NZ760009A - Means and methods for counteracting myeloproliferative or lymphoproliferative disorders - Google Patents
Means and methods for counteracting myeloproliferative or lymphoproliferative disorders Download PDFInfo
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- NZ760009A NZ760009A NZ760009A NZ76000914A NZ760009A NZ 760009 A NZ760009 A NZ 760009A NZ 760009 A NZ760009 A NZ 760009A NZ 76000914 A NZ76000914 A NZ 76000914A NZ 760009 A NZ760009 A NZ 760009A
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Abstract
The invention provides a human AML-specific binding compound (AT14-013) that is able to bind a cell surface component of AML cells. The anti-AML antibody is able to bind AML primary blasts of at least three different French-American-British (FAB) classifications, namely M0, M4, and M5. The invention further relates to its method of use for diagnosing and treating acute myeloid leukaemia (AML).
Description
Title: Means and methods for counteracting myeloproliferative or
lymphoproliferative disorders
This application is a divisional of New Zealand patent ation 722155,
which is the al phase entry in New d of PCT international application
shed as ), filed 17 December 2014, all
of which are incorporated herein by reference.
The invention relates to the fields of biology, logy, medicine and
cancer therapy, in particular therapy against myeloproliferative or
lymphoproliferative disorders. More in particular, the invention relates to
antibodies against acute myeloid leukemia cells.
Acute myeloid leukemia (AML) is a high risk ancy with five year
survival rates of 40 - 50% in patients younger than 60 years of age. For patients
over 65 years of age outcomes are even worse, with only less than 20% of patients
obtaining durable remissions. Allogeneic stem cell transplantation is frequently
applied in the treatment of acute leukemia. It was initially designed to rescue
patients from otherwise lethal myeloablative chemotherapy but was subsequently
found to be complicated by alloreactive immune response d complications
(graft versus host disease; GvHD). T cell depletion of grafts before reinfusion
averted GvHD but the observation that T cell depleted graft recipients, r to
monozygotic twin donor transplant recipients, experienced much higher rates of
relapse made it increasingly clear that the success of allogeneic SCT is dependent
on the induction of an anti-leukemic immune response (graft versus leukemia
(GvL)). This has led to the pment of strategies to apply allogeneic stem cell
transplantation without myeloablative conditioning (reduced intensity stem cell
transplantation, RIST), to reduce cytotoxicity and to allow allogeneic SCT in a
larger group of patients ing older patients and heavily pretreated patients.
Preparative regimens in RIST are aimed at decimating the ents adaptive
immune system to prevent graft rejection, without complete ablation of the
recipients bone marrow thereby reducing early SCT toxicity. ing
transplantation, donor stem cells lly replace stem cells of the recipient and
full donor chimerism is usually achieved within three months after SCT. Although
allogeneic SCT is ve in significant numbers of ts, and much progress
has been made in the supportive care of SCT recipients, still 15-30% of patients die
as a result of transplantation related complications such as GvHD and ious
complications (arising as a result of slow immune recovery following SCT or as a
complication of immunosuppressive therapy of GvHD).
Hence, although SCT is potentially curative when potent graft versus
leukemia (GvL) responses are induced, its therapeutic success is limited by antihost
immune responses leading to GvHD which causes high morbidity and
mortality.
About 70% of SCT patients develop GvHD at one point following SCT, with
target organs including the skin, liver, intestine and lung. GvHD is treated with
local or systemic immunosuppressive therapy including corticosteroids. A
icant number of GvHD patients is not responsive to d therapy, and
about half of these patients also respond poorly to alternative (and partly still
experimental) measures such as mesenchymal stromal cell (MSC) lantation
or T cell ting therapy such as anti-tumor necrosis factor α (anti-TNFα;
infliximab). Extensive and long-term immunosuppressive therapy is undesirable,
since it may hamper the development of therapeutic antileukemia responses.
Indeed, when an AML relapse occurs while the patient is still on
immunosuppressive therapy, the first step is to rapidly taper
immunosuppressants. This may induce a curative GvL response, often at the
expense of GvHD. In ed patients on immunosuppressive therapy who do not
respond to tapering of suppressants or relapsed patients in whom
immunosuppressants were already phased out before relapse occurred but who had
not developed GvHD, tumor load reduction by chemotherapy, followed by
increasing doses of donor- lymphocyte ons (DLI) may lead to durable
remission of disease. While the absence of diagnostic tests to demonstrate the
presence of a robust GvL makes it difficult to estimate exactly how often allogeneic
SCT induce GvL responses in y or ed AML, some studies provided
compelling evidence of the induction of such a response in a considerable number of
patients. For example, Schmid and colleagues demonstrated potent GvL ses
by DLI in 50% of a small group of AML patients with disease relapse who were in
second remission following chemotherapy (Schmid et al., 2007). Schlenk and
colleagues convincingly showed the additive value of allogeneic SCT in primary
AML, with a doubling of the 5-year disease free survival in patients with high risk
AML who received an allogeneic SCT from a sibling, compared with patients who
did not have a suitable sibling donor (Schlenk et al., 2008). Thus, GvL responses
often occur at the cost of GvHD, and the observation that T cell depletion from the
graft reduced GvHD incidence but increased disease relapse rates suggested that
both are ily mediated by T cell dependent immune responses t
recipient-antigens.
In view of the high GvHD incidence after allogeneic stem cell
transplantation, resulting in death of 15-30% of the patients, as well as the fact
that a suitable donor is not always available for a given patient, alternative
ent approaches are needed. It is an object of the present invention to provide
alternative means and s for racting and/or preventing AML; and/or to
at least e with public with a useful choice.
SUMMARY OF THE INVENTION
In a first aspect the present invention provides an isolated, synthetic or
recombinant human antibody, or a functional part or a functional equivalent
thereof, which is able to bind acute myeloid leukemia (AML) cells and which
comprises:
- a heavy chain CDR1 sequence comprising the sequence of SEQ ID NO: 209;
- a heavy chain CDR2 sequence comprising the sequence of SEQ ID NO: 213;
- a heavy chain CDR3 sequence comprising the sequence of SEQ ID NO: 217;
- a light chain CDR1 sequence comprising the sequence of SEQ ID NO: 221;
- a light chain CDR2 sequence comprising the sequence of SEQ ID NO: 225; and
- a light chain CDR3 sequence comprising the sequence of SEQ ID NO: 229.
In a second aspect the present ion es an isolated, synthetic or
recombinant nucleic acid molecule with a length of at least 15 nucleotides, or a
functional equivalent thereof, or a vector, encoding at least the six CDR sequences
of an antibody or functional part or functional equivalent ing to the first
aspect.
In a third aspect the present invention provides a nucleic acid molecule or
functional equivalent thereof, or a , encoding an antibody or functional part
or functional lent according to the first aspect.
In a fourth aspect the t invention provides an isolated or recombinant
cell, or a non-human animal, comprising a nucleic acid molecule or functional
equivalent or vector according to second or third aspect, with the proviso that said
cell is not present in a human being.
In a fifth aspect the t invention provides a composition comprising an
antibody or functional part or functional equivalent according to the first aspect, or
a nucleic acid molecule or functional equivalent or vector according to the second or
third aspect, or a cell according to the fourth aspect.
In a sixth aspect the present invention provides a use of an antibody or
functional part or functional equivalent according to the first aspect, or a nucleic
acid molecule or functional equivalent or a vector according to the second or third
aspect, or a cell according to the fourth aspect, in the manufacture of a medicament
or prophylactic agent.
In a seventh aspect the present invention provides a use of an antibody
or onal part or functional lent according to the first aspect, or a nucleic
acid le or functional equivalent or a vector ing to the second or third
aspect, or a cell according to the fourth aspect, in the manufacture of a
medicament or lactic agent for treating or preventing acute myeloid
leukemia (AML).
In an eighth aspect the present invention provides a use of an antibody
or functional part or functional lent according to the first , or a nucleic
acid molecule or functional equivalent or a vector according to the second or third
aspect, or a cell according to the fourth aspect, in the manufacture of a diagnostic
agent for acute myeloid leukemia (AML).
In a ninth aspect the t ion provides a method for producing an
dy or functional part or functional equivalent according to the first aspect,
the method comprising providing a cell with a nucleic acid molecule or functional
equivalent or a vector according to the second or third aspect, and allowing said cell
to translate said nucleic acid molecule or functional equivalent or vector, thereby
producing said antibody or functional part or functional equivalent according to the
first aspect, with the proviso that said method is not carried out in a human being.
In a tenth aspect the present invention es a method for determining
whether AML cells are t in a sample comprising:
- contacting said sample with an antibody or functional part or onal
equivalent according to the first aspect, and
- allowing said antibody or functional part or functional equivalent to bind AML
cells, if t, and
- determining r or not AML cells are bound to said antibody or functional
part or functional equivalent, thereby determining whether or not AML cells are
present in said sample.
In an eleventh aspect the present invention provides a method for
determining whether an AML patient has a Graft versus Leukemia response,
comprising contacting a sample from said AML patient with an antibody or
functional part or functional equivalent according to the first aspect, and ng
said antibody or functional part or functional equivalent to bind AML cells, if
present, and determining whether or not AML cells are bound to said antibody or
onal part or functional equivalent, thereby determining whether or nor said
individual has a GvL response, whereby the absence of AML cells is indicative for a
GvL response.
In a twelfth aspect the present invention provides a bispecific or
multispecific binding compound, comprising an antibody or a functional part
or a functional equivalent according to the first aspect and an
immunomodulatory molecule.
In a thirteenth aspect the t invention provides a synthetic or
recombinant antibody, or a functional part or a functional equivalent
thereof, which comprises one Fab fragment of an antibody ing to the
first aspect, and one Fab fragment of r antibody.
In a fourteenth aspect the present invention provides a chimeric
antigen receptor (CAR) T cell sing an antibody or a functional part or
a functional equivalent according to the first aspect.
DETAILED DESCRIPTION
The present invention es patient-derived, AML-specific, human
antibodies that are able to bind intact AML cells. Importantly, the antibodies are
derived from human AML patients that received an allogeneic SCT and are in
complete remission indicating that the antibodies are effective against AML.
Indeed, in the Examples it has been demonstrated that antibodies described herein
are able to bind intact AML cells. The AML-specific human antibodies bed
herein are, therefore, particularly suitable for use in anti AML y. For
instance, an antibody described herein is provided with a toxic moiety. After
administration, AML cells will be bound and/or internalised and the toxic moiety
will exert its toxic effect on the AML cell. Alternatively, in some embodiments
antibody-dependent cell-mediated cytotoxicity (ADCC) or ment-dependent
cytotoxicity (CDC) is induced using an dy described herein, optionally bound
to an immunomodulatory compound. The fact that human antibodies are bed,
or functional parts or functional equivalents of human antibodies, diminishes the
chance of side effects in human beings, which is an important age. Hence,
with the antibodies described herein, novel AML treatment options have become
available which can be used in addition to existing AML therapy, or as an
alternative.
An important embodiment of the present disclosure describes human AML-
specific antibodies that are capable of diminishing proliferation of AML cells.
Administration of such antibodies to an AML patient will counteract AML cells
without the need to add additional (toxic) moieties to the dy. A particularly
preferred embodiment describes human AML-specific antibodies that are capable
of diminishing proliferation of AML cells ndently from antibody-dependent
ediated xicity , complement-dependent cytotoxicity (CDC),
apoptosis or phagocytosis by tumor-associated myeloid cells such as hages
or dendritic cells. Preferably, such antibodies are capable of diminishing
proliferation of AML cells (essentially) independently from any other immune cells,
or complement or apoptosis. As shown in the Examples, such antibodies are
e of directly diminishing or inhibiting AML cell growth, or are even capable
of killing AML cells, in the absence of immune cells like NK cells or macrophages,
and in the absence of complement. This embodiment is in contrast to prior art
approaches such as for instance described in Majeti et al., 2009 and gham et
al., 2012, wherein a CD47-specific antibody is used in order to enable macrophage
phagocytosis of tumor cells. describes the use of C-type lectin-like
molecule-1 (CLL-1)-specific antibodies, which are capable of inducing CDC against
AML cells in vitro in the presence of rabbit complement. Bakker et al., 2004,
however, does not envision that antibody-dependent cell-mediated cytotoxicity or
complement-dependent cytotoxicity via CLL-1 will be an effective targeting
mechanism, and proposes toxin-conjugated CLL-1 antibodies for use against AML.
discloses the use of dies specific for IL1RAP for inducing
antibody-dependent cell-mediated cytotoxicity against AML cells, by recruiting NK
cells which induce AML cell death. Hence, the above described prior art focuses on
the use of antibodies in order to activate other immune cells, which may not
provide the desired results in immune compromised individuals such as AML
patients who have undergone chemotherapy with or without an allogeneic SCT.
describes anti-CXCR4 antibodies that were raised in
enic transchromosomic mice expressing human antibody genes. The obtained
antibodies bind a broad range of hematopoietic cells and are capable of inhibiting
growth of an AML cell line in vitro. These anti-CXCR4 antibodies induce apoptosis
of various AML cell lines.
D33 onal antibody y against AML has also been
described (Walther et al., 2012). For instance, CD33-specific antibodies coupled to a
drug conjugate are described.
describes a humaneered anti-EphA3 antibody that is
capable of inducing sis of AML cells. Furthermore, Biernacki et al., 2010 and
Wu et al., 2000 describe antibodies obtained from AML patients that are specific
for ellular antigens such as RAB38, TBCE, DUSP12 and RAFTK.
In conclusion, several publications describe AML antibodies that bind
intracellular antigens. Such antibodies are not a first choice for combating living,
intact AML cells in vivo due to the unreachability of such intracellular s
when the AML cells are intact. Other publications describe antibodies that bind a
broad range of hematopoietic cells, for instance through CD33, CXCR4, CD47 or
CLL-1. Antibodies which bind a broad range of (hematopoietic) cells involve the
risk of severe side-effects.
Several antibodies described in the art act by inducing phagocytosis, ADCC
or CDC, g that other immune cells or complement components are required
for counteracting AML cells. The use of such antibodies is, therefore, limited in
immune compromised individuals. Moreover, some of the described antibodies are
not human, which involves a high risk of side s. Other publications (such as
and ) describe antibodies that induce apoptosis
of AML cells.
In contrast to the above-mentioned publications, the present inventors have
taken a different approach. d of artificially producing antibodies against a
ent that is present on, amongst other , AML cells, the ors have
ed antibodies from human AML patients who, after ing an allogeneic
SCT, mounted a potent graft versus leukemia response as they maintained in
complete remission.These antibodies are capable of specifically binding intact AML
cells. Hence, instead of using artificially developed antibodies, the t
inventors have elegantly taken advantage of the natural immune defenses elicited
in human AML patients after receiving an allogeneic SCT. The humoral response
found in nature results in a very adequate selection and outgrowth of B-cells that
produce effective antibodies in vivo. The dies described herein, or functional
parts or functional derivatives thereof, are therefore particularly suitable for
binding AML cells in AML patients. The fact that the dies are specific for
intact AML cells, instead of intracellular antigens, makes them particularly
suitable for AML therapy.
As described, an antibody or functional part or functional equivalent
described herein can be coupled to a toxic moiety. Such toxic moiety will then be
directed to AML cells in vivo through the binding of the antibody to AML cells. One
embodiment of the present sure describes human antibodies, or onal
parts or functional equivalents f, which are able to bind intact AML cells and
which are able to diminish proliferation of AML cells. Such antibody obviates the
need of ng the antibody to an additional, toxic component (although the use
of a toxic moiety can still be useful for providing additional anti-AML s). In
one particularly preferred embodiment, human antibodies, or functional parts or
functional equivalents thereof, described herein are able to bind intact AML cells
and which are able to diminish proliferation of AML cells independently from
antibody-dependent cell-mediated cytotoxicity (ADCC), ment-dependent
cytotoxicity (CDC) or phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells. Preferably, such antibodies are capable of
diminishing proliferation of AML cells independently from, or essentially
independently from, other immune cells or complement components. The use of
such dies, having a strong anti-AML activity, is preferred, especially in
immune-compromised duals. One preferred aspect of the present sure
therefore describes an isolated, synthetic or recombinant human antibody, or a
functional part or a functional equivalent thereof, which is able to bind a cell
surface component of acute myeloid leukemia (AML) cells and which is able to
diminish proliferation of AML cells independently from, or essentially
independently from, antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC) or phagocytosis by tumor-associated
myeloid cells such as macrophages or dendritic cells. Preferably, such antibodies
are capable of diminishing eration of AML cells (essentially) independently
from any other immune cells or ment. As shown in the Examples, at least
antibodies AT12-023, AT12-025 and AT13-024 have these characteristics. These
are, therefore, preferred antibodies according to the present disclosure.
In a further preferred ment, human antibodies, or functional parts or
functional equivalents thereof, described herein are able to bind intact AML cells
and which are able to diminish proliferation of AML cells independently from
apoptosis. As shown in the Examples, at least dies AT12-023, AT13-031 and
AT13-037 have this characteristic. This is clear from the fact that these antibodies
can induce death of AML cells in the presence of apoptosis inhibitors such as the
pan caspase inhibitors Q-VD-OPh or Z-VAD-fmk, and from the fact that most of
these antibodies (except antibody AT13-031) maintain their cytotoxic ties at
4ºC. According to the present disclosure, antibodies such as AT12-023, AT13-031
and AT13-037 kill their AML target cells via necrosis (such as oncosis or
tosis). This provides the advantage over currently known apoptosis-inducing
antibodies that the necrosis-inducing antibodies bed herein will enhance a
patient’s immune response more than sis-inducing antibodies. This is due to
the fact that necrosis causes more exposure of the cell contents to an individual’s
immune system. Further described is therefore an isolated, synthetic or
recombinant human antibody, or a functional part or a functional equivalent
thereof, which is able to bind a cell e component of acute myeloid leukemia
(AML) cells and which is able to sh proliferation of AML cells independently
from, or essentially independently from, apoptosis.
In a further preferred embodiment, human antibodies, or functional parts or
functional equivalents thereof, are described which are able to diminish
proliferation of AML cells independently from both apoptosis and the abovementioned
immune cells and complement. Further described is therefore an
isolated, synthetic or inant human antibody, or a onal part or a
functional lent thereof, which is able to bind a cell surface component of
acute myeloid leukemia (AML) cells and which is able to diminish proliferation of
AML cells essentially independently from dy-dependent cell-mediated
cytotoxicity (ADCC), ment-dependent cytotoxicity (CDC), apoptosis, or
phagocytosis by macrophages or dendritic cells.
An antibody or functional part or onal equivalent described herein is
preferably capable of binding intact AML cells. ably, such antibody or
functional part or onal equivalent is able to bind a cell surface component
that is specific for AML cells. This typically means that non-hematopoietic cells or
non-malignant hematopoietic cells, such as for instance hepatocytes, colon cells,
fibroblasts, endothelial cells, healthy peripheral blood mononuclear cells (PBMC)
and healthy bone marrow cells, are not, or to a significantly lesser extent,
recognized as compared to the binding affinity of an antibody or functional part or
onal equivalent described herein for AML cells. This means that the binding
of an antibody or functional part or functional equivalent described herein to nonhematopoietic
cells or non-malignant hematopoietic cells is typically in the same
range as binding of an irrelevant control antibody to these cells (wherein the
control antibody is not specific for said cells). Some reactivity towards other types
of malignant hematopoietic cells is, however, embraced within the term “AML-
specific”. For instance, it is shown in Table 4 and Figure 6 that antibodies AT13-
031 and 23 are capable of binding patient-derived B- non-Hodgkin
ma cells. A use of antibody AT13-031 or antibody AT12-023, or a functional
part or functional tive of any of these antibodies, for the preparation of a
medicament or prophylactic agent against B- non-Hodgkin lymphoma is therefore
also described, as well as dy AT13-031 or dy AT12-023, or a functional
part or functional derivative of any of these antibodies, for use in a method for at
least in part treating or preventing B- dgkin lymphoma. Similarly,
antibodies AT13-024, AT13-031 and AT12-019 are capable to bind lymphomaand
/or le myeloma cell lines (Table 4). A use of antibody AT13-024 or
antibody AT13-031 or antibody AT12-019, or a functional part or functional
derivative of any of these antibodies, for the preparation of a medicament or
prophylactic agent against lymphoma and/or myeloma is therefore also described,
as well as antibody AT13-024 or antibody AT13-031 or antibody AT12-019, or a
functional part or functional derivative of any of these dies, for use in a
method for at least in part treating or preventing lymphoma and/or myeloma.
As used herein, the term “AML cells” embraces natural AML cells, such as
primary AML blasts that are present in AML patients, as well as AML cell lines
such as for ce THP-1, Mono-Mac 6 and Molm13.
The term “antibody” as used herein, refers to an immunoglobulin
protein comprising at least a heavy chain variable region (VH), paired with a light
chain variable region (VL), that is specific for a target epitope.
A “functional part of an antibody” is defined herein as a part that has at
least one shared property as said antibody in kind, not necessarily in . Said
functional part is capable of g the same antigen as said antibody, albeit not
necessarily to the same extent. In one ment a functional part of an antibody
comprises at least a heavy chain variable domain (VH). Non-limiting examples of a
functional part of an dy are a single domain antibody, a single chain
antibody, a nanobody, an unibody, a single chain variable fragment (scFv), a Fab
fragment and a F(ab')2 fragment.
A ional equivalent of an antibody” is defined herein as an artificial
g compound, comprising at least one CDR sequence of an antibody,
preferably a heavy chain CDR3 sequence. Said functional equivalent preferably
comprises the heavy chain CDR3 sequence of an antibody, as well as the light
chain CDR3 sequence of said antibody. More preferably, said functional equivalent
comprises the heavy chain CDR1, CDR2 and CDR3 sequences of an antibody, as
well as the light chain CDR1, CDR2 and CDR3 sequences of said antibody. A
functional equivalent of an antibody is for instance produced by altering an
antibody such that at least an antigen-binding property of the resulting compound
is essentially the same in kind, not necessarily in . This is done in many
ways, for instance through conservative amino acid tution, y an
amino acid residue is substituted by another residue with generally similar
properties (size, hydrophobicity, etc), such that the l functioning of the
antibody is ially not affected.
As is well known by the skilled person, a heavy chain of an antibody is the
larger of the two types of chains making up an immunoglobulin molecule. A heavy
chain comprises a constant domain and a variable domain, which variable domain
is involved in antigen binding. A light chain of an antibody is the smaller of the two
types of chains making up an immunoglobulin molecule. A light chain ses a
constant domain and a variable domain. The variable domain is often, but not
always, together with the variable domain of the heavy chain involved in antigen
binding.
Complementary-determining regions (CDRs) are the hypervariable regions
present in heavy chain variable domains and light chain variable domains. In case
of whole antibodies, the CDRs 1-3 of a heavy chain and the CDRs 1-3 of the
connected light chain together form the antigen-binding site.
As used herein, the term “an antibody or functional part or functional
equivalent according to the invention” is also referred to as “a binding compound
ing to the invention”.
The term “cell surface component of AML cells” means any ent that
is at least in part present in or on the cell surface of AML cells, or a component
that is attached to an AML cell e. Non-limiting examples of cell surface
components of AML cells are (trans)membrane proteins, glycoproteins, and any
compound attached thereto.
The terms “specific for” and "capable of specifically g" are used herein
interchangeably and refer to the interaction between an antibody, or functional
part or functional equivalent thereof, and its epitope. This means that said
antibody, or onal part or onal lent thereof, preferentially binds to
said epitope over other antigens or amino acid sequences. Thus, although the
antibody, functional part or equivalent may non-specifically bind to other antigens
or amino acid sequences, the binding affinity of said antibody or functional part or
functional equivalent for its e is significantly higher than the non-specific
binding affinity of said antibody or functional part or functional equivalent for
other antigens or amino acid sequences.
An antibody or functional part or functional equivalent described herein
that is able to bind a particular epitope of AML cells can also be specific for other,
non-AML cells if said epitope of AML cells is also present on other cells (for
instance other leukemic cells, myeloma cells or lymphoma cells). In that case an
antibody referred to herein as being specific for AML cells is also specific for said
other cells comprising the same epitope. Preferably, human AML dies and
functional parts and functional equivalents thereof, as described herewith, do not
significantly bind non-hematopoietic cells and lignant poietic cells.
ng affinity” refers to the strength of the total sum of the noncovalent
interactions between a single binding site of an antibody or onal part or
functional equivalent and its binding partner (e.g., an antigen). Unless indicated
otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity
which reflects a 1:1 interaction between members of a binding pair (e.g., antibody
and antigen). The affinity can generally be represented by the equilibrium
iation constant (KD), which is calculated as the ka to kd ratio, see, e.g., Chen,
Y., et al., (1999) J. Mol Biol 5-881. Affinity can be ed by common
methods known in the art, such as for instance a surface plasmon resonance (SPR)
assay such as BiaCore or IBIS-iSPR instrument at IBIS Technologies BV (Hengelo,
the Netherlands) or solution phase assays, such as Kinexa. Preferably an antibody
described herein has a binding affinity for an e at or on the cell surface of
AML cells characterized by a dissociation constant (KD) of at most 100 nM, more
preferably at most 50 nM, more ably at most 25 nM, more preferably at most
10 nM, more preferably at most 5 nM, more preferably at most 2 nM, more
preferably at most 1 nM, more preferably at most 0.5 nM, more preferably at most
0.3 nM, more preferably at most 0.1 nM.
The percentage of identity of an amino acid or nucleic acid sequence, or the
term “% sequence identity”, is defined herein as the percentage of residues in a
candidate amino acid or nucleic acid sequence that is identical with the residues in
a reference ce after aligning the two sequences and introducing gaps, if
necessary, to achieve the maximum percent identity. Methods and computer
programs for the alignment are well known in the art, for example "Align 2".
In a ularly preferred embodiment, an antibody or functional part or
functional equivalent according to the present disclosure is described which is able
to induce death of primary AML blasts. Since primary AML blasts are directly
d from an AML patient, as opposed to commercially available cell lines, the
activity of an antibody against such AML blasts is even more indicative for an in
vivo situation. As shown in the Examples, at least antibodies AT13-024 and AT12-
025 have this characteristic. These dies are, therefore, preferred. In a
particularly preferred ambodiment, an antibody or functional part or functional
equivalent according to the present disclosure is described which is able to induce
death of primary AML blasts ndently from, or essentially independently
from, antibody-dependent cell-mediated cytotoxicity (ADCC), complementdependent
cytotoxicity (CDC), apoptosis, and/or phagocytosis by macrophages or
dendritic cells. ably, such binding nds are capable of ng death
of AML blasts essentially independently from other immune cells, or complement
or apoptosis. Antibodies AT13-024 and AT12-025 also have this preferred
characteristic.
As used herein, the term “essentially independently from ADCC, CDC or
phagocytosis by tumor-associated d cells such as macrophages or dendritic
cells” means that ADCC, CDC or phagocytosis by tumor-associated myeloid cells
such as macrophages or dendritic cells are not required for an anti-AML effect
induced by a binding compound described herein, even though in an in vivo
situation ADCC, CDC or phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells may also occur. Hence, in this embodiment, ADCC,
CDC and phagocytosis by tumor-associated myeloid cells such as macrophages or
dendritic cells is not ed, but not essential either. Likewise, the term
“essentially independently from other immune cells or complement” means that a
binding compound bed herein is, in principle, capable of exerting an anti-
AML effect without the presence of other immune cells or complement, even
though in an in vivo situation other immune cells or complement may also have an
anti-AML activity. The term “essentially independently from apoptosis” means that
the anti-AML effect is exerted by a binding compound described herein via a
mechanism other than apoptosis. Preferably, said anti-AML effect is exerted via
necrosis.
In a further preferred embodiment, an antibody or functional part or
functional equivalent ing to the present disclosure is described which is able
to diminish proliferation of AML cells in vitro within 7 days, preferably within 5
days, more preferably within 3 days and even more preferably within 1 day. This
indicates a quick therapeutic effect.
The present disclosure describes isolated, tic and inant human
antibodies, and functional parts or onal equivalents thereof, which are able to
bind intact AML cells. Said AML cells preferably belong to a French-American-
British (FAB) fication selected from the group consisting of M5, M0, M1, M2,
M3 and M4. More preferably, said AML cells belong to the FAB fication M5 or
M1 or M0 or M4, ably M5. These are the FAB classifications commonly found
in AML patients.
In a further preferred embodiment, isolated, synthetic and recombinant human
antibodies, or functional parts or functional equivalents thereof, are described
which are able to bind different AML cells of at least two, preferably at least three,
more preferably at least four different FAB classifications. Such antibodies are
useful for ent AML patients having different FAB classifications, so that
these antibodies are broadly applicable. As shown in Table 3, antibody AT12-025 is
capable of binding AML cells of at least two FAB classifications (M5 + M1). This is,
therefore, a preferred antibody according to the present disclosure. Antibodies
24, AT12-019, AT13-023, and 22 are capable of g AML cells of
at least three FAB classifications ( M5 + M0 + M1). Antibody AT13-031 is also
capable of binding AML cells of at least three FAB classifications ( M5 + M1 + M4).
These dies are, therefore, even more preferred. Furthermore, antibody AT12-
023 is capable of binding AML cells of at least four FAB classifications (M5 + M0 +
M1 + M4). This antibody is, therefore, even more broadly applicable and is,
therefore, particularly preferred.
In one particularly red embodiment, an antibody or functional part or
functional equivalent according to the present disclosure is described wherein said
antibody is of the IgG isotype, preferably IgG1 or IgG3. This is beneficial for
medical applications in humans.
Tables 1A and 1B, and Figure 1, provide an overview of the variable heavy
and light chain sequences, as well as the dual CDR sequences, of antibodies
AT12-023, AT12-025, AT13-024, AT12-019, 22, AT13-023, AT13-031, AT12-
020, AT13-033, AT13-034, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014,
and AT14-016. These are red antibodies according to the present
disclosure, obtained from three human AML patients. The terms 023”,
“AT12-025”, “AT13-024”, “AT12-019”, “AT13-022”, “AT13-023”, “AT13-031”, “AT12-
020”, “AT13-033”, “AT13-034”, “AT13-035”, 036”, 037”, “AT14-013”,
“AT14-014”, “AT14-015” and “AT14-016” as used herein encompass all antibodies
and functional parts and functional equivalents having at least the heavy and light
chain CDR1-3 regions, preferably the variable heavy chain and light chain
sequences, of these antibodies as depicted in Tables 1A and 1B and Figure 1, such
as for ce isolated and/or purified dies or recombinantly produced
antibodies.
As used herein, any reference to “Table 1” includes a nce to Table 1A
and/or Table 1B.
Based on the antibodies depicted in Table 1 and Figure 1, it is possible to
e an antibody or functional part or functional equivalent f comprising
at least one CDR ce of an antibody depicted in Table 1 or Figure 1, which is
specific for AML cells. bed is therefore an isolated, recombinant and/or
tic antibody or a functional part or functional equivalent thereof comprising
at least one CDR sequence of an antibody as depicted in Table 1. Said CDR
sequence is preferably a CDR3 sequence of an antibody according to the present
disclosure. Preferably, binding compounds are described which comprise at least
two CDRs, more preferably at least three CDRs, of the heavy and light chains of
the same antibody indicated in Table 1 or Figure 1. Hence, preferably at least two
or three CDRs of the heavy and light chains of antibody AT12-023, AT12-025,
AT13-024, AT12-019, AT13-022, AT13-023, AT13-031, AT12-020, AT13-033, AT13-
034, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 or 16
are jointly present in one binding compound according to the present disclosure.
Preferably, a binding compound described herein comprises all three heavy chain
CDRs and all three light chain CDRs of the same antibody depicted in Table 1 or
Figure 1. Optionally, at least one of said CDR sequences is zed, thereby
generating a variant binding compound, preferably in order to improve binding
efficacy, selectivity, or stability. This is for instance done by mutagenesis
procedures where after the stability and/or binding efficacy of the resulting
compounds are preferably tested and an improved AML-specific binding compound
is selected. A skilled person is well e of generating variants comprising at
least one altered CDR sequence according to the present disclosure. For instance,
conservative amino acid substitution is applied. Examples of conservative amino
acid substitution include the substitution of one hobic residue such as
isoleucine, valine, leucine or methionine for another hydrophobic residue, and the
substitution of one polar e for another polar residue, such as the substitution
of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
ably, an antibody or functional part or functional equivalent is described
comprising a CDR ce which is at least 80% identical to a CDR sequence as
depicted in Table 1 or Figure 1, so that the favourable AML-binding and/or AML-
killing characteristic of an AML-specific antibody as depicted in Table 1 or Figure 1
is maintained or even improved. Variant binding compounds comprising an amino
acid sequence which is at least 80% identical to a CDR sequence as depicted in
Table 1 or Figure 1 are therefore also within the scope of the present invention.
Preferably, said binding compounds comprise heavy chain and light chain CDR 1-3
ces which are at least 80% identical to the heavy and light chain CDR 1-3
sequences of the same antibody depicted in Table 1 or Figure 1. Preferably, the
CDR sequences differ in no more than three, preferably in no more than two,
preferably in no more than one amino acid from the al CDR sequences of an
antibody according to the present disclosure.
Besides optimizing CDR sequences in order to improve binding cy or
stability, at least one sequence in at least one of the framework regions can be
optimized. This is ably done in order to improve binding efficacy or stability.
Framework sequences are for instance optimized by mutating a nucleic acid
molecule encoding such framework sequence where after the characteristics of the
resulting antibody – or functional part or functional equivalent – are preferably
tested. This way, it is possible to obtain improved g compounds. In a
preferred embodiment, human germline sequences are used for framework s
in antibodies according to the present disclosure. The use of human germline
sequences minimizes the risk of immunogenicity of said antibodies, because these
sequences are less likely to contain c alterations which are unique to
individuals from which the framework regions are derived, and may cause an
immunogenic se when d to another human individual. Further
described is ore a synthetic or recombinant antibody or functional part or
functional equivalent according to the present disclosure, comprising at least one
non-natural mutation in a framework region. Additionally, or alternatively, a
tic or recombinant antibody or functional part or onal equivalent
according to the present disclosure is described that comprises at least one nonnatural
on in a constant region. By a “non-natural mutation” is meant that
the resulting amino acid sequence does not occur in nature. Instead, it has been
artificially produced. In one embodiment, an IgG3 Fc region of antibody AT12-023,
AT12-025, AT13-024, AT13-022, AT12-020, 14, AT14-015 or AT14-016 is at
least partly replaced by an IgG1 Fc region. This typically increases the stability
and half life of the resulting immunoglobulin.
A binding compound described herein preferably comprises a human
variable region. More preferably, said binding nd comprises a human
constant region and a human variable . Most preferably, said g
compound is a human antibody. The use of human AML-specific antibodies is
advantageous over the use of non-human antibodies. The in vivo use of non-human
antibodies for diagnosis and/or treatment of human diseases is hampered by a
number of s. In ular, the human body may recognize man
antibodies as foreign, which will result in an immunogenic response against the
non-human antibodies, resulting in adverse side effects and/or rapid clearance of
the antibodies from the circulation. A human antibody diminishes the chance of
side-effects when administered to a human dual and often results in a longer
ife in the circulation because of reduced clearance when compared to nonhuman
antibodies. In r embodiment a binding compound described herein is
a humanized antibody. In another embodiment a binding nd described
herein is a chimeric antibody. In a chimeric antibody, sequences of interest, such as
for instance an additional binding site of interest, are included into a binding
compound according to the present disclosure.
Further, binding nds described herein are preferably monoclonal
antibodies. A monoclonal antibody is an antibody consisting of a single molecular
species. Monoclonal antibodies can be produced in large quantities by monoclonal
antibody-producing cells or recombinant DNA technology.
Hence, variant binding compounds based on the preferred antibodies
ed in Table 1 and Figure 1 can also be ted, using techniques known in
the art such as for instance mutagenesis. Typically, sequence variations between
80 and 99% are tolerated while maintaining a certain antigen specificity. Binding
compounds described herein comprising a sequence that has at least 80% sequence
identity to at least a CDR sequence of any of the antibodies of Table 1 or Figure 1
are ore also described herein. Since the antigen specificity of an antibody is
typically ted by the CDR3 sequences, a variant antibody described herein
preferably comprises at least a heavy chain CDR3 sequence having at least 80%
sequence identity with a heavy chain CDR3 ce as depicted in Table 1 or
Figure 1. Said variant antibody preferably comprises a heavy chain CDR3 sequence
and a light chain CDR3 sequence having at least 80% sequence identity with the
heavy and light chain CDR3 sequences of the same antibody, selected from the
group consisting of AT12-023, AT12-025, AT13-024, 19, AT13-022, AT13-
023, AT13-031, AT12-020, AT13-033, AT13-034, AT13-035, AT13-036, AT13-037,
AT14-013, 14, AT14-015 and AT14-016.
Further described is, therefore, an isolated, synthetic or recombinant
antibody, or a functional part or a functional equivalent f, which comprises
at least a heavy chain CDR3 sequence having at least 80% sequence identity with a
sequence selected from the group consisting of SEQ ID NOs: 27-39 and 0,
and a light chain CDR3 sequence having at least 80% sequence identity with a
sequence ed from the group ting of SEQ ID NOs: 66-78 and 229-232.
These are the CDR3 sequences of antibodies AT12-023, AT12-025, AT13-024,
AT12-019, AT13-022, AT13-023, AT13-031, AT12-020, AT13-033, AT13-034, AT13-
035, AT13-036, AT13-037, AT14-013, 14, AT14-015 and AT14-016 depicted
in Table 1 and Figure 1. Preferably, said sequence identity is at least 85%, more
preferably at least 86%, more preferably at least 87%, more preferably at least
88%, more preferably at least 89%, more preferably at least 90%, more preferably
at least 91%, more preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, more preferably at least 95%, more preferably at least
96%, more preferably at least 97%, more preferably at least 98%, more preferably
at least 99%, more preferably 100%. As said before, a binding compound described
herein preferably comprises a heavy and light chain CDR3 sequence having at
least 80% sequence identity with the heavy and light chain CDR3 sequences of the
same antibody, selected from Table 1 or Figure 1.
Typically, at least 1, 2 or 3 amino acid residues of a given CDR sequence
may vary while retaining the same kind of binding activity (in kind, not necessarily
in ). Hence, a binding compound bed herein preferably contains a
heavy chain and light chain CDR3 sequence wherein at most 3, preferably at most
2, more preferably at most 1 amino acid deviates from a heavy and light chain CDR
3 sequence from the same antibody selected from the group consisting of AT12-023,
AT12-025, AT13-024, AT12-019, 22, AT13-023, AT13-031, AT12-020, AT13-
033, 34, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015
and AT14-016.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 1-13 and 209-212; and/or
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence ty with a sequence selected from the group consisting of SEQ ID
NOs: 14-26 and 213-216; and/or
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group ting of SEQ ID
NOs: 27-39 and 217-220; and/or
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 40-52 and 221-224; and/or
- a light chain CDR2 sequence sing a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 53-65 and 225-228; and/or
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with a sequence ed from the group consisting of SEQ ID
NOs: 66-78 and 229-232.
These are the heavy and light chain CDR ces of antibodies 23, AT12-
025, AT13-024, AT12-019, AT13-022, AT13-023, AT13-031, AT12-020, AT13-033,
AT13-034, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 and
AT14-016, depicted in Table 1A and 1B and in Figure 1.
The above ned heavy and light chain CDR 1 to 3 sequences are
ably from the same antibody selected from Table 1 or Figure 1. Preferably,
said antibody or functional part or equivalent comprises heavy chain CDR1, CDR2
and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are at
least 85%, more preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more preferably at least
90%, more preferably at least 91%, more ably at least 92%, more preferably
at least 93%, more preferably at least 94%, more preferably at least 95%, more
preferably at least 96%, more preferably at least 97%, more preferably at least
98%, more preferably at least 99%, more preferably 100% identical to the above
recited CDR sequences (SEQ ID NOs 1-78 and 209-232).
In a preferred embodiment, the heavy chain CDR1 and CDR2 and CDR3
sequences, as well as the light chain CDR1 and CDR2 and CDR3 ces, of the
same antibody, ed from the group consisting of antibodies 23, AT12-
025, 24, AT12-019, AT13-022, AT13-023, AT13-031, AT12-020, AT13-033,
AT13-034, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 and
AT14-016 are present in a given binding compound according to the present
disclosure.
A preferred antibody described herein is antibody AT12-023. This antibody
is preferred because it is capable of efficiently binding and killing cells of the AML
cell line THP-1, as shown in the Examples and Figures 7 and 8. This antibody is,
therefore, particularly suitable for AML therapy and/or diagnosis. Interestingly,
AT12-023 is of the IgG3 e and belongs to the VH4-34 family, which is a
family of VH sequences known for their potential killing properties (Bhat et al,
1997). Antibody AT12-023 is also capable of efficiently g primary AML blasts
of at least four different FAB classifications (Table 3). The heavy chain CDR1,
CDR2 and CDR3 sequences, and the light chain CDR1, CDR2 and CDR3 sequences
of antibody AT12-023 are SEQ ID NOs 1, 14, 27, 40, 53 and 66, respectively, as
ed in Table 1. Also bed is an isolated, synthetic or recombinant
antibody, or a functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 85%
sequence identity with SEQ ID NO: 1; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 85%
sequence identity with SEQ ID NO: 14; and
- a heavy chain CDR3 ce comprising a ce which has at least 85%
sequence identity with SEQ ID NO: 27; and
- a light chain CDR1 sequence comprising a sequence which has at least 85%
sequence identity with SEQ ID NO: 40; and
- a light chain CDR2 sequence sing a sequence which has at least 85%
sequence identity with SEQ ID NO: 53; and
- a light chain CDR3 sequence comprising a ce which has at least 85%
sequence identity with SEQ ID NO: 66. Preferably, said sequence identities are at
least 86%, more preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more preferably at least
91%, more preferably at least 92%, more preferably at least 93%, more preferably
at least 94%, more preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more preferably at least
99%, more preferably 100%. As described herein before, at least 1, 2 or 3 amino
acid residues in the recited CDR sequences may vary while retaining the same
kind of binding activity (in kind, not necessarily in amount). Hence, said heavy and
light chain CDR 1, 2 and 3 sequences preferably contain dy AT12
derived CDR sequences that deviate in no more than three, preferably no more
than two, more preferably no more than one amino acid from the recited AT12-023
CDR sequences.
Another preferred antibody described herein is antibody AT12-025. This
antibody is preferred because it is capable of efficiently binding and killing cells of
the AML cell line THP-1, as well as patient-derived y AML blasts, as shown
in the es and Figures 8 and 10. This antibody is, therefore, ularly
suitable for AML therapy and/or diagnosis. Interestingly, AT12-025 is of the IgG3
e and belongs to the VH4-34 family, which is a family of VH sequences
known for their potential killing properties (Bhat et al, 1997). The heavy chain
CDR1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3
sequences of antibody AT12-025 are SEQ ID NOs 2, 15, 28, 41, 54 and 67,
tively, as depicted in Table 1. Also described is an isolated, synthetic or
recombinant antibody, or a functional part or a functional equivalent thereof,
which ses:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 2; and
- a heavy chain CDR2 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 15; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 28; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 41; and
- a light chain CDR2 ce comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 54; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 67. Again, said sequence identities are
preferably at least 85%, more preferably at least 86%, more preferably at least
87%, more preferably at least 88%, more preferably at least 89%, more preferably
at least 90%, more ably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more preferably at least
95%, more preferably at least 96%, more preferably at least 97%, more preferably
at least 98%, more preferably at least 99%, more ably 100%. As bed
above, at least 1, 2 or 3 amino acid residues in the recited AT12-025 CDR
sequences may vary while retaining the same kind of binding activity (in kind, not
necessarily in amount). Hence, the above mentioned heavy and light chain CDR 1,
2 and 3 ces preferably n CDR sequences that deviate in no more than
three, preferably no more than two, more preferably no more than one amino acid
from the recited AT12-025 CDR sequences.
Another preferred antibody described herein is dy AT13-024. This
antibody is preferred because it is capable of binding and killing patient-derived
primary AML blasts, as shown in the Examples and Figure 10. This antibody is,
therefore, particularly suitable for AML therapy and/or diagnosis. Interestingly,
AT13-024 is of the IgG3 isotype and belongs to the VH3-30 family. The heavy chain
CDR 1, CDR2 and CDR3 sequences and the light chain CDR1, CDR2 and CDR3
sequences of antibody AT13-024 are SEQ ID NOs 3, 16 29, 42, 55 and 68,
respectively, as depicted in Table 1. Also described is an isolated, synthetic or
inant antibody, or a functional part or a functional equivalent thereof,
which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 3; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 16; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 29; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 42; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 55; and
- a light chain CDR3 sequence sing a sequence which has at least 80%
sequence ty with SEQ ID NO: 68. Again, said sequence identities are
preferably at least 85%, more preferably at least 86%, more preferably at least
87%, more preferably at least 88%, more preferably at least 89%, more preferably
at least 90%, more ably at least 91%, more preferably at least 92%, more
preferably at least 93%, more preferably at least 94%, more preferably at least
95%, more ably at least 96%, more preferably at least 97%, more ably
at least 98%, more preferably at least 99%, more preferably 100%. As described
above, at least 1, 2 or 3 amino acid residues in the recited AT13-024 CDR
ces may vary while retaining the same kind of binding activity (in kind, not
necessarily in amount). Hence, the above mentioned heavy and light chain CDR 1,
2 and 3 sequences preferably contain CDR sequences that deviate in no more than
three, preferably no more than two, more preferably no more than one amino acid
from the recited AT13-024 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 4; and
- a heavy chain CDR2 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 17; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 30; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 43; and
- a light chain CDR2 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 56; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 69. These are the CDR sequences of antibody
AT12-019. Antibody AT12-019, derived from a human AML patient in complete
remission, is e of efficiently binding intact AML cells, which makes a binding
compound containing AT12derived CDR sequences particularly suitable for
AML therapy and/or diagnosis. Such binding compound is for instance used as an
antibody-drug conjugate (ADC), such that a toxic compound will be directed to
AML cells, or it is used by inducing CDC and/or ADCC. Alternatively, or
additionally, such binding compound can be used to mark AML cells for specific
ytosis by tumor-associated myeloid cells such as macrophages or dendritic
cells, which cells can subsequently induce an AML specific immune response.
Again, said sequence identities are preferably at least 85%, more preferably at
least 86%, more preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more preferably at least
91%, more preferably at least 92%, more preferably at least 93%, more preferably
at least 94%, more preferably at least 95%, more preferably at least 96%, more
ably at least 97%, more ably at least 98%, more preferably at least
99%, more preferably 100%. As bed above, at least 1, 2 or 3 amino acid
residues in the recited AT12-019 CDR sequences may vary while retaining the
same kind of binding activity (in kind, not necessarily in amount). Hence, the above
mentioned heavy and light chain CDR 1, 2 and 3 ces preferably contain CDR
sequences that deviate in no more than three, preferably no more than two, more
preferably no more than one amino acid from the recited AT12-019 CDR ces.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a onal equivalent thereof, which comprises:
- a heavy chain CDR1 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 5; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 18; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 31; and
- a light chain CDR1 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 44; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 57; and
- a light chain CDR3 sequence sing a sequence which has at least 80%
sequence ty with SEQ ID NO: 70. These are the CDR sequences of antibody
22. Interestingly, dy AT13-022 is of the IgG3 isotype. Antibody AT13-
022, derived from a human AML patient in complete remission, is capable of
specifically binding intact AML cells, which makes a binding compound containing
AT13derived CDR sequences particularly suitable for AML therapy and/or
diagnosis. Such binding compound is for instance used as an antibody-drug
conjugate (ADC), such that a toxic compound will be directed to AML cells, or it is
used by inducing CDC and/or ADCC. Alternatively, or additionally, such binding
compound can be used to mark AML cells for ic phagocytosis by tumorassociated
myeloid cells such as macrophages or dendritic cells, which cells can
subsequently induce an AML specific immune response. Again, said sequence
identities are preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more preferably at least
89%, more preferably at least 90%, more preferably at least 91%, more preferably
at least 92%, more preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more preferably at least
97%, more ably at least 98%, more preferably at least 99%, more preferably
100%. As described above, at least 1, 2 or 3 amino acid residues in the recited
AT13-022CDR sequences may vary while retaining the same kind of binding
activity (in kind, not necessarily in amount). Hence, the above ned heavy
and light chain CDR 1, 2 and 3 ces ably contain CDR sequences that
deviate in no more than three, preferably no more than two, more ably no
more than one amino acid from the recited AT13-022 CDR sequences.
Also decribed is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 6; and
- a heavy chain CDR2 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 19; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 32; and
- a light chain CDR1 sequence comprising a ce which has at least 80%
ce identity with SEQ ID NO: 45; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 58; and
- a light chain CDR3 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 71. These are the CDR sequences of antibody
23. Interestingly, AT13-023 belongs to the VH4-34 family, which is a family
of VH sequences known for their potential g properties (Bhat et al, 1997).
Antibody AT13-023, derived from a human AML patient in complete remission, is
capable of ically binding intact AML cells, which makes a binding compound
containing AT13derived CDR sequences particularly suitable for AML
therapy and/or diagnosis. Such binding compound is for instance used as an
antibody-drug conjugate (ADC), such that a toxic compound will be directed to
AML cells, or it is used by inducing CDC and/or ADCC. Alternatively, or
additionally, such binding compound can be used to mark AML cells for ic
phagocytosis by tumor-associated myeloid cells such as macrophages or dendritic
cells, which cells can uently induce an AML specific immune response.
Again, said sequence identities are preferably at least 85%, more preferably at
least 86%, more preferably at least 87%, more preferably at least 88%, more
preferably at least 89%, more preferably at least 90%, more preferably at least
91%, more preferably at least 92%, more preferably at least 93%, more preferably
at least 94%, more preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more preferably at least
99%, more preferably 100%. As described above, at least 1, 2 or 3 amino acid
residues in the d AT13-023 CDR sequences may vary while retaining the
same kind of binding activity (in kind, not necessarily in amount). Hence, the above
mentioned heavy and light chain CDR 1, 2 and 3 sequences preferably contain CDR
sequences that deviate in no more than three, preferably no more than two, more
ably no more than one amino acid from the recited AT13-023 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 7; and
- a heavy chain CDR2 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 20; and
- a heavy chain CDR3 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 33; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 46; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 59; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence ty with SEQ ID NO: 72. These are the CDR sequences of antibody
AT13-031. Interestingly, AT13-031 belongs to the VH4-34 family, which is a family
of VH sequences known for their potential killing properties (Bhat et al, 1997).
Antibody AT13-031, derived from a human AML patient in complete remission, is
capable of specifically binding intact AML cells. Moreover, this dy is
preferred e it is capable of efficiently binding and killing cells of the AML
cell line THP-1. Hence, a binding compound containing AT13derived CDR
sequences is particularly suitable for AML therapy and/or diagnosis. Such binding
compound is for instance used as an antibody-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by ng CDC and/or
ADCC. Alternatively, or additionally, such binding compound can be used to mark
AML cells for ic phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
specific immune response. Again, said sequence identities are preferably at least
85%, more preferably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more ably at least 97%, more preferably at least 98%, more
ably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT13-031 CDR sequences may vary while
ing the same kind of g activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT13-031 CDR sequences.
Also described is an isolated, tic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 8; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 21; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 34; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 47; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 60; and
- a light chain CDR3 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 73. These are the CDR ces of antibody
AT12-020. Interestingly, antibody AT12-020 is of the IgG3 e. Antibody AT12-
020, derived from a human AML patient in complete remission, is capable of
specifically binding intact AML cells, which makes a binding compound containing
AT12derived CDR sequences particularly suitable for AML therapy and/or
diagnosis. Such binding compound is for instance used as an antibody-drug
conjugate (ADC), such that a toxic compound will be directed to AML cells, or it is
used by inducing CDC and/or ADCC. Alternatively, or additionally, such binding
compound can be used to mark AML cells for specific phagocytosis by tumorassociated
d cells such as macrophages or dendritic cells, which cells can
subsequently induce an AML specific immune response. Again, said sequence
identities are preferably at least 85%, more preferably at least 86%, more
preferably at least 87%, more preferably at least 88%, more preferably at least
89%, more preferably at least 90%, more preferably at least 91%, more preferably
at least 92%, more preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more preferably at least
97%, more preferably at least 98%, more preferably at least 99%, more ably
100%. As described above, at least 1, 2 or 3 amino acid residues in the recited
AT12-020 CDR sequences may vary while retaining the same kind of binding
activity (in kind, not necessarily in amount). Hence, the above mentioned heavy
and light chain CDR 1, 2 and 3 sequences preferably contain CDR sequences that
deviate in no more than three, preferably no more than two, more preferably no
more than one amino acid from the recited AT12-020 CDR sequences.
Also described is an isolated, tic or recombinant antibody, or a
onal part or a functional equivalent f, which comprises:
- a heavy chain CDR1 sequence comprising a ce which has at least 80%
ce identity with SEQ ID NO: 9; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 22; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 35; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 48; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 61; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 74. These are the CDR sequences of dy
AT13-033. Antibody AT13-033, derived from a human AML patient in complete
remission, is capable of specifically binding intact AML cells. Moreover, this
antibody is preferred e it is capable of efficiently binding and killing cells of
the AML cell line THP-1. Hence, a binding nd containing 33-derived
CDR sequences particularly suitable for AML therapy and/or diagnosis. Such
binding compound is for instance used as an antibody-drug conjugate (ADC), such
that a toxic compound will be directed to AML cells, or it is used by inducing CDC
and/or ADCC. Alternatively, or additionally, such binding compound can be used to
mark AML cells for ic phagocytosis by tumor-associated d cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
specific immune response. Again, said sequence identities are preferably at least
85%, more preferably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more ably at least 95%, more preferably
at least 96%, more preferably at least 97%, more ably at least 98%, more
preferably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT13-033 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
ably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT13-033 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 10; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 23; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 36; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 49; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 62; and
- a light chain CDR3 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 75. These are the CDR ces of dy
AT13-034. Antibody AT13-034, derived from a human AML patient in complete
remission, is capable of ically binding intact AML cells, which makes a
binding compound containing AT13derived CDR sequences particularly
suitable for AML therapy and/or diagnosis. Such binding compound is for instance
used as an antibody-drug conjugate (ADC), such that a toxic nd will be
directed to AML cells, or it is used by inducing CDC and/or ADCC. Alternatively, or
additionally, such g compound can be used to mark AML cells for ic
phagocytosis by tumor-associated myeloid cells such as macrophages or dendritic
cells, which cells can subsequently induce an AML specific immune se.
Again, said sequence identities are preferably at least 85%, more ably at
least 86%, more ably at least 87%, more preferably at least 88%, more
ably at least 89%, more preferably at least 90%, more preferably at least
91%, more preferably at least 92%, more preferably at least 93%, more preferably
at least 94%, more preferably at least 95%, more preferably at least 96%, more
preferably at least 97%, more preferably at least 98%, more preferably at least
99%, more preferably 100%. As described above, at least 1, 2 or 3 amino acid
residues in the recited AT13-034 CDR sequences may vary while retaining the
same kind of binding activity (in kind, not necessarily in amount). Hence, the above
mentioned heavy and light chain CDR 1, 2 and 3 sequences preferably contain CDR
sequences that deviate in no more than three, preferably no more than two, more
preferably no more than one amino acid from the recited 34 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 11; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 24; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence ty with SEQ ID NO: 37; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 50; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 63; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 76. These are the CDR sequences of dy
AT13-035. Antibody AT13-035, derived from a human AML patient in complete
remission, is capable of specifically binding intact AML cells. Moreover, this
antibody is preferred because it is e of efficiently binding and killing cells of
the AML cell line THP-1. Hence, a binding nd containing AT13derived
CDR sequences particularly suitable for AML therapy and/or diagnosis. Such
binding compound is for instance used as an antibody-drug conjugate (ADC), such
that a toxic compound will be directed to AML cells, or it is used by inducing CDC
and/or ADCC. Alternatively, or additionally, such binding compound can be used to
mark AML cells for ic phagocytosis by tumor-associated myeloid cells such as
macrophages or tic cells, which cells can subsequently induce an AML
specific immune response. Again, said sequence ties are preferably at least
85%, more preferably at least 86%, more preferably at least 87%, more ably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more ably at least 92%, more ably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
ably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT13-035 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT13-035 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 12; and
- a heavy chain CDR2 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 25; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 38; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 51; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 64; and
- a light chain CDR3 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 77. These are the CDR sequences of antibody
AT13-036. Antibody AT13-036, d from a human AML patient in complete
ion, is capable of specifically g intact AML cells. Moreover, this
antibody is preferred because it is capable of efficiently binding and killing cells of
the AML cell line THP-1. Hence, a binding compound containing AT13derived
CDR sequences ularly suitable for AML therapy and/or diagnosis. Such
binding compound is for instance used as an antibody-drug conjugate (ADC), such
that a toxic compound will be directed to AML cells, or it is used by inducing CDC
and/or ADCC. Alternatively, or additionally, such binding compound can be used to
mark AML cells for specific phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can uently induce an AML
specific immune response. Again, said sequence ties are preferably at least
85%, more preferably at least 86%, more ably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited 36 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT13-036 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
ce identity with SEQ ID NO: 13; and
- a heavy chain CDR2 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 26; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 39; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 52; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 65; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 78. These are the CDR sequences of antibody
AT13-037. Antibody AT13-037, derived from a human AML patient in complete
ion, is capable of ically binding intact AML cells. Moreover, this
antibody is preferred because it is capable of efficiently binding and g cells of
the AML cell line THP-1, as well as patient-derived primary AML blasts, as shown
in the Examples. Hence, a binding compound containing AT13derived CDR
ces ularly suitable for AML therapy and/or diagnosis. Such binding
compound is for instance used as an antibody-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by inducing CDC and/or
ADCC. Alternatively, or additionally, such binding compound can be used to mark
AML cells for ic ytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
ic immune response. Again, said sequence identities are preferably at least
85%, more preferably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more ably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT13-037 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT13-037 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence ty with SEQ ID NO: 209; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 213; and
- a heavy chain CDR3 ce comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 217; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 221; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 225; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 229. These are the CDR sequences of antibody
AT14-013. dy AT14-013, derived from a human AML patient in complete
remission, is capable of specifically binding primary AML blasts of at least three
different FAB classifications, which makes a g compound ning AT14-
013-derived CDR sequences particularly suitable for AML y and/or
diagnosis. Such binding compound is for instance used for diminishing proliferation
of AML cells, preferably independently from apoptosis and/or independently from
ADCC, CDC or phagocytosis by macrophages or dendritic cells. Preferably, said
binding compound is used for inducing death of AML cells. In some embodiments,
such g compound is used as an dy-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by inducing CDC and/or
ADCC. Alternatively, or additionally, such binding compound can be used to mark
AML cells for specific phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
specific immune response. Again, said sequence identities are preferably at least
85%, more ably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more ably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT14-013 CDR sequences may vary while
retaining the same kind of g activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
ably contain CDR sequences that deviate in no more than three, preferably
no more than two, more ably no more than one amino acid from the recited
13 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional lent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 210; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 214; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 218; and
- a light chain CDR1 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 222; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 226; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 230. These are the CDR sequences of antibody
AT14-014. stingly, antibody AT14-014 is of the IgG3 isotype. Antibody
AT14-014, derived from a human AML patient in complete remission, is capable of
specifically binding intact AML cells, which makes a binding nd containing
AT14derived CDR sequences ularly suitable for AML therapy and/or
diagnosis. Such binding compound is for instance used for diminishing proliferation
of AML cells, preferably independently from apoptosis and/or independently from
ADCC, CDC or phagocytosis by macrophages or dendritic cells. Preferably, said
binding compound is used for inducing death of AML cells. In some embodiments,
such binding compound is used as an antibody-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by inducing CDC and/or
ADCC. atively, or additionally, such binding compound can be used to mark
AML cells for specific phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
specific immune response. Again, said sequence identities are preferably at least
85%, more ably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more ably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably 100%. As described above, at least 1, 2 or
3 amino acid residues in the recited AT14-014 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, ably
no more than two, more preferably no more than one amino acid from the recited
AT14-014 CDR sequences.
Also bed is an ed, tic or recombinant antibody, or a
functional part or a onal equivalent f, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 211; and
- a heavy chain CDR2 sequence comprising a ce which has at least 80%
sequence identity with SEQ ID NO: 215; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 219; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 223; and
- a light chain CDR2 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 227; and
- a light chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 231. These are the CDR sequences of antibody
AT14-015. Interestingly, antibody AT14-015 is of the IgG3 isotype. Antibody
AT14-015, derived from a human AML patient in complete remission, is capable of
specifically binding intact AML cells, which makes a binding compound containing
AT14derived CDR sequences particularly suitable for AML therapy and/or
diagnosis. Such binding compound is for instance used for diminishing proliferation
of AML cells, ably independently from apoptosis and/or ndently from
ADCC, CDC or phagocytosis by macrophages or dendritic cells. Preferably, said
binding compound is used for inducing death of AML cells. In some embodiments,
such binding compound is used as an antibody-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by inducing CDC and/or
ADCC. Alternatively, or additionally, such g compound can be used to mark
AML cells for specific phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can subsequently induce an AML
ic immune response. Again, said sequence identities are ably at least
85%, more preferably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more ably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more ably 100%. As described above, at least 1, 2 or
3 amino acid es in the recited AT14-015 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that deviate in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT14-015 CDR sequences.
Also described is an isolated, synthetic or recombinant antibody, or a
functional part or a functional equivalent thereof, which comprises:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 212; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 216; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence ty with SEQ ID NO: 220; and
- a light chain CDR1 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 224; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with SEQ ID NO: 228; and
- a light chain CDR3 sequence sing a sequence which has at least 80%
sequence identity with SEQ ID NO: 232. These are the CDR sequences of antibody
AT14-016. Interestingly, antibody AT14-016 is of the IgG3 isotype. Antibody
AT14-016, derived from a human AML patient in complete remission, is capable of
specifically binding intact AML cells, which makes a binding compound containing
AT14derived CDR sequences ularly suitable for AML therapy and/or
sis. Such binding compound is for instance used for diminishing proliferation
of AML cells, preferably independently from apoptosis and/or independently from
ADCC, CDC or phagocytosis by macrophages or dendritic cells. Preferably, said
binding compound is used for inducing death of AML cells. In some embodiments,
such binding nd is used as an antibody-drug conjugate (ADC), such that a
toxic compound will be directed to AML cells, or it is used by inducing CDC and/or
ADCC. Alternatively, or additionally, such binding compound can be used to mark
AML cells for specific phagocytosis by tumor-associated myeloid cells such as
macrophages or dendritic cells, which cells can uently induce an AML
specific immune response. Again, said sequence identities are preferably at least
85%, more preferably at least 86%, more preferably at least 87%, more preferably
at least 88%, more preferably at least 89%, more ably at least 90%, more
preferably at least 91%, more preferably at least 92%, more preferably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more ably at least 98%, more
ably at least 99%, more preferably 100%. As bed above, at least 1, 2 or
3 amino acid residues in the recited AT14-016 CDR sequences may vary while
retaining the same kind of binding activity (in kind, not necessarily in amount).
Hence, the above mentioned heavy and light chain CDR 1, 2 and 3 sequences
preferably contain CDR sequences that e in no more than three, preferably
no more than two, more preferably no more than one amino acid from the recited
AT14-016 CDR sequences.
Preferably, an antibody described hereincomprises a variable heavy chain
sequence and/or a variable light chain sequence as depicted in Table 1 or Figure 1,
or a ce which has at least 80% sequence identity thereto. As shown in Table
1, the variable heavy chain sequences of antibodies AT12-023, AT12-025, AT13-
024, AT12-019, AT13-022, 23, AT13-031, AT12-020, AT13-033, 34,
AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 and AT14-016 are
SEQ ID NOs 79-91 and 233-236, respectively. The variable light chain sequences of
antibodies AT12-023, AT12-025, AT13-024, AT12-019, AT13-022, AT13-023, AT13-
031, AT12-020, AT13-033, AT13-034, AT13-035, AT13-036, AT13-037, AT14-013,
AT14-014, AT14-015 and AT14-016 are SEQ ID NOs 92-104 and 237-240,
respectively. Also described is, therefore, an antibody or functional part or
equivalent thereof according to the present disclosure, comprising a variable heavy
chain ce having at least 80% sequence identity with a sequence selected
from the group consisting of SEQ ID NOs: 79-91 and 233-236, and/or comprising a
variable light chain ce having at least 80% sequence identity with a
sequence selected from the group consisting of SEQ ID NOs: 92-104 and 237-240,
or sequences that are at least 85%, more preferably at least 86%, more preferably
at least 87%, more preferably at least 88%, more ably at least 89%, more
preferably at least 90%, more preferably at least 91%, more ably at least
92%, more preferably at least 93%, more preferably at least 94%, more preferably
at least 95%, more preferably at least 96%, more preferably at least 97%, more
preferably at least 98%, more preferably at least 99%, or even 100% identical to
any one of these heavy chain or light chain ces. The higher the identity, the
more closely an antibody resembles an antibody depicted in Figure 1. Preferably, a
binding nd described hereincomprises the variable heavy chain sequence of
any one of the antibodies depicted in Figure 1, together with the variable light
chain sequence of the same antibody, or heavy and light chain sequences that are
at least 80%, preferably at least 85%, or at least 86%, or at least 87%, or at least
88%, or at least 89%, or at least 90%, or 91%, or at least 92%, or at least 93%, or at
least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at
least 99%, o.
For instance, antibody 23 has the variable heavy chain sequence of
SEQ ID NO: 79 and the variable light chain sequence of SEQ ID NO: 92 as
depicted in Table 1. A binding compound according to the present disclosure,
having a variable heavy chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 79 is therefore preferably bed.
ably, said binding compound also comprises a variable light chain sequence
having at least 80%, preferably at least 85%, or at least 86%, or at least 87%, or at
least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at
least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at
least 98%, or at least 99%, sequence identity with SEQ ID NO: 92.
Antibody AT12-025 has the variable heavy chain sequence of SEQ ID NO:
80 and the variable light chain sequence of SEQ ID NO: 93 as depicted in Table 1.
r described is ore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
ce identity with SEQ ID NO: 80. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 93.
dy AT13-024 has the variable heavy chain sequence of SEQ ID NO:
81 and the variable light chain sequence of SEQ ID NO: 94 as depicted in Table 1.
Further described is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 81. Preferably, said binding compound also
ses a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 94.
Antibody AT12-019 has the le heavy chain sequence of SEQ ID NO:
82 and the variable light chain sequence of SEQ ID NO: 95 as depicted in Table 1.
Further described is therefore a binding compound according to the present
disclosure, having a le heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 82. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, ably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 95.
Antibody AT13-022 has the le heavy chain sequence of SEQ ID NO:
83 and the variable light chain sequence of SEQ ID NO: 96 as depicted in Table 1.
Further described is therefore a binding compound ing to the present
disclosure, having a variable heavy chain ce having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 83. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 96.
Antibody AT13-023 has the variable heavy chain sequence of SEQ ID NO:
84 and the variable light chain sequence of SEQ ID NO: 97 as depicted in Table 1.
Further described is ore a binding compound according to the t
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 84. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 97.
Antibody AT13-031 has the variable heavy chain sequence of SEQ ID NO:
85 and the variable light chain ce of SEQ ID NO: 98 as depicted in Table 1.
r described is therefore a g compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 85. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
ty with SEQ ID NO: 98.
Antibody AT12-020 has the le heavy chain sequence of SEQ ID NO:
86 and the variable light chain sequence of SEQ ID NO: 99 as depicted in Table 1.
Further described is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 86. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 99.
Antibody AT13-033 has the variable heavy chain sequence of SEQ ID NO:
87 and the variable light chain ce of SEQ ID NO: 100 as depicted in Table 1.
Further provided is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 87. Preferably, said binding compound also
comprises a le light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 100.
Antibody AT13-034 has the variable heavy chain sequence of SEQ ID NO:
88 and the variable light chain sequence of SEQ ID NO: 101 as depicted in Table 1.
Further described is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 88. Preferably, said g compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 101.
Antibody AT13-035 has the variable heavy chain ce of SEQ ID NO:
89 and the variable light chain ce of SEQ ID NO: 102 as depicted in Table 1.
Further described is ore a g compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 89. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 102.
dy AT13-036 has the variable heavy chain ce of SEQ ID NO:
90 and the variable light chain sequence of SEQ ID NO: 103 as depicted in Table 1.
Further described is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 90. Preferably, said binding compound also
comprises a variable light chain ce having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 103.
dy AT13-037 has the variable heavy chain ce of SEQ ID NO:
91 and the variable light chain sequence of SEQ ID NO: 104 as depicted in Table 1.
Further described is therefore a binding compound ing to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 91. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 104.
Antibody AT14-013 has the variable heavy chain sequence of SEQ ID NO:
233 and the le light chain sequence of SEQ ID NO: 237 as depicted in Table
1. Further described is therefore a binding compound according to the present
disclosure, having a variable heavy chain ce having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 233. Preferably, said binding compound also
comprises a le light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 237.
Antibody AT14-014 has the variable heavy chain sequence of SEQ ID NO:
234 and the variable light chain sequence of SEQ ID NO: 238 as depicted in Table
1. r described is therefore a binding compound ing to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 234. Preferably, said binding nd also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 238.
dy AT14-015 has the variable heavy chain sequence of SEQ ID NO:
235 and the variable light chain sequence of SEQ ID NO: 239 as depicted in Table
1. Further desribed is therefore a binding compound according to the present
sure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 235. ably, said binding compound also
comprises a le light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 239.
Antibody AT14-016 has the variable heavy chain sequence of SEQ ID NO:
236 and the variable light chain sequence of SEQ ID NO: 240 as depicted in Table
1. Further bed is therefore a binding compound according to the present
disclosure, having a variable heavy chain sequence having at least 80%, preferably
at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at
least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at
least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%
sequence identity with SEQ ID NO: 236. Preferably, said binding compound also
comprises a variable light chain sequence having at least 80%, preferably at least
85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least
90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least
95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, sequence
identity with SEQ ID NO: 240.
In one particularly preferred embodiment, an antibody or functional part or
functional equivalent according to the present sure is described, which is able
to bind snRNP200. snRNP200, also known as U5-snRNP, is a protein complex that
is part of the spliceosome in all eukaryotic cells. Normally, snRNP200 is located in
the nucleus. However, the present disclosure describes the sing insight that
00 is also t on the surface of AML cells. This antigen is bound by at
least antibodies AT12-023, AT13-031 and AT13-037, and functional parts and
functional equivalents thereof. Hence, snRNP200 is an important target for anti-
AML therapy and 00-specific antibodies described hereinare therefore
particularly suitable for counteracting these cells.
Also described is an isolated, tic or recombinant nucleic acid le
with a length of at least 15 nucleotides, or a functional equivalent thereof, encoding
at least one CDR sequence of an antibody or functional part or functional
equivalent according to the disclosure. Preferably a nucleic acid molecule described
hereinhas a length of at least 30 nucleotides, more preferably at least 50
nucleotides, more preferably at least 75 nucleotides. A nucleic acid molecule
described hereinis for instance isolated from a B-cell which is capable of producing
an antibody according to the disclosure. Said B-cell preferably es antibody
AT12-023, AT12-025, AT13-024, AT12-019, 22, AT13-023, AT13-031, AT12-
020, AT13-033, AT13-034, AT13-035, AT13-036, AT13-037 AT14-013, AT14-014,
AT14-015, or AT14-016. In a preferred embodiment a nucleic acid molecule
ng at least the heavy chain CDR3 ce and the light chain CDR3
sequence of an dy according to the present disclosure is described.
As used herein the term “an isolated, synthetic or inant nucleic acid
molecule with a length of at least 15 nucleotides, or a functional lent thereof,
encoding at least one CDR sequence of an antibody or functional part or functional
lent according to the ion” is herein also referred to as “a nucleic acid
molecule or functional equivalent according to the ion”.
As used herein, a nucleic acid molecule or nucleic acid sequence of the
ion preferably comprises a chain of nucleotides, more preferably DNA, cDNA
or RNA. In other embodiments a nucleic acid molecule or nucleic acid sequence of
the invention comprises other kinds of nucleic acid structures such as for instance
a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a
ribozyme. Such other nucleic acid structures are referred to as functional
equivalents of a nucleic acid sequence. The term “functional equivalent of a nucleic
acid molecule” thus encompasses a chain comprising non-natural nucleotides,
modified nucleotides and/or non-nucleotide building blocks which exhibit the same
function as natural nucleotides.
Nucleic acid sequences encoding heavy chain and light chain CDRs of
antibodies AT12-023, AT12-025, 24, AT12-019, AT13-022, AT13-023, AT13-
031, AT12-020, AT13-033, AT13-034, AT13-035, 36, AT13-037, AT14-013,
AT14-014, AT14-015 and AT14-016 are depicted in Table 1. Nucleic acid molecules
encoding a heavy or light chain CDR of an antibody described hereinwhich differ
from the CDR nucleic acid sequences ed in Table 1 but have nucleic acid
codons which encode the same amino acids of said heavy or light chain CDR are
also encompassed by the sure. Such nucleic acid molecules for instance
comprise nucleic acid sequences that have been codon zed for a producer cell,
such as for instance E.coli or Chinese hamster ovary (CHO) cells, NSO cells (a
mouse a) or 293(T) cells, enabling high scale production of binding
compounds according to the present disclosure. It should be noted that antibody
production can be done by any recombinant antibody production system; the four
producer cell systems mentioned here are only a few examples of the many systems
that are available to date. As used herein, the term “codon” means a triplet of
tides (or functional equivalents thereof) that encode a specific amino acid
e. The term “codon zed” means that one or more codons from the
original, human nucleic acid sequence are replaced by one or more codons that are
preferred by a certain antibody production system. These replacement codons
preferably encode the same amino acid residue as the original human codon that
has been replaced. Alternatively, one or more replacement codons encode a
different amino acid residue. This preferably results in conservative amino acid
substitution, although this is not necessary. Typically, in constant regions and
framework regions one or more amino acid substitutions are generally allowed. In
CDR regions, preferably codons are used that encode the same amino acid residue
as the original human codon that has been replaced.
Furthermore, nucleic acid molecules encoding a heavy or light chain CDR
which is not identical to, but based on, a CDR sequence of an antibody depicted in
Table 1 are also encompassed by the present disclosure, as long as the resulting
CDR has at least 80% sequence identity with a CDR sequence depicted in Table 1.
Further bed is, ore, a nucleic acid molecule or onal
equivalent thereof or a vector, comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
-182 and 4. Preferably, the resulting CDR differs in no more than
three, preferably in no more than two, preferably in only one amino acid from the
original CDR sequence of an antibody according to the present disclosure.
Preferred c acid molecules or functional lents or vectors
described hereincomprise:
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to a sequence which is selected from the group consisting of SEQ
ID NO's:105-117 and 241-244, and/or
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to a sequence which is selected from the group consisting of SEQ ID
18-130 and 245-248, and/or
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to a sequence which is ed from the group consisting of SEQ ID
NO's:131-143 and 249-252, and/or
- a light chain CDR1 encoding sequence which has at least 80% ce
identity to a sequence which is selected from the group consisting of SEQ ID
NO's:144-156 and 253-256, and/or
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to a sequence which is selected from the group consisting of SEQ ID
57-169 and 257-260, and/or
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to a ce which is selected from the group consisting of SEQ ID
70-182 and 261-264.
Said sequence identities are preferably at least 85%, more preferably at
least 90%, most preferably at least 95% sequence, most preferably 100%. A nucleic
acid molecule described hereinpreferably comprises heavy chain CDR 1-3 -encoding
sequences and light chain CDR 1-3 -encoding sequences from the same antibody,
n said dy is selected from the group consisting of AT12-023, AT12-025,
AT13-024, AT12-019, AT13-022, 23, AT13-031, AT12-020, AT13-033, AT13-
034, AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 and
AT14-016, and heavy chain CDR 1-3 -encoding sequences and light chain CDR 1-3 -
encoding sequences that have at least 80% sequence identity thereto.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 105, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 118, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
ty to SEQ ID NO: 131, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 144, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 157, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 170. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT12-023. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. ably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
ably in only one amino acid from the original CDR ces of dy
AT12-023.
Further described is a nucleic acid molecule or functional lent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence ty to SEQ ID NO: 106, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 119, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
ty to SEQ ID NO: 132, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 145, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 158, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 171. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT12-025. Again, said sequence identities are
preferably at least 80%, more ably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. ably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT12-025.
Further described is a nucleic acid le or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 107, and
- a heavy chain CDR2 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 120, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 133, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 146, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 159, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 172. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-024. Again, said sequence identities are
ably at least 80%, more preferably at least 85%, more ably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-024.
Further bed is a nucleic acid molecule or functional equivalent or one
or more s that comprise(s):
- a heavy chain CDR1 ng nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 108, and
- a heavy chain CDR2 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 121, and
- a heavy chain CDR3 encoding ce which has at least 80% sequence
ty to SEQ ID NO: 134, and
- a light chain CDR1 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 147, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 160, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 173. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT12-019. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the al CDR sequences of antibody
AT12-019.
r described is a nucleic acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 109, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 122, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 135, and
- a light chain CDR1 encoding sequence which has at least 80% ce
ty to SEQ ID NO: 148, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 161, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 174. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-022. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-022.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 ng nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 110, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 123, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 136, and
- a light chain CDR1 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 149, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 162, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 175. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 ces of antibody AT13-023. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most ably at least 95%, most ably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-023.
r described is a nucleic acid molecule or functional lent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 111, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 124, and
- a heavy chain CDR3 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 137, and
- a light chain CDR1 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 150, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 163, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 176. The d SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody 31. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR ces differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-031.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that se(s):
- a heavy chain CDR1 ng nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 112, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 125, and
- a heavy chain CDR3 encoding ce which has at least 80% sequence
identity to SEQ ID NO: 138, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 151, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 164, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 177. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT12-020. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR ces differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT12-020.
Further described is a nucleic acid le or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 113, and
- a heavy chain CDR2 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 126, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
ty to SEQ ID NO: 139, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 152, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 165, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 178. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-033. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most ably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
ably in only one amino acid from the original CDR ces of antibody
AT13-033.
Further described is a nucleic acid molecule or functional lent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
ce identity to SEQ ID NO: 114, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 127, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 140, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 153, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 166, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 179. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-034. Again, said sequence identities are
ably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR ces differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of dy
AT13-034.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
ce identity to SEQ ID NO: 115, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 128, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 141, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 154, and
- a light chain CDR2 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 167, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 180. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-035. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR ces differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR ces of antibody
AT13-035.
Further described is a nucleic acid molecule or functional lent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 116, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 129, and
- a heavy chain CDR3 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 142, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 155, and
- a light chain CDR2 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 168, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 181. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-036. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-036.
Further described is a nucleic acid le or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 117, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 130, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 143, and
- a light chain CDR1 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 156, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
ty to SEQ ID NO: 169, and
- a light chain CDR3 ng sequence which has at least 80% sequence
ty to SEQ ID NO: 182. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT13-037. Again, said sequence ties are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
AT13-037.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that se(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence ty to SEQ ID NO: 241, and
- a heavy chain CDR2 encoding sequence which has at least 80% ce
identity to SEQ ID NO: 245, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 249, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 253, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 257, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 261. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT14-013. Again, said ce identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most ably 100%. Preferably, the ing
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of antibody
Further described is a nucleic acid molecule or functional equivalent or one
or more s that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 242, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 246, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 250, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 254, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 258, and
- a light chain CDR3 encoding sequence which has at least 80% ce
ty to SEQ ID NO: 262. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody AT14-014. Again, said ce identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the al CDR sequences of antibody
AT14-014.
Further described is a nucleic acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 243, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 247, and
- a heavy chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 251, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 255, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 259, and
- a light chain CDR3 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 263. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 ces of antibody AT14-015. Again, said sequence identities are
preferably at least 80%, more ably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR ces of antibody
AT14-015.
Further described is a c acid molecule or functional equivalent or one
or more vectors that comprise(s):
- a heavy chain CDR1 encoding nucleic acid sequence which has at least 80%
sequence identity to SEQ ID NO: 244, and
- a heavy chain CDR2 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 248, and
- a heavy chain CDR3 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 252, and
- a light chain CDR1 encoding sequence which has at least 80% sequence
identity to SEQ ID NO: 256, and
- a light chain CDR2 encoding sequence which has at least 80% sequence
ty to SEQ ID NO: 260, and
- a light chain CDR3 ng sequence which has at least 80% sequence
identity to SEQ ID NO: 264. The recited SEQ ID NOs are the heavy and light chain
CDR1-3 sequences of antibody 16. Again, said sequence identities are
preferably at least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95%, most preferably 100%. Preferably, the resulting
CDR sequences differ in no more than three, preferably in no more than two,
preferably in only one amino acid from the original CDR sequences of dy
AT14-016.
A preferred nucleic acid molecule or vector(s) described hereinencode(s) at
least the variable heavy chain sequence and/or the variable light chain sequence of
an antibody or functional part or onal equivalent according to the present
disclosure. Preferably, said nucleic acid molecule(s) or vector(s) encode(s) at least
the variable heavy chain sequence and the variable light chain sequence of an
antibody or functional part or functional equivalent according to the present
disclosure. In one embodiment, a nucleic acid molecule or functional equivalent or
one or more s described herein which se(s) a sequence which has at
least 80% ce identity with a sequence selected from the group consisting of
SEQ ID NOs 183-195 and 265-268 and/or a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs 196-208 and 269-272. Said sequence identities are preferably at least 85%,
more ably at least 86%, more preferably at least 87%, more preferably at
least 88%, more preferably at least 89%, more preferably at least 90%, more
preferably at least 91%, more preferably at least 92%, more ably at least
93%, more preferably at least 94%, more preferably at least 95%, more preferably
at least 96%, more preferably at least 97%, more preferably at least 98%, more
preferably at least 99%, more preferably 100%.
More preferably, a c acid molecule or a functional equivalent or one or
more vectors according to the present sure comprise(s) a variable heavy chain
encoding sequence as well as a variable light chain encoding sequence which
resemble the le heavy and light chain encoding sequences of the same
antibody depicted in Table 1. Thus, in a preferred embodiment a c acid
molecule or functional equivalent or vector(s) described comprise(s) the
variable heavy chain ng sequence of antibody AT12-023, 25, AT13-
024, AT12-019, AT13-022, AT13-023, AT13-031, AT12-020, AT13-033, AT13-034,
AT13-035, AT13-036, AT13-037, AT14-013, AT14-014, AT14-015 or AT14-016, and
the variable light chain encoding sequence of the same antibody, or sequences that
are at least 80%, more preferably at least 85%, more ably at least 86%, more
preferably at least 87%, more preferably at least 88%, more preferably at least
89%, more preferably at least 90%, more preferably at least 91%, more ably
at least 92%, more preferably at least 93%, more preferably at least 94%, more
preferably at least 95%, more preferably at least 96%, more preferably at least
97%, more preferably at least 98%, more preferably at least 99%, identical thereto.
In some embodiments, nucleic acid molecules and onal equivalents
f and vectors are described that encode an dy or functional part or
functional equivalent according to the present disclosure. Further described is
therefore a nucleic acid molecule, or functional equivalent thereof, or one or more
vectors, that encode(s) antibody AT12-023, AT12-025, 24, AT12-019, AT13-
022, AT13-023, AT13-031, AT12-020, AT13-033, AT13-034, AT13-035, AT13-036,
AT13-037, AT14-013, AT14-014, AT14-015 or AT14-016, or functional part or
functional equivalent thereof. In some embodiments, said nucleic acid molecule or
functional equivalent or vector(s) is/are codon optimized for a non-human
recombinant expression system.
Further bed is a vector comprising a nucleic acid molecule or
functional equivalent according to the present disclosure. As used herein “a vector
sing a nucleic acid molecule or functional equivalent according to the
invention” is also referred to as “a vector ing to the invention”. These terms
ass one or more vector(s) according to the invention, comprising one or more
nucleic acid molecule(s) or functional equivalent(s) according to the invention. As
used herein, the singular term “a” encompasses the term “one or more”.
Methods for constructing s comprising one or more nucleic acid
molecule(s) or functional equivalent(s) described herein are well known in the art.
Non-limiting examples of vectors suitable for generating a vector described herein
are retroviral and lentiviral vectors. Such vectors are suitable for a variety of
applications. For instance, one or more vectors described herein comprising a
therapeutically cial nucleic acid sequence described hereinis/are suitable for
prophylactic or therapeutic applications against AML. Administration of such
vector(s) to an individual, preferably a human, in need thereof results in expression
of said prophylactic or therapeutic nucleic acid sequence in vivo resulting in at
least partial treatment or laxis against AML. Said vector(s) can also be used
in applications involving in vitro expression of a nucleic acid molecule of interest,
for instance for (commercial) tion of antibodies or onal equivalents
according to the present disclosure. Also described is therefore an isolated or
recombinant cell, or a non-human animal, comprising at least one nucleic acid
le or functional equivalent, or at least one vector, according to the t
disclosure.
A nucleic acid molecule or a vector described hereinis particularly useful for
generating antibodies or functional parts or functional equivalents ing to the
present disclosure, which are specific for AML. This is for instance done by
introducing such nucleic acid molecule or (s) into a cell so that the cell’s
nucleic acid translation machinery will produce the encoded antibodies or
functional parts or functional equivalents. In one embodiment, a nucleic acid
le or vector encoding a heavy and/or light chain described herein is
expressed in so called er cells, such as for instance E.coli, CHO, NSO or
293(T) cells, some of which are adapted to commercial antibody production. Of
note, any recombinant antibody production system is suitable; these four producer
cell systems mentioned are only a few examples of the many systems that are
available to date. As described herein before, in such cases it is preferred to use
nucleic acid molecules wherein the original human sequences as described herein
are codon optimized for the producer cell. Proliferation of said producer cells
results in a producer cell line capable of producing binding compounds ing to
the present disclosure. Preferably, said producer cell line is suitable for producing
antibodies for use in . Hence, said producer cell line is preferably free of
pathogenic agents such as pathogenic organisms. Most preferably, antibodies
consisting of human sequences are generated using at least one nucleic acid
molecule or vector according to the present disclosure.
An isolated or recombinant antibody producing cell capable of producing a
binding compound described herein is therefore also described. Such cell typically
ses at least one nucleic acid molecule or vector according to the present
disclosure, which preferably contains a nucleic acid sequence that is codon
optimized for said cell. An antibody producing cell is defined herein as a cell which
is capable of producing and/or secreting antibodies or functional parts or functional
equivalents thereof, and/or which is capable of developing into a cell which is
e of producing and/or secreting antibodies or functional parts or functional
equivalents thereof. An antibody ing cell bed herein is preferably a
producer cell which is adapted to commercial antibody production. As explained
above, said producer cell is preferably suitable for ing antibodies for use in
humans. A method for producing an antibody or functional part or functional
equivalent according to the present disclosure is therefore also described, said
method comprising providing a cell, ably an antibody producing cell, with a
c acid le or functional equivalent or a vector according to the present
disclosure, and allowing said cell to translate said nucleic acid molecule or
functional equivalent or vector, thereby producing said antibody or functional part
or functional equivalent according to the present disclosure. A method described
herein ably further ses a step of harvesting, purifying and/or isolating
said antibody or functional part or functional equivalent according to the t
disclosure. Obtained binding compounds described herein are preferably used in
human therapy, optionally after onal purifying, ion or processing steps.
Another aspect of the present disclosure describes an antibody or functional
part or functional equivalent described herein, which is coupled to another
compound. In one ment, a binding compound described herein is coupled to
another therapeutic moiety, such as a chemotherapeutic drug or other toxic
compound or radioactive compound, to form a so called “antibody-drug conjugate”.
In another embodiment, a moiety that is coupled to a binding compound described
herein is an immunomodulatory molecule such as for instance a CD3-specific
antibody. Such CD3-specific antibody is e of binding T cells and, if coupled to
a binding compound bed herein, it will target T cells to AML cells, thereby
enhancing an anti-leukemic T-cell response. This provides an even stronger anti-
AML effect. One preferred embodiment of the present disclosure ore
bes a bispecific or multispecific binding nd, comprising an AML-
specific binding compound described herein and an immunomodulatory molecule,
preferably a CD3-specific binding compound. Another preferred embodiment
describes an anti-AML compound, said compound comprising a binding compound
ing to the t disclosure, which is specific for AML cells, and a toxic
moiety. In some other ments, a binding compound described herein is
coupled to a label. This allows detection of myeloproliferative cells, such as AML
cells, using such labeled binding compound. Other embodiments describe a binding
compound described herein that is coupled to another AML-binding compound. In
some embodiments, such other AML-binding nd is also a binding compound
according to the present disclosure. Described is therefore a compound comprising
two binding compounds described herein that are d to each other. This is,
however, not necessary since a binding compound described herein can also be
coupled to other AML-binding compounds, such as currently known antibodies that
bind AML cells. Bispecific compounds described herein allow, for instance, for
increased binding of AML cells, especially when the two coupled binding
compounds are ic for different epitopes on AML cells. Such bispecific
compound is thus very le for therapeutic or stic applications. It is also
possible to use bispecific compounds described herein in assays wherein different
AML cells are bound to the same bispecific binding compound.
In one embodiment, a synthetic or recombinant antibody is bed, or a
onal part or a functional equivalent thereof, which comprises one Fab
fragment of an antibody according to the present disclosure, and one Fab fragment
of another antibody according to the present disclosure. The resulting binding
compound is specific for AML cells, but each Fab arm will typically bind its own
epitope. In some embodiments, the epitopes recognized by the Fab fragments are
different from each other. In another embodiment, the epitopes are the same. The
Fab arms may bind the epitopes with different affinity. Alternatively, the Fab arms
bind their epitopes with essentially the same affinity, meaning that the KD of the
Fab arms differ no more than 30%, preferably no more than 20% or no more than
% from each other.
Said other moiety, for example a chemotherapeutic agent or a CD3-specific
antibody, is preferably coupled to a binding compound described herein via a linker
such as for instance an acid-labile hydrazone linker, or via a peptide linker like
citruline-valine, or through a thioether linkage, or by sortase catalized
transamidation, which is described in detail in WO 87994.
Sortase catalized transamidation involves engineering of a e
recognition site (LPETGG) on the heavy chain of an antibody, preferably on the C-
terminal part of the heavy chain, and on the moiety to be coupled to said antibody.
The antibody and the moiety further typically contain a GGGGS sequence and a
tag for purification es, such as a HIS tag. Subsequently sortase mediated
midation is performed followed by click try linkage. In a sortase
zed transaminidation, "click chemistry linkage" typically involves chemical
coupling of, for instance, an alkyne-containing reagent and, for instance, an ontaining
reagent which are added by sortase through addition of glycines to the
sortase motif on the heavy chain of the antibody and to a sortase motif on the
moiety (such as a protein, peptide or antibody) to be coupled to the antibody. In one
embodiment, the present disclosure therefore bes an antibody described
herein wherein a sortase recognition site (LPETGG) is engineered on the heavy
chain of the antibody, ably on the C-terminal part of the heavy chain, the
antibody preferably further ning a GGGGS sequence and a purification tag,
such as a HIS tag.
In r ment a binding compound described herein is coupled to
another moiety via a thioether linkage. In such case, one or more cysteines are
preferably incorporated into a binding compound according to the invention.
Cysteines contain a thiol group and, therefore, incorporation of one or more
cysteines into a binding compound according to the present sure, or
replacement of one or more amino acids by one or more cysteines of a binding
compound according to the present disclosure, enable coupling of said binding
compound to another moiety. Said one or more cysteines are preferably introduced
into a binding compound described herein at a position where it does not
significantly nce folding of said binding compound, and does not significantly
alter antigen binding or effector function. The present disclosure therefore also
describes a binding nd described herein wherein at least one amino acid
other than cysteine has been replaced by a ne.
In one embodiment, an AML-specific binding compound described herein is coupled
to at least one other ecific binding compound according to the t
disclosure. Such bispecific or multispecific binding compound provides a stronger
anti-AML effect.
Binding compounds described herein are suitable for use against
myeloproliferative disorders such as AML, or acute leukemia’s that ped from
myelodysplastic syndrome, chronic myeloid leukemia, myelofibrosis or other, nonmalignant
myeloproliferative syndromes. As some of the antibodies also bind to
non-myeloid, lymphoproliferative malignancies such as multiple myeloma and B-
non Hodgkin lymphoma (B-NHL), they are suitable for use against these disorders
as well. Binding compounds described herein are ore particularly suitable for
use as a medicine or lactic agent. Preferably, binding compounds described
herein are used which consist of human sequences, in order to reduce the chance of
adverse side s when human individuals are treated. Such human sequences
can be isolated from a human or synthetically or recombinantly produced based on
the sequence of human antibodies, optionally using codon optimized nucleic acid
sequences that encode the same amino acids as the original human nucleic acid
ce. Described is therefore an antibody or functional part or onal
equivalent described herein for use as a medicament and/or prophylactic agent.
Said antibody preferably comprises an antibody selected from the group consisting
of AT12-023, AT12-025, AT13-024, AT12-019, AT13-022, AT13-023, 31,
AT12-020, AT13-033, 34, AT13-035, AT13-036, AT13-037, 13,
14, AT14-015 and AT14-016. Also described is a nucleic acid molecule or
functional equivalent thereof described herein or a vector described herein
comprising such nucleic acid or functional equivalent, or a cell ing to the
present disclosure, for use as a medicament and/or prophylactic agent. When (a
vector comprising) one or more nucleic acid molecule(s) or functional equivalent(s)
described herein is/are administered, the nucleic acid molecule(s) or functional
equivalent(s) will be translated in situ by the host’s machinery into a binding
compound according to the present disclosure. ed binding compounds
described herein are capable of preventing and/or racting myeloproliferative
disorders such as AML and lymphoproliferative disorders such as for ce
lymphoma, B-NHL and multiple myeloma. Further described is therefore an
antibody or functional part or functional lent according to the present
disclosure, or a nucleic acid molecule or functional equivalent according to the
present disclosure, or a vector or a cell according to the present disclosure, for use
in a method for at least in part treating or preventing a myeloproliferative or
lymphoproliferative disorder. As bed herein before, such disorder can be
treated or ted by using cytotoxic binding compounds according to the present
disclosure. As demonstrated in the Examples, at least dies AT13-033, AT13-
035, AT13-036, AT13-037, AT12-023, AT12-025 and AT13-031 have cytotoxic
activity. Further described is therefore an antibody ed from the group
consisting of antibodies AT13-033, AT13-035, AT13-036, AT13-037, AT12-023,
AT12-025 and AT13-031, and functional parts and functional equivalents f,
or a nucleic acid molecule or functional equivalent or one or more vectors ng
therefore, for use in a method for at least in part treating or preventing a
roliferative or lymphoproliferative disorder. Preferably, said disorder is
AML.
In some embodiments, a binding compound described herein is coupled to a
therapeutic moiety, such as a chemotherapeutic drug or other toxic compound or a
radioactive compound or an immunomodulatory molecule such as for instance a
ecific antibody, to form a so called “antibody-drug conjugate” or a “chimeric
antigen receptor (CAR) T cell”, respectively, which is able to counteract a
myeloproliferative or lymphoproliferative disorder.
In some embodiments, said lymphoproliferative disorder is d with one
or more antibodies selected from the group consisting of antibodies AT12-019,
AT12-023, AT12-025, AT13-024 and AT13-031, and functional parts and functional
equivalents thereof. Further described is therefore an antibody ed from the
group consisting of dies AT12-019, AT12-023, AT12-025, AT13-024 and
AT13-031, and functional parts and functional equivalents thereof, or a nucleic acid
molecule or functional equivalent or one or more vectors encoding therefore, for use
in a method for at least in part ng or preventing a proliferative
disorder. Preferably, said lymphoproliferative disorder is ma, B-NHL or
le myeloma.
A binding nd according to the present disclosure, or a nucleic acid
le or functional equivalent f according to the present disclosure, or at
least one vector or cell according to the present disclosure, is preferably used for at
least in part treating and/or preventing AML. As used herein the term “at least in
part treating and/or preventing AML” includes counteracting AML tumor growth
and/or alleviating symptoms resulting from the presence of AML cells in a patient.
Also bed is therefore a use of an antibody or functional part or functional
equivalent according to the present disclosure, or of a nucleic acid molecule or
functional equivalent according to the present disclosure, or of at least one vector
or cell according to the present disclosure, for the preparation of a medicament
and/or prophylactic agent for at least in part treating and/or preventing AML.
Further described is an antibody or functional part or functional equivalent
according to the present disclosure, or a nucleic acid le or functional
equivalent according to the present disclosure, or at least one vector or cell
according to the present disclosure, for use in a method for at least in part treating
and/or preventing AML.
Preferred antibodies for use in any of the recited s are antibodies
AT12-023, 25, AT13-024, AT12-019, AT13-022, AT13-023, AT13-031, AT12-
020, AT13-033, AT13-034, AT13-035, AT13-036, 37, AT14-013, AT14-014,
AT14-015 and AT14-016.
The present disclosure further describes a composition comprising an
antibody or functional part or functional equivalent according to the present
disclosure. A composition comprising a nucleic acid le or functional
equivalent described herein is also bed, as well as a composition comprising a
vector or a cell according to the present disclosure. In some embodiments a
composition described herein ses at least two antibodies, onal parts or
functional lents according to the present disclosure.
A composition described herein preferably comprises a pharmaceutical
composition. Said pharmaceutical composition preferably also comprises a
pharmaceutical acceptable carrier, t and/or excipient. Non-limiting examples
of suitable carriers for instance comprise keyhole limpet yanin (KLH),
serum albumin (e.g. BSA or RSA) and ovalbumin. In one preferred ment
said le carrier comprises a solution, like for example saline. A
pharmaceutical composition described herein is preferably suitable for human use.
The present disclosure further describes a method for at least in part
treating and/or preventing a myeloproliferative or lymphoproliferative disorder,
comprising administering to an individual in need thereof a therapeutically
effective amount of an antibody or onal part or functional equivalent
according to the present disclosure, and/or a nucleic acid molecule or functional
equivalent thereof according to the present disclosure, and/or a vector or cell
according to the present sure, and/or a composition according to the present
disclosure. As used herein, an “individual” or ct” is a human or an animal,
ably a human AML patient. Said ition is preferably a pharmaceutical
composition according to the present disclosure.
A binding compound and/or composition described herein is particularly
suitable for administering to immune compromised individuals with an increased
risk of cations, such as individuals that have undergone chemotherapy,
particularly infants and elderly people. A binding compound, or a nucleic acid
molecule or functional equivalent thereof, or a vector, and/or a pharmaceutical
composition described herein is preferably administered via one or more ions.
Typical doses of administration of a binding compound described herein are
n 0.1 and 10 mg per kg body weight.
A binding compound described herein is also particularly suitable for
diagnostic use. For instance, if an individual, preferably a human, is suspected of
suffering from a myeloproliferative disorder such as AML or a lymphoproliferative
disorder such as B-NHL or multiple a, a sample, such as a blood or tissue
, from said individual can be tested for the presence of myeloproliferative
cells or lymphoproliferative cells, using a binding compound according to the
present disclosure. ably, said sample is mixed with a binding compound
according to the present disclosure, which will specifically bind to
roliferative cells. Myeloproliferative cells or lymphoproliferative cells bound
to a binding compound described herein can be ed from the sample and/or
detected using any method known in the art, for example, but not limited to,
ion using magnetic beads, streptavidin-coated beads, or isolation through the
use of secondary antibodies immobilized on a column. Alternatively, or
additionally, a binding compound bed herein is labeled in order to be able to
detect said antibody, for instance, but not limited to, fluorescently labeled,
enzymatically labeled, or radioactively labeled. Alternatively, a binding compound
described herein is detected using a labeled secondary antibody which is directed
against said binding compound. If binding of said antibody is detected, it is
indicative for the presence of myeloproliferative or lymphoproliferative cells.
Therefore also described is an dy or functional part or functional equivalent
according to the present sure, or a nucleic acid molecule or functional
equivalent according to the present sure, or a vector or a cell according to the
present sure, for use in detecting myeloproliferative cells or
lymphoproliferative cells. Also described is an antibody or functional part or
functional equivalent according to the present disclosure, or a nucleic acid molecule
or functional equivalent according to the t disclosure, or a vector or a cell
according to the present disclosure, for use in diagnosis of a myeloproliferative
disorder or proliferative disorder. Said myeloproliferative disorder is
preferably AML. In some embodiments, said lymphoproliferative disorder is
lymphoma, B-NHL or multiple myeloma. Lymphoproliferative cells are preferably
detected with one or more antibodies selected from the group consisting of AT12-
019, AT12-023, AT12-025, AT13-024 and AT13-031, and functional parts and
functional equivalents thereof.
A use of an antibody or functional part or functional equivalent according to
the t disclosure, or use of a nucleic acid molecule or functional equivalent
according to the present sure, or use of a vector or a cell according to the
present disclosure, for determining r a sample comprises myeloproliferative
cells or lymphoproliferative cells is also bed, as well as a method for detecting
myeloproliferative cells or lymphoproliferative cells using an dy or
functional part or functional equivalent according to the present disclosure. Also
described is a method for determining r myeloproliferative cells or
lymphoproliferative cells are present in a sample comprising:
- contacting said sample with an antibody or functional part or functional
equivalent according to the present disclosure, and
- allowing said antibody or functional part or functional equivalent to bind
myeloproliferative cells or lymphoproliferative cells, if present, and
- determining whether or not myeloproliferative cells or lymphoproliferative cells
are bound to said antibody or functional part or functional equivalent, thereby
determining whether or not myeloproliferative cells or lymphoproliferative cells
are present in said .
In a red embodiment, said myeloproliferative cells are AML cells. In
r preferred embodiment, said proliferative cells are lymphoma,
B-NHL or multiple myeloma cells. Lymphoproliferative cells are preferably
detected with one or more antibodies selected from the group consisting of AT12-
019, AT12-023, AT12-025, AT13-024 and AT13-031, and functional parts and
functional equivalents thereof.
In a further ment it is determined whether an individual is suffering
from a myeloproliferative or lymphoproliferative disorder. Also described is
therefore a method for determining whether an individual is suffering from a
myeloproliferative disorder or a lymphoproliferative disorder, comprising:
- contacting a sample from said individual with an antibody or functional part or
functional equivalent according to the present disclosure, and
- ng said antibody or functional part or functional equivalent to bind
myeloproliferative cells or lymphoproliferative cells, if present, and
- determining r or not myeloproliferative cells or lymphoproliferative cells
are bound to said dy or functional part or functional equivalent, thereby
determining whether or nor said individual is suffering from a myeloproliferative
disorder or a lymphoproliferative disorder. Preferably said individual is a human.
In some embodiments, said myeloproliferative disorder is AML. In other
embodiments, said lymphoproliferative disorder is lymphoma, B-NHL or multiple
myeloma.
As described herein before, the t disclosure es the surprising
insight that snRNP200 is t on the surface of AML cells, whereas snRNP200
is normally located in the nucleus only. Hence, snRNP200 is an important target
for anti-AML therapy. Moreover, antibodies AT12-023 and AT13-031, which are
specific for snRNP200, also bind B-NHL cells and multiple myeloma cells. This
indicates that surface expression of snRNP200 also occurs on B-NHL cells and
multiple myeloma cells. Now that this insight has been provided, many
applications have become le. For instance, snRNP200-specific binding
compounds can now be used for treating or preventing myeloproliferative or
lymphoproliferative disorders. Further described is therefore a use of an
snRNP200-specific binding compound for the preparation of a medicament for the
treatment or prophylaxis of a myeloproliferative or lymphoproliferative disorder,
such as AML, B-NHL or multiple myeloma. An snRNP200-specific binding
compound for use in a method for at least in part treating or preventing a
roliferative or lymphoproliferative disorder is also described, as well as a
method for at least in part treating and/or preventing a roliferative or
lymphoproliferative disorder, comprising administering to an individual in need
thereof a therapeutically effective amount of an snRNP200-specific binding
compound.
Novel detection methods have also become available. Since snRNP200 is
normally only present in the nucleus, but s to be also present at the surface
of myeloproliferative and lymphoproliferative cells, these cells can now be detected
and distinguished from healthy cells by determining whether 00 is present
at their e. Further described is therefore a method for determining r a
myeloid cell or lymphoid cell is a myeloproliferative cell or lymphoproliferative cell,
the method sing determining whether 00 is t on the surface of
said cell, wherein the presence of snRNP200 on the surface of said cell indicates
that said cell is myeloproliferative or lymphoproliferative. Also described is a
method for identifying myeloproliferative or lymphoproliferative cells, comprising
ing the presence of snRNP200 on the surface of said cells. As used herein, the
expression “present at the surface of a cell” or “present on the surface of a cell”
means that at least part of snRNP200 is present on, or within, the cell surface, or
associated therewith.
In some embodiments, a ontaining sample of an individual is typed.
Such sample, which typically contains lymphoid cells and/or myeloid cells, is in
some embodiments tested for the presence of snRNP200 on the e of these
cells. If snRNP200 appears to be present on the surface of cells, the sample is typed
as containing myeloproliferative or lymphoproliferative cells. Further described is
therefore a method for typing a myeloid cell-containing sample or a lymphoid cellcontaining
sample of an individual, the method comprising determining whether
snRNP200 is present on the surface of cells of said sample. If this is the case, it
indicates that myeloproliferative cells or lymphoproliferative cells are present in
said sample.
In some embodiments, the individual is suffering from, or suspected of
suffering from, a myeloproliferative or lymphoproliferative disorder. This is
however not necessary, since such typing method can also be part of a general
screening test, for instance for health checks.
Said sample can be any sample that contains d and/or lymphoid cells,
such as for instance a bone marrow sample, a tissue sample or a lymph fluid
sample. Preferably, said sample ses peripheral blood mononuclear cells,
since a blood sample is easily obtainable with little discomfort for the individual.
The present disclosure es the insight that at least a subpopulation of
patients who suffer from a roliferative or lymphoproliferative er
produce antibodies that are specific for snRNP200. The presence of such antibodies
in a sample from an individual is thus indicative for a myeloproliferative or
lymphoproliferative disorder. Further described is therefore a method for
ining whether an individual is suffering from a myeloproliferative or
proliferative disorder, comprising determining whether a sample from said
individual ses dies that are specific for snRNP200. In some
embodiments such method comprises the steps of:
- contacting a sample from said individual with snRNP200 or an epitope thereof;
- allowing said snRNP200 or epitope to bind snRNP200-specific antibodies from
said , if present, and
- ining whether or not said snRNP200 or epitope is bound to snRNP200-
specific antibodies, wherein binding of said 00 or epitope to snRNP200-
specific dies indicates that said individual is suffering from a
myeloproliferative or lymphoproliferative disorder.
Screening assays as described herein can be performed using methods
such as for instance enzyme-linked sorbent assays (ELISA), radio-immuno
assays (RIA), Western Blot assays and immunohistochemical staining assays.
These assays are well known in the art and therefore need no further explanation.
Variations or adaptations of ELISA, RIA, Western blot assay and
immunohistochemical staining assay are also known in the art.
Also described is a method for determining whether a patient suffering from
a myeloproliferative or lymphoproliferative disorder has an improved chance of a
ve outcome of treatment with an antibody, onal part or functional
equivalent according to the present sure, as compared to the mean population
of patients ing from a myeloproliferative or lymphoproliferative disorder, the
method comprising ining whether snRNP200 is present on the surface of
myeloproliferative cells or lymphoproliferative cells of said patient. If this is the
case, snRNP200-specific antibodies such as AT12-023, AT13-031 and AT13-037 are
particularly suitable for counteracting such myeloproliferative or
lymphoproliferative disorder. Therefore, if it is known that myeloproliferative or
lymphoproliferative cells of an individual express snRNP200 at their surface, the
chance of succesfull treatment is increased. Such method described herein
preferably comprises the steps of:
- contacting a roliferative cell- or lymphoproliferative cell-containing sample
from said individual with an antibody or functional part or onal equivalent
that is specific for snRNP200;
- allowing said antibody or functional part or functional equivalent to bind
myeloproliferative cells or lymphoproliferative cells of said sample, and
- determining whether or not said a snRNP200-specific antibody or functional part
or functional equivalent is bound to myeloproliferative cells or lymphoproliferative
cells of said sample, wherein g of said snRNP200-specific antibody or
functional part or functional equivalent to roliferative cells or
lymphoproliferative cells of said sample indicates that said t has an
improved chance of a positive outcome of ent with an antibody, functional
part or functional equivalent according to the present disclosure, as compared to
the mean population of patients suffering from a myeloproliferative or
lymphoproliferative disorder. Said ent preferably comprises the use of
antibodies AT12-023, AT13-031 or AT13-037, or functional parts or functional
equivalents thereof, since at least these antibodies are able to bind snRNP200.
Further described is therefore an antibody according to the present disclosure, or
an antibody for use as described herein, or a use or a method according to the
present disclosure, wherein said antibody is AT12-023, AT13-031, AT13-037, or a
functional part or a functional equivalent f.
If the use of antibody AT12-023, AT13-031 and/or AT13-037 is
contemplated, it is preferably determined beforehand if a certain patient contains
malignant cells that express snRNP200 at their surface. Some embodiments
therefore describe a method for determining whether a patient suffering from a
roliferative disorder or lymphoproliferative disorder is a candidate for
treatment with antibody 23, AT13-031 or AT13-037, or a onal part or
a functional equivalent thereof, the method comprising determining whether
snRNP200 is t on the surface of myeloproliferative cells or
lymphoproliferative cells of said patient.
In a method according to the present disclosure, said myeloproliferative
disorder is preferably AML and said myeloproliferative cells are preferably AML
cells. Moreover, said lymphoproliferative er is preferably lymphoma, B- non-
n lymphoma or multiple a and said lymphoproliferative cells are
preferably lymphoma, B- non-Hodgkin lymphoma or multiple myeloma cells.
The above-mentioned procedures for detecting myeloproliferative or
lymphoproliferative cells using binding compounds described herein are for
instance particularly suitable for determining whether a patient suffering from a
myeloproliferative disorder or lymphoproliferative disorder who has received
medical treatment, such as for instance an AML patient who has been treated
t AML, for instance an AML patient who has received immunotherapy such
as a stem cell transplantation or donor-lymphocyte infusion, has a GvL response.
Until to date, there are no diagnostic tools to test for the presence of a potent GvL
response in a treated patient. Such stic tool is much needed, for instance
e: 1) It will allow early identification of allogeneic SCT recipients at high
risk for e, at a time-point before e has occurred y ng
earlier interventions such as tapering of immunosuppressants or donor-lymphocyte
infusions; 2) It will allow titrating such donor lymphocyte infusions until anti-
leukemia antibodies do appear; and 3) It will offer hope for allogeneic SCT
recipients at a time they often suffer from one of many SCT-related complications
when the presence of a potent GvL response can be trated. Nowadays
patients have to wait and see whether or not a relapse occurs, and there is no way
to predict relapse of disease. The availability of a test for determing r a
patient has a GvL response will therefore greatly improve the clinical care of SCT
patients, affecting prognosis and quality of life. Further described is, therefore, a
use of a binding compound bed herein for determining r a sample is
tive for a GvL response. Also described is a use of a binding compound
described herein for determining whether or not an AML patient has a GvL
response, and a use of a g compound described herein for determining
whether a treatment against a myeloproliferative or lymphoproliferative disease,
such as for instance anti-AML therapy, anti-B-NHL therapy or anti-multiple
myeloma therapy, is ceous. This is for instance done by determining whether
a sample (for instance a blood or tissue sample) from a patient (that has for
instance ed a SCT or DLI or any other form of immunotherapy) contains
myeloproliferative or lymphoproliferative cells, for instance AML cells, B-NHL cells
or multiple myeloma cells. The absence of myeloproliferative or lymphoproliferative
cells indicates that said patient has a GvL response. Further described is,
therefore, a method for determining whether a patient suffering from a
myeloproliferative disorder or a lymphoproliferative disorder who has received
immunotherapy against said disorder has a GvL response, the method comprising
contacting a sample from said patient with an antibody or functional part or
functional equivalent according to the present disclosure, and allowing said
antibody or functional part or functional equivalent to bind myeloproliferative or
lymphoproliferative cells, if present, and ining whether or not
myeloproliferative cells or lymphoproliferative cells are bound to said antibody or
functional part or functional equivalent, thereby determining whether or not said
individual has a GvL response, whereby the absence of myeloproliferative or
lymphoproliferative cells is indicative for a GvL response and the presence of
myeloproliferative or lymphoproliferative cells is tive for a lack of, or
insufficient ective), GvL response. In some embodiments, said
lymphoproliferative disorder is ma, B-NHL, or multiple a. In some
embodiments, said myeloproliferative disorder is AML. Also described is, therefore,
a method for determining whether an AML patient that has received anti-AML
therapy has a GvL response, the method comprising contacting a sample
from said AML patient with an antibody or functional part or functional equivalent
according to the present disclosure, and allowing said antibody or functional part
or functional equivalent to bind AML cells, if present, and determining whether or
not AML cells are bound to said antibody or functional part or functional
equivalent, thereby determining whether or nor said dual has a GvL
response, whereby the absence of AML cells is indicative for a GvL se and
the presence of AML response is indicative for a lack of, or insufficient
(ineffective), GvL response. Preferably said individual is a human. Alternatively,
binding compounds described herein that are labeled with a detectable moiety,
such as for instance with a copper compound, are administered to an AML patient
that has received anti-AML immunotherapy such as a SCT or DLI, and it is
subsequently determined whether said labeled antibody is bound to AML cells of
said patient in vivo, for instance using a PET scan. The absence of bound binding
nds is indicative for a GvL response and the presence of bound binding
compounds is indicative for a lack of, or insufficient ective), GvL response.
It is also possible to determine the amount of antibody belonging to the
VH4-34 family before and after anti-AML immunotherapy. If the amount of
antibody belonging to the VH4-34 family, which is a family of VH sequences known
for their potential killing ties (Bhat et al, 1997), is icantly raised after
immunotherapy, it indicates that a GvL se is t. Further described is,
therefore, a method for determining whether an AML patient that has received
anti-AML immunotherapy has a GvL response, the method comprising
determining the amount of antibody belonging to the VH4-34 family before and
after anti-AML immunotherapy, and determining whether the amount of antibody
belonging to the VH4-34 family is significantly raised after immunotherapy. If this
is the case, it is concluded that a GvL se is present in said patient. If this is
not the case, it is concluded that a GvL response is g, or not sufficient.
The term “comprising” as used in this specification and claims means
“consisting at least in part of”. When interpreting statements in this specification, and
claims which include the term “comprising”, it is to be understood that other features
that are additional to the features prefaced by this term in each statement or claim may
also be present. Related terms such as “comprise” and “comprised” are to be
interpreted in similar manner.
In this specification where reference has been made to patent specifications,
other external documents, or other sources of information, this is generally for the
purpose of ing a context for discussing the features of the invention. Unless
specifically stated otherwise, nce to such external documents is not to be
construed as an admission that such documents, or such sources of information, in any
jurisdiction, are prior art, or form part of the common l knowledge in the art.
In the description in this specification reference may be made to subject matter
that is not within the scope of the claims of the t application. That subject matter
should be y fiable by a person skilled in the art and may assist in g into
practice the invention as defined in the claims of this application.
The invention is further explained in the following examples. These
examples do not limit the scope of the invention, but merely serve to clarify the
invention.
Brief description of the drawings
Figure 1. Sequences of dies AT12-023, AT12-025, AT13-024, AT12-019,
22, AT13-023, AT13-031, AT12-020, AT13-033, AT13-034, AT13-035, AT13-
036, AT13-037, AT14-013, AT14-014, AT14-015 and AT14-016.
Figure 2. 36 year old patient with AML, who obtained complete remission after the
first cycle of induction chemotherapy and received a myeloablative (MA) allogeneic
stem cell lantation (SCT) from her tched brother (sib) as
consolidation therapy. AML relapse occurred however 5 months posttransplantation.
Re-induction chemotherapy resulted again in complete remission
and in severe, grade IV graft versus host disease (GvHD) of the liver and grade I
GvHD of the skin. The observation that she has remained in te remission for
more than three years now implies that she has developed a potent graft versus
leukemia response. B lymphocytes were isolated from a tomy t
obtained about 1,5 years after the one cycle of re-induction herapy.
Figure 3. a) Supernatant of one of the minicultures (20 or 40 cells per well) binding
to the AML cell line THP-1. b) B cells from this miniculture were plated into 1
cell/well solutions and supernatants again screened for binding to THP-1. THP-1
express HLA-DR, that was used in this ing experiment as a positive control.
As a negative control an influenza-specific monoclonal antibody was used.
Figure 4. Two es of the AML specific antibodies identified. AT12-023 (a)
and AT13-031 (b) bind to AML cell lines THP-1 and MonoMac6, but not to healthy
peripheral blood mononuclear cells (PBMC), endothelial cells (human umbilical
vein endothelial cells: HUVEC), the T cell line Jurkat, primary fibroblasts,
hepatocytes (liver hepatocellular oma cell line: HepG2) or the colon
adenocarcinoma cell line HT-29. The MRSA-mAb F1 is an in-house generated
human IgG3 mAb that is used as l antibody.
Figure 5. Antibodies derived from donor 59 bind to the AML cell lines THP-1 and
MonoMac6, and to primary leukemic blasts isolated from newly diagnosed AML
patients (ranging from FAB classification M0-M4). See also table 3 for a more
detailed overview.
Figure 6. Some of the antibodies also bind to other hematological malignancies.
Here, binding of AT12-023 (antibodies isolated from supernatant of B cell clone)
and AT13-031 binant antibody) to B-non Hodgkin lymphoma (B-NHL) cells
that were freshly isolated from a lymphoma patient is shown. The dies did
not bind to non-malignant B cells (not shown in this experiment). The RSV
antibody Palivizumab was used as a negative control, and Rituximab, a CD20-
antibody specifically binding B-cell lymphoma, as a positive control. See for more
details of antibody binding to other hematologic malignancies table 4.
Figure 7. THP-1 cells were cultered in medium alone or with antibodies added at a
concentration of 5 μg/ml at day 0. In the presence of AT12-023, THP-1 cells showed
significant growth inhibition. ed are total cell numbers in culture.
Figure 8. When added at day 0 of the culture, antibodies AT12-023 and AT12-025
strongly d the viability of THP-1 cells. ity is depicted as the proportion
of cells double-negative for cell-death markers Annexin V and 7AAD.
Figure 9. To quantify the amount of THP-1 cells specifically killed by AT12-023 we
used a calcein AM release assay. Briefly, before THP-1 cells were ted with
AT12-023, the THP-1 cells were loaded with calcein AM, that is released when cells
are dying and the cell membranbe becomes instable. Lysis was read out after 4
hours of incubation and was related to the background death of n-loaded cells
as measured with an irrelevant antibody.
Figure 10. a) Besides g of THP-1 cells, AT12-025 does also induce death of
primary tumor cells, similar to AT13-024. Leukemic blasts, isolated from a patient
with AML-M5 at diagnosis, were incubated with AT12-025 or AT13-024 (5 ug/ml)
for 24 hours at 37°C, after which cells were stained with 7AAD and AnnexinV to
determine the amount of dead cells (7AAD and Annexin V double-positive).
b) In addition, Lactate dehydrogenase (LDH) can be used as a marker to determine
dying cells. Leukemic blasts, ed from a patient with AML-M0, were incubated
with AT13-024 or the RSV dy palivizumab for 18 hrs, after which LDH
release was measured. These two experiments show that AT13-024, but not
palivizumab, induced death of leukemic blasts and that this property was not
restricted to one type of AML but es different FAB classifications (M0 to M5).
Figure 11. Donor 58 was diagnosed with AML-M5, for which she ed three
cycles of chemotherapy. She remained in complete remission for about a year, after
which the AML relapsed. She received one cycle of re-induction chemotherapy,
followed by an allogeneic d intensity stem cell transplantation (RIST) from a
matched unrelated donor (MUD). She developed GvHD grade I of the liver and has
remained in complete ion for almost 4 years now, implicating that she has
ped a potent graft versus leukemia response. B cells were isolated from a
phlebotomy product obtained from this patient about 3 years post-SCT.
Figure 12. dies derived from donor 58 bind to the AML cell lines THP-1 and
MonoMac6. Palivizumab, the commercially available RSV specific antibody, was
used as a negative control.
Figure 13. al history of a third patient with a potent GvL response. Donor
101 was diagnosed with an intermediate-risk AML (no cytogenetic or molecular
abnormalities; FAB classification AML-M5) at the age of 49 years. He received two
s of chemotherapy and one course of consolidation chemotherapy followed by
an autologous HSCT, as there was no HLA-matched sibling stem cell donor
available. Fourteen months after the first diagnosis his disease relapsed. He
obtained complete remission after one cycle of high-dose bine, after which he
received a rediced-intensity allogeneic HSCT with a matched unrelated donor
MUD). Six weeks later he developed acute GvHD of skin, liver and intestine
(stage 1; grade II) that responded well to corticosteroid therapy. B cells were
isolated from a phlebotomy product obtained from this patient 38 months after the
allogeneic HSCT. He has maintained durable remission until today.
Figure 14. Antibodies derived from donor 101 bind to the AML cell lines THP-1 and
, but not to fibroblasts, monocytes, B cells and T cells. An in-house
produced human antibody against CD30 was used as a ve control. AT14-013
showed minor reactivity with fibroblasts, compared to its high binding to the AML
cell lines. See tables 7 and 8 for an overview of the binding capacities of the
antibodies derived from donor 101.
Figure 15. AML-specific antibody AT14-013 (donor 101) binds primary leukemic
blasts isolated from newly diagnosed AML patients (FAB classifications M0-M5).
Negative control: in-house generated CD30 antibody. See table 7 for an overview of
the y AML blast binding capacities of the antibodies derived from donor 101.
Figure 16. Antibody AT13-037 induces death of primary blasts. Leukemic blasts,
isolated from an AML patient at diagnosis 8; AML-M5), were incubated with
antibodies purified from the supernatants of the parental B cell clone (sAT13-037)
or with the recombinant dy (rAT13-037) at a tration of 5 ug/ml for 4
hours at 37°C, after which cells were stained with the death cell marker Dapi. To
fy death of cells a standard amount of beads was added. Recombinant
nza specific rAT10-002 was used as a negative control.
Figure 17. Cytotoxic dies d a non-apoptotic death y.
a) Phase contrast imaging of THP-1 cells. THP-1 cells were incubated with the noncytotoxic
AML-specific antibody AT13-023 (left panel) or the cytotoxic antibody
AML-specific AT13-037 (right panel). Interaction was visualized using time-lapse
imaging and demonstrated swelling of target cells after which cells died. Stills were
taken after 4 hours of incubation, with blue arrows indicating large cells that are
dead. b) Double staining with DiOC6 and PI showed that cytotoxic antibodies do
not induce apoptosis. THP-1 cells were ted with the cytotoxic AML-specific
dy AT12-023, with diclofenac (that induces apoptosis in THP-1 cells) or with
medium only. Cells undergoing apoptosis first loose their mitochondrial ne
potential (loss of DiOC6 stain) after which they become permeable (PI positive), as
can be seen after incubation of THP-1 cells with diclofenac. THP-1 cells incubated
with AT12-023 showed increased membrane permeability (PI+) but maintained
mitochondrial membrane potential (DioC6+), indicating the induction of a nonapoptotic
cell death pathway. c) To confirm the non-apoptotic nature of the death
pathway induced we tested the involvement of caspases in cell death induction.
Cell death of THP-1 cells by AML-specific antibodies could not be prevented with
the pan caspase inhibitors Q-VD-OPh (left panel) or Z-VAD-fmk (right .
Figure 18. Cell death induced by AML-specific antibodies also occured at 4°C,
suggesting the involvement of a passive process. This was true for all antibodies
except AT13-031, which is cytotoxic at 37°C only (not shown). Negative controls
included the AML-specific non-cytotoxic antibodies AT13-034, AT12-019, AT13-
022, AT13-023 and AT13-024.
Figure 19. (a) Incubation of target cells with cytochalasin D did not inhibit binding
of the antibodies. THP-1 cells were ted with cytochalasin D, after which
AML-specific dies AT12-023, AT13-031 and AT13-037 were added. (b) Preincubation
of the target cells with the membrane stabilizing agent cytocholasin D
did protect the THP-1 cells against cell death by the ecific cytotoxic
dies.
Figure 20. Target verification of AT12-023, AT13-031 and AT13-037. a) THP-1
membrane lysate was incubated with AT12-023 and AT13-031, with the influenzaspecific
antibody AT10-002 or with marker alone. Western blot is including
anti-human snRNP200 (and HMGB1 as a negative control) revealed specific
binding of AT12-023 and AT13-031 to 00. b) AML specific antibodies AT12-
019, AT12-023, AT13-031 and AT13-037 or commercially available snRNP200-
specific dies snRNP200 453 and snRNP200 454 were coated on an ELISA
plate, incubated with a snRNP200-flag construct for capturing and with anti-flag
HRP for detection. AT12-023, AT13-031 and AT13-037 specifically bound
snRNP200, whereas negative controls and for example AT12-019 did not.
Figure 21. THP-1 cells express snRNP200 on their membrane. THP-1 cells and
Jurkat cells were stained with a commercially available anti-mouse
snRNP200 antibody. Intracellular staining of Jurkat cells and THP-1 cells showed
nuclear staining, as expected (left panels). r, membrane staining of
snRNP200 was restricted to THP-1 cells (right panels).
Examples
Example 1
MATERIALS & METHODS
Patient and healthy human materials
Study protocols were approved by the Medical Ethical Committee of the Academic
Medical Centre. All participants signed informed consent. We selected two patients
who had received an allogeneic stem cell transplantation (myeloablative sibling
lant for donor 59 and a matched ted donor- reduced intensity stem cell
transplantation for donor 58) for AML and who, based on their clinical ies,
can be d to have generated strong graft versus leukemia responses. These
two SCT recipients donated 500 ml peripheral blood each, the products of one of
many phlebotomies they underwent for post-transfusion hyperferritinemia. In
on, patients admitted to our clinic with newly diagnosed AML consented to
donate 2-5 ml bone marrow or blood containing AML blasts to be used for antibody
binding assays. Healthy bone marrow was donated by patients undergoing
thoracotomy for cardiac surgery in our ute. B-non Hodgkin’s lymphoma cells
were obtained as rest material from biopsies of ts with newly diagnosed B-
non Hodgkin’s lymphoma. These B-non Hodgkin’s lymphoma cells were used to test
for binding of our antibodies as described below. Rest material of human umbilical
vein elial cells (HUVECS), that were freshly isolated at the Department of
Ophthalmology (AMC, Amsterdam, The Netherlands) were directly used for
binding assays as described below. For purity, cells were co stained with and
selected for anti human CD14+.
clear cells were ed from peripheral blood and bone marrow of healthy
individuals and AML patients by Ficoll separation. Extra markers like CD45,
CD33, CD34, CD14, CD3 and CD19 were used to isolate specific cell populations
(AML blasts, monocytes, T and B lymphocytes, respectively, obtained from blood
bank donors).
Isolation of B cells
We ed B cells from peripheral blood by Ficoll separation and CD22 MACS
microbeads (Miltenyi Biotech). Subsequently we sorted these cells for CD27- or
CD27+ CD19+CD3−IgM−IgA− (naïve or memory IgG cells, tively) and
CD19+CD3−CD27+IgG−IgA− (memory IgM cells) on a FACSAria (Becton
Dickinson).
Cell culture
We maintained B cells (2×105 ml) in IMDM (Gibco) culture medium
containing 8% FBS (HyClone) and penicillin/streptomycin (Roche), supplemented
with recombinant mouse IL-21 (50 ng/ml, in house produced) and co-cultured them
on γ-irradiated (50Gy) mouse L cell fibroblasts stably sing CD40L (CD40L-L
cells, 105 cells/ml). The cultures were tested routinely for the presence of
mycoplasma by PCR (data not shown).
Retroviral transduction
Retroviral transduction was performed as described in Kwakkenbos ea, Nat Med
2010. Briefly, naïve and memory IgG and memory IgM B cells were cultured and
activated during 36 hours on CD40L-L cells in the presence of mIL-21. Then, BCL6
and Bcl-xL retroviral ucts that include the marker gene for GFP were used to
transduce the B cells as described before (Diehl ea, J Immunol 2008), with the
addition that we centrifuged cells and virus at room temperature for 60 min at 360
× g (1800 RPM). Transduction efficiency ranged from 69-90%.
Culture of target cell lines
Target AML cell lines THP-1 (ATCC; density of 2×105 cells/ml to 1×106 cells/ml)
and Mono-Mac6 (a kind gift from Dr. Hamann of the Experimental Immunology
department, density of 2×105 cells/ml to 2×106 ml) were maintained in RPMI
1640 (Gibco) culture medium, containing 10% FBS (HyClone) and
penicillin/streptomycin (Roche). Culture medium of Mono-Mac6 was enriched with
non-essential amino acids (Invitrogen), 1 mM sodium te (Invitrogen) and 10
µg/ml human insulin ). The AML cell line Molm13 was maintained at a
density of 5×105 cells/ml to 1,5×106 ml in RPMI 1640 (Gibco) culture ,
containing 20% FBS (HyClone) and penicillin/streptomycin (Roche). The liver cell
lines HepG2 and Huh7 and the colon cell line HT29 (a kind gift from the Tytgat
Institute, AMC, Amsterdam, The Netherlands) were cultured in DMEM (Gibco)
culture medium, containing 8% FBS (HyClone) and penicillin/streptomycin
(Roche). The acute T-cell leukemia cell line Jurkat (ATCC) was maintained in
RPMI 1640 (Gibco) culture medium, containing 10% FBS (HyClone) and
penicillin/streptomycin (Roche) at a y of 1×105 cells/ml to 2×106 ml. Skin
y fibroblasts (a kind gift of the University of Leiden, The Netherlands) were
cultured twice a week in DMEM (Gibco) culture medium, containing 10% FBS
(HyClone) and penicillin/streptomycin (Roche). Cells were passaged no more then
10 times. The diffuse large cell B cell lymphoma (DLBCL) cell lines OCI-Ly1 and
OCI-Ly7 (DSMZ and ATCC, respectively) were ined in IMDM (Gibco)
culture medium containing 8% FBS (HyClone) and penicillin/streptomycin (Roche).
The cultures were maintained at 106 cells/ml. Finally, the multiple myeloma
(MM) cell lines U266 and 29 were maintained and cultured as described for
THP-1 cells.
Generation of AML-specific clones
Transduced naïve and memory IgG and IgM B cells of each t were seeded at
concentrations of 20 or 40 cells per well (hereafter named microcultures) and
expanded with IL-21 and CD40L. Supernatants of expanded B cell micro cultures
were then screened for antibody binding to leukemia cell lines (THP-1, MonoMac6
and the cell lines ed in Table 4), to liver- and colon cell lines, and some
supernatants also to the primary blasts isolated from patients with AML- M0, M1,
M4 and M5, by FACS, using human-IgG-PE (Southern Biotech) or human IgG H+L
AF647 (Life Technologies) as a secondary antibody. Several in-house generated
antibodies were used as negative control dies, such as anti-CD30 (expressed
on activated B and T lymphocytes), anti-CD33 (expressed on monocytes, myeloid
progenitor cells and myeloid leukaemias), D25 (against RSV; described in
), AT10-002, AT10-004 (against influenza; described in
), and F1 (against MRSA; described in ). In
on, some commercially available antibodies were used, such as Rituximab
(anti-CD20), Palivizumab (anti-RSV), mumab (anti-EGFR) and HLA-DR.
Microcultures binding to AML cell lines but not to liver- and colon cell lines were
selected and seeded at a concentration of 1 cell/well and their supernatants tested
again for specificity for AML cell lines. Clones with supernatants ically
binding AML cell lines and not liver- or colon cell lines, or healthy PBMC and bone
marrow were selected for sequencing. Clones were expanded under normal culture
conditions in the ce of FBS IgG low serum (Hyclone) and antibodies purified
from the supernatants of these cultures as described below for the recombinant
antibodies.
Cloning of AML-specific antibodies
To produce recombinant antibodies we isolated total RNA with the ® mini
kit (Qiagen), generated cDNA, performed PCR and cloned the heavy and light
chain le regions into the pCR2.1 TA cloning vector (Invitrogen). To rule out
e transcriptase or DNA polymerase induced mutations, we performed several
independent cloning experiments. To produce recombinant mAb we cloned heavy
and light variable regions of each antibody in frame with human IgG1 or IgG3 and
Kappa constant regions into a pcDNA3.1 (Invitrogen) based vector and transiently
transfected 293T cells. We ed recombinant antibodies from the culture
supernatant with Protein A or G, depending on the Ig subtype of the clone.
In vitro activity of AML-specific antibodies
To measure the in vitro effect of AML-specific antibodies on AML cells we used
several approaches. First, THP-1 cells (2x104) were seeded in flat bottom 96 well
plates (Costar). AML or control -antibodies were added on day 1, in a concentration
of 5-10 μg/ml. Numbers of cells per well were counted daily. ion of cell death
on both THP-1 cells and primary leukemic cells by AML-specific antibodies was
measured in parallel by costaining an aliquot of cells for Annexin V (BD
ngen) and 7- Aminoactinomycin D (7-AAD; Beckman Coulter), negative
cells being the viable cells. In addition, we used a specific lysis assay.
Target cells (THP-1) were labeled with Calcein AM (Becton Dickinson), a green
fluorescent dye that is ed from the cytoplasm when cells die. Briefly, 2
million THP-1 cells were incubated with 2 ml of 2 μM Calcein AM for 30 minutes at
37°C. AML or control antibodies were added for 4 hours, after which green
scence was measured using a fluoresence plate reader. The proportion of
specific lysis was calculated as (experimental value-low control) / (high control-low
control) x 100, where low control means the spontaneous release of Calcein AM by
unaffected cells and high control means the m amount of released n
AM when all cells are lysed. In addition to the Calcein AM release assay we applied
the lactate dehydrogenase (LDH) release assay to measure the killing activity of
AML specific antibodies. LDH is released by damaged cells, from the cytosol.
Leukemic , isolated at diagnosis from a patient with AML-M0, were
incubated with AT13-024 at a maximum concentration of 10 μg/ml per 10.000
blasts. LDH release is measured after adding Reaction mixture and Stop on
(Roche Diagnostics/Applied Science) according to the manufacturer’s protocol on an
ELISA . The percent cytotoxicity is calculated as the (experimental value-
low control) / (high control-low control) x 100, where low control means the
spontaneous release of LDH by unaffected cells and high control means the
m amount of LDH when all cells are lysed. To quantify death of target cells
induced by ecific antibodies we used a ased leukemia cell lysis
assay as previously described (Schmiedel et al, 2013). In brief, FACS calibration
beads (Accudrop Fluorescent Beads, BD Biosciences) were added to the cells in a
50/50 ratio after which a standard amount of beads was acquired with FACS. As an
equal assay volume was ascertained by the calibration beads, the amount of dead
or vanished cells can then be calculated as followes: 100 - ((Dapi negative cells in
respective treatment/Dapi negative cells in control) × 100).
Finally, to discern between apoptotic and optotic pathways of cell death
induced by our antibodies we co-stained target cells with DiOC6 and propidium
iodide (PI). DiOC6 ihexyloxacabocyanine iodide) is a ermeant, greenfluorescent
, lipophilic dye that is selective for the mitochondria of live cells, when
used at low concentrations. Apoptotic cells will lose their mitochondrial membrane
potential (loss of DiOC6 stain) before the cell membrane becomes permeable (PI
positive); necrotic cells become PI positive before they loose their mitochondrial
membrane potential (and become DiOC6-negative). The methods used for staining
were adapted from Hugh J. M et al., 2004. In brief, THP-1 cells were stained with
40nM of DiOC6 (Invitrogen) for 20 minutes at 37°C. PI was added after which the
samples were analyzed immediately by flow cytometry.
Flow cytometry
Stained cells were analyzed on FACSAria (BD), FACSCanto (BD), FACS
LSRFortessa X-20 (BD) and Guava (Millipore) tometers and flow cytometry
data were processed with FlowJo software (Tree Star).
Phase contrast imaging
THP-1 cells were added to a two chambered Cover glass system (LabTek). Using a
phase contrast-imaging microscope (in-house manufactured), four fields of interest
per chamber were chosen. The software was set to take a picture of every field of
interest every other minute. Just prior to the run, the THP-1 binding, non-cytotoxic
antibody 23 and the THP-1 binding, cytotoxic antibody AT13-037 were
added to their respective wells at a concentration of 10 ug/ml.
Target verification: Immunoprecipitation and flow cytometry
THP-1 cells were lysed and precleared with an irrelevant antibody use
generated RSV antibody D25) and beads to remove non specific binding proteins.
Precleared lysate was then incubated with bead-labeled AML-specific antibodies or
with the influenza specific antibody 02 as a negative control (3 hrs at 4°C).
dy-incubated lysates were washed five times, bound proteins were eluted
from the THP-1 lysate and then run on an SDS-PAGE gel. Proteins were blotted
and stained with Ponseau S to reveal total protein. The blot was blocked with BSA
and incubated with mouse-anti-snRNP200 (Millipore, clone 3B6.1) or Rabbit-anti-
HMGB1 (Abcam) for Western blot analysis. Intracelllar staining for snRNP200 was
performed after fixing and permeabilizing THP-1 cells with methanol (Sigma) and
permeabilization buffer containing triton X-100 (Sigma), EDTA ) and BSA
(Roche), followed by incubation with rabbit-anti-human snRNP200 antibody
(Sigma) overnight at 4°C.
Target mation: snRNP200 ELISA
HEK 293T cells were ected with an sion vector containing the fulllength
open reading frame of snRNP200 with an N-terminal FLAG tag. At 2 days
after transfection cells were harvested and lysed in lysis buffer containing se
inhibitors. This lysate was cleared and protein concentration was measured. AML
specific antibodies AT12-019, 23, AT13-031 and AT13-037 or commercially
ble snRNP200 ic antibodies (Bethyl labs) were then coated on an
ELISA plate. The lysate was added at a tration of 3 μg/ml for capturing.
After extensive washing, captured snRNP200-flag was detected with a monoclonal
mouse anti-flag HRP antibody (Sigma-Aldrich, clone M2).
RESULTS
tion of AML-specific antibodies
In our project we use a unique technology that was recently developed in our
laboratory (WO 67046; incorporated herein by reference) that allows for
selection of lly occurring leukemia-specific B cells and the antibodies
produced by these cells. We employed this technology to generate leukemia-specific
B cell lines from two AML patients who developed potent GvL responses in parallel
with severe GvHD of the liver and skin. With our B cell immortalization technique,
it became possible to identify B cell responses of leukemia ts and to create
ia-specific B cell lines that allow functional studies of the antibodies these
cell lines produce and allow generation of larger numbers of dies, as
compared to other methods known in the art, with diagnostic and therapeutic
potential.
First, we applied our technique as described in to perform a set of
pilot experiments, where we isolated and immortalized B lymphocytes from a
carefully selected patient (donor 59) who developed a potent GvL response
following extensive GvHD of liver and skin. This patient was diagnosed with AML
at the age of 36 years. She obtained complete remission after the first of two
courses induction chemotherapy after which she received a myeloablative, HLA-
d g donor SCT as consolidation therapy. Only 5 months after SCT,
shortly after immunosuppressive y had been tapered, her AML relapsed.
With one cycle of high-dose cytarabine she obtained complete remission again, after
which she spontaneously, before the scheduled donor-lymphocyte infusion,
developed stage III GvHD of the liver and stage II GvHD of the skin. She was
successfully treated with high-dose corticosteroid therapy but during tapering of
therapy hepatic GvHD relapsed twice. In the end, corticosteroid therapy was
successfully phased out about 6 months before the current experiments were
d (1,5 yrs after AML relapse) and she has remained in complete remission
from her leukemia for more than three years now (Figure 2).
From this patient, we ted B cell clones using our que to immortalize B
cells. Briefly, PBMC were ed from the peripheral blood by ficoll
centrifugation, and B lymphocytes sorted by FACS. Isolated B cytes were
then cultured with IL21 and CD40L and transduced with BCL6 and BCL-xL, with
a transduction efficiency of around 70%. Using this technique, unique B cell clones
that concurrently express the B cell or and secrete monoclonal antibodies
were generated. Immortalized CD27- naïve and CD27+ memory IgG+ B cells were
seeded at concentrations of 20 or 40 cells per well and expanded with IL21 and
CD40L. Supernatants of expanded B cell micro cultures were then screened for
antibody binding to leukemia-, liver- and colon cell lines by FACS, using human-
IgG-PE as a secondairy detection antibody.
As the patient had been sed with AML-M5 according to the French-
American-British (FAB) classification (Bennett et al., 1976), we selected the THP-1
cell line that morphologically and phenotypically is roughly similar to AML-M5 for
these screening essays (Tsuchiya et al., 1988). In addition, because the patient
suffered from extensive GvHD of the liver we screened for the identification of
liver-binding B cell clones using the liver cell line HepG2. As a negative control,
and to select out cross-binding B cell clones we used the colon cell lines HT-29 and
LSTR. Micro cultures with the highest binding to either of these cell lines were
then sorted and single cells were deposited in 96 well s, expanded and the
supernatants of these expanded clones were again screened for binding against
leukemia cell lines (Figure 3).
Properties of AML-specific antibodies
With this approach we generated eight B cell lines from this one patient, producing
antibodies that bind to THP-1 (Figure 3) but not liver or colon cell lines, lasts,
endothelial cells (human umbilical vein endothelial cells or HUVEC), healthy
PBMC or bone marrow (Table 2 and Figure 4). These are antibodies AT12-019,
AT12-023, AT12-025, 22, AT13-023, AT13-024, AT13-031 and AT12-020.
The heavy and light chain variable s of these antibodies are ed in
Table 1 and Figure 1.
Interestingly, the antibodies are of the IgG1 and IgG3 isotypes, and l of the
DNA sequences showed somatic hypermutations. Antibodies AT12-023, 25,
AT13-023 and AT13-031 belong to the VH4-34 family, which is a family of VH
sequences known for their potential killing ties (Bhat et al, 1997). The
antibodies are of donor-origin, as confirmed by microchimerism analyses.
We then determined the breadth of binding within the spectrum of AML subtypes,
and tested the generated AML-specific B cell clones for binding to other AML FAB
classification types. Selected leukemia-specific clones also bound to the AML-M5
cell line MonoMac6 (Table 3) (see for review of AML cell lines (Drexler and
Minowada, 1998)).
Our AML-specific antibodies also bound to freshly isolated AML blasts of newly
diagnosed patients (Table 3 and Figure 5).
Furthermore, some antibodies also bound other hematopoietic malignancy -cell
lines, such as the diffuse large cell B cell lymphoma cell lines 1 and OCILy7
and the multiple myeloma cell lines U266 and NCI-H929, and/or patientderived
hematologic tumor cells (Table 4 and Figure 6).
In vitro activity of AML specific dies
While testing the specificity of the AML-binding antibodies, we made a striking
observation. Three of the eight antibodies spontaneously induced death of the
leukemic cells they were binding to. THP-1 cells were killed by AT12-023 and
AT12-025, and primary AML blasts ed from patients diagnosed with AML
were killed by AT12-025 and AT12-024 (Table 5 and Figures 7-10).
Figure 7 shows rapid killing of THP-1 cells by AT12-023. Dying blasts expressed 7-
AAD and Annexin V. Strikingly, while co-culture of THP-1 with AT12-025 at first
did not affect total cell numbers in the cultures (Figure 7), this antibody ed
to also induce death of THP-1 cells, albeit with a little delay compared to AT12-023
e 8). The observation that some of the antibodies show direct killing activity
against leukemic cells is highly ng and has to the best of our knowledge not
been reported before.
mation of our findings by ting AML-specific B cell lines from a second
patient
About 0.05 to 0.1% of the B cells that we screened from the first patient were found
to be AML-specific. To confirm our findings, we selected a second patient with
relapsed AML who obtained durable remission after an allogeneic SCT (donor 58;
Figure 11). Also this t developed GvHD of the skin and liver for which she
was d with oral steroids. After ds were phased out, about one year after
the SCT, PBMC were isolated, B cells immortalized, and B cell lines screened for
binding to AML cell lines as described above. Five B cell clones have been
generated from this patient that bind to the AML cell lines THP-1 and MonoMac6
(Figure 12), but not to liver or gut cell lines, fibroblasts or healthy PBMC (Table 6).
These clones produce antibodies AT13-033, AT13-034, AT13-035, AT13-036 and
AT13-037. Interestingly, all these antibodies are of the IgG3 isotype. The sequences
of the heavy and light chain variable regions of these antibodies are also depicted
in Table 1 and Figure 1. These data confirm the achievability of our project; despite
the low frequencies of the B cells we are aiming at, we were able to isolate them
and to generate robust antibody-producing B cell lines that were specific for AML.
Discussion
These preliminary date are highly exciting, as - to the best of our knowledge - we
are the first to generate several human, non-chimeric, leukemia-specific B
lymphocyte clones from patients that developed a robust, lasting GvL response
against AML. The observation that some of these antibodies neously show
g activity against leukemic blasts in vitro is highly exciting and very
promising. In addition, the observation that some of the antibodies have a germline
sequence and are of the IgG3 subclass, a type of antibody that can be induced
without T cell help, ts further attention as these findings demonstrate that
‘natural’ antibodies generated in a T-cell-independent way affect GvL responses in
SCT patients. Importantly, these data offer proof of concept that using this
technique, we can select leukemia-specific B cell clones.
Example 2
AML-specific antibodies from a third patient
To further confirm our approach and to show this anti-AML response is not only
reserved for female patients, we selected a male patient as well. This patient
(Figure 13), donor 101, was diagnosed with an intermediate-risk AML (no
cytogenetic or molecular abnormalities; FAB classification ) at the age of
49 years. He received two courses of chemotherapy (cytarabine, idarubicine,
amsacrine) and one course of consolidation chemotherapy (busulphan,
cyclophosphamide) followed by an autologous HSCT, as there was no HLA-matched
sibling stem cell donor available. Fourteen months after the first sis his
disease relapsed. He obtained complete ion after one cycle of ose
cytarabine, after which he received a reduced intensity allogeneic HSCT of a
matched, ted donor MUD). Six weeks later he developed acute GvHD
of skin, liver and intestine (stage 1; grade II) that responded well to corticosteroid
therapy. B cells were isolated from a phlebotomy t obtained from this
patient 38 months post-SCT.
Using the same methods as bed in Example 1, four B cell clones have been
generated from this patient that bind to the AML cell lines THP-1 and/or Molm13
(Figure 14 and Table 7) but not to (fetal) liver or gut cell lines, fibroblasts or
healthy PBMC (Table 8). These clones produce antibodies AT14-013 (IgG1), AT14-
014 (IgG3), AT14-015 (IgG3) and 16 (IgG3). Interestingly, again the
majority of the antibodies is of the IgG3 isotype. The sequences of the heavy and
light chain variable s of these antibodies show high amounts of somatic
hypermutations and are depicted in Table 1B and Figure 1. Clone AT14-013 shows
very minor reactivity with the hepatocellular oma cell line Huh7, but not to
the hepatocellular carcinoma cell line HepG2 or healthy fetal liver cells. Also minor
reactivity to fibroblasts is observed, however, the binding of this antibody to these
tissue specific cells, compaired to the binding to primary leukemic blasts is
negligible (Figure 14).
It was also tested whether the four antibodies of donor 101 could bind primary
AML blasts (Materials & s: see e 1). Figure 15 and Table 7 show
that antibody AT14-013 appears to bind primary blasts of at least three different
FAB classifications (AML-M0, M4 and M5). dies AT14-015 and AT14-016
are able to bind primary AML blasts of the M4 fication.
Example 3
In vitro activity of AML-specific antibodies of donors 59, 58 and 101
In Example 1, the breadth of binding of the antibodies of donor 59 within the
spectrum of AML subtypes was determined (Table 3). Using the same methods,
this has also been determined for the antibodies of donor 58. The results are shown
in Table 9A: all five antibodies of donor 58 (AT13-033, AT13-034, AT13-035,
AT13-036 and AT13-037) bound to the AML-M5 cell line THP-1, as well as to the
AML-M5 cell line MonoMac6. These AML-specific dies did not significantly
bind to freshly isolated AML blasts of newly diagnosed patients of FAB
classification M0, M1 or M4. AT13-034 could only weakly bind M0 blasts. This
tes that these antibodies have a specificity for (at least) AML M5 blasts.
In Example 1, it was also determined that antibodies AT12-023 and AT12-025 of
donor 59 killed THP-1 cells (Table 5 and Figures 7-9). Subsequently, we have
tested whether the other antibodies of donor 59, as well as the antibodies of donor
58, could also kill THP-1 cells, using the same Materials & Methods as in Example
1. As shown in Tables 9B-10, in addition to 23 and AT12-025 also dy
AT13-031 (donor 59), as well as dies AT13-033, AT13-035, AT13-036 and
AT13-037 (donor 58) appear to be able to kill THP-1 cells.
The capability of inducing death of primary AML blasts was also tested (Materials
& Methods: see Example 1). r to some of the antibodies obtained from donor
59, some of the antibodies derived from donor 58 are also capable of inducing cell
death of primary AML cells. This is shown in Figure 16 for antibody AT13-037, in
both forms (purified B cell supernatant (sAT13-037) and recombinant produced
antibody (rAT13-037); both in the IgG3 confirmation). The leukemic cells are
derived from the bone marrow of a newly diagnosed AML patient, FAB
classification AML-M5.
Example 4
In vitro activity of AML antibodies is rapid and involves a non-apoptotic cell death
pathway
To investigate the y through which several of the above-mentioned
antibodies induced death of their target cells, we visualized death of target cells
with time-lapse microscopy (Figure 17a). We observed that within a few minutes
after incubation with antibody 37, cells started to swell and then died. This
suggested that antibodies according to the invention activated pathways other than
the classical apoptosis pathways, as apoptotic cells shrink rather than swell. To
further investigate this we performed double staining with the cell death markers
DiOC6 and propidiumiodide (PI). Cells engaged to undergo apoptosis first loose
mitochondrial charge (which can be visualized by the loss of DiOC6 binding) before
membrane permeability increases (visualized by propidium iodide (PI) staining),
while cells oing necrosis loose their membrane integrity (PI positive) but not
immediately the ondrial membrane potential. As diclofenac is known to
induce sis of THP-1 cells, we used diclofenac as a positive l.
Diclofenac-treated, apoptotic THP-1 cells became DiOC6-negative (lost
mitochondrial membrane potential), but maintained membrane integrity. Upon
incubation with the AML-specific cytotoxic antibody AT12-023 ondrial
membrane potential was maintained (DiOC6-positive) while the membrane
permeability of THP-1 cells increased (PI-positive). Thus, AML-antibody treated
THP-1 cells became PI-positive while maintaining ondrial membrane
potential (Figure 17b, right panel) indicating the tion of a non-apoptotic
death pathway. , death of THP-1 cells by cytotoxic AML-specific antibody
treatment could not be ted by incubating the cells with the pan caspase
inhibitors Z-VAD-fmk or h (Figure 17c).
Cell death mediated through interference with the target cell membrane
Non-apoptotic death pathways include necroptosis and oncosis. Necroptosis is,
similar to apoptosis, an active cell death pathway that depends on the activation of
defined molecular pathways including the activation of receptor-interacting n
kinase 3 (RIPK3). To investigate whether AML-antibody-induced cell death was
dependent on the activation of intracellular molecular pathways we tested whether
antibody-induced cytotoxicity was ent of ature. We found that under
these experimental conditions the xic ties of at least antibodies
AT13-033, AT13-035, AT13-036, AT13-037, AT12-023 and AT12-025 were equally
potent at 4°C as compared to their activities at 37°C (Figure 18), suggesting that at
least these antibodies induced cell death by a e s. Of note, the cytotoxic
activity of antibody AT13-031 was significantly more potent at 37°C as compared to
4°C. However, since antibody AT13-031 does induce death of AML cells in the
presence of apoptosis inhibitors (such as the pan caspase inhibitors Q-VD-OPh or
fmk), it is clear that antibody AT13-031 is also able to diminish
proliferation of AML cells independently from apoptosis.
Oncosis is a mode of cell death characterized by swelling of the cell, through
selective membrane injury, resulting in increased ne permeability and
ultimately cell death. Oncosis-inducing antibodies have been described that
mediate cell death by forming large pores in the membrane through destabilization
of the membrane (Hernandez et al, 2011). Cytocholasin D is an actin
polymerization inhibitor that can stabilize the cytoskeleton. Treatment of the
target cell line THP-1 with alasin D did not prevent dy binding to
these cells (Figure 19a), however, it did prevent death of the target cells (Figure
19b ). Thus, membrane isation protects the target cells against the cytotoxic
activity of our antibodies.
Example 5
One of the targets recognized by our antibodies is snRNP200
We then set out to determine the target recognized by several AML-specific
antibodies. For this we used immunoprecipitation of cell membrane lysates of THP-
1 cells with 23 and AT13-031. We found a clear protein band at 250 kD that
was sent for mass-spectometry analysis which revealed snRNP200 as the target
antigen which could be confirmed by Western blot analysis (Figure 20a). We
developed an ELISA, to verify snRNP200 as the target of some of the AML
antibodies. With this ELISA we found that in addition to AT12-023 and AT13-031,
AT13-037 specifically izes snRNP200 (Figure 20b). Other AML specific
antibodies like AT12-019 did not recognize snRNP200 as target antigen.
snRNP200 is part of the osome in all eukaryotic cells and is therefore
expected to be located in the nucleus. To confirm that AML cells express this
protein on their cell surface we stained the cell membrane of THP-1 cells with a
commercially available, nRNP200 antibody. Figure 21 shows that the anti
00 antibody is indeed binding to the membrane of the AML cells, but not to
the Jurkat cell line. As expected, as snRNP200 is a nuclear protein, it did bind
intracellularly to the Jurkat and THP-1 cell lines (Figure 21).
All eukaryotic cells have snRNP200 (also known as U5-snRNP) protein in the
nucleus, as part of the spliceosome (Kattah, 2010). The spliceosome consists of a
number of ns. At least one of these proteins, U1-snRNP, has been described
to be expressed on apoptotic cells in patients with systemic lupus erythematosus
(SLE) and mixed connective tissue disease (MCTD) leading to mmune
responses (Kattah, 2010).
We propose that 00 expression by AML cells has triggered an allo-immune
se (graft vs leukemia response) that has kept the patients in durable
remission. Therefore, this protein can now be used as a new AML .
Moreover, since dies AT12-023 and AT13-031 specifically recognize
snRNP200 and also recognize B-NHL cells, snRNP200 can now also be used as a
target for B-NHL.
Table 1A. red anti-AML antibodies according to the invention (CDR numbering according to Kabat et al. 1991)
SEQ ID Antibody Identity Sequence
1 AT12-023 Heavy GYYWS
chain
CDR1
2 AT12-025 Heavy GYYWS
chain
CDR1
3 AT13-024 Heavy SYGMH
chain
CDR1
4 AT12-019 Heavy SYAMS
chain
CDR1
AT13-022 Heavy SYGMH
chain
CDR1
6 AT13-023 Heavy GYFWT
chain
CDR1
7 31 Heavy GYYWS
chain
CDR1
8 AT12-020 Heavy TYSMN
chain
CDR1
9 AT13-033 Heavy NYGMH
chain
CDR1
AT13-034 Heavy SHAIH
chain
CDR1
11 AT13-035 Heavy SYGMH
chain
CDR1
12 AT13-036 Heavy SYSMN
chain
CDR1
13 AT13-037 Heavy TYGMH
chain
CDR1
14 AT12-023 Heavy STNYNPSLKS
chain
CDR2
AT12-025 Heavy EINHSGSTNYNPSLKS
chain
CDR2
16 AT13-024 Heavy FIRYDGSNKYFADSVRG
chain
CDR2
17 AT12-019 Heavy TIRASGGSTSYADSVKG
chain
CDR2
18 AT13-022 Heavy ISYDGSNKYYADSVKG
chain
CDR2
19 AT13-023 Heavy ETVHSGGTNYNPSLKS
chain
CDR2
AT13-031 Heavy EINHSGSTNYNPSLKS
chain
CDR2
21 AT12-020 Heavy SISSSSGYIYYADSVKG
chain
CDR2
22 AT13-033 Heavy VISHDGSKTYYGHSVKG
chain
CDR2
23 AT13-034 Heavy LIWYDGSNNYYADSVKG
chain
CDR2
24 AT13-035 Heavy VISYDGSNKYYADSVKG
chain
CDR2
AT13-036 Heavy SISSSSTYIYYADSVKG
chain
CDR2
26 AT13-037 Heavy VIWYDGSNTYYADSVKG
chain
CDR2
27 AT12-023 Heavy GRSTSPLDYYYYYMDV
chain
CDR3
28 AT12-025 Heavy GSMARPKPFDY
chain
CDR3
29 AT13-024 Heavy DPQERIYYSDTSGYLDY
chain
CDR3
AT12-019 Heavy SPAMIRGVRGGDYFDY
chain
CDR3
31 AT13-022 Heavy DGKGIVVIYYYYGMDV
chain
CDR3
32 AT13-023 Heavy DY
chain
CDR3
33 AT13-031 Heavy GPRGMYSSSSGDY
chain
CDR3
34 AT12-020 Heavy DGTFSYYYYMDV
chain
CDR3
AT13-033 Heavy AGLNYYGNLLSNYFYYGMDV
chain
CDR3
36 AT13-034 Heavy ARDGCTGGSCCYFDN
chain
CDR3
37 AT13-035 Heavy AKDSYYYGSGRRWGYYFDY
chain
CDR3
38 AT13-036 Heavy GRDGYSLYPRGYHYGMDV
chain
CDR3
39 AT13-037 Heavy ARGRGYSAQGNRNRAYYFDY
chain
CDR3
40 AT12-023 Light QGDFLRSYYAS
chain
CDR1
41 25 Light RASQSISRYLN
chain
CDR1
42 AT13-024 Light RASQSISSWLA
chain
CDR1
43 AT12-019 Light RASQAFSSYLV
chain
CDR1
44 AT13-022 Light SGDKLGDKYAC
chain
CDR1
45 AT13-023 Light RASQGIRNVLG
chain
CDR1
46 AT13-031 Light RNDLG
chain
CDR1
47 AT12-020 Light RASQDISSSLA
chain
CDR1
48 33 Light TGTSSDIGGYNYVS
chain
CDR1
49 AT13-034 Light RASQSISNNLG
chain
CDR1
50 AT13-035 Light QGDSLRSYYAS
chain
CDR1
51 AT13-036 Light TGTSSDVGGYNYVS
chain
CDR1
52 AT13-037 Light RASQSVSSNLA
chain
CDR1
53 AT12-023 Light GKNKRPS
chain
CDR2
54 AT12-025 Light AASSLQS
chain
CDR2
55 AT13-024 Light KASSLES
chain
CDR2
56 AT12-019 Light ATSTLQG
chain
CDR2
57 AT13-022 Light S
chain
CDR2
58 AT13-023 Light AASSLQS
chain
CDR2
59 AT13-031 Light AAVSLQS
chain
CDR2
60 AT12-020 Light AASTLQS
chain
CDR2
61 AT13-033 Light EVTKRPS
chain
CDR2
62 AT13-034 Light GASTRAT
chain
CDR2
63 35 Light GKNNRPS
chain
CDR2
64 AT13-036 Light DVNDRPS
chain
CDR2
65 AT13-037 Light GAFTRVT
chain
CDR2
66 AT12-023 Light NSRDRSGNHLV
chain
CDR3
67 AT12-025 Light QQSYSTPRT
chain
CDR3
68 AT13-024 Light QQYNTYPYT
chain
CDR3
69 AT12-019 Light QQYYSYPPT
chain
CDR3
70 AT13-022 Light QAWDSSTVVF
chain
CDR3
71 23 Light LQHNSHPRT
chain
CDR3
72 AT13-031 Light LQHNSYPRT
chain
CDR3
73 AT12-020 Light QQYYSYPPT
chain
CDR3
74 AT13-033 Light SSYAGSNDLL
chain
CDR3
75 AT13-034 Light QQYNNWPRLT
chain
CDR3
76 35 Light NSRDSSGNHVV
chain
CDR3
77 AT13-036 Light SSYTRSNTVI
chain
CDR3
78 AT13-037 Light QQYNDRPPYT
chain
CDR3
79 AT12-023 Heavy QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISV
chain DTSKNQFSLKLSSVTAADTAVYYCARGRSTSPLDYYYYYMDVWAKGTTVTVSS
80 AT12-025 Heavy QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISV
chain DTSKNQFSLKLSSVTAADTAVYYCARGSMARPKPFDYWGQGTLVTVSS
81 AT13-024 Heavy QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYFADSVRGRFTIS
chain RDNSKNTLFLQMNSLRAEDTAVYYCAKDPQERIYYSDTSGYLDYWGQGTLVTVSS
82 AT12-019 Heavy EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIRASGGSTSYADSVKGRFTISR
chain DNSQSRLYLQMNSLTAEDTAVYYCAKSPAMIRGVRGGDYFDYWGQGTLVTVSS
83 AT13-022 Heavy QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTIS
chain RDNSKNTLYLQMNSLRAEDTAVYYCAKDGKGIVVIYYYYGMDVWGQGTTVTVSS
84 AT13-023 Heavy WGAGLLKPSETLSLTCAVYGGSFSGYFWTWIRQPPGKGLEWIGETVHSGGTNYNPSLKSRVTISV
chain DTSKNQFSLRLNSVTAADTAVYYCVRGLNSPFDYWGQGTLVTVSS
85 31 Heavy QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISV
chain DTSKKQFSLKLSSVTAADTAVYYCARGPRGMYSSSSGDYWGQGTLVTVSS
86 AT12-020 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSSGYIYYADSVKGRFTISR
chain DNAKNSLYLQMNSLRAEDTAVYYCARDGTFSYYYYMDVWGKGTTVTVSS
87 33 Heavy QVQLVESGGGVVQPGRSLRLSCAVSGLSFRNYGMHWVRQAPGKGLEWVAVISHDGSKTYYGHSVKGRFTI
chain SRDKSKTMLFLQMNSLRPEDTAVYYCAKAGLNYYGNLLSNYFYYGMDVWGQGTTVTVSS
88 AT13-034 Heavy QVHLVESGGGVVQPGTSLRLSCAASEFTFSSHAIHWVRQAPGKGLEWVALIWYDGSNNYYADSVKGRFTIS
chain RDSSKNTVHLQMNSLRVEDTAVYYCARDGCTGGSCCYFDNWGQGTLVTVSS
89 AT13-035 Heavy QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTIS
chain RDNSKNTLYLQMNSLRAEDTAVYYCAKDSYYYGSGRRWGYYFDYWGQGTLVTVSS
90 AT13-036 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSTYIYYADSVKGRFTISR
chain DNARNSLYLQMNSLRAEDTAVYYCARRREVGRDGYSLYPRGYHYGMDVWGQGTTVTVSS
91 AT13-037 Heavy QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWYDGSNTYYADSVKGRFTI
chain SRDNSKNTLYLQIKSLRAEDTAVYYCARGRGYSAQGNRNRAYYFDYWGQGTLVTVSS
92 AT12-023 Light SSELTQDPAVSVALGQTVRITCQGDFLRSYYASWYQQKPGQAPVLVIFGKNKRPSGIPDRFSGSSSGNTASL
chain TITGAQAEDEADYYCNSRDRSGNHLVFGGGTKLTVL
93 AT12-025 Light DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
chain TISSLQPEDFATYYCQQSYSTPRTFGPGTKVDIK
94 AT13-024 Light DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGTGSGTEFTL
chain TISSLQPDDFATYYCQQYNTYPYTFGQGTKLEIK
95 AT12-019 Light AIRLTQSPSSVSASTGDRVTITCRASQAFSSYLVWYQQKPGKAPNLLIYATSTLQGGVPSRFSGSGSGTDFTL
chain TISNLQSEDFATYYCQQYYSYPPTFGQGTKLEIK
96 AT13-022 Light SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATL
chain TISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVL
97 AT13-023 Light DIQMTQSPSSLSASVGDRVTITCRASQGIRNVLGWYQQKPGKAPKCLIYAASSLQSGVPSRFSGSGSGTEFTL
chain TISSLQPEDFATYYCLQHNSHPRTFGQGTKVEIK
98 AT13-031 Light DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAAVSLQSGVPSRFSGSGSGTEFT
chain LTISSLQPEDFATYYCLQHNSYPRTFGQGTKLEIK
99 AT12-020 Light AIRMTQSPSSFSASTGDRVTITCRASQDISSSLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTL
chain TISCLQSEDFATYYCQQYYSYPPTFGQGTRLEIK
100 AT13-033 Light PPSASGSPGQSVTISCTGTSSDIGGYNYVSWYQHHPGKAPKLMIYEVTKRPSGVPDRFSGSKSGNT
chain GLQAEDEAHYYCSSYAGSNDLLFGGGTKLTVL
101 AT13-034 Light EVVMTQSPATLSVSPGERATLSCRASQSISNNLGWYQQKPGQAPRLLIYGASTRATGIPGRFSGSGSGTEFT
chain LTIYSLQSEDFAVYYCQQYNNWPRLTFGGGTKVEIK
102 AT13-035 Light SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASL
chain AEDEADYYCNSRDSSGNHVVFGGGTKLTVL
103 AT13-036 Light QSALTQPASVSGSPRQSITISCTGTSSDVGGYNYVSWYQQLPGKAPKLMIYDVNDRPSGVSIRFSGSKSGNT
chain ASLTISGLQAEDEADYYCSSYTRSNTVIFGGGTKLTVL
104 AT13-037 Light EIVMTQSPATLSVSPGERVILSCRASQSVSSNLAWYQQKPGQPPRLLIYGAFTRVTGVPARFSGSGSGTEFTL
chain TISSLQSEDFAVYYCQQYNDRPPYTFGQGTKLEIK
105 AT12-023 Heavy ggt tac tac tgg agc
chain
CDR1
106 AT12-025 Heavy ggt tat tac tgg agc
chain
CDR1
107 AT13-024 Heavy agc tat ggc atg cac
chain
CDR1
108 AT12-019 Heavy agc tat gcc atg agt
chain
CDR1
109 AT13-022 Heavy agc tat ggc atg cac
chain
CDR1
110 23 Heavy ggt tac ttc tgg acc
chain
CDR1
111 AT13-031 Heavy ggt tac tac tgg agc
chain
CDR1
112 AT12-020 Heavy acc tat agc atg aac
chain
CDR1
113 AT13-033 Heavy aat tat ggc atg cac
chain
CDR1
114 AT13-034 Heavy tcc cat gcc ata cac
chain
CDR1
115 AT13-035 Heavy agc tat ggc atg cac
chain
CDR1
116 AT13-036 Heavy agt tat agc atg aac
chain
CDR1
117 AT13-037 Heavy acc tat ggc atg cac
chain
CDR1
118 AT12-023 Heavy gaa atc aat cat agt gga agc acc aac tac aac ccg tcc ctc aag agt
chain
CDR2
119 AT12-025 Heavy gaa atc aat cat agt gga agc acc aac tac aac ccg tcc ctc aag agt
chain
CDR2
120 AT13-024 Heavy ttt ata cgg tat gat gga agt aat aaa tac ttt gca gac tcc gtg agg ggc
chain
CDR2
121 AT12-019 Heavy act att agg gct agt ggt ggt agc aca agc tac gca gac tcc gtg aag ggc
chain
CDR2
122 AT13-022 Heavy ata tca tat gat gga agt aat aaa tac tat gca gac tcc gtg aag ggc
chain
CDR2
123 23 Heavy gaa acc gtt cat agt gga ggc acc aac tac aac ccg tcc ctc aag agt
chain
CDR2
124 AT13-031 Heavy gaa atc aat cat agt gga agc acc aac tac aac ccg tcc ctc aag agt
chain
CDR2
125 AT12-020 Heavy tcc att agt agt agt agt ggt tac ata tac tac gca gac tca gtg aag ggc
chain
CDR2
126 AT13-033 Heavy gtc att tcg cat gat gga agt aag aca tac tat gga cac tcc gtg aag ggc
chain
CDR2
127 34 Heavy ctt ata tgg tat gat gga agt aat aat tat tat gca gac tcc gtg aag ggc
chain
CDR2
128 AT13-035 Heavy gtt ata tca tat gat gga agt aat aaa tac tat gca gac tcc gtg aag ggc
chain
CDR2
129 AT13-036 Heavy tcc att agt agt agt agt act tac ata tac tac gca gac tca gtg aag ggc
chain
CDR2
130 AT13-037 Heavy gtt ata tgg tat gat gga agt aat aca tac tat gca gac tcc gtg aag ggc
chain
CDR2
131 AT12-023 Heavy ggc cgt agt acc agc ccg ctc gac tac tac tac tac tac atg gac gtc
chain
CDR3
132 AT12-025 Heavy ggc tca atg gca aga ccc aag cca ttt gac tac
chain
CDR3
133 AT13-024 Heavy gcg aaa gat ccc caa gag cgt att tat tac tct gat act agt ggt tac ctt gac tac
chain
CDR3
134 AT12-019 Heavy tct cct gct atg att cgg gga gtt agg ggg ggt gac tac ttt gac tac
chain
CDR3
135 AT13-022 Heavy gat ggg aag ggg att gta gtt att tac tac tac tac ggt atg gac gtc
chain
CDR3
136 AT13-023 Heavy ggc ctt aac agc ccc ttt gac tac
chain
CDR3
137 AT13-031 Heavy ccc cgg ggc atg tat agc agc tcg tcc ggg gac tac
chain
CDR3
138 AT12-020 Heavy gat ggg act ttc tcc tac tac tac tac atg gac gtc
chain
CDR3
139 AT13-033 Heavy gcc ggg ttg aac tac tat gga aac cta tta tca aac tac ttc tac tac gga atg gac gtc
chain
CDR3
140 AT13-034 Heavy gcg aga gat ggt tgt act ggt ggt agc tgc tgc tat ttt gac aac
chain
CDR3
141 AT13-035 Heavy gcg aaa gac tcg tat tac tat ggt tcg ggg aga cga tgg ggc tac tac ttt gac tac
chain
CDR3
142 AT13-036 Heavy gcg aga agg agg gag gtc ggt aga gat ggc tac agt ttg tac ccc cgg ggg tac cac tac ggt atg gac gtc
chain
CDR3
143 AT13-037 Heavy gcg aga ggc cgt gga tat agt gcc caa ggg aat cgg aat agg gct tac tac ttt gac tac
chain
CDR3
144 AT12-023 Light caa gga gac ttc ctc aga agc tat tat gca agc
chain
CDR1
145 25 Light cgg gca agt cag agc att agc agg tat tta aat
chain
CDR1
146 AT13-024 Light cgg gcc agt cag agt att agt agc tgg ttg gcc
chain
CDR1
147 AT12-019 Light cgg gcg agt cag gct ttt agc agt tat tta gtc
chain
CDR1
148 AT13-022 Light tct gga gat aaa ttg ggg gat aaa tat gct tgc
chain
CDR1
149 AT13-023 Light cgg gca agt cag ggc att aga aat gtt tta ggc
chain
CDR1
150 AT13-031 Light cgg gca agt cag ggc att aga aat gat tta ggc
chain
CDR1
151 20 Light cgg gcg agt cag gat att agc agt tct tta gcc
chain
CDR1
152 AT13-033 Light act ggg acc agc agt gac att ggt ggt tat aac tat gtc tcc
chain
CDR1
153 AT13-034 Light agg gcc agt cag agc att agc aac aac tta ggc
chain
CDR1
154 AT13-035 Light caa gga gac agc ctc aga agc tat tat gca agc
chain
CDR1
155 AT13-036 Light act gga acc agc agt gac gtt ggt ggt tat aac tat gtc tcc
chain
CDR1
156 AT13-037 Light agg gcc agt cag agt gtt agc agc aac tta gcc
chain
CDR1
157 AT12-023 Light ggt aaa aac aag cgg ccc tca
chain
CDR2
158 AT12-025 Light gct gca tcc agt ttg caa agt
chain
CDR2
159 AT13-024 Light aag gcg tct agt tta gaa agt
chain
CDR2
160 AT12-019 Light gct aca tcc act ttg caa ggt
chain
CDR2
161 AT13-022 Light caa gat agc aag cgg ccc tca
chain
CDR2
162 AT13-023 Light gct gca tcc agt ttg caa agt
chain
CDR2
163 AT13-031 Light gct gca gtc agt ttg caa agt
chain
CDR2
164 AT12-020 Light gct gca tcc act ttg caa agt
chain
CDR2
165 AT13-033 Light gag gtc act aag cgg ccc tca
chain
CDR2
166 34 Light ggt gca tcc acc agg gcc act
chain
CDR2
167 AT13-035 Light ggt aaa aac aac cgg ccc tca
chain
CDR2
168 AT13-036 Light gat gtc aat gat cgg ccc tca
chain
CDR2
169 AT13-037 Light ggt gca ttc acg agg gtc act
chain
CDR2
170 AT12-023 Light aac tcc cgg gac cgc agt ggt aac cac ctg gtg
chain
CDR3
171 AT12-025 Light caa cag agt tac agt acc cct cgc act
chain
CDR3
172 AT13-024 Light caa cag tat aat act tac ccg tac act
chain
CDR3
173 AT12-019 Light caa cag tat tat agt tac cct ccg act
chain
CDR3
174 AT13-022 Light cag gcg tgg gac agc agc act gtg gta ttc
chain
CDR3
175 AT13-023 Light cta cag cat aat agt cac ccc cgg acg
chain
CDR3
176 AT13-031 Light cta cag cat aat agt tac cct cgg act
chain
CDR3
177 AT12-020 Light caa cag tat tat agt tac cct ccg acg
chain
CDR3
178 AT13-033 Light agc tca tat gca ggc agc aac gat ttg cta
chain
CDR3
179 AT13-034 Light caa caa tat aat aac tgg cct cgg ctc act
chain
CDR3
180 35 Light aac tcc cgg gac agc agt ggt aac cat gtg gta
chain
CDR3
181 AT13-036 Light agc tca tat aca aga agc aac act gtg ata
chain
CDR3
182 AT13-037 Light cag cag tac aat gac cgg ccc ccg tac act
chain
CDR3
183 AT12-023 Heavy cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc
chain agt ggt tac tac tgg agc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att ggg gaa atc aat cat agt gga agc acc aac
tac aac ccg tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc
gcg gac acg gct gtg tat tac tgt gcg agg ggc cgt agt acc agc ccg ctc gac tac tac tac tac tac atg gac gtc tgg gcc aaa
ggg acc acg gtc acc gtc tcc tca
184 AT12-025 Heavy cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc
chain agt ggt tat tac tgg agc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att ggg gaa atc aat cat agt gga agc acc aac
tac aac ccg tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc aag aac cag ttc tcc ctg aag ctg agc tct gtg acc gcc
gcg gac acg gct gtg tat tac tgt gcg aga ggc tca atg gca aga ccc aag cca ttt gac tac tgg ggc cag gga acc ctg gtc acc
gtc tcc tca
185 AT13-024 Heavy cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc
chain agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca ttt ata cgg tat gat gga agt aat aaa
tac ttt gca gac tcc gtg agg ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg ttt ctg caa atg aac agc ctg aga
gct gag gac acg gct gtg tat tac tgt gcg aaa gat ccc caa gag cgt att tat tac tct gat act agt ggt tac ctt gac tac tgg
ggc cag gga acc ctg gtc acc gtc tcc tca
186 AT12-019 Heavy gag gtg cac ctg ttg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt
chain agc agc tat gcc atg agt tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc tca act att agg gct agt ggt ggt agc aca
agc tac gca gac tcc gtg aag ggc cgg ttc acc atc tcc aga gac aat tcc cag agc agg ttg tat ctg caa atg aac agt ctg aca
gcc gag gac acg gcc gta tat tac tgt gcg aaa tct cct gct atg att cgg gga gtt agg ggg ggt gac tac ttt gac tac tgg ggc
cag gga acc ctg gtc acc gtc tcc tca
187 22 Heavy cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc
chain agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca gtt ata tca tat gat gga agt aat aaa
tac tat gca gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg aac agc ctg aga
gct gag gac acg gct gtg tat tac tgt gcg aaa gat ggg aag ggg att gta gtt att tac tac tac tac ggt atg gac gtc tgg ggc
caa ggg acc acg gtc acc gtc tcc tca
188 AT13-023 Heavy cag gta cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc
chain agt ggt tac ttc tgg acc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att ggg gaa acc gtt cat agt gga ggc acc aac
tac aac ccg tcc ctc aag agt cga gtc acc ata tca gtc gac acg tcc aag aac cag ttc tcc ctg agg ctg aac tct gtg acc gcc
gcg gac acg gct gtg tat tac tgt gtg aga ggc ctt aac agc ccc ttt gac tac tgg ggc cag gga acc cta gtc acc gtc tcc tca
189 31 Heavy cag gtg cag cta cag cag tgg ggc gca gga ctg ttg aag cct tcg gag acc ctg tcc ctc acc tgc gct gtc tat ggt ggg tcc ttc
chain agt ggt tac tac tgg agc tgg atc cgc cag ccc cca ggg aag ggg ctg gag tgg att ggg gaa atc aat cat agt gga agc acc aac
tac aac ccg tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc aag aag cag ttc tcc ctg aag ctg agc tct gtg acc gcc
gcg gac acg gct gtg tat tat tgt gcg aga ggc ccc cgg ggc atg tat agc agc tcg tcc ggg gac tac tgg ggc cag gga acc ctg
gtc acc gtc tcc tca
190 AT12-020 Heavy gag gtg cag ctg gtg gag tct ggg gga ggc ctg gtc aag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc
chain agt acc tat agc atg aac tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc tca tcc att agt agt agt agt ggt tac ata
tac tac gca gac tca gtg aag ggc cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat ctg caa atg aac agc ctg aga
gcc gag gac acg gct gtg tat tac tgt gcg aga gat ggg act ttc tcc tac tac tac tac atg gac gtc tgg ggc aaa ggg acc acg
gtc acc gtc tcc tca
191 AT13-033 Heavy cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tcc ctg aga ctc tcc tgt gca gtc tct gga ctc agt ttc
chain agg aat tat ggc atg cac tgg gtc cgc cag gct ccc ggc aag ggg ctg gag tgg gtg gca gtc att tcg cat gat gga agt aag aca
tac tat gga cac tcc gtg aag ggc cga ttc acc ata tcc aga gac aaa tcc aag act atg ttg ttt ctc caa atg aac agc ctg aga
cct gag gac acg gct gtt tat tac tgt gcg aaa gcc ggg ttg aac tac tat gga aac cta tta tca aac tac ttc tac tac gga atg
gac gtc tgg ggc caa ggg acc aca gtc acc gtc tcg tca
192 AT13-034 Heavy cag gtg cac ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg acg tcc ctg aga ctc tcc tgt gca gcg tct gaa ttc acc ttc
chain agt tcc cat gcc ata cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca ctt ata tgg tat gat gga agt aat aat
tat tat gca gac tcc gtg aag ggc cga ttc acc atc tcc aga gac agt tcc aag aac acg gtg cat ctg caa atg aac agc ctg aga
gtc gag gac acg gct gtg tat tac tgt gcg aga gat ggt tgt act ggt ggt agc tgc tgc tat ttt gac aac tgg ggc cag gga acc
cta gtc acc gtc tcc tcg
193 AT13-035 Heavy cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc
chain agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca gtt ata tca tat gat gga agt aat aaa
tac tat gca gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat ctg caa atg aac agc ctg aga
gct gag gac acg gct gtg tat tac tgt gcg aaa gac tcg tat tac tat ggt tcg ggg aga cga tgg ggc tac tac ttt gac tac tgg
ggc cag gga acc ctg gtc acc gtc tcc tca
194 AT13-036 Heavy gag gtg cag ctg gtg gag tct ggg gga ggc ctg gtc aag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc
chain agt agt tat agc atg aac tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc tca tcc att agt agt agt agt act tac ata
tac tac gca gac tca gtg aag ggc cga ttc acc atc tcc aga gac aac gcc agg aac tca ctg tat ctg caa atg aac agc ctg aga
gcc gag gac acg gct gtg tat tat tgt gcg aga agg agg gag gtc ggt aga gat ggc tac agt ttg tac ccc cgg ggg tac cac tac
ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca
195 AT13-037 Heavy cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc
chain agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctt gag tgg gtg gca gtt ata tgg tat gat gga agt aat aca
tac tat gca gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac aca ctg tat ctg caa ata aag agc ctg aga
gcc gag gac acg gct gtc tat tac tgt gcg aga ggc cgt gga tat agt gcc caa ggg aat cgg aat agg gct tac tac ttt gac tac
tgg ggc cag gga acc ctg gtc acc gtc tcc tca
196 AT12-023 Light tct tct gag ctg act cag gac cct gct gtg tct gtg gcc ttg gga cag aca gtc agg atc aca tgc caa gga gac ttc ctc aga agc
chain tat tat gca agc tgg tac cag cag aag cca gga cag gcc cct gta ctt gtc atc ttt ggt aaa aac aag cgg ccc tca ggg atc cca
gac cga ttc tct ggc tcc agc tca gga aac aca gct tcc ttg acc atc act ggg gct cag gcg gaa gat gag gct gac tat tac tgt
aac tcc cgg gac cgc agt ggt aac cac ctg gtg ttc ggc gga ggg acc aag ctg acc gtc cta
197 AT12-025 Light gac atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta gga gac aga gtc acc atc act tgc cgg gca agt cag agc att
chain agc agg tat tta aat tgg tat cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc tat gct gca tcc agt ttg caa agt ggg gtc
cca tca agg ttc agt ggc agt gga tct ggg aca gat ttc act ctc acc atc agc agt ctg caa cct gaa gat ttt gca act tac tac tgt
caa cag agt tac agt acc cct cgc act ttc ggc cct ggg acc aaa gtg gat atc aaa
198 AT13-024 Light gac atc cag atg acc cag tct cct tcc acc ctg tct gca tct gta gga gac aga gtc acc atc act tgc cgg gcc agt cag agt att
chain agt agc tgg ttg gcc tgg tat cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc tat aag gcg tct agt tta gaa agt ggg gtc
cca tca agg ttc agc ggc act gga tct ggg aca gaa ttc act ctc acc atc agc agc ctg cag cct gat gat ttt gca act tat tac tgc
caa cag tat aat act tac ccg tac act ttt ggc cag ggg acc aag ctg gag atc aaa
199 19 Light gcc atc cgg ttg acc cag tct cca tcc tca gtc tct gca tct aca gga gac aga gtc acc atc act tgt cgg gcg agt cag gct ttt agc
chain agt tat tta gtc tgg tat cag caa aaa cca ggg aaa gcc cct aac ctc ctg atc tac gct aca tcc act ttg caa ggt ggg gtc cca
tca agg ttc agc ggc agt gga tct ggg aca gat ttc act ctc acc atc agc aac ctg cag tct gaa gat ttt gca act tat tac tgt
caa cag tat tat agt tac cct ccg act ttt ggc cag ggg acc aag ttg gag atc aaa
200 AT13-022 Light tcc tat gag ctg act cag cca ccc tca gtg tcc gtg tcc cca gga cag aca gcc agc atc acc tgc tct gga gat aaa ttg ggg gat
chain aaa tat gct tgc tgg tat cag cag aag cca ggc cag tcc cct gtg ctg gtc atc tat caa gat agc aag cgg ccc tca ggg atc cct
gag cga ttc tct ggc tcc aac tct ggg aac aca gcc act ctg acc atc agc ggg acc cag gct atg gat gag gct gac tat tac tgt
cag gcg tgg gac agc agc act gtg gta ttc ggc gga ggg acc aag ctg acc gtc cta
201 AT13-023 Light gac atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta gga gac aga gtc acc atc act tgc cgg gca agt cag ggc att
chain aga aat gtt tta ggc tgg tat cag cag aaa cca ggg aaa gcc cct aag tgc ctg atc tat gct gca tcc agt ttg caa agt ggg gtc
cca tca agg ttc agc ggc agt gga tct ggg aca gaa ttc act ctc aca atc agc agc ctg cag cct gaa gat ttt gca act tat tac
tgt cta cag cat aat agt cac ccc cgg acg ttc ggc caa ggg acc aag gtg gaa atc aaa
202 AT13-031 Light gac atc cag atg acc cag tct cca tcc tcc ctg tct gca tct gta gga gac aga gtc acc atc act tgc cgg gca agt cag ggc att
chain aga aat gat tta ggc tgg tat cag cag aaa cca ggg aaa gcc cct aag cgc ctg atc tat gct gca gtc agt ttg caa agt ggg gtc
cca tca agg ttc agc ggc agt gga tct ggg aca gaa ttc act ctc aca atc agc agc ctg cag cct gaa gat ttt gca act tat tac
tgt cta cag cat aat agt tac cct cgg act ttt ggc cag ggg acc aag ctg gag atc aaa
203 AT12-020 Light gcc atc cgg atg acc cag tct cca tcc tca ttc tct gca tct aca gga gac aga gtc acc atc act tgt cgg gcg agt cag gat att
chain agc agt tct tta gcc tgg tat cag caa aaa cca ggg aaa gcc cct aag ctc ctg atc tat gct gca tcc act ttg caa agt gga gtc
cca tca agg ttc agc ggc agt gga tct ggg aca gac ttc act ctc acc atc agc tgc ctg cag tct gaa gat ttt gca act tat tac tgt
caa cag tat tat agt tac cct ccg acg ttc ggc caa ggg acc agg ttg gaa atc aaa
204 33 Light cag tct gcc ctg act cag cct ccc tcc gcg tcc ggg tct cct ggt cag tca gtc acc atc tcc tgt act ggg acc agc agt gac att ggt
chain ggt tat aac tat gtc tcc tgg tac caa cac cac cca ggc aaa gcc ccc aaa ttg atg att tat gag gtc act aag cgg ccc tca ggg
gtc cct gat cgt ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc gtc tct gga ctc cag gct gag gat gag gct cat tat tac
tgc agc tca tat gca ggc agc aac gat ttg cta ttc ggc gga ggg acc aag ctg acc gtc ctg
205 AT13-034 Light gaa gta gtg atg acg cag tct cca gcc acc ctg tct gtg tct cca ggg gaa aga gcc acc ctc tcc tgc agg gcc agt cag agc att
chain agc aac aac tta ggc tgg tat cag cag aaa cct ggc cag gct ccc agg ctc ctc atc tac ggt gca tcc acc agg gcc act ggt atc
cca ggc agg ttc agt ggc agt ggg tct ggg aca gag ttc act ctc acc atc tac agc ctg cag tct gag gat ttt gca gtt tat tac tgt
caa caa tat aat aac tgg cct cgg ctc act ttc ggc gga ggg acc aag gtg gag atc aaa
206 AT13-035 Light tct tct gag ctg act cag gac cct gct gtg tct gtg gcc ttg gga cag aca gtc agg atc aca tgc caa gga gac agc ctc aga agc
chain tat tat gca agc tgg tac cag cag aag cca gga cag gcc cct gta ctt gtc atc tat ggt aaa aac aac cgg ccc tca ggg atc cca
gac cga ttc tct ggc tcc agc tca gga aac aca gct tcc ttg acc atc act ggg gct cag gcg gaa gat gag gct gac tat tac tgt
aac tcc cgg gac agc agt ggt aac cat gtg gta ttc ggc gga ggg acc aag ctg acc gtc cta
207 AT13-036 Light cag tct gcc ctg act cag cct gcc tcc gtg tct ggg tct cct aga cag tcg atc acc atc tcc tgc act gga acc agc agt gac gtt ggt
chain ggt tat aac tat gtc tcc tgg tac caa caa ctc cca ggc aaa gcc ccc aaa ctc atg att tat gat gtc aat gat cgg ccc tca ggg
gtt tct att cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc atc tct ggg ctc cag gct gag gac gag gct gat tat tac
tgc agc tca tat aca aga agc aac act gtg ata ttc ggc gga ggg acc aaa ctg acc gtc cta
208 AT13-037 Light gaa ata gtg atg acg cag tct cca gcc acc ctg tct gtg tct cca ggg gaa agg gtc atc ctc tcc tgc agg gcc agt cag agt gtt
chain agc agc aac tta gcc tgg tac cag cag aaa cct ggc cag cct ccc agg ctc ctc atc tat ggt gca ttc acg agg gtc act ggt gtc
cca gcc agg ttc agt ggc agt ggg tct ggg aca gaa ttc act ctc acc atc agc agc ctg cag tct gaa gat ttt gca gtt tat tac tgt
cag cag tac aat gac cgg ccc ccg tac act ttt ggc cag ggg acc aag ctg gag atc aaa
Table 1B. Preferred anti-AML antibodies ing to the invention (CDR numbering according to Kabat et al. 1991)
SEQ ID Antibody Identity ce
209 AT14-013 Heavy SPNWWT
chain
CDR1
210 AT14-014 Heavy DAWMS
chain
CDR1
211 AT14-015 Heavy DFAMS
chain
CDR1
212 AT14-016 Heavy SYAMT
chain
CDR1
213 AT14-013 Heavy EIYYGGRVSYNSALRS
chain
CDR2
214 AT14-014 Heavy HINTKVDGGTTEYAAPVKG
chain
CDR2
215 AT14-015 Heavy FIRTKANDGTTEYAASVKG
chain
CDR2
216 AT14-016 Heavy SISGSGGSTYYADSVRG
chain
CDR2
217 AT14-013 Heavy AGQKNIGCGYSSCFISWFDT
chain
CDR3
218 AT14-014 Heavy TTEAIYDSSGYFHDY
chain
CDR3
219 AT14-015 Heavy ASDPFMTTDYYYYYMDV
chain
CDR3
220 AT14-016 Heavy AKGYVGCSGGNCYSGGAFDI
chain
CDR3
221 13 Light KSSQTILQRSNHLNYLA
chain
CDR1
222 AT14-014 Light KSSRSVLYSSNNKNYLA
chain
CDR1
223 AT14-015 Light TGTSSDVGGYNSVS
chain
CDR1
224 AT14-016 Light GGNNIGSESVH
chain
CDR1
225 AT14-013 Light WASTRES
chain
CDR2
226 AT14-014 Light WASIRES
chain
CDR2
227 AT14-015 Light L
chain
CDR2
228 AT14-016 Light YDTDRPS
chain
CDR2
229 AT14-013 Light HQYYTTPQT
chain
CDR3
230 AT14-014 Light QQYSRPPT
chain
CDR3
231 AT14-015 Light SSYGGTVLF
chain
CDR3
232 AT14-016 Light QVWDNTSDHPVVF
chain
CDR3
233 AT14-013 Heavy QGRLQESGPGLVKPSETLTLTCAVSGGSSVSSPNWWTWVRQAPGKGLEWIGEIYYGGRVSYNSALRSRVTI
chain SSDRSKEEFSLKLRSVTAADTAIYYCAGQKNIGCGYSSCFISWFDTWGQGIAVTVSS
234 AT14-014 Heavy EVQLVESGGGLVKPGGSLRLSCAASGFTFSDAWMSWVRQAPGKGLEWVGHINTKVDGGTTEYAAPVKGR
chain FTISRDDSKNSLYLHMDSLKTEDTAVYYCTTEAIYDSSGYFHDYWGQGSLVTVSS
235 15 Heavy EVQLVESGGGLAQPGRSLRLSCTASGFRFGDFAMSWVRQAPGKGLEWVGFIRTKANDGTTEYAASVKGRF
chain IISRDDSKSIAYLQMNSLKTEDTAVYYCASDPFMTTDYYYYYMDVWGKGTTVTVSS
236 AT14-016 Heavy EVQVLESGGDSVQPGGSLRLSCAASGFTFSSYAMTWVRQAPGKGLKWVSSISGSGGSTYYADSVRGRFTISR
chain DNSKNTLYVQMNSLRAEDTAVYYCAKGYVGCSGGNCYSGGAFDIWGQGTVVTVSS
237 AT14-013 Light DIVMTQSPDSLAVSLGERATIACKSSQTILQRSNHLNYLAWYQQKPGQPPKVLIYWASTRESGVPDRFSGSG
chain SGTDFTLTINSLQAEDVAVYYCHQYYTTPQTFGQGTKVEIK
238 AT14-014 Light DIVMTQSPDSLAVSLGERATINCKSSRSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASIRESGVPDRFSGSG
chain SGTDFTLTINSLQAEDVAVYYCQQYSRPPTFGQGTKVEIK
239 AT14-015 Light PPSASGSPGQSVTISCTGTSSDVGGYNSVSWYQHHPGKAPKLMIYEVYKRPLGVPDRFSGSKSGN
chain SGLQAEDEAYYYCSSYGGTVLFGGGTKLTVL
240 AT14-016 Light SYVLTQPPSVSVAPGKTARITCGGNNIGSESVHWYQQKPGQAPVVVIYYDTDRPSGIPERFSGSNSGNTATL
TISRVEAGDEADYYCQVWDNTSDHPVVFGGGTKLTVL
chain
241 AT14-013 Heavy agt cct aac tgg tgg act
chain
CDR1
242 AT14-014 Heavy gac gcc tgg atg agc
chain
CDR1
243 AT14-015 Heavy gat ttt gct atg agt
chain
CDR1
244 AT14-016 Heavy agc tat gcc atg acc
chain
CDR1
245 AT14-013 Heavy gaa atc tat tat ggt ggg aga gtg agc tac aac tcg gcc ctc agg agt
chain
CDR2
246 AT14-014 Heavy cat att aac acc aaa gtt gat ggt ggg aca aca gag tac gct gca ccc gtg aaa ggc
chain
CDR2
247 AT14-015 Heavy ttc att aga acc aaa gct aat gat ggg aca aca gaa tac gcc gcg tct gtg aaa ggc
chain
CDR2
248 AT14-016 Heavy agt att agt ggt agt ggt ggt agc aca tac tac gca gac tcc gtg agg ggc
chain
CDR2
249 AT14-013 Heavy gcg ggt caa aaa aat att ggc tgt ggt tac agc agt tgc ttt atc agt tgg ttc gac acc
chain
CDR3
250 14 Heavy acc aca gag gcg ata tat gat agt agt ggt tat ttc cat gac tat
chain
CDR3
251 AT14-015 Heavy gct agc gat ccc ttc atg act aca gac tat tac tac tac tac atg gac gtc
chain
CDR3
252 AT14-016 Heavy gcg aaa gga tat gtg ggg tgt agt ggt ggg aac tgc tac tcg ggg ggt gct ttt gat atc
chain
CDR3
253 AT14-013 Light aag tcc agc cag act att tta caa agg tcc aac cat ttg aac tac tta gct
chain
CDR1
254 AT14-014 Light aag tcc agc cgg agt gtt tta tac agc tcc aac aat aag aac tac tta gct
chain
CDR1
255 AT14-015 Light act ggg acc agc agt gac gtt ggt ggt tat aac tct gtc tcc
chain
CDR1
256 AT14-016 Light ggg ggg aac aac att gga agt gaa agt gtt cac
chain
CDR1
257 AT14-013 Light tgg gca tct acc cgg gaa tcc
chain
CDR2
258 14 Light tgg gca tct atc cgg gaa tcc
chain
CDR2
259 AT14-015 Light gag gtc tat aag cgg ccc tta
chain
CDR2
260 AT14-016 Light tat gat acc gac cgg ccc tca
chain
CDR2
261 AT14-013 Light cac caa tat tat act act ccg cag act
chain
CDR3
262 AT14-014 Light cag caa tat tct cgt cct ccg acg
chain
CDR3
263 AT14-015 Light agc tca tat gga ggc acc gtg cta ttc
chain
CDR3
264 AT14-016 Light cag gtg tgg gat aac act agt gat cat cct gtg gta ttc
chain
CDR3
265 AT14-013 Heavy cag ggg cga ctg cag gag tcg ggc cca gga ctg gtg aag cct tcg gag acc ctg acc ctc acg tgc gct gtg tcc ggt ggc tcc tcc
chain gtc agc agt cct aac tgg tgg act tgg gtc cgc cag gcc ccc ggg aag ggg ctg gag tgg att gga gaa atc tat tat ggt ggg aga
gtg agc tac aac tcg gcc ctc agg agt cga gtc acc att tca tca gac agg tcc aaa gag gag ttc tcc ctg aaa ctg agg tct gtg
acc gcc gcg gac acg gcc ata tat tat tgt gcg ggt caa aaa aat att ggc tgt ggt tac agc agt tgc ttt atc agt tgg ttc gac
acc tgg gga cag gga att gcg gtc acc gtc tcc tca
266 AT14-014 Heavy gag gtg cag ctg gtg gag tct ggg gga ggt ttg gta aag cct ggg ggg tcc ctt aga ctc tcc tgt gca gcc tct gga ttc act ttc
chain agt gac gcc tgg atg agc tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt ggc cat att aac acc aaa gtt gat ggt ggg
aca aca gag tac gct gca ccc gtg aaa ggc aga ttc acc atc tca aga gat gat tca aaa aat tcg ctg tat ctg cac atg gac agc
ctg aaa acc gag gac aca gcc gtg tat tac tgt acc aca gag gcg ata tat gat agt agt ggt tat ttc cat gac tat tgg ggc cag
gga tcc ctg gtc acc gtc tcc tca
267 15 Heavy gag gtg cag ctg gtg gag tcg ggg gga ggc ttg gca cag cca ggg cgg tcc ctg aga ctc tcc tgt aca gct tct gga ttc agg ttt
chain ggt gat ttt gct atg agt tgg gtc cgc cag gct cca ggg aag gga ctg gag tgg gta ggt ttc att aga acc aaa gct aat gat ggg
aca aca gaa tac gcc gcg tct gtg aaa ggc aga ttc atc atc tca aga gat gat tcc aaa agt atc gcc tat ctg caa atg aac agc
ctg aaa acc gag gac aca gcc gtt tat tac tgt gct agc gat ccc ttc atg act aca gac tat tac tac tac tac atg gac gtc tgg
ggc aaa ggg acc acg gtc acc gtc tcc tca
268 AT14-016 Heavy gag gtg caa gtg ttg gag tct ggg gga gac tcg gta cag cct ggg ggg tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttt
chain agc agc tat gcc atg acc tgg gtc cgc cag gct cca ggg aag ggg ctg aaa tgg gtc tca agt att agt ggt agt ggt ggt agc aca
tac tac gca gac tcc gtg agg ggc cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat gtg cag atg aac agc ctg aga
gcc gag gac acg gcc gta tat tac tgt gcg aaa gga tat gtg ggg tgt agt ggt ggg aac tgc tac tcg ggg ggt gct ttt gat atc
tgg ggc caa ggg aca gtg gtc acc gtc tct tca
269 AT14-013 Light gac atc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg ggc gag agg gcc acc atc gcc tgc aag tcc agc cag act att
chain tta caa agg tcc aac cat ttg aac tac tta gct tgg tac cag cag aaa cca gga cag cct cct aaa gtg ctc att tat tgg gca tct
acc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc aac agc ctg cag gct gag
gat gtg gca gtt tat tac tgt cac caa tat tat act act ccg cag act ttt ggc cag ggg acc aag gtg gag atc aaa
270 AT14-014 Light gac atc gtg atg acc cag tct cca gac tcc ctg gct gtg tct ctg ggc gag agg gcc acc atc aac tgc aag tcc agc cgg agt gtt
chain tta tac agc tcc aac aat aag aac tac tta gct tgg tac cag cag aaa cca gga cag cct cct aag ctg ctc att tac tgg gca tct
atc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg aca gat ttc act ctc acc atc aac agc ctg cag gct gaa
gat gtg gca gtt tat tac tgt cag caa tat tct cgt cct ccg acg ttc ggc caa ggg acc aag gtg gaa atc aaa
271 15 Light cag tct gcc ctg act cag cct ccc tcc gcg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc act ggg acc agc agt gac gtt ggt
chain ggt tat aac tct gtc tcc tgg tac caa cat cac cca ggc aaa gcc ccc aaa ctc atg att tat gag gtc tat aag cgg ccc tta ggg
gtc cct gat cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc gtc tct ggg ctc cag gct gag gat gag gct tat tat tac
tgc agc tca tat gga ggc acc gtg cta ttc ggc gga ggg acc aag ctg acc gtc cta
272 AT14-016 Light tcc tat gtg ctg act cag cca ccc tca gtg tca gtg gcc cca gga aag acg gcc cgg att acc tgt ggg ggg aac aac att gga agt
chain gaa agt gtt cac tgg tac cag cag aag cca ggc cag gcc cct gtg gtg gtc atc tat tat gat acc gac cgg ccc tca ggg atc cct
gag cgc ttc tct ggc tcc aac tct ggg aac acg gcc acc ctg acc atc agc agg gtc gaa gcc ggg gat gag gcc gac tat tac tgt
cag gtg tgg gat aac act agt gat cat cct gtg gta ttc ggc gga ggg acc aag ctg acc gtc cta
Tabel 2
mAbare specificforAML
mAbs Cell lines Prim cells
Name Ig class Mel1 Mel2 Mel3 BJ FB Col1 Col2 Col3 Vero HUVEC HepG2 PBMC BM
19 IgG1 κ - - - - - - - - - - - - -
AT13-023 IgG1 κ - - - - - - - - - - - - -
31 IgG1 κ - - - - - -
AT12-020 IgG3 κ - - - - - - - - - - - - -
AT12-023 IgG3 λ - - - - - - - - - - - - -
AT12-025 IgG3 κ - - - - - - - - - - - - -
AT13-022 IgG3 λ - - - - - - - - - - - - -
AT13-024 IgG3 κ - - - - - - - - - - - - -
Mel1: Mel 126.2; Mel2: Mel BLM; Mel3: Mel WBO; BJ: Fibroblast cell line; FB:
primary fibroblasts (skin); Col1: Colo205; Col2: Caco-2; Col3 HT29; Vero: nonhuman
kidney; PBMC: peripheral blood mononuclear cells; BM: bone marrow.
Tabel 3
ew AML-specific mAbsderived from a patient
with a potent GvLresponse (donor 59)
mAbs AML Primary AML*
Name Ig class CD27 SHM VH/VL THP-1 MM6 M0 M1 M1 M1 M4
AT12-019 IgG1 κ + 10/ 9 ++ + + + ++ +/- -
AT13-023 IgG1 κ + 8/4 ++ ++ +
AT13-031 IgG1 κ + 1/1 ++ + - + - - +/-
AT12-020 IgG3 κ + 4/2 ++
AT12-023 IgG3 λ - Germline/6 +++ ++ + ++ ++ +/- ++
IgG3 κ - 1/1 ++ ++ - +/- + - -
AT13-022 IgG3 λ + Germline ++ ++ ++
AT13-024 IgG3 κ - 6/3 ++ ++ ++ ++ ++
MM6: MonoMac6; M0: donor 77; M1 donor 69, 79, 86 respectively; M4 donor 78
* AML according to the FAB classification; SHM: number of somatic hypermutations
Tabel 4
SomemAbbind otherhematologictumors
mAbs Other hematologic tumor cell lines Primary tumors
Name Ig class 1 OCI-Ly7 U266 NCI-H929 NHL pt ALL pt
AT12-019 IgG1 κ + - - - - -
AT13-023 IgG1 κ
AT13-031 IgG1 κ ++ + - +/- ++ -
AT12-020 IgG3 κ
AT12-023 IgG3 λ - - +/- - ++ -
AT12-025 IgG3 κ - - + - - -
22 IgG3 λ
AT13-024 IgG3 κ ++ + - - - -
1 and OCI-Ly7: diffuse large B cell lymphoma cell lines; U266 and NCI-H929: multiple
myeloma cell lines; NHL pt: B-non Hodgkin lymphoma cells freshly isolated from a newly
diagnosed patient ; ALL pt: B-acute lymphatic leukemia cells freshly isolated from a newly
diagnosed patient.
Tabel 5
SomemAbshow in vitro activity
mAbs Killing
Name Ig class CD27 SHM VH/VL THP-1 M0/5
AT12-019 IgG1 κ + 10/ 9 no no
AT13-023 IgG1 κ + 8/4
AT13-031 IgG1 κ + 1/1
AT12-020 IgG3 κ + 4/2
AT12-023 IgG3 λ - Germline/6 yes
AT12-025 IgG3 κ - 1/1 yes yes
AT13-022 IgG3 λ + Germline
AT13-024 IgG3 κ - 6/3 no yes
23, AT13-031, AT12-020 and AT13-022 were not yet tested for in vitro activity
Tabel 6
2nd AML patientwithGvLresponse (donor 58)
mAbs AML Not binding to
Clone CD27 Ig class SHM VH/VL THP-1 MM6 PBMC Caco HT-29 HepG2
AT13-033 + IgG3 λ 18/5 ++ ++ - - - -
AT13-034 + IgG3 κ 14/6 ++ ++ - - - -
AT13-035 - IgG3 λ 0/0 ++ ++ - - - IgG3 λ 4/9 ++ ++ - - - -
AT13-037 + IgG3 κ 4/8 ++ ++ - - - -
SHM: number of somatic hypermutations
135135
Table 7
Overview of AM L-specific mAbs d from a 3rd
patient with a potent GvL response (donor 101)
AT14-013 +++ +++
AT14-014 ++ -
AT14-015
AT14-016 ++ -
M1: donor 77; M4: donor BL-046; M51: donor BL-034, M52: donor BL-038
* AML according to the FAB classification; SHM: number of somatic hypermutations
136136
Table 8
mAb are specific for AML (donor 101)
“s-l—C-—ellli-nes-—BMC5 P__imarycells
AT14-014
AT14-015
FB: primary fibroblasts (skin); Col: Caco-Z; Liv1: Huh7 liver cell line; Livz: HepGZ liver cell
line PBMC: peripheral blood mononuclear cells;
137137
Tabel 9A
Overview of AM L-specific mAbs derived from a 2'“
patient with a potentAGvL response (donor 58)
mAbs Primary AML*
AT13-033
AT13-034
AT13-036
AT13-037
MM6: MonoMac6; M0: donor 77, BL-030; M1 donor 69, respectively; M4 donor 78
* AML according to the FAB classification; SHM: number of somatic hypermutations
138138
Tabel 98
Some mAb show in vitro activity (donor 59)
THP-l
AT12-019 10/ 9
AT13-023 8/4
AT13-031 1/1
AT12-020 4/2
AT12-023 Germline/6
AT12-025 1/1
AT13-022 ne
AT13-024 6/3
* Only at 37 C; nd
= not determined
139139
Tabel 10
Some mAb show in vitro activity (donor 58)
AT13-O33
AT13-034
AT13-036
AT13-037
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The following numbered paragraphs define particular aspects of the present
invention:
1. An isolated, synthetic or recombinant human antibody, or a functional
part or a functional equivalent f, which is able to bind a cell surface
component of acute myeloid leukemia (AML) cells and which is able to diminish
proliferation of AML cells essentially independently from antibody-dependent cellmediated
cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC),
apoptosis, or phagocytosis by macrophages or dendritic cells.
2. An antibody or functional part or functional equivalent ing to
paragraph 1, which is able to bind a cell e ent that is specific for AML
cells.
3. An antibody or functional part or functional lent according to
paragraph 1 or 2, which is able to induce death of primary AML blasts essentially
independently from antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), apoptosis, or phagocytosis by
hages or dendritic cells.
4. An antibody or functional part or functional lent according to any
one of paragraphs 1-3, which is able to diminish proliferation of AML cells in vitro
within 3 days.
. An antibody or onal part or functional equivalent according to any
one of paragraphs 1-4, wherein said AML cells belong to a French-American-
British (FAB) classification selected from the group consisting of M5, M0, M1, M2,
M3 and M4.
6. An antibody or functional part or functional equivalent according to any
one of paragraphs 1-5, wherein said AML cells belong to the FAB classification M5
or M1 or M0 or M4, preferably M5.
7. An antibody or functional part or onal equivalent according to any
one of paragraphs 1-6, which is able to bind a cell surface ent of different
AML cells of at least two, preferably at least three, more preferably at least four
FAB classifications.
8. An antibody or functional part or functional equivalent according to any
one of paragraphs 1-7, which is able to bind snRNP200.
9. An antibody or functional part or functional equivalent according to any
one of paragraphs 1-8, wherein said antibody is of the IgG isotype, ably IgG1
or IgG3.
. An isolated, synthetic or recombinant antibody, or a functional part or a
onal equivalent thereof, which comprises at least a heavy chain CDR3
sequence having at least 80% sequence identity with a sequence selected from the
group consisting of SEQ ID NOs: 27-39, and a light chain CDR3 sequence having at
least 80% sequence ty with a sequence selected from the group consisting of
SEQ ID NOs: 66-78.
11. An isolated, synthetic or recombinant antibody, or a functional part or a
onal equivalent f, which ses:
- a heavy chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 1-13; and
- a heavy chain CDR2 sequence comprising a sequence which has at least 80%
sequence ty with a sequence selected from the group consisting of SEQ ID
NOs: 14-26; and
- a heavy chain CDR3 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 27-39; and
- a light chain CDR1 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 40-52; and
- a light chain CDR2 sequence comprising a sequence which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 53-65; and
- a light chain CDR3 sequence sing a ce which has at least 80%
sequence identity with a sequence selected from the group consisting of SEQ ID
NOs: 66-78.
12. An antibody or functional part or functional equivalent according to
paragraph 10 or 11, comprising a variable heavy chain sequence having at least
80% sequence identity with a sequence selected from the group consisting of SEQ
ID NOs: 79-91.
13. An antibody or functional part or functional equivalent ing to any
one of paragraphs 10-12, comprising a variable light chain sequence having at least
80% sequence identity with a sequence selected from the group consisting of SEQ
ID NOs: 92-104.
14. An antibody or functional part or functional equivalent according to any
one of aphs 1-13, which is coupled to another compound.
. An antibody or functional part or functional lent ing to
paragraph 14, wherein said other compound is a detectable label, a
chemotherapeutic drug, a toxic moiety, an immunomodulatory molecule, another
AML-specific binding compound, or a radioactive compound.
16. An ed, synthetic or recombinant nucleic acid molecule with a length
of at least 15 nucleotides, or a functional equivalent thereof, encoding at least one
CDR sequence of an dy or functional part or functional equivalent according
to any one of paragraphs 1-15.
17. A nucleic acid molecule or functional equivalent according to paragraph
16, which encodes at least the variable heavy chain sequence and/or the le
light chain ce of an antibody or functional part or functional equivalent
according to any one of paragraphs 1-15
18. A nucleic acid molecule or functional equivalent according to paragraph
16 or 17, comprising a sequence which has at least 80% sequence identity with a
sequence selected from the group consisting of SEQ ID NOs: 2
19. A nucleic acid molecule or functional equivalent according to any one of
paragraphs 16-18, comprising a sequence which has at least 80% sequence identity
with a sequence selected from the group consisting of SEQ ID NOs: 183-195 and/or
comprising a sequence which has at least 80% sequence identity with a ce
selected from the group consisting of SEQ ID NOs: 196-208.
. A c acid molecule or functional equivalent thereof, encoding an
antibody or functional part or functional equivalent ing to any one of
paragraphs 1-15.
21. A nucleic acid molecule according to any one of paragraphs 16-20, which
comprises cDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), or a
DNA/RNA helix.
22. A vector comprising a nucleic acid molecule or functional equivalent
according to any one of aphs 16-21.
23. An isolated or recombinant cell, or a non-human , comprising a
nucleic acid molecule or functional equivalent according to any one of paragraphs
16-21 or a vector according to paragraph 22.
24. A composition comprising an antibody or functional part or functional
equivalent according to any one of paragraphs 1-15, or a nucleic acid molecule or
functional lent according to any one of paragraphs 16-21, or a vector
according to aph 22, or a cell ing to paragraph 23.
. A composition according to paragraph 24, wherein said composition is a
pharmaceutical composition which comprises a ceutically acceptable
carrier, diluent or excipient.
26. A composition according to paragraph 25, which ses at least two
antibodies, functional parts or functional equivalents according to any one of
aphs 1-15.
27. An antibody or functional part or onal equivalent according to any
one of paragraphs 1-15, or a nucleic acid le or functional equivalent
according to any one of paragraphs 16-21, or a vector according to paragraph 22, or
a cell according to paragraph 23, for use as a medicament or prophylactic agent.
28. An antibody or functional part or functional equivalent according to any
one of paragraphs 1-15, or a c acid molecule or functional equivalent
according to any one of paragraphs 16-21, or a vector according to paragraph 22, or
a cell according to paragraph 23, for use in a method for at least in part treating or
preventing a myeloproliferative or lymphoproliferative disorder.
29. An antibody or functional part or functional equivalent according to any
one of paragraphs 1-15, or a nucleic acid molecule or functional equivalent
ing to any one of paragraphs 16-21, or a vector according to paragraph 22, or
a cell according to paragraph 23, for use in diagnosis of a myeloproliferative or
lymphoproliferative disorder.
. An antibody or onal part or functional equivalent or nucleic acid
molecule or functional equivalent or vector or cell for use according to paragraph 28
or 29, n said myeloproliferative disorder is acute myeloid leukemia (AML).
31. An antibody or functional part or functional equivalent or nucleic acid
molecule or functional equivalent or vector or cell for use ing to paragraph 28
or 29, wherein said proliferative disorder is lymphoma, B- non-Hodgkin
lymphoma or multiple myeloma.
32. Use of an antibody or functional part or onal equivalent according
to any one of paragraphs 1-15, or a nucleic acid molecule or functional equivalent
according to any one of paragraphs 16-21, or a vector according to paragraph 22, or
a cell according to paragraph 23, for determining whether a sample comprises
myeloproliferative or lymphoproliferative cells.
33. A method for producing an antibody or functional part or functional
equivalent according to any one of paragraphs 1-15, the method comprising
providing a cell with a nucleic acid molecule or functional equivalent or a vector
according to any one of paragraphs 16-22, and allowing said cell to translate said
nucleic acid molecule or functional equivalent or , thereby producing said
antibody or functional part or functional equivalent according to any one of
aphs 1-15, the method preferably further comprising harvesting, purifying
and/or isolating said antibody or onal part or functional lent according
to any one of paragraphs 1-15.
34. A method for ing roliferative or lymphoproliferative cells
using an antibody or functional part or functional equivalent according to any one
of paragraphs 1-15.
. A method for determining whether a myeloid cell or lymphoid cell is a
myeloproliferative cell or proliferative cell, the method comprising
determining whether snRNP200 is present on the surface of said cell, wherein the
presence of snRNP200 on the surface of said cell indicates that said cell is
myeloproliferative or proliferative.
36. A method for identifying myeloproliferative or lymphoproliferative cells,
comprising detecting the presence of snRNP200 on the e of said cells.
37. A method for determining r myeloproliferative or
lymphoproliferative cells are present in a sample comprising:
- contacting said sample with an antibody or functional part or functional
equivalent according to any one of paragraphs 1-15, and
- allowing said antibody or functional part or functional equivalent to bind
myeloproliferative cells or lymphoproliferative cells, if present, and
- determining whether or not myeloproliferative cells or lymphoproliferative cells
are bound to said antibody or functional part or functional equivalent, thereby
determining whether or not myeloproliferative cells or lymphoproliferative cells are
present in said sample.
38. A use according to paragraph 32 or a method according to any one of
aphs 33-37, wherein said myeloproliferative cells are AML cells.
39. A use according to paragraph 32 or a method according to any one of
paragraphs 33-37, wherein said proliferative cells are lymphoma, B- non-
Hodgkin lymphoma or le myeloma cells.
40. A method for at least in part treating and/or preventing a
myeloproliferative or lymphoproliferative disorder, comprising administering to an
individual in need thereof a therapeutically effective amount of an antibody or
functional part or functional equivalent according to any one of paragraphs 1-15, or
a nucleic acid molecule or functional equivalent according to any one of paragraphs
16-21, or a vector according to paragraph 22, or a cell according to paragraph 23, or
a composition ing to any one of paragraphs 24-26.
41. A method for determining whether an individual is ing from a
roliferative or lymphoproliferative er, comprising:
- contacting a sample from said individual with an antibody or functional part or
functional equivalent ing to any one of paragraphs 1-15, and
- allowing said antibody or functional part or functional lent to bind
myeloproliferative cells or lymphoproliferative cells, if present, and
- determining whether or not myeloproliferative cells or lymphoproliferative cells
are bound to said antibody or functional part or functional equivalent, thereby
determining whether or nor said individual is suffering from a myeloproliferative
disorder or a proliferative disorder.
42. A method for ining whether an individual is suffering from a
myeloproliferative or proliferative disorder, comprising determining
whether a sample from said individual comprises antibodies that are ic for
snRNP200.
43. A method according to paragraph 42, the method comprising:
- contacting a sample from said individual with snRNP200 or an epitope thereof;
- allowing said snRNP200 or epitope to bind snRNP200-specific antibodies from
said sample, if present, and
- determining whether or not said snRNP200 or epitope is bound to snRNP200-
specific antibodies, wherein binding of said snRNP200 or epitope to 00-
specific antibodies indicates that said individual is suffering from a
myeloproliferative or lymphoproliferative disorder.
44. A method for typing a myeloid cell-containing sample or a id cellcontaining
sample of an individual, the method comprising determining whether
snRNP200 is present on the surface of cells in said sample.
45. A method for determining whether a patient ing from a
myeloproliferative or lymphoproliferative disorder has an improved chance of a
positive outcome of treatment with an antibody, functional part or onal
equivalent according to any one of paragraphs 1-15, as compared to the mean
population of patients suffering from a myeloproliferative or lymphoproliferative
disorder, the method comprising determining whether snRNP200 is present on the
surface of myeloproliferative cells or lymphoproliferative cells of said patient.
46. A method according to paragraph 45, the method comprising:
- contacting a myeloproliferative cell- or lymphoproliferative cell-containing sample
from said dual with an dy or functional part or functional equivalent
that is specific for snRNP200;
- allowing said antibody or onal part or onal equivalent to bind
myeloproliferative cells or lymphoproliferative cells of said sample, and
- determining whether or not said snRNP200-specific dy or functional part or
functional equivalent is bound to myeloproliferative cells or lymphoproliferative
cells of said sample, wherein binding of said snRNP200-specific antibody or
onal part or functional equivalent to myeloproliferative cells or
lymphoproliferative cells of said sample indicates that said patient has an
improved chance of a positive e of treatment with an antibody, functional
part or functional equivalent according to any one of paragraphs 1-15, as compared
to the mean population of patients suffering from a myeloproliferative or
lymphoproliferative disorder.
47. A method according to any one of paragraphs 40-46, wherein said
roliferative disorder is AML and wherein said roliferative cells are
AML cells.
48. A method according to any one of paragraphs 40-46, wherein said
lymphoproliferative disorder is lymphoma, B- non-Hodgkin ma or multiple
myeloma and wherein said lymphoproliferative cells are lymphoma, B- non-
Hodgkin lymphoma or multiple myeloma cells.
49. An antibody according to any one of paragraphs 1-15, or an antibody for
use according to any one of paragraphs 27-31, or a use according to 32, or a method
according to any one of paragraphs 33-48, n said antibody is selected from
the group consisting of AT12-023, AT13-031, AT13-037, AT13-024, AT12-025 and
AT12-019, and functional parts and functional equivalents thereof.
50. A method for ining whether a patient suffering from a
myeloproliferative disorder or lymphoproliferative disorder is a candidate for
treatment with antibody AT12-023, AT13-031 and/or AT13-037, or a functional
part or a functional equivalent thereof, the method comprising ining
whether snRNP200 is present on the surface of roliferative cells or
proliferative cells of said patient.
51. Use of an antibody or functional part or functional equivalent according
to any one of paragraphs 1-15 for determining whether an AML patient has a Graft
versus Leukemia response.
Claims (41)
1. An isolated, synthetic or recombinant human antibody, or a functional part or a functional equivalent thereof, which is able to bind acute myeloid leukemia (AML) cells and which comprises: - a heavy chain CDR1 sequence comprising the sequence of SEQ ID NO: 209; - a heavy chain CDR2 ce comprising the ce of SEQ ID NO: 213; - a heavy chain CDR3 sequence comprising the ce of SEQ ID NO: 217; - a light chain CDR1 sequence comprising the sequence of SEQ ID NO: 221; - a light chain CDR2 sequence comprising the sequence of SEQ ID NO: 225; and - a light chain CDR3 sequence comprising the ce of SEQ ID NO: 229.
2. An antibody or functional part or functional equivalent according to claim 1, wherein said AML cells belong to a French-American-British (FAB) classification selected from the group consisting of M5, M0, M1, M2, M3 and M4.
3. An antibody or functional part or functional equivalent according to any one of claims 1-2, wherein said AML cells belong to the FAB classification M5 or M1 or M0 or M4.
4. An antibody or functional part or functional equivalent ing to any one of claims 1-3, which is able to bind a cell surface component of different AML cells of at least two, or at least three, or at least four FAB classifications.
5. An antibody or functional part or functional equivalent according to any one of claims 1-4, wherein said dy is of the IgG isotype.
6. An antibody or functional part or functional equivalent according to any one of claims 1-5, wherein said antibody is of the IgG1 or IgG3 isotype.
7. An antibody or functional part or functional equivalent according to any one of claims 1-6, comprising a le heavy chain sequence having at least 80% sequence identity with the sequence of SEQ ID NO: 233.
8. An antibody or functional part or onal equivalent according to any one of claims 1-7, comprising a variable light chain sequence having at least 80% ce identity with the sequence of SEQ ID NO: 237.
9. An antibody or functional part or functional equivalent according to any one of claims 1-8, which is coupled to another compound.
10. An antibody or functional part or functional equivalent ing to claim 9, wherein said other compound is a detectable label, a herapeutic drug, a toxic moiety, an immunomodulatory le, another AML-specific binding compound, or a radioactive compound.
11. An isolated, synthetic or recombinant nucleic acid molecule with a length of at least 15 nucleotides, or a functional equivalent thereof, or a vector, encoding at least the six CDR sequences of an antibody or functional part or functional equivalent according to any one of claims 1-10.
12. A nucleic acid molecule or functional equivalent or vector according to claim 11, which encodes at least a variable heavy chain sequence and/or a variable light chain sequence of an antibody or functional part or functional equivalent according to any one of claims 1-10.
13. A nucleic acid molecule or functional equivalent or vector according to claim 11 or 12, sing a sequence which has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 241, 245, 249, 253, 257 and 261.
14. A c acid molecule or functional equivalent or vector according to any one of claims 11-13, comprising a ce which has at least 80% sequence identity with the sequence of SEQ ID NO: 265; and/or comprising a sequence which has at least 80% sequence identity with the sequence of SEQ ID NO: 269.
15. A nucleic acid molecule or functional equivalent thereof, or a , encoding an antibody or functional part or functional equivalent according to any one of claims 1-10.
16. A nucleic acid le or functional equivalent or vector according to any one of claims 11-15, which comprises cDNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), or a DNA/RNA helix.
17. An isolated or recombinant cell, or a non-human animal, comprising a nucleic acid le or functional lent or vector according to any one of claims 11-16, with the proviso that said cell is not present in a human being.
18. A composition comprising an antibody or functional part or functional equivalent according to any one of claims 1-10, or a nucleic acid molecule or onal equivalent or vector according to any one of claims 11-16, or a cell ing to claim 17.
19. A composition according to claim 18, wherein said composition is a pharmaceutical composition which comprises a pharmaceutically acceptable carrier, diluent or excipient.
20. A composition according to claim 19, which comprises at least two antibodies, functional parts or functional equivalents according to any one of claims 1-10.
21. Use of an antibody or functional part or functional equivalent ing to any one of claims 1-10, or a c acid molecule or functional equivalent or a vector according to any one of claims 11-16, or a cell according to claim 17, in the manufacture of a medicament or prophylactic agent.
22. Use of an antibody or functional part or functional equivalent according to any one of claims 1-10, or a nucleic acid molecule or functional lent or a vector according to any one of claims 11-16, or a cell according to claim 17, in the cture of a medicament or prophylactic agent for treating or preventing acute d leukemia (AML).
23. Use of an antibody or functional part or functional equivalent according to any one of claims 1-10, or a nucleic acid molecule or functional equivalent or a vector according to any one of claims 11-16, or a cell according to claim 17, in the cture of a diagnostic agent for acute myeloid leukemia (AML).
24. A method for producing an antibody or functional part or functional equivalent ing to any one of claims 1-10, the method comprising providing a cell with a nucleic acid molecule or functional equivalent or a vector according to any one of claims 11-16, and allowing said cell to ate said nucleic acid molecule or functional equivalent or vector, thereby producing said antibody or functional part or functional equivalent according to any one of claims 1-10, with the proviso that said method is not carried out in a human being.
25. A method according to claim 24, wherein the method further ses harvesting, purifying and/or isolating said antibody or functional part or functional equivalent according to any one of claims 1-10.
26. A method for determining r AML cells are present in a sample comprising: - contacting said sample with an antibody or onal part or functional equivalent according to any one of claims 1-10, and - allowing said antibody or onal part or functional equivalent to bind AML cells, if present, and - determining whether or not AML cells are bound to said antibody or functional part or functional equivalent, thereby determining whether or not AML cells are present in said sample.
27. A method for determining whether an AML patient has a Graft versus Leukemia response, comprising contacting a sample from said AML patient with an antibody or functional part or functional equivalent according to any one of claims 1-10, and allowing said dy or functional part or functional equivalent to bind AML cells, if present, and determining r or not AML cells are bound to said antibody or functional part or functional equivalent, thereby determining whether or nor said individual has a GvL response, whereby the absence of AML cells is indicative for a GvL response.
28. A bispecific or multispecific binding compound, comprising an antibody or a functional part or a functional equivalent according to any one of claims 1-8 and an immunomodulatory molecule.
29. A bispecific or multispecific binding compound according to claim 28, wherein said immunomodulatory molecule comprises a CD3-specific binding compound.
30. A synthetic or inant antibody, or a functional part or a functional equivalent thereof, which comprises one Fab fragment of an dy according to any one of claims 1-8, and one Fab fragment of another antibody.
31. A chimeric antigen receptor (CAR) T cell comprising an antibody or a functional part or a functional lent according to any one of claims 1-
32. An antibody or functional part or functional lent according to any one of claims 1-10 substantially as herein described with nce to any example thereof.
33. A nucleic acid molecule or functional equivalent or vector ing to any one of claims 11-16 substantially as herein described with nce to any example thereof.
34. A cell as d in claim 17 ntially as herein described with reference to any example f, with the proviso that said cell is not present in a human being.
35. A non-human animal as claimed in claim 17 substantially as herein described.
36. A composition as claimed in any one of claims 18-20 substantially as herein described with reference to any example thereof.
37. A use as claimed in any one of claims 21-23 substantially as herein described with reference to any example thereof.
38. A method as claimed in any one of claims 24-27 substantially as herein described with reference to any example thereof.
39. A bispecific or multispecific binding compound as claimed in claim 28 or 29 substantially as herein described.
40. A synthetic or recombinant antibody, or a onal part or a onal equivalent thereof as claimed in claim 30 substantially as herein described.
41. A chimeric antigen receptor (CAR) T cell as claimed in claim 31 substantially as herein described.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP13197882 | 2013-12-17 | ||
EP13197882.7 | 2013-12-17 | ||
NZ72215514 | 2014-12-17 |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ760009A true NZ760009A (en) | 2021-03-26 |
NZ760009B2 NZ760009B2 (en) | 2021-06-29 |
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