HUMANIZED ANTI CXCR4 ANTIBODIES FOR THE TREATMENT OF CANCER
The present invention relates to a novel antibody, in particular a humanized
monoclonal antibody, able to bind specifically to chemokine receptors (CXCR) as well
as the amino and nucleic acid sequences coding for such an antibody. From one aspect,
the invention relates to a novel antibody, derived compounds or functional fragments,
able to bind specifically to the CXCR4 and having strong anti-tumor activities. The
invention also comprises the use of such an antibody as a drug for the preventive and/or
therapeutic treatment of cancer, as well as in the procedures or kits related to cancer
diagnosis. Finally, the invention comprises compositions comprising such an antibody
in combination or conjugation with other anti-cancer compound(s), such as antibodies,
toxins, cytotoxic/cytostatic, and the use of same for the prevention and/or treatment of
certain cancers.
Chemokines are small, secreted peptides that control the migration of leukocytes
along a chemical gradient of ligand, known as chemokine gradient, especially during
immune reactions (Zlotnick A. et al., 2000). They are divided into two major
subfamilies, CC and CXC, based on the position of their NH2 -terminal cysteine
residues, and bind to G protein coupled receptors, whose two major sub families are
designated CCR and CXCR. More than 50 human chemokines and 18 chemokine
receptors have been discovered so far.
Many cancers have a complex chemokine network that influences the immunecell
infiltration of tumor, as well as tumor cell growth, survival, migration and
angiogenesis. Immune cells, endothelial cells and tumor cells themselves express
chemokine receptors and can respond to chemokine gradients. Studies of human cancer
biopsy samples and mouse cancer models show that cancer cell chemokine-receptor
expression is associated with increase metastatic capacity. Malignant cells from
different cancer types have different profiles of chemokine-receptor expression, but
Chemokine receptor 4 (CXCR4) is most commonly found. Cells from at least 23
different types of human cancers of epithelial, mesenchymal and haematopoietic origin
express CXCR4 receptor (Balkwill F. et al., 2004).
Chemokine receptor 4 (also known as fusin, CD184, LESTR or HUMSTR)
exists as two isoforms comprising 352 or 360 amino acids. Residue Asnl l is
glycosylated, residue Tyr is modified by the addition of a sulfate group and Cys 109
and 186 are bond with a disulfide bridge on the extracellular part of the receptor (Juarez
J . et al., 2004).
This receptor is expressed by different kind of normal tissues, na'ive, nonmemory
T-cells, regulatory T cells, B-cells, neutrophils, endothelial cells, primary
monocytes, dendritic cells, Natural Killer cells, CD34+ hematopoietic stem cells and at
a low level in heart, colon, liver, kidneys and brain. CXCR4 plays a key role in
leukocytes trafficking, B cell lymphopoiesis and myelopoiesis.
CXCR4 receptor is over-expressed in a large number of cancers including but
not limited to colon (Ottaiano A. et al., 2004), breast (Kato M. et al., 2003), prostate
(Sun Y.X. et al., 2003), lungs [small-cell- and non-small-cell- carcinoma (Phillips R.J.
et al., 2003)], ovary (Scotton C.J. et al., 2002), pancreas (Koshiba T. et al., 2000),
kidneys, brain (Barbero S et al., 2002), glioblastoma and lymphomas.
The unique ligand of CXCR4 receptor described so far is the Stromal-cell-
Derived Factor- (SDF-1) or CXCL12. SDF-1 is secreted in large amount in lymph
nodes, bone marrow, liver, lungs and to a less extent by kidneys, brain and skin.
CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage
inflammatory protein II (vMIP-II) encoded by human herpesvirus type III.
CXCR4/SDF-1 axis plays a key role in cancer and is implicated directly in
migration, invasion leading to metastases. Indeed, cancer cells express CXCR4
receptor, they migrate and enter the systemic circulation. Then cancer cells are arrested
in vascular beds in organs that produce high levels of SDF-1 where they proliferate,
induce angiogenesis and form metastatic tumors (Murphy PM., 2001). This axis is also
involved in cell proliferation via activation of Extracellular-signal-Regulated Kinase
(ERK) pathway (Barbero S. et al., 2003) and angiogenesis (Romagnani P., 2004).
Indeed, CXCR4 receptor and its ligand SDF-1 clearly promote angiogenesis by
stimulating VEGF-A expression which in turns increases expression of CXCR4/SDF-1
(Bachelder R.E. et al., 2002). It is also known that tumor associated macrophages
(TAM) accumulated in hypoxic areas of tumors and are stimulated to co-operate with
tumor cells and promote angiogenesis. It was observed that hypoxia up-regulated
selectively expression of CXCR4 in various cell types including TAM (Mantovani A. et
al., 2004). It has been recently demonstrated that CXCR4/SDF-1 axis regulates the
trafficking/homing of CXCR4+ hematopoietic stem/progenitor cells (HSC) and could
play a role in neovascularization. Evidence indicates that besides HSC, functional
CXCR4 is also expressed on stem cells from other tissues (tissue-committed stem cells
= TCSCs) so SDF-1 may play a pivotal role in chemottracting CXCR4+ TCSCs
necessary for organ/tissue regeneration but these TCSC may also be a cellular origin of
cancer development (cancer stem cells theory). A stem cell origin of cancer was
demonstrated for human leukemia and recently for several solid tumors such as brain
and breast. There are several examples of CXCR4+ tumors that may derive from the
normal CXCR4+ tissue/organ-specific stem cells such as leukemias, brain tumors, small
cell lung cancer, breast cancer, hepatoblastoma, ovarian and cervical cancers (Kucia M.
et al., 2005).
Targeting cancer metastases by interfering with CXCR4 receptor was
demonstrated in vivo using a monoclonal antibody directed against CXCR4 receptor
(Muller A. et al., 2001). Briefly, it was shown that a monoclonal antibody directed
against CXCR4 receptor (Mab 173 R&D Systems) decreased significantly the number
of lymph node metastases in an orthotopic breast cancer model (MDA-MB231) in SCID
mice. Another study (Phillips R.J et al, 2003) also showed the critical role of SDF-
1/CXCR4 axis in metastases in an orthotopic lung carcinoma model (A549) using
polyclonal antibodies against SDF-1 but in this study there was no effect neither on
tumor growth nor on angiogenesis. Several other studies described also the inhibition of
either metastasis in vivo using siRNAs duplexes of CXCR4 (Liang Z. et al., 2005)
biostable CXCR4 peptide antagonists (Tamamura H. et al., 2003) or tumor growth in
vivo using small molecule antagonist of CXCR4 like AMD 3100 (Rubin J.B. et al,
2003; De Falco V. et al, 2007) or Mab (patent WO2004/059285 A2). Thus, CXCR4 is
a validated therapeutic target for cancers.
Chemokine receptor 2 (CXCR2), another chemokine receptor is also described
as an interesting target in oncology. Indeed, CXCR2 transmits an autocrine cell growth
signal in several tumor cell types and can also affect tumor growth indirectly by
promoting angiogenesis (Tanaka T. et al. 2005).
CXCR2 chemokine receptor encompasses 360 amino acids. It is expressed
mainly in endothelial cells and specialy during neovascularization. Several chemokines
bind CXCR2 receptor: CXCL5, -6, -7, IL-8, GRO-a, -b and g which belong to ERL+
pro-angiogenic chemokines. The CXCR2 receptor share sequence homologies with
CXCR4 receptor: 37% sequence identity and 48% sequence homology. The
CXCR2/ligands axis is involved in several tumor growth mechanisms such
as metastasis (Singh RK. et al, 1994) cell proliferation (Owen J.D. et al., 1997) and in
ERL+ chemokines-mediated angiogenesis (Strieter R.M. et al., 2004; Romagnani et al.,
2004). Finally, tumor-associated macrophages and neutrophils are key elements of
inflammatory-induced tumor growth and chemokines such as CXCL5, IL-8 and GRO-a
initiate neutrophils recruitment.
Dimerization has emerged as a common mechanism for regulating the function
of G-protein-coupled receptors, among these are chemokine receptors (Wang J . and
Norcross M., 2008). Homo- and heterodimerization in response to chemokine binding
has been shown to be required for the initiation and the alteration of signaling by a
number of chemokine receptors. Growing evidence supports the concept that receptor
dimers or oligomers are probably the basic functional unit of chemokine receptors.
Chemokine receptor dimers are found in the absence of ligands and chemokines induce
conformational changes of receptor dimers. CXCR4 is known to form homodimers but
also heterodimers, for examples with the d-opioid receptor (DOR) (Hereld D., 2008) or
CCR2 (Percherancier Y. et al., 2005). In the latter example, peptides derived from the
transmembrane domains of CXCR4 inhibited activation by blocking the ligand-induced
conformational transitions of the dimer (Percherancier Y. et al., 2005). Another study
showed that CXCR4-TM4 peptide, a synthetic peptide of the transmembrane region of
CXCR4, decreases energy transfer between protomers of CXCR4 homodimers and
inhibits SDF-1-induced migration and actin polymerization in malignant cells (Wang J .
et al., 2006). More recently, it was also described that CXCR7 formed functional
heterodimers with CXCR4 and enhanced SDF-1 -induced signaling (Sierro F. et al.,
2007). Other examples of constitutive heterodimers include studies showing CXCR1
and CXCR2 interact as well as forming respective homodimers. No interactions were
noted for either of them with another GPCR (alpha(l A)-adrenoreceptor), indicating the
specificity of CXCR1 and CXCR2 interaction (Wilson S. et al., 2005).
As previously mentioned, CXCR4 and CXCR2 receptors are interesting tumor
targets. Interfering with those receptors should inhibit tumor growth and metastases in a
very efficient way, by decreasing tumor cell proliferation, angiogenesis, tumor cell
migration and invasion, neutrophils and macrophages recruitment by tumors and by
inhibiting CXCR4 cancer stem cells.
One of the inventive aspects of the present invention is to generate a humanized
monoclonal antibody inducing CXCR4 dimers conformational changes. The invention
encompasses the CXCR4 Mab hz515H7 (or fragments thereof) able to bind and to
induce conformational changes of both CXCR4 homodimers and CXCR4/CXCR2
heterodimers, and having strong anti-tumor activities. Hz515H7 induces conformational
changes on CXCR4 homodimers but also on CXCR4/CXCR2 heterodimers. This new
property should be of interest for cancer therapy application given the important roles of
these two chemokine receptors in cancer.
Targeting both homo- and hetero-dimers of receptors has already been found to
increase Mab therapeutic effect. Indeed, it has been demonstrated for example, that a
Mab (h7C10) targeting both IGF-1R and insulin/IGF- 1 hybrid receptors was more
potent to inhibit tumor growth in vivo than a Mab targeting solely IGF-1R (Pandini G.,
2007).
Moreover the anti-CXCR4 Mab hz515H7 is a silent antagonists for CXCR4, it
does not change basal signal in in vitro assays but inhibits signaling induced by SDF-1
in different assays (GTPyS binding, Ca release) and is also able to inhibit SDF-1
induced tumor cells migration in vitro.
Molecules acting as either partial agonists or inverse agonists exhibit intrinsic
activity in the absence of ligands. These types of molecules stabilize, respectively a
high-affinity or a low-affinity GPCR state, even in the absence of ligand, thereby
activating or inhibiting downstream signaling cascades (Galandin et al., 2007; Kenakin,
2004).
In case of the hz515H7 Mab, it behaves as a silent antagonist, without any
intrinsic activity at CXCR4 receptor in the absence of SDF-1. This pharmacological
feature is likely to be associated with less adverse side-effects as compared to partial or
inverse agonists, as already observed for opioid receptor ligands (Bosier and Hermans,
2007). Indeed, the functional activity of hz515H7 Mab is totally dependent on the
presence of SDF-1 and no modulation of CXCR4 receptor activity will be observed in
tissues and organs where SDF-1 ligand is not expressed, secreted or provided by the
blood flow. Thus, hz515H7 Mab is likely to be less toxic as compared to other CXCR4
receptor ligands with positive or negative efficacy. In addition, silent antagonists are the
minority species in the pharmacological space (Wurch et al, 99, Kenakin, 2004).
Surprisingly, for the first time, inventors have managed to generate a humanized
antibody capable of binding to CXCR4 but also capable of inducing conformational
changes of the CXCR4 homodimers and/or heterodimers. More particularly, the
humanized antibody of the invention is capable of inducing conformational changes of
the CXCR4 homodimers but also of the CXCR4/CXCR2 heterodimers.
In the following specification, the plural expression "CXCR4 dimers" must be
understood as encompassing the CXCR4 homodimers and also the CXCR4/CXCR2
heterodimers.
It must be mentioned at this stage that such humanized antibodies have never
been described in the prior art. Moreover, it must be mentioned that the existence of
CXCR4/CXCR2 heterodimers was never described.
A part of the invention is the discovery of the existence of a heterodimer formed
by CXCR4 and CXCR2.
So, in a particular aspect, the present invention discloses an isolated complex
comprising or consisting of the CXCR4/CXCR2 heterodimer.
CXCR4 compound part of said CXCR4/CXCR2 heterodimer complex is one of
the two human CXCR4 isoforms selected from the group consisting of:
- the chemokine (C-X-C motif) receptor 4 isoform b [Homo sapiens] having the
sequence as depicted under the Genbank accession number NP 003458 (SEQ ID
No. 1);
- the chemokine (C-X-C motif) receptor 4 isoform a [Homo sapiens] having the
sequence as depicted under the Genbank accession number NP_001008540 SEQ ID
No. 2);
- an alternate transcriptional splice variant or a natural variant thereof having at least
95 % identity with one of these b or a isoforms having the SEQ ID No. 1 or 2; and
- a fragment thereof capable of being specifically recognizing by its natural ligand
stromal cell-derived factor-1 (SDF-1) and having preferably at least 100, 150 and 200
amino acid length.
CXCR2 compound part of said CXCR4/CXCR2 heterodimer complex is
selected from the group consisting of:
- the interleukin 8 receptor beta [Homo sapiens] having the sequence as depicted under
the Genbank accession number NP 001548 (SEQ ID No. 3);
- an alternate transcriptional splice variant or a natural variant thereof having at least
95 % identity with this interleukin 8 receptor beta having the SEQ ID No. 3; and
- a fragment thereof capable of being specifically recognizing by IL-8 and having
preferably at least 100, 150 and 200 amino acid length.
In this particular aspect, the present invention also discloses an isolated RNA or
DNA encoding for a polypeptide comprising said CXCR4/CXCR2 heterodimer
complex.
This invention further discloses a nucleic construct, preferably an expression
vector, such as a plasmid, encoding said CXCR4/CXCR2 heterodimer complex.
The invention further comprises a composition comprising at least one nucleic
construct, preferably an expression vector, such as a plasmid, encoding the part CXCR4
of said CXCR4/CXCR2 heterodimer complex, and a second construct, preferably an
expression vector, such as a plasmid, encoding the part CXCR2 of said CXCR4/CXCR2
heterodimer complex.
In this aspect, the invention further discloses a method for the preparation of a
recombinant host cell which expresses said CXCR4/CXCR2 heterodimer complex,
wherein this method comprises a step of transforming said host cell:
a) with a nucleic construct, preferably an expression vector, such as a plasmid, encoding
said CXCR4/CXCR2 heterodimer complex; or
b) with at least one nucleic construct, preferably an expression vector, such as a
plasmid, encoding the part CXCR4 of said CXCR4/CXCR2 heterodimer complex, and a
second construct, preferably an expression vector, such as a plasmid, encoding the part
CXCR2 of said CXCR4/CXCR2 heterodimer complex.
Said host cell is an eukaryotic cell, such as a mammalian cell.
The nucleic construct(s) encoding said CXCR4/CXCR2 heterodimer complex
encode also for a first marker which is associated (particularly by covalent coupling)
with the CXCR4 sequence, such as the luc marker, and for a second marker which is
associated (particularly by covalent coupling) with the CXCR2 sequence, such as the
GFP marker (i.e. for BRET analysis).
The invention also discloses a method for selecting a compound having an anti¬
cancer activity or which can be used for the preparation of a composition for the
treatment of cancer, characterized in that said method comprises the step of:
a) contacting a recombinant host cell of the present invention which expresses said
CXCR4/CXCR2 heterodimer complex, with the compound to be tested; and
b) determining whether this compound is capable of modulating, preferably inhibiting,
the activity of this CXCR4/CXCR2 heterodimer complex in the recombinant host cell.
In a first aspect, a subject of the present invention is a process for the generation
and the selection of humanized antibodies according to the invention.
In a first aspect the invention concerns a process for the selection of an anti
CXCR4 humanized antibody, or one of its functional fragments or derivatives, capable
to inhibit both ligand-dependent and ligand-independent activation of CXCR4, said
process comprising the following steps:
i) screening the generated humanized antibodies and selecting antibodies
capable to bind specifically to CXCR4 and also to modulate CXCR4 activation;
ii) testing the selected antibodies of step i) and selecting antibodies capable to
induce CXCR4 homodimers conformational change, and then
iii) testing the selected antibodies of step ii) and selecting antibodies capable to
induce CXCR4/CXCR2 heterodimers conformational change.
By the expression "to modulate", it must be understood an increase or an
inhibition. Preferably, the selected antibodies of the invention must inhibit the CXCR4
activation.
As it was explained before, the induction of CXCR4 dimers conformational
changes is a capital aspect of the invention as such antibodies will present a real interest
for a larger population of patients.
The generation of the antibody can be realized by any method known by the
man skilled in the art, such as for example, from recombinant humanized antibodies
designed from the sequenced CDRs of murine antibodies secreted by a fusion of a
myeloma cell with spleen cells from immunized mice or other species compatible with
the selected myeloma cells [Kohler & Milstein, 1975, Nature, 256:495-497]. The
immunized animals could include transgenic mice with human immunoglobulin loci
which then directly produce human antibodies. Another possible embodiment could
consist in using phage display technologies to screen libraries.
The screening step i) can be realized by any method or process known by the
man skilled in the art. As non limitative examples, can be mentioned ELISA, BIAcore,
immunohistochemistry, FACS analysis and functional screens. A preferred process
consists in a screen by FACS analysis on CXCR4 transfectant and on at least a tumoral
cell line to be sure that the produced antibodies will be able to also recognize the native
receptor on tumor cells. This process will be described more precisely in the following
examples.
By the expression "to modulate CXCR4 activation" it is intended to modulate at
least one of the activities depicted in the examples 4, 5, 7 and 11 for the murine
antibody, 16, 17 and 19 for the chimeric one and 27, 24 and 28 for the humanized one
below:
Preferably to modulate:
- The specific binding at cellular membranes of the ligand SDF-1 on the receptor
CXCR4 (see examples 4, 16, 27), particularly by competition on eukaryotic transformed
cell membrane, such as CHO-Kl membranes, stably expressing human wild type
CXCR4 receptor;
- The specific binding at cellular membranes of the GTPyS on the receptor CXCR4 (see
examples 5, 17, 24), particularly on eukaryotic transformed cells membrane, such as
NIH-3T3 cells, stably and constitutively expressing wild-type CXCR4 receptor
membranes;
- The CXCR4-mediated inhibition of cAMP production (see Example 7); and
- The CXCR4 receptor-mediated mobilization of intracellular calcium stores (see
Examples 11, 19, 28).
More preferably, this modulation of at least one of these activities is an
inhibition of the activity.
In a preferred embodiment of the steps ii) and iii) of selection of the process of
the invention, said steps ii) and iii) consist in evaluating antibodies by BRET analysis
on cells expressing CXCR4-RLuc/CXCR4-YFP and CXCR4-Rluc/CXCR2-YFP,
respectively, and selecting antibodies capable to inhibit at least 40 %, preferably 45 %,
50 %, 55 % and most preferably 60 % of the BRET signal.
The technology BRET is a technology known as being representative of the
protein dimerization [Angers et al., PNAS, 2000, 97:3684-89].
The technology BRET, used in the steps ii) and iii) of the process, is well known
by the man skilled in the art and will be detailed in the following examples. More
particularly, BRET (Bioluminescence Resonance Energy Transfer) is a non-radiative
energy transfer occurring between a bioluminescent donor (Renilla Luciferase (Rluc))
and a fluorescent acceptor, a mutant of GFP (Green Fluorescent Protein) or YFP
(Yellow fluorescent protein). In the present case EYFP (Enhanced Yellow Fluorescent
Protein) was used. The efficacy of transfer depends on the orientation and the distance
between the donor and the acceptor. Then, the energy transfer can occur only if the two
molecules are in close proximity (1-10 nm). This property is used to generate proteinprotein
interaction assays. Indeed, in order to study the interaction between two
partners, the first one is genetically fused to the Renilla Luciferase and the second one
to the yellow mutant of the GFP. Fusion proteins are generally, but not obligatory,
expressed in mammalian cells. In presence of its membrane permeable substrate
(coelenterazine), Rluc emits blue light. If the GFP mutant is closer than 10 nm from the
Rluc, an energy transfer can occur and an additional yellow signal can be detected. The
BRET signal is measured as the ratio between the light emitted by the acceptor and the
light emitted by the donor. So the BRET signal will increase as the two fusion proteins
are brought into proximity or if a conformational change brings Rluc and GFP mutant
closer.
If the BRET analysis consists in a preferred embodiment, any method known by
the man skilled in the art can be used to measure CXCR4 dimers conformational
changes. Without limitation, the following technologies can be mentioned: FRET
(Fluorescence Resonance Energy Transfer), HTRF (Homogenous Time resolved
Fluorescence), FLIM (Fluorescence Lifetime Imaging Microscopy) or SW-FCCS single
wavelength fluorescence cross-correlation spectroscopy).
Other classical technologies could also be used, such as Coimmunoprecipitation,
Alpha screen, Chemical cross-linking, Double-Hybrid, Affinity
Chromatography, ELISA or Far western blot.
In a particular aspect of the process according to the invention, step ii) consists
in evaluating antibodies by BRET analysis on cells expressing both CXCR4-
RLuc/CXCR4-YFP and selecting antibodies capable to inhibit at least 40 %, of the
BRET signal.
In another particular aspect of the process according to the invention, step iii)
consists in evaluating antibodies by BRET analysis on cells expressing both CXCR4-
RLuc/CXCR2-YFP and selecting antibodies capable to inhibit at least 40 %, of the
BRET signal.
In a second aspect, a subject of the invention is an isolated humanized antibody,
or one of its functional fragments or derivatives, being obtained by said process. Said
humanized antibody or one of its said fragments or derivatives, is capable of binding
specifically to the human CXCR4 and, if necessary, preferably moreover capable of
inhibiting the natural attachment of its ligand, said humanized antibody being also
capable to induce CXCR4 dimers conformational changes.
The expressions "functional fragments and derivatives" will be defined in details
later in the present specification.
It must be understood here that the invention does not relate to the antibodies in
natural form, that is to say they are not in their natural environment but that they have
been able to be isolated or obtained by purification from natural sources, or else
obtained by genetic recombination, or by chemical synthesis, and that they can then
contain unnatural amino acids as will be described further on.
More particularly, according to another aspect of the invention, it is described an
antibody, or one of its functional fragments or derivatives, said humanized antibody
being characterized in that it comprises at least one complementary determining region
CDR, as defined according to IMGT, chosen from CDRs comprising the amino acid
sequence SEQ ID Nos. 4 to 9.
According to a second aspect, the invention relates to an isolated humanized
antibody, or a derived compound or functional fragment of same, comprising at least
one CDR chosen among the CDRs of sequences SEQ ID Nos. 4, 5, 6, 7, 8 or 9 or at
least one CDR, as defined according to IMGT, whose sequence has at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequences
SEQ ID No. 4, 5, 6, 7, 8 or 9.
A "functional fragment" of an antibody means in particular an antibody
fragment with the same specificity to CXCR4 as the parental antibody, such as
fragments Fv, scFv (sc=single chain), Fab, F(ab') , Fab', scFv-Fc or diabodies, or any
fragment whose half-life has been increased. Such functional fragments will be
described in detail later in the present description.
A "derived compound" or "derivative" of an antibody means in particular a
binding protein composed of a peptide scaffold and at least one of the CDRs of the
original antibody in order to preserve its ability to recognize CXCR4. Such derived
compounds, well-known to a person skilled in the art, will be described in more detail
later in the present description. In another embodiment of the invention, the derived
compound or derivative can comprise at least 2, preferably at least 3, more preferably 4,
still more preferably 5 or, most preferably, 6 CDRs of the original antibody.
More preferably, the invention comprises the humanized antibodies, their
derived compounds or their functional fragments, according to the present invention,
obtained by genetic recombination or chemical synthesis.
According to a preferred embodiment, the humanized antibody according to the
invention, or its derived compounds or functional fragments, is characterized in that it
consists of a monoclonal antibody.
"Monoclonal antibody" is understood to mean an antibody arising from a nearly
homogeneous antibody population. More particularly, the individual antibodies of a
population are identical except for a few possible naturally-occurring mutations which
can be found in minimal proportions. In other words, a monoclonal antibody consists of
a homogeneous antibody arising from the growth of a single cell clone (for example a
hybridoma, a eukaryotic host cell transfected with a DNA molecule coding for the
homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule coding
for the homogeneous antibody, etc.) and is generally characterized by heavy chains of
one and only one class and subclass, and light chains of only one type. Monoclonal
antibodies are highly specific and are directed against a single antigen. In addition, in
contrast with preparations of polyclonal antibodies which typically include various
antibodies directed against various determinants, or epitopes, each monoclonal antibody
is directed against a single epitope of the antigen.
It must be understood here that the invention does not relate to humanized
antibodies in natural form, i.e., they are not taken from their natural environment but are
isolated or obtained by purification from natural sources or obtained by genetic
recombination or chemical synthesis and thus they can carry unnatural amino acids as
will be described below.
More particularly, according to a preferred embodiment of the invention, the
humanized antibody, or its derived compounds or functional fragments, is characterized
in that it comprises a heavy chain comprising at least one CDR chosen among the CDRs
of amino acid sequences SEQ ID Nos. 4, 5 or 6, or at least one CDR whose sequence
has at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequences SEQ ID Nos. 4, 5 or 6; or it comprises a light chain comprising at least
one CDR chosen among the CDRs of amino acid sequences SEQ ID No. 7, 8 or 9, or at
least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95% and 98%
identity after optimal alignment with sequences SEQ ID No. 7, 8 or 9.
In a preferred manner, the humanized antibodies of the invention, or one of their
derived compounds or functional fragments, are characterized in that they comprise a
heavy chain comprising the following three CDRs, respectively CDR-H1, CDR-H2 and
CDR-H3, wherein:
- CDR-H1 comprises the sequence SEQ ID No. 4, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 4;
- CDR-H2 comprises the sequences SEQ ID No. 5, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 5; and
- CDR-H3 comprises the sequences SEQ ID No. 6, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 6.
According to a particular embodiment, antibodies, or one of their derived
compounds or functional fragments, are characterized in that they comprise a heavy
chain comprising the CDR-H1 of the sequence SEQ ID No. 4, the CDR-H2 of the
sequence SEQ ID No. 5 and the CDR-H3 of the sequence SEQ ID No. 6.
Even more preferably, the antibodies of the invention, or one of their derived
compounds or functional fragments, are characterized in that they comprise a light chain
comprising the following three CDRs, respectively CDR-L1, CDR-L2 and CDR-L3,
wherein:
- CDR-L1 comprises the sequence SEQ ID No. 7, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 7;
- CDR-L2 comprises the sequences SEQ ID No. 8, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 8; and
- CDR-L3 comprises the sequence SEQ ID No. 9, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98%, identity after optimal alignment with sequence
SEQ ID No. 9.
According to a particular embodiment, antibodies, or one of their derived
compounds or functional fragments, are characterized in that they comprise a light chain
comprising the CDR-L1 of the sequence SEQ ID No. 7, the CDR-L2 of the sequence
SEQ ID No. 8 and the CDR-L3 of the sequence SEQ ID No. 9.
In the present description, the terms "polypeptides", "polypeptide sequences",
"peptides" and "proteins attached to antibody compounds or to their sequences" are
interchangeable.
It must be understood here that the invention does not relate to antibodies in
natural form, i.e., they are not taken from their natural environment but are isolated or
obtained by purification from natural sources or obtained by genetic recombination or
chemical synthesis and thus they can carry unnatural amino acids as will be described
below.
The IMGT unique numbering has been defined to compare the variable domains
whatever the antigen receptor, the chain type, or the species [Lefranc M.-P.,
Immunology Today 18, 509 (1997) / Lefranc M.-P., The Immunologist, 7, 132-136
(1999) / Lefranc, M.-P., Pommie, C , Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,
Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the
IMGT unique numbering, the conserved amino acids always have the same position, for
instance cystein 23 (lst-CYS), tryptophan 4 1 (CONSERVED-TRP), hydrophobic
amino acid 89, cystein 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or JTRP).
The IMGT unique numbering provides a standardized delimitation of the
framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT:
66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions:
CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps
represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and
separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique
numbering is used in 2D graphical representations, designated as IMGT Colliers de
Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q.
and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in
IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and
MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].
Three heavy chain CDRs and 3 light chain CDRs exist. The term CDR or CDRs
is used here in order to indicate, according to the case, one of these regions or several,
or even the whole, of these regions which contain the majority of the amino acid
residues responsible for the binding by affinity of the antibody for the antigen or the
epitope which it recognizes.
In the sense of the present invention, the "percentage identity" between two
sequences of nucleic acids or amino acids means the percentage of identical nucleotides
or amino acid residues between the two sequences to be compared, obtained after
optimal alignment, this percentage being purely statistical and the differences between
the two sequences being distributed randomly along their length. The comparison of
two nucleic acid or amino acid sequences is traditionally carried out by comparing the
sequences after having optimally aligned them, said comparison being able to be
conducted by segment or by using an "alignment window". Optimal alignment of the
sequences for comparison can be carried out, in addition to comparison by hand, by
means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J.
Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman
(1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computer software using
these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the
comparison software BLAST NR or BLAST P).
The percentage identity between two nucleic acid or amino acid sequences is
determined by comparing the two optimally-aligned sequences in which the nucleic acid
or amino acid sequence to compare can have additions or deletions compared to the
reference sequence for optimal alignment between the two sequences. Percentage
identity is calculated by determining the number of positions at which the amino acid
nucleotide or residue is identical between the two sequences, preferably between the
two complete sequences, dividing the number of identical positions by the total number
of positions in the alignment window and multiplying the result by 100 to obtain the
percentage identity between the two sequences.
For example, the BLAST program, "BLAST 2 sequences" (Tatusova et al.,
"Blast 2 sequences - a new tool for comparing protein and nucleotide sequences",
FEMS Microbiol., 1999, Lett. 174:247-250) available on the site
http://www.ncbi.nlm.nih.gov/gorf7bl2.html, can be used with the default parameters
(notably for the parameters "open gap penalty": 5, and "extension gap penalty": 2; the
selected matrix being for example the "BLOSUM 62" matrix proposed by the program);
the percentage identity between the two sequences to compare is calculated directly by
the program.
For the amino acid sequence exhibiting at least 80%, preferably 85%, 90%, 95%
and 98% identity with a reference amino acid sequence, preferred examples include
those containing the reference sequence, certain modifications, notably a deletion,
addition or substitution of at least one amino acid, truncation or extension. In the case of
substitution of one or more consecutive or non-consecutive amino acids, substitutions
are preferred in which the substituted amino acids are replaced by "equivalent" amino
acids. Here, the expression "equivalent amino acids" is meant to indicate any amino
acids likely to be substituted for one of the structural amino acids without however
modifying the biological activities of the corresponding antibodies and of those specific
examples defined below.
Equivalent amino acids can be determined either on their structural homology
with the amino acids for which they are substituted or on the results of comparative tests
of biological activity between the various antibodies likely to be generated.
As a non-limiting example, table 1 below summarizes the possible substitutions
likely to be carried out without resulting in a significant modification of the biological
activity of the corresponding modified antibody; inverse substitutions are naturally
possible under the same conditions.
Table 1
It is known by those skilled in the art that in the current state of the art the
greatest variability (length and composition) between the six CDRs is found at the three
heavy-chain CDRs and, more particularly, at CDR-H3 of this heavy chain.
Consequently, it will be evident that the preferred characteristic CDRs of the antibodies
of the invention, or of one of their derived compounds or functional fragments, will be
the three CDRs of the heavy chain.
Another embodiment of the invention discloses an antibody, or its derived
compounds or functional fragments, comprising:
a heavy chain comprising the following three CDRs:
CDR-H1 of the sequence SEQ ID No. 4 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No. 4;
CDR-H2 of the sequence SEQ ID No. 5 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No. 5;
CDR-H3 of the sequence SEQ ID No. 6 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No 6;
and a light chain comprising the following three CDRs:
CDR-L1 of the sequence SEQ ID No. 7 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No. 7;
CDR-L2 of the sequence SEQ ID No. 8 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No. 8;
CDR-L3 of the sequence SEQ ID No. 9 or of a sequence with at least 80%, preferably
85%, 90%, 95% and 98% identity after optimal alignment with sequence SEQ ID No. 9.
Another aspect of the invention relates to the functional fragments of the
humanized antibody described above. As it will be obvious for the man skilled in the
art, a functional fragment is necessarily a binding fragment, i.e. a fragment capable of
binding to the same target as the parental antibody. A functional fragment, moreover,
retains the function of the parental antibody. In particular, a functional fragment of the
invention is capable of modulating, the activation of CXCR4. More preferably, a
functional fragment according to the invention is capable of inhibiting the activation of
CXCR4. In one embodiment, the CXCR4 activation is ligand dependent; in another
embodiment, the said CXCR4 activation is ligand independent.
In a preferred embodiment, the functional fragments of the invention retain the
function of the parental antibody consisting in modulating, the activity of the
CXCR4/CXCR2 heterodimer complex. Preferably, the said functional fragments retain
the capacity of inhibiting the activity of the CXCR4/CXCR2 heterodimer complex.
More particularly, the invention targets an antibody, or its derived compounds or
functional fragments, characterized in that said functional fragment is selected among
the fragments Fv, Fab, (Fab')2, Fab', scFv, scFv-Fc and diabodies, or any fragment
whose half-life has been increased such as PEGylated fragments.
Such functional fragments of the antibody according to the invention consist, for
example, of the fragments Fv, scFv (sc=simple chain), Fab, F(ab') 2, Fab', scFv-Fc or
diabodies, or any fragment whose half-life has been increased by chemical
modification, such as the addition of polyalkylene glycol such as polyethylene glycol
(PEGylation) (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG,
F(ab') 2-PEG and Fab'-PEG), or by incorporation in a liposome, microspheres or PLGA,
said fragments possessing at least one of the characteristic CDRs of the humanized
antibodies of the invention which is notably capable of exerting in a general manner
activity, even partial, of the antibody from which it arises.
Preferably, said functional fragments will comprise or include a partial sequence
of the variable heavy or light chain of the antibody from which they are derived, said
partial sequence being sufficient to retain the same binding specificity as the antibody
from which it arises and sufficient affinity, preferably at least equal to 1/100, more
preferably at least 1/10 of that of the antibody from which it arises.
Such a functional fragment will contain at least five amino acids, preferably 6, 7,
8, 10, 15, 25, 50 or 100 consecutive amino acids of the sequence of the antibody from
which it arises.
Preferably, these functional fragments will be of the types Fv, scFv, Fab, F(ab') 2,
F(ab'), scFv-Fc or diabodies, which generally have the same binding specificity as the
antibody from which they result. According to the present invention, fragments of the
antibody of the invention can be obtained from the antibodies described above by
methods such as enzyme digestion, including pepsin or papain, and/or by cleavage of
the disulfide bridges by chemical reduction. The antibody fragments can be also
obtained by recombinant genetics techniques also known to a person skilled in the art or
by peptide synthesis by means, for example, of automatic peptide synthesizers such as
those sold by Applied BioSystems, etc.
More particularly, the invention relates to different humanized antibody
variants of the murine 515H7 Mab, referred in the present specification as i) HZ515H7,
Hz515H7 or hz515H7 Mab and ii) HZ515H7-2, Hz515H7-2 or hz515H7-2 Mab or iii)
any combination of a light chain and/or a heavy chain of the said antibody variants..
For more clarity, table 2a below summarizes the various amino acid sequences
corresponding to the CDRs of the humanized variants (also referred as forms) hz515H7
and hz515H7-2 of the invention; table 2b summarizes the various amino acid sequences
corresponding to the variable domains and the full length sequences of the various
variants of the humanized form hz515H7 of the invention; and table 2c summarizes the
various amino acid sequences corresponding to the variable domains and the full length
sequences of the humanized form hz515H7-2 of the invention.
Table 2a
Antibody Heavy chain Light chain SEQ ID NO.
Hz515H7
CDR-H1 - 4
CDR-H2 - 5
CDR-H3 - 6
CDR(s) - CDR-L1 7
- CDR-L2 8
- CDR-L3 9
Table 2b
Antibody
Heavy chain Light chain SEQ ID NO.
Hz515H7
VH1 - 10
VH1 D76N _ 1 1
VH1 V48L D76N - 12
VH2 - 13
- VL1 14
Variable Domains - VL1 T59A E61D 15
- VL2 16
- VL2.1 17
- VL2.2 18
- VL2.3 19
- VL3 20
Complete Sequences VH1 - 2 1
(without signal peptide) VH1 D76N - 22
VH1 V48L D76N - 23
VH2 - 24
- VL1 25
- VL1 T59A E61D 26
- VL2 27
- VL2.1 28
- VL2.2 29
- VL2.3 30
- VL3 3 1
Table 2c
Antibody
Heavy chain Light chain SEQ ID NO.
Hz515H7-2
"consensus" - 85
- "consensus" 86
Variable Domains
VH1 - 87
- VL1 88
"consensus" - 89
Complete Sequences - "consensus" 90
(without signal peptide) VH1 - 9 1
- VL1 92
As an example, for the avoidance of doubt, the expression "VH1" is similar to
the expressions "VH Variant 1", "VH variant 1", "VH Var 1" or "VH var 1). The
obtention of "consensus" sequences is described in Example 22.
Yet another specific aspect of the present invention relates to a humanized
antibody, or its derived compounds or functional fragments, characterized in that the
constant regions of the light-chain and the heavy-chain derived from human antibody
are, respectively, the lambda or kappa region and the gamma- 1, gamma-2 or gamma-4
region.
The invention describes the murine hybridoma secreting a monoclonal antibody
filed with the French collection for microorganism cultures (CNCM, Institut Pasteur,
Paris, France) on June 25, 2008, under number 1-4019. Said hybridoma was obtained by
the fusion of Balb/C immunized mice splenocytes and cells of the myeloma Sp 2/O-Ag
14 lines.
The murine monoclonal antibody, here referred to as 515H7 is secreted by the
hybridoma filed with the CNCM on June 25, 2008, under number 1-4019.
The invention also describes chimeric antibodies.
A chimeric antibody is one containing a natural variable region (light chain and
heavy chain) derived from an antibody of a given species in combination with constant
regions of the light chain and the heavy chain of an antibody of a species heterologous
to said given species.
These antibodies, or chimeric fragments of same, can be prepared by using the
techniques of recombinant genetics. For example, the chimeric antibody could be
produced by cloning recombinant DNA containing a promoter and a sequence coding
for the variable region of a nonhuman monoclonal antibody of the invention, notably
murine, and a sequence coding for the human antibody constant region. A chimeric
antibody according to the invention coded by one such recombinant gene could be, for
example, a mouse-human chimera, the specificity of this antibody being determined by
the variable region derived from the murine DNA and its isotype determined by the
constant region derived from human DNA. Refer to Verhoeyn et al. (BioEssays, 8:74,
1988) for methods for preparing chimeric antibodies.
In another embodiment, the invention relates to a chimeric antibody heavy chain
(referred as c515H7 VH) comprising a variable region of sequence selected SEQ ID No.
83.
In another embodiment, the invention relates to a chimeric antibody light chain
(referred as c515H7 VL) comprising a variable region of sequence SEQ ID No. 84.
In a particular preferred embodiment, the chimeric antibody, or a derived
compound or functional fragment of same, of the invention comprises a heavy chain
sequence comprising the amino acid sequence SEQ ID No. 83, and a light chain
sequence comprising the amino acid sequence SEQ ID No.84.
A "humanized antibody", as used herein, refers to an antibody that comprises at
least one heavy chain or one light chain, said heavy or light chain containing CDR
regions derived from an antibody of nonhuman origin, the other parts of the antibody
molecule being of human origin (for example, the said other parts may be derived from
one (or several) human antibodies). By the expression "humanized antibody", the
present invention thus comprises antibodies with only one humanized chain, the second
being a chimeric or murine chain. Preferably, a "humanized antibody" of the invention
comprises two humanized chains, i.e. both the heavy chain and the light chain are
humanized.
The humanized antibodies of the invention or fragments of same can be prepared
by techniques known to a person skilled in the art (such as, for example, those described
in the documents Singer et al., J. Immun., 150:2844-2857, 1992; Mountain et
al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et al.,
Bio/Technology, 10:169-175, 1992). Such humanized antibodies are preferred for their
use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment
in vivo. Other humanization techniques, also known to a person skilled in the art, such
as, for example, the "CDR grafting" technique described by PDL in patents EP 0 451
216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US
5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and
5,877,293 can also be cited.
The invention relates to the humanized antibodies arising from the murine
antibody 515H7 described above.
In a preferred manner, constant regions of the light-chain and the heavy-chain
derived from human antibody are, respectively, the lambda or kappa and the gamma- 1,
gamma- or gamma-4 region.
More particularly, the invention relates to a humanized antibody heavy chain
comprising i) a framework region homologous to corresponding framework region of a
human antibody heavy chain, and ii) CDRs homologous to corresponding CDRs of an
antibody derived from a different mammalian species, wherein said CDRs consist of
CDR-H1, CDR-H2 and CDR-H3 comprising respectively the sequences SEQ ID Nos.
4, 5 and 6.
In other words, the invention relates to a humanized antibody heavy chain
having CDRs consisting of CDR-H1, CDR-H2 and CDR-H3, said CDR-H1, CDR-H2
and CDR-H3 comprising respectively the sequences SEQ ID Nos. 4, 5 and 6. It will be
obvious, for the man skilled in the art, that different germlines can be selected for the
humanization of the antibody 515H7, resulting then in different forms of humanized
515H7. More particularly, in a preferred non limitative embodiment, different germlines
can be used for the sequence coding the v genes whereas the same germlines will be
conserved for the j-genes.
For the present invention, germlines which could be used are selected on the
bases of two complementary criteria:
- the length in amino acids for each CDRs must be identical between the
murine CDR and the equivalent in the Germline ; and
- the germline sequence in amino acids must have at least 70 % of
identity with the parental murine sequence.
For the avoidance of doubt, it is reminded here that the present invention
encompasses two preferred non limitative humanized forms (also referred as versions)
of the same antibody 515H7. The first one, referred as Hz515H7, consists of a
humanized antibody obtained with the germlines IGHV3-49*04 (SEQ ID No. 77) and
IGHJ4*01 (SEQ ID No. 81) for the heavy chain and IGKV4-1*01 (SEQ ID No. 78) and
IGKJ1*01 (SEQ ID No. 82) for the light chain. The second one, referred as Hz515H7-2,
consists of a humanized antibody obtained with the germlines IGHV3-73*01 (SEQ ID
No. 79) and IGHJ4*01 (SEQ ID No. 81) for the heavy chain and IGKV2D-40*01 (SEQ
ID No. 80) and IGKJ1*01 (SEQ ID No. 82) for the light chain.
In another embodiment, the invention relates to a humanized antibody heavy
chain comprising a variable region of sequence selected from the group consisting of
SEQ ID Nos. 10, 11, 12, 13, 85 or 87.
In another embodiment, the invention relates to the humanized antibody
H515H7 heavy chain comprising a variable region of sequence selected from the group
consisting of SEQ ID Nos. 10, 11, 12 or 13.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 heavy chain comprising a variable region of sequence SEQ ID No. 85.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 heavy chain comprising a variable region of sequence SEQ ID No. 87.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 heavy chain variable region of sequence SEQ ID No. 87 which
comprises one or more amino acid substitution(s) selected from the group consisting of
H35S, V48L, R50F, A61D, D76N and A81L.
In still another embodiment, the invention relates to a humanized antibody
heavy chain comprising the complete sequence selected from the group consisting of
SEQ ID Nos. 21, 22, 23, 24, 89 or 91.
In still another embodiment, the invention relates to a humanized antibody
heavy chain comprising the complete sequence selected from the group consisting of
SEQ ID Nos. 21, 22, 23 or 24.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 heavy chain comprising the complete sequence SEQ ID No. 89.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 heavy chain comprising the complete sequence SEQ ID No. 91.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 heavy chain of sequence SEQ ID No. 9 1 which comprises one or
more amino acid substitution(s) selected from the group consisting of H35S, V48L,
R50F, A61D, D76N and A81L.
More particularly, the invention relates to a humanized antibody light chain
comprising i) a framework region homologous to corresponding framework region of a
human antibody light chain, and ii) CDRs homologous to corresponding CDRs of an
antibody derived from a different mammalian species, wherein said CDRs consist of
CDR-L1, CDR-L2 and CDR-L3 comprising respectively the sequences SEQ ID Nos. 7,
8 and 9.
In other words, the invention relates to a humanized antibody light chain having
CDRs consisting of CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDRL3
comprising respectively the sequences SEQ ID Nos. 7, 8 and 9.
In another embodiment, the invention relates to a humanized antibody light
chain comprising a variable region of sequence selected from the group consisting of
SEQ ID Nos. 14, 15, 16, 17, 18, 19, 20, 86 or 88.
In another embodiment, the invention relates to the humanized antibody
H515H7 light chain comprising a variable region of sequence selected from the group
consisting of SEQ ID Nos. 14, 15, 16, 17, 18, 19 or 20.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 light chain comprising a variable region of sequence SEQ ID No. 86.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 light chain comprising a variable region of sequence SEQ ID No. 88.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 light chain variable region of sequence SEQ ID No. 88 which
comprises one or more amino acid substitution(s) selected from the group consisting of
L9S, I21M, D40A, L43Q, Y59A, A61D, D66A, S69T, G74E, D76Y and/orV89L.
In still another embodiment, the invention relates to a humanized antibody light
chain comprising the complete sequence selected from the group consisting of SEQ ID
Nos. 25, 26, 27, 28, 29, 30, 31, 90 or 92.
In another embodiment, the invention relates to the humanized antibody
H515H7 light chain comprising the complete sequence selected from the group
consisting of SEQ ID Nos. 25, 26, 27, 28, 29, 30 or 31.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 light chain comprising the complete sequence SEQ ID No. 90.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 light chain comprising the complete sequence SEQ ID No. 92.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 light chain of sequence SEQ ID No. 92 which comprises one or
more amino acid substitution(s) selected from the group consisting of L9S, 12 1M,
D40A, L43Q, Y59A, A61D, D66A, S69T, G74E, D76Y and/orV89L.
More particularly, the invention relates to a humanized antibody, or a derived
compound or functional fragment of same, characterized in that it comprises heavy and
light chains each having i) framework regions homologous to corresponding framework
regions of a human antibody, and ii) CDRs homologous to corresponding CDRs of an
antibody derived from a different mammalian species, wherein said CDRs consist of
CDR-Hl, CDR-H2 and CDR-H3 of the heavy chain comprising respectively the
sequences SEQ ID Nos. 4, 5 and 6, and CDR-L1, CDR-L2 and CDR-L3 of the light
chain comprising respectively the sequences SEQ ID Nos. 7, 8 and 9.
In other words, the invention relates to a humanized antibody, or a derived
compound or functional fragment of same, characterized in that the said humanized
antibody comprises heavy and light chains, said heavy chain having CDRs consisting of
CDR-Hl, CDR-H2 and CDR-H3, and the said light chain having CDRs consisting of
CDR-L1, CDR-L2 and CDR-L3, wherein said CDR-Hl, CDR-H2 and CDR-H3
comprise respectively the sequences SEQ ID Nos. 4, 5 and 6, and said CDR-L1, CDRL2
and CDR-L3 comprise respectively the sequences SEQ ID Nos. 7, 8 and 9.
In another embodiment, the invention relates to a humanized antibody, or a
derived compound or functional fragment of same, comprising a heavy chain variable
region of sequence selected from the group consisting of SEQ ID Nos. 10, 11, 12, 13,
83, 85 or 87 and a light chain variable region of sequence selected from the group
consisting of SEQ ID Nos. 14, 15, 16, 17, 18, 19, 20, 84, 86 or 88.
In another embodiment, the invention relates to the humanized antibody
H515H7 comprising a heavy chain variable region selected from the group consisting of
SEQ ID Nos. 10, 11, 12 or 13 and a light chain variable region of sequence selected
from the group consisting of SEQ ID Nos. 14, 15, 16, 17, 18, 19 or 20.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain variable region of sequence SEQ ID No. 85 and a
light variable region of sequence SEQ ID No. 86.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain variable region of sequence SEQ ID No. 87 and a
light variable region of sequence SEQ ID No. 88.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain variable region of sequence SEQ ID No. 85 and a
light variable region of sequence SEQ ID No. 88.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain variable region of sequence SEQ ID No. 87 and a
light variable region of sequence SEQ ID No. 86.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 comprising a heavy chain variable region of sequence SEQ ID No.
87 which comprises one or more amino acid substitution(s) selected from the group
consisting of H35S, V48L, R50F, A61D, D76N and A81L and light chain variable
region of sequence SEQ ID No. 88 which comprises one or more amino acid
substitution(s) selected from the group consisting of D40A, L43Q, Y59A, A61D, S69T,
G74E and D76Y.
In still another embodiment, the invention relates to a humanized antibody
comprising a humanized heavy chain, combined with a chimeric light chain.
In still another embodiment, the invention relates to a humanized antibody
comprising a chimeric heavy chain, combined with a humanized light chain.
More particularly, the invention describes a humanized antibody, or a derived
compound or functional fragment of same, comprising a heavy chain variable region of
sequence SEQ ID No. 83 and a light chain variable region of sequence selected from the
group consisting of SEQ ID Nos. 14, 15, 16, 17, 18, 19, 20, 86 or 88.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL1 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 1 .
In a preferred embodiment, the invention relates to the humanized antibody
C515H7 VH / Hz515H7 VL1 T59A E61D or a derived compound or functional
fragment of same, comprising a heavy chain variable region of sequence SEQ ID No.
83, and a light chain variable region of sequence SEQ ID No. 15.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL2 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 16.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL2.1 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 17.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL2.2 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 18.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL2.3 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 19.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7 VL3 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 20.
In a preferred embodiment, the invention relates to a humanized antibody or a
derived compound or functional fragment of same, comprising a heavy chain variable
region of sequence SEQ ID No. 83, and a light chain variable region of sequence SEQ
ID No. 86.
In a preferred embodiment, the invention relates to the humanized antibody
c515H7 VH / Hz515H7-2 VL1 or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 83, and a light chain
variable region of sequence SEQ ID No. 88.
In another embodiment, the invention describes a humanized antibody, or a
derived compound or functional fragment of same, comprising a heavy chain variable
region of sequence selected from the group consisting of SEQ ID Nos. 10, 1, 12, 13,
85 or 87 and a light chain variable region of sequence SEQ ID No. 84.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VH1 / c515H7 VL or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 10, and a light chain
variable region of sequence SEQ ID No. 84.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VH1 D76N / c515H7 VL or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 1, and a light
chain variable region of sequence SEQ ID No. 84.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VHl V48L D76N / c515H7 VL or a derived compound or functional
fragment of same, comprising a heavy chain variable region of sequence SEQ ID No.
12, and a light chain variable region of sequence SEQ ID No. 84.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VH2 / c515H7 VL or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 13, and a light chain
variable region of sequence SEQ ID No. 84.
In a preferred embodiment, the invention relates to a humanized antibody or a
derived compound or functional fragment of same, comprising a heavy chain variable
region of sequence SEQ ID No. 85, and a light chain variable region of sequence SEQ
ID No. 84.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VH1 / c515H7 VL or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light chain
variable region of sequence SEQ ID No. 84.
In still another embodiment, the invention relates to a humanized antibody, or a
derived compound or functional fragment of same, comprising a heavy chain of
sequence selected from the group consisting of SEQ ID Nos. 21, 22, 23, 24, 89 or 9 1
and a light chain of sequence selected from the group consisting of SEQ ID Nos. 25, 26,
27, 28, 29, 30, 31, 90 or 92.
In another embodiment, the invention relates to the humanized antibody
H515H7 comprising a heavy chain of sequence selected from the group consisting of
SEQ ID Nos. 21, 22, 23 or 24 and a light chain of sequence selected from the group
consisting of SEQ ID Nos. 25, 26, 27, 28, 29, 30 or 31.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain of sequence SEQ ID No. 89 and a light chain of
sequence SEQ ID No. 90.
In another embodiment, the invention relates to the humanized antibody
H515H7-2 comprising a heavy chain of sequence SEQ ID No. 9 1 and a light chain of
sequence SEQ ID No. 92.
In still another embodiment, the invention also relates to the humanized
antibody H515H7-2 comprising a heavy chain of sequence SEQ ID No. 9 1 which
comprises one or more amino acid substitution(s) selected from the group consisting of
H35S, V48L, R50F, A61D, D76N and A81L and light chain of sequence SEQ ID No.
92 which comprises one or more amino acid substitution(s) selected from the group
consisting of D40A, L43Q, Y59A, A61D, S69T, G74E and D76Y.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VH1 D76 VL2, or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 11, and a light chain
variable region of sequence SEQ ID No. 16.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VH1 D76N VL2, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 22, and a light chain of
sequence SEQ ID No. 27.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VH D76N VL2.1, or a derived compound or functional fragment
of same, comprising a heavy chain variable region of sequence SEQ ID No. 11, and a
light chain variable region of sequence SEQ ID No. 17.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl D76N VL2.1, or a derived compound or functional fragment
of same, comprising a heavy chain of sequence SEQ ID No. 22, and a light chain of
sequence SEQ ID No. 28.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl D76N VL2.2, or a derived compound or functional fragment
of same, comprising a heavy chain variable region of sequence SEQ ID No. 11, and a
light chain variable region of sequence SEQ ID No. 18.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl D76N VL2.2, or a derived compound or functional fragment
of same, comprising a heavy chain of sequence SEQ ID No. 22, and a light chain of
sequence SEQ ID No. 29.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl D76N VL2.3, or a derived compound or functional fragment
of same, comprising a heavy chain variable region of sequence SEQ ID No. 1, and a
light chain variable region of sequence SEQ ID No. 19.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl D76N VL2.3, or a derived compound or functional fragment
of same, comprising a heavy chain of sequence SEQ ID No. 22, and a light chain of
sequence SEQ ID No. 30.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N VL1, or a derived compound or functional
fragment of same, comprising a heavy chain variable region of sequence SEQ ID No.
12, and a light chain variable region of sequence SEQ ID No. 14.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N VLl, or a derived compound or functional
fragment of same, comprising a heavy chain of sequence SEQ ID No. 23, and a light
chain of sequence SEQ ID No. 25.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N VLl T59A E61D, or a derived compound or
functional fragment of same, comprising a heavy chain variable region of sequence
SEQ ID No. 12, and a light chain variable region of sequence SEQ ID No. 15.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N VLl T59A E61D, or a derived compound or
functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 23, and
a light chain of sequence SEQ ID No. 26.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl VLl, or a derived compound or functional fragment of same,
comprising a heavy chain variable region of sequence SEQ ID No. 10, and a light chain
variable region of sequence SEQ ID No. 14.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl VLl, or a derived compound or functional fragment of same,
comprising a heavy chain of sequence SEQ ID No. 21, and a light chain of sequence
SEQ ID No. 25.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VHl / Hz515H7-2 VLl, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 10, and a light
chain variable region of sequence SEQ ID No. 88.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VHl D76N / Hz515H7-2 VLl, or a derived compound or functional fragment
of same, comprising a heavy chain variable region of sequence SEQ ID No. 11, and a
light chain variable region of sequence SEQ ID No. 88.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N / Hz515H7-2 VLl, or a derived compound or
functional fragment of same, comprising a heavy chain variable region of sequence
SEQ ID No. 12, and a light chain variable region of sequence SEQ ID No. 88.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VH2 / Hz515H7-2 VLl, or a derived compound or functional
fragment of same, comprising a heavy chain variable region of sequence SEQ ID No.
13, and a light chain variable region of sequence SEQ ID No. 88.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VHl / Hz515H7-2 VLl, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 21, and a light chain of
sequence SEQ ID No. 92.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7 VHl D76N / Hz515H7-2 VLl, or a derived compound or functional fragment
of same, comprising a heavy chain of sequence SEQ ID No. 22, and a light chain of
sequence SEQ ID No. 92.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VHl V48L D76N / Hz515H7-2 VLl, or a derived compound or
functional fragment of same, comprising a heavy chain of sequence SEQ ID No. 23, and
a light chain of sequence SEQ ID No. 92.
In another preferred embodiment, the invention relates to the humanized
antibody Hz515H7 VH2 / Hz515H7-2 VLl, or a derived compound or functional
fragment of same, comprising a heavy chain of sequence SEQ ID No. 24, and a light
chain of sequence SEQ ID No. 92.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VLl, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 14.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VLl T59A E61D, or a derived compound or functional
fragment of same, comprising a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 15.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 16.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.1, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 17.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.2, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 18.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.3, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 19.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL3, or a derived compound or functional fragment of
same, comprising a heavy chain variable region of sequence SEQ ID No. 87, and a light
chain variable region of sequence SEQ ID No. 20.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VLl, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 25.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VLl T59A E61D, or a derived compound or functional
fragment of same, comprising a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 26.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 27.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.1, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 28.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.2, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 29.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL2.3, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 30.
In a preferred embodiment, the invention relates to the humanized antibody
Hz515H7-2 VHl / Hz515H7 VL3, or a derived compound or functional fragment of
same, comprising a heavy chain of sequence SEQ ID No. 91, and a light chain of
sequence SEQ ID No. 31.
It must be understood that the above exemplified VH / VL combinations are not
limitative. The man skilled in the art could of course, without undue burden and without
applying inventive skill, rearrange all the VH and VL disclosed in the present
specification. The skilled person could thus obtain all the humanized antibodies
corresponding to all the combinations of all the VH and the VL disclosed in the present
application.
A novel aspect of the present invention relates to an isolated nucleic acid
characterized in that it is selected among the following nucleic acids (including any
degenerate genetic code):
a) a nucleic acid, DNA or RNA, coding for a humanized antibody heavy
chain, or for a derived compound or functional fragment of same,
according to the present invention;
b) a nucleic acid, DNA or RNA, coding for a humanized antibody light
chain, or for a derived compound or functional fragment of same,
according to the present invention;
c) a nucleic acid, DNA or RNA, coding for a humanized antibody, or for
a derived compound or functional fragment of same, according to the
present invention;
d) a nucleic acid complementary to a nucleic acid as defined in a), b) or
c);
e) a nucleic acid of at least 18 nucleotides capable of hybridizing under
highly stringent conditions with at least a heavy chain comprising the
nucleic acid sequences SEQ ID No. 38 to 41,49 to 52, 93 or 95
preferably with at least one of the 3 CDRs thereof according to the
IMGT or to the abat CDR numbering;
f a nucleic acid of at least 18 nucleotides capable of hybridizing under
highly stringent conditions with at least a light chain comprising the
nucleic acid sequences SEQ ID No. 42 to 48, 53 to 59, 94 or 96
preferably with at least one of the 3 CDRs thereof according to the
IMGT or to the Kabat CDR numbering.
The invention also relates to an isolated nucleic acid molecule comprising a
nucleic acid sequence encoding a heavy chain variable region of a humanized antibody,
said heavy chain variable region nucleotide sequence comprising a CDR-H1 nucleotide
sequence of SEQ ID No. 32; a CDR-H2 nucleotide sequence of SEQ ID No. 33; and a
CDR-H3 nucleotide sequence of SEQ ID No. 34.
The invention also relates to an isolated nucleic acid molecule comprising a
nucleic acid sequence encoding a light chain variable region of a humanized antibody,
said light chain variable region nucleotide sequence comprising a CDR-L1 nucleotide
sequence of SEQ ID No. 35 or 60; a CDR-L2 nucleotide sequence of SEQ ID No. 36 or
61; and a CDR-L3 nucleotide sequence of SEQ ID No. 37 or 62.
The invention also relates to an isolated nucleic acid molecule comprising a
nucleic acid sequence encoding a heavy chain variable region and a light chain variable
region of a humanized antibody,
said heavy chain variable region nucleotide sequence comprising a CDR-H1
nucleotide sequence of SEQ ID No. 32; a CDR-H2 nucleotide sequence of SEQ ID No.
33; and a CDR-H3 nucleotide sequence of SEQ ID No. 34;
said light chain variable region nucleotide sequence comprising a CDR-L1
nucleotide sequence of SEQ ID No. 35 or 60; a CDR-L2 nucleotide sequence of SEQ
ID No. 36 or 61; and a CDR-L3 nucleotide sequence of SEQ ID No. 37 or 62.
Table 3a below summarizes the optimized nucleotide sequences corresponding
to the CDRs of the antibody hz515H7 of the invention; table 3b summarizes the various
optimized nucleotide sequences corresponding to the variable domains and the full
length sequences of the various variants of the humanized antibody hz515H7 of the
invention. Table 3c summarizes the various optimized nucleotide sequences
corresponding to the variable domains and the full length sequences the humanized
version hz515H7-2 of the invention.
Table 3a
Table 3b
Antibody Heavy chain Light chain SEQ Hz515H7 ID NO.
VH1 - 38
Variable VH1 D76N - 39
Domains V l V48L D76N - 40
VH2 - 4 1
- VL1 42
- VL1 T59A E61D 43
- VL2 44
- VL2.1 45
- VL2.2 46
- VL2.3 47
- VL3 48
VH1 - 49
VH1 D76N - 50
VH1 V48L D76N - 5 1
VH2 - 52
Complete - VL1 53
Sequences - VL1 T59A E61D 54
(without signal
peptide) - VL2 55
- VL2.1 56
- VL2.2 57
- VL2.3 58
- VL3 59
Table 3c
The expression "optimized sequence" means that the codons encoding the amino
acids constitutive of the protein of interest (herein the antibody variable domains) have
been optimized for a better recognition by the translation machinery in a dedicated cell
type, herewith mammalian cells. With this respect, the amino acid sequence of the given
protein encoded by the optimized sequence is identical to that of the non-optimized
sequence, but the nucleotide sequence is different. Optimization also include G/C
content adaptation and prevention of stable RNA secondary structure (see as example
Kim et al., 1997 Genel99(l-2):293-301).
For example, the nucleotide sequence of the murine CDR-H1 (SEQ ID No. 71)
has been optimized and corresponds to the nucleotide sequence of the humanized CDRHl
(SEQ ID No. 32) wherein the codons ggg, act and gat (coding for the residues Gly,
Thr and Asp, respectively) have been replaced by the codons ggc, acc and gac,
respectively (also coding for the residue Gly, Thr and Asp, respectively).
Concerning CDR-H2 and CDR-H3 (SEQ ID Nos. 72 and 73, respectively), they
have also been optimized and correspond to the optimized CDRs of SEQ ID Nos. 33
and 34, respectively.
It is the same for the three CDRs of the light chain (SEQ ID Nos. 74, 75 and 76,
respectively) with two humanized optimized forms corresponding to the VL1, VL2 and
VL3 (SEQ ID Nos. 35, 36 and 37, respectively) and to the VL2.1, VL2.2 and VL2.3
(SEQ ID Nos. 60, 6 and 62, respectively).
The following table 4 summarizes the original CDRs, i.e. the murine non
optimized sequences.
Table 4
The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence",
"polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide
sequence", used interchangeably in the present description, mean a precise sequence of
nucleotides, modified or not, defining a fragment or a region of a nucleic acid,
containing unnatural nucleotides or not, and being either a double-strand DNA, a singlestrand
DNA or transcription products of said DNAs.
It should also be included here that the present invention does not relate to
nucleotide sequences in their natural chromosomal environment, i.e., in a natural state.
The sequences of the present invention have been isolated and/or purified, i.e., they
were sampled directly or indirectly, for example by a copy, their environment having
been at least partially modified. Isolated nucleic acids obtained by recombinant
genetics, by means, for example, of host cells, or obtained by chemical synthesis should
also be mentioned here.
"Nucleic sequences exhibiting a percentage identity of at least 80%, preferably
85%, 90%, 95% and 98%, after optimal alignment with a preferred sequence" means
nucleic sequences exhibiting, with respect to the reference nucleic sequence, certain
modifications such as, in particular, a deletion, a truncation, an extension, a chimeric
fusion and/or a substitution, notably punctual. Preferably, these are sequences which
code for the same amino acid sequences as the reference sequence, this being related to
the degeneration of the genetic code, or complementarity sequences that are likely to
hybridize specifically with the reference sequences, preferably under highly stringent
conditions, notably those defined below.
Hybridization under highly stringent conditions means that conditions related to
temperature and ionic strength are selected in such a way that they allow hybridization
to be maintained between two complementarity DNA fragments. On a purely
illustrative basis, the highly stringent conditions of the hybridization step for the
purpose of defining the polynucleotide fragments described above are advantageously
as follows.
DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)
prehybridization at 42°C for three hours in phosphate buffer (20 mM, pH 7.5)
containing 5X SSC (IX SSC corresponds to a solution of 0.15 M NaCl + 0.015 M
sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10X Denhardt's,
5% dextran sulfate and 1% salmon sperm DNA; (2) primary hybridization for 20 hours
at a temperature depending on the length of the probe (i.e.: 42°C for a probe >100
nucleotides in length) followed by two 20-minute washings at 20°C in 2X SSC + 2%
SDS, one 20-minute washing at 20°C in 0.1X SSC + 0.1% SDS. The last washing is
carried out in 0.1X SSC + 0.1% SDS for 30 minutes at 60°C for a probe >100
nucleotides in length. The highly stringent hybridization conditions described above for
a polynucleotide of defined size can be adapted by a person skilled in the art for longer
or shorter oligonucleotides, according to the procedures described in Sambrook, et al.
(Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd edition,
2001).
The invention also relates to a vector comprising a nucleic acid as described in
the invention.
The invention notably targets cloning and/or expression vectors that contain
such a nucleotide sequence.
The vectors of the invention preferably contain elements which allow the
expression and/or the secretion of nucleotide sequences in a given host cell. The vector
thus must contain a promoter, translation initiation and termination signals, as well as
suitable transcription regulation regions. It must be able to be maintained in a stable
manner in the host cell and may optionally have specific signals which specify secretion
of the translated protein. These various elements are selected and optimized by a person
skilled in the art according to the host cell used. For this purpose, the nucleotide
sequences can be inserted in self-replicating vectors within the chosen host or be
integrative vectors of the chosen host.
Such vectors are prepared by methods typically used by a person skilled in the
art and the resulting clones can be introduced into a suitable host by standard methods
such as lipofection, electroporation, heat shock or chemical methods.
The vectors are, for example, vectors of plasmid or viral origin. They are used to
transform host cells in order to clone or express the nucleotide sequences of the
invention.
The invention also comprises host cells transformed by or comprising a vector as
described in the present invention.
The host cell can be selected among prokaryotic or eukaryotic systems such as
bacterial cells, for example, but also yeast cells or animal cells, notably mammal cells.
Insect or plant cells can also be used.
The invention also relates to animals, other than man, that have a transformed
cell according to the invention.
Another aspect of the invention relates to a method for the production of an
antibody according to the invention, or one of its functional fragments, characterized in
that said method comprises the following steps:
a) the culture in a medium of and the suitable culture conditions for a host cell
according to the invention; and
b) the recovery of said antibody, or one of its functional fragments, thus
produced from the culture medium or from said cultured cells.
The transformed cells according to the invention are of use in methods for the
preparation of recombinant polypeptides according to the invention. Methods for the
preparation of polypeptide according to the invention in recombinant form,
characterized in that said methods use a vector and/or a cell transformed by a vector
according to the invention, are also comprised in the present invention. Preferably, a cell
transformed by a vector according to the invention is cultured under conditions that
allow the expression of the aforesaid polypeptide and recovery of said recombinant
peptide.
As already mentioned, the host cell can be selected among prokaryotic or
eukaryotic systems. In particular, it is possible to identify the nucleotide sequences of
the invention that facilitate secretion in such a prokaryotic or eukaryotic system. A
vector according to the invention carrying such a sequence can thus be used
advantageously for the production of recombinant proteins to be secreted. Indeed, the
purification of these recombinant proteins of interest will be facilitated by the fact that
they are present in the supernatant of the cellular culture rather than inside host cells.
The polypeptides of the invention can also be prepared by chemical synthesis.
One such method of preparation is also an object of the invention. A person skilled in
the art knows methods for chemical synthesis, such as solid-phase techniques (see
notably Steward et al, 1984, Solid phase peptides synthesis, Pierce Chem. Company,
Rockford, 1 1 1, 2nd ed.) or partial solid-phase techniques, by condensation of fragments
or by conventional synthesis in solution. Polypeptides obtained by chemical synthesis
and capable of containing corresponding unnatural amino acids are also comprised in
the invention.
The antibodies, or the derived compounds or functional fragments of same,
likely to be obtained by the method of the invention are also comprised in the present
invention.
According to still another aspect, the present invention relates to an antibody as
described above, characterized in that it is, in addition, capable of specifically binding to
a human chemokine family receptor and/or capable of specifically inhibiting the
signaling of such a receptor.
According to a novel embodiment, the invention relates to an antibody, or its
derived compounds or functional fragments, consisting of an antibody that is bispecific
in the sense that it comprises a second motif capable of interacting with any receptor
implicated in the development of tumors, such as, for example, VEGFR, VEGF, EGFR,
IGF-1R, HER2neu, HGF, cMET, FGF, tetraspanins, integrins, CXCR4 (other than the
antibody of the present invention, i.e. targeting another epitope), CXCR7 or CXCR2.
The bispecific or bifunctional antibodies constitute a second generation of
monoclonal antibodies in which two different variable regions are combined in the same
molecule (Hollinger and Bohlen, 1999, Cancer and metastasis, rev. 18:41 1-419). Their
utility was demonstrated in both diagnostic and therapeutic domains relative to their
capacity to recruit new effector functions or to target several molecules on the surface of
tumor cells; such antibodies can be obtained by chemical methods (Glennie MJ et al,
1987, J. Immunol. 139, 2367-2375; Repp R. et al, 1995, J . Hemat, 377-382) or
somatic methods (Staerz U.D. and Bevan M.J., 1986, PNAS 83, 1453-1457; Suresh
M.R. et al, 1986, Method Enzymol., 121:210-228) but also, preferentially, by genetic
engineering techniques that make it possible to force heterodimerization and thus
facilitate the purification of the antibody sought (Merchand et al, 1998, Nature
Biotech., 16:677-681).
These bispecific antibodies can be constructed as whole IgG, bispecific Fab'2,
Fab'PEG, diabodies or bispecific scFv, but also as a tetravalent bispecific antibody in
which two binding sites are present for each antigen targeted (Park et al, 2000, Mol.
Immunol., 37(1 8): 1123-30) or the fragments of same as described above.
In addition to an economic advantage given that the production and
administration of a bispecific antibody are cheaper than the production of two specific
antibodies, the use of such bispecific antibodies has the advantage of reducing the
treatment's toxicity. Indeed, the use of a bispecific antibody makes it possible to
decrease the overall quantity of circulating antibodies and, consequently, possible
toxicity.
In a preferred embodiment of the invention, the bispecific antibody is a bivalent
or tetravalent antibody.
Lastly, the present invention relates to the antibody described above, or its
derived compounds or functional fragments, for use as a drug.
The invention also relates to a pharmaceutical composition comprising as an
active ingredient a compound consisting of an antibody of the invention, or one of its
derived compounds or functional fragments. Preferably, said antibody is supplemented
by an excipient and/or a pharmaceutically acceptable carrier.
The invention also relates to a composition characterized in that it comprises, in
addition, as a combination product for use in a simultaneous, separated or extended
fashion, an anti-tumor antibody other that an antibody directed against CXCR4.
According to still another embodiment, the present invention also relates to a
pharmaceutical composition as described above that comprises at least a second
antitumor compound selected among the compounds capable of specifically inhibiting
the tyrosine kinase activity of receptors such as IGF-IR, EGFR, HER2/neu, cMET,
VEGFR or VEGF, or any other antitumor compound known to a person skilled in the
art.
In a second preferred aspect of the invention, said second compound can be
selected among the antibodies antiEGFR, antilGF-IR, antiHER2/neu, anticMET,
VEGFR, VEGF, etc., isolated, or their functional fragments and derived compounds,
capable of inhibiting the proliferative and/or anti-apoptotic and/or angiogenic and/or
inductive activity of metastatic dissemination promoted by said receptors.
Also suitable for mention are antiCD20 antibodies such as a rituximab,
ibritumomab or tositumomab; antiCD33 antibodies such as gemtuzumab or lintuzumab;
antiCD22 antibodies such as epratuzumab; antiCD52 antibodies such as alemtuzumab;
antiEpCAM antibodies such as edrecolomab, Ch 17-1A or IGN-101; antiCTP21 or 16
antibodies such as Xactin; antiDNA-Ag antibodies such as I-Cotara TNT-1;
antiMUCl antibodies such as pemtumomab or R1150; antiMUC18 antibodies such as
ABX-MAl; antiGD3 antibodies such as mitumomab; antiECA antibodies such as
CeaVac or labetuzumab; antiCA125 antibodies such as OvaRex; antiHLA-DR
antibodies such as apolizumab; antiCTLA4 antibodies such as MDX-010; antiPSMA
antibodies such as MDX-070, In & Y-J591, 1 Lu J591, J591-DM1; antiLewis Y
antibodies such as IGN311; antiangiogenesis antibodies such as AS1405 and
90YmuBCl; antiTrail-Rl antibodies such as TRAIL RlmAb or TRAIL R2mAb.
Another embodiment complementary to the invention consists of a composition
as described above comprised of, in addition, as a combination or conjugaison product
for simultaneous, separated or extended use, a cytotoxic/cytostatic agent.
"Simultaneous use" means the administration of both compounds of the
composition comprised in a single dosage form.
"Separated use" means administration, at the same time, of both compounds of
the composition, comprised in distinct dosage forms.
"Extended use" means the successive administration of both compounds of the
composition, each comprised in a distinct dosage form.
Generally, the composition according to the invention considerably increases
cancer treatment effectiveness. In other words, the therapeutic effect of the antibody of
the invention is enhanced in an unexpected way by the administration of a cytotoxic
agent. Another major subsequent advantage produced by a composition of the invention
relates to the possibility of using lower effective doses of the active ingredient, thus
making it possible to avoid or reduce the risks of the appearance of side effects, in
particular the effect of the cytotoxic agent. Moreover, this composition makes it
possible to achieve the expected therapeutic effect more quickly.
"Therapeutic anticancer agent" or "cytotoxic agent" means a substance which,
when it is administered to a patient, treats or prevents the development of cancer in the
patient. Non-limiting examples of such agents include "alkylating" agents,
antimetabolites, antitumor antibiotics, mitotic inhibitors, inhibitors of chromatin
functioning, antiangiogenics, antiestrogens, antiandrogens and immunomodulators.
Such agents, for example, are cited in VIDAL, on the page devoted to
compounds related to oncology and hematology under the heading "Cytotoxic"; the
cytotoxic compounds cited by reference to this document are cited herein as preferred
cytotoxic agents.
"Alkylating agent" refers to any substance that can bind covalently with or can
alkylate any molecule, preferentially a nucleic acid (e.g., DNA), within a cell. Examples
of such alkylating agents include nitrogen mustards such as mechlorethamine,
chlorambucil, melphalan, chlorhydrate, pipobroman, prednimustine, disodium
phosphate or estramustine; oxazaphosphorines such as cyclophosphamide, altretamine,
trofosfamide, sulfofosfamide or ifosfamide; aziridines or ethylene-imines such as
thiotepa, triethyleneamine or altetramine; nitrosoureas such as carmustine,
streptozocine, fotemustine or lomustine; alkyl sulfonates such as busulfan, treosulfan or
improsulfan; triazenes such as dacarbazine; or platinum complexes such as cisplatine,
oxaliplatine or carboplatine.
"Antimetabolite" refers to a substance that blocks growth and/or cellular
metabolism by interfering with certain activities, generally DNA synthesis. Examples of
antimetabolites include methotrexate, 5-fluorouracile, floxuridine, 5-
fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-
mercaptopurine (6-MP), 6-thioguanine (6-TG), chlorodesoxyadenosine, 5-azacytidine,
gemcitabine, cladribine, deoxycoformycin and pentostatin.
"Antitumor antibiotic" refers to a compound that can prevent or inhibit the
synthesis of DNA, RNA and/or proteins. Examples of such antitumor antibiotics include
doxorubicin, daunorubicin, idarubicin valrubicin, mitoxantrone, dactinomycin,
mithramycin, plicamycin, mitomycin C, bleomycin and procarbazine.
"Mitotic inhibitors" prevent the normal progression of the cell cycle and mitosis.
In general, microtubule inhibitors or "taxoids" such as paclitaxel and docetaxel are
capable of inhibiting mitosis. The vinca alkaloids, such as vinblastine, vincristine,
vindesine and vinorelbine, are also capable of inhibiting mitosis.
"Chromatin inhibitors" or "topoisomerase inhibitors" are substances that inhibit
the normal functioning of proteins that shape chromatin, such as topoisomerases I and
II. Examples of such inhibitors include, for topoisomerase I, camptothecine and its
derivatives, such as irinotecan or topotecan; for topoisomerase II, etoposide, etiposide
phosphate and teniposide.
An "antiangiogenic" is any drug, compound, substance or agent that inhibits the
growth of the blood vessels. Examples of antiangiogenics include, without being
limiting, razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-
27023A, halofuginone, COL-3, neovastat, BMS-275291, thalidomide, CDC 501,
DMXAA, L-651582, squalamine, endostatine, SU5416, SU6668, interferon-alpha,
EMD121974, interleukin-12, IM862, angiostatin and vitaxin.
"Antiestrogen" or "estrogen antagonist" refers to any substance that decreases,
antagonizes or inhibits estrogen action. Examples of such agents are tamoxifene,
toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole and exemestane.
"Antiandrogen" or "androgen antagonist" refers to any substance that reduces,
antagonizes or inhibits androgen action. Examples of antiandrogens include flutamide,
nilutamide, bicalutamide, sprironolactone, cyproterone acetate, finasteride and
cimitidine.
Immunomodulators are substances that stimulate the immune system. Examples
of immunomodulators include interferon, interleukins such as aldesleukin, OCT-43,
denileukin diftitox or interleukine-2, tumor necrosis factors such as tasonermine, or
other types of immunomodulators such as lentinan, sizofiran, roquinimex, pidotimod,
pegademase, thymopentine, poly I:C or levamisole in combination with 5-fluorouracil.
For further details, a person skilled in the art can refer to the manual published
by the French Association of Therapeutic Chemistry Teachers titled "Therapeutic
chemistry, vol. 6, Antitumor drugs and perspectives in the treatment of cancer, TEC and
DOC edition, 2003 [in French]".
In a particularly preferred embodiment, said composition of the invention as a
combination product is characterized in that said cytotoxic agent is bound chemically to
said antibody for use simultaneously.
In a particularly preferred embodiment, said composition is characterized in that
said cytotoxic/cytostatic agent is selected among the spindle inhibitors or stabilizers,
preferably vinorelbine and/or vmflunine and/or vincristine.
In order to facilitate binding between said cytotoxic agent and the antibody
according to the invention, spacer molecules can be introduced between the two
compounds to bind, such as the poly(alkylene)glycol polyethyleneglycol or the amino
acids; or, in another embodiment, said cytotoxic agents' active derivatives, into which
have been introduced functions capable of reacting with said antibody, can be used.
These binding techniques are well-known to a person skilled in the art and will not be
discussed in more detail in the present description.
Other EGFR inhibitors include, without being limiting, monoclonal antibodies
C225 and antiEGFR 22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell
Genesys), EMD-7200 (Merck gaA) or compounds ZD-1834, ZD-1838 and ZD-1839
(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787
(Novartis), CP 701 (Cephalon), flunomide (Pharmacia/Sugen), CI-1033 (Warner
Lambert Parke Davis), CI-1033/PD 183, 805 (Warner Lambert Parke Davis), CL-387,
785 (Wyeth-Ayerst), BBR-1611 (Boehringer Mannheim GMBH/Roche), Naamidine A
(Bristol-board Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer
Ingelheim), OLX-103 (Merck & Co), VRCTC-310 (Ventech Research), EGF fusion
toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808 (Imperial Cancer
Research Fund), RG-50864 (INSERM), LFM-A12 (Parker Hughes Center Cancer),
WHI-P97 (Parker Hughes Center Cancer), GW-282974 (Glaxo), KT-8391 (Kyowa
Hakko) or the "EGFR vaccine" (York Medical/Centro of Immunologia Molecular).
Another aspect of the invention relates to a composition characterized in that at
least one of said antibodies, or of the derived compounds or functional fragments of
same, is combined or conjugated with a cellular toxin and/or a radioisotope.
Preferably, said toxin or said radioisotope is capable of preventing the growth or
proliferation of the tumor cell, notably of completely inactivating said tumor cell.
Also preferably, said toxin is an enterobacteria toxin, notably Pseudomonas
exotoxin A.
The radioisotopes preferentially combined with therapeutic antibodies are
radioisotopes that emit gamma rays, preferentially iodine 131 , yttrium90, gold199,
palladium100, copper67, bismuth217 and antimony2 . Radioisotopes that emit alpha and
beta rays can also be used in therapy.
"Toxin or radioisotope combined with at least one antibody of the invention, or a
functional fragment of same" refers to any means that makes it possible to bind said
toxin or said radioisotope to that at least one antibody, notably by covalent binding
between the two compounds, with or without the introduction of the binding molecule.
Examples of agents that allow chemical (covalent), electrostatic, or non-covalent
bonding of all or part of the conjugate's elements include, in particular, benzoquinone,
carbodiimide and more particularly EDC (l-ethyl-3-[3-dimethyl-aminopropyl]-
carbodiimide-hydrochloride), dimaleimide, dithiobis-nitrobenzoic (DTNB) acid, Nsuccinimidyl
S-acetyl thio-acetate (SATA), bridging agents with one or more groups,
with one or more phenylaside groups, reacting with ultraviolet (UV) rays, most
preferentially N-[-4 (azidosalicylamino)butyl]-3'-(2'-pyridyldithio)-propionamide
(APDP), N-succinimid-yl 3(2-pyridyldithio) propionate (SPDP) and 6-hydrazinonicotinamide
(HYNIC).
Another form of binding, notably for radioisotopes, can consist of the use of
bifunctional ion chelating agents.
Examples of such chelators include the chelators derived from EDTA
(ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaacetic acid) which
were developed to bind metals, particularly radioactive metals, with immunoglobulins.
Thus, DTPA and its derivatives can be substituted on the carbon chain by various
groups in such a way as to increase the stability and the rigidity of the ligand-metal
complex (Krejcarek et al, 1977; Brechbiel et al, 1991; Gansow, 1991; US patent
4,831,175).
For example, DTPA (diethylenetriaminepentaacetic acid) and its derivatives,
which long have been widely used in drug and biology either in its free form or in a
complex with a metal ion, exhibit the remarkable characteristic of forming stable
chelates with metal ions which can be coupled with proteins of therapeutic or diagnostic
interest, such as antibodies, for the development of radio-immuno conjugates for cancer
therapy (Meases et al, 1984; Gansow et al, 1990).
Also preferably, said at least one antibody of the invention forming said
conjugate is selected among its functional fragments, notably fragments that have lost
their Fc component, such as scFv fragments.
The present invention also comprises the use of the composition for the
preparation of a drug intended for the prevention or the treatment of cancer.
The present invention also relates to the use of an antibody, or a derived
compound or functional fragment of same, preferably humanized, and/or of a
composition according to the invention for the preparation of a drug for inhibiting the
growth of tumor cells. Generally, the present invention relates to the use of an antibody,
or a derived compound or functional fragment of same, preferably humanized, and/or of
a composition, for the preparation of a drug for cancer prevention or treatment.
Preferred cancers that can be prevented and/or treated include prostate cancer,
osteosarcoma, lung cancer, breast cancer, endometrial cancer, colon cancer, multiple
myeloma, ovarian cancer, pancreatic cancer or any other cancer.
The invention also concerns the use of an antibody, or a derived compound or
functional fragment of same, and/or of a composition as above described for the
preparation of a drug for modulating CXCR4 activity in a cell.

CLAIMS
1. A humanized antibody heavy chain having CDRs consisting of CDR-H1,
CDR-H2 and CDR-H3, said CDR-H1, CDR-H2 and CDR-H3 comprising respectively
the sequences SEQ ID Nos. 4, 5 and 6.
2. A humanized antibody heavy chain according to claim 1, characterized in that
it comprises a variable region of sequence selected from the group consisting of SEQ ID
Nos. 10, 11, 12, 13, 85 or 87.
3. A humanized antibody heavy chain according to claim 1 or 2, characterized in
that it comprises the complete sequence selected from the group consisting of SEQ ID
Nos. 21, 22, 23, 24, 89 or 91.
4. A humanized antibody light chain having CDRs consisting of CDR-L1, CDRL2
and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 comprising respectively the
sequences SEQ ID Nos. 7, 8 and 9.
5. A humanized antibody light chain according to claim 4, characterized in that
it comprises a variable region of sequence selected from the group consisting of SEQ ID
Nos. 14, 15, 16, 17, 18, 19, 20, 86 or 88.
6. A humanized antibody light chain according to claim 4 or 5, characterized in
that it comprises the complete sequence selected from the group consisting of SEQ ID
Nos. 25, 26, 27, 28, 29, 30, 31, 90 or 92.
7. A humanized antibody, or a derived compound or functional fragment of
same, characterized in that the said humanized antibody comprises heavy and light
chains, said heavy chain having CDRs consisting of CDR-H1, CDR-H2 and CDR-H3,
and the said light chain having CDRs consisting of CDR-L1, CDR-L2 and CDR-L3,
wherein said CDR-H1, CDR-H2 and CDR-H3 comprise respectively the sequences
SEQ ID Nos. 4, 5 and 6, and said CDR-L1, CDR-L2 and CDR-L3 comprise
respectively the sequences SEQ ID Nos. 7, 8 and 9.
8. A humanized antibody, or a derived compound or functional fragment of
same, according to claim 7, characterized in that it comprises a heavy chain variable
region of sequence selected from the group consisting of SEQ ID Nos. 10, 11, 12, 13,
83, 85 or 87 and a light chain variable region of sequence selected from the group
consisting of SEQ ID Nos. 14, 15, 16, 17, 18, 19, 20, 84, 86 or 88.
9. A humanized antibody, or a derived compound or functional fragment of
same, according to claim 7 or 8, characterized in that it comprises a heavy chain of
sequence selected from the group consisting of SEQ ID Nos. 21, 22, 23, 24, 89 or 9 1
and a light chain of sequence selected from the group consisting of SEQ ID Nos. 25, 26,
27, 28, 29, 30, 31, 90 or 92.
10. A humanized antibody, or a derived compound or functional fragment of
same, according to claim 7, selected from the group consisting of:
- humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
11, and a light chain variable region of sequence SEQ ID No. 16;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 22, and a light
chain of sequence SEQ ID No. 27;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
11, and a light chain variable region of sequence SEQ ID No. 7;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 22, and a light
chain of sequence SEQ ID No. 28;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
11, and a light chain variable region of sequence SEQ ID No. 18;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 22, and a light
chain of sequence SEQ ID No. 29;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
11, and a light chain variable region of sequence SEQ ID No. 19;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 22, and a light
chain of sequence SEQ ID No. 30;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
12, and a light chain variable region of sequence SEQ ID No. 14;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 23, and a light
chain of sequence SEQ ID No. 25;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
12, and a light chain variable region of sequence SEQ ID No. 15;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 23, and a light
chain of sequence SEQ ID No. 26;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
10, and a light chain variable region of sequence SEQ ID No. 14;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 21, and a light
chain of sequence SEQ ID No. 25;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
10, and a light chain variable region of sequence SEQ ID No. 88;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 21, and a light
chain of sequence SEQ ID No. 92;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
11, and a light chain variable region of sequence SEQ ID No. 88;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 22, and a light
chain of sequence SEQ ID No. 92;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
1 , and a light chain variable region of sequence SEQ ID No. 88;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 23, and a light
chain of sequence SEQ ID No. 92;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
13, and a light chain variable region of sequence SEQ ID No. 88;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 24, and a light
chain of sequence SEQ ID No. 92;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 14;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 25;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 15;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 26;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 16;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 27;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 17;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 28;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 18;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 29;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 19;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 30;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain variable region of sequence SEQ ID No.
87, and a light chain variable region of sequence SEQ ID No. 20;
- a humanized antibody, or a derived compound or functional fragment of same,
characterized in that it comprises a heavy chain of sequence SEQ ID No. 91, and a light
chain of sequence SEQ ID No. 31.
11. An isolated nucleic acid molecule characterized in that it is selected among
the following nucleic acids:
a) a nucleic acid, DNA or RNA, coding for a humanized antibody heavy chain, or for a
derived compound or functional fragment of same, according to one of the claims 1, 2
or 3;
b) a nucleic acid, DNA or RNA, coding for a humanized antibody light chain, or for a
derived compound or functional fragment of same, according to one of the claims 4, 5
or 6;
c) a nucleic acid, DNA or RNA, coding for a humanized antibody, or for a derived
compound or functional fragment of same, according to one of the claims 7 to 10;
d) a nucleic acid complementary to a nucleic acid as defined in a), b) or c);
e) a nucleic acid of at least 8 nucleotides capable of hybridizing under highly stringent
conditions with at least one of the 3 CDRs of a heavy chain comprising the nucleic acid
sequences SEQ ID Nos. 38 to 41, 49 to 52, 93 or 95; and
f a nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent
conditions with at least one of the 3 CDRs of a light chain comprising the nucleic acid
sequences SEQ ID Nos. 42 to 48, 53 to 59, 94 or 96.
12. An isolated nucleic acid molecule according to claim 11 comprising a
nucleic acid sequence selected from the group consisting of:
- a nucleic acid sequence encoding a heavy chain variable region of a humanized
antibody, said heavy chain variable region nucleotide sequence comprising a CDR-H1
nucleotide sequence of SEQ ID No. 32, a CDR-H2 nucleotide sequence of SEQ ID No.
33 and a CDR-H3 nucleotide sequence of SEQ ID No. 34;
- a nucleic acid sequence encoding a light chain variable region of a humanized
antibody, said light chain variable region nucleotide sequence comprising a CDR-L1
nucleotide sequence of SEQ ID No. 35 or 60, a CDR-L2 nucleotide sequence of SEQ
ID No. 36 or 61, and a CDR-L3 nucleotide sequence of SEQ ID No. 37 or 62; and
- a nucleic acid sequence encoding a nucleic acid sequence encoding a heavy chain
variable region and a light chain variable region of a humanized antibody,
i) said heavy chain variable region nucleotide sequence comprising a CDR-H1
nucleotide sequence of SEQ ID No. 32, a CDR-H2 nucleotide sequence of SEQ ID No.
33 and a CDR-H3 nucleotide sequence of SEQ ID No. 34; and
ii) said light chain variable region nucleotide sequence comprising a CDR-L1
nucleotide sequence of SEQ ID No. 35 or 60, a CDR-L2 nucleotide sequence of SEQ
ID No. 36 or 61, and a CDR-L3 nucleotide sequence of SEQ ID No. 37 or 62.
13. A vector composed of a nucleic acid according to any of the claims 11 and
12.
14. A host cell comprising a vector according to claim 13.
15. A transgenic animal, except for man, comprising a cell transformed by a
vector according to claim 14.
16. A method for producing a humanized antibody, or a derived compound or
functional fragment of same, characterized in that said method comprises the following
steps:
- the culture in a medium of and the suitable culture conditions for a host cell
according to claim 14; and
- the recovery of said antibody, or one of its functional fragments, thus produced
from the culture medium or from said cultured cells.
17. A humanized antibody, or a derived compound or functional fragment of
same, according to one of the claims 7 to 10, for use as a drug.
18. A composition comprising as an active ingredient a compound consisting of
a humanized antibody heavy chain according to claim 1 to 3 and/or a humanized
antibody light chain according to claims 4 to 6.
19. A composition according to claim 18 comprising as an active ingredient a
compound consisting of a humanized antibody, or a derived compound or functional
fragment of same, according to one of the claims 7 to 10 and 17.
20. A composition according to claim 18 or 19, characterized in that it
comprises, in addition, as a combination product for use in a simultaneous, separated or
extended fashion, an anti-tumor antibody other that an antibody directed against
CXCR4.
21. A composition according to one of the claims 18 to 20, characterized in that
it comprises, in addition, as a combination or conjugation product for use in a
simultaneous, separated or extended fashion, a cytotoxic/cytostatic agent, a cellular
toxin and/or a radioisotope.
22. A composition according to one of the claims 18 to 21, for use as a drug.
23. A humanized antibody heavy chain according to any of the claims 1 to 3
and/or a humanized antibody light chain according to any of the claims 4 to 6 and/or a
humanized antibody, or a derived compound or functional fragment of same, according
to any of the claims 7 to 10 or 17, and/or of a composition according to any of the
claims 18 to 22, for the prevention or treatment of cancer.
24. A humanized antibody heavy and/or light chain(s) and/or a humanized
antibody, or a derived compound or functional fragment of same, and/or a composition
according to claim 23, characterized in that said cancer is a cancer selected among
prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, multiple
myeloma, ovarian cancer, pancreatic cancer and colon cancer.
25. A process of detecting in vitro the presence and/or the location of a CXCR4
expressing tumor in a subject, wherein said process comprises the steps of:
(a) contacting a sample from the subject with a humanized antibody heavy chain
according to one of the claims 1 to 3 and/or a humanized antibody light chain according
to one of the claims 4 to 6 and/or a humanized antibody, or a derived compound or
functional fragment of same, according to one of the claims 7 to 10 or 17; and
(b) detecting the binding of said antibody with the sample.
26. A kit comprising at least a humanized antibody heavy chain according to one
of the claims 1 to 3 and/or a humanized antibody light chain according to one of the
claims 4 to 6 and/or a humanized antibody, or a derived compound or functional
fragment of same, according to one of the claims 7 to 10 or 17, said antibody being
preferably labeled.