Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF ELIMINATING HEMATOPOIETIC STEM
CELLS/HEMATOPOIETIC PROGENITORS (HSC/HP) IN A PATIENT USING
BI-SPECIFIC ANTIBODIES
CROSS-REFERENCE TO RELATED APPLICATIONS
[1] This application claims priority to U.S. Patent Application No.
16/506,764,
filed July 9, 2019, which is a continuation-in-part of U.S. Patent Application
No.
16/091,139, filed October 4, 2018, which is a 35 U.S.C. 371 national stage
filing of
International Application No. PCT/U52017/025951, filed April 4, 2017, which
claims the
benefit of priority to U.S. Provisional Application No. 62/317,906, filed
April 4, 2016,
entitled "Method of Eliminating Hematopoietic Stem Cells/Hematopoietic
Progenitors
(HSC/HP) in a Patient Using Bi-Specific Antibodies." The entire contents of
each of the
aforementioned applications are incorporated by reference herein in their
entireties.
SEQUENCE LISTING
[2] The instant application contains a sequence Listing which has been
submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said
ASCII copy, created on July 7, 2020, is named 128557-00119 SL.txt and is
30,539 bytes in
size.
FIELD OF THE INVENTION
[3] The described invention generally relates to hematopoietic cell
transplantation,
therapeutic antibody preparations and their uses.
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BACKGROUND OF THE INVENTION
Hematopoietic Stem Cells
[4] The hematopoietic stem cell is the common ancestor of all blood
cells. As
multipotent cells, they can differentiate into multiple cell lineages, but not
all the lineages
derived from the three germ layers. Hematopoietic stem cell differentiation
gives rise to the
lymphoid and myeloid cell lineages, the two major branches of hematopoiesis.
(Kondo, M.
"Lymphoid and myeloid lineage commitment in multipotent hematopoietic
progenitors,"
Immunol. Rev. 2010 Nov; 238(1): 37-46). Lymphoid lineage cells include T, B,
and
natural killer (NK) cells. The myeloid lineage includes megakaryocytes and
erythrocytes
(MegE) as well as different subsets of granulocytes (neutrophils, eosinophils
and basophils),
monocytes, macrophages, and mast cells (GM), which belong to the myeloid
lineage (Id.
citing Kondo M, et al. Biology of hematopoietic stem cells and progenitors:
implications for
clinical application. Ann. Rev Immunol. 2003;21:759-806., Weissman IL.
Translating stem
and progenitor cell biology to the clinic: barriers and opportunities. Science
(New York, NY.
2000 Feb 25;287(5457):1442-6; see also Iwaskaki, H. and Akashi, K. "Myeloid
lineage
commitment from the hematopoietic stem cell," Immunity 26(6) June 2007,726-
40).
151 HSCs present self-renewal potential and differentiation capacity
into blood
lineages; i.e., when stem cells divide, 50% of the daughter cells, on average,
are committed
with a cell lineage, while the remaining 50% do not differentiate. The process
maintains the
same number of stem cells by asymmetric cell division, so that each dividing
stem cell
originates one new stem cell and one differentiated cell. In contrast, in
symmetric division,
the stem cells originate 100% of identical stem cells. (Gordon, M. Stem cells
and
haemopoiesis. In: Hoffbrand, V., Catovsky, D., Tuddenham, E.G., 5th ed.
Blackwell
Publishing, (2005): Differential niche and Wnt requirements during acute
myeloid leukemia,
pp. 1-12. New York.).
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[6] The lymphoid and myeloid lineages are separable at the progenitor
level.
Common lymphoid progenitors (CLPs) can differentiate into all types of
lymphocytes
without noticeable myeloid potential under physiological conditions (Kondo M,
Scherer DC,
Miyamoto T, King AG, Akashi K, Sugamura K, et al. Cell-fate conversion of
lymphoid-
committed progenitors by instructive actions of cytokines. Nature. 2000 Sep
21;407(6802):383-6), although some myeloid related genes might be detected in
CLPs,
depending on the experimental conditions (Delogu A, Schebesta A, Sun Q,
Aschenbrenner
K, Perlot T, Busslinger M. Gene repression by Pax5 in B cells is essential for
blood cell
homeostasis and is reversed in plasma cells. Immunity. 2006 Mar;24(3):269-81).
171 Similarly, common myeloid progenitors (CNIPs) can give rise to all
classes of
myeloid cells with no or extensively low levels of B-cell potential (Akashi K,
Traver D,
Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives
rise to all
myeloid lineages. Nature. 2000 Mar 9;404(6774):193-7). Another cell type,
dendritic cells
(DCs), is not clearly grouped either in lymphoid or myeloid lineage, because
DC can arise
from either CLPs or CNIPs (Manz MG, Traver D, Miyamoto T, Weissman IL, Akashi
K.
Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood.
2001 Jun
1;97(11):3333-41, Traver D, Akashi K, Manz M, Merad M, Miyamoto T, Engleman
EG, et
al. Development of CD8alpha-positive dendritic cells from a common myeloid
progenitor.
Science (New York, NY. 2000 Dec 15;290(5499):2152-4). CMPs can proliferate and
differentiate into megakaryocyte-erythrocyte (MegE) progenitors and
granulocyte-monocyte
(GM) progenitors, which further give rise to megakaryocytes, erythrocytes,
granulocytes,
monocytes and others. (Iwasaki H, Akashi K. Myeloid lineage commitment from
the
hematopoietic stem cell. Immunity. 2007;26:726-740).
[8] It is likely that differences in the expression levels of
transcription factors
determine the lineage affiliation of a differentiating cell. The transcription
factors PU.1 and
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GATA-1 have been implicated in myeloid and erythroid/megakaryocyte lineage
differentiation, respectively (Gordon, M. Stem cells and haemopoiesis. In:
Hoffbrand, V.,
Catovsky, D., Tuddenham, E.G., 5th ed. Blackwell Publishing, (2005):
Differential niche and
Wnt requirements during acute myeloid leukemia, pp. 1-12. New York.).
Characterization of HSCs
[9] HSCs are undifferentiated and resemble small lymphocytes. A large
fraction
of HSCs is quiescent, in the GO phase of the cell cycle, which protects them
from the action
of cell cycle-dependent drugs. The quiescent state of stem cells is maintained
by
transforming growth factor-0 (TGF-0). The activity of TGF-0 is mediated by
p53, a tumor
suppressor gene that regulates cell proliferation and targets the cyclin-
dependent kinase
inhibitor p21 (Gordon, M. Stem cells and haemopoiesis. In: Hoffbrand, V.,
Catovsky, D.,
Tuddenham, E.G., 5th ed. Blackwell Publishing, (2005): Differential niche and
Wnt
requirements during acute myeloid leukemia, pp. 1-12. New York.). Quiescence
of HSCs is
critical not only for protecting the stem cell compartment and sustaining stem
cell pools
during long periods of time, but also for minimizing the accumulation of
replication-
associated mutations. Many of the intrinsic transcriptional factors that
maintain HSCs
quiescence are found to be associated with leukemias. For example, chromosomal
translocations resulting in the fusion of Fox0s and myeloid/lymphoid or mixed
lineage
leukemia have been reported in acute myeloid leukemias (See, e.g., Sergio
Paulo Bydlowski
and Felipe de Lara Janz (2012). Hematopoietic Stem Cell in Acute Myeloid
Leukemia
Development, Advances in Hematopoietic Stem Cell Research, Dr. Rosana Pelayo
(Ed.),
ISBN: 978-953-307-930-1).
[10] The majority of normal HSCs are present among the CD34+/CD38-/CD90+
bone marrow cell fractions with some HSCs also observed among CD34-/Lin-
cells.
CD34+/CD38+ cell fractions contain some HSCs endowed with short-term
repopulating
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activity. Other recognized markers include the tyrosine kinase receptor c-kit
(CD117)
coupled with a lack of terminal differentiation markers such as CD4 and CD8
(Rossi et al.,
Methods in Molecular Biology (2011) 750(2): 47-59).
Classification of HSCs.
1111 The hematopoietic stem cell pool can be subdivided into three main
groups:
(1) short-term HSCs, capable of generating clones of differentiating cells for
only 4-6
weeks; (2) intermediate-term HSCs, capable of sustaining a differentiating
cell progeny for
6-8 months before becoming extinct; and (3) long-term HSCs, capable of
maintaining
hematopoiesis indefinitely. (Testa U. Annals of Hematology (2011) 90(3): 245-
271).
Hematopoiesis
[12] Hematopoiesis is a highly coordinated process wherein HSCs differentiate
into
mature blood cells supported by a specialized regulatory microenvironment,
consisting of
components which control the fate specification of stem and progenitor cells,
as well as
maintaining their development by supplying the requisite factors ("niche").
The term "bone
marrow (BM) niche" as used herein refers to a well-organized architecture
composed of
elements (e.g., osteoblasts, osteoclasts, bone marrow endothelial cells,
stromal cells,
adipocytes and extracellular matrix proteins (ECM)) that play an essential
role in the
survival, growth and differentiation of diverse lineages of blood cells. The
bone marrow
niche is an important post-natal microenvironment in which HSCs proliferate,
mature and
give rise to myeloid and lymphoid progenitors.
[13] Bone marrow (BM) is present in the medullary cavities of all animal
bones. It
consists of a variety of precursor and mature cell types, including
hematopoietic cells (the
precursors of mature blood cells) and stromal cells (the precursors of a broad
spectrum of
connective tissue cells), both of which appear to be capable of
differentiating into other cell
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types. The mononuclear fraction of bone marrow contains stromal cells,
hematopoietic
precursors, and endothelial precursors.
[14] Unlike secondary lymphoid organs such as spleen with distinct gross
structures including red and white pulp, BM has no clear structural features,
except for the
endosteum that contains osteoblasts. The endosteum region comes in contact
with calcified
hard bones and provides a special microenvironment which is necessary for the
maintenance
of HSC activity (Kondo M, Immunology Reviews (2010) 238(1): 37-46; Sergio
Paulo
Bydlowski and Felipe de Lara Janz (2012). Hematopoietic Stem Cell in Acute
Myeloid
Leukemia Development, Advances in Hematopoietic Stem Cell Research, Dr. Rosana
Pelayo (Ed.), ISBN: 978-953-307-930-1).
[15] Within the niche, HSCs are believed to receive support and growth signals
originating from several sources, including: fibroblasts, endothelial and
reticular cells,
adipocytes, osteoblasts and mesenchymal stem cells (MSCs). The main function
of the
niche is to integrate local changes in nutrients, oxygen, paracrine and
autocrine signals and
to change HSCs quiescence, trafficking, and/ or expansion in response to
signals from the
systemic circulation (Broner, F. & Carson, M C. Topics in bone biology.
Springer. 2009; 4:
pp. 2-4. New York, USA.).
[16] Although the nature of true MSCs remains misunderstood, CXC chemokine
ligand 12 (CXCL12) - expressing CD146 MSCs were recently reported to be self-
renewing
progenitors that reside on the sinusoidal surfaces and contribute to
organization of the
sinusoidal wall structure, produce angiopoietin-1 (Ang-1), and are capable of
generating
osteoblasts that form the endosteal niche (Konopleva, MY, & Jordan, CT,
Biology and
Therapeutic Targeting (2011) 9(5): 591-599). These CXCL12 reticular cells may
serve as a
transit pathway for shuttling HSCs between the osteoblastic and vascular
niches where
essential but different maintenance signals are provided.
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[17] Cytokines and chemokines produced by bone marrow MSCs concentrate in
particular niches secondary to varying local production and through the
effects of cytokine-
binding glycosaminoglycans. Of these, CXCL12/stromal cell-derived factor-1
alpha
positively regulates HSCs homing, while transforming growth factors FMS-like
tyrosine
kinase 3 (F1t3) ligand and Ang-1 act as quiescence factors (See, e.g., Sergio
Paulo
Bydlowski and Felipe de Lara Janz (2012). Hematopoietic Stem Cell in Acute
Myeloid
Leukemia Development, Advances in Hematopoietic Stem Cell Research, Dr. Rosana
Pelayo (Ed.), ISBN: 978-953-307-930-1). CXCL12-CXCR4 signaling is involved in
homing
of HSCs into BM during ontogeny as well as survival and proliferation of
colony-forming
progenitor cells. The CXCR4-selective antagonist-induced mobilization of HSCs
into the
peripheral blood further indicates a role for CXCL12 in retaining HSCs in
hematopoietic
organs.
[18] BM engraftment involves subsequent cell-to-cell interactions through the
BMSC-produced complex extracellular matrix. Thus, vascular cell adhesion
molecule-1
(VCAM-1) or fibronectin is critical for adhesion to the BM derived MSCs. In
this way, the
control of hematopoietic stem cell proliferation kinetics is critically
important for the
regulation of correct hematopoietic cell production. These control mechanisms
could be
classified as intrinsic or extrinsic to the stem cells, or a combination of
both (See, e.g.,
Sergio Paulo Bydlowski and Felipe de Lara Janz (2012). Hematopoietic Stem Cell
in Acute
Myeloid Leukemia Development, Advances in Hematopoietic Stem Cell Research,
Dr.
Rosana Pelayo (Ed.), ISBN: 978-953-307-930-1).
[19] HSC self-renewal and differentiation can be controlled by external
factors
(extrinsic control), such as cell-cell interactions in the hematopoietic
microenvironment or
cytokines, such as SCF (stem cell factor) and its receptor c-kit, Flt-3
ligand, TGF-f3, TNF-a
and others. Cytokines regulate a variety of hematopoietic cell functions
through the
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activation of multiple signal transduction pathways. The major pathways
relevant to cell
proliferation and differentiation are the Janus kinase (Jak)/signal
transducers and activators
of transcription (STATs), the mitogen-activated protein (MAP) kinase and the
phosphatidylinositol (PI) 3-kinase pathways (Sergio Paulo Bydlowski and Felipe
de Lara
Janz (2012). Hematopoietic Stem Cell in Acute Myeloid Leukemia Development,
Advances
in Hematopoietic Stem Cell Research, Dr. Rosana Pelayo (Ed.), ISBN: 978-953-
307-930-1).
[20] In addition, expression of other transcription factors, such as, stem
cell
leukemia (SCL) hematopoietic transcription factor; GATA-2; and gene products
involved in
cell cycle control, such as the cyclin dependent kinase inhibitors (CKIs) p16,
p21 and p27
have been shown to be essential for hematopoietic cell development from the
earliest stages
(intrinsic control), (Sergio Paulo Bydlowski and Felipe de Lara Janz (2012).
Hematopoietic
Stem Cell in Acute Myeloid Leukemia Development, Advances in Hematopoietic
Stem Cell
Research, Dr. Rosana Pelayo (Ed.), ISBN: 978-953-307-930-1).
[21] Notch-1-Jagged pathway may serve to integrate extracellular signals with
intracellular signaling and cell cycle control. Notch-1 is a surface receptor
on hematopoietic
stem cell membranes that binds to its ligand. Jagged, on stromal cells. This
results in
cleavage of the cytoplasmic portion of Notch-1, which can then act as a
transcription factor
(Gordon, M. Stem cells and haemopoiesis. In: Hoffbrand, V., Catovsky, D.,
Tuddenham,
E.G., 5th ed. Blackwell Publishing, (2005): Differential niche and Wnt
requirements during
acute myeloid leukemia, pp. 1-12. New York.).
Disorders that are treated using Bone Marrow (BM)/Hematopoietic Stem Cell
(HSC)
transplantation
[22] Disorders that are treated using Bone Marrow (BM)/Hematopoietic Stem Cell
(HSC) transplantation include, without limitation, Acute Myeloid Leukemia
(AML), Acute
Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic
Myeloid
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Leukemia (CML), peripheral T cell lymphoma, follicular lymphoma, diffuse large
B cell
lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, neuroblastoma, non-malignant
inherited and acquired marrow disorders (e.g. sickle cell anemia, beta-
thalassemia major,
refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic
severe aplastic
anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, Fanconi
anemia,
amegakaryocytosis, or congenital thrombocytopenia), multiple myeloma, and
Severe
Combined Immunodeficiency (SCID).
Hematopoietic Malignancies
[23] Most hematopoietic malignancies comprise functionally heterogeneous
cells,
with only a subset, known as cancer stem cells, responsible for tumor
maintenance. Cancer
stem cells are so named because they possess qualities reminiscent of normal
tissue stem
cells including self-renewal, prolonged survival, and the ability to give rise
to cells with
more differentiated characteristics (Jones RJ and Armstrong SA, Biol Blood
Marrow
Transplant. 2008 Jan; 14 (Supplement 1): 12-16).
[24] A transforming event in hematopoietic stem cells can produce several
different
malignancies, including, without limitation, chronic myeloid leukemia,
myelodysplastic
syndrome, acute myeloid leukemia, and probably even acute lymphocytic
leukemia,
depending on the degree of differentiation associated with the oncogenic hit
(Jones RJ and
Armstrong SA, Biol Blood Marrow Transplant. 2008 Jan; 14 (Supplement 1): 12-
16).
[25] The cancer stem cell concept is based on the idea that tumors of a
specific
tissue often appear to "attempt" to recapitulate the cellular heterogeneity
found in the tissues
of origin, and thus there are cells in the tumor that are stem-cell like
giving rise to the varied
cell types. A fundamental test for this hypothesis is whether tumor cells can
be separated
into those that have the ability to regenerate the tumor, and those that do
not possess this
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ability. This cellular hierarchy has been most clearly demonstrated in acute
myelogenous
leukemias where some AMLs possess cells with a unique immunophenotype that are
able to
initiate leukemias in immunodeficient mice whereas most cells are unable to
initiate
leukemia development. Furthermore, the cells that initiate leukemias also give
rise to cells
that have lost tumor-initiating activity and thus recapitulate the cellular
heterogeneity found
in the original tumor (Lapidot T et al., Nature. 1994; 367: 645-648; Bonnet D
et al., Nat
Med. 1997; 3: 730-737).
Acute Myeloid Leukemia
[26] Acute myeloid leukemia (AML) is a clonal disorder characterized by arrest
of
differentiation in the myeloid lineage coupled with an accumulation of
immature progenitors
in the bone marrow, resulting in hematopoietic failure (Pollyea DA et al.,
British Journal of
Haematology (2011) 152(5): 523-542). There is wide patient-to-patient
heterogeneity in the
appearance of the leukemic blasts. The discovery of leukemia-initiating cells
in acute
myeloid leukemias (AMLs) started with the discovery that the large majority of
AML blasts
do not proliferate and only a small minority is capable of forming new
colonies (Testa U,
Annals of Hematology (2011) 90(3): 245-271). A common feature to all AML cases
is the
arrested aberrant differentiation leading to an accumulation of more than 20%
blast cells in
the bone marrow (Gilliland, DG and Tallman MS, Cancer Cell (2002) 1(5): 417-
420).
[27] More than 80% of myeloid leukemias are associated with at least one
chromosomal rearrangement (Pandolfi PP, Oncogene (2001) 20(40): 5726-5735),
and over
100 different chromosomal translocations have been cloned (Gilliland, DG and
Tallman MS,
Cancer Cell (2002) 1(5): 417-420). These translocations frequently involve
genes encoding
transcription factors that have been shown to play an important role in
hematopoietic lineage
development. Thus, alteration of the transcriptional machinery appears to be a
common
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PCT/US2020/041095
mechanism leading to arrested differentiation (Pandolfi PP, Oncogene (2001)
20(40): 5726-
5735; Tenen DG, Nature Reviews of Cancer (2003) 3(2): 89-101).
[28] Clinical investigation and experimental animal models suggest that at
least
two genetic alterations are required for the clinical manifestation of acute
leukemia.
According to the model proposed by Gilliland & Tallman (Cancer Cell (2002)
1(5): 417-
420), cooperation between class I activating mutations and class II mutations
that induce
termination of differentiation give rise to AML. The class I mutations, such
as mutations in
the receptor tyrosine kinase genes FLT3 and KIT, RAS family members, and loss
of
function of neurofibromin 1, confer proliferative and/or survival advantage to
hematopoietic
progenitors, typically as a consequence of aberrant activation of signal
transduction
pathways. The class II mutations lead to a halt in differentiation via
interference with
transcription factors or co-activators (Frankfurt 0 et al., Current Opinion in
Oncology
(2007) 19(6): 635-649).
[29] While the leukemia stem cell (LSC) appears to share many of the cell
surface
markers previously identified for HSC such as CD34, CD38, HLA-DR, and CD71,
several
groups have reported surface markers that are differentially expressed in the
two
populations.
[30] For example, CD90 or Thy-1 has been described as potentially specific of
the
LSC compartment. Thy-1 is downregulated in normal hematopoiesis as the most
primitive
stem cells progress toward the progenitor stage. (Hope KJ et al., Archives of
Medical
Research (2003) 34(6): 507-514).
[31] The interaction between CXCL12 (stromal cell-derived factor-1 alpha) and
its
receptor CXCR4 on leukemic progenitor cells contributes to their homing to the
bone
marrow microenvironment. CXCR4 levels are significantly elevated in leukemic
cells from
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patients with AML, and CXCR4 expression is associated with poor outcome
(Konopleva
MY and Jordan CT, Biology and Therapeutic Targeting (2011) 29(5): 591-599).
[32] Constitutive activation of the nuclear factor kappa f3 (NF-k13) pathway
in
primary human AML stem cells provided evidence that NF-k13 plays a significant
role in the
overall survival of LSCs as well as AML cell types in general. (Konopleva MY
and Jordan
CT, Biology and Therapeutic Targeting (2011) 29(5): 591-599).
[33] FLT3, a member of the class III tyrosine kinase receptor family, is
expressed
in normal hematopoietic progenitors as well as in leukemic blasts, and it
plays an important
role in cell proliferation, differentiation, and survival. Activation of the
FLT3 receptor by
the FLT3 ligand leads to receptor dimerization and phosphorylation, and
activation of
downstream signaling pathways, including the Janus kinase (JAK) 2 signal
transducer
(JAK2), signal transducer and activator of transcription (STAT) 5, and mitogen-
activated
protein kinase (MAPK) pathways. Mutations in the FLT3 gene, found in
approximately 40%
of patients with AML, are believed to promote its autophosphorylation and
constitutive
activation, leading to ligand-independent proliferation (Frankfurt 0 et al.,
Current Opinion
in Oncology (2007) 19(6): 635-649).
Lymphoid Malignancies
[34] Self-renewal capacity in most tissues is lost as cells progress through
their
normal stages of differentiation; for example, myeloid lineage blood cells
beyond the level
of hematopoietic stem cells no longer possess self-renewal capacity. A notable
exception to
differentiation-associated loss of self-renewal is the lymphoid system, where
self-renewal
capacity is preserved until the memory lymphocyte stage in order to maintain
life-long
immune memory (Fearon DT et al., Science. 2001; 293: 248-250; Luckey CJ et
al., Proc
Natl Acad Sci U S A. 2006; 103: 3304-3309). Somatic hypermutation serves as a
marker for
the stage of differentiation at which B cell malignancies arise. In general,
the presence of
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somatic hypermutation identifies a tumor as having arisen in germinal center
or post-
germinal center B cells, while the absence of mutation identifies pre-germinal
center B cells.
In contrast to myeloid malignancies but consonant with the lineage's preserved
self-renewal
capacity, immunoglobulin (Ig) mutation patterns suggest that B cell
malignancies can arise
from cells throughout the stages of B cell differentiation (Lapidot T et al.,
Nature. 1994;
367: 645-648; Bonnet D and Dick JE, Nat Med. 1997; 3: 730-737; Jones RJ et
al., J Natl
Cancer Inst. 2004; 96: 583-585).
[35] Multiple myeloma (MM) has generally been considered a disease of
malignant
plasma cells with many of the clinical consequences of the disease resulting
from the plasma
cell bulk. However, normal plasma cells are terminally differentiated and lack
self-renewal
capacity and it has been clear for over 30 years that only a minority of cells
from mouse and
human MM were clonogenic. These rare clonogenic cells have been termed "tumor
stem
cells" (Park CH et al., J Natl Cancer Inst. 1971; 46: 411-422; Hamburger AW
and Salmon
SE, Science. 1977; 197: 461-463). MINI plasma cells arise from a small
population of self-
renewing cancer stem cells that resemble memory B cells. Not only do these
clonotypic B
cells circulate in most patients but they also are resistant to many standard
anti-MNI agents,
and thus appear to be responsible for most disease relapses (Matsui WH et al.,
Blood. 2004;
103: 2332-2336; Kukrej a A et al., J Exp Med. 2006; 203: 1859-1865; Jones RJ
and
Armstrong SA, Biol Blood Marrow Transplant. 2008 Jan; 14 (Supplement 1): 12-
16).
[36] Reed-Sternberg (RS) cells, the hallmark of Hodgkin's lymphoma (HL), are
the
only blood cells other than plasma cells to occasionally express CD138
(Carbone A et al.,
Blood. 1998; 92: 2220-2228). It has been shown that HL cell lines include a
small
population of cells that lack the RS markers CD15 and CD30 present on the rest
of the cells,
while expressing markers consistent with a memory B cell phenotype (Newcom SR
et al.,
Int J Cell Cloning. 1988; 6:417-431; Jones RJ et al., Blood. 2006; 108: 470).
This small
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subpopulation of phenotypic memory B cells possessed all of the clonogenic
capacity within
the HL cell lines. Most HL patients, including those with early stage disease,
harbor
circulating memory B cells with the same clonal Ig gene rearrangement as the
patients' RS
cells (Jones RJ et al., Blood. 2006; 108: 470; Jones RJ and Armstrong SA, Biol
Blood
Marrow Transplant. 2008 Jan; 14 (Supplement 1): 12-16). These data suggest
that these
clonotypic memory B cells likely represent the HL stem cells.
[37] Hematopoietic stem cells (HSCs) are used in bone marrow transplantation
for
treatment of hematological malignancies as well as nonmalignant disorders
(Warner et al,
Oncogene (2004) 23(43): 7164-7177). Until researchers discovered which
cellular
components were responsible for the engraftment of the donor hematopoietic and
immune
systems in marrow-ablated patients, bone marrow (BM) had been transplanted as
an
unfractionated cell pool for many years (See, e.g., Sergio Paulo Bydlowski and
Felipe de
Lara Janz (2012). Hematopoietic Stem Cell in Acute Myeloid Leukemia
Development,
Advances in Hematopoietic Stem Cell Research, Dr. Rosana Pelayo (Ed.), ISBN:
978-953-
307-930-1).
[38] Preparation or conditioning of a patient for bone marrow/hematopoietic
stem
cell (BM/HSC) transplant is a critical element of the procedure. It serves two
main
purposes: (1) it provides adequate immunosuppression of the patient and clears
sufficient
niche space in the bone marrow for the transplanted HSC, which allows
transplanted cells to
engraft in the recipient; and (2) it often helps to eradicate the source of
the malignancy.
[39] Conditioning of patients has traditionally been achieved by administering
maximally tolerated doses of a cocktail of chemotherapeutic agents with or
without
radiation. Components of the cocktail are often chosen to have non-overlapping
toxicities.
All preparative regimens currently in use are toxic and have severe side
effects that can be
life threatening. Among these side effects are mucositis, nausea and vomiting,
alopecia,
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diarrhea, rash, peripheral neuropathies, infertility, pulmonary toxicities and
hepatic
toxicities. Many of these side effects are especially dangerous for older and
sick patients,
and often become a decisive component in deciding whether a patient will
receive a
transplant.
[40] Thus, a need exists to prepare or condition patients eligible for bone
marrow/hematopoietic stem cell (BM/HSC) transplant without these toxicities.
The
described invention provides compositions and methods for eliminating
hematopoietic stem
cells/hematopoietic progenitors (HSC/HP) in a patient using bi-specific
antibodies that bind
to human tyrosine kinase receptor FLT3/FLK2 receptor protein and to CD3
receptor protein
expressed on T-cells.
SUMMARY OF THE INVENTION
[41] According to one aspect, the described invention provides a method for
preparing or conditioning a patient in need thereof for hematopoietic cell
transplantation
comprising: providing a recombinant single chain bi-specific antibody that
binds to both
human FLT3 and human CD3, and administering a therapeutic amount of a
pharmaceutical
composition comprising the bi-specific antibody to the patient; wherein the
therapeutic
amount is effective: to reduce by at least 90% a level in peripheral blood of
a cell
population expressing one or more of CD45, CD3, FLT3, CD19, CD33, and to
reduce
toxicity of protocols for preparing or conditioning the patient.
[42] According to one embodiment, an amino acid sequence of a heavy chain of
an
antigen-binding portion of the bispecific antibody that binds FLT3 is SEQ ID
NO: 1 and an
amino acid sequence of a light chain of the antigen-binding portion of the
bispecific
antibody that binds FLT3 is SEQ ID NO: 2. According to another embodiment, the
bi-
specific antibody comprises a monoclonal antibody that reacts with a subunit
of human
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CD3. According to another embodiment, the bi-specific antibody or antigen-
binding portion
thereof comprises an isotype selected from the group consisting of an
immunoglobulin G
(IgG), an IgM, an IgE, an IgA, and an IgD isotype.
[43] According to one embodiment, the effective amount comprises 0.01 mg/kg to
mg/kg, better 0.05 mg/kg to 2 mg/kg, better 0.1 mg/kg to 0.5 mg/kg, better 0.1
mg/kg to
0.3 mg/kg, better 0.1 mg/kg.
[44] According to one embodiment, the patient in need thereof is suffering
from
acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic
myeloid
leukemia (CLL), CML, peripheral T cell lymphoma, follicular lymphoma, diffuse
large B
cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, neuroblastoma, a non-
malignant inherited and acquired marrow disorder, multiple myeloma, or SCID.
According
to another embodiment, the non-malignant inherited and acquired marrow
disorder is
selected from sickle cell anemia, beta-thalassemia major, refractory Diamond-
Blackfan
anemia, myelodysplastic syndrome, idiopathic severe aplastic anemia,
paroxysmal nocturnal
hemoglobinuria, pure red cell aplasia, Fanconi anemia, amegakaryocytosis, and
congenital
thrombocytopenia.
[45] According to one embodiment, the composition further comprises an
antitumor agent.
[46] According to one embodiment, the bispecific antibody is a humanized
antibody.
[47] According to another aspect, the described invention provides a method
for
preparing a recombinant single chain bi-specific antibody that binds to both
human FLT3
and human CD3 comprising: joining a C-terminus of an Fab antigen-binding
fragment of an
Flt3 monoclonal antibody to a CH2 domain of IgGl, and joining to the CH2
domain of the
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IgG1 a single chain variable fragment (ScFv) of a monoclonal antibody that
reacts with a
subunit of human CD3 (UCHT1).
[48] According to another aspect, the described invention provides a
recombinant
single chain bi-specific antibody that binds to both human FLT3 and human CD3
comprising: a C-terminus of an Fab antigen-binding fragment of an Flt3
monoclonal
antibody that is joined to a CH2 domain of IgGl, and a single chain variable
fragment
(ScFv) of a monoclonal antibody that reacts with a subunit of human CD3
(UCHT1) joined
to the CH2 domain of the IgG1 .
[49] According to one embodiment, an amino acid sequence of a heavy chain
binding domain of the Fab antigen-binding fragment is SEQ ID NO: 1 (H3113) and
an
amino acid sequence of a light chain binding domain of the Fab antigen-binding
fragment is
SEQ ID NO: 2 (L3133).
[50] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 5 and an amino acid sequence of a
heavy chain
of the antigen-binding portion of the antibody or fragment thereof that binds
to human
FLT3/FLK2 receptor protein is SEQ ID NO: 7.
[51] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 9 and an amino acid sequence of a
heavy chain
of the antigen-binding portion of the antibody or fragment thereof that binds
to human
FLT3/FLK2 receptor protein is SEQ ID NO: 11.
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[52] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 13 and an amino acid sequence of a
heavy
chain of the antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 15.
[53] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 17 and an amino acid sequence of a
heavy
chain of the antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 19.
[54] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 21, and an amino acid sequence of a
heavy
chain of the antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 23.
[55] According to another aspect, the described invention provides a
monoclonal
antibody or antigen binding fragment thereof, wherein an amino acid sequence
of a light
chain of an antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 25, and an amino acid sequence of a
heavy
chain of the antigen-binding portion of the antibody or fragment thereof that
binds to human
FLT3/FLK2 receptor protein is SEQ ID NO: 27.
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[56] According to some embodiments, a half maximal effective concentration
(EC50) of the antibody or fragment thereof is between 1 ng/mL (6.25 pM) and
2,000 ng/mL
(12.5nM). According to some embodiments, the half maximal effective
concentration
(EC50) of the antibody or antigen-binding fragment thereof is between 10 ng/mL
(62.5 pM)
and 200 ng/mL (1.25nM). According to some embodiments, FLT3 antibody binding
to
human FLT3/FLK2 receptor protein on a cell is effective for the cell to
internalize the bound
antibody or antigen-binding fragment.
BRIEF DESCRIPTION OF THE DRAWINGS
[57] FIGS. 1A, 1B, 1C. FIGS. 1A and 1B: native fluorescence of amino acids
such
as phenylalanine, tyrosine and tryptophan. FIG. 1C: measurement of purity of
the
synthesized antibody.
[58] FIGS. 2A, 2B, 2C, 2D. Administering bi-specific antibodies that bind to
FLT3/FLK2 expressed by HSC/HP and CD3 expressed by T-cells reduces the level
of
chimerism in the peripheral blood in the humanized immune-compromised mice.
FIG. 2A:
an example of flow cytometry analysis of the peripheral blood of a humanized
NOG mouse
before (Control; upper row) and three weeks after application of CD3-FLT3 bi-
specific
antibodies. From left to right: analyses of the amounts of human hCD45+ cells
(% of total
CD45+ cells), human hCD3+ cells (% of total hCD45+ cells; T-cells), human
hCD19+ cells
(% of total hCD45+ cells; B-cells), human hCD33+ cells (% of total hCD45+
cells; Myeloid
cells). FIG. 2B: effect of bi-specific antibodies administration on the level
of chimerism in
the peripheral blood in the humanized mice (n=27). FIG. 2C: effect of bi-
specific antibodies
administration on the levels of T-cells (% hCD3+ cells of total hCD45+ cells),
B-cells (%
hCD19+ cells of total hCD45+ cells) and myeloid lineages (% hCD33+ cells of
total
hCD45+ cells) in the peripheral blood (n=27). FIG. 2D: reduced effect of bi-
specific
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antibodies application in a humanized immune-compromised mouse (labeled with
asterisks
in C) with the reduced amount of human hCD3+ cells (n=3).
[59] FIGS. 3A and 3B. Screening of culture supernatants from clonally expanded
hybridomas. FIG. 3A: fluorescence intensity histograms obtained from flow
cytometry
analysis of supernatants of nine positive hybridoma clones. The supernatants
show immune-
reactivity against FLT3/FLK2 expressed by REH (human B cell precursor leukemia
cells,
established from the peripheral blood of a 15 year old girl with ALL at first
relapse) cells.
FIG. 3B: a table showing median fluorescence intensity (MFI) of the histograms
in FIG. 3A.
All nine clones reacted with REH cells that express human FLT3/FLK2 receptor
proteins.
[60] FIGS. 4A and 4B. Screening of purified monoclonal antibodies from
expanded hybridomas. FIG. 4A: fluorescence intensity histograms obtained from
flow
cytometry analysis of purified monoclonal antibodies from nine positive
hybridoma clones.
The supernatants show immune-reactivity to the human FLT3/FLK2 receptor
protein
expressed by 5P2/0 cells. Monoclonal antibodies were non-reactive with wild-
type 5P2/0
cells that do not express human FLT3/FL2 receptor protein. FIG. 4B: a table
showing
median fluorescence intensity (MFI) of the histograms in FIG. 4A. All nine
clones reacted
with 5P2/0 cells that express human FLT3/FLK2 receptor proteins and did not
react with
wild-type 5P2/0 cells.
[61] FIGS. 5A, 5B, 5C, 5D, and 5E. Affinity of anti-human FLT3/FLK2
antibodies determined by Effective Concentration (EC) curve using flow
cytometry. FIG.
5A: antibody clone Ab2-81. FIG. 5B: antibody clone Ab1-23DA. FIG. 5C: antibody
clone
Ab3-16HA. FIG. 5D: antibody clone Ab0-30A. FIG. 5E: antibody clone Ab1-18New.
[62] FIG. 6. Time Course of Anti-FLT3/FLK2 Antibody Internalization. Mean
fluorescent intensity (MFI) of monoclonal mouse anti-human CD135 antibodies
was
detected with a secondary Alexa Fluor 488 plotted versus time for the live Reh
cell
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population. Internalization assays were conducted at 37 C in parallel with
control cells that
were kept on ice at 4 C for 10, 30, 60 and 120 minutes. The percent change in
MFI for each
antibody (clones 123D, A281A, 330A and 316HA) was graphed versus time at 4 C
and
37 C in triplicate over 2 hours, with MFI at 10 minutes set to 100%.
DETAILED DESCRIPTION OF THE INVENTION
Glossary
[63] The term "activation" or "lymphocyte activation" refers to stimulation of
lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells
resulting in
synthesis of RNA, protein and DNA and production of lymphokines; it is
followed by
proliferation and differentiation of various effector and memory cells. For
example, a
mature B cell can be activated by an encounter with an antigen that expresses
epitopes that
are recognized by its cell surface immunoglobulin Ig). The activation process
may be a
direct one, dependent on cross-linkage of membrane Ig molecules by the antigen
(cross-
linkage-dependent B cell activation) or an indirect one, occurring most
efficiently in the
context of an intimate interaction with a helper T cell ("cognate help
process"). T-cell
activation is dependent on the interaction of the TCR/CD3 complex with its
cognate ligand,
a peptide bound in the groove of a class I or class II MHC molecule. The
molecular events
set in motion by receptor engagement are complex. Among the earliest steps
appears to be
the activation of tyrosine kinases leading to the tyrosine phosphorylation of
a set of
substrates that control several signaling pathways. These include a set of
adapter proteins
that link the TCR to the ras pathway, phospholipase Cyl, the tyrosine
phosphorylation of
which increases its catalytic activity and engages the inositol phospholipid
metabolic
pathway, leading to elevation of intracellular free calcium concentration and
activation of
protein kinase C, and a series of other enzymes that control cellular growth
and
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differentiation. Full responsiveness of a T cell requires, in addition to
receptor engagement,
an accessory cell-delivered costimulatory activity, e.g., engagement of CD28
on the T cell
by CD80 and/or CD86 on the antigen presenting cell (APC). The soluble product
of an
activated B lymphocyte is immmunoglobulins (antibodies). The soluble product
of an
activated T lymphocyte is lymphokines.
[64] The term "administer" as used herein means to give or to apply. The term
"administering" as used herein and its various grammatical forms includes in
vivo
administration, as well as administration directly to tissue ex vivo.
Antibodies:
[65] Antibodies are serum proteins the molecules of which possess small areas
of
their surface that are complementary to small chemical groupings on their
targets. These
complementary regions (referred to as the antibody combining sites or antigen
binding sites)
of which there are at least two per antibody molecule, and in some types of
antibody
molecules ten, eight, or in some species as many as 12, may react with their
corresponding
complementary region on the antigen (the antigenic determinant or epitope) to
link several
molecules of multivalent antigen together to form a lattice.
[66] The basic structural unit of a whole antibody molecule consists of four
polypeptide chains, two identical light (L) chains (each containing about 220
amino acids)
and two identical heavy (H) chains (each usually containing about 440 amino
acids). The
two heavy chains and two light chains are held together by a combination of
noncovalent
and covalent (disulfide) bonds. The molecule is composed of two identical
halves, each
with an identical antigen-binding site composed of the N-terminal region of a
light chain and
the N-terminal region of a heavy chain. Both light and heavy chains usually
cooperate to
form the antigen binding surface.
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[67] Human antibodies show two kinds of light chains, lc and X.; individual
molecules of immunoglobulin generally are only one or the other. In normal
serum, 60% of
the molecules have been found to have lc determinants and 30 percent X.. Many
other species
have been found to show two kinds of light chains, but their proportions vary.
For example,
in the mouse and rat, X. chains comprise but a few percent of the total; in
the dog and cat, lc
chains are very low; the horse does not appear to have any lc chain; rabbits
may have 5 to
40% X., depending on strain and b-locus allotype; and chicken light chains are
more
homologous to X. than K.
[68] In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG, and
IgM,
each with its own class of heavy chain ¨ a (for IgA), 6 (for IgD), c (for
IgE), y (for IgG) and
11 (for IgM). In addition, there are four subclasses of IgG immunoglobulins
(IgGl, IgG2,
IgG3, IgG4) having yl, y2, y3, and y4 heavy chains respectively. In its
secreted form, IgM
is a pentamer composed of five four-chain units, giving it a total of 10
antigen binding sites.
Each pentamer contains one copy of a J chain, which is covalently inserted
between two
adjacent tail regions.
[69] All five immunoglobulin classes differ from other serum proteins in that
they
show a broad range of electrophoretic mobility and are not homogeneous. This
heterogeneity ¨ that individual IgG molecules, for example, differ from one
another in net
charge ¨ is an intrinsic property of the immunoglobulins.
[70] An "antigenic determinant" or "epitope" is an antigenic site on a
molecule.
Sequential antigenic determinants/epitopes essentially are linear chains. In
ordered
structures, such as helical polymers or proteins, the antigenic
determinants/epitopes
essentially would be limited regions or patches in or on the surface of the
structure involving
amino acid side chains from different portions of the molecule which could
come close to
one another. These are conformational determinants.
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[71] The principle of complementarity, which often is compared to the fitting
of a
key in a lock, involves relatively weak binding forces (hydrophobic and
hydrogen bonds,
van der Waals forces, and ionic interactions), which are able to act
effectively only when the
two reacting molecules can approach very closely to each other and indeed so
closely that
the projecting constituent atoms or groups of atoms of one molecule can fit
into
complementary depressions or recesses in the other. Antigen-antibody
interactions show a
high degree of specificity, which is manifest at many levels. Brought down to
the molecular
level, "specificity" means that the combining sites of antibodies to an
antigen have a
complementarity not at all similar to the antigenic determinants of an
unrelated antigen.
Whenever antigenic determinants of two different antigens have some structural
similarity,
some degree of fitting of one determinant into the combining site of some
antibodies to the
other may occur, and that this phenomenon gives rise to cross-reactions. Cross
reactions are
of major importance in understanding the complementarity or specificity of
antigen-antibody
reactions. Immunological specificity or complementarity makes possible the
detection of
small amounts of impurities/contaminations among antigens
[72] "Monoclonal antibodies" (mAbs) can be generated by fusing mouse spleen
cells from an immunized donor with a mouse myeloma cell line to yield
established mouse
hybridoma clones that grow in selective media. A "hybridoma cell" is an
immortalized
hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell
with a myeloma
cell. "In vitro immunization", which refers to primary activation of antigen-
specific B cells
in culture, is another well-established means of producing mouse monoclonal
antibodies.
[73] Diverse libraries of immunoglobulin heavy (VH) and light (Vic and VX.)
chain
variable genes from peripheral blood lymphocytes also can be amplified by
polymerase
chain reaction (PCR) amplification. Genes encoding single polypeptide chains
in which the
heavy and light chain variable domains are linked by a polypeptide spacer
(single chain Fv
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or scFv) can be made by randomly combining heavy and light chain V-genes using
PCR. A
combinatorial library then can be cloned for display on the surface of
filamentous
bacteriophage by fusion to a minor coat protein at the tip of the phage.
[74] The technique of guided selection is based on human immunoglobulin V gene
shuffling with rodent immunoglobulin V genes. The method entails (i) shuffling
a repertoire
of human X, light chains with the heavy chain variable region (VH) domain of a
mouse
monoclonal antibody reactive with an antigen of interest; (ii) selecting half-
human Fabs on
that antigen (iii) using the selected X, light chain genes as "docking
domains" for a library of
human heavy chains in a second shuffle to isolate clone Fab fragments having
human light
chain genes; (v) transfecting mouse myeloma cells by electroporation with
mammalian cell
expression vectors containing the genes; and (vi) expressing the V genes of
the Fab reactive
with the antigen as a complete IgGl, X. antibody molecule in the mouse
myeloma.
[75] The term "antibody-dependent cell mediated cytotoxicity (ADCC)" as used
herein is triggered when antibody bound to the surface of a cell interacts
with Fc receptors
on a natural killer (NK) cells. NK cells express the receptor FcyRIII (CD16),
which
recognizes the IgG1 and IgG3 subclasses. The killing mechanism is analogous to
that of
cytotoxic T cells, involving the release of cytoplasmic granules containing
perforin and
granzymes (see below).
[76] CD3 (TCR complex) is a protein complex composed of four distinct chains.
In mammals, the complex contains a CD3y chain, a CD3 6 chain, and two CD3E
chains,
which associate with the T cell receptor (TCR) and the -chain to generate an
activation
signal in T lymphocytes. Together, the TCR, the -chain and CD3 molecules
comprise the
TCR complex. The intracellular tails of CD3 molecules contain a conserved
motif known as
the immunoreceptor tyrosine-based activation motif (ITAM), which is essential
for the
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signaling capacity of the TCR. Upon phosphorylation of the ITAM, the CD3 chain
can bind
ZAP70 (zeta associated protein), a kinase involved in the signaling cascade of
the T cell.
[77] The term "binding" and its various grammatical forms means a lasting
attraction between chemical substances. Binding specificity involves both
binding to a
specific partner and not binding to other molecules. Functionally important
binding may
occur at a range of affinities from low to high, and design elements may
suppress undesired
cross-interactions. Post-translational modifications also can alter the
chemistry and structure
of interactions. "Promiscuous binding" may involve degrees of structural
plasticity, which
may result in different subsets of residues being important for binding to
different partners.
"Relative binding specificity" is a characteristic whereby in a biochemical
system a
molecule interacts with its targets or partners differentially, thereby
impacting them
distinctively depending on the identity of individual targets or partners.
[78] The term "contact" and its various grammatical forms as used herein
refers to
a state or condition of touching or of immediate or local proximity.
Contacting a
composition to a target destination, such as, but not limited to, an organ, a
tissue, or a cell,
may occur by any means of administration known to the skilled artisan.
[79] The term "half maximal effective concentration" (EC50), as used herein
means
an antibody concentration that induces a response halfway between the baseline
and
maximum after an exposure time.
[80] The term "hematopoietic-cell transplantation" (HCT) is used herein to
refer to
blood and marrow transplantation (BMT), a procedure that involves infusion of
cells
(hematopoietic stem cells; also called hematopoietic progenitor cells) to
reconstitute the
hematopoietic system of a patient.
[81] The term "lymphocyte" refers to a small white blood cell formed in
lymphatic
tissue throughout the body and in normal adults making up about 22-28% of the
total
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number of leukocytes in the circulating blood that plays a large role in
defending the body
against disease. Individual lymphocytes are specialized in that they are
committed to
respond to a limited set of structurally related antigens. This commitment,
which exists
before the first contact of the immune system with a given antigen, is
expressed by the
presence on the lymphocyte's surface membrane of receptors specific for
determinants
(epitopes) on the antigen. Each lymphocyte possesses a population of
receptors, all of which
have identical combining sites. One set, or clone, of lymphocytes differs from
another clone
in the structure of the combining region of its receptors and thus differs in
the epitopes that it
can recognize. Lymphocytes differ from each other not only in the specificity
of their
receptors, but also in their functions.
[82] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-
cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-
cells),
B lymphocytes
[83] B-lymphocytes are derived from hematopoietic cells of the bone marrow. A
mature B-cell can be activated with an antigen that expresses epitopes that
are recognized by
its cell surface. The activation process may be direct, dependent on cross-
linkage of
membrane Ig molecules by the antigen (cross-linkage-dependent B-cell
activation), or
indirect, via interaction with a helper T-cell, in a process referred to as
cognate help. In
many physiological situations, receptor cross-linkage stimuli and cognate help
synergize to
yield more vigorous B-cell responses. (Paul, W. E., "Chapter 1: The immune
system: an
introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-
Raven
Publishers, Philadelphia (1999)).
[84] Cross-linkage dependent B-cell activation requires that the antigen
express
multiple copies of the epitope complementary to the binding site of the cell
surface receptors
because each B-cell expresses Ig molecules with identical variable regions.
Such a
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requirement is fulfilled by other antigens with repetitive epitopes, such as
capsular
polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-
dependent B-
cell activation is a major protective immune response mounted against these
microbes.
(Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental
Immunology,
4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia
(1999)).
[85] Cognate help allows B-cells to mount responses against antigens that
cannot
cross-link receptors and, at the same time, provides costimulatory signals
that rescue B cells
from inactivation when they are stimulated by weak cross-linkage events.
Cognate help is
dependent on the binding of antigen by the B-cell's membrane immunoglobulin
(Ig), the
endocytosis of the antigen, and its fragmentation into peptides within the
endosomal/lysosomal compartment of the cell. Some of the resultant peptides
are loaded
into a groove in a specialized set of cell surface proteins known as class II
major
histocompatibility complex (WIC) molecules. The resultant class II/peptide
complexes are
expressed on the cell surface and act as ligands for the antigen-specific
receptors of a set of
T-cells designated as CD4+ T-cells. The CD4+ T-cells bear receptors on their
surface
specific for the B-cell's class II/peptide complex. B-cell activation depends
not only on the
binding of the T cell through its T cell receptor (TCR), but this interaction
also allows an
activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-
cell (CD40)
signaling B-cell activation. In addition, T helper cells secrete several
cytokines that regulate
the growth and differentiation of the stimulated B-cell by binding to cytokine
receptors on
the B cell. (Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,
Philadelphia
(1999)).
[86] During cognate help for antibody production, the CD40 ligand is
transiently
expressed on activated CD4+ T helper cells, and it binds to CD40 on the
antigen-specific B
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cells, thereby tranducing a second costimulatory signal. The latter signal is
essential for B
cell growth and differentiation and for the generation of memory B cells by
preventing
apoptosis of germinal center B cells that have encountered antigen.
Hyperexpression of the
CD40 ligand in both B and T cells is implicated in the pathogenic autoantibody
production
in human SLE patients. (Desai-Mehta, A. et al., "Hyperexpression of CD40
ligand by B and
T cells in human lupus and its role in pathogenic autoantibody production," J.
Clin. Invest.,
97(9): 2063-2073 (1996)).
T-lymphocytes
[87] T-lymphocytes derive from precursors in hematopoietic tissue, undergo
differentiation in the thymus, and are then seeded to peripheral lymphoid
tissue and to the
recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide
range of
immunologic functions. These include the capacity to help B cells develop into
antibody-
producing cells, the capacity to increase the microbicidal action of
monocytes/macrophages,
the inhibition of certain types of immune responses, direct killing of target
cells, and
mobilization of the inflammatory response. These effects depend on their
expression of
specific cell surface molecules and the secretion of cytokines. (Paul, W. E.,
"Chapter 1: The
immune system: an introduction," Fundamental Immunology, 4th Edition, Ed.
Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia (1999)).
[88] T cells differ from B cells in their mechanism of antigen recognition.
Immunoglobulin, the B cell's receptor, binds to individual epitopes on soluble
molecules or
on particulate surfaces. B-cell receptors see epitopes expressed on the
surface of native
molecules. Antibody and B-cell receptors evolved to bind to and to protect
against
microorganisms in extracellular fluids. In contrast, T cells recognize
antigens on the surface
of other cells and mediate their functions by interacting with, and altering,
the behavior of
these antigen-presenting cells (APCs). There are three main types of antigen-
presenting
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cells in peripheral lymphoid organs that can activate T cells: dendritic
cells, macrophages
and B cells. The most potent of these are the dendritic cells, whose only
function is to
present foreign antigens to T cells. Immature dendritic cells are located in
tissues
throughout the body, including the skin, gut, and respiratory tract. When they
encounter
invading microbes at these sites, they endocytose the pathogens and their
products, and carry
them via the lymph to local lymph nodes or gut associated lymphoid organs. The
encounter
with a pathogen induces the dendritic cell to mature from an antigen-capturing
cell to an
antigen-presenting cell (APC) that can activate T cells. APCs display three
types of protein
molecules on their surface that have a role in activating a T cell to become
an effector cell:
(1) MEW proteins, which present foreign antigen to the T cell receptor; (2)
costimulatory
proteins which bind to complementary receptors on the T cell surface; and (3)
cell-cell
adhesion molecules, which enable a T cell to bind to the antigen-presenting
cell (APC) for
long enough to become activated. ("Chapter 24: The adaptive immune system,"
Molecular
Biology of the Cell, Alberts, B. et al., Garland Science, NY, 2002).
[89] T-cells are subdivided into two distinct classes based on the cell
surface
receptors they express. The majority of T cells express T cell receptors (TCR)
consisting of
a and 0 chains. A small group of T cells express receptors made of y and 6
chains. Among
the a/f3 T cells are two important sublineages: those that express the
coreceptor molecule
CD4 (CD4+ T cells); and those that express CD8 (CD8+ T cells). These cells
differ in how
they recognize antigen and in their effector and regulatory functions.
[90] CD4+ T cells are the major regulatory cells of the immune system. Their
regulatory function depends both on the expression of their cell-surface
molecules, such as
CD40 ligand whose expression is induced when the T cells are activated, and
the wide array
of cytokines they secrete when activated.
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[91] T cells also mediate important effector functions, some of which are
determined by the patterns of cytokines they secrete. The cytokines can be
directly toxic to
target cells and can mobilize potent inflammatory mechanisms.
[92] In addition, T cells particularly CD8+ T cells, can develop into
cytotoxic T-
lymphocytes (CTLs) capable of efficiently lysing target cells that express
antigens
recognized by the CTLs. (Paul, W. E., "Chapter 1: The immune system: an
introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers,
Philadelphia (1999)).
[93] T cell receptors (TCRs) recognize a complex consisting of a peptide
derived
by proteolysis of the antigen bound to a specialized groove of a class II or
class I MHC
protein. The CD4+ T cells recognize only peptide/class II complexes while the
CD8+ T
cells recognize peptide/class I complexes. (Paul, W. E., "Chapter 1: The
immune system:
an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven
Publishers, Philadelphia (1999)).
[94] The TCR's ligand (i.e., the peptide/MHC protein complex) is created
within
antigen-presenting cells (APCs). In general, class II MHC molecules bind
peptides derived
from proteins that have been taken up by the APC through an endocytic process.
These
peptide-loaded class II molecules are then expressed on the surface of the
cell, where they
are available to be bound by CD4+ T cells with TCRs capable of recognizing the
expressed
cell surface complex. Thus, CD4+ T cells are specialized to react with
antigens derived
from extracellular sources. (Paul, W. E., "Chapter 1: The immune system: an
introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers,
Philadelphia (1999)).
[95] In contrast, class I MHC molecules are mainly loaded with peptides
derived
from internally synthesized proteins, such as viral proteins. These peptides
are produced
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from cytosolic proteins by proteolysis by the proteosome and are translocated
into the rough
endoplasmic reticulum. Such peptides, generally nine amino acids in length,
are bound into
the class I MHC molecules and are brought to the cell surface, where they can
be recognized
by CD8+ T cells expressing appropriate receptors. This gives the T cell
system, particularly
CD8+ T cells, the ability to detect cells expressing proteins that are
different from, or
produced in much larger amounts than, those of cells of the remainder of the
organism (e.g.,
vial antigens) or mutant antigens (such as active oncogene products), even if
these proteins
in their intact form are neither expressed on the cell surface nor secreted.
(Paul, W. E.,
"Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th
Edition,
Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
[96] T cells can also be classified based on their function as helper T cells;
T cells
involved in inducing cellular immunity; suppressor T cells; and cytotoxic T
cells.
Helper T cells
[97] Helper T cells are T cells that stimulate B cells to make antibody
responses to
proteins and other T cell-dependent antigens. T cell-dependent antigens are
immunogens in
which individual epitopes appear only once or a limited number of times such
that they are
unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so
inefficiently. B
cells bind the antigen through their membrane Ig, and the complex undergoes
endocytosis.
Within the endosomal and lysosomal compartments, the antigen is fragmented
into peptides
by proteolytic enzymes and one or more of the generated peptides are loaded
into class II
MHC molecules, which traffic through this vesicular compartment. The resulting
peptide/class II MHC complex is then exported to the B-cell surface membrane.
T cells with
receptors specific for the peptide/class II molecular complex recognize this
complex on the
B-cell surface. (Paul, W. E., "Chapter 1: The immune system: an introduction,"
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Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers,
Philadelphia (1999)).
[98] B-cell activation depends both on the binding of the T cell through its
TCR
and on the interaction of the T-cell CD40 ligand (CD4OL) with CD40 on the B
cell. T cells
do not constitutively express CD4OL. Rather, CD4OL expression is induced as a
result of an
interaction with an APC that expresses both a cognate antigen recognized by
the TCR of the
T cell and CD80 or CD86. CD80/CD86 is generally expressed by activated, but
not resting,
B cells so that the helper interaction involving an activated B cell and a T
cell can lead to
efficient antibody production. In many cases, however, the initial induction
of CD4OL on T
cells is dependent on their recognition of antigen on the surface of APCs that
constitutively
express CD80/86, such as dendritic cells. Such activated helper T cells can
then efficiently
interact with and help B cells. Cross-linkage of membrane Ig on the B cell,
even if
inefficient, may synergize with the CD4OL/CD40 interaction to yield vigorous B-
cell
activation. The subsequent events in the B-cell response, including
proliferation, Ig
secretion, and class switching (of the Ig class being expressed) either depend
or are
enhanced by the actions of T cell-derived cytokines. (Paul, W. E., "Chapter 1:
The
immune system: an introduction," Fundamental Immunology, 4th Edition, Ed.
Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia (1999)).
[99] CD4+ T cells tend to differentiate into cells that principally secrete
the
cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that mainly
produce IL-2,
IFN-y, and lymphotoxin (TH1 cells). The TH2 cells are very effective in
helping B-cells
develop into antibody-producing cells, whereas the TH1 cells are effective
inducers of
cellular immune responses, involving enhancement of microbicidal activity of
monocytes
and macrophages, and consequent increased efficiency in lysing microorganisms
in
intracellular vesicular compartments. Although the CD4+ T cells with the
phenotype of
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TH2 cells (i.e., IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, TH1
cells also have the
capacity to be helpers. (Paul, W. E., "Chapter 1: The immune system: an
introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers,
Philadelphia (1999)).
T cells involved in Induction of Cellular Immunity
[100] T cells also may act to enhance the capacity of monocytes and
macrophages to
destroy intracellular microorganisms. In particular, interferon-gamma (IFN- 0
) produced by
helper T cells enhances several mechanisms through which mononuclear
phagocytes destroy
intracellular bacteria and parasitism including the generation of nitric oxide
and induction of
tumor necrosis factor (TNF) production. The TH1 cells are effective in
enhancing the
microbicidal action because they produce IFN-y. By contrast, two of the major
cytokines
produced by TH2 cells, IL-4 and IL-10, block these activities. (Paul, W. E.,
"Chapter 1: The
immune system: an introduction," Fundamental Immunology, 4th Edition, Ed.
Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia (1999)).
Suppressor or Regulatory T (Treg) cells
[101] A controlled balance between initiation and downregulation of the immune
response is important to maintain immune homeostasis. Both apoptosis and T
cell anergy (a
tolerance mechanism in which the T cells are intrinsically functionally
inactivated following
an antigen encounter (Scwartz, R. H., "T cell anergy," Annu. Rev. Immunol.,
21: 305-334
(2003)) are important mechanisms that contribute to the downregulation of the
immune
response. A third mechanism is provided by active suppression of activated T
cells by
suppressor or regulatory CD4+ T (Treg) cells. (Reviewed in Kronenberg, M. et
al.,
"Regulation of immunity by self-reactive T cells," Nature 435: 598-604
(2005)). CD4+
Tregs that constitutively express the IL-2 receptor alpha (IL-2R0) chain (CD4+
CD25+) are
a naturally occurring T cell subset that are anergic and suppressive. (Taams,
L. S. et 1.,
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"Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and
apoptosis-
prone population," Eur. J. Immunol., 31: 1122-1131 (2001)). Depletion of
CD4+CD25+
Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of
these Tregs
prevents development of autoimmune disease. Human CD4+CD25+ Tregs, similar to
their
murine counterpart, are generated in the thymus and are characterized by the
ability to
suppress proliferation of responder T cells through a cell-cell contact-
dependent mechanism,
the inability to produce IL-2, and the anergic phenotype in vitro. Human
CD4+CD25+ T
cells can be split into suppressive (CD25high) and nonsuppressive (CD25low)
cells,
according to the level of CD25 expression. A member of the forkhead family of
transcription factors, FOXP3, has been shown to be expressed in murine and
human
CD4+CD25+ Tregs and appears to be a master gene controlling CD4+CD25+ Treg
development. (Battaglia, M. et al., "Rapamycin promotes expansion of
functional
CD4+CD25+Foxp3+ regulator T cells of both healthy subjects and type 1 diabetic
patients,"
J. Immunol., 177: 8338-8347 (200)).
Cytotoxic T Lymphocytes (CTL)
[102] The CD8+ T cells that recognize peptides from proteins produced within
the
target cell have cytotoxic properties in that they lead to lysis of the target
cells. The
mechanism of CTL-induced lysis involves the production by the CTL of perforin,
a
molecule that can insert into the membrane of target cells and promote the
lysis of that cell.
Perforin-mediated lysis is enhanced by a series of enzymes produced by
activated CTLs,
referred to as granzymes. Many active CTLs also express large amounts of fas
ligand on
their surface. The interaction of fas ligand on the surface of CTL with fas on
the surface of
the target cell initiates apoptosis in the target cell, leading to the death
of these cells. CTL-
mediated lysis appears to be a major mechanism for the destruction of virally
infected cells.
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Priming
[103] The term "unprimed cells" (also referred to as virgin, naïve, or
inexperienced
cells) as used herein refers to T cells and B cells that have generated an
antigen receptor
(TCR for T cells, BCR for B cells) of a particular specificity, but have never
encountered the
antigen. The term "priming" as used herein refers to the process whereby T
cells and B cell
precursors encounter the antigen for which they are specific.
[104] For example, before helper T cells and B cells can interact to produce
specific
antibody, the antigen-specific T cell precursors must be primed. Priming
involves several
steps: antigen uptake, processing, and cell surface expression bound to class
II MHC
molecules by an antigen presenting cell, recirculation and antigen-specific
trapping of helper
T cell precursors in lymphoid tissue, and T cell proliferation and
differentiation. Janeway,
CA, Jr., "The priming of helper T cells, Semin. Immunol. 1(1): 13-20 (1989).
Helper T
cells express CD4, but not all CD4 T cells are helper cells. Id. The signals
required for
clonal expansion of helper T cells differ from those required by other CD4 T
cells. The
critical antigen-presenting cell for helper T cell priming appears to be a
macrophage; and the
critical second signal for helper T cell growth is the macrophage product
interleukin 1 (IL-
O. Id. If the primed T cells and/or B cells receive a second, co-stimulatory
signal, they
become activated T cells or B cells.
[105] The term "transplantation" as used herein, refers to removal and
transfer of
cells, a tissue or an organ from one part or individual to another.
[106] According to one aspect, the described invention provides a recombinant
bispecific antibody that binds to both human Flt3 and human CD3. According to
some
embodiments, the Flt3 antibody binds to a FLT3/FLK2 receptor protein.
According to some
embodiments, the FLT3/FLK2 receptor protein is a mammalian protein. According
to some
embodiments, the FLT3/FLK2 receptor protein is human. According to some
embodiments,
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the FLT3/FLK2 receptor protein is native. According to some embodiments, the
FLT3/FLK2 receptor protein is in a modified form. According to some
embodiments, the
FLT3/FLK2 receptor protein is in a denatured form. According to some
embodiments, the
FLT3/FLK2 receptor protein is in an unmodified form. According to some
embodiments,
the Flt3 antibody is selected from the group consisting of a monoclonal
antibody, a
polyclonal antibody, an antibody fragment and a synthetic antibody mimic.
According to
some embodiments, the Flt3 antibody is a monoclonal antibody. According to
some
embodiments, the FLt3 monoclonal antibody is selected from the group
consisting of a
synthetic antibody and an engineered antibody. According to some embodiments,
the
synthetic antibody is a recombinant antibody. According to some embodiments,
the
recombinant antibody is a single-chain variable fragment (scFv) antibody.
According to
some embodiments, the single chain antibody comprises a C terminus of an Fab
fragment of
an Flt3 antibody that is joined to a CH2 domain of an IgG1 . According to some
embodiments, the CH2 domain of IgG1 is joined to a single chain variable
fragment (ScFv)
of an antibody that reacts with a subunit of human CD3. According to some
embodiments,
the single chain variable fragment is a monoclonal antibody. According to some
embodiments, the subunit of human CD3 is UCHT1. According to some embodiments,
the
engineered antibody is a chimeric antibody. According to some embodiments, the
engineered antibody is a humanized antibody.
[107] According to some embodiments, the FLT3 antibody binding to Flt3 is
effective to block the binding of an FLT3 ligand to FLT3/FLK2 receptor
protein. According
to some embodiments, the FLT3 antibody binding to Flt3 on the cell is
effective for the cell
to internalize the bound antibody.
[108] According to some embodiments, the Flt3 antibody has a half maximal
effective concentration (EC50) between about 1 ng/mL (6.25 pM) and about 2,000
ng/mL
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(12.5 nM). According to some embodiments, the Flt3 antibody has a half maximal
effective
concentration (EC50) between about 10 ng/mL (62.5 pM) and about 200 ng/mL
(1.25 nM).
According to some embodiments, the bispecific antibody that binds to both
human Flt3 and
human CD3 is effective to eliminate one or more of hematopoietic stem cells
(HPC), early
hematopoietic progenitors (HP), and cancer cells. According to some
embodiments, one or
more of the HPC, HP, and cancer cells express FLT3. According to some
embodiments, a
subject in need thereof is a patient that qualifies for, will be receiving or
is receiving
BM/HPCPC transplantation. Examples of the cancer cells include, without
limitation, blast
cells of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), blast-
crisis
phase of chronic myeloid leukemia (BC-CML) and chronic lymphocytic leukemia
(CLL).
According to some embodiments, the bispecific antibody is effective to
condition patients
undergoing bone marrow (BM)/hematopoietic stem cell (HSC) transplantation.
According
to some embodiments, the HSC/HP transplantation is for treating a
hematological
malignancy or hyperproliferative disorder, e.g., Acute Myeloid Leukemia (AML),
Acute
Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Chronic
Myeloid
Leukemia (CML), peripheral T cell lymphoma, follicular lymphoma, diffuse large
B cell
lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, neuroblastoma, non-malignant
inherited and acquired marrow disorders (e.g. sickle cell anemia, beta-
thalassemia major,
refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic
severe aplastic
anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, Fanconi
anemia,
amegakaryocytosis, or congenital thrombocytopenia), multiple myeloma, or
Severe
Combined Immunodeficiency (SCID).
[109] According to another aspect, a method for preparing a recombinant single
chain bi-specific antibody that binds to both human FLT3 and human CD3
comprises
joining a C-terminus of an Fab fragment of an Flt3 monoclonal antibody to a
CH2 domain of
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IgGl, and joining to the CH2 domain of the IgG1 a single chain variable
fragment (ScFv) of
a monoclonal antibody that reacts with a subunit of human CD3 (UCHT1).
[110] According to another aspect, the described invention provides for a
method of
eliminating hematopoietic stem cells/hematopoietic progenitors (HSC/HP) in
patients in
need thereof. According to some embodiments, the method comprises
administering to said
patients a bi-specific antibody that specifically binds to HSC/HP and to T-
cells.
Specifically, the bi-specific antibody binds to human FLT3 expressed by HSC/HP
and to
human CD3 expressed by T cells. The simultaneous binding of the antibody
redirects T-
cells to specifically eliminating the HSC/HP of the patients.
[111] The method provides also the administration of an effective amount of
the
specific antibody to the patient. The effective amount goes from 0.01 mg/kg to
10 mg/kg,
better 0.05 mg/kg to 2mg/kg, better 0.1mg/kg to 0.5mg/kg, better 0.1mg/kg to
0.3mg/kg,
better 0.1mg/kg.
[112] According to some embodiments, the bi-specific antibody that binds to
primate and human CD3 is a humanized antibody.
[113] According to some embodiments, the bi-specific antibody or antigen-
binding
portion thereof comprises amino acid sequences of FLT3 antibody.
[114] According to some embodiments, the bi-specific antibody or antigen-
binding
portion thereof comprises amino acid sequences of CD3 antibody.
[115] According to some embodiments, the bi-specific antibody or antigen-
binding
portion thereof comprises an isotype selected from the group consisting of: an
immunoglobulin G (IgG), an IgM, an IgE, an IgA, or an IgD isotype.
[116] According to another aspect, the invention also provides for a method of
eliminating HSC/HP in a patient in need thereof, wherein the HSC/HP express
FLT3. The
method comprises selecting a patient in need of eliminating HSC/HP and
administering to
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the patient a therapeutically effective amount of a pharmaceutical composition
comprising a
bi-specific antibody specifically binding to human FLT3 expressed by HSC/HP
and to
human CD3 expressed by T-cells, wherein the bi-specific antibody redirects T-
cells to kill
HSC/HP of the patient.
[117] The patients in need of eliminating HSC/HP are patients suffering from
Acute
Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic
Lymphocytic
Leukemia (CLL), Chronic Myeloid Leukemia (CML), peripheral T cell lymphoma,
follicular lymphoma, diffuse large B cell lymphoma, Hodgkin lymphoma, non-
Hodgkin
lymphoma, non-hematological malignancies such as neuroblastoma, non-malignant
inherited and acquired marrow disorders (e.g. sickle cell anemia, beta-
thalassemia major,
refractory Diamond-Blackfan anemia, myelodysplastic syndrome, idiopathic
severe aplastic
anemia, paroxysmal nocturnal hemoglobinuria, pure red cell aplasia, Fanconi
anemia,
amegakaryocytosis, or congenital thrombocytopenia), multiple myeloma, Severe
Combined
Immunodeficiency (SCID) and other disorders that are treated using Bone Marrow
(BM)/Hematopoietic Stem Cell (HSC) transplantation.
[118] The pharmaceutical composition comprises the antibody and
pharmaceutically
acceptable carriers, diluents or excipients. The carriers are selected from
for example, one
or more of water, saline, phosphate buffered saline, dextrose, glycerol,
ethanol and the like,
as well as combinations thereof. Pharmaceutically acceptable carriers can
further comprise
minor amounts of auxiliary substances such as wetting or emulsifying agents,
preservatives
or buffers, which enhance the shelf life or effectiveness of the binding
proteins. The
pharmaceutical compositions can, as is well known in the art, be formulated so
as to provide
rapid, sustained or delayed release of the active ingredient after
administration (Mishra, M.
K. (2016). Handbook of encapsulation and controlled release. Boca Raton, CRC
Press,
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Taylor & Francis Group, CRC Press is an imprint of the Taylor & Francis Group,
an
Informa business, incorporated herein by reference in its entirety).
[119] The pharmaceutical composition may further comprise another component
such as T-cells or an antitumor agent. Antitumor agents administered in
conjunction with
the antibody include any agents which destroy or damage a tumor or malignant
cells.
[120] The antitumor agent is selected from the group consisting of suitable
anti-
neoplastic agents that are known to those skilled in the art and include
anthracyclines (e.g.
daunomycin and doxorubicin), auristatin, methotrexate (MTX), vindesine,
neocarzinostatin,
cis-platinum, chlorambucil, cytosine arabinoside, 5-fluorouridine, melphalan,
ricin and
calicheamicin including combination chemotherapy such with doxorubicin,
bleomycin,
vinblastine, and dacarbazine (ABVD), BEACOPP or escalated BEACOPP (bleomycin,
etoposide, doxorubicin,cyclophosphamide, vincristine, procarbazine, and
prednisone) and
Stanford V (doxorubicin, vinblastine, mechlorethamine, vincristine, bleomycin,
etoposide,
and prednisone). The antitumor agent can also be immunotherapy (e.g. anti-CD20
antibody
rituximab), immunotoxins (e.g. Brentuximab vedotin (SGN-35) is an immunotoxin
comprised of a CD-30 directed antibody linked to the antitubulin agent
monomethyl
auristatin E (MMAE)), adoptive immunotherapy (cytotoxic T lymphocytes),
programmed
death 1 (PD-1) blockade (eg, nivolumab, pembrolizumab).
[121] According to another aspect, the invention further provides for a method
of
testing of bi-specific antibodies redirecting T-cells to kill HSC/HP in an
animal model in
vivo, wherein said animal model is immune-compromised humanized mice with a
chimeric
mouse-human hematopoietic system, wherein said humanized mice are created by
transplantation of human HSC/HP or transplantation of human post-natal
hemogenic
endothelial cells into said myeloablated immune-compromised mice.
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[122] The bi-specific antibody of the present invention has been synthesized
according to the method described in Durben et al. (Molecular Therapy, vol.
23, no. 4 April
2015), which is incorporated herein by reference in its entirety.
[123] The FLT3 antibody sequence used is described in the U.S. Patent No.
9,023,996 to Grosse-Hovest et al, also incorporated herein by reference in its
entirety.
[124] It is to be understood that while the invention is described in
conjunction of
the preferred embodiments thereof, those skilled in the art are aware that
other embodiments
could be made without departing from the spirit of the invention.
[125] Where a range of values is provided, it is understood that each
intervening
value, to the tenth of the unit of the lower limit unless the context clearly
dictates otherwise,
between the upper and lower limit of that range and any other stated or
intervening value in
that stated range is encompassed within the invention. The upper and lower
limits of these
smaller ranges which may independently be included in the smaller ranges is
also
encompassed within the invention, subject to any specifically excluded limit
in the stated
range. Where the stated range includes one or both of the limits, ranges
excluding either
both of those included limits are also included in the invention.
[126] Unless defined otherwise, all technical and scientific terms used herein
have
the same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although any methods and materials similar or equivalent to
those
described herein can also be used in the practice or testing of the described
invention,
exemplary methods and materials have been described. All publications
mentioned herein
are incorporated herein by reference to disclose and described the methods
and/or materials
in connection with which the publications are cited.
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[127] It must be noted that as used herein and in the appended claims, the
singular
forms "a", "and", and "the" include plural references unless the context
clearly dictates
otherwise.
EXAMPLES
[128] The following examples are put forth so as to provide those of ordinary
skill in
the art with a complete disclosure and description of how to make and use the
described
invention, and are not intended to limit the scope of what the inventors
regard as their
invention nor are they intended to represent that the experiments below are
all or the only
experiments performed. Efforts have been made to ensure accuracy with respect
to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors and
deviations should be
accounted for. Unless indicated otherwise, parts are parts by weight,
molecular weight is
weight average molecular weight, temperature is in degrees Centigrade, and
pressure is at or
near atmospheric.
[129] To better illustrate the invention the following examples are given.
[130] Example 1: Antibody Synthesis Description
Background Information
[131] Fabsc is a recombinant bispecific antibody format. The Fabsc format for
a
bispecific antibody targeting FLT3 (using 4G8 clone) and CD3 (using UCHT1
antibody
sequence, also referred to as huxCD3v1) is as follows: C-terminus of Fab
fragment of Flt3
mAb will be joined to the CH2 domain of IgGl, followed by the ScFv of UCHT1.
[132] Sequences of 4G8 clone and UCHT1 were obtained from Patent No. US
9,023,996 B2 and Patent No. 6,054,297, respectively, incorporated herein by
reference in
their entirety.
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Scope of Experiment
[133] Gene synthesis of 4G8 and UCHT1 variable heavy and light chain sequences
based on the format described in the background.
[134] Molecular construction of IgG expression vectors.
[135] 0.1 liter premium transient production in HEK293 cells.
[136] Custom purification (KappaSelect and protein L-columns and eluted at pH
2.3).
[137] Protein aggregation analysis by SE-HPLC.
Target Deliverables
[138] All purified protein from 0.1 liter production.
[139] Study Report including: Certificate of Analysis, CE-SDS analysis, SE-
HPLC
analysis report.
[140] Depending on yield obtained after expression and purification, client
will
determine if produced antibody is to be used for the following analytical
steps:
[141] Test purity, monomer content, and aggregation by SE-HPLC (0.1 mg).
[142] Association and dissociation using varying concentrations of antigen and
calculation of kD by ForeBio Octet QKe (0.2 mg).
Results
[143] The Fabsc antibody was cloned into a high expression mammalian vector
system and a small-scale (0.1 liter) premium transient production was
completed in HEK293
cells. The protein was purified by Protein L purification and 20.17 mg of
protein was
obtained. Yields were reported and client confirmed that SE-HPLC should be
performed.
The antibody was determined to be 92% non-aggregated monomer by SE-HPLC.
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Vector Construction and Transient Production
Molecular Construction of Expression Vector
[144] DNA Studio gene synthesized and cloned programmed sequences into one of
high expression mammalian vectors. Completed constructs were sequence
confirmed before
proceeding to transfection.
Table A
Construct Name Fabsc HC Fabsc LC
Internal code H3113 L3113
Antibody details Human IgG1 Fab Human kappa
Notes Sequences were codon optimized
Small-Scale Transient Transfection
[145] HEK293 cells were seeded in a shake flask one day before transfection,
and
were grown using serum-free chemically defined media. The DNA expression
constructs
were transiently transfected into 0.1 liter of suspension HEK293 cells using
standard
operating procedure for transient transfection. After 20 hours, cells were
sampled to obtain
the viability and viable cell count, and titer was measured (Octet QKe,
ForteBio). The
culture was harvested at day 5 and additional readings were taken.
Protein L Affinity Purification
[146] The conditioned media for Fabsc was harvested and clarified from the
transient transfection production run by centrifugation and filtration. The
supernatant was
run over a Protein L column and eluted with a low pH buffer. Filtration using
a 0.2 p.m
membrane filter was performed before aliquoting. After purification and
filtration, the
protein concentration was calculated from the 0D280 and the extinction
coefficient. See
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Table 1 for a summary of yields and aliquots. CE- SDS analysis was performed
(LabChip
GXII, Perkin Elmer) and the electropherogram was plotted and is shown in FIG.
1A and 1B.
SE-HPLC Analysis
[147] 5 [IL of purified antibody was injected into a MAbPac SEC-1, 5 p.m, 4 x
300
mm column with a flow of 0.2 mL/min for 25 minutes. The protein eluted at the
expected
time with 92% in its non- aggregated form. The chromatogram and specifications
of the SE-
HPLC can be reviewed in FIG. 1C.
[148] See Table 1 for a summary of aggregation level.
SE-HPLC
Conc. Vol. No. of Total Yield
Protein Name Lot # HC# LC# analysis 0/0
(mg/mL) (mL) Vials (mg)
monomeric)
4622-848799 0.5 7 19.07
Fabsc H3113 L3113 5.45 >92
4622-848799 0.2 1 1.09
Table 1. Final yields and aliquots
Project Summary
[149] The Fabsc antibody was cloned into LakePharma's high expression
mammalian vector system and a small-scale (0.1 liter) premium transient
production was
completed in HEK293 cells. The protein was purified by Protein L purification
and 20.17
mg of protein was obtained and 19.07 mg was delivered. The antibody was
determined to be
92% non-aggregated by SE-HPLC. See Table 1 for a summary of yields and
aliquots.
Protein Purification Results
[150] Process Summary and Specifications
[151] Protein L affinity chromatography
[152] 0.21Am sterile filtering
Protein Name Fabsc
Lot # 4622-848799
Extinction Coefficient 1.67 mg/ml 1 cm-1-
(used for concentration calculation)
Protein concentration 5.45 mg/ml
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Protein Name Fabsc
Volume 0.50 ml
Total Protein 2.72 mg
Endotoxin Not Measured
Physical state Liquid
Buffer 230 mM HEPES, 115 mM NaCl, 58 mM
na0Ac, pH 7.0
[153] Table 2
Test SE-HPLC
SR # 3916
Sample ID Fabsc (PP4622)
Date 2015-12-09
Scientist SW
[154] Table 3
Methods
Time Peak
Peak # Peak Size Peak ID
(min)
(kDa) Area /0
1 11.2 ¨230 5.8 Aggregate
2 12.7 ¨100 91.8 Monomer
3 13.9 ¨40 2.4 Fragment
4
6
[155] Table 4
Column MabPac SEC-1, 5 p.m, 4x 300 mm
Mobile Phase 50 mM Sodium Phosphate, 300 mM NaCl, pH 6.2
Isocratic 0-25 min
Flow Rate (mL/min) 0.2
Injection Volume 5
(1ut)
[156] Table 5
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[157] Example 2: Preparation or Conditioning of a Patient for Bone
Marrow/Hematopoietic Stem Cell (BM/HSC) Transplant.
[158] Preparation or conditioning of a patient for bone marrow/hematopoietic
stem
cell (BM/HSC) transplant is a critical element of the procedure. It serves two
main purposes:
(1) It provides adequate immunosuppression of the patient and clears
sufficient niche space
in the bone marrow for the transplanted HSC. This allows transplanted cells to
engraft in the
recipient; (2) It often helps to eradicate the source of malignancy.
[159] Conditioning of patients has traditionally been achieved by
administering
maximally tolerated doses of a cocktail of chemotherapeutical agents with or
without
radiation. Components of the cocktail are often chosen to have non-overlapping
toxicities.
All preparative regiments that are currently in use are toxic and have severe
side effects that
can be life threatening. Among these side effects are mucositis, nausea and
vomiting,
alopecia, diarrhea, rash, peripheral neuropathies, infertility, pulmonary and
hepatic
toxicities. Many of these side effects are especially dangerous for older and
sick patients and
often become a decisive component in deciding whether a patient will receive a
transplant.
[160] To eliminate use of chemotherapeutic agents for conditioning of the
patients
undergoing BM/HSC transplantations we developed a method of selective
elimination of
hematopoietic stem cells/hematopoietic progenitors (HSC/HP) using redirected T-
cell
killing. This method is based on the use of bi-specific antibodies that bind
to a target (FLT3)
on the surface of HSC/HP and also to a target (CD3) on a surface of T-cells,
recruiting T-
cells against HSC/HP.
[161] As a proof of principle that the developed method is useful for
eliminating
HSC/HP we tested bi-specific (FLT3xCD3) antibodies designed for killing of
leukemic
blasts in primary peripheral blood mononuclear cells of acute myeloid leukemia
(AML)
patients(Durben, Schmiedel et al. 2015).
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[162] Females (4-6 weeks of age) of immune-compromised NOG (NOD.Cg-
Prkdcscid Il2rgtm1Sug/JicTac) mice were used for transplantation of human
CD34+
HSC/HP from umbilical cord blood (CB). Mononuclear cell fraction of CB was
separated
using Ficoll-Paque (GE Healthcare Life Sciences) by gradient density
centrifugation.
Briefly, CB treated with anti-coagulant was mixed with phosphate-buffered
saline (PBS) in
the 1:1 ratio and overlaid (35m1 of the mixture) on a layer of Ficoll-Paque
(10m1) in 50m1
conical centrifuge tubes. The tubes were then spun at a speed of 400 x g. A
monocyte
lymphocyte layer was carefully removed and cells obtained from that layer were
washed
twice with PBS.
[163] CD34+ HSC/HP were isolated by negative selection with platelet depletion
(Stemcell Technologies). Unwanted cells were targeted for removal with
Tetrameric
Antibody Complexes recognizing CD2, CD3, CD11b, CD11 c, CD14, CD16, CD19,
CD24,
CD56, CD61, CD66b, glycophorin A and dextran-coated magnetic particles. The
labeled
cells were separated using an EasySepTM magnet without the use of columns.
[164] CD34+ HSC/HP were re-suspended in PBS at 10,000-50,000 cells per 200
11.1
for transplantation into a myeloablated NOG mouse.
[165] Mice were myeloablated using Busulfan (10 mg/kg) via intra-peritoneal
injection twenty-four hours before the transplantation. CD34+ HSC/HP were
transplanted
by tail vein injection of 200 1 of cell suspension (n=52). Eighteen (18) weeks
post-
transplantation peripheral blood of transplanted mice was tested for the
presence of human
CD45+ cells. Mice with the level of chimerism AO% (% of human CD45+ cellsA0%
of
total CD45+ cells) were selected for further experimentation (n=27; FIG. 1A,
FIG. 1B). The
level of chimerism was tested in the peripheral blood and calculated as
follows:
%hCD45+
_________________________________________ * 100%
%hCD45+ + %mCD45+
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[166] Peripheral blood of the selected mice was also tested for the presence
of
human B-cells (hCD19+), human T-cells (hCD3+) and human cells belonging to
myeloid
lineages (hCD33+). A majority of mice exhibited robust development of all
three lineages
(FIG. 1A, FIG. 1B). Some mice (n=3) were deficient for the development of CD3+
cells
(FIG. 1B asterisks, FIG. 1D). These T-cell deficient mice were used as an
internal control
group within the experiment.
Protein sequence of insert
Fabsc HC [H3113] (SEQ ID NO: 1):
MEW SWVFLFFL SVTTGVH SQVQLQQP GAELVKPGASLKL S CKS SGYTFTSYWMHWVRQRPGHGLEW
IGEIDP SD SYKDYNQKFKDKATLTVDRS SNTAYMHLS SLTSDD SAVYYCARAITTTPFDFWGQGTTLT
VS SASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNS GALT S GVHTFPAVLQ S SGLYSLS
SVVTVP SS SLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTSPP SPAPPVAGP SVFLFPPKPKDTLMIS
RTPEVTCVVVGVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKQLP SPIEKTI SKAKGGGGAGGGGEVQLVE S GGGLVQPGGSLRL S CAASGYSFTGYTMNWV
RQAPGKGLEWVALINPYKGVTTYAD SVKGRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARS GYYG
D SDWYFDVWGQGTLVTVS S GGGGS GGGGS GGGGSD IQMTQ SP S SL S AS VGDRVTITCRAS
QDIRNYL
NWYQQKPGKAPKLLIYYTSRLESGVPSRFS GS GS GTDYTLTIS SLQPEDFATYYCQQGNTLPWTFGQGT
KVEIKR*
Fabsc LC [L3113] (SEQ ID NO: 2):
METDTLLLWVLLLWVP GS TGDIVL TQSPATL SVTPGD SVSLS CRASQ SI SNNLHWYQQKSHE SPRLLIK
YASQSIS GIP SRF S G S GS GTDFTL SINSVETEDFGVYF CQQ SNTWPYTFGGGTKLEIKRTVAAP
SVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQD SKD STY SL S STLTLSKADYEK
HKVYACEVTHQGLS SPVTKSFNR GEC*
Signal Peptide
Variable heavy
Variable light
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DNA sequence of insert
Fabsc HC [H3113] (SEQ ID NO: 3):
ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCAGGTGCAG
CTGCAGCAGCCTGGTGCCGAGCTCGTGAAACCTGGCGCCTCCCTGAAGCTGTCCTGCAAGTCCTCC
GGCTACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGAGGCCTGGCCACGGACTGGAATG
GATCGGCGAGATCGACCCCTCCGACTCCTACAAGGACTACAACCAGAAGTTCAAGGACAAGGCCA
CCCTGACCGTGGACAGATCCTCCAACACCGCCTACATGCACCTGTCCTCCCTGACCTCCGACGACT
CCGCCGTGTACTACTGCGCCAGAGCCATCACAACCACCCCCTTCGATTTCTGGGGCCAGGGCACCA
CACTGACAGTGTCCTCCGCTTCCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGT
CCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCG
TGTCCTGGAACTCTGGCGCTCTGACATCCGGCGTGCACACCTTCCCTGCTGTGCTGCAGTCTAGCG
GCCTGTACTCCCTGTCCAGCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCT
GCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGA
CAAGACCCACACCAGCCCTCCAAGCCCTGCTCCTCCTGTGGCTGGCCCTAGCGTGTTCCTGTTCCC
TCCAAAGCCCAAGGATACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTCGTGGGAG
TGTCTCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCC
AAGACCAAGCCTAGAGAGGAACAGTACCAGTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT
GCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGCAGCTGCCCAGC
CCCATCGAAAAGACCATCTCCAAGGCTAAGGGCGGAGGCGGAGCTGGTGGTGGCGGAGAAGTGC
AGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCC
AGCGGCTACTCTTTCACCGGCTATACCATGAATTGGGTGCGCCAGGCCCCTGGAAAGGGCCTGGA
ATGGGTGGCCCTGATCAACCCCTACAAGGGCGTGACCACCTACGCCGACTCCGTGAAGGGCCGGT
TCACCATCTCCGTGGACAAGTCCAAGAATACCGCTTACCTGCAGATGAACTCCCTGCGGGCCGAG
GACACCGCTGTGTATTACTGTGCTAGATCCGGCTACTACGGCGACAGCGATTGGTACTTCGACGTG
TGGGGACAGGGAACCCTCGTGACTGTGTCATCAGGCGGCGGTGGTTCTGGCGGAGGGGGATCTGG
GGGCGGTGGATCCGATATCCAGATGACCCAGTCCCCCAGCTCCCTGTCTGCCTCTGTGGGCGACAG
AGTGACCATCACCTGTCGGGCCTCTCAGGACATCCGGAACTACCTGAACTGGTATCAGCAGAAGC
CCGGCAAGGCCCCCAAGCTGCTGATCTACTACACCTCCCGGCTGGAAAGCGGCGTGCCCTCCAGA
TTCTCCGGCTCTGGCTCTGGAACCGACTATACCCTGACCATCTCTAGCCTGCAGCCCGAGGACTTC
GCCACCTACTACTGCCAGCAGGGCAACACCCTGCCCTGGACCTTTGGCCAGGGAACAAAGGTGGA
AATCAAGCGGTGA
Fabsc LC [L3113] (SEQ ID NO: 4):
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGCTCCACCGGAGACATC
GTGCTGACCCAGTCTCCCGCCACCCTGTCTGTGACCCCTGGCGACTCTGTGTCCCTGTCCTGCAGA
GCCTCCCAGTCCATCTCCAACAACCTGCACTGGTATCAGCAGAAGTCCCACGAGAGCCCTCGGCT
GCTGATTAAGTACGCCAGCCAGTCTATCTCCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGG
CACCGACTTCACCCTGTCCATCAACTCCGTGGAAACCGAGGACTTCGGCGTGTACTTCTGCCAGCA
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GTCCAACACCTGGCCCTACACCTTTGGCGGAGGCACCAAGCTGGAAATCAAGCGGACCGTGGCCG
CCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGT
GCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAG
AGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCA
GCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCAC
CAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGG GGCGAGTGCTAA
[167] Example 3: Generation and Characterization of Monoclonal Antibodies
Directed Against Flt3/FLK2 Human Receptor Protein
[168] Cells from the murine myeloma cell line SP2/0 were transduced with
lentivirus expressing full coding sequence of human FT3/FLK2 receptor protein
and a
selection marker for puromycin resistance. Transduced cells were selected in
the presence of
puromycin in vitro. Cells selected and verified for expression of the human
FLT3/FLK2
protein cells (SP2/0-Hu-FLT3) were used as antigen.
[169] Eight week-old Balb/c mice were immunized three times with 107 SP2/0-Hu-
FLT3 cells by intraperitoneal injection every 5 days in order to generate
antibodies specific
to FLT3/FLK2 protein. The development of antibodies was tested by screening
blood serum
of the immunized mice for binding of the FLT3/FLK2 antigen using flow
cytometry.
[170] Approximately 3 weeks after the first immunization, spleens of immunized
mice were collected and used for isolation of splenocytes. Isolated
splenocytes we fused
with SP2/0 cells and selected for the hybrid phenotype (hybridomas). Hybidomas
were
cultured in vitro and supernatants from the culture of the hybridomas were
screened for the
presence of anti-FLT3/FLK2 antibodies by flow cytometry (FIGS. 3A and 3B).
Nine
hybridoma clones demonstrated production of anti-FLT3/FLK2 antibodies. These
hybridoma clones were expanded for isolation of monoclonal antibodies. The
isolated
monoclonal anti-FLT3/FLK2 antibodies were purified and tested for their
selectivity (FIGS.
4A and 4B).
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[171] Example 4: Characterization of Specificity of Monoclonal Antibodies to
the Human FLT3/FLK2 Receptor Protein
[172] Specificity of the monoclonal antibodies was determined by evaluating
their
affinity to the FLT3/FLK2 antigen. To define affinity of the anti-human
FLT3/FLK2
antibodies, an Effective Concentration (EC) curve was built using flow
cytometry. Nine
monoclonal antibody clones were used to stain human REH cells that
endogenously express
human FLT3/FLK2. The concentration of the clones ranged from 1 ng/ml (6.25 pM)
to
10,000 ng/ml (62.5 nM). Five clones with EC50 ranging from around 70 ng/ml
(437.5 pM)
to 1566 ng/ml (9.79 nM) (FIGS. 5A, 5B, 5C, 5D, and 5E) were chosen for
sequencing.
Sequencing of the clones revealed that clones 1-23DA and 1-18 had the same
amino acid
sequences. The sequences of the clones are shown below.
MHC1692 - 1-23DA SEQUENCES:
Amino Acid Sequence in FASTA format (MHC1692LC.2\,M13F) ¨ Light Chain (SEQ ID
NO: 5)
> MHC1692LC.2\;M13F
DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLHSGVP
KRFSGSRSGSDYSLTISRLESEDVADYYCLQYASYPFTFGSGTKLEIR
Nucleotide Sequence in FASTA format (MHC1692LC.2\,M13F) ¨ Light Chain (SEQ ID
NO:
> MHC1692LC.2\;M13F
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAG
TCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCA
GCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTACAT
TCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTACTCTCTCA
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CCATCAGCAGGCTTGAGTCTGAAGATGTTGCAGACTATTACTGTCTACAATATGC
TAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAGA
Amino Acid Sequence in FASTA format (MHC1692HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 7)
> MHC1692HC.1 \;Ml3F
QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSTMGVGWIRQPSGKGLEWLLHILWNDS
KYYNPALKSRLTISKDTYNKQVFLKIANVDTADTATYYCARIVYYSTYVGYFDVWG
AGTTVTVSS
Nucleotide Sequence in FASTA format (MHC1692HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 8)
> MHC1692HC.1 \;Ml3F
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCA
GTCTGACTTGTTCTTTCTCTGGGTTTTCTCTGAGCACTTCTACTATGGGTGTAGGC
TGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGTTACACATTTTGTGGA
ATGATAGTAAGTATTATAACCCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGG
ATACCTACAACAAGCAGGTATTCCTCAAGATCGCCAATGTGGACACTGCAGATA
CTGCCACATACTACTGTGCTCGAATAGTTTACTACTCTACCTACGTCGGGTACTTC
GATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
M11C1693 - 3-16HA SEQUENCES:
Amino Acid Sequence in FASTA format (MHC1693LC.1\;1\413F) ¨ Light Chain (SEQ
ID
NO: 9)
>MHC1693LC.1\;Ml3F
DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQSPKLLIYAVSNQ
GSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK
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Nucleotide Sequence in FASTA format (MHC1693LC.1\;1\413F) ¨ Light Chain (SEQ
ID NO:
1))
>MHC1693LC.1\;Ml3F
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG
CCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTAT
GAACTGGTTCCAACAGAAACCAGGACAGTCACCCAAACTCCTCATCTATGCTGTA
TCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACA
GACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTCT
GTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAA
TCAAA
Amino Acid Sequence in FASTA format (MHC1693HC.3\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 11)
>MHC1693HC.3\;Ml3F
EVQLQQSGAELVRPGALVKLSCKGSGFNIKDYYIHWVKQRPEQGLEWIGRIDPENDI
TMYDPKFQGKASITADTSSNTAYLQLSSLTSEDTAVYYCARNGNFFAYWGQGTLVT
VSA
Nucleotide Sequence in FASTA format (MHC1693HC.3\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 12)
>MHC1693HC.3\;Ml3F
GAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTC
AAGTTGTCCTGCAAAGGTTCTGGCTTCAACATTAAAGACTACTATATACACTGGG
TGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGAGA
ATGATATTACTATGTATGACCCGAAGTTCCAGGGCAAGGCCAGTATAACAGCAG
ACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACA
CTGCCGTCTATTACTGTGCTAGAAATGGTAATTTCTTTGCTTACTGGGGCCAAGG
GACTCTGGTCACTGTCTCTGCA
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M11C1695 - 3-30A SEQUENCES:
Amino Acid Sequence in FASTA format (MHC1695LC.8\;1\413F) ¨ Light Chain (SEQ
ID
NO: 13)
>MHC1695LC.8\;Ml3F
DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLNSGVP
RRFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPFTFGSGTKLEIK
Nucleotide Sequence in FASTA format (MHC1695LC.8\;1\413F) ¨ Light Chain (SEQ
ID NO:
>MHC1695LC.8\;Ml3F
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAG
TCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCA
GCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTAAAT
TCTGGTGTCCCAAGAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCA
CCATCAGCAGCCTTGAGTCTGAAGATTTTGCAGACTATTACTGTCTACAATATGC
TAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA
Amino Acid Sequence in FASTA format (MHC1695HC.3\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 15)
>MHC1695HC.3\;Ml3F
QVTLKESGPGILQP SQTLSLTC SF SGF SLSTSHMGVGWIRQPSGKGLEWLLHILWNDS
VYYNPALKSRLTISKDTYNKQVFLKIANVDTADTATYYCARIVYYGISYVGYFDVW
GAGTTVTVSS
Nucleotide Sequence in FASTA format (MHC1695HC.3\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 16)
>MHC1695HC.3\;Ml3F
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CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCA
GTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTCACATGGGTGTAGGC
TGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGTTACACATTTTGTGGA
ATGATAGTGTGTACTATAACCCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGG
ATACCTACAACAAGCAGGTATTCCTCAAGATCGCCAATGTGGACACTGCAGATA
CTGCCACATACTACTGTGCTCGAATAGTTTACTACGGTATTAGTTACGTCGGGTA
CTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
M11C1696 - 2-8I4 SEQUENCES:
Amino Acid Sequence in FASTA format (MHC1696LC.3\;1\413F) ¨ Light Chain (SEQ
ID
NO: 17)
> MHC1696LC .3 \;Ml3F
DTVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYGFQSISGIP
SRFSGSGSGTDFTLRINSVETEDFGMYFCQQTNSWPLTFGAGTKLELK
Nucleotide Sequence in FASTA format (MHC1696LC.3\;1\413F) ¨ Light Chain (SEQ
ID NO:
1I;)
> MHC1696LC .3 \;Ml3F
GATACTGTGCTAACTCAATCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGCG
TCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTACACTGGTATCA
ACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGGTTTCCAGTCCATC
TCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCA
GAATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGACTAA
CAGCTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA
Amino Acid Sequence in FASTA format (MHC1696HC.2\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 19)
> MHC1696HC.2\;M13F
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EIQLQ Q S GPELVKP GA S VKV S CKA S GY SF ID YNMYWVKQ SHGK SLEWIGYINPYNG
GT SNNQKFKDKATLTVDK S S STAFMHLNSLTSEDSAVYYCARGTTGDYWGQGTTLT
VS S
Nucleotide Sequence in FASTA format (MHC1696HC.2\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 20)
> MHC1696HC.2\;M13F
GAGATCCAGCTGCAGCAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTG
AAGGTATCCTGCAAGGCTTCTGGTTACTCATTCATTGACTACAACATGTACTGGG
TGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTAATCCTTACA
ATGGTGGTACTAGCAACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTTG
ACAAGTCCTCCAGCACAGCCTTCATGCATCTCAACAGCCTGACATCTGAGGACTC
TGCAGTCTATTACTGTGCAAGAGGTACTACGGGTGACTACTGGGGCCAAGGCAC
CACTCTCACAGTCTCCTCA
M11C2279 - 1B11.E7 SEQUENCES:
Amino Acid Sequence in FASTA format (MHC2279LC.5\;1\413F) ¨ Light Chain (SEQ
ID
NO: 21)
> MHC2279LC.5\;M13F
DIVLTQ SPA SLAV SLGQRATIS CRA SES VDNYGISFMNWF Q QKP GQPPKLLIYAA SNQ
GS GVPARF S GS GS GTDF SLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK
Nucleotide Sequence in FASTA format (MHC2279LC.5\;1\413F) ¨ Light Chain (SEQ
ID NO:
> MHC2279LC.5\;M13F
GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG
CCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTAT
GAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGC
ATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGAC
AGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTC
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TGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA
ATCAAA
Amino Acid Sequence in FASTA format (MHC2279HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 23)
> MHC2279HC.1\;M13F
QVQLQ QP GAELVMP GA S VKL SCKA S GYTF T S YWIVII-IWVKQRP GQ GLEWIGEIDP SD
SYTNYNQKFKGKATLTVDKS S STAYMQLS SLTSEDSAVYYCARSAYYSKRDDYWG
QGTTLTVS S
Nucleotide Sequence in FASTA format (MHC2279HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 24)
> MHC2279HC.1\;M13F
CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGATGCCTGGGGCTTCAGTGA
AGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGT
GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGA
TAGTTATACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGA
CAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCT
GCGGTCTATTACTGTGCAAGATCAGCCTACTATAGTAAAAGGGATGACTACTGGG
GCCAAGGCACCACTCTCACAGTCTCCTCA
M11C1694 - 1-18BA SEQUENCES:
Amino Acid Sequence in FASTA format (MHC1694LC.2\;1\413F) ¨ Light Chain (SEQ
ID
NO: 25)
> MHC1694LC.2\;M13F
DIQMTQ SP S SLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLHSGVP
KRF SGSRSGSDYSLTISRLESEDVADYYCLQYASYPFTFGSGTKLEIR
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Nucleotide Sequence in FASTA format (MHC1694LC.2\;1\413F) ¨ Light Chain (SEQ
ID NO:
> MHC1694LC.2\;M13F
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAG
TCAGTCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCA
GCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGCCGCATCCACTTTACAT
TCTGGTGTCCCAAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTACTCTCTCA
CCATCAGCAGGCTTGAGTCTGAAGATGTTGCAGACTATTACTGTCTACAATATGC
TAGTTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAGA
Amino Acid Sequence in FASTA format (MHC1694HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 27)
> MHC1694HC.1 \;Ml3F
QVTLKES GP GILQP S Q TL SLTC SF S GF SLSTSTMGVGWIRQPSGKGLEWLLHILWNDS
KYYNPALKSRLTISKDTYNKQVFLKIANVDTADTATYYCARIVYYSTYVGYFDVWG
AGTTVTVS S
Nucleotide Sequence in FASTA format (MHC1694HC.1\;1\413F) ¨ Heavy Chain (SEQ
ID
NO: 28)
> MHC1694HC.1 \;Ml3F
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCA
GTCTGACTTGTTCTTTCTCTGGGTTTTCTCTGAGCACTTCTACTATGGGTGTAGGC
TGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGTTACACATTTTGTGGA
ATGATAGTAAGTATTATAACCCAGCCCTGAAGAGCCGGCTCACAATCTCCAAGG
ATACCTACAACAAGCAGGTATTCCTCAAGATCGCCAATGTGGACACTGCAGATA
CTGCCACATACTACTGTGCTCGAATAGTTTACTACTCTACCTACGTCGGGTACTTC
GATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA
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Sample ID KD (M) Kon (1/Ms) Kdis (1/s) Full XA2 Full RA2
MHC1694:1-18BA 5.76E-12 2.46E+ 6 1.42E- 5 0.0165 0.9962
MHC2279:1B11.E7 2.78E-1 1.32E+ 6 3.66E- 4 0.026 0.9777
MHC1696:2-81A 5.03E-11 2.21E+ 6 1.11E- 4 0.0216 0.9882
Table 6
[173] Example 5: Characterization of Internalization of Monoclonal Antibodies
to the Human FLT3/FLK2 Receptor Protein
[174] Internalization of the monoclonal antibodies against FLT3/FLK2 (Example
3)
was quantified by an internalization assay.
[175] Briefly, 2X (4 g/m1) working stocks of antibody were prepared on ice for
antibodies 281A, 330A, 316HA and 123DA in staining buffer (lx phosphate
buffered saline
(PBS) with 2% bovine calf serum - BCS). A 4 pg/m1 stock of anti-human CD135
(FLT3/FLK2) antibody (BioLegend #313302, Clone BV10A4H2) and a 4 g/m1 isotype
control (BioLegend #400102, Clone MOPC-21) were prepared as positive and
negative
CD135 staining controls, respectively. Reh cells, a human cell line that
expresses CD135,
were washed and re-suspended in staining buffer at a concentration of 2x106
cells/ml.
Primary antibodies were added 1:1 with equal volume of cells for a final
concentration of
2 g/ml. Cells were stained in 15 mL centrifuge tubes for ease of washing.
Next, cells were
incubated on ice for 30 minutes, then washed three times in 5 ml of PBS to
remove unbound
primary antibodies. The stained cells were re-suspended in complete culture
media
(RPMI1640 containing glutamine and 2% BCS) and divided into parallel 96 well
plates at
100 1 per well, with separate plates for each time point in triplicate wells.
One set of plates
was transferred to a 37 C incubator, 5% CO2, and a second set of plates was
kept at 4 C.
Incubation times were 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours and 4
hours.
Following incubation, plates were washed in lx PBS. Cells were then stained
with an anti-
mouse IgG Alexa 488 secondary antibody for 30 minutes on ice in the dark at a
1:800
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dilution (Jackson Immuno #115545164). Triplicate control wells containing
unstained cells
and cells stained with secondary antibody alone were also prepared. After
incubation with
the secondary antibody, cells were washed a final time in lx PBS containing 2%
BCS and
stained with 7AAD immediately prior to FACS.
[176] Stained cells were analyzed by flow cytometry on a Beckman Coulter
Cytoflex at a sample flow rate of 60 1/minute. 10,000 events were captured for
each well
and FCS files were evaluated using FloJo software, Version 10. Mean
fluorescent intensity
(MFI) was calculated for the live cell population for Alexa 488 and the change
in MFIs for
each antibody were graphed versus time at 4 C and 37 C.
[177] As shown in FIG. 6, all clones exhibited internalization, with clones
330A and
123DA displaying the most rapid internalization (FIG. 6). Without being
limited by theory,
it is hypothesized that the internalization property of anti-FLT3/FLK2
antibodies (clones
330A, 123DA, 316HA and 281A) render them effective as vehicles (e.g., Antibody-
Drug-
Conjugates ¨ ADC) to deliver drugs/toxins inside targeted cells.
[178] While the present invention has been described with reference to the
specific
embodiments thereof it should be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spirit and
scope of the invention. In addition, many modifications may be made to adopt a
particular
situation, material, composition of matter, process, process step or steps, to
the objective
spirit and scope of the present invention. All such modifications are intended
to be within
the scope of the claims appended hereto.
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