Interleukin-11 Compositions and Methods of Use
Related Applications
[1] The present application claims priority from Provisional Application No.
60/742,658, filed December 6, 2005 and Provisional Application No. 60/842,294 filed
September 5, 2006, both entitled "Interleukin-11 Compositions and Methods of Use".
Each of the provisional applications is incorporated herein by reference in its entirety.
Background of the Invention
[2] Platelets are important for maintaining hemostasis and for initiating blood clot
formation at sites of injury. Platelets also release growth factors at the site of clot
formation that, among several functions, speed the healing process. In patients suffering
from depressed levels of platelets (a condition known as thrombocytopenia), the inability to
form clots is the most immediate consequence. Severe thrombocytopenia results in a
typical pattern of bleeding: multiple petechiae in the skin, often most evident on the lower
legs; scattered small ecchymoses at sites of minor trauma; mucosal bleeding including
nosebleed, gingival bleeding, bleeding in the gastrointestinal and genitourinary tracts; and
excessive bleeding after surgery. Heavy gastrointestinal bleeding and bleeding of the
central nervous system (CNS) may be life-threatening.
[3] Thrombocytopenia manifests itself if either one of the steps in the
thrombopoietic process is interfered with resulting in failed platelet production, abnormal
platelet distribution, increased platelet destruction, and/or increased platelet consumption.
The differentiation and proliferation of hematopoietic cells may be interfered with by either
congenital or acquired causes, and these causes can vary widely. For example, congenital
amegakaryocytic hypoplasia can selectively decrease production of megakaryocytes, the
cells responsible for platelet production, thus resulting in thrombocytopenia. Low levels of
circulating platelets may also occur after exposure to or treatment with a chemical agent or
drug. Such drug-induced thrombocytopenia is generally treated by partial or complete
withdrawal of the offending agent.
[4] Thrombocytopenia can be a potentially fatal complication of many therapies for
cancer including gamma irradiation, therapeutic exposure to radiation, cytotoxic

chemotherapeutic drag treatment, and bone marrow transplantation. The diagnosis of
thrombocytopenia in cancer patients is often complicated by the fact that such patients are
often treated with multiple drugs and may also receive procedures that can enhance the
toxicity of the drugs. Drug-induced thrombocytopenia can therefore limit the benefits of
chemotherapy for potentially curable malignancies by preventing appropriate
administration of drugs at the optimal doses and schedule, which can lead to an increase in
cancer morbidity or even mortality.
Summary of the Invention
[5] The present invention encompasses the recognition that certain
chemotherapeutic agents, and in particular conjugates of protein targeting moieties with
cytotoxic agents, pose particular risks with respect to development of thrombocytopenia.
The invention provides a new system for the management of patients suffering from
thrombocytopenia induced by administration of such agent. In particular, the present
invention provides pharmaceutical compositions and methods that are useful for the
prevention and/or treatment of thrombocytopenia, such as drug-induced thrombocytopenia
(for example thrombocytopenia induced by chemotherapeutics, particularly conjugate
chemotherapeutics). Inventive pharmaceutical compositions and methods of treatment may
also be used for preventing or treating thrombocytopenia associated with liver damage
(e.g., drug-induced liver damage). Alternatively or additionally, the inventive
pharmaceutical compositions and methods of treatment may be used for preventing or
treating thrombocytopenia associated with bone marrow destruction (e.g., drug-induced
bone marrow destruction).
[6] More specifically, in one aspect, the present invention provides methods of
alleviating thrombocytopenia in a subject comprising a step of: administering a
therapeutically effective amount of interleukin-11 to the subject suffering from or
susceptible to thrombocytopenia, wherein thrombocytopenia is associated with
administering to the subject a conjugate comprising a targeting moiety and a cytotoxic
drug. Interleukin-11 used in the methods of the present invention may be, for example,
recombinant human interleukin-11.

[7] In certain embodiments of the methods of the present invention, the step of
administering comprises administering a therapeutically effective amount of interleukin-11
to a subject suffering from cancer or a cancerous condition.
[8] In certain embodiments, the targeting moiety in the conjugate whose
administration results in thrombocytopenia, comprises an antibody, such as an anti-CD22
antibody, an anti-CD33 antibody, an anti-Lewis Y antibody, an anti-5T4 antibody, an anti-
CD30 antibody, or any combinations thereof. The cytotoxic drug in the conjugate may be
a calicheamicin, a calicheamicin derivative, an esperamicin, or an esperamicin derivative.
For example, the conjugate may be an anti-CD22 antibody-calicheamicin conjugate, an
anti-CD33 antibody-calicheamicin conjugate, an anti-Lewis Y antibody-calicheamicin
conjugate, an anti-5T4 antibody-calicheamicin conjugate, or an anti-CD30 antibody-
calicheamicin conjugate.
[9] In certain embodiments of the methods of the present invention, interleukin-11
is administered prior to administration of the conjugate.
[10] In certain embodiments, administration of interleukin-11 according to methods
of the invention prevents, reduces, slows down or stops thrombocytopenia in the subject.
Thrombocytopenia produced by administration of the conjugate may be, at least partly,
resulting from bone marrow destruction. Alternatively or additionally, thrombocytopenia
produced by administration of the conjugate may be, at least partly, resulting from liver
damage. In such embodiments, administration of interleukin-11 may prevent, reduce, slow
down or stop liver damage and/or liver damage-related inflammation in the subject,
[11] In another aspect, the present invention provides pharmaceutical compositions
comprising a therapeutically effective amount of interleukin-11, at least one conjugate
whose administration results in thrombocytopenia, and at least one physiologically
acceptable carrier, wherein the conjugate comprises a targeting moiety and a cytotoxic
drug. In certain embodiments, interleukin-11 included in a pharmaceutical composition of
the present invention, comprises recombinant human interleukin-11. In some
embodiments, interleukin-11, at least one conjugate and at least one physiologically
acceptable carrier are combined as one or more preparations for simultaneous or sequential
administration of interleukin-11 and the conjugate.

[12] The targeting moiety in the conjugate may be an antibody (e.g., an anti-CD22
antibody, an anti-CD33 antibody, an anti-Lewis Y antibody, an anti-5T4 antibody, an anti-
CD30 antibody, or any combinations thereof), and the cytotoxic drug may be a
calicheamicin, a calicheamicin derivative, an esperamicin, or an esperamicin derivative.
For example, the conjugate included in an inventive pharmaceutical composition may be an
anti-CD22 antibody-calicheaniicin conjugate, an anti-CD33 antibody-calicheamicin
conjugate, an anti-Lewis Y antibody-calicheamicin conjugate, an anti-5T4 antibody-
calicheamicin conjugate, or an anti-CD30 antibody-calicheamicin conjugate.
[13] In certain embodiments, administration of a pharmaceutical composition of the
present invention to a subject prevents, reduces or stops thrombocytopenia in the subject.
As mentioned above, the subject may suffer from cancer or a cancerous condition.
Thrombocytopenia produced by administration of the conjugate may be, at least partly,
resulting from bone marrow destruction. Alternatively or additionally, thrombocytopenia
produced by administration of the conjugate may be, at least partly, resulting from liver
damage. In such embodiments, administration of a pharmaceutical composition of the
present invention may prevent, reduce, slow down or stop liver damage and/or liver
damage-related inflammation in the subject.
[14] In another aspect, the present invention provides a kit comprising: interleukm-
11 and at least one conjugate whose administration results in thrombocytopenia. The
conjugate comprises a targeting moiety (e.g., an antibody as described above) and a
cytotoxic drug (e.g., a calicheamicin, a calicheamicin derivative, an esperamicin or an
esperamicin derivative). Thrombocytopenia that results from administration of the
conjugate may result, at least partly, from liver damage. Alternatively or additionally,
thrombocytopenia that results from administration of the conjugate may result, at least
partly, from bone marrow destruction.
[15] These and other objects, advantages and features of the present invention will
become apparent to those of ordinary skill in the art having read the following detailed
description.

Brief Description of the Drawing
[16] Figure 1 is a graph showing the effects of EL-11 on CMC-544-induced
thrombocytopenia in nude mice. The results presented on this figure were obtained as
described in Example 1. Nude mice were administered vehicle alone, CMC-544
(200 g/kg) alone, IL-11 alone, IL-11 (125 ug/kg sc) after administration of CMC-544
(200 g/kg bid), or IL-11 (250 g/kg sc) before and after administration of CMC-544
(200 g/kg).
[17] Figure 2 is a graph showing the effects of IL-11 on CMC-544-induced
thrombocytopenia in nude mice. The results presented on this figure were obtained as
described in Example 1. Nude mice were administered vehicle alone, CMC-544
(160 g/kg) alone, IL-11 (250 g/kg sc) after administration of CMC-544 (160 g/kg), or
IL-11 (250 g/kg sc) before and after administration of CMC-544 (160 ug/kg).
[18] Figure 3 is a graph showing the effects of intravenous administration of CMC-
544 on the platelet counts of cynomolgus macaques. The results presented were obtained
in an experiment where 9 monkeys were administered one dose of CMC-544 at 4.28 mg/m2
(25 g/kg calicheamicin) per week for four weeks (CMC-544 administration is designated
by arrows in the graph). The platelet counts were followed as a function of time.
[19] Figure 4 is a graph showing the effects of IL-11 on CMC-544-induced
thrombocytopenia in cynomolgus macaques. Concentrations of platelets are plotted as a
function of the day of the procedure. Details of the experiments reported in this graph are
described in Example 2. Four (4) test monkeys were administered IL-11 (represented by
dark symbols in the graph) and four (4) control monkeys were administered the vehicle
alone (represented by open symbols in the graph) following a similar administration
schedule.
[20] Figure 5 presents two graphs showing the effects of IL-11 on CMC-544-
induced increase in liver enzymes (ALT) in cynomolgus macaques. Details of the
experiments are described in Example 2. The results presented on Fig. 5(A) were obtained
for four (4) control monkeys that received CMC-544 and the vehicle alone. The results
presented on Fig. 5(B) were obtained for four (4) test monkeys that received CMC-544 and
IL-11.

[21] Figure 6 presents two graphs showing the effects of IL-11 on CMC-544-related
increased peripheral blood neutrophil counts in cynomolgus macaques. Details of the
experiments are described in Example 2. The results presented on Fig. 6(A) were obtained
for four (4) control monkeys that received CMC-544 and the vehicle alone. The results
presented on Fig. 6(B) were obtained for four (4) test monkeys that received CMC-544 and
IL-11.
[22] Figure 7 presents two graphs showing the effects of IL-11 on CMC-544-related
decreased serum albumin in cynomolgus macaques. Details of the experiments are
described in Example 2. The results presented on Fig. 7(A) were obtained for four (4)
control monkeys that received CMC-544 and the vehicle alone. The results presented on
Fig. 7(B) were obtained for four (4) test monkeys that received CMC-544 and IL-11.
[23] Figure 8 presents two graphs showing the effects of IL-11 on CMC-544-related
decrease in red cell mass (hemoglobin) in cynomolgus macaques. Details of the
experiments are described in Example 2. The results presented on Fig. 8(A) were obtained
for four (4) control monkeys that received CMC-544 and the vehicle alone. The results
presented on Fig. 8(B) were obtained for four (4) test monkeys that received CMC-544 and
IL-11.
[24] Figure 9 presents two graphs showing the effects of administration of IL-11 in
combination with CMC-544 on alkaline phosphatase in cynomolgus macaques. Details of
the experiments are described in Example 2. The results presented on Fig. 9(A) were
obtained for four (4) control monkeys that received CMC-544 and the vehicle alone. The
results presented on Fig. 9{B) were obtained for four (4) test monkeys that received
CMC-544 and IL-11.
[25] Figure 10 presents two graphs showing the effects of administration of PBS
(vehicle), CMC-544 or Carboplatin on circulating platelets (Fig. 10(A)) and circulating
thrombopoietin (Tpo) (Fig. 10(B)) levels in nude mice. Details of the experiments are
described in Example 5.
[26] Figure 11 presents a graph allowing comparison of the effects of administration
of PBS (vehicle), CMC-544 or Carboplatin on circulating platelets levels and circulating
thrombopoietin levels in nude mice. Details of the experiments are described in
Example 5.

[27] Figure 12 presents two graphs showing the effects of administration of CMC-
544 (Fig. 12(A)) and CMC-544 + NEUMEGA (Fig. 12(B)) on platelet count (from pre-
study to day 10) in cynomolgus macaques treated as described in the Second Study of
Example 2.
[28] Figure 13 presents two graphs showing the effects of administration of CMC-
544 (Fig. 13(A)) and CMC-544 + NEUMEGA (Fig. 13(B)) on AST (from pre-study to day
14) in cynomolgus macaques treated as described in the Second Study of Example 2.
Detailed Description of Certain Preferred Embodiments
[29] As mentioned above, the present invention encompasses the recognition that
conjugate chemotherapeutics can pose particular risks for the development of
thrombocytopenia in patients. Such conjugates generally include a protein-targeting
moiety linked to a cytotoxic agent. According to the present invention, the protein-
targeting moiety may target the conjugate chemotherapeutic for preferential metabolism in
the liver, where the cytotoxic agent may induce damage that results in or exacerbates
thrombocytopenia. Conjugates containing antibodies or other heavily glycosylated proteins
may be particularly problematic in this regard.
[30] The present invention encompasses the finding that administration of
interleukin-11 can block drug-induced decrease in platelets circulating in the peripheral
blood (i.e., thrombocytopenia). In addition, administration of IL-11 was found to block
drug-induced liver damage, and to limit liver damage-related inflammation. Accordingly,
the present invention provides pharmaceutical compositions and methods for the
prevention and/or treatment of drug-induced thrombocytopenia, such as that resulting from
liver damage and/or bone marrow destruction.
[31] In the context of the present invention, a conjugate comprises a targeting moiety
and a cytotoxic drug. Conjugates whose administration results in thrombocytopenia
include any of a variety of conjugates of drugs known or suspected to cause, bring about, or
stimulate the occurrence of thrombocytopenia or to be associated with one or more
symptoms of thrombocytopenia. According to the present invention, administration pf
IL-11 prior to, following, or concomitant with administration of one or more of such
conjugates alleviates thrombocytopenia. Specifically, such administration prevents,

reduces, delays, treats or stops thrombocytopenia. As will be appreciated by one skilled in
the art, administration of EL-11 may prevent, reduce or stop thrombocytopenia by
preventing, reducing or stopping increased platelet destruction or consumption and/or by
preventing, reducing or stopping decreased platelet production by the bone marrow.
I. Thrombocyfopenia Resulting From Conjugate Administration
[32] In the context of the present invention, thrombocytopenia, in a broad sense,
refers to a physiological condition in mammals usually characterized by an abnormally low
blood platelet count, typically resulting in easy bruising and abnormal bleeding from
capillaries. In humans, the platelet count in the circulating blood is normally between 150
and 400 million per milliliter of blood (or 150 to 400 x 109/L).
[33] Platelets are produced in the bone marrow by large cells called megakaryocytes
via a process called endomitosis (Y. Nagata et al, J. Cell Biol., 1997,139:449-457; L. Roy
et al, Blood, 2001, 97: 2238-2247). In response to a decreased circulating platelet count,
the rate of the endomitotic process increases, and the number of megakaryocytes in the
bone marrow may increase up to 3-fold (L.A. Harker, J. Clin. Invest., 1968, 47: 458-465).
This mechanism, in turn, causes the production and release into the circulation of
additional platelets. In contrast, in response to an elevated circulating platelet count, the
endomitotic rate decreases, and the number of megakaryocytes in the bone marrow may
decrease by 50%. The exact physiological feedback mechanism by which the mass of
circulating platelets regulates the endomitotic rate and number of bone marrow
megakaryocytes is not fully understood. However, the major circulating factor involved in
the feedback loop, and thus in platelet production, is thought to be thrombopoietin
(K. Kaushansky et al, Proc. Natl. Acad. Sci. USA, 1995, 92: 3234-3238; K. Kaushansky,
Thromb. Haemost, 1995, 74: 521-525), which is produced by hepatocytes in the liver (F.J.
de Sauvage et al, Nature, 1994, 369; 533-538; R. Sungaran et al, Blood, 1997, 89: 101-
107; S. Nomura et al, Exp. Hematol., 1997, 25: 565-572). Thus, in the context of the
present invention, thrombocytopenia can result from bone marrow destruction, liver
damage, or a combination of the two.
[34] In some embodiments, administration of a conjugate results, at least in part, in
liver damage, which ultimately results in thrombocytopenia. Conjugates whose
administration results in liver damage generally include those conjugated cytotoxic agents

whose administration causes, brings about or stimulates liver damage or is correlated with
one or more symptoms of liver damage. In certain embodiments, such conjugates have a
targeting moiety that is or includes a protein; in some embodiments the targeting moiety is
or includes an antibody.
[35] Liver damage according to the present invention includes not only degeneration
or necrosis of liver parenchyma cells (hepatocytes) (e.g., which results from damage or
injury caused by a certain factor), but also undesirable phenomena caused by biological
reaction to the damage or injury, such as mobilization, infiltration, activation of Kupffer
cells, leukocytes, and the like, swelling of the liver, fibrosis of the liver tissue, etc, which
can occur alone or in combination. Liver injury, defect or dysfunction that is entirely or
less than entirely caused, brought about or stimulated by administration of one or more
conjugates is considered to be at least partly drug-induced. Thus, before administration of
one or more conjugates, a subject may have a healthy liver (i.e., a liver showing no
detectable sign of injury, defect, damage or dysfunction) or, alternatively, the subject may
exhibit some existing level of liver injury/damage/defect/dysfunction (e.g., due to diseases,
viruses, chemicals, drugs, or other factors).
[36] In certain embodiments, administration of IL-11 prior to, following, or
concomitant with administration of one or more conjugates prevents, reduces, delays, treats
or stops thrombocytopenia resulting, at least in part, from conjugate-induced liver damage.
[37] In some embodiments, administration of a conjugate results, alternatively (or in
some cases additionally), at least in part, in bone marrow destruction, which produces
thrombocytopenia. Conjugates whose administration results in bone marrow destruction
include those conjugated cytotoxic agents whose administration causes, brings about,
stimulates bone marrow destruction or is correlated with one or more symptoms of bone
marrow destruction. Bone marrow destruction (i.e., bone marrow damage, defect or
dysfunction) includes any condition affecting the bone marrow that results in abnormally
low platelet production.
[38] According to the present invention, administration of IL-11 prior to, following,
or concomitant with administration of one or more conjugates prevents, reduces, delays,
treats or stops thrombocytopenia resulting, at least partly, from bone marrow destruction.
In certain embodiments, administration of IL-11 according to the present invention

prevents, reduces or stops decreased production of platelets due to bone marrow
destruction. Before administration of the conjugate inducing bone marrow destruction, a
subject may have a healthy bone marrow (i.e., a bone marrow showing no detectable sign
of destruction, damage, defect or dysfunction) or alternatively, the subject may exhibit
some existing level of bone marrow destruction/defect/dysfunction (e.g., due to cancer,
such as leukemia or lymphoma; viral infection or aplastic anemia or caused by toxic
chemicals, radiation therapy or previous chemotherapy).
II. Conjugates
[39] A conjugate generally is a molecule resulting from the bonding of at least two
other molecules. The bonding between the two molecules may be covalent or non-
covalent. As already mentioned above, a conjugate herein comprises a targeting moiety
and a cytotoxic drug.
[40] Targeting moieties are entities that have some degree of attraction for a target of
interest when comprised in a conjugate. A targeting moiety often exhibits high affinity
and/or specificity for the target, i.e., it specifically and/or efficiently recognizes, interacts
with, binds to, or labels the target under the conditions or circumstances of its exposure to
the target. A target may be a specific tissue or organ in the body, a specific type of cells or
a specific cell component (e.g., cell surface receptor or antigen). Targeting moieties may
desirably be stable, non-toxic entities that retain their properties under in vitro and/or in
vivo conditions. The interaction between a targeting moiety and a target may be covalent
or non-covalent. Most often, the interaction between a targeting moiety and a target is non-
covalent. Examples of non-covalent interactions include, but are not limited to,
hydrophobic interactions, electrostatic interactions, dipole interactions, van der Waals
interactions, and hydrogen bonding. Irrespective of the nature of the interaction, the
binding between a target and a targeting moiety within a conjugate is preferably selective,
specific, and strong enough to allow the drug to play its role (e.g., exert its anti-cancer
activity if the cytotoxic drug is a chemotherapeutics).
[41] Within a conjugate, a cytotoxic drug may be associated with the targeting
moiety in any of a variety of ways. In many embodiments, the drug is covalently attached
to the targeting moiety. As will be appreciated by those skilled in the art, the drug and

targeting moiety may be attached to each, other either directly or indirectly (e.g., through a
linker).
[42] In certain embodiments, the cytotoxic drug and targeting moiety are directly,
covalently linked to each other. The direct covalent binding can be through a linkage such
as an amide, ester, carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, or
carbonate linkage. The covalent binding can be achieved by taking advantage of functional
groups present on the drug and the targeting moiety. Suitable functional groups that can be
used to attach the two moieties together include, but are not limited to, amines, anhydrides,
hydroxy groups, carboxy groups, and thiols. An activating agent, such as a carbodiimide,
can be used to form a direct linkage. A wide range of activating agents are known in the
art and are suitable for linking a drug and a targeting moiety.
[43] In other embodiments, the cytotoxic drug and targeting moiety are indirectly
covalently linked to each other via a linker group. This can be accomplished by using any
number of stable bifunctional agents well known in the art, including homofunctional and
heterofunctional linkers (see, for example, Pierce Catalog and Handbook). The use of a
bifunctional linker differs from the use of an activating agent in that the former results in a
linking moiety being present in the resulting conjugate, whereas the latter results in a direct
coupling between the two moieties involved in the reaction. The role of the bifunctional
linker may be to allow the reaction between two otherwise inert moieties. Alternatively or
additionally, the bifunctional linker, which becomes part of the reaction product, may be
selected such that it confers some degree of conformational flexibility to the conjugate.
Alternatively or additionally, the bifunctional linker may be selected such that the linkage
formed between the drug and the targeting moiety is hydrolysable (for examples of such
linkers, see e.g. U.S. Pat. Nos. 5,773,001; 5,739,116 and 5,877,296). Such linkers are
preferably used when higher activity of the drug is observed after hydrolysis of the
targeting moiety. Exemplary mechanisms by which a drug is cleaved from the targeting
moiety (e.g., antibody) include hydrolysis in the acidic pH of the lysosomes (hydrazones,
acetals, and cis-aconitate-like amides), peptide cleavage by lysosomal enzymes (the
capthepsins and other lysosomal enzymes), and reduction of disulfides.
[44] One example of a suitable conjugate relies on the conjugation of hydrazides and
other nucleophiles to the aldehydes generated by oxidation of the carbohydrates that
naturally occur on antibodies. Hydrazone-containing conjugates can be made with

introduced carbonyl groups that provide the desired drug-release properties. Conjugates
can also be made with a linker that has a disulfide at one end, an alkyl chain in the middle,
and a hydrazine derivative at the other end. The anthracyclines are one example of
cytotoxins that can be conjugated to antibodies using this technology.
[45] Linkers containing functional groups other than hydrazones have the potential
to be cleaved in the acidic milieu of the lysosomes. For example, conjugates can be made
from thiol-reactive linkers that contain a site other than a hydrazone that is cleavable
intracellularly, such as esters, amides, and acetals/ketals. Camptothecin is one cytotoxic
agent that can be conjugated using these linkers. Ketals made from a 5 to 7-member ring
ketone and that has one of the oxygen atoms attached to the cytotoxic agent and the other
to a linker for antibody attachment also can be used. The anthracyclines are again an
example of a suitable cytotoxic agent for use with these linkers.
[46] Another example of class of pH sensitive linkers are the cis-aconitates, which
have a carboxylic acid group juxtaposed to an amide group. The carboxylic acid
accelerates amide hydrolysis in the acidic lysosomes. Linkers that achieve a similar type of
hydrolysis rate acceleration with several other types of structures can also be used. The
maytansinoids are an example of cytotoxin that can be conjugated with linkers attached at
C-9.
[47] Another potential release method for drug conjugates is the enzymatic
hydrolysis of peptides by the lysosomal enzymes. In one example, a peptide is attached via
an amide bond to para-aminobenzyl alcohol and then a carbamate or carbonate is made
between the benzyl alcohol and the cytotoxic agent. Cleavage of the peptide leads to the
collapse, or self-immolation, of the aminobenzyl carbamate or carbonate. The cytotoxic
agents exemplified with this strategy include anthracyclines, taxanes, mitomycin C, and the
auristatins. hi one example, a phenol can also be released by collapse of the linker instead
of the carbamate. In another variation, disulfide reduction is used to initiate the collapse of
a para-mercaptobenzyl carbamate or carbonate.
[48] Many cytotoxic agents have little, if any, solubility in water and that can limit
drug loading on the conjugate due to aggregation of the conjugate. One approach to
overcoming this is to add solubilizing groups to the linker. Conjugates made with a linker
consisting of PEG and a dipeptide can be used, including those having a PEG di-acid, thiol-

acid, or maleimide-acid attached to the targeting moiety (e.g., antibody), a dipeptide spacer,
and an amide bond to the amine of an anthracycline or a duocarmycin analogue. Another
example is conjugates that are made with a PEG-containing linker disulfide bonded to a
cytotoxic agent and amide bonded to an antibody. Approaches that incorporate PEG
groups may be beneficial in overcoming aggregation and limits in drug loading.
A. Targeting Antibodies
[49] In certain embodiments, the conjugate comprises an antibody as targeting
moiety. These types of conjugates are commonly referred to as immunoconjugates, with
those conjugates having a radioisotope as the drug, referred to as radioimmunoconjugates
and those having a chemotherapeutic agent as the drug, referred to as
chemoimmunoconjugates. Generally speaking, an antibody for these purposes may be any
immunoglobulin (i.e., an intact immunoglobulin molecule, an active portion of an
immunoglobulin molecule, etc), that binds to a specific epitope. The term encompasses
monoclonal antibodies and antibody compositions with polyepitopic specificity (i.e.,
polyclonal antibodies).
[50] Targeting antibodies can be from almost any mammalian species (e.g., mouse,
human, primate, dog, etc) and can be produced by various methods well known in the art
(e.g., murine antibodies via hybridomas, human antibodies via hybridomas from transgenic
mice, etc).
[51] Examples of antibodies that may be used in the formation of conjugates useful
in the present invention include monoclonal antibodies (mAbs), for example, chimeric
antibodies, humanized antibodies, primatized antibodies, resurfaced antibodies, human
antibodies and biologically active fragments thereof. As already mentioned above, the
term antibody is used broadly to refer to both antibody molecules and a variety of antibody
derived molecules. Such antibody-derived molecules generally comprise at least one
complementarity determining region (CDR) from either a heavy chain or light chain
variable region, including molecules such as Fab fragments, F(ab')2 fragments, Fd
fragments, Fabc fragments, Sc antibodies (single chain antibodies), diabodies, individual
antibody light single chains, individual antibody heavy chains, chimeric fusions between
antibody chains and other molecules, and the like. Antibodies mimetics having binding

affinity for an antigen but not having one or more traditional CDRs also can be used in the
formation of conjugates useful in the present invention.
[52] In certain embodiments, a targeting antibody of a conjugate is directed against
one or more cell surface antigens expressed on target cells and/or tissues in proliferative
disorders such as cancer.
[53] Examples of specific antibodies directed against cell surface antigens on target
cells include without limitation, antibodies against CD22 antigen, which is over-expressed
on most B-cell lymphomas; G5/44, a humanized form of a murine anti-CD22 monoclonal
antibody; antibodies against cell surface antigen CD33, which is prevalent on certain
human myeloid tumors especially acute myeloid leukemia (see, for example U.S. Pat.
Appln. Nos. 2004-0192900 and 2004-0082764, each of which is incorporated herein by
reference in its entirety); hP67.6, a humanized form of the anti-CD33 murine antibody (see,
U.S. Pat. No. 5,773,001, which is incorporated herein by reference in its entirety); an
antibody against PEM antigen found on many tumors of epithelial origin designated
mP67.6 (see, for example, I.D. Bernstein et al., J. Clin. Invest, 1987, 79: 1153-1159 and
I.D. Bernstein et al, J. Immunol., 1992, 128: 867-881, each of which is incorporated herein
by reference in its entirety), and a humanized antibody against the Lewis Y carbohydrate
antigen overexpressed on many solid tumors and designated hu3S193 (see, for example,
U.S. Pat. No. 6,310,185, which is incorporated herein by reference in its entirety).
[54] Other suitable antibodies include antibodies directed against the 5T4 oncofetal
antigen. The 5T4 antigen is a 72 kDa highly glycosylated transmembrane glycoprotein
comprising a 42 kDa non-glycosylated core (Hole et al, Br. J. Cancer, 1988, 57: 239-246;
Hole et al, Int. J. Cancer, 1990, 45: 179-184; WO 89/07947; U.S. Pat. No. 5,869,053, each
of which is incorporated herein by reference in its entirety). 5T4 includes an extracellular
domain characterized by two leucine-rich repeats (LRRs) and an intervening hydrophilic
region, which is an accessible antigen for targeted therapy (Myers et al., J. Biol. Chem.,
1994, 269: 9319-9324). Other suitable antibodies include antibodies directed against CD30
antigen, which is over-expressed on a variety of hematologic malignancies. CD30 is an
attractive target for cancer therapy because it has minimal expression on normal tissues.
SGN-30 is an example of an anti-CD30 antibody that has been shown to induce direct anti-
cancer activity towards tumor cells expressing CD30 (A. Forero et al, J. Clin. Oncol.,
2005: Vol. 23, No. 16S: 6601).

[55] In addition, there are several commercially available antibodies directed against
cell surface antigens, such as rituximab (Rituxan™) and trastuzumab (Herceptin™), which
may be used as targeting moieties in chemotherapeutic conjugates. Rituximab (Rituxan )
is a chimeric anti-CD20 antibody used to treat various B-cell lymphomas and trastuzumab
(Herceptin™) is a humanized anti-Her2 antibody used to treat breast cancer.
[56] In certain embodiments of the invention, a targeting moiety of a
chemotherapeutic conjugate is an anti-CD22 antibody, an anti-CD33 antibody, an anti-
Lewis Y antibody, an anti-CD30 antibody, or an anti-5T4 antibody.
B. Cytotoxic Drugs
[57] Suitable cytotoxic drugs include any of a large variety of substances, molecules,
compounds, agents, or factors that are toxic to living cells. Administered as a conjugate to
a patient, a suitable cytotoxic drug is associated with thrombocytopenia.
Thrombocytopenia may result, at least partly, from drug-induced liver damage.
Alternatively or additionally, thrombocytopenia may result at least partly, from drug-
induced bone marrow destruction.
[58] As will be appreciated by one of ordinary skill in the art, a cytotoxic drug may
be a synthetic or a natural compound; a single molecule or a complex of different
molecules. Suitable cytotoxic drugs can belong to any of various classes of compounds
including, but not limited to, small molecules, peptides, saccharides, steroids, antibodies,
fusion proteins, antisense polynucleotides, ribozymes, small interfering RNAs,
peptidomimetics, and the like. When cytotoxic drugs are used in the treatment of cancer or
a cancerous condition, they can be found among the following classes of anti-cancer drugs:
alkylating agents, anti-metabolite drugs, anti-mitotic antibiotics, alkaloidal anti-tumor
agents, hormones and anti-hormones, interferons, non-steroidal anti-inflammatory drugs,
and various other anti-tumor agents.
[59] Examples of suitable drugs for use in immunoconjugates include the taxanes,
maytansines, CC-1065 and the duocarmycins, the calicheamicins and other enediynes, and
the auristatins. Other examples include the anti-folates, vinca alkaloids, and the
anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT),
radioisotopes, photosensitizers such as those employed in photodynamic therapy, can also

be used in immunoconjugates. In addition, conjugates can be made using secondary
carriers as the cytotoxic agent, such as liposomes or polymers, for example.
[60] In certain embodiments, the cytotoxic drug belongs to the enediyne family of
antibiotics. As a family, the enediyne antibiotics are the most potent, anti-tumor agents
discovered so far. Some members are 1000 times more potent than adriamycin, one of the
most effective, clinically used anti-tumor antibiotics (Y.S. Zhen et al., J. Antibiot., 1989,
42: 1294-1298).
[61] In certain embodiments, the cytotoxic drug is a member of the enediyne family
of calicheamicins. Originally isolated from a broth extract of the soil microorganism
Micromonospora echinospora ssp. calichensis, the calicheamicins were detected in a
screen for potent DNA damaging agents (M.D. Lee et al, J. Am. Chem. Soc, 1987, 109:
3464-3466; M.D. Lee et al, J. Am. Chem. Soc, 1987, 109: 3466-3468; W.M. Maiese et
al, J. Antibiot., 1989, 42: 558-563; M.D. Lee etal, J. Antibiot., 1989,42:1070-1087).
[62] Calicheamicins are characterized by a complex, rigid bicyclic enediyne allylic
trisulfide core structure linked through glycosyl bonds to an oligosaccharide chain. The
oligosaccharide portion contains a number of substituted sugar derivatives, and a
substituted tetrahydropyran ring. The enediyne containing core (or aglycone) and
carbohydrate portions of calicheamicins have been reported to carry out different roles in
the biological activity of these molecules. Without wishing to be bound by any particular
theory, we note that it is generally believed that the core portion cleaves DNA, whereas the
oligosaccharide portion of the calicheamicins serves as a recognition and delivery system
and guides the drug to a double-stranded DNA minor groove in which the drug anchors
itself. Once positioned in the minor groove of DNA, the enediyne core undergoes an
electronic rearrangement (Bergman cyclization) to form a transient 1,4-benzenoid
diradical. Formation of the diradical intermediate can be triggered by the presence of
reducing agents such as NADPH or dithiothrietol. The diradical species provides the
thermodynamic driving force for the DNA cleaving reaction by promoting hydrogen atom
abstraction from the deoxyriboses. Reaction of the resulting deoxyribose carbon-centered
radicals with molecular oxygen initiates a process that results in both single-strand and
double-strand DNA cleavages ("Enediyne Antibiotics as Antitumor Agents", Doyle and
Borders, 1995, Marcel-Dekker: New York; N. Zein et al, Science, 1988, 240: 1198-1201;
N. Ikemoto et al, Proc. Natl. Acad. Sci., USA, 1995, 92: 10506-10510; A.G. Myers et al.,

J. Am. Chem. Soc., 1994, 116: 1255-1271: M.D. Lee et al, Acc. Chem. Res., 1991, 24:
235-243; Y. Xu et al, Biochemistry, 1997, 36: 14975-14984). Double-stranded DNA
cleavage is a type of damage that is usually non-repairable oi non-easily repairable for the
cell and is most often lethal.
[63] Because of their chemical and biological properties, several analogues of the
calicheamicins have been tested in preclinical models as potential anti-tumor agents. Their
development as single agent therapies has not been pursued because of delayed toxicities
that limit the therapeutic dose range for treatment. However, their potency makes them
particularly useful for conjugate-targeted chemotherapy, where limits in expression of the
conjugate target can require high potency in order to achieve effectiveness (C. Liu and
R.V.J. Chri, Exp. Opin. Invest. Drugs, 1997, 6: 169-172; R.V.J. Chari et al., Cancer Res.,
1995, 55: 4079-4084).
[64] Accordingly, in certain embodiments of the present invention, the
chemotherapeutic comprises an antibody-calicheamicin conjugate.
[65] It will be appreciated that the term "calicheamicin" can refer to any member of
the family of antibacterial and anti-tumor agents known as calicheamicins, for example, as
described in U.S. Pat. Nos. 4,970,198 and 5,108,912 (each of which is incorporated herein
by reference in its entirety). Analogs or derivatives of calicheamicins, such as, for
example, N-acyl derivatives described in U.S. Pat. No. 5,079,233 (which is incorporated
herein by reference in its entirety); disulfide analogs of calicheamicin (e.g., as described in
U.S. Pat. Nos. 5,606,040; and 5,770,710, each of which is incorporated herein by reference
in its entirety); dihydro derivatives (e.g., as described in U.S. Pat. No. 5,037,651, which is
incorporated herein by reference rn its entirety); and N-acetylated derivatives (e.g., as
described in U.S. Pat. No. 5,079,233, which is incorporated herein by reference in its
entirety) can be employed.
[66] Several antibody-calicheamicin conjugates have been prepared and tested for
their anti-tumor properties (L.M, Hinmarn et al., Cancer Res., 1993, 53: 3336-3342; P.R.
Hamann et al., Bioconj. Chem., 2005, 16: 346-353; N.K. Damle and P. Frost, Curr. Opin.
Pharmacol., 2003, 3: 386-390). In certain embodiments, an antibody-calicheamicin
conjugate can be included in an inventive pharmaceutical composition or used in an
inventive method of treatment; such conjugates include those conjugates described in U.S.

Pat. No. 5,773,001; 5,739,116; 5,712,374; 5,714,586; and 5,877,296; PCT application WO
03/092623, each of which is incorporated herein by reference in its entirety.
[67] In certain embodiments of the present invention, the antibody-calicheamicin
conjugate is CMC-544. CMC-544 is targeted to CD22 expressed by B-lymphoid
malignancies. CMC-544 comprises a humanized IgG4 anti-CD22 monoclonal antibody
(mAb), G5/44, covalently linked to N-acetyl--calicheamicin dimethyl hydrazide
(CalichDMH) via an acid-labile 4-(4'-acetylphenoxy) butanoic acid linker. CMC-544 can
be prepared, for example, as described in J.F. DiJoseph et al., Blood, 2004,103: 1807-1814
and U.S. Pat. Appln. Nos. 2004-0082764A1 and 2004-0192900A1, each of which is
incorporated herein by reference in its entirety.
[68] In other embodiments, the antibody-calicheamicin conjugate is CMD-193,
which is described in U.S. Pat. Appln. No. 10/080,587 (incorporated herein by reference in
its entirety). CMD-193 is Af-acetyl--calicheamicin dimethyl hydrazide covalently attached
to the anti-Lewis Y antibody G193 with the average loading of calicheamicin conjugate
from about 5 to about 7 moles of calicheamicin per mole of antibody and the low
conjugated fraction (LCF) of the conjugate less than about 10%.
[69] hi other embodiments, the antibody-calicheamicin conjugate is MYLOTARG®,
also known as, CMA-676, CMA, or gemtuzumab ozogamicin (see, for example,
E.L. Sievers et al., Blood, 1999, Blood, 93: 3678-3584, and U.S. Pat. Nos. 5,712,374;
5,714,586; 5,739,116; 5,767,285; 5,773,001; 5,877,296 and U.S. Pat. Application No.
2004-0152632, each of which is incorporated herein by reference). MYLOTARG® is
currently approved for the treatment of acute myeloid leukemia in elderly patients. The
conjugate consists of an antibody against CD33 that is bound to calicheamicin by means of
an acid-hydrolysable linker. The disulfide analog of the semi-synthetic N-acetyl-
y-calicheamicin is used in the conjugation (U.S. Pat. No. 5,606,040 and 5,770,710).
[70] In other embodiments, the antibody-calicheamicin conjugate is CME-548 (see,
for example, U.S. Pat. Appln. No. 11/221,902 (incorporated herein by reference in its
entirety)).
[71] In certain other embodiments, the cytotoxic drug belongs to the enediyne family
of esperamicins. Esperamicins have been identified in cultures of Actinomadura
verrucosospora (M. Konishi et al., J, Antibiot, 1985, 38: 1605-1609), and the elucidation

of their structures has been reported (J. Golik et al., J. Am. Chem. Soc., 1987, 109: 3461-
3462; J. Golik et al, J. Am. Chem. Soc., 1987,109: 3462-3464). The mechanism by which
these molecules produce cytotoxicity was investigated and found to be similar to that of
calicheamicins and involve the participation of a diradial species which leads to the
formation of single- and double-stranded DNA breaks (B.H. Long et al., Proc. Natl. Acad.
Sci. USA, 1989, 86:2-6).
[72] In certain embodiments of the present invention, the chemotherapeutic
conjugate is an antibody-esperamicin conjugate. As will be appreciated, the term
"esperamicin" can refer to any member of the esperamicin family of antibacterial and anti-
tumor agents known in the art; analogs or derivatives of such esperamicins may also be
employed (see, for example, U.S. Pat. Nos. 4,675,187; 4,539,203; 4,554,162; and
4,837,206, each of which is incorporated herein by reference in its entirety).
III. Interleukin-ll
[73] The present invention provides methods of administration of, and
pharmaceutical compositions comprising, interleukin-11 (IL-11).
[74] Interleukin-ll is a member of a family of growth factors that includes growth
hormone, granulocyte colony-stimulating factor (G-CSF), and other growth factors. IL-11
is also a member of a family of cytokines that includes IL-6, leukemia inhibitory factor
(LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), which all signal
through a common receptor subunit, gpl30 (S. Neben and K. Turner, Stem Cells, 1993, 11:
156-162). IL-11, which is naturally produced by bone marrow stromal cells, is a
thrombopoietic growth factor that, in conjunction with other factors, stimulates the
proliferation of hematopoietic stem cells and megakaryocyte progenitor cells and induces
maturation resulting in increased platelet production.
[75] Typically, inventive methods and compositions utilize IL-11 in an active form,
and often in an active form substantially free from association with other mammalian
proteins or proteinaceous materials.
[76] Interleukin-ll (or IL-11), as used in accordance with the present invention, is
generally an isolated protein comprising the entire polypeptide sequence of wild-type or
mutant DL-11 or an active fragment thereof. A protein or polypeptide may be considered

isolated by virtue of its origin or manipulation, for example if (a) it is present in a host cell
as the expression product of a portion of an expression vector; or (b) it is linked to a protein
or chemical moiety other than that to which it is linked in nature; or (c) it does not occur in
nature. Alternatively or additionally, an isolated polypeptide or protein may be one that is
produced or prepared (including by chemical synthesis), by the hand of man. Those of
ordinary skill in the art will appreciate that a wild-type polypeptide or protein has a normal
amino acid sequence found in nature, whereas a mutant polypeptide or protein has an
amino acid sequence that is identical to that of the wild type at most positions but that
includes one or more differences (e.g., substitutions, additions, deletions, alterations or
combinations thereof) at precise locations. A mutant can have more than one difference
but, as can be appreciated by those of ordinary skill in the art, overall sequence similarity to
the wild-type is maintained.
[77] In certain embodiments, IL-11 utilized in accordance with the present invention
has the sequence of naturally occurring human IL-11 or of other mammals (e.g., mouse,
rat, rabbit, monkey, dog, cat, pig, cow, horse and the like). The molecular cloning and
characterization of murine interleukin-11 has been reported by J.C. Norris et al., (Exp.
Hematol., 1996, 24: 1369-1376, which is incorporated herein by reference in its entirety).
The cDNA sequence and the amino acid sequence (single letter code) of primate (healthy
macaque monkey) and human clones of the IL-11 polypeptide can be found in U.S. Pat.
Nos. 5,371,193; 5,700,664; 5,854,028 and 6,066,317 (each of which is Incorporated herein
by reference in its entirety). The primate nucleotide sequence comprises 1100 base pairs,
including a 5' non-coding sequence of 72 bases and a 3' non-coding sequence of 431
bases. The human nucleotide sequence similarly contains a single long reading frame of
597 nucleotides. Both the primate and human IL-11 proteins have a molecular mass of
approximately 19,000 daltons; are 178 amino acids in length; and are non-glycosylated. A
polynucleotide that encodes primate or human IL-11 has been disclosed in U.S. Pat.
No. 5,215,895 (which is incorporated herein by reference in its entirety).
[78] Thus, in certain embodiments, IL-11 included in a pharmaceutical composition
or used in a method of treatment of the present invention comprises murine IL-11. In other
embodiments, the IL-11 comprises primate IL-11. In still other embodiments, IL-11
comprises human IL-11. In some embodiments, the type of IL-11 protein utilized matches
that of the target species (i.e., species of subjects to undergo treatment with IL-11). For

instance, in some embodiments, human IL-11 is used in compositions and methods for
treatment of humans.
[79] In other embodiments, the amino acid sequence of IL-11 to be used in the
compositions and methods of the present invention is sufficiently homologous to naturally
occurring IL-11 (e.g., to one or more sequences published in references cited above and/or
listed in an established database such as GenBank, SwissProt, etc). Typically, polypeptides
or proteins are considered sufficiently homologous to naturally occurring IL-11 if they
share overall sequence identity of at least 35% with the IL-11. In certain embodiments, the
sequence identity is at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or better.
Calculations of the percent homology or identity of two amino acid sequences can be
performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can
be introduced in one or both of a first and a second amino acid sequence for optimal
alignment and non-homologous sequences can be disregarded for comparison purposes).
In certain embodiments, the length of a reference sequence aligned for comparison
purposes is at least 30%, 40%, 60%, 80%, 90%, 95% or more, e.g., 96%, 97%, 98%, 99%
or 100% of the length of the reference sequence. The amino acid residues at corresponding
amino acid positions are then compared. When a position in the first sequence is occupied
by the same amino acid residue as the corresponding position in the second sequence, then
the molecules are identical (or homologous) at that position. The percent identity between
the two sequences is a function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[80] The comparison of sequences and determination of percent identity between
two sequences can be accomplished using a mathematical algorithm. For example, the
percent identity between two amino acid sequences may be determined using the
Needleman and Wunsch algorithm (J. Mol. Biol., 1970, 48: 444-453), which has been
incorporated into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The percent
identity between two amino acid sequences can also be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the

ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty
of 12 and a gap penalty of 4.
[81] Compared to the amino acid sequence of naturally occurring IL-11, a
sufficiently homologous sequence may include one or more of conservative substitutions,
additions, alterations or deletions of one or more selected amino acid residues. As will be
understood by those of ordinary skill in the art, conservative substitutions generally
represent substitutions that are physically or functionally similar to the corresponding
reference residue, e.g., that have a similar size, shape, electric charge, chemical properties,
including the ability to form covalent or hydrogen bonds, or the like. In some
embodiments, conservative substitutions are those fulfilling the criteria defined for an
"accepted point mutation" by Dayhoff et al. ("Atlas of Protein Sequence and Structure",
1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).
Techniques for such replacement, insertion or deletions of individual residues or sets of
residues are well known in the art (see, for example, U.S. Pat. No. 4,518,584).
[82] In some embodiments of the invention, the amino acid sequence of IL-11 to be
used in the compositions and methods of the present invention is an active fragment of the
naturally occurring sequence of IL-11 or an active fragment of a sequence that is
sufficiently homologous to the naturally occurring sequence of IL-11.
[83] Active fragments of IL-11 generally have a sequence that is identical to or
sufficiently homologous with IL-11 but includes fewer amino acids than the full length
protein, and retains the ability to block thrombocytopenia, liver damage and/or liver
damage-related inflammation. Specifically, an active fragment of IL-11, for purposes of
the present invention, may be one that retains the ability to prevent, slow down, reduce or
stop thrombocytopenia; prevent, slow down, reduce or stop liver damage; limit liver-
damage-related inflammation; or any combination thereof, when administered to a subject.
Typically, an active fragment may comprise a domain or motif of the full length protein
having the activity. An active fragment can be of a polypeptide which is, for example, 10,
25, 50, 100, 150, 175, 177, 178, 180, 185, 190, 195, 200 or more amino acids in length.
When applied to IL-11, the term "active fragment" refers to any peptide comprising an
amino acid sequence sufficiently homologous to or derived from the amino acid sequence
of naturally occurring IL-11, which includes fewer amino acids than the full length protein,
and retains the ability to prevent, slow down, reduce or stop thrombocytopenia; prevent,

slow down, reduce or stop liver damage; limit liver-damage-related inflammation; or any
combination thereof, when administered to a subject.
[84] Particular active fragments of IL-11 have amino acid sequences substantially
identical to a portion of the amino acid sequence of the naturally occurring IL-11 sequence.
Such substantially identical sequences contain significant number of amino acid residues
that are (i) identical to, or (ii) conservative substitutions of aligned amino acid residues
such that they include relevant structural domain and/oT functional activity of IL-11. For
example, amino acid sequences that contain a common structural domain showing at least
about 60% or 65% identity, at least 75% identity, or at least 85%, 90%, 95%, 96%, 97%,
98%, 99%, or 100% identity with a relevant domain of IL-11 may be substantially
identical.
[85] Interleukin-11 to be used in the compositions and methods of the present
invention may be obtained by any suitable method. Methods of producing polypeptides or
proteins are well known in the art. For example, IL-11 can be obtained as a homogeneous
protein purified from a mammalian cell line secreting it; or it can be chemically
synthesized. Alternatively, interleukin-11 can be produced via recombinant techniques to
enable large quantity production of pure, active IL-11 useful for therapeutic applications
(as described in U.S. Pat. Nos. 5,215,895; 5,31,193; 5,700,664; 5,854,028 and 6,066,317,
each of which is incorporated by reference in its entirety).
[86] For example, IL-11 included in an inventive pharmaceutical composition or
used in an inventive method of treatment may be produced in a host cell by recombinant
DNA methods. Host cells may be mammalian or non-mammalian cells. Suitable
mammalian cells include, but are not limited to, non-human mammalian tissue culture cells
such as Chinese Hamster Ovary (CHO) cells, monkey COS cells, and mouse fibroblast
NHI3T3 cells; and human tissue culture cells such as HeLa cells, HL-60 cells, kidney 293
cells, and epidermal S431 cells! Non-mammalian host cells include bacteria cells such as
Escherichia coli, Bacillus subtilis, attenuated strains of Salmonella typhimurium, and the
like; yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Kluyveromyces strains, Candida, or any yeast strain capable of expressing proteins; insect
cells such as Spodoptera frugiperda.

[87] In certain embodiments, IL-11 included in an inventive pharmaceutical
composition or used in an inventive method of treatment is human IL-11 produced in
Escherlchia coli (E. coli) by recombinant DNA methods. The protein produced by this
method is 177 ammo acids in length and differs from the 178 ammo acid length of native
IL-11 only in lacking the amino-terminal proline residue. This alteration was not found to
result in measurable differences in bioactivity in either in vitro or in vivo (U.S. Pat. No.
6,066,317).
[88] Recombinant human IL-11 produced by this method is called Oprelvekin-
(S.V. Sitaraman and A.T. Gewirtz, Curr. Opin. Invest. Drugs, 2001, 2: 1395-1400).
Oprelvekin, is the active ingredient in NEUMEGA® (Wyeth), the first and only platelet
growth factor commercially available so far. In November 1997, the FDA cleared
Oprelvekin for the prevention of severe thrombocytopenia and the reduction of the need for
platelet transfusions following myelosuppressive chemotherapy in susceptible patients with
non-myeloid malignancies (J.A. Kaye, Stem Cells, 1996, 14 Suppl. 1: 256-260).
NEUMEGA® (Oprelvekin) can help prevent progressively lower platelet counts caused by
chemotherapy. In particular, treatment with NEUMEGA® may help cancer patients keep
their chemotherapy planned dose on time (PDOT), thereby avoiding dose reduction-and
dose delays, and may help reduce the need for platelet transfusion. NEUMEGA
(Oprelvekin) has also shown potent thrombopoietic activity in animal models of
compromised hematopoiesis, including moderately to severely myelosuppressed mice and
non-human primates. In these models, NEUMEGA® improved platelet nadirs and
accelerated platelet recoveries compared to controls.
IV. Methods of Treatment
[89] In one aspect, the present invention relates to methods and/or systems for the
management of drug-induced thrombocytopenia including thrombocytopenia resulting, at
least partly, from drug-induced liver damage and thrombocytopenia resulting, at least
partly, from drug-induced bone marrow destruction. Specifically, the invention provides
methods and/or systems for alleviating thrombocytopenia (i.e., for preventing, reducing,
slowing down or stopping thrombocytopenia).
[90] In general, methods of prevention are aimed at delaying or preventing the onset
of a medical condition. In such methods, therapy is typically administered prior to the

onset of the condition for a prophylactic action. Methods of treatment, in general, are
aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the
symptoms of a medical condition; (2) bringing about amelioration of one or more
symptoms of the condition; and/or (3) curing the condition. In such methods, therapy is
typically administered after initiation of the condition, for a therapeutic action.
[91] Methods of the present invention include the step of: administering a
therapeutically effective amount of interleukin-11 to a subject in need thereof. Appropriate
subjects or individuals receiving inventive therapy include humans or another mammals
(e.g., mice, rats, rabbits, dogs, cats, cattle, swine, sheep, horses, or primates) that are or can
be afflicted with, or are susceptible to, a disease or disorder (e.g., thrombocytopenia, liver
damage or bone marrow destruction) but may or may not have the disease or disorder or
symptoms of the disease or disorder. In some embodiments, the subject is sometimes a
human patient.
[92] Methods of the present invention often involve administration of a
therapeutically effective amount of a particular agent. A therapeutically effective amount
is an amount sufficient to achieve (in principle, for a subject of comparable characteristics,
such as species, body type, size, extent of disease or disorder, degree or type of symptoms,
history of responsiveness, and/or overall health) an intended biological or medical response
or therapeutic benefit in a tissue, system or subject. For example, a desirable response may
include one or mote of: delaying or preventing the onset of a medical condition, disease or
disorder, slowing down or stopping the progression, aggravation, or deterioration of the
symptoms of the condition, bringing about ameliorations of the symptoms of the condition,
and curing the condition.
[93] A therapeutically effective amount of a chemotherapeutic is typically an amount
sufficient to achieve an intended delay, reduction, or amelioration of tumor progress. A
therapeutically effective amount of IL-11 may be different depending on the desired
response. For instance, an amount of IL-11 effective to prevent thrombocytopenia may be
different from an amount of IL-11 effective to treat thrombocytopenia, and either may be
different from amounts to prevent or treat liver damage or bone marrow destruction.
Similarly, an amount of IL-11 effective to prevent thrombocytopenia induced by a first
drug may be different from an amount of IL-11 effective to prevent thrombocytopenia
induced by a second, different drug, etc.

[94] Also, it will be appreciated that, when combinations of therapeutic agents are
administered, the amount of any individual agent required in the combination may be
different from the amount required of that same agent to achieve its therapeutic effect
alone. In some cases, synergies between or among therapeutic agents used in a
combination may reduce amounts required; in other cases, inhibitory interactions may
increase amounts required. Thus, in general, therapeutically effective amounts of a
combination of agents may utilize different absolute amounts of the agents than constitute
therapeutically effective amounts of the agents individually,
[95] Methods of the present invention may be used to prevent the onset of
thrombocytopenia (e.g., in a subject undergoing or intending to undergo therapy involving
a therapeutic conjugate whose administration results in, or may result in
thrombocytopenia). Alternatively, methods of the present invention may be used to treat
thrombocytopenia (e.g., in a subject that has received and/or is receiving therapy involving
a therapeutic conjugate whose administration results in thrombocytopenia). It will be
appreciated that different platelet counts may warrant a thrombocytopenia diagnosis for
different mammalian species. In humans, the platelet count in the circulating blood is
normally between 150 and 400 million per milliliter of blood (or 150 to 400 x 109/L).
[96] Laboratory tests used to diagnose thrombocytopenia (or to assess the effects of
the methods of treatment of the present invention) include full blood count. A cell count
analysis may be performed manually, by viewing a slide prepared with a sample of the
patient's blood under a microscope, or automatically, by using a automated analyzer (e.g., a
Coulter Model S-plus instrument). A full blood count usually provides information on the
concentrations of different cells present in the blood, including platelets.
A. Liver Damage
[97] Certain embodiments of the presenf invention provide methods and
compositions for administration to a subject suffering from or susceptible to
thrombocytopenia resulting from drug-induced liver damage. A subject may be suffering
from or susceptible to liver damage if that subject that has been diagnosed with liver
damage (e.g., has been tested and found to have liver damage); is suspected of having liver
damage (e.g., presents one or more symptoms indicative of liver damage, has one or more
risk factors, or is being screened for liver damage); is clinically known to have a tendency

to suffer from liver damage (e.g., has a history of liver damage); or has received, is
receiving or is about to receive a treatment involving a therapeutic agent whose
administration results in liver damage. Individuals that have previously undergone therapy
for liver damage may also be considered to be suffering from or susceptible to liver
damage.
[98] Liver damage may include any injury, defect or dysfunction of the liver caused
by a particular factor (e.g., a conjugate) or a combination of factors. For instance, liver
damage includes, but is not limited to, degeneration or necrosis of liver parenchyma cells;
mobilization, infiltration, activation of Kupffer cells, leukocytes, and the like; swelling of
the liver; fibrosis of the liver tissue; as well as any biological reaction or condition that
results from the damage (including, but not limited to, inflammation and splenomegaly).
[99] In certain embodiments, the methods and pharmaceutical compositions of the
present invention are used to prevent or treat drug-induced liver damage that results in
thrombocytopenia. For example, the inventive methods and pharmaceutical compositions
may be used to prevent the onset of drug-induced liver damage (e.g., in a subject to be
submitted to a treatment involving a conjugate whose administration results in liver
damage). Alternatively or additionally, inventive methods and pharmaceutical
compositions may be used to treat drug-induced liver damage (e.g., in a subject that has
received a treatment involving a conjugate whose administration results in liver damage).
[100] Liver damage may be diagnosed, according to established techniques. For
example, liver damage is often diagnosed using a set of clinical biochemistry laboratory
blood assays designed to provide information about the state of the subject's liver. Since
the liver produces most of the plasma proteins in the body, measuring the amount of total
protein and/or the amount of albumin (the main constituent of total protein and a protein
made specifically by the liver) in the blood gives information regarding the functioning
state of the liver. When the liver is damaged, it may fail to produce blood clotting factors:
the prothrombin time may be measured to diagnose disorders of blood clotting, usually
bleeding, resulting from liver damage. Serum bilirubin concentration may also be
measured as an indication of the patient's liver state. Bilirubin is the major breakdown
product that results from the destruction of old red blood cells (as well as other sources). It
is removed from the blood by the liver, chemically modified by a process called
conjugation, secreted into the bile, passed into the intestine and to some extent reabsorbed

from the intestine. Many different liver diseases and conditions can cause the serum
bilirubin concentrations to be elevated. Blood assays may also be performed to measure
one or more of alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate
transaminase (AST), and gamma ghrtamyl transpeptidase (GGT). ALT is an enzyme
present in hepatocytes. When a hepatic cell is damaged, it leaks this enzyme into the
blood, where it can be measured. ALT rises dramatically in acute liver damage, such as
viral hepatitis or acetaminophen overdose. Elevations are often measured in multiples of
the upper limit of normal (ULN). ALP is an enzyme present in the cells lining the biliary
ducts of the liver. If there is an obstruction in the bile duct (e.g., gallstones), ALP levels in
the plasma will rise. AST is similar to ALT in that it is another enzyme associated with
liver parenchymal cells, that is raised in acute liver damage. GGT is an enzyme whose
levels may be elevated with even minor, sub-clinical levels of liver dysfunction.
[101] Alternatively or additionally, these laboratory tests for the diagnosis of liver
damage may be used to assess the effects of the methods of the present invention.
[102] In certain embodiments of the present invention, liver damage is associated with
(i.e., includes, is accompanied by or results in) inflammation. Inflammation is a natural
consequence of injury of adult tissue and the body's initial attempt at healing itself. During
the early phase of inflammation, neutrophils and macrophages are attracted to the site of
injury/damage. Once activated, they produce large amounts of reactive oxygen species
(ROS) through an oxygen-consuming respiratory burst. One purpose of these cell products
is to destroy damaged tissue, kill invading organisms and prevent infection. Despite this
beneficial effect, prolonged production of high levels of ROS can impair healing and cause
severe additional tissue damage and deterioration.
[103] Methods and pharmaceutical compositions of the present invention may be used
to prevent or limit inflammation associated with drug-induced liver damage. For example,
inventive methods and pharmaceutical compositions may be used to limit the extent or
degree of inflammation otherwise observed after administration of a conjugate producing
liver damage.
[104] The extent of inflammation and/or the effects of inventive methods and
compositions on inflammation may be evaluated using any of a variety of suitable tests
known in the art. Such tests include assays measuring the erythrocyte sedimentation rate

(ESR), which serves as a convenient way to screen for any inflammatory process in the
body; assays measuring C-reactive protein (CRP, whose production by the liver increases
up to a thousand fold in response to insult); and assays measuring neutrophil counts.
B. Bone Marrow Destruction
[105] Certain embodiments of the present invention relate to methods and
compositions for administration to a subject suffering from or susceptible to
thrombocytopenia resulting from drug-induced bone marrow destruction. A subject may
be suffering from or susceptible to bone marrow destruction if that subject has been
diagnosed with bone marrow destruction (e.g., has been tested and found to present bone
marrow damage, defect or dysfunction); is suspected of having bone marrow destruction
(e.g., presents one or more symptoms of bone marrow destruction); or has received, is
receiving or is about to receive a treatment involving a conjugate whose administration
results in bone marrow destruction.
[106] Bone marrow destruction may include any damage, defect or dysfunction of the
bone marrow caused by a particular factor (e.g., conjugate) or a combination of factors that
results in decreased or defective platelet production. For instance, bone marrow
destruction includes, but is not limited to, degeneration or necrosis of megakaryocytes,
depressed production of megakaryocytes, and alteration or damage of bone marrow
producing an unfavorable environment for platelet production from megakaryocytes.
[107] In certain embodiments, the inventive methods and pharmaceutical
compositions are used to prevent or treat drug-induced bone marrow destruction that results
in thrombocytopenia. For example, methods and pharmaceutical compositions of the
present invention may be used to prevent drug-induced bone marrow destruction (e.g., in a
subject to be submitted to a treatment involving a conjugate whose administration results in
bone marrow destruction) or to treat drug-induced bone marrow destruction (e.g., in a
subject that has received a treatment involving a conjugate whose administration results in
bone marrow destruction).
[108] Bone marrow destruction may be diagnosed according to established
techniques. For example, bone marrow destruction may be diagnosed by assessing the
cellularity and morphology of residual erythroid cells in a bone marrow aspirate or biopsy.

Bone marrow activity can also be determined by radiographic methods or imaging methods
including magnetic resonance imaging (MRI) and positron emission tomography (PET).
C. Selection of Subjects
[109] In certain embodiments, subjects suitable to receive a treatment according to the
present invention include individuals suffering from drug-induced thrombocytopenia;
individuals clinically known to have a tendency to suffer from drug-induced
thrombocytopenia; individuals that have received, are receiving or are about to receive a
treatment involving a conjugate therapeutic agent whose administration results in
thrombocytopenia. Suitable subjects may or may not have previously received traditional
treatment for the condition.
[110] Before administration of an inventive therapy or composition, a subject may be
tested for thrombocytopenia, liver damage, inflammation, and/or bone marrow destruction,
using one or more of the methods described above. The same or similar methods may be
used to determine the effects of the inventive treatment/pharmaceutical composition on the
subject.
[111] Subjects suffering from or susceptible to drug-induced thrombocytopenia may
be suffering from cancer or a cancerous condition. In some embodiments,
thrombocytopenia in the subject results, at least partly, from chemotherapeutic conjugate-
induced liver damage. Alternatively or additionally, thrombocytopenia results, at least
partly, from chemotherapeutic conjugate-induced bone marrow destruction.
[112] In general, cancer or cancerous condition refers to or describes a physiological
condition in mammals that is typically characterized by unregulated cell growth. Examples
of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and
leukemia. More particularly, examples of such cancers include squamous cell carcinoma,
small-cell lung cancer, non-small cell lung cancer, pancreatic cancer, glioblastoma
multiform, melanoma, multiple myeloma, non-Hodgkin's lymphoma, esophageal/oral
cancer, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder
cancer, hepatoma, breast cancer, colon and rectal cancer, bone cancer, renal cancer,
myeloid, lymphocytic, myelocytic, and lymphoblastic leukemias, and head and neck
cancer, to name a few.

[113] In some embodiments, subjects suitable to receive an. inventive therapy and/or a
pharmaceutical composition include cancer patients suffering from or susceptible to
thrombocytopenia. Such cancer patients may include individuals diagnosed with cancer
(e.g., tested and found to have cancer), individuals suspected of having cancer (e.g.,
presenting one or more symptoms indicative of cancer, having one or more risk factors, or
being screened for cancer). Alternatively or additionally, cancer patients may include
individuals that have previously undergone therapy for cancer.
D. Dosage and Administration
[114] Administration of IL-11 (and/or other agent), according to methods of the
present invention, may consist of a single dose or a plurality of doses over a period of time.
Administration of interleukin-11 prior to administration of CMC-544 results in prevention
of CMC-544-related liver damage and thrombocytopenia (see Example 1), and in reduction
of CMC-544-related inflammation (see Example 2). Accordingly, in certain embodiments,
IL-11 is administered prior to administration of the conjugate that produces
thrombocytopenia. Alternatively or additionally, IL-11 may be administered concurrently
with administration of the conjugate and/or following administration of the conjugate.
[115] Inventive administrations may be carried out in any convenient manner such as
by injection (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) or oral
administration.
[116] Depending on the route of administration, effective doses may be calculated
according to the body weight, body surface area, or organ size. Optimization of the
appropriate dosages can readily be made by one skilled in the art, for example, in light of
pharmacokinetic data observed in pre-clinical studies or human clinical trials. The final
dosage regimen will generally be determined by the attending physician, who may consider
various factors which modify the action of the drugs, e.g., the drug's specific activity, the
severity of the damage and the responsiveness of the patient, the age, condition, body
weight, sex and diet of the patient, the severity of any present infection, time of
administration, and other clinical factors. As studies are conducted, further information
will emerge regarding the appropriate dosage levels and duration of treatment.
[117] It will also be appreciated that, in the methods of the present invention, IL-11
and/or other therapeutic agents (e.g., conjugates) can be employed in combination therapies

(i.e., can be administered concurrently with, prior to, or subsequent to one or more desired
therapies of medical procedures). The particular combination of therapies (therapeutics or
procedures) to employ in a combination regimen will usually take into account
compatibility of the desired therapeutics and/or procedures and the desired therapeutic
effect to be achieved.
[118] For example, in those embodiments where the conjugate is used in the treatment
of cancer, methods of the present invention can be employed in combination with other
procedures including surgery, radiotherapy (e.g., Y-radiation, neuron beam radiotherapy,
electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive
isotopes), endocrine therapy, hyperthermia and cryotherapy.
[119] Alternatively or additionally, methods of the present invention can be employed
in combination with other agents, for example to attenuate one or more adverse effects
(e.g., antiemetics, pain relievers, anti-nausea drugs), other approved chemotherapeutic
drugs, including, but not limited to, alkylating drugs (mechlorethamine, chlorambucil,
Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (Methotrexate), purine
antagonists and pyrimidine antagonists (6-Mercaptopurine, 5-Fluorouracil, Cytarabile,
Gemcitabine), spindle poisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),
podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin,
Mitomycin), nitrosoureas (Carmustine, Lomustine), inorganic ions (Cisplatin, Carboplatin),
enzymes (Asparaginase), and hormones (Tamoxifen, Leuprolide, Flutamide, and
Megestrol), to name a few. For a more comprehensive discussion of updated cancer
therapies, see The Merck Manual, 17th Ed. 1999, the cancer therapeutics sections of which
are hereby incorporated by reference.
[120] Methods of the present invention can also be employed together with one or
more combinations of cytotoxic agents as part of a treatment regimen, wherein the
combination of cytotoxic agents is selected, for example, from CHOPP
(cyclophosphamide, doxorubicin, vincristine, prednisone, and procarbazine); CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone); COP (cyclophosphamide,
vincristine, and prednisone); CAP-BOP (cyclophosphamide, doxorubicin, procarbazine,
bleomycin, vincristine, and prednisone); m-BACOD (methotrexate, bleomycin,
doxorubicin, cyclophosphamide, vincristine, dexamethasone, and leucovorin); ProMACE-
MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin,

mechloethamine, vincristine, prednisone, and procarbazine); ProMACE-CytaBOM
(prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin,
cytarabine, bleomycin, and vincristine); MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, prednisone, bleomycin, and leucovorin); MOPP
(mechloethamine, vincristine, prednisone, and procarbazine); ABVD
(adriamycin/doxorubicin, bleomycin, vinblastine, and dacarbazine); MOPP
(mechloethamine, vincristine, prednisone and procarbazine) alternating with ABV
(adriamycin/doxorubicin, bleomycin, and vinblastine); MOPP (mechloethamine,
vincristine, prednisone, and procarbazine) alternating with ABVD
(adriamycin/doxorubicin, bleomycin, vinblastine, and dacarbazine); ChlVPP
(chlorambucil, vinblastine, procarbazine, and prednisone); IMVP-16 (ifosfamide,
methotrexate, and etoposide); MME (methyl-gag, ifosfamide, methotrexate, and
etoposide); DHAP (dexamethasone, high-dose cytaribine, and cisplatin); ESHAP
(etoposide, methylpredisolone, high-dose cytarabine, and cisplatin); CEPP(B)
(cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin); CAMP
(lomustine, mitoxantrone, cytarabine, and prednisone); CVP-1 (cyclophosphamide,
vincristine, and prednisone), ESHOP (etoposide, methylpredisolone, high-dose cytarabine,
vincristine and cisplatin); EPOCH (etoposide, vincristine, and doxorubicin for 96 hours
with bolus doses of cyclophosphamide and oral prednisone), ICE (ifosfamide,
cyclophosphamide, and etoposide), CEPP(B) (cyclophosphamide, etoposide, procarbazine,
prednisone, and bleomycin), CHOP-B (cyclophosphamide, doxorubicin, vincristine,
prednisone, and bleomycin), CEPP-B (cyclophosphamide, etoposide, procarbazine, and
bleomycin), and P/DOCE (epirubicin or doxorubicin, vincristine, cyclophosphamide, and
prednisone).
[121] Alternatively or additionally, methods of the present invention can be employed
together with therapies involving administration of one or more of bioactive agents selected
from the group consisting of antibodies, growth factors (e.g., Tumor-Necrosis Factor
(TNF), Colony Stimulating Factor (CSF), Granulocyte-Colony Stimulating Factor (G-CSF)
or Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)), hormones (e.g.,
estrogens, androgens, progestins, and corticosteroids), cytokines, anti-hormones, xanthines,
interleukins (e.g., IL-2), and interferons.

E. Mechanisms
[122] Kinetics of platelet decrease provide a rough guide to the mechanism of
thrombocytopenia; although the kinetics of platelet reduction is a highly variable criterion
and can be dependent on the chemical entity used. For example, mice treated with
cyclophosphamide have been shown to develop a platelet nadir on day 3 (G. Hangoc et al.,
Blood, 1993, 81: 965-972); and mice treated with carboplatin and irradiation exhibit a nadir
on day 9 (J.P. Leonard et al., Blood, 1994, 83: 1499-1506). Based on the present
Examples, carboplatin alone gave a nadir of 11. All of these agents affect platelet
production, but they have very different effects on the timing of the platelet nadir. Similar
effects have been reported in monkeys. Carboplatin was found to give a platelet nadir on
day 13-14 (F.J. Schlerman et al., Stem Cell, 1996, 14: 517-532), but ACNU (i.e., 3-[(4-
amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride)
does not produce a nadir until day 21 (M. Saitoh et al, J. Interferon and Cytokine Res.,
2000, 20: 539-545). In humans, CHOP has been shown to produce a nadir around day 9.
Despite the differences, thrombocytopenia produced by these classical chemotherapeutic
agents, all of which are bone marrow toxins, is thought to result from disruption of platelet
production in the bone marrow.
[123] The mechanisms of thrombopoiesis are also very complex and can be impacted
in many different ways. Many chemotherapeutic agents have direct effects on
megakaryocytes and on human megakaryocytic progenitors (CFU-megs). Since
megakaryocytes do not divide, they may not necessarily stop producing platelets
immediately when hit by a cycle-specific toxin. If the effects of the chemical agent are
directed more toward progenitors and early-megakaryocytes, the effect on peripheral
platelets is slower and the nadir occurs later. If the effects are directly on megakaryocytes
and affect proteins as well as DNA, the effects on circulating platelets can occur more
rapidly.
[124] Alternatively, CMC-544 (and other similar protein conjugates) may have a clear
impact on the liver (transaminases released) and on TPO production in the liver (reduced
TPO levels). TPO production seems to slow very soon after CMC-544 administration.
This would be expected to result in an immediate reduction in stimulation of
megakaryocytes and an associated reduction of platelet production. However, since the

half life of platelet is around 5 days in humans, the kinetics suggest that this is not the sole
mechanisms of thrombocytopenia.
[125] The effects of oprelvekin (IL-11) observed in the present Examples are
consistent with its known effects as a liver protective agent (in animals, see for example,
W.L. Trepicchio et al., Toxicol. Pathol., 2001, 29: 242-249; and in humans, see for
example, R. Ghalib et al., Hepatology, 2003, 37: 1165-1171). Its ability to ameliorate the
platelet drop associated with CMC-544 treatment, when IL-11 is given prophylactically but
not when it is given concomitantly, is also consistent with the known kinetics of platelet
products induced by IL-11. Administration of IL-11 6 hours after CMC-544 administration
may have been too late to have full protective effect on the liver, but it may have some
positive effect nevertheless.
[126] Thrombocytopenia associated with treatment using CMC-544 (and other similar
protein conjugates) may result, at least in part, from disruption of TPO production
secondary to liver damage and is distinct from that caused by conventional
chemotherapeutic agents that disrupt platelet production by damaging bone marrow cells.
V. Pharmaceutical Compositions and Kits
[127] In another aspect, the present invention provides pharmaceutical compositions
comprising a therapeutically effective amount of interleukin-11 (IL-11) and at least one
physiologically acceptable carrier or excipient.
[128] A physiologically acceptable carrier or excipient generally is a carrier medium
or an excipient that does not block the effectiveness of the biological activity of the active
ingredient(s) of the composition and that is not excessively toxic to the host at the
concentrations at which it is administered. The term includes solvents, dispersion media,
coatings, antibacterial agents, isotonic agents, absorption delaying agents, and the like.
The use of such media and agents for the formulation of pharmaceutically active
substances is well known in the art (see, for example, "Remington's Pharmaceutical
Sciences", E.W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, PA, which is
incorporated herein by reference in its entirety).
[129] In certain embodiments, IL-11 is the only active ingredient in an inventive
pharmaceutical composition. In other embodiments, the pharmaceutical composition

further comprises one or more other therapeutic agents (e.g., one or more conjugates). In
still other embodiments, the pharmaceutical composition further comprises a combination
of therapeutic agents. One or more conjugates included in an inventive pharmaceutical
composition may induce liver damage and/or bone marrow destruction.
A. Formulations
[130] In certain embodiments, a pharmaceutical composition of the present invention
is administered in such amounts and for such time as necessary to achieve a desired result.
For example, a pharmaceutical composition can be administered in such amounts and for
such time that it prevents, reduces, slows down or stops drug-induced thrombocytopenia in
a subject (e.g., thrombocytopenia resulting from administration of a conjugate). More
specifically, an inventive pharmaceutical composition can be administered in such amounts
and for such time that it prevents, reduces, slows down or stops abnormally high
destruction of circulating platelets and/or decreased platelet production by the bone
marrow.
[131] A pharmaceutical composition of the present invention may also be
administered in such amounts and for such time that it prevents, reduces, slows down or
stops thrombocytopenia resulting, at least partly, from drug-induced liver damage.
Alternatively or additionally, a pharmaceutical composition may be administered in such
amounts and for such time that is prevents, reduces, slows down or stops thrombocytopenia
resulting, at least partly, from drug-induced bone marrow destruction.
[132] In certain pharmaceutical compositions, interleukin-11 (IL-11), one or more
conjugate(s) and one or more physiologically acceptable carriers or excipients are
combined in one or more preparations for simultaneous or sequential administration of IL-
11 and the conjugate(s). More specifically, an inventive composition may be formulated in
such a way that IL-11 and the conjugate(s) can be administered at the same time or
independently from each other (e.g., the composition can comprise one or more
preparations in individual containers).
[133] Pharmaceutical compositions, according to the present invention, may be
administered using any amount and any route of administration effective for preventing,
slowing down, reducing or stopping thrombocytopenia that would otherwise be observed in
the absence of IL-11 administration.

[134] As already mentioned above, the exact amount of pharmaceutical composition
to be administered may vary from subject to subject, depending on the species, age, and
general condition of the subject, the severity of the condition, the particular therapeutic
agent, its mode of administration, the severity of thrombocytopenia and/or liver damage or
bone marrow destruction it induces, and the like.
[135] Desirable optimal pharmaceutical formulations can be determined depending
upon the route of administration and desired dosage. Such formulations may influence the
physical state, stability, rate of in vivo release, and rate of in vivo clearance of the
administered compounds.
[136] Pharmaceutical compositions of the present invention may be formulated in
dosage unit form for ease of administration and uniformity of dosage. The expression
"dosage unit form", typically refers to a physically discrete unit of IL-11 alone, conjugate
alone, or combination of IL-11 and conjugate appropriate for the patient to be treated. It
will be understood, however, that the total daily usage of the compositions of the present
invention will generally be decided by an attending physician within the scope of sound
medical judgment.
[137] After formulation with one or more appropriate physiologically acceptable
carriers or excipients in a desired dosage(s), pharmaceutical compositions of the present
invention can be administered to humans or other mammals by any suitable route. For
example, pharmaceutical compositions of the present invention may be administered orally,
parenterally, intravenously, intraperitoneally, intramuscularly or subcutaneously,
depending on the condition being treated (e.g., thrombocytopenia resulting from bone
marrow destruction or thrombocytopenia resulting from liver damage) by administration of
the at least one therapeutic agent.
[138] Injectable preparations, for example, sterile injectable aqueous or oleaginous
suspensions, may be formulated according to established procedures, for example, using
suitable dispersing or wetting agents, and suspending agents. Sterile injectable
preparations may also be sterile injectable solutions, suspensions or emulsions in a non-
toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that may be employed are water,
Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed

oils are conventionally employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids
such as oleic acid may also be used in the preparation of injectables.
[139] Inventive injectable formulations can be sterilized, for example, by filtration
through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of
sterile solid compositions which can be dissolved or dispersed in sterile water or other
sterile injectable medium prior to use.
[140] In order to prolong the effect of a drug, it is often desirable to slow the
absorption of the drug from subcutaneous or intramuscular injection. This may be
accomplished, for example, through the use of a liquid suspension of crystalline or
amorphous material with poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend upon crystal size and
crystalline form. Alternatively, delayed absorption of a parenterally administered drug
form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable
depot forms are made by forming microencapsule matrices of the drug in biodegradable
polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer
and the nature of the particular polymer employed, the rate of drug release can be
controlled. Examples of other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the
drug in liposomes or microemulsions which are compatible with body tissues.
[141] Liquid dosage forms for oral administration include, but are not limited to,
pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups,
and elixirs. In addition to the active ingredients, the liquid dosage forms may contain inert
diluents commonly used in the art such as, for example, water or other solvents,
solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene
glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, corn, germ, olive,
castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and
fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral
compositions can also include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents.

[142] Solid dosage forms for oral administration include capsules, tablets, pills,
powders, and granules. In such solid dosage forms, the active compound may be mixed
with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose,
sucrose, glucose, mannitol, and silicic acid; (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; (c)
humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e)
solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary
ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol
monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as
talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form
may also comprise buffering agents.
[143] Solid compositions of a similar type may also be employed as fillers in soft and
hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high
molecular weight polyethylene glycols and the like. The solid dosage forms of tablets,
dragees, capsules, pills, and granules can be prepared with coatings and shells such as
enteric coatings and other coatings well known in the pharmaceutical formulating art.
They may optionally contain opacifying agents and can also be of a composition that they
release the active ingredient(s) only, or preferentially, in a certain part of the intestinal
tract, optionally, in a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. Solid compositions of a similar type may
also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
[144] Active compounds (e.g., IL-11 and/or one or more conjugates) can also be in
micro-encapsulated form with one or more excipients as noted above. The solid dosage
forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and
shells such as enteric coatings, release controlling coatings and other coatings well known
in the pharmaceutical formulating art. In such solid dosage forms the active compound
may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such
dosage forms may also comprise, as is common practice, additional substances other than

inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate
and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms
may also comprise buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
Examples of embedding compositions which can be used include polymeric substances and
waxes.
B. Kits
[145] In still another aspect, the present invention provides pharmaceutical packs or
kits comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles)
containing one or more ingredients of the inventive pharmaceutical compositions, for
example, allowing for the simultaneous or sequential administration of interleukin-11 and
conjugate(s).
[146] In certain embodiments, an inventive kit includes one or more additional
approved therapeutic agents for use as a combination therapy (e.g., one or more anti-cancer
drugs as described above). Optionally associated with such container(s) can be a notice in
the form prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceutical products, which notice reflects approval by the agency of manufacture, use
or sale for human administration.
[147] Different ingredients may be supplied in solid (e.g., lyophilized) or liquid form.
Each ingredient will generally be suitable as aliquoted in its respective container or
provided in a concentrated form. Kits may also include media for the reconstitution of
lyophilized ingredients. The individual containers of the kit are preferably maintained in
close confinement for commercial sale.
Examples
[148] The following examples describe some of the preferred modes of making and
practicing the present invention. However, it should be understood that these examples are
for illustrative purposes only and are not meant to limit the scope of the invention.
Furthermore, unless the description in an Example is presented in the past tense, the text,

like the rest of the specification, is not intended to suggest that experiments were actually
performed or data were actually obtained.
Example 1: Effect of IL-11 on CMC-544 induced Thrombocytopenia in Nude
Mice
Experimental Design:
[149] The effect of IL-11 (NEUMEGA®) on CMC-544-induced thrombocytopenia is
shown in the present example. The initial IL-11 dose was administered i.p. daily (250
g/kg) beginning up to 2 days before, or BID (125 g/kg) beginning up to 8 hours after
CMC-544 administration (considered Day 0). IL-11 was given daily for up to 8 days post
CMC-544 administration. On Day 0, mice were bled for baseline platelet values and then
dosed with vehicle or CMC-544 at 4 g/ mouse i.p. Higher or lower doses of CMC-544
were also administered. Blood was sampled at various time points up to 3 days post drug
administration.
Procedures:
[150] A 25 gauge needle was inserted into the tail vein of the mouse and then
withdrawn allowing for a drop of blood to seep out. A 5 L sample of blood was collected
for analysis. Platelet values were quantitated using a dual threshold Beckman Coulter Zl
Particle Counter (Fullerton, CA). Threshold values were set to measure mouse platelets
according to manufacturer specification.
[151] As shown on Figures 1 and 2, administration of IL-11 prior to administration of
CMC-544 prevents CMC-544-induced reduction in platelet count in nude mice.
Example 2: Effects of IL-11 on CMC-544 induced Thrombocytopenia in
Monkeys
A - First Study
Experimental Design:
[152] Ten (10) monkeys (cynomolgus macaques) were bled initially on Day -9 in
order to select eight (8) of them to be put on study that have the more normal blood values.

The selected monkeys were divided into 2 groups of 4 each. One group of test monkeys
was pre-dosed with IL-11 for 5 days prior to receiving CMC-544. The other group of
monkeys (or control monkeys) was administered a vehicle control of sterile saline. This
provided the appropriate control for the stress involved in dosing the monkeys with IL-11
in order to discern between potential vehicle and/or IL-11 side effects (i.e., effects on blood
or hematology parameters) if they were to occur. Both groups received CMC-544 on Day
1. Dosing with IL-11 in the test group also took place on the day of and continue for 4
days after CMC-544 administration. The test group received a total of 10 doses of IL-11.
Both groups of monkeys were monitored for 6 days after CMC-544 at which time they
were euthanized.
[153] Blood was drawn for analysis on Day -11 (pre-test), Day -5 (time of first IL-11
administration), Day 1 (time of CMC-544 administration), Days 3, 4, 5 (time of platelet
nadir for CMC-544), and Day 7 (end of the study). For each day that requires a blood
sample collection and drug administration, blood was drawn before IL-11 or CMC-544
administration. On Day 1, IL-11 was administered immediately after CMC-544
administration.
[154] More specifically, the test group of monkeys were submitted to the following
procedures: on Day -11: drawing of 6 mL of blood; on Day -5: drawing of 2 mL of blood,
administration of IL-11; on Day -4: administration of IL-11; on Day -3: administration of
IL-11; on Day -2: administration of IL-11; on Day -1: administration of IL-11; on Day 1
(drawing of 2 mL of blood, administration of IL-11 and CMC-544); on Day 2:
administration of IL-11; on Day 3: drawing of 2 mL of blood, administration of IL-11; on
Day 4: drawing of 6 mL of blood, administration of IL-11; on Day 5: drawing of 2 mL of
blood, administration of IL-11; on Day 7: drawing of 6 mL of blood, euthanasia, and
necrospy.
Procedures:
[155] Drug Administration: IL-11 was administered at 125 g/kg (as a solution) by
subcutaneous injection. IL-11 was reconstituted fresh with sterile water and administered
within 3 hours of reconstitution. The dose volume was 0.25 mL/kg. IL-11 was
administered after any scheduled blood samples were taken. CMC-544 was administered
at 25 g/kg (dose based on Calicheamicin DMH content) intravenously as a solution. Dose

volume was 1 mL/kg. Monkeys were anesthetized with ketamine (10 mg/kg) before iv
injection of CMC-544 through an indwelling catheter. CMC-544 was administered after
any scheduled blood samples were taken and immediately before IL-11 administration.
[156] Blood Sampling: On Days -11, 4 and 7, a total of 6 mL of blood were collected
according to the following procedure: 3 mL were collected in a red top tube (serum) for
clinical chemistry analysis and 3 mL in a lavender top tube (EDTA anti-coagulant) for
CBC (Complete Blood Count). On Days -5, 1, 3, and 5, 2 mL of EDTA treated blood (in
lavender top tubes) were collected for platelet determinations.
[157] Total blood collected per monkey were 8 mL the first week, 12 mL the second
week, and 6 mL the third week. The total blood volume of a monkey is estimated to be
5.4% of body weight (54 mL/kg). Therefore, monkeys weighting > 2.5 kg had less than
10% of blood volume withdrawn per week.
Ill Effects :
[158] Monkeys were monitored daily for health and a staff veterinarian was notified
of their condition if ill effects were observed. Food consumption was monitored daily.
[159] IL-11 (125 mg/kg s.c.) has been previously administered to monkeys with no
adverse events reported (F.J. Schlerman et al., "The effect of rhIL-11 on Platelet
Aggregation in vitro and ex vivo following Subcutaneous Administration to Non-Human
Primates for 14 Days", Genetics Institute Report # P98034-14; A.G. Bree et al.,
"Thrombopoietic Activity of Recombinant Human Interleukin-11 Alone or in Combination
with Recombinant Human Granulocyte-Colony Stimulating Factor in a Novel
Myelosuppressed Non-Human Primate Model", Genetics Institute Report # P99029-14;
A.G. Bree et al., "Thrombopoietic Activity of a Normal or Short Course of Recombinant
Human Interleukin-11 in Combination with a Normal or Delayed Course of Recombinant
Human Granulocyte-Colony Stimulating Factor in a One-Cycle Myelosuppressed Non-
Human Primate Model, Genetics Institute Report # P99030-14).
[160] Previous studies with CMC-544 at the dose of 25 mg/kg reported reduced food
consumption, ernesis (75% of monkeys at day 4 post CMC-544 administration) and fecal
alterations such as liquid, mucoid, soft and/or reduced feces (beginning day 4 through day
7 in > 50% of monkeys). Fresh fruit and anti-diarrheal agents (Kao-pectate) may be
provided for inappetance and for fecal changes, respectively. Monkeys exhibiting

unanticipated adverse effects (other than the 3 symptoms reported above) were reported to
the attending veterinarian for consultation.
Euthanasia:
[161] Monkeys were killed by an overdose of barbiturates (100 mg/kg i.v.).
Necropsy:
[162] Post-mortem tissue samples of liver, bone marrow (stern brae), lung (a section
from each side of the lung), and spleen were collected and fixed in 10% formalin. Bone
marrow smears were collected from an open rib.
Hematology:
[163] Hematology and coagulation parameters were evaluated twice at pretest and
dosing phase days 1, 3, 4, 5, and 7. Mean platelet volume (MPV) was evaluated during
these dosing phase intervals only. In animals administered CMC-544 only, compound-
related alterations in individual animals relative to their pretest values occurred in counts of
platelets (PLT), neutrophils (NEU), monocytes (MONO), and eosinophils (EOS). In
animals administered rhuIL-11 (NEUMEGA®), rhuIL-11-related changes were observed in
parameters of red cell mass (hemoglobin, HGB, hematocit, HCT, and red blood cell count,
RBC).
[164] These decreases in PLT (31% to 69% relative to the first pretest count) were
observed in all CMC-544 only-administered animals.on dosing phase days 3, 4, 5 and
(except for one animal) day 7, with the nadir generally observed at day 4 or 5 post-CMC-
544 dosing. In these same animals, slight increases in PLT (30% to 126%) on dosing phase
day 1 preceded these subsequent decreases. In contrast, all monkeys given rhuIL-11 prior
to CMC-544, had slightly to mildly increased PLT at all dosing phase intervals (48% to
272%, relative to the first pretest value). These increases were peaked at day 4, 5 or 7 in
these animals. These differences in PLT between groups support that rhuIL-11 pre-
treatment prevents or ameliorates CMC-544-related platelet count decreases.
[165] In males administered rhuIL-11 pretreatment, MPV was also generally greater
at all dosing phase intervals than that of males administered CMC-544 alone. However,
this pattern was not seen with the single female administered rhuIL-11 pretreatment

compared with that given CMC-544 alone; thus, no conclusion pertaining to compound-
related effects on MPV were made.
[166] In all animals administered CMC-544 only, mild to moderate increases in NEU
above pretest counts (85% to 212% relative to the first pretest value) were observed at one
or more dosing phase intervals. Similarly, increases above pretest counts in MONO (39%
to 386%) and EOS (50% to 538%, relative to the first pretest value) occurred during the
dosing phase in these same animals. In contrast, dosing phase increases above pretest
values occurred in only 1 of 4 animals given rhuIL-11 pretreatment, (the single female) in
NEU (164% to 284%) and EOS (143%) at day 4 and/or 5, and in 2 of 4 animals (male or
female) given rhuIL-11 pretreatment in MONO (93% to 329%). These differences
between groups in incidence of increased leukocyte counts suggests amelioration or partial
prevention of an inflammatory response to animals given rhuIL-11 prior to CMC-544. The
change in NEU correlated with slight increased extramedullary hematopoiesis in the spleen
in some individual animals given CMC-544 alone.
[167] In all animals given rhuIL-11 prior to CMC-544, decreases in parameters of red
cell mass (hemoglobin, hematocrit, red blood cell count) were observed at most to all
dosing phase intervals (males: 12% to 32%; female: 22% to 42% relative to the first pretest
values). These changes generally exceeded those at most intervals for animals given CMC-
544 alone and were an anticipated effect of high dosage rhuIL-11 in monkeys. No notable
differences in reticulocyte counts or red cell indices between animals given CMC-544 with
or without rhuIL-11, and no correlation between these decreases in red cell mass and
histologic bone marrow hypocellularity were observed.
[168] Differences in other hematology parameters in rhuIL-11 administered animals
compared with CMC-544 only-administered animals were not noteworthy due to their
broadly overlapping absolute values and/or magnitude.
Clinical Chemistry:
[169] Serum chemistry parameters were evaluated once at pretest and days 4 and 7.
In animals administered CMC-544 only, compound-related increases (relative to pretest
values) occurred in alanine aminotransferase (ALT). In one animal administered CMC-544
alone, and all animals administered rhuIL-11 (NEUMEGA®), increases in alkaline
phosphatase (AP) also occurred; these compound-related increases were considered partly

related to rhuIL-11 pretreatment. In all animals, decreases in albumin (ALB) occurred; the
magnitude of these decreases was consistently greater with, and related to rhuIL-11
administration.
[170] The increases in ALT (68% to 750%) occurred in 3 of 4 animals given CMC-
544 only. These increases occurred at days 4 and/or 7 relative to each animal's pretest
concentration and suggested minor to mild CMC-544-related liver injury. Supporting the
occurrence of liver injury and correlating with the changes in ALT in these animals were
microscopic mild multifocal hepatocellular necrosis (primarily centrilobular) and single
cell necrosis in the monkey with the greatest increase in ALT (8.5 fold, female), and mild
single cell necrosis in another animal (male) with a slight (1.7-fold) increase in ALT. The
occurrence of these ALT increases in animals administered CMC-544 alone and not in
those given rhuIL-11 pretreatment provided limited (due to small number of animals to
evaluate) support for amelioration of liver injury with rhuIL-11 pretreatment.
[171] Increases in AP occurred at both dosing phase intervals (relative to pretest
concentrations) in one animal administered CMC-544 (122% to 242%) and all animals
given rhuIL-11 pretreatment (276% to 589%). The greater incidence and magnitude of AP
increase in rhuIL-11 administered animals and CMC-544 administered animals indicated
the change is partly rhuIL-11-related. Similar increases in AP in monkeys following
rhuIL-11 administration has been previously observed.
[172] Decreases in ALB that were mild to moderate (17% to 45%) occurred at both
dosing phase intervals in all animals administered rhuIL-11 pretreatment. These decreases
corresponded with the decreases in red cell mass in these animals, as previously reported in
monkeys given rhuIL-11. In contrast, decreases in ALB occurred in only some animals
administered CMC-544 alone, and at one or both dosing phase intervals and were slight to
mild (up to 24%). The greater incidence and magnitude of these decreases in animals
administered rhuIL-11 relative to those given CMC-544 alone indicated the decrease is
partly related to rhuIL-11 and notably exacerbated or superseded with rhuIL-11
administration in animals given CMC-544.
[173] Differences in other serum chemistry values in compound-administered animals
compared with controls were attributed to random variation due to their minor magnitude,
direction, absence of a dosage-related pattern, and/or general overlap of individual values.

B - Second Study
[174] A second study was carried out, the experimental design of which is presented
in Table 1. Briefly, 8 female monkeys were used, 4 of them were administered a single
dose i.v. of CMC-544 (on day 1; 25 g/kg) and the other 4 received a single dose i.v. of
CMC-544 (on day 1; 25 g/kg) and 5 subcutaneous doses of NEUMEGA (125 g/kg/day)
on Days 1-5. Hematology studies were performed, as described above, twice prestudy and
on dosing days 1 through 8, day 10, day 12 and day 14 (always prior to NEUMEGA
administration). Coagulation and serum chemistry tests were performed, as described
above, twice prestudy and on dosing days 7 and 14.
[175] Figure 12 shows the changes in platelet counts observed in both groups of
monkeys. The onset and magnitude of platelet count decreases were found to be
comparable between animals administered CMC-544 with or without NEUMEGA. Nadir
occurred at post-dose day 3 or 4 (CMC-544 alone: 70% to 87%; CMC-544 + NEUMEGA:
75% to 84% below last pretest value). Essentially complete resolution of these decreases
(within 90% of the second pretest value) occurred by day 7 or 8 in all animals administered
CMC-544 + NEUMEGA, but only one animal given CMC-544 alone.

[176] A slight increase in AST was observed at day 14 in all animals, and at day 7 in
most animals administered CMC-544 alone (39-124% above last pre-study value) (See
Figure 13). The corresponding increase in ALT occurred in 2 of these four animals (82%-
198%). Increases in these liver-associated serum enzymes that were of generally lower
magnitude occurred at these intervals in 1 or 2 of 4 animals administered CMC-544 +
NEUMEGA (AST: 35-79%; ALT: 66%).

[177] A slight to mild decrease in reticulocyte count was observed in all animals (8-
56%) generally on days 3-5. These decreases were associated with slight to mild decreases
in red cell mass (up to 22%) beginning on day 4. The incidence and onset between animals
administered CMC-544 alone and animals administered CMC-544 + NEUMEGA were
similar in magnitude, and thus, are considered to be primarily CMC-544-related (as well as
partly blood collection-related).
[178] A slight to mild increase in GLOB was observed for both groups of animals
(CMC-544 alone: 17-42%; CMC-544 + NEUMEGA: 19-52%) at both day 7 and day 14
without a consistent pattern of resolution in either group.
[179] A transient moderate increase in fibrinogen was observed at day 7 in all animals
administered CMC-544 + NEUMEGA (197% to 327% above pre-study values. These
fibrinogen increases showed rapid partial resolution relative to pretest values by day 14.
Comparable increases in fibrinogen have been observed in previous studies in monkeys
administered NEUMEGA. These increases were attributed to the transient acute phase
reaction induced by IL-11.
[180] A slight decrease in albumin (17% to 23%) was observed at day 7 in all animals
administered NEUMEGA and a partial resolution occurred by day 14. This slight decrease
is attributed to a transient acute phase response. Although a plasma volume expansion may
have also contributed.
[181] The results obtained in this Second Study have shown that resolution of CMC-
544-related platelet count decreases occurred slightly earlier in monkeys given NEUMEGA
for 5 days beginning 6 hours after the single CMC-544 dose, than in the majority of
animals given CMC-544 alone. CMC-related increases in liver-associated serum enzymes,
AST and ALT occurred with somewhat lower magnitude and incidence in animals
administered CMC-544 + NEUMEGA than animals administered CMC-544 alone.
NEUMEGA-related changes (decrease in albumin, transient increase in fibrinogen)
observed in the present study were consistent with those previously reported.

Example 3: Effect of Calicheamicin Conjugates on Platelet Levels in the Mouse
Experimental Design:
[182] On Day 0, mice will be bled for baseline platelet values. Mice will then be
dosed with vehicle, CMC-544 or other calicheamicin conjugates at 4 g /mouse i.v. or i.p..
Higher or lower doses may also be administered. Blood will be sampled 72 hours (Day 3),
96 hours (Day 4), and 168 hours (Day 8) post drug administration.
Procedures:
[183] A 25 gauge needle will be inserted into the tail vein of the mouse and then
withdrawn allowing for a drop of blood to seep out. A 5 L sample of the blood will be
collected for analysis. Blood will be sampled on Day 0, before drug administration, and on
Days 3, 4, and 8, for a total of 4 collections of 5 L each (total of 20 L).
[184] Dosing: CMC-544 or other conjugates of calicheamicin will be administered
once either intraperitonealy or intravenously at a dose of 4 g of calicheamicin DMH. The
dose volume will be 200 L for either route of administration.
[185] Blood Collection: 5 L of blood will be collected from the tail vein.
[186] Pain or Distress: No pain or distress is anticipated but if it occurs, the attending
veterinarian will be consulted.
Animal Toxicity or Ill Effects: No overt toxic effects of CMC-544 have been observed in
mice when administered at a dose of 4 ug or less.
Animal Disposition:
[187] The study will be terminated after the final blood collection time point on Day 8
post-drug administration. Mice will be euthanized by CO2 inhalation.
Example 4: Study of CMC-544 in Patients with B-Cell Non-Hodgkin's
Lymphoma (NHL)
[188] CMC-544, is an antibody-targeted chemotherapy agent composed of a
monoclonal antibody, which specifically targets the CD22 antigen, conjugated to
calicheamicin, a potent cytotoxic antitumor antibiotic. Malignant cells of mature B-

lymphocyte lineage express CD22; CMC-544 may be useful for treating lymphomas of B-
cell origin.
[189] Methods: A phase I, dose escalation trial of CMC-544 is ongoing in patients
with relapsed or refractory B-cell NHL across 13 European and US sites. CMC-544 is
administered intravenously every 3-4 weeks at doses of 0.4, 0.8, 1.34, 1.8, and 2.4 mg/m2.
Standard safety and pharmacokinetic data and preliminary efficacy data (assessed using the
International Workshop to Standardize Response Criteria for NHL) are being collected.
[190] Enrollment of patients with any type of B-cell NHL, except for Burkitt's and
lymphoblastic lymphomas, is allowed in the dose escalation phase (1-6 patients per cohort).
After the maximum tolerated dose (MTD) was identified, 15 patients with follicular
lymphoma and 15 patients with diffuse large B-cell lymphoma will be enrolled in an
expanded MTD cohort.
[191] Results: As of June 2005, 34 patients (8 women, median 71 years old; 26 men,
median 62 years old; number of previous treatments, median 4, range 2-11) were enrolled.
Dose escalation was based on 1st cycle safety evaluations of patient cohorts. Dose-limiting
toxicities (DTLs) were reported for dose levels of 1.34 mg/m2 (grade 4 thrombocytopenia,
2/11 patients), 1.8 mg/m2 (bleeding requiring platelet transfusion, 1/6 patients), and 2.4
mg/m2 (grade 4 thrombocytopenia, 1/6 patients; grade 4 neutropenia for 7 days, 1/6
patients).
[192] Thus, the MTD, the dose level prior to the one where > 33% DTLs occurred,
was 1.8 mg/m2. The most common drug-related adverse events (AEs, all grades) were:
thrombocytopenia (65%), asthenia (47%), nausea (41%), neutropenia (29%), elevated liver
function tests (27%), anorexia (14%), and epistaxis (12%). Grade 3-4 AEs that occurred
with a frequency  10% included: thrombocytopenia (38%), asthenia (12%), and
neutropenia (12%). The nadir of the thrompocytopenia was 9 ± 2 days and platelet counts
recovered spontaneously. No major bleeding episodes were reported. At the 1.8 and 2.4
mg/m2 dose levels, data suggested a dose-dependent component of platelet recovery to
baseline levels and dose delays were required between successive doses. CMC-544 and
total calicheamicin exposures in serum increased with dose. Clearance after 2nd and 3rd
doses decreased approximately 8-14 fold compared with 1st dose, and half-life increased
from approximately 1 day to 4 days. Available data suggested an association of peak

CMC-544 exposures and/or total calicheamicin levels with decreases in platelet counts.
Accordingly, patients in the expanded MTD cohort receive 1.8 mg/m2 CMC-544 every 4
weeks. Preliminary antitumor activity was observed in most cohorts. Complete and/or
partial responses were observed in the 0.8 mg/m2 (1/3 patients), 1.34 mg/m2 (3/9 patients),
1.8 mg/m2 (2/5 patients), and 2.4 mg/m2 (2/5 patients) cohorts, and in the 1st 6 patients in
the expanded MTD cohort (4/6 patients).
Example 5: Comparison of the Effects of CMC-544 and Carboplatin on
Platelets and Thrombopoietin in the Mouse
1193] Vehicle (PBS), Carboplatin (125 mg/kg i.p.) or CMC-544 (200 g/kg i.p) was
each administered to nude mice on Day 0. Blood was collected as described above on Day
0, Day 4, Day 7, Day 11 and Day 13 for measurement of circulating platelets and
circulating thrombopoietin (TPO) levels.
[194] As shown on Figures 10 and 11, which present the results obtained in these
experiments, CMC-544 was found to cause thrombocytopenia in mice with nadir on Day 3
or 4 after treatment; while Carboplatin causes thrombocytopenia with nadir on Day 10 or
11 after treatment.
[195] Conventional chemotherapy is known to cause thrombocytopenia accompanied
by increased levels of circulating thrombopoietin (TPO). In the present experiments,
administration of Carboplatin was found to cause increased levels of circulating
thrombopoietin (TPO) while a significant reduction in the levels of circulating TPO was
observed in the case of CMC-544, suggesting that CMC-544 may inhibit the production of
TPO. CMC-544-mediated inhibition of TPO production may be related to its effects on the
liver function.
Example 6: Thrombocytopenia induced by CMC-544 and its Amelioration
Using Oprelvekin (NEUMEGA®)
[196] CMC-544 has demonstrated significant anti-tumor activity in pre-clinical and
clinical trials in B-Non-Hodgkin's Lymphoma (B-NHL) patients (J.F. DiJoseph et al.,
Blood, 2004, 103: 1807-1814; J.F. DiJoseph et al., Clin. Cancer Res., 2004, 10: 8620-8629;
J.F. DiJoseph et al., Cancer Immunol. Immunother., 2005, 54: 11-24; J.F. DiJoseph et al.,

Clin. Cancer Res., 2006, 12: 242-249; A Advani et al., Blood, 2005, 106: 230). In these
trials, thrombocytopenia was reported as the most common dose-limiting adverse event.
The present non-clinical study provides insights into the observed thrombocytopenia
associated with CMC-544 treatment of B-NHL patients.
[197] In vitro, CMC-544 neither bound to nor cause aggregation of platelets in
platelet-rich plasma of human or murine origin. When administered to mice, a single dose
of CMC-544 resulted in a 50% to 75% reduction in the number of circulating platelets with
a nadir on day 3 or 4. The platelet values returned to normal at the latest by day 12.
Similar effects were also observed in cynomolgus macaques with nadirs on day 4 or 5.
Unconjugated anti-CD22 antibody, G5/44 (humanized IgG4 antibody) had no effect on
circulating platelets. CMC-544-induced thrombocytopenia in mice was associated with a
concomitant reduction in circulating levels of thrombopoietin (TPO) with a nadir on day 4.
In contrast, treatment with a known cytotoxic chemotherapeutic agent, carboplatin, caused
thrombocytopenia in mice but with a nadir on day 11. Unlike that with CMC-544,
carboplatin-induced thrombocytopenia was associated with an increase in the levels of
circulating TPO.
[198] Subcutaneous pre-treatment of mice with oprelvekin (NEUMEGA® / rhIL-11)
(250 g/kg) significantly reduced the magnitude of the CMC-544-associated
thrombocytopenia. When evaluated in cynomolgus macaques, daily subcutaneous pre-
treatment with oprelvekin (125 g/kg) for 5 days completely prevented CMC-544-
associated thrombocytopenia but also suppressed elevations in serum hepatic
aminotransferases associated with CMC-544 treatment alone. Concurrent administration of
oprelvekin with CMC-544 failed to prevent the initial thrombocytopenia but did accelerate
complete recovery of platelets counts to pre-treatment levels and also suppressed elevations
in circulating serum hepatic aminotransferases.
[199] This study further provides evidence that the prophylactic use of oprelvekin can
ameliorate CMC-544-induced thrombocytopenia, potentially augmenting the therapeutic
benefit of CMC-544 in patient with B-NHL.
[200] Some of the results reported in this section have been presented at the American
Society of Hematology annual meeting, Dec 9-12, 2006 , Orlando, Fl in the following
abstract: "Thrombocytopenia induced by CMC-544 and its amelioration using oprelvekin

(NEUMEGA®/recombinant human interleukin-11", by John F DiJoseph, Dana Walker,
Maureen M Dougher, Doug C Armellino, Dave Clarke and Nitin K Damle.
Other Embodiments
[201] Other embodiments of the invention will be apparent to those skilled in the art
from a consideration of the specification or practice of the invention disclosed herein. It is
intended that the specification and examples be considered as exemplary only, with the true
scope of the invention being indicated by the following claims.

Claims
What is claimed is:
1. A method of alleviating thrombocytopenia in a subject, the method comprising a
step of: administering a therapeutically effective amount of interleukin-11 to the
subject, wherein thrombocytopenia is associated with administering to the subject a
conjugate comprising a targeting moiety and a cytotoxic drug.
2. The method of claim 1, wherein the subject is suffering from cancer or a cancerous
condition.
3. The method of claim 1 or 2, wherein the targeting moiety comprises an antibody.
4. The method of claim 3, wherein the antibody is an anti-CD22 antibody, an anti-
CD33 antibody, an anti-Lewis Y antibody, an anti-5T4 antibody, an anti-CD30
antibody, or any combinations thereof.
5. The method of claim 1 or 2, wherein the cytotoxic drug is a calicheamicin, a
calicheamicin derivative, an esperamicin, or an esperamicin derivative.
6. The method of claim 1 or 2, wherein the conjugate is an anti-CD22 antibody-
calicheamicin conjugate.
7. The method of claim 1 or 2, wherein the conjugate is an anti-CD33 antibody-
calicheamicin conjugate.
8. The method of claim 1 or 2, wherein the conjugate is an anti-Lewis Y antibody-
calicheamicin conjugate.
9. The method of claim 1 or 2, wherein the conjugate is an antt-5T4 antibody-
calicheamicm conjugate.
10. The method of claim 1 or 2, wherein the conjugate is an anti-CD30 antibody-
calicheamicin conjugate.
11. The method of claim 1 or 2, wherein interleukin-11 comprises recombinant human
interleukin-11.

12. The method of claim 1 or 2, wherein interleukin-11 is administered prior to
administration of the conjugate.
13. The method of claim 1 or 2, wherein said method prevents, reduces, slows down or
stops thrombocytopenia in the subject.
14. The method of claim 1 or 2, wherein thrombocytopenia is at least partly resulting
from bone marrow destruction.
15. The method of claim 14, wherein the targeting moiety comprises an antibody.
16. The method of claim 15, wherein the antibody is an anti-CD22 antibody, an anti-
CD33 antibody, an anti-Lewis Y antibody, an anti-5T4 antibody, an anti-CD30
antibody, or any combinations thereof.
17. The method of claim 14, wherein the cytotoxic drug is a calicheamicin, a
calicheamicin derivative, an esperamicin, or an esperamicin derivative.
18. The method of claim 14, wherein the conjugate is an anti-CD22 antibody-
calicheamicin conjugate.
19. The method of claim 14, wherein the conjugate is an anti-CD33 antibody-
calicheamicin conjugate.
20. The method of claim 14, wherein the conjugate is an anti-Lewis Y antibody-
calicheamicin conjugate.
21. The method of claim 14, wherein the conjugate is an anti-5T4 antibody-
calicheamicin conjugate.
22. The method of claim 14 wherein the conjugate is an anti-CD30 antibody-
calicheamicin conjugate.
23. The method of claim 14, wherein interleukin-11 comprises recombinant human
interleukin-11.
24. The method of claim 14, wherein interleukin-11 is administered prior to
administration of the conjugate.

25. The method of claim 14, wherein said method prevents, reduces, slows down or
stops thrombocytopenia at least partly resulting from bone marrow destruction.
26. The method of claim 1 or 2, wherein thrombocytopenia is at least partly resulting
from liver damage.
27. The method of claim 26, wherein the targeting moiety comprises an antibody.
28. The method of claim 27, wherein the antibody is an anti-CD22 antibody, an anti-
CD33 antibody, an anti-Lewis Y antibody, an anti-5T4 antibody, an anti-CD30
antibody, or any combinations thereof.
29. The method of claim 26, wherein the cytotoxic drug is a calicheamicin, a
calicheamicin derivative, an esperamicin, or an esperamicin derivative.
30. The method of claim 26, wherein the conjugate is an anti-CD22 antibody-
calicheamicin conjugate.
31. The method of claim 26, wherein the conjugate is an anti-CD33 antibody-
calicheamicin conjugate.
32. The method of claim 26, wherein the conjugate is an anti-Lewis Y antibody
calicheamicin conjugate.
33. The method of claim 26, wherein the conjugate is an anti-5T4 antibody-
calicheamicin conjugate.
34. The method of claim 26 wherein the conjugate is an anti-CD30 antibody-
calicheamicin conjugate.
35. The method of claim 26, wherein interleukin-11 comprises recombinant human
interleukin-11.
36. The method of claim 26, wherein interleukin-11 is administered prior to
administration of the conjugate.
37. The method of claim 26, wherein said method prevents, reduces, slows down or
stops thrombocytopenia at least partly resulting from liver damage.

38. The method of claim 37, wherein said method further prevents, reduces, slows
down or stops liver damage in the subject.
39. The method of claim 37, wherein said method further prevents, reduces, slows
down or stops liver-damage related inflammation in the subject.
40. A pharmaceutical composition comprising a therapeutically effective amount of
interleukin-11, at least one conjugate whose administration results in
thrombocytopenia, and at least one. physiologically acceptable carrier, wherein the
conjugate comprises a targeting moiety and a cytotoxic drug.
41. The pharmaceutical composition of claim 40, wherein the targeting moiety
comprises an antibody.
42. The pharmaceutical composition of claim 41, wherein the antibody is an anti-CD22
antibody, an anti-CD33 antibody, an anti-Lewis antibody, an anti-5T4 antibody, an
anti-CD30 antibody, or any combinations thereof.
43. The pharmaceutical composition of claim 40, wherein the cytotoxic drug is a
calicheamicin, a calicheamicin derivative, an esperamicin, or an esperamicin
derivative.
44. The pharmaceutical composition of claim 40, wherein the conjugate is an anti-
CD22-antibody-calicheamicin conjugate.
45. The pharmaceutical composition of claim 40, wherein the conjugate is an anti-
CD33-antibody-calicheamicin conjugate.
46. The pharmaceutical composition of claim 40, wherein the conjugate is an anti-
Lewis Y antibody-calicheamicin conjugate.
47. The pharmaceutical composition of claim 40, wherein the conjugate is an anti-5T4
antibody-calecheamicin conjugate.
48. The method of claim 26 wherein the conjugate is an anti-CD30 antibody-
calicheamicin conjugate.

49. The pharmaceutical composition of claim 40, wherein interleukin-11 comprises
recombinant human interleukin-11.
50. The pharmaceutical composition of claim 40 or 49, wherein interleukin-11, the at
least one conjugate, and physiologically acceptable carrier are combined as one or
more preparations for simultaneous or sequential administration of interleukin-11
and the at least one conjugate.
51. The pharmaceutical composition of claim 40, wherein administration of said
composition to a subject prevents, reduces or stops thrombocytopenia in the subject.
52. The pharmaceutical composition of claim 51, wherein the subject suffers from
cancer or a cancerous condition.
53. The pharmaceutical composition of claim 40, wherein thrombocytopenia produced
by administration of the at least one conjugate results, at least partly, from bone
marrow destruction.
54. The pharmaceutical composition of claim 40, wherein thrombocytopenia produced
by administration of the at least one conjugate results, at least partly, from liver
damage.
55. A kit comprising:
interleukin-11; and
at least one conjugate whose administration to a subject results in
thrombocytopenia in the subject, wherein the conjugate comprises a
targeting moiety and a cytotoxic drug.
56. The kit of claim 55, wherein the targeting moiety comprises an antibody.
57. The kit of claim 56, wherein the antibody is an anti-CD22 antibody, an anti-CD33
antibody, an anti-Lewis antibody, an anti-5T4 antibody, an anti-CD30 antibody, or
any combinations thereof.
58. The kit of claim 55, wherein the cytotoxic drug is a calicheamicin, a calicheamicin
derivative, an esperamicin, or an esperamicin derivative.

59. The kit of claim 55, wherein the conjugate is an anti-CD22-antibody-calicheamicin
conjugate.
60. The kit of claim 55, wherein the conjugate is an anti-CD33-antibody-calicheamicin
conjugate.
61. The kit of claim 55, wherein the conjugate is an anti-Lewis Y antibody-
calicheamicin conjugate.
62. The kit of claim 55, wherein the conjugate is an anti-5T4-antibody-calicheamicin
conjugate.
63. The method of claim 55 wherein the conjugate is an anti-CD30 antibody-
calicheamicin conjugate.
64. The kit of claim 55, wherein interleukin-11 comprises recombinant human
interleukin-11.
65. The kit of claim 55, wherein thrombocytopenia produced by the at least one
conjugate results, at least partly, from bone marrow destruction.
66. The kit of claim 55, wherein thrombocytopenia produced by the at least one
conjugate results, at least partly, from liver damage.

The present invention provides methods for the treatment and/or prevention of thrombocytopenia including thrombocytopenia
associated with drug-induced liver damage and thrombocytopenia associated with drug-induced bone marrow destruction.
The methods of treatment of the invention include administration of interleukin-11 to a subject suffering from or susceptible to thrombocytopenia
and/or receiving or about to receive a treatment involving a conjugate therapeutic agent whose administration results in
thrombocytopenia. Also provided are pharmaceutical compositions and kits useful for carrying out such methods of treatment.