Note: Descriptions are shown in the official language in which they were submitted.
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Anti-Adrenomedullin (ADM) antibody or anti-ADM antibody fragment or anti-ADM
non-
Ig scaffold for regulating the fluid balance in a patient having a chronic or
acute disease
Field of the invention
Subject matter of the present invention is an anti-Adrenomedullin (ADM)
antibody or an anti-
adrenomedullin antibody fragment or anti-adrenomedullin non-Ig scaffold for
regulating the
fluid balance in a patient having a chronic or acute disease or acute
condition.
Subject matter of the present invention is a method for regulating the fluid
balance in a patient
having a chronic or acute disease or acute condition.
Background
The peptide adrenomedullin (ADM) was described for the first time in 1993
(Kitamura, K., et
al., "Adrenornedullin: A Novel Hypotensive Peptide Isolated From Human
Pheochromocytoma",
Biochemical and Biophysical Research Communications, Vol. 192 (2), pp. 553-560
(1993)) as a
novel hypotensive peptide comprising 52 amino acids, which had been isolated
from a human
pheochromocytome; SEQ ID No.: 21. In the same year, cDNA coding for a
precursor peptide
comprising 185 amino acids and the complete amino acid sequence of this
precursor peptide
were also described. The precursor peptide, which comprises, inter alia, a
signal sequence of 21
amino acids at the N-teiminus, is referred to as "preproadrenomedullin" (pre-
proADM). In the
present description, all amino acid positions specified usually relate to the
pre-proADM which
comprises the 185 amino acids. The peptide adrenomedullin (ADM) is a peptide
which
comprises 52 amino acids (SEQ ID NO: 21) and which comprises the amino acids
95 to 146 of
pre-proADM, from which it is formed by proteolytic cleavage. To date,
substantially only a few
fragments of the peptide fragments formed in the cleavage of the pre-proADM
have been more
exactly characterized, in particular the physiologically active peptides
adrenomedullin (ADM)
and "PAMP", a peptide comprising 20 amino acids (22-41) which follows the 21
amino acids of
the signal peptide in pre-proADM. The discovery and characterization of ADM in
1993 triggered
intensive research activity, the results of which have been summarized in
various review articles,
in the context of the present description, reference being made in particular
to the articles to be
found in an issue of "Peptides" devoted to ADM in particular (Editorial,
Takahashi, K.,
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"Adrenotnedullin: from a pheochromocytoma to the eyes", Peptides, Vol. 22, p.
1691 (2001))
and (Eto, T., "A review of the biological properties and clinical implications
of adrenomedullin
and proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and
vasodilating peptides",
Peptides, Vol. 22, pp. 1693-1711(200!)). A further review is (Hinson, et al.,
"Adrenomedullin, a
Multifunctional Regulatory Peptide", Endocrine Reviews, Vol. 21(2), pp. 138-
167 (2000)). In
the scientific investigations to date, it has been found, inter alia, that ADM
may be regarded as a
polyfunctional regulatory peptide. It is released into the circulation in an
inactive form extended
by glycine (Kitamura, K., et aL, "The intermediate form of glycine-extended
adrenomedullin is
the major circulating molecular foriii in human plasma", Biochem. Biophys,
Res. Commun., Vol.
244(2), pp. 551-555 (1998). Abstract Only). There is also a binding protein
(Pio, R., et al.,
"Complement Factor H is a Serum-binding Protein for adrenomedullin, and the
Resulting
Complex Modulates the Bioactivities of Both Partners", The Journal of
Biological Chemistry,
Vol. 276(15), pp. 12292-12300 (2001)) which is specific for ADM and probably
likewise
modulates the effect of ADM. Those physiological effects of ADM as well as of
PAMP which
are of primary importance in the investigations to date were the effects
influencing blood
pressure.
Hence, ADM is an effective vasodilator, and thus it is possible to associate
the hypotensive
effect with the particular peptide segments in the C-terminal part of ADM. It
has furthermore
been found that the above-mentioned further physiologically active peptide
PAMP formed from
pre-proADM likewise exhibits a hypotensive effect, even if it appears to have
an action
mechanism differing from that of ADM (cf in addition to the abovementioned
review articles
(Eto, T., "A review of the biological properties and clinical implications of
adrenomedullin and
proadrenomedullin N-terminal 20 peptide (PAMP), hypotensive and vasodilating
peptides",
Peptides, Vol. 22, pp. 1693-1711 (2001)) and (Hinson, et aL, "Adrenornedullin,
a
Multifunctional Regulatory Peptide", Endocrine Reviews, Vol. 21(2), pp. 138-
167 (2000)) also
(Kuwasako, K., et al., "Purification and characterization of PAMP-12 (PAMP-20)
in porcine
adrenal medulla as a major endogenous biologically active peptide", FEBS Lett,
Vol. 414(1), pp.
105-110 (1997). Abstract only), (Kuwasaki, K., et al., "Increased plasma
proadrenomedullin N-
terminal 20 peptide in patients with essential hypertension", Ann. Clin.
Biochem., Vol. 36 (Pt.
5), pp. 622-628 (1999). Abstract only) or (Tsuruda, T., et aL, "Secretion of
proadrenomedullin
N-termina120 peptide from cultured neonatal rat cardiac cells", Life Sci.,
Vol. 69(2), pp. 239-245
(2001). Abstract only) and EP-A2 0 622 458). It has furthermore been found
that the
concentrations of ADM which can be measured in the circulation and other
biological liquids,
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are in a number of pathological states, significantly above the concentrations
to be found in
healthy control persons. Thus, the ADM level in patients with congestive heart
failure,
myocardial infarction, kidney diseases, hypertensive disorders, Diabetes
mellitus, in the acute
phase of shock and in sepsis and septic shock are significantly increased,
although to different
extents. The PAMP concentrations are also increased in some of said
pathological states, but the
plasma levels are reduced relative to ADM ((Eto, T., "A review of the
biological properties and
clinical implications of adrenomedullin and proadrenomedullin N-terminal 20
peptide (PAMP),
hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693-
1711(2001)); page 1702). It
is furthermore known that unusually high concentrations of ADM are to be
observed in sepsis,
.10 and the highest concentrations in septic shock (cf. (Eto, T., "A review
of the biological properties
and clinical implications of adrenomedullin and proadrenornedullin N-terminal
20 peptide
(PAMP), hypotensive and vasodilating peptides", Peptides, Vol. 22, pp. 1693-
1711(2001)) and
(Hirata, et al., "Increased Circulating Adrenomedullin, a Novel Vasodilatory
Peptide, in Sepsis",
Journal of Clinical Endocrinology and Metabolism, Vol. 81(4), pp. 1449-1453
(1996)), (Ehlenz,
K., et al., "High levels of circulating adrenomedullin in severe illness:
Correlation with C-
reactive protein and evidence against the adrenal medulla as site of origin",
Exp Clin Endocrinol
Diabetes, Vol. 105, pp. 156-162 (1997)), (Tomoda, Y., et at., "Regulation of
adrenomedullin
secretion from cultured cells", Peptides, Vol. 22, pp. 1783-1794 (2001)),
(Ueda, S., et at.,
"Increased Plasma Levels of Adrenomedullin in Patients with Systemic
Inflammatory Response
Syndrome", Am. T. Respir. Crit. Care Med., Vol. 160, pp. 132-136 (1999)) and
(Wang, P.,
"Adrenomedullin and cardiovascular responses in sepsis", Peptides, Vol. 22,
pp. 1835-1840
(2001))).
Known in the art is further a method for identifying adrenomedullin
irnmunoreactivity in
biological liquids for diagnostic purposes and, in particular within the scope
of sepsis diagnosis,
cardiac diagnosis and cancer diagnosis. According to the invention, the
midregional partial
peptide of the proadrenomedullin, which contains amino acids (45-92) of the
entire
preproadrenomedullin, is measured, in particular, with an immunoassay which
works with at
least one labeled antibody that specifically recognizes a sequence of the mid-
proADM
(W02004/090546).
WO-Al 2004/097423 describes the use of an antibody against adrenomedullin for
diagnosis,
prognosis, and treatment of cardiovascular disorders. Treatment of diseases by
blocking the
ADM receptor are also described in the art, (e.g. WO-Al 2006/027147,
PCT/EP2005/012844)
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said diseases may be sepsis, septic shock, cardiovascular diseases,
infections, dermatological
diseases, endocrinological diseases, metabolic diseases, gastroenterological
diseases, cancer,
inflammation, hematological diseases, respiratory diseases, muscle skeleton
diseases,
neurological diseases, urological diseases.
It is reported for the early phase of sepsis that ADM improves heart function
and the blood
supply in liver, spleen, kidney and small intestine. ADM-neutralizing
antibodies neutralize the
before mentioned effects during the early phase of sepsis (Wang, P.,
"Adrenomedullin and
cardiovascular responses in sepsis", Peptides, Vol. 22, pp. 1835-1840 (2001).
In the later phase of sepsis, the hypodynamical phase of sepsis, ADM
constitutes a risk factor
that is strongly associated with the mortality of patients in septic shock.
(Schiitz et al.,
"Circulating Precursor levels of endothelin-1 and adrenomedullin, two
endothelium-derived,
counteracting substances, in sepsis", Endothelium, 14:345-351, (2007)).
Methods for the
diagnosis and treatment of critically ill patients, e.g. in the very late
phases of sepsis, and the use
of endothelin and endothelin agonists with vasoconstrictor activity for the
preparation of
medicaments for the treatment of critically ill patients have been described
in WO-Al
2007/062676. It is further described in WO-Al 2007/062676 to use, in place of
endothelin and/or
endothelin agonists, or in combination therewith, adrenomedullin antagonists,
i.e. molecules
which prevent or attenuate the vasodilating action of adrenomedulin, e.g. by
blocking its relevant
receptors, or substances preventing the binding of adrenomedullin to its
receptor (e.g. specific
binders as e.g. antibodies binding to adrenomedullin and blocking its receptor
bindings sites;
"immunological neutralization"). Such use, or combined use, including a
subsequent or
preceding separate use, has been described in certain cases to be desirable
for example to
improve the therapeutic success, or to avoid undesirable physiological stress
or side effects.
Thus, it is reported that neutralizing ADM antibodies may be used for the
treatment of sepsis in
the late stage of sepsis.
Administration of ADM in combination with ADM-binding-Protein-1 is described
for treatment
of sepsis and septic shock in the art. It is assumed that treatment of septic
animals with ADM and
ADM-binding-Protein-1 prevents transition to the late phase of sepsis. It has
to be noted that in a
living organism ADM binding protein (complement factor H) is present in the
circulation of said
organism in high concentrations (Pio et al.: Identification, characterization,
and physiological
actions of factor H as an Adrenoniedullin binding Protein present in Human
Plasma; Microscopy
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Res. and Technique, 55:23-27 (2002) and Martinez et al.; Mapping of the
Adrenomedullin-
Binding domains in Human Complement factor H; Hypertens Res Vol. 26, Suppl
(2003), S56-
59).
5 In accordance with the invention the ADM-binding-Protein-1 may be also
referred to as ADM-
binding-Protein-I (complement factor H).
Patients having a chronic or acute disease or acute condition, especially
patients at the ICU
(Intensive Care Unit), may suffer from fluid imbalance. This may cause severe
adverse events
such as kidney failure and mortality.
It was the subject of the present invention to provide a medicament for
regulating the fluid
balance and/or improving the fluid balance of such patients.
The expression "regulating fluid balance" with the context of the instant
invention is directed to
any correction of a manifested ¨ imbalance ¨ of a patient's fluid balance due
to an underlying
chronic or acute disease or acute condition. Said correction is in favour of
re-establishing
nomotension in said patients. The person skilled in the art is fully aware
that blood pressure in
general, as well as hyper- and hypotension is closely related to the fluid
balance of a patient.
Fluid balance is the balance of the input and the output of fluids in the body
to allow metabolic
processes to function. Dehydration is defined as a 1% or greater loss of body
mass as a result of
fluid loss. The three elements for assessing fluid balance and hydration
status are: clinical
assessment, body weight and urine output; review fluid balance charts and
review of blood
chemistry. All this is very well known to a man skilled in the art (Alison
Shepherd, Nursing
Tomes 19.07.11/Vol 107 No 28, pages 12 to 16).
Thus, in one embodiment a person in need of regulating the fluid balance
and/or improving the
fluid balance of such patients is a person that has a 1% or greater loss of
body mass as a result of
fluid loss. The fluid balance may be assessed according to Scales and
Pilsworth (2008) Nursing
Standard 22:47, 50-57. For instance, normal urine output is in the range of
0.5 to 2 ml/kg of body
weight per hour. The minimum acceptable urine output for a patient with normal
renal function
is 0.5 ml/kg per hour. All these standards may be used to assess whether a
patient is in need for
regulating the fluid balance and/or improving the fluid balance.
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Throughout the specification the "antibodies", or "antibody fragments" or "non-
Ig scaffolds" in
accordance with the invention are capable to bind ADM, and thus are directed
against ADM, and
thus can be referred to as "anti-ADM antibodies", "anti-ADM antibody
fragments", or "anti-
ADM non-Ig scaffolds".
In another embodiment of the invention the anti-ADM antibodies, anti-ADM
antibody
fragments, or anti-ADM non-Ig scaffolds in accordance with the invention are
capable to bind
circulating ADM, and thus are directed against circulating ADM.
In another embodiment of the invention an anti-Adrenomedullin (ADM) antibody
or an anti-
ADM antibody fragment thereof or and ADM non-Ig scaffold is to be used in
combination with
fluids administered intravenously, wherein said combination is for use in
therapy of an acute
disease or acute condition of a patient for the regulation of fluid balance.
In another embodiment of the invention an anti-Adrenomedullin (ADM) antibody
or an anti-
ADM antibody fragment thereof or an ADM non-Ig scaffold is to be used in
combination with
vasopressor agents, e.g. catecholarnine, wherein said combination is for use
in therapy of an
acute disease or acute condition of a patient for the regulation of fluid
balance.
Subject matter of the present invention is an anti-adrenomedullin (ADM)
antibody or an anti-
ADM antibody fragment or an anti-ADM non-Ig scaffold for regulating the fluid
balance in a
patient having a chronic or acute disease or acute condition.
Subject matter of the present invention is a method for regulating the fluid
balance in a patient
having a chronic or acute disease or acute condition. According to the
invention said patient is a
patient in need of regulating the fluid balance.
Subject matter of the present invention is an anti-ADM antibody or anti-ADM
antibody fragment
or an anti-ADM non-Ig scaffold for use in therapy of an acute disease or acute
condition of a
patient for the regulation of fluid balance.
An anti-adrenomedullin (ADM) antibody is an antibody that binds specifically
to ADM, anti-
adrenomedullin antibody fragment is a fragment of an anti-ADM antibody,
wherein said
fragment binds specifically to ADM. An anti-ADM non-Ig scaffold is a non-Ig
scaffold that
binds specifically to ADM.
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Specifically binding to ADM allows binding to other antigens as well. This
means, this
specificity would not exclude that the antibody may cross-react with other
polypeptides that
against it has been raised.
Patient in status of fluid imbalance may get fluid administered intravenously
as a standard
measure of care, especially in an ICU setting. It is, however, desirable to
reduce or avoid the
additional fluid administration because of complications that might occur as
e.g. the occurrence
of edema (acroedema). Edema means swelling caused by fluid in the body's
tissues. It may occur
in feet and legs, but can involve the entire body and can involve organs as
e.g. lung, heart, eye.
Thus, anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig
scaffold may be
administered at a point of time when the patient is in need of fluid
administration. According to
the invention said patient is a patient in need of regulating the fluid
balance.
Thus, subject matter of the present invention is also an anti-ADM antibody or
anti-ADM
antibody fragment or anti-ADM non-Ig scaffold for use in therapy of an acute
disease or acute
condition of a patient for the regulation of fluid balance which includes but
is not limited to the
prevention or reduction of edema.
The anti-ADM antibody or the anti-ADM antibody fragment or anti-ADM non-Ig
scaffold may
be also administered preventively before the patient exhibits any signs of
fluid imbalance. This
might be the case if the patient has a chronic or acute disease or acute
condition where fluid
imbalance problems may be expected, e.g. comprising severe infections as e.g.
meningitis,
Systemic inflammatory Response-Syndrom (SIRS), sepsis; other diseases as
diabetes, cancer,
acute and chronic vascular diseases as e.g. heart failure, myocardial
infarction, stroke,
atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g.
kidney dysfunction,
liver dysfunction, burnings, surgery, traumata, poisoning, damages by
chemotherapy. Especially
useful is the antibody or fragment or scaffold according to the present
invention for reducing the
risk of mortality during sepsis and septic shock, i.e. late phases of sepsis.
In the following clinical criteria for SIRS, sepsis, severe sepsis, septic
shock will be defined.
1) Systemic inflammatory host response (SIRS) characterized by at least two of
the following
symptoms
= patients exhibit hypotension (mean arterial pressure is < 65 mm Hg)
= elevated serum lactate level being > 4 mmol/L
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= blood glucose > 7.7 mmol/L (in absence of diabetes)
= central venous pressure is not within the range 8-12 mm Hg
= urine output is < 0.5 mL x x hrI
= central venous (superior vena cava) oxygen saturation is < 70% or mixed
venous <65%
= heart rate is > 90 beats/min
= temperature < 36 C or > 38 C
= respiratory rate > 20/min
= white cell count < 4 or > 12 x 109/L (leucocytes); > 10% immature
neutrophils
2) Sepsis
Following at least two of the symptoms mentioned under 1), and additionally a
clinical suspicion
of new infection, being it:
= cough/sputum/chest pain
= abdominal pain/distension/diarrhoea
= line infection
= endocarditis
= dysuria
= headache with neck stiffness
= cellulitis/wound/joint infection
= positive microbiology for any infection
3) Severe sepsis
Provided that sepsis is manifested in patient, and additionally a clinical
suspicion of any organ
dysfunction, being it:
= blood pressure systolic < 90/mean; < 65namHG
= lactate > 2 mmol/L
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= Bilirubine > 341.anol/L
= urine output < 0.5 mL/kg/h for 2h
= creatinine > 177 punol/L
= platelets < 1 00x1 09/L
= Sp02 > 90% unless 02 given
4) Septic shock
At least one sign of end-organ dysfunction as mentioned under 3) is
manifested. Septic shock is
indicated, if there is refractory hypotension that does not respond to
treatment and intravenous
fluid administration alone is insufficient to maintain a patient's blood
pressure from becoming
hypotensive also provides for an administration of an anti-ADM antibody or an
anti-ADM
antibody fragment or an anti-ADM non-Ig scaffold in accordance with the
present invention.
Thus, acute disease or acute conditions may be selected from the group but are
not limited to the
group comprising severe infections as e.g. meningitis, Systemic inflammatory
Response-
Syndrom (SIRS), or sepsis; other diseases as diabetes, cancer, acute and
chronic vascular
diseases as e.g. heart failure, myocardial infarction, stroke,
atherosclerosis; shock as e.g. septic
shock and organ dysfunction as e.g. kidney dysfunction, liver dysfunction,
bumings, surgery,
traumata, poisoning, damages induced by chemotherapy. Especially useful is the
antibody or
fragment or scaffold according to the present invention for reducing the risk
of mortality during
sepsis and septic shock, i.e. late phases of sepsis.
In one embodiment of the present invention the patient is not suffering from
SIRS, a severe
infection, sepsis, shock as e.g. septic shock, Said severe infection denotes
e.g. meningitis,
Systemic inflammatory Response-Syndrome (SIRS), sepsis, severe sepsis, and
shock as e.g.
septic shock. In this regard, a severe sepsis is characterized in that sepsis
is manifested in said
patient, and additionally a clinical suspicion of any organ dysfunction is
present, being it:
* blood pressure systolic < 90/mean; < 65mmHG
* lactate > 2 mmol/L
= Bilirubine > 341,1mol/L
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= urine output < 0.5 mL/kg/h for 2h
= creatinine >177 umol/L
= platelets < 1 00x 1 09/L
= Sp02 > 90% unless 02 given
5
In another embodiment said acute disease or acute condition is not sepsis, or
not severe sepsis, or
not SIRS, or not shock, or not septic shock.
In another embodiment said acute disease or acute condition is not sepsis.
In another embodiment said acute disease or acute condition is selected from
the group
10 comprising meningitis, diabetes, cancer, acute and chronic vascular
diseases as e.g. heart failure,
myocardial infarction, stroke, atherosclerosis; shock as e.g. septic shock and
organ dysfunction
as e.g. kidney dysfunction, liver dysfunction, burnings, surgery, traumata,
poisoning, damages
induced by chemotherapy.
Fluid balance/ Fluid therapy
In an acute hospital setting, being it e.g. a setting in the ICU, commonly the
fluid balance is
monitored carefully by the clinical staff since this provides for particular
infothiation on a
patient's actual state of hydration, and thus for renal and cardiovascular
function.
If, however, acute fluid loss is greater than fluid gain, the patient is
referred to as being in
negative fluid balance. In this case, physiological fluid is often given
intravenously by a
physician to compensate for that loss.
In contrast, a positive fluid balance where fluid gain is greater than fluid
loss may provides for
information to a problem with either the renal or cardiovascular system.
This particularly means in context with e.g. SIRS, sepsis, severe sepsis and
septic shock, that
also blood pressure is low (commonly referred to as hypotension), and the
filtration rate in the
kidneys will lessen, thus causing less fluid reabsorption and less urine
output.
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The ten" "fluid therapy" in general denotes the therapeutic administration of
fluids (such as
physiologic saline solution or water for injection (WFI)) to a patient as a
treatment or
preventative measure. It can be administered via intravenous, intraperitoneal,
intraosseous,
subcutaneous and oral routes.
Fluid therapy is indicated either when there is a loss of fluid or there is a
risk of loss of fluid due
to an underlying disease or condition. The severity of the fluid loss, and the
compartment from
which it has been lost, influences the choice of fluid and the speed at which
it needs to be
administered. If fluid therapy is performed as a treatment then it is
necessary to diagnose and
treat the underlying disease or condition. Fluid therapy is routinely
indicated in case of
hypotension, hypovolemia, metabolic disorders, decreased oxygen delivery,
SIRS, sepsis, severe
sepsis, shock, and septic shock.
However, it should be emphasized that the medicaments provided by the present
invention, being
anti-ADM antibodies, anti-ADM antibody fragments, or anti-ADM non-Ig scaffolds
are only
intended to be used for sake of regulating the fluid balance and thus not for
any methods of
primary treatment to a chronic or acute disease or condition itself. This
means the present
invention does not provide for a therapy of healing/curing e.g. meningitis,
Systemic
inflammatory Response-Syndrom (SIRS), or sepsis, or severe sepsis; other
diseases as diabetes,
cancer, acute and chronic vascular diseases as e.g. heart failure, myocardial
infarction, stroke,
atherosclerosis; shock as e.g. septic shock and organ dysfunction as e.g.
kidney dysfunction,
liver dysfunction, buntings, surgery, traumata, poisoning, or damages induced
by
chemotherapywithin the scope of the invention.
The fluid regulating effect of the anti-ADM antibody or the anti-ADM antibody
fragment or
anti-ADM non-Ig scaffold is thus supporting the primary therapy of said
chronic or acute disease
or acute condition. In case of a chronic or acute disease or acute condition
like severe infections
as e.g. meningitis, Systemic inflammatory Response-Syndrom (SIRS), sepsis or
the like the
primary therapy would be e.g. the administration of antibiotics. The anti-ADM
antibody or the
anti-ADM antibody fragment or anti-ADM non-Ig scaffold would regulate the
fluid balance and
would help to prevent worsening of the critical condition of said patient
until the e.g. antibiotic
administration takes effect. As before mentioned the anti-ADM antibody or the
anti-ADM
antibody fragment or anti-ADM non-IG scaffold may be administered in a
preventive way or in a
therapeutic way, this means in order to prevent fluid imbalance problems or in
order to reduce
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fluid imbalance when fluid imbalance problems are present in said patient.
Edema is included in
the Willi fluid imbalance problems.
It should be emphasized that in accordance with the invention the patients may
have a chronic or
acute disease or acute condition as primary or underlying disease such as e.g.
cancer, or diabetes
mellitus. However, those primary or underlying diseases are not prima fade
targeted by the
therapeutic treatment according to the invention. By contrast, the therapeutic
treatment pursuant
to the invention is solely directed against acute symptoms that are diagnosed
or indicated for
fluid therapy.
Thus, the invention does not provide for a primary therapy for cancer,
diabetes mellitus,
meningitis, Systemic inflammatory Response-Syndrom (SIRS), sepsis or the like,
but for a
therapy of patients that suffer from fluid imbalance that is due to an acute
disease or acute
condition, and thus they are in need of fluid administration.
In one embodiment of the invention an antiADM antibody or an anti-ADM antibody
fragment or
an anti-ADM non-Ig scaffold is to be used in combination with fluids
administered
intravenously, wherein said combination is for use in therapy of an acute
disease or acute
condition of a patient for the regulation of fluid balance of said patient.
In one embodiment of the invention said patient having a chronic or acute
disease or condition
being in need for regulation of fluid balance is characterized by the need of
said patient to get
intravenous fluids. In another embodiment of the invention said patient having
a chronic or acute
disease or condition being in need for regulation of fluid balance is
characterized by the risk of
said patient of getting edema or by the presence of edema in said patient.
Subject matter of the invention in one specific embodiment is, thus, an anti-
Adrenomedullin
(ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig
scaffold for
use in therapy of a patient in need of intravenous fluids or for use in
therapy of a patient having a
risk of getting edema or by the presence of edema in said patient.
In another embodiment of the invention an anti-Adrenomedullin (ADM) antibody
or an anti-
adrenornedullin antibody fragment or anti-ADM non-Ig scaffold is to be used in
combination
with vasopressor agents, e.g. catecholarnine, wherein said combination is for
use in therapy of an
acute disease or acute condition of a patient for regulation of fluid balance.
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In one embodiment of the invention said patient having a chronic or acute
disease or condition
being in need for regulation of fluid balance is characterized by the need of
said patient to get
vasopressor agents, e.g. catecholamine, administration.
Subject matter of the invention in one specific embodiment is, thus, an anti-
Adrenomed-ullin
(ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-
Ig scaffold
for use in therapy of a patient in need of a vasopressor agent, e.g.
catecholamine treatment.
A patient in need of improvement of fluid balance may be characterized by a
capillary leakage
and may be a urine output 0.5 ¨ 1 cc/kg per hour.
Furthermore, in one embodiment of the invention an anti-Adrenomedullin (ADM)
antibody or an
anti-adrenomedullin antibody fragment or an anti-ADM non-Ig scaffold is
monospecific.
Monospecific anti-Adrenomedullin (ADM) antibody or monospecific anti-
adrenornedullin
antibody fragment or monospecific anti-ADM non-Ig scaffold means that said
antibody or
antibody fragment or non-Ig scaffold binds to one specific region encompassing
at least 5 amino
acids within the target ADM. Monospecific anti-Adrenomedullin (ADM) antibody
or
monospecific anti-adrenomedullin antibody fragment or monospecific anti-ADM
non-Ig scaffold
are anti-Adrenomedullin (ADM) antibodies or anti-adrenomedullin antibody
fragments or anti-
ADM non-Ig scaffolds that all have affinity for the same antigen.
In a specific and preferred embodiment the present invention provides for a
monospecific anti-
Adrenomedullin (ADM) antibody or monospecific anti-adrenom.edullin antibody
fragment or
monospecific anti-ADM non-Ig scaffold, characterized in that said antibody or
antibody
fragment or non-Ig scaffold binds to one specific region encompassing at least
4 amino acids
within the target ADM.
In another special embodiment the anti-ADM antibody or the antibody fragment
binding to
ADM is a monospecific antibody. Monospecific means that said antibody or
antibody fragment
binds to one specific region encompassing preferably at least 4, or at least 5
amino acids within
the target ADM. Monospecific antibodies or fragments are antibodies or
fragments that all have
affinity for the same antigen. Monoclonal antibodies are monospecific, but
monospecific
antibodies may also be produced by other means than producing them from a
common germ cell.
An antibody according to the present invention is a protein including one or
more polypeptides
substantially encoded by immunoglobulin genes that specifically binds an
antigen. The
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14
recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma
(IgGi, IgG2,
IgG3, IgG4), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as
well as the
myriad immunoglobulin variable region genes. Full-length immunoglobulin light
chains are
generally about 25 Kd or 214 amino acids in length. Full-length immunoglobulin
heavy chains
are generally about 50 Kd or 446 amino acid in length. Light chains are
encoded by a variable
region gene at the N112-terminus (about 110 amino acids in length) and a kappa
or lambda
constant region gene at the COOH--tetiainus. Heavy chains are similarly
encoded by a variable
region gene (about 116 amino acids in length) and one of the other constant
region genes.
The basic structural unit of an antibody is generally a tetramer that consists
of two identical pairs
of immunoglobulin chains, each pair having one light and one heavy chain. In
each pair, the light
and heavy chain variable regions bind to an antigen, and the constant regions
mediate effector
functions. Immunoglobulins also exist in a variety of other forms including,
for example, Fv,
Fab, and (Fabt)2, as well as bifunctional hybrid antibodies and single chains
(e.g., Lanzavecchia
et at., Eur. J. Immunol. 17:105,1987; Huston et at., Proc. Nail. Acad. Sci.
U.S.A., 85:5879-5883,
1988; Bird et at., Science 242:423-426, 1988; Hood et al., Immunology,
Benjamin, N.Y., 2nd
ed., 1984; Hunkapiller and Hood, Nature 323:15-16,1986). An immunoglobulin
light or heavy
chain variable region includes a framework region interrupted by three
hypervariable regions,
also called complementarity determining regions (CDR's) (see, Sequences of
Proteins of
Immunological Interest, E. Kabat et al., U.S. Department of Health and Human
Services, 1983).
As noted above, the CDRs are primarily responsible for binding to an epitope
of an antigen. An
immune complex is an antibody, such as a monoclonal antibody, chimeric
antibody, humanized
antibody or human antibody, or functional antibody fragment, specifically
bound to the antigen.
Chimeric antibodies are antibodies whose light and heavy chain genes have been
constructed,
typically by genetic engineering, from immunoglobulin variable and constant
region genes
belonging to different species. For example, the variable segments of the
genes from a mouse
monoclonal antibody can be joined to human constant segments, such as kappa
and gamma 1 or
gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid
protein composed of
the variable or antigen-binding domain from a mouse antibody and the constant
or effector
domain from a human antibody, although other mammalian species can be used, or
the variable
region can be produced by molecular techniques. Methods of making chimeric
antibodies are
well known in the art, e.g., see U.S. Patent No, 5,807,715. A "humanized"
immunoglobulin is an
immunoglobulin including a human framework region and one or more CDRs from a
non-
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human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human
immunoglobulin
providing the CDRs is tenned a "donor" and the human immunoglobulin providing
the
framework is termed an "acceptor." In one embodiment, all the CDRs are from
the donor
immunoglobulin in a humanized immunoglobulin. Constant regions need not be
present, but if
5 they are, they must be substantially identical to human immunoglobulin
constant regions, i.e., at
least about 85-90%, such as about 95% or more identical. Hence, all parts of a
humanized
immunoglobulin, except possibly the CDRs, are substantially identical to
corresponding parts of
natural human immunoglobulin sequences. A "humanized antibody" is an antibody
comprising a
humanized light chain and a humanized heavy chain immunoglobulin. A humanized
antibody
10 binds to the same antigen as the donor antibody that provides the CDRs.
The acceptor
framework of a humanized immunoglobulin or antibody may have a limited number
of
substitutions by amino acids taken from the donor framework. Humanized or
other monoclonal
antibodies can have additional conservative amino acid substitutions which
have substantially no
effect on antigen binding or other immunoglobulin functions. Exemplary
conservative
15 substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn,
gln; ser, thr; lys, arg; and phe,
tyr. Humanized immunoglobulins can be constructed by means of genetic
engineering (e.g., see
U.S. Patent No. 5,585,089). A human antibody is an antibody wherein the light
and heavy chain
genes are of human origin. Human antibodies can be generated using methods
known in the art.
Human antibodies can be produced by immortalizing a human B cell secreting the
antibody of
interest. Immortalization can be accomplished, for example, by EBV infection
or by fusing a
human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human
antibodies can
also be produced by phage display methods (see, e.g., Dower et aL, PCT
Publication No.
W091/17271; McCafferty et aL, PCT Publication No. W092/001047; and Winter, PCT
Publication No. W092/20791), or selected from a human combinatorial monoclonal
antibody
library (see the Morphosys website). Human antibodies can also be prepared by
using transgenic
animals carrying a human immunoglobulin gene (for example, see Lonberg et al.,
PCT
Publication No. W093/12227; and Kucherlapati, PCT Publication No. W091/10741).
Thus, the anti-ADM antibody may have the foimats known in the art. Examples
are human
antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies,
CDR-grafted
antibodies. In a preferred embodiment antibodies according to the present
invention are
recombinantly produced antibodies as e.g. IgG, a typical full-length
immunoglobulin, or
antibody fragments containing at least the F-variable domain of heavy and/or
light chain as e.g.
chemically coupled antibodies (fragment antigen binding) including but not
limited to Fab-
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fragments including Fab minibodies, single chain Fab antibody, monovalent Fab
antibody with
epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized with the
CH3 domain;
bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid
of a heterologous
domain, e.g. via dimerization of dHLX domains,e.g. Fab-dHLX-FSx2; F(ab`)2-
fragments, scFv-
fragments, multimerized multivalent or/and multispecifie scFv-fragments,
bivalent and/or
bispecific diabodies, BITE (bispecific T-cell engager), trifunctional
antibodies, polyvalent
antibodies, e.g. from a different class than G; single-domain antibodies, e.g.
nanobodies derived
from camelid or fish immunoglobulines and numerous others.
In addition to anti-ADM antibodies other biopolymer scaffolds are well known
in the art to
complex a target molecule and have been used for the generation of highly
target specific
biopolymers. Examples are aptamers, spiegelmers, anticalins and conotoxins.
For illustration of
antibody formats please see Fig. la, lb and lc.
In a preferred embodiment the ADM antibody format is selected from the group
comprising Fv
fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and
scFv-Fc Fusion
protein. In another preferred embodiment the antibody format is selected from
the group
comprising scFab fragment, Fab fragment, scFv fragment and bioavailability
optimized
conjugates thereof, such as PEGylated fragments. One of the most preferred
formats is the scFab
foiniat.
Non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics
as they are
capable to bind to ligands or antigenes. Non-Ig scaffolds may be selected from
the group
comprising tetranectin-based non-Ig scaffolds (e.g. described in US
2010/0028995), fibronectin
scaffolds (e.g. described in EP 1266 025; lipocalin-based scaffolds ((e.g.
described in WO
2011/154420); ubiquitin scaffolds (e.g. described in WO 2011/073214),
transferring scaffolds
(e.g. described in US 2004/0023334), protein A scaffolds (e.g. described in EP
2231860),
ankrin repeat based scaffolds (e.g. described in WO 2010/060748),
microproteins, preferably
rnicroproteins forming a cystine knot) scaffolds (e.g. described in EP
2314308), Fyn SH3
domain based scaffolds (e.g. described in WO 2011/023685) EGFR-A-domain based
scaffolds
(e.g. described in WO 2005/040229) and Kunitz domain based scaffolds (e.g.
described in EP
1941867).
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In one embodiment of the invention antibodies according to the present
invention may be
produced as follows:
A Balb/c mouse was immunized with ADM-100pg Peptide-BSA-Conjugate at day 0 and
14
(emulsified in 100111 complete Freund's adjuvant) and 50pg at day 21 and 28
(in 1001i1
incomplete Freund's adjuvant). Three days before the fusion experiment was
performed, the
animal received 50 jig of the conjugate dissolved in 100p1 saline, given as
one intraperitoneal and
one intravenous injection.
Spenoeytes from the immunized mouse and cells of the myeloma cell line SP2/0
were fused with
lml 50% polyethylene glycol for 30s at 37 C. After washing, the cells were
seeded in 96-well
cell culture plates. Hybrid clones were selected by growing in HAT medium
[RPMI 1640 culture
medium supplemented with 20% fetal calf serum and HAT-Supplement. After two
weeks the
HAT medium is replaced with HT Medium for three passages followed by returning
to the
normal cell culture medium.
The cell culture supernatants were primary screened for antigen specific IgG
antibodies three
weeks after fusion. The positive tested microcultures were transferred into 24-
well plates for
propagation. After retesting, the selected cultures were cloned and recloned
using the limiting-
dilution technique and the isotypes were determined (see also Lane, R.D.
(1985). A short-
duration polyethylene glycol fusion technique for increasing production of
monoclonal antibody-
secreting hybridomas. J. Immunol. Meth. 81: 223-228; Ziegler, B. et al. (1996)
Glutamate
decarboxylase (GAD) is not detectable on the surface of rat islet cells
examined by
cytofluorometry and complement-dependent antibody-mediated cytotoxicity of
monoclonal
GAD antibodies, Horm. Metab. Res. 28: 11-15).
Antibodies may be produced by means of phage display according to the
following procedure:
The human naive antibody gene libraries HAL7/8 were used for the isolation of
recombinant
single chain F-Variable domains (scFv) against adrenomedullin peptide. The
antibody gene
libraries were screened with a panning strategy comprising the use of peptides
containing a
biotin tag linked via two different spacers to the adrenomedullin peptide
sequence. A mix of
panning rounds using non-specifically hound antigen and streptavidin bound
antigen were used
to minimize background of non-specific binders. The eluted phages from the
third round of
panning have been used for the generation of monoclonal seFv expressing E.coli
strains.
Supernatant from the cultivation of these clonal strains has been directly
used for an antigen
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ELISA testing (see Hust, M., Meyer, T., Voedisch, B., Milker, T., Thie, H., El-
Ghezal, A.,
Kirsch, M.1., Schatte, M., Helmsing, S., Meier, D., Schilunann, T., Dubel, S.,
2011. A human
scFv antibody generation pipeline for proteome research. Journal of
Biotechnology 152, 159-
170; Schiitte, M., ThuHier, P., Pelat, T., Wezler, X., Rosenstoek, P., Hinz,
D., Kirsch,
M.I.,Hasenberg, M., Frank, R., Schirrmann, T., Gunzer, M., Hust, M., Diibel,
S., 2009.
Identification of a putative Crf splice variant and generation of recombinant
antibodies for the
specific detection of Aspergillus fumigatus. PLUS One 4, e6625).
Humanization of murine antibodies may be conducted according to the following
procedure:
For humanization of an antibody of murine origin the antibody sequence is
analyzed for the
structural interaction of framework regions (FR) with the complementary
determining regions
(CDR) and the antigen. Based on structural modeling an appropriate FR of human
origin is
selected and the murine CDR sequences are transplanted into the human FR.
Variations in the
amino acid sequence of the CDRs or FRs may be introduced to regain structural
interactions,
which were abolished by the species switch for the FR sequences. This recovery
of structural
interactions may be achieved by random approach using phage display libraries
or via directed
approach guided by molecular modeling (see Almagro JC, Fransson J., 2008.
Humanization of
antibodies. Front Biosci. 2008 Jan 1;13:1619-33).
In a preferred embodiment the ADM antibody format is selected from the group
comprising Fv
fragment, scFv fragment, Fab fragment, scFab fragment, F(ab)2 fragment and
scFv-Fe Fusion
protein. In another preferred embodiment the antibody format is selected from
the group
comprising scFab fragment, Fab fragment, say fragment and bioavailability
optimized
conjugates thereof, such as PEGylated fragments. One of the most preferred
formats is scFab
format.
In another preferred embodiment, the anti-ADM antibody, anti-ADM antibody
fragment, or anti-
ADM non-Ig scaffold is a full length antibody, antibody fragment, or non-Ig
scaffold.
In a preferred embodiment the anti-ADM antibody or an anti-adrenomedullin
antibody fragment
or an anti-ADM non-1g scaffold is directed to and can bind to an epitope of at
least 5 amino acids
in length contained in ADM.
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In a more preferred embodiment the anti-ADM antibody or an anti-adrenomedullin
antibody
fragment or an anti-ADM non-Ig scaffold is directed to and can bind to an
epitope of at least 4
amino acids in length contained in ADM.
In one specific embodiment of the invention the anti-Adrenomedullin (ADM)
antibody or anti-
ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold
binding to
adrenomedullin is provided for use in therapy of an acute disease or acute
condition of a patient
wherein said antibody or fragment or scaffold is not ADM-binding-Protein-1
(complement factor
H).
In one specific embodiment of the invention the anti-Adrenomedullin (ADM)
antibody or anti-
ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold
binding to
adrenomedullin is provided for use in therapy of an acute disease or acute
condition of a patient
wherein said antibody or antibody fragment or non-Ig scaffold binds to a
region of preferably at
least 4, or at least 5 amino acids within the sequence of aa 1-42 of mature
human ADM:
SEQ ID No 24
YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVA.
In one specific embodiment of the invention the anti-Adrenomedullin (ADM)
antibody or anti-
ADM antibody fragment binding to adrenomedullin or anti-ADM non-Ig scaffold
binding to
adrenomedullin is provided for use in therapy of an acute disease or acute
condition of a patient
wherein said antibody or fragment or scaffold binds to a region of preferably
at least 4, or at least
5 amino acids within the sequence of aa 1-21 of mature human ADM:
SEQ ID No 23
YRQSMNNFQGLRSFGCRFGTC.
In a preferred embodiment of the present invention said anti-ADM antibody or
an anti-
antibody fragment or anti-ADM non-Ig scaffold binds to a region of ADM of
preferably at least 4, or at least 5 amino acids that is located in the N-
terminal part (aa 1-21) of
adrenomedullin, (see Fig. 2).
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In a preferred embodiment the anti-adrenomedullin antibody or an anti-
adrenomedullin antibody
fragment or anti-adrenomedullin non-1g scaffold is directed to and can bind to
an epitope of at
least 5 amino acids in length contained in ADM, preferably in human ADM.
5 In a more preferred embodiment the anti-adrenomedullin antibody or an
anti-adrenomedullin
antibody fragment or anti-adrenomedullin non-Ig scaffold is directed to and
can bind to an
epitope of at least 4 amino acids in length contained in ADM, preferably in
human ADM.
10 In another preferred embodiment said anti-ADM antibody or an anti-
adrenomedullin antibody
fragment or anti-ADM non-Ig scaffold recognizes and binds to the N-teiminal
end (aa 1) of
adrenomedullin. N-terminal end means that the amino acid 1, that is "Y" of SEQ
ID No. 21 or
23; is mandatory for antibody binding. Said antibody or fragment or scaffold
would neither bind
N-teitainal extended nor N-terminal modified adrenomedullin nor N-terminal
degraded
15 adrenomedullin.
In another specific embodiment pursuant to the invention the herein provided
anti-ADM
antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold does not
bind to the C-
terminal portion of ADM, i.e. the aa 43 ¨ 52 of ADM (SEQ ID NO: 25):
20 PRSICISPOGY-NH2
(SEQ ID NO:25)
In one specific embodiment it is preferred to use an anti-ADM antibody or an
anti-
adrenomedullin antibody fragment or anti-ADM non-1g scaffold according to the
present
invention, wherein said adrenomedullin antibody or said adrenomedullin
antibody fragment or
non-Ig scaffold is an ADM stabilizing antibody or an adrenomedullin
stabilizing antibody
fragment or an adrenomedullin stabilizing non-Ig scaffold that enhances the
half life (t112; half
retention time) of adrenomedullin in serum, blood, plasma at least 10 %,
preferably at least 50
%, more preferably >50 %, most preferably >100%.
The half life (half retention time) of ADM may be deteimined in human plasma
in absence and
presence of an ADM stabilizing antibody or an adrenomedullin stabilizing
antibody fragment or
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an adrenomedullin stabilizing non-Ig scaffold, respectively, using an
immunoassay for the
quantification of ADM.
The following steps may be conducted:
- ADM may be diluted in human citrate plasma in absence and presence of an ADM
stabilizing antibody or an adrenomedullin stabilizing antibody fragment or an
adrenomedullin stabilizing non-Ig scaffold, respectively, and may be incubated
at 24 C
- Aliquots are taken at selected time points (e.g. within 24 hours) and
degradation of ADM
may be stopped in said aliquots by freezing at -20 C
- The quantity of ADM may be determined by a hADM immunoassay directly, if the
selected assay is not influenced by the stabilizing antibody. Alternatively,
the aliquot may
be treated with denaturing agents (like HC1) and, after clearing the sample
(e.g. by
centrifugation) the pH can be neutralized and the ADM-quantified by an ADM
immunoassay. Alternatively, non-immunoassay technologies (e.g. rpHPLC) can be
used
for ADM-quantification
- The half life of ADM is calculated for ADM incubated in absence and
presence of an
ADM stabilizing antibody or an adrenomedullin stabilizing antibody fragment or
an
adrenomedullin stabilizing non-Ig scaffold, respectively,
-
The enhancement of half life is calculated for the stabilized ADM in
comparison to ADM
that has been incubated in absence of an ADM stabilizing antibody or an
adrenomedullin
stabilizing antibody fragment or an adrenomedullin stabilizing non-Ig
scaffold.
A two-fold increase of the half life of ADM is an enhancement of half life of
100%.
Half Life (half retention time) is defined as the period over which the
concentration of a
specified chemical or drug takes to fall to half its baseline concentration in
the specified fluid or
blood.
An assay that may be used for the deteunination of the Half life (half
retention time) of
adrenomedullin in serum, blood, plasma is described in Example 3.
For some diseases blocking of ADM may be beneficial to a certain extent.
However, it might
also be detrimental if ADM is totally neutralized as a certain amount of ADM
may be required
for several physiological functions. In many reports it was emphasized that
the administration of
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ADM may be beneficial in certain diseases. In contrast thereto in other
reports ADM was
reported as being life threatening when administered in certain conditions.
In a specific embodiment said anti-ADM antibody, anti-ADM antibody fragment or
anti-ADM
non-Ig scaffold is a non-neutralizing antibody, fragment or non-Ig scaffold. A
neutralizing anti-
ADM antibody, anti-ADM antibody fragment or anti-ADM non-Ig scaffold would
block the
bioactivity of ADM to nearly 100%, to at least more than 90%, preferably to at
least more than
95%.
In contrast, a non-neutralizing anti-ADM antibody, or anti-ADM antibody
fragment or anti-
ADM non-Ig scaffold blocks the bioactivity of ADM less than 100%, preferably
to less than
95%, preferably to less than 90%, more preferred to less than 80 % and even
more preferred to
less than 50 %. This means that the residual bioactivity of ADM bound to the
non-neutralizing
anti-ADM antibody, or anti-ADM antibody fragment or anti-ADM non-Ig scaffold
would be
more than 0%, preferably more than 5 %, preferably more than 10 %, more
preferred more than
%, more preferred more than 50 %.
In this context (a) molecule(s), being it an antibody, or an antibody fragment
or a non-Ig scaffold
with "non-neutralizing anti-ADM activity", collectively termed here for
simplicity as "non-
20
neutralizing" anti-ADM antibody, antibody fragment, or non-Ig scaffold, that
e.g. blocks the
bioactivity of ADM to less than 80 %, is defined as
a molecule or molecules binding to ADM, which upon addition to a culture of an
eukaryotic cell line, which expresses functional human recombinant ADM
receptor composed of CRLR (calcitonin receptor like receptor) and RAMP3
(receptor-activity modifying protein 3), reduces the amount of cAMP produced
by
the cell line through the action of parallel added human synthetic ADM
peptide,
wherein said added human synthetic ADM is added in an amount that in the
absence of the non-neutralizing antibody to be analyzed, leads to half-maximal
stimulation of cAMP synthesis, wherein the reduction of cAMP by said
molecule(s) binding to ADM takes place to an extent, which is not more than
80%, even when the non-neutralizing molecule(s) binding to ADM to be analyzed
is added in an amount, which is 10-fold more than the amount, which is needed
to
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obtain the maximal reduction of cAMP synthesis obtainable with the non-
neutralizing antibody to be analyzed.
The same definition applies to the other ranges; 95%, 90%, 50% etc.
In a specific embodiment according to the present invention an anti-ADM
antibody or an anti-
adrenomedullin antibody fragment or anti-ADM non-Ig scaffold is used, wherein
said antibody
or an adrenomedullin antibody fragment blocks the bioactivity of ADM to less
than 80 %,
preferably less than 50% (of baseline values). This is in the sense of
blocking the circulating
ADM of no more than 80% or no more than 50%, respectively.
It has been understood that said limited blocking of the bioactivity of ADM
occurs even at
excess concentration of the antibody, fragment or scaffold, meaning an excess
of the antibody,
fragment or scaffold in relation to ADM. Said limited blocking is an intrinsic
property of the
ADM binder itself. This means that said antibody, fragment or scaffold has a
maximal inhibition
of 80% or 50% respectively. In a preferred embodiment said anti-ADM antibody,
anti-ADM
antibody fragment or anti-ADM non-Ig scaffold would block the bioactivity of
ADM to at least 5
%.
The stated above means that approximately 20% or 50% or even 95% residual ADM
bioactivity
remains present, respectively.
Thus, in accordance with the present invention the provided anti-ADM
antibodies, anti-ADM
antibody fragments, and anti-ADM non-Ig scaffolds do not neutralize the
respective circulating
ADM bioactivity.
The bioactivity is defined as the effect that a substance takes on a living
organism or tissue or
organ or functional unit in vivo or in vitro (e.g. in an assay) after its
interaction. In case of ADM
bioactivity this may be the effect of ADM in a human recombinant
Adrenomedullin receptor
cAMP functional assay. Thus, according to the present invention bioactivity is
defined via an
Adrenomedullin receptor cAMP functional assay. The following steps may be
performed in
order to determine the bioactivity of ADM in such an assay:
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- Dose response curves are performed with ADM in said human recombinant
Adrenomedullin receptor cAMP functional assay.
- The ADM-concentration of half-maximal cAMP stimulation may be calculated.
- At constant half-maximal cAMP-stimulating ADM-concentrations dose
response curves
(up to 100ug/m1 final concentration) are performed by an ADM stabilizing
antibody or
an adrenomedullin stabilizing antibody fragment or an adrenomedullin
stabilizing non-1g
scaffold, respectively,
A maximal inhibition in said ADM bioassay of 50% means that said anti-ADM
antibody or said
anti-adrenomedullin antibody fragment or said anti-adrenomedullin non-Ig
scaffold, repectively,
blocks the bioactivity to 50% of baseline values. A maximal inhibition in said
ADM bioassay of
80% means that said anti-ADM antibody or said anti-adrenomedullin antibody
fragment or said
anti-adrenomedullin non-Ig scaffold, respectively, blocks the bioactivity of
ADM to 80%. This is
in the sense of blocking the ADM bioactivity to not more than 80%. This means
approximately
20% residual ADM bioactivity remains present.
However, by the present specification and in the above context the expression
"blocks the
bioactivity of ADM" in relation to the herein disclosed anti-ADM antibodies,
anti-ADM
antibody fragments, and anti-ADM non-Ig scaffolds should be understood as mere
decreasing
the bioactivity of ADM, preferably decreasing circulating ADM bioactivity from
100% to 20%
remaining ADM bioactivity at maximum, preferably decreasing the ADM
bioactivity from 100%
to 50% remaining ADM bioactivity; but in any case there is ADM bioactivity
remaining that can
be deteimined as detailed above.
The bioactivity of ADM may be determined in a human recombinant Adrenomedullin
receptor
cAMP functional assay (Adrenornedullin Bioassay) according to Example 2.
In a preferred embodiment a modulating anti-ADM antibody or a modulating anti-
ADM
adrenomedullin antibody fragment or a modulating anti-ADM adrenomedullin non-
Ig scaffold is
used in therapy of acute disease or acute condition of a patient for
regulation of fluid balance.
Such a modulating anti-ADM antibody or a modulating anti-ADM adrenomedullin
antibody
fragment or a modulating anti-ADM adrenomedullin non-Ig scaffold may be
especially useful in
the treatment of sepsis. A modulating anti-ADM antibody or a modulating anti-
ADM
adrenomedullin antibody fragment or a modulating anti-adrenomedullin non-Ig
scaffold
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enhances the bioactivity of ADM in the early phase of sepsis and reduces the
damaging effects
of ADM in the late phase of sepsis.
A "modulating" antibody or a modulating adrenomedullin antibody fragment or a
modulating
5 adrenomedullin non-Jg scaffold is an antibody or an adrenomedullin
antibody fragment or non-Ig
scaffold that enhances the half life (t1/2 half retention time) of
adrenomedullin in serum, blood,
plasma at least 10 %, preferably at least, 50 %, more preferably >50 %, most
preferably >100%
and blocks the bioactivity of ADM to less than 80 %, preferably less than 50
%. These values
related to half-life and blocking of bioactivity have to be understood in
relation to the before-
10 mentioned assays and definitions in order to determine these values.
It should be emphasized that blocking the ADM bioactivity is in the sense of
no more than 80%,
and thus 20% residual ADM bioactivity. The same applies to blocking the ADM
bioactivity to
no more than 50%, and thus residual 50% ADM bioactivity.
Such a modulating anti-ADM antibody or a modulating anti-ADM adrenomedullin
antibody
fragment or a modulating anti-adrenomedullin non-Ig scaffold offers the
advantage that the
dosing of the administration is facilitated. The combination of partially
blocking or partially
reducing Adrenomedullin bioactivity and increase of the in vivo half life
(increasing the
Adrenomedullin bioactivity) leads to beneficial simplificity of anti-
Adrenomedullin antibody or
an anti-adrenomedullin antibody fragment or anti-adrenomedullin non-Ig
scaffold dosing. In a
situation of excess of endogenous Adrenomedullin (maximal stimulation, late
sepsis phase,
shock, hypodynamic phase) the activity lowering effect is the major impact of
the antibody or
fragment or scaffold, limiting the (negative) effect of Adrenomedullin. In
case of low or normal
endogenous Adrenomedullin concentrations, the biological effect of anti-
Adrenomedullin
antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig
scaffold is a
combination of lowering (by partially blocking) and increase by increasing the
Adrenomedullin
half life. If the half life effect is stronger than the blocking effect, the
net biological activity of
endogenous Adrenomedullin is beneficially increased in early phases of sepsis
(low
Adrenomedullin, hyperdynamic phase). Thus, the non-neutralizing and modulating
anti-
Adrenomedullin antibody or anti-adrenomedullin antibody fragment or anti-
adrenomedullin non-
Ig scaffold acts like an ADM bio activity buffer in order to keep the
bioactivity of ADM within a
certain physiological range.
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Thus, the dosing of the anti-ADM antibody/fragment/scaffold in e.g. sepsis may
be selected from
an excessive concentration, because both sepsis phases (early and late)
benefit from excessive
anti-ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-
Ig scaffold
treatment in case of a modulating effect. Excessive means: The anti-
Adrenomedullin antibody
or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold
concentration is
higher than endogenous Adrenomedullin during late phase (shock) of e.g.
sepsis. This means, in
case of a modulating anti-ADM antibody or modulating anti-ADM antibody
fragment or
modulating anti-ADM scaffold dosing in sepsis may be as follows:
The concentration of Adrenomedullin in septic shock is 226+1-66 fmol/nal
(Nishio et al.,
"Increased plasma concentrations of adrenomedullin correlate with relaxation
of vascular tone in
patients with septic shock.", Crit Care Med, 1997, 25(6):953-7), an equirnolar
concentration of
antibody or fragment or scaffold is 42.51.tg/1 blood, (based on 6 1 blood
volume / 80kg body
weight) 3.2ug/kg body weight. Excess means at least double (mean) septic shock
Adrenomedullin concentration, at least > 3pg anti-Adrenomedullin antibody or
an anti-
adrenomedullin antibody fragment or anti-ADM non-Ig scaffold / kg body weight,
preferred at
least 6.4 jig anti-Adrenomedullin antibody or an anti-adrenomedullin antibody
fragment or anti-
ADM non-Ig scaffold /kg body weight. Preferred > lOgg / kg, more preferred
>20jtg/kg, most
preferred >10Oug anti-Adrenomedullin antibody or an anti-adrenomedullin
antibody fragment or
anti-ADM non-Ig Scaffold / kg body weight. This may apply to other severe and
acute
conditions than septic shock as well.
In a specific embodiment of the invention the anti-ADM antibody is a
monoclonal antibody or an
anti-ADM antibody fragment thereof. In one embodiment of the invention the
anti-ADM
antibody or the anti-ADM antibody fragment is a human or humanized antibody or
derived
therefrom. In one specific embodiment one or more (murine) CDR's are grafted
into a human
antibody or antibody fragment.
Subject matter of the present invention in one aspect is a human CDR-grafted
antibody or
antibody fragment thereof that binds to ADM, wherein the human CDR-grafted
antibody or
antibody fragment thereof comprises an antibody heavy chain (H chain)
comprising
SEQ ID NO:1
GYTFSRYW
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27
SEQ ID NO: 2
ILPGSGST
and/or
SEQ ID NO: 3
TEGYEYDGFDY
and/or further comprises an antibody light chain (L chain) comprising:
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
and/or
SEQ ID NO: 6
FQGSHIPYT.
In one specific embodiment of the invention subject matter of the present
invention is a human
monoclonal antibody that binds to ADM or an antibody fragment thereof wherein
the heavy
chain comprises at least one CDR selected from the group comprising:
SEQ ID NO: 1
GYTFSRYW
SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
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and wherein the light chain comprises at least one CDR selected from the group
comprising:
SEQ ID No: 4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
In a more specific embodiment of the invention subject matter of the invention
is a human
monoclonal antibody that binds to ADM or an antibody fragment thereof wherein
the heavy
chain comprises the sequences
SEQ ID NO: I
GYTFSRYW
SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
and wherein the light chain comprises the sequences
SEQ ID NO: 4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
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In a very specific embodiment the anti-ADM antibody has a sequence selected
from the group
comprising: SEQ ID NO 7, 8,9, 10, 11, 12, 13 and 14.
The anti-ADM antibody or anti-adrenomedullin antibody fragment or anti-ADM non-
Ig scaffold
according to the present invention exhibits an affinity towards human ADM in
such that affinity
constant is greater than 10-7 M, preferred 10-8 M, preferred affinity is
greater than 10-9 M, most
preferred higher than 10-10 M A person skilled in the art knows that it may be
considered to
compensate lower affinity by applying a higher dose of compounds and this
measure would not
lead out-of-the-scope of the invention. The affinity constants may be
determined according to the
method as described in Example 1.
In a preferred embodiment the anti-ADM antibody or the anti-ADM antibody
fragment or the
anti-ADM non-Ig scaffold is used for reducing the risk of mortality during
said chronic or acute
disease or acute condition of a patient.
Chronic or acute disease or acute condition according to the present invention
may be a disease
or condition selected from the group comprising severe infections as e.g.
meningitis, Systemic
inflammatory Response-Syndrom (SIRS), sepsis; other diseases as diabetes,
cancer, acute and
chronic vascular diseases as e.g. heart failure, myocardial infarction,
stroke, artheriosclerosis;
shock as e.g. septic shock and organ dysfunction as e.g. kidney dysfunction,
liver dysfunction or
capillary leakage, trauma, poisoning, surgery. Especially useful is the anti-
ADM antibody or
anti-ADM antibody fragment or anti-ADM non-Ig scaffold according to the
present invention for
reducing the risk of mortality during sepsis and septic shock, i.e. late
phases of sepsis.
Hereto it should be emphasized that the patient may be has a chronic or acute
disease or
condition as primary and underlying disease as outlined in the above
paragraph; however, the
anti-ADM antibody or anti-ADM antibody fragment or anti-ADM non-Ig scaffold
pursuant to
the invention are not intended for primary therapy of said diseases, but
rather for regulating the
fluid balance of a patient that is in need of administration of fluids, which
can thus be considered
as an acute disease or acute condition besides the primary disease. Moreover,
said need for fluid
administration may be associated with a primary underlying disease but this is
not mandatory
within the scope of the instant invention.
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Thus, in one embodiment the anti-ADM antibody or an anti-adrenomedullin
antibody fragment
or anti-ADM non-Ig scaffold is used in therapy of an acute disease or acute
condition of a patient
according to the present invention wherein said patient is an ICU patient. In
another embodiment
the anti-ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM
non-Ig
5 scaffold is used in therapy of an acute disease of a patient according to
the present invention,
wherein said patient is critically ill. Critically ill means that the patient
is having a disease or
state in which death is possible or imminent.
Subject of the present invention is further an anti-ADM antibody or an anti-
adrenomedullin
10 antibody fragment or anti-ADM non-Ig scaffold for use in therapy of an
acute disease of a
patient according to the present invention, wherein said antibody or fragment
is to be used in
combination of ADM binding protein. ADM binding protein is also naturally
present in the
circulation of said patient.
15 It should be emphasized that the tenn ADM binding protein also denotes
ADM-binding-protein-
1 (complement factor H), which however is not a non-neutralizing and
modulating anti-ADM
antibody, anti-ADM antibody fragment, or anti-ADM non-Ig scaffold as in
accordance with the
invention.
20 Subject of the present invention is further an anti-ADM antibody or an
anti-adrenomedullin
antibody fragment or anti-ADM non-Ig scaffold for use in therapy of an acute
disease or acute
condition of a patient according to the present invention wherein said
antibody or fragment or
scaffold is to be used in combination with farther active ingredients.
25 Subject matter of the invention is also an anti-Adrenomedullin (ADM)
antibody or an anti-
adrenornedullin antibody fragment or an anti-ADM non-Ig scaffold to be used in
combination
with a primary medicament wherein said combination is for use in therapy of an
acute disease or
acute condition of a patient for regulating the fluid balance of said patient.
Primary medicament means a medicament that acts against the primary cause of
said disease or
30 condition. Said primary medicament may be antibiotics in case of
infections.
It should be emphasized that said primary cause is related to the primary and
underlying disease
or condition, and is not related to the acute disease or acute condition that
is associated with fluid
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imbalance of a patient, for which the herein provided therapy of regulating
the fluid balance is
intended.
In a specific embodiment of the before mentioned combinations said
combinations are to be used
in combination with vasopressors e.g. catecholamine wherein said further
combination is for use
in therapy of an acute disease or condition of a patient for regulating the
fluid balance.
In one embodiment of the invention said patient having a chronic or acute
disease or chronic
condition being in need for regulating the fluid balance is characterized by
the need of the patient
to get administration of vasopressors e.g. of catecholamine.
It should be emphasized that said patient is having a chronic or acute disease
or chronic
condition such as cancer, or diabetes, and thus this can be considered as
primary, underlying
disease, but in addition said patient is in acute need for regulating the
fluid balance that is may be
due to another acute disease or acute condition such as e.g. SIRS, sepsis,
severe sepsis, or shock,
or septic shock.
Subject matter of the invention in one specific embodiment is, thus, an anti-
Adrenomedullin
(ADM) antibody or an anti-adrenomedullin antibody fragment or an anti-ADM non-
Ig scaffold
to be used in combination with ADM binding protein and/or further active
ingredients for use in
therapy of a patient in need of a treatment of vasopressors e.g. catecholamine
treatment.
In a specific embodiment of the above mentioned combinations said combinations
are to be used
in combination with fluids administered intravenously, wherein said
combination is for use in
therapy of an acute disease or condition of a patient for regulating the fluid
balance.
In one embodiment of the invention said patient having a chronic or acute
disease or acute
condition being in need for regulating the fluid balance is characterized by
the need of the patient
to get intravenous fluids.
Subject matter of the invention in one specific embodiment is, thus, an anti-
Adrenomedullin
(ADM) antibody or an anti-adrenomedullin antibody fragment or anti-ADM non-Ig
scaffold in
combination with ADM binding protein and/or further active ingredients for use
in therapy of a
patient in need of intravenous fluids.
Said anti-ADM antibody or an anti-adrenomedullin antibody fragment or anti-ADM
non-Ig
scaffold or combinations thereof with ADM binding protein and/or further
active ingredients
may be used in combination with vasopressors e.g. catecholamine and/or with
fluids
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administered intravenously for use in therapy of an acute disease or acute
condition of a patient
for regulating the fluid balance.
Subject matter of the invention is also an anti-ADM antibody or an anti-
adrenomedullin antibody
Subject of the present invention is further a pharmaceutical formulation
comprising an anti-
Subject of the present invention is further a pharmaceutical formulation
according to the present
invention wherein said pharmaceutical formulation is a solution, preferably a
ready-to-use
15 solution.
Said pharmaceutical formulation may be administered intra-muscular. Said
pharmaceutical
foimulation may be administered intra-vascular. Said pharmaceutical
formulation may be
administered via infusion.
It should be emphasized that the pharmaceutical formulation in accordance with
the invention as
may be administered intra-muscular, intra-vascular, or via infusion is
preferably administered to
a patient for regulating the systemic fluid balance with the proviso that said
patient is in need of
regulating the fluid balance.
Therefore, in another embodiment of the present invention the pharmaceutical
formulation
according to the present invention is to be administered to a patient for
regulating the systemic
fluid balance with the proviso that said patient is in need of regulating the
fluid balance.
any correction of a manifested ¨ imbalance ¨ of a patient's fluid balance due
to an underlying
chronic or acute disease or acute condition. Said correction is in favour of
re-establishing
nonnotension in said patients. The person skilled in the art is fully aware
that blood pressure in
general, as well as hyper- and hypotension is closely related to the fluid
balance of a patient.
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Fluid balance is the balance of the input and the output of fluids in the body
to allow metabolic
processes to function. Dehydration is defined as a 1% or greater loss of body
mass as a result of
fluid loss. The three elements for assessing fluid balance and hydration
status are: clinical
assessment, body weight and urine output; review fluid balance charts and
review of blood
chemistry. All this is very well known to a man skilled in the art (Alison
Shepherd, Nursing
Tomes 19.07.11Nol 107 No 28, pages 12 to 16).
Thus, in one embodiment a person in need of regulating the fluid balance
and/or improving the
fluid balance of such patients is a person that has a 1% or greater loss of
body mass as a result of
fluid loss. The fluid balance may be assessed according to Scales and
Pilsworth (2008) Nursing
Standard 22:47, 50-57. For instance, normal urine output is in the range of
0.5 to 2 ml/kg of body
weight per hour. The minimum acceptable urine output for a patient with normal
renal function
is 0.5 ml/kg per hour. All these standards may be used to assess whether a
patient is in need for
regulating the fluid balance and/or improving the fluid balance.
In another embodiment subject of the present invention is further a
phaunaceutical formulation
according to the present invention wherein said pharmaceutical formulation is
in a dried state to
be reconstituted before use.
In another embodiment subject of the present invention is further a
pharmaceutical formulation
according to the present invention wherein said pharmaceutical formulation is
in a freeze-dried
state.
Further embodiments within the scope of the present invention are set out
below:
I. Adrenomedullin ADM antibody or an adrenomedullin antibody fragment for use
in
therapy of a chronic or acute disease of a patient for the regulation of
liquid balance.
2. ADM antibody or an adrenomedullin antibody fragment according to claim 1
wherein the
antibody format is selected from the group comprising Fv fragment, scFv
fragment, Fab
fragment, seFab fragment, (Fab)2 fragment and seFv-Fe Fusion protein.
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3. ADM antibody or an adrenomedullin antibody fragment according claim 1 or 2
wherein
said antibody or fragment binds to the N-terminal part (aa 1-21) of
adrenomedullin.
4. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
3, wherein said antibody or fragment recognizes and binds to the N-terminal
end (aal) of
adrenomedullin.
5. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
4, wherein said antibody or fragment is an ADM stabilizing antibody or ADM
stabilizing
a antibody fragment that enhances the ti/2 half retention time of
adrenomedullin in serum,
blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %,
most
preferably >100 %.
6. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
5, wherein said antibody or fragment blocks the bioactivity of ADM to less
than 80 %,
preferably less than 50%.
7. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a
chronic or
acute disease of a patient according to any of claims 1 to 6 wherein said
disease is
selected from the group comprising sepsis, diabetis, cancer, heart failure,
shock and
kidney dysfunction.
8. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a
chronic or
acute disease of a patient according to any of claims 1 to 7 wherein said
patient is an ICU
patient.
9. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a
chronic or
acute disease of a patient according to any of claims 1 to 7 wherein said
antibody or
fragment is a modulating antibody or fragment that enhances the t112 half
retention time of
adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50
%, more
preferably >50 %, most preferably >100 A) and that blocks the bioactivity of
ADM to less
than 80 %, preferably less than 50%.
10. Pharmaceutical formulation comprising an antibody or fragment according to
any of
claims 1 to 9.
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11. Pharmaceutical formulation according to claim 10 wherein said
pharmaceutical
founulation is a solution, preferably a ready-to-use solution.
5
12. Pharmaceutical formulation according to claim 10 wherein said
pharmaceutical
formulation is in a freeze-dried state.
13. Phannaceutical formulation according to any of claims 10 to 11, wherein
said
pharmaceutical formulation is administered intra-muscular.
14. Pharmaceutical formulation according to any of claims 10 to 11, wherein
said
pharmaceutical formulation is administered intra-vascular.
15. Phamaceutical formulation according to claim 14, wherein said
pharmaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin ADM antibody or an adrenomedullin antibody fragment an ADM
non-
scaffold for use in therapy of a chronic or acute disease or acute condition
of a patient
for the regulation of fluid balance.
2. ADM antibody or an adrenomedullin antibody fragment or ADM non-1G scaffold
according to claim 1 wherein said ADM antibody or an adrenomedullin antibody
fragment or ADM non-TG scaffold is a non-neutralizing ADM antibody or a non-
neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-IG
scaffold.
3. Adrenornedullin ADM antibody or an adrenomedullin antibody fragment or an
ADM
non-Ig scaffold for use in therapy of a chronic or acute disease or acute
condition
according to claim 1 or 2 for preventing or reducing edema in said patient.
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4. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 3 wherein the antibody format is selected from
the group
comprising Fv fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2
fragment
and scFv-Fc Fusion protein.
5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 4, wherein said antibody or fragment or
scaffold binds to
the N-terminal part (aa 1-21) of adrenomedullin.
6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 5, wherein said antibody or fragment scaffold
recognizes
and binds to the N-terminal end (aal) of adrenomedullin.
7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 6, wherein said antibody or fragment or
scaffold is an
ADM stabilizing antibody or ADM stabilizing antibody fragment or ADM
stabilizing
non-IG scaffold that enhances the half life (ti/2 half retention time) of
adrenomedullin in
serum, blood, plasma at least 10 %, preferably at least 50 %, more preferably
>50 %,
most preferably >100 %.
8. ADM antibody or an adrenornedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 7, wherein said antibody or fragment blocks
the
bioactivity of ADM to less than 80 %, preferably less than 50%.
9. ADM antibody or an adrenomedullin antibody fragment or ADM non-JO scaffold
for use
in therapy of a chronic or acute disease of a patient according to any of
claims 1 to 8
wherein said disease is selected from the group comprising SIRS, sepsis,
diabetis, cancer,
heart failure, shock and kidney dysfunction
10. ADM antibody or an adrenornedullin antibody fragment according to any of
claims 1 to
9, wherein said antibody or fragment is a human monoclonal antibody or
fragment that
binds to ADM or an antibody fragment thereof wherein the heavy chain comprises
the
sequences
SEQ ID NO: 1
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GYTFSRYW
SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
and wherein the light chain comprises the sequences
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
11. A human monoclonal antibody or fragment that binds to ADM or an antibody
fragment
thereof according to claim 10 wherein said antibody or fragment comprises a
sequence
selected from the group comprising:
SEQ ID NO: 7 (AM-VH-C)
QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG
SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGEDYW
GQGTTLTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
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LTSGVHTFPAVLQS S GLYS LS SVVTVP S SSLGTQTYICNVNHKPSNTKVDKRVEP
KHHHHHH
SEQ ID NO: 8 (AM-VHI)
QVQLVQSGAEVKKP GS SVKVS CKASGYTFSRYWISWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTV SS ASTKGP SVFP LAP SSKSTSGGTAALGCLVKDYFPEP VTVSWNS
GALTS GVHTFPAVLQS SGLYSLS SVVTVPSS SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 9 (AM-VH2-E40)
QVQLVQSGAEVKKPGS SVKVS CKASGYTFSRYWIEWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SASTKGP SVFP LAP S SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTS GVHTFPAVLQSSGLYSLS SVVTVPS S S LGTQTYICNVNHKP SNTKVDKRV
EP KHHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP
GS GSTNYAQKF QGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SA STKGP SVFP LAP S SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTS GVHTFP AVLQS S G LY SLS SVVTVP SS SLGTQTYICNVN HKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: II (AM-VH4-T26-E40-E55)
QVQLVQSGAEVKKP GS SVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGEILP
GS GSTNYAQKF QGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYS LS SVVTVP SS SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 12 (AM-VL-C)
DVLLSQTPLS LPVSLGDQATIS CRS SQ S IVYSNGNTYLEWYLQKPGQS PKLLIYRV
SNRFS GVPDRFS GS GS GTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK
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RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 13 (AM-VL1)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEK_HKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
12. ADM antibody or an adrenomedullin antibody fragment or ADM non-JO
scaffold for use
in therapy of a chronic or acute disease of a patient according to any of
claims 1 to 9
wherein said patient is an ICU patient.
13. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold for use
in therapy of a chronic or acute disease of a patient according to any of
claims 1 to 12
wherein said antibody or fragment or scaffold is a modulating antibody or
fragment or
scaffold that enhances the half life (t1/2 half retention time) of
adrenomedullin in serum,
blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %,
most
preferably >100 % and that blocks the bioactivity of ADM to less than 80 %,
preferably
less than 50%.
14. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold for use
in therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 13
to be used in combination with catecholamine and/ or fluids administered
intravenously.
15. ADM antibody or adrenomedullin antibody fragment or ADM non-TG scaffold
for use in
therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 13 or
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a combination according to claim 12 to be used in combination with ADM binding
protein and/or further active ingredients.
16. Pharmaceutical formulation comprising an antibody or fragment or
scaffold according to
5 any of claims Ito 15.
17. Phainiaceutical formulation according to claim 16 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
10 18. Pharmaceutical formulation according to claim 16 wherein said
pharmaceutical
formulation is in a freeze-dried state.
19. Pharmaceutical formulation according to any of claims 16 to 17, wherein
said
pharmaceutical formulation is administered intra-muscular.
20. Pharmaceutical formulation according to any of claims 16 to 17, wherein
said
pharmaceutical fat _____ Ululation is administered intra-vascular.
21. Pharmaceutical formulation according to claim 20, wherein said
pharmaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
I. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for
use in
therapy of a chronic or acute disease of a patient for stabilizing the
circulation.
2. ADM antibody or an adrenomedullin antibody fragment according to claim I
wherein
said antibody or fragment reduces the catecholamine requirement of said
patient,
3. ADM antibody or an adrenomedullin antibody fragment according to claim 1 or
2
wherein the antibody format is selected from the group comprising Fv fragment,
scFv
fragment, Fab fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion
protein.
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4. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 3
wherein said antibody or fragment binds to the N-terminal part (aa 1-21) of
adrenomedullin.
5. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
4, wherein said antibody or fragment recognizes and binds to the N-terminal
end (aal) of
adrenomedullin.
6. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
5, wherein said antibody or fragment is an ADM stabilizing antibody that
enhances the
t1/2 half retention time of adrenomedullin in serum, blood, plasma at least 10
%,
preferably at least, 50 %, more preferably > 50 %, most preferably >100 %.
7. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
6, wherein said antibody or fragment blocks the bioactivity of ADM to less
than 80 %,
preferably less than 50 %.
8. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
7, wherein said antibody or fragment is a modulating ADM antibody or a
modulating
adrenomedullin antibody fragment that enhances the t1/2 half retention time of
adrenomedullin in serum, blood, plasma at least 10 %, preferably at least, 50
%, more
preferably > 50 %, most preferably >100 % and that blocks the bioactivity of
ADM to
less than 80 %, preferably less than 50 %:
9. ADM antibody or an adrenomedullin antibody fragment for use in therapy of a
chronic or
acute disease of a patient according to any of the claims 1 to 8 wherein said
disease is
selected from the group comprising sepsis, diabetis, cancer, acute and chronic
vascular
diseases as e.g. heart failure, shock as e.g. septic shock and organ
dysfunction as e.g.
kidney dysfunction.
10. Pharmaceutical formulation comprising an antibody according to any of
claims 1 to 9.
11. Pharmaceutical foimulation according to claim 10 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
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12. Pharmaceutical formulation according to claim 10 wherein said
phaimaceutical
formulation is in a freeze-dried state.
13. Pharmaceutical formulation according to any of claims 10 to 11, wherein
said
pharmaceutical formulation is administered intra-muscular.
14. Pharmaceutical formulation according to any of claims 10 to 11, wherein
said
pharmaceutical formulation is administered intra-vascular.
15. Pharmaceutical formulation according to claim 14, wherein said
phainiaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment
or an ADM
non-IG scaffold for use in therapy of a chronic or acute disease or condition
of a patient for
stabilizing the circulation.
2. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to claim 1 wherein said antibody or fragment or scaffold reduces the
vasopressor
requirement, e.g. catecholamine requirement of said patient.
3. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to claim 1 or 2 wherein said ADM antibody or an adrenomedullin
antibody
fragment or ADM non-IG scaffold is a non-neutralizing ADM antibody or a non-
neutralizing adrenomedullin antibody fragment or a non-neutralizing ADM non-IG
scaffold.
4. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 3
wherein the antibody format is selected from the group comprising Fv fragment,
scFv
fragment, Fab fragment, scFab fragment, (Fab)2 fragment and seFv-Fc Fusion
protein.
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5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 4 wherein said antibody or fragment or
scaffold binds to the
N-terminal part (aa 1-21) of adrenomedullin.
6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 5, wherein said antibody or fragment or
scaffold recognizes
and binds to the N-terminal end (aal) of adrenomedullin_
7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 6, wherein said antibody or fragment or
scaffold is an ADM
stabilizing antibody or fragment or scaffold that enhances the half life (t1/2
half retention
time) of adrenomedullin in serum, blood, plasma at least 10 %, preferably at
least, 50 %,
more preferably > 50 %, most preferably >100 %.
8. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 7, wherein said antibody or fragment or
scaffold blocks the
bioactivity of ADM to less than 80 %, preferably less than 50 A.
9. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 8, wherein said antibody or fragment or
scaffold is a
modulating ADM antibody or a modulating adrenomedullin antibody fragment or
scaffold
that enhances the half life (t1/2 half retention time) of adrenomedullin in
serum, blood,
plasma at least 10 Vo, preferably at least, 50 %, more preferably > 50 %, most
preferably
>100 % and that blocks the bioactivity of ADM to less than 80 %, preferably
less than 50
%:
10. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
9, wherein said antibody or fragment is a human monoclonal antibody or
fragment that
binds to ADM or an antibody fragment thereof wherein the heavy chain comprises
the
sequences
SEQ ID NO: I
GYTFSRYW
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SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
and wherein the light chain comprises the sequences
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
11. A human monoclonal antibody or fragment that binds to ADM Or an
antibody fragment
thereof according to claim 10 wherein said antibody or fragment comprises a
sequence
selected from the group comprising:
SEQ ID NO: 7 (AM-VH-C)
QVQLQQSGAELMKPGASVKISCKATGYTTSRYWIEWVKQRPGHGLEWIGEILPG
SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGEDYW
GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KHHHHHH
SEQ ID NO: 8 (AM-VHI)
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QVQLVQ SGAEVKKP GS SVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP
GS GSTNY AQKFQ GRVTITADESTS TAYMELS SLRS ED TAVYY CTE GYEYDGFDY
WGQGTTVTVS SA STKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTS GVHTFPAVLQS SGLYS LS S VVTVP S S SLGTQTYICNVNHICPSNTICVD KRV
5 EPICHHHHHH
SEQ ID NO: 9 (AM-VH2-E40)
QVQ LVQ SGAEVKKP GS SVKVS CKA SGYTF SRYWIEWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
10 GALTS GVHTFPAVLQS SGLY S LS S VVTVP S S SLGTQTYICNVNHKPSNTKVDKRV
EP KHHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQSGAEVICKP GS SVKVS CICATGYTFSRYWIS WVRQAPGQGLEWMGEILP
GS GS TNYAQKFQGRVTITADESTS TAYMELS SLRSED TAVYYCTEGY EYDGFDY
Is WGQGTTVTVS SAS TKGPSVFPLAP S SKS TSGGTAALGCLVKDYFPEPVTVSWNS
GALT S GVHTFPAVLQS S G LYS LS S VVTVP S S SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 11 (AM-VH4-T26-E40-E55)
QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYWIEWVRQAPGQGLEWMGE1LP
20 GS GS TNYAQKFQGRVTITADESTS TAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SAS TKGPSVFPLAP S SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDICRV
EPICHHHHHH
SEQ ID NO: 12 (AM-VL-C)
25 DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK
RTVAAP SVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSICD S TYS LS S TLTL SKADYEKHKVYACEVTHQ GL S SPVTKSFNRGEC
SEQ ID NO: 13 (AM-VLI)
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DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
12. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
for use
in therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 11
wherein said disease is selected from the group comprising SIRS, sepsis,
diabetis, cancer,
acute and chronic vascular diseases as e.g. heart failure, shock as e.g.
septic shock and
organ dysfunction as e.g kidney dysfunction.
13. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
for use
in therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 12
to be used in combination with catecholamine and/ or fluids administered
intravenously.
14. ADM antibody or adrenomedullin antibody fragment or ADM non-JO scaffold
for use in
therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 13 or a
combination according to claim 10 to be used in combination with ADM binding
protein
and/or further active ingredients.
15. Pharmaceutical formulation comprising an antibody or fragment or non-IG
scaffold
according to any of claims Ito 14.
16. Pharmaceutical formulation according to claim 15 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
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17. Pharmaceutical formulation according to claim 15 wherein said
pharmaceutical
formulation is in a freeze-dried state.
18. Pharmaceutical formulation according to any of claims 15 to 16, wherein
said
pharmaceutical formulation is administered intra-muscular.
19. Pharmaceutical formulation according to any of claims 14 to 16, wherein
said
pharmaceutical formulation is administered intra-vascular.
20. Pharmaceutical formulation according to claim 16, wherein said
pharmaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
1) Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a
treatment of
a chronic or acute disease wherein said antibody or said fragment is an ADM
stabilizing
antibody or fragment that enhances the t112 half retention time of
adrenomedullin in serum,
blood, plasma at least 10 %, preferably at least, 50 %, more preferably >50 %,
most
preferably 100 % and/or wherein said antibody blocks the bioactivity of ADM to
less than
80 %, preferably to less than 50 A.
2) Adrenomedullin antibody or an adrenomedullin antibody fragment for use
in a treatment of
a chronic or acute disease wherein said antibody or said fragment is a
modulating ADM
antibody or fragment that enhances the tii2 half retention time of
adrenomedullin in serum,
blood, plasma at least 10 %, preferably at least, 50 A, more preferably >50
A, most
preferably 100 % and that blocks the bioactivity of ADM to less than 80 %,
preferably to
less than 50 %.
3) Adrenomedullin antibody or an adrenomedullin antibody fragment for use
in a treatment of
a chronic or acute disease according to claim 1 or 2, wherein said antibody or
fragment
binds to the N-terminal part (aa 1-21) of adrenomedullin.
4) Adrenomedullin antibody or an adrenomedullin antibody fragment for use in a
treatment of
a chronic or acute disease wherein said antibody or said fragment according to
claim 3
binds to the N-teiiiiinal end of adrenomedullin.
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5) Adrenomedullin antibody or an adrenomedullin antibody fragment for use
in use in a
treatment of a chronic or acute disease according to any of claims 1 to 4,
wherein said
antibody or said fragment is an ADM stabilizing antibody or fragment that
enhances the tin
half retention time of adrenomedullin in serum, blood, plasma at least 10 %,
preferably at
least, 50 %, more preferably >50 %, most preferably 100 %.
6) Adrenomedullin antibody or an adrenomedullin antibody fragment for use
in a treatment of
a chronic or acute disease according to any of claims 1 to 5, wherein said
antibody or said
fragment blocks the bioactivity of ADM to less than 80 %, preferably to less
than 50 %.
7) Adrenomedullin antibody or an adrenomedullin antibody fragment according
to any of the
claims 1 to 6 for use in a treatment of a chronic or acute disease wherein
said disease is
selected from the group comprising SIRS, sepsis, septic shock, diabetis,
cancer, heart
failure, shock, organ failure, kidney dysfunction, acute liquid dysbalance,
and low blood
pressure.
8) Adrenomedullin antibody or an adrenomedullin antibody fragment according
to any of the
claims 1 to 7 for use in a treatment of a chronic or acute disease wherein
said disease is
septic shock or sepsis.
9) Adrenomedullin antibody or an adrenomedullin antibody fragment for use
in a treatment of
a chronic or acute disease according to any of the claims 1 to 8 wherein said
antibody or
fragment regulates the liquid balance of said patient.
10) Adrenomedullin antibody or an adrenomedullin antibody fragment for use in
a treatment of
a chronic or acute disease according to any of the claims 1 to 9 wherein said
antibody or
fragment used for prevention of organ dysfunction or organ failure.
11) Adrenomedullin antibody or an adrenomedullin antibody fragment for use in
a treatment of
a chronic or acute disease according to claim 10 wherein said antibody or
fragment is used
for prevention of kidney dysfunction or kidney failure.
12) Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for
use in a
treatment of a chronic or acute disease in a patient according to claims 1 to
11 wherein said
antibody or fragment is used for stabilizing the circulation.
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13) ADM antibody or an adrenomedullin antibody fragment for use in a
treatment of a chronic
or acute disease in a patient according to claim 12 wherein said antibody or
fragment
reduces the catecholamine requirement of said patient.
14) ADM antibody or an adrenomedullin antibody fragment for use in a
treatment of a chronic
or acute disease in a patient according to any of claims I to 13 for the
reduction of the
mortality risk for said patient.
15) ADM antibody or an adrenomedullin antibody fragment for use in a
treatment of a chronic
or acute disease in a patient according to any of claims 1 to 14 wherein said
antibody or
fragment may be administered in a dose of at least 3 jig / Kg body weight.
Da
16) Pharmaceutical composition comprising an antibody or fragment according to
any of
claims 1 to 15.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
Ig
scaffold wherein said antibody or said fragment or scaffold is a non-
neutralizing
antibody.
2. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
Ig
scaffold wherein said antibody or said fragment or scaffold is an ADM
stabilizing
antibody or fragment or scaffold that enhances the half life (t112 half
retention time) of
adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50
%, more
preferably >50 %, most preferably 100 % and/or wherein said antibody or
fragment or
scaffold blocks the bioactivity of ADM to less than 80 %, preferably to less
than 50 %.
3. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
Ig
scaffold wherein said antibody or said fragment is a modulating ADM antibody
or
fragment or scaffold that enhances the half life (t112 half retention time) of
adrenomedullin
in serum, blood, plasma at least 10 %, preferably at least, 50 %, more
preferably >50 %,
most preferably 100 % and that blocks the bioactivity of ADM to less than 80
%,
preferably to less than 50 %.
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4. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
1g
scaffold according to claim 1 or 2, wherein said antibody or fragment or
scaffold binds to
the N-terminal part (aa 1-21) of adrenomedullin.
5. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
Ig
5 scaffold wherein said antibody or said fragment or scaffold according to
claim 3 binds to
the N-teiminal end of adrenomedullin.
6. Adrenomedullin antibody or an adrenomedullin antibody fragment ADM non-Ig
scaffold
according to any of claims 1 to 4, wherein said antibody or said fragment or
said scaffold
is an ADM stabilizing antibody or fragment that enhances the t112 half
retention time of
10 adrenomedullin in serum, blood, plasma at least 10 %, preferably at
least, 50 %, more
preferably >50 %, most preferably 100 %.
7. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
Ig
scaffold according to any of the claims 1 to 6 for use as an active
pharmaceutical
substance.
15 8. Adrenomedullin antibody or an adrenomedullin antibody fragment ADM
non-Ig scaffold
according to any of the claims 1 to 7 for use in a treatment of a chronic or
acute disease
or acute condition wherein said disease or condition is selected from the
group
comprising severe infections as e.g. meningitis, systemic inflammatory
Response-
Syndrome (SIRS) sepsis; other diseases as diabetes, cancer, acute and chronic
vascular
20 diseases as e.g. heart failure, myocardial infarction, stroke,
atherosclerosis; shock as e.g.
septic shock and organ dysfunction as e.g. kidney dysfunction, liver
dysfunction,
bumings, surgery, traumata.
9. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM non-
1g
scaffold according to any of the claims 1 to 8 for use in a treatment of a
chronic or acute
25 disease or acute condition wherein said disease is septic shock or
sepsis.
10. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
9, wherein said antibody or fragment is a human monoclonal antibody or
fragment that
binds to ADM or an antibody fragment thereof wherein the heavy chain comprises
at
least one of the sequences:
30 SEQ ID NO: 1
GYTFSRYW
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SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
And/or wherein the light chain comprises the at least one of the sequences
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
11. A human monoclonal antibody or fragment that binds to ADM or an
antibody fragment
thereof according to claim 10 wherein said antibody or fragment comprises a
sequence
selected from the group comprising:
SEQ ID NO: 7 (AM-VH-C)
QVQLQQSGAELMKPGASVKISCKATGYTESRYWIEWVKQRPGHGLEWIGEILPG
SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW
GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KHHHHHH
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SEQ ID NO: 8 (AM-VH1)
QVQLVQS GAEVKKPGS SVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP
GS GSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTV S SAS TKGP SVFPLAP SSKST S GGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQS SGLYS LS SVVTVPS S SLGTQTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 9 (AM-VH2-E40)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP
GS GSTNYAQKFQGRVTITADESTSTAYMELS S LRS ED TAVYYCTEGYEYDGFDY
WGQGTTVTVS SAS TKGP SVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTS GVHTFP AVLQ S SGLY S LS SVVTVP S S SLGT QTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQS GAEVKKP GS SVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP
GS GSTNYAQKFQGRVTITADESTSTAYMELS SLRS ED TAVYYCTEGYEYDGFDY
WGQGTTVTVS SASTKGP SVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQ SSGLYSLS SVVTVP SS SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 11 (AM-VH4-T26-E40-E55)
QVQLVQS GAEVKKP G S S VKVS CKATGYTF SRYWIEWVRQAP GQ G LEWMGEILP
GS GSTNYAQKFQGRVTITADESTSTAYMELS SLRS ED TAVYYCTEGYEYDGFDY
WGQGTTVTVS SASTKGP SVFPLAP S SKS TS GGTAALGCLVKDYFP EPVTV SWNS
GALTSGVHTFPAVLQS S GLYS LS SVVTVP S S SLGTQTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 12 (AM-VL-C)
DVLLS QTP LS LPV S LGDQATIS CRS SQSIVYSNGNTYLEWYLQKPGQSPKLLIYRV
SNRF SGVPDRFS G SGSGTDFTLKISRVEAED LGVYYCFQG SHIP YTFGGGTKLEIK
RTVAAP SVFIFPP S DEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQS GNS QE
SVTEQD SKD S TY S LS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRG EC
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SEQ ID NO: 13 (AM-VL1)
DVVMTQSPLSLPVTLGQPAS1SCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTEGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQE
I 0 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTFIQGLSSPVTKSFNRGEC
12. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to any of the claims 1 to 11 for regulating the fluid
balance in a patient
having a chronic or acute disease or acute condition. .
13. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to any of the claims 1 to 11 for preventing or reducing
organ
dysfunction or organ failure in a patient having in a chronic or acute disease
or acute
condition.
14. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to claim 10 wherein organ is kidney or liver.
15. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or
ADM non-
Ig scaffold according to claims 1 to 14 for stabilizing the circulation in a
patient having a
chronic or acute disease or acute condition.
16. ADM antibody or an adrenomedullin antibody fragment or ADM non-Ig
scaffold for use
in a treatment of a chronic or acute disease in a patient according to claim
15 wherein
said antibody or fragment reduces the catecholamine requirement of said
patient.
17. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to any of the claims 1 to 16 to be used in combination with
vasopressors e.g. catecholamine.
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18. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to any of the claims 1 to 17 to be used in combination with
intravenous fluid administration.
19. Adrenomedullin antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold according to any of the claims 1 to 18 to be used in combination with
an TNF-
alpha-antibody.
20. ADM antibody or an adrenomedullin antibody fragment or non-lg-scaffold
according to
any of claims 1 to 19 for use in a treatment of a patient in need thereof
wherein said
antibody or fragment may be administered in a dose of at least 3 pg / Kg body
weight.
21. Pharmaceutical composition comprising an antibody or fragment or
scaffold according to
any of claims Ito 20.
22. ADM antibody or an adrenomedullin antibody fragment or non-Ig-scaffold
according to
any of claims 1 to 20 for use in a treatment of a chronic or acute disease or
chronic
condition.
23. ADM antibody or an adrenomedullin antibody fragment or non-Ig-scaffold
according to
claim 22 wherein said disease is sepsis.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin ADM antibody or an adrenomedullin antibody fragment for
use in
therapy of a severe chronical or acute disease of a patient for the reduction
of the
mortality risk for said patient.
2. ADM antibody or an adrenomedullin antibody fragment according to claim 1
wherein the
antibody format is selected from the group comprising Fv fragment, say
fragment, Fab
fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion protein.
3. ADM antibody or an adrenomedullin antibody fraguaent according claim 1
or 2 wherein
said antibody or fragment binds to the N-terminal part (aa 1-21) of
adrenomedullin.
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4. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
3, wherein said antibody or fragment recognizes and binds to the N-terminal
end (aal) of
adrenomedullin.
5 5. ADM antibody or an adrenomedullin antibody fragment according to
any of claims I to
4, wherein said antibody or fragment is an ADM stabilizing antibody or
fragment that
enhances the tl /2 half retention time of adrenomedullin in serum, blood,
plasma at least
10 %, preferably at least, 50 %, more preferably > 50 %, most preferably > 100
%.
10 6. ADM antibody or an adrenomedullin antibody fragment according to
any of claims 1 to
5, wherein said antibody or fragment blocks the bioactivity of ADM to less
than 80 %,
preferably less than 50%.
7. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical
15 or acute disease of a patient according to any of claims 1 to 6 wherein
said disease is
selected from the group comprising sepsis, diabetis, cancer, heart failure,
shock and
kidney dysfunction.
8. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical
20 or acute disease of a patient according to any of claims 1 to 7 wherein
said patient is an
ICU patient.
9. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical
or acute disease of a patient according to any of claims 1 to 8 wherein the
mortality risk
25 is reduced by preventing adverse event wherein the latter are selected
from the group
comprising SIRS, sepsis, septic shock, organ failure, kidney failure, liquid
dysbalance
and low blood pressure.
10. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical
30 or acute disease of a patient according to any of claims I to 8 wherein
said antibody or
fragment is to be used in combination of ADM binding protein.
11. Pharmaceutical founulation comprising an antibody or fragment according
to any of
claims 1 to 10.
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12. Pharmaceutical foimulation according to claim 11 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
13. Pharmaceutical foimulation according to claim 11 wherein said
pharmaceutical
formulation is in a freeze-dried state.
14. Pharmaceutical formulation according to any of claims 11 to 12, wherein
said
pharmaceutical foimulation is administered intra-muscular.
15. Pharmaceutical formulation according to any of claims 11 to 12, wherein
said
pharmaceutical formulation is administered intra-vascular.
16. Pharmaceutical formulation according to claim 15, wherein said
pharmaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM
non-
Ig scaffold for use in therapy of a severe chronical or acute disease or acute
condition of a
patient for the reduction of the mortality risk for said patient wherein said
antibody or
fragment or scaffold is a non-neutralizing ADM antibody or a non-neutralizing
adrenomedullin antibody fragment or a non-neutralizing ADM non-Ig scaffold.
2. ADM antibody or an adrenomedullin antibody fragment according to claim I
wherein the
antibody format is selected from the group comprising Fv fragment, scFv
fragment, Fab
fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion protein.
3. ADM antibody or an adrenomedullin antibody fragment or an ADM non-1g
scaffold
according claim 1 or 2 wherein said antibody or fragment or scaffold binds to
the N-
terminal part (aa 1-21) of adrenomedullin.
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4. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold
according to any of claims 1 to 3, wherein said antibody or fragment or
scaffold
recognizes and binds to the N-terminal end (aal) of adrenomedullin.
5. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold
according to any of claims I to 4, wherein said antibody or fragment or
scaffold is an
ADM stabilizing antibody or fragment or scaffold that enhances the half life
(t1/2 half
retention time) of adrenomedullin in serum, blood, plasma at least 10 %,
preferably at
least, 50 %, more preferably > 50 %, most preferably > 100 %.
6. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold
according to any of claims 11 to 5, wherein said antibody or fragment or
scaffold blocks
the bioactivity of ADM to less than 80 %, preferably less than 50%.
7. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold for
use in therapy of a chronical or acute disease of a patient according to any
of claims 1 to
6 wherein said disease is selected from the group comprising severe infections
as e.g.
meningitis, Systemic inflammatory Response-Syndrom (SIRS,) sepsis; other
diseases as
diabetis, cancer, acute and chronic vascular diseases as e.g. heart failure,
myocardial
infarction, stroke, atherosclerosis; shock as e.g. septic shock and organ
dysfunction as
e.g. kidney dysfunction, liver dysfunction; bumings, surgery, traumata.
8. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold for
use in therapy of a chronical or acute disease of a patient according to any
of claims 1 to
7 wherein said disease is selected from the group comprising SIRS, a severe
infection,
sepsis, shock e.g. septic shock.
9. ADM antibody or an adrenomedullin antibody fragment or an ADM non-Ig
scaffold for
use in therapy of a chronical or acute disease or acute condition of a patient
according to
any of claims 1 to 8 wherein said patient is an ICU patient. ADM antibody or
an
adrenomedullin antibody fragment or an ADM non-Ig scaffold for use in therapy
of a
chronical or acute disease or acute condition of a patient according to any of
claims I to 9
wherein the mortality risk is reduced by preventing an adverse event wherein
the latter
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are selected from the group comprising SIRS, sepsis, shock as e.g. septic
shock, acute
and chronic vascular diseases as e.g. acute heart failure, myocardial
infarction, stroke;
organ failure as e.g, kidney failure, liver failure, fluid dysbalance and low
blood pressure.
10. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to
9, wherein said antibody or fragment is a human monoclonal antibody or
fragment that
binds to ADM or an antibody fragment thereof wherein the heavy chain comprises
the
sequences
SEQ ID NO: 1
GYTFSRYW
SEQ ID NO: 2
ILPGSGST
SEQ ID NO: 3
TEGYEYDGFDY
and wherein the light chain comprises the sequences
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
RVS
SEQ ID NO: 6
FQGSHIPYT.
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12. A human monoclonal antibody or fragment that binds to ADM or an
antibody fragment
thereof according to claim 10 wherein said antibody or fragment comprises a
sequence
selected from the group comprising:
SEQ ID NO: 7 (AM-VH-C)
QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG
SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW
GQGTTLTVS SAS TKGP SVFP LAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LT SGVHTFPAVLQ S S GLYSLS SVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEP
KHHHHHH
SEQ ID NO: 8 (AM-VH1)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQ GTTVTVS SAS TKGPSVFP LAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQ S S GLY S LS SVVTVP S SSLGTQTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 9 (AM-VH2-E40)
QVQLVQSGAEVKKP GSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALT S GVHTFPAVLQSSGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQSGAEVKKPGSSVKVSCKATGYTF SRYWISWVRQAPGQGLEWMGEILP
GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGEDY
WGQGTTVTVSSASTKGP SVFP LAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALT SGVHTFPAVLQ S SGLYSLS SVVTVP SS SLGTQTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 11 (AM-VH4-T26-E40-E55)
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QVQLVQSGAEVKKPGSSVKVSCKATGYTESRYWIEWVRQAPGQGLEWMGEILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV
5 EPKHHHHHH
SEQ ID NO: 12 (AM-VL-C)
DVLLSQTPLSLPVSLGDQATISCRSSQSIVYSNGNTYLEWYLQKPGQSPKWYRV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTEGGGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
10 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 13 (AM-VL1)
DVVMTQSPLSLPVTLGQPAS1SCRSSQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
15 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
20 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC
13. ADM antibody or an adrenomedullin antibody fragment or ADM non-IC
scaffold for use
in therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 12
to be used in combination with vasopressors e.g. catecholamine and/ or fluids
25 administered intravenously.
14. ADM antibody or adrenomedullin antibody fragment or ADM non-IC scaffold
for use in
therapy of a chronic or acute disease of a patient according to any of the
claims 1 to 13 or
a combination according to claim 10 to be used in combination with ADM binding
30 protein and/or further active ingredients.
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15. Pharmaceutical formulation comprising an antibody or fragment or
scaffold according to
any of claims 1 to 14.
16. Pharmaceutical formulation according to claim 15 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
17. Pharmaceutical formulation according to claim 15 wherein said
pharmaceutical
formulation is in a freeze-dried state.
18. Phainiaceutical formulation according to any of claims 15 to 16,
wherein said
pharmaceutical formulation is administered intra-muscular.
19. Pharmaceutical formulation according to any of claims 15 to 16, wherein
said
pharmaceutical formulation is administered intra-vascular.
20. Pharmaceutical fotinulation according to claim 19, wherein said
pharmaceutical
formulation is administered via infusion.
21. ADM antibody or an Adrenomedullin antibody fragment or AM non-Ig
scaffold, wherein
said antibody or fragment or scaffold binds to the N-terminal part (aa 1-21)
of
Adrenomedullin in, preferably human ADM.
22. Antibody or fragment or scaffold according to claim 2, wherein said
antibody or
fragment or scaffold recognizes and binds to the N-terminal end (aa 1) of
Adrenomedullin.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment for
use in
therapy of a chronical or acute disease of a patient for prevention of organ
dysfunction or
organ failure.
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2. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical or
acute disease according to claim I wherein said organ is kidney.
3. ADM antibody or an adrenomedullin antibody fragment according to claim 1
wherein the
antibody format is selected from the group comprising Fv fragment, scFv
fragment, Fab
fragment, scFab fragment, (Fab)2 fragment and scFv-Fc Fusion protein.
4. ADM antibody or an adrenomedullin antibody fragment according any of
claims 1 to 3
wherein said antibody or fragment binds to the N-terutinal part (aa 1-21) of
adrenomedullin.
5. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 4,
wherein said antibody or fragment recognizes and binds to the N-terminal end
(aal) of
adrenornedullin.
6. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 5,
wherein said antibody or said fragment is an ADM stabilizing antibody or
fragment that
enhances the t1/2 half retention time of adrenomedullin in serum, blood,
plasma at least 10
%, preferably at least 50 %, more preferably >50 %, most preferably >100%.
7. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 6,
wherein said antibody blocks the bioactivity of ADM to less than 80 %,
preferably less
than 50%.
8. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical or
acute disease of a patient according to any of claims I to 7 wherein said
disease is selected
from the group comprising sepsis, diabetis, cancer, heart failure, and shock.
9. ADM antibody or an adrenomedullin antibody fragment for use in therapy
of a chronical or
acute disease of a patient according to any of claims I to 8 wherein said
patient is an ICU
patient.
10. ADM antibody or an adrenomedullin antibody fragment for use in therapy of
a chronical or
acute disease of a patient according to any of claims I to 9 wherein said
antibody or
fragment is a modulating antibody or fragment that enhances the t1/2 half
retention time of
adrenomedullin in serum, blood, plasma at least 10 %, preferably at least 50
A, more
preferably >50 %, most preferably >100% and that blocks the bioactivity of ADM
to less
than 80 %, preferably less than 50%.
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11. Pharmaceutical formulation comprising an antibody or fragment according
to any of claims
Ito 10.
12. Pharmaceutical formulation according to claim 11 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
13. Phaimaceutical formulation according to claim 11 wherein said
pharmaceutical
formulation is in a freeze-dried state.
14. Pharmaceutical formulation according to any of claims 11 to 12, wherein
said
pharmaceutical formulation is administered intra-muscular.
15. Pharmaceutical formulation according to any of claims 11 to 12, wherein
said
pharmaceutical formulation is administered intra-vascular.
16. Pharmaceutical formulation according to claim 15, wherein said
pharmaceutical
formulation is administered via infusion.
Further embodiments within the scope of the present invention are set out
below:
1. Adrenomedullin (ADM) antibody or an adrenomedullin antibody fragment or ADM
non-Ig
scaffold for use in therapy of a chronical or acute disease or acute condition
of a patient for
prevention or reduction of organ dysfunction or prevention of organ failure in
said patient.
2. ADM antibody or an adrenomedullin antibody fragment or ADM non-Ig
scaffold for use in
therapy of a chronical or acute disease or acute disease according to claim 1
wherein said
organ is kidney or liver.
3. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold according
to claim 1 or 2 wherein said ADM antibody or an adrenomedullin antibody
fragment or
ADM non-IG scaffold is a non-neutralizing ADM antibody or a non-neutralizing
adrenomedullin antibody fragment or a non-neutralizing ADM non-1G scaffold
4. ADM antibody or an adrenomedullin antibody fragment or ADM non-JO
scaffold according
to any of claims 1 or 3 wherein the antibody format is selected from the group
comprising Fv
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fragment, scFv fragment, Fab fragment, scFab fragment, (Fab)2 fragment and
scFv-Fc
Fusion protein.
5. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold according
any of claims 1 to 4 wherein said antibody or fragment or scaffold binds to
the N-terminal
part (aa 1-21) of adrenomedullin.
6. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold according
to any of claims 1 to 5, wherein said antibody or fragment Or scaffold
recognizes and binds to
the N-terminal end (aal) of adrenomedullin.
7. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG scaffold
according to any of claims 1 to 6, wherein said antibody or said fragment or
scaffold is an
ADM stabilizing antibody or fragment or scaffold that enhances the half life
(t1/2 half
retention time) of adrenomedullin in serum, blood, plasma at least 10 %,
preferably at least
50 %, more preferably >50 %, most preferably >100%.
8. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold according
to any of claims 1 to 7, wherein said antibody or fragment or scaffold blocks
the bioactivity
of ADM to less than 80 %, preferably less than 50%.
9. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold for use in
therapy of a chronical or acute disease or acute condition of a patient
according to any of
claims 1 to 8 wherein said disease is selected from the group comprising
sepsis, diabetis,
cancer, heart failure, and shock.
10. ADM antibody or an adrenomedullin antibody fragment according to any of
claims 1 to 9,
wherein said antibody or fragment is a human monoclonal antibody or fragment
that binds to
ADM or an antibody fragment thereof wherein the heavy chain comprises the
sequences
SEQ ID NO: 1
GYTFSRYW
SEQ ID NO: 2
ILPGSGST
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SEQ ID NO: 3
TEGYEYDGFDY
5 and wherein the light chain comprises the sequences
SEQ ID NO:4
QSIVYSNGNTY
SEQ ID NO: 5
10 RVS
SEQ ID NO: 6
FQGSHIPYT.
15 11. A human monoclonal antibody or fragment that binds to ADM or an
antibody fragment
thereof according to claim 10 wherein said antibody or fragment comprises a
sequence
selected from the group comprising:
SEQ ID NO: 7 (AM-VH-C)
20 QVQLQQSGAELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPG
SGSTNYNEKFKGKATITADTSSNTAYMQLSSLTSEDSAVYYCTEGYEYDGFDYW
GQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP
KHHHHHH
25 SEQ ID NO: 8 (AM-VH1)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWISWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
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GALTSGVHTFPAVLQ S SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 9 (AM-VH2-E40)
QVQLVQSGAEVKKPGS SVKVSCKASGYTF SRYWIEWVRQAPGQGLEWMGRILP
GSGSTNYAQKFQGRVTITADESTSTAYMELS SLRS ED TAVYYCTEGYEYD GFDY
WGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALT S GVHTFPAVLQ S SGLYS LS SVVTVP S SSLGTQTYICNVNHKP SNTKVDKRV
EPKHHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQSGAEVKKPGS SVKVSCKATGYTFSRYWISWVRQAPGQGLEWMGEILP
G SG STNYAQKFQ GRVTITADES TSTAYMELS SLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SAS TKGP SVFP LAP S SKSTS GGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQS S GLY S LS SVVTVPS S SLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 11 (AM-VH4-T26-E40-E55)
QVQLVQS GAEVKKP GS SVKVSCKATGYITSRYWIEWVRQAPGQGLEWMGEILP
GSGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDY
WGQGTTVTVS SASTKGP SVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV
EPKHHHHHH
SEQ ID NO: 12 (AM-VL-C)
DVLLS QTP LS LP VS LGD QATIS CRS SQSIVY SNGNTYLEWYLQKPGQSPKLLIYRV
SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFGGGTKLEIK
RTVAAP SVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQE
SVTEQD SKDSTYS LS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
SEQ ID NO: 13 (AM-VL1)
DVVMTQS P LS LPVTLGQPASIS CRS SQSIVYSNGNTYLNWFQQRPGQSPRRLIYRV
SNRD SGVPD RF S GSGS GTDFTLKIS RVEAED VGVYYCF QGSHIPYTFGQGTKLEIK
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RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSQSIVYSNGNTYLEWFQQRPGQSPRRLIYRV
SNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIK
RTVAAPSVFIFFPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC
12. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold for use
in therapy of a chronical or acute disease of a patient according to any of
claims 1 to 11
wherein said antibody or fragment or scaffold is a modulating antibody or
fragment or
scaffold that enhances the half life (t1/2 half retention time) of
adrenomedullin in serum,
blood, plasma at least 10 %, preferably at least 50 %, more preferably >50 %,
most
preferably >100% and that blocks the bioactivity of ADM to less than 80 %,
preferably
less than 50%.
13. ADM antibody or an adrenomedullin antibody fragment or ADM non-IG
scaffold for
use in therapy of a chronic or acute disease or acute condition of a patient
according to
any of the claims 1 to 12 to be used in combination with vasopressors
e.g.catecholamine
and/ or fluids administered intravenously.
14. ADM antibody or adrenomedullin antibody fragment or ADM non-IG scaffold
for use
in therapy of a chronic or acute disease or acute condition of a patient
according to any of
the claims 1 to 13 or a combination according to claim 13 to be used in
combination with
ADM binding protein and/or further active ingredients.
15. Pharmaceutical formulation comprising an antibody or fragment according
to any of
claims Ito 13.
16. Phaimaceutical formulation according to claim 14 wherein said
pharmaceutical
formulation is a solution, preferably a ready-to-use solution.
17. Pharmaceutical formulation according to claim 14 wherein said
pharmaceutical
formulation is in a freeze-dried state.
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18. Pharmaceutical formulation according to any of claims 14 to 15, wherein
said
pharmaceutical formulation is administered intra-muscular.
19. Phaimaceutical formulation according to any of claims 14 to 15, wherein
said
pharmaceutical formulation is administered intra-vascular.
20. Pharmaceutical formulation according to claim 18, wherein said
pharmaceutical
folmulation is administered via infusion.
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EXAMPLES
It should be emphasized that the antibodies, antibody fragments and non-Ig
scaffolds of the
example portion in accordance with the invention are binding to ADM, and thus
should be
considered as anti-ADM antibodies/antibody fragments/non-Ig scaffolds.
Example I
Generation of Antibodies and determination of their affinity constants
Several human and murine antibodies were produced and their affinity constants
were
determined (see tables 1 and 2).
Peptides/ conjugates for Immunization:
Peptides for immunization were synthesized, see Table 1, OPT Technologies,
Berlin, Germany)
with an additional N-terminal Cystein (if no Cystein is present within the
selected ADM-
sequence) residue for conjugation of the peptides to Bovine Serum Albumin
(BSA). The
peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-
science, Bonn,
Germany). The coupling procedure was perfoinied according to the manual of
Perbio.
The murine antibodies were generated according to the following method:
A Balb/c mouse was immunized with 10011g Peptide-BSA-Conjugate at day 0 and 14
(emulsified in 100 1 complete Freund's adjuvant) and 501.ig at day 21 and 28
(in 1001.11
incomplete Freund's adjuvant). Three days before the fusion experiment was
performed, the
animal received 50 jig of the conjugate dissolved in 100111 saline, given as
one intraperitoneal and
one intra-venous injection.
Spenocytes from the immunized mouse and cells of the myeloma cell line SP2/0
were fused with
1 tril 50% polyethylene glycol for 30s at 37 C. After washing, the cells were
seeded in 96-well
cell culture plates. Hybrid clones were selected by growing in HAT medium
[RPMI 1640 culture
medium supplemented with 20% fetal calf serum and HAT-Supplement]. After two
weeks the
HAT medium is replaced with HT Medium for three passages followed by returning
to the
nolinal cell culture medium.
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The cell culture supernatants were primary screened for antigen specific IgG
antibodies three
weeks after fusion. The positive tested microcultures were transferred into 24-
well plates for
propagation. After retesting, the selected cultures were cloned and recloned
using the limiting-
dilution technique and the isotypes were determined.
5 (see also Lane, R.D. "A short-duration polyethylene glycol fusion
technique for increasing
production of monoclonal antibody-secreting hybridomas", J. Immunol. Meth. 81:
223-228;
(1985), Ziegler, B. et al. "Glutamate deearboxylase (GAD) is not detectable on
the surface of rat
islet cells examined by cytofluorometry and complement-dependent antibody-
mediated
cytotoxicity of monoclonal GAD antibodies", Horm. Metab. Res. 28: 11-15,
(1996)).
Mouse monoclonal antibody production:
Antibodies were produced via standard antibody production methods (Marx et al,
Monoclonal
Antibody Production, ATLA 25, 121, 1997,) and purified via Protein A. The
antibody purities
were > 95% based on SDS gel electrophoresis analysis.
Human Antibodies
Human Antibodies were produced by means of phage display according to the
following
procedure:
The human naive antibody gene libraries HAL7/8 were used for the isolation of
recombinant
single chain F-Variable domains (scFv) against adrenomedullin peptide. The
antibody gene
libraries were screened with a panning strategy comprising the use of peptides
containing a
biotin tag linked via two different spacers to the adrenomedullin peptide
sequence. A mix of
panning rounds using non-specifically bound antigen and streptavidin bound
antigen were used
to minimize background of non-specific binders. The eluted phages from the
third round of
panning have been used for the generation of monoclonal scFv expressing E.coli
strains.
Supernatant from the cultivation of these clonal strains has been directly
used for an antigen
ELISA testing (see also Hust, M., Meyer, T., Voedisch, B., Rulker, T., Thie,
H., EI-Ghezal, A.,
Kirsch, MI, Schlitte, M., Hehnsing, S., Meier, D., Schimnann, T., Diibel, S.,
2011. A human
scFv antibody generation pipeline for proteome research. Journal of
Biotechnology 152, 159-
170; Schiitte, M., Thullier, P., Pelat, T., Wezler, X., Rosenstock, P., Hinz,
D., Kirsch,
M.I.,Hasenberg, M., Frank, R., Schilmann, T., Gunzer, M., Hust, M., Diibel,
S., 2009.
Identification of a putative Crf splice variant and generation of recombinant
antibodies for the
specific detection of Aspergillus fumigatus. PLoS One 4, e6625).
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Positive clones have been selected based on positive ELISA signal for antigen
and negative for
streptavidin coated micro titer plates. For further characterizations the scFv
open reading frame
has been cloned into the expression plasmid pOPE107 (Bust et al., J. Biotechn.
2011), captured
from the culture supernatant via immobilised metal ion affinity chromatography
and purified by
a size exclusion chromatography.
Affinity Constants
To determine the affinity of the antibodies to Adrenornedullin, the kinetics
of binding of
Adrenomedullin to immobilized antibody was determined by means of label-free
surface
plasrnon resonance using a Biacore 2000 system (GE Healthcare Europe GmbH,
Freiburg,
Germany). Reversible immobilization of the antibodies was performed using an
anti-mouse Pc
antibody covalently coupled in high density to a CM5 sensor surface according
to the
manufacturer's instructions (mouse antibody capture kit; GE Healthcare).
(Lorenz et al.,"
Functional Antibodies Targeting IsaA of Staphylococcus aureus Augment Host
Immune
Response and Open New Perspectives for Antibacterial Therapy"; Antimicrob
Agents
Chemother. 2011 January; 55(1): 165-173.)
The monoclonal antibodies were raised against the below depicted ADM regions
of human and
rnurine ADM, respectively. The following table represents a selection of
obtained antibodies
used in further experiments. Selection was based on target region:
Table 1:
Sequence Antigen/Immunogen ADM Designation Affinity
Number Region constants
Kd (M)
SEQ ID: 15 YRQSMNNFQGLRSFGCRFGTC 1-21 NT-H 5.9 x 10-
9
SEQ ID: 16 CTVQKLAHQIYQ 21-32 MR-H 2 x 10-9
SEQ ID: 17 CAPRSKISPQGY-NH2 C-42-52 CT-H-9
1.1 x 10
SEQ ID: 18 YRQSMNQGSRSNGCRFGTC 1-19 NT-M 19 x 10-9
SEQ ID: 19 CTFQKLAHQIYQ 19-31 MR-M 4.5 x 10-
1
=SEQ ID: 20 CAPRNKISPQGY-NH2 C-40-50
CT-M x 10-9
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The following is a list of further obtained monoclonal antibodies:
List of anti-ADM-antibodies
Table 2:
Target Source Klone number Affinity max inhibition
(M) bioassay (/o) (see
example 2)
NT-M Mouse ADM/63 5.8x10-9 45
Mouse ADM/364 2.2x10-8 48
Mouse ADM/365 3.0x10-8
Mouse ADM/366 1.7x10-8
Mouse ADM/367 1.3x10-8
Mouse ADM/368 L9 x10-8
Mouse ADM/369 2.0 x10-8
Mouse ADM/370 1.6 x10-8
Mouse ADM/371 2.0 x10-'
Mouse ADM/372 2.5 x10-8
Mouse ADM/373 1.8 x10-8
Mouse ADM/377 1.5 x10-8
Mouse ADM/378 2.2 x10-8
Mouse ADM/379 1.6 x10-8
Mouse ADM/380 L8 x10-;
Mouse ADM/381 2.4 x10-8
Mouse ADM/382 1.6 x10-8
Mouse ADM/383 1.8 x10-8
Mouse ADM/384 1.7 x10
Mouse ADM/385 1.7 x10-'
Mouse ADM/403 1.2 x10-8
Mouse ADM/395 1.2 x10-8
Mouse ADM/396 3.0 x10-8
Mouse ADM/397 1.5x10-8
MR-M Mouse ADM/38 4.5x104 68
MR-M Mouse ADM/39 5.9 x10-9 72
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CT-M Mouse ADM/65 9.0x10-9 100
CT-M Mouse ADM/66 1.6x10-8 100
NT-H Mouse ADM/33 5.9x10-8 38
NT-H Mouse ADM/34 1.6x10-8 22
MR-H Mouse ADM/41 1.2x10-8 67
MR-H Mouse ADM/42 <1x10-8
MR-H Mouse ADM/43 2.0x10-9 73
MR-H Mouse ADM/44 <1x10-8
CT-H Mouse ADM/15 <1x108
CT-H Mouse ADM/16 1.1x10-9 100
CT-H Mouse ADM/17 3.7x10-9 100
CT-H Mouse ADM/18 <1x10-8
hADM Phage display ADM/A7 <1x10-g
Phage display ADM/B7 <1x10-8
Phage display ADM/C7 <1x10-8
Phage display ADM/G3 <1x10-8
Phage display ADM/B6 <1x10-8
Phage display ADM/B11 <1x10-8
Phage display ADM/D8 <1x10-8
Phage display ADM/D11 <1x10-8
Phage display ADM/G12 <1x10-8
Generation of antibody fragments by enzymatic digestion:
The generation of Fab and F(ab)2 fragments was done by enzymatic digestion of
the murine full
length antibody NT-M. Antibody NT-M was digested using a) the pepsin-based
F(ab)2
Preparation Kit (Pierce 44988) and b) the papain-based Fab Preparation Kit
(Pierce 44985). The
fragmentation procedures were performed according to the instructions provided
by the supplier.
Digestion was carried out in ease of F(ab)2-fragmentation for 8h at 37 C. The
Fab-fragmentation
digestion was carried out for 16h, respectively.
Procedure for Fab Generation and Purification:
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The immobilized papain was equilibrated by washing the resin with 0.5 ml of
Digestion Buffer
and centrifuging the column at 5000 x g for 1 minute. The buffer was discarded
afterwards. The
desalting column was prepared by removing the storage solution and washing it
with digestion
buffer, centrifuging it each time afterwards at 1000 x g for 2 minutes. 0.5m1
of the prepared IgG
sample where added to the spin column tube containing the equilibrated
Immobilized Papain.
Incubation time of the digestion reaction was done for 16h on a tabletop
rocker at 37 C. The
column was centrifuged at 5000 x g for 1 minute to separate digest from the
Immobilized
Papain. Afterwards the resin was washed with 0.5m1 PBS and centrifuged at 5000
x g for 1
minute. The wash fraction was added to the digested antibody that the total
sample volume was
1.0m1. The NAb Protein A Column was equilibrated with PBS and IgG Elution
Buffer at room
temperature. The column was centrifuged for 1 minute to remove storage
solution (contains
0.02% sodium azide) and equilibrated by adding 2m1 of PBS, centrifuge again
for 1 minute and
the flow-through discarded. The sample was applied to the column and
resuspended by
inversion. Incubation was done at room temperature with end-over-end mixing
for 10 minutes.
The column was centrifuged for 1 minute, saving the flow-through with the Fab
fragments.
(References: Coulter, A. and Harris, R. (1983). J. Irnmunol. Meth. 59, 199-
203.; Lindner I. et al.
(2010) {alpha.}2-Macroglobulin inhibits the malignant properties of
astrocytoma cells by
impeding {beta}-catenin signaling. Cancer Res. 70, 277-87.; Kaufmann B. et al.
(2010)
Neutralization of West Nile virus by cross-linking of its surface proteins
with Fab fragments of
the human monoclonal antibody CR4354. PNAS. 107, 18950-5.; Chen X. et al.
(2010)
Requirement of open headpiece conformation for activation of leukocyte
integrin 042. PNAS.
107, 14727-32.; Uysal H. et al. (2009) Structure and pathogenicity of
antibodies specific for
citrullinated collagen type II in experimental arthitis. J. Exp. Med. 206, 449-
62.; Thomas G. M.
et al. (2009) Cancer cell-derived microparticles bearing P-selectin
glycoprotein ligand I
accelerate thrombus formation in vivo. J. Exp. Med. 206, 1913-27.; Kong F. et
al. (2009)
Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol.
185, 1275-84.)
Procedure for generation and purification of F(ab`)? Fragments:
The immobilized Pepsin was equilibrated by washing the resin with 0.5 ml of
Digestion Buffer
and centrifuging the column at 5000 x g for 1 minute. The buffer was discarded
afterwards. The
desalting column was prepared by removing the storage solution and washing it
with digestion
buffer, centrifuging it each time afterwards at 1000 x g for 2 minutes. 0.5ml
of the prepared IgG
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sample where added to the spin column tube containing the equilibrated
Immobilized Pepsin.
Incubation time of the digestion reaction was done for 16h on a tabletop
rocker at 37 C. The
column was centrifuged at 5000 x g for 1 minute to separate digest from the
Immobilized
Papain. Afterwards the resin was washed with 0.5rnL PBS and centrifuged at
5000 x g for 1
5 minute. The wash fraction was added to the digested antibody that the
total sample volume was
1.0m1. The NAb Protein A Column was equilibrated with PBS and IgG Elution
Buffer at room
temperature. The column was centrifuged for 1 minute to remove storage
solution (contains
0.02% sodium azide) and equilibrated by adding 2mL of PBS, centrifuge again
for 1 minute and
the flow-through discarded. The sample was applied to the column and
resuspended by
10 inversion. Incubation was done at room temperature with end-over-end
mixing for 10 minutes.
The column was centrifuged for 1 minute, saving the flow-through with the Fab
fragments.
(References: Mariani, M., et at. (1991). A new enzymatic method to obtain high-
yield F(ab")2
suitable for clinical use from mouse IgGl. Mollmmunol. 28: 69-77.;Beale, D.
(1987). Molecular
fragmentation: Some applications in immunology. Exp Comp Immunol 11:287-96.;
Ellerson,
15 J.R., et at. (1972). A fragment corresponding to the CH2 region of
immunoglobulin G (IgG) with
complement fixing activity. FEBS Letters 24(3):318-22.; Kerbel, R.S. and
Elliot, B.E. (1983).
Detection of Fc receptors. Meth Enzymol 93:113-147.; Kulkami, P.N., et at.
(1985). Conjugation
of methotrexate to IgG antibodies and their F(ab")2 fragments and the effect
of conjugated
methotrexate on tumor growth in vivo. Cancer Immunol Immunotherapy 19:211-4.;
Lamoyi, E.
20 (1986). Preparation of F(ab")2 Fragments from mouse IgG of various
subclasses. Meth Enzyrnol
121:652-663.; Parham, P., et at. (1982). Monoclonal antibodies: purification,
fragmentation and
application to structural and functional studies of class I MHC antigens. J
Immunol Meth
53:133-73.; Raychaudhuri, G., et at. (1985). Human lgG1 and its Fe fragment
bind with different
affinities to the Fe receptors on the human U937, HL-60 and ML-1 cell lines.
Mal Immunol
25 22(9):1009-19.; Rousseaux, J., et at. (1980). The differential enzyme
sensitivity of rat
immunoglobulin G subclasses to papain an pepsin. Mol Immunol 17:469-82.;
Rousseaux, J., et
al. (1983). Optimal condition for the preparation of Fab and F(ab")2 fragments
from monoclonal
IgG of different rat IgG subclasses. J Immunol Meth 64:141-6.; Wilson, K.M.,
et at. (1991).
Rapid whole blood assay for HIV-I seropositivity using an Fab-peptide
conjugate. J hninunol
30 Meth 138:111-9.)
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NT-H-Antibody Fragment Humanization
The antibody fragment was humanized by the CDR-grafting method (Jones, P. T.,
Dear, P. H.,
Foote, J., Neuberger, M. S., and Winter, G. (1986) Replacing the
complementarity-determining
regions in a human antibody with those from a mouse. Nature 321, 522-525).
The following steps where done to achieve the humanized sequence:
Total RNA extraction: Total RNA was extracted from NT-H hybridomas using the
Qiagen kit.
First-round RT-PCR: QIAGEN OneStep RT-PCR Kit (Cat No. 210210) was used. RT-
PCR
was performed with primer sets specific for the heavy and light chains. For
each RNA sample,
12 individual heavy chain and 11 light chain RT-PCR reactions were set up
using degenerate
forward primer mixtures covering the leader sequences of variable regions.
Reverse primers are
located in the constant regions of heavy and light chains. No restriction
sites were engineered
into the primers.
Reaction Setup: 5x QIAGEN OneStep RT-PCR Buffer 5.0 il, dNTP Mix (containing
10 mM of
each dNTP) 0.8 111, Primer set 0.5 pi, QIAGEN OneStep RT-PCR Enzyme Mix 0.8
Template RNA 2.0 tl, RNase-free water to 20.0 pi, Total volume 20.0 jil
PCR condition: Reverse transcription: 50 C, 30 min; Initial PCR activation: 95
C, 15 min
Cycling: 20 cycles of 94 C, 25 sec; 54 C, 30 sec; 72 C, 30 sec; Final
extension: 72 C, 10 min
Second-round semi-nested PCR: The RT-PCR products from the first-round
reactions were
further amplified in the second-round PCR. 12 individual heavy chain and 11
light chain RT-
PCR reactions were set up using semi-nested primer sets specific for antibody
variable regions.
Reaction Setup: 2x PCR mix 10 ill; Primer set 2 pl; First-round PCR product 8
1.11; Total volume
20 pl; Hybridoma Antibody Cloning Report
PCR condition: Initial denaturing of 5 min at 95 C; 25 cycles of 95 C for 25
sec, 57 C for 30
sec, 68 C for 30 sec; Final extension is 10 min 68 C.
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After PCR is finished, run PCR reaction samples onto agarose gel to visualize
DNA fragments
amplified.After sequencing more than 15 cloned DNA fragments amplified by
nested RT-PCR,
several mouse antibody heavy and light chains have been cloned and appear
correct. Protein
sequence alignment and CDR analysis identifies one heavy chain and one light
chain. After
alignment with homologous human framework sequences the resulting humanized
sequence for
the variable heavy chain is the following: see figure 6 (As the amino acids on
positions 26, 40
and 55 in the variable heavy chain and amino acid on position 40 in the
variable light are critical
to the binding properties, they may be reverted to the murine original. The
resulting candidates
are depicted below) (Padlan, E. A. (1991) A possible procedure for reducing
the immunogenicity
of antibody variable domains while preserving their ligand-binding properties.
Mol. Immunol.
28, 489-498.; Harris, L. and Bajorath, J. (1995) Profiles for the analysis of
immunoglobulin
sequences: Comparison of V gene subgroups. Protein Sci. 4, 306-310.).
Annotation for the antibody fragment sequences (SEQ ID NO: 7-14): bold and
underline are the
CDR 1, 2, 3 in chronologically arranged; italic are constant regions; hinge
regions are
highlighted with bold letters and the histidine tag with bold and italic
letters; framework point
mutation have a grey letter-background.
SEQ ID NO: 7 (AM-VH-C)
QV QLQQSGAELMKP GAS VKISCKATGYTF SRYWIEWVKQRP GHGLEWIGEILPGSGST
NYNEKFKGKATITADTSSNTA YMQLSSLTSEDSAVYYCTEGYEYDGFDYWGQGTTLTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNTINHKPSNTKVDKRVEPKHHHHHH
SEQ ID NO: 8 (AM-VH1)
QVQLVQSGAEVKKPGS SVKVS CKASGYTFSRYWISWVRQAP GQGLEWMGRILPGS GS
TNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNIIKPSNTKVDKRVEPERHHHIIH
SEQ ID NO: 9 (AM-VH2-E40)
QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWWWVRQAPGQGLEWMGRILPGSGS
TNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKIIHHHHH
SEQ ID NO: 10 (AM-VH3-T26-E55)
QVQLVQSGAEVKKP G S S VKVS CIWGYTFSRYWI SWVRQAP GQ GLEWMGOL PGSGS
TNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSA
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STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEP1KHHHHHH
SEQ ID NO: 11 (AM-VH4-T26-E40-E55)
QVQLVQSGAEVKKPGSSVKVSCKATGYTFSRYW*WVRQAPGQGLEWMG[EILPGSGS
TNYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVIITFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKHHHHHH
SEQ ID NO: 12 (AM-VL-C)
DVLLSQTPLSLPVSLGDQATISCRSSOSIVYSNGNTYLEWYLQIUGQSPKWYRVSNRF
SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCKIGS1111PYTFGGGTKLEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LS STLTLSKADYEKHKVYACEVTHQ GLS SP VTKSFNRGEC
SEQ ID NO: 13 (AM-VL1)
DVVMTQSPLSLPVTLGQPASISCRSS QSIVYSNGNTYLNWFQQRPGQSPRRLIYRVSNRD
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHIPYTFGQGTKLEIKRTVAAP SV
FIFPP SDEQLKSGTA S VVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKD STYS
LS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
SEQ ID NO: 14 (AM-VL2-E40)
DVVMTQSPLSLPVTLGQPASISCRSSOSIVYSNGNTYDEWFQQRPGQSPRRLIYRVSNRD
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFOGSHIPYTEGQGTKLEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LS S TLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
Example 2
Effect of selected anti-ADM-antibodies on anti-ADM-bioactivity
The effect of selected ADM-antibodies on ADM-bioaetivity was tested in an
human recombinant
Adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay).
Testing of antibodies targeting human or mouse adrenomedullin in human
recombinant
Adrenomedullin receptor cAMP functional assay (Adrenomedullin Bioassay)
Materials:
Cell line: CHO-K1
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Receptor: Adrenornedullin (CRLR + RAMP3)
Receptor Accession Number Cell line: CRLR: U17473; RAMP3: M001016
CHO-Kl cells expressing human recombinant adrenomedullin receptor (FAST-027C)
grown
prior to the test in media without antibiotic were detached by gentle flushing
with PBS-EDTA (5
mM EDTA), recovered by centrifugation and resuspended in assay buffer (KRH: 5
mM KC1,
1.25 mM MgSO4, 124 mM NaC1, 25 mM HEPES, 13.3 mM Glucose, 1.25 mM KH2PO4, 1.45
mM CaC12, 0.5 g/l BSA).
Dose response curves were performed in parallel with the reference agonists
(hADM or
mADM).
Antagonist test (96we11):
For antagonist testing, 6 ill of the reference agonist (human (5 ,63nM) or
mouse (0,67nM)
adrenomedullin) was mixed with 6 ill of the test samples at different
antagonist dilutions; or with
6 ul buffer. After incubation for 60 min at room temperature, 12 p.1 of cells
(2,500 cells/well)
were added. The plates were incubated for 30 min at room temperature. After
addition of the
lysis buffer, percentage of DeltaF will be estimated, according to the
manufacturer specification,
with the HTRF kit from Cis-Bio International (cat n 62AM2 PEB). hADM 22-52 was
used as
reference antagonist.
Antibodies testing cAMP-HTRF assay
The anti-h-ADM antibodies (NT-H, MR-H, CT-H) were tested for antagonist
activity in human
recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in the
presence of
5.63nM Human ADM 1-52, at the following final antibody concentrations:
100ttghtil, 20 g/ml,
41.tg/ml, 0.8p,g/ml, 0.16ggiml.
The anti-m-ADM antibodies (NT-M, MR-M, CT-M) were tested for antagonist
activity in
human recombinant adrenomedullin receptor (FAST-027C) cAMP functional assay in
the
presence of 0.67nM Mouse ADM 1-50, at the following final antibody
concentrations:
100pgiml, 20).tgiml, 0.811g/ml, 0.16ng/ml. Data were plotted relative
inhibition vs.
antagonist concentration (see figs. 3a to 31). The maximal inhibition by the
individual antibody is
given in table 3.
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Table 3:
Antibody Maximal inhibition of ADM bioactivity (ADM-
Bioassay) (%)
NT-H 38
MR-H 73
CT-H 100
NT-M FAB 26
NT-M FAB2 28
NT-M 45
MR-M 66
CT-M 100
Non specific mouse IgG 0
Example 3
5 Data for stabilization of hADM by the anti-ADM antibody
The stabilizing effect of human ADM by human ADM antibodies was tested using a
hADM
immunoassay.
10 Immunoassay for the quantification of human Adrenomedullin
The technology used was a sandwich coated tube luminescence immunoassay, based
on
Acridinium ester labelling.
15 Labelled compound (tracer): 100lig (100u1) CT-H (lmg/ ml in PBS, pH 7.4,
AdrenoMed
AGGermany) was mixed with 101.d Acridiniurn NHS-ester (lmg/ ml in
acetonitrile, InVent
GmbH, Germany) (EP 0353971) and incubated for 20min at room temperature.
Labelled CT-H
was purified by Gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad
Laboratories, Inc., USA)
The purified CT-H was diluted in (300 mmol/L potassiumphosphate, 100 mmol/L
NaC1, 10
20 mmol/L Na-EDTA, 5 g/L Bovine Serum Albumin, pH 7.0). The final
concentration was approx.
800.000 relative light units (RLU) of labelled compound (approx. 2Ong labeled
antibody) per
200 !IL. Acridiniurnester chemilumineseenee was measured by using an AutoLumat
LB 953
(Berthold Technologies GmbH & Co. KG).
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Solid phase: Polystyrene tubes (Greiner Bio-One International AG, Austria)
were coated (18h at
room temperature) with MR-H (AdrenoMed AG, Germany) (1.5 pg MR-H/0.3 mL 100
ininol/L
NaCl, 50 mmol/L TRIS/HCI, pH 7,8). After blocking with 5% bovine serum
albumine, the tubes
were washed with PBS, pH 7.4 and vacuum dried.
Calibration:
The assay was calibrated, using dilutions of hADM
(SACHEM AG, Switzerland) in 250 mrnol/L NaC1, 2 g/L Triton X-100, 50 g/L
Bovine Serum
Albumin, 20 tabs/L Protease Inhibitor Cocktail (Roche Diagnostics AG,
Switzerland))
hADM Immunoassay:
50 pl of sample (or calibrator) was pipetted into coated tubes, after adding
labeleld CT-H
(2000), the tubes were incubated for 4h at 4 C. Unbound tracer was removed by
washing 5
times (each 1m1) with washing solution (20mM PBS, pH 7.4, 0.1 % Triton X-100).
Tube-bound chemilumineseenee was measured by using the LB 953
Figure 4 shows a typical hADM dose/ signal curve. And an hADM dose signal
curve in the
presence of 100 pglmL antibody NT-H.
NT-H did not affect the described hADM immunoassay.
Stability of human Adrenomedullin:
Human ADM was diluted in human Citrate plasma (final concentration lOnM) and
incubated at
24 C. At selected time points, the degradation of hADM was stopped by
freezing at -20 C. The
incubation was performed in absence and presence of NT-H (100n/m1). The
remaining hADM
was quantified by using the hADM immunoassay described above.
Figure 5 shows the stability of hADM in human plasma (citrate) in absence and
in the presence
of NT-H antibody. The half life of hADM alone was 7,8b and in the presence of
NT-H, the half
life was 18,3h. (2.3 times higher stability).
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Example 4
Sepsis Mortality (early treatment)
Animal model
12-15 week old male C57B1/6 mice (Charles River Laboratories, Germany) were
used for the
study. Peritonitis had been surgically induced under light isofluran
anesthesia. Incisions were
made into the left upper quadrant of the peritoneal cavity (natmal location of
the eecum). The
cecum was exposed and a tight ligature was placed around the cecum with
sutures distal to the
insertion of the small bowel. One puncture wound was made with a 24-gauge
needle into the
cecum and small amounts of cecal contents were expressed through the wound.
The cecum was
replaced into the peritoneal cavity and the laparotomy site was closed.
Finally, animals were
returned to their cages with free access to food and water. 500g1 saline were
given s.c. as fluid
replacement.
Application and dosage of the compound (NT-M, MR-M, CT-M)
Mice were treated immediately after CLP (early treatment). CLP is the
abbreviation for cecal
ligation and puncture (CLP).
Study groups
Three compounds were tested versus: vehicle and versus control compound
treatment. Each
group contained 5 mice for blood drawing after 1 day for BUN (serum blood urea
nitrogen test)
determination. Ten further mice per each group were followed over a period of
4 days.
Group Treatment (10u1/ g bodyweight) dose/ Follow-Up:
I NT-M, 0.2 mg/ml survival over 4 days
2 MR-M, 0.2 mg/m1 survival over 4 days
3 CT-M, 0.2 mg/ml survival over 4 days
4 non-specific mouse IgG, 0.2 mg/ml survival over 4 days
5 control - PBS 100/g bodyweight survival over 4 days
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Clinical chemistry
Blood urea nitrogen (BUN) concentrations for renal function were measured
baseline and day 1
after CLP. Blood samples were obtained from the cavernous sinus with a
capillary under light
ether anaesthesia. Measurements were performed by using an AU 400 Olympus
Multianalyser.
The 4-day mortality is given in table 4. The average BUN concentrations are
given in table 5.
Table 4:
4 day mortality survival (%)
PBS 0
non-specific mouse IgG 0
CT-M 10
MR-M 30
NT-M 70
Table 5:
Average from 5 animals BUN pre CL? (rnM) BUN day 1 (mM)
PBS 8.0 23.2
non-specific mouse IgG 7.9 15.5
CT-M 7.8 13.5
MR-M 8.1 24.9
NT-M 8.8 8.2
It can be seen from Table 4 that the NT-M antibody reduced mortality
considerably. After 4 days
70 % of the mice survived when treated with NT-M antibody. When treated with
MR-M
antibody 30 % of the animals survived and when treated with CT-M antibody 10 %
of the
animals survived after 4 days. In contrast thereto all mice were dead after 4
days when treated
with unspecific mouse IgG. The same result was obtained in the control group
where PBS
(phosphate buffered saline) was administered to mice.
The blood urea nitrogen or BUN test is used to evaluate kidney function, to
help diagnose kidney
disease, and to monitor patients with acute or chronic kidney dysfunction or
failure.
The results of the S-BUN Test revealed that the NT-M antibody was the most
effective to protect
the kidney.
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Sepsis Mortality (late treatment)
Animal model
12-15 week old male C57B1/6 mice (Charles River Laboratories, Germany) were
used for the
study. Peritonitis had been surgically induced under light isofluran
anesthesia. Incisions were
made into the left upper quadrant of the peritoneal cavity (normal location of
the cecum). The
cecum was exposed and a tight ligature was placed around the cecum with
sutures distal to the
i0 insertion of the small bowel. One puncture wound was made with a 24-
gauge needle into the
cecum and small amounts of cecal contents were expressed through the wound.
The cecum was
replaced into the peritoneal cavity and the laparotomy site was closed.
Finally, animals were
returned to their cages with free access to food and water. 500g1 saline were
given s.c. as fluid
replacement.
Application and dosage of the compound (NT-M FAB2)
NT-M FAB2 was tested versus: vehicle and versus control compound treatment.
Treatment was
performed after full development of sepsis, 6 hours after CLP (late
treatment). Each group
contained 4 mice and were followed over a period of 4 days.
Group Treatment (101.11/ g bodyweight) dose/ Follow-Up:
Study groups
1 NT-M, FAB2 0.2 mg/m1 survival over 4 days
2 control : non-specific mouse IgG, 0.2 mg/m1 survival over 4 days
3 vehicle: - PBS 10111/g bodyweight survival over 4 days
Table 6:
4 day mortality survival (%)
PBS 0
Non-specific mouse IgG 0
NT-M FAB2 75
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It can be seen from Table 6 that the NT-M FAB 2 antibody reduced mortality
considerably. After
4 days 75 'A of the mice survived when treated with NT-M FAB 2 antibody. In
contrast thereto
all mice were dead after 4 days when treated with non-specific mouse IgG. The
same result was
obtained in the control group where PBS (phosphate buffered saline) was
administered to mice.
5
Example 5
Incremental effect of anti-ADM antibody in CLP-animals on top of antibiotic
treatment
and circulation stabilization via catecholamines as well as regulation of
fluid balance.
Animal model
In this study male C57B1/6 mice (8-12 weeks, 22-30g) were utilized. A
polymicrobial sepsis
induced by cecal ligation and puncture (CLP) was used as the model for
studying septic shock
((Albuszies G, et al: Effect of increased cardiac output on hepatic and
intestinal rnicrocirculatory
blood flow, oxygenation, and metabolism in hyperdynamic murine septic shock.
Crit Care Med
2005;33:2332-8), (Albuszies G, et al: The effect of iNOS deletion on hepatic
gluconeogenesis in
hyperdynamic murine septic shock. Intensive Care Med 2007;33:1094-101), (Barth
E, et al: Role
of NOS in the reduced responsiveness of the myocardium to catecholamines in a
hyperdynamic,
murine model of septic shock. Crit Care Med 2006;34:307-13), (Baumgart K, et
al: Effect of
SOD-1 over-expression on myocardial function during resuscitated murine septic
shock.
Intensive Care Med 2009;35:344-9),
(Baumgart K, et al: Cardiac and metabolic effects of hypothermia and inhaled
H2S in
anesthetized and ventilated mice. Crit Care Med 2010;38:588-95), (Simkova V,
eta!: The effect
of SOD-1 over-expression on hepatic gluconeogenesis and whole-body glucose
oxidation during
resuscitated, norrnotensive murine septic shock. Shock 2008;30:578-84),
(Wagner F, et al.:
Inflammatory effects of hypothermia and inhaled H2S during resuscitated,
hyperdynamic murine
septic shock. Shock, im Druck), (Wagner F, et al: Effects of intravenous H2S
after murine blunt
chest trauma: a prospective, randomized controlled trial. Crit Care 2011,
submittes for
publication)).
After weighing, mice were anesthetized by intraperitoneal injection of 120
ng/g Ketamin, 1.25
ng/g Midazolam and 0.25 tig/g Fentanyl. During the surgical procedure, body
temperature was
kept at 37-38 C. A 1 cm midline abdominal section was performed to get access
to the cecum.
The cecum then was ligated with 3-0 silk tie close to the basis and a single
puncture with a 18-
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gauge needle was applied. The cecum was returned and the incision was closed
again (4-0 tie).
For the compensation of perioperative loss of liquids, 0.5 ml lacted Ringer's
solution with l nig
Buprenorphin as analgetic was injected subcutaneously in dorsal dermis. For
antibiosis the mice
received Cefthaxon 30ug/g and Clindamycin 30ug/g subcutaneously via the lower
extremities.
After CLP surgery the animal were kept in an adequately heated environment
with water and
food ad libitum.
The covering of liquid requirements were ensured by a dorsal subcutaneous
injections with 0.5
ml lactated ringer's solution with 4 ug/g glucose and Buprenorphin lug/g,
which were applied in
an 8 hour cycle, after short term anesthesia by isofluran. In addition,
antibiosis was maintained
by subcutaneous injections of Ceiliiaxon 30p.g/g and Clindamycin 30[Ig/g via
the lower
extremities.
Dosing of test substances
Early treatment
Immediately after the CLP surgery and closing of the incision, the test
substance antibody NT-M
was applied in a concentration of 500 ug/ml in phosphate buffered saline (PBS)
via injection into
the penis vein for a dose of 2rng per kg body weight (dose volume 88-120 ill)
(5 animals).
Late treatment
After full Sepsis development, 15.5h after CLP surgery, animals were
anesthetized as described
above and NT-M was applied in a concentration of 500 g/ml in phosphate
buffered saline
(PBS) via injection into the penis vein for a dose of 2Ing per kg body weight
(dose volume 88-
120 ul) (3 animals).
The control group (6 animals) received a corresponding amount of the vehicle
PBS solution
without antibody (4111/g, 88-120 pl) immediately after CLP surgery.
Study groups and experimental setting
Murine septic shock model under intensive care monitoring:
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Three groups with 3, 5 and 6 animals were monitored. Group 1 (5 animals)
received the antibody
NT-M 15.5h after CLP, group 2 received the antibody NT-M immediately after CLP
surgery and
group 3 received a comparable amount of PBS (4 1/g). 16 hour incubation post
CLP (to allow
the polymicrobial sepsis to progress), the experiment was continued with
monitoring and
interventions comparable to an intensive medical care regime. Therefore, after
weighing the
animals were anesthetized as described in the CLP surgery part (except the
late treated animals,
which were anesthized before treatment). Body temperature was maintained at 37-
38 C for the
rest of the experiment. After a tracheotomy and intubation, respiration was
monitored and
supported by laboratory animal lung ventilator Flexivent , (Emka Technologies,
Fi02 0,5, PEEP
10 H20, VT 8111/g, 1:E 1:1,5, AF 70-140 depending on temperature).
Anesthesia was maintained throughout the experiment via the cannulated vena
jugularis extema
dextra with a continuous infusion of Ketamin 30 ug/gxh and Fentanyl 0.3
ug/gxh. Furthermore,
the right aorta carotis communis was cannulated for continuous monitoring of
heart rate and the
mean arterial pressure (MAP). The mean arterial pressure was maintained at MAP
> 65 mmHg
via intravenous (V. jugularis) infusion of colloids (80 ullgxh, Hextend ) and,
if needed,
Noradrenalin dissolved in colloids as vasopressor. Blood samples (120 ul) were
taken via the
cannulated A. carotis at 0 and 4 hours for determination of creatinine. The
bladder was punctured
and urine was collected via a bladder catheter. The experiment was either
terminated after 6
hours or prior to this, if the MAP > 65 nunHg (V. jugularis) could not be
maintained with the
vasorpressor dosing.
Measured parameters
The following parameters were measured and analyzed: Total consumption of
noradrenalin (ug
NA/g), consumption rate of noradrenalin (jig NA/g/h), total volume of urine
collected during the
experiment, creatinine concentration (ug/mL) at the end of the experiment and
mean creatinine
clearance (uL/tnin).
Table 7:
Total consumption of consumption rate
of
Noradrenalin (jig NA/g) Noradrenalin (jig NA/g/h)
(Average) (Average)
Control (mouse IgG) (N=6) 0.17 ug/g 0.032 ug/h/g
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NT-M (N=5) early treatment 0.07 iug/g 0.012gg/h/g
Relative change (early treatment, 59% 62.5%
amelioration) (59%) (62.5%)
NT-M (N=3) late treatment 0.04 nig 0.0075 ug/h/g
Relative change (late treatment, 76,5% 76,5%
amelioration) (76.5%) (76.5%)
The catecholamine requirement was measured after administration of either non
specific mouse
IgG to a total of 6 mice as control group, NT-murine antibody to a group of 5
mice immediately
after CLP (early treatment) or NT-murine antibody to a group of 3 mice 15.5h
after CLP (late
treatment).
The reduction of the catecholamine requirement is a measure for the
stabilization of the
circulation. Thus, the data show that the ADM antibody, especially the NT-M
antibody, leads to
a considerable stabilization of the circulation and to a considerable
reduction of the
eatecholamine requirement. The circulation-stabilizing effect was given in
early treatment
(immediately after CLP) and treatment after full sepsis development (late
treatment) (see fig. 7).
Regulation of Fluid Balance
More positive fluid balance both early in resuscitation and cumulatively over
4 days is associated
with an increased risk of mortality in septic shock. The control of the liquid
balance is of utmost
importance for the course of disease of patients having sepsis. (s. Boyd eta!,
2011). Controlling
the liquid balance of critical ill patients remains as a substantial challenge
in intensive care
medicine. As can be seen in table 8 treatment of mice after CLP (experimental
procedures see
"Animal Model") with NT-M antibody lead to an enhancement of the total volume
of urine
excreted. The urine secreted was approx. three times higher in NT-M-treated
animals compared
to non-treated mice. The positive treatment effect was given in early- and in
late treatment. The
fluid balance was improved by about 20-30%, also in both, early and late
treatment. Thus, the
data show that the use of ADM antibody, especially the use of NT ADM antibody,
is favorable
for regulating the fluid balance in patients. (see table 8 and figures 8 and
9).
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Table 8
Urine average Fluid balance average
volume/ g body volume/ g body weight
weight
Control (mouse IgG) 0.042 ml/g 0,23 mug
(N=6)
NT-M early (N=5) 0.12 ml 0,18 ml/g
Relative change early + 186% -21.7%%
treatment
NT-M late (N=3) 0325 ml 0,16 ml/g
Relative change late + 198% -30,5%
treatment
Improvement of kidney function
The combination of acute renal failure and sepsis is associated with a
70 percent mortality, as compared with a 45 percent mortality among patients
with
acute renal failure alone. (Schrier and Wang, "Mechanisms of Disease Acute
Renal Failure and
Sepsis"; The New England Journal of Medicine; 351:159-69; 2004). Creatinine
concentration
and creatinine clearance are standard laboratory parameters for monitoring
kidney (dys)function
(Jacob, "Acute Renal Failure", Indian J. Anaesth.; 47 (5): 367-372; 2003).
Creatinine and
creatinine clearance data from above described animal experiment (early
treatment) are given in
Table 9.
Table 9
Kidney function:
creatinine mean creatinine
concentration clearance (AL/min)
(ggimL)
control mouse IgG (MW) 2.6 tg/ml. 174 Wrnin
NT-M (MW) 1.5 ng/ml 373 ill/min
Relative change -42% +114%
(amelioration)
(42%) (114%)
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In comparision to control septic animals, the creatinine concentration was
lowered by 42% and
the creatinine clearance was improved by more than 100% as a result of NT-M
treatment (Table
9). The data show that the administration of ADM-antibody, especially NT-M,
leads to an
5 improvement of kidney function.
Improvement of liver inflammatory status
Liver tissue for control and early treated animals was homogenized and lysed
in lysing buffer.
10 For cell extract preparation, cells were resuspended, lysed on ice, and
centrifuged. The
supernatant (protein extract) was stored at -80 'C. Activation of nuclear
factor kappa-light-chain
gene enhancer in B cells (NF-KB) was determined as previously described using
an
electrophoretic mobility shift assay (EMSA)1,2. Cell extracts (10tig) were
incubated on ice with
poly-doxy-inosinic-deoxy-cytidylie acid (poly-dl-dC) and 32P-labeled double
stranded
15 oligonueleotide (Biomers, Ulm, Germany) containing the NF-KB (HIV icBsite)
( 5'-
GGATCCTCAACAGAGGGGACTTTCCGAGGCCA-3'). Complexes were separated in native
polyaerylamide gels, dried and exposed to X-ray films. A phosphorimager and
image analyzer
software (AIDA Image Analyzer; Raytest) was used to quantify the radioactively
labeled NF-kB
by densitometry. For comparison between individual gels, the intensity of each
band was related
20 to that of simultaneously loaded control animals which had not undergone
surgical
instrumentation and CLP. Therefore, the EMSA data are expressed as fold
increase over control
values. Statistics: All data are presented as median (range) unless otherwise
stated differences
between the two groups were analyzed with the Mann-Whitney rank sum test for
unpaired
samples. Results: The animals treated with NT-M presented with significantly
attenuated liver
25 tissue NF-kB activation (2.27 (1.97-2.53)) compared to vehicle animals
(2.92 (2.50-3.81))
(p<0.001) (see figure 10).
References:
1. Wagner F, Wagner K, Weber S, Stahl B, Knoferl MW, Huber-Lang M, Seitz DH,
Asfar P,
30 Calzia E, Senftleben U, Gebhard F, Georgieff M, Radermacher P, Hysa V:
Inflammatory effects
of hypothermia and inhaled H2S during resuscitated, hyperdynamic murine septic
shock. Shock
2011;35(4):396-402
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2. Wagner F, Scheuerle A, Weber S, Stahl 13, McCook 0, Knoferl MW, Huber-Lang
M. Seitz
DH, Thomas J, Asfar P, SzabO C, M011er P, Gebhard F, Georgieff M, Calzia E,
Radermacher P,
Wagner K: Cardiopulmonary, histologic, and inflammatory effects of intravenous
Na2S after
blunt chest trauma-induced lung contusion in mice. J. Trauma 2011; 71(6):1659-
67
Example 6
In vivo side effect determination of antibody NT-M
12-15 week old male C57B1/6 mice (Charles River Laboratories, Germany) were
used for the
study. 6 mice were treated with (10u1/ g bodyweight) dose of NT-M, 0.2 mg/ml.
As control, 6
mice were treated with (10 1/g body weight) PBS. Survival and physical
condition was
monitored for 14 days. The mortality was 0 in both groups, there were no
differences in physical
condition between NT-M and control group.
Example 7
Gentamicin-induced nephrotoxieity
A non-septic acute kidney injury model has been established, which makes use
of the
nephrotoxicity induced by Gentainicin (Chiu PJS. Models used to assess renal
functions. Drug
Develop Res 32:247-255, 1994.). This model was used to assess whether
treatment with anti-
Adreno-medullin antibody can improve kidney function.
The experiment was performed as follows:
Effect of a NT-M on Gentamicin-Induced Nephrotoxieity in Rats
Study Design:
Test Cone Dosage Ratsd
Group Article Route mg/m1 ml/kg mg/kg (Male)
1 Gentarnicina + IV 8
vehicle'' NA x
2 Gentamicina + IV 8
NT-M X 4'
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aGentarnicin at 120 mg/kg intramuscularly for 7 days (days 0-6).
bVehicle; injected intravenously (i.v.) 5 min before gentamicin on Day 0,
followed by
injections on Days 2, 4, and 6.
eNT-M at 4 mg/kg was injected intravenously (i.v.) 5 min before gentamicin on
Day 0,
followed by 2 mg/kg i.v. on Days 2, 4, and 6.
dPlasma samples were collected in EDTA tubes (Days 1 and 3 before Test and
Control
article: 100 pl; Day 7:120 [d. 24h urine collection on ice is initiated after
gentamicin on
Day 0, followed by Days 2 and 6; blood collection on days 1, 3, and 7.
Groups of 8 male Sprague-Dawley rats weighing 250 20 g were employed.
Animals were
challenged with gentamicin at 120 mg/kg i.m. for seven consecutive days
(Groups 1 and 2). Test
compound (anti-adrenomedullin antibody NT-M) and vehicle (phosphate buffered
saline) were
injected intravenously 5 mm before gentamicin on day 0, followed by injection
on days 2, 4, and
6. Body weights and clinical signs were monitored daily. Twenty-four (24) hour
urine collections
on ice were performed on Days 0, 2, and 6. Urine specimens were assayed for
concentrations of
Na+ and K+, and creatinine. Blood samples for clinical chemistry were
collected on Days 1
(before gentamicin), 3 (before gentamicin), and 7. Serum electrolytes (Na+ and
K+), creatinine,
and BUN were the primary analytes that were monitored for assessing renal
function. Plasma
samples were collected in EDTA tubes (Days 1 and 3:100
Day 7:120 0). Creatinine
clearance was calculated. Urine volume, urinary electrolytes, and creatinine
are expressed as
amount excreted per 100 g of animal body weight. All animals were sacrificed
on Day 7.
Kidneys were weighed.
Urine collection. The animals were placed in individual cages where urine was
collected for 24 h
on Day 0, Day 2, and Day 6. Urine volume, urinary Na+, K+, and creatinine were
measured.
Endogenous creatinine clearance was calculated as follows:
CCr (m1/24 h) = [UCr (mg/m1) x V (m1/24 h)] / SCr (mg/ml)
24-hr urinary excretion of sodium (Na+) was calculated as follows:
UNaV (j.i.Eq/24 h) = UNa ( Eq/m1) x V (m1/24 h)
24-hr urinary excretion of NAG and NGALwas similarly calculated.
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The fractional excretion of Na+ (FENa), or percentage of the filtered sodium
that is excreted into
the final urine, is a measure of tubular Na+reabsorptive function. It was
computed as follows:
FENa (%) ¨100 X [UNa Oi.Eq/m1) x V (m1/24 h)] / PNa (11Eq/m1) X Ccr (m1/24 h)
Treatment with anti-Adrenomedullin antibody improved several measures of
kidney function on
day 7 as compared to vehicle: serum creatinine 1.01 mg/dL (NT-M) vs 1.55 mg/dL
(vehicle)
(Fig. 11), BUN 32.08 mg/dL(NT-M) vs. 52.41 mg/dL (vehicle) (Fig. 12),
endogenous creatinine
clearance 934A3 rnL/24 h (NT-M) vs. 613.34 inL/24 h (vehicle) (Fig. 13),
fractional secretion of
Na+ 0.98 % (NT-M) vs. 1.75 % (vehicle) (Fig. 14).
0 Example 8
In the mice CLP model described above, the effect of treatment with anti-
adrenomedullin
antibody NT-M on several parameters of kidney function was investigated.
NT-M caused a three- and two-fold higher diuresis and creatinine clearance,
respectively,
ultimately resulting in lower creatinine, urea, and NGAL blood concentrations
at the end of the
experiment (see Table 10). Moreover, keratinocyte-derived chem.okine (KC)
concentrations in
the kidney were significantly lowered by treatment with NT-M (Fig. 15).
Table 10: Parameters of kidney function in the vehicle- (n=11) and NT-M-
treated (n-9) animals.
Blood concentrations were measured in samples taken at the end of the
experiment. NGAL ¨
neutrophil gelatinase-associated lipocalin. All data are median (quartiles).
Vehicle NT-M p-Value
Urine output hut =g-1.1-fli 4.4 (3.5;16.5) 15.2 (13.9;22.5)
0.033
Creatinine clearance [pl.min-l] 197 (110;301) 400 (316;509) 0.006
Creatinine [..ig=mL-1] 1.83 (1.52;3.04) 1.28
(1.20;1.52) 0.010
Urea [J_ig.m.L-1] 378 (268;513) 175 (101;184) 0.004
NGAL 16 (15;20) 11 (10;13) 0.008
-
The experiments were performed as follows:
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Creatinine, urea, and neutrophil gelatinase-associated lipocalin (NGAL)
Blood NGAL concentrations were measured using a commercial ELISA (mouse NGAL,
RUO
042, BioPorto Diagnostics A/S, Denmark, Gentofte). Urea and creatinine
concentrations were
measured with a capillary column (Optima-5MS, Macherey-Nagel, Daren, Germany)
gas
chromatography/mass spectrometry system (Agilent 5890/5970, Boblingen,
Germany) using
2H3-creatinine (CDN isotopes, Pointe-Claire, QU, Canada) and methyl-urea
(FlukaChemikalien,
Buchs, Switzerland) as internal standards. After deproteinization with
acetonitrile, centrifugation
and evaporation to dryness, the supernatant was reconstituted in formic acid,
and extracted over
a weak anion exchange column (WCX, Phenomenex, Aschaffenburg, Germany).
Acetonitrile
plus N,O-Bis(trimethylsilyl)trifluoroacetamide
and N-(tert-butyldimethylsily1)-N-
methyltrifluoroacetamide allowed foimation of the urea tert-butyl-
dimethylsilyl- and the
creatininetrimethylsilyl-derivatives, respectively. Ions rn/z 231 and 245, and
m/z 329 and 332
were monitored for urea and creatinine analytes and internal standards,
respectively. From the
urine output and the plasma and urine creatinine concentrations creatinine
clearance was
calculated using the standard formula.
Sample preparation
The kidney which was stored at -80 C was disrupted with a homogenizer in PBS
and lysed with
a 2-fold concentrated buffer for a whole cell lysate (100 mM Tris pH 7,6; 500
mM NaCl; 6 mM
EDTA; 6 mM EGTA; I % Triton-X-100; 0,5 % NP 40; 10 % Glycerol; Protease-
Inhibitors (13-
Glycerolphosphate 2 mM; DTT 4 mM; Leupeptine 20 1.111/1; Natriumorthovanadate
0,2 mM)) and
subsequently centrifuged. The whole cell lysate was obtained out of the
supernatant; the pellet
consisting of cell remnants was discarded. The amount of protein was
determined
photometrically with a commercially available protein assay (Bio-Rad,
Hercules, CA) and the
specimens were adjusted in the way that the final protein concentration was 4
pg/[il. The samples
for the Multiplex- and EIVISA analysis were diluted 1:1 with EMSA buffer (10
mM Hepes; 50
mM KCI; 10 % Glycerol; 0,1 mM EDTA; 1 mM DTT), the samples for the immuno
blots 1:1
with 2-fold Sample Buffer (2 % SDS; 125 mM Tris-HCL (pH 6,8 at 25 C); 10 %
Glycerol; 50
mM DTT; 0,01 % Bromophenol blue).
Levels of keratinocyte-derived chemokine (KC) concentrations were determined
using a mouse
multiplex cytokine kit (Bio-Plex Pro Cytokine Assay, Bio-Rad, Hercules, CA),
the assay was
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performed by using the Bio-plex suspension array system with the
manufacturer's instructions
(see also Wagner F, Wagner K, Weber S, Stahl B, Knoferl MW, Huber-Lang M,
Seitz DH, Asfar
P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Rademacher P, Hysa V.
Inflammatory
effects of hypothetuna and inhaled H2S during resuscitated, hyperdynamie
murine septic shock.
5 Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl B,
McCook 0, Knoferl
MW, Huber-Lang M, Seitz DH, Thomas .1., Asfar P, Szabo C, Möller P, Gebhard F,
Georgieff M,
Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic, and
inflammatory effects of
intravenous Na2S after blunt chest trauma-induced lung contusion in mice. I
Trauma
2011;71:1659-1667). In brief, the appropriate cytokine standards and samples
were added to a
10 filter plate. The samples were incubated with antibodies chemically
attached to fluorescent-
labeled micro beads. Thereafter, premixed detection antibodies were added to
each well, and
subsequently, streptavidin-phycoerythrin was added. Beads were then re-
suspended, and the
cytokines reaction mixture was quantified using the Bio-Plex protein array
reader. Data were
automatically processed and analyzed by Bio-Plex Manager Software 4.1 using
the standard
15 curve produced from recombinant cytokine standards. Levels below the
detection limit of the
assays were set to zero for statistical purposes.
Example 9
20 In the mice CLP model described above, the effect of treatment with anti-
adrenomedullin
antibody NT-M on the liver was investigated.
NT-M caused a significant lowering of keratinocyte-derived chemokine (KC)
concentrations in
the liver (Fig. 16).
Measurement of keratinocyte-derived chernokine (KC) was done analogous to
example 8
25 (kidney).
Example 10
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In the mice CLP model described above, the effect of treatment with anti-
adrenomedullin
antibody NT-M on several cytokines and chernokinesin the blood circulation
(plasma) was
investigated.
Cytokine and chemokine concentrations
Plasma levels of tumor necrosis factor (TNF)-a, interleukin (IL)-6, monocyte
chemoattractant
protein (MCP)-1, and keratinocyte-derived chemokine (KC) concentrations were
determined
using a mouse multiplex cytokine kit (Bio-Plex Pro Cytokine Assay, Bio-Rad,
Hercules, CA),
the assay was performed by using the Bio-plex suspension array system with the
manufacturer's
instructions (see also Wagner F, Wagner K, Weber 5, Stahl B, Knoferl MW, Huber-
Lang M,
Seitz DH, Asfar P, Calzia E, Senftleben U, Gebhard F, Georgieff M, Rademiacher
P, Hysa V.
Inflammatory effects of hypothermia and inhaled H2S during resuscitated,
hyperdynamic murine
septic shock. Shock 2011;35:396-402; and Wagner F, Scheuerle A, Weber S, Stahl
B, McCook
0, Knaferl MW, Huber-Lang M, Seitz DH, Thomas .1, Asfar P, Szabo C, Moller P,
Gebhard F,
Georgieff M, Calzia E, Radermacher P, Wagner K. Cardiopulmonary, histologic,
and
inflammatory effects of intravenous Na2S after blunt chest trauma-induced lung
contusion in
mice. .1 Trauma 2011;71:1659-1667). In brief, the appropriate cytokine
standards and samples
were added to a filter plate. The samples were incubated with antibodies
chemically attached to
fluorescent-labeled micro beads. Thereafter, premixed detection antibodies
were added to each
well, and subsequently, streptavidin-phycoerythrin was added. Beads were then
re-suspended,
and the cytokines reaction mixture was quantified using the Bio-Plex protein
array reader. Data
were automatically processed and analyzed by Bio-Plex Manager Software 4.1
using the
standard curve produced from recombinant cytokine standards. Levels below the
detection limit
of the assays were set to zero for statistical purposes.
Plasma levels and kidney tissue concentrations of tumor necrosis factor (TNF)-
a, interleukin
(1L)-6 and IL-10, monocyte chemoattractant protein (MCP)-1, and keratinocyte-
dervived
chemokine (KC) were determined using a commercially available "Multiplex
Cytokine Kit"
(Bio-Plex Pro Precision Pro Cytokine Assay, Bio-Rad, Hercules, CA), which
allows to collect
several parameters out of one single sample. The individual work steps of the
assay were
performed according to the manufacturer's instructions (see also Wagner F,
Wagner K, Weber S,
Stahl B, Knoferl MW, Huber-Lang M, Seitz DH, Astor P, Calzia E, Senftleben U,
Gebhard F,
Georgieff M, Radennacher P, Hysa V. Inflammatory effects of hypotheimia and
inhaled H2S
during resuscitated, hyperdynamic murine septic shock. Shock 2011;35:396-402;
and Wagner F,
Scheuerle A, Weber 5, Stahl B, McCook 0, Knoterl MW, Huber-Lang M, Seitz DH,
Thomas J,
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Asfar P, Szabo C, Mailer P, Gebhard F, Georgieff M, Calzia E, Radelniacher P,
Wagner K.
Cardiopulmonary, histologic, and inflammatory effects of intravenous Na2S
after blunt chest
trauma-induced lung contusion in mice. J Trauma 2011;71:1659-1667).
In brief, the fluorescence-labed microspheres ("beads") were added to a 96-
well plate, followed
by two washing steps, the addition of internal standards and the addition of
plasma- and kidney
homogenate samples. During the subsequent incubation the single cytokines bind
to the
antibodies attached to polystyrene-beads. After the addition of the cytokine-
specific biotin-
labeled antibodies, which are for the detection of the single cytokines, and
an additional
incubation time, subsequently phycoerythrin-labeled streptavidine was added.
After an additional
incubation time, beads were then resuspended, and the plates could be measured
with a specific
flow cytometer (Bio-Plex suspension array system, Bio-Rad, Hercules, CA). Data
were
automatically processed and analyzed by Bio-Plex Manager Software 4.1 using
the standard
curve produced from recombinant eytokine standards. For the plasma levels the
concentration
was provided in pg * mL-1, the concentration of the kidney homogenates were
converted to the
appropriate protein concentration and provided in pg * mg-1 protein.
NT-M caused a significant lowering of plasma concentrations of IL-6 (Fig. 17),
IL-10 (Fig. 18),
keratinocyte-derived chemokine (KC) (Fig. 19), monoeyte chemoattractant
protein-1 (MCP-1)
(Fig. 20), TNF-alpha (Fig. 21).
Example 11
Ischemia/Reperfusion-Induced Acute Kidney Injury
Another non-septic acute kidney injury model has been established, where acute
kidney injury is
induced by ischemia/reperfusion (Nakamoto M, Shapiro JI, Shanley PF, Chan L,
and Schrier
RW. In vitro and in vivo protective effect of atriopeptin III on ischemie
acute renal failure. J
ClinInvest 80:698-705, 1987., Chintala MS, Bernardino V, and Chiu PIS. Cyclic
GMP but not
cyclic AMP prevents renal platelet accumulation following ischernia-
reperfitsion in anesthetized
rats. J PharmacolExpTher 271:1203-1208, 1994). This model was used to assess
whether
treatment with anti-adrenomedullin antibody can improve kidney function.
The experiment was perfokined as follows:
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Effect of a NT-M on Acute Kidney Injury Induced by Ischemia/Reperfusion in
Rats
Study Design:
Test Cone Dosage Rats
Group Article Route mg/nil ml/kg mg/kg (Male)
1 I-R + vehicle' IV 5 NA x 3 8
2 I-R + NT-M IV 5 x 3b
8
a vehicle; injected intravenously (i.v.) 5 min before reperfusion on day 0,
followed by
injections on days 1 and 2.
bl\IT-M at 4 mg/kg was injected intravenously (i.v.) 5 min before reperfusion
on day 0,
followed by 2 mg/kg i.v. each on days 1 and 2.
Trine collection on days -1, 0, 1 and 2, with blood chemistry and urine
analysis on days
0, 1, 2 and 3, respectively. Plasma samples were collected in EDTA tubes (Days
0
(immediate before surgery), 1, 2: 100 jtl, before vehicle or TA; Day 3:120
Clinical observations: daily before surgery, following surgery and throughout
treatment.
Groups of 8 male Sprague-Dawley rats weighing 250 to 280 g were used. The
animals were kept
on a I2-hr light/dark cycle and receive a standard diet with distilled water
ad libitum. The
animals receive fluid supplements (0.9% NaCl and 5% dextrose/1:1, 10 ml/kg
p.o.) 30 min prior
to surgery (day 0). The rats were anaesthetized with pentobarbital (50 mg/kg,
i.p.). The
abdominal cavity was exposed via a midline incision, followed by intravenous
administration of
heparin (100 U/kg, i.v.) and both renal arteries were occluded for 45 min by
using vascular
clamps. Immediately after removal of the renal clips, the kidneys were
observed for additional 1
min to ensure color change indicating blood reperfusion. The test compound (NT-
M) and vehicle
(phosphate buffered saline) were injected intravenously 5 min before
reperfusion, followed by
daily injection on days 1 and 2.
Urine collection. The 24-h urine collection on ice was initiated at 24h before
ischemia/reperfusion on day -1 (-24h to Oh), and day 0 (0-24h), day 1 (24-48h)
and day 2 (48-
72h) after reperfusion,
Blood collection: 0.4 ml blood was collected through the tail vein into EDTA
tubes at Oh (before
I RI surgery), 24h (before vehicle or TA), 48h (before vehicle or TA) and 72h
for determination
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of plasma creatininefNa+/K+, and BUN; 2 ml blood was collected through venal
cava
terminally.
The animals were placed in individual cages where urine was collected for 24 h
day -1 (-24h-Oh),
day 0 (0-24h), day 1 (24-48h) and day 2 (48-72h) after reperfusion on day 0.
Urine volume,
urinary Na+, K+, and creatinine were measured.
The creatinine clearance (CCr) was calculated as follows:
CCr (m1/24 h) = [UCr (mg/m1) x V (m1/24 h)] / PCr (mg/m1)
The 24-hr urinary excretion of sodium (Na+) was calculated as follows:
UNaV (p.Eq/24 h) = UNa (uEq/m1) x V (m1/24 h)
ID The fractional excretion of Na+ (FENa), or percentage of the filtered
sodium that is excreted into
the final urine, is a measure of tubular Na+ reabsorptive function. It was
computed as follows:
FENa (%) =100 x [UNa (uEq/m1) x V (m1/24 h)] / PNa (uEq/m1) X CCr (ml/24 h)
Treatment with anti-Adrenomedullin antibody improved several measures of
kidney function:
Blood urea nitrogen (BUN) showed a strong increase in the vehicle group (0 h:
17.49 nag/dL, 24
h: 98.85 mg/dL, 48 h: 109.84 mg/dL, 72 h: 91.88 mg/dL), which was less
pronounced with NT-
M treatment (0 h: 16.33 mg/dL, 24 h: 84.2 mg/dL, 48 h: 82.61 mg/dL, 72 h:
64.54 mg/dL) (Fig.
22).
Serum creatinine developed similarity: Vehicle group (0 h: 0.61 mg/dL, 24 h:
3.3 mg/dL, 48 It:
3.16 mg/dL, 72 h: 2.31 mg/dL), NT-M group: (0 h: 0.59 mg/dL, 24 h: 2.96 mg/dL,
48 h: 2.31
mg/dL, 72 h: 1.8 mg/dL) (Fig. 23).
The endogenous creatinine clearance dropped massively on day one and
thereafter improved
better in the NT-M group than in the vehicle group. Vehicle group: (0 h:
65.17rnL/h, 24 h:
3.5mL/h, 48 h: 12.61mL/h, 72 h: 20.88mL/h), NT-M group:(0 h: 70.11mL/h, 24 h:
5.84mL/h, 48
h: 21.23mIlh, 72 h: 26.61mL/h) (Fig. 24).
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FIGURE DESCRIPTION
Fig. la:
Illustration of antibody formats ¨ Fv and scFv-Variants
Fig lb:
Illustration of antibody formats ¨ heterologous fusions and bifunctional
antibodies
Fig lc:
Illustration of antibody formats ¨ bivalental antibodies and bispecific
antibodies
Fig. 2:
hADM 1-52 (SEQ ID No. 21)
mADM 1-50 (SEQ ID No. 22)
aa 1-21 of human ADM (SEQ ID No. 23)
aa 1-42 of human ADM (SEQ ID No. 24)
aa 43-52 of human ADM (SEQ ID No. 25)
aa 1-14 of human ADM (SEQ ID NO: 26)
aa 1-10 of human ADM (SEQ ID NO: 27)
aa 1-6 of human ADM (SEQ ID NO: 28)
aa 1-32 of human mature human ADM (SEQ ID NO: 29)
aa 1-40 of mature murine ADM (SEQ ID NO: 30)
aa 1-31 of mature murine ADM (SEQ ID NO: 31)
Fig. 3:
a: Dose response curve of human ADM. Maximal cAMP stimulation was adjusted to
100%
activation
b: Dose/ inhibition curve of human ADM 22-52 (ADM-receptor antagonist) in the
presence of
5.63nM hADM.
c: Dose/ inhibition curve of CT-H in the presence of 5.63 nM hADM.
d: Dose/ inhibition curve of MR-H in the presence of 5.63 nM hADM.
e: Dose/ inhibition curve of NT-H in the presence of 5_63 nM hADM.
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f: Dose response curve of mouse ADM. Maximal cAMP stimulation was adjusted to
100%
activation
g: Dose/ inhibition curve of human ADM 22-52 (ADM-receptor antagonist) in the
presence of
0,67 nM mADM.
h: Dose/ inhibition curve of CT-M in the presence of 0,67 nM mADM.
i: Dose/ inhibition curve of MR-M in the presence of 0,67 nM mADM.
j: Dose/ inhibition curve of NT-M in the presence of 0,67 nM mADM.
k: shows the inhibition of ADM by F(ab)2 NT-M and by Fab NT-M
1: shows the inhibition of ADM by F(ab)2 NT-M and by Fab NT-M
Fig. 4:
This figure shows a typical hADM dose/ signal curve. And an hADM dose signal
curve in the
presence of 100 pg/mL antibody NT-H.
Fig. 5:
This figure shows the stability of hADM in human plasma (citrate) in absence
and in the
presence of NT-H antibody.
Fig. 6:
Alignment of the Fab with homologous human framework sequences
Fig. 7:
This figure shows the Noradrenalin requirements for early and late treatment
with NT-M
Fig. 8:
This figure shows urine production after early and late treatment with NT-M
Fig. 9:
This figure shows the fluid balance after early and late treatment with NT-M
Fig. 10:
Liver tissue activation of nuclear factor kappa-light-chain gene enhancer in B
cells (NF-KB)
analyzed by electophoretic mobility shift assay (EMSA). # depicts p<0.001 vs.
vehicle.
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Fig. 11:
Development of serum creatinine over time. Mean +/- SEM are shown.
Fig. 12:
Development of blood urea nitrogen (BUN) over time. Mean +/- SEM are shown.
Fig. 13:
Development of endogenous creatinine clearance over time. Mean +/- SEM are
shown.
Fig. 14:
Development of fractional secretion of Na + over time. Mean +/- SEM are shown.
Fig. 15:
Keratinocyte-derived chernokine (KC) levels determined in relation to the
total kidney protein
extracted. The white box-plot shows results obtained with vehicle, the grey
box-plot shows
results obtained after treatment with NT-M.
Fig. 16:
Keratinocyte-derived ehemokine (KC) levels determined in relation to the total
liver protein
extracted. The white box-plot shows results obtained with vehicle, the grey
box-plot shows
results obtained after treatment with NT-M.
Fig. 17:
Plasma 1L-6 levels. The white box-plot shows results obtained with vehicle,
the grey box-plot
shows results obtained after treatment with NT-M.
Fig. 18:
Plasma IL-10 levels. The white box-plot shows results obtained with vehicle,
the grey box-plot
shows results obtained after treatment with NT-M.
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Fig. 19:
Plasma keratinocyte-derived chernokine (KC) levels. The white box-plot shows
results obtained
with vehicle, the grey box-plot shows results obtained after treatment with NT-
M.
Fig. 20:
Plasma monoeyte chemoattractant protein- l (MCP-l) levels. The white box-plot
shows results
obtained with vehicle, the grey box-plot shows results obtained after
treatment with NT-M.
Fig. 21:
Plasma TNF-alpha levels. The white box-plot shows results obtained with
vehicle, the grey box-
plot shows results obtained after treatment with NT-M.
Fig. 22:
Development of blood urea nitrogen (BUN) over time. Mean +/- SEM are shown.
Fig. 23:
Development of serum creatinine over time. Mean +7- SEM are shown.
Fig. 24:
Development of endogenous creatinine clearance over time. Mean +1- SEM are
shown.
