Other
secretion signals of interest also include mammalian signal sequences, which may be
heterologous to the protein being secreted, or may be a native sequence for the protein
being secreted. Signal sequences include pre-peptide sequences, and in some instances
may include propeptide sequences. Many such signal sequences are known in the art,
including the signal sequences found on immunoglobulin chains, e.g., K28 preprotoxin
sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human
Ig heavy chain, human Ig light chain, and the like. For example, see Hashimoto et. al.
Protein Eng 11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998).
Transcription may be increased by inserting a transcriptional activator sequence
into the vector. These activators are cis-acting elements of DNA, usually about from 10 to
300 bp, which act on a promoter to increase its transcription. Transcriptional enhancers are
relatively orientation and position independent, having been found 5' and 3' to the
transcription unit, within an intron, as well as within the coding sequence itself. The
enhancer may be spliced into the expression vector at a position 5' or 3' to the coding
sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells may also contain sequences
necessary for the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from 3' to the translation termination codon, in
untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of
the mRNA.
Construction of suitable vectors containing one or more of the above-listed
components employs standard ligation techniques or PCR/recombination methods.
Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form
desired to generate the plasmids required or via recombination methods. For analysis to
confirm correct sequences in plasmids constructed, the ligation mixtures are used to
transform host cells, and successful transformants selected by antibiotic resistance (e.g.
ampicillin or Zeocin) where appropriate. Plasmids from the transformants are prepared,
analyzed by restriction endonuclease digestion and/or sequenced.
As an alternative to restriction and ligation of fragments, recombination methods
based on att sites and recombination enzymes may be used to insert DNA sequences into a
vector. Such methods are described, for example, by Landy (1989) Ann.Rev.Biochem.
58:913-949; and are known to those of skill in the art. Such methods utilize intermolecular
DNA recombination that is mediated by a mixture of lambda and E.coli –encoded
recombination proteins. Recombination occurs between specific attachment (att) sites on
the interacting DNA molecules. For a description of att sites see Weisberg and Landy
(1983) Site-Specific Recombination in Phage Lambda, in Lambda II, Weisberg, ed.(Cold
Spring Harbor, NY:Cold Spring Harbor Press), pp. 211-250. The DNA segments flanking
the recombination sites are switched, such that after recombination, the att sites are hybrid
sequences comprised of sequences donated by each parental vector. The recombination
can occur between DNAs of any topology.
Att sites may be introduced into a sequence of interest by ligating the sequence
of interest into an appropriate vector; generating a PCR product containing att B sites
through the use of specific primers; generating a cDNA library cloned into an appropriate
vector containing att sites; and the like.
Folding, as used herein, refers to the three-dimensional structure of polypeptides
and proteins, where interactions between amino acid residues act to stabilize the structure.
While non-covalent interactions are important in determining structure, usually the proteins
of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two
cysteine residues. For naturally occurring proteins and polypeptides or derivatives and
variants thereof, the proper folding is typically the arrangement that results in optimal
biological activity, and can conveniently be monitored by assays for activity, e.g. ligand
binding, enzymatic activity, etc.
In some instances, for example where the desired product is of synthetic origin,
assays based on biological activity will be less meaningful. The proper folding of such
molecules may be determined on the basis of physical properties, energetic considerations,
modeling studies, and the like.
The expression host may be further modified by the introduction of sequences
encoding one or more enzymes that enhance folding and disulfide bond formation, i.e.
foldases, chaperonins, etc. Such sequences may be constitutively or inducibly expressed in
the yeast host cell, using vectors, markers, etc. as known in the art. Preferably the
sequences, including transcriptional regulatory elements sufficient for the desired pattern of
expression, are stably integrated in the yeast genome through a targeted methodology.
For example, the eukaryotic PDI is not only an efficient catalyst of protein
cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity.
Co-expression of PDI can facilitate the production of active proteins having multiple
disulfide bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain
binding protein); cyclophilin; and the like. In one embodiment of the disclosure, each of
the haploid parental strains expresses a distinct folding enzyme, e.g. one strain may express
BIP, and the other strain may express PDI or combinations thereof.
The terms "desired protein" or "desired antibody" are used interchangeably and
refer generally to a parent antibody specific to a target, i.e., CGRP or CGRP receptor or a
chimeric or humanized antibody or a binding portion thereof derived therefrom as
described herein. The term “antibody” is intended to include any polypeptide chain-
containing molecular structure with a specific shape that fits to and recognizes an epitope,
where one or more non-covalent binding interactions stabilize the complex between the
molecular structure and the epitope. The archetypal antibody molecule is the
immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all
sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other
avians, etc., are considered to be “antibodies.” A preferred source for producing antibodies
useful as starting material according to the disclosure is rabbits. Numerous antibody
coding sequences have been described; and others may be raised by methods well-known
in the art. Examples thereof include chimeric antibodies, human antibodies and other non-
human mammalian antibodies, humanized antibodies, single chain antibodies (such as
scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks),
small-modular immunopharmaceuticals (SMIPs), and antibody fragments such as Fabs,
Fab', F(ab') and the like. See Streltsov VA, et al., Structure of a shark IgNAR antibody
variable domain and modeling of an early-developmental isotype, Protein Sci. 2005
Nov;14(11):2901-9. Epub 2005 Sep 30; Greenberg AS, et al., A new antigen receptor gene
family that undergoes rearrangement and extensive somatic diversification in sharks,
Nature. 1995 Mar 9;374(6518):168-73; Nuttall SD, et al., Isolation of the new antigen
receptor from wobbegong sharks, and use as a scaffold for the display of protein loop
libraries, Mol Immunol. 2001 Aug;38(4):313-26; Hamers-Casterman C, et al., Naturally
occurring antibodies devoid of light chains, Nature. 1993 Jun 3;363(6428):446-8; Gill DS,
et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin
Biotechnol. 2006 Dec;17(6):653-8. Epub 2006 Oct 19.
For example, antibodies or antigen binding fragments may be produced by
genetic engineering. In this technique, as with other methods, antibody-producing cells are
sensitized to the desired antigen or immunogen. The messenger RNA isolated from
antibody producing cells is used as a template to make cDNA using PCR amplification. A
library of vectors, each containing one heavy chain gene and one light chain gene retaining
the initial antigen specificity, is produced by insertion of appropriate sections of the
amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is
constructed by combining the heavy chain gene library with the light chain gene library.
This results in a library of clones which co-express a heavy and light chain (resembling the
Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry
these genes are co-transfected into a host cell. When antibody gene synthesis is induced in
the transfected host, the heavy and light chain proteins self-assemble to produce active
antibodies that can be detected by screening with the antigen or immunogen.
Antibody coding sequences of interest include those encoded by native
sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are
not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant
polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino
acid substitutions can be conservative amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding
by substitution or deletion of one or more cysteine residues that are not necessary for
function. Variants can be designed so as to retain or have enhanced biological activity of a
particular region of the protein (e.g., a functional domain, catalytic amino acid residues,
etc). Variants also include fragments of the polypeptides disclosed herein, particularly
biologically active fragments and/or fragments corresponding to functional domains.
Techniques for in vitro mutagenesis of cloned genes are known. Also included in the
subject disclosure are polypeptides that have been modified using ordinary molecular
biological techniques so as to improve their resistance to proteolytic degradation or to
optimize solubility properties or to render them more suitable as a therapeutic agent.
Chimeric antibodies may be made by recombinant means by combining the
variable light and heavy chain regions (V and V ), obtained from antibody producing cells
of one species with the constant light and heavy chain regions from another. Typically
chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in
order to produce an antibody with predominantly human domains. The production of such
chimeric antibodies is well known in the art, and may be achieved by standard means (as
described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its
entirety). It is further contemplated that the human constant regions of chimeric antibodies
of the disclosure may be selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8,
IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19
constant regions.
Humanized antibodies are engineered to contain even more human-like
immunoglobulin domains, and incorporate only the complementarity-determining regions
of the animal-derived antibody. This is accomplished by carefully examining the sequence
of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting
them to the structure of the human antibody chains. Although facially complex, the
process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated
fully herein by reference.
In addition to entire immunoglobulins (or their recombinant counterparts),
immunoglobulin fragments comprising the epitope binding site (e.g., Fab’, F(ab’) , or other
fragments) may be synthesized. “Fragment,” or minimal immunoglobulins may be
designed utilizing recombinant immunoglobulin techniques. For instance “Fv”
immunoglobulins for use in the present disclosure may be produced by synthesizing a
fused variable light chain region and a variable heavy chain region. Combinations of
antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities.
In another embodiment of the disclosure, SMIPs (small molecule
immunopharmaceuticals), camelbodies, nanobodies, and IgNAR are encompassed by
immunoglobulin fragments.
Immunoglobulins and fragments thereof may be modified post-translationally,
e.g. to add effector moieties such as chemical linkers, detectable moieties, such as
fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive
materials, chemiluminescent moieties and the like, or specific binding moieties, such as
streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions
of the present disclosure. Examples of additional effector molecules are described infra.
A polynucleotide sequence "corresponds" to a polypeptide sequence if
translation of the polynucleotide sequence in accordance with the genetic code yields the
polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide
sequence), one polynucleotide sequence "corresponds" to another polynucleotide sequence
if the two sequences encode the same polypeptide sequence.
A "heterologous" region or domain of a DNA construct is an identifiable
segment of DNA within a larger DNA molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene,
the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA
in the genome of the source organism. Another example of a heterologous region is a
construct where the coding sequence itself is not found in nature (e.g., a cDNA where the
genomic coding sequence contains introns, or synthetic sequences having codons different
than the native gene). Allelic variations or naturally-occurring mutational events do not
give rise to a heterologous region of DNA as defined herein.
A "coding sequence" is an in-frame sequence of codons that (in view of the
genetic code) correspond to or encode a protein or peptide sequence. Two coding
sequences correspond to each other if the sequences or their complementary sequences
encode the same amino acid sequences. A coding sequence in association with appropriate
regulatory sequences may be transcribed and translated into a polypeptide. A
polyadenylation signal and transcription termination sequence will usually be located 3' to
the coding sequence. A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a downstream (3'
direction) coding sequence. Promoter sequences typically contain additional sites for
binding of regulatory molecules (e.g., transcription factors) which affect the transcription
of the coding sequence. A coding sequence is "under the control" of the promoter sequence
or "operatively linked" to the promoter when RNA polymerase binds the promoter
sequence in a cell and transcribes the coding sequence into mRNA, which is then in turn
translated into the protein encoded by the coding sequence.
Vectors are used to introduce a foreign substance, such as DNA, RNA or
protein, into an organism or host cell. Typical vectors include recombinant viruses (for
polynucleotides) and liposomes (for polypeptides). A "DNA vector" is a replicon, such as
plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as
to bring about the replication of the attached segment. An "expression vector" is a DNA
vector which contains regulatory sequences which will direct polypeptide synthesis by an
appropriate host cell. This usually means a promoter to bind RNA polymerase and initiate
transcription of mRNA, as well as ribosome binding sites and initiation signals to direct
translation of the mRNA into a polypeptide(s). Incorporation of a polynucleotide sequence
into an expression vector at the proper site and in correct reading frame, followed by
transformation of an appropriate host cell by the vector, enables the production of a
polypepide encoded by said polynucleotide sequence.
"Amplification" of polynucleotide sequences is the in vitro production of
multiple copies of a particular nucleic acid sequence. The amplified sequence is usually in
the form of DNA. A variety of techniques for carrying out such amplification are
described in a review article by Van Brunt (1990, Bio/Technol., 8(4):291-294). Polymerase
chain reaction or PCR is a prototype of nucleic acid amplification, and use of PCR herein
should be considered exemplary of other suitable amplification techniques.
The general structure of antibodies in vertebrates now is well understood
(Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). Antibodies consist of two
identical light polypeptide chains of molecular weight approximately 23,000 daltons (the
“light chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the
“heavy chain”). The four chains are joined by disulfide bonds in a “Y” configuration
wherein the light chains bracket the heavy chains starting at the mouth of the “Y”
configuration. The “branch” portion of the “Y” configuration is designated the F region;
the stem portion of the “Y” configuration is designated the F region. The amino acid
sequence orientation runs from the N-terminal end at the top of the “Y” configuration to
the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable
region having specificity for the antigen that elicited it, and is approximately 100 amino
acids in length, there being slight variations between light and heavy chain and from
antibody to antibody.
The variable region is linked in each chain to a constant region that extends the
remaining length of the chain and that within a particular class of antibody does not vary
with the specificity of the antibody (i.e., the antigen eliciting it). There are five known
major classes of constant regions that determine the class of the immunoglobulin molecule
(IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and ε (gamma, mu, alpha, delta,
or epsilon) heavy chain constant regions). The constant region or class determines
subsequent effector function of the antibody, including activation of complement (Kabat,
E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436,
Holt, Rinehart, Winston (1976)), and other cellular responses (Andrews, D. W., et al.,
Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S., et al., Immunology, 48:
187 (1983)); while the variable region determines the antigen with which it will react.
Light chains are classified as either κ (kappa) or λ (lambda). Each heavy chain class can be
prepared with either kappa or lambda light chain. The light and heavy chains are
covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded
to each other by covalent disulfide linkages when the immunoglobulins are generated either
by hybridomas or by B cells.
The expression “variable region” or “VR” refers to the domains within each pair
of light and heavy chains in an antibody that are involved directly in binding the antibody
to the antigen. Each heavy chain has at one end a variable domain (V ) followed by a
number of constant domains. Each light chain has a variable domain (V ) at one end and a
constant domain at its other end; the constant domain of the light chain is aligned with the
first constant domain of the heavy chain, and the light chain variable domain is aligned
with the variable domain of the heavy chain.
The expressions “complementarity determining region,” “hypervariable region,”
or “CDR” refer to one or more of the hyper-variable or complementarity determining
regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See
Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of
Health, Bethesda, Md., (1987)). These expressions include the hypervariable regions as
defined by Kabat et al. (“Sequences of Proteins of Immunological Interest,” Kabat E., et
al., US Dept. of Health and Human Services, 1983) or the hypervariable loops in 3-
dimensional structures of antibodies (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)).
The CDRs in each chain are held in close proximity by framework regions and, with the
CDRs from the other chain, contribute to the formation of the antigen binding site. Within
the CDRs there are select amino acids that have been described as the selectivity
determining regions (SDRs) which represent the critical contact residues used by the CDR
in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).
The expressions “framework region” or “FR” refer to one or more of the
framework regions within the variable regions of the light and heavy chains of an antibody
(See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid
sequence regions interposed between the CDRs within the variable regions of the light and
heavy chains of an antibody.
Anti-CGRP Antibodies and Binding Fragments Thereof Having Binding Activity for
CGRP
Antibody Ab1
The present disclosure broadly contemplates inhibition or prevention of
photophobia in a subject in need thereof, e.g., a migraine sufferer or another photophobia
associated disorder by administering an effective amount of a CGRP/CGRP receptor
inhibitor polypeptide, e.g., an anti-CGRP or an anti-CGRP receptor antibody or fragment
thereof or a fragment of CGRP or a CGRP receptor which is capable of effective treatment
or prevention of photophobia. This may be determined e.g., using appropriate in vivo
models such as the transgenic mice model disclosed in Example 8.
In one exemplary embodiment, the disclosure includes chimeric antibodies
derived from Ab1 having binding specificity to CGRP and possessing a variable light chain
sequence comprising the sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYDNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLGSYDCSSGDCFVFGGGTEVVV
KR (SEQ ID NO: 1).
The disclosure also includes chimeric antibodies having binding specificity to
CGRP and possessing a light chain sequence comprising the sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYDNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLGSYDCSSGDCFVFGGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 2).
The disclosure further includes chimeric antibodies having binding specificity to
CGRP and possessing a variable heavy chain sequence comprising the sequence set forth
below:
QSLEESGGRLVTPGTPLTLTCTVSGLDLSSYYMQWVRQAPGKGLEWIGVIGINDNT
YYASWAKGRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 3).
The disclosure also includes chimeric antibodies having binding specificity to
CGRP and possessing a heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGLDLSSYYMQWVRQAPGKGLEWIGVIGINDNT
YYASWAKGRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 4).
The disclosure further contemplates antibodies comprising one or more of the
polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ
ID NO: 2, and/or one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO:
9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or
the heavy chain sequence of SEQ ID NO: 4, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy
and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP. In one embodiment of the present disclosure, antibody
fragments decribed herein comprise, or alternatively consist of, the polypeptide sequence
of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment of the present disclosure,
antibody fragments described herein comprise, or alternatively consist of, the polypeptide
sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP comprise, or alternatively consist of, one or more of
the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ
ID NO: 2.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP comprise, or alternatively consist of, one or more of
the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of
SEQ ID NO: 4.
The present disclosure also contemplates antibody fragments which include one
or more of the antibody fragments described herein. In one embodiment of the present
disclosure, fragments of the antibodies having binding specificity to CGRP comprise, or
alternatively consist of, one, two, three or more, including all of the following antibody
fragments: the variable light chain region of SEQ ID NO: 1; the variable heavy chain
region of SEQ ID NO: 3; the complementarity-determining regions (SEQ ID NO: 5; SEQ
ID NO: 6; and SEQ ID NO: 7) of the variable light chain region of SEQ ID NO: 1; and the
complementarity-determining regions (SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO:
) of the variable heavy chain region of SEQ ID NO: 3.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody is Ab1, comprising, or alternatively consisting of, SEQ ID NO: 2 and
SEQ ID NO: 4, and having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab1, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 1 and the variable heavy chain
sequence of SEQ ID NO: 3. This embodiment of the present disclosure further
contemplates additions, deletions, and variants of SEQ ID NO: 1 and/or SEQ ID NO: 3 in
said Fab while retaining binding specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments may for potential treatment or prevention of photophobia be produced by
enzymatic digestion (e.g., papain) of Ab1. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab1 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab2
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
R (SEQ ID NO: 11).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 12).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 13).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 14).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12,
and/or one or more of the polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and
SEQ ID NO: 20 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 13 or the
heavy chain sequence of SEQ ID NO: 14, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof for potential treatment or prevention of photophobia comprise, or
alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP for potential treatment or prevention of photophobia. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 13 or SEQ
ID NO: 14.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 11; the variable heavy chain region of SEQ ID NO: 13; the
complementarity-determining regions (SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO:
17) of the variable light chain region of SEQ ID NO: 11; and the complementarity-
determining regions (SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20) of the
variable heavy chain region of SEQ ID NO: 13.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab2,
comprising, or alternatively consisting of, SEQ ID NO: 12 and SEQ ID NO: 14, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab2, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 11 and the variable heavy chain sequence of SEQ ID NO: 13. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 11 and/or SEQ ID NO: 13 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab2. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab2 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab3
In a preferred embodiment, the present disclosure includes humanized antibodies
having binding specificity to CGRP and possessing a variable light chain sequence
comprising the sequence set forth below. As disclosed in Example 8 this antibody has been
demonstrated in a transgenic mouse light aversion behavioral model to effectively inhibit
CGRP-associated photophobia:
VLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIY
STSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGT
KVEIKR (SEQ ID NO: 21).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 22).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 23).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGIN
DNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 24).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22,
and/or one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and
SEQ ID NO: 30 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the
heavy chain sequence of SEQ ID NO: 24, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 21 or SEQ ID NO: 22.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 23 or SEQ
ID NO: 24.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP comprise, or alternatively consist of, one or more of
the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ
ID NO: 22.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 21; the variable heavy chain region of SEQ ID NO: 23; the
complementarity-determining regions (SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO:
27) of the variable light chain region of SEQ ID NO: 21; and the complementarity-
determining regions (SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30) of the
variable heavy chain region of SEQ ID NO: 23.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for treatment or prevention of photophobia is Ab3, comprising, or
alternatively consisting of, SEQ ID NO: 22 and SEQ ID NO: 24, and having at least one of
the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments
having binding specificity for CGRP. With respect to antibody Ab3, the Fab fragment
includes the variable light chain sequence of SEQ ID NO: 21 and the variable heavy chain
sequence of SEQ ID NO: 23. This embodiment of the present disclosure further
contemplates additions, deletions, and variants of SEQ ID NO: 21 and/or SEQ ID NO: 23
in said Fab while retaining binding specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab3. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab3 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab4
In one embodiment, the present disclosure includes chimeric antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTL
ASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVV
VKR (SEQ ID NO: 31).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a light
chain sequence comprising the sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTL
ASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 32).
The present disclosure further includes chimeric antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGAT
YYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 33).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGAT
YYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 34).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32,
and/or one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and
SEQ ID NO: 40 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the
heavy chain sequence of SEQ ID NO: 34, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP for potential treatment or prevention of photophobia. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 31 or SEQ ID NO: 32.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 33 or SEQ
ID NO: 34.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 31; the variable heavy chain region of SEQ ID NO: 33; the
complementarity-determining regions (SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO:
37) of the variable light chain region of SEQ ID NO: 31; and the complementarity-
determining regions (SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40) of the
variable heavy chain region of SEQ ID NO: 33.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab4,
comprising, or alternatively consisting of, SEQ ID NO: 32 and SEQ ID NO: 34, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab4, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 31 and the variable heavy chain sequence of SEQ ID NO: 33. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 31 and/or SEQ ID NO: 33 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab4. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab4 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab5
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
R (SEQ ID NO: 41).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 42).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 43).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 44).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42,
and/or one or more of the polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and
SEQ ID NO: 50 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 43 or the
heavy chain sequence of SEQ ID NO: 44, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 41 or SEQ ID NO: 42.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 43 or SEQ
ID NO: 44.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 41; the variable heavy chain region of SEQ ID NO: 43; the
complementarity-determining regions (SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO:
47) of the variable light chain region of SEQ ID NO: 41; and the complementarity-
determining regions (SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50) of the
variable heavy chain region of SEQ ID NO: 43.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab5,
comprising, or alternatively consisting of, SEQ ID NO: 42 and SEQ ID NO: 44, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab5, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 41 and the variable heavy chain sequence of SEQ ID NO: 43. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 41 and/or SEQ ID NO: 43 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab5. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab5 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab6
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
R (SEQ ID NO: 51).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 52).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 53).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGIN
GATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 54).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52,
and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and
SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the
heavy chain sequence of SEQ ID NO: 54, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 52.
In another embodiment of the present disclosure, antibody fragments described herein for
potential treatment or prevention of photophobia comprise, or alternatively consist of, the
polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 51; the variable heavy chain region of SEQ ID NO: 53; the
complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO:
57) of the variable light chain region of SEQ ID NO: 51; and the complementarity-
determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the
variable heavy chain region of SEQ ID NO: 53.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab6,
comprising, or alternatively consisting of, SEQ ID NO: 52 and SEQ ID NO: 54, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab6, the Fab fragment for potential treatment or prevention of
photophobia includes the variable light chain sequence of SEQ ID NO: 51 and the variable
heavy chain sequence of SEQ ID NO: 53. This embodiment of the present disclosure
further contemplates additions, deletions, and variants of SEQ ID NO: 51 and/or SEQ ID
NO: 53 in said Fab while retaining binding specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab6. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab6 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab7
In one embodiment, the present disclosure includes chimeric antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEVV
VKR (SEQ ID NO: 61).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a light
chain sequence comprising the sequence set forth below:
QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 62).
The present disclosure further includes chimeric antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
QEQLKESGGRLVTPGTSLTLTCTVSGIDLSNHYMQWVRQAPGKGLEWIGVVGING
RTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 63).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a heavy
chain sequence comprising the sequence set forth below:
QEQLKESGGRLVTPGTSLTLTCTVSGIDLSNHYMQWVRQAPGKGLEWIGVVGING
RTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ
ID NO: 64).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62,
and/or one or more of the polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and
SEQ ID NO: 70 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 63 or the
heavy chain sequence of SEQ ID NO: 64, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof for potential treatment or prevention of photophobia comprise, or
alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein for potential
treatment or prevention of photophobia comprise, or alternatively consist of, the
polypeptide sequence of SEQ ID NO: 61 or SEQ ID NO: 62. In another embodiment of
the present disclosure, antibody fragments described herein comprise, or alternatively
consist of, the polypeptide sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP for potential treatment or prevention of
photophobia comprise, or alternatively consist of, one, two, three or more, including all of
the following antibody fragments: the variable light chain region of SEQ ID NO: 61; the
variable heavy chain region of SEQ ID NO: 63; the complementarity-determining regions
(SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67) of the variable light chain region
of SEQ ID NO: 61; and the complementarity-determining regions (SEQ ID NO: 68; SEQ
ID NO: 69; and SEQ ID NO: 70) of the variable heavy chain region of SEQ ID NO: 63.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab7,
comprising, or alternatively consisting of, SEQ ID NO: 62 and SEQ ID NO: 64, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab7, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 61 and the variable heavy chain sequence of SEQ ID NO: 63. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 61 and/or SEQ ID NO: 63 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab7. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab7 or Fab fragments thereof for potential
treatment or prevention of photophobia may be produced via expression in mammalian
cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as
yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab8
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYNYNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSTGDCFVFGGGTKVEIK
R (SEQ ID NO: 71).
The present disclosure also includes humanized antibodies having binding
specificity to CGRP for potential treatment or prevention of photophobia and possessing a
light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQSVYNYNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSTGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 72).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSNHYMQWVRQAPGKGLEWVGVVGIN
GRTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 73).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDLSNHYMQWVRQAPGKGLEWVGVVGIN
GRTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 74).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72,
and/or one or more of the polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and
SEQ ID NO: 80 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 73 or the
heavy chain sequence of SEQ ID NO: 74, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof for potential treatment or prevention of photophobia comprise, or
alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 71 or SEQ ID NO: 72.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 73 or SEQ
ID NO: 74.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies for potential treatment or prevention of photophobia having binding specificity
to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the
following antibody fragments: the variable light chain region of SEQ ID NO: 71; the
variable heavy chain region of SEQ ID NO: 73; the complementarity-determining regions
(SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77) of the variable light chain region
of SEQ ID NO: 71; and the complementarity-determining regions (SEQ ID NO: 78; SEQ
ID NO: 79; and SEQ ID NO: 80) of the variable heavy chain region of SEQ ID NO: 73.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab8,
comprising, or alternatively consisting of, SEQ ID NO: 72 and SEQ ID NO: 74, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab8, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 71 and the variable heavy chain sequence of SEQ ID NO: 73. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 71 and/or SEQ ID NO: 73 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab8. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab8 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab9
In one embodiment, the present disclosure includes chimeric antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQNVYNNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVV
VKR (SEQ ID NO: 81).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a light
chain sequence comprising the sequence set forth below:
QVLTQTPSPVSAAVGSTVTINCQASQNVYNNNYLAWYQQKPGQPPKQLIYSTSTL
ASGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVV
VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 82).
The present disclosure further includes chimeric antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGIGLSSYYMQWVRQSPGRGLEWIGVIGSDGKT
YYATWAKGRFTISKTSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSS
(SEQ ID NO: 83).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGTPLTLTCTVSGIGLSSYYMQWVRQSPGRGLEWIGVIGSDGKT
YYATWAKGRFTISKTSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSSAST
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 84).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82,
and/or one or more of the polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and
SEQ ID NO: 90 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 83 or the
heavy chain sequence of SEQ ID NO: 84, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof for potential treatment or prevention of photophobia comprise, or
alternatively consist of, combinations of one or more of the CDRs, the variable heavy and
variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody having
binding specificity to CGRP for potential treatment or prevention of photophobia. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 81 or SEQ ID NO: 82.
In another embodiment of the present disclosure, antibody fragments described herein for
potential treatment or prevention of photophobia comprise, or alternatively consist of, the
polypeptide sequence of SEQ ID NO: 83 or SEQ ID NO: 84.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP comprise, or alternatively consist of, one or more of
the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which
correspond to the complementarity-determining regions (CDRs, or hypervariable regions)
of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ
ID NO: 82.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP for potential treatment or prevention of
photophobia comprise, or alternatively consist of, one, two, three or more, including all of
the following antibody fragments: the variable light chain region of SEQ ID NO: 81; the
variable heavy chain region of SEQ ID NO: 83; the complementarity-determining regions
(SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87) of the variable light chain region
of SEQ ID NO: 81; and the complementarity-determining regions (SEQ ID NO: 88; SEQ
ID NO: 89; and SEQ ID NO: 90) of the variable heavy chain region of SEQ ID NO: 83.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab9,
comprising, or alternatively consisting of, SEQ ID NO: 82 and SEQ ID NO: 84, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab9, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 81 and the variable heavy chain sequence of SEQ ID NO: 83. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 81 and/or SEQ ID NO: 83 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab9. In another embodiment of the present
disclosure, anti-CGRP antibodies for potential treatment or prevention of photophobia such
as Ab9 or Fab fragments thereof may be produced via expression in mammalian cells such
as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells
(for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
Antibody Ab10
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
R (SEQ ID NO: 91).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 92).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSS (SEQ ID NO: 93).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 94).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92,
and/or one or more of the polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and
SEQ ID NO: 100 which correspond to the complementarity-determining regions (CDRs, or
hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 93 or the
heavy chain sequence of SEQ ID NO: 94, or combinations of these polypeptide sequences.
In another embodiment of the present disclosure, the antibodies described herein or
fragments thereof comprise, or alternatively consist of, combinations of one or more of the
CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 91 or SEQ ID NO: 92.
In another embodiment of the present disclosure, antibody fragments described herein for
potential treatment or prevention of photophobia comprise, or alternatively consist of, the
polypeptide sequence of SEQ ID NO: 93 or SEQ ID NO: 94.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable light chain sequence
of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92.
In a further embodiment of the present disclosure, fragments of the antibody
having binding specificity to CGRP for potential treatment or prevention of photophobia
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-
determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence
of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 91; the variable heavy chain region of SEQ ID NO: 93; the
complementarity-determining regions (SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO:
97) of the variable light chain region of SEQ ID NO: 91; and the complementarity-
determining regions (SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100) of the
variable heavy chain region of SEQ ID NO: 93.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab10,
comprising, or alternatively consisting of, SEQ ID NO: 92 and SEQ ID NO: 94, and having
at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab10, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 91 and the variable heavy chain sequence of SEQ ID NO: 93. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 91 and/or SEQ ID NO: 93 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab10. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab10 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab11
In one embodiment, the present disclosure includes chimeric antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQTASPVSPAVGSTVTINCRASQSVYYNNYLAWYQQKPGQPPKQLIYSTSTLA
SGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSNGDCFVFGGGTEVVV
KR (SEQ ID NO: 101).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a light
chain sequence comprising the sequence set forth below:
QVLTQTASPVSPAVGSTVTINCRASQSVYYNNYLAWYQQKPGQPPKQLIYSTSTLA
SGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSNGDCFVFGGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 102).
The present disclosure further includes chimeric antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGGSLTLTCTVSGIDVTNYYMQWVRQAPGKGLEWIGVIGVNGK
RYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
(SEQ ID NO: 103).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a heavy
chain sequence comprising the sequence set forth below:
QSLEESGGRLVTPGGSLTLTCTVSGIDVTNYYMQWVRQAPGKGLEWIGVIGVNGK
RYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID
NO: 104).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102,
and/or one or more of the polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109;
and SEQ ID NO: 110 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 103
or the heavy chain sequence of SEQ ID NO: 104, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof for potential treatment or prevention of photophobia comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy
and variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 101 or SEQ ID NO: 102.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 103 or SEQ
ID NO: 104.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO:
104.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 101; the variable heavy chain region of SEQ ID NO: 103; the
complementarity-determining regions (SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID
NO: 107) of the variable light chain region of SEQ ID NO: 101; and the complementarity-
determining regions (SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110) of the
variable heavy chain region of SEQ ID NO: 103.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab11,
comprising, or alternatively consisting of, SEQ ID NO: 102 and SEQ ID NO: 104, and
having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab11, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 101 and the variable heavy chain sequence of SEQ ID NO: 103. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 101 and/or SEQ ID NO: 103 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab11. In another embodiment of the present
disclosure, anti-CGRP antibodies such as Ab11 or Fab fragments thereof may be produced
via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or
microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and
other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab12
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCRASQSVYYNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSNGDCFVFGGGTKVEIK
R (SEQ ID NO: 111).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCRASQSVYYNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSNGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 112).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDVTNYYMQWVRQAPGKGLEWVGVIGVN
GKRYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSS (SEQ ID NO: 113).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIDVTNYYMQWVRQAPGKGLEWVGVIGVN
GKRYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 114).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112,
and/or one or more of the polypeptide sequences of SEQ ID NO: 118; SEQ ID NO: 119;
and SEQ ID NO: 120 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 113
or the heavy chain sequence of SEQ ID NO: 114, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof for potential treatment or prevention of photophobia comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy
and variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 111 or SEQ ID NO: 112.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 113 or SEQ
ID NO: 114.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 115; SEQ ID NO: 116; and SEQ ID NO: 117 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO:
114.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 111; the variable heavy chain region of SEQ ID NO: 113; the
complementarity-determining regions (SEQ ID NO: 115; SEQ ID NO: 116; and SEQ ID
NO: 117) of the variable light chain region of SEQ ID NO: 111; and the complementarity-
determining regions (SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120) of the
variable heavy chain region of SEQ ID NO: 113.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab12,
comprising, or alternatively consisting of, SEQ ID NO: 112 and SEQ ID NO: 114, and
having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab12, the Fab fragment includes the variable light chain sequence
of SEQ ID NO: 111 and the variable heavy chain sequence of SEQ ID NO: 113. This
embodiment of the present disclosure further contemplates additions, deletions, and
variants of SEQ ID NO: 111 and/or SEQ ID NO: 113 in said Fab while retaining binding
specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab12. In another embodiment of the present
disclosure, anti-CGRP antibodies for potential treatment or prevention of photophobia such
as Ab12 or Fab fragments thereof may be produced via expression in mammalian cells
such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast
cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable
Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab13
In one embodiment, the present disclosure includes chimeric antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQPPKRLIYDASKL
ASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEVVV
KR (SEQ ID NO: 121).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a light
chain sequence comprising the sequence set forth below:
AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQPPKRLIYDASKL
ASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEVVV
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 122).
The present disclosure further includes chimeric antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
QSVEESGGGLVQPEGSLTLTCTASGFDFSSNAMWWVRQAPGKGLEWIGIIYNGDG
STYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTVS
S (SEQ ID NO: 123).
The present disclosure also includes chimeric antibodies for potential treatment
or prevention of photophobia having binding specificity to CGRP and possessing a heavy
chain sequence comprising the sequence set forth below:
QSVEESGGGLVQPEGSLTLTCTASGFDFSSNAMWWVRQAPGKGLEWIGCIYNGD
GSTYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 124).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122,
and/or one or more of the polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129;
and SEQ ID NO: 130 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 123
or the heavy chain sequence of SEQ ID NO: 124, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof comprise, or alternatively consist of, combinations of one or
more of the CDRs, the variable heavy and variable light chain sequences, and the heavy
and light chain sequences set forth above, including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein comprise, or
alternatively consist of, the polypeptide sequence of SEQ ID NO: 121 or SEQ ID NO: 122.
In another embodiment of the present disclosure, antibody fragments described herein
comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 123 or SEQ
ID NO: 124.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO:
124.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies for potential treatment or prevention of photophobia having binding specificity
to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the
following antibody fragments: the variable light chain region of SEQ ID NO: 121; the
variable heavy chain region of SEQ ID NO: 123; the complementarity-determining regions
(SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127) of the variable light chain
region of SEQ ID NO: 121; and the complementarity-determining regions (SEQ ID NO:
128; SEQ ID NO: 129; and SEQ ID NO: 130) of the variable heavy chain region of SEQ
ID NO: 123.
In a particularly preferred embodiment of the present disclosure, the chimeric
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab13,
comprising, or alternatively consisting of, SEQ ID NO: 122 and SEQ ID NO: 124, and
having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab13, the Fab fragment for potential treatment or prevention of
photophobia includes the variable light chain sequence of SEQ ID NO: 121 and the
variable heavy chain sequence of SEQ ID NO: 123. This embodiment of the present
disclosure further contemplates additions, deletions, and variants of SEQ ID NO: 121
and/or SEQ ID NO: 123 in said Fab while retaining binding specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab13. In another embodiment of the present
disclosure, anti-CGRP antibodies for potential treatment or prevention of photophobia such
as Ab13 or Fab fragments thereof may be produced via expression in mammalian cells
such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast
cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable
Pichia species include, but are not limited to, Pichia pastoris.
Antibody Ab14
In one embodiment, the present disclosure includes humanized antibodies for
potential treatment or prevention of photophobia having binding specificity to CGRP and
possessing a variable light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
R (SEQ ID NO: 131).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a light chain sequence comprising the sequence set forth below:
QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKVPKQLIYSTSTL
ASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 132).
The present disclosure further includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a variable heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSS (SEQ ID NO: 133).
The present disclosure also includes humanized antibodies for potential
treatment or prevention of photophobia having binding specificity to CGRP and possessing
a heavy chain sequence comprising the sequence set forth below:
EVQLVESGGGLVQPGGSLRLSCAVSGIGLSSYYMQWVRQAPGKGLEWVGVIGSD
GKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTC
PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(SEQ ID NO: 134).
The present disclosure further contemplates antibodies for potential treatment or
prevention of photophobia comprising one or more of the polypeptide sequences of SEQ
ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132,
and/or one or more of the polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139;
and SEQ ID NO: 140 which correspond to the complementarity-determining regions
(CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 133
or the heavy chain sequence of SEQ ID NO: 134, or combinations of these polypeptide
sequences. In another embodiment of the present disclosure, the antibodies described
herein or fragments thereof for potential treatment or prevention of photophobia comprise,
or alternatively consist of, combinations of one or more of the CDRs, the variable heavy
and variable light chain sequences, and the heavy and light chain sequences set forth above,
including all of them.
The present disclosure also contemplates fragments of the antibody for potential
treatment or prevention of photophobia having binding specificity to CGRP. In one
embodiment of the present disclosure, antibody fragments described herein for potential
treatment or prevention of photophobia comprise, or alternatively consist of, the
polypeptide sequence of SEQ ID NO: 131 or SEQ ID NO: 132. In another embodiment of
the present disclosure, antibody fragments described herein for potential treatment or
prevention of photophobia comprise, or alternatively consist of, the polypeptide sequence
of SEQ ID NO: 133 or SEQ ID NO: 134.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.
In a further embodiment of the present disclosure, fragments of the antibody for
potential treatment or prevention of photophobia having binding specificity to CGRP
comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID
NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the
complementarity-determining regions (CDRs, or hypervariable regions) of the variable
heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO:
134.
The present disclosure also contemplates antibody fragments for potential
treatment or prevention of photophobia which include one or more of the antibody
fragments described herein. In one embodiment of the present disclosure, fragments of the
antibodies having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following antibody fragments: the variable light
chain region of SEQ ID NO: 131; the variable heavy chain region of SEQ ID NO: 133; the
complementarity-determining regions (SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID
NO: 137) of the variable light chain region of SEQ ID NO: 131; and the complementarity-
determining regions (SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140) of the
variable heavy chain region of SEQ ID NO: 133.
In a particularly preferred embodiment of the present disclosure, the humanized
anti-CGRP antibody for potential treatment or prevention of photophobia is Ab14,
comprising, or alternatively consisting of, SEQ ID NO: 132 and SEQ ID NO: 134, and
having at least one of the biological activities set forth herein.
In a further particularly preferred embodiment of the present disclosure, antibody
fragments for potential treatment or prevention of photophobia comprise, or alternatively
consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
With respect to antibody Ab14, the Fab fragment for potential treatment or prevention of
photophobia includes the variable light chain sequence of SEQ ID NO: 131 and the
variable heavy chain sequence of SEQ ID NO: 133. This embodiment of the present
disclosure further contemplates additions, deletions, and variants of SEQ ID NO: 131
and/or SEQ ID NO: 133 in said Fab while retaining binding specificity for CGRP.
In one embodiment of the present disclosure described herein (infra), Fab
fragments for potential treatment or prevention of photophobia may be produced by
enzymatic digestion (e.g., papain) of Ab14. In another embodiment of the present
disclosure, anti-CGRP antibodies for potential treatment or prevention of photophobia such
as Ab14 or Fab fragments thereof may be produced via expression in mammalian cells
such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast
cells (for example diploid yeast such as diploid Pichia) and other yeast strains. Suitable
Pichia species include, but are not limited to, Pichia pastoris.
In another embodiment, antibody fragments for potential treatment or prevention
of photophobia may be present in one or more of the following non-limiting forms: Fab,
Fab', F(ab') , Fv and single chain Fv antibody forms. In a preferred embodiment, the anti-
CGRP antibodies for potential treatment or prevention of photophobia described herein
further comprises the kappa constant light chain sequence comprising the sequence set
forth below:
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO: 283).
In another preferred embodiment, the anti-CGRP antibodies described herein for
potential treatment or prevention of photophobia further comprises the gamma-1 constant
heavy chain polypeptide sequence comprising the sequence set forth below:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PGK (SEQ ID NO: 284).
In another embodiment, the present disclosure contemplates an isolated anti-
CGRP antibody for potential treatment or prevention of photophobia comprising a V
polypeptide sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103,
113, 123, or 133, or a variant thereof; and further comprising a V polypeptide sequence
selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or 131, or a
variant thereof, wherein one or more of the framework residues (FR residues) in said V
H or
V polypeptide has been substituted with another amino acid residue resulting in an anti-
CGRP antibody that specifically binds CGRP for potential treatment or prevention of
photophobia. The present disclosure contemplates humanized and chimeric forms of these
antibodies. The chimeric antibodies for potential treatment or prevention of photophobia
may include an Fc derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9,
IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant
regions.
In one embodiment of the present disclosure, the antibodies or V or V
polypeptides originate or are selected from one or more rabbit B cell populations prior to
initiation of the humanization process referenced herein.
In another embodiment of the present disclosure, the anti-CGRP antibodies and
fragments thereof do not have binding specificity for CGRP-R. In a further embodiment of
the present disclosure, the anti-CGRP antibodies and fragments thereof inhibit the
association of CGRP with CGRP-R. In another embodiment of the present disclosure, the
anti-CGRP antibodies and fragments thereof inhibit the association of CGRP with CGRP-R
and/or additional proteins and/or multimers thereof, and/or antagonizes the biological
effects thereof.
As stated herein, antibodies and fragments thereof may be modified post-
translationally to add effector moieties such as chemical linkers, detectable moieties such
as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive
materials, and chemiluminescent moieties, or functional moieties such as for example
streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
Antibodies or fragments thereof may also be chemically modified to provide
additional advantages such as increased solubility, stability and circulating time (in vivo
half-life) of the polypeptide, or decreased immunogenicity (See U.S. Pat. No. 4,179,337).
The chemical moieties for derivitization may be selected from water soluble polymers such
as polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The antibodies and
fragments thereof may be modified at random positions within the molecule, or at
predetermined positions within the molecule and may include one, two, three or more
attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between about 1
kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene
glycol, some molecules will weigh more, some less, than the stated molecular weight) for
ease in handling and manufacturing. Other sizes may be used, depending on the desired
therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on
biological activity, the ease in handling, the degree or lack of antigenicity and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branched polyethylene glycols are
described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem.
Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750
(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of
which are incorporated herein by reference.
There are a number of attachment methods available to those skilled in the art,
See e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), See
also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF
using tresyl chloride). For example, polyethylene glycol may be covalently bound through
amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive
groups are those to which an activated polyethylene glycol molecule may be bound. The
amino acid residues having a free amino group may include lysine residues and the N-
terminal amino acid residues; those having a free carboxyl group may include aspartic acid
residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups
may also be used as a reactive group for attaching the polyethylene glycol molecules.
Preferred for therapeutic purposes is attachment at an amino group, such as attachment at
the N-terminus or lysine group.
As suggested above, polyethylene glycol may be attached to proteins via linkage
to any of a number of amino acid residues. For example, polyethylene glycol can be linked
to polypeptides via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or
cysteine residues. One or more reaction chemistries may be employed to attach
polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid,
glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine,
histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).
Alternatively, antibodies or fragments thereof may have increased in vivo half
lives via fusion with albumin (including but not limited to recombinant human serum
albumin or fragments or variants thereof (See, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2,
1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein
incorporated by reference in their entirety)) or other circulating blood proteins such as
transferrin or ferritin. In a preferred embodiment, polypeptides and/or antibodies of the
present disclosure (including fragments or variants thereof) are fused with the mature form
of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in
FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its
entirety. Polynucleotides encoding fusion proteins of the present disclosure are also
encompassed herein.
Regarding detectable moieties, further exemplary enzymes include, but are not
limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-
galactosidase and luciferase. Further exemplary fluorescent materials include, but are not
limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone,
dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary
chemiluminescent moieties include, but are not limited to, luminol. Further exemplary
bioluminescent materials include, but are not limited to, luciferin and aequorin. Further
exemplary radioactive materials include, but are not limited to, Iodine 125 ( I), Carbon 14
14 35 3 32
( C), Sulfur 35 ( S), Tritium ( H) and Phosphorus 32 ( P).
Regarding functional moieties, exemplary cytotoxic agents include, but are not
limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-
fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil,
melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea,
cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin,
mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin
(paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin
(adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and
bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin,
mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids,
vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin,
pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide,
emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol,
puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-
(DDD)), interferons, and mixtures of these cytotoxic agents.
Further cytotoxic agents include, but are not limited to, chemotherapeutic agents
such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-
fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin.
Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas
toxin may be conjugated to the humanized or chimeric antibodies, or binding fragments
thereof, to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci.
USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick,
et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).
Other cytotoxic agents include cytotoxic ribonucleases as described by
Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the present disclosure also relate
to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably
coupled to the antibody, or binding fragments thereof, with or without the use of a
complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32
32 47 67 67 88
( P), Scandium-47 ( Sc), Copper-67 ( Cu), Gallium-67 ( Ga), Yttrium-88 ( Y),
90 125 131 153
Yttrium-90 ( Y), Iodine-125 ( I), Iodine-131 ( I), Samarium-153 ( Sm), Lutetium-
177 186 188
177 ( Lu), Rhenium-186 ( Re) or Rhenium-188 ( Re), and alpha-emitters such as
211 212 212 213
Astatine-211 ( At), Lead-212 ( Pb), Bismuth-212 ( Bi) or -213 ( Bi) or Actinium-
225 ( Ac).
Methods are known in the art for conjugating an antibody or binding fragment
thereof to a detectable moiety and the like, such as for example those methods described by
Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al,
J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407
(1982).
Embodiments described herein further include variants and equivalents that are
substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs,
camelbodies, nanobodies, IgNAR, polypeptides, variable regions and CDRs set forth
herein. These may contain, e.g., conservative substitution mutations, (i.e., the substitution
of one or more amino acids by similar amino acids). For example, conservative
substitution refers to the substitution of an amino acid with another within the same general
class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with
another basic amino acid, or one neutral amino acid by another neutral amino acid. What is
intended by a conservative amino acid substitution is well known in the art.
In another embodiment, the present disclosure contemplates polypeptide
sequences having at least 90% or greater sequence homology to any one or more of the
polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein.
More preferably, the present disclosure contemplates polypeptide sequences having at least
95% or greater sequence homology, even more preferably at least 98% or greater sequence
homology, and still more preferably at least 99% or greater sequence homology to any one
or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set
forth herein. Methods for determining homology between nucleic acid and amino acid
sequences are well known to those of ordinary skill in the art.
In another embodiment, the present disclosure further contemplates the above-
recited polypeptide homologs of the antibody fragments, variable regions and CDRs set
forth herein further having anti-CGRP activity. Non-limiting examples of anti-CGRP
activity are set forth herein, for example, in paragraphs [0329]-[0350] infra.
In another embodiment, the present disclosure further contemplates the
generation and use of anti-idiotypic antibodies that bind any of the foregoing sequences. In
an exemplary embodiment, such an anti-idiotypic antibody could be administered to a
subject who has received an anti-CGRP antibody to modulate, reduce, or neutralize, the
effect of the anti-CGRP antibody. Such anti-idiotypic antibodies could also be useful for
treatment of an autoimmune disease characterized by the presence of anti-CGRP
antibodies. A further exemplary use of such anti-idiotypic antibodies is for detection of the
anti-CGRP antibodies of the present disclosure, for example to monitor the levels of the
anti-CGRP antibodies present in a subject’s blood or other bodily fluids.
The present disclosure also contemplates anti-CGRP antibodies for potential
treatment or prevention of photophobia comprising any of the polypeptide or
polynucleotide sequences described herein substituted for any of the other polynucleotide
sequences described herein. For example, without limitation thereto, the present dislcosure
contemplates antibodies comprising the combination of any of the variable light chain and
variable heavy chain sequences described herein, and further contemplates antibodies
resulting from substitution of any of the CDR sequences described herein for any of the
other CDR sequences described herein.
Additional Exemplary Embodiments of the Invention
In another embodiment, the present disclosure contemplates one or more anti-
human anti-CGRP antibodies or antibody fragments thereof for potential treatment or
prevention of photophobia which specifically bind to the same or overlapping linear or
conformational epitope(s) and/or competes for binding to the same ovelappping linear or
conformational epitope(s) on an intact human CGRP polypeptide or fragment thereof as an
anti-human CGRP antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,
Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14. In a preferred embodiment, the anti-human
CGRP antibody or fragment thereof specifically binds to the same ovelappping linear or
conformational epitope(s) and/or competes for binding to the same ovelappping linear or
conformational epitope(s) on an intact human CGRP polypeptide or a fragment thereof as
Ab3, Ab6, Ab13, or Ab14, and most preferably Ab3.
A preferred embodiment of the present disclosure is directed to chimeric or
humanized antibodies and fragments thereof (including Fab fragments) having binding
specificity for CGRP and inhibiting biological activities mediated by the binding of CGRP
to the CGRP receptor especially for treatment or prevention of photophobia. In a
particularly preferred embodiment of the present disclosure, the chimeric or humanized
anti-CGRP antibodies are selected from Ab3, Ab6, Ab13, or Ab14, or more preferably
Ab3.
A preferred embodiment of the present disclosure is directed to methods of
screening antibodies and fragments thereof (including Fab fragments) having binding
specificity to human Calcitonin Gene Related Peptide (hereinafter “CGRP”) in animal
models to determine the in vivo effects thereof, especially their ability to antagonize the
adverse side effects of CGRP and to treat conditions involving excess CGRP especially
their ability to treat or prevent photophobia, e.g., in migraine.
A more specific preferred embodiment of the present disclosure involves a
method of assessing the potential in vivo efficacy of a candidate CGRP/CGRP receptor
inhibitor polypeptide, e.g., an anti-CGRP or anti-CGRP antibody or antibody fragment
comprising determining whether the antibody inhibits light aversive behavior in a rodent
administered CGRP compared to a rodent administered CGRP in the absence of the
candidate anti-CGRP antibody or antibody fragment.
A more specific preferred embodiment of the present disclosure involves a
method of assessing the potential in vivo efficacy of a candidate anti-CGRP antibody or
antibody fragment to treat a neurological condition characterized by increased CGRP levels
and photophobia.
Another more specific preferred embodiment of the present disclosure involves a
method of assessing the potential in vivo efficacy of a candidate anti-CGRP antibody or
antibody fragment to treat a CGRP associated disorder associated with photophobia such as
migraine or chronic migraine, (with or without aura), or conditions such as weight loss,
cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis
associated with cancer or tumor survival, diarrhea, hemiplagic migraines, cluster
headaches, migrainous neuralgia, chronic headaches, tension headaches, general
headaches, hot flashes, chronic paroxysomal hemicrania, secondary headaches due to an
underlying structural problem in the head or neck, cranial neuralgia, sinus headaches (such
as for example associated with sinusitis), allergy-induced headaches or migraines, pain,
headache-free migraine, abdominal migraine, inflammatory pain, post-operative incision
pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer
pain, fracture pain, chronic pain, osteoporotic fracture pain, pain resulting from burn,
osteoporosis, gout joint pain, abdominal pain, pain associated with sickle cell crises, and
other nociceptic pain, as well as hepatocellular carcinoma, breast cancer, liver cirrhosis,
menstrual pain, neurogenic pain, neuropathic pain, nociceptic pain, trigeminal neuralgia,
post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarialgia, reflex
sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower
back pain, diabetic neuropathy, sciatica, or pain or visceral pain associated with: gastro-
esophageal reflux, dyspepsia, irritable bowel syndrome, irritable colon, spastic colon,
mucous colitis, inflammatory bowel disease, Crohn’s disease, ileitis, ulcerative colitis,
renal colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis,
pancreatitis, renal colic, dysmenorrhea, cystitis, including interstitial cystitis (IC), surgery
associated with the ileus, diverticulitis, peritonitis, pericarditis, hepatitis, appendicitis,
colitis, cholecystitis, endometriosis, chronic and/or acute pancreatitis, myocardial
infarction, kidney pain, pleural pain, prostatitis, pelvic pain, trauma to an organ, chronic
nociceptive pain, chronic neuropathic pain, chronic inflammatory pain, fibromyalgia,
breakthrough pain and persistent pain.Still another preferred embodiment of the present
disclosure involves a method of determining a suitable therapeutic dosage or dosage
regimen of a candidate anti-CGRP antibody or antibody fragment in humans in order to
treat a photophobia- associated condition selected from those identified herein based on the
effects of said antibody or antibody fragment in a light aversive behavioral rodent animal
model described in detail infra. Common causes of photophobia include migraine
headaches, cataracts, or severe ophthalmologic diseases such as uveitis or corneal abrasion.
A more extensive list of disorders associated with photophobia includes eye related causes
such as Achromatopsia, Aniridia, Anticholinergic drugs may cause photophobia by
paralyzing the iris sphincter muscle, Aphakia (absence of the lens of the eye), Buphthalmos
(abnormally narrow angle between the cornea and iris), Cataracts, Cone dystrophy,
Congenital abnormalities of the eye, Viral conjunctivitis ("pink eye") Corneal abrasion,
Corneal dystrophy, Corneal ulcer, disruption of the corneal epithelium, such as that caused
by a corneal foreign body or keratitis, Ectopia lentis, Endophthalmitis, Eye trauma caused
by disease, injury, or infection such as chalazion, episcleritis, glaucoma, keratoconus, or
optic nerve hypoplasia, Hydrophthalmos, or congenital glaucoma Iritis, Optic neuritis,
Pigment dispersion syndrom, Pupillary dilation (naturally or chemically induced), Retinal
detachment, Scarring of the cornea or sclera and Uveitis. In addition photophobia has
nervous-system-related or urological causes including: Autism spectrum disorders, Chiari
malformation, Dyslexia, Encephalitis including Myalgic encephalomyelitis aka Chronic
fatigue syndrome, Meningitis, Subarachnoid haemorrhage, Tumor of the posterior cranial
fossa, as well as other causes such as Ankylosing spondylitis, Albinism, Ariboflavinosis,
Benzodiazepines (long term use of or withdrawal from benzodiazepines), Chemotherapy,
Chikungunya, Cystinosis, Ehlers-Danlos syndrome, Hangover, Influenza, Infectious
Mononucleosis, Magnesium deficiency, Mercury poisoning, Migraine, Rabies, and
Tyrosinemia type II, also known as "Richner-Hanhart syndrome". Additionally it is known
that photophobia is elevated in depression, bipolar disorder and agoraphobia.
Further another preferred embodiment of the present disclosure relates to
methods of assessing based on results in a rodent CGRP animal model a suitable
therapeutic dosage or dosage regimen of the candidate anti-CGRP antibody or antibody
fragment in humans.
Other preferred embodiments the present disclosure are directed to screening
assays and therapeutic usage of specific antibodies and fragments thereof having binding
specificity for CGRP for treatment or prevention of photophobia, in particular antibodies
having desired epitopic specificity, high affinity or avidity and/or functional properties. In
preferred embodiments this present disclosure relates to assays and usage of the antibodies
described herein, comprising the sequences of the V , V and CDR polypeptides described
herein, and the polynucleotides encoding them. A preferred embodiment of the present
disclosure is directed to chimeric or humanized antibodies and fragments thereof (including
Fab fragments) capable of binding to CGRP and/or inhibiting the biological activities
mediated by the binding of CGRP to the CGRP receptor (“CGRP-R”).
In a further embodiment of the present disclosure is contemplated a method of
reducing, treating or preventing diseases or disorders associated with CGRP by affecting
those biological activities mediated via CGRP, especially inhibiting or preventing
photophobia thereby avoiding the adverse biological activities mediated via binding of
CGRP to CGRP-R. In one embodiment, the disease or disorder associated with
photophobia is migraine, headache, pain, or other conditions aforementioned which are
associated with photophobia. A further non-limiting listing of diseases and disorders
associated with CGRP is described herein.
Another preferred embodiment of the present disclosure contemplates the use of
Fab polypeptide sequences for the treatment of migraines and headaches and especially for
treatment or prevention of photophobia in a patient. Non-limiting types of migraines and
headaches that may be treated using Fab polypeptide sequences are described elsewhere in
this disclosure.
In another embodiment of the present disclosure, the anti-human CGRP antibody
for treatment or prevention of photophobia is an antibody which specifically binds to the
same ovelappping linear or conformational epitopes on an intact CGRP polypeptide or
fragment thereof that is (are) specifically bound by Ab3, Ab6, Ab13, or Ab14 as
ascertained by epitopic mapping using overlapping linear peptide fragments which span the
full length of the native human CGRP polypeptide.
The present disclosure is also directed to an anti-CGRP antibody for treatment or
prevention of photophobia that binds with the same CGRP epitope and/or competes with an
anti-CGRP antibody for binding to CGRP as an antibody or antibody fragment disclosed
herein, including but not limited to an anti-CGRP antibody selected from Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14, preferably Ab6,
Ab10, Ab12, or Ab3. As mentioned, common causes of photophobia include migraine
headaches, cataracts, or severe ophthalmologic diseases such as uveitis or corneal abrasion.
A more extensive list of disorders associated with photophobia includes eye related causes
such as Achromatopsia, Aniridia, Anticholinergic drugs may cause photophobia by
paralyzing the iris sphincter muscle, Aphakia (absence of the lens of the eye), Buphthalmos
(abnormally narrow angle between the cornea and iris), Cataracts, Cone dystrophy,
Congenital abnormalities of the eye, Viral conjunctivitis ("pink eye") Corneal abrasion,
Corneal dystrophy, Corneal ulcer, disruption of the corneal epithelium, such as that caused
by a corneal foreign body or keratitis, Ectopia lentis, Endophthalmitis, Eye trauma caused
by disease, injury, or infection such as chalazion, episcleritis, glaucoma, keratoconus, or
optic nerve hypoplasia, Hydrophthalmos, or congenital glaucoma Iritis, Optic neuritis,
Pigment dispersion syndrom, Pupillary dilation (naturally or chemically induced), Retinal
detachment, Scarring of the cornea or sclera and Uveitis.
In addition photophobia has nervous-system-related or urological causes
including: Autism spectrum disorders, Chiari malformation, Dyslexia, Encephalitis
including Myalgic encephalomyelitis aka Chronic fatigue syndrome, Meningitis,
Subarachnoid haemorrhage, Tumor of the posterior cranial fossa, as well as other causes
such as Ankylosing spondylitis, Albinism, Ariboflavinosis, Benzodiazepines (long term use
of or withdrawal from benzodiazepines), Chemotherapy, Chikungunya, Cystinosis, Ehlers-
Danlos syndrome, Hangover, Influenza, Infectious Mononucleosis, Magnesium deficiency,
Mercury poisoning, Migraine, Rabies, and Tyrosinemia type II, also known as "Richner-
Hanhart syndrome". Additionally it is known that photophobia is elevated in depression,
bipolar disorder and agoraphobia.
In another embodiment, the present disclosure is also directed to an isolated anti-
CGRP antibody or antibody fragment for treatment or prevention of photophobia
comprising one or more of the CDRs contained in the V polypeptide sequences selected
from: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133, or a variant thereof,
and/or one or more of the CDRs contained in the V polypeptide sequences selected from:
1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or 131, or a variant thereof.
In one embodiment of the present disclosure, the anti-human CGRP antibody for
treatment or prevention of photophobia discussed in the two prior paragraphs comprises at
least 2 complementarity determining regions (CDRs) in each the variable light and the
variable heavy regions which are identical to those contained in an anti-human CGRP
antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11,
Ab12, Ab13, or Ab14.
In a preferred embodiment, the anti-human CGRP antibody discussed above for
treatment or prevention of photophobia comprises at least 2 complementarity determining
regions (CDRs) in each the variable light and the variable heavy regions which are
identical to those contained in Ab3 or Ab6. In another embodiment, all of the CDRs of the
anti-human CGRP antibody discussed above are identical to the CDRs contained in an anti-
human CGRP antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,
Ab10, Ab11, Ab12, Ab13, or Ab14. In a preferred embodiment of the present disclosure,
all of the CDRs of the anti-human CGRP antibody discussed above are identical to the
CDRs contained in an anti-human CGRP antibody selected from Ab3, Ab10, Ab12 or Ab6.
The present disclosure further contemplates that the one or more anti-human
CGRP antibodies discussed above for treatment or prevention of photophobia are
aglycosylated or minimally glycosylated, e.g., lack N-glycosylation and comprise some O-
glycosylation such as some 1 or more mannose residues; e.g., that contain an Fc region that
has been modified to alter effector function, half-life, proteolysis, and/or glycosylation; are
human, humanized, single chain or chimeric; and are a humanized antibody derived from a
rabbit (parent) anti-human CGRP antibody.
The present disclosure further contemplates one or more anti-human CGRP
antibodies for treatment or prevention of photophobia wherein the framework regions
(FRs) in the variable light region and the variable heavy regions of said antibody
respectively are human FRs which are unmodified or which have been modified by the
substitution of one or more human FR residues in the variable light or heavy chain region
with the corresponding FR residues of the parent rabbit antibody, and wherein said human
FRs have been derived from human variable heavy and light chain antibody sequences
which have been selected from a library of human germline antibody sequences based on
their high level of homology to the corresponding rabbit variable heavy or light chain
regions relative to other human germline antibody sequences contained in the library.
In one embodiment of the present disclosure, the anti-human CGRP antibody or
fragment for treatment or prevention of photophobia specifically binds to CGRP expressing
human cells and/or to circulating soluble CGRP molecules in vivo, including CGRP
expressed on or by human cells in a patient with a disease associated with cells that express
CGRP.
In another embodiment, the disease is selected from photophobia or light
aversion associated with one or more of: migraines (with or without aura), menstrual
headache, menstrual migraine, menopausal migraine or another hormonally related
migraine, hemiplegic migraines, cluster headaches, migrainous neuralgia, chronic
headaches, tension headaches, general headaches, migraines associated with hot flashes,
chronic paroxysomal hemicrania, secondary headaches due to an underlying structural
problem in the head or neck, cranial neuralgia, sinus headaches (such as for example
associated with sinusitis), allergy-induced headaches or migraines, headache-free migraine,
and abdominal migraine.
The present disclosure further contemplates anti-human CGRP antibodies or
fragments for treatment or prevention of photophobia directly or indirectly attached to a
detectable label or therapeutic agent.
The present disclosure also contemplates one or more nucleic acid sequences
which result in the expression of an anti-human CGRP antibody or antibody fragment for
treatment or prevention of photophobia as set forth above, including those comprising, or
alternatively consisting of, yeast or human preferred codons. The present disclosure also
contemplates vectors (including plasmids or recombinant viral vectors) comprising said
nucleic acid sequence(s). The present disclosure also contemplates host cells or
recombinant host cells expressing at least one of the antibodies set forth above, including a
mammalian, yeast, bacterial, and insect cells. In a preferred embodiment, the host cell is a
yeast cell. In a further preferred embodiment, the yeast cell is a diploidal yeast cell. In a
more preferred embodiment, the yeast cell is a Pichia yeast.
The present disclosure also contemplates a method of treatment comprising
administering to a patient with a disease or condition associated with CGRP expressing
cells a therapeutically effective amount of at least one anti-human CGRP antibody or
fragment described herein for treatment or prevention of photophobia. The present
disclosure also contemplates that the treatment method may involve the administration of
two or more anti-CGRP antibodies or fragments thereof and disclosed herein. If more than
one antibody is administered to the patient, the multiple antibodies may be administered
simultaneously or concurrently, or may be staggered in their administration. The diseases
that may be treated are presented in the non-limiting list set forth above and elsewhere
herein. In a preferred embodiment, the disease associated with photophobia is selected
from migraine, headache, pain, diarrhea, cancer pain or neuropathic pain. In another
embodiment the treatment further includes the administration of another therapeutic agent
or regimen selected from chemotherapy, radiotherapy, cytokine administration or gene
therapy.
In a non-limiting embodiment of the present disclosure, another therapeutic
agent or regimen includes opioids, analgesics such as NSAIDs, Taxol (paclitaxel) or its
derivatives, platinum compounds such as carboplatin or cisplatin, anthrocyclines such as
doxorubicin, alkylating agents such as cyclophosphamide, anti-metabolites such as 5-
fluorouracil, or etoposide.
The present disclosure further contemplates a method of in vivo imaging which
detects the presence of cells which express CGRP comprising administering a
diagnostically effective amount of at least one anti-human CGRP antibody. In one
embodiment, said administration further includes the administration of a radionuclide or
fluorophore that facilitates detection of the antibody at CGRP expressing disease sites. In a
further embodiment, the results of said in vivo imaging method are used to facilitate the
design of an appropriate therapeutic regimen, including therapeutic regimens including
radiotherapy, chemotherapy or a combination thereof.
The anti-CGRP activity of the anti-CGRP antibodies of the present disclosure,
and fragments thereof having binding specificity to CGRP for treatment or prevention of
photophobia, may also be described by their strength of binding or their affinity for CGRP.
In one embodiment of the present disclosure, the anti-CGRP antibodies of the present
disclosure, and fragments thereof having binding specificity to CGRP, bind to CGRP with
-7 -7 -8 -8
a dissociation constant (K ) of less than or equal to 5x10 M, 10 M, 5x10 M, 10 M,
-9 -9 -10 -10 -11 -11 -12 -12 -13
5x10 M, 10 M, 5x10 M, 10 M, 5x10 M, 10 M, 5x10 M, 10 M, 5x10 M,
or 10 M. Preferably, the anti-CGRP antibodies and fragments thereof bind CGRP with a
-11 -12 -12
dissociation constant of less than or equal to 10 M, 5x10 M, or 10 M. In another
embodiment of the present disclosure, the anti-CGRP antibodies described herein, and
fragments thereof having binding specificity to CGRP, bind to a linear or conformational
CGRP epitope.
In another embodiment of the present disclosure, the anti-CGRP activity of the
anti-CGRP antibodies described herein, and fragments thereof having binding specificity to
-4 -1 -5 -1 -5 -1
CGRP, bind to CGRP with an off-rate of less than or equal to 10 S , 5x10 S , 10 S ,
-6 -1 -6 -1 -7 -1 -7 -1
5x10 S , 10 S , 5x10 S , or 10 S .
In a further embodiment of the present disclosure, the anti-CGRP activity of the
anti-CGRP antibodies described herein, and fragments thereof having binding specificity to
CGRP, exhibit anti-CGRP activity by preventing, ameliorating or reducing the symptoms
of, or alternatively treating, diseases and disorders associated with CGRP especially for
treatment or prevention of photophobia. Non-limiting examples of diseases and disorders
associated with CGRP and conditions associated with photophobia are set forth herein.
Polynucleotides Encoding Anti-CGRP Antibody Polypeptides
Antibody Ab1
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 1:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTA
CTACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGG
AGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 141).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 2:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTA
CTACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGG
AGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC
CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA
CAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 142).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 3:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCA
ATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTA
TTAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AGAGCCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 143).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 4:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGCACAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTATTAATG
ATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGAGCC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 144).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 145; SEQ ID NO: 146;
and SEQ ID NO: 147 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 148; SEQ ID NO: 149;
and SEQ ID NO: 150 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 141 encoding the light chain variable sequence
of SEQ ID NO: 1; the polynucleotide SEQ ID NO: 142 encoding the light chain sequence
of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 143 encoding the heavy chain variable
sequence of SEQ ID NO: 3; the polynucleotide SEQ ID NO: 144 encoding the heavy chain
sequence of SEQ ID NO: 4; polynucleotides encoding the complementarity-determining
regions (SEQ ID NO: 145; SEQ ID NO: 146; and SEQ ID NO: 147) of the light chain
variable sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2; and
polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 148;
SEQ ID NO: 149; and SEQ ID NO: 150) of the heavy chain variable sequence of SEQ ID
NO: 3 or the heavy chain sequence of SEQ ID NO: 4.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab1, the polynucleotides encoding the full length Ab1 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 142 encoding the light chain sequence of SEQ
ID NO: 2 and the polynucleotide SEQ ID NO: 144 encoding the heavy chain sequence of
SEQ ID NO: 4.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab1 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab1 or Fab fragments thereof may be
produced via expression of Ab1 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab2
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 11:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 151).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 12:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 152).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 13:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATCAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 153).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 14:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATCA
ATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 154).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 155; SEQ ID NO: 156;
and SEQ ID NO: 157 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 11 or the light chain sequence of SEQ ID NO: 12.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 158; SEQ ID NO: 159;
and SEQ ID NO: 160 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 151 encoding the light chain variable sequence
of SEQ ID NO: 11; the polynucleotide SEQ ID NO: 152 encoding the light chain sequence
of SEQ ID NO: 12; the polynucleotide SEQ ID NO: 153 encoding the heavy chain variable
sequence of SEQ ID NO: 13; the polynucleotide SEQ ID NO: 154 encoding the heavy
chain sequence of SEQ ID NO: 14; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 155; SEQ ID NO: 156; and SEQ ID NO: 157) of the
light chain variable sequence of SEQ ID NO: 11 or the light chain sequence of SEQ ID
NO: 12; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 158; SEQ ID NO: 159; and SEQ ID NO: 160) of the heavy chain variable sequence of
SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab2, the polynucleotides encoding the full length Ab2 antibody comprise, or alternatively
consist of,the polynucleotide SEQ ID NO: 152 encoding the light chain sequence of SEQ
ID NO: 12 and the polynucleotide SEQ ID NO: 154 encoding the heavy chain sequence of
SEQ ID NO: 14.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab2 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab2 or Fab fragments thereof may be
produced via expression of Ab2 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab3
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 21:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 161).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 22:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATGATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTAGGCAGTTATGATTGTAGTAGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 162).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 23:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATCAATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 163).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 24:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGACTCGACCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATCA
ATGATAACACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 164).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 165; SEQ ID NO: 166;
and SEQ ID NO: 167 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 168; SEQ ID NO: 169;
and SEQ ID NO: 170 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 161 encoding the light chain variable sequence
of SEQ ID NO: 21; the polynucleotide SEQ ID NO: 162 encoding the light chain sequence
of SEQ ID NO: 22; the polynucleotide SEQ ID NO: 163 encoding the heavy chain variable
sequence of SEQ ID NO: 23; the polynucleotide SEQ ID NO: 164 encoding the heavy
chain sequence of SEQ ID NO: 24; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 165; SEQ ID NO: 166; and SEQ ID NO: 167) of the
light chain variable sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID
NO: 22; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 168; SEQ ID NO: 169; and SEQ ID NO: 170) of the heavy chain variable sequence of
SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab3, the polynucleotides encoding the full length Ab3 antibody comprise, or alternatively
consist of,the polynucleotide SEQ ID NO: 162 encoding the light chain sequence of SEQ
ID NO: 22 and the polynucleotide SEQ ID NO: 164 encoding the heavy chain sequence of
SEQ ID NO: 24.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments for treatment or prevention of photophobia may be produced by enzymatic
digestion (e.g., papain) of Ab3 following expression of the full-length polynucleotides in a
suitable host. In another embodiment of the present disclosure, anti-CGRP antibodies for
treatment or prevention of photophobia such as Ab3 or Fab fragments thereof may be
produced via expression of Ab3 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab4
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 31:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACAACTGATCTATGATG
CATCCACTCTGGCGTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTAACGATGCTGCCGCTTAC
TACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 171).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 32:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAACAACTGATCTATGATG
CATCCACTCTGGCGTCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGGCGTGCAGTGTAACGATGCTGCCGCTTAC
TACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 172).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 33:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGTTCCGTCTCTGGCATCGACCTCAGTGGCTACTACATGAA
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTA
TTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCGGGCACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 173).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 34:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGTTCCGTCTCTGGCATCGACCTCAGTGGCTACTACATGAACTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTATTAATG
GTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCGGGCACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 174).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 175; SEQ ID NO: 176;
and SEQ ID NO: 177 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 178; SEQ ID NO: 179;
and SEQ ID NO: 180 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 171 encoding the light chain variable sequence
of SEQ ID NO: 31; the polynucleotide SEQ ID NO: 172 encoding the light chain sequence
of SEQ ID NO: 32; the polynucleotide SEQ ID NO: 173 encoding the heavy chain variable
sequence of SEQ ID NO: 33; the polynucleotide SEQ ID NO: 174 encoding the heavy
chain sequence of SEQ ID NO: 34; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 175; SEQ ID NO: 176; and SEQ ID NO: 177) of the
light chain variable sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID
NO: 32; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 178; SEQ ID NO: 179; and SEQ ID NO: 180) of the heavy chain variable sequence of
SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab4, the polynucleotides encoding the full length Ab4 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 172 encoding the light chain sequence of SEQ
ID NO: 32 and the polynucleotide SEQ ID NO: 174 encoding the heavy chain sequence of
SEQ ID NO: 34.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab4 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab4 or Fab fragments thereof may be
produced via expression of Ab4 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab5
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 41:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 181).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 42:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 182).
In another embodiment of the present disclosure, polynucleotides described
hrein comprise, or alternatively consist of, the following polynucleotide sequence encoding
the variable heavy chain polypeptide sequence of SEQ ID NO: 43:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACAT
GAACTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 183).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 44:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACATGAACTG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATTA
ATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 184).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 185; SEQ ID NO: 186;
and SEQ ID NO: 187 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 188; SEQ ID NO: 189;
and SEQ ID NO: 190 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 181 encoding the light chain variable sequence
of SEQ ID NO: 41; the polynucleotide SEQ ID NO: 182 encoding the light chain sequence
of SEQ ID NO: 42; the polynucleotide SEQ ID NO: 183 encoding the heavy chain variable
sequence of SEQ ID NO: 43; the polynucleotide SEQ ID NO: 184 encoding the heavy
chain sequence of SEQ ID NO: 44; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 185; SEQ ID NO: 186; and SEQ ID NO: 187) of the
light chain variable sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID
NO: 42; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 188; SEQ ID NO: 189; and SEQ ID NO: 190) of the heavy chain variable sequence of
SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab5, the polynucleotides encoding the full length Ab5 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 182 encoding the light chain sequence of SEQ
ID NO: 42 and the polynucleotide SEQ ID NO: 184 encoding the heavy chain sequence of
SEQ ID NO: 44.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab5 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab5 or Fab fragments thereof may be
produced via expression of Ab5 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab6
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 51:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 191).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 52:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATCATAACACCTACCT
GGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATGATG
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTACTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 192).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 53:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACAT
GAACTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTATTAATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 193).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 54:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTGGCTACTACATGAACTG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTATTA
ATGGTGCCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 194).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 195; SEQ ID NO: 196;
and SEQ ID NO: 197 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 198; SEQ ID NO: 199;
and SEQ ID NO: 200 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 191 encoding the light chain variable sequence
of SEQ ID NO: 51; the polynucleotide SEQ ID NO: 192 encoding the light chain sequence
of SEQ ID NO: 52; the polynucleotide SEQ ID NO: 193 encoding the heavy chain variable
sequence of SEQ ID NO: 53; the polynucleotide SEQ ID NO: 194 encoding the heavy
chain sequence of SEQ ID NO: 54; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 195; SEQ ID NO: 196; and SEQ ID NO: 197) of the
light chain variable sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID
NO: 52; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 198; SEQ ID NO: 199; and SEQ ID NO: 200) of the heavy chain variable sequence of
SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab6, the polynucleotides encoding the full length Ab6 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 192 encoding the light chain sequence of SEQ
ID NO: 52 and the polynucleotide SEQ ID NO: 194 encoding the heavy chain sequence of
SEQ ID NO: 54.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab6 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab6 or Fab fragments thereof may be
produced via expression of Ab6 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab7
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 61:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATTACAACTACCT
TGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGACTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 201).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 62:
CAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATTACAACTACCT
TGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGATTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGACTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 202).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 63:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGA
CATCCCTGACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAACCACTACA
TGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCGTT
GGTATTAATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCAT
CTCCAGAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGGCTGACAACCG
AGGACACGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACC
CTGGTCACCGTCTCGAGC (SEQ ID NO: 203).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 64:
CAGGAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACATCCCT
GACACTCACCTGCACCGTCTCTGGAATCGACCTCAGTAACCACTACATGCAAT
GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGAGTCGTTGGTATT
AATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAG
AACCTCGTCGACCACGGTGGATCTGAAAATGACCAGGCTGACAACCGAGGACA
CGGCCACCTATTTCTGTGCCAGAGGGGACATCTGGGGCCCAGGCACCCTGGTC
ACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG
TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA
CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT
CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGT
GGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG
AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ
ID NO: 204).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 205; SEQ ID NO: 206;
and SEQ ID NO: 207 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 208; SEQ ID NO: 209;
and SEQ ID NO: 210 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 201 encoding the light chain variable sequence
of SEQ ID NO: 61; the polynucleotide SEQ ID NO: 202 encoding the light chain sequence
of SEQ ID NO: 62; the polynucleotide SEQ ID NO: 203 encoding the heavy chain variable
sequence of SEQ ID NO: 63; the polynucleotide SEQ ID NO: 204 encoding the heavy
chain sequence of SEQ ID NO: 64; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 205; SEQ ID NO: 206; and SEQ ID NO: 207) of the
light chain variable sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID
NO: 62; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 208; SEQ ID NO: 209; and SEQ ID NO: 210) of the heavy chain variable sequence of
SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab7, the polynucleotides encoding the full length Ab7 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 202 encoding the light chain sequence of SEQ
ID NO: 62 and the polynucleotide SEQ ID NO: 204 encoding the heavy chain sequence of
SEQ ID NO: 64.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab7 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab7 or Fab fragments thereof may be
produced via expression of Ab7 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab8
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 71:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTACAATTACAACTACCTT
GCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTAC
ATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTA
CTGTCTGGGCAGTTATGATTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGAGG
AACCAAGGTGGAAATCAAACGT (SEQ ID NO: 211).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 72:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTACAATTACAACTACCTT
GCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTAC
ATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGAC
AGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTA
CTGTCTGGGCAGTTATGATTGTAGTACTGGTGATTGTTTTGTTTTCGGCGGAGG
AACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCAC
CTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACAC
AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAA
GAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 212).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 73:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTAACCACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCGTTG
GTATCAATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 213).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 74:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACCTCAGTAACCACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCGTTGGTATCA
ATGGTCGCACATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCTAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 214).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 215; SEQ ID NO: 216;
and SEQ ID NO: 217 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 218; SEQ ID NO: 219;
and SEQ ID NO: 220 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 211 encoding the light chain variable sequence
of SEQ ID NO: 71; the polynucleotide SEQ ID NO: 212 encoding the light chain sequence
of SEQ ID NO: 72; the polynucleotide SEQ ID NO: 213 encoding the heavy chain variable
sequence of SEQ ID NO: 73; the polynucleotide SEQ ID NO: 214 encoding the heavy
chain sequence of SEQ ID NO: 74; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 215; SEQ ID NO: 216; and SEQ ID NO: 217) of the
light chain variable sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID
NO: 72; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 218; SEQ ID NO: 219; and SEQ ID NO: 220) of the heavy chain variable sequence of
SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab8, the polynucleotides encoding the full length Ab8 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 212 encoding the light chain sequence of SEQ
ID NO: 72 and the polynucleotide SEQ ID NO: 214 encoding the heavy chain sequence of
SEQ ID NO: 74.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab8 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab8 or Fab fragments thereof may be
produced via expression of Ab8 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab9
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 81:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTATAATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACGTCCACTCTGGCATCTGGGGTCTCATCGCGATTCAGAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTT
ACTACTGTCTAGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCG
GAGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 221).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 82:
CAAGTGCTGACCCAGACTCCATCCCCCGTGTCTGCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTATAATAACAACTACC
TAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCT
ACGTCCACTCTGGCATCTGGGGTCTCATCGCGATTCAGAGGCAGTGGATCTGG
GACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTT
ACTACTGTCTAGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCG
GAGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATC
TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGC
CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGA
AACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC
ACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 222).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 83:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACC
CCTGACACTCACCTGCACAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCA
GTGGGTCCGCCAGTCTCCAGGGAGGGGGCTGGAATGGATCGGAGTCATTGGTA
GTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCC
AAGACCTCGTCGACCACGGTGGATCTGAGAATGGCCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTACCAGAGGGGACATCTGGGGCCCGGGGACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 223).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 84:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGAC
ACTCACCTGCACAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAGTGGGT
CCGCCAGTCTCCAGGGAGGGGGCTGGAATGGATCGGAGTCATTGGTAGTGATG
GTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAAGACC
TCGTCGACCACGGTGGATCTGAGAATGGCCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTACCAGAGGGGACATCTGGGGCCCGGGGACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 224).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 225; SEQ ID NO: 226;
and SEQ ID NO: 227 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 228; SEQ ID NO: 229;
and SEQ ID NO: 230 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 221 encoding the light chain variable sequence
of SEQ ID NO: 81; the polynucleotide SEQ ID NO: 222 encoding the light chain sequence
of SEQ ID NO: 82; the polynucleotide SEQ ID NO: 223 encoding the heavy chain variable
sequence of SEQ ID NO: 83; the polynucleotide SEQ ID NO: 224 encoding the heavy
chain sequence of SEQ ID NO: 84; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 225; SEQ ID NO: 226; and SEQ ID NO: 227) of the
light chain variable sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID
NO: 82; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 228; SEQ ID NO: 229; and SEQ ID NO: 230) of the heavy chain variable sequence of
SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab9, the polynucleotides encoding the full length Ab9 antibody comprise, or alternatively
consist of, the polynucleotide SEQ ID NO: 222 encoding the light chain sequence of SEQ
ID NO: 82 and the polynucleotide SEQ ID NO: 224 encoding the heavy chain sequence of
SEQ ID NO: 84.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab9 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab9 or Fab fragments thereof may be
produced via expression of Ab9 polynucleotides in mammalian cells such as CHO, NSO or
HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid
yeast such as diploid Pichia) and other yeast strains. Suitable Pichia species include, but
are not limited to, Pichia pastoris.
Antibody Ab10
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 91:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 231).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 92:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 232).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 93:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTAGTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 233).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 94:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTAGTG
ATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 234).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 235; SEQ ID NO: 236;
and SEQ ID NO: 237 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 238; SEQ ID NO: 239;
and SEQ ID NO:240 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 231 encoding the light chain variable sequence
of SEQ ID NO: 91; the polynucleotide SEQ ID NO: 232 encoding the light chain sequence
of SEQ ID NO: 92; the polynucleotide SEQ ID NO: 233 encoding the heavy chain variable
sequence of SEQ ID NO: 93; the polynucleotide SEQ ID NO: 234 encoding the heavy
chain sequence of SEQ ID NO: 94; polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 235; SEQ ID NO: 236; and SEQ ID NO: 237) of the
light chain variable sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID
NO: 92; and polynucleotides encoding the complementarity-determining regions (SEQ ID
NO: 238; SEQ ID NO: 239; and SEQ ID NO: 240) of the heavy chain variable sequence of
SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab10, the polynucleotides encoding the full length Ab10 antibody comprise, or
alternatively consist of, the polynucleotide SEQ ID NO: 232 encoding the light chain
sequence of SEQ ID NO: 92 and the polynucleotide SEQ ID NO: 234 encoding the heavy
chain sequence of SEQ ID NO: 94.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab10 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab10 or Fab fragments thereof may
be produced via expression of Ab10 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
Antibody Ab11
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 101:
CAGGTGCTGACCCAGACTGCATCCCCCGTGTCTCCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTATTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 241).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 102:
CAGGTGCTGACCCAGACTGCATCCCCCGTGTCTCCAGCTGTGGGAAG
CACAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTATTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGG
ACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTAC
TACTGTCTAGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGA
GGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 242).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 103:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATC
CCTGACACTCACCTGCACAGTCTCTGGAATCGACGTCACTAACTACTATATGCA
ATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTG
TGAATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCC
AAAACCTCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGA
CACGGCCACCTATTTCTGTGCCAGAGGCGACATCTGGGGCCCGGGGACCCTCG
TCACCGTCTCGAGC (SEQ ID NO: 243).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 104:
CAGTCGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGAGGATCCCTGAC
ACTCACCTGCACAGTCTCTGGAATCGACGTCACTAACTACTATATGCAATGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAGTCATTGGTGTGAATG
GTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACC
TCGTCGACCACGGTGGATCTGAAAATGACCAGTCTGACAACCGAGGACACGGC
CACCTATTTCTGTGCCAGAGGCGACATCTGGGGCCCGGGGACCCTCGTCACCG
TCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCA
AGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA
CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT
CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC
CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG
TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC
ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG
AACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA
CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID
NO: 244).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 245; SEQ ID NO: 246;
and SEQ ID NO: 247 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 248; SEQ ID NO: 249;
and SEQ ID NO: 250 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 241 encoding the light chain variable sequence
of SEQ ID NO: 101; the polynucleotide SEQ ID NO: 242 encoding the light chain
sequence of SEQ ID NO: 102; the polynucleotide SEQ ID NO: 243 encoding the heavy
chain variable sequence of SEQ ID NO: 103; the polynucleotide SEQ ID NO: 244
encoding the heavy chain sequence of SEQ ID NO: 104; polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 245; SEQ ID NO: 246; and SEQ ID
NO: 247) of the light chain variable sequence of SEQ ID NO: 101 or the light chain
sequence of SEQ ID NO: 102; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 248; SEQ ID NO: 249; and SEQ ID NO: 250) of the
heavy chain variable sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID
NO: 104.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab11, the polynucleotides encoding the full length Ab11 antibody comprise, or
alternatively consist of, the polynucleotide SEQ ID NO: 242 encoding the light chain
sequence of SEQ ID NO: 102 and the polynucleotide SEQ ID NO: 244 encoding the heavy
chain sequence of SEQ ID NO: 104.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab11 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab11 or Fab fragments thereof may
be produced via expression of Ab11 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
Antibody Ab12
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 111:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTACTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 251).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 112:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCGGGCCAGTCAGAGTGTTTACTATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTAATGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 252).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 113:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGACGTCACTAACTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTGTGAATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTGCCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 253).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 114:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGACGTCACTAACTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTGTGA
ATGGTAAGAGATACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTGCCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC
TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG
GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA
GCCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC
AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC
CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 254).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 255; SEQ ID NO: 256;
and SEQ ID NO: 257 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 112.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 258; SEQ ID NO: 259;
and SEQ ID NO: 260 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 251 encoding the light chain variable sequence
of SEQ ID NO: 111; the polynucleotide SEQ ID NO: 252 encoding the light chain
sequence of SEQ ID NO: 112; the polynucleotide SEQ ID NO: 253 encoding the heavy
chain variable sequence of SEQ ID NO: 113; the polynucleotide SEQ ID NO: 254
encoding the heavy chain sequence of SEQ ID NO: 114; polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 255; SEQ ID NO: 256; and SEQ ID
NO: 257) of the light chain variable sequence of SEQ ID NO: 111 or the light chain
sequence of SEQ ID NO: 112; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 258; SEQ ID NO: 259; and SEQ ID NO: 260) of the
heavy chain variable sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID
NO: 114.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab12, the polynucleotides encoding the full length Ab12 antibody comprise, or
alternatively consist of, the polynucleotide SEQ ID NO: 252 encoding the light chain
sequence of SEQ ID NO: 112 and the polynucleotide SEQ ID NO: 254 encoding the heavy
chain sequence of SEQ ID NO: 114.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab12 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab12 or Fab fragments thereof may
be produced via expression of Ab12 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
Antibody Ab13
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 121:
GCCATCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGA
GACACAGTCACCATCAATTGCCAGGCCAGTGAGAGTCTTTATAATAACAACGC
CTTGGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATG
ATGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCGGTGGGTCT
GGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGACGATGCTGCCAC
TTACTACTGTGGAGGCTACAGAAGTGATAGTGTTGATGGTGTTGCTTTCGCCGG
AGGGACCGAGGTGGTGGTCAAACGT (SEQ ID NO: 261).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 122:
GCCATCGTGATGACCCAGACTCCATCTTCCAAGTCTGTCCCTGTGGGA
GACACAGTCACCATCAATTGCCAGGCCAGTGAGAGTCTTTATAATAACAACGC
CTTGGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATG
ATGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCGGTGGGTCT
GGGACACAGTTCACTCTCACCATCAGTGGCGTGCAGTGTGACGATGCTGCCAC
TTACTACTGTGGAGGCTACAGAAGTGATAGTGTTGATGGTGTTGCTTTCGCCGG
AGGGACCGAGGTGGTGGTCAAACGTACGGTGGCTGCACCATCTGTCTTCATCT
TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC
CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCA
CAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 262).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 123:
CAGTCGGTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGAT
CCCTGACACTCACCTGCACAGCCTCTGGATTCGACTTCAGTAGCAATGCAATGT
GGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTAC
AATGGTGATGGCAGCACATACTACGCGAGCTGGGTGAATGGCCGATTCTCCAT
CTCCAAAACCTCGTCGACCACGGTGACTCTGCAACTGAATAGTCTGACAGTCG
CGGACACGGCCACGTATTATTGTGCGAGAGATCTTGACTTGTGGGGCCCGGGC
ACCCTCGTCACCGTCTCGAGC (SEQ ID NO: 263).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 124:
CAGTCGGTGGAGGAGTCCGGGGGAGGCCTGGTCCAGCCTGAGGGATCCCTGAC
ACTCACCTGCACAGCCTCTGGATTCGACTTCAGTAGCAATGCAATGTGGTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGATGCATTTACAATGGTG
ATGGCAGCACATACTACGCGAGCTGGGTGAATGGCCGATTCTCCATCTCCAAA
ACCTCGTCGACCACGGTGACTCTGCAACTGAATAGTCTGACAGTCGCGGACAC
GGCCACGTATTATTGTGCGAGAGATCTTGACTTGTGGGGCCCGGGCACCCTCGT
CACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC
CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT
ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 264).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 265; SEQ ID NO: 266;
and SEQ ID NO: 267 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 268; SEQ ID NO: 269;
and SEQ ID NO: 270 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 261 encoding the light chain variable sequence
of SEQ ID NO: 121; the polynucleotide SEQ ID NO: 262 encoding the light chain
sequence of SEQ ID NO: 122; the polynucleotide SEQ ID NO: 263 encoding the heavy
chain variable sequence of SEQ ID NO: 123; the polynucleotide SEQ ID NO: 264
encoding the heavy chain sequence of SEQ ID NO: 124; polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 265; SEQ ID NO: 266; and SEQ ID
NO: 267) of the light chain variable sequence of SEQ ID NO: 121 or the light chain
sequence of SEQ ID NO: 122; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 268; SEQ ID NO: 269; and SEQ ID NO: 270) of the
heavy chain variable sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID
NO: 124.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab13, the polynucleotides encoding the full length Ab13 antibody comprise, or
alternatively consist of, the polynucleotide SEQ ID NO: 262 encoding the light chain
sequence of SEQ ID NO: 122 and the polynucleotide SEQ ID NO: 264 encoding the heavy
chain sequence of SEQ ID NO: 124.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab13 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab13 or Fab fragments thereof may
be produced via expression of Ab13 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
Antibody Ab14
The present disclosure is further directed to polynucleotides encoding antibody
polypeptides having binding specificity to CGRP. In one embodiment of the present
disclosure, polynucleotides described herein comprise, or alternatively consist of, the
following polynucleotide sequence encoding the variable light chain polypeptide sequence
of SEQ ID NO: 131:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGT (SEQ ID NO: 271).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
light chain polypeptide sequence of SEQ ID NO: 132:
CAAGTGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGAC
AGAGTCACCATCAATTGCCAGGCCAGTCAGAATGTTTACAATAACAACTACCT
AGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCAACTGATCTATTCTA
CATCCACTCTGGCATCTGGGGTCCCATCTCGTTTCAGTGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATT
ACTGTCTGGGCAGTTATGATTGTAGTCGTGGTGATTGTTTTGTTTTCGGCGGAG
GAACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTC
CCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTG
AATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCT
CCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 272).
In another embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 133:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACAT
GCAATGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTG
GTAGTGATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATC
TCCAGAGACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGC
TGAGGACACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGA
CCCTCGTCACCGTCTCGAGC (SEQ ID NO: 273).
In one embodiment of the present disclosure, polynucleotides described herein
comprise, or alternatively consist of, the following polynucleotide sequence encoding the
heavy chain polypeptide sequence of SEQ ID NO: 134:
GAGGTGCAGCTTGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCT
GAGACTCTCCTGTGCAGTCTCTGGAATCGGCCTCAGTAGCTACTACATGCAATG
GGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGAGTCATTGGTAGTG
ATGGTAAGACATACTACGCGACCTGGGCGAAAGGCCGATTCACCATCTCCAGA
GACAATTCCAAGACCACGGTGTATCTTCAAATGAACAGCCTGAGAGCTGAGGA
CACTGCTGTGTATTTCTGTACCAGAGGGGACATCTGGGGCCAAGGGACCCTCG
TCACCGTCTCGAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCT
CCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG
TGAATCACAAGCCCAGCAACACCAAGGTGGACGCGAGAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGG
TCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG
CCGCGGGAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA
AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGA
ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC
GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACA
AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
(SEQ ID NO: 274).
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 275; SEQ ID NO: 276;
and SEQ ID NO: 277 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the light chain variable sequence
of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.
In a further embodiment of the present disclosure, polynucleotides encoding
antibody fragments having binding specificity to CGRP comprise, or alternatively consist
of, one or more of the polynucleotide sequences of SEQ ID NO: 278; SEQ ID NO: 279;
and SEQ ID NO: 280 which correspond to polynucleotides encoding the complementarity-
determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence
of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.
The present disclosure also contemplates polynucleotide sequences including
one or more of the polynucleotide sequences encoding antibody fragments described
herein. In one embodiment of the present disclosure, polynucleotides encoding antibody
fragments having binding specificity to CGRP comprise, or alternatively consist of, one,
two, three or more, including all of the following polynucleotides encoding antibody
fragments: the polynucleotide SEQ ID NO: 271 encoding the light chain variable sequence
of SEQ ID NO: 131; the polynucleotide SEQ ID NO: 272 encoding the light chain
sequence of SEQ ID NO: 132; the polynucleotide SEQ ID NO: 273 encoding the heavy
chain variable sequence of SEQ ID NO: 133; the polynucleotide SEQ ID NO: 274
encoding the heavy chain sequence of SEQ ID NO: 134; polynucleotides encoding the
complementarity-determining regions (SEQ ID NO: 275; SEQ ID NO: 276; and SEQ ID
NO: 277) of the light chain variable sequence of SEQ ID NO: 131 or the light chain
sequence of SEQ ID NO: 132; and polynucleotides encoding the complementarity-
determining regions (SEQ ID NO: 278; SEQ ID NO: 279; and SEQ ID NO: 280) of the
heavy chain variable sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID
NO: 134.
In a preferred embodiment of the present disclosure, polynucleotides described
herein comprise, or alternatively consist of, polynucleotides encoding Fab (fragment
antigen binding) fragments having binding specificity for CGRP. With respect to antibody
Ab14, the polynucleotides encoding the full length Ab14 antibody comprise, or
alternatively consist of, the polynucleotide SEQ ID NO: 272 encoding the light chain
sequence of SEQ ID NO: 132 and the polynucleotide SEQ ID NO: 274 encoding the heavy
chain sequence of SEQ ID NO: 134.
Another embodiment of the present disclosure contemplates these
polynucleotides incorporated into an expression vector for expression in mammalian cells
such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells
such as the yeast Pichia. Suitable Pichia species include, but are not limited to, Pichia
pastoris. In one embodiment of the present disclosure described herein (infra), Fab
fragments may be produced by enzymatic digestion (e.g., papain) of Ab14 following
expression of the full-length polynucleotides in a suitable host. In another embodiment of
the present disclosure, anti-CGRP antibodies such as Ab14 or Fab fragments thereof may
be produced via expression of Ab14 polynucleotides in mammalian cells such as CHO,
NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for
example diploid yeast such as diploid Pichia) and other yeast strains. Suitable Pichia
species include, but are not limited to, Pichia pastoris.
In one embodiment, the present disclosure is directed to an isolated
polynucleotide comprising a polynucleotide encoding an anti-CGRP V antibody amino
acid sequence selected from SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113,
123, or 133, or encoding a variant thereof wherein at least one framework residue (FR
residue) has been substituted with an amino acid present at the corresponding position in a
rabbit anti-CGRP antibody V polypeptide or a conservative amino acid substitution.
In another embodiment, the present disclosure is directed to an isolated
polynucleotide comprising the polynucleotide sequence encoding an anti-CGRP V
antibody amino acid sequence of 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or
131, or encoding a variant thereof wherein at least one framework residue (FR residue) has
been substituted with an amino acid present at the corresponding position in a rabbit anti-
CGRP antibody V polypeptide or a conservative amino acid substitution.
In yet another embodiment, the present disclosure is directed to one or more
heterologous polynucleotides comprising a sequence encoding the polypeptides contained
in SEQ ID NO:1 and SEQ ID NO:3; SEQ ID NO:11 and SEQ ID NO:13; SEQ ID NO:21
and SEQ ID NO:23; SEQ ID NO:31 and SEQ ID NO:33; SEQ ID NO:41 and SEQ ID
NO:43; SEQ ID NO:51 and SEQ ID NO:53, SEQ ID NO:61 and SEQ ID NO:63; SEQ ID
NO:71 and SEQ ID NO:73; SEQ ID NO:81 and SEQ ID NO:83; SEQ ID NO:91 and SEQ
ID NO:93; SEQ ID NO:101 and SEQ ID NO:103; SEQ ID NO:111 and SEQ ID NO:113;
SEQ ID NO:121 and SEQ ID NO:123; or SEQ ID NO:131 and SEQ ID NO:133.
In another embodiment, the present disclosure is directed to an isolated
polynucleotide that expresses a polypeptide containing at least one CDR polypeptide
derived from an anti-CGRP antibody wherein said expressed polypeptide alone specifically
binds CGRP or specifically binds CGRP when expressed in association with another
polynucleotide sequence that expresses a polypeptide containing at least one CDR
polypeptide derived from an anti-CGRP antibody wherein said at least one CDR is selected
from those contained in the V or V polypeptides of SEQ ID NO: 1, 3, 11, 13, 21, 23, 31,
33, 41, 43, 51, 53, 61, 63, 71, 73, 81, 83, 91, 93, 101, 103, 111, 113, 121, 123, 131, or SEQ
ID NO:133.
Host cells and vectors comprising said polynucleotides are also contemplated.
The present disclosure further contemplates vectors comprising the
polynucleotide sequences encoding the variable heavy and light chain polypeptide
sequences, as well as the individual complementarity-determining regions (CDRs, or
hypervariable regions), as set forth herein, as well as host cells comprising said vector
sequences. In one embodiment of the present disclosure, the host cell is a yeast cell. In
another embodiment of the present disclosure, the yeast host cell belongs to the genus
Pichia.
B-cell Screening and Isolation
In one embodiment, the present disclosure contemplates the preparation and
isolation of a clonal population of antigen-specific B cells that may be used for isolating at
least one CGRP antigen-specific cell, which can be used to produce a monoclonal antibody
against CGRP, which is specific to a desired CGRP antigen, or a nucleic acid sequence
corresponding to such an antibody. Methods of preparing and isolating said clonal
population of antigen-specific B cells are taught, for example, in U.S. patent publication
no. US 2007/0269868 to Carvalho-Jensen et al., the disclosure of which is herein
incorporated by reference in its entirety. Methods of preparing and isolating said clonal
population of antigen-specific B cells are also taught herein in the examples. Methods of
“enriching” a cell population by size or density are known in the art. See, e.g., U.S. Patent
,627,052. These steps can be used in addition to enriching the cell population by antigen-
specificity.
Methods of Humanizing Antibodies
In another embodiment, the present disclosure contemplates methods for
humanizing antibody heavy and light chains. Methods for humanizing antibody heavy and
light chains which may be applied to anti-CGRP antibodies are taught, for example, in U.S.
patent application publication no. US 2009/0022659 to Olson et al., and in U.S. patent no.
7,935,340 to Garcia-Martinez et al., the disclosures of each of which are herein
incorporated by reference in their entireties.
Methods of Producing Antibodies and Fragments thereof
In another embodiment, the present disclosure contemplates methods for
producing anti-CGRP antibodies and fragments thereof. Methods for producing anti-
CGRP antibodies and fragments thereof secreted from polyploidal, preferably diploid or
tetraploid strains of mating competent yeast are taught, for example, in U.S. patent
application publication no. US 2009/0022659 to Olson et al., and in U.S. patent no.
7,935,340 to Garcia-Martinez et al., the disclosures of each of which are herein
incorporated by reference in their entireties.
Other methods of producing antibodies are well known to those of ordinary skill
in the art. For example, methods of producing chimeric antibodies are now well known in
the art (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al.; Morrison et al.,
P.N.A.S. USA, 81:8651-55 (1984); Neuberger, M.S. et al., Nature, 314:268-270 (1985);
Boulianne, G.L. et al., Nature, 312:643-46 (1984), the disclosures of each of which are
herein incorporated by reference in their entireties).
Likewise, other methods of producing humanized antibodies are now well
known in the art (See, for example, U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370 to Queen et al; U.S. Patent Nos. 5,225,539 and 6,548,640 to Winter; U.S. Patent
Nos. 6,054,297, 6,407,213 and 6,639,055 to Carter et al; U.S. Patent No. 6,632,927 to
Adair; Jones, P.T. et al, Nature, 321:522-525 (1986); Reichmann, L., et al, Nature,
332:323-327 (1988); Verhoeyen, M, et al, Science, 239:1534-36 (1988), the disclosures of
each of which are herein incorporated by reference in their entireties).
Antibody polypeptides of the present disclosure having CGRP binding
specificity may also be produced by constructing, using conventional techniques well
known to those of ordinary skill in the art, an expression vector containing an operon and a
DNA sequence encoding an antibody heavy chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a non-human cell source,
preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of
the antibody chain is derived from a human cell source.
A second expression vector is produced using the same conventional means well
known to those of ordinary skill in the art, said expression vector containing an operon and
a DNA sequence encoding an antibody light chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a non-human cell source,
preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of
the antibody chain is derived from a human cell source.
The expression vectors are transfected into a host cell by convention techniques
well known to those of ordinary skill in the art to produce a transfected host cell, said
transfected host cell cultured by conventional techniques well known to those of ordinary
skill in the art to produce said antibody polypeptides.
The host cell may be co-transfected with the two expression vectors described
above, the first expression vector containing DNA encoding an operon and a light chain-
derived polypeptide and the second vector containing DNA encoding an operon and a
heavy chain-derived polypeptide. The two vectors contain different selectable markers, but
preferably achieve substantially equal expression of the heavy and light chain polypeptides.
Alternatively, a single vector may be used, the vector including DNA encoding both the
heavy and light chain polypeptides. The coding sequences for the heavy and light chains
may comprise cDNA, genomic DNA, or both.
The host cells used to express the antibody polypeptides may be either a
bacterial cell such as E. coli, or a eukaryotic cell such as P. pastoris. In one embodiment of
the present disclosure, a mammalian cell of a well-defined type for this purpose, such as a
myeloma cell, a Chinese hamster ovary (CHO) cell line, a NSO cell line, or a HEK293 cell
line may be used.
The general methods by which the vectors may be constructed, transfection
methods required to produce the host cell and culturing methods required to produce the
antibody polypeptides from said host cells all include conventional techniques. Although
preferably the cell line used to produce the antibody is a mammalian cell line, any other
suitable cell line, such as a bacterial cell line such as an E. coli-derived bacterial strain, or a
yeast cell line, may alternatively be used.
Similarly, once produced the antibody polypeptides may be purified according to
standard procedures in the art, such as for example cross-flow filtration, ammonium
sulphate precipitation, affinity column chromatography and the like.
The antibody polypeptides described herein may also be used for the design and
synthesis of either peptide or non-peptide mimetics that would be useful for the same
therapeutic applications as the antibody polypeptides of the present disclosure. See, for
example, Saragobi et al, Science, 253:792-795 (1991), the contents of which is herein
incorporated by reference in its entirety.
Screening Assays
The present disclosure also includes screening assays designed to assist in the
identification of diseases and disorders associated with CGRP especially conditions
associated with photophobia such as migraine, other headache and pain conditions,
depression, bipolar disorder, agoraphobia and others in patients exhibiting symptoms of
photophobia or a CGRP associated disease or disorder.
In one embodiment of the present disclosure, the anti-CGRP antibodies
described herein, or CGRP binding fragments thereof, are used to detect the presence of
CGRP in a biological sample obtained from a patient exhibiting symptoms of a disease or
disorder associated with CGRP especially one associated with photophobia. The presence
of CGRP, or elevated levels thereof when compared to pre-disease levels of CGRP in a
comparable biological sample, may be beneficial in diagnosing a disease or disorder
associated with CGRP.
Another embodiment of the present disclosure provides a diagnostic or screening
assay to assist in diagnosis of diseases or disorders associated with CGRP and photophobia
in patients exhibiting symptoms of a CGRP associated disease or disorder identified herein,
comprising assaying the level of CGRP expression in a biological sample from said patient
using a post-translationally modified anti-CGRP antibody or binding fragment thereof.
The anti-CGRP antibody or binding fragment thereof may be post-translationally modified
to include a detectable moiety such as set forth previously in the disclosure.
The CGRP level in the biological sample is determined using a modified anti-
CGRP antibody or binding fragment thereof as set forth herein, and comparing the level of
CGRP in the biological sample against a standard level of CGRP (e.g., the level in normal
biological samples). The skilled clinician would understand that some variability may exist
between normal biological samples, and would take that into consideration when
evaluating results. In one embodiment of the present disclosure, the anti-CGRP antibodies
described herein may be used to correlate CGRP expression levels with a particular stage
of cancerous development. One skilled in the art would be able to measure CGRP in
numerous subjects in order to establish ranges of CGRP expression that correspond to
clinically defined stages of cancerous development. These ranges will allow the skilled
practitioner to measure CGRP in a subject diagnosed with a cancer and correlate the levels
in each subject with a range that corresponds to a stage of said cancer. One skilled in the
art would understand that by measuring CGRP in the patient at different intervals, the
progression of the cancer can be determined.
The above-recited assay may also be useful in monitoring a disease or disorder,
where the level of CGRP obtained in a biological sample from a patient believed to have a
CGRP associated disease or disorder especially one associated with photophobia is
compared with the level of CGRP in prior biological samples from the same patient, in
order to ascertain whether the CGRP level in said patient has changed with, for example, a
treatment regimen.
The present disclosure is also directed to a method of in vivo imaging which
detects the presence of cells which express CGRP comprising administering a
diagnostically effective amount of a diagnostic composition. Said in vivo imaging is useful
for the detection or imaging of CGRP expressing tumors or metastases, for example, and
can be useful as part of a planning regimen for the design of an effective cancer treatment
protocol. The treatment protocol may include, for example, one or more of radiation,
chemotherapy, cytokine therapy, gene therapy, and antibody therapy, as well as an anti-
CGRP antibody or fragment thereof.
The present disclosure further describes a kit for detecting binding of an anti-
CGRP antibody described herein to CGRP. In particular, the kit may be used to detect the
presence of a CGRP specifically reactive with an anti-CGRP antibody of the present
disclosure or an immunoreactive fragment thereof. The kit may also include an antibody
bound to a substrate, a secondary antibody reactive with the antigen and a reagent for
detecting a reaction of the secondary antibody with the antigen. Such a kit may be an
ELISA kit and can comprise the substrate, primary and secondary antibodies when
appropriate, and any other necessary reagents such as detectable moieties, enzyme
substrates, and color reagents, for example as described herein. The diagnostic kit may
also be in the form of an immunoblot kit. The diagnostic kit may also be in the form of a
chemiluminescent kit (Meso Scale Discovery, Gaithersburg, MD). The diagnostic kit may
also be a lanthanide-based detection kit (PerkinElmer, San Jose, CA).
A skilled clinician would understand that a biological sample includes, but is not
limited to, sera, plasma, urine, saliva, mucous, pleural fluid, synovial fluid and spinal fluid.
Methods of Ameliorating or Reducing Symptoms of, or Treating, or Preventing, Diseases
and Disorders Associated with, CGRP
In another embodiment of the present disclosure, anti-CGRP antibodies
described herein, or fragments thereof, are useful for ameliorating or reducing the
symptoms of, or treating, or preventing, diseases and disorders associated with CGRP
especially for treatment or prevention of photophobia. In a preferred embodiment the anti-
CGRP antibodies or antibody fragments will be shown to be efficacious (block adverse
side effects associated with excess circulating CGRP including light aversive behavior) in
the rodent animal model disclosed in Example 8.
Anti-CGRP antibodies described herein, or fragments thereof, as well as
combinations, can also be administered in a therapeutically effective amount to patients in
need of treatment of diseases and disorders associated with CGRP for treatment or
prevention of photophobia in the form of a pharmaceutical composition as described in
greater detail below.
In another embodiment of the present disclosure, anti-CGRP antibodies
described herein, or fragments thereof, are useful for ameliorating or reducing the
symptoms of, or treating, or preventing, migraines (with or without aura), weight loss,
cancer or tumors, angiogenesis associated with cancer or tumor growth, angiogenesis
associated with cancer or tumor survival, pain, hemiplagic migraines, cluster headaches,
migrainous neuralgia, chronic headaches, tension headaches, general headaches, headache-
free migraine, abdominal migraine, hot flashes, chronic paroxysomal hemicrania,
secondary headaches due to an underlying structural problem in the head or neck, cranial
neuralgia, sinus headaches (such as for example associated with sinusitis), and allergy-
induced headaches or migraines.
In another embodiment of the present disclosure, anti-CGRP antibodies
described herein, or fragments thereof and/or with a second agent, are useful for
ameliorating or reducing the symptoms of, or treating, or preventing, photophobia
associated with the following non-limiting listing of diseases and disorders: neurogenic,
neuropathic or nociceptic pain. Neuropathic pain may include, but is not limited to,
trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrual
pain, ovarialgia, reflex sympathetic dystrophy and neurogenic pain. In other preferred
embodiments, anti-CGRP antibodies described herein, or fragments thereof and/or with a
second agent, are useful for ameliorating or reducing the symptoms of, or treating, or
preventing, photophobia associated with osteoarthritis or rheumatoid arthritis pain, lower
back pain, diabetic neuropathy, sciatica, and other neuropathic pain.
In another embodiment of the present disclosure, anti-CGRP antibodies
described herein, or fragments thereof and/or with a second agent, are useful for
ameliorating or reducing the symptoms of, or treating, or preventing, photophobia
associated with the following non-limiting listing of diseases and disorders: visceral pain or
more specifically associated with gastro-esophageal reflux, dyspepsia, irritable bowel
syndrome, inflammatory bowel disease, Crohn’s disease, ileitis, ulcerative colitis, renal
colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, or
pancreatitis.
Administration
In one embodiment of the present disclosure, the anti-CGRP antibodies or
fragments described herein, or anti-CGRP-R antibodies or fragments thereof, as well as
combinations of said antibodies or antibody fragments, for treatment or prevention of
photophobia, are administered to a subject at a concentration of between about 0.1 and
100.0 mg/kg of body weight of recipient subject. In a preferred embodiment of the present
disclosure, the anti-CGRP antibodies described herein, or CGRP binding fragments
thereof, as well as combinations of said antibodies or antibody fragments, are administered
to a subject at a concentration of about 0.4 mg/kg of body weight of recipient subject. In a
preferred embodiment of the present disclosure, the anti-CGRP antibodies described
herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or
antibody fragments, are administered to a recipient subject with a frequency of once every
twenty-six weeks or less, such as once every sixteen weeks or less, once every eight weeks
or less, once every four weeks or less, once every two weeks or less, once every week or
less, or once daily or less.
Fab fragments for treatment or prevention of photophobia may be administered
every two weeks or less, every week or less, once daily or less, multiple times per day,
and/or every few hours. In one embodiment of the present disclosure, a patient receives
Fab fragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses of 1 to 6 times a
day, or in a sustained release form, effective to obtain desired results.
It is to be understood that the concentration of the antibody or Fab administered
to a given patient for treatment or prevention of photophobia may be greater or lower than
the exemplary administration concentrations set forth above in paragraphs [0566] and
A person of skill in the art would be able to determine an effective dosage and
frequency of administration for treatment or prevention of photophobia through routine
experimentation, for example guided by the disclosure herein and the teachings in
Goodman, L. S., Gilman, A., Brunton, L. L., Lazo, J. S., & Parker, K. L. (2006). Goodman
& Gilman's the pharmacological basis of therapeutics. New York: McGraw-Hill; Howland,
R. D., Mycek, M. J., Harvey, R. A., Champe, P. C., & Mycek, M. J. (2006). Pharmacology.
Lippincott's illustrated reviews. Philadelphia: Lippincott Williams & Wilkins; and Golan,
D. E. (2008). Principles of pharmacology: the pathophysiologic basis of drug therapy.
Philadelphia, Pa., [etc.]: Lippincott Williams & Wilkins.
In another embodiment of the present disclosure, the anti-CGRP antibodies
described herein, or CGRP binding fragments thereof, as well as combinations of said
antibodies or antibody fragments, for treatment or prevention of photophobia are
administered to a subject in a pharmaceutical formulation.
A “pharmaceutical composition” refers to a chemical or biological composition
suitable for administration to a mammal. Such compositions may be specifically
formulated for administration via one or more of a number of routes, including but not
limited to buccal, epicutaneous, epidural, inhalation, intraarterial, intracardial,
intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal,
intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository,
subcutaneous, subdermal, sublingual, transdermal, and transmucosal. In addition,
administration can occur by means of injection, powder, liquid, gel, drops, or other means
of administration.
In one embodiment of the present disclosure, the anti-CGRP antibodies
described herein, or CGRP binding fragments thereof, as well as combinations of said
antibodies or antibody fragments, for treatment or prevention of photophobia may be
optionally administered in combination with one or more active agents. Such active agents
include analgesic, anti-histamine, antipyretic, anti-inflammatory, antibiotic, antiviral, and
anti-cytokine agents. Active agents include agonists, antagonists, and modulators of TNF-
α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN- α, IFN- γ, BAFF, CXCL13, IP-10,
VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including
antibodies reactive against any of the foregoing, and antibodies reactive against any of their
receptors. Active agents also include but are not limited to 2-Arylpropionic acids,
Aceclofenac, Acemetacin, Acetylsalicylic acid (Aspirin), Alclofenac, Alminoprofen,
Amoxiprin, Ampyrone, Arylalkanoic acids, Azapropazone, Benorylate/Benorilate,
Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesium salicylate,
Clofezone, COX-2 inhibitors, Dexibuprofen, Dexketoprofen, Diclofenac, Diflunisal,
Droxicam, Ethenzamide, Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen,
Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam,
Indometacin, Indoprofen, Kebuzone, Ketoprofen, Ketorolac, Lornoxicam, Loxoprofen,
Lumiracoxib, Magnesium salicylate, Meclofenamic acid, Mefenamic acid, Meloxicam,
Metamizole, Methyl salicylate, Mofebutazone, Nabumetone, Naproxen, N-Arylanthranilic
acids, Nerve Growth Factor (NGF), Oxametacin, Oxaprozin, Oxicams, Oxyphenbutazone,
Parecoxib, Phenazone, Phenylbutazone, Phenylbutazone, Piroxicam, Pirprofen, profens,
Proglumetacin, Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate, Salicylamide,
Salicylates, Substance P, Sulfinpyrazone, Sulindac, Suprofen, Tenoxicam, Tiaprofenic
acid, Tolfenamic acid, Tolmetin, and Valdecoxib.
An anti-histamine can be any compound that opposes the action of histamine or
its release from cells (e.g., mast cells). Anti-histamines include but are not limited to
acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine,
cetirizine, cetirizine analogues, chlorpheniramine, clemastine, CS 560, cyproheptadine,
desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609,
hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole,
phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.
Antibiotics include but are not limited to Amikacin, Aminoglycosides,
Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin, Azlocillin,
Aztreonam, Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor, Cefadroxil,
Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime,
Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime,
Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins,
Chloramphenicol, Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin,
Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin, Dirithromycin,
Doripenem, Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol,
Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid, Gatifloxacin, Geldanamycin,
Gentamicin, Glycopeptides, Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin,
Lincomycin, Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem,
Meticillin, Metronidazole, Mezlocillin, Minocycline, Monobactams, Moxifloxacin,
Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin,
Oxacillin, Oxytetracycline, Paromomycin, Penicillin, Penicillins, Piperacillin,
Platensimycin, Polymyxin B, Polypeptides, Prontosil, Pyrazinamide, Quinolones,
Quinupristin, Rifampicin, Rifampin, Roxithromycin, Spectinomycin, Streptomycin,
Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfonamides,
Teicoplanin, Telithromycin, Tetracycline, Tetracyclines, Ticarcillin, Tinidazole,
Tobramycin, Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin,
Trovafloxacin, and Vancomycin.
Active agents also include Aldosterone, Beclometasone, Betamethasone,
Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosterone acetate, Dexamethasone,
Fludrocortisone acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone,
Prednisolone, Prednisone, Steroids, and Triamcinolone. Any suitable combination of these
active agents is also contemplated.
In preferred embodiments, the subject antibodies and antibody fragments may be
administered in a therapeutic regimen that includes compounds typically used to treat
migraines, including migraines associated with photophobia. Examples hereof include
analgesics such as NSAIDs. Examples include those afore-mentioned such as Ibuprofen,
naproxen, sumatriptan, Paracetamol/acetaminophen, either alone or in combination with
metoclopramide, and caffeine.
Triptans such as sumatriptan are commonly used as are Ergotamines such as
Ergotamine. In addition, corticosteroids may be used.
Also, antimimetics may help relieve symptoms of nausea and help prevent
vomiting, which can diminish the effectiveness of orally taken analgesics. In addition,
some antiemetics, such as metoclopramide, are prokinetics and help gastric emptying,
which is often impaired during episodes of migraine. Three combination antiemetic and
analgesic preparations used for migraines include (aspirin with metoclopramide),
(paracetamol/codeine for analgesia, with buclizine as the antiemetic) and
paracetamol/metoclopramide.
A “pharmaceutical excipient” or a “pharmaceutically acceptable excipient” is a
carrier, usually a liquid, in which an active therapeutic agent is formulated. In one
embodiment of the present disclosure, the active therapeutic agent is a humanized antibody
described herein, or one or more fragments thereof. The excipient generally does not
provide any pharmacological activity to the formulation, though it may provide chemical
and/or biological stability, and release characteristics. Exemplary formulations can be
found, for example, in Remington’s Pharmaceutical Sciences, 19 Ed., Grennaro, A., Ed.,
1995 which is incorporated by reference.
As used herein “pharmaceutically acceptable carrier” or “excipient” includes any
and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents that are physiologically compatible. In one embodiment,
the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable
for intravenous, intraperitoneal, intramuscular, or sublingual administration.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile injectable solutions or
dispersions. The use of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or agent is incompatible
with the active compound, use thereof in the pharmaceutical compositions of the present
disclosure is contemplated. Supplementary active compounds can also be incorporated into
the compositions.
Pharmaceutical compositions typically must be sterile and stable under the
conditions of manufacture and storage. The present disclosure contemplates that the
pharmaceutical composition is present in lyophilized form. The composition can be
formulated as a solution, microemulsion, liposome, or other ordered structure suitable to
high drug concentration. The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The present disclosure further
contemplates the inclusion of a stabilizer in the pharmaceutical composition. The proper
fluidity can be maintained, for example, by the maintenance of the required particle size in
the case of dispersion and by the use of surfactants.
In many cases, it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including in
the composition an agent which delays absorption, for example, monostearate salts and
gelatin. Moreover, the alkaline polypeptide can be formulated in a time release
formulation, for example in a composition which includes a slow release polymer. The
active compounds can be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including implants and
microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are known to those skilled in the art.
For each of the recited embodiments, the compounds can be administered by a
variety of dosage forms. Any biologically-acceptable dosage form known to persons of
ordinary skill in the art, and combinations thereof, are contemplated. Examples of such
dosage forms include, without limitation, reconstitutable powders, elixirs, liquids,
solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible
granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot
implants, injectables (including subcutaneous, intramuscular, intravenous, and
intradermal), infusions, and combinations thereof.
The above description of various illustrated embodiments of the invention is not
intended to be exhaustive or to limit the invention to the precise form disclosed. While
specific embodiments of, and examples for, the invention are described herein for
illustrative purposes, various equivalent modifications are possible within the scope of the
invention, as those skilled in the relevant art will recognize. The teachings provided herein
of the invention can be applied to other purposes, other than the examples described above.
These and other changes can be made to the invention in light of the above
detailed description. In general, in the following claims, the terms used should not be
construed to limit the invention to the specific embodiments disclosed in the specification
and the claims. Accordingly, the invention is not limited by the disclosure, but instead the
scope of the invention is to be determined entirely by the following claims.
The invention may be practiced in ways other than those particularly described
in the foregoing description and examples. Numerous modifications and variations of the
invention are possible in light of the above teachings and, therefore, are within the scope of
the appended claims.
Certain teachings related to methods for obtaining a clonal population of
antigen-specific B cells were disclosed in U.S. Provisional patent application no.
60/801,412, filed May 19, 2006, the disclosure of which is herein incorporated by reference
in its entirety.
Certain teachings related to humanization of rabbit-derived monoclonal
antibodies and preferred sequence modifications to maintain antigen binding affinity were
disclosed in International Application No. , corresponding to
International Publication No. WO/2008/144757, entitled “Novel Rabbit Antibody
Humanization Methods and Humanized Rabbit Antibodies”, filed May 21, 2008, the
disclosure of which is herein incorporated by reference in its entirety.
Certain teachings related to producing antibodies or fragments thereof using
mating competent yeast and corresponding methods were disclosed in U.S. Patent
application no. 11/429,053, filed May 8, 2006, (U.S. Patent Application Publication No.
US2006/0270045), the disclosure of which is herein incorporated by reference in its
entirety.
Certain anti-CGRP antibody polynucleotides and polypeptides are disclosed in
the sequence listing accompanying this patent application filing, and the disclosure of said
sequence listing is herein incorporated by reference in its entirety.
The entire disclosure of each document cited (including patents, patent
applications, journal articles, abstracts, manuals, books, or other disclosures) in the
Background of the Invention, Detailed Description, and Examples is herein incorporated by
reference in their entireties.
The following examples are put forth so as to provide those of ordinary skill in
the art with a complete disclosure and description of how to make and use the subject
invention, and are not intended to limit the scope of what is regarded as the invention.
Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts,
temperature, concentrations, etc.) but some experimental errors and deviations should be
allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is
average molecular weight, temperature is in degrees centigrade; and pressure is at or near
atmospheric.
EXAMPLES
Example 1 Preparation of Antibodies that Bind CGRP
By using the antibody selection protocol described herein, one can generate an
extensive panel of antibodies.
Immunization Strategy
Rabbits were immunized with human CGRPα (American Peptides, Sunnyvale
CA and Bachem, Torrance CA). Immunization consisted of a first subcutaneous (sc)
injection of 100 μg of antigen mixed with 100 μg of KLH in complete Freund’s adjuvant
(CFA) (Sigma) followed by two boosts, two weeks apart each containing 50 μg antigen
mixed with 50 μg in incomplete Freund’s adjuvant (IFA) (Sigma). Animals were bled on
day 55, and serum titers were determined by ELISA (antigen recognition) and by inhibition
of CGRP driven cAMP increase in SK-N-MC.
Antibody Selection Titer Assessment
To identify and characterize antibodies that bind to human CGRPα, antibody-
containing solutions were tested by ELISA. Briefly, neutravidin coated plates (Thermo
Scientific), were coated with N-term biotinylated human CGRPα (50μL per well, 1μg/mL)
diluted in ELISA buffer (0.5% fish skin gelatin in PBS pH 7.4,) either for approximately
1hr at room temperature or alternatively overnight at 4°C. The plates were then further
blocked with ELISA buffer for one hour at room temperature and washed using wash
buffer (PBS,0.05% tween 20). Serum samples tested were serially diluted using ELISA
buffer. Fifty microliters of diluted serum samples were transferred onto the wells and
incubated for one hour at room temperature for one hour. After this incubation, the plate
was washed with wash buffer. For development, an anti-rabbit specific Fc-HRP (1:5000
dilution in ELISA buffer) was added onto the wells and incubated for 45 min at RT. After
a 3x wash step with wash solution, the plate was developed using TMB substrate for two
minutes at room temperature and the reaction was quenched using 0.5M HCl. The well
absorbance was read at 450 nm.
Titer determination of serum samples by functional activity (Inhibition of CGRP driven
cAMP levels)
To identify and characterize antibodies with functional activity, an inhibition of
CGRP driven increase of cAMP levels assay was done using electrochemiluminescence
(Meso Scale Discovery, MSD). Briefly, antibody preparations to be tested were serially
diluted in MSD assay buffer (Hepes, MgCl2, pH 7.3, 1mg/mL blocker A, Meso Scale
Discovery) in a 96 well round bottom polystyrene plate (Costar). To this plate, human
CGRPα was added (10ng/mL final concentration) diluted in MSD assay buffer and
incubated for one hour at 37C. Appropriate controls were used as suggested by the assay-
kit manufacturer. Human neuroepithelioma cells (SK-N-MC, ATCC) were detached using
an EDTA solution (5mM in PBS) and washed using growth media (MEM, 10% FBS,
antibiotics) by centrifugation. The cell number was adjusted to 2 million cells per mL in
assay buffer, and IBMX (3-Isobutyl-1Methylxanthine, Sigma) was added to a final
concentration of 0.2mM right before loading cells onto cAMP assay plate. After the
antibody human CGRPα solution was incubated for one hour 20 microliters of solution
containing cells were transferred to the cAMP assay plate. All tested samples were run in
duplicates with appropriate controls. Ten microliters of cells were added to the wells and
the plate was incubated for 30 minutes with shaking at room temperature. While cells were
being incubated with the CGRP solution, the stop solution was prepared by making a 1:200
solution of TAG labeled cAMP (MSD) in lysis buffer (MSD). To stop the cells-CGRP
incubation, 20 microliters of stop solution was added to the cells and the plate was
incubated for one hour with shaking at room temperature. The read buffer (MSD) was
diluted four times with water and 100 microliters were added to all wells on the plate. The
plate was then read using a Sector Imager 2400 (MSD) and the Prism software was used
for data fit and IC50 determination.
Tissue Harvesting
Once acceptable titers were established, the rabbit(s) were sacrificed. Spleen,
lymph nodes, and whole blood were harvested and processed as follows:
Spleen and lymph nodes were processed into a single cell suspension by
disassociating the tissue and pushing through sterile wire mesh at 70 μm (Fisher) with a
plunger of a 20 cc syringe. Cells were collected in PBS. Cells were washed twice by
centrifugation. After the last wash, cell density was determined by trypan blue. Cells were
centrifuged at 1500 rpm for 10 minutes; the supernatant was discarded. Cells were
resuspended in the appropriate volume of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS
(Hyclone) and dispensed at 1 ml/vial. Vials were stored at -70°C in a slow freezing
chamber for 24 hours and stored in liquid nitrogen.
Peripheral blood mononuclear cells (PBMCs) were isolated by mixing whole
blood with equal parts of the low glucose medium described above without FBS. 35 ml of
the whole blood mixture was carefully layered onto 8 ml of Lympholyte Rabbit
(Cedarlane) into a 45 ml conical tube (Corning) and centrifuged 30 minutes at 2500 rpm at
room temperature without brakes. After centrifugation, the PBMC layers were carefully
removed using a glass Pasteur pipette (VWR), combined, and placed into a clean 50 ml
vial. Cells were washed twice with the modified medium described above by
centrifugation at 1500 rpm for 10 minutes at room temperature, and cell density was
determined by trypan blue staining. After the last wash, cells were resuspended in an
appropriate volume of 10% DMSO/FBS medium and frozen as described above.
B cell selection, enrichment and culture conditions
On the day of setting up B cell culture, PBMC, splenocyte, or lymph node vials
were thawed for use. Vials were removed from LN2 tank and placed in a 37°C water bath
until thawed. Contents of vials were transferred into 15 ml conical centrifuge tube
(Corning) and 10 ml of modified RPMI described above was slowly added to the tube.
Cells were centrifuged for 5 minutes at 2K RPM, and the supernatant was discarded. Cells
were resuspended in 10 ml of fresh media. Cell density and viability was determined by
trypan blue.
a) The following protocol was used for Ab1 and Ab13
Cells were pre-mixed with the biotinylated human CGRPα as follows. Cells
were washed again and resuspended at 1E07 cells/80 μL medium. Biotinylated human
CGRPα was added to the cell suspension at the final concentration of 5 ug/mL and
incubated for 30 minutes at 4°C. Unbound biotinylated human CGRPα was removed
performing two 10 ml washes using PBF [Ca/Mg free PBS (Hyclone), 2 mM
ethylenediamine tetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA) (Sigma-
biotin free)]. After the second wash, cells were resuspended at 1E07 cells/80 μl PBF and
μl of MACS® streptavidin beads (Miltenyi Biotech, Auburn CA) per 10E7 cells were
added to the cell suspension. Cells and beads were incubated at 4°C for 15 minutes and
washed once with 2 ml of PBF per 10E7 cells.
b) The following protocol was used for Ab4, Ab7, Ab9 and Ab11:
Biotinylated human CGRPα was pre-loaded onto the streptavidin beads as
follows. Seventy five microliters of streptavidin beads (Milteny Biotec, Auburn CA) were
mixed with N-terminally biotinylated huCGRPα (10ug/ml final concentration) and 300 μl
PBF. This mixture was incubated at 4°C for 30 min and unbound biotinylated human
CGRPα was removed using a MACS® separation column (Miltenyi Biotec, with a 1ml
rinse to remove unbound material. Then material was plunged out, then used to resuspend
cells from above in 100ul per 1E7 cells, the mixture was then incubated at 4°C for 30min
and washed once with 10 ml of PBF.
For both a) and b) protocols the following applied: After washing, the cells were
resuspended in 500 μl of PBF and set aside. A MACS® MS column (Miltenyi Biotec,
Auburn CA) was pre-rinsed with 500 ml of PBF on a magnetic stand (Milteni). Cell
suspension was applied to the column through a pre-filter, and unbound fraction was
collected. The column was washed with 2.5 ml of PBF buffer. The column was removed
from the magnet stand and placed onto a clean, sterile 1.5 ml eppendorf tube. 1 ml of PBF
buffer was added to the top of the column, and positive selected cells were collected. The
yield and viability of positive cell fraction was determined by trypan blue staining. Positive
selection yielded an average of 1% of the starting cell concentration.
A pilot cell screen was established to provide information on seeding levels for
the culture. Plates were seeded at 10, 25, 50, 100, or 200 enriched B cells/well. In
addition, each well contained 50K cells/well of irradiated EL-4.B5 cells (5,000 Rads) and
an appropriate level of activated rabbit T cell supernatant (See U.S. Patent Application
Publication No. 20070269868)(ranging from 1-5% depending on preparation) in high
glucose modified RPMI medium at a final volume of 250 μl/well. Cultures were incubated
for 5 to 7 days at 37°C in 4% CO .
B-Cell culture screening by antigen-recognition (ELISA)
To identify wells producing anti-human CGRPα antibodies, the same protocol as
described for titer determination of serum samples by antigen-recognition (ELISA) was
used with the following changes. Briefly, neutravidin coated plates were coated with a
mixture of both N- and C- terminally biotinylated human CGRPα (50μL per well, 1μg/mL
each). B-cell supernatant samples (50μL) were tested without prior dilution.
Identification of functional activity in B-cell supernatants using CGRP driven cAMP
production
To determine functional activity contained in B-cell supernatants, a similar
procedure to that described for the determination of functional titer of serum samples was
used with the following modifications. Briefly, B-cell supernatant (20μL) were used in
place of the diluted polyclonal serum samples.
Isolation of antigen-specific B-cells
Plates containing wells of interest were removed from -70 °C, and the cells from
each well were recovered using five washes of 200 microliters of medium (10% RPMI
complete, 55μM BME) per well. The recovered cells were pelleted by centrifugation and
the supernatant was carefully removed. Pelleted cells were resuspended in 100 μl of
medium. To identify antibody expressing cells, streptavidin coated magnetic beads (M280
dynabeads, Invitrogen) were coated with a combination of both N- and C- terminal
biotinylated human CGRPα. Individual biotinylated human CGRPα lots were optimized
by serial dilution. One hundred microliters containing approximately 4x10E7 coated beads
were then mixed with the resuspended cells. To this mixture 15 microliters of goat anti-
rabbit H&L IgG-FITC (Jackson Immunoresearch) diluted 1:100 in medium were added.
Twenty microliters of cell/beads/anti-rabbit H&L suspension were removed and
microliter droplets were dispensed on a one-well glass slide previously treated with
Sigmacote (Sigma) totaling 35 to 40 droplets per slide. An impermeable barrier of paraffin
oil (JT Baker) was used to submerge the droplets, and the slide was incubated for 90
minutes at 37°C in a 4% CO2 incubator in the dark.
Specific B cells that produce antibody can be identified by the fluorescent ring
around produced by the antibody secretion, recognition of the bead-associated biotinylated
antigen, and subsequent detection by the fluorescent-IgG detection reagent. Once a cell of
interest was identified it was recovered via a micromanipulator (Eppendorf). The single
cell synthesizing and exporting the antibody was transferred into a microcentrifuge tube,
frozen using dry ice and stored at -70°C.
Amplification and sequence determination of Antibody Sequences From Antigen-Specific
B Cells
Antibody sequences were recovered using a combined RT-PCR based method
from a single isolated B-cell. Primers containing restriction enzymes were designed to
anneal in conserved and constant regions of the target immunoglobulin genes (heavy and
light), such as rabbit immunoglobulin sequences, and a two-step nested PCR recovery was
used to amplify the antibody sequence. Amplicons from each well were analyzed for
recovery and size integrity. The resulting fragments are then digested with AluI to
fingerprint the sequence clonality. Identical sequences displayed a common fragmentation
pattern in their electrophoretic analysis. The original heavy and light chain amplicon
fragments were then digested using the restriction enzyme sites contained within the PCR
primers and cloned into an expression vector. Vector containing subcloned DNA
fragments were amplified and purified. Sequence of the subcloned heavy and light chains
were verified prior to expression.
Recombinant Production of Monoclonal Antibody of Desired Antigen Specificity and/or
Functional Properties
To determine antigen specificity and functional properties of recovered
antibodies from specific B-cells, vectors driving the expression of the desired paired heavy
and light chain sequences were transfected into HEK-293 cells.
Antigen-recognition of recombinant antibodies by ELISA
To characterize recombinant expressed antibodies for their ability to bind to
human-CGRPα, antibody-containing solutions were tested by ELISA. All incubations were
done at room temperature. Briefly, Immulon IV plagtes (Thermo Scientific), were coated
with a CGRPα containing solution (1ut/mL in PBS) for 2 hours. CGRPα-coated plates
were then washed three times in wash buffer (PBS, 0.05% Tween-20). The plates were
then blocked using a blocking solution (PBS, 0.5% fish skin gelatin, 0.05% Tween-20) for
approximately one hour. The blocking solution was then removed and the plates were then
incubated with a dilution series of the antibody being tested for approximately one hour.
At the end of this incubation, the plate was washed three times with wash buffer and
further incubated with a secondary antibody containing solution (Peroxidase conjugated
affinipure F(ab’)2 fragment goat anti-human IgG, Fc fragment specific (Jackson
Immunoresearch) for approximately 45 minutes and washed three times. At that point a
substrate solution (TMB peroxidase substrate, BioFx) and incubated for 3 to 5 minutes in
the dark. The reaction was stopped by addition of a HCl containing solution (0.5M) and
the plate was read at 450 nm in a plate-reader.
Results: Figures 15-18 demonstrate that anti-CGRP antibodies Ab1-Ab14 bind
to and recognize CGRPα.
Functional characterization of recombinant antibodies by modulation of CGRP driven
intracellular cAMP levels and cross reactivity to rats
To characterize recombinant expressed antibody for their ability to inhibit
CGRPα mediated increased cellular levels of cAMP assay, an electrochemiluminescence
assay-kit (Meso Scale Discovery, MSD) was used. Briefly, antibody preparations to be
tested were serially diluted in MSD assay buffer (Hepes, MgCl2, pH 7.3, 1mg/mL blocker
A,Meso Scale Discovery) in a 96 well round bottom polystyrene plate (Costar). To this
plate, human CGRPα was added (25ng/mL final concentration) diluted in MSD assay
buffer and incubated for one hour at 37°C. Appropriate controls were used as suggested by
the assay-kit manufacturer. Human neuroepithelioma cells (SK-N-MC, ATCC) were
detached using an EDTA solution (5mM) and washed using growth media (MEM, 10%
FBS, antibiotics) by centrifugation. The cell number was adjusted to 2 million cells per
mL in assay buffer, and IBMX (3-Isobutyl-1Methylxanthine, 50mM Sigma) was added to a
final concentration of 0.2mM right before loading cells onto cAMP assay plate. The
antibody human CGRPα solution was incubated for one hour after which 20 microliters of
solution containing cells were transferred to the cAMP assay plate. All tested samples
were run in duplicates with appropriate controls. Ten microliters of cells were added to the
wells and the plate was incubated for 30 minutes with shaking. While cells were being
incubated with the CGRP solution, the stop solution was prepared by making a 1:200
solution of TAG labeled cAMP (MSD) in lysis buffer (MSD). To stop the cells-CGRP
incubation, 20 microliters of stop solution was added to the cells and the plate was
incubated for one hour with shaking. The read buffer (MSD) was diluted four times with
water and 100 microliters were added to all wells on the plate. The plate was then read
using a Sector Imager 2400 (MSD) and the Prism software was used for data fit and IC50
determination.
To test for the ability of recombinant antibodies to antagonize human CGRPβ a
similar assay was performed with the substitution of the CGRP agonist (CGRPβ 10ng/mL
final concentration). Evaluation of the recombinant antibodies to recognize and inhibit rat
CGRP-mediated cAMP generation was conducted using rat CGRP (5ng/mL final
concentration) and the rat L6 cell line (ATCC).
Results: Figures 19-37 demonstrate that anti-CGRP antibodies Ab1-Ab14
inhibit CGRPα, CGRPβ, and rat CGRP mediated increased cellular levels of cAMP.
Example 2: Enzymatic Production of Fab Fragments
Papain digestions were conducted using immobilized papain (Thermo/Pierce) as
per manufacturer’s instructions. Briefly, purified antibodies were incubated in a
cystein/HCl-containing buffer with immobilized papain at 37°C with gentle rocking. The
digestion was monitored by taking an aliquot and analyzing using SDS-PAGE for cleavage
of the heavy chain. To stop the reaction, the immobilized papain was spun out and washed
using 50 mM Tris pH 7.5 and filtered. Undigested full length antibody and Fc fragments
were removed by using a MabSelectSure (GE) column.
Example 3 Yeast Cell Expression
Construction of Pichia pastoris expression vectors for heavy and light chain.
The humanized light and heavy chain fragments were commercially synthesized
and subcloned into a pGAP expression vector. The pGAP expression vector uses the GAP
promoter to drive expression of the immunoglobulin chain and the human serum albumin
(HSA) leader sequence for export. In addition, this vector contains common elements such
as a bacterial origin of replication, and a copy of the kanamycin resistance gene which
confers resistance to the antibiotic G418 in P. pastoris. G418 provides a means of
selection for strains that contain the desired expression vector integrated into their genome.
Transformation of expression vectors into haploid met1 and lys3 host strains of Pichia
pastoris
All methods used for transformation of haploid P. pastoris strains and
manipulation of the P. pastoris sexual cycle were done as described in Pichia Protocols
(Methods in Molecular Biology Higgings, DR, and Cregg, JM, Eds. 1998. Humana Press,
Totowa, NJ). Prior to transformation each vector was linearized within the GAP promoter
sequences to direct the integration of the vector into the GAP promoter locus of the P.
pastoris genome. Haploid strains were transfected using electroporation and successful
transformants were selected on YPDS (yeast extract, peptone dextrose with sorbitol) G418
agar plates. Copy numbers of heavy and light chain genes were determined for haploid
strains by Southern blot analysis. Haploid strains were then mated and selected for their
ability to grow in the absence of the amino acid markers (i.e., Lys and Met). Resulting
diploid clones were then subjected to a final Southern blot to confirm copy numbers of
heavy and light chain genes. A clone expressing the antibody of interest was selected using
biolayer interferometry Protein-A biosensors to monitor expression (Octet, ForteBio).
Example 4 Expression of Ab3, Ab6 and Ab14 in Pichia pastoris
Three Pichia strains for expression of full-length antibody were made. For all
the full length antibody expressing strains, haploids strains were created and subsequently
mated. One haploid strain expressed full-length light chain sequence and another haploid
strain expressed the full-length heavy chain sequence. Each diploid strain was used to
generate a research cell bank and used for expression in a bioreactor.
First an inoculum was expanded using the research cell bank using medium
comprised of the following nutrients (%w/v): yeast extract 3%, anhydrous dextrose 4%,
YNB 1.34%, Biotin 0.004% and 100 mM potassium phosphate. To generate the inoculum
for the fermenters, the cell bank was expanded for approximately 24 hours in a shaking
incubator at 30ºC and 300 rpm. A 10% inoculum was then added to Labfors 2.5L working
volume vessels containing 1 L sterile growth medium. The growth medium was comprised
of the following nutrients: potassium sulfate 18.2 g/L, ammonium phosphate monobasic
36.4 g/L, potassium phosphate dibasic 12.8 g/L, magnesium sulfate heptahydrate 3.72 g/L,
sodium citrate dihydrate 10 g/L, glycerol 40 g/L, yeast extract 30 g/L, PTM1 trace metals
4.35 mL/L, and antifoam 204 1.67 mL/L. The PTM1 trace metal solution was comprised
of the following components: cupric sulfate pentahydrate 6 g/L, sodium iodide 0.08 g/L,
manganese sulfate hydrate 3 g/L, sodium molybdate dihyrate 0.2 g/L, boric acid 0.02 g/L,
cobalt chloride 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2
g/L, and sulfuric acid 5 mL/L.
The bioreactor process control parameters were set as follows: Agitation 1000
rpm, airflow 1.35 standard liter per minute, temperature 28ºC and pH was controlled at six
using ammonium hydroxide. No oxygen supplementation was provided.
Fermentation cultures were grown for approximately 12 to 16 hours until the
initial glycerol was consumed as denoted by a dissolved oxygen spike. The cultures were
starved for approximately three hours after the dissolved oxygen spike. After this
starvation period, a bolus addition of ethanol was added to the reactor to reach 1% ethanol
(w/v). The fermentation cultures were allowed to equilibrate for 15 to 30 minutes. Feed
addition was initiated 30 minutes post-ethanol bolus and set at a constant rate of 1 mL/min
for 40 minutes, then the feed pump was controlled by an ethanol sensor keeping the
concentration of ethanol at 1% for the remainder of the run using an ethanol sensing probe
(Raven Biotech). The feed was comprised of the following components: yeast extract 50
g/L, dextrose 500 g/L, magnesium sulfate heptahydrate 3 g/L, and PTM1 trace metals 12
mL/L. For fermentation of the full length Ab6 and Ab14, sodium citrate dihydrate (0.5g/L)
was also added to the feed. The total fermentation time was approximately 90 hours.
Example 5 Methods of Humanizing Antibodies
Methods of humanizing antibodies have been described previously in issued
U.S. Patent No. 7935340, the disclosure of which is incorporated herein by reference in its
entirety. In some instances, a determination of whether additional rabbit framework
residues are required to maintain activity is necessary. In some instances the humanized
antibodies still requires some critical rabbit framework residues to be retained to minimize
loss of affinity or activity. In these cases, it is necessary to change single or multiple
framework amino acids from human germline sequences back to the original rabbit amino
acids in order to have desired activity. These changes are determined experimentally to
identify which rabbit residues are necessary to preserve affinity and activity. This is now
the end of the variable heavy and light chain humanized amino acid sequence.
Example 6 Inhibition of CGRP Binding to its Cellular Receptor
To characterize recombinantly expressed antibodies for their ability to inhibit
CGRP binding to its cellular receptor, a radioligand-binding assay was performed as
previously described [Elshourbagy et al, Endocrinology 139:1678 (1998); Zimmerman et
al, Peptides, 16:421 (1995)]. Membrane preparations of recombinant human CGRP
receptors, calcitonin receptor-like receptor and RAMP1 (Chemiscreen, Millipore) were
used. Antibody dilutions were preincubated with I radiolabeled human CGRPα
(0.03nM) for 30 minutes at room temperature. Non-specific binding was estimated in the
presence of 0.1μM human CGRPα. Membranes were filtered and washed. The filters
were then counted to determine I radiolabeled human CGRPα specifically bound.
Results: Figure 38 demonstrates that anti-CGRP antibodies Ab1-Ab13 inhibit
CGRP binding to its cellular receptor.
Example 7 Inhibition of Neurogenic Vasodilation by Anti-CGRP Antibodies in Rats
CGRP is a potent vasodilator (Nature 313: 54-56 (1985) and Br J. Clin.
Pharmacol. 26(6):691-5. (1988)). A pharmacodynamic assay to measure CGRP receptor
antagonist activity non-invasively was used to characterize anti-CGRP antibodies. The
model relied on changes in dermal blood flow measured using a laser Doppler imaging
following the topical application of a capsaicin solution. Capsaicin activates the transient
receptor potential vanilloid type 1 receptor (TRPV-1), producing neurogenic inflammation
and vasodilatation via the local release of vasoactive mediators including CGRP and
substance P (Br. J. Pharmacol. 110: 772-776 (1993)).
On the day prior to the vasodilatation assay, animals were dosed with the test
agent or control via IP (intraperitoneal). Following dosing, the animals were shaved and
depilated in the lower back region of their dorsal side, in an area approximately 2x6cm.
The animals were then returned to their cages overnight. On the day of test, approximately
24 hours post dosing, animals were anesthetized with isoflurane gas and placed on a
temperature controlled heating pad and fitted with a nose cone for continuous delivery of
isoflurane. A laser doppler imager was used for the observation of vasodilatation. A beam
of coherent red light generated by a 633 nm helium-neon laser was directed to the shaved
area, a rectangle (2x6 cm), and scanned at a medium resolution mode. A baseline Doppler
scan was obtained first and the location of O-ring placement predetermined by identifying
two similar low flux areas. Two rubber Orings (~1cm in diameter) were placed in the
selected regions and a baseline scan was performed. Immediately after completion of the
scan, 1mg of capsaicin in 5 μL of an ethanol:acetone solution (1:1) was applied within each
of the two O-rings Doppler scans were repeated at 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5,
, 27.5 and 30 minutes after the application of capsaicin. Percent change from baseline
mean Flux within each of the two O-rings, was plotted as the results of vasodilatation due
to capsaicin.
In order to test recombinantly expressed antibodies for their ability to inhibit
CGRP binding to its cellular receptor, a radioligand-binding assay was performed as
previously described.
Results: Figures 39 and 40 demonstrates that anti-CGRP antibodies Ab3 and
Ab6 reduced vasodilation in this model following capsaicin administration.
Example 8 Inhibition of Light Aversion or Photophobia by Systemic (IP) Injection of
Anti-CGRP Antibody in Transgenic Nestin/Ramp1 Mice
As discussed supra, one of the hallmarks of migraines is photophobia, or
increased sensitivity to light [Mulleners et al, Headache 41: 31-39 (2001); Recober et al, J.
Neuroscience 29:8798:8804 (2009)]. It is also known that migraineurs, but not non-
migraineurs, are sensitive to CGRP-induced headache [reviewed in Neurology 22:241-246
(2009)]. CGRP binds to a G protein coupled receptor called CLR (calcitonin like receptor)
that works concomitantly with the receptor activity-modifying protein 1 (RAMP1) in
mediating CGRP binding and signaling. In-vitro, the activity of CGRP is strongly
enhanced by overexpression of the RAMP1 subunit of the CGRP receptor [(J. Neurosci.
27:2693-2703 (2007)]. To study light aversion behavior in mice, a nestin/human-RAMP1
transgenic mouse model was developed [Recober et al, J. Neuroscience 29: 8798-8804
(2009); Russo et al, Mol. Cell. Pharmacol., 1:264-270 (2009)]. These mice when exposed
to CGRP present symptoms associated with migraines in particular light aversion (ibid).
This protocol is detailed below.
To test the ability of anti-CGRP antibodies to block CGRP-induced light
aversion or photophobia, mice are housed under standard conditions in groups of 2-5 per
cage with a 12 hour light cycle (lights on at 0500 CST)/0600 CDT and off at 1700
CST/1800 CDT) and access to water and food ad libitum. The mice used in the studies are
comprised in mice colonies of genotype nestin/hRAMP1 that contain two transgene alleles
Tg(Nes-cre)1Kln/J and Tg(RAMP1) alleles (B6;SJL-Tg(Nes-cre)1Kln Tg(RAMP1). Nes-
cre was introduced in these mice by an intercross involving mice obtained from The
Jackson Laboratory (stock 003771) on a B6 genetic background yielding mice.
The control mice used in the protocol are littermates that are either non-
transgenic, or single transgenic (not expressing hRAMP1) containing either transgene:
nestin-cre or Cx1-GFP-hRAMP1. The stock colony is maintained by backcrossing CX1-
GFP-hRAMP1 mice with non-transgenic littermates in the barrier facility. For behavior
studies, the colony is maintained by crossing CX1-GFP-hRAMP1 single transgenic with
nestin-cre mice in non-barrier facilities. All of these mice are cared for by animal care and
procedures approved by the University of Iowa Animal Care and Use Committee and
further are performed in accordance with the standard set by the National Institutes of
Health.
The materials and equipment used in this protocol include a light-dark box and
testing chambers comprising a plexiglass open field (27 x 27 x 20.3 cm) containing 16
beam infrared arrays (Med Associates Inc., St. Albans, VT). The light/dark box is divided
in two equally sized zones by a dark insert that is opaque to visible light. There is an
opening (5.2 x 6.8 cm) in the dark insert that allows the mouse to freely move between the
two zones. This testing chamber is placed inside a sound-attenuating cubicle (56 x 38 x 36
cm) with a fan for ventilation (Med Associates Inc.). There are six chambers for the overall
system that integrates with a computer containing software for recording and data
collection (Med Associates Inc.).
The software used to monitor results is Activity Monitor v 6.02 (Med Associates
Inc.). The software settings used for recording comprise: Resolution (ms): 50, Box Size: 3,
Resting Delay (ms): 500, Ambulatory Trigger: 3, Session Type: C, Session Time (min): 20,
Block Interval (sec): 300, and Compressed File: DEFAULT.ZIP.
In the protocol, the light source for each chamber is an LED panel, which was
installed to the ceiling of the sound-attenuating cubicle. The LED panel contains 36
collimated - 1 watt LED bulbs (5500k Daylight White) (LEDwholesalers, Burlingame,
CA). To control light intensity, each LED panel is connected to a dimmable LED driver
(LINEARdrive; eldoLED America Inc., San Jose, CA) leading to a potential range of light
intensity from ~300 to 27,000 lux. The standard light intensity is ~1000-1200 lux unless
otherwise stated. Alternatively, lower light intensities have been achieved by using layers
of wax paper to filter the light leading to an intensity of ~55 lux.
The injectors used are hand-made by inserting a stripped 30 gauge x ½” needle
into non-radiopaque polyethylene tubing (inner diameter .38 mm; outer diameter 1.09 mm).
Using the tubing described above, a stopper (~1cm in length) is placed over the needle
leaving approximately 2.5 mm of the bevel uncovered. These injectors are connected to a
µL Hamilton syringe.
The mice are injected with rat α-CGRP (Sigma) diluted in Dulbecco phosphate-
buffered saline (D-PBS) (Hyclone). The total dose delivery is 0.5 nmol. For example, 250
or 500 µg CGRP is diluted in 250 or 500 µL sterile PBS for a final concentration of 1
µg/µL. The CGRP is stored at-20°C and aliquots are freeze-thawed at most one time. The
PBS is stored at 4°C.
The mice are administered one of the anti-CGRP antibodies disclosed herein
(Ab3), vehicle or a control antibody, which are stored at 4°C prior to administration. In this
protocol prior to the administration of the CGRP i.e., approximately 24 hours prior to
testing, the mice are weighed and then receive an intraperitoneal (ip) injection of either:
vehicle, control antibody, or CGRP-binding antibody at a dosage of 30 mg/kg. The mice
are also screened to detect any abnormal physical conditions that could affect the assay
such as a missing eye, cataracts, or other abnormalities such as grooming, etc. The day
after antibody administration, mice are transported in cages from animal housing on a cart
and then the mice are placed in the behavior room for acclimation at least 1 hour prior to
any injection or testing. Any coverings required for transport are removed from the cages
and normal light conditions (standard overhead fluorescent lighting) are turned on during
acclimation and remain on for the remainder of the procedure. In addition, all equipment
that produces sound including anesthetic devices, light/dark chambers, and LED panels are
turned on during acclimation and remain until testing is complete. Typically there is
minimal human presence in the room during acclimation.
After acclimation each mouse is placed in an induction chamber and
administered 3.5% isoflurane. After the mouse is anesthetized, it is transferred to a nose
cone maintaining 3.5% isoflurane administration, so that it remains anesthetized during
injection. Thereafter drug administration is effected using the injector by direct injection
into the right lateral ventricle through the intact scalp aiming at 1 mm posterior to bregma
and 1 mm right from the midline.
Typically for consistency all the injections are performed by the same person
after a period of training yielding a success rate of >90% as demonstrated by injections of
dye into the ventricles. The drugs injected are either 2.0 µL vehicle (D-PBS) µL or 2.0 µg
CGRP in 2.0 µL vehicle (1 µg/µL) administered as a direct intracerebroventricular
injection into the right lateral ventricle of the brain through the intact scalp aiming at 1 mm
posterior to bregma and 1 mm right from the midline as described before [Recober et al, J.
Neuroscience 29: 8798-8804 (2009)] After all 2.0 µL is delivered, the needle remains in
place for 10 sec and then removed. The time of injection is then recorded.
After injection the mice are allowed to recover for 30 minutes prior to testing in
an empty, uncovered cage containing a paper towel for bedding. During recovery, the
following is recorded: diarrhea, excessive urination, bleeding post-injection, abnormal
behavior such as lack of movement, seizures, etc. After a 30 minute recovery testing is
effected. Each mouse is placed along the back wall (furthest from the opening between the
two zones) in the light zone approximately in the center. This triggers the recording to
begin. Up to six mice are tested at one time (one mouse per chamber). During testing the
shelf with the chamber is pushed back into the cabinet and the doors closed. The software
records mouse movement for 20 minutes. After the recording is completed, each mouse is
removed and placed back in home cage for transport back to animal housing.
Results
Using this protocol an anti-CGRP antibody developed by Alder
Biopharmaceuticals (Ab3) was tested to determine its potential suitability for treating
migraine, particularly chronic migraine in human subjects and more particularly for
treatment or prevention of CGRP-associated photophobia. The results of these studies are
shown in Figure 41 and Figure 42. Figure 41 contains data that compares the effect of ICV
injection of CGRP in hRAMP1 tg mice and control littermate mice. The data reveals that
the CGRP administration results in decreased time in light behavior in the hRAMP1 tg
mice relative to their control littermates.
Figure 42 contains data which compares the effect of systemic (IP) injection of
anti-CGRP antibody (Ab3) in vehicle, vehicle alone, and control antibody in vehicle in
nestin/RAMP1 mice which are administered these moieties intraperitoneally at 30 mg/kg
about 24 hours prior to administration of CGRP. The data in the left side of the graph is the
total time in light (seconds) for the first 10 minutes, and the data on the right side of the
graph is the total time in light (seconds) for the first 20 minutes measured after CGRP
injection (administered via ICV injection) and the recovery period. Light intensity in light
zone was approximately 1x10 lx. The data reveal that the mice who received the anti-
CGRP antibody Ab3 according to the present disclosure had a statistically significant
increase in the amount of time spent in the light relative to the mice who received the
controls.
These results indicate that Ab3 inhibits CGRP-associated photophobia or light
aversion and should be well suited for treating migraine or other disorders that involve
photophobia, especially CGRP related photophobia. Based on this, it is anticipated that
other anti-CGRP antibodies including others disclosed herein may behave similarly. These
results further indicate that the subject light aversion behavior assay may be used to assess
the potential therapeutic efficacy (ability to antagonize effects of CGRP in vivo) of
candidate of anti-CGRP antibodies and antibody fragments. This was unanticipated as it
was unforseeable that a large polypeptide such as an anti-CGRP antibody would go
through the blood-brain barrier and inhibit photophobia or light aversion.
The results reveal that the excess CGRP that induces light aversive behavior in
mice is reduced by the systemic administration of anti-CGRP antibody suggesting that the
antibody is able to bind a sufficient amount of the circulating CGRP to counteract the light
aversive behavior. These results suggest that the anti-CGRP antibody may be crossing the
blood-brain barrier and thereby inhibiting the neurological effects of CGRP, in particular
migraine associated photophobia and pain.
This is the first demonstration that the subject animal light aversive behavior
assay may be used to assess therapeutic efficacy of a polypeptide such as an anti-CGRP
antibody or anti-CGRP antibody fragment. In addition these results suggest that this animal
model potentially may be useful in determining effective dosages of a candidate anti-CGRP
antibody or antibody fragment, effective modes of administration, as well as a suitable
dosage regimen.
The term “comprising” as used in this specification and claims means
“consisting at least in part of”. When interpreting statements in this specification, and
claims which include the term “comprising”, it is to be understood that other features that
are additional to the features prefaced by this term in each statement or claim may also be
present. Related terms such as “comprise” and “comprised” are to be interpreted in similar
manner.
In this specification where reference has been made to patent specifications,
other external documents, or other sources of information, this is generally for the purpose
of providing a context for discussing the features of the invention. Unless specifically
stated otherwise, reference to such external documents is not to be construed as an
admission that such documents, or such sources of information, in any jurisdiction, are
prior art, or form part of the common general knowledge in the art.
In the description in this specification reference may be made to subject
matter that is not within the scope of the claims of the current application. That subject
matter should be readily identifiable by a person skilled in the art and may assist in putting
into practice the invention as defined in the claims of this application.
Claims (64)
1). A method of assessing the potential in vivo efficacy of a candidate anti-CGRP antibody or anti-CGRP antibody fragment, anti-CGRP receptor antibody or anti-CGRP receptor antibody fragment, or polypeptide comprising a fragment of CGRP or CGRP receptor for treating photophobia or light aversion, comprising determining whether the candidate anti- CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising a fragment of CGRP or CGRP receptor inhibits light aversive behavior in a nestin/Ramp1 rodent administered CGRP compared to a rodent administered CGRP in the absence of the candidate anti-CGRP antibody or antibody fragment, anti- CGRP receptor antibody or antibody fragment, or polypeptide comprising a fragment of CGRP or CGRP receptor.
2). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising the fragment of CGRP or CGRP receptor may be used to treat photophobia or light aversion associated with a neurological condition characterized by increased CGRP levels.
3). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising the fragment of CGRP or CGRP receptor may be used to treat photophobia or light aversion associated with one or more of the following: chronic migraine, hemiplegic migraines, cluster headaches, migrainous neuralgia, chronic headaches, tension headaches, general headaches, hot flashes, chronic paroxysmal hemicrania, secondary headaches due to an underlying structural problem in the head or neck, cranial neuralgia, sinus headaches, allergy-induced headaches, menopausal migraine, menstrual migraine or headache, headache-free migraine, abdominal migraine, and other migraine conditions. 18 1
4). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or the polypeptide comprising the fragment of CGRP or CGRP receptor may be used to treat photophobia or light aversion associated with an ocular disorder selected from one or more of the following: achromatopsia, aniridia, photophobia caused by an anticholinergic drug, aphakia, buphthalmos, cone dystrophy, congenital abnormalities of the eye, viral conjunctivitis, corneal abrasion, corneal dystrophy, corneal ulcer, disruption of the corneal epithelium, ectopia lentis, endophthalmitis, eye trauma caused by disease, eye trauma caused by injury, eye trauma caused by infection, episcleritis, glaucoma, keratoconus, optic nerve hypoplasia, hydrophthalmos, congenital glaucoma iritis, optic neuritis, pigment dispersion syndrome, pupillary dilation (naturally or chemically induced), retinal detachment, scarring of the cornea, sclera, and uveitis.
5). The method of claim 4, wherein the eye trauma caused by infection is chalazion.
6). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising the fragment of CGRP or CGRP receptor may be used to treat photophobia or light aversion associated with one or more of the following: autism spectrum disorders, Chiari malformation, Dyslexia, encephalitis , meningitis, subarachnoid haemorrhage, tumor of the posterior cranial fossa, ankylosing spondylitis, albinism, ariboflavinosis, benzodiazepines (long term use of or withdrawal from benzodiazepines), chemotherapy, chikungunya, cystinosis, Ehlers-Danlos syndrome, hangover, influenza, infectious mononucleosis, magnesium deficiency, mercury poisoning, migraine, rabies, and Tyrosinemia type II.
7). The method of claim 6, wherein the encephalitis is myalgic encephalomyelitis.
8). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising the fragment of CGRP or CGRP receptor, may be used to treat photophobia or light aversion associated with one or more of the following: migraine (with 18 2 or without aura), iritis, uveitis, meningitis, depression, bipolar disorder, cluster headache or anther trigeminal autonomic cephalalgia or blepharospasm, depression, agoraphobia and bipolar disorder.
9). The method of claim 1, which is used to assess whether the candidate anti-CGRP antibody or antibody fragment, anti-CGRP receptor antibody or antibody fragment, or polypeptide comprising the fragment of CGRP or CGRP receptor may be used to treat photophobia or light aversion associated with migraine headaches.
10). The method of any one of claims 1 to 9, wherein the candidate is an anti-CGRP antibody or antibody fragment.
11). The method of claim 10, wherein the candidate is an anti-CGRP antibody.
12). The method of claim 10, wherein the candidate is an anti-CGRP antibody fragment.
13). The method of any one of claims 1 to 9, wherein the candidate is an anti-CGRP receptor antibody.
14). The method of any one of claims 1 to 9, wherein the candidate is an anti-CGRP receptor antibody fragment.
15). The method of any one of claims 1 to 9, wherein the candidate is a polypeptide comprising a fragment of CGRP.
16). The method of any one of claims 1 to 9, wherein the candidate is a polypeptide comprising a fragment of CGRP receptor.
17). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment is an anti-human CGRP antibody or antibody fragment which specifically binds to the same or overlapping linear or conformational epitope(s) and/or competes for binding to the same or overlapping linear or conformational epitope(s) on an intact CGRP polypeptide or fragment thereof as an anti-human CGRP antibody comprising a variable light (V ) chain selected from SEQ ID NOs. 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 18 3 121 and 131, and a variable light (V ) chain selected from SEQ ID NOs. 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 and 133 or an anti-human CGRP antibody or antibody fragment comprising a V chain comprising the same complementarity determining regions (CDRs) as any of said V chains or an anti-human CGRP antibody or antibody fragment comprising a V chain comprising the same CDRs as any of said V chains.
18). The method of claim 17, wherein the candidate anti-CGRP antibody or antibody fragment specifically binds to the same or overlapping linear or conformational epitope(s) and/or competes for binding to the same or overlapping linear or conformational epitope(s) on an intact human CGRP polypeptide or a fragment thereof as an anti-human CGRP antibody comprising a V chain selected from SEQ ID NOs. 11, 21, 41, 51, 121 and 131, and a V chain selected from SEQ ID NOs. 13, 23, 43, 53, 123 and 133 or an anti-human CGRP antibody or antibody fragment comprising a V chain comprising the same complementarity determining regions (CDRs) as any of said V chains or an anti-human CGRP antibody or antibody fragment comprising a V chain comprising the same CDRs as any of said V chains.
19). The method of claim 18, wherein the candidate anti-CGRP antibody or antibody fragment specifically binds to the same or overlapping linear or conformational epitope(s) and/or competes for binding to the same or overlapping linear or conformational epitope(s) on an intact human CGRP polypeptide or a fragment thereof as an anti-human CGRP antibody comprising a V chain selected from SEQ ID NOs. 11, 21, 41 or 51, and a V chain selected from SEQ ID NOs. 13, 23, 43 and 53 or an anti-human CGRP antibody or antibody fragment comprising a VL chain comprising the same CDRs as any of said VL chains or an anti-human CGRP antibody or antibody fragment comprising a V chain comprising the same CDRs as any of said V chains.
20). The method of any one of claims 1 to 10, 12, 14 and 17 to 19, wherein the candidate is an antibody fragment.
21). The method of claim 20, wherein the antibody fragment is selected from a Fab fragment, a Fab’ fragment, and a F(ab’)2 fragment. 18 4
22). The method of claim 21, wherein said fragment is a Fab fragment.
23). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment comprises a variable light chain comprising the CDR 1 sequence of SEQ ID NO:25, the CDR 2 sequence of SEQ ID NO:26, and the CDR 3 sequence of SEQ ID NO:27, and/or a variable heavy chain comprising the CDR 1 sequence of SEQ ID NO:28, the CDR 2 sequence of SEQ ID NO:29, and the CDR 3 sequence of SEQ ID NO:30.
24). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment comprises a variable light chain comprising the CDR 1 sequence of SEQ ID NO:55, the CDR 2 sequence of SEQ ID NO:56, and the CDR 3 sequence of SEQ ID NO:57, and/or a variable heavy chain comprising the CDR 1 sequence of SEQ ID NO:58, the CDR 2 sequence of SEQ ID NO:59, and the CDR 3 sequence of SEQ ID NO:60.
25). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment comprises at least 2 complementarity determining regions (CDRs) in each of the variable light and the variable heavy regions which are identical to those contained in an anti-human CGRP antibody comprising a V chain selected from SEQ ID NOs. 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131, and a V chain selected from SEQ ID NOs. 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 and 133.
26). The method of claim 25, wherein the candidate anti-CGRP antibody or antibody fragment comprises at least 2 complementarity determining regions (CDRs) in each of the variable light and the variable heavy regions which are identical to those contained in an anti-human CGRP antibody comprising a V chain selected from SEQ ID NOs. 11, 21, 41, 51, 121 and 131, and a V chain selected from SEQ ID NOs. 13, 23, 43, 53, 123 and 133.
27). The method of any one of claims 1 to 10, 12, 14 and 17 to 26, wherein the candidate anti-CGRP antibody or antibody fragment is entirely non-glycosylated or lacks N- glycosylation or contains only mannose residues. 18 5
28). The method of any one of claims 1 to 10, 12, 14 and 17 to 27, wherein the candidate anti-CGRP antibody or antibody fragment contains an Fc region that has been modified to alter effector function, half-life, proteolysis, and/or glycosylation.
29). The method of any one of claims 1 to 10, 12, 14 and 17 to 28, wherein the candidate anti-CGRP antibody or antibody fragment is a humanized, single chain or chimeric antibody.
30). The method of any one of claims 1 to 10, 12, 14 and 17 to 19, wherein the candidate anti-CGRP antibody or antibody fragment specifically binds to CGRP expressing human cells and/or to circulating soluble CGRP molecules in vivo.
31). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment comprises a V polypeptide sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, and 133, or a variant thereof which possesses at least 90% sequence identity therewith; and further comprising a V polypeptide sequence selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 or 131, or a variant thereof which possesses at least 90% sequence identity therewith, wherein one or more of the framework (FR) or CDR residues in said V or V polypeptide has been substituted with another amino acid residue resulting in an anti-CGRP antibody or antibody fragment that specifically binds CGRP.
32). The method of claim 31, wherein one or more of said FR residues are substituted with an amino acid present at the corresponding site in a parent rabbit anti-CGRP antibody from which the complementarity determining regions (CDRs) contained in said V or V polypeptides have been derived or by a conservative amino acid substitution.
33). The method of claim 32, wherein said candidate anti-CGRP antibody or antibody fragment is humanized.
34). The method of claim 32, wherein said candidate anti-CGRP antibody or antibody fragment is chimeric. 18 6
35). The method of claim 32, wherein said candidate anti-CGRP antibody comprises a single chain antibody.
36). The method of claim 34, wherein said chimeric antibody comprises a human F .
37). The method of claim 36, wherein said human F is derived from IgG1, IgG2, IgG3, or IgG4.
38). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment inhibits the association of CGRP with CGRP-R and/or multimers thereof, one or more additional proteins in a CGRP-CGRP-R complex, and/or antagonizes the biological effects thereof.
39). The method of claim 21, wherein the candidate anti-CGRP antibody or antibody fragment comprises a polypeptide sequence having at least 90% or greater homology to two of the polypeptide sequences recited therein.
40). The method of claim 21, wherein the candidate anti-CGRP antibody or antibody fragment comprises a polypeptide sequence having at least 95% or greater homology to any one of the polypeptide sequences recited therein.
41). The method of claim 21, wherein the candidate anti-CGRP antibody or antibody -4 -1 -5 -1 fragment binds to CGRP with an off-rate (K ) of less than or equal to 10 S , 5x10 S , -5 -1 -6 -1 -6 -1 -7 -1 -7 -1 10 S , 5x10 S , 10 S , 5x10 S , or 10 S .
42). The method of claim 21, wherein the candidate anti-CGRP antibody or antibody fragment inhibits the production of CGRP with CGRP-R and/or multimers thereof, and/or the production of CGRP with CGRP-R and one or more additional proteins in a complex.
43). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment binds to the same or overlapping CGRP epitope as an anti-human CGRP antibody comprising a V chain selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131, and a V chain selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, and 133 or an anti-human CGRP antibody or antibody 18 7 fragment comprising a V chain comprising the same CDRs as any of said V chains and a V chain comprising the same CDRs as any of said V chains.
44). The method of claim 10, wherein the candidate anti-CGRP antibody or fragment comprises one or more of the CDRs contained in the V polypeptide sequences selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, and 133 and/or one or more of the CDRs contained in the V polypeptide sequences selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131.
45). The method of claim 10, wherein the candidate anti-CGRP antibody or fragment comprises at least 2 of the CDRs contained in the V chain selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, and 133 and/or at least 2 of the CDRs contained in the V chain selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131.
46). The method of claim 10, wherein the candidate anti-CGRP antibody or fragment comprises all 3 of the CDRs contained in the V chain selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, and 133 and/or all 3 of the CDRs contained in the V polypeptide sequences selected from: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 and 131.
47). The method of claim 10, wherein the candidate anti-CGRP antibody or antibody fragment comprises variable light chain CDR1, CDR2, and CDR3 polypeptide sequences and variable heavy chain CDR1, CDR2, and CDR3 polypeptide sequences selected from the following: V V V V V V L L L H H H CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 A Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 5 No: 6 No: 7 No: 8 No: 9 No: 10 B Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 15 No: 16 No: 17 No: 18 No: 19 No: 20 C Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 25 No: 26 No: 27 No: 28 No: 29 No: 30 D Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID 18 8 No: 35 No: 36 No: 37 No: 38 No: 39 No: 40 E Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 45 No: 46 No: 47 No: 48 No: 49 No: 50 F Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 55 No: 56 No: 57 No: 58 No: 59 No: 60 G Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 65 No: 66 No: 67 No: 68 No: 69 No: 70 H Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 75 No: 76 No: 77 No: 78 No: 79 No: 80 I Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 85 No: 86 No: 87 No: 88 No: 89 No: 90 J Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 95 No: 96 No: 97 No: 98 No: 99 No: 100 K Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 105 No: 106 No: 107 No: 108 No: 109 No: 110 L Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 115 No: 116 No: 117 No: 118 No: 119 No: 120 M Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 125 No: 126 No: 127 No: 128 No: 129 No: 130 N Seq ID Seq ID Seq ID Seq ID Seq ID Seq ID No: 135 No: 136 No:137 No: 138 No: 139 No: 140
48). The method of claim 47, wherein said candidate anti-CGRP antibody fragment is an scFv, Fab, Fab', or F(ab')2 fragment.
49). The method of claim 48, wherein said candidate anti-CGRP antibody fragment is a Fab fragment.
50). The method of claim 47, wherein said candidate anti-CGRP antibody or fragment comprises a variable light chain polypeptide sequence and a variable heavy chain polypeptide sequence selected from the following: Variable Light Variable Heavy Chain Chain A Seq ID No: 1 Seq ID No: 3 B Seq ID No: 11 Seq ID No: 13 C Seq ID No: 21 Seq ID No: 23 D Seq ID No: 31 Seq ID No: 33 E Seq ID No: 41 Seq ID No: 43 F Seq ID No: 51 Seq ID No: 53 18 9 G Seq ID No: 61 Seq ID No: 63 H Seq ID No: 71 Seq ID No: 73 I Seq ID No: 81 Seq ID No: 83 J Seq ID No: 91 Seq ID No: 93 K Seq ID No: 101 Seq ID No: 103 L Seq ID No: 111 Seq ID No: 113 M Seq ID No: 121 Seq ID No: 123 N Seq ID No: 131 Seq ID No: 133
51). The method of claim 47, wherein said candidate anti-CGRP antibody or fragment comprises a light chain polypeptide sequence and a heavy chain polypeptide sequence selected from the following: Light Chain Heavy Chain Ab1 Seq ID No: 2 Seq ID No: 4 Ab2 Seq ID No: 12 Seq ID No: 14 Ab3 Seq ID No: 22 Seq ID No: 24 Ab4 Seq ID No: 32 Seq ID No: 34 Ab5 Seq ID No: 42 Seq ID No: 44 Ab6 Seq ID No: 52 Seq ID No: 54 Ab7 Seq ID No: 62 Seq ID No: 64 Ab8 Seq ID No: 72 Seq ID No: 74 Ab9 Seq ID No: 82 Seq ID No: 84 Ab10 Seq ID No: 92 Seq ID No: 94 Ab11 Seq ID No: 102 Seq ID No: 104 Ab12 Seq ID No: 112 Seq ID No: 114 Ab13 Seq ID No: 122 Seq ID No: 124 Ab14 Seq ID No: 132 Seq ID No: 134
52). The method of 47, wherein said candidate anti-CGRP antibody or fragment comprises a variable light chain having an amino acid sequence at least 90% identical to one of the variable light chain polypeptide sequences of SEQ ID NOS: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, or 131, and a variable heavy chain having an amino acid sequence at least 90% identical to one of the variable heavy chain polypeptide sequences of SEQ ID NOS: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 or 133. 19 0
53). The method of claim 47, wherein said candidate anti-CGRP antibody or fragment comprises a variable light chain having an amino acid sequence at least 95% identical to one of the variable light chain polypeptide sequences of SEQ ID NOS: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, and 131, and a variable heavy chain having an amino acid sequence at least 95% identical to one of the variable heavy chain polypeptide sequences of SEQ ID NOS: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 and 133, .
54). The method of claim 47, wherein said candidate anti-CGRP antibody or fragment is chimeric or humanized.
55). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment is entirely non-glycosylated or lacks N-glycosylation or comprises only mannose residues.
56). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment comprises a human constant domain.
57). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment is an IgG1, IgG2, IgG3 or IgG4 antibody.
58). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment contains an Fc region that has been modified to alter at least one of effector function, half-life, proteolysis, and/or glycosylation.
59). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment has an Fc region that contains a mutation that alters or eliminates all glycosylation or reduces or eliminates N-glycosylation.
60). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment is directly or indirectly attached to a detectable label or therapeutic agent.
61). The method of claim 47, wherein said candidate anti-CGRP antibody or antibody fragment further comprises an effector moiety. 19 1
62). The method of claim 61, wherein said effector moiety is a detectable moiety or a functional moiety.
63). The method of claim 62, wherein said detectable moiety is a fluorescent dye, an enzyme, a substrate, a bioluminescent material, a radioactive material, or a chemiluminescent material.
64). The method of claim 62, wherein said functional moiety is streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, or a radioactive material.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ732970A NZ732970A (en) | 2011-05-20 | 2012-05-21 | Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US201161488660P | 2011-05-20 | 2011-05-20 | |
US61/488,660 | 2011-05-20 | ||
US201161496860P | 2011-06-14 | 2011-06-14 | |
US61/496,860 | 2011-06-14 | ||
NZ618638A NZ618638B2 (en) | 2011-05-20 | 2012-05-21 | Use of anti-cgrp antibodies and antibody fragments to prevent or inhibit photophobia or light aversion in subjects in need thereof, especially migraine sufferers |
Publications (2)
Publication Number | Publication Date |
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NZ717700A NZ717700A (en) | 2018-11-30 |
NZ717700B2 true NZ717700B2 (en) | 2019-03-01 |
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