U.S. patent application number 12/810006 was filed with the patent office on 2011-02-24 for immunoglobulin and/or toll-like receptor proteins associated with myelogenous haematological proliferative disorders and uses thereof.
This patent application is currently assigned to Cellerant Therapeutics, Inc.. Invention is credited to Holger Karsunky.
Application Number | 20110044894 12/810006 |
Document ID | / |
Family ID | 41114735 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110044894 |
Kind Code |
A1 |
Karsunky; Holger |
February 24, 2011 |
Immunoglobulin and/or Toll-Like Receptor Proteins Associated with
Myelogenous Haematological Proliferative Disorders and Uses
Thereof
Abstract
The disclosure relates to methods and compositions effective in
the diagnosis, prognosis and treatment of human hematopoietic
cancers. In particular, the disclosure provides tumor-associated
genes that encode for members of the immunoglobulin (Ig) and/or
toll-like receptor superfamilies that are differentially expressed
in hematopoietic tumor cells of myeloid origin compared with other
cells, e.g., normal stem cells.
Inventors: |
Karsunky; Holger; (Redwood
City, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Cellerant Therapeutics,
Inc.
San Carlos
CA
|
Family ID: |
41114735 |
Appl. No.: |
12/810006 |
Filed: |
March 26, 2009 |
PCT Filed: |
March 26, 2009 |
PCT NO: |
PCT/US09/38462 |
371 Date: |
September 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61039701 |
Mar 26, 2008 |
|
|
|
Current U.S.
Class: |
424/1.11 ;
424/133.1; 424/173.1; 435/375; 435/6.16; 506/7; 530/387.3;
530/389.1; 530/389.6 |
Current CPC
Class: |
A61K 39/39558 20130101;
A61K 45/06 20130101; G01N 2800/52 20130101; A61K 2039/505 20130101;
C07K 16/28 20130101; Y10T 436/143333 20150115; C07K 16/2866
20130101; A61P 35/02 20180101; G01N 33/57426 20130101; G01N
33/56966 20130101; A61P 35/00 20180101; C07K 2317/73 20130101; G01N
2333/726 20130101 |
Class at
Publication: |
424/1.11 ;
530/389.6; 530/389.1; 530/387.3; 424/133.1; 424/173.1; 435/375;
506/7; 435/6 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C07K 16/28 20060101 C07K016/28; C07K 16/18 20060101
C07K016/18; A61K 39/395 20060101 A61K039/395; C12N 5/09 20100101
C12N005/09; C40B 30/00 20060101 C40B030/00; C12Q 1/68 20060101
C12Q001/68; A61P 35/00 20060101 A61P035/00; A61P 35/02 20060101
A61P035/02 |
Claims
1. An antibody, wherein said antibody specifically binds to an
immunoglobin (Ig) superfamily or toll-like receptor superfamily
protein selected from the group consisting of: CD84; lymphocyte
antigen 86 (Ly86); CD180 (RP105); hepatitis A virus cellular
receptor 2 (HAVCR2); leukocyte immunoglobulin-like receptor,
subfamily A with TM domain, member 1 (LILRA1); leukocyte
immunoglobulin-like receptor, subfamily A with TM domain, member 2
(LILRA2); neuronal growth regulator 1 (NEGR1); and toll-like
receptor (TLR2).
2. The antibody of claim 1, wherein said antibody specifically
binds to an expression product of a gene selected from the group
consisting of: CD84; lymphocyte antigen 86 (Ly86); and CD180
(RP105).
3. The antibody of claim 2, wherein said antibody is a humanized
monoclonal antibody.
4. The antibody of claim 3, wherein said antibody specifically
binds to hematopoietic tumor cells (HTCs) of myeloid origin.
5. The antibody of claim 4, wherein said binding inhibits
proliferation and/or mediates destruction of said HTCs.
6. A pharmaceutical composition comprising an antibody of any one
of claims 2-5.
7. The pharmaceutical composition of claim 6, further comprising a
radioisotope or radionuclide.
8. The pharmaceutical composition of claim 6, further comprising a
bioactive compound.
9. The pharmacuetical composition of claim 8, wherein the bioactive
compound is a cytotoxic agent.
10. The pharmacuetical composition of claim 6, wherein said
composition comprises at least two antibodies and wherein said at
least two antibodies specifically bind to at least two different
expression products.
11. A method of mediating destruction of HTCs of myeloid origin,
comprising contacting said HTCs with a composition comprising an
antibody of any one of claims 2-5.
12. The method of claim 11, wherein said contacting inhibits the
proliferation of said HTCs.
13. A method of treating a patient with a myelogenous
haematological proliferative disorder characterized by
over-expression of at least one member of the Ig superfamily or TLR
superfamily associated with HTCs selected from the group consisting
of: CD84; lymphocyte antigen 86 (Ly86); CD180 (RP105); hepatitis A
virus cellular receptor 2 (HAVCR2); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 1 (LILRA1); leukocyte
immunoglobulin-like receptor, subfamily A with TM domain, member 2
(LILRA2); neuronal growth regulator 1 (NEGR1); and toll-like
receptor (TLR2), wherein the patient is administered a composition
comprising an antibody of any one of claims 2-5.
14. The method of claim 13, wherein said administering depletes
said HTCs in said patient.
15. The method of claim 13, wherein said haematological
proliferative disorder is AML.
16. A method of diagnosing a myelogenous haematological
proliferative disorder in a patient, comprising: obtaining a test
sample from said patient; and detecting in said test sample a level
of an expression product corresponding to a member of the Ig
superfamily or TLR superfamily associated with HTCs selected from
the group consisting of: CD84; lymphocyte antigen 86 (Ly86); CD180
(RP105); hepatitis A virus cellular receptor 2 (HAVCR2); leukocyte
immunoglobulin-like receptor, subfamily A with TM domain, member 1
(LILRA1); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 2 (LILRA2); neuronal growth regulator 1 (NEGR1);
and toll-like receptor (TLR2); wherein the level of said expression
product in said sample in comparison with a control is indicative
of said haematological proliferative disorder.
17. The method of claim 16, wherein said expression product is a
mRNA.
18. The method of claim 16, wherein the levels of at least two
different expression products are detected.
19. The method of claim 16, wherein said detecting uses a
microarray.
20. The method of claim 16, wherein the myelogenous haematological
proliferative disorder diagnosed is AML.
21. A method of monitoring the efficacy of treating a myelogenous
haematological proliferative disorder in a patient, comprising:
obtaining a test sample from said patient at two or more time
points during said treatment; and detecting in each of said test
samples a level of an expression product corresponding to a member
of the Ig superfamily or TLR superfamily associated with HTCs
selected from the group consisting of: CD84; lymphocyte antigen 86
(Ly86); CD180 (RP105); hepatitis A virus cellular receptor 2
(HAVCR2); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 1 (LILRA1); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 2 (LILRA2); neuronal
growth regulator 1 (NEGR1); and toll-like receptor (TLR2); wherein
a reduction in the levels of said expression product over time
indicates a positive response to treatment.
22. The method of claim 21, wherein said expression product is a
mRNA.
23. The method of claim 21, wherein the levels of at least two
different expression products are detected at two or more of said
time points.
24. The method of claim 21, wherein said detecting uses a
microarray.
25. The method of claim 21, wherein the myelogenous haematological
proliferative disorder monitored is AML.
26. A method of mediating destruction of HTCs of myeloid origin,
comprising contacting said HTCs with a composition comprising a
small molecule agonist or antagonist.
27. A method of treating a patient with a haematological
proliferative disorder characterized by over-expression of at least
one Ig superfamily or TLR superfamily member associated with HTCs
selected from the group consisting of: CD84; lymphocyte antigen 86
(Ly86); CD180 (RP105); hepatitis A virus cellular receptor 2
(HAVCR2); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 1 (LILRA1); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 2 (LILRA2); neuronal
growth regulator 1 (NEGR1); and toll-like receptor (TLR2), wherein
the patient is administered a composition comprising a small
molecule agonist or antagonist.
28. The method of claim 27, wherein said haematological
proliferative disorder is AML.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119 of U.S. application Ser. No. 61/039,701 (filed
Mar. 26, 2008), which is incorporated herein by reference in its
entirety.
1. TECHNICAL FIELD
[0002] The present disclosure relates generally to agents capable
of specifically targeting cancer stem cell markers and methods of
using the agents, particularly in diagnostic and therapeutic
treatments. In particular, the present disclosure provides proteins
belonging to the immunoglobulin (Ig) superfamily or the toll-like
receptor (TLR) superfamily as novel cancer stem cell targets that
are expressed extracellularly and that are targeted by antibodies
and other agents disclosed herein.
2. BACKGROUND
[0003] The cells of the hematopoietic system arise from multipotent
progenitors, the hematopoietic stem cells (HSCs), which progress
through a series of developmental programs to ultimately form the
terminally differentiated cells of the myeloid or lymphoid lineage.
It is believed that in the initial stages of hematopoiesis, HSCs
commit to two distinguishable oligopotent but developmentally
restricted progenitor cell types, the common lymphoid progenitors
(CLPs) and the common myeloid progenitor (CMPs). T lymphocytes, B
lymphocytes, natural killer (NK) cells, and lymphoid dendritic
cells develop from corresponding progenitor cells derived from the
CLPs whereas erythroid cells, megakaryocytes, granulocytes,
macrophages, and myeloid dendritic cells develop from their
corresponding progenitor cells derived from CMPs. Cell populations
at each stage of differentiation are distinguishable from other
cell populations in the hematopoietic pathway based on programmed
expression of a unique set of cell markers.
[0004] Although HSCs are capable of self renewal--cell division
that results in at least one of the daughter cells having the same
characteristics as the parent cell--the progenitor cells committed
to the lymphoid or myeloid lineages lose their potential to
self-renew. That is, mitotic cell division of the committed
progenitors leads to differentiated progeny rather than generation
of a cell with the same proliferative and differentiation capacity
as the parent cell. This loss of self-renewal potential is seen in
the ability of committed progenitors cells to maintain
hematopoiesis only for a limited time period (i.e., short term
reconstitution) following transplantation of the progenitor cells
into an immunocompromised animal, as compared to an HSC, which can
completely regenerate and maintain hematopoiesis during the life of
the host animal (i.e., long term reconstitution).
[0005] It has been observed, however, that in certain disease
states of the hematopoietic system, dysregulation of cellular
regulatory pathways may lead to progenitor cells that acquire the
ability to self-renew. For instance, acute myeloid leukemia (AML,
also called acute myelogenous leukemia) is a myeloproliferative
disorder marked, in part, by infiltration of bone marrow by
abnormal hematopoietic cells. Indeed, the stem cell nature of
cancer was first shown in AML (Lapidot et al., 1994 Nature
17:645-8). AML is categorized into different subtypes based on
morphological features and cytochemical staining properties, and
although the self-renewal characteristic in most types of AML is
attributable to leukemic cells having cell marker phenotypes
consistent with HSCs (Bonnet, D. and Dick, J. E., Nat. Med.
3(7):730-737 (1997)), the chromosomal abnormality associated with
the AML M3 subtype is observed in cell populations with a cell
marker phenotype characteristic of more differentiated cells of the
myeloid lineage (CD34.sup.-, CD38.sup.+) whereas the HSC population
in M3 does not carry the translocation (Turhan, A. G. et al., Blood
79:2154-2161 (1995)).
[0006] Gain of self-renewing characteristic in the committed
progenitor cell population is also suggested in chronic myeloid
leukemia (CML, also called chronic myelogenous leukemia, or chronic
granulocytic leukemia), a disease commonly associated with the
Philadelphia chromosome, which is a balanced translocation between
chromosomes 9 and 22, t(9;22). The translocation produces a fusion
between the bcr and c-abl genes and results in expression of a
chimeric protein BCR-ABL with increased tyrosine kinase activity.
Although the HSC population in CML typically contains the
chromosomal abnormality, the BCR-ABL fusion protein is mainly
expressed in the committed cells of myelomonocytic lineage rather
than the HSCs, indicating that committed cells in the myeloid
lineage may be the source of the leukemic cells rather than the
HSCs. Additional evidence for the committed myeloid cells as being
the source of the leukemic clones in CML comes from studies of
controlled expression of BCR-ABL in transgenic animals. Use of
promoters active specifically in myeloid progenitor cells to force
expression of BCR-ABL in committed cells but not in HSCs produces
disease characteristic of CML in these transgenic animal models
(Jaiswal, S. et al., Proc. Natl. Acad. Sci. USA 100:10002-10007
(2003)).
[0007] Although myeloproliferative disorders, such as AML and CML
are typically associated with cytogenetic abnormalities, the
cytogenetic defect may not be solely responsible for the
proliferative trait. In some instances, the chromosomal abnormality
is observed in normal cells, which suggests that accumulation of
additional mutations in either the HSCs or committed myeloid cells
is required for full manifestation of the disease state. Even in
CML, the disorder displays a multiphasic course, beginning from a
chronic phase, which after 3-5 years and up to 10 years, leads to
an accelerated or blastic phase similar to AML. The time period
required to transition from the chronic phase (less than 5% blasts
or promyelocytes) to the blastic phase (>30% blasts in the
peripheral blood or bone marrow) may reflect the time needed to
accumulate the mutations responsible for conversion of the chronic
phase to the more aggressive blastic phase. For the most part,
however, the leukemic cells appear to retain the cell marker
phenotypes detectable in normal progenitor cells.
[0008] Treatments for proliferative disorders normally rely on the
sensitivity of proliferating cells to cytotoxic or cytostatic
chemotherapeutic agents. For instance, busulfan, a bifunctional
alkylating agent, and hydroxyurea, an inhibitor of ribonucleoside
diphosphate, affect DNA synthesis and stability, resulting in
toxicity to dividing cells. Other therapeutic agents of similar
activity include cytosine arabinoside (cytarabine) and
daunorubicin. However, the effects of these agents are
non-discriminatory and as a result they have serious side effects
due to toxicity to normal dividing cells.
[0009] Another treatment used in patients with haematological
malignancies is bone marrow transplant (BMT), where the recipient's
hematopoietic cells are eliminated with radiation and/or
chemotherapy (e.g., cyclophosphamide), and the hematopoietic system
reconstituted by transplant of healthy hematopoietic stem cells.
Typically, the transplant uses HLA matched allogeneic bone marrow
cells from a family member (HLA-identical) or a serologically
matched altruistic donor (MUD). Approximately, <50% of
recipients find a donor, with exactly matching histocompatibility.
Transplant with less well matched donors marketed increases the
transplant related morbidity and mortality. This therapeutic
approach has limited application because of its dependence on the
availability of suitable donors and because the treatments show
better outcome for patients in the chronic or early phase of the
disease as compared to acute or late stages.
[0010] Antibody therapy for cancer involves the use of antibodies,
or antibody fragments, against an antigen to target
antigen-expressing tumor cells. Because antibody therapy targets
cells expressing a particular antigen, there is a possibility of
cross-reactivity with normal cells and can lead to detrimental
results. Substantial efforts have been directed to finding
tumor-specific antigens. Tumor-specific antigens are found almost
exclusively on tumors or are expressed at a greater level in tumor
cells than the corresponding normal cells. Thus, tumor-specific
antigens provide targets for antibody targeting of cancer, or other
disease-related, cells expressing the antigen, as well as providing
markers for diagnosis, for example, by identifying increased levels
of expression. In immunotherapy approaches, antibodies specific to
such tumor-specific antigens can be conjugated to cytotoxic
compounds or can be used alone in immunotherapy.
[0011] Immunotherapy as a treatment option against hematpoietic
cancers, such as AML, is limited by the lack of tumor-associated
antigens that are tumor-specific and that are shared among diverse
patients. It is desirable to find other therapeutic agents that
take advantage of the developmental origins of the leukemic cells
by exploiting the common characteristics between leukemic cells and
normal cell populations in the myeloid lineage. This approach would
provide treatments that can supplement traditional therapies for
myeloid leukemias, or that can be used as an alternative treatment
to directly target the stem cell fractions of leukemic cells. This
approach also provides additional diagnostic and prognostic
strategies, as well as strategies for monitoring the efficacy of a
therapeutic regimen.
[0012] Generally, therapeutic treatment is more effective when
tailored to a specific type of hematopoietic cancer. Predicting and
determining efficacy of a treatment regime over time is also
valuable in terms of clinical management. It is thus desirable to
find tumor-specific markers that can be used in more efficient and
accurate diagnosis and prognosis of myeloiod leukemic disorders,
such as AML.
[0013] Members of the immunoglobulin (Ig) superfamily of proteins
are cell surface and soluble proteins that are involved in the
recognition, binding or adhesion processes of cells. Members
include cell surface antigen receptors, co-receptors,
co-stimulatory molecules of the immune system, molecules involved
in antigen presentation to lymphocytes, cell adhesion molecules,
some cytokine receptors, and intracellular muscle proteins.
[0014] Proteins belonging to the toll-like receptor (TLR)
superfamily of proteins are generally pattern recognition receptors
that recognize that bind to different microbial components. Binding
of TLRs to pathogen-associated molecular patterns (PAMP) induces
the production of reactive oxygen and nitrogen intermediates,
initiation of the proinflammatory cytokine network, and
upregulation of costimulatory molecules of the immune system.
3. SUMMARY
[0015] The present invention provides methods and compositions
effective in the diagnosis and treatment of human hematopoietic
cancers of myeloid origin. As described herein, the following
markers of the immunoglobulin (Ig) superfamily or toll-like
receptor (TLR) superfamily have been found to be associated with
hematopoietic tumor cells (HTCs) of myeloid origin: CD84;
lymphocyte antigen 86 (Ly86); CD180 (RP105); hepatitis A virus
cellular receptor 2 (HAVCR2); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 1 (LILRA1); leukocyte
immunoglobulin-like receptor, subfamily A with TM domain, member 2
(LILRA2); neuronal growth regulator 1 (NEGR1); and toll-like
receptor (TLR2).
[0016] In preferred embodiments, the disclosed members of the Ig
superfamily or TLR superfamily are differentially expressed in HTCs
of myeloid origin compared to normal HSCs. In some embodiments, the
disclosed members of the Ig superfamily or TLR superfamily are
differentially expressed by at least about 2 fold. In other
embodiments, the members of the Ig superfamily or TLR superfamily
are differentially expressed by at least about 3 fold. In other
embodiments, the members of the Ig superfamily or TLR superfamily
are differentially expressed by at least about 5 fold, etc. The
present disclosure provides agents specifically directed to these
markers that find use in therapeutic and diagnostic
applications.
[0017] The following members of the Ig superfamily or TLR
superfamily are over-expressed on the surface of myelogenous HTCs:
CD84; CD180 (RP105); hepatitis A virus cellular receptor 2
(HAVCR2); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 1 (LILRA1); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 2 (LILRA2); neuronal
growth regulator 1 (NEGR1); and toll-like receptor (TLR2). Agents
specifically directed to these markers can specifically bind HTCs
of myeloid origin by virtue of binding to the surface-expressed
marker.
[0018] The following extracellulary-expressed markers are
over-expressed and found on the surface of myelogenous HTCs by
virtue of binding other membrane proteins:lymphocyte antigen 86
(Ly86). Compositions that specifically target the disclosed
myelogenous HTC markers (e.g., members of the Ig superfamily or TLR
superfamily disclosed herein), interfering with the expression
thereof or binding to the expressed products, are provided herein,
as well as methods of using the same in the diagnosis, prognosis
and treatment of haematological proliferative disorders
characterized by such markers. The compositions include antibodies
that specifically bind one or more of the extracellularly-expressed
antigens associated with myelogenous HTCs that can inhibit their
proliferation and/or mediate their destruction. The invention
further provides immortal cell lines that produce one or more such
antibodies.
[0019] In one aspect, the invention provides antibodies that
specifically bind to one or more of CD84; lymphocyte antigen 86
(Ly86); CD180 (RP105); hepatitis A virus cellular receptor 2
(HAVCR2); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 1 (LILRA1); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 2 (LILRA2); neuronal
growth regulator 1 (NEGR1); and toll-like receptor (TLR2)
associated with HTCs of myeloid origin. In some embodiments, the
antibody is a monoclonal antibody, for example, an antibody which
is produced from a hybridoma cell line. In preferred embodiments,
the monoclonal antibody specifically binds to hematopoietic tumor
cells of myeloid origin including, without limitation, chronic
myeloid leukemia (CML) blasts, acute myeloid leukemia (AML) blasts,
as well as to cells from the KG-1a, Pfeiffer, MOLT-3, GA-10, Ramos,
and Jurkat cell lines. In another embodiment, the monoclonal
antibody specifically binds to AML blasts.
[0020] In preferred embodiments, the invention provides antibodies
that specifically bind to one or more of the disclosed members of
the Ig superfamily or TLR superfamily associated with myelogenous
HTCs and thereby inhibit their proliferation and/or mediate their
destruction, but do not mediate destruction of normal hematopoietic
stem cells. In a preferred embodiment, the antibody is a monoclonal
antibody. In some embodiments, the antibody is an IgG isotype or a
humanized antibody. In one embodiment, the humanized antibody is
from a transgenic animal that includes a human immunoglobulin
gene.
[0021] In another embodiment, the invention provides an antibody
complex having at least one antibody that specifically binds to one
or more of the disclosed members of the Ig superfamily or TLR
superfamily associated with HTCs of myeloid origin. In a preferred
embodiment, the antibody complex comprises a multimer comprising a
monoclonal antibody that binds to one of the disclosed members of
the Ig superfamily or TLR superfamily.
[0022] In alternative embodiments, the antibodies of the present
invention include detectable moieties, radioactive compounds (e.g.
radioisotopes or radionuclides), or bioactive compounds (e.g. drugs
or small molecules). In some embodiments, the bioactive compound is
a cytotoxic agent.
[0023] In another embodiment, the invention provides small
molecules, which bind, preferably specifically, to one or more of
the members of the Ig superfamily or TLR superfamily polypeptides
disclosed herein. In preferred embodiments, the small molecule is a
small organic molecule, including small organic molecules known in
the art as being an agonist or antagonist of a polypeptide
corresponding to a member of the Ig superfamily or TLR superfamily
as disclosed herein. Small molecules known to bind polypeptides
corresponding to other HTC markers disclosed herein can also find
use in the subject therapeutic, prognostic and/or diagnostic
applications.
[0024] Optionally, the small molecule is conjugated to a growth
inhibitory agent or cytotoxic agent such as a toxin, including, for
example, a maytansinoid or calicheamicin, an antibiotic, a
radioactive isotope, a nucleolytic enzyme, or the like. The small
molecules that find use in the therapeutic methods of the instant
invention preferably induce death of a cell to which they bind. For
diagnostic purposes, the small molecules can be detectably labeled
and/or attached to a solid support.
[0025] The subject agents and antibodies directed to the disclosed
members of the Ig superfamily or TLR superfamily have significant
therapeutic and diagnostic utilities and in additional aspects
pharmaceutical compositions, methods and kits are provided
employing the subject agents and antibodies for use in diagnosing
and treating haematological proliferative disorders characterized
by the presence of one or more of the disclosed Ig superfamily or
TLR superfamily members such as, e.g., acute myelogenous leukemia,
acute myelomonocytic leukemia, chronic myelogenous leukemia and
acute myeloid leukemia.
[0026] In one aspect, the present disclosure provides methods of
using antibodies to target one or more of the disclosed members of
the Ig superfamily or TLR superfamily. In the present teachings,
the antibodies provide a basis for immunotherapeutic approaches in
treating disorders involving HTCs of myeloid origin, for example,
myeloproliferative disorders such as chronic myeloid leukemia (CML)
and acute myeloid leukemia (AML).
[0027] In one embodiment, the present invention provides methods of
inhibiting the proliferation of HTCs of myeloid origin by
contacting the HTCs with a composition comprising an antibody or
other agent directed to one or more of the disclosed members of the
Ig superfamily or TLR superfamily. In another embodiment, the
present invention provides methods of mediating the destruction of
HTCs of myeloid origin by contacting the HTCs with a composition
comprising an antibody or other agent directed to one or more of
the disclosed members of the Ig superfamily or TLR superfamily. In
one embodiment, the antibody is a monoclonal antibody that
specifically binds an epitope on a disclosed member of the Ig
superfamily or TLR superfamily or a portion thereof. In another
embodiment, the composition comprises an antibody complex.
[0028] In another embodiment, a method of depleting HTCs
over-expressing one or more of the disclosed members of the Ig
superfamily or TLR superfamily in a subject in need thereof is
provided in which the subject is administered a composition
comprising an antibody or antibody complex as described herein. In
yet another embodiment, the present invention provides a method of
treating a patient with a myelogenous haematological proliferative
disorder characterized by over-expression in HTCs of one or more of
the disclosed members of the Ig superfamily or TLR superfamily
where the patient is administered a composition that includes an
antibody or antibody complex or other agent as described
herein.
[0029] In some embodiments, the methods of the present invention
are suitable for treating a haematological proliferative disorder
of myeloid origin, including myoproliferative disorders such as,
for example, chronic myeloid leukemia (CML) and/or acute myeloid
leukemia (AML).
[0030] In another aspect, the present invention provides diagnostic
methods for hematological proliferative disorders of myeloid
origin, where the level of an expression product corresponding to
one or more of the disclosed members of the Ig superfamily or TLR
superfamily is detected. In one embodiment, the expression product
is a transcription product, such as RNA. Methods of detecting the
level of RNA include utilizing a specific hybridization probe or an
array of such probes. In another embodiment, the expression product
is a translation product such as one or more of the Ig superfamily
or TLR superfamily associated with HTCs disclosed herein. Methods
of detecting the level of an antigen include utilizing antibodies
of the instant disclosure. The RNA or antigen level can be compared
to control levels, e.g., levels obtained from samples of normal
HSCs.
[0031] In another embodiment, the present invention provides
prognostic methods for predicting the efficacy of treating a
haematological proliferative disorder of myeloid origin, where the
level of an expression product corresponding to one or more of the
disclosed members of the Ig superfamily or TLR superfamily is
detected and wherein the expression product level is correlated
with a treatment outcome. In one embodiment, the expression product
is RNA and lower expression levels correlate with more favorable
outcomes.
[0032] In still another embodiment the present invention provides
methods for monitoring the efficacy of treating a haematological
proliferative disorder of myeloid origin, where the level of an
expression product corresponding to one or more of the disclosed
members of the Ig superfamily or TLR superfamily is detected at
various time points; and where a change in the level is correlated
with treatment outcome. In one embodiment, the expression product
is RNA and decreasing levels indicate a positive response to
treatment.
[0033] In some embodiments, the methods of the present invention
are suitable for diagnosis, prognosis and monitoring of a
haematological proliferative disorder of myeloid origin, such as
myoproliferative disorders. The present invention provides methods
of diagnosis, prognosis and monitoring of chronic myeloid leukemia
(CML) and/or acute myeloid leukemia (AML). In a particularly
preferred embodiment, the haematological proliferative disorder is
AML and the level of RNA or antigen is detected using a test sample
comprising AML HTCs, which is compared to control levels obtained
from a control sample of normal HSCs.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows the results of double sorting
Lin.sup.-CD34.sup.+CD90.sup.+CD45RA.sup.-CD38.sup.- and
Lin.sup.-CD34.sup.+CD90.sup.+CD45RA.sup.-CD38.sup.+ cells from 3
samples taken from 3 individual mobilized peripheral blood (MPB)
donors that are not afflicted with AML.
[0035] FIG. 2 shows the results of double sorting
Lin-CD34+CD90+CD45RA-CD38- and Lin-CD34+CD90+CD45RA-CD38+ cells
from 3 samples taken from peripheral blood of 3 individual patients
diagnosed with AML.
[0036] FIG. 3 shows representative results of incubating peripheral
blood cells with an antibody specific for (A) Ly86, (B) CD84, and
(C) CD180. The samples were taken from an individual diagnosed with
AML or an individual mobilized peripheral blood (MPB) donors not
afflicted with AML.
5. DETAILED DESCRIPTION OF EMBODIMENTS
5.1 Definitions
[0037] For the following descriptions, the technical and scientific
terms used herein will have the meanings commonly understood by one
of ordinary skill in the art, unless specifically defined
otherwise. Accordingly, the following terms are intended to have
the following meanings:
[0038] The terms "cancer stem cell (CSC) markers" or "cancer stem
cell (CSC) targets" as well as "hematopoietic tumor cell (HTC)
markers" or "hematopoietic tumor cell (HTC) targets" refer to genes
and their expression products, such as mRNA and polypeptides, that
have been found to be associated with HTCs by virture, for example,
of increased expression and/or biological activity. For example, in
the case of the CD180 marker disclosed herein, CD180 mRNA
transcribed from the CD180 gene is found at higher levels in
samples comprising AML HTCs as compared with samples comprising
normal HSCs. An HTC associated with a given marker is referred to
herein as a "marker+ HTC."
[0039] "HTCs of myeloid origin" particularly refers to cancer stem
cells derived from cells of the myeloid (nonlymphoid) lineages,
including monocytes, macrophages, neutrophils, basophils,
eosinophils, erythrocytes, megakaryocytes/platelets, dendritic
cells and the like. HTCs of myeloid origin will be those found in
myeloid leukemias, such as AML and CML, where it is believed that
progenitor cells committed to myeloid lineages regain self-renewing
characteristics. Various forms of leukemia, for example, appear to
have their origins in a small population of HSCs or committed
myeloid progenitor cells in which the cells acquire a combination
of mutations that give rise to the malignant phenotype. The terms
"HTCs of myeloid origin" and "myeloid HTCs" or "myelogenous HTCs"
are used herein interchangeably.
[0040] "Hematopoietic stem cell" or "HSC" generally refers to
clonogenic, self renewing pluripotent cells, capable of ultimately
differentiating into all cell types of the hematopoietic system,
including B cells T cells, NK cells, lymphoid dendritic cells,
myeloid dendritic cells, granulocytes, macrophages, megakaryocytes,
and erythroid cells. As with other cells of the hematopoietic
system, HSCs are typically defined by the presence of a
characteristic set of cell markers.
[0041] "Marker phenotyping" refers to identification of markers or
antigens on cells for determining its phenotype (e.g.,
differentiation state and/or cell type). This may be done by
immunophenotyping, which uses antibodies that recognize antigens
present on a cell. The antibodies may be monoclonal or polyclonal,
but are generally chosen to have minimal crossreactivity with other
cell markers. It is to be understood that certain cell
differentiation or cell surface markers are unique to the animal
species from which the cells are derived, while other cell markers
will be common between species. These markers defining equivalent
cell types between species are given the same marker identification
even though there are species differences in structure (e.g., amino
acid sequence). Cell markers include cell surfaces molecules, also
referred to in certain situations as cell differentiation (CD)
markers, and gene expression markers. The gene expression markers
are those sets of expressed genes indicative of the cell type or
differentiation state. In part, the gene expression profile will
reflect the cell surface markers, although they may include
non-cell surface molecules.
[0042] Lineage markers are cell surface antigens that can be used
for immunophenotyping cells of a particular developmental lineage.
For example, a set of `Lin` antigens comprising CD2, CD3, CD4, CD5,
CD8, NK1.1, B220, TER-119, Gr-1 can be used to identify mature
murine blood cells. Cells that do not express these marker
antigens, or express them at very low levels, are said to be
lineage marker negative (Lin.sup.-). The monoclonal antibody
cocktails directed against these lineage markers can be used to
remove cells expressing these antigens from source tissues (for
example, bone marrow, umbilical cord blood, mobilized peripheral
blood, fetal liver, and the like). This negative selection
procedure yields a population of cells that is enriched for
primitive hematopoietic stem cells or very early progenitor cells
or precursor cells that do (not yet) express these markers (see,
for example: KTLS cells). These cells are called lineage negative
cells, abbreviated Lin.sup.- cells. Several subpopulations of
lineage negative cells have been identified that are enriched for
hematopoietic stem cells. They include Lin.sup.-CD34.sup.+ cells
(Krause et al, 1994), Lin.sup.-Sca.sup.-1+c-Kit.sup.+Thy1.sup.low
cells (Fleming et al, 1993) and human CD34.sup.+CD38.sup.- cell
populations.
[0043] "Agent" refers to any molecule specifically directed to one
or more of the disclosed HTC markers and that can act to inhibit,
hinder and/or suppress a biological activity of the HTC marker in a
haematological proliferative disorder and/to to mediate destruction
of the HTCs. Agents include any molecule that specifically
interacts with an HTC marker gene and/or expression product,
including for example, antibodies that specifically bind to an
antigen corresponding to a HTC marker to inhibit HTC proliferation
and/or mediate their destruction; antisense molecules that
interfere with the expression of an HTC marker; or molecules that
interfere with a biological activity mediated by the HTC marker,
such as by sterically inhibiting interaction between an HTC marker
and its ligand to interfere with activation of a cancer stem cell
signal transduction pathway. The molecule may be one known in the
art, e.g., small molecule agonists or antagonists directed towards
one or more of the HTC markers disclosed herein. An antibody that
specifically binds to an antigen corresponding to an HTC marker
disclosed herein is referred to as a "marker specific
antibody."
[0044] A "small molecule", "small molecule compound", or "small
organic molecule" refers to a molecule having a molecular weight
usually less than about 2000 daltons, alternatively less than about
1500, about 750, about 500, about 250 or about 200 daltons in size,
wherein such molecules are known in the art to be capable of
binding (preferably specifically binding) to a polypeptide
corresponding to an HTC marker disclosed herein.
[0045] With regard to the binding of a small molecule compound to a
target molecule, the term "specifically interacts with" or
"specifically binds" or is "directed to or towards" a particular
product means binding that is measurably different from a
non-specific interaction. Specific binding can be measured, for
example, by methods known in the art, e.g., using competition
assays with a control molecule that is similar to the target, for
example, an excess of non-labeled target. A small molecule compound
that specifically binds a target can have a Kd for the target of at
least about 10.sup.-4 M, alternatively at least about 10.sup.-5 M,
alternatively at least about 10.sup.-6 M, alternatively at least
about 10.sup.-7 M, alternatively at least about 10.sup.-8 M,
alternatively at least about 10.sup.-9M, alternatively at least
about 10.sup.-10 M, alternatively at least about 10.sup.-11 M,
alternatively at least about 10.sup.-12 M, or greater. In one
embodiment, the term "specific binding" refers to binding where a
small molecule compound binds to its particular target without
substantially binding to any other polypeptide or
macromolecule.
[0046] "Antibody" refers to a composition comprising a protein that
binds specifically to a corresponding antigen and has a common,
general structure of immunoglobulins. The term antibody
specifically covers polyclonal antibodies, monoclonal antibodies,
dimers, multimers, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments, so long as they exhibit the
desired biological activity. Antibodies may be murine, human,
humanized, chimeric, or derived from other species. Typically, an
antibody will comprise at least two heavy chains and two light
chains interconnected by disulfide bonds, which when combined form
a binding domain that interacts with an antigen. Each heavy chain
is comprised of a heavy chain variable region (V.sub.H) and a heavy
chain constant region (C.sub.H). The heavy chain constant region is
comprised of three domains, C.sub.H1, C.sub.H2 and C.sub.H3, and
may be of the mu, delta, gamma, alpha or epsilon isotype.
Similarly, the light chain is comprised of a light chain variable
region (V.sub.L) and a light chain constant region (C.sub.L). The
light chain constant region is comprised of one domain, C.sub.L,
which may be of the kappa or lambda isotype. The V.sub.H and
V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system. The heavy chain constant region
mediates binding of the immunoglobulin to host tissue or host
factors, particularly through cellular receptors such as the Fc
receptors (e.g., Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII, etc.).
As used herein, antibody also includes an antigen binding portion
of an immunoglobulin that retains the ability to bind antigen.
These include, as examples, F(ab), a monovalent fragment of V.sub.L
C.sub.L and V.sub.H C.sub.H antibody domains; and F(ab).sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region. The term antibody also
refers to recombinant single chain Fv fragments (scFv) and
bispecific molecules such as, e.g., diabodies, triabodies, and
tetrabodies (see, e.g., U.S. Pat. No. 5,844,094).
[0047] Antibodies may be produced and used in many forms, including
antibody complexes. As used herein, the term "antibody complex" or
"antibody complexes" is used to mean a complex of one or more
antibodies with another antibody or with an antibody fragment or
fragments, or a complex of two or more antibody fragments. Antibody
complexes include multimeric forms of antibodies directed to the
disclosed HTC markers such as homoconjugates and heteroconjugates
as well as other cross-linked antibodies as described herein.
[0048] "Antigen" is to be construed broadly and refers to any
molecule, composition, or particle that can bind specifically to an
antibody. An antigen has one or more epitopes that interact with
the antibody, although it does not necessarily induce production of
that antibody.
[0049] The terms "cross-linked", "cross-linking" and grammatical
equivalents thereof, refer to the attachment of two or more
antibodies to form antibody complexes, and may also be referred to
as multimerization. Cross-linking or multimerization includes the
attachment of two or more of the same antibodies (e.g.
homodimerization), as well as the attachment of two or more
different antibodies (e.g. heterodimerization). Those of skill in
the art will also recognize that cross-linking or multimerization
is also referred to as forming antibody homoconjugates and antibody
heteroconjugates. Such conjugates may involve the attachment of two
or more monoclonal antibodies of the same clonal origin
(homoconjugates) or the attachment of two or more antibodies of
different clonal origin (also referred to as heteroconjugates or
bispecific). Antibodies may be crosslinked by non-covalent or
covalent attachment. Numerous techniques suitable for cross-linking
will be appreciated by those of skill in the art. Non-covalent
attachment may be achieved through the use of a secondary antibody
that is specific to the primary antibody species. For example, a
goat anti-mouse (GAM) secondary antibody may be used to cross-link
a mouse monoclonal antibody. Covalent attachment may be achieved
through the use of chemical cross-linkers.
[0050] "Epitope" refers to a determinant capable of specific
binding to an antibody. Epitopes are chemical features generally
present on surfaces of molecules and accessible to interaction with
an antibody. Typical chemical features are amino acids and sugar
moieties, having three-dimensional structural characteristics as
well as chemical properties including charge, hydrophilicity, and
lipophilicity. Conformational epitopes are distinguished from
non-conformational epitopes by loss of reactivity with an antibody
following a change in the spatial elements of the molecule without
any change in the underlying chemical structure.
[0051] "Humanized antibody" refers to an immunoglobulin molecule
containing a minimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances. Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, a humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the framework (FR) regions are those of a
human immunoglobulin consensus sequence. A humanized antibody will
also encompass immunoglobulins comprising at least a portion of an
immunoglobulin constant region (Fc), generally that of a human
immunoglobulin (Jones et al., Nature 321:522-525 (1986); Reichmann
et al, Nature 332:323-329 (1988)).
[0052] "Immunogen" refers to a substance, compound, or composition
which stimulates the production of an immune response.
[0053] The term "immunoglobulin locus" refers to a genetic element
or set of linked genetic elements that comprise information that
can be used by a B cell or B cell precursor to express an
immunoglobulin peptide. This peptide can be a heavy chain peptide,
a light chain peptide, or the fusion of a heavy and a light chain
peptide. In the case of an unrearranged locus, the genetic elements
are assembled by a B cell precursor to form the gene encoding an
immunoglobulin peptide. In the case of a rearranged locus, a gene
encoding an immunoglobulin peptide is contained within the
locus.
[0054] "Isotype" refers to an antibody class defined by its heavy
chain constant region. Heavy chains are generally classified as
gamma, mu, alpha, delta, epsilon and designated as IgG, IgM, IgA,
IgD, and IgE. Variations within each isotype are categorized into
subtypes, for example subtypes of IgG are divided into IgG.sub.1,
IgG.sub.2, IgG.sub.3, and IgG.sub.4, while IgA is divided into
IgA.sub.1 and IgA.sub.2. The IgY isotype is specific to birds.
[0055] "Monoclonal antibody" or "monoclonal antibody composition"
refers to a preparation of antibody molecules of single molecular
composition. A monoclonal antibody composition displays a single
binding specificity and affinity for a particular epitope.
[0056] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable and/or
constant regions (if present) derived from human germline
immunoglobulin sequences. In one embodiment, the human monoclonal
antibodies are produced by a hybridoma which includes a B cell
obtained from a transgenic non-human animal, e.g., a transgenic
mouse, having a genome comprising a human heavy chain transgene and
a light chain transgene fused to an immortalized cell.
[0057] "Single chain Fv" or "scFv" refers to an antibody comprising
the V.sub.H and V.sub.L regions of an antibody, wherein these
domains are present in a single polypeptide chain. Generally, a
scFv further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains which enables the scFv to form the desired
structure for antigen binding.
[0058] "Specifically immunoreactive" or antibody that "specifically
binds" refers to a binding reaction of the antibody that is
determinative of the presence of the antigen in a heterogeneous
population of antigens. The antibody may be described as being
"directed to" or "directed against" the particular antigen. Under a
designated immunoassay condition, the antibody binds to the antigen
at least two times, and typically 10-1000 times or more over
background. "Specifically immunoreactive" or "antibody that
specifically binds" also refers to an antibody that is capable of
binding to an antigen with sufficient affinity such that the
antibody is useful in targeting a cell having the antigen bound to
its surface or in targeting the soluble antigen itself. In such
embodiments, the extent of non-specific binding is the amount of
binding at or below background and will typically be less than
about 10%, preferably less than about 5%, and more preferably less
than about 1% as determined by fluorescence activated cell sorting
(FACS) analysis or radioimmunoprecipitation (RIA), for example.
[0059] "Recombinant antibody" refers to all antibodies prepared and
expressed, created or isolated by recombinant techniques. These
include antibodies obtained from an animal that is transgenic for
the immunoglobulin locus, antibodies expressed from a recombinant
expression vector, or antibodies created, prepared, and expressed
by splicing of any immunoglobulin gene sequence to other nucleic
acid sequences.
[0060] The term "associated with" as in, for example, members of
the immunoglobulin (Ig) superfamily or toll-like receptor (TLR)
superfamily being "associated with" hematopoietic tumor cells
(HTCs), refers to the case where the HTC marker genes (e.g., the
genes of the disclosed members of the immunoglobulin (Ig)
superfamily or toll-like receptor (TLR) superfamily) are
differentially expressed in HTCs as opposed to other mammalian
cells, e.g., normal HSCs. That is, a transcription product, such as
RNA, and/or a translation product, such as a polypeptide,
corresponding to the gene has been found at differential levels in
one or more samples comprising HTCs compared with one or more
samples comprising other mammalian cells, e.g., normal HSCs.
[0061] "Expression" or "expressing" as used herein refers to both
transcriptional and translational processes directed by a gene.
That is, the terms refer to the process of converting genetic
information encoded in a nucleic acid sequence (gene) into RNA
(e.g, mRNA rRNA, tRNA, snRNA, etc) through transcription of the
gene; and/or converting genetic information into protein through
translation of mRNA. Similarly, "expression product" as used herein
refers to a transcription or translation product of a gene, and
includes, e.g., RNA (mRNA, tRNA, rRNA, snRNA, etc.,) as well as
polypeptides (intracellular, extracellular or surface expressed
proteins).
[0062] "Differential expression", "differentially expressed",
"differential levels", and the like, as used herein refers to a
difference in the level of an expression product corresponding to a
marker in HTCs in comparison to other mammalian cells, e.g., normal
HSCs in particular. The difference can be expressed as an
expression ratio or signal ratio, obtained from the quotient of the
level of expression of an expression product in HTCs over the level
of expression of the same expression product in HSCs. "Differential
expression" generally refers to a difference in expression levels
of at least about 2 fold, at least about 3 fold, at least about 5
fold, at least about 7 fold, at least about 10 fold, or at least
about 15 fold. In particularly preferred embodiments, the
difference in expression levels is at least about 20 fold, at least
about 30 fold, at least about 40 fold, or at least about 50 fold.
In still more preferred embodiments, the difference in expression
levels is at least about 70 fold, at least about 100 fold, at least
about 200 fold, or as much as nearly 300 fold, 400 fold or 500
fold.
[0063] The term "extracellularly-expressed" refers to the case
where expression products are found outside of a cell, whether
existing entirely outside of the plasma membrane of a cell (as in
the case of secreted, soluble products); or existing partly outside
of a cell as in the case of some membrane- (or surface-) expressed
products. Membrane-bound proteins may be integral or peripheral, so
long as at least a portion of the protein is accessible to
antibodies outside the cell. The term membrane-bound includes
membrane-expressed products as well as receptor bound products that
become associated with surface membranes by virtue of binding to a
membrane-bound receptor.
[0064] "Subject" or "patient" are used interchangeably and refer
to, except where indicated, mammals such as humans and non-human
primates, as well as rabbits, rats, mice, goats, pigs, and other
mammalian species.
[0065] "Haematological proliferative disorder" or "hematopoietic
proliferative disorder" refers to a condition characterized by the
clonal proliferation of one or more hematopoietic cells of the
myeloid and/or lymphoid lineage. "Hematological proliferative
disorder of the myeloid lineage" refers to conditions characterized
by the clonal proliferation primarily of one or more hematopoietic
cells of the myeloid lineage, rather than the lymphoid lineage. It
is to be noted that herein the term "of myeloid origin" is used
interchangeably with the adjectives "myeloid" or "myelogenous."
Myelogenous hematopoietic proliferative disorders include, e.g.,
the general classes of (a) dysmyelopoietic disease, (b) acute
myeloproliferative leukemia and (c) chronic myeloproliferative
disease. Each general class is further categorized into different
subtypes, as is known in the art.
5.2 Hybridomas and Monoclonal Antibodies
[0066] The teachings of the present disclosure provide hybridoma
cell lines and monoclonal antibodies that specifically bind to one
or more of the following members of the immunoglobulin (Ig)
superfamily or toll-like receptor (TLR) superfamily: CD84;
lymphocyte antigen 86 (Ly86); CD180 (RP105); hepatitis A virus
cellular receptor 2 (HAVCR2); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 1 (LILRA1); leukocyte
immunoglobulin-like receptor, subfamily A with TM domain, member 2
(LILRA2); neuronal growth regulator 1 (NEGR1); and toll-like
receptor (TLR2).
[0067] The following members of the Ig superfamily or TLR
superfamily are over-expressed on the surface of HTCs of myeloid
origin: CD84; CD180 (RP105); hepatitis A virus cellular receptor 2
(HAVCR2); leukocyte immunoglobulin-like receptor, subfamily A with
TM domain, member 1 (LILRA1); leukocyte immunoglobulin-like
receptor, subfamily A with TM domain, member 2 (LILRA2); neuronal
growth regulator 1 (NEGR1); and toll-like receptor (TLR2). The
invention provides anti-CD84 antibodies, anti-CD180 antibodies,
anti-HAVCR2 antibodies, anti-LILRA1 antibodies, anti-LILRA2,
anti-NEGR1 antibodies, and anti-TLR2 antibodies, where the
antibodies are preferably humanized monoclonal antibodies.
[0068] CD84 is a transmembrane protein of the Ig SLAM superfamily.
Seven alternative splice forms are known, generally found on mature
B cells, a subset of T cells that comprises mostly
CD4.sup.+CD45RA.sup.+, monocytes, thymocytes, dendritic cells and
platelets; and a subset of CD34.sup.+ cells. The polypeptide is
normally involved in cell adhesion and homophilic interactions and
serves to enhance IFN-.gamma. secretion from lymphocytes as well as
inducing platelet production. Signaling through CD84 occurs via
homotypic interactions and involves phosphorylation and recruitment
of SAP and EAT-2, which can lead to IFN.gamma. production and T
cell activation. CD84 is also known as Hly9-beta, Ly9B, SLAMF5 and
DKFZp781E2378. Although normally-occurring splice forms are widely
expressed on hematopoietic cells, HTCs (e.g., HTCs from AML
patients) may express a different splice version that is expressed
more restrictively.
[0069] The present invention provides anti-CD84 antibodies,
preferably monoclonal antibodies, that can specifically bind to
CD84 antigen, e.g., CD84 antigens exposed on the surface of HTCs of
myeloid origin. In preferred embodiments, the anti-CD84 monoclonal
antibodies specifically target the CD84 splice form expressed by
myelogenous HTCs (referred to herein as an AML-expressed isoform of
CD84), as opposed to those expressed in healthy CSC samples. As
discussed in more detail below, the anti-CD84 antibodies preferably
bind such myelogenous HTCs, thereby inhibiting their proliferation
and/or mediating their destruction. One of skill in the art will
further recognize that antibodies known in the art can find use in
the methods disclosed in the instant invention. For example, one or
more anti-CD84 antibodies commercially available from Abcam, AbD
Serotec, ABR-Affinity BioReagents, Biolegend, BDBiosciences,
eBioscience, GeneTex, Lifespan Biosciences, R&D Systems,
Raybiotech and Santa Cruz Antibodies, may also find use with
respect to diagnostic and/or therapeutic applications taught
herein.
[0070] CD180, also known as LY64, LY78, MGC126233, MGC126234 and
RP105, is a type I transmembrane molecule of the TLR family that
physically associates with secreted Ly86 (discussed below). CD180
is normally expressed partly on the cell surface of B cells,
monocytes/macrophages, and dendritic cells, and its cytoplasmic
domain shares homology with the type 1 IL-1 receptor. CD180
generally functions as a signal transduction molecule, playing an
important role in the regulation of B cell growth and death. More
specifically, the CD180/Ly86 complex functions in concert with TLR4
to mediate B cell recognition, as well as LPS signaling. For
example, MHR73-11 activates B cells, leading to increases in cell
size, expression of the costimulatory molecule CD80 and DNA
synthesis. Further, ligation of CD180 protects B cells from
irradiation- and dexamethasone-induced apoptosis. CD180 is also
believed to potentially play a role in Systemic Lupus Erythematosus
(SLE).
[0071] The present invention provides anti-CD180 antibodies,
preferably monoclonal antibodies, that can specifically bind to
CD180 antigen, e.g., CD180 polypeptide exposed on the surface of
HTCs of myeloid origin. As discussed in more detail below, the
anti-CD180 antibodies preferably bind such myelogenous HTCs,
thereby inhibiting their proliferation and/or mediating their
destruction. One of skill in the art will further recognize that
antibodies known in the art can find use in the methods disclosed
in the instant invention. For example, one or more anti-CD180
antibodies commercially available from Abcam, AbD Serotec, AnaSpec,
Atlas Antibodies, Biolegend, BDBiosciences, Cell Sciences,
eBioscience, GeneTex, Lifespan Biosciences, Raybiotech and ProSci
may also find use with respect to diagnostic and/or therapeutic
applications taught herein.
[0072] HAVCR2, also known as TIM3, KIM-3, TIMD3, and FLJ14428, is a
T helper cell type 1-specific cell surface protein that regulates
macrophage activation and enhances the severity of experimental
autoimmune encephalomyelitis in mice. The present invention
provides anti-HAVCR2 antibodies, preferably monoclonal antibodies,
that can specifically bind to HAVCR2 antigen, e.g., HAVCR2
polypeptide exposed on the surface of HTCs of myeloid origin. As
discussed in more detail below, the anti-HAVCR2 antibodies
preferably bind such myelogenous HTCs, thereby inhibiting their
proliferation and/or mediating their destruction. One of skill in
the art will further recognize that antibodies known in the art can
find use in the methods disclosed in the instant invention. For
example, one or more anti-HAVCR2 antibodies commercially available
from IMGENIX, Novus Biologicals, Lifespan Biosciences, and Atlas
Antibodies, may also find use with respect to diagnostic and/or
therapeutic applications taught herein.
[0073] LILRA1 is also known as CD125, CDw125, HSIL5R3, and
MGC26560. The present invention provides anti-LILRA1 antibodies,
preferably monoclonal antibodies, that can specifically bind to
LILRA1 antigen, e.g., LILRA1 polypeptide exposed on the surface of
HTCs of myeloid origin. As discussed in more detail below, the
anti-LILRA1 antibodies preferably bind such myelogenous HTCs,
thereby inhibiting their proliferation and/or mediating their
destruction. One of skill in the art will further recognize that
antibodies known in the art can find use in the methods disclosed
in the instant invention. For example, one or more anti-LILRA1
antibodies commercially available from LifeSpan BioSciences may
also find use with respect to diagnostic and/or therapeutic
applications taught herein.
[0074] LILRA2, also known as ILT1, LIR7, CD85H, and LIR-7, is
expressed predominantly on monocytes and B cells and at lower
levels on dendritic cells and natural killer (NK) cells. Many, if
not all, leukocyte immunoglobulin-like receptors in subfamily A
lack an immunoreceptor tyrosine-based inhibitory motif (ITIM) and
may initiate stimulatory cascades.
[0075] The present invention provides anti-LILRA2 antibodies,
preferably monoclonal antibodies, that can specifically bind to
LILRA2 antigen, e.g., LILRA2 polypeptide exposed on the surface of
HTCs of myeloid origin. As discussed in more detail below, the
anti-LILRA2 antibodies preferably bind such myelogenous HTCs,
thereby inhibiting their proliferation and/or mediating their
destruction. One of skill in the art will further recognize that
antibodies known in the art can find use in the methods disclosed
in the instant invention. For example, one or more anti-LILRA2
antibodies commercially available from eBioscience and Novus
Biologicals may also find use with respect to diagnostic and/or
therapeutic applications taught herein.
[0076] NEGR1, also known as Ntra, KILON, IGLON4, DMML2433, and
MGC46680. The present invention provides anti-NEGR1 antibodies,
preferably monoclonal antibodies, that can specifically bind to
NEGR1 antigen, e.g., NEGR1 polypeptide exposed on the surface of
HTCs of myeloid origin. As discussed in more detail below, the
anti-CD180 antibodies preferably bind such myelogenous HTCs,
thereby inhibiting their proliferation and/or mediating their
destruction. One of skill in the art will further recognize that
antibodies known in the art can find use in the methods disclosed
in the instant invention. For example, one or more anti-NEGR1
antibodies commercially available from AbNova and Novus Biologicals
may also find use with respect to diagnostic and/or therapeutic
applications taught herein.
[0077] TLR2, also known TIL4 and CD282, is a member of the
toll-like receptor (TLR) family. It is expressed most abundantly in
peripheral blood leukocytes and mediates host responses to
gram-positive bacteria and yeast via NF-kappaB. The present
invention provides anti-TLR2 antibodies, preferably monoclonal
antibodies, that can specifically bind to TLR2 antigen, e.g., TLR2
polypeptide exposed on the surface of HTCs of myeloid origin. As
discussed in more detail below, the anti-TLR2 antibodies preferably
bind such myelogenous HTCs, thereby inhibiting their proliferation
and/or mediating their destruction. One of skill in the art will
further recognize that antibodies known in the art can find use in
the methods disclosed in the instant invention. For example, one or
more anti-TLR2 antibodies commercially available from AbCam,
Abnova, ABR-Affinity Bioreagents/Thermo Scientific BioLegend,
LifeSpan BioSciences and Novus Biologicals may also find use with
respect to diagnostic and/or therapeutic applications taught
herein. TLR2 agonists include many microbial molecules
(lipoglycans, lipopolysaccharide, lipoteichoic acids,
peptidoglycans, zymosan) and synthetic lipoproteins.
[0078] Ly86, also known as LY86, DJ80N2.1, MD-1, MD1 and MMD-1, is
a secreted product highly expressed by B cells and monocytes.
Secreted Ly86 forms a complex with CD180 (discussed above), as well
as with TLR4, thereby becoming membrane bound. Such binding
generally mediates LPS signaling.
[0079] The present invention provides anti-Ly86 antibodies,
preferably monoclonal antibodies, that can specifically bind to
Ly86 antigen, e.g., Ly86 polypeptide existing on the surface of
myelogenous HTCs by virtue of binding to CD180 (and TLR4). As
discussed in more detail below, the anti-Ly86 antibodies preferably
bind such myelogenous HTCs, thereby inhibiting their proliferation
and/or mediating their destruction. One of skill in the art will
further recognize that antibodies known in the art can find use in
the methods disclosed in the instant invention. For example, one or
more anti-Ly86 antibodies commercially available from Abcam,
Abgent, Abnova Corporation, ABR-Affinity BioReagents, GeneTex,
Lifespan Biosciences, Novus Biologicals and R&D Systems may
also find use with respect to diagnostic and/or therapeutic
applications taught herein.
[0080] Monoclonal antibodies of the instant disclosure specifically
bind myelogenous HTCs by virtue of specific binding to its target
antigen. In preferred embodiments, the monoclonal antibody (or a
derivative thereof) is specifically immunoreactive with cells of
myeloid origin the hematopoietic system, such as
granulocyte/macrophage progenitors (GMP), KG-1a, K-562, Jurkat, CML
blasts, and AML blasts. For example, in some such embodiments,
specific binding to HTCs, by virtue of a member of the Ig
superfamily or TLR superfamily disclosed herein, mediates
destruction of hematopoietic tumor cells of myeloid origin.
[0081] Antibodies can be produced readily by one skilled in the
art. The general methodology for making monoclonal antibodies by
hybridomas is now well known to the art. See, e.g., M. Schreier et
al., Hybridoma Techniques (Cold Spring Harbor Laboratory);
Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas
(Elsevier Biomedical Press). As described above, the present
disclosure provides methods of producing the monoclonal antibodies
or derivatives thereof. In some embodiments, these methods comprise
cultivating a hybridoma cell under suitable conditions, wherein the
antibody is produced and obtaining the antibody and/or derivative
thereof from the cell and/or from the cell culture medium. A
specific example of making the monoclonal antibodies of the instant
invention is also provided in the Examples below.
[0082] The antibodies can be purified by methods known to the
skilled artisan. Purification methods include, among others,
selective precipitation, liquid chromatography, HPLC,
electrophoresis, chromatofocusing, and various affinity techniques.
Selective precipitation may use ammonium sulfate, ethanol (Cohn
precipitation), polyethylene glycol, or others available in the
art. Liquid chromatography mediums, include, among others, ion
exchange medium DEAE, polyaspartate), hydroxylapatite, size
exclusion (e.g., those based on crosslinked agarose, acrylamide,
dextran, etc.), hydrophobic matrixes (e.g., Blue Sepharose).
Affinity techniques typically rely on proteins that interact with
the immunoglobulin Fc domain. Protein A from Staphylococcus aureas
can be used to purify antibodies that are based on human .gamma.1,
.gamma.2, or .gamma.4 heavy chains (Lindmark et al., J. Immunol.
Meth. 62:1-13 (1983)). Protein G from C and G streptococci is
useful for all mouse isotypes and for human. .gamma.3 (Guss et al.,
EMBO J. 5:15671575 (1986)). Protein L, a Peptostreptococcus magnus
cell-wall protein that binds immunoglobulins (Ig) through k
light-chain interactions (BD Bioscience/ClonTech. Palo Alto,
Calif.), is useful for affinity purification of Ig subclasses IgM,
IgA, IgD, IgG, IgE and IgY. Recombinant forms of these proteins are
also commercially available. If the antibody contains metal binding
residues, such as phage display antibodies constructed to contain
histidine tags, metal affinity chromatography may be used. When
sufficient amounts of specific cell populations are available,
antigen affinity matrices may be made with the cells to provide an
affinity method for purifying the antibodies.
[0083] The present invention provides the antibodies described
herein, as well as corresponding antibody fragments and
antigen-binding portions. The terms "antibody fragment" or
"antigen-binding portion" of an antibody (or simply "antibody
portion") of the present invention, as used herein, refers to one
or more fragments of an antibody that retain the ability to
specifically bind to an antigen. It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antibody fragment" or "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H,
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and
C.sub.H1 domains; (iv) a Fv fragment consisting of the V.sub.L and
V.sub.H domains of a single arm of an antibody, (v) a dAb fragment
(Ward et al., (1989) Nature 341:544-546), which consists of a
V.sub.H domain; and (vi) an isolated complementarity determining
region (CDR), and (vii) bispecific single chain Fv dimers
(PCT/US92/09965). Furthermore, although the two domains of the Fv
fragment, V.sub.L and V.sub.H, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in
which the V.sub.L and V.sub.H regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies. The antibody fragments may be modified. For
example, the molecules may be stabilized by the incorporation of
disulphide bridges linking the VH and VL domains (Reiter et al.,
1996, Nature Biotech. 14:1239-1245).
[0084] The present disclosure further provides fragments of the
antibodies disclosed herein. Immunoglobulin molecules can be
cleaved into fragments. The antigen binding region of the molecule
can be divided into either F(ab').sub.2 or Fab fragments. The
F(ab').sub.2 fragment is divalent and is useful when the Fc region
is either undesirable or not a required feature. The Fab fragment
is univalent and is useful when an antibody has a very high avidity
for its antigen. Eliminating the Fc region from the antibody
decreases non-specific binding between the Fc region and Fc
receptor bearing cells. To generate Fab or F(ab).sub.2 fragments,
the antibodies are digested with an enzyme. Proteases that cleave
at the hinge region of an immunoglobulin molecule preserve the
disulfide bond(s) linking the F(ab) domain such that they remain
together following cleavage. A suitable protease for this purpose
is pepsin. For producing F(ab) fragments, proteases are chosen such
that cleavage occurs above the hinge region containing the
disulfide bonds that join the heavy chains but which leaves intact
the disulfide bond linking the heavy and light chain. A suitable
protease for making F(ab) fragments is papain. The fragments are
purified by the methods described above, with the exception of
affinity techniques requiring the intact Fc region (e.g., Protein A
affinity chromatography).
[0085] Antibody fragments can be produced by limited proteolysis of
antibodies and are called proteolytic antibody fragments. These
include, but are not limited to, the following: F(ab').sub.2
fragments, Fab' fragments, Fab'-SH fragments, and Fab fragments.
"F(ab').sub.2 fragments" are released from an antibody by limited
exposure of the antibody to a proteolytic enzyme, e.g., pepsin or
ficin. A F(ab').sub.2 fragment comprises two "arms," each of which
comprises a variable region that is directed to and specifically
binds a common antigen. The two Fab' molecules are joined by
interchain disulfide bonds in the hinge regions of the heavy
chains; the Fab' molecules may be directed toward the same
(bivalent) or different (bispecific) epitopes. "Fab' fragments"
contain a single anti-binding domain comprising a Fab and an
additional portion of the heavy chain through the hinge region.
"Fab'-SH fragments" are typically produced from F(ab').sub.2
fragments, which are held together by disulfide bond(s) between the
H chains in an F(ab').sub.2 fragment. Treatment with a mild
reducing agent such as, by way of non-limiting example,
beta-mercaptoethylamine, breaks the disulfide bond(s), and two Fab'
fragments are released from one F(ab').sub.2 fragment. Fab'-SH
fragments are monovalent and monospecific. "Fab fragments" (i.e.,
an antibody fragment that contains the antigen-binding domain and
comprises a light chain and part of a heavy chain bridged by a
disulfide bond) are produced by papain digestion of intact
antibodies. A convenient method is to use papain immobilized on a
resin so that the enzyme can be easily removed and the digestion
terminated. Fab fragments do not have the disulfide bond(s) between
the H chains present in a F(ab').sub.2 fragment.
[0086] "Single-chain antibodies" are one type of antibody fragment.
The term single chain antibody is often abbreviated as "scFv" or
"sFv." These antibody fragments are produced using molecular
genetics and recombinant DNA technology. A single-chain antibody
consists of a polypeptide chain that comprises both a V.sub.H and a
V.sub.L domains which interact to form an antigen-binding site. The
V.sub.H and V.sub.L domains are usually linked by a peptide of 10
to 25 amino acid residues.
[0087] The term "single-chain antibody" further includes but is not
limited to a disulfide-linked Fv (dsFv) in which two single-chain
antibodies (each of which may be directed to a different epitope)
linked together by a disulfide bond; a bispecific sFv in which two
discrete scFvs of different specificity is connected with a peptide
linker; a diabody (a dimerized sFv formed when the V.sub.H domain
of a first sFv assembles with the V.sub.L domain of a second sFv
and the V.sub.L domain of the first sFv assembles with the V.sub.H
domain of the second sFv; the two antigen-binding regions of the
diabody may be directed towards the same or different epitopes);
and a triabody (a trimerized sFv, formed in a manner similar to a
diabody, but in which three antigen-binding domains are created in
a single complex; the three antigen binding domains may be directed
towards the same or different epitopes).
[0088] "Complementary determining region peptides" or "CDR
peptides" are another form of an antibody fragment. A CDR peptide
(also known as "minimal recognition unit") is a peptide
corresponding to a single complementarity-determining region (CDR),
and can be prepared by constructing genes encoding the CDR of an
antibody of interest. Such genes are prepared, for example, by
using the polymerase chain reaction to synthesize the variable
region from RNA of antibody-producing cells. See, for example,
Larrick et al., Methods: A Companion to Methods in Enzymology
2:106, 1991.
[0089] In "cysteine-modified antibodies," a cysteine amino acid is
inserted or substituted on the surface of antibody by genetic
manipulation and used to conjugate the antibody to another molecule
via, e.g., a disulfide bridge. Cysteine substitutions or insertions
for antibodies have been described (see U.S. Pat. No. 5,219,996).
Methods for introducing Cys residues into the constant region of
the IgG antibodies for use in site-specific conjugation of
antibodies are described by Stimmel et al. (J. Biol. Chem.
275:330445-30450, 2000).
[0090] The present disclosure further provides humanized and
non-humanized antibodies. Humanized forms of non-human (e.g.,
mouse) antibodies are chimeric antibodies that contain minimal
sequence derived from non-human immunoglobulin. Generally,
humanized antibodies are non-human antibodies that have had the
variable-domain framework regions swapped for sequences found in
human antibodies. The humanized antibodies may be human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or nonhuman primate having the desired
specificity, affinity, and capacity. In some instances, framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, humanized antibodies
may comprise residues that are not found in the recipient antibody
or in the donor antibody. These modifications are made to further
refine antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of
a human immunoglobulin sequence. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin.
[0091] Generally, in a humanized antibody, the entire antibody,
except the CDRs, is encoded by a polynucleotide of human origin or
is identical to such an antibody except within its CDRs. The CDRs,
some or all of which are encoded by nucleic acids originating in a
non-human organism, are grafted into the beta-sheet framework of a
human antibody variable region to create an antibody, the
specificity of which is determined by the engrafted CDRs. The
creation of such antibodies is described in, e.g., WO 92/11018,
Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science
239:1534-1536. Humanized antibodies can also be generated using
mice with a genetically engineered immune system. Roque et al.,
2004, Biotechnol. Prog. 20:639-654.
[0092] The present disclosure further provides humanized and
non-humanized antibodies. Humanized forms of non-human (e.g.,
mouse) antibodies are chimeric antibodies that contain minimal
sequence derived from non-human immunoglobulin. Generally,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a hypervariable region of the recipient are
replaced by residues from a hypervariable region of a non-human
species (donor antibody) such as mouse, rat, rabbit or nonhuman
primate having the desired specificity, affinity, and capacity. In
some instances, framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin.
[0093] It can be desirable to modify the antibodies of the
invention with respect to effector function, so as to enhance,
e.g., the effectiveness of the antibody in treating cancer. For
example, cysteine residue(s) can be introduced into the Fc region,
thereby allowing interchain disulfide bond formation in this
region. The homodimeric antibody thus generated can have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J.
Immunol., 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity can also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53:2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and can thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3:219-230 (1989).
[0094] In preferred embodiments, the antibodies described herein
specifically bind to an antigen corresponding to one or more of the
disclosed members of the Ig superfamily or TLR superfamily present
on the cell surface of HTCs that arose from progenitor cell
populations in the myeloid lineage of the hematopoietic system.
Differentiation in the myeloid lineage leads to formation of
terminally differentiated cells that include, among others,
megakaryocytes, erythroid cells, macrophages, basophils,
eosinophils, neutrophils and myeloid dendritic cells. These cells
originate from hematopoietic stem cells (HSC), which differentiate
through a series of progenitor cell populations displaying
progressively restricted differentiation potential. The HSCs and
the progenitor cell populations are identifiable from each other
based on, among other distinguishing characteristics, their
capacity to differentiate into specific cell subsets and the
presence of a particular set of cellular markers that is specific
to the cell population. In some embodiments, the monoclonal
antibodies of the present disclosure are directed to progenitor
cells of the myeloid lineage that are marker+ HTCs. In some
embodiments, the monoclonal antibodies in the present disclosure
are directed to committed myeloid progenitor cells that are marker+
HTCs.
5.2.1 Modified Antibodies
[0095] In another aspect, the present invention provides modified
antibodies that are derived from an antibody that specifically
binds an antigen corresponding to an HTC marker disclosed herein.
Modified antibodies also include recombinant antibodies as
described herein.
[0096] Numerous types of modified or recombinant antibodies will be
appreciated by those of skill in the art. Suitable types of
modified or recombinant antibodies include without limitation,
engineered murine monoclonal antibodies (e.g. murine monoclonal
antibodies, chimeric monoclonal antibodies, humanized monoclonal
antibodies), domain antibodies (e.g. Fab, Fv, V.sub.H, scFV, and
dsFv fragments), multivalent or multispecific antibodies (e.g.
diabodies, minibodies, miniantibodies, (scFV).sub.2, tribodies, and
tetrabodies), and antibody conjugates as described herein.
[0097] In one aspect, the present invention includes domain
antibodies. "Domain antibodies" are functional binding domains of
antibodies, corresponding to the variable regions of either the
heavy (VH) or light (VL) chains of human antibodies. Domain
antibodies may have a molecular weight of approximately 13 kDa, or
less than one-tenth the size of a full antibody. They are well
expressed in a variety of hosts including bacterial, yeast, and
mammalian cell systems. In addition, domain antibodies are highly
stable and retain activity even after being subjected to harsh
conditions, such as freeze-drying or heat denaturation. See, for
example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197;
US Serial No. 2004/0110941; European Patent 0368684; U.S. Pat. No.
6,696,245, WO04/058821, WO04/003019 and WO03/002609. In one
embodiment, the domain antibody of the present invention is a
single domain. Single domain antibodies may be prepared, for
example, as described in U.S. Pat. No. 6,248,516, incorporated
herein by reference in its entirety. In some embodiments, the
present invention provides domain antibodies derived from an
antibody that specifically binds to an antigen corresponding to one
of the members of the Ig superfamily or TLR superfamily disclosed
herein.
[0098] In another aspect, the present invention includes
multi-specific antibodies. Multi-specific antibodies include
bispecific, trispecific, etc. antibodies. Bispecific antibodies can
be produced via recombinant means, for example by using leucine
zipper moieties (i.e., from the Fos and Jun proteins, which
preferentially form heterodimers; Kostelny et al., 1992, J.
Immunol. 148:1547) or other lock and key interactive domain
structures as described in U.S. Pat. No. 5,582,996. Additional
useful techniques include those described in U.S. Pat. No.
5,959,083; and U.S. Pat. No. 5,807,706. In one embodiment, the
present invention provides multi-specific antibodies that include
an antibody that specifically binds an antigen corresponding to a
member of the Ig superfamily or TLR superfamily disclosed herein.
In another embodiment, the multispecific antibody is
bispecific.
[0099] Bispecific antibodies are also sometimes referred to as
"diabodies." These are antibodies that bind to two (or more)
different antigens. Also known in the art are triabodies (a
trimerized sFv, formed in a manner similar to a diabody, but in
which three antigen-binding domains are created in a single
complex; the three antigen binding domains may be directed towards
the same or different epitopes) or a tetrabodies (four
antigen-binding domains created in a single complex where the four
antigen binding domains may be directed towards the same or
different epitopes), and the like. Dia-, tria- and tetrabodies can
be manufactured in a variety of ways known in the art (Holliger and
Winter, 1993, Current Opinion Biotechnol. 4:446-449), e.g.,
prepared chemically or from hybrid hybridomas. In addition, such
antibodies and fragments thereof may be constructed by gene fusion
(Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804;
Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448,
each of which is incorporated herein by reference in their
entirety). In one embodiment, the diabody, triabody, or tetrabody
is derived from an antibody that specifically binds an antigen
corresponding to a member of the Ig superfamily or TLR superfamily
disclosed herein. In a preferred embodiment, the diabody, triabody,
or tetrabody is derived from two or more monoclonal antibodies that
specifically bind to different antigens, each antigen corresponding
to different members of the Ig superfamily or TLR superfamily
disclosed herein.
[0100] In another embodiment, the present invention provides
minibodies, which are minimized antibody-like proteins that include
a scFV joined to a CH3 domain, that are derived from an antibody
that specifically binds an antigen corresponding to a member of the
Ig superfamily or TLR superfamily disclosed herein. Minibodies can
be made as described in the art (Hu et al., 1996, Cancer Res.
56:3055-3061).
[0101] In another embodiment, the present invention provides
binding domain-immunglobulin fusion proteins, where the binding
domain specifically binds an antigen corresponding to a member of
the Ig superfamily or TLR superfamily disclosed herein. The fusion
protein may include a marker specific binding domain polypeptide
fused to an immunoglobulin hinge region polypeptide, which is fused
to an immunoglobulin heavy chain CH2 constant region polypeptide
fused to an immunoglobulin heavy chain CH3 constant region
polypeptide. Under the present invention, marker specific antibody
fusion proteins can be made by methods appreciated by those of
skill in the art (See published U.S. Patent Application Nos.
20050238646, 20050202534, 20050202028, 2005020023, 2005020212,
200501866216, 20050180970, and 20050175614, each of which is
incorporated herein by reference in their entirety).
[0102] In another embodiment, the present invention provides a
heavy-chain protein (V.sub.HH) derived from a marker specific
antibody. Naturally-occurring heavy chain antibodies (e.g.
camelidae antibodies having no light chains) have been utlitized to
develop antibody-derived therapeutic proteins that typically retain
the structure and functional properties of naturally-occurring
heavy-chain antibodies. They are known in the art as
Nanobodies.RTM.. Under the present invention, heavy chain proteins
(V.sub.HH) derived from a marker specific heavy chain antibody may
be made by methods appreciated by those of skill in the art (See
published U.S. Patent Application Nos. 20060246477, 20060211088,
20060149041, 20060115470, and 20050214857, each of which is
incorporated herein by reference in their entirety).
[0103] In one aspect, the present invention provides a modified
antibody that is a human antibody. In one embodiment, the marker
specific antibodies described herein are fully human antibodies.
"Fully human antibody" or "complete human antibody" refers to a
human antibody having only the gene sequence of an antibody derived
from a human chromosome. The anti-human marker specific complete
human antibody can be obtained by a method using a human
antibody-producing mouse having a human chromosome fragment
containing the genes for a heavy chain and light chain of a human
antibody [see Tomizuka, K. et al., Nature Genetics, 16, p. 133-143,
1997; Kuroiwa, Y. et al., Nuc. Acids Res., 26, p. 3447-3448, 1998;
Yoshida, H. et al., Animal Cell Technology: Basic and Applied
Aspects vol. 10, p. 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S.
eds.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc.
Natl. Acad. Sci. USA, 97, 722-727, 2000] or obtained by a method
for obtaining a human antibody derived from a phage display
selected from a human antibody library (see Wormstone, I. M. et
al., Investigative Ophthalmology & Visual Science. 43(7), p.
2301-8, 2002; Carmen, S. et al., Briefings in Functional Genomics
and Proteomics, 1 (2), p. 189-203, 2002; Siriwardena, D. et al.,
Ophthalmology, 109(3), p. 427-431, 2002).
[0104] In one aspect, the present invention provides a marker
specific antibody that is an antibody analog, sometimes referred to
as "synthetic antibodies." For example, a variety of recent work
utilizes either alternative protein scaffolds or artificial
scaffolds with grafted CDRs. Such scaffolds include, but are not
limited to, mutations introduced to stabilize the three-dimensional
structure of the binding protein as well as wholly synthetic
scaffolds consisting for example of biocompatible polymers. See,
for example, Korndorfer et al., 2003, Proteins: Structure,
Function, and Bioinformatics, Volume 53, Issue 1:121-129. Roque et
al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptide
antibody mimetics ("PAMs") can be used, as well as work based on
antibody mimetics utilizing fibronection components as a
scaffold.
5.2.2 Cross-Linked Antibodies
[0105] In one aspect, the present invention provides cross-linked
antibodies that include two or more antibodies described herein
attached to each other to form antibody complexes. Cross-linked
antibodies are also referred to as antibody multimers,
homoconjugates, and heteroconjugates. It has been observed in the
art that the multimerization of an antibody previously observed to
have no signalling activity can result in a multimerized antibody
with potent signalling activity. This has been particularly noted
in the field of anti-tumor agents. For example, it has been
reported that the IgG-IgG homodimerization of anti-CD19, anti-CD20,
anti-CD21, anti-CD22, and anti-Her-2 monoclonal antibodies confers
potent anti-tumor ability to such homodimers (Ghetie, M. et al.
(1997) Proc. Natl. Acad. Sci., USA, Vol. 94, pp-7509-7514
incorporated herein by reference in its entirety). In addition, the
homodimerization of monoclonal antibodies known to have anti-tumor
activity, such as Rituximab.RTM., can lead to an increase in
effectiveness as an anti-tumor agent (Ghetie, M. (2001) Blood, Vo.
97; 5: 1392-1398 incorporated herein by reference in its
entirety).
[0106] In some embodiments, the antibody complexes provided herein
include multimeric forms of antibodies directed to one or more of
the antigens corresponding to one or more of the members of the Ig
superfamily or TSR superfamily disclosed herein. For example,
antibodies complexes of the invention may take the form of antibody
dimers, trimers, or higher-order multimers of monomeric
immunoglobulin molecules. Crosslinking of antibodies can be done
through various methods know in the art. For example, crosslinking
of antibodies may be accomplished through natural aggregation of
antibodies, through chemical or recombinant linking techniques or
other methods known in the art. For example, purified antibody
preparations can spontaneously form protein aggregates containing
antibody homodimers, and other higher-order antibody multimers. In
one embodiment, the present invention provides homodimerized
antibodies that specifically bind to an antigen corresponding to an
HTC marker disclosed herein.
[0107] Antibodies can be cross-linked or dimerized through linkage
techniques known in the art (see Ghetie et al. (1997) supra; Ghetie
et al. (2001) supra). Non-covalent methods of attachment may be
utilized. In a specific embodiment, crosslinking of antibodies can
be achieved through the use of a secondary crosslinker antibody.
The crosslinker antibody can be derived from a different animal
compared to the antibody of interest. For example, a goat
anti-mouse antibody (Fab specific) may be added to a mouse
monoclonal antibody to form a heterodimer. This bivalent
crosslinker antibody recognizes the Fab or Fc region of the two
antibodies of interest forming a homodimer.
[0108] In one embodiment of the present invention, an antibody that
specifically binds to an antigen corresponding to a member of the
Ig superfamily or TLR superfamily disclosed herein is cross-linked
using a goat anti-mouse antibody (GAM). In another embodiment, the
GAM crosslinker recognizes the Fab or Fc region of two antibodies,
each of which specifically binds the same or two different antigens
corresponding to the same or different members of the Ig
superfamily or TLR superfamily disclosed herein.
[0109] Methods for covalent or chemical attachment of antibodies
may also be utilized. Chemical crosslinkers can be homo or
heterobifunctional and will covalently bind with two antibodies
forming a homodimer. Cross-linking agents are well known in the
art; for example, homo- or hetero-bifunctional linkers as are well
known (see the 2006 Pierce Chemical Company Crosslinking Reagents
Technical Handbook; Hermanson, G. T., Bioconjugate Techniques,
Academic Press, San Diego, Calif. (1996); Aslam M. and Dent A H.,
Bioconjugation: protein coupling techniques for the biomedical
sciences, Houndsmills, England: Macmillan Publishers (1999);
Pierce: Applications Handbook & Catalog, Perbio Science,
Ermbodegem, Belgium (2003-2004); Haughland, R. P., Handbook of
Fluorescent Probes and Research Chemicals Eugene, 9.sup.th Ed.,
Molecular Probes, Oreg. (2003); and U.S. Pat. No. 5,747,641; all
references incorporated herein by reference) Those of skill in the
art will appreciate the suitability of various functional groups on
the amino acid(s) of an antibody for modification, including
cross-linking. Suitable examples of chemical crosslinkers used for
antibody crosslinking include, but not limited to, SMCC
[succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate], SATA
[N-succinimidyl S-acethylthio-acetate], hemi-succinate esters of
N-hydroxysuccinimide; sulfo-N-hydroxy-succinimide;
hydroxybenzotriazole, and p-nitrophenol; dicyclohexylcarbodiimide
(DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ECD), and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI)
(see, e.g., U.S. Pat. No. 4,526,714, the disclosure of which is
fully incorporated by reference herein). Other linking reagents
include glutathione,
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),
onium salt-based coupling reagents, polyoxyethylene-based
heterobifunctional cross-linking reagents, and other reagents
(Haitao, et al., Organ Lett 1:91-94 (1999); Albericio et al., J
Organic Chemistry 63:9678-9683 (1998); Arpicco et al., Bioconjugate
Chem. 8:327-337 (1997); Frisch et al., Bioconjugate Chem. 7:180-186
(1996); Deguchi et al., Bioconjugate Chem. 10:32-37 (1998); Beyer
et al., J. Med. Chem. 41:2701-2708 (1998); Drouillat et al., J.
Pharm. Sci. 87:25-30 (1998); Trimble et al., Bioconjugate Chem.
8:416-423 (1997)).
[0110] Exemplary protocols for the formation of antibody homodimers
is given in U.S. Patent Publication 20060062786, and Ghetie et al.,
(1997) supra, which are hereby incorporated by reference in their
entirety. In a preferred embodiment, the chemical cross-linker used
is an SMCC or SATA crosslinker.
[0111] In addition, the antibody-antibody conjugates of this
invention can be covalently bound to each other by techniques known
in the art such as the use of the heterobifunctional cross-linking
reagents, GMBS (maleimidobutryloxy succinimide), and SPDP
(N-succinimidyl 3-(2-pyridyldithio)propionate) [see, e.g., Hardy,
"Purification And Coupling Of Fluorescent Proteins For Use In Flow
Cytometry", Handbook Of Experimental Immunology, Volume 1,
Immunochemistry, Weir et al. (eds.), pp. 31.4-31.12 4th Ed.,
(1986), and Ledbetter et al. U.S. Pat. No. 6,010,902, each of which
is incorporated herein by reference in their entirety].
[0112] In addition, antibodies may be linked via a thioether
cross-link as described in U.S. Patent Publication 20060216284,
U.S. Pat. No. 6,368,596, which is incorporated herein by reference.
As will be appreciated by those skilled in the art, antibodies can
be crosslinked at the Fab region. In some embodiments, it is
desirable that the chemical crosslinker not interact with the
antigen-binding region of the antibody as this may affect antibody
function.
5.2.3 Conjugated Antibodies
[0113] The antibodies disclosed herein can be conjugated to
inorganic or organic compounds, including, by way of example and
not limitation, other proteins, nucleic acids, carbohydrates,
steroids, and lipids (see for example Green, et al., Cancer
Treatment Reviews, 26:269-286 (2000). The compound may be
bioactive. Bioactive refers to a compound having a physiological
effect on the cell as compared to a cell not exposed to the
compound. A physiological effect is a change in a biological
process, including, by way of example and not limitation, DNA
replication and repair, recombination, transcription, translation,
secretion, membrane turnover, cell adhesion, signal transduction,
cell death, and the like. A bioactive compound includes
pharmaceutical compounds.
[0114] In one aspect, the antibodies are conjugated to or modified
to carry a detectable compound. Conjugating antibodies to
detectable enzymes, fluorochromes, or ligands provides a signal for
visualization or quantitation of the target antigen. Antibodies may
be labeled with various enzymes to provide highly specific probes
that both visualize the target and amplify the signal by acting on
a substrate to produce a colored or chemiluminescent product.
Horseradish peroxidase, alkaline phosphatase, glucose oxidase, and
.beta.-galactosidase are the commonly used enzymes for this
purpose. Fluorochromes, such as fluorescein isothiocyanate,
tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, and
Cy5, provide a colored reagent for visualization and detection.
Suitable fluorescent compounds are described in Haughland, R. P.,
Handbook of Fluorescent Probes and Research Chemicals Eugene, 9th
Ed., Molecular Probes, Oreg. (2003).
[0115] In another aspect, the conjugated compounds are chelating
ligands, or macrocyclic organic chelating compounds, particularly
metal chelating compounds used to image intracellular ion
concentrations or used as contrast agents for medical imaging
purposes. Chelating ligands are ligands that can bind with more
than one donor atom to the same central metal ion. Chelators or
their complexes have found applications as MRI contrast agents,
radiopharmaceutical applications, and luminescent probes.
Conjugates of chelating compounds useful for assessing
intracellular ion concentrations may be voltage sensitive dyes and
non-voltage sensitive dyes. Exemplary dye molecules for measuring
intracellular ion levels include, by way of example and not
limitation, Quin-2; Fluo-3; Fura-Red; Calcium Green; Calcium Orange
550 580; Calcium Crimson; Rhod-2 550 575; SPO; SPA; MQAE; Fura-2;
Mag-Fura-2; Mag-Fura-5; Di-4-ANEPPS; Di-8-ANEPPS; BCECF; SNAFL-1;
SBFI; and SBFI.
[0116] In another embodiment, the ligands are chelating ligands
that bind paramagnetic, superparamagnetic or ferromagnetic metals.
These are useful as contrast agents for medical imaging and for
delivery of radioactive metals to selected cells. Metal chelating
ligands, include, by way of example and not limitation,
diethylenetriaminepenta acetic acid (DTPA); diethylenetriaminepenta
acetic acid bis(methylamide); macrocyclic tetraamine
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA); and porphyrins (see, e.g., The Chemistry of Contrast Agents
in Medical Magnetic Resonance Imaging, Merbach A. E. and Toth E.,
Ed., Wiley Interscience (2001)). Paramagnetic metal ions, which are
detectable in their chelated form by magnetic resonance imaging,
include, for example, iron(III), gadolinium(III), manganese (II and
III), chromium(III), copper(II), dysprosium(III), terbium(III),
holmium (III), erbium (III), and europium (III). Paramagnetic metal
ions particularly useful as magnetic resonance imaging contrast
agents comprise iron(III) and gadolinium(III) metal complexes.
Other paramagnetic, superparamagnetic or ferromagnetic are well
known to those skilled in the art.
[0117] In another embodiment, the metal-chelate comprises a
radioactive metal. Radioactive metals may be used for diagnosis or
as therapy based on delivery of small doses of radiation to a
specific site in the body. Targeted metalloradiopharmaceuticals are
constructed by attaching the radioactive metal ion to a metal
chelating ligand, such as those used for magnetic imaging, and
delivering the chelate-complex to cells. An exemplary radioactive
metal chelate complex is DTPA (see, e.g., U.S. Pat. No.
6,010,679).
[0118] In a further aspect, the conjugated compounds are peptide
tags used for purposes of detection, particularly through the use
of antibodies directed against the peptide. Various tag
polypeptides and their respective antibodies are well known in the
art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol.
8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol.
5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D
(gD) tag and its antibody (Paborsky et al., Protein Engineering
3:547-553 (1990)). Other tag polypeptides include the Flag-peptide
(Hopp et al., BioTechnology 6:1204-1210 (1988)); the KT3 epitope
peptide (Martin et al., Science 255:192-194 (1992)); tubulin
epitope peptide (Skinner et al., J. Biol. Chem. 266:15163-15166
(1991)); and the 17 gene 10 protein peptide tag (Lutz-Freyermuth et
al., Proc. Natl. Acad. Sci. USA 87:6393-6397 (1990)).
[0119] In another embodiment, the conjugated compounds may comprise
toxins that cause cell death, or impair cell survival when
introduced into a cell. A suitable toxin is campylobacter toxin CDT
(Lara-Tejero, M., Science 290:354-57 (2000)). Expression of the
CdtB subunit, which has homology to nucleases, causes cell cycle
arrest and ultimately cell death. Another exemplary toxin is
diptheria toxin (and similar Pseudomonas exotoxin), which functions
by ADP ribosylating ef-2 (elongation factor 2) molecule in the cell
and preventing translation. Entry of the diptheria toxin A subunit
induces cell death in cells containing the toxin fragment. Other
useful toxins include cholera toxin and pertussis toxin (catalytic
subunit-A ADP ribosylates the G protein regulating adenylate
cyclase), pierisin from cabbage butterflys, an inducers of
apoptosis in mammalian cells (Watanabe, M., Proc. Natl. Acad. Sci.
USA 96:10608-13 (1999)), ribosome inactivating toxins (e.g., ricin
A chain, Gluck, A. et al., J. Mol. Biol. 226:411-24 (1992)); and
nigrin (Munoz, R. et al., Cancer Lett. 167: 163-69 (2001)).
[0120] Bioactive compounds suitable for delivery by the
compositions herein, include, among others, chemotherapeutic
compounds, including by way of example and not limitation,
vinblastin, bleomycin, taxol, cis-platin, adriamycin, and
mitomycin. Exemplary chemotherapeutic agents suitable for the
present purposes are compounds acting on DNA synthesis and
stability. For example, anti-neoplastic agents of the anthracyclin
class of compounds act by causing strand breaks in the DNA and are
used as standard therapy against cancer. Exemplary anti-neoplastic
agents of this class are daunorubicin and doxorubicin. Coupling of
these compounds to proteins, including antibodies, are described in
Langer, M. et al., J. Med. Chem. 44(9):1341-1348 (2001) and King,
H. D. et al., Bioconjug. Chem. 10:279-288 (1999)). By attaching or
linking the antineoplastic agents to the antibodies, the compounds
are delivered to HTCs of myeloid origin with a high degree of
specificity and promote killing of the targeted cells.
[0121] Other classes of antitumor agents are the enediyne family of
antibiotics, representative members of which include
calicheamicins, neocarzinostatin, esperamincins, dynemicins,
kedarcidin, and maduropeptin (see, e.g., Smith, A. L. and Nicolaou,
K. C., J. Med. Chem. 39:2103-2117 (1996)). Similar to doxorubicin
and daunorubicin, the antitumor activity of these agents resides in
their ability to create strand breaks in the cellular DNA.
Conjugates to antibodies have been used to deliver these molecules
into those tumor cells expressing antigens recognized by the
antibody and shown to have potent antitumor activity with reduced
unwanted toxicity as compared to the unconjugated compounds
(Hinman, L. M. et al., Cancer Res. 53:3336-3342 (1993)).
Conjugating the enediyne compounds to the compositions described
herein provides another method of targeting HTCs of myeloid
origin.
[0122] Radioactive compounds are useful as signals (e.g., tracers)
or used to provide a therapeutic effect by their delivery to a cell
targeted (e.g., in the form of radiopharmaceutical preparations)
and may be attached to the antibodies by methods described below.
Useful radioactive nuclides include, by way of example and not
limitation, .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.51Cr,
.sup.57Co .sup.59Fe, .sup.67Ga, .sup.82Rb, .sup.89Sr, .sup.99Tc,
.sup.111In, .sup.123I, .sup.125I, .sup.129I, .sup.131I,
.sup.186Re.
[0123] The conjugation of compounds to antibodies is well know to
the skilled artisan, and typically takes advantage of functional
groups present on or introduced onto the antibodies and compound.
Functional groups include, among others, hydroxyl, amino, thio,
imino, and carboxy moieties. Reaction between functional groups may
be aided by coupling reagents and crosslinking agents. Crosslinking
agents and linkers and corresponding methods for conjugation are
described in Hermanson, G. T., Bioconjugate Techniques, Academic
Press, San Diego, Calif. (1996); Aslam M. and Dent A H.,
Bioconjugation: protein coupling techniques for the biomedical
sciences, Houndsmills, England: Macmillan Publishers (1999);
Pierce: Applications Handbook & Catalog, Perbio Science,
Ermbodegem, Belgium (2003-2004); Haughland, R. P., Handbook of
Fluorescent Probes and Research Chemicals Eugene, 9.sup.th Ed.,
Molecular Probes, Oreg. (2003); and U.S. Pat. No. 5,747,641; all
references incorporated herein by reference. Exemplary coupling or
linking reagents include, by way of example and not limitation,
hemi-succinate esters of N-hydroxysuccinimide;
sulfo-N-hydroxy-succinimide; hydroxybenzotriazole, and
p-nitrophenol; dicyclohexylcarbodiimide (DCC),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ECD), and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI)
(see, e.g., U.S. Pat. No. 4,526,714) the disclosure of which is
fully incorporated by reference herein. Other linking reagents
include glutathione,
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),
onium salt-based coupling reagents, polyoxyethylene-based
heterobifunctional cross-linking reagents, and other reagents that
facilitate the coupling of antibodies to organic drugs and peptides
and other ligands (Haitao, et al., Organ Lett 1:91-94 (1999);
Albericio et al., J Organic Chemistry 63:9678-9683 (1998); Arpicco
et al., Bioconjugate Chem. 8:327-337 (1997); Frisch et al.,
Bioconjugate Chem. 7:180-186 (1996); Deguchi et al., Bioconjugate
Chem. 10:32-37 (1998); Beyer et al., J. Med. Chem. 41:2701-2708
(1998); Drouillat et al., J. Pharm. Sci. 87:25-30 (1998); Trimble
et al., Bioconjugate Chem. 8:416-423 (1997)).
[0124] Techniques for conjugating therapeutic compounds to
antibodies are also described in Amon et al., "Monoclonal
Antibodies for Immunotargeting of Drugs in Cancers Therapy," in
Monoclonal Antibodies and Cancer Therapy, Reisfeld et al., ed., pp
243-256, Alan R. Liss, Inc. (1985); Thorpe, et al. "The Preparation
and Cytotoxic Properties of Antibody Toxin Conjugates," Immunol.
Rev. 62:119-58 (1982); and Pietersz, G. A., "The linkage of
cytotoxic drugs to monoclonal antibodies for the treatment of
cancer," Bioconjugate Chemistry 1(2):89-95 (1990), all references
incorporated herein by reference.
6. Antisense Molecules and Small Molecule Compounds
[0125] Another aspect of the present invention relates to antisense
and sense molecules comprising a singe-stranded nucleic acid
sequence (either RNA or DNA) that can bind to mRNA (sense) or DNA
(antisense) target sequences corresponding to a member of the Ig
superfamily or TLR superfamily disclosed herein. Antisense or sense
oligonucleotides, according to the present invention, comprise a
fragment of the coding region of DNA of the gene corresponding to a
member of the Ig superfamily or TLR superfamily. Such a fragment
generally comprises at least about 14 nucleotides, preferably from
about 14 to about 30 nucleotides. Antisense or sense RNA or DNA
molecules are generally at least about 5 nucleotides in length,
alternatively at least about 15, at least about 30, at least about
50, at least about 100, at least about 150, at least about 200, at
least about 300, at least about 400, at least about 500, at least
about 700, at least about 800, or at least about 1000 nucleotides
in length. The ability to derive an antisense or a sense
oligonucleotide, based upon a cDNA sequence encoding a given
protein is described in, for example, Stein and Cohen (Cancer Res.
48:2659, 1988) and van der Krol et al. (BioTechniques 6:958,
1988).
[0126] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. Such methods are encompassed by the present invention. The
antisense oligonucleotides thus may be used to block the expression
of proteins corresponding to a member of the Ig superfamily or TLR
superfamily, wherein those marker proteins may play a role in the
induction and/or persistance of myeloid leukemias in mammals.
[0127] Preferred intragenic sites for antisense binding include the
region incorporating the translation initiation/start codon
(5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and
5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of
the gene. These regions refer to a portion of the mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation or
termination codon. Other preferred regions for antisense binding
include: introns; exons; intron-exon junctions; the open reading
frame (ORF) or "coding region," which is the region between the
translation initiation codon and the translation termination codon;
the 5' cap of an mRNA which comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage and includes 5' cap structure itself as well
as the first 50 nucleotides adjacent to the cap; the 5'
untranslated region (5'UTR), the portion of an mRNA in the 5'
direction from the translation initiation codon, and thus including
nucleotides between the 5' cap site and the translation initiation
codon of an mRNA or corresponding nucleotides on the gene; and the
3' untranslated region (3'UTR), the portion of an mRNA in the 3'
direction from the translation termination codon, and thus
including nucleotides between the translation termination codon and
3' end of an mRNA or corresponding nucleotides on the gene.
[0128] Antisense or sense oligonucleotides further comprise
oligonucleotides having modified sugar-phosphodiester backbones (or
other sugar linkages, such as those described in WO 91/06629) and
wherein such sugar linkages are resistant to endogenous nucleases.
Such oligonucleotides with resistant sugar linkages are stable in
vivo (i.e., capable of resisting enzymatic degradation) but retain
sequence specificity to be able to bind to target nucleotide
sequences. Oligonucleotides having modified backbones include those
that retain a phosphorus atom in the backbone and those that do not
have a phosphorus atom in the backbone. In other embodiments, both
the sugar and the internucleoside linkage, i.e., the backbone, of
the nucleotide units are replaced with different groups. A further
modification includes Locked Nucleic Acids (LNAs) in which the
2'-hdroxyl group is linked to the 3' or 4' carbon atom of the sugar
ring to form a bycyclic sugar moiety. The sense or antisense
oligonucleotide molecules may also include nucleobase (base)
modifications or substitutions.
[0129] The antisense and sense compounds used in accordance with
this invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
modified oligonucleotides. The compounds of the invention may also
be admixed, encapsulated, conjugated or otherwise associated with
other molecules, molecule structures or mixtures of compounds, as
for example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption.
[0130] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A. DCT5B and DCT5C (see WO 90/13641).
[0131] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell. Alternatively, a sense or an
antisense oligonucleotide may be introduced into a cell containing
the target nucleic acid sequence by formation of an
oligonucleotide-lipid complex, as described in WO 90/10448. The
sense or antisense oligonucleotide-lipid complex is preferably
dissociated within the cell by an endogenous lipase.
[0132] In another embodiment, the invention provides small
molecules, which bind, preferably specifically, to one or more
members of the Ig superfamily or TLR superfamily disclosed herein.
In preferred embodiments, the small molecule is a small organic
molecule, including small organic molecules known in the art as
being an agonist or antagonist of a polypeptide corresponding to a
member of the Ig superfamily or TLR superfamily disclosed
herein.
[0133] In some embodiments, the small molecule is conjugated to a
growth inhibitory agent or cytotoxic agent such as a toxin. Such
toxins include, for example, a maytansinoid or calicheamicin, an
antibiotic, a radioactive isotope, a nucleolytic enzyme, or the
like. The small molecules that find use in the therapeutic methods
of the instant invention preferably induce death of a cell to which
they bind. As detailed above, binding to HTCs of myeloid origin may
occur by virtue of binding to a surface-expressed marker disclosed
herein; or by virtue of binding to the surface-expressed marker,
which itself is bound to its receptor on myelogenous HTCs.
7. Pharmaceutical Compositions
[0134] In the preparation of pharmaceutical compositions comprising
the antibodies and/or antisense molecules and/or small molecule
agonists or antagonists described in the teachings herein, a
variety of vehicles and excipients and routes of administration may
be used, as will be apparent to the skilled artisan. Representative
formulation technology is taught in, inter alia, Remington: The
Science and Practice of Pharmacy, 19th Ed., Mack Publishing Co.,
Easton, Pa. (1995) and Handbook of Pharmaceutical Excipients, 3rd
Ed, Kibbe, A. H. ed., Washington D.C., American Pharmaceutical
Association (2000); hereby incorporated by reference in their
entirety.
[0135] The pharmaceutical compositions will generally comprise a
pharmaceutically acceptable carrier and a pharmacologically
effective amount of the antibodies, or mixture of antibodies, or
suitable salts thereof. Use of a mixture of monoclonal antibodies
specific to a progenitor cell population as a therapeutic has a
number of advantages. Abnormally proliferating cells have a
tendency to mutate, and thus may lose the antigen recognized by a
single type of monoclonal antibody. Moreover, antigen density of a
single target antigen in the targeted cell could be low such that
there is insufficient triggering of the signals necessary for
destruction of the cell by the immune system. The present
disclosure addresses these issues by providing multiple members of
the immunoglobulin (Ig) superfamily or toll-like receptor (TLR)
superfamily associated with HTCs of myeloid origin that can be
targeted by a mixture of different monoclonal antibodies.
[0136] For known small molecule agonists or antagonists,
pharmaceutical compositions can similarly be prepared based on
known characteristics of the molecules.
[0137] The pharmaceutical composition may be formulated as powders,
granules, solutions, suspensions, aerosols, solids, pills, tablets,
capsules, gels, topical cremes, suppositories, transdermal patches,
and other formulations known in the art.
[0138] For the purposes described herein, pharmaceutically
acceptable salts of the antibodies is intended to include any art
recognized pharmaceutically acceptable salts including organic and
inorganic acids and/or bases. Examples of salts include sodium,
potassium, lithium, ammonium, calcium, as well as primary,
secondary, and tertiary amines, esters of lower hydrocarbons, such
as methyl, ethyl, and propyl. Other salts include organic acids,
such as acetic acid, propionic acid, pyruvic acid, maleic acid,
succinic acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, salicylic acid, etc.
[0139] As used herein, "pharmaceutically acceptable carrier"
comprises any standard pharmaceutically accepted carriers known to
those of ordinary skill in the art in formulating pharmaceutical
compositions. Thus, the antibodies, by themselves, such as being
present as pharmaceutically acceptable salts, or as conjugates, may
be prepared as formulations in pharmaceutically acceptable
diluents; for example, saline, phosphate buffer saline (PBS),
aqueous ethanol, or solutions of glucose, mannitol, dextran,
propylene glycol, oils (e.g., vegetable oils, animal oils,
synthetic oils, etc.), microcrystalline cellulose, carboxymethyl
cellulose, hydroxylpropyl methyl cellulose, magnesium stearate,
calcium phosphate, gelatin, polysorbate 80 or the like, or as solid
formulations in appropriate excipients.
[0140] The pharmaceutical compositions will often further comprise
one or more buffers (e.g., neutral buffered saline or phosphate
buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or
dextrans), mannitol, proteins, polypeptides or amino acids such as
glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite,
butylated hydroxytoluene, butylated hydroxyanisole, etc.),
bacteriostats, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminium hydroxide), solutes that render the
formulation isotonic, hypotonic or weakly hypertonic with the blood
of a recipient, suspending agents, thickening agents and/or
preservatives. Alternatively, compositions of the present invention
may be formulated as a lyophilizate.
[0141] While any suitable carrier known to those of ordinary skill
in the art may be employed in the compositions of this invention,
the type of carrier will typically vary depending on the mode of
administration. Antibody compositions may be formulated for any
appropriate manner of administration, including for example, oral,
nasal, mucosal, intravenous, intraperitoneal, intradermal,
subcutaneous, and intramuscular administration.
[0142] For parenteral administration, the compositions can be
administered as injectable dosages of a solution or suspension of
the substance in a physiologically acceptable diluent with a
pharmaceutical carrier that can be a sterile liquid such as sterile
pyrogen free water, oils, saline, glycerol, polyethylene glycol or
ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents, surfactants, pH buffering substances and the
like can be present in compositions. Other components of
pharmaceutical compositions are those of petroleum, animal,
vegetable, or synthetic origin, for example, non-aqueous solutions
of peanut oil, soybean oil, corn oil, cottonseed oil, ethyl oleate,
and isopropyl myristate. Antibodies can be administered in the form
of a depot injection or implant preparation which can be formulated
in such a manner as to permit a sustained release of the active
ingredient. An exemplary composition comprises antibody at 5 mg/ml,
formulated in aqueous buffer consisting of 50 mM L-histidine, 150
mM NaCl, adjusted to pH 6.0 with HCl.
[0143] Typically, the compositions are prepared as injectables,
either as liquid solutions or suspensions; solid or powder forms
suitable for reconstitution with suitable vehicles, including by
way example and not limitation, sterile pyrogen free water, saline,
buffered solutions, dextrose solution, etc., prior to injection.
The preparation also can be emulsified or encapsulated in liposomes
or micro particles such as polylactide, polyglycolide, or
copolymers, as further discussed below (see, e.g., Langer, Science
249:1527 (1990) and Hanes, Advanced Drug Delivery Rev. 28:97-119
(1997)).
[0144] Additionally, the compositions may also be introduced or
encapsulated into the lumen of liposomes for delivery and for
extending their life time ex vivo or in vivo. As known in the art,
liposomes can be categorized into various types: multilamellar
(MLV), stable plurilamellar (SPLV), small unilamellar (SUV) or
large unilamellar (LUV) vesicles. Liposomes can be prepared from
various lipid compounds, which may be synthetic or naturally
occurring, including phosphatidyl ethers and esters, such as
phosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine,
phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such
as cholesterol; cerebrosides; sphingomyelin; glycerolipids; and
other lipids (see, e.g., U.S. Pat. No. 5,833,948).
[0145] Cationic lipids are also suitable for forming liposomes.
Generally, the cationic lipids have a net positive charge and have
a lipophilic portion, such as a sterol or an acyl or diacyl side
chain. Preferably, the head group is positively charged. Typical
cationic lipids include 1,2-dioleyloxy-3-(trimethylamino)propane;
N-[1-(2,3,-ditetradecycloxy)propyl]-N,N-dimethyl-N-N-hydroxyethylammonium
bromide; N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy
ethylammonium bromide; N-[1-(2,3-dioleyloxy)
propyl]-N,N,N-trimethylammonium chloride;
3-[N--(N',N'-dimethylaminoethane) carbamoyl] cholesterol; and
dimethyldioctadecylammonium.
[0146] Liposomes also include vesicles derivatized with a
hydrophilic polymer, as provided in U.S. Pat. Nos. 5,013,556 and
5,395,619, hereby incorporated by reference, (see also, Kona, K. et
al., J. Controlled Release 68: 225-35 (2000); Zalipsky, S. et al.,
Bioconjug. Chem. 6: 705-708 (1995)) to extend the circulation
lifetime in vivo. Hydrophilic polymers for coating or derivation of
the liposomes include polyethylene glycol, polyvinylpyrrolidone,
polyvinylmethyl ether, polyaspartamide, hydroxymethyl cellulose,
hydroxyethyl cellulose, and the like.
[0147] Liposomes are prepared by ways well known in the art (see,
e.g., Szoka, F. et al., Ann. Rev. Biophys. Bioeng. 9: 467-508
(1980)). One typical method is the lipid film hydration technique
in which lipid components are mixed in an organic solvent followed
by evaporation of the solvent to generate a lipid film. Hydration
of the film in aqueous buffer solution, preferably containing the
subject antibodies, results in an emulsion, which is sonicated or
extruded to reduce the size and polydispersity. Other methods
include reverse-phase evaporation (see, e.g., Pidgeon, C. et al.,
Biochemistry 26: 17-29 (1987); Duzgunes, N. et al., Biochim.
Biophys. Acta. 732: 289-99 (1983)), freezing and thawing of
phospholipid mixtures, and ether infusion.
[0148] In another embodiment, the carriers are in the form of
microparticles, microcapsules, micropheres and nanoparticles, which
may be biodegradable or non-biodegradable (see, e.g.,
"Microencapsulates: Methods and Industrial Applications," in Drugs
and Pharmaceutical Sciences, Benita, S. ed, Vol 73, Marcel Dekker
Inc., New York (1996); incorporated herein by reference). As used
herein, nnicroparticles, microspheres, microcapsules and
nanoparticles mean a particle, which is typically a solid,
containing the substance to be delivered. The substance is within
the core of the particle or attached to the particle's polymer
network. Generally, the difference between microparticles (or
microcapsules or microspheres) and nanoparticles is one of size. As
used herein, microparticles have a particle size range of about 1
to about >1000 microns. Nanoparticles have a particle size range
of about 10 to about 1000 nm.
[0149] A variety of materials are useful for making microparticles.
Non-biodegradable microcapsules and microparticles include, but not
limited to, those made of polysulfones, poly(acrylonitrile-co-vinyl
chloride), ethylene-vinyl acetate,
hydroxyethylmethacrylate-methyl-methacrylate copolymers. These are
useful for implantation purposes where the encapsulated composition
diffuses out from the capsules. In another aspect, the
microcapsules and microparticles are based on biodegradable
polymers, preferably those that display low toxicity and are well
tolerated by the immune system. These include protein based
microcapsulates and microparticles made from fibrin, casein, serum
albumin, collagen, gelatin, lecithin, chitosan, alginate or
poly-amino acids such as poly-lysine. Biodegradable synthetic
polymers for encapsulating may comprise polymers such as
polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)
(PLGA), poly(caprolactone), polydioxanone trimethylene carbonate,
polyhybroxyalkonates (e.g., poly(.beta.-hydroxybutyrate)),
poly(.gamma.-ethyl glutamate), poly(DTH iminocarbony (bisphenol A
iminocarbonate), poly (ortho ester), and polycyanoacrylate. Various
methods for making microparticles containing the subject
compositions are well known in the art, including solvent removal
process (see, e.g., U.S. Pat. No. 4,389,330); emulsification and
evaporation (Maysinger, D. et al., Exp. Neuro. 141: 47-56 (1996);
Jeffrey, H. et al., Pharm. Res. 10: 362-68 (1993)), spray drying,
and extrusion methods.
[0150] Another type of carrier is nanoparticles, which are
generally suitable for intravenous administrations. Submicron and
nanoparticles are generally made from amphiphilic diblock,
triblock, or multiblock copolymers as is known in the art. Polymers
useful in forming nanoparticles include, but are limited to,
poly(lactic acid) (PLA; see Zambaux et al., J. Control Release 60:
179-188 (1999)), poly(lactide-co-glycolide), blends of
poly(lactide-co-glycolide) and polycarprolactone, diblock polymer
poly(l-leucine-block-l-glutamate), diblock and triblock poly(lactic
acid) (PLA) and poly(ethylene oxide) (PEO) (De Jaeghere, F. et al.,
Pharm. Dov. Technol.; 5: 473-83 (2000)), acrylates, arylamides,
polystyrene, and the like. As described for microparticles,
nanoparticles may be non-biodegradable or biodegradable.
Nanoparticles may be also be made from poly(alkylcyanoacrylate),
for example poly(butylcyanoacrylate), in which the therapeutic
composition is absorbed onto the nanoparticles and coated with
surfactants (e.g., polysorbate 80). Methods for making
nanoparticles are similar to those for making microparticles and
include, among others, emulsion polymerization in continuous
aqueous phase, emulsification-evaporation, solvent displacement,
and emulsification-diffusion techniques (see, e.g., Kreuter, J.
Nano-particle Preparation and Applications, in Microcapsules and
Nanoparticles in Medicine and Pharmacy, pg. 125-148, (M. Donbrow,
ed.) CRC Press, Boca Rotan, Fla. (1991); incorporated herein by
reference).
[0151] The pharmaceutical compositions described herein may be
presented in unit-dose or multi-dose containers, such as sealed
ampoules or vials. Such containers are typically sealed in such a
way to preserve the sterility and stability of the formulation
until use. In general, formulations may be stored as suspensions,
solutions or emulsions in oily or aqueous vehicles, as indicated
above. Alternatively, a pharmaceutical composition may be stored in
a freeze-dried condition requiring only the addition of a sterile
liquid carrier immediately prior to use.
8. Use of Antibodies and Other Agents
8.1 Therapeutic Use of Antibodies and Small Molecules
[0152] Methods of immunotargeting cancer cells using antibodies or
antibody fragments are well known in the art. U.S. Pat. No.
6,306,393 describes the use of anti-CD22 antibodies in the
immunotherapy of B-cell malignancies, and U.S. Pat. No. 6,329,503
describes immunotargeting of cells that express serpentine
transmembrane antigens. Antibodies described herein (including
humanized or human monoclonal antibodies or fragments or other
modifications thereof, optionally conjugated to cytotoxic agents)
can be introduced into a patient such that the antibody binds to
cancer cells or their secreted expression products and mediates the
destruction of the cells and the tumor and/or inhibits the growth
of the cells or the tumor. Without intending to limit the
disclosure, mechanisms by which such antibodies can exert a
therapeutic effect may include complement-mediated cytolysis,
antibody-dependent cellular cytotoxicity (ADCC), modulating the
physiologic function of the tumor antigen, inhibiting binding or
signal transduction pathways, modulating tumor cell differentiation
or proliferation, altering tumor angiogenesis factor profiles,
modulating the secretion of immune stimulating or tumor suppressing
cytokines and growth factors, modulating cellular adhesion, and/or
by inducing apoptosis. The antibodies can also be conjugated to
toxic or other therapeutic agents, such as radioligands or
cytosolic toxins, discussed in detail above, and may also be used
therapeutically to deliver the toxic or therapeutic agent directly
to tumor cells.
[0153] In preferred embodiments, the compositions have applications
to the treatment of conditions or diseases involving myeloid cells
of the hematopoietic system. The present disclosure further
provides methods of using the antibodies to target myeloid leukemic
stem cells. For example, the disclosure provides methods of using
the antibodies to treat disorders involving cells of the myeloid
lineages. Various diseases have origins in the committed progenitor
cell populations, or involve progenitor cells by differentiation of
diseased cells through the myeloid pathway.
[0154] By "treatment" herein is meant therapeutic or prophylactic
treatment, or a suppressive measure for the disease, disorder or
undesirable condition. Treatment encompasses administration of the
subject antibodies in an appropriate form prior to the onset of
disease symptoms and/or after clinical manifestations, or other
manifestations, of the disease to reduce disease severity, halt
disease progression, or eliminate the disease. Prevention of the
disease includes prolonging or delaying the onset of symptoms of
the disorder or disease, preferably in a subject with increased
susceptibility to the disease.
[0155] The antibodies described herein are particularly applicable
to the treatment of myeloproliferative disorders, also referred to
generally as hematopoietic malignancies, which are proliferative
disorders involving cells of the myeloid lineage. The term
malignancy refers to growth and proliferation of one or more clones
of abnormal cells. Leukemia typically describes a condition in
which abnormal cells are present in the bone marrow and peripheral
blood. Myeloproliferative disorders, also called myeloid leukemias
or myelogenous leukemias, are categorized into three general groups
of conditions: dysmyelopoietic disorder, acute myeloproliferative
leukemia, and chronic myeloproliferative disorder.
[0156] Myelodysplastic Syndromes (MDS) include a group of
closely-related blood formation disorders, in which the bone marrow
shows qualitative and quantitative changes suggestive of a
preleukemic process, but having a chronic course that does not
necessarily terminate as acute leukemia. A variety of terms,
including preleukemia, refractory anemia, refractory
dysmyelopoietic anemia, smoldering or subacute leukemia,
dysmyelopoietic syndrome (DMPS), and myelodysplasia, have all been
used to describe MDS. These conditions are all characterized by a
cellular marrow with impaired maturation (dysmyelopoiesis) and a
reduction in the number of blood cells. DMPS is characterized by
presence of megablastoids, megarkaryocyte dysplasia, and an
increase in number of abnormal blast cells, reflective of enhanced
granulocyte maturation process. Patients with DMPS show chromosomal
abonormalities similar to those found in acute myeloid leukemia and
progress to acute myeloid leukemia in a certain fraction of
afflicted patients (Kardon, N. et al., Cancer 50(12):2834-2838
(1982)).
[0157] Acute myeloproliferative leukemia (AML), also known as acute
nonlymphocytic leukemia, acute myelocytic leukemia, acute
myeloblastic leukemia, and acute granulocytic leukemia, is
characterized by the presence of abnormal hematopoietic progenitor
cells that have been blocked at an undifferentiated or partially
differentiated stage of maturation, and thus are unable to
differentiate into myeloid, erythroid, and/or megakaryocytic cell
lines. The abnormal cells block differentiation of normal
progenitor cells in the bone marrow, resulting in thrombocytopenia,
anaemia, and granulocytopenia. Diagnosis of AML is made when at
least 30% of nucleated cells in the bone marrow are blasts. Acute
myeloid leukemia is further divided into subtypes M1 to M7 based on
morphology of the proliferating cells and cytochemical staining
properties.
[0158] Chronic myeloproliferative disorders are a collection of
conditions characterized by increased number of mature and immature
granulocytes, erythrocytes, and platelets. Chronic
myeloproliferative disorders can transition to other forms within
this group, with a tendency to terminate in acute myeloid leukemia.
Specific diseases within this group include polycythemia vera,
chronic myeloid leukemia, agnogenic myeloid leukemia, essential
thrombocythemia, and chronic neutrophilic leukemia.
[0159] The therapeutic preparations can use non-modified marker
specific antibodies or antibodies conjugated with a therapeutic
compound, such as a toxin or cytotoxic molecule, depending on the
functionality of the antibody. Generally, when non-modified
antibodies are used, they will typically have a functional Fc
region. By "functional Fc region" herein is meant a minimal
sequence for effecting the biological function of Fc, such as
binding to Fc receptors, particularly Fc.gamma.R (e.g.,
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII). Without being bound
by theory, it is believed that the Fc region may affect the
effectiveness of anti-tumor monoclonal antibodies by binding to Fc
receptors immune effector cells and modulating cell mediated
cytotoxicity, endocytosis, phagocytosis, release of inflammatory
cytokines, complement mediated cytotoxicity, and antigen
presentation. In this regard, polyclonal antibodies, or mixtures of
monoclonals, will be advantageous because they will bind to
different epitopes (of a single antigen or of different antigens
corresponding to different members of the Ig superfamily or TLR
superfamily) and thus have a higher density of Fc on the cell
surface as compared to when a single monoclonal antibody is used.
Of course, to enhance their effectiveness in depleting targeted
cells, or where non-modified antibodies are not therapeutically
effective, antibodies conjugated to toxins or cytotoxic agents may
be used. Thus, not only are the antibodies useful as therapeutic
molecules themselves, they also find utility in targeted delivery
of therapeutic molecules to myeloid cells.
[0160] Alternatively, where the antibodies exhibit a direct effect
on antigen and/or cell function, enhancement of the Fc receptor
functionality may be less significant. For example, this may be the
case where binding of the marker specific antibody sterically
inhibits interaction between antigen and its corresponding
ligand.
[0161] The antibody compositions may be used either alone or in
combination with other therapeutic agents to increase efficacy of
traditional treatments for myeloid leukemias, or to target abnormal
cells not targeted by the antibodies. Combining the antibody
therapy method with a chemotherapeutic, radiation or surgical
regimen may be preferred in patients that have not received
chemotherapeutic treatment, whereas treatment with the antibody
therapy may be indicated for patients who have received one or more
chemotherapies. Additionally, antibody therapy can also enable the
use of reduced dosages of concomitant chemotherapy, particularly in
patients that do not tolerate the toxicity of the chemotherapeutic
agent very well. Furthermore, antibody treatment of cancer patients
with tumors resistant to chemotherapeutic agents might induce
sensitivity and responsiveness to these agents in combination.
[0162] In one aspect, the antibodies are used adjunctively with
therapeutic cytotoxic agents, including, by way of example and not
limitation, busulfan, thioguanine, idarubicin, cytosine
arabinoside, 6-mercaptopurine, doxorubicin, daunorubicin,
etoposide, and hydroxyurea. Other agents useful as adjuncts to
antibody therapy are compounds directed specifically to an abnormal
cellular molecule found in the disease state. These agents can be
disease specific. For example, for treating chronic myeloid
leukemia arising from BCR-ABL activity, one class of useful
compounds are inhibitors of abl kinase activity, such as Imatinib,
an inhibitor of bcr-abl kinase, and antisense oligonucleotides
against bcr (e.g., Oblimersen). Other agents include, among others,
interferon-alpha, humanized anti-CD52, deacetylase inhibitor
FR901228 (depsipeptide), and the like.
[0163] In another aspect, isotopes are attached to the antibodies
and/or fragments for therapeutic purposes. By "isotope" is meant
atoms with the same number of protons and hence of the same element
but with different numbers of neutrons (e.g., .sup.1H vs. .sup.2H
or D). The term "isotope" includes "stable isotopes", e.g.
non-radioactive isotopes, as well as "radioactive isotopes", e.g.
those that decay over time, and radioactive radionuclides. In one
embodiment, the antibodies and/or fragments are labeled with a
radioisotope, which are useful in radioimmunotherapy. Suitable
radioisotopes include without limitation an alpha-emitter, a
beta-emitter, and an Auger electron-emitter (Behrt, T. et al.,
(2000). Eur. J. Nuclear Med. vol. 27 (7):753-765; Vallabhajosula,
S. et al., J. Nucl. Med. (2005) Apr; 46(4):634-41). Such
radioisotopes include without limitation [65]Zinc, [140]neodymium,
[177]lutetium, [179]lutetium, [176m]lutetium, [67]gallium,
[159]gallium, [161]terbium, [153]samarium, [169]erbium,
[175]ytterbium, [161]holmium, [166]holmium, [167]thulium,
[142]praseodymium, [143]praseodymium, [145]praseodymium,
[149]promethium, [150]europium, [165]dysprosium, [111]indium,
[131]iodine, [125]iodine, [123]iodine, [88]yttrium and [90]yttrium.
Suitable radioactive radionuclides include without limitation,
.sup.67Cu, .sup.90Y, .sup.125I, .sup.131I, .sup.186Re, .sup.188Re,
.sup.211At, .sup.212Bi. These and other uses of the antibodies will
be apparent to those of ordinary skill in the art in light of the
disclosures provided herein.
[0164] Known small molecule agonists or antagonists can also find
use in methods of treating of one or more of the myelogenous
hematological proliferative disorders described above. Generally,
the methods comprise administration of a therapeutically effective
amount of the small molecule (or mixture of small molecules).
Similarly as detailed above with respect to immunotherapies, the
therapeutic composition can use the non-modified small molecule or,
optionally, the small molecule is conjugated to a toxin or
cytotoxic molecule or growth inhibitory agent. For example, the
small molecule may be conjugated to a maytansinoid or
calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic
enzyme, or the like.
[0165] Binding of the small molecule to a HTC of myeloid origin
preferably induce cell death. Cell death may be mediated by the
conjugated cytotoxic molecule or by the physiological response
induced by the binding of the small molecule itself.
8.1.1. Administration and Dosages
[0166] The amount of the compositions needed for achieving a
therapeutic effect will be determined empirically in accordance
with conventional procedures for the particular purpose. Generally,
for administering the compositions ex vivo or in vivo for
therapeutic purposes, the compositions are given at a
pharmacologically effective dose. By "pharmacologically effective
amount" or "pharmacologically effective dose" is meant an amount
sufficient to produce the desired physiological effect or amount
capable of achieving the desired result, particularly for treating
or retreating the disorder or disease condition, including reducing
or eliminating one or more symptoms or manifestations of the
disorder or disease. As an illustration, administration of
antibodies to a patient suffering from a myeloproliferative
disorder provides a therapeutic benefit not only when the
underlying disease is eradicated or ameliorated, but also when the
there is a decrease in the severity or duration of the symptoms
associated with the disease. Therapeutic benefit also includes
halting or slowing the progression of the underlying disease or
disorder, regardless of whether improvement is realized.
[0167] The amount administered to the subject will vary depending
upon the agent being administered, the purpose of the
administration, such as prophylaxis or therapy, the state of the
subject, the manner of administration, the number of
administrations, the intervals between administrations, and the
like. These can be determined empirically by those skilled in the
art and may be adjusted based on the extent of the therapeutic
response. Factors to consider in determining an appropriate dose
include, but are not limited to, size and weight of the subject,
the age and sex of the subject, the severity of the symptoms, the
stage of the disease, method of delivery of the agents, half-life
of the agents, efficacy of the agents, and what other therapy
regimes have been or will be administered. Stage of the disease to
consider includes whether the disease is acute or chronic,
relapsing or remitting phase, as well as the progressiveness of the
disease. Determining the dosages and times of administration for a
therapeutically effective amount are well within the skill of the
ordinary person in the art, in light of the disclosures provided
herein.
[0168] For any compositions of the present disclosure, the
therapeutically effective dose is readily determined by methods
well known in the art. For example, an initial effective dose can
be estimated from cell culture or other in vitro assays. For
example, Sliwkowsky, M. X. et al., Semin. Oncol. 26(suppl. 12)
60-70 (1999) describes in vitro measurements of antibody dependent
cellular cytoxicity. A dose can then be formulated in animal models
to generate a circulating concentration or tissue concentration,
including that of the IC.sub.50 as determined by the cell culture
assays. A suitable animal model for leukemia is described in detail
in the Examples below.
[0169] In addition, the toxicity and therapeutic efficacy are
generally determined by cell culture assays and/or experimental
animals, typically by determining a LD.sub.50 (lethal dose to 50%
of the test population) and ED.sub.50 (therapeutically
effectiveness in 50% of the test population). The dose ratio of
toxicity and therapeutic effectiveness is the therapeutic index.
Preferred are compositions, individually or in combination,
exhibiting high therapeutic indices. Determination of the effective
amount is well within the skill of those in the art, particularly
given the detailed disclosure provided herein. Guidance is also
found in standard reference works, for example Fingl and Woodbury,
General Principles In: The Pharmaceutical Basis of Therapeutics pp.
1-46 (1975), and the references cited therein.
[0170] To achieve an initial tolerizing dose, consideration is
given to the possibility that the antibodies may be immunogenic in
humans and in non-human primates. The immune response may be
biologically significant and may impair the therapeutic efficacy of
the antibody even if the antibody is partly or chiefly comprised of
human immunoglobulin sequences such as, for example, in the case of
a chimeric or humanized antibody. Within certain embodiments, an
initial high dose of antibody is administered such that a degree of
immunological tolerance to the therapeutic antibody is established.
The tolerizing dose is sufficient to prevent or reduce the
induction of an antibody response to repeat administration of the
marker specific antibody. Preferred ranges for the tolerizing dose
are between 10 mg/kg body weight to 50 mg/kg body weight,
inclusive. More preferred ranges for the tolerizing dose are
between 20 and 40 mg/kg, inclusive. Still more preferred ranges for
the tolerizing dose are between 20 and 25 mg/kg, inclusive.
[0171] Within these therapeutic regimens, the therapeutically
effective dose of antibodies is preferably administered in the
range of 0.1 to 10 mg/kg body weight, inclusive. More preferred
second therapeutically effective doses are in the range of 0.2 to 5
mg/kg body weight, inclusive. Still more preferred therapeutically
effective doses are in the range of 0.5 to 2 mg/kg, inclusive.
Within alternative embodiments, the subsequent therapeutic dose or
doses may be in the same or different formulation as the tolerizing
dose and/or may be administered by the same or different route as
the tolerizing dose.
[0172] For the purposes of this invention, the methods of
administration are chosen depending on the condition being treated,
the form of the subject antibodies or other agents, and the
pharmaceutical composition. Administration of the antibody
compositions or small molecule compositions can be done in a
variety of ways, including, but not limited to, continuously,
subcutaneously, intravenously, orally, topically, transdermal,
intraperitoneal, intramuscularly, and intravesically. For example,
microparticle, microsphere, and microencapsulate formulations are
useful for oral, intramuscular, or subcutaneous administrations.
Liposomes and nanoparticles are additionally suitable for
intravenous administrations. Administration of the pharmaceutical
compositions may be through a single route or concurrently by
several routes. For instance, intraperitoneal administration can be
accompanied by intravenous injections. Preferably the therapeutic
doses are administered intravenously, intraperitonealy,
intramuscularly, or subcutaneously. In some embodiments, the small
molecule compositions can be administered orally.
[0173] The compositions may be administered once or several times.
In some embodiments, the compositions may be administered once per
day, a few or several times per day, or even multiple times per
day, depending upon, among other things, the indication being
treated and the judgement of the prescribing physician.
[0174] Administration of the compositions may also be achieved
through sustained release or long-term delivery methods, which are
well known to those skilled in the art. By "sustained release or"
"long term release" as used herein is meant that the delivery
system administers a pharmaceutically therapeutic amount of subject
agents for more than a day, preferably more than a week, and most
preferable at least about 30 days to about 60 days, or longer. Long
term release systems may comprise implantable solids or gels
containing the antibodies, such as biodegradable polymers described
above (Brown, D. M. et al., Anticancer Drugs 7: 507-513 (1996));
pumps, including peristaltic pumps and fluorocarbon propellant
pumps; osmotic and mini-osmotic pumps; and the like.
[0175] The method of the invention contemplates the administration
of marker specific monoclonal antibodies, as well as combinations
of different mAbs. As discussed above, two or more monoclonal
antibodies may provide an improved effect compared to a single
antibody. For example, a combination of a monoclonal antibody with
another monoclonal antibody that binds a different antigen, e.g.,
an antigen corresponding to a member of the Ig superfamily or TLR
superfamily, may provide an improved effect compared to a single
antibody. Such mAb "cocktails" may have certain advantages in as
much as they contain mAbs, which exploit different effector
mechanisms or combine directly cytotoxic mAbs with mAbs that rely
on immune effector functionality. Specific mAbs in combination may
exhibit synergistic therapeutic effects. Some methods of the
invention also contemplate administration of one or more known
small molecule agonists or antagonists, alone or in combination
with one or more mAbs described herein. The small molecule in
combination with specific mAbs may exhibit synergistic therapeutic
effects.
8.2 Diagnostic Use of Antibodies and Other Agents
[0176] The present invention further provides methods to identify
the presence of an antigen using the compositions of the present
invention, optionally conjugated or otherwise associated with a
suitable label. Such methods comprise incubating a test sample with
one or more of the marker specific antibodies of the present
invention and assaying for antibodies that bind to components
within the test sample. Conditions for incubating the antibody with
a test sample may vary. Incubation conditions depend on the format
employed, the detection methods employed, and the antibody used in
the assay. One skilled in the art will recognize that any one of
the commonly available immunological assay formats can readily be
adapted to employ antibodies of the present invention (see Chard,
T., An Introduction to Radioimmunoassay and Related Techniques,
Elsevier Science Publishers, Amsterdam, The Netherlands (1986);
Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic
Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);
Tijssen, P., Practice and Theory of immunoassays: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1985).
[0177] The sample to be assessed can be any sample that contains an
expression product (e.g., RNA transcript or extracellular protein).
A "test sample" generally refers to a sample obtained or derived
from a patient afflicted with, or suspected of being afflicted
with, a hematological proliferative disorder of myeloid origin. The
test samples of the present invention include cells, protein or
membrane extracts of cells, or biological fluids. Samples can
comprise brain, blood, serum, plasma, lymphatic fluid, bone marrow,
plasma, lymph, urine, tissue, mucus, sputum, saliva or other cell
samples. The test sample used will vary based on the assay format,
nature of the detection method and the tissues, cells or extracts
used and the sample to be assayed. Methods for preparing protein
extracts or membrane extracts of cells are well known in the art
and can be readily adapted in order to obtain a sample which is
compatible with the system utilized.
[0178] In preferred embodiments, the test sample is obtained from
peripheral blood, particularly from mobilized peripheral blood
(MPB). The sample initially obtained from a subject is generally
enriched for stem cells. Hematopoietic stem cells can be identified
by the presence or absence of certain surface markers, as described
above, including, e.g., CD34.sup.+; CD38.sup.-; as well as
Lin.sup.- and/or CD90.sup.+.
[0179] The present invention provides diagnostic methods for
hematological proliferative disorders of myeloid origin, where the
level of an expression product of one or more of the disclosed
members of the Ig superfamily or TLR superfamily is detected.
"Detected," and its grammatical variations, refers to assessing,
measuring, reading, or otherwise determining a value for the level
or amount of expression product corresponding to one or more of the
members of the Ig superfamily or TLR superfamily disclosed herein.
An expression product "corresponds to" a designated Ig or TLR
superfamily member when it is derived therefrom via transcription
and/or translation of the gene encoding the designated member.
Expression levels refer to the amount of expression of the
expression product, as described herein. A value for an expression
level is also referred to as a "signal." The values for expression
levels can be absolute or relative values, e.g., values provided in
comparison to control levels. The values for expression levels can
be raw values, or values that are optionally rescaled, filtered
and/or normalized. The approach used will depend, for example, on
the nature of the expression product (e.g., RNA or polypeptide) as
well as specific characteristics of the product, and the intended
use for the data.
[0180] For example, in one embodiment, the expression product is a
transcription product, such as RNA. RNA includes, e.g, mRNA rRNA,
tRNA, snRNA, and the like, including any nucleic acid molecule that
is transcribed from a gene. The nucleic acid molecule levels
measured can be derived directly from the gene or, alternatively,
from a corresponding regulatory gene or regulatory sequence
element. Additionally, variants of genes and gene expression
products including, for example, spliced variants and polymorphic
alleles, can be detected.
[0181] Methods of detecting the level of an RNA transcript include,
for example, utilizing a specific hybridization probe or an array
of such probes. In a preferred embodiment, the probe comprises a
polynucleotide sequence that can hybridize to all or a portion of
the transcribed RNA sequence.
[0182] The stringency conditions allowing hybridization can be high
to moderate to low. As used herein, conditions of moderate
stringency refer to those known to the ordinarily skilled artisian,
e.g., as defined by Sambrook et al. Molecular Cloning: A Laboratory
Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory
Press, (1989). Moderate stringency conditions include use of a
prewashing solution for nitrocellulose filters 5.times.SSC, 0.5%
SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50%
formamide, 6.times.SSC at 42.degree. C. (or other similar
hybridization solution, such as Stark's solution, in 50% formamide
at 42.degree. C.), and washing conditions of about 60.degree. C.,
0.5.times.SSC, 0.1% SOS. High stringency conditions usually
involve, for example, hybridization conditions as above, with
washing at 68.degree. C., 0.2.times.SSC, 0.1% SDS. The skilled
artisan will recognize that the temperature and wash solution salt
concentration can be adjusted as necessary according to factors
such as the length of the probe. The hybridization probe can be of
any length and usually consists of at least about 5 nucleotides, at
least about 10, at least about 15, at least about 20, or at least
about 30 nucleotides. Longer lengths are suitable to lower
stringency conditions.
[0183] The probe may include natural (i.e. A, G, U, C, or T) or
modified bases (7-deazaguanosine, inosine, etc.). In addition, the
bases in probes may be joined by a linkage other than a
phosphodiester bond, so long as the bond does not interfere with
hybridization. Thus, probes may be peptide nucleic acids in which
the constituent bases are joined by peptide bonds rather than
phosphodiester linkages.
[0184] In some embodiments, more than one hybridization probe is
used, e.g., two, three, four, five, 10 or more probes, up to,
including and beyond as many probes as members of the Ig
superfamily or TLR superfamily disclosed herein. In some
embodiments, different probes are directed to different RNA
products, e.g., RNA products corresponding to two or more different
members of the Ig superfamily or TLR superfamily disclosed herein.
For example, probes for detecting the expression level of two,
three, four, five, or more of the members of the Ig superfamily or
TLR superfamily may be used.
[0185] In preferred embodiments, the probes are immobilized, e.g.,
on an array, in different known locations. An array refers to a
solid support with a surface to which a plurality of different
nucleic acid sequences (probes) are attached. The array can be
prepared either synthetically or biosynthetically, and can assume a
variety of formats, e.g., libraries of compounds tethered to resin
beads, silica chips, or other solid supports, as well as libraries
of nucleic acids prepared by spotting nucleic acids onto a
substrate. The solid support can be any material or group of
materials having a rigid or semi-rigid surface or surfaces.
Generally, at least one surface of the solid support will be
substantially flat, although in some case the array will include
wells, raised regions, pins, etched trenches, or the like. Although
a planar array surface is preferred, the array may be fabricated on
a surface of virtually any shape or even a multiplicity of
surfaces. Solid support(s) can also take the form of beads, resins,
fibers such as fiber optics, gels, microspheres, or other geometric
configurations. See U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153,
6,040,193 and 5,800,992, which are hereby incorporated in their
entirety for all purposes.
[0186] Such arrays are generally termed "microarrays", or
colloquially "chips", and have been described in the art, for
example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195,
6,040,193, 5,424,186 and Fodor et al., Science, 251:767-777 (1991),
each of which is incorporated by reference in its entirety for all
purposes. These arrays may generally be produced using mechanical
synthesis methods or light directed synthesis methods, which can
incorporate a combination of photolithographic methods and solid
phase synthesis methods. Techniques for the synthesis of these
arrays using mechanical synthesis methods are described in, e.g.,
U.S. Pat. No. 5,384,261, incorporated herein by reference in its
entirety for all purposes. Commercially available probes and arrays
can be used, including, for example, Affymetrix human U133 Plus 2.0
Array.
[0187] In a preferred embodiment, the expression product is mRNA
and the mRNA levels are obtained by contacting the sample with a
suitable microarray, and determining the extent of hybridization of
the nucleic acid in the sample to the probes on the microarray.
[0188] For example, mRNA levels can be obtained from a GeneChip.TM.
probe array or Microarray (Affymetrix, Inc.) (U.S. Pat. Nos.
5,631,734, 5,874,219, 5,861,242, 5,858,659, 5,856,174, 5,843,655,
5,837,832, 5,834,758, 5,770,722, 5,770,456, 5,733,729, 5,556,752,
all of which are incorporated herein by reference in their
entirety), and the expression levels can be calculated with
software (e.g., Affymetrix GENECHIP.TM. software). Briefly, nucleic
acids (e.g., mRNA) from a sample which has been subjected to
particular stringency conditions hybridize to the probes on the
chip. The nucleic acid to be analyzed (e.g., mRNA corresponding to
an HTC marker) is isolated, amplified and labeled with a detectable
label, (e.g., .sup.32P or fluorescent label) prior to hybridization
to the arrays. Once hybridization occurs, the arrays are inserted
into a scanner which can detect patterns of hybridization. The
hybridization data are collected as light emitted from the labeled
groups, which are now bound to the probe array. The probes that
perfectly match the mRNA corresponding to an HTC marker produce a
stronger signal than those that have mismatches. Since the sequence
and position of each probe on the array are known, by
complementarity, the identity of the nucleic acid applied to the
probe is determined. Quantitation from the hybridization of labeled
mRNA/DNA microarray can be performed by scanning the microarrays to
measure the amount of hybridization at each position on the
microarray. This can be performed with, for example, an Affymetrix
scanner (Affymetrix, Santa Clara, Calif.). Microarrays are only one
method of detecting RNA levels. Other methods known in the art or
developed in the future can be used with the present invention.
[0189] In another embodiment, the expression product is a
translation product, e.g. one or more of the Ig superfamily or TLR
superfamily proteins disclosed herein. The Ig superfamily or TLR
superfamily proteins include any polypeptide or derivative thereof
including, e.g., peptides, glycoproteins, lipoproteins and nucleic
acid-protein complexes.
[0190] Techniques for protein detection and quantitation are known
in the art. For example, antibodies specific for the protein or
polypeptide can be obtained using methods which are routine in the
art, and the specific binding of such antibodies to protein or
polypeptide expression products can be detected and measured.
Methods of detecting the level of proteins preferably involve
utilizing antibodies of the instant disclosure, as discussed above.
In some embodiments, small molecules known to bind to a polypeptide
corresponding to a disclosed member of the immunoglobulin (Ig)
superfamily or toll-like receptor (TLR) superfamily can similarly
be detectably labeled and/or attached to a solid support.
[0191] In some embodiments, more than one antibody is used, e.g.,
two or more mAbs. In some embodiments, different mAbs are directed
to a proteinaceous product expressed from two or more different
members of the Ig superfamily or TLR superfamily disclosed herein.
For example, antibodies for detecting the expression level of two,
three, four, five, or more of the members of the Ig superfamily or
TLR superfamily disclosed herein may be used. In some embodiments,
both RNA and protein levels are detected in a given sample or
multiple samples from an individual.
[0192] The RNA or antigen level can be compared to control levels,
e.g., levels obtained using normal HSCs. A control sample
comprising one or more normal HSCs can be obtained, e.g., from an
individual not afflicted with a hematological proliferative
disorder, e.g, not afflicted with a hematological proliferative
disorder of myeloid origin. Preferably, the control sample is
obtained in a manner similar to that used to obtain the test
sample, e.g, being obtained from the same organs, tissues or fluids
(as detailed above with respect to test samples); and sorted to
enrich for corresponding cell types. Similarly, the level of RNA or
antigen preferably is assessed in the control sample in a manner
similar to that used to obtain the test values. Methods are known
in the art for permitting direct comparison of test and control
levels, and a specific example of such comparison is detailed below
in Example 1. In some embodiments, control levels are provided by
previously-obtained data, such as from control samples that have
been detected in prior assays; from published data; and/or from
accessible data bases.
[0193] Diagnosis is based on a correlation between the level of a
given expression product in a test sample compared to that in a
control sample. For example, as detailed in Example 1 below, mRNA
levels of the members of the Ig superfamily or TLR superfamily
disclosed herein are higher in AML HTC samples as compared to
normal HSC samples. Preferably, the difference in expression levels
is at least about 2 fold, at least about 3 fold, at least about 5
fold, at least about 7 fold, at least about 10 fold or at least
about 15 fold. In particularly preferred embodiments, the
difference in expression levels is at least about 20 fold, at least
about 30 fold, at least about 40 fold or at least about 50 fold. In
still more preferred embodiments, the difference in expression
levels is at least about 70 fold, at least about 100 fold, at least
about 200, fold or as much as nearly 300 fold, 400 fold or 500
fold. For example, CD84 mRNA levels in AML HTCs is over 15 times
that in normal HSCs (see Table 1). Accordingly, the information
provided by the present disclosure, alone or in conjunction with
other test results, aids in sample classification and diagnosis of
hematological proliferative disorders of myeloid origin, such as
AML.
[0194] In some embodiments, more than one test sample and/or more
than one control sample are obtained and/or detected. For example,
as illustrated in Example 1 below, all of 3 normal samples showed
low levels of expression of each HTC marker, while at least 2 out
of 3 AML samples showed high expression levels. Alternatively, AML
samples over-expressed the cytokine receptor by at least about 5
fold compared to normal HSCs. Diagnosis may take into account the
difference in expression levels between more than one test sample
and/or more than one control sample. In some embodiments, repeat
assays are performed using a given sample.
[0195] In some embodiments, expression of more than one HTC marker
can be detected. The members of the Ig superfamily or TLR
superfamily disclosed herein can be detected simultaneously. For
example, in some embodiments, two, three, four, five, or more of
the members of the Ig superfamily or TLR superfamily disclosed
herein are detected. The detection of numerous genes can provide a
more accurate evaluation of the sample. The correlation between
expression product levels and a given disease, such as AML, can be
determined using a variety of methods. Methods of classifying
samples are described, for example, in U.S. patent application Ser.
No. 09/544,627, filed Apr. 6, 2000 by Golub et al., the teachings
of which are incorporated herein by reference in their
entirety.
[0196] The present invention also provides prognostic methods for
predicting the efficacy of treating a haematological proliferative
disorder of myeloid origin, where the level of an expression
product of one or more of the disclosed members of the Ig
superfamily or TLR superfamily is detected and wherein the
expression product level is correlated with a treatment outcome.
"Treatment outcome" as used herein refers to the efficacy of a
treatment with respect to a disease, that is, the response of the
disease to a particular treatment. The levels of expression
products can be used to detect the likelihood that a given disorder
will respond well to a particular treatment, or to determine which
of a number of treatment options is more preferable. In some
embodiments, the treatment is one disclosed herein, e.g., one or
more of the therapeutic uses of the antibodies described herein. In
some embodiments, the treatment is a treatment otherwise known or
used or to be used in the art; or a combination of such treatments
with one or more of those disclosed herein.
[0197] The disclosure above regarding, for example, test and
control samples; stem cell sorting; use of one or more members of
the Ig superfamily or TLR superfamily; detection of RNA and/or
antigen levels; correlation of data and so forth, as provided in
the case of diagnostic methods, also applies to the prognostic
methods disclosed herein.
[0198] In a preferred embodiment, the expression product is RNA and
RNA levels in test samples are compared to control levels. In a
particularly preferred embodiment, the expression product is mRNA
and the mRNA levels are obtained by contacting the sample with a
suitable microarray, and determining the extent of hybridization of
the nucleic acid in the sample to the probes on the microarray.
[0199] In particularly preferred embodiments, the expression
product is mRNA and lower mRNA levels correlate with more favorable
treatment outcomes. For example, a CD84 expression level that is
about 10 times higher than control levels indicates a more
favorable outcome than where the CD84 expression level is about 30
times higher. In some embodiments, the expression levels of more
than one member of the Ig superfamily or TLR superfamily is be
detected; and lower expression levels of multiple markers is
further indication of a more favorable treatment outcome.
[0200] The present invention also provides methods for monitoring
the efficacy of treating a haematological proliferative disorder of
myeloid origin, where the level of an expression product of one or
more of the disclosed members of the Ig superfamily or TLR
superfamily is detected at various time points and correlated with
treatment outcome. The various time points can include, for
example, time of diagnosis, times prior to commencing a treatment
regime; times at intervals during a treatment regime; and times
after the conclusion of treatment regime. The time intervals can
include several hours; a day; 2, 3, or 4 days; a week; 2, 3, 4, 5
or 6 weeks; a month, 2, 3, 4, 5, or 6 months, a year, or several
years post-initiation of a treatment regime. In some embodiments,
the treatment is one disclosed herein, e.g., one or more of the
therapeutic uses of the antibodies described herein. In some
embodiments, the treatment is a treatment otherwise known or used
or to be used in the art; or a combination of such treatments with
one or more of those disclosed herein.
[0201] The disclosure above regarding, for example, test and
control samples; stem cell sorting; use of one or more members of
the Ig superfamily or TLR superfamily; detection of RNA and/or
protein levels; correlation of data and so forth, as provided in
the case of diagnostic methods, also applies to the monitoring
methods disclosed herein.
[0202] In a preferred embodiment, the expression product is RNA and
RNA levels in test samples are compared to control levels. In a
particularly preferred embodiment, the expression product is mRNA
and the mRNA levels are obtained by contacting the sample with a
suitable microarray, and determining the extent of hybridization of
the nucleic acid in the sample to the probes on the microarray.
[0203] Monitoring the efficacy of a treatment is useful, e.g. in
facilitating clinical management of the disease, e.g., where
decisions must be made as to whether to continue a treatment course
or advance to other treatment options. Whether a myeloid leukemia
is responding positively to a treatment can be determined based on
changes in the level of a given expression product (or products) in
test samples taken at various time points, e.g., before and after
administration of a particular treatment.
[0204] A shift in expression product levels from a level correlated
with HTCs of myeloid origin towards a level correlated with normal
HSCs is evidence of an effective therapeutic regime. In some
embodiments, a reduction in the expression product level
corresponding to one or more of the members of the Ig superfamily
or TLR superfamily disclosed herein indicates a positive response
to treatment. The reduction indicates a trend towards levels seen
in normal HSC samples. For example, as detailed in Example 1 below,
the expression product level in AML HTCs is higher than that in
normal HSCs for each of the members of the Ig superfamily or TLR
superfamily disclosed herein. A reduction in expression level in
one or more of the disclosed HTC markers, e.g., in test samples
obtained after the commencement of treatment, would indicate a
positive response.
[0205] In preferred embodiments, the expression level for one or
more HTC markers is reduced at least about 5 fold, at least about 7
fold, at least about 10 fold or at least about 15 fold, during the
course of treatment. In particularly preferred embodiments, the
expression level for one or more HTC markers is reduced at least
about 20 fold, at least about 30 fold, at least about 40 fold or at
least about 50 fold, during the course of treatment. In still more
preferred embodiments, the expression level for one or more HTC
markers is reduced at least about 70 fold, at least about 100 fold,
at least about 200 fold, or as much as nearly about 300 fold, about
400 fold or about 500 fold, during the course of treatment. For
example, CD84 mRNA levels in AML HTCs is 15 times that in normal
HSCs (see Table 1). A reduction to 10, or just 2 times the CFD mRNA
levels in normal HSCs is evidence of an effective therapeutic
regime.
[0206] In some embodiments, the methods of the present invention
are suitable for diagnosis prognosis and/or monitoring of a
haematological proliferative disorder of myeloid origin, such as
myoproliferative disorders. The present invention also provides
methods of diagnosis, prognosis and monitoring of a
myoproliferative disorder that is chronic myeloid leukemia (CML)
and/or acute myeloid leukemia (AML). In a particularly preferred
embodiment, the myelogenous haematological proliferative disorder
is AML and the level of RNA or antigen is detected using a test
sample comprising AML HTCs, compared to control levels obtained
from a control sample of normal HSCs.
9. Kits
[0207] In another aspect of the present invention, kits are
provided which contain one or more of the necessary reagents to
carry out methods of the present invention. Specifically, some
embodiments provide a compartment kit having one or more
containers, which comprise: (a) a first container comprising one of
the marker specific antibodies or complexes of the present
invention, e.g., a first monoclonal antibody that specifically
binds a first antigen corresponding to one of the members of the Ig
superfamily or TLR superfamily disclosed herein; and (b) one or
more other containers comprising one or more of the following: wash
reagents, reagents capable of detecting presence of the antibody,
and/or another marker specific antibody or complex of the present
invention, e.g., a second monoclonal antibody that specifically
binds a second antigen corresponding to a different member of the
Ig superfamily or TLR superfamily disclosed herein. In some
embodiments, kits include additional containers, e.g, comprising
third, fourth, fifth, etc., antibodies directed to antigens
corresponding to a third, fourth, fifth, etc., member of the Ig
superfamily or TLR superfamily disclosed herein. In some
embodiments, additional containers include one or more known small
molecule agonists or antagonists that find use in the prognostic,
diagnostic and/or therapeutic methods taught herein.
[0208] The kit typically contains containers which may be formed
from a variety of materials such as glass or plastic, and can
include, for example, bottles, vials, syringes, and test tubes. A
compartment kit includes any kit in which reagents are contained in
separate containers. Such containers include small glass
containers, plastic containers or strips of plastic or paper. Such
containers allow efficient transfer of reagents from one
compartment to another compartment, such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. One skilled in the art will readily
recognize that the disclosed antibodies of the present invention
can be readily incorporated into one of the established kit
formats, which are well known in the art.
[0209] Provided herein are kits which include a composition
described herein. In some embodiments the kit comprises a
hybridoma, complex, antibody and/or mixtures of antibodies
disclosed herein. In some embodiments, kits for therapeutic
applications are provided, such as a kit housing a pharmaceutical
formulation, e.g., one or more of the pharmaceutical compositions
described herein. In some embodiments, the kits contain at least
one additional reagent, including other antibodies, other
monoclonal antibodies directed to HSCs, other agents described
herein, committed progenitor cells, polyclonal antibodies, or
mixtures of the antibodies as reagents for detecting myeloid cell
types. Frozen or fixed forms of HSCs, CMPs, GMP and/or MEPs
reactive with the antibodies and reagents form additional contents
of the kits.
[0210] In some embodiments, the kit is a diagnostic kit for use in
detecting test samples. The kit can include a control antibody that
does not react with the antigen to be assayed, along with a marker
specific antibody or antigen-binding fragment thereof which
specifically binds to an antigen corresponding to a member of the
Ig superfamily or TLR superfamily disclosed herein. Further, such a
kit can include a means for detecting the binding of said antibody
to the antigen (for example, the antibody may be conjugated to a
fluorescent compound such as fluorescein or rhodamine which can be
detected by flow cytometry).
[0211] In preferred embodiments, the diagnostic kit includes a
substantially isolated antibody that specifically binds an antigen
corresponding to an HTC marker (e.g., a member of the Ig
superfamily or TLR superfamily disclosed herein), as well as means
for detecting antigen-antibody binding. In some embodiments, the
antibody is attached to a solid support. In some embodiments, the
antibody is a monoclonal antibody. The detecting means of the kit
can include a second, labeled monoclonal antibody. Alternatively,
or in addition, the detecting means can include a labeled,
competing antigen.
[0212] In one diagnostic configuration, the test sample is reacted
with a solid phase reagent having a surface-bound antigen, where
the antigen corresponds to one (or more) of the members of the Ig
superfamily or TLR superfamily disclosed herein. After washing to
removing unbound components, the reagent can be reacted with
reporter-labeled anti-human antibody to determine the amount of
anti-antigen antibody bound to the solid support. Such methods are
well known and have been extensively described in the art. The
reporter label can be an enzyme, for example, which is detected by
incubating the solid phase with a suitable fluorometric,
luminescent or calorimetric substrate, as is standard in the
art.
[0213] The solid surface bearing bound antigens and/or antibodies,
as described above, can be prepared by known techniques for
attaching protein material to solid support material. Suitable
solid support materials include, for example and without
limitation, polymeric beads, dip sticks, 96-well plate or filter
material. In some embodiments, a small molecule known to bind to a
polypeptide corresponding to a disclosed HTC marker can be attached
to a solid support, based on its chemical structure by methods
known in the art.
[0214] A text label typically accompanies the kit, and includes any
writing or recorded material, which may be electronic or computer
readable form (e.g., disk, optical disc, or tape) providing
instructions or other information for using one or more of the
contents of the kit. In some embodiments, the label indicates that
the contents are used for diagnosing or treating the disorder of
choice, such as a hematopoietic proliferative disorder of myeloid
origin, according to one or more of the methods described herein.
In some embodiments, AML represents the disorder to be diagnosed
and/or treated using the contents of the kit. In some embodiments,
CML represents the disorder to be diagnosed and/or treated using
the contents of the kit.
10. EXAMPLES
10.1 Example 1
Identification of Members of the Immunoglobulin (Ig) Superfamily or
Toll-Like Receptor (TLR) Superfamily Associated with HTCs
[0215] HTC markers were identified by comparing RNA transcript
levels in normal HSCs and in AML CSCs for a variety of genes using
microarrays. Specifically, data was obtained for test samples
comprising AML Lin.sup.-CD34.sup.+CD38.sup.- cells, where three AML
samples were taken from peripheral blood;
Lin.sup.-CD34.sup.+CD38.sup.- and Lin.sup.-CD34.sup.+CD38.sup.+
cells were double sorted. The sorting strategy produced cells which
were also over 90% CD90.sup.-. The sorting strategy produced
purities greater than 98%.
[0216] To allow for direct comparison, data was then obtained from
control samples comprising normal HSCs. A sample was taken from
mobilized peripheral blood (MPB) of each of three individual donors
and Lin.sup.-CD34.sup.+CD90.sup.+CD45RA.sup.-CD38.sup.- and
Lin.sup.- CD34.sup.+CD90.sup.+CD45RA.sup.-CD38.sup.+ cells were
double sorted. The sorting strategy produced a purity of over
99%.
[0217] For both sorting strategies, the Lineage (Lin) cocktail
included antibodies against CD2, CD3, CD11b, CD15, CD19, CD41, and
CD235a. Exemplary dot plots of control samples and AML samples are
shown in FIGS. 1 and 2, respectively.
[0218] Total RNA was then extracted, the RNA was reverse
transcribed and in vitro transcribed to ultimately yield
fluorochrome labeled cRNA probes from the transcripts. Transcript
levels were detected using Affymetrix whole Human Genome U133 Plus
2.0 Array. Briefly, the gene array was hybridized and read out.
Signal intensities, probe set ID# and absence/presence score for
each probe set was tabulated using MS Excel.
[0219] The signal values corresponding to RNA transcript levels in
AML test samples and control samples
(Lin.sup.-CD34.sup.+CD90.sup.+CD45RA.sup.-CD38.sup.- normal HSCs)
were then directly compared using MS Excel and Fisher's t-test.
[0220] HTC marker genes were then selected based on the following
criteria: (1) significance of signal intensity difference between
test and control samples cohorts p<0.05; (2) genes not expressed
in all 3 normal HSC samples (scored `absent` by Affymetrix
software); (3) genes expressed in at least 2 out of 3 AML samples
(scored `present` by Affymetrix software); or (4) AML/HSC signal
ratio .gtoreq.5 and (5) genes whose expression was known to be
extracellular, encode an Ig domain and/or be involved in Toll-like
receptor signaling.
[0221] Tables 1 and 2 below shows comparison for 8 genes that were
differentially expressed in at least 2 out of the 3 of the
CD38.sup.- or CD38.sup.+, respectively, AML CSC samples compared to
all three of the normal HSC samples. "Pos. samples" refers to
positive samples, scored as `present` by Affymetrix software.
TABLE-US-00001 TABLE 1 Normal CD38.sup.- HSC sample CD38.sup.- AML
CSC sample Gene Pos. samples Average signal Pos. samples Average
signal Signal detected intensity detected intensity ratio CD84 0/3
11 2/3 162 15 CD180 0/3 5 2/3 52 10 HAVCR2 0/3 13 3/3 142 11 LILRA1
0/3 19 2/3 57 3 LILRA2 0/3 28 2/3 89 3 Ly86 0/3 30 3/3 333 11 NEGR1
0/3 12 2/3 65 5 TLR2 2/3 79 3/3 481 6
TABLE-US-00002 TABLE 2 Normal CD38.sup.+ HSC sample CD38.sup.+ AML
CSC sample Gene Pos. samples Average signal Pos. samples Average
signal Signal detected intensity detected intensity ratio CD84 3/3
112 3/3 317 3 CD180 0/3 13 3/3 188 14 HAVCR2 0/3 20 3/3 193 10
LILRA1 0/3 13 2/3 44 3 LILRA2 3/3 66 3/3 282 4 Ly86 0/3 15 3/3 952
63 NEGR1 0/3 21 2/3 128 6 TLR2 2/3 115 3/3 977 8
[0222] Comparison of published data by Gal et al. ("Gene expression
profiles of AML derived stem cells; similarity to hematopoietic
stem cells." Leukemia 2006, 20: 2147-2154, incorporated herein by
reference in its entirety) with control samples described herein
corroborates use of Ly86/MD1, CD84, LILRA1 and CD180 as cancer stem
cell targets (See U.S. Patent Application No. 61/039,701,
incorporated herein by reference).
[0223] As shown in FIGS. 3A-3C, Ly86, CD84 and CD180 expression by
AML CSCs is detectable by flow cytometric analysis using clone 1H3
antibody from Abnova (Walnut, Calif.) to detect CD84; clone
CD84.1.21 from BioLegend (San Diego, Calif.) to detect CD84; or
clone MHR73-11 from BioLegend to detect CD180. In contrast,
expression of these markers by normal HSCs is undetectable by flow
cytometric analysis (FIG. 3).
10.2 Example 2
Production and In Vivo Efficacy of Monoclonal Antibodies
10.2.1. Preparation of Monoclonal Antibodies that Specifically Bind
a Member of the Immunoglobulin (Ig) Superfamily or Toll-Like
Receptor (TLR) Superfamily Associated with HTCs
[0224] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986). Immunogens that may be employed include a purified
polypeptide corresponding to CD84; lymphocyte antigen 86 (Ly86);
CD180 (RP105); HAVCR2; LILRA1; LILRA2, NEGR1; or TLR2, as well as
fusion proteins containing the same. Alternatively, cells
expressing recombinant an AML-expressed isoform of CD84; Ly86;
CD180; HAVCR2; LILRA1; LILRA2, NEGR1; or TLR2, on the cell surface,
may be used. Selection of the immunogen can be made by the skilled
artisan without undue experimentation.
[0225] Mice, such as Balb/c, are immunized with the selected
immunogen, emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect antibodies directed to the HTC marker polypeptide.
[0226] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of the immunogen corresponding to an HTC
marker. Three to four days later, the mice are sacrificed and the
spleen cells harvested. The spleen cells are then fused (using 35%
polyethylene glycol) to a selected murine myeloma cell line such as
P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate
hybridoma cells which can then be plated in 96 well tissue culture
plates containing HAT (hypoxanthine, amininopterin, and thymidine)
medium to inhibit proliferation of non-fused cells, myeloma
hybrids, and spleen cell hybrids.
[0227] The hybridoma cells then can be screened in an ELISA for
reactivity against the immunogen corresponding to the HTC marker.
Determination of "positive" hybridoma cells secreting the desired
monoclonal antibodies directed against the HTC marker polypeptide
is within the skill in the art.
[0228] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the monoclonal antibodies directed to polypeptide
corresponding to the HTC marker. Alternatively, the hybridoma cells
can be grown in tissue culture flasks or roller bottles.
Purification of the monoclonal antibodies produced in the ascites
can be accomplished using ammonium sulfate precipitation, followed
by gel exclusion chromatography, as known in the art.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be employed, also as known
in the art.
10.2.2. In Vivo Efficacy of Monoclonal Antibodies Against Myeloid
Leukemia
[0229] In vivo models of human cancer are useful to determine
preclinical efficacy of candidate therapeutic agents. For
monoclonal antibodies, studies in appropriate animal models help
evaluate target cell lysis and tumor eradication under
physiological conditions in vivo. Several groups have described
engraftment of CML chronic phase (CP), accelerated phase (AP),
and/or blast phase (BP) and AML cells into SCID and NOD/SCID mice.
In general, generation of chimeric animals showing engraftment of
human CML cells is more consistent in NOD/SCID mice (See Dazzi, F
et al, Blood 92: 1390-1396 (1998); Wang, J. C. Y. et al, Blood 91:
2406-2414 (1998); Dick, J et al Blood 87: 1539-1548 (1996); Bonnet,
D et al, Blood 106: 4086-4092 (2005)). In vivo efficacy of
monoclonal antibodies against CML and/or normal GMP and not HSC can
be determined using the NOD/SCID human CML model.
[0230] Xenograft animals can be generated as described by Dazzi et
al. Briefly, NOD/SCID mice are bred in house or purchased from a
commercial supplier (Jackson Laboratories) and housed under
pathogen-free conditions. Prior to injection of cells, animals are
irradiated (250 cGy, x-ray source). Cryopreserved cells from a CML
or AML patient obtained from peripheral blood, mobilized peripheral
blood, or bone marrow are analyzed by flow cytometry to determine
the percentage of CD34.sup.+ cells in the sample. Samples
containing 1 to 10.times.10.sup.6 CD34.sup.+ cells are injected IV
into the conditioned mouse in a total volume of 1 mL.
Alternatively, CD34.sup.+ cells can be sorted from the sample by
FACS prior to transplantation. A subset of the animals is
sacrificed weekly and bone marrow and spleen analyzed for human
CD34.sup.+ cells.
[0231] Patient samples with engraftment potential are selected for
use in antibody efficacy studies. For efficacy studies, CML/AML
cells are transplanted and the test monoclonal antibody or control
antibody will be injected on a schedule. Alternate schedules
include once to 3 times per week, 1-3 injections per week for 1-4
weeks, or 1-2 per week for 1-4 months. Injections can be
intravenous by tail vein injection, intraperitoneal, subcutaneous,
or intramuscular. Following completion of the treatment schedule,
animals are sacrificed and tissues collected for analysis.
Peripheral blood, spleen and bone marrow can be evaluated by FACS
analysis for the presence of human phenotypic CML cancer stem
cells, CD34.sup.+ marker.sup.+ HTCs, detectable in the bone marrow
and spleen at the conclusion of the treatment. Philadelphia (Ph)
chromosome can be assayed by PCR to determine whether the cells are
CML or normal.
[0232] As an example, eleven mice can be transplanted with CML
sample (MISIRB 31104 750), 5.times.10.sup.6 cells/mouse. Mice can
be conditioned with 250rad TBI (x-ray source, Faxitron CP160), and
anti-asialo GM1. The anti-asialo GM1 is injected by intraperitoneal
injection on days 0, 5 and 11. At 4 weeks post transplant half the
mice in each group will begin receiving intraperitoneal or
intravenous injections of a clone of a marker specific mAb,
described herein, or control antibody, 250-1000 mg/dose, two times
a week for 4 weeks. Additionally, a group of 40 mice are also
transplanted with CML (MISIRB 31104 750) as above. Antibody
administration begins at the time of transplant. Mice are injected
by intraperitoneal injection with 0.5-1 mg/dose of antibody, twice
a week for 8 weeks. Alternatively, CML cells isolated from
previously engrafted mice will be serially transplanted. Some of
these secondary recipients will be treated with the marker specific
mAb clone at time of transplantation by intraperitoneal injection
with 0.5-1 mg/dose of antibody, twice a week for 8 weeks. Following
the above treatments with the marker specific mAb clone, the mice
are analyzed by flow cytometry for tumor burden, expression of CD34
and expression of the specific HTC marker gene. The number and
frequency of human cells in the bone marrow and spleen will be
determined for all mice surviving to the end of the study. Human
cells (marker.sup.+) will be sorted by FACS from both groups of
mice for serial transplantation to determine if cells with
functional cancer stem cell potential are present.
[0233] For secondary transplant of CML cells,
CD34.sup.+marker.sup.+ cells can be sorted from the bone marrow and
spleen of several mice for transplantation. NOD/SCID analysis for
the secondary transplant can be performed 8 or 10 weeks post
transplant with the CD34 compartment of bone marrow.
[0234] Determination of whether the marker specific monoclonal
antibody clone can eliminate or reduce tumor burden in mice
transplanted with primary human AML blast crisis cells. Twenty-five
mice can be transplanted with AML cells injected at
10.times.10.sup.6 CD34.sup.+marker.sup.+ cells/mouse. Cells are
injected intravenously into the tail vein or the post lateral
aspect of the orbital cavity. Mice are conditioned with 250rad TBI
(x-ray source, Faxitron CP160), and anti-asialo GM1. The
anti-asialo GM1 is injected IP on days 0, 5 and 11. Beginning 4
weeks post transplant, mice are randomized into 2 groups, therapy
and control. The group receiving the therapeutic is injected I.P.
with the marker specific mAb clone. Antibody will be administered
by intraperitoneal injection 2 times a week, for 4 weeks. Antibody
concentration will be 1 mg per injection (a total of 2
mg/mouse/week). Volume will vary depending on the antibody lot
used. Mouse IgG will be used as the control article; it will be
prepared in the same diluent and injected at the same concentration
and volume as the monoclonal antibody. Mice will be sacrificed 2-3
days following the last injection of marker specific antibody
clone. Bone marrow and spleen will be isolated and counted. Tissues
will be analyzed by FACS for expression of CD34 and the specific
HTC marker gene. The number and frequency of human cells in the
bone marrow and spleen will be determined for all mice surviving to
the end of the study. Human cells will be sorted by FACS from both
groups of mice for serial transplantation to determine if cells
with functional cancer stem cell potential are present
post-antibody treatment. Alternatively, mice will begin antibody
treatment at the time of AML cell transplant. These mice will be
injected with 0.5 mg of antibody 2 times a week for 8 weeks.
Analysis will proceed as described above.
[0235] In addition, the efficacy of the maker specific mAb clone
can be tested for efficacy in leukemia in vivo models using human
cell lines expressing the corresponding antigen. Immunocompromised
animals will be inoculated with human leukemia cell lines
recognized by the marker specific mAb clone. The efficacy of the
clone will be tested using multiple cell lines. The cell lines used
should maintain a primitive phenotype in vivo with sustained
expression of the epitope recognized by the marker specific mAb
clone. Such cell lines will be identified and used as appropriate.
Each cell line will be characterized using different routes of
administration; intravenous, subcutaneous or intraperitoneal
injection. NOD/SCID mice will be tested for tumor engraftment at
the site of injection and subsequent invasion of bone marrow and
spleen. Animals will be treated with the marker specific mAb clone
beginning at the time of tumor inoculation or following tumor
engraftment with injections starting 1-4 weeks post cell
administration. Antibody will be administered by intraperitoneal
injection 2 times a week, for 4 weeks. Mouse IgG will be used as
the control article, injected at the same concentration and volume
as the marker specific mAb clone. Test cells will be administered
at a single site. Animals are weighed weekly and observed for
clinical signs of toxicity and death for the duration of treatment.
Animals are observed and palpated for the formation of nodules at
the site of injection twice weekly. Detected nodules will be
measured in two dimensions and findings recorded. The injection
site is exposed at the end of study and tumor removed and measured
and weighed. In addition, a sample of bone marrow, spleen and tumor
mass are to be removed for phenotyping by FACS. These tissues will
be disassociated and prepared for analysis by flow cytometry.
Tissues will be screened for expression of one or more of the human
HTC markers and the marker specific mAb clone.
[0236] Using the above described in vivo models, it can be shown
that treatment with monoclonal antibody compositions of the present
invention is effective in reducing tumor size, ameliorating one or
more symptoms and/or prolonging survival of mice in the therapy
group.
[0237] The foregoing descriptions of specific embodiments of the
present invention have been presented for purposes of illustration
and description. They are not intended to be exhaustive or to limit
the invention to the precise forms disclosed, and obviously many
modifications and variations are possible in light of the above
teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best
utilize the invention and various embodiments with various
modifications as are suited to the particular use contemplated. It
is intended that the scope of the invention be defined by the
Claims appended hereto and their equivalents.
[0238] All patents, patent applications, publications, and
references cited herein are expressly incorporated by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
* * * * *