ANTIGEN BINDING PROTEIN AND ITS USE AS ADDRESSING
PRODUCT FOR THE TREATMENT OF CANCER
The present invention relates to a novel antigen binding protein, in particular a
monoclonal antibody, capable of binding specifically to the protein Axl as well as the
amino and nucleic acid sequences coding for said protein. From one aspect, the
invention relates to a novel antigen binding protein, or antigen binding fragments,
capable of binding specifically to Axl and, by inducing internalization of Axl, being
internalized into the cell. The invention also comprises the use of said antigen binding
protein as an addressing product in conjugation with other anti-cancer compounds, such
as toxins, radio-elements or drugs, and the use of same for the treatment of certain
cancers.
"Axl" (also referred to as "Ufo", "Ark" or "Tyro7") was cloned from patients
with chronic myeloid leukemia as an oncogene triggering the transformation when overexpressed
by mouse NIH3T3. It belongs to a family of receptor tyrosine kinases (RTKs)
called the TAM (Tyro3, Axl, Mer) family, which includes Tyro3 (Rse, Sky, Dtk, Etk,
Brt, Tif), Axl, and Mer (Eyk, Nyk, Tyro-12) [Lemke G. Nat. Rev. Immunol. (2008). 8,
327-336].
The human protein Axl is a 894 amino acids protein which sequence is
represented in the sequence listing as SEQ ID NO. 29. Amino acids 1-25 corresponding
to the signal peptide, the human protein Axl, without the said peptide signal, is
represented in the sequence listing as SEQ ID NO. 30.
Gas6, originally isolated as growth arrest-specific gene, is the common ligand
for the members of the TAM family [Varnum B.C. et al. Nature (1995).373, 623-626].
Gas6 exhibits the highest affinity for Axl, followed by Tyro3 and finally by Mer
[Nagata K. et al. J . Biol. Chem. (1996).271, 30022-30027]. Gas6 consists in a g -
carboxyglutamate (Gla)-rich domain that mediates binding to phospholipid membranes,
four epidermal growth factor-like domains, and two laminin G-like (LG) domains
[Manfioletti G., Brancolini,C, Avanzi,G. & Schneider,C. Mol. Cell Biol. (1993). 13,
4976-4985]. As many other RTKs, ligand binding results in receptor dimerization and
autophosphorylation of tyrosine residues (tyrosine residues 779, 821 and 866 for the
receptor Axl) which serve as docking sites for a variety of intracellular signaling
molecules [Linger R.M. Adv. Cancer Res. (2008). 100, 35-83]. Moreover, the Axl
receptor can be activated through a ligand-independent process. This activation can
occur when the Axl receptor is overexpressed.
Gas6/Axl signaling has been shown to regulate various cellular processes
including cell proliferation, adhesion, migration and survival in a large variety of cells
in vitro [Hafizi S. & Dahlback,B. FEBS J . (2006).273, 5231-5244]. In addition, the
TAM receptors are involved in the control of innate immunity; they inhibit the
inflammatory responses to pathogens in dendritic cells (DCs) and macrophages. They
also drive phagocytosis of apoptotic cells by these immune cells and they are required
for the maturation and killing activity of natural killer (NK) cells [Lemke G. Nat. Rev.
Immunol. (2008).8, 327-336].
Weakly expressed on normal cells, it is predominantly observed in fibroblasts,
myeloid progenitor cells, macrophages, neural tissues, cardiac and skeletal muscle
where it supports mainly cell survival. The Gas6/Axl system plays an important role in
vascular biology by regulating vascular smooth muscle cell homeostasis [Korshunov
V.A., Mohan, A.M., Georger, M.A. & Berk, B.C. Circ. Res. (2006).98, 1446-1452;
Korshunov V.A., Daul, M., Massett, M.P. & Berk, B.C. Hypertension (2007).50, 1057-
1062].
In tumor cells, Axl plays an important role in regulating cellular invasion and
migration. Over-expression of Axl is associated not only with poor prognosis but also
with increased invasiveness of various human cancers as reported for breast, colon,
esophageal carcinoma, hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma,
ovarian, prostate, rhabdomyosarcoma, renal, thyroid and uterine endometrial cancer
[Linger R.M. Adv. Cancer Res. (2008). 100, 35-83 and Verma A. Mol. Cancer Ther.
(201 1). 10, 1763-1773, for reviews]. In breast cancer, Axl appears to be a strong effector
of the Epithelial-to-mesenchymal transition (EMT); EMT program contributes actively
to migration and dissemination of cancer cells in the organism [Thiery J.P. Curr. Opin.
Cell Biol. (2003).15, 740-746].
Axl has also been shown to regulate angiogenesis. Indeed knockdown of Axl in
endothelial cells impaired tube formation and migration [Holland S.J. et al. Cancer Res.
(2005). 65, 9294-9303] as well as disturbed specific angiogenic signaling pathways [Li
Y. et al. Oncogene (2009).28, 3442-3455].
More recently several studies on a range of cellular models described the
involvement of an Axl overexpression in drug resistance phenomena. The following
table 1 summarized these studies.
Table 1
Reference Cancer type Therapeutic agent Cellular model
Macleod et al., 2005 Ovarian cancer Cisplatin PE01/PE01CDDP
Imatinib
Mahadevan et al.,
GIST inhibitor of c - GIST882 >GIST-R
2007
kit/PDGFR
CL- 1 clones
Lay et al., 2007 NSCLC Doxorubicin
CL1-5F4 clones
Hong et al., 2008 AML Doxoru bid n/Cisplati n U937
Lapatinib
HER2 (+) BT474
Liu et al., 2009 Breast Cancer (HER1 and HER2
(J4)
inhibitor)
Temozolomide
G12
Keating et al., 2010 Astrocytoma Carboplatin
A172
Vincristin
Ye et al. , 2010
NSCLC Erlotinib HCC827
Complete references cited in table 1 above are as follow:
- Macleod, K. et al. Cancer Res. (2005).65, 6789-6800
- Mahadevan D. et al. Oncogene (2007).26, 3909-3919
- Lay J.D. et al. Cancer Res. (2007).67, 3878-3887
- Hong C.C. et al. Cancer Lett. (2008).268, 314-324
- Liu L. et al. Cancer Res. (2009).69, 6871-6878
- Keating A.K. et al. Mol. Cancer Ther. (2010).9, 1298-1307
- Ye X. et al. Oncogene (2010).29, 5254-5264
In such a context the Axl RTK is considered as an interesting target in oncology.
Several groups already developed anti-tumoral strategies targeting the gas6/Axl axis,
either using naked monoclonal antibodies or targeted small molecules [Verma A. Mol.
Cancer Ther. (201 1). 10, 1763-1773].
In a first embodiment, the invention relates to an antigen binding protein, or an
antigen binding fragment thereof, which i) specifically binds to the human protein Axl,
and ii) is internalized following its binding to said human protein Axl.
More generally, the invention relates to the use of the protein Axl for the
selection of an antigen binding protein, or an antigen binding fragment thereof, capable
of being internalized following its binding to the said target Axl. More particularly, the
said target is the extracellular domain of Axl.
In this particular aspect, the present invention is thus directed to an in vitro
method for the screening of a compound, or a binding fragment thereof, capable of
delivering or internalizing a molecule of interest into mammalian cells, said molecule of
interest being covalently linked to said compound, wherein said method comprises the
following steps of:
a) selecting a compound which is capable of specifically binding the Axl
protein, or the extracellular domain (ECD) thereof, or an epitope thereof ;
b) optionally, covalently linking said molecule of interest, or a control
molecule, to said compound selected in step a) to form a complex;
c) contacting said compound selected in step a), or said complex obtained in
step b), with a mammal cell, preferably viable cell, expressing at its surface
the Axl protein, or a functional fragment thereof;
d) determining whether said compound, or said molecule of interest or said
complex, has been intracellularly delivered or internalized into said
mammalian cell; and
e) selecting said compound as a compound capable of delivering or
internalizing a molecule of interest into a viable mammalian cell .
In a preferred embodiment, said compound capable of delivering or internalizing a
molecule of interest into a viable mammalian cell is a protein (also designated herein
polypeptide or peptide) or a protein-like compound comprising a peptidic structure,
particularly an amino-acid sequence of at least 5, 10, 15 or more amino acids residues,
said amino-acid residue(s) can be glycosylated.
When said compound capable of delivering or internalizing a molecule of
interest into a viable mammalian cell is a protein or a protein-like compound, said
compound is also called herein an "antigen binding protein", said antigen binding
protein, or binding fragment thereof, can:
- i) specifically binds to the protein Axl, preferably the human Axl protein, and
- ii) is internalized into a mammalian cell following its binding to said protein Axl when
said Axl protein is expressed at the surface of said mammalian cell.
In a preferred embodiment, said mammalian viable cell is a human cell, preferably
a cell naturally expressing the Axl protein receptor.
In a particular embodiment, the mammalian viable cells in step c) are mammalian
cells which express recombinant Axl protein(s) at their surface.
In an also preferred embodiment, said molecule of interest is a cytotoxic molecule
(also designated herein as cytoxic or cytostatic agent).
In an also preferred embodiment, said molecule of interest is covalently linked to
said compound capable of binding the Axl protein using a linker, more preferably a
peptidic linker, more preferably a cleavable peptidic linker, more preferably a linker
which can be cleaved by natural intracellular compounds contained in the mammalian
cell, particularly in the cytosol of said mammalian cell.
In an also preferred embodiment, said compound capable of binding the Axl
protein is an antibody, or functional binding fragment thereof, which is specifically
directed against the Axl protein, or against an epitope thereof located into the Axl EDC
domain.
The selection step of e) can be realized by any method known by the person
skilled in the art for the evaluation of the intracellular delivering or internalization.
Assay or test capable of demonstrating or evaluating the presence, absence, or the
activity of said compound capable of specifically binding the Axl protein, or of said
complex formed by said compound and said molecule of interest, or of said molecule of
interest which is covalently linked to said compound, are well known by the skilled
person (see some examples of such test or assay disclosed hereinafter, without limiting
these tests to these following test examples).
More particularly, these tests or assays can be realized by FACS,
Immunofluorescence, flow cytometry, western-blot, cytotoxicity/cytostatic evaluations,
etc. . .
In this aspect, the present invention is also directed to an in vitro method for the
preparation of a cytotoxic or cytostatic complex capable of delivering a cytotoxic
compound into a mammalian cell, preferably a viable cell, said method comprising the
step of:
- covalently linked a cytotoxic agent to a compound which is:
- i) capable of specifically binding the Axl protein, preferably the human Axl
protein, and
- ii) is internalized into a mammalian cell following its binding to said protein
Axl when said Axl protein is expressed at the surface of said mammalian cell.
Preferably said compound is a protein-like protein, more preferably an antibody
which is specifically directed against the Axl protein, or against an epitope thereof
located into the Axl EDC domain, or a functional binding fragment of said antibody.
In preferred embodiment, said cytotoxic agent is covalently linked to the said
anti-Axl antibody or functional fragment thereof, using a linker, more preferably a
peptidic linker, more preferably a cleavable peptidic linker, more preferably a linker
which can be cleaved, as non limitative example by natural intracellular compounds.
Like the other members of the TAM family, the Axl extracellular domain (ECD)
has an organization closed to those of cell adhesion molecules. Axl ECD i s
characterized by a combination of two immunoglobulin-like domains followed by two
adjacent fibronectin type Ill-like domains [O'Bryan J.P. et al. Mol. Cell Biol.
(1991). 11, 5016-5031]. The two N-terminal immunoglobulin-like domains are sufficient
for Gas6 ligand binding [Sasaki T. et al. EMBO J . (2006).25, 80-87].
The ECD of the human protein Axl is a 451 amino acids fragment,
corresponding to amino acids 1-451 of the sequence SEQ ID NO. 29, which sequence is
represented in the sequence listing as SEQ ID NO. 31. Amino acids 1-25 corresponding
to the signal peptide, the ECD of the human protein Axl without the signal peptide
corresponds to the amino acids 26-451 of the sequence SEQ ID NO.29, represented by
the sequence SEQ ID NO. 32.
To date different modes of internalization have been identified. They orientate
the becoming the internalized proteins or proteic complex in the cell. After endocytosis,
most membranes proteins or lipids returns to the cell surface (recycling), but some
membrane components are delivered to late endosomes or the Golgi [Maxfield F.R. &
McGraw, T.E. Nat. Rev. Mol. Cell Biol. (2004).5, 121-132].
In a preferred embodiment, the invention relates to an antigen binding protein,
or an antigen binding fragment thereof, which i) specifically binds to the human protein
Axl , and ii) is internalized following its binding to said human protein Axl, said antigen
binding protein comprising at least an amino acid sequence selected from the group
consisting of SEQ ID NOs. 1 to 14, or any sequence exhibiting at least 80%, preferably
85%, 90%, 95% and 98% identity with SEQ ID NOs. 1 to 14.
In a most preferred embodiment, the invention relates to an antigen binding
protein, or an antigen binding fragment thereof, which
i) specifically binds to the human protein Axl, preferably having the sequence
SEQ ID NO. 29 or 30 or natural variant sequence thereof, and
ii) is internalized following its binding to said human protein Axl,
said antigen binding protein comprising at least an amino acid sequence selected
from the group consisting of SEQ ID NOs. 1to 14.
A "binding protein" or "antigen binding protein" is a peptidic chain having a
specific or general affinity with another protein or molecule (generally referred as
antigen). Proteins are brought into contact and form a complex when binding is
possible. The antigen binding protein of the invention can preferably be, without
limitation, an antibody, a fragment or derivative of an antibody, a protein or a peptide.
By "antigen binding fragment" of an antigen binding protein according to the
invention, it is intended to indicate any peptide, polypeptide, or protein retaining the
ability to specifically bind to the target (also generally referred as antigen) of the antigen
binding protein and comprising an amino acid sequence of at least 5 contiguous amino
acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino
acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino
acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino
acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid
residues, at least contiguous 80 amino acid residues, at least contiguous 90 amino acid
residues, at least contiguous 100 amino acid residues, at least contiguous 125 amino
acid residues, at least 150 contiguous amino acid residues, at least contiguous 175
amino acid residues, at least contiguous 200 amino acid residues, or at least contiguous
250 amino acid residues of the amino acid sequence of the antigen binding protein.
In a preferred embodiment wherein the antigen binding protein is an antibody,
such "antigen binding fragments" are selected in the group consisting of Fv, scFv (sc
for single chain), Fab, F(ab') 2, Fab', scFv-Fc fragments or diabodies, or any fragment of
which the half-life time would have been increased by chemical modification, such as
the addition of poly(alkylene) glycol such as poly(ethylene) glycol ("PEGylation")
(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') 2-PEG or Fab'-PEG)
("PEG" for Poly(Ethylene) Glycol), or by incorporation in a liposome, said fragments
having at least one of the characteristic CDRs of the antibody according to the
invention. Preferably, said "antigen binding fragments" will be constituted or will
comprise a partial sequence of the heavy or light variable chain of the antibody from
which they are derived, said partial sequence being sufficient to retain the same
specificity of binding as the antibody from which it is descended and a sufficient
affinity, preferably at least equal to 1/100, in a more preferred manner to at least 1/10,
of the affinity of the antibody from which it is descended, with respect to the target.
Such a functional fragment will contain at the minimum 5 amino acids, preferably 10,
15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody from which
it is descended.
The term "epitope" is a region of an antigen that is bound by an antigen binding
protein, including antibodies. Epitopes may be defined as structural or functional.
Functional epitopes are generally a subset of the structural epitopes and have those
residues that directly contribute to the affinity of the interaction. Epitopes may also be
conformational, that is, composed of non-linear amino acids. In certain embodiments,
epitopes may include determinants that are chemically active surface groupings of
molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl
groups, and, in certain embodiments, may have specific three-dimensional structural
characteristics, and/or specific charge characteristics.
In the present application, the epitope is localized into the extracellular domain
of the human protein Axl.
According to a preferred embodiment of the invention, the antigen binding
protein, or an antigen binding fragment thereof, specifically binds to an epitope
localized into the human protein Axl extracellular domain, preferably having the
sequence SEQ ID NO. 3 1 or 32 or natural variant sequence thereof.
By "specifically binding", "specifically binds", or the like, it is intended that the
antigen binding protein, or antigen-binding fragment thereof, forms a complex with an
antigen that is relatively stable under physiologic conditions. Specific binding can be
characterized by an equilibrium dissociation constant of at least about 1.10 6 M or less.
Methods for determining whether two molecules specifically bind are well known in the
art and include, for example, equilibrium dialysis, surface plasmon resonance, and the
like. For the avoidance of doubt, it does not mean that the said antigen binding fragment
could not bind or interfere, at a low level, to another antigen. Nevertheless, as a
preferred embodiment, the said antigen binding fragment binds only to the said antigen.
In this sense, "EC 50" refers to 50% effective concentration. More precisely the
term half maximal effective concentration (EC 50) corresponds to the concentration of a
drug, antibody or toxicant which induces a response halfway between the baseline and
maximum after some specified exposure time. It is commonly used as a measure of
drug's potency. The EC50 of a graded dose response curve therefore represents the
concentration of a compound where 50% of its maximal effect is observed. The EC50 of
a quantal dose response curve represents the concentration of a compound where 50%>
of the population exhibits a response, after specified exposure duration. Concentration
measures typically follow a sigmoidal curve, increasing rapidly over a relatively small
change in concentration. This can be determined mathematically by derivation of the
best-fit line.
As a preferred embodiment, the EC50 determined in the present invention
characterized the potency of antibody binding on the Axl ECD exposed o human
tumor ceils. The EC50 parameter is determined using FACS analysis. The EC50
parameter reflects the antibody concentration for which 50% of the maximal binding on
the human Axl expressed on human tumor cells is obtained. Each EC50 value was
calculated as the midpoint of the dose response curve using a four-parameter regression
curve fitting program (Prism Software). This parameter has been selected as to be
representative of physiological/pathological conditions.
In an embodiment of the invention, the antigen binding protein, or an antigen
binding fragment thereof, binds to its epitope with an EC50 of at least 10 9 M,
preferentially between 10 9 and 10 12 M.
Another embodiment of the invention is a process or method for the selection of
an antigen binding protein, or an antigen binding fragment thereof, capable of being
intracellularly internalizing into a mammalian cell, preferably into a human cell,
preferably a viable cell, comprising the steps of:
- i) selecting antigen binding protein which specifically binds to Axl, preferably to its
EDC domain or to an epitope thereof; and
- ii) selecting said antigen binding protein from previous step i) which is internalized
into a mammalian cell following their binding to an Axl protein expressed at the surface
of said mammalian cell.
In a particular embodiment, said mammalian cell naturally expresses the Axl
protein receptor at their surface or are mammalian cells which express recombinant Axl
protein at their surface, preferably human cells.
Such method or process can comprise the steps of i) selecting antigen binding
protein which specifically bind to Axl with an EC50 of at least 10 9 M and ii) selecting
antigen binding protein from previous step which are internalized following their
binding to Axl. The selection step of ii) can be realized by any method known by the
person skilled in the art for the evaluation of the internalization. More particularly, tests
can be realized by FACS, Immunofluorescence, flow cytometry, western-blot,
cytotoxicity evaluations, etc. ..
Another characteristic of the antigen binding protein according to the invention
is that it does not have any significant activity on the proliferation of tumor cells. More
particularly, as illustrated in the following examples, the antigen binding protein
according to the invention does not have any significant in vitro activity on the
proliferation SN12C model.
In oncology, there are multiple mechanisms by which mAbs can exert
therapeutic efficacy, but often their activity is not sufficient to produce a lasting benefit.
Hence several strategies have been employed to enhance their activity particularly by
combining them with drugs as chemotherapeutic agents. As an efficient alternative to
combination protocols, immunotoxins become a novel therapeutic option for treating
cancer [Beck A. et al. Discov. Med. (2010). 10, 329-339; Alley S.C. et al. J . Pharmacol.
Exp. Ther. (2009).330, 932-938]. Antibody-drug conjugates (ADCs) represent one
approach where the ability to harness mAbs specificity and target the delivery of a
cytotoxic agent to the tumor may significantly enhance both mAbs and drug activities.
Ideally the mAb will specifically bind to an antigen with substantial expression on
tumor cells but limited expression on normal cells.
The present invention focused on a specific anti-Axl binding protein, and more
particularly on a specific anti-Axl antibody, presenting a high ability to be internalized
following Axl binding. Such antigen binding protein is interesting as one of the
immuno-drug-conjugate components, so it addresses the linked cytotoxic into the
targeted cancer cells. Once internalized the cytotoxic triggers cancer cell death.
Important keys to success with immunoconjugate therapy are thought to be the
target antigen specificity and the internalization of the antigen-binding protein
complexes into the cancer cells. Obviously non-internalizing antigens are less effective
than internalizing antigens to delivers cytotoxic agents. Internalization processes are
variable across antigens and depend on multiple parameters that can be influenced by
binding proteins. Cell-surface RTKs constitute an interesting antigens family to
investigate for such an approach.
In the biomolecule, the cytotoxic brings the cytotoxic activity and the used
antigen binding protein brings its specificity against cancer cells, as well as a vector for
entering within the cells to correctly address the cytotoxic.
Thus to improve the immunoconjugate molecule, the carrier-binding protein
must exhibit high ability to internalize into the targeted cancer cells. The efficiency with
which the binding proteins mediated internalisation differs significantly depending on
the epitope targeted. Selection of potent internalizing anti-Axl binding proteins requires
various experimental data studying not only Axl downregulation but also following
anti-Axl binding proteins becoming into the cells.
In a preferred embodiment, the internalization of the antigen binding protein
according to the invention can be evaluated preferably by immunofluorescence (as
exemplified hereinafter in the present application) or any method or process known by
the person skilled in the art specific for the internalization mechanism.
In another preferred embodiment, as the complex Axl-antigen binding protein,
according to the invention, is internalized after the binding of the binding protein of the
invention to the ECD of said Axl, a reduction in the quantity of Axl at the surface of the
cells is induced. This reduction can be quantified by any method known by the person
skilled in the art (western-blot, FACS, immunofluorescence, etc.).
In an embodiment of the invention, this reduction, thus reflecting the
internalization, can be preferably measured by FACS and expressed as the difference or
delta between the Mean Fluorescence Intensity (MFI) measured on untreated cells with
the MFI measured with cells treated with the antigen binding protein according to the
invention.
As non limitative example of the present invention, this delta is determined
based on MFIs obtained with untreated cells and cells treated with the antigen binding
protein of the invention as described in example 9 using i) human renal tumor SN12C
cells after a 24 hour incubation period with the antigen binding protein of the invention
and ii) a secondary antibody labelled with Alexa488. This parameter is defined as
calculated with the following formula:
D (MFI2 4h untreated cells - MFI24 antigen binding protein treated cells)
This difference between MFIs reflects the Axl downregulation as MFIs are proportional
of Axl expressed on the cell-surface.
In a more preferred and advantageous aspect, the antigen binding protein, or an
antigen binding fragment thereof, of the invention consists of a monoclonal antibody,
preferably an isolated Mab, triggering a D (MFI24 untreated cells - MFI24 treated
cells) of at least 200, preferably of at least 300.
The antigen binding protein, or an antigen binding fragment thereof,
according to the invention, induces a reduction of MFI of at least 200.
In more details, the above mentioned delta can be measured according to the
following process, which must be considered as an illustrative and non limitative
example:
a) Treating and incubating tumoral cells of interest with the antigen
binding protein of the invention;
b) Treating the treated cells of step a) and, in parallel, untreated cells
with the antigen binding protein of the invention,
c) Measuring the MFI (representative of the quantity of Axl present at
the surface) for the treated and the non treated cells with a secondary
labeled antibody capable of binding to the antigen binding protein,
and
d) Calculating the delta as the subtraction of the MFI obtained with the
treated cells from the MFI obtained with the non treated cells.
The terms "antibody", "antibodies" or "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal antibodies, preferably
isolated Mab, (e.g., full length or intact monoclonal antibodies), polyclonal antibodies,
multivalent antibodies or multispecific antibodies (e.g., bispecific antibodies so long as
they exhibit the desired biological activity).
More particularly, such molecule consists of a glycoprotein comprising at least
two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain comprises a heavy chain variable region (or domain) (abbreviated herein as
HCVR or VH) and a heavy chain constant region. The heavy chain constant region
comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain
variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
The light chain constant region comprises one domain, CL. The VH and VL regions can
be further subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1,
FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (e.g. effector cells) and the first
component (Clq) of the classical complement system.
Antibodies in the sense of the invention also include certain antibody fragments,
thereof. The said antibody fragments exhibit the desired binding specificity and affinity,
regardless of the source or immunoglobulin type (i.e., IgG, IgE, IgM, IgA, etc.), i.e.,
they are capable of binding specifically the Axl protein with an affinity comparable to
the full-length antibodies of the invention.
In general, for the preparation of monoclonal antibodies or their functional
fragments, especially of murine origin, it is possible to refer to techniques which are
described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726,
1988) or to the technique of preparation from hybridomas described by Kohler and
Milstein (Nature, 256:495-497, 1975).
The term "monoclonal antibody" or "Mab" as used herein refers to an antibody
molecule that is directed against a specific antigen and which may be produced by a
single clone of B cells or hybridoma. Monoclonal antibodies may also be recombinant,
i.e. produced by protein engineering. 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. The invention relates to antibodies isolated or obtained
by purification from natural sources or obtained by genetic recombination or chemical
synthesis.
A preferred embodiment of the invention is an antigen binding protein, or an
antigen binding fragment thereof, comprising or consisting of an antibody, said
antibody comprising the three light chain CDRs comprising the sequences SEQ ID
NOs. 1, 2 and 3, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95%
and 98% identity with SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRs
comprising the sequences SEQ ID NOs. 4, 5 and 6, or any sequence exhibiting at least
80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs. 4, 5 and 6.
In a more preferred embodiment of the invention, the antigen binding protein, or
an antigen binding fragment thereof, consists of an antibody, said antibody comprising
the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3; and the
three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6.
In a preferred aspect, by CDR regions or CDR(s), it is intended to indicate the
hypervariable regions of the heavy and light chains of the immunoglobulins as defined
by IMGT. Without any contradictory mention, the CDRs will be defined in the present
specification according to the IMGT numbering system.
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)].
It must be understood that, without contradictory specification in the present
specification, complementarity-determining regions or CDRs, mean the hypervariable
regions of the heavy and light chains of immunoglobulins as defined according to the
IMGT numbering system.
Nevertheless, CDRs can also be defined according to the Kabat numbering
system (Kabat et al, Sequences of proteins of immunological interest, 5th Ed., U.S.
Department of Health and Human Services, NIH, 1991, and later editions). There are
three heavy-chain CDRs and three light-chain CDRs. Here, the terms "CDR" and
"CDRs" are used to indicate, depending on the case, one or more, or even all, of the
regions containing the majority of the amino acid residues responsible for the
antibody's binding affinity for the antigen or epitope it recognizes.
According to the Kabat numbering system, the present invention relates to an
antigen binding protein, or an antigen binding fragment thereof, consisting of an
antibody, said antibody comprising the three light chain CDRs, as defined according to
Kabat numbering system, comprising the sequences SEQ ID NOs. 9, 10 and 11, or any
sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with
SEQ ID NOs. 9, 10 and 11; and the three heavy chain CDRs, as defined according to
Kabat numbering system, comprising the sequences SEQ ID NOs. 12, 13 and 14, or any
sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with
SEQ ID NOs. 12, 13 and 14.
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. 174:247-2 5 0 ) avai l a b l e o n the s ite
http://www.ncbi.nlm.nih.gov/gorf/bl2.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 9 8% 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 antigen binding proteins likely to b e
generated.
As a non-limiting example, table 2 below summarizes the possible substitutions
likely to be carried out without resulting in a significant modification of the biological
activity of the corresponding modified antigen binding protein; inverse substitutions are
naturally possible under the same conditions.
Table 2
An embodiment of the invention relates to an antigen binding protein, or an
antigen binding fragment thereof, comprising a light chain variable domain of sequence
SEQ ID NO. 7, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and
98% identity with SEQ ID NO. 7; and the three heavy chain CDRs comprising the
sequences SEQ ID NOs. 4, 5 and 6, or any sequence exhibiting at least 80%>, preferably
85%, 90%, 95% and 98% identity with SEQ ID NOs. 4, 5 and 6.
According to a preferred embodiment of the invention, the antigen binding
protein, or an antigen binding fragment thereof, comprises a light chain variable domain
of sequence SEQ ID NO. 7, or any sequence exhibiting at least 80%> identity with SEQ
ID NO.7; and the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5
and 6.
According to another preferred embodiment of the invention, the antigen
binding protein, or an antigen binding fragment thereof, comprises a light chain variable
domain of sequence SEQ ID NO. 7, or any sequence exhibiting at least 80%> identity
with SEQ ID NO.7.
Another embodiment of the invention relates to an antigen binding protein,
or an antigen binding fragment thereof, comprising the three light chain CDRs
comprising the sequences SEQ ID NOs. 1, 2 and 3, or any sequence exhibiting at least
80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NOs.l, 2 and 3; and a
heavy chain variable domain of sequence SEQ ID NO. 8, or any sequence exhibiting at
least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 8.
According to a preferred embodiment of the invention, the antigen binding
protein, or an antigen binding fragment thereof comprises the three light chain CDRs
comprising the sequences SEQ ID NOs. 1, 2 and 3; and a heavy chain variable domain
of sequence SEQ ID NO. 8, or any sequence exhibiting at least 80%> identity with SEQ
ID NO.8.
According to another preferred embodiment of the invention, the antigen
binding protein, or an antigen binding fragment thereof comprises a heavy chain
variable domain of sequence SEQ ID NO. 8, or any sequence exhibiting at least 80%>
identity with SEQ ID NO.8.
Another embodiment of the invention relates to an antigen binding protein,
or an antigen binding fragment thereof, comprising a light chain variable domain of
sequence SEQ ID NO. 7, or any sequence exhibiting at least 80%>, preferably 85%>,
90%, 95% and 98% identity with SEQ ID NO. 7; and a heavy chain variable domain of
sequence SEQ ID NO. 8, or any sequence exhibiting at least 80%>, preferably 85%>,
90%, 95% and 98% identity with SEQ ID NO. 8.
According to a preferred embodiment of the invention, the antigen binding
protein, or an antigen binding fragment thereof, comprises a light chain variable domain
of sequence SEQ ID NO. 7, or any sequence exhibiting at least 80%> identity with SEQ
ID NO. 7 and a heavy chain variable domain of sequence SEQ ID NO. 8, or any
sequence exhibiting at least 80%> identity with SEQ ID NO. 8.
For more clarity, table 3a below summarizes the various amino acid sequences
corresponding to the antigen binding protein of the invention (with Mu. = murine).
Table 3a
A specific aspect of the present invention relates to a murine antibody, or its
derived compounds or antigen binding fragments, characterized in that said antibody
also comprises light-chain and heavy-chain constant regions derived from an antibody
of a species heterologous with the mouse, notably man.
Another specific aspect of the present invention relates to a chimeric antibody,
or its derived compounds or antigen binding fragments, characterized in that said
antibody also comprises light-chain and heavy-chain constant regions derived from an
antibody of a species heterologous with the mouse, notably human.
Yet another specific aspect of the present invention relates to a humanized
antibody, or its derived compounds or antigen binding 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.
Another aspect of the invention is an antigen binding protein consisting of
the monoclonal antibody 1613F12 derived from the hybridoma 1-4505 deposited at the
CNCM, Institut Pasteur, France, on the 28 July 201 1, or an antigen binding fragment
thereof.
According to another aspect, the invention relates to a murine hybridoma
capable of secreting an antigen binding protein according to the invention, notably the
hybridoma of murine origin filed with the French collection for microorganism cultures
(CNCM, Pasteur Institute, Paris, France) on July 28, 201 1, under number 1-4505. Said
hybridoma was obtained b y the fusion o f B alb/C immunized mice
splenocytes/lymphocytes and cells of the myeloma Sp 2/0- Ag 14 cell line.
According to another aspect, the invention relates to a murine hybridoma
capable of secreting an antibody comprising the three light chain CDRs comprising the
sequences SEQ ID NOs. 1, 2 and 3; and the three heavy chain CDRs comprising the
sequences SEQ ID NOs. 4, 5 and 6, said hybridoma being filed at the CNCM, Pasteur
Institute, Paris, France, on July 28, 201 1, under number 1-4505. Said hybridoma was
obtained by the fusion of Balb/C immunized mice splenocytes/lymphocytes and cells of
the myeloma Sp 2/O-Ag 14 cell line.
An object of the invention is the murine hybridoma 1-4505 deposited at the
CNCM, Institut Pasteur, France, on the 28 July 2011.
The antigen binding protein of the invention also comprises chimeric or
humanized 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.
The 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 aspect, the invention describes a binding protein which consists of a
chimeric antibody.
In a particular preferred embodiment, the chimeric antibody, or an antigen
binding fragment of same, of the invention comprises a light chain variable domain
sequence comprising the amino acid sequence SEQ ID NO. 7, and in that it comprises a
heavy chain variable domain sequence comprising the amino acid sequence SEQ ID
NO. 8.
In another aspect, the invention describes a binding protein which consists of a
humanized antibody.
"Humanized antibodies" means an antibody that contains CDR regions derived
from an antibody of nonhuman origin, the other parts of the antibody molecule being
derived from one (or several) human antibodies. In addition, some of the skeleton
segment residues (called FR) can be modified to preserve binding affinity (Jones et al,
Nature, 321:522-525, 1986; Verhoeyen et al, Science, 239:1534-1536, 1988;
Riechmann et al, Nature, 332:323-327, 1988).
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 0 451 261, 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.
In addition, the invention also relates to humanized antibodies arising from the
murine antibodies 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-2 or gamma-4 region.
In a preferred embodiment, the invention relates to an antigen binding protein
consisting of a humanized antibody, or an antigen binding fragment, which comprises a
light chain variable domain comprising the sequence SEQ ID NO. 36, or any sequence
exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.
36; and the three heavy chain CDRs comprising the sequences SEQ ID NO. 4, 5 and 6.
Another embodiment of the invention relates to an antigen binding protein, or an
antigen binding fragment thereof, comprising a light chain variable domain of sequence
selected in the group consisting of SEQ ID NO. 37 to 47, or any sequence exhibiting at
least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 37 to 47; and
the three heavy chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID NO. 36 or 37 to 47", its is intended to designate the sequences
exhibiting the three light chain CDRs SEQ ID NOs. 1, 2 and 3 and, in addition,
exhibiting at least 80%, preferably 85%, 90%, 95% and 98% , identity with the full
sequence SEQ ID NO. 36 or 37 to 47 outside the sequences corresponding to the CDRs
(i.e. SEQ ID NO. 1, 2 and 3).
For more clarity, table 3b below summarizes the various amino acid sequences
corresponding to the humanized antigen binding protein light chain (VL) of the
invention (with Hz. = humanized)
Table 3b
Version SEQ ID NO.
consensus 36
VL1 37
VL1 12V 38
VL1 M4I 39
VL2.1 40
Hzl613F12 VL VL2.1 V49T 4 1
VL2.1 P50N 42
VL2.2 43
VL2.2 V49T 44
VL2.2 P50N 45
VL2.3 46
VL3 47
In an embodiment of the invention, the antigen binding protein, or an antigen
binding fragment thereof, comprises a light chain variable domain selected in the group
consisting of:
i) a light chain variable domain of sequence SEQ ID NO. 7 or any sequence
exhibiting at least 80% identity with SEQ ID NO. ,
ii) a light chain variable domain of sequence SEQ ID NO. 36 or any sequence
exhibiting at least 80% identity with SEQ ID NO. 36; and
iii) a light chain variable domain of sequence SEQ ID NO. 37 to 47 or any
sequence exhibiting at least 80% identity with SEQ ID NO. 37 to 47.
In a preferred embodiment, the invention relates to an antigen binding protein
consisting of a humanized antibody, or an antigen binding fragment, which comprises a
heavy chain variable domain comprising the sequence SEQ ID NO. 48, or any sequence
exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.
48; and the three light chain CDRs comprising the sequences SEQ ID NO. 1, 2 and 3.
Another embodiment of the invention relates to an antigen binding protein, or an
antigen binding fragment thereof, comprising a heavy chain variable domain of
sequence selected in the group consisting of SEQ ID NO. 49 to 68, or any sequence
exhibiting at least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO.
49 to 68; and the three light chain CDRs comprising the sequences SEQ ID NOs. 1, 2
and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and 98%
identity with SEQ ID NO. 48 and 49 to 68", its is intended to designate the sequences
exhibiting the three heavy chain CDRs SEQ ID NOs. 4, 5 and 6 and, in addition,
exhibiting at least 80%, preferably 85%, 90%, 95% and 98% , identity with the full
sequence SEQ ID NO. 48 and 49 to 68 outside the sequences corresponding to the
CDRs (i.e. SEQ ID NO. 4, 5 and 6).
For more clarity, table 3c below summarizes the various amino acid sequences
corresponding to the humanized antigen binding protein heavy chain (VH) of the
invention (with Hz. = humanized)
Table 3c
In an embodiment of the invention, the antigen binding protein, or an antigen
binding fragment thereof, comprises a heavy chain variable domain selected in the
group consisting of:
i) a heavy chain variable domain of sequence SEQ ID NO. 8 or any sequence
exhibiting at least 80% identity with SEQ ID NO.8;
ii) a heavy chain variable domain of sequence SEQ ID NO. 48 or any sequence
exhibiting at least 80% identity with SEQ ID NO. 48; and
iii) a heavy chain variable domaine of sequence SEQ ID NO. 49 to 68 or any
sequence exhibiting at leat 80% identity with SEQ ID NO. 49 to 68.
In an embodiment of the invention, the antigen binding protein, or an antigen
binding fragment thereof, comprises a light chain variable domain of sequence
SEQ ID NO. 36, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95%
and 98% identity with SEQ ID NO. 36; and a heavy chain variable domain of sequence
SEQ ID NO. 48, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95%
and 98% identity with SEQ ID NO. 48.
In another embodiment of the invention, the antigen binding protein, or an
antigen binding fragment thereof, comprises a light chain variable domain of sequence
selected in the group consisting of SEQ ID NO. 37 to 47, or any sequence exhibiting at
least 80%, preferably 85%, 90%, 95% and 98% identity with SEQ ID NO. 37 to 47; and
a heavy chain variable domain of sequence selected in the group consisting of SEQ ID
NO. 49 to 68, or any sequence exhibiting at least 80%, preferably 85%, 90%, 95% and
98% identity with SEQ ID NO. 49 to 68.
In an embodiment of the invention, the antigen binding protein, or an antigen
binding fragment thereof, comprises:
i) a light chain variable domain of sequence SEQ ID NO. 7, 36 or 37 to 47 or
any sequence exhibiting at least 80%> identity with SEQ ID NO.7, 36 or 37 to 47; and
ii) a heavy chain variable domain of sequence SEQ ID NO. 8, 48 or 49 to 68 or
any sequence exhibiting at least 80% identity with SEQ ID NO.8, 48 or 49 to 68.
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 coding for an antigen binding protein, or for an antigen
binding fragment of same, according to the invention;
b) a nucleic acid comprising:
- a nucleic acid sequence selected from the group consisting of SEQ ID NOs.
15 to 28 and 69 to 99, or
- a nucleic acid sequence comprising the 6 nucleic acid sequences SEQ ID
NOs.: 15 to 20, or
- a nucleic acid sequence comprising the two nucleic acid sequences SEQ ID
NOs.: 21, 22, or the two nucleic acid sequences selected from one part from SEQ ID
NOs.: 69 to 79 and for the other part from SEQID NOs: 80 to 99;
c) a nucleic acid complementary to a nucleic acid as defined in a) or b); and
d) a nucleic acid, preferably having at least 18 nucleotides, capable of
hybridizing under highly stringent conditions with a nucleic acid sequence as defined in
part a) or b), or with a sequence with at least 80%, preferably 85%, 90%>, 95% and 98%>
identity after optimal alignment with a nucleic acid sequence as defined in part a) or b),.
Table 4a below summarizes the various nucleotide sequences concerning the
binding protein of the invention (with Mu. = Murine).
Table 4a
For more clarity, table 4b below summarizes the various nucleotide sequences
corresponding to the humanized antigen binding protein light chain (VL) of the
invention (with Hz. = humanized)
Table 4b
For more clarity, table 4c below summarizes the various nucleotide sequences
corresponding to the humanized antigen binding protein heavy chain (VH) of the
invention (with Hz. = humanized)
Table 4c
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.
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 i s 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 isolated 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
(with the exception of human). Insect or plant cells can also be used.
The invention also relates to animals, other than human, that have a transformed
cell according to the invention.
Another aspect of the invention relates to a method for the production of an
antigen binding protein according to the invention, or an antigen binding fragment
thereof, characterized in that said method comprises the following steps:
a) the culture in a medium with the suitable culture conditions for a host cell
according to the invention; and
b) the recovery of the antigen binding protein, or one of its antigen binding
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 antigen binding proteins according to the invention.
Methods for the preparation of antigen binding proteins 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 antigen binding
protein and recovery of said recombinant protein.
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 antigen binding protein 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 aί , 1984, Solid phase peptides synthesis, Pierce Chem.
Company, Rockford, 111, 2nd ed., pp 71-95) 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 antigen binding protein, or the antigen binding fragments of same, likely to
be obtained by the method of the invention are also comprised in the present invention.
According to a particular aspect, the invention concerns an antigen binding
protein, or an antigen binding fragment thereof, as above described for use as an
addressing product for delivering a cytotoxic agent at a host target site, said host target
site consisting of an epitope localized into the protein Axl extracellular domain,
preferably the human protein Axl extracellular domain, more preferably the human
protein Axl extracellular domain having the sequence SEQ ID NO. 3 1 or 32, or natural
variant sequence thereof.
In a preferred embodiment, said host target site is a target site of a mammalian
cell, more preferably of a human cell, more preferably cells which naturally or by way
of genetical recombination, express the Axl protein.
The invention relates to an immunoconjugate comprising the antigen binding
protein as described in the present specification conjugated to a cytotoxic agent.
In the sense of the present invention, the expression "immunoconjugate" or
"immuno-conjugate" refers generally to a compound comprising at least an addressing
product physically linked with a one or more therapeutic agent(s), thus creating a highly
targeted compound.
In a preferred embodiment, such therapeutic agents consist of cytotoxic agents.
By "cytotoxic agent" or "cytotoxic", it is intended an agent which, when
administered to a subject, treats or prevents the development of cell proliferation,
preferably the development of cancer in the subject's body, by inhibiting or preventing a
cellular function and/or causing cell death.
Many cytotoxic agents have been isolated or synthesized and make it possible to
inhibit the cells proliferation, or to destroy or reduce, if not definitively, at least
significantly the tumour cells. However, the toxic activity of these agents is not limited
to tumour cells, and the non-tumour cells are also effected and can be destroyed. More
particularly, side effects are observed on rapidly renewing cells, such as haematopoietic
cells or cells of the epithelium, in particular of the mucous membranes. By way of
illustration, the cells of the gastrointestinal tract are largely effected by the use of such
cytotoxic agents.
One of the aims of the present invention is also to be able to provide a cytotoxic
agent which makes it possible to limit the side effects on normal cells while at the same
time conserving a high cytotoxicity on tumour cells.
More particularly, the cytotoxic agent may preferably consist of, without
limitation, a drug (i.e "antibody-drug conjugate"), a toxin (i.e. "immunotoxin" or
"antibody-toxin conjugate"), a radioisotope (i.e. "radioimmunoconjugate" or "antibodyradioisotope
conjugate"), etc.
In a first preferred embodiment of the invention, the immunoconjugate consists
of a binding protein linked to at least a drug or a medicament. Such an
immunoconjugate is referred as an antibody-drug conjugate (or "ADC") when the
binding protein is an antibody, or an antigen binding fragment thereof.
In a first embodiment, such drugs can be described regarding their mode of
action. As non limitative example, it can be mentioned alkylating agents such as
nitrogen mustard, alkyle-sulfonates, nitrosourea, oxazophorins, aziridines or imineethylenes,
anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin function
inhibitors, anti-angiogenesis agents, anti-estrogens, anti-androgens, chelating agents,
Iron absorption stimulant, Cyclooxygenase inhibitors, Phosphodiesterase inhibitors,
DNA inhibitors, DNA synthetis inhibitors, Apopstotis stimulants, Thymidylate
inhibitors, T cell inhibitors, Interferon agonists, Ribonucleoside triphosphate reductase
inhibitors, Aromatase inhibitors, Estrogen receptor antagonists, Tyrosine kinase
inhibitors, Cell cycle inhibitors, Taxane, Tubulin inhibitors, angiogenesis inhibitors,
macrophage stimulants, Neurokinin receptor antagonists, Cannabinoid receptor
agonists, Dopamine receptor agonsists, granulocytes stimulating factor agonists,
Erythropoietin receptor agonists, somatostatin receptor agonists, LHRH agonists,
Calcium sensitizers, VEGF receptor antagonists, interleukin receptor antagonists,
osteoclast inhibitors, radical formation stimulants, endothelin receptor antagonists,
Vinca alkaloid, anti-hormone or immunomodulators or any other new drug that fullfills
the activity criteria of a cytotoxic or a toxin.
Such drugs are, for example, cited in the VIDAL 2010, on the page devoted to
the compounds attached to the cancerology and hematology column "Cytotoxics", these
cytotoxic compounds cited with reference to this document are cited here as preferred
cytotoxic agents.
More particularly, without limitation, the following drugs are preferred
according to the invention : mechlorethamine, chlorambucol, melphalen, chlorydrate,
pipobromen, prednimustin, disodic-phosphate, estramustine, cyclophosphamide,
altretamine, trofosfamide, sulfofosfamide, ifosfamide, thiotepa, triethylenamine,
altetramine, carmustine, streptozocin, fotemustin, lomustine, busulfan, treosulfan,
improsulfan, dacarbazine, cis-platinum, oxaliplatin, lobaplatin, heptaplatin, miriplatin
hydrate, carboplatin, methotrexate, pemetrexed, 5-fluoruracil, floxuridine, 5-
fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-
mercaptopurine (6-MP), nelarabine, 6-thioguanine (6-TG), chlorodesoxyadenosine, 5-
azacytidine, gemcitabine, cladribine, deoxycoformycin, tegafur, pentostatin,
doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin,
mithramycin, plicamycin, mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel,
vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide,
valrubicin, amrubicin hydrochloride, pirarubicin, elliptinium acetate, zorubicin,
epirubicin, idarubicin and teniposide, razoxin, marimastat, batimastat, prinomastat,
tanomastat, ilomastat, CGS-27023A, halofuginon, COL-3, neovastat, thalidomide,
CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferonalpha,
EMD121974, interleukin-12, IM862, angiostatin, tamoxifen, toremifene,
raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole, exemestane, flutamide,
nilutamide, sprironolactone, cyproterone acetate, finasteride, cimitidine, bortezomid,
Velcade, bicalutamide, cyproterone, flutamide, fulvestran, exemestane, dasatinib,
erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, retinoid, rexinoid,
methoxsalene, methylaminolevulinate, aldesleukine, OCT-43, denileukin diflitox,
interleukin-2, tasonermine, lentinan, sizofilan, roquinimex, pidotimod, pegademase,
thymopentine, poly I:C, procodazol, Tic BCG, corynebacterium parvum, NOV-002,
ukrain, levamisole, 131 1-chTNT, H-101, celmoleukin, interferon alfa2a, interferon
alfa2b, interferon gammala, interleukin-2, mobenakin, Rexin-G, teceleukin, aclarubicin,
actinomycin, arglabin, asparaginase, carzinophilin, chromomycin, daunomycin,
leucovorin, masoprocol, neocarzinostatin, peplomycin, sarkomycin, solamargine,
trabectedin, streptozocin, testosterone, kunecatechins, sinecatechins, alitretinoin,
belotecan hydrocholoride, calusterone, dromostanolone, elliptinium acetate, ethinyl
estradiol, etoposide, fluoxymesterone, formestane, fosfetrol, goserelin acetate, hexyl
aminolevulinate, histrelin, hydroxyprogesterone, ixabepilone, leuprolide,
medroxyprogesterone acetate, megesterol acetate, methylprednisolone,
methyltestosterone, miltefosine, mitobronitol, nadrolone phenylpropionate,
norethindrone acetate, prednisolone, prednisone, temsirrolimus, testolactone,
triamconolone, triptorelin, vapreotide acetate, zinostatin stimalamer, amsacrine, arsenic
trioxide, bisantrene hydrochloride, chlorambucil, chlortrianisene, cisdiamminedichloroplatinium,
cyclophosphamide, diethylstilbestrol,
hexamethylmelamine, hydroxyurea, lenalidomide, lonidamine, mechlorethanamine,
mitotane, nedaplatin, nimustine hydrochloride, pamidronate, pipobroman, porfimer
sodium, ranimustine, razoxane, semustine, sobuzoxane, mesylate, triethylenemelamine,
zoledronic acid, camostat mesylate, fadrozole HC1, nafoxidine, aminoglutethimide,
carmofur, clofarabine, cytosine arabinoside, decitabine, doxifluridine, enocitabine,
fludarabne phosphate, fluorouracil, ftorafur, uracil mustard, abarelix, bexarotene,
raltiterxed, tamibarotene, temozolomide, vorinostat, megastrol, clodronate disodium,
levamisole, ferumoxytol, iron isomaltoside, celecoxib, ibudilast, bendamustine,
altretamine, mitolactol, temsirolimus, pralatrexate, TS-1, decitabine, bicalutamide,
flutamide, letrozole, clodronate disodium, degarelix, toremifene citrate, histamine
dihydrochloride, DW-166HC, nitracrine, decitabine, irinoteacn hydrochloride,
amsacrine, romidepsin, tretinoin, cabazitaxel, vandetanib, lenalidomide, ibandronic
acid, miltefosine, vitespen, mifamurtide, nadroparin, granisetron, ondansetron,
tropisetron, alizapride, ramosetron, dolasetron mesilate, fosaprepitant dimeglumine,
nabilone, aprepitant, dronabinol, TY-10721, lisuride hydrogen maleate, epiceram,
defibrotide, dabigatran etexilate, filgrastim, pegfilgrastim, reditux, epoetin,
molgramostim, oprelvekin, sipuleucel-T, M-Vax, acetyl L-carnitine, donepezil
hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate,
icodextrin, leuprorelin, metbylphenidate, octreotide, amlexanox, plerixafor,
menatetrenone, anethole dithiolethione, doxercalciferol, cinacalcet hydrochloride,
alefacept, romiplostim, thymoglobulin, thymalfasin, ubenimex, imiquimod, everolimus,
sirolimus, H-101, lasofoxifene, trilostane, incadronate, gangliosides, pegaptanib
octasodium, vertoporfin, minodronic acid, zoledronic acid, gallium nitrate, alendronate
sodium, etidronate disodium, disodium pamidronate, dutasteride, sodium
stibogluconate, armodafinil, dexrazoxane, amifostine, WF-10, temoporfm, darbepoetin
alfa, ancestim, sargramostim, palifermin, R-744, nepidermin, oprelvekin, denileukin
diftitox, crisantaspase, buserelin, deslorelin, lanreotide, octreotide, pilocarpine,
bosentan, calicheamicin, maytansinoids and ciclonicate.
For more detail, the person skilled in the art could refer to the manual edited by
the "Association Francaise des Enseignants de Chimie Therapeutique" and entitled
"traite de chimie therapeutique, vol. 6, Medicaments antitumoraux et perspectives dans
le traitement des cancers, edition TEC & DOC, 2003".
In a second preferred embodiment of the invention, the immunoconjugate
consists of a binding protein linked to at least a radioisotope. Such an immunoconjugate
is referred as an antibody-radioisotope conjugate (or "ARC") when the binding protein
is an antibody, or an antigen binding fragment thereof.
For selective destruction of the tumor, the antibody may comprise a highly
radioactive atom. A variety of radioactive isotopes are available for the production of
ARC such as, without limitation, At211 , C13 , N15, O17 , Fl19 , I123 , I131 , I125 , In111 , Y90,
Re186 , Re188 , Sm153 , tc m, Bi212, P32, Pb212, radioactive isotopes of Lu, gadolinium,
manganese or iron.
Any methods or processes known by the person skilled in the art can be used to
incorporate such radioisotope in the ARC (see, for example "Monoclonal Antibodies in
Immunoscintigraphy", Chatal, CRC Press 1989). As non limitative example, tc m or
I123 , Re186 , Re188 and In111 can be attached via a cysteine residue. Y can be attached via
a lysine residue. I123 can be attached using the IODOGEN method (Fraker et al (1978)
Biochem. Biophys. Res. Commun. 80: 49-57).
Several examples can be mentioned to illustrate the knowledge of the person
skilled in the art in the field of ARC such as Zevalin® which is an ARC composed of an
anti-CD20 monoclonal antibody and In111 or Y90 radioisotope bound by a thiourea
linker-chelator (Wiseman et at (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et
al (2002) Blood 99(12):4336-42; Witzig et at (2002) J . Clin. Oncol. 20(10):2453-63;
Witzig et al (2002) J . Clin. Oncol. 20(15):3262-69) ; or Mylotarg® which is composed
of an anti-CD33 antibody linked to calicheamicin, (US 4,970,198; 5,079,233;
5,585,089; 5,606,040; 5,693,762; 5,739,1 16; 5,767,285; 5,773,001). More recently, it
can also be mentioned the ADC referred as Adcetris (corresponding to the Brentuximab
vedotin) which has been recently accepted by the FDA in the treatment of Hodgkin's
lymphoma (Nature, vol. 476, pp380-381, 25 August 201 1).
In a third preferred embodiment of the invention, the immunoconjugate consists
of a binding protein linked to at least a toxin. Such an immunoconjugate is referred as
an antibody-toxin conjugate (or "ATC") when the binding protein is an antibody, or an
antigen binding fragment thereof.
Toxins are effective and specific poisons produced by living organisms. They
usually consist of an amino acid chain which can vary molecular weight between a
couple of hundred (peptides) and one hundred thousand (proteins). They may also be
low-molecular organic compounds. Toxins are produced by numerous organisms, e.g.,
bacteria, fungi, algae and plants. Many of them are extremely poisonous, with a toxicity
that is several orders of magnitude greater tha the nerve agents.
Toxins used in ATC can include, without limitation, all kind of toxins which
may exert their cytotoxic effects by mechanisms including tubulin binding, DNA
binding, or topoisomerase inhibition.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alphasarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,
PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis
inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes.
Small molecule toxins, such as dolastatins, auristatins, a trichothecene, and
CC1065, and the derivatives of these toxins that have toxin activity, are also
contemplated herein. Dolastatins and auristatins have been shown to interfere with
microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have
anticancer and antifungal activity.
"Linker", "Linker Unit", or "link" means a chemical moiety comprising a
covalent bond or a chain of atoms that covalently attaches a binding protein to at least
one cytotoxic agent.
Linkers may be made using a variety of bifunctional protein coupling agents
such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(Nmaleimidomethyl)
cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-
(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-
diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-
dinitrobenzene). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation
of cyctotoxic agents to the addressing system. Other cross-linker reagents may be
BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC,
SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate)
which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, 111.,
U.S.A).
The linker may be a "non cleavable" or "cleavable".
In a preferred embodiment, it consists in a "cleavable linker" facilitating release
of the cytotoxic agent in the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing linker may be used.
The linker is, in a preferred embodiment, cleavable under intracellular conditions, such
that cleavage of the linker releases the cytotoxic agent from the binding protein in the
intracellular environment.
For example, in some embodiments, the linker is cleavable by a cleaving agent
that is present in the intracellular environment (e.g., within a lysosome or endosome or
caveolea). The linker can be, for example, a peptidyl linker that i s cleaved b y an
intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or
endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at
least three amino acids long. Cleaving agents can include cathepsins B and D and
plasmin, all o f which are known to hydrolyze dipeptide drug derivatives resulting in the
release of active drug inside target cells. For example, a peptidyl linker that is cleavable
by the thiol-dependent protease cathepsin-B, which i s highly expressed in cancerous
tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker). In specific
embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit
linker or a Phe-Lys linker. One advantage of using intracellular proteolytic release of
the cytotoxic agent is that the agent is typically attenuated when conjugated and the
serum stabilities of the conjugates are typically high.
In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to
hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydro lyzable under
acidic conditions. For example, an acid-labile linker that i s hydro lyzable in the
lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,
orthoester, acetal, ketal, or the like) can be used. Such linkers are relatively stable under
neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or
5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable
linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via
an acylhydrazone bond.
In yet other embodiments, the linker is cleavable under reducing conditions (e.g.,
a disulfide linker). A variety of disulfide linkers are known in the art, including, for
example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate),
SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-
pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-
(2-pyridyl-dithio)toluene)- , SPDB and SMPT.
As non limitative example of non-cleavable or "non reductible" linkers, it can be
mentioned the immunoconjugate Trastuzumab-DM 1 (TDM 1) which combines
trastuzumab with a linked chemotherapy agent, maytansine (Cancer Research 2008; 68:
(22). November 15, 2008).
In a preferred embodiment, the immunoconjugate of the invention may be
prepared by any method known by the person skilled in the art such as, without
limitation, i) reaction of a nucleophilic group of the antigen binding protein with a
bivalent linker reagent followed by reaction with the cytotoxic agent or ii) reaction of a
nucleophilic group of a cytotoxic agent with a bivalent linker reagent followed by
reaction with the nucleophilic group of the antigen binding protein.
Nucleophilic groups on antigen binding protein include, without limitation, Nterminal
amine groups, side chain amine groups, e.g. lysine, side chain thiol groups, and
sugar hydroxyl or amino groups when the antigen binding protein is glycosylated.
Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form
covalent bonds with electrophilic groups on linker moieties and linker reagents
including, without limitation, active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; alkyl and benzyl halides such as haloacetamides;
aldehydes, ketones, carboxyl, and maleimide groups. The antigen binding protein may
have reducible interchain disulfides, i.e. cysteine bridges. The antigen binding proteins
may be made reactive for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically,
two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into
the antigen binding protein through any reaction known by the person skilled in the art.
As non limitative example, reactive thiol groups may be introduced into the antigen
binding protein by introducing one or more cysteine residues.
Immunoconjugates may also be produced by modification of the antigen binding
protein to introduce electrophilic moieties, which can react with nucleophilic
substituents on the linker reagent or cytotoxic agent. The sugars of glycosylated antigen
binding protein may be oxidized to form aldehyde or ketone groups which may react
with the amine group of linker reagents or cytotoxic agent. The resulting imine Schiff
base groups may form a stable linkage, or may be reduced to form stable amine
linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated
antigen binding protein with either galactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can react with
appropriate groups on the drug. In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate, resulting in
production of an aldehyde in place of the first amino acid.
In certain preferred embodiments, the linker unit may have the following general
formula:
-Ta-Ww-Yywherein:
-T- is a stretcher unit;
a is 0 or 1;
-W- is an amino acid unit;
w is independently an integer ranging from 1to 12;
-Y- is a spacer unit;
y is 0, 1 or 2.
The stretcher unit (-T-), when present, links the antigen binding protein to an
amino acid unit (-W-). Useful functional groups that can be present on the antigen
binding protein, either naturally or via chemical manipulation, include sulfhydryl,
amino, hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl.
Suitable functional groups are sulfhydryl and amino. Sulfhydryl groups can be
generated by reduction of the intramolecular disulfide bonds of the antigen binding
protein, if present. Alternatively, sulfhydryl groups can be generated by reaction of an
amino group of a lysine moiety of the antigen binding protein with 2-iminothiolane or
other sulfhydryl generating reagents. In specific embodiments, the antigen binding
protein is a recombinant antibody and is engineered to carry one or more lysines. More
preferably, the antigen binding protein can be engineered to carry one or more Cysteines
(cf. ThioMabs).
In certain specific embodiments, the stretcher unit forms a bond with a sulfur
atom of the antigen binding protein. The sulfur atom can be derived from a sulfhydryl (-
-SH) group of a reduced antigen binding protein.
In certain other specific embodiments, the stretcher unit is linked to the antigen
binding protein via a disulfide bond between a sulfur atom of the antigen binding
protein and a sulfur atom of the stretcher unit.
In other specific embodiments, the reactive group of the stretcher contains a
reactive site that can be reactive to an amino group of the antigen binding protein. The
amino group can be that of an arginine or a lysine. Suitable amine reactive sites include,
but are not limited to, activated esters such as succinimide esters, 4-nitrophenyl esters,
pentafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and
isothiocyanates.
In yet another aspect, the reactive function of the stretcher contains a reactive
site that is reactive to a modified carbohydrate group that can be present on the antigen
binding protein. In a specific embodiment, the antigen binding protein is glycosylated
enzymatically to provide a carbohydrate moiety (to be noticed that, when the antigen
binding protein is an antibody, said antibody is generally naturally glycosylated). The
carbohydrate may be mildly oxidized with a reagent such as sodium periodate and the
resulting carbonyl unit of the oxidized carbohydrate can be condensed with a stretcher
that contains a functionality such as a hydrazide, an oxime, a reactive amine, a
hydrazine, a thiosemicarbazide, a hydrazine carboxylate, or an arylhydrazide.
The amino acid unit (-W-) links the stretcher unit (-T-) to the Spacer unit (-Y-) if
the spacer unit is present, and links the stretcher unit to the cytotoxic agent if the spacer
unit is absent.
As above mentioned, -Ww- may be a dipeptide, tripeptide, tetrapeptide,
pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,
undecapeptide or dodecapeptide unit
In some embodiments, the amino acid unit may comprise amino acid residues
such as, without limitation, alanine, valine, leucine, isoleucine, methionine,
phenylalanine, tryptophan, proline, lysine protected with acetyl or formyl, arginine,
arginine protected with tosyl or nitro groups, histidine, ornithine, ornithine protected
with acetyl or formyl and citrulline. Exemplary amino acid linker components include
preferably a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: Val-Cit, Ala-Val, Lys-Lys, Cit-Cit, Val-Lys, Ala-
Phe, Phe-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N -tosyl-Arg,
Phe-N -Nitro-Arg.
Exemplary tripeptides include: Val-Ala-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-
Ala-Asn, Phe-Phe-Lys, Gly-Gly-Gly, D-Phe-Phe-Lys, Gly-Phe-Lys.
Exemplary tetrapeptide include: Gly-Phe-Leu-Gly (SEQ ID NO. 33), Ala-Leu-
Ala-Leu (SEQ ID NO. 34).
Exemplary pentapeptide include: Pro-Val-Gly-Val-Val (SEQ ID NO. 35).
Amino acid residues which comprise an amino acid linker component include
those occurring naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker components can be designed
and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for
example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
The amino acid unit of the linker can be enzymatically cleaved by an enzyme
including, but not limited to, a tumor-associated protease to liberate the cytotoxic agent.
The amino acid unit can be designed and optimized in its selectivity for
enzymatic cleavage by a particular tumor-associated protease. The suitable units are
those whose cleavage is catalyzed by the proteases, cathepsin B, C and D, and plasmin.
The spacer unit (-Y-), when present, links an amino acid unit to the cytotoxic
agent. Spacer units are of two general types: self-immolative and non self-immolative.
A non self-immolative spacer unit is one in which part or all of the spacer unit remains
bound to the cytotoxic agent after enzymatic cleavage of an amino acid unit from the
immunoconjugate. Examples of a non self-immolative spacer unit include, but are not
limited to a (glycine-glycine) spacer unit and a glycine spacer unit. To liberate the
cytotoxic agent, an independent hydrolysis reaction should take place within the target
cell to cleave the glycine-drug unit bond.
In another embodiment, a non self-immolative the spacer unit (-Y-) is -Gly-.
In one embodiment, the immunoconjugate lacks a spacer unit (y=0).
Alternatively, an imunoconjugate containing a self-immolative spacer unit can release
the cytotoxic agent without the need for a separate hydrolysis step. In these
embodiments, -Y- is a p-aminobenzyl alcohol (PAB) unit that is linked to -Ww- via the
nitrogen atom of the PAB group, and connected directly to -D via a carbonate,
carbamate or ether group.
Other examples of self-immolative spacers include, but are not limited to,
aromatic compounds that are electronically equivalent to the PAB group such as 2-
aminoimidazol-5-methanol derivatives and ortho or para-aminobenzylacetals. Spacers
can be used that undergo facile cyclization upon amide bond hydrolysis, such as
substituted and unsubstituted 4-aminobutyric acid amides, appropriately substituted
bicyclo[2.2.1] and bicyclo[2.2.2] ring systems and 2-aminophenylpropionic acid
amides.
I n an alternate embo diment , the s p a c e r unit i s a branched
bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional
cytotoxic agents.
Finally, the invention relates to an immunoconjugate as above described for use
in the treatment of cancer.
Cancers can be preferably selected through Axl-related cancers including
tumoral cells expressing or over-expressing whole or part of the protein Axl at their
surface.
More particularly, said cancers are breast, colon, esophageal carcinoma,
hepatocellular, gastric, glioma, lung, melanoma, osteosarcoma, ovarian, prostate,
rhabdomyosarcoma, renal, thyroid, uterine endometrial cancer and any drug resistance
phenomena. Another object of the invention is a pharmaceutical composition
comprising the immunoconjugate as described in the specification.
More particularly, the invention relates to a pharmaceutical composition
comprising the immunoconjugate of the invention with at least an excipient and/or a
pharmaceutical acceptable vehicle.
In the present description, the expression "pharmaceutically acceptable vehicle"
or "excipient" is intended to indicate a compound or a combination of compounds
entering into a pharmaceutical composition not provoking secondary reactions and
which allows, for example, facilitation of the administration of the active compound(s),
an increase in its lifespan and/or in its efficacy in the body, an increase in its solubility
in solution or else an improvement in its conservation. These pharmaceutically
acceptable vehicles and excipients are well known and will be adapted by the person
skilled in the art as a function of the nature and of the mode of administration of the
active compound(s) chosen.
Preferably, these immunoconjugates will be administered by the systemic route,
in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal
or subcutaneous route, or by the oral route. In a more preferred manner, the composition
comprising the immunoconjugates according to the invention will be administered
several times, in a sequential manner.
Their modes of administration, dosages and optimum pharmaceutical forms can
be determined according to the criteria generally taken into account in the establishment
of a treatment adapted to a patient such as, for example, the age or the body weight of
the patient, the seriousness of his/her general condition, the tolerance to the treatment
and the secondary effects noted.
Other characteristics and advantages of the invention appear in the continuation
of the description with the examples and the figures whose legends are represented
below.
FIGURE LEGENDS
Figure 1: in vitro cytotoxicity assay using Mab-zap conjugated secondary
antibody on SN12C cells.
Figures 2A, 2B and 2C: Binding specificity of 1613F12 on the immobilized
rhAxl-Fc protein (2A), rhDtk-Fc (2B) or rhMer-Fc (2C) proteins by ELISA.
Figure 3 : FACS analysis of the 1613F12 binding on human tumor cells
Figure 4 : ELISA on the immobilized rmAxl-Fc protein ("rm" for murine
recombinant).
Figure 5 : 1613F12 binding on COS7 cells as determined by indirect labelling
protocol using flow cytometry method.
Figure 6 : Competition ELISA of Gas6 binding using 1613F12.
Figure 7 : Epitope binding analysis by western Blot using SN12C cell lysate. NH
(no heat); NR (no reduction); H (heat); R (reduction). GAPDH detection attests to the
correct sample loading on the gel.
Figures 8A and 8B: Study of Axl downregulation after 1613F12 binding on
SN12C cells by Western Blot with Figure 8A- Western blot image representative of the
3 independent experiments performed (The western blot analysis was performed after a
4 h and 24 h incubation of the 1613F12 on SN12C cells) ; and Figure 8B- Optical
density quantification of the presented film using "QuantityOne" software.
Figures 9A, 9B and 9C: Immunofluorescence microscopy of SN12C cells after
incubation with the 1613F12 Figure 9A- Photographs of the mlgGl isotype control
conditions both for the membrane and the intracellular staining. Figure 9B- Membrane
staining. Figure 9C- Intracellular staining of both Axl receptor using the 1613F12 and
of the early endosome marker EEA1. Image overlays are presented bellow and colocalizations
visualized are indicated by the arrows.
Figure 10: Effect of 1613F12 on in vitro SN12C cells proliferation compared to
the effect of the mlgGl isotype control antibody.
Figures 11A-1 1K: Direct cytotoxicity assays of the 1613F12-saporin
immuno conjugate using various human tumor cell lines. A- SN12C , B-Calu-1, CA172,
D-A431, E-DU145, F-MDA-MB435S, G-MDA-MB23 1, H-PC3, 1-NCI-H226, JNCI-
H125, K-Pancl.
Figure 12: ELISA experiments studying binding on rhAxl-Fc protein of both
ml613F12 and hzl613F12 antibodies.
Figure 13: Binding comparison of the murine, chimeric and humanized 1613F12
antibodies on SN12C cells.
Figure 14: Direct cytotoxicity assay in presence of both mouse and humanized
1613F12-saporin immunoconjugate and of the isotype controls using SN12C human
renal tumor cell line.
Figure 15: Direct cytotoxicity assay in presence of both mouse and humanized
1613F12-saporin immunoconjugate and of the isotype controls using Calu-1 human
lung carcinoma cell line.
EXAMPLES
In the following examples, the expressions 1613F12 or ml613F12 antibody refer
to a murine form of the 1613F12 antibody. Humanized forms of the 1613F12 antibody
are named hzl613F12.
In the same way, isotype control antibody used consists of a murine IgGl
referred as 9G4. It means that, in the following examples, the expressions mlgGl
control and 9G4 are similar.
Example 1: Axl receptor internalization
As an immunoconjugate approach is more efficient when the targeted antigen is
an internalizing protein, Axl receptor internalization using Mab-Zap cytotoxicity assay
on human tumor cell lines was studied. More precisely, the Mab-Zap reagent is a
chemical conjugate including an affinity purified goat anti-mouse IgG and the
ribosome-inactivating protein, saporin. If internalization of the immune complex occurs,
saporin breaks away from the targeting agent and inactivates the ribosomes, resulting in
protein synthesis inhibition and, ultimately, cell death. Cell viability determination after
72 hours of incubation with the 1613F12 or with mlgGl isotype control antibody on
Axl-positive cells allows concluding on the 1613F12 induced Axl receptor
internalization.
For this example highly Axl-positive cells, as determined using Qifikit reagent
(Dako), were used. Data are presented in the following table 5.
Table 5
In the following example, the SN12C cells were used as non limitative example.
Any other cell line expressing appropriate level of Axl receptor on its cell surface could
be used.
Concentration ranges of the 1613F12 or the mlgGl isotype control antibody
were pre-incubated with 100 ng of Mab-Zap (Advanced targeting systems) secondary
antibody in cell culture medium for 30 min at RT. These mixtures were loaded on subconfluent
SN12C cells plated in white 96-well plate microplate. Plates were incubated
for 72 h at 37°C in presence of 5% C0 2. Cell viability was determined using a Cell Titer
Glo cell proliferation method according to the manufacturer's instructions (Promega).
Several controls are performed: i) without any secondary immunoconjugate and ii)
without primary antibody. In parallel, assays are performed with a mlgGl isotype
control.
Obtained results are represented in the Figure 1.
The 1613F12 shows a maximal cytotoxic effect on the SN12C cells of ~36 %.
No cytotoxic effect was observed in presence of the 9G4 antibody, considered as mlgGl
isotype control in the experiment. No cytotoxicity was observed in wells containing
only primary antibodies (data not shown). Thus the Axl receptor appears to be a
convenient antigen to target for an immunoconjugate approach as the immune complex
comprising Axl-1613F12-MabZap triggers an effective cytotoxicity of the targeted
cells.
Example 2 : Generation of an antibody against rhAxl ECD.
To generate murine monoclonal antibodies (Mabs) against human extracellular
domain (ECD) of the Axl receptor, 5 BALB/c mice were immunized 5-times s.c. with
15-20. 106 CHO-Axl cells and twice with 20 mg of the rh Axl ECD. The first
immunization was performed in presence of Complete Freund Adjuvant (Sigma, St
Louis, MD, USA). Incomplete Freund adjuvant (Sigma) was added for following
immunizations.
Three days prior to the fusion, immunized mice were boosted with both 20. 106
CHO-Axl cells and 20 mg of the rhAxl ECD with IFA.
To generate hybridomas, splenocytes and lymphocytes were prepared by
perfusion of the spleen and by mincing of the proximal lymph nodes, respectively,
harvested from 1 out of the 5 immunized mice (selected after sera titration) and fused to
SP2/0-Agl4 myeloma cells (ATCC, Rockville, MD, USA). The fusion protocol is
described by Kohler and Milstein (Nature, 256:495-497, 1975). Fused cells are then
subjected to HAT selection. In general, for the preparation of monoclonal antibodies or
their functional fragments, especially of murine origin, it is possible to refer to
techniques which are described in particular in the manual "Antibodies" (Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor NY, pp. 726, 1988).
Approximately 10 days after the fusion, colonies of hybrid cells were screened.
For the primary screen, supernatants of hybridomas were evaluated for the secretion of
Mabs raised against the Axl ECD protein using an ELISA. In parallel, a FACS analysis
was performed to select Mabs able to bind to the cellular form of Axl present on the cell
surface using both wt CHO and Axl expressing CHO cells (ATCC).
As soon as possible, selected hybridomas were cloned by limit dilution and
subsequently screened for their reactivity against the Axl ECD protein. Cloned Mabs
were then isotyped using an Isotyping kit (cat #5300.05, Southern Biotech,
Birmingham, AL, USA). One clone obtained from each hybridoma was selected and
expanded.
ELISA assays are performed as followed either using pure hybridoma
supernatant or, when IgG content in supernatants was determined, titration was realized
starting at 5mg/ml. Then a ½ serial dilution was performed in the following 11 rows.
Briefly, 96-well ELISA plates (Costar 3690, Corning, NY, USA) were coated 50
mΐ/well of the rh Axl-Fc protein (R and D Systems, cat N° 154-AL) or rhAxl ECD at 2
mg/ml in PBS overnight at 4°C. The plates were then blocked with PBS containing 0.5%
gelatin (#22151, Serva Electrophoresis GmbH, Heidelberg, Germany) for 2 h at 37°C.
Once the saturation buffer discarded by flicking plates, 50 mΐ of pure hybridoma cell
supernatants or 50 mΐ of a 5 mg/ml solution were added to the ELISA plates and
incubated for 1 h at 37°C. After three washes, 50 mΐ horseradish peroxidase-conjugated
polyclonal goat anti-mouse IgG (#1 15-035-164, Jackson Immuno-Research
Laboratories, Inc., West Grove, PA, USA ) was added at a 1/5000 dilution in PBS
containing 0.1% gelatin and 0.05% Tween 20 (w:w) for 1 h at 37°C. Then, ELISA
plates were washed 3-times and the TMB (#UP664782, Uptima, Interchim, France)
substrate was added. After a 10 min incubation time at room temperature, the reaction
was stopped using 1M sulfuric acid and the optical density at 450 nm was measured.
For the selection by flow cytometry, 105 cells (CHO wt or CHO-Axl) were
plated in each well of a 96 well-plate in PBS containing 1% BSA and 0.01% sodium
azide (FACS buffer) at 4°C. After a 2 min centrifugation at 2000 rpm, the buffer was
removed and hybridoma supernatants or purified Mabs ( 1 mg/ml) to be tested were
added. After 20 min of incubation at 4°C, cells were washed twice and an Alexa 488-
conjugated goat anti-mouse antibody 1/500° diluted in FACS buffer (#A1 1017,
Molecular Probes Inc., Eugene, USA) was added and incubated for 20 min at 4°C. After
a final wash with FACS buffer, cells were analyzed by FACS (Facscalibur, Becton-
Dickinson) after addition of propidium iodide to each tube at a final concentration of 40
mg/ml. Wells containing cells alone and cells incubated with the secondary Alexa 488-
conjugated antibody were included as negative controls. Isotype controls were used in
each experiment (Sigma, ref M90351MG). At least 5000 cells were assessed to
calculate the mean value of fluorescence intensity (MFI).
More precisely, the fusion was performed with 300. 106 of harvested splenocytes
and 300. 106 myeloma cells ( 1:1 ratio). Two hundred cells of the resulting cell
suspension were then plated at 2.106 cell/ml in 30 96-well plates.
A first screen (around Day 14 after fusion) both by ELISA on the rhAxl ECD
protein and by FACS analysis using the both wt CHO and Axl expressing CHO cells
allowed to select 10 hybridomas presenting optical densities (ODs) above 1 on the rh
AxlECD coating and MFI bellow 50 on wt CHO cells and above 200 on CHO-Axl
cells.
These 10 hybridomas were expanded and cloned by limit dilution. One 96-well
plate was prepared for each code. Nine days after plating, supernatants from cloning
plates were first screened by ELISA for their binding specificity for the extracellular
domain of the rh AxlECD protein. Three clones of each code were expanded and
isotyped. Once produced the anti-Axl antibodies were further studied for their ability to
be internalized following Axl binding on the cell-surface.
Example 3 : Axl binding specificity
In this example, the binding of the 1613F12 was first studied on the rhAxl-Fc
protein. Then, its binding on the two other members of the TAM family, rhDtk-Fc and
rhMer-Fc, was studied.
Briefly, the recombinant human Axl-Fc (R and D systems, cat ° 154AL/CF),
rhDtk (R and D Systems, cat N° 859-DK) or rhMer-Fc (R and D Systems, cat N° 891-
MR) proteins were coated overnight at 4°C to Immulon II 96-well plates and, after a 1 h
blocking step with a 0.5% gelatine solution, 1613F12 purified antibody was added for
an additional 1 h at 37°C at starting concentration of 5 mg/ml (3.33 10 M). Then ½
serial dilutions were done over 12 columns. Plates were washed and a goat anti-mouse
(Jackson) specific IgG-HRP was added for 1 h at 37°C. Reaction development was
performed using the TMB substrate solution. The isotype control antibody mlgGl and
the commercial anti-Axl Mab 154 antibody were also used in parallel. Coating controls
were performed in presence of a goat anti-human IgG Fc polyclonal serum labelled with
HRP (Jackson, ref 109-035-098) and/or in presence of a HRP-coupled anti-Histidine
antibody (R and D Systems, ref : MAB050H). Results are represented in Figures 2A, 2B
and 2C, respectively.
This example shows that the 1613F12 antibody only binds to the rhAxl-Fc
protein and does not bind on the two other members of the TAM family, rhDtk or
rhMer. No cross-specificity of binding of the 1613F12 antibody is observed between
TAM members. No non specific binding was observed in absence of primary antibody
(diluant). No binding was observed in presence of the isotype control antibody.
Example 4 : 1613F12 recognized the cellular form of Axl expressed on tumor
cells.
Cell surface Axl expression level on human tumor cells was first established
using a commercial Axl antibody (R and D Systems, ref: MAB154) in parallel of
calibration beads to allow the quantification of Axl expression level. Secondly, binding
of the cell-surface Axl was studied using the 1613F12.
For cell surface binding studies, two fold serial dilutions of a 10 mg/ml (6.66 10 8
M) primary antibody solution (1613F12, MAB154 antibody or mlgGl isotype control
9G4 Mab) are prepared and are applied on 2.105 cells for 20 min at 4°C. After 3 washes
in phosphate-buffered saline (PBS) supplemented with 1% BSA and 0.01% NaN3, cells
were incubated with secondary antibody Goat anti-mouse Alexa 488 (1/500° dilution)
for 20 minutes at 4°C. After 3 additional washes in PBS supplemented with 1% BSA
and 0.1% NaN3, cells were analyzed by FACS (Facscalibur, Becton-Dickinson). At
least 5000 cells were assessed to calculate the mean value of fluorescence intensity.
For quantitative ABC determination using MAB154 antibody, QIFIKIT®
calibration beads are used. Then, the cells are incubated, in parallel with the QIFIKIT®
beads, with Polyclonal Goat Anti-Mouse Immunoglobulins/FITC, Goat F(ab')2, at
saturating concentration. The number of antigenic sites on the specimen cells is then
determined by interpolation of the calibration curve (the fluorescence intensity of the
individual bead populations against the number of Mab molecules on the beads.
4.1. Quantification of cell-surface Axl expression level
Axl expression level on the surface of human tumor cells was determined by flow
cytometry using indirect immunofluorescence assay (QIFIKIT® method (Dako,
Denmark), a quantitative flow cytometry kit for assessing cell surface antigens. A
comparison of the mean fluorescence intensity (MFI) of the known antigen levels of the
beads via a calibration graph permits determination of the antibody binding capacity
(ABC) of the cell lines.
Table 6 presents Axl expression level detected on the surface of various human
tumor cell lines (SN12C, Calu-1, A172, A431, DU145, MDA-MB435S, MDA-MB231,
PC3, NCI-H226, NCI-H125, MCF7, Panel) (ATCC, NCI) as determined using
QIFIKIT® using the commercial antibody MAB154 (R and D Systems). Values are
given as Antigen binding complex (ABC).
Table 6
Results obtained with a commercial Axl monoclonal antibody (MAB154) showed that
Axl receptor is expressed at various levels depending of the considered human tumor
cell.
4.2. Axl detection by 1613F12 on human tumor cells
More specifically, Axl binding was studied using the 1613F12.
1613F12 dose response curves were prepared. MFIs obtained using the various
human tumor cells were then analysed with Prism software. Data are presented in
Figure 3.
Data indicate that the 1613F12 binds specifically to the membrane Axl receptor
as attested by the saturation curve profiles. However different intensities of labelling
were observed, revealing variable levels of cell-surface Axl receptor on human tumor
cells. No binding of Axl receptor was observed using MCF7 human breast tumor cell
line.
Example 5: 1613F12 inter-species crosspecificity
To address the species cross-specificity of the 1613F12, two species were
considered: mouse and monkey. First the binding on the recombinant mouse (rm) Axl
receptor is studied by ELISA (Figure 4). Then, flow cytometry experiments were
performed using monkey COS7 cells as these cells express the Axl receptor on their
surface (Figure 5). The COS7 cell line was obtained by immortalizing a CV-1 cell line
derived from kidney cells of the African green monkey with a version of the SV40
genome that can produce large T antigen but has a defect in genomic replication.
rmAxl-Fc ELISA
Briefly, the recombinant mouse Axl-Fc (R and D systems, cat N° 854-AX /CF)
proteins were coated overnight at 4°C to Immulon II 96-well plates and, after a 1 h
blocking step with a 0.5% gelatine solution, the 1613F12 purified antibody was added
for one additional hour at 37°C at starting concentration of 5 mg/ml (3.33 10 8 M). Then
½ serial dilutions were done over 1 columns. Plates were then washed and a goat antimouse
(Jackson) specific IgG HRP was added for 1 h at 37°C. Reaction development
was performed using the TMB substrate solution. The mlgGl isotype control and the
commercial antibody Mab 154 are also used in parallel. Coating controls are performed
in presence of a goat anti-human IgG Fc polyclonal serum coupled with HRP (Jackson,
ref 109-035-098) and/or in presence of a HRP-coupled anti-Histidine antibody (R and D
Systems, ref : MAB050H).
Results are represented in Figure 4. This figure shows that the 1613F12 does not
bind to the murine Axl ECD domain. No specific binding is observed in the absence of
primary antibody (diluant).
FACS COS7
For 1613F12 cellular binding studies using COS7 cells, 2.105 cells were
incubated with an antibody concentration range prepared by ½ serial dilution (12 points)
of a 10 mg/ml (6,66 10 8 M) antibody solution of 1613F12 or mlgGl isotype control
Mab for 20 min at 4°C. After 3 washes in phosphate-buffered saline (PBS)
supplemented with 1% BSA and 0.01% NaN3, cells were incubated with secondary
antibody goat anti-mouse Alexa 488 (dilution 1/500) for 20 minutes at 4°C. After 3
additional washes in PBS supplemented with 1% BSA and 0.1% NaN3, cells were
analyzed by FACS (Facscalibur, Becton-Dickinson). At least 5000 cells were assessed
to calculate the mean value of fluorescence intensity. Data are analyzed using Prism
software.
Results are represented in Figure 5. The titration curve established on COS7
cells using 1613F12 confirms that 1613F12 is able to recognize the monkey cellular
form of the Axl receptor expressed on the surface of the COS7 cells. Plateau is reached
for 1613F12 concentrations above 0.625 mg/ml (4.2 10 10 M). No binding is observed in
presence of the mlgGl isotype control.
This example illustrates the fact that the 1613F12 does not cross-react with the
mouse Axl receptor. In contrast it strongly binds to the monkey Axl receptor expressed
on the surface of COS7 cells.
Example 6: Gas6 Competition experiments performed in presence of the
1613F12
To further characterize the 1613F12, Gas6 competition assays were performed.
In this assay, the free rhAxl-Fc protein and the 1613F12 are incubated to form antigenantibody
complex and then the complexes are loaded on Gas6-coated surface in the
assay plate. The unbound antibody-antigen complexes are washed out before adding
enzyme-linked secondary antibody against the human Fc portion of the rhAxl-Fc
protein. The substrate is then added and the antigen concentration can be determined by
the signal strength elicited by the enzyme-substrate reaction.
Briefly reaction mixture comprising the rhAxl-Fc protein in the presence or not
of the anti-Axl Mabs to be tested, are prepared on a separate saturated (0.5% gelatin in
PBS IX) plate. Serial 1: 2 dilutions (starting from 80 mg/ml on 12 columns) of murine
anti-Axl antibodies are performed. Then 0.5 mg/ml of the rhAxl-Fc protein is added (R
and D Systems, ref. 154AL/CF), except to the negative control line that contains only
ELISA diluant (0.1% gelatin, 0.05% Tween 20 in PBS IX). After homogenisation, the
competition samples are loaded on Gas6-coated plates with a 6 mg/ml rhGas6 solution
in PBS (R and D Systems cat N° 885-GS-CS / CF). After incubation and several
washes, bound rhAxl-Fc proteins are detected using a goat anti-Human IgG-HRP
(Jackson, ref. 109-035-098). Once bound, the TMB substrate is added to the plates. The
reaction is stopped by addition of 1M H2SO4 acid solution and the obtained optical
densities read at 450 nm using a microplate reader instrument.
This experiment (Figure 6) shows that the 1613F12 is able to compete with the
rhAxl-Fc binding on its immobilized ligand. Competition with Gas6 binding occurs in
presence of 1613F12 antibody concentrations above 2.5 mg/ml (1.67 10 8 M). No more
binding of the rhAxl-Fc on the immobilized Gas6 is observed in presence of a 1613F12
concentration above 10 mg/ml (6.67 10 8 M). The 1613F12 blocks Gas6 binding to
rhAxl-Fc.
Example 7: Epitope recognition by Western Blot
To determine if the 1613F12 recognizes a linear or a conformational epitope,
western blot analysis was done using SN12C cell lysates. Samples were differently
treated to be in reducing or non reducing conditions. If a band is visualized with
reduced sample, the tested antibody targets a linear epitope of the ECD domain; If not,
it is raised against a conformation epitope of the Axl ECD.
SN12C cells were seeded in RPMI + 10 % heat inactivated FBS + 2 mM Lglutamine
at 5.104 cells /cm2 in T162 cm2 flasks for 72h at 37°C in a 5% C0 2
atmosphere. Then the cells were washed twice with phosphate buffered saline (PBS)
and lysed with 1.5 ml of ice-cold lysis buffer [50 mM Tris-HCl (pH7.5); 150 mM NaCl;
1% Nonidet P40; 0.5% deoxycholate; and 1 complete protease inhibitor cocktail tablet
plus 1% antiphosphatases]. Cell lysates were shaken for 90 min at 4°C and cleared at
15 000 rpm for 10 min. Protein concentration was quantified using BCA. Various
samples were loaded. First 10 mg of whole cell lysate (10 mg in 20 mΐ) were prepared in
reducing conditions (lx sample buffer (BIORAD) + l x reducing agent (BIORAD)) and
loaded on a SDS-PAGE after 2 min incubation at 96°C. Secondly two other samples of
10 mg of whole cell lysate were prepared in non-reducing conditions (in l x sample
buffer (BIORAD) only). Prior to be loaded on the SDS-PAGE gel, one of these two last
samples is heated 2 min incubation at 96°C; the other one is kept on ice. After
migration, the proteins are transferred to nitrocellulose membrane. Membranes were
saturated for 1 h at RT with TBS-tween 20 0.1% (TBST), 5% non-fat milk and probed
with the 1613F12 at 10 mg/ml overnight at 4°C. Antibodies were diluted in Trisbuffered
saline-0.1% tween 20 (v/v) (TBST) with 5% non-fat dry milk. Then
membranes were washed with TBST and incubated with peroxydase-conjugated
secondary antibody (dilution 1/1000) for 1 h at RT. Immunoreactive proteins were
visualized with ECL (Pierce #32209). After Axl visualization, membranes were washed
once again with TBST and incubated for 1 h at RT with mouse anti-GAPDH antibody
(dilution 1/200 000). Then membranes were washed in TBST and incubated with
peroxydase-conjugated secondary antibodies, for l h at RT. Membranes were washed
and GAPDH was revealed using ECL.
Results are represented in Figure 7.
The 1613F12 mainly recognizes a conformational epitope as a specific band is
essentially observed in non-reduced conditions. However a faint signal is detected in
the denaturating migrating condition of the SN12C cell lysate indicating 1613F12 is
able to weakly bind to a linear epitope.
Example 8: Measurement of Axl down-regulation triggered by the 1613F12
by Western Blot.
In the following example, the human renal cell carcinoma cell line SN12C
(ATCC) was selected to address the activity of Axl antibodies on Axl receptor
expression. The SN12C cell line overexpresses the Axl receptor. The Axl downregulation
was studied by Western-Blot on whole cell extracts in Figures 8A-8B.
SN12C cells were seeded in RPMI + 10 % heat inactivated FBS + 2 mM Lglutamine
at 6.104 cells/cm2 in six-well plates for 48 h at 37°C in a 5% C0 2 atmosphere.
After two washes with phosphate buffer saline (PBS), cells were serum-starved in a
medium containing either 800 ng/ml recombinant mouse gas6 ligand (R and D Systems,
ref: 986-GS/CF) or 10 mg/ml of a mlgGl isotype control antibody (9G4) or 10 mg/ml of
the Axl antibody of the present invention and incubated for 4 h or 24 additional hours.
Then the medium was gently removed and cells washed twice with cold PBS. Cells
were lysed with 200 mΐ of ice-cold lysis buffer [50 mM Tris-HCl (pH7.5); 150 mM
NaCl; 1% Nonidet P40; 0.5% deoxycholate; and 1 complete protease inhibitor cocktail
tablet plus 1% antiphosphatases]. Cell lysates were shaken for 90 min at 4°C and
cleared at 15 000 rpm for 10 min. Protein concentration was quantified using BCA
method. Whole cell lysates (10 mg in 20 mΐ) were separated by SDS-PAGE and
transferred to nitrocellulose membrane. Membranes were saturated for 1 h at RT with
TBS-Tween 20 0.1% (TBST), 5% non-fat milk and probed with a commercial M02 Axl
antibody at 0.5 m ΐ (AbNova H00000558-M02) overnight at 4°C. Antibodies were
diluted in Tris-buffered saline-0.1% tween 20 (v/v) (TBST) with 5% non-fat dry milk.
Then membranes were washed with TBST and incubated with peroxydase-conjugated
secondary antibody (dilution 1/1000) for 1 h at RT. Immunoreactive proteins were
visualized with ECL (Pierce #32209). After Axl visualization, membranes were washed
once again with TBST and incubated for 1 h at RT with mouse anti-GAPDH antibody
(dilution 1/200000). Then membranes were washed in TBST and incubated with
peroxydase-conjugated secondary antibodies, for l h at RT. Membranes were washed
and GAPDH was revealed using ECL. Band intensity was quantified by densitometry.
Results presented in Figures 8A and 8B are representative of 3 independent
experiments and demonstrate that 1613F12 is able to down-regulate Axl in an Axloverexpressing
human tumor cell line. At 4 h, the 1613F12 triggers a 66 % Axl downregulation,
and up to 87 % after a 24 hour incubation with the 1613F12.
Example 9: Flow cytometry study of the 1613F12 effect on cell surface Axl
expression
Flow cytometry technique allows labelling of cell-surface Axl receptor. The use
of this technique can highlight the effect of antibodies on the membrane Axl expression.
Human renal tumor SN12C cells that express high levels of Axl were used in this
example.
SN12C tumor cell line was cultured in RMPI1640 with 1% L-glutamine and
10% of FCS for 3 days before experiment. Cells were then detached using trypsin and
plated in 6-multiwell plate in RPMI1640 with 1% L-glutamine and 5% FBS. The next
day, antibodies of interest were added at 10 mg/ml. Untreated wells were also included.
The cells are incubated at 37°C, 5% C0 2. Twenty four hours later, cells were washed
with PBS, detached and incubated with the same antibodies of interest in FACS buffer
(PBS, 1% BSA, 0.0 1% sodium azide). Untreated wells were also stained with the same
antibody in order to compare the signal intensity obtained with the same Mab on the
treated and the non-treated cells. Cells were incubated for 20 minutes at 4°C and
washed three times with FACS buffer. An Alexa 488-labeled goat anti-mouse IgG
antibody was incubated for 20 minutes and cells were washed three times before FACS
analysis on propidium iodide negative cell population.
Two parameters are determined: (i) the difference of the fluorescent signal
detected on the surface of untreated (no Ab) cells compared to the Ab-treated cells at
T24 h and (ii) the percentage of remaining Axl on the cell surface. The percentage of
remaining Axl is calculated as follows:
% remaining Axl = (MFI A b 24 h / MFI o Ab 24 h) x 100
Data from one representative experiment are presented in Table 7. The results
were reproduced in three independent experiments.
The difference of MFI between the staining of a Mab in the untreated cells and
the treated condition with the same antibody reflects a down-regulation of the Axl
protein on the surface of the cells due to the binding of the considered Mab. Conditions
without antibody gave similar results to conditions in presence of the isotype control
antibody (m9G4).
Table 7
The data demonstrate that the mean fluorescence intensity detected on the
surface of the cells treated with 1613F12 for 24 hours is reduced (-514) compared to the
MFIs obtained with untreated cells labelled with the 1613F12. After a 24 h incubation
with the 1613F12 antibody, 45.2 % of the cell-surface Axl receptor remains at the
SN12C cell-surface.
Example 10: 1613F12 internalization study using fluorescent
immunocytochemistry labelling.
Complementary internalization results are obtained by confocal microscopy
using indirect fluorescent labelling method.
Briefly, SN12C tumor cell line was cultured in RMPI1640 with 1% L-glutamine
and 10 % of FCS for 3 days before experiment. Cells were then detached using trypsin
and plated in 6-multiwell plate containing coverslide in RPMI1640 with 1 % Lglutamine
and 5 % FCS. The next day, the 1613F12 was added at 10 mg/ml. Cells
treated with an irrelevant antibody were also included. The cells were then incubated for
1 h and 2 h at 37°C, 5% C0 2. For T 0 h, cells were incubated for 30 minutes at 4°C to
determine antibody binding on cell surface. Cells were washed with PBS and fixed with
paraformaldehyde for 15 minutes. Cells were rinsed and incubated with a goat antimouse
IgG Alexa 488 antibody for 60 minutes at 4°C to identify remaining antibody on
the cell surface. To follow antibody penetration into the cells, cells were fixed and
permeabilized with saponin. A goat anti-mouse IgG Alexa 488 (Invitrogen) was used to
stained both the membrane and the intracellular antibody. Early endosomes were
identified using a rabbit polyclonal antibody against EEA1 revealed with a goat antirabbit
IgG-Alexa 555 antibody (Invitrogen). Cells were washed three times and nuclei
were stained using Draq5. After staining, cells were mounted in Prolong Gold mounting
medium (Invitrogen) and analyzed by using a Zeiss LSM 510 confocal microscope.
Photographs are presented in Figures 9A-9C.
Images were obtained by confocal microscopy. In presence of the mlgGl
isotype control (9G4), neither membrane staining nor intracellular labelling is observed
(Figure 9A). A progressive loss of the membrane anti-Axl labelling is observed as soon
as after 1 h incubation of the SN12C cells with the 1613F12 (Figure 9B). Intracellular
accumulation of the 1613F12 antibody is clearly observed at 1 h and 2 h (Figure 9C).
Intracellular antibody co-localizes with EEA1 , an early endosome marker. These
photographs confirm the internalization of the 1613F12 into SN12C cells.
Example 11: in vitro anti-Axl mediated anti-tumoral activity.
SN12C proliferation assay
Ten thousand SN12C cells per well were seeded in FCS-free medium on 96 well
plates over night at 37°C in a 5% C0 2 atmosphere. The next day, cells were preincubated
with 10 mg/ml o f each antibody for l h at 37°C. Cells were treated with or
without rmGas6 (R and D Systems, cat N°986-GS/CF), by adding the ligand directly to
the well, and then left to grown for 72h. Proliferation was measured following H
thymidine incorporation.
Data are presented in figure 10. No effect was observed with the 1613F12 which
is silent when added to SN12C cells.
Example 12: Cytotoxicity potency of 1613F12-saporin immunoconjugate in
various human tumor cell lines
In the present example, is documented the cytotoxicity potency of the saporin
coupled-1613F12. For this purpose direct in vitro cytotoxicity assays using a large panel
of human tumor cell lines were performed (Figures 11A-1 1K). This tumor cell line
panel offers various cell surface Axl expressions.
Briefly, 5000 cells were seeded in 96 well culture plates in 100 mΐ of 5 % FBS
adequate culture medium (DO). After 24 hours incubation in a 5% C0 2 atmosphere at
37°C, a range of concentration o f the immunoconjugate (1613F12-saporin or 9G4-
saporin or the naked 1613F12 or 9G4) is applied to the cells. Culture plates are then
incubated at 37°C in a humidified 5% C0 2 incubator for 72 hours.
At D4, the cell viability is assessed using the CellTiter-Glo® Luminescent Cell
Viability kit (Promega Corp., Madison, Wis.) that allows determining the number of
viable cells in culture based on quantification o f the ATP present, an indicator o f
metabolically active cells. Luminescent emissions are recorded by a luminometer
device.
From luminescence output is calculated the percentage of cytotoxicity using the
following formula:
%cytotoxicity = 100- [(RLU Ab-saP x 100)/ RLU No AJ
On Figures 11A-l IK are put together graphs presenting cytotoxicity percentage
in function of the immunoconjugate concentration obtained in distinct in vitro cell
cytotoxicity assays with (A) SN12C, (B) Calu-1, (C) A172, (D) A431, (E) DU145, (F)
MDA-MB-435S, (G) MDA-MB-231, (H) PC3, (I) NCI-H226, (J) NCI-H125 or (K)
Panel tumor cells treated with a range of 1613F12-saporin immunoconjugate
concentrations.
Figures 11A-l IK shows that the 1613F12-saporin immunoconjugate triggered
cytotoxicity in these different human tumor cell lines. The potency of the resulting
cytotoxicity effect depends on the human tumor cell line.
Example 13: Humanization of the 1613F12 antibody variable domains
The use of mouse antibodies (Mabs) for therapeutic applications in humans
generally results in a major adverse effect, patients raise a human anti-mouse antibody
(HAMA) response, thereby reducing the efficacy of the treatment and preventing
continued administration. One approach to overcome this problem is to humanize
mouse Mabs by replacing mouse sequences by their human counterpart but without
modifying the antigen binding activity. This can be achieved in two major ways: (i) by
construction of mouse/human chimeric antibodies where the mouse variable regions are
joined to human constant regions (Boulianne et al., 1984) and (ii) by grafting the
complementarity determining regions (CDRs) from the mouse variable regions into
carefully selected human variable regions and then joining these "re-shaped human"
variable regions to human constant regions (Riechmann et al., 1988).
13.1. Design of humanized version of the 1613F12 antibody
13.1.1 Humanization of the light chain variable domain VL
As a preliminary step, the nucleotide sequence of the 1613F12 VL was
compared to the murine germline gene sequences part of the IMGT database
(http://www.imgt.org). Murine IGKV16-104*01 and IGKJ5*01 germline genes were
identified. In order to identify the best human candidate for the CDR grafting, the
human germline gene displaying the best identity with the 1613F12 VL murine
sequence has been searched. With the help of the IMGT database analyses tools, a
possible acceptor human V regions for the murine 1613F12 VL CDRs was identified:
IGKV1-27*01 and IGKJ4*02. In order to perform the humanization to the light chain
variable domain each residue which is different between the human and mouse
sequences was given a priority rank order. These priorities (1-4) were used to create 11
different humanized variants of the light chain variable region with up to 14
backmutations.
FR1-IMGT CDR1 -IMGT FR2-IMGT CD
1613F12VL DVQITQSPSYLATSPGETITINCRAS KSI SKY LAWYQEKPGKTNKLLIY SG
Horns ap IGKV1-2 7*01 DIQMTQSPSSLSASVGDRVTITCRAS QGI. .. SNY LAWYQQKPGKVPKLLIY AA
V I Y AT P ETI N E TN
Priority 1 1 3 34 4 433 2 3 33
hzl613F12 (VL1 ) DIQMTQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKVPKLLIY SG
hzl613F12 (VL1I2V) DVQMTQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKVPKLLIY SG
hzl613F12 (VL1M4I) DIQITQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKVPKLLIY SG
hzl613F12 (VL2 1 ) DVQITQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKVPKLLIY SG
hzl613F12 (VL2 .1V49T) DVQITQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKTPKLLIY SG
hzl613F12 (VL2 .1P50N) DVQITQSPSSLSASVGDRVTITCRAS KSI SKY LAWYQQKPGKVNKLLIY SG
hzl613F12 (VL2 2 ) DVQITQSPSSLSASVGDRVTINCRAS KSI SKY LAWYQQKPGKVPKLLIY SG
hzl613F12 (VL2 .2V49T) DVQITQSPSSLSASVGDRVTINCRAS KSI SKY LAWYQQKPGKTPKLLIY SG
hzl613F12 (VL2 .2P50N) DVQITQSPSSLSASVGDRVTINCRAS KSI SKY LAWYQQKPGKVNKLLIY SG
hzl613F12 (VL2 3 ) DVQITQSPSSLSASVGDRVTINCRAS KSI SKY LAWYQEKPGKTNKLLIY SG
z 613F12 (VL3 ) DVQITQSPSYLAASVGDTITINCRAS KSI SKY LAWYQEKPGKTNKLLIY SG
R2-IMGT FR3-IMGT
1613F12VL S TLQSGVP. SRFSGSG. .SGTDFTLTI SSLE PEDFAMYFC
Horns ap IGKV1-2 7*01 S TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
E F M F
Priority 4 4 4 2
hzl613F12 (VL1 ) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL1 I2V) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL1M4I) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL2 1 ) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL2 .1V49T) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL2 .1P50N) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYYC
hzl613F12 (VL2 2 ) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYFC
hzl613F12 (VL2 .2V49T) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYFC
hzl613F12 (VL2 .2P50N) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYFC
hzl613F12 (VL2 3 ) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYFC
hzl613F12 (VL3 ) s TLQSGVP. SRFSGSG. .SGTDFTLTI SSLQPEDVATYFC
CDR3-IMGT FR4-IMGT
1613F12VL QQHHEYPLT FGAGTELELK
Horns ap IGKJ4*0i LT FGGGTKVEIK
A EL L
Priority 3 33 4
hzl613F12 (VL1) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL1I2V) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL1M4I) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .1) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .1V49T) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .1P50N) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .2) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .2V49T) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .2P50N) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL2 .3) QQHHEYPLT FGGGTKVEIK
hzl613F12 (VL3) QQHHEYPLT FGAGTELEIK
13.1.2 Humanization of the heavy chain variable domain VH
In order to identify the best human candidate for the CDR grafting, the mouse
and human germline genes displaying the best identity with the 1613F12 VH were
searched. The nucleotide sequence of 1613F12 VH was aligned with both mouse and
human germline gene sequences by using the sequence alignment software "IMGT
QUEST" which is part of the IMGT database. Alignments of amino acid sequences
were also performed to verify the results of the nucleotide sequence alignment using the
"Align X" software of the VectorNTI package. The alignment with mouse germline
genes showed that the mouse germline V-gene IGHV14-3*02 and J-gene IGHJ2*01 are
the most homologue mouse germline genes. Using the IMGT database the mouse Dgene
germline IGHD1-1*01 was identified as homologous sequence. In order to select
an appropriate human germline for the CDR grafting, the human germline gene with the
highest homology to the 1613F12 VH murine sequence was identified. With the help of
IMGT databases and tools, the human IGHVl-2*02 germline gene and human
IGHJ5*01 J germline gene were selected as human acceptor sequences for the murine
1613F12 VH CDRs. In order to perform the humanization to the heavy chain variable
domain each residue which is different between the human and mouse sequences was
given a priority rank order (1-4). These priorities were used to create 20 different
humanized variants of the heavy chain variable region with up to 18 backmutations,
FR1-IMGT CDR1 -IMGT FR2-IMGT CD
(1-26) (27 -38) (39-55) (
1613F12 EVHLQQSGA ELVKP GA SVKLSCTAS GFNI ....RDTY IHWVKQRPEQGLEWI GR LD
Homsap IGHV1-2 k02 QVQLVQSGA EVKKPGASVKVSCKAS GYTF ....TGYY MHWVRQAPGQGLEWMGW IN
E H Q LV L T I K R E I R
Priority 3 2 3 33 3 3 1 3 4 4 3 2
hzl613F12 (VH1 QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY MHWVRQAPGQGLEWMGW LD
hzl613F12 (VH1M3 9I) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH1W55RN66K) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY MHWVRQAPGQGLEWMGR LD
hzl613F12 (VH1I84S) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY MHWVRQAPGQGLEWMGW LD
hzl613F12 (VH1S85N) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY MHWVRQAPGQGLEWMGW LD
hzl613F12 (VH1I84NS85N) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY MHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 1 ) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 1Q3H) QVHLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 1W55R) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGR LD
hzl613F12 (VH2 1N66K) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 1W55RN66K) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGR LD
hzl613F12 (VH2 1R80S) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 1N66KR80S) QVQLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 2 ) QVHLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 2M89L) QVHLVQSGA EVKKPGASVKVSCKAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 3 ) QVQLQQSGA EVKKPGASVKLSCTAS GFNI ....RDTY IHWVRQAPGQGLEWMGW LD
hzl613F12 (VH2 3W55R) QVQLQQSGA EVKKPGASVKLSCTAS GFNI ....RDTY IHWVRQAPGQGLEWMGR LD
hzl613F12 (VH2 3Q3HW55R) QVHLQQSGA EVKKPGASVKLSCTAS GFNI ....RDTY IHWVRQAPGQGLEWMGR LD
hzl613F12 (VH2 4 ) QVQLQQSGA EVKKPGASVKLSCTAS GFNI ....RDTY IHWVRQAPGQGLEWIGR LD
z 613F12 (VH3 EVHLQQSGA ELVKPGASVKLSCTAS GFNI ....RDTY IHWVKQAPGQGLEWIGR LD
R2-IMGT FR3-IMGT
56-65) (66-104)
1613F12 PA. .NGHT KYGPNFQ GRATMTSDTSSNTAYLQLSSLTSEDTAVYYC
Homsap IGHV1-2 *02 PN. .SGGT NYAQKFQ GRVTMTRDTSISTAYMELSRLRSDDTAVYYC
K GPN A S SN LQ S T E
Prority 2 344 4 2 11 33 4 4 4
hzl613F12 (VH1 ) PA. .NGHT NYAQKFQ GRVTMTRDTSISTAYMELSRLRSDDTAVYYC
hzl613F12 (VH1M3 9I) PA. .NGHT NYAQKFQ GRVTMTRDTSISTAYMELSRLRSDDTAVYYC
hzl613F12 (VH1W55RN66K) PA. .NGHT KYAQKFQ GRVTMTRDTSISTAYMELSRLRSDDTAVYYC
hzl613F12 (VH1 I84S) PA. .NGHT NYAQKFQ GRVTMTRDTSSSTAYMELSRLRSDDTAVYYC
hzl613F12 (VH1 S85N) PA. .NGHT NYAQKFQ GRVTMTRDTSINTAYMELSRLRSDDTAVYYC
hzl613F12 (VH1 I84NS85N) PA. .NGHT NYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 1 ) PA. .NGHT NYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 1Q3H ) PA. .NGHT NYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .1W55R) PA. .NGHT NYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .1N66K) PA. .NGHT KYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .1W55RN66K) PA. .NGHT KYAQKFQ GRVTMTRDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .1R80S) PA. .NGHT NYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .1N66KR80S) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 2 ) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .2M89L) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYLELSRLRSDDTAVYYC
hzl613F12 (VH2 3 ) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .3W55R) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 .3Q3HW55R) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYMELSRLRSDDTAVYYC
hzl613F12 (VH2 4 ) PA. .NGHT KYAQKFQ GRVTMTSDTSSNTAYLELSRLRSDDTAVYYC
hzl613F12 (VH3 ) PA. .NGHT KYGQKFQ GRVTMTSDTSSNTAYLQLSRLRSDDTAVYYC
CDR3-IMGT
1613F12VH ARGAYYYGS SGLFYFDY WGQGTTLSVSS
Homsap IGHJ5*01 WGQGTLVTVSS
TLS
Prority 444
z1613F12 (VH1 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
z1613F12 (VH1M3 9I) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH1W55RN66K) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH1I84S) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH1S85N) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH1 I84NS85N) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 1 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 1Q3H ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .1W55R) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .1N66K) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .1W55RN66K) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .1R80S) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .1N66KR80S) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 2 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .2 M89L) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 3 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .3W55R) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 .3Q3HW55R) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH2 4 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
hz1613F12 (VH3 ) ARGAYYYGS SGLFYFDY WGQGTLVTVSS
13.2. Validation of the hz!613F12 vs. ml613F12
In order to establish whether the humanized 1613F12 was comparable to its
murine 1613F12 form, binding experiments were performed both by ELISA using
rhAxl-Fc protein assays and by FACS using SN12C cells. In complement, direct in vitro
cytotoxicity assays were performed using SN12C human renal tumor cells and Calu-1
human lung carcinoma cell line.
First ELISA experiments were realized. In the assay, 96 well plates (Immulon II,
Thermo Fisher) were coated with a 5 mg/ml of the 1613F12 solution in l x PBS,
overnight at 4°C. After a saturation step, a range of rh Axl-Fc protein (R and D
Systems, ref: 154-AL) concentration (from 5 mg/ml to 0.02 mg/ml) is incubated for 1
hour at 37°C on the coated plates. For the revelation step, a biotinylated-Axl antibody
(in house product) was added at 0.85 mg/ml for 1 hour at 37°C. This Axl antibody
belongs to a distinct epitopic group. Then an avidin-horseradish peroxidase solution at
1/2000° in diluent buffer is added to the wells. Then the TMB substrate solution is
added for 5 min. After addition of the peroxydase stop solution, the absorbance at 405
nm was measured with a microplate reader.
Figure 12 shows that both murine and humanized 1613F12 antibodies binds
similarly the rhAxl-Fc protein.
For FACS analysis, SN12C cells were cultured in RPMI 1640 + 2 mM Lglutamine
+ 10% serum. Cells were detached using trypsin and cell concentration was
adjusted at 1 x 106 cells / ml in FACS buffer. A volume of 100 x of cell suspension
was incubated with increasing concentrations of either isotype controls or anti-Axl
antibodies for 20 min. at 4°C. Cells were then washed three times with FACS buffer and
incubated for 20 min. more using either anti-mouse IgG Alexa488 secondary antibody
or anti-human IgG Alexa488 secondary antibody at 4°C in the dark. Cells were washed
three times with FACS buffer and resuspended with 100 mΐ of FACS buffer before
adding propidium iodide.
Cells were incubated with increasing concentration of either isotype control or
anti-Axl antibodies. ml613F12 corresponds to the murine 1613F12, cl613F12,
corresponds to the chimeric 1613F12 and the hzl613F12 corresponds to the humanized
antibody. EC50S were determined using Prism software.
As illustrated in Figure 13, the humanized form of the 1613F12 bound SN12C
cells with equivalent EC50 to the chimeric and the murine form of the 1613F12. Those
results indicated that the hzl613F12 recognized Axl antigen with similar binding
properties to the murine 1613F12.
Experimental procedures of the direct in vitro cytotoxicity assay were previously
described in example 12. In the present example, four saporin-immunoconjugates were
prepared: m9G4-saporin, ch9G4-saporin, 1613F12-saporin and hzl613F12-saporin and
tested in two cellular models (human SN12C renal tumor cells and human Calu-1 lung
carcinoma cells).
Figure 14 shows that both m9G4-saporin and ch9G4-saporin isotype controls
were silent, and that the humanized Axl 1613F12-saporin antibody triggers similar
cytotoxic effects on SN12C cells than the mouse 1613F12-saporin immunoconjugate.
Figure 15 shows that the humanized 1613F12-saporin immunoconjugate triggers
similar cytotoxic effects on Calu-1 cells than the mouse 1613F12-saporin
immunoconjugate. In contrast, both m9G4-saporin and ch9G4-saporin isotype controls
showed weak activity (~ 10 % max cytotoxicity) for antibody concentrations above 10 9
M.
Example 14: Binding kinetics of 1613F12 to human Axl ECD
Affinity measurement of 1613F12 was then determined using Biacore. A
Biacore X is used to measure the binding kinetics of 1613F12 on human Axl ECD.
The instrument based on the optical phenomenon of surface plasmon resonance
(SPR) used by Biacore systems enables the detection and measurement of proteinprotein
interactions in real time, without the use of labels.
Briefly, the experiments were realized using a sensor chip CM5 as the biosensor.
Rabbit IgGs were immobilized on the flow cells 1 and 2 (FC1 and FC2) of a CM5
sensor chip at a level of 9300-10000 response units (RU) using amine coupling
chemistry to capture antibodies.
Binding is evaluated using multiple cycles. Each cycle of measure is performed
using a flow rate of 30m1 h h in a HBS-EP buffer. Then the Axl antibody to test is
captured on the chip for 1 min on FC2 only to reach a mean capture value of 311.8 RU
(SD=5.1 RU) for the 1613F12. The analyte (Axl ECD antigen) is injected starting at
200 nM and using two-fold serial dilutions to measure rough ka and kd in real time.
At the end of each cycle, the surfaces are regenerated by injecting a lOmM
glycine hydrochloride pH1.5 solution to eliminate the antibody-antigen complexes and
the capture antibody as well. The considered signal corresponds to the difference of the
signals observed between FC1 and FC2 (FC2-FC1). Association rates (ka) and
dissociation rates (kd) were calculated using a one-to-one Langmuir binding model. The
equilibrium dissociation constant (KD) is determined as the ka/kd ratio. The
experimental values were analyzed in the Biaevaluation software version 3.0. A c2
analysis will be performed to assess the accuracy of the data.
Data are summarized in the following Table 8.
Table 8
To produce the human extracellular domain (ECD) of Axl, the human cDNAs
coding for the human soluble AXL receptor was first cloned into the pCEP4 expression
vector by PCR. The purified product was then digested with restriction enzymes Hindlll
and BamHI and ligated into pCEP4 expression vector which had been precut with the
same enzymes. Finally, the identified recombinant plasmid pCEP[AXL]His6 was further
confirmed by DNA sequencing.
Then suspension adapted cells HEK293E were cultivated in Ex-cell 293 (SAFC
Biosciences) medium with 4 mM glutamine. All transfections were performed using
linear 25 kDa polyethyleneimine (PEI). The transfected cells were maintained at 37°C
in an incubateur shaker with 5% C0 2 and with agitation at 120 rpm for 6 days. The cells
were collected by centrifugation, and the supernatant containing the recombinant Histagged
protein was treated for purification on a Ni-NTA agarose column.
PCT
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CLAIMS
1. An antigen binding protein, or an antigen binding fragment thereof,
which:
i) specifically binds to the human protein Axl, preferably having the
sequence SEQ ID NOS. 29 or 30 or natural variant sequence thereof, and
ii) is internalized following its binding to said human protein Axl,
said antigen binding protein being characterized in that it comprises at least
an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 14 or
36 to 68.
2. The antigen binding protein, or an antigen binding fragment thereof,
according to claim 1, characterized in that it specifically binds to an epitope localized
into the human protein Axl extracellular domain, preferably having the sequence SEQ
ID NO. 3 1 or 32 or natural variant sequence thereof.
3. The antigen binding protein, or an antigen binding fragment thereof,
according to claim 1, characterized in that it induces a reduction of MFI of at least 200.
4. The antigen binding protein, or an antigen binding fragment thereof,
according any of the preceding claims, characterized in that it consists of a monoclonal
antibody.
5. An antigen binding protein, or an antigen binding fragment thereof,
characterized in that it consists of an antibody, said antibody comprising the three light
chain CDRs comprising the sequences SEQ ID NOs. 1, 2 and 3; and the three heavy
chain CDRs comprising the sequences SEQ ID NOs. 4, 5 and 6.
6. The antigen binding protein, or an antigen binding fragment thereof,
according to claim 5, characterized in that it comprises a light chain variable domain
selected in the group consisting of:
i) a light chain variable domain of sequence SEQ ID NO. 7 or any sequence
exhibiting at least 80% identity with SEQ ID NO.7,
ii) a light chain variable domain of sequence SEQ ID NO. 36 or any
sequence exhibiting at least 80%> identity with SEQ ID NO. 36; and
iii) a light chain variable domain of sequence SEQ ID NO. 37 to 47 or any
sequence exhibiting at least 80% identity with SEQ ID NO. 37 to 47.
7. The antigen binding protein, or an antigen binding fragment thereof,
according to claim 5, characterized in that comprises a heavy chain variable domain
selected in the group consisting of:
i) a heavy chain variable domain of sequence SEQ ID NO. 8 or any sequence
exhibiting at least 80% identity with SEQ ID NO.8;
ii) a heavy chain variable domain of sequence SEQ ID NO. 48 or any
sequence exhibiting at least 80%> identity with SEQ ID NO. 48; and
iii) a heavy chain variable domaine of sequence SEQ ID NO. 49 to 68 or any
sequence exhibiting at leat 80% identity with SEQ ID NO. 49 to 68.
8. The antigen binding protein, or an antigen binding fragment thereof,
according to claim 5, 6 or 7, characterized in that it comprises:
i) a light chain variable domain of sequence SEQ ID NO. 7, 36 or 37 to 47
or any sequence exhibiting at least 80%> identity with SEQ ID NO.7, 36 or 37 to 47; and
ii) a heavy chain variable domain of sequence SEQ ID NO. 8, 48 or 49 to 68
or any sequence exhibiting at least 80% identity with SEQ ID NO.8, 48 or 49 to 68.
9. The antigen binding protein according to claim 5, characterized in that it
consists of the monoclonal antibody 1613F12 derived from the hybridoma 1-4505
deposited at the CNCM, Institut Pasteur, France, on the 28 July 201 1, or an antigen
binding fragment thereof.
10. The murine hybridoma 1-4505 deposited at the CNCM, Institut Pasteur,
France, on the 28 July 201 1.
11. The antigen binding protein, or an antigen binding fragment thereof,
according to any of the claims 1 to 9, for use as an addressing product for delivering a
cytotoxic agent at a host target site, said host target site consisting of an epitope
localized into the protein Axl extracellular domain, preferably the human protein Axl
extracellular domain, more preferably the human protein Axl extracellular domain
having the sequence SEQ ID NO. 3 1 or 32 or natural variant sequence thereof.
12. An immunoconjugate comprising the antigen binding protein, or an
antigen binding fragment thereof, according to any of the claims 1 to 9 and 11
conjugated to a cytotoxic agent.
13. The immunoconjugate of claim 12 for use in the treatment of cancer.
14. Pharmaceutical composition comprising the immunoconjugate of claim
13 and at least an excipient and/or a pharmaceutical acceptable vehicle.