IGF-1 R ANTIBODY AND ITS USE AS ADDRESSING VEHICLE FOR THE TREATMENT OF CANCER
The present invention relates to a novel antibody, in particular a monoclonal
antibody, capable of binding to IGF-IR, as well as the amino and nucleic acid
sequences coding for said antibody. From one aspect, the invention relates to a novel
antibody, or an antigen binding fragment thereof, capable of binding to IGF-IR and, by
inducing internalization of IGF-IR, being internalized into the cell. The invention also
comprises the use of said antibody as an addressing product or vehicle 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.
The insulin-like growth factor 1 receptor called IGF-IR (also called IGF1R or
IGF-IR) is a receptor with tyrosine kinase activity having 70% homology with the
insulin receptor IR. IGF-IR is a glycoprotein of molecular weight approximately
350,000. It is a hetero-tetrameric receptor of which each half -linked by disulfide
bridges- is composed of an extracellular a-subunit and of a transmembrane b-subunit.
IGF-IR binds IGF1 and IGF2 with a very high affinity (Kd # 1 nM) but is equally
capable of binding to insulin with an affinity 100 to 1000 times lower. Conversely, the
IR binds insulin with a very high affinity although the IGFs only bind to the insulin
receptor with a 100 times lower affinity. The tyrosine kinase domains of IGF-IR and of
IR have a very high sequence homology although the zones of weaker homology
respectively concern the cysteine-rich region situated on the a-subunit and the Cterminal
part of the b-subunit. The sequence differences observed in the a-subunit are
situated in the binding zone of the ligands and are therefore at the origin of the relative
affinities of IGF-IR and of IR for the IGFs and insulin respectively. The differences in
the C-terminal part of the b-subunit result in a divergence in the signalling pathways of
the two receptors; IGF-IR mediating mitogenic, differentiation and anti-apoptosis
effects, while the activation of the IR principally involves effects at the level of the
metabolic pathways.
The cytoplasmic tyrosine kinase proteins are activated by the binding of the
ligand to the extracellular domain of the receptor. The activation of the kinases in its
turn involves the stimulation of different intra-cellular substrates, including IRS-1, IRS-
2, She and Grb 10. The two major substrates of IGF-IR are IRS and She which mediate,
by the activation of numerous effectors downstream, the majority of the growth and
differentiation effects connected with the attachment of the IGFs to this receptor. The
availability of substrates can consequently dictate the final biological effect connected
with the activation of the IGF-IR. When IRS-1 predominates, the cells tend to
proliferate and to transform. When She dominates, the cells tend to differentiate. It
seems that the route principally involved for the effects of protection against apoptosis
is the phosphatidyl-inositol 3-kinases (PI 3-kinases) route.
The role of the IGF system in carcinogenesis has become the subject of intensive
research in the last ten years. This interest followed the discovery of the fact that in
addition to its mitogenic and antiapoptosis properties, IGF-IR seems to be required for
the establishment and the maintenance of a transformed phenotype. In fact, it has been
well established that an overexpression or a constitutive activation of IGF-IR leads, in a
great variety of cells, to a growth of the cells independent of the support in media
devoid of foetal calf serum, and to the formation of tumors in nude mice. This in itself is
not a unique property since a great variety of products of overexpressed genes can
transform cells, including a good number of receptors of growth factors. However, the
crucial discovery which has clearly demonstrated the major role played by IGF-IR in
the transformation has been the demonstration that the IGF-IR cells, in which the gene
coding for IGF-IR has been inactivated, are totally refractory to transformation by
different agents which are usually capable of transforming the cells, such as the E5
protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T
antigen of SV 40, activated ras or the combination of these two last factors.
IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs
amplify the tumor growth via their attachment to IGF-IR. Other arguments in favor of
the role of IGF-IR in carcinogenesis come from studies using murine monoclonal
antibodies directed against the receptor or using negative dominants of IGF-IR.
Actually, murine monoclonal antibodies directed against IGF-IR inhibit the
proliferation of numerous cell lines in culture and the growth of tumor cells in vivo. It
has likewise been shown that a negative dominant of IGF-IR is capable of inhibiting
tumor proliferation.
In such a context IGF-IR has been considered for a long time as an interesting
target in oncology. A large number of projects targeting IGF-IR (humanized or human
antibodies or small molecules) have been initiated to develop IGF-IR antibodies for the
treatment of cancers and more than 70 clinical trials have been performed in various
indications. Nevertheless, at this date, none of these projects have been successful and
there are no IGF-IR antibodies on the market despite the frequent overexpression of this
target described for many patients in a wide series of indications.
Moreover, a series of clinical trials involving anti-IGF-lR antibodies combined
to anti-EGFR antibodies in order to target both EGFR and IGF-IR, have failed as none
of these antibodies were able to treat KRAS mutant patients.
As a consequence, IGF-IR is not considered now as a major target and, in the
research of potential therapeutic antibodies, IGF-IR appears no longer considered as of
particular interest.
Nevertheless, it must also be noticed that endeavours to generate IGF-IR
antibodies were focussed on naked antibodies, i.e. antibodies useful by their intrinsic
properties. In this sense, IGF-IR is considered as a target not suitable for the generation
of an immunoconjugate such as an antibody-drug conjugate (referred as "ADC") as
IGF-IR is described as a target also widely expressed by normal cells, including blood
vessels. In this sense, it can be noticed that the most recent IGF-IR antibody, i.e.
AVE1642, is developed as a naked antibody not armed with a drug. It is the same with
the other IGF-IR antibodies currently in development and with all those which failed in
clinical trials.
In one aspect, the present invention tends to remedy these issues and is describing
an IGF-IR antibody capable of binding to IGF-IR in a specific manner such as it is
suitable to be used armed with a drug. More particularly, the invention relates to an
IGF-IR antibody presenting particular properties such as it is a perfect candidate for
being used armed in the context of an immunoconjugate.
In a first embodiment, the invention relates to an antibody, or an antigen binding
fragment thereof, which i) binds to human IGF-IR, and ii) is internalized following its
binding to said human IGF- 1R.
The terms "antibody", "antibodies", "ab", "Ab" or "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal antibodies, isolated,
engineered, chemically synthesized, or recombinant antibodies (e.g., full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific
antibodies (e.g., bispecific antibodies) and also antibody fragment, so long as they
exhibit the desired biological activity. In an embodiment, the invention relates to a
recombinant antibody.
More particularly, such a 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.
By "IGF-1R binding fragment" or "antigen binding fragment" of an antibody
according to the invention, it is intended to indicate any peptide, polypeptide, or protein
retaining the ability to bind to the target (also generally referred as antigen) of the
antibody.
In an embodiment, 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 into 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. More preferably, said "antigen binding fragments"
will be constituted of or will comprise at least the three CDRs CDR-Hl, CDR-H2 and
CDR-H3 of the heavy variable chain and the three CDRs CDR-L1, CDR-L2 and CDRL3
of the light variable chain of the antibody from which they are derived.
By "binding", "binds", or the like, it is intended that the antibody, or any 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 lxlO 6 M or less. Methods for determining
whether two molecules 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 antibody could not bind or interfere, at a low level,
to another antigen. Nevertheless, as an embodiment, the said antibody binds only to the
said antigen.
As used in the present specification, the expression "IGF-1R antibody" should
be interpreted as similar to "anti-IGF-lR antibody" and means an antibody capable of
binding to IGF- 1R.
In an embodiment of the present application, the epitope of the antibody is
localized into the extracellular domain of the human IGF-1R (also referred as IGF-1R
ECD).
In a particular embodiment, the antibody, or any antigen binding fragment
thereof, is capable of binding to IGF-1R with an EC50 comprised between lOxlO 10 to
lxlO 10 , and more preferentially between 8xl0 10 to 2xlO 10M.
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 IGF-IR ECD exposed on human
tumor cells. The EC50 parameter is determined using FACS analysis. The EC50
parameter reflects the antibody concentration for which 50% the ma al binding on
the human IGF-IR 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.
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 other words
conformational epitopes are composed of non-sequential 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 a particular embodiment, the present invention relates to a method for selecting
an internalizing antibody, or an internalizing IGF-IR binding fragment thereof, which
binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) and is internalized
following to its binding to IGF-IR, said method comprising the step of selecting an
antibody:
i) which binds to an IGF-IR of SEQ ID N°52, and
ii) which does not bind to an IGF-IR of SEQ ID N°52 with an amino acid
other than Histidine at position 494 of SEQ ID N°52 or with an Aspartic acid (ASP) at
position 491, preferably which does not bind to an IGF-IR of SEQ ID N°52 with an
amino acid other than Histidine at position 494 of SEQ ID N°52 and Aspartic acid
(ASP) at position 491.
In a more particular embodiment, the present invention relates to a method for
selecting an internalizing antibody, or an internalizing IGF-IR binding fragment
thereof, which binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) and
is internalized following to its binding to IGF-IR, said method comprising the steps of:
1) selecting an antibody:
i) that binds to an IGF-IR of SEQ ID N°52, and
ii) that does not bind to an IGF-IR of SEQ ID N°52 with an amino acid other
than Histidine at position 494 of SEQ ID N°52, or with an Aspartic acid (ASP) at
position 491, preferably which does not bind to an IGF-IR of SEQ ID N°52 with an
amino acid other than Histidine at position 494 of SEQ ID N°52 and Aspartic acid
(ASP) at position 491,
and, then, from such an antibody,
2) selecting an internalizing antibody, or an IGF-IR binding fragment thereof, which
percentage of internalization following to its binding to IGF-IR is at least of 40%,
preferably at least 50%, at least 60%>, at least 70%>, or at least 80%>.
In another particular embodiment, the present invention relates to a method for
selecting an internalizing antibody, or an internalizing IGF-IR binding fragment
thereof, which binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) and
is internalized following to its binding to IGF-IR, said method comprising the steps of:
1) selecting an internalizing antibody, or an IGF-IR binding fragment thereof,
which percentage of internalization following to its binding to IGF-IR is at least
of 40%, preferably at least 50%, at least 60%, at least 70%, or at least 80%,
2) and, then, from such an antibody, selecting an antibody:
i) that binds to an IGF-IR of SEQ ID N°52, and
ii) that does not bind to an IGF-IR of SEQ ID N°52 with an amino acid
other than Histidine at position 494 of SEQ ID N°52, or with an Aspartic acid (ASP) at
position 491, preferably which does not bind to an IGF-IR of SEQ ID N°52 with an
amino acid other than Histidine at position 494 of SEQ ID N°52 and Aspartic acid
(ASP) at position 491.
In a method according to the invention, the step of selecting an antibody upon its
characteristics of internalization and of binding, or not binding, to IGF-IR may be
performed in any successive order.
According to a particular embodiment, the present invention relates to an
internalizing antibody, or an internalizing IGF-IR binding fragment thereof, which
binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR), such as obtained
by one of the above cited methods according to the invention.
In another particular embodiment, the present invention relates to an
internalizing antibody, or an internalizing IGF-IR binding fragment thereof, which
binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) of SEQ ID N°52
and is internalized following to its binding to IGF-IR, and which does not bind to an
IGF-IR of SEQ ID N°82 or 92, preferably SEQ ID N°82 and 92.
For an antibody according to the present invention, SEQ ID N°52 corresponds to
the amino acid sequence of the human IGF-IR receptor, wherein there is a Histidine at
position 494, i.e. wild-type IGF-IR, whereas SEQ ID N°82 corresponds to the mutated
amino acid sequence of the human IGF-IR receptor, wherein there is an Arginine at
position 494, and whereas SEQ ID N°92 corresponds to the mutated amino acid
sequence of the human IGF-IR receptor, wherein there is an Alanine at position 491 .
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 of SEQ
ID N°52.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 of SEQ
ID N°52, with said epitope comprising an amino acid sequence of at least 8 amino
acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Aspartic acid amino acid at position 491 of
SEQ ID N°52, with said epitope comprising an amino acid sequence of at least 8 amino
acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 and the
Aspartic acid amino acid at position 491 of SEQ ID N°52, with said epitope comprising
an amino acid sequence of at least 8 amino acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 of SEQ
ID N°52, with said epitope comprising an amino acid sequence of 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 amino acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises Histidine amino acid at position 494 of SEQ ID
N°52, with said epitope comprising an amino acid sequence of at least 8 amino acids,
wherein said epitope comprises an amino acid sequence chosen in the group consisting
of:
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 487 to the amino acid at
position 494 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 488 to the amino acid at
position 495 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 489 to the amino acid at
position 496 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 490 to the amino acid at
position 497 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 491 to the amino acid at
position 498 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 492 to the amino acid at
position 499 of SEQ ID N°52, and
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 493 to the amino acid at
position 500 of SEQ ID N°52.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Aspartic acid amino acid at position 491 of
SEQ ID N°52, with said epitope comprising an amino acid sequence of 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises Aspartic acid amino acid at position 491 of SEQ
ID N°52, with said epitope comprising an amino acid sequence of at least 8 amino
acids, wherein said epitope comprises an amino acid sequence chosen in the group
consisting of:
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 484 to the amino acid at
position 491 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 485 to the amino acid at
position 492 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 486 to the amino acid at
position 493 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 487 to the amino acid at
position 494 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 488 to the amino acid at
position 495 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 489 to the amino acid at
position 496 of SEQ ID N°52, and
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 490 to the amino acid at
position 497 of SEQ ID N°52.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 and the
Aspartic acid at position 491 of SEQ ID N°52, with said epitope comprising an amino
acid sequence of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
In a more particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, wherein the epitope of
said internalizing antibody comprises the Histidine amino acid at position 494 and the
Aspartic acid at position 491 of SEQ ID N°52, with said epitope comprising an amino
acid sequence of at least 8 amino acids, wherein said epitope comprises an amino acid
sequence chosen in the group consisting of:
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 487 to the amino acid at
position 494 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 488 to the amino acid at
position 495 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 489 to the amino acid at
position 496 of SEQ ID N°52,
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 490 to the amino acid at
position 497 of SEQ ID N°52 and
- an amino acid sequence identical to, or exhibiting at least 80% identity with,
the amino acid sequence from the amino acid at position 491 to the amino acid at
position 498 of SEQ ID N°52.
In another particular embodiment, the present invention relates to an
internalizing antibody, or an internalizing IGF-IR binding fragment thereof, which
binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) of SEQ ID N°52
and is internalized following to its binding to IGF-IR, and which does not bind to an
IGF-IR of SEQ ID N°82, or wherein the epitope of said internalizing antibody
comprises the Histidine amino acid at position 494 and/or the Aspartic acid amino acid
at position 491 of SEQ ID N°52, wherein the percentage of internalization of said
antibody following to its binding to IGF-IR is of at least 40%, at least 50%, at least
60%, at least 70%, or at least 80%. The percentage of internalization of an antibody, or
of an antigen-binding fragment thereof, may be determined by any method known by a
person skilled in the art, such as, for example, a method described in the present
specification.
In a particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, according to the
invention, wherein said amino acid other than Histidine at position 494 of SEQ ID N°52
is Arginine (SEQ ID N°82).
In a particular embodiment, the present invention relates to an internalizing
antibody, or an internalizing IGF-IR binding fragment thereof, according to the
invention, wherein said amino acid other than Aspartic acid at position 491 of SEQ ID
N°52 is Alanine (SEQ ID N°92)
According to an embodiment, the invention relates to an antibody, or an antigen
binding fragment thereof, which binds to the human Insulin like Growth Factor 1
Receptor (IGF-IR) and which is internalized following its binding to IGF-IR, wherein
said antibody is selected from:
i) an antibody comprising three heavy chain CDRs with CDR-H2 of sequence SEQ ID
No. 2 and CDR-H3 of sequence SEQ ID No. 3, and three light chain CDRs with CDRL2
of sequence SEQ ID No. 5;
ii) an antibody which competes for binding to IGF-IR with the antibody of i); and
iii) an antibody which binds to the same epitope of IGF-IR as does the antibody of i).
The competition for binding to IGF-IR can be determined by any methods or
techniques known by the person skilled in the art such as, without limitation,
radioactivity, Biacore, ELISA, Flow cytometry, etc, or according to a method such as
described in the present specification.
The determination of the binding to the same epitope can be determined by any
methods or techniques known by the person skilled in the art such as, without
limitation, radioactivity, Biacore, ELISA, Flow cytometry, etc, or according to a method
such as described in the present specification.
As above mentioned, and contrary to the general knowledge, the present
invention focuses on specific IGF-IR antibodies presenting a high ability to be
internalized following IGF-IR binding. As used herein, an antibody that "is
internalized" or that "internalized" (the two expressions being similar) is one that is
taken up by (meaning it "enters") the cell upon binding to IGF-IR on a mammalian cell.
Such an antibody is interesting as one of the immuno-drug-conjugate components, so it
addresses or directs the linked cytotoxic into the targeted cells, preferably cancer cells.
Once internalized the cytotoxic triggers cancer cell death.
Preferably, the antibodies according to the invention are all presenting the same
sequences for the CDR-H2, CDR-H3 and CDR-L2, the other 3 CDRs being different.
This observation seems coherent as it is part of the general knowledge that, regarding
the binding specificity of an antibody, the CDR-H3 is described as being the most
important and the most implicated with the recognition of the epitope.
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
antibodies.
In the immunoconjugate, the cytotoxic brings the cytotoxic activity and the used
antibody 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, the antibody can exhibit high ability to internalize into the targeted
cancer cells. The efficiency with which the antibody mediated internalisation differs
significantly depending on the epitope targeted. Selection of potent internalizing IGF-
1R antibodies requires various experimental data studying not only IGF-1R
downregulation but also following IGF-1R antibody internalization into the cells.
In one embodiment, the internalization of the antibody according to the
invention can be evaluated 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.
The complex IGF-lR/antibody is internalized after the binding of the antibody to
the ECD of said IGF-1R, a reduction in the quantity of IGF-1R at the surface of the
cells is induced. This reduction can be quantified by any method known by the person
skilled in the art such as, as non limitative examples, western-blot, FACS,
immunofluorescence and the like.
In one embodiment, 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 at 4°C with the MFI measured at 37°C
after 4 hours incubation with the antibody.
As non limitative example, this delta is determined based on MFIs obtained with
untreated cells and cells treated with the antibody using i) breast cancer cells MCF7
after a 4 hour incubation period with the antibody herein described and ii) a secondary
antibody labelled with Alexa488. This parameter is defined as calculated with the
following formula: A(MFI4°c- MFl37°c).
This difference between MFIs reflects the IGF-1R downregulation as MFIs are
proportional of IGF-1R expressed on the cell-surface.
In an advantageous aspect, the antibodies, or any antigen binding fragment
thereof, consist of monoclonal antibodies triggering a A(MFI4°c- MFl3 c ) on MCF7 of
at least 280, preferably of at least 400.
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 antibody of
the invention in either cold (4°C) or warm (37°C) complete culture
medium;
b) Treating the treated cells of step a) and, in parallel, untreated cells
with a secondary antibody,
c) Measuring the MFI (representative of the quantity of IGF-1R present
at the surface) for the treated and the non treated cells with a
secondary labeled antibody capable of binding to the antibody of the
invention, 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.
From this delta MFI, an internalization percentage can be determined as:
100x(MFI 4°c-MFI37 c) / MFI 4°c
The antibodies, or any antigen binding fragment thereof, according to the
invention, present on MCF7 a internalization percentage comprised between 70% and
90%, preferentially between 75% and 87%.
A particular advantage of the antibodies herein described relies on their rate of
internalization.
It is generally known that, for an immunoconjugate, it is desirable that the used
antibodies exhibit a rapid rate of internalization, preferably within 24 hours from
administration of the antibody in vivo and, more preferably within 12 hours and, even
more preferably within 6 hours.
In the present invention, the internalization rate, also referred as cell surface
bound antibody decrease or cell surface antibody decay, is expressed as tl/2 (half life)
and corresponds as the time necessary to obtain a decrease of 50% of the AMFI (this
aspect will be clearly understood regarding the following examples).
A particular advantage is that the antibodies of the invention have a tl/2
comprised between 5 and 25 minutes, and preferentially between 10 and 20 minutes.
A particular embodiment of the invention relates to an antibody comprising the
three heavy chain CDRs of sequences SEQ ID Nos. 1, 2 and 3 and the three light chain
CDRs of sequences SEQ ID Nos. 4, 5 and 6.
An embodiment is an antibody, or an antigen binding fragment thereof,
comprising the three heavy chain CDRs comprising, or consisting of, the sequences
SEQ ID Nos. 1, 2 and 3, or any sequence exhibiting at least 80%, preferably 85%>, 90%>,
95% or 98% identity with SEQ ID Nos. 1, 2 and 3; and the three light chain CDRs
comprising or consisting of the sequences SEQ ID Nos. 4, 5 and 6, or any sequence
exhibiting at least 80%, preferably 85%, 90%, 95% or 98% identity with SEQ ID Nos.
4, 5 and 6.
In another embodiment, the antibody, or any antigen binding fragment thereof,
comprises the three heavy chain CDRs comprising or consisting of the sequences SEQ
ID Nos. 1, 2 and 3; and the three light chain CDRs comprising or consisting of the
sequences SEQ ID Nos. 4, 5 and 6.
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.
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. In order to simplify
the reading of the present application, the CDRs according to Kabat are not defined.
Nevertheless, it would be obvious for the person skilled in that art, using the definition
of the CDRs according to IMGT, to define the CDRs according to Kabat.
In the sense of the present invention, the "percentage identity" between two
sequences of nucleic acids or amino acids means the percentage of identical nucleotides
or amino acid residues between the two sequences to be compared, obtained after
optimal alignment, this percentage being purely statistical and the differences between
the two sequences being distributed randomly along their length. The comparison of
two nucleic acid or amino acid sequences is traditionally carried out by comparing the
sequences after having optimally aligned them, said comparison being able to be
conducted by segment or by using an "alignment window". Optimal alignment of the
sequences for comparison can be carried out, in addition to comparison by hand, by
means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J.
Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman
(1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means of computer software using
these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by the
comparison software BLAST NR or BLAST P).
The percentage identity between two nucleic acid or amino acid sequences is
determined by comparing the two optimally-aligned sequences in which the nucleic acid
or amino acid sequence to compare can have additions or deletions compared to the
reference sequence for optimal alignment between the two sequences. Percentage
identity is calculated by determining the number of positions at which the amino acid
nucleotide or residue is identical between the two sequences, preferably between the
two complete sequences, dividing the number of identical positions by the total number
of positions in the alignment window and multiplying the result by 100 to obtain the
percentage identity between the two sequences.
For example, the BLAST program, "BLAST 2 sequences" (Tatusova et al,
"Blast 2 sequences - a new tool for comparing protein and nucleotide sequences",
FEMS Microbiol, 1999, Lett. 174:247-250) available on the site
http://www.ncbi.nlm.nih.gov/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%
or 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 be
generated.
As a non-limiting example, table 1 below summarizes the possible substitutions
likely to be carried out without resulting in a significant modification of the biological
activity of the corresponding modified antigen binding protein; inverse substitutions are
naturally possible under the same conditions.
Table 1
A particular aspect of the invention is that the antibody, or any antigen binding
fragment thereof, does not bind to the Insulin receptor (IR). This aspect is of interest as
the antibody herein described will not have any negative impact on the IR, meaning the
Insulin metabolism.
In another embodiment, still another advantageous aspect of the antibody of the
invention is that it is capable of binding not only to the human IGF-IR but also to the
monkey IGF-IR, and more particularly to the cynomolgus IGF-IR. This aspect is also
of interest as it will facilitate the toxicity and clinical trials.
In another embodiment, the antibody of the invention consists of a monoclonal
antibody.
The term "monoclonal antibody" or "Mab" as used herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e. the individual
antibodies of the population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single epitope. Such monoclonal antibody may be
produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also
be recombinant, i.e. produced by protein engineering. Monoclonal antibodies may also
be isolated from phage antibody libraries. 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 an antibody isolated or obtained
by purification from natural sources or obtained by genetic recombination or chemical
synthesis.
In one embodiment, the monoclonal antibody herein includes murine, chimeric
and humanized antibody, such as described after.
The antibody can be derived from an hybridoma of murine origin filed within
the French collection for microorganism cultures (CNCM, Pasteur Institute, Paris,
France), said hybridoma being obtained by the fusion of Balb/C immunized mice
splenocytes/lymphocytes and cells of the myeloma Sp 2/0- Ag 14 cell line.
In another embodiment, the antibody of the invention consists of a recombinant
antibody. The term "recombinant antibody" refers to an antibody that results from the
expression of recombinant DNA within living cells. A recombinant antibody of the
invention is obtained by using laboratory methods of genetic recombination, well
known by a person skilled in the art, creating DNA sequences that would not be found
in biological organisms.
In another embodiment, the antibody of the invention consists of a chemically
synthesized antibody.
In an embodiment, the IGF-IR antibody of the invention consists of a murine
antibody, then referred as m[name of the antibody].
In an embodiment, the IGF-IR antibody consists of a chimeric antibody, then
referred as c[name of the antibody].
In an embodiment, the IGF-IR antibody consists of a humanized antibody, then
referred as hz[name of the antibody].
For the avoidance of doubt, in the following specification, the expressions "IGF-
1R antibody" and "[name of the antibody]" are similar and include (without contrary
specification) the murine, the chimeric and the humanized versions of the said IGF-IR
antibody and said "[name of the antibody]". When necessary, the prefix m- (murine), c-
(chimeric) or hz- (humanized) is used.
In another embodiment, the antibody of the invention is selected from:
a) an antibody comprising the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2
and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2
and 3 and the three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;
c) an antibody comprising the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2
and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12; and
d) an antibody comprising the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2
and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.
For more clarity, the following table 2 illustrates the CDR sequences, defined
according to IMGT, for the preferred antibodies.
Table 2
It will be obvious for the man skilled in the art that any combination of the 6
CDRs as above described should be considered as part of the present invention.
As can be observed from this table 2, all the antibodies described in the table
have the same sequences for the CDR-H2, CDR-H3 and CDR-L2, this property being of
particular interest as above described.
A specific aspect relates to a murine (m) antibody, or any antigen binding
fragments, characterized in that said antibody also comprises light-chain and heavychain
constant regions derived from an antibody of a species heterologous with the
mouse, notably man.
Another specific aspect relates to a chimeric (c) antibody, or any antigen binding
fragments, characterized in that said antibody also comprises light-chain and heavychain
constant regions derived from an antibody of a species heterologous with the
mouse, notably human.
In an embodiment of the invention, the antibody consists of a chimeric antibody.
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.
In a preferred, but not limitative, embodiment, the antibody of the invention is
selected from:
a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13
or any sequence exhibiting at least 80% identity with SEQ ID No. 13 and the three light
chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising, or consisting of, a heavy chain variable domain of sequence
SEQ ID No. 14 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98%
identity with SEQ ID No. 14 and the three light chain CDRs of sequences SEQ ID Nos.
10, 5 and 11;
c) an antibody comprising , or consisting of, a heavy chain variable domain of sequence
SEQ ID No. 15 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98%
identity with SEQ ID No. 15 and the three light chain CDRs of sequences SEQ ID Nos.
9, 5 and 12;
d) an antibody comprising, or consisting of, a heavy chain variable domain of sequence
SEQ ID No. 16 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98%
identity with SEQ ID No. 16 and the three light chain CDRs of sequences SEQ ID Nos.
9, 5 and 11; and
e) an antibody comprising, or consisting of, a heavy chain variable domain of sequence
SEQ ID No. 17 or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98%
identity with SEQ ID No. 1 and the three light chain CDRs of sequences SEQ ID Nos.
9, 5 and 12.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% or 98%
identity with SEQ ID No. 13 to 17", its is intended to designate, respectively, a
sequence exhibiting the three heavy chain CDRs SEQ ID Nos. 1, 2 and 3 and, in
addition, exhibiting at least 80%, preferably 85%, 90%, 95% or 98%, identity with the
full sequence SEQ ID Nos. 13 to 17 outside the sequences corresponding to the CDRs
(i.e. SEQ ID No. 1, 2 and 3), wherein "outside the sequences corresponding to the
CDRs" is intended for "excepting the sequences corresponding to the CDRs".
In another preferred, but not limitative, embodiment, the antibody of the
invention is selected from:
a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 18 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 18 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 19 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 19 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
c) an antibody comprising a light chain variable domain of sequence SEQ ID No. 20 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 20 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
d) an antibody comprising a light chain variable domain of sequence SEQ ID No. 2 1 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 2 1 and the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and 3; and
e) an antibody comprising a light chain variable domain of sequence SEQ ID No. 22 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 22 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% or 98%
identity with SEQ ID No. 18 to 22", its is intended to designate the sequences exhibiting
the three light chain CDRs SEQ ID Nos. 4, 5 and 6 and, in addition, exhibiting at least
80%, preferably 85%, 90%, 95% or 98% , identity with the full sequence SEQ ID No.
18 to 22 outside the sequences corresponding to the CDRs (i.e. SEQ ID No. 4, 5 and 6).
An embodiment of the invention relates to an antibody selected from:
a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 13
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 13 and a light chain variable domain of sequence SEQ ID No. 18 or any sequence
exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 18;
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 14
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 14 and a light chain variable domain of sequence SEQ ID No. 19 or any sequence
exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO. 19;
c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 15
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 15 and a light chain variable domain of sequence SEQ ID No. 20 or any sequence
exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 20;
d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 16
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 16 and a light chain variable domain of sequence SEQ ID No. 2 1 or any sequence
exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 2 1; and
e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 1
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 1 and a light chain variable domain of sequence SEQ ID No. 22 or any sequence
exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 22.
Chimeric antibodies herein described can be also characterized by the constant
domain and, more particularly, said chimeric antibodies can be selected or designed
such as, without limitation, IgGl, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably,
in the context of the present invention, said chimeric antibodies are IgGl or IgG4.
An embodiment of the invention relates to a chimeric antibody comprising
variable domains VH and VL as above described in the format IgGl. More preferably,
said chimeric antibody comprises a constant domain for the VH of sequence SEQ ID
No. 43 and a Kappa domain for the VL of sequence SEQ ID No. 45.
An embodiment of the invention relates to a chimeric antibody comprising
variable domains VH and VL as above described in the format IgG4. More preferably,
said chimeric antibody comprises a constant domain for the VH of sequence SEQ ID
No. 44 and a Kappa domain for the VL of sequence SEQ ID No. 45.
In another preferred, but not limitative, embodiment, the antibody of the
invention is selected from:
a) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 23
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 23 and a light chain of sequence SEQ ID No. 28 or any sequence exhibiting at least
80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 28;
b) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 24
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 24 and a light chain of sequence SEQ ID No. 29 or any sequence exhibiting at least
80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 29;
c) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 25
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 25 and a light chain of sequence SEQ ID No. 30 or any sequence exhibiting at least
80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 30;
d) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 26
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 26 and a light chain of sequence SEQ ID No. 3lor any sequence exhibiting at least
80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 31; and
e) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 27
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 27 and a light chain of sequence SEQ ID No. 32 or any sequence exhibiting at least
80%,85%, 90%, 95% or 98% identity with SEQ ID No. 32.
For more clarity, the following table 3 illustrates the sequences of the VH and
VL, respectively, for the preferred chimeric antibodies.
Table 3
Yet another specific aspect of the present invention relates to a humanized
antibody, or an antigen binding fragment thereof, 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.
In an embodiment of the invention, the antibody 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.
The humanized antibodies or fragments of same can be prepared by techniques
known to a person skilled in the art. Such humanized antibodies are preferred for their
use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment
in vivo. Other humanization techniques, also known to a person skilled in the art, such
as, for example, the "CDR grafting" technique described by PDL in patents EP 0 451
216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101, US 6,180,370, US
5,585,089 and US 5,693,761. US patents 5,639,641 or 6,054,297, 5,886,152 and
5,877,293 can also be cited.
As a particular embodiment of the invention, and as it will be explicated in more
details in the following examples, it is herein described an antibody consisting of the
hz208F2. Such humanization can also be applied to the other antibodies part of the
present invention.
In a preferred embodiment, the antibody according to the present invention
comprises a heavy chain variable domain (VH) having:
i) the CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7, 2 and 3,
respectively,
ii) the FR1, FR2 and FR3 derived from the human germline IGHV1-46*01 (SEQ ID
No. 46), and
iii) the FR4 derived from the human germline IGHJ4*01 (SEQ ID No. 48).
In a preferred embodiment, the antibody according to the present invention
comprises a light chain variable domain (VL) having:
i) the CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5 and 11,
respectively,
ii) the FR1, FR2 and FR3 derived from the human germline IGKV1-39*01 (SEQ ID
No. 47), and
iii) the FR4 derived from the human germline IGKJ4*01 (SEQ ID No. 49).
In a preferred, but not limitative, embodiment of the invention, the antibody
comprises:
a) a heavy chain having CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID Nos. 7,
2 and 3, respectively, and FR1, FR2 and FR3 derived from the human germline IGHV1-
46*01 (SEQ ID No. 46), and the FR4 derived from the human germline IGHJ4*01
(SEQ ID No. 48); and
b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID Nos. 9, 5
and 11, respectively, and FR1, FR2 and FR3 derived from the human germline IGKV1-
39*01 (SEQ ID No. 47), and the FR4 derived from the human germline IGKJ4*01
(SEQ ID No. 49).
In an embodiment, the antibody according to the invention comprises a heavy
chain variable domain (VH) of sequence SEQ ID No. 33 and a light chain variable
domain (VL) of sequence SEQ ID No. 35. Said humanized antibody will be called
thereinafter hz208F2 ("Variant" or "Var." 1).
In another embodiment, the antibody according to the present invention
comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein
said sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from the
residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95.
By the expressions "back-mutation" or "back mutation" it is meant a mutation or
replacement of the human residue present in the germline by the corresponding residue
initially present in the murine sequence.
In another embodiment, the antibody according to the present invention
comprises a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein
said sequence SEQ ID No. 33 comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
or 17 back-mutations selected from the residues 20, 34, 35, 38, 48, 50, 59, 61, 62, 70,
72, 74, 76, 77, 79, 82 and 95.
For more clarity, the following table 4 illustrates the preferred back-mutations.
Table 4
In an embodiment, the antibody according to the present invention comprises a
light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said sequence
SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues 22, 53,
55, 65, 71, 72, 77 and 87.
In an embodiment, the antibody according to the present invention comprises a
light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said sequence
SEQ ID No. 35 comprises 2, 3, 4, 5, 6, 7 or 8 back-mutations selected from the residues
22, 53, 55, 65, 71, 72, 77 and 87.
In another embodiment, the antibody according to the present invention
comprises:
a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein said
sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from the residues
20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95; and
b) a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues
22, 53, 55, 65, 71, 72, 77 and 87.
For more clarity, the following table 5 illustrates the preferred back-mutations.
Table 5
In such an embodiment, the antibody according to the invention comprises all
the back-mutations above mentioned and corresponds to an antibody comprising a
heavy chain variable domain (VH) of sequence SEQ ID No. 34 and a light chain
variable domain (VL) of sequence SEQ ID No. 36. Said humanized antibody will be
called thereinafter hz208F2 ("Variant" or "Var." 3).
In another embodiment, all the humanized forms comprised between the Variant
1 and the Variant 3 are also encompassed by the present invention. In other words, the
antibody according to the invention corresponds to an antibody comprising a heavy
chain variable domain (VH) of "consensus" sequence SEQ ID No. 4 1 and a light chain
variable domain (VL) of "consensus" sequence SEQ ID No. 42. Said humanized
antibody, as a whole, will be called thereinafter hz208F2 ("Variant" or "Var." 2).
In a preferred, but not limitative, embodiment, the antibody of the invention is
selected from:
a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 33
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 33 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 34
or any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 34 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and
c) an antibody comprising a heavy chain variable domain of sequence selected from
SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at least
80%, 85%, 90%, 95% or 98% identity with SEQ ID No.56, 62, 64, 66, 68, 70, 72, 74,
76, 78 80; and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% or 98%
identity with SEQ ID No. 33, 34, 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80", its is
intended to designate the sequences exhibiting the three heavy chain CDRs SEQ ID
Nos. 1, 2 and 3 and, in addition, exhibiting at least 80%>, preferably 85%, 90%>, 95% or
98%, identity with the full sequence SEQ ID No. 33,34, 56, 62, 64, 66, 68, 70, 72, 74,
76, 78 80 outside the sequences corresponding to the CDRs (i.e. SEQ ID Nos. 1, 2 and
3).
In a preferred, but not limitative, embodiment, the antibody of the invention is
selected from:
a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 35 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 35 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and
b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 36 or
any sequence exhibiting at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID
No. 36 and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and
c) an antibody comprising a light chain variable domain of sequence selected from
SEQ ID Nos. 57 and 60 or any sequence with at least 80%, 85%, 90%, 95% or 98%
identity with SEQ ID No. 57 or 60; and the three heavy chain CDRs of sequences SEQ
ID Nos. 7, 2 and 3.
By "any sequence exhibiting at least 80%, preferably 85%, 90%, 95% or 98%
identity with SEQ ID No. 35,36, 57 or 60", its is intended to designate the sequences
exhibiting the three light chain CDRs SEQ ID Nos. 4, 5 and 6 and, in addition,
exhibiting at least 80%, preferably 85%, 90%, 95% or 98%, identity with the full
sequence SEQ ID Nos. 35,36, 57 or 60 outside the sequences corresponding to the
CDRs (i.e. SEQ ID Nos. 4, 5 and 6).
Humanized antibodies herein described can be also characterized by the constant
domain and, more particularly, said humanized antibodies can be selected or designed
such as, without limitation, IgGl, IgG2, IgG3, IgM, IgA, IgD or IgE. More preferably,
in the context of the present invention, said humanized antibodies are IgGl or IgG4.
An embodiment of the invention relates to a humanized antibody comprising
variable domains VH and VL as above described in the format IgGl. More preferably,
said humanized antibody comprises a constant domain for the VH of sequence SEQ ID
No. 43 and a Kappa domain for the VL of sequence SEQ ID No. 45.
An embodiment of the invention relates to a humanized antibody comprising
variable domains VH and VL as above described in the format IgG4. More preferably,
said humanized antibody comprises a constant domain for the VH of sequence SEQ ID
No. 44 and a Kappa domain for the VL of sequence SEQ ID No. 45.
Still another embodiment of the invention relates to an antibody selected from:
a) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 37
or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID
No. 37 and a light chain comprising, or consisting of, sequence SEQ ID No. 39 or any
sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No.
39;
b) an antibody comprising, or consisting of, a heavy chain of sequence SEQ ID No. 38
or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID
No. 38 and a light chain comprising, or consisting of, sequence SEQ ID No. 40 or any
sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No.
40; and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No. 56, 62, 64, 66, 68, 70,
72, 74, 76, 78 or 80 and a light chain variable domain of sequence selected from
SEQ ID Nos. 57 60 or any sequence with at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 57 or 60.For more clarity, the following table 6a illustrates
non limitative examples of sequences of the VH and VL for the variant 1 (Var. 1) and
the variant 3 (Var. 3) of the humanized antibody hz208F2. It also comprises the
consensus sequence for the variant 2 (Var. 2).
Table 6a
In another preferred, but not limitative, embodiment, the antibody of the
invention is selected from:
a) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No.56, 62, 64, 66, 68, 70, 72,
74, 76, 78 or 80; and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 and60 or any sequence with at least 80%, 85%, 90%, 95%, or
98%o identity with SEQ ID No. 57 or 60; and the three heavy chain CDRs of sequences
SEQ ID Nos. 7, 2 and 3; and
c) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 and60 or any sequence with at least 80%, 85%, 90%, 95%, or
98% identity with SEQ ID No. 57 or 60; and a heavy chain variable domain of sequence
selected from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence
with at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No. 56, 62, 64, 66, 68,
70, 72, 74, 76, 78 or 80.
Still another embodiment of the invention relates to an antibody selected from an
antibody comprising or consisting of:
a) a heavy chain of sequence selected from SEQ ID Nos. 58, 63, 65, 67, 69, 71,
73, 75, 77, 79 and 8 1 or any sequence with at least 80%,preferably 85%, 90%, 95% or
98% identity with SEQ ID Nos. 58, 63, 65, 67, 69, 71, 73, 75, 77, 79 or 81; and
b) a light chain of sequence selected from SEQ ID Nos. 59 and 6 1 or any
sequence with at least 80%, preferably 85%, 90%, 95% or 98% identity with SEQ ID
Nos. 59 or 61. Still another embodiment of the invention relates to an antibody selected
from:a) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence
exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No. 56, 62, 64,
66, 68, 70, 72, 74, 76, 78 or 80;and a light chain variable domain of sequence SEQ ID
No. 57 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with
SEQ ID No. 57; and
b) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 64, 68 and 78 or any sequence exhibiting at least 80%>, 85%>,
90%, 95%, or 98% identity with SEQ ID No. 56, 64, 68 or 78 and a light chain variable
domain of sequence SEQ ID No. 60 or any sequence exhibiting at least 80%> identity
with SEQ ID No. 60
Still another embodiment of the invention relates to an antibody selected from:
a) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 58 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 58 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
b) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 58 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 58 and a light chain of sequence SEQ ID No. 6 1 or any
sequence exhibiting at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID No.
61;
c) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 63 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 63 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
d) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 65 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 65 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
e) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 65 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 65 and a light chain comprising, or consisting of, sequence
SEQ ID No. 6 1 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 61;
f an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 67 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 67 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
g) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 69 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 69 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
h) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 69 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 69 and a light chain comprising, or consisting of, sequence
SEQ ID No. 6 1 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 61;
i) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 7 1 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 1 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
j ) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 73 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 73 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
k) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 75 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 75 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
1) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 77 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 77 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
m) an antibody comprising, or consisting of,a heavy chain of sequence
SEQ ID No. 79 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 79 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59;
n) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 79 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 79 and a light chain comprising, or consisting of, sequence
SEQ ID No. 6 1 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 61; and
o) an antibody comprising, or consisting of, a heavy chain of sequence
SEQ ID No. 8 1 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 8 1 and a light chain comprising, or consisting of, sequence
SEQ ID No. 59 or any sequence exhibiting at least 80%, 85%, 90%, 95%, or 98%
identity with SEQ ID No. 59.
For more clarity, the following table 6b illustrates non limitative examples of
sequences of the VH and VL (vaiable domain and full length) for different variants of
the humanized antibody hz208F2.
Table 6b
Heavy Chain Light chain SEQ ID NO.
Variable domain (VH) 56
hz208F2 Variable domain (VL) 57
H037/L01 8 Full length 58
Full length 59
Variable domain (VH) 56
Hz208F2 Variable domain (VL) 60
H037/L021 Full length 58
Full length 6 1
Variable domain (VH) 62
Hz208F2 Variable domain (VL) 57
H047/L01 8 Full length 63
Full length 59
Variable domain (VH) 64
Hz208F2 Variable domain (VL) 57
H049/L01 8 Full length 65
Full length 59
Variable domain (VH) 64
Hz208F2 Variable domain (VL) 60
H049/L021 Full length 65
Full length 6 1
Variable domain (VH) 66
Hz208F2 Variable domain (VL) 57
H05 1/L01 8 Full length 67
Full length 59
Variable domain (VH) 68
Hz208F2 Variable domain (VL) 57
H052/L01 8 Full length 69
Full length 59
Variable domain (VH) 68
Hz208F2 Variable domain (VL) 60
H052/L021 Full length 69
Full length 6 1
Variable domain (VH) 70
Hz208F2 Variable domain (VL) 57
H057/L018 Full length 7 1
Full length 59
Variable domain (VH) 72
Hz208F2 Variable domain (VL) 57
H068/L018 Full length 73
Full length 59
Variable domain (VH) 74
Hz208F2 Variable domain (VL) 57
H070/L018 Full length 75
Full length 59
Variable domain (VH) 76
Hz208F2 Variable domain (VL) 57
H071/L018 Full length 77
Full length 59
Variable domain (VH) 78
Hz208F2 Variable domain (VL) 57
H076/L018 Full length 79
Full length 59
Variable domain (VH) 78
Hz208F2 Variable domain (VL) 60
H076/L021 Full length 79
Full length 6 1
Variable domain (VH) 80
Hz208F2 Variable domain (VL) 57
H077/L018 Full length 8 1
Full length 59
Another aspect of the present invention is an antibody selected from:
i) an antibody produced by the hybridoma 1-4757, 1-4773, 1-4775, 1-4736 or 1-4774
deposited at the CNCM, Collection Nationale de Culture de Microorganismes, Institut
Pasteur, 25, rue du Docteur Roux, 75724 Paris, France on the 30 May 2013, 26 June
2013, 26 June 2013, 24 April 2013 and 26 June 2013, respectively,
ii) an antibody which competes for binding to IGF-1R with the antibody of i); and
iii) an antibody which binds to the same epitope of IGF-1R as does the antibody of i).
According to another aspect, the invention relates to a murine hybridoma
selected from the hybridoma 1-4757, 1-4773, 1-4775, 1-4736 and 1-4774 deposited at the
CNCM, Institut Pasteur France on the 30 May 2013, 26 June 2013, 26 June 2013, 24
April 2013 and 26 June 2013, respectively.
A novel aspect of the present invention relates to an isolated nucleic acid
coding for an antibody, or for an antigen binding fragment thereof, according to the
invention.
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.
The invention also relates to a vector comprising a nucleic acid coding for an
antibody, or for an antigen binding fragment thereof, according to the invention.
The invention notably targets cloning and/or expression vectors that contain
such a nucleotide sequence.
The vectors 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 relates to isolated host cells transformed by or comprising a
vector as above described.
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.
Another aspect relates to a method for the production of an antibody 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 antibody, or one of its antigen binding fragments, thus
produced from the culture medium or from said cultured cells.
The transformed cells are of use in methods for the preparation of recombinant
antibodies according to the invention. Methods for the preparation of antibodies in
recombinant form using a vector and/or a cell transformed by a vector according to the
invention, are also comprised in the present specification. Preferably, a cell transformed
by a vector as above described is cultured under conditions that allow the expression of
the aforesaid antibody and recovery of said antibody.
As already mentioned, the host cell can be selected among prokaryotic or
eukaryotic systems. In particular, it is possible to identify the nucleotide sequences 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 antibody 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 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 antibody, or any antigen binding fragments of same, likely to be obtained by
the method above described are also comprised in the present invention.
According to a particular aspect, the invention concerns an antibody, or an
antigen binding fragment thereof, as above described for use as an addressing vehicle
for delivering a cytotoxic agent at a host target site, said host target site consisting of an
epitope localized into IGF-IR, preferably the IGF-IR extracellular domain, more
preferably the human IGF-IR (SEQ ID No. 50) and still more preferably the human
IGF-IR extracellular domain (SEQ ID No. 51), and still more preferably to the Nterminal
of the human IGF-IR extracellular domain (SEQ ID No. 52), or any 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 genetic recombination, express IGF-IR.
Another aspect of the invention is an antibody-drug conjugate comprising the
antibody, or an antigen binding fragment thereof, as above described, conjugated to a
cytotoxic agent.
The invention relates to an immunoconjugate comprising the antibody as
described in the present specification conjugated to a cytotoxic agent.
The expressions "immunoconjugate" or "immuno-conjugate" refer generally to a
compound comprising at least an addressing product, such as an antibody, 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 abnormal 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 affected 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 affected 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, the immunoconjugate consists of an antibody
linked to at least a drug or a medicament. Such an immunoconjugate is referred as an
antibody-drug conjugate (or "ADC").
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 agonists, 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 or medicaments 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
amino levulinate, histrelin, hydroxyprogesterone, ixabepilone, leuprolide,
medroxyprogesterone acetate, megesterol acetate, methylprednisolone,
methyltestosterone, miltefosine, mitobronitol, nadrolone phenylpropionate,
norethindrone acetate, prednisolone, prednisone, temsirrolimus, testo lactone,
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,
defrbrotide, 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, armodafmil, 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, ciclonicate and pyrrolobenzodiazepines,
particularly those disclosed in the PCT application published under number
WO201 1/130598.
In another embodiment, the immunoconjugate consists of an antibody linked to
at least a radioisotope. Such an immunoconjugate is referred as an antibodyradioisotope
conjugate (or "ARC").
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. 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.
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 ; 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.
In another embodiment, the immunoconjugate consists of an antibody linked to
at least a toxin. Such an immunoconjugate is referred as an antibody-toxin conjugate (or
"ATC").
Toxins are effective and specific poisons produced by living organisms. They
usually consist of an amino acid chain which can vary i 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, wit a toxicity
that is several orders of magnitude greater than 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, particularly the
monomethylauristatine E (MMAE), 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 an antibody 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 o f 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 o f the linker releases the cytotoxic agent from the antibody 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 is cleaved by 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 is 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 hydrolyzable under
acidic conditions. For example, an acid-labile linker that is hydrolyzable 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-DMl (TDM1) which combines
trastuzumab with a linked chemotherapy agent, maytansine.
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 antibody 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 antibody.
Nucleophilic groups on antibody include, without limitation, N-terminal 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 antibody may have reducible interchain disulfides, i.e. cysteine bridges.
The antibody 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 antibody through any reaction known by the person skilled in the art.
As non limitative example, reactive thiol groups may be introduced into the antibody by
introducing one or more cysteine residues.
Immunoconjugates may also be produced by modification of the antibody to
introduce electrophilic moieties, which can react with nucleophilic substituents on the
linker reagent or cytotoxic agent. The sugars of glycosylated antibody 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 antibody with either galactose oxidase or
sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the
antibody that can react with appropriate groups on the drug. In another embodiment,
antibodies 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 1 to 12;
-Y- is a spacer unit;
y is 0, 1 or 2.
The stretcher unit (-T-), when present, links the antibody to an amino acid unit (-
W-). Useful functional groups that can be present on the antibody, 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 antibody, if present. Alternatively, sulfhydryl groups can be generated by
reaction of an amino group of a lysine moiety of the antibody with 2-iminothiolane or
other sulfhydryl generating reagents. In specific embodiments, the antibody is a
recombinant antibody and is engineered to carry one or more lysines. More preferably,
the antibody 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 antibody. The sulfur atom can be derived from a sulfhydryl (—SH) group of
a reduced antibody.
In certain other specific embodiments, the stretcher unit is linked to the antibody
via a disulfide bond between a sulfur atom of the antibody 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 antibody. 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
antibody. In a specific embodiment, the antibody is glycosylated enzymatically to
provide a carbohydrate moiety. 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, Ala-Ala, Val-Ala, 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. 53), Ala-Leu-
Ala-Leu (SEQ ID NO. 54).
Exemplary pentapeptide include: Pro-Val-Gly-Val-Val (SEQ ID NO. 55).
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.
In an alternate embodiment, the spacer unit is a branched
bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporate additional
cytotoxic agents.
The drug loading also referred as the Drug-Antibody ratio (DAR) is the average
number of PBD drugs per cell binding agent.
In the case of an antibody IgGl isotype, where the drugs are bound to cysteines
after partial antibody reduction, drug loading may range from 1 to 8 drugs (D) per
antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the
antibody.
In the case of an antibody IgG2 isotype, where the drugs are bound to cysteines
after partial antibody reduction, drug loading may range from 1 to 12 drugs (D) per
antibody, i.e. where 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 drug moieties are covalently
attached to the antibody.
Compositions of ADC include collections of cell binding agents, e.g. antibodies,
conjugated with a range of drugs, from 1 to 8 or 1 to 12.
Where drugs are bound to lysines, drug loading may range from 1 to 80 drugs
(D) per cell antibody, although an upper limit of 40, 20, 10 or 8 may be preferred.
Compositions of ADC include collections of cell binding agents, e.g. antibodies,
conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.
The average number of drugs per antibody in preparations of ADC from
conjugation reactions may be characterized by conventional means. The quantitative
distribution of ADC in terms of drug ratio may also be determined. For some antibodydrug
conjugates, drug ratio may be limited by the number of attachment sites on the
antibody. For example, an antibody may have only one or several cysteine thiol groups,
or may have only one or several sufficiently reactive thiol groups through which a
linker may be attached. Higher drug loading, e.g. drug ratio >5, may cause aggregation,
insolubility, toxicity, or loss of cellular permeability of certain antibody-drug
conjugates.
Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
Antibodies 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 antibodies through the reaction of lysines with 2-iminothiolane (Traut's
reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be
introduced into the antibody (or fragment thereof) by engineering one, two, three, four,
or more cysteine residues (e.g., preparing mutant antibodies comprising one or more
non-native cysteine amino acid residues). US 7521541 teaches engineering antibodies
by introduction of reactive cysteine amino acids.
Cysteine amino acids may be engineered at reactive sites in an antibody and
which do not form intrachain or intermolecular disulfide linkages (Junutula, et al,
2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729;
US 7521541; US 7723485; WO2009/052249). The engineered cysteine thiols may react
with linker reagents or the drug-linker reagents of the present invention which have
thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form
ADC with cysteine engineered antibodies and the PBD drug moieties. The location of
the drug moiety can thus be designed, controlled, and known. The drug loading can be
controlled since the engineered cysteine thiol groups typically react with thiol-reactive
linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to
introduce a cysteine amino acid by substitution at a single site on the heavy or light
chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can
be achieved with near homogeneity of the conjugation product ADC.
In addition, the invention also relates to an immunoconjugate or an antibodydrug
conjugate as above described for use as a medicament.
Also, the invention further relates to an immunoconjugate or an antibody-drug
conjugate as above described for use in the treatment of cancer.
The invention relates to antibody-drug conjugate as above described for use as a
medicament. In a particular embodiment, the invention relates to antibody-drug
conjugate as above described for use in the treatment of cancer. In a more particular
embodiment, the invention relates to antibody-drug conjugate as above described for
use in the treatment of IGF-1R expressing cancer, or IGF-1R related cancers.
IGF-IR related cancers include tumoral cells expressing or over-expressing
whole or part of the IGF-IR at their surface.
More particularly, said cancers are breast, colon, esophageal carcinoma,
hepatocellular, gastric, glyoma, lung, melanoma, osteosarcoma, ovarian, prostate,
rhabdomyosarcoma, renal, thyroid, uterine endometrial cancer and any drug resistance
phenomena.
In another aspect, the present invention relates to the use of an antibody-drug
conjugate according to the invention for the treatment of an IGF-IR expressing cancer.
Another object of the invention is a pharmaceutical composition comprising an
antibody according to the invention or an antibody-drug conjugate, or
immunoconjugate, as described in the specification.
More particularly, the invention relates to a pharmaceutical composition
comprising an antibody according to the invention or an antibody-drug conjugate, or the
immunoconjugate above described and at least an excipient and/or a pharmaceutically
acceptable vehicle.
The invention concerns a pharmaceutical composition comprising the antibody
or the antibody-drug conjugate above described, and 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 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.
In another aspect, the present invention relates to a pharmaceutical composition
comprising an antibody according to the invention or an antibody-drug conjugate, or the
immunoconjugate above described and at least an excipient and/or a pharmaceutically
acceptable vehicle for use in the treatment of cancer. In a more particular aspect, the
present invention relates to a pharmaceutical composition comprising an antibody
according to the invention or an antibody-drug conjugate, or the immunoconjugate
above described and at least an excipient and/or a pharmaceutically acceptable vehicle
for use in the treatment of an IGF-1R expressing cancer.
The invention also relates to a method for the treatment of cancer in a subject,
and in particular for the treatment of an IGF-1R expressing cancer, comprising
administering to said subject an effective amount of at least an antibody-drug conjugate
according to the invention. The present invention further relates to a method for the
treatment of cancer in a subject, and in particular for the treatment of an IGF-1R
expressing cancer, comprising administering to said subject an effective amount of a
pharmaceutical composition according to the invention.
In another embodiment, the present invention relates to a method of delivering a
drug or a medicament to an IGF-1R expressing cancer cell in a subject, comprising
administering to said subject an effective amount of at least the antibody-drug conjugate
according to the invention or a pharmaceutical composition according to the invention.
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: Example of Biacore binding profile obtained with 3 antibodies on
hIGF-lR ECD captured by an anti-His-Tag antibody.
Figure 2 : Epitope mapping scheme defined from the panel of 15 anti-hlGF-lR
monoclonal antibodies witch defined 5 epitope groups. The numbering of the groups is
not linked to a position regarding the sequence nor the 3D structure of the antigen.
Figures 3A to 3C: Antibody binding to the human native IGF-1R by FACS
analyses. Figure 3A represents the titration curve, on MCF-7 cell line, of one chimeric
anti-IGF-lR Ab representative for each epitope clustering group. MFI represents the
mean of fluorescent intensity. Figure 3B represents the EC50 of both murine and
chimeric anti-IGF-lR antibodies on the MCF-7 cell line. Figure 3C represents the Bmax
of chimeric anti-IGF-lR antibodies on MCF-7 cell line.
Figures 4A and 4B: Evaluation of hIGF-lR recognition using transfected vs non
transfected cells. Figure 4A represents titration curves of one chimeric anti-IGF-lR Ab
representative of each epitope clustering group on IGF-1R+ cell line. MFI represents the
mean of fluorescent intensity. Figure 4B represents the binding of one chimeric anti-
IGF-1R Ab representative of each epitope clustering group on the human IGF-1R cell
line. MFI represents the mean of fluorescent intensity
Figures 5A and 5B: Evaluation of the specificity of Abs to hIGF-lR vs hIR
using transfected cells. Figure 5A represents the binding of murine anti-IGF-lR Ab on
the hIR+ transfected cell line. Figure 5B represents the binding of chimeric anti-IGF-lR
Ab on the IR+ cell line. MFI represents the mean of fluorescent intensity. In panel A
and B the commercially available anti-hIR antibody described as GR05 (Calbiochem)
has been introduced as a positive control.
Figure 6 : Binding of murine anti-IGF-lR Ab on the IM-9 cell line. MFI
represents the mean of fluorescent intensity. The GR05 anti-hIR Mab was introduced as
a positive control.
Figures 7A, 7B and 7C: Evaluation of recognition of the monkey IGF-1R.
Figure 7A represents the titration curves of one chimeric anti-IGF-lR Ab representative
of each epitope clustering group on the COS-7 cell line. MFI represents the mean of
fluorescent intensity. Figure 7B represents the EC50 of both murine and chimeric anti-
IGF-1R antibodies on COS-7 cell line. Figure 7C represents the EC50 of chimeric anti-
IGF-1R antibodies on both hIGF-lR + transfected cells and COS-7 cells. GR1 1L
(Calbiochem) was introduced as a positive control.
Figure 8 : Comparison of c208F2 binding on either hIGF-lR ECD or
Cynomolgus monkey IGF-1R ECD using a Biacore assay. Sensorgrams obtained on a
SPR technology based Biacore XI00 using a CM5 sensorchip activated with more the
11000 RU of mouse anti-Tag His antibody chemically grafted to the carboxymethyl
dextran matrix. The experiment is run at a flow rate of 30 mΐ/min at 25°C using the
HBS-EP+ as the running and samples diluting buffer. The figure shows the
superposition of 4 independent sensorgrams aligned on the x-axis at the beginning of
the first injection of the analytes and on the y-axis by the baseline defined just before
this first injection. The sensorgrams obtained with the capture of the human based
sequence of the recombinant soluble IGF-1R are marked by diamonds. The sensorgrams
obtained with the capture of the cynomolgus based sequence of the recombinant soluble
IGF-1R are marked by triangles. White symbols correspond to the blank cycles (5
injections of the running buffer) and black symbols correspond to the injections of the
growing range of concentrations of c208F2 (5, 10, 20, 40 and 80 nM).
Figure 9 : Evaluation of the intrinsic effect of anti-hlGF-lR antibodies on the
receptor phosphorylation compared to IGF1 .
Figure 10: Inhibition of IGF-1R phosphorylation in response to IGF-1 by murine
anti-hlGF-lR.
Figure 11: Cell surface binding of anti-IGF-lR antibodies is down-regulated at
37°C. MCF-7 cells were incubated at 4°C or 37°C for 4 h with 10 m ΐ of each Ab.
The figure represents the AMFI.
Figures 12A and 12B: Antibody surface decay. Cell surface bound antibody was
assessed after 10, 20, 30, 60 and 120 min at 37°C. Figure 12A represents the % of
remaining IGF-1R in comparison to the signal intensity measured at 4°C. Figure 12B
represents Half Life calculation using Prims Software and using exponential decay
fitting.
Figure 13: Kinetic of antibody internalization evaluated by FACS analyses. Cells
were incubated with 10 mg/ml of murine Abs for 0, 30 or 60 min at 37°C. Cells were
permeabilized or not and incubated with a secondary anti-mouse IgG-Alexa 488.
Membrane corresponds to the signal intensity w/o permeabilization. Total correspond to
the signal intensity after cell permeabilization and cytoplasmic corresponds to
internalized Ab. The name of each evaluated antibody is depicted on the top of each
graph.
Figures 14A and 14B: Imaging Ab internalization. Figure 14A: MCF-7 cells
incubated with m208F2 for 20 min. at 4°C and washed before incubation [a)] at 37°C
for 15 [b)], 30 [c)] and 60 [d)] min. Cells were fixed and permeabilized. The m208F2
Ab was revealed using an anti-mouse IgG Alexa488 and Lamp-1 was revealed with a
rabbit anti-Lamp- 1 antibody and with a secondary anti-rabbit IgG Alexa 555. Figure
14B (Part I to III): MCF-7 cells were incubated for 30 minutes at 37°C with each of the
other anti-hlGF-lR murine antibody to be tested and then stained as described above.
Colocalization was identified using the colocalization highliter plug-in of the Image J
software.
Figure 15: Involvement of the lysosome pathway in antibody degradation
Figure 16: Evaluation of the binding of anti-hlG-lR murine antibodies at
different pH. The EC50s of the binding of the different antibodies was evaluated on
MCF-7 using buffer with different pH ranging from 5 to 8.
Figure 17: Evaluation of the ability of the selected anti-IGF-lR Abs to induce
cytotoxicity on a Fab-ZAP assay. A) MCF-7 cells were incubated with increasing
concentrations of the chimeric anti-IGF-lR antibodies in combination with the human
Fab-ZAP kit. Cell viability was measured using CellTiter-Glo® luminescent cell
viability assay. The c9G4 chimeric antibody was used as irrelevant antibody. B) IC50S
from results depicted in A).
Figure 18: Correlation between i) cytotoxic potency, ii) influence of pH on the
Ab/IGF-IR binding, iii) effect of Abs on the IGF- 1-induced phosphorylation of IGF-1R
and iv) antibody clustering.
Figures 19A to 19D: Binding characteristic of the first humanized form of the
c208F2 Mab. Binding properties of the hz208F2 VH3/VL3 mAb was evaluated on the
human cell line MCF-7 (Figure 19A), on the monkey cell line COS-7 (Figure 19B) and
on the transfected murine cell line expressing the human insulin receptor (Figure 19C).
The binding of both the murine and the chimeric 208F2 mAbs was evaluated in parallel.
The anti-hIR antibody clone GR05 was used to verify the expression of the hIR on the
transfected cell line (Figure 19D).
Figures 20A to 20D: ELISA validation of the AF305-NA polyclonal antibody
that has been used for IHC assays. Figure 20A: Binding to hIGF-lR, Figure 20B:
Binding to human recombinant IR. No recognition of hIR EDC and of cellular IR
expressed by transfected cells (Figure 20D) compared to the control Ab GR05 on these
hIR transfected cells (Figure 20C).
Figure 21: Validation of hIGF-lR staining on FFPE sections from xenografts
expressing various levels of hIGF-lR. Hs746T was introduced as a negative control
Figures 22A and 22B: Evaluation of hIGF-lR expression on normal FFPE tissue
sections. Placenta sections were used as a positive control for normal tissues while
positive tumor xenograft tissues were introduced in each run in order to calibrate hlGF-
1R expression.
Figure 23: Evaluation of hIGF-lR expression on NSCL FFPE tissue sections.
Four cases which are representative for the strong staining observed in the large panel of
tissue analyzed.
Figure 24: Evaluation of hIGF-lR expression on breast cancer FFPE tissue
sections. Three cases which are representative for the strong staining observed in the
tested panel of tissue analyzed.
Figure 25: Evaluation of hIGF-lR expression on FFPE tissue sections from
various tumors.
Figure 26: Superposition of sensorgrammes obtained with a SPR based Biacore
XI00 device at a temperature of 25°C with a CM5 sensor chip activated on both
flowcells with aroud 12.000 RU of a mouse anti-TagHis monoclonal antibodies
chemically grafted to the carboxymethyldextran matrix using a HBS-EP+ as the running
buffer at a flow rate of 30 mΐ/min. Each sensorgrammes (the first one marked by
triangles and the second one marked by diamonds) correspond to a complete cycle:
1- Injection during one minute of a solution of recombinant h-IGF-
1R (10 mg/ml) on the second flowcell.
2- For the first sensorgramme: 5 injections of running buffer during
90s each
For the second sensorgramme: five injections in the growing range of
concentrations of the anti-IGF-lR c208F2 antibody solutions during 90 s each.
3- A delay of 300 s for the determination of the dissociation kinetic
rates.
4- A regeneration of the surface by an injection during 45 s of a 10
mM Glycine, HC1 pH 1.5 buffer.
Figure 27: The sensorgramme corresponding to the subtraction of the blank
sensorgramme (5 injections of HBS-EP+) to the sensorgramme obtained with the
growing range of concentrations of the anti-IGF-lR c208F2 solutions is presented in
grey. The theoretical sensorgramme corresponding to the 1:1 model with the following
parameters: ko = (1.206 ± 0.036) x 106 M V , koff = (7.81 ± 0.18) x 10 5 s 1 , Rmax =
307.6 ± 0.3 RU is presented by a thin black line. The calculated concentrations of
c208F2 are reported on the graph: only the highest concentration (24 nM) is considered
as a constant).
Figure 28: The dissociation constants correspond to the mean of the four
experiments run for each antibody and correspond to the ratio: k0ff/ko x 10 12 to be
express in the pM unit. The error bars correspond to the standard error (n=4).
Figure 29: The half-lives correspond to the mean of the four experiments run for
each antibody and correspond to the ratio: Ln(2)/koff/3600 to be express in the h unit.
The error bars correspond to the standard error (n=4).
Figure 30: Superposition of two sensorgrams corresponding to two cycle of an
experiment running on a Biacore XI00 device at a flow rate of 30m1/h h and at 25°C.
The first step of the cycle correspond the injection of a solution of c208F2
antibody at the concentration of 10 mg/ml during 60s on the second flowcell of a CM5
sensor chip activated by the grafting of more than 10,500 RU of a mouse anti-human
IgG Fc monoclonal antibody chemically linked to the carboxymethyldextran matrix by
its amine functions. The second step correspond to the injection of the extracellular
domain of either h-IGF-lR (plain diamonds) or m-IGF-lR (empty diamonds) solutions
of crude cell medium culture supernatants during 120 s with a delay of 120 s . The
double headed arrows indicate the positions of measure of the antibody capture level
and the IGF-IR binding level used in this study.
Figure 31: Histograms representing the ratio between IGF-IR binding level
obtained for each chimerical h/m IGF-IR constructs and the level of c208F2 captured
on second flowcell of the sensorchip during the corresponding cycle.
Figures 32A and B: Histograms representing the EC50 of hz208F2 H076/L024
for pH 5 to pH 8, Acidic pH decreases binding capacity of the humanized IGF-IR
antibodies hz208F2 H076/L024 (A) and hz208F2 (H077/L018 (B).
Figures 33: Binding of Hz208F2 (10mg/ml) on either 170 RU of the wild-type of
a soluble version of the h-IGFIR (black diamond) or on 120 RU of the mutant C29
(Asp491>Ala) of this receptor. Each receptor is captured by their C-terminal 66His Tag
on a CM5 sensor chip. The experiment was run with a Biacore XI00 device at 25°C at a
flow rate of 30m1 h h using the classical HBS-EP+ as the running buffer.
EXAMPLES
All hybridomas mentioned in the present invention have been deposited at the
CNCM (Institut Pasteur, France) and are identified in the following table 7.
Table 7
Example 1: Generation of IGF-1R antibodies
To generate murine monoclonal antibodies (Mabs) against human extracellular
domain (ECD) of the human IGF-1 receptor (hIGF-lR), 5 BALB/c mice were
immunized 3-times s.c. with 10 mg of the rhIGF-lR protein (R&D Systems, Cat N°391-
GR). As an alternative, three additional immunizations with 10 mg of the murine
extracellular domain (ECD) of IGF-1R (R&D Systems, Cat N° 6630-GR /Fc) were
performed on some animals. The first immunization was done 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 10 mg of the rhIGF-lR protein. Then 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 of all mice) 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
rhIGF-lR ECD protein by FACS analysis using human breast MCF7 tumor cells
(ATCC) and/or monkey COS7 cells (African green monkey kidney-SV40 transformed)
which express monkey IGF-1R on their cell surface. More precisely, for the selection
by flow cytometry, 105 cells (either MCF7 or COS7) 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.

CLAIMS
1. A method for selecting an internalizing antibody, or an internalizing IGFIR
binding fragment thereof, which binds to the human Insulin like Growth Factor 1
Receptor (IGF-IR) and is internalized following to its binding to IGF-IR, said method
comprising the steps of selecting an antibody:
i) that binds to an IGF-IR of SEQ ID N°52, and
ii) that does not bind to an IGF-IR of SEQ ID N°52 with an amino acid
other than Histidine at position 494 or other than an Aspartic acid at position 491 of
SEQ ID N°52.
2. The method for selecting an internalizing antibody, or an internalizing
IGF-IR binding fragment thereof, according to claim 1, wherein said antibody does not
bind to an IGF-IR of SEQ ID N°52 with an amino acid other than Histidine at position
494 and with an amino acid other than an Aspartic acid at position 491 of SEQ ID
N°52.
3. The method according to claim 1 or 2, further comprising a step of selecting
an internalizing antibody, or an IGF-IR binding fragment thereof, which percentage of
internalization following to its binding to IGF-IR is at least of 40%.
4. An internalizing antibody, or an internalizing IGF-IR binding fragment
thereof, which binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR),
such as obtained by a method according to one of claims 1 to 3.
5. An internalizing antibody, or an internalizing IGF-IR binding fragment
thereof, which binds to the human Insulin like Growth Factor 1 Receptor (IGF-IR) of
SEQ ID N°52 and is internalized following to its binding to IGF-IR, and which does
not bind to an IGF-IR of SEQ ID N°82 an/or SEQ ID N°92.
6. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to claim 5, wherein the epitope of said internalizing antibody
comprises the Histidine amino acid at position 494 of SEQ ID N°52 and/or the Aspartic
acid amino acid at position 491 of SEQ ID N°52.
7. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to claim 6, wherein said epitope comprises an amino acid sequence
of at least 8 amino acids.
8. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to any one of claims 4 to 7, which percentage of internalization
following to its binding to IGF-1R is of at least 40%.
9. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to any one of claims 4 to 8, wherein said amino acid other than
Histidine at position 494 of SEQ ID N°52 is Arginine and/or said amino acid other than
an Aspartic acid at position 491 of SEQ ID N°52 is Alanine.
10. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to any one of claims 4 to 9, characterized in that it comprises six
CDRs, wherein at least one of said CDRs is chosen in the group consisting of: CDR-H2
of SEQ ID N°2, CDR-H3 of SEQ ID N°3 and CDR-L2 of SEQ ID N°5.
11. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to claim 10, characterized in that it comprises six CDRs, wherein
three of said CDRs are CDR-H2 of SEQ ID N°2, CDR-H3 of SEQ ID N°3 and CDR-L2
of SEQ ID N°5.
12. The internalizing antibody, or an internalizing IGF-1R binding fragment
thereof, according to any one of claims 4 to 11, characterized in that it comprises the
three heavy chain CDRs of sequence SEQ ID N°l, 2 and 3, and the three light chain
CDRs of sequence SEQ ID N°4, 5 and 6.
13. The internalizing antibody, or an IGF-1R binding fragment thereof,
according to any one of claims 4 to 12, characterized in that it consists of a monoclonal
antibody, a recombinant monoclonal antibody, or an IGF-1R binding fragment thereof.
14. The internalizing antibody of any one of claims 4 to 13, characterized in that
it is selected from:
a) an antibody comprising the three heavy chain CDRs of sequences SEQ ID
Nos. 7, 2 and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising the three heavy chain CDRs of sequences SEQ ID
Nos. 7, 2 and 3 and the three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;
c) an antibody comprising the three heavy chain CDRs of sequences SEQ ID
Nos. 7, 2 and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12;
and
d) an antibody comprising the three heavy chain CDRs of sequences SEQ ID
Nos. 8, 2 and 3 and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.
15. The internalizing antibody of claim 14, characterized in that it consists of a
chimeric antibody.
16. The internalizing antibody of claim 15, characterized in that it is selected
from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 13or any sequence exhibiting at least 80% identity with SEQ ID No. 13
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 14 or any sequence exhibiting at least 80% identity with SEQ ID No. 14
and the three light chain CDRs of sequences SEQ ID Nos. 10, 5 and 11;
c) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 15 or any sequence exhibiting at least 80% identity with SEQ ID No.
15and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12;
d) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 16 or any sequence exhibiting at least 80% identity with SEQ ID No. 16
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and
e) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 17 or any sequence exhibiting at least 80% identity with SEQ ID No. 17
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 12.
17. The internalizing antibody of claim 15, characterized in that it is selected
from:
a) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 18 or any sequence exhibiting at least 80% identity with SEQ ID No. 18
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 19 or any sequence exhibiting at least 80% identity with SEQ ID No. 19
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
c) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 20 or any sequence exhibiting at least 80% identity with SEQ ID No. 20
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
d) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 2 1 or any sequence exhibiting at least 80% identity with SEQ ID No. 2 1
and the three heavy chain CDRs of sequences SEQ ID Nos. 8, 2 and 3; and
e) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 22 or any sequence exhibiting at least 80% identity with SEQ ID No. 22
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.
18. The internalizing antibody of claim 15, characterized in that it is selected
from:
a) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 23 or any sequence exhibiting at least 80% identity with SEQ ID No. 23
and a light chain of sequence SEQ ID No. 28 or any sequence exhibiting at least 80%
identity with SEQ ID No. 28;
b) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 24 or any sequence exhibiting at least 80% identity with SEQ ID No. 24
and a light chain of sequence SEQ ID No. 29 or any sequence exhibiting at least 80%
identity with SEQ ID No. 29;
c) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 25 or any sequence exhibiting at least 80% identity with SEQ ID No. 25
and a light chain of sequence SEQ ID No. 30 or any sequence exhibiting at least 80%
identity with SEQ ID No. 30;
d) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 26 or any sequence exhibiting at least 80% identity with SEQ ID No. 26
and a light chain of sequence SEQ ID No. 3 1 or any sequence exhibiting at least 80%
identity with SEQ ID No. 31; and
e) an antibody comprising or consisting of a heavy chain of sequence
SEQ ID No. 27 or any sequence exhibiting at least 80% identity with SEQ ID No. 27
and a light chain of sequence SEQ ID No. 32 or any sequence exhibiting at least 80%
identity with SEQ ID No. 32.
19. The internalizing antibody of claim 14, characterized in that it consists of a
humanized antibody.
20. The internalizing antibody of claim 19, characterized in that it comprises:
a) a heavy chain having CDR-H1, CDR-H2 and CDR-H3 of sequences SEQ ID
Nos. 7, 2 and 3, respectively, and FRl, FR2 and FR3 derived from the human germline
IGHV1-46*01 (SEQ ID No. 44), and the FR4 derived from the human germline
IGHJ4*01 (SEQ ID No. 46); and
b) a light chain having CDR-L1, CDR-L2 and CDR-L3 of sequences SEQ ID
Nos. 9, 5 and 11, respectively, and FRl, FR2 and FR3 derived from the human germline
IGKV1-39*01 (SEQ ID No. 45), and the FR4 derived from the human germline
IGKJ4*01 (SEQ ID No. 47).
21. The internalizing antibody of claim 20, characterized in that it is selected
from:
a) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 33 or any sequence exhibiting at least 80% identity with SEQ ID No. 33
and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11;
b) an antibody comprising a heavy chain variable domain of sequence
SEQ ID No. 34 or any sequence exhibiting at least 80% identity with SEQ ID No.
34and the three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11; and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 or any sequence with at
least 80% identity with SEQ ID No.56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80; and the
three light chain CDRs of sequences SEQ ID Nos. 9, 5 and 11.
22. The internalizing antibody of claim 20, characterized in that it is selected
from:
a) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 35 or any sequence exhibiting at least 80% identity with SEQ ID No. 35
and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3;
b) an antibody comprising a light chain variable domain of sequence
SEQ ID No. 36 or any sequence exhibiting at least 80% identity with SEQ ID No.
36and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3; and
c) an antibody comprising a light chain variable domain of sequence selected
from SEQ ID Nos. 57 and 60 or any sequence with at least 80% identity with SEQ ID
Nos. 57 or 60; and the three heavy chain CDRs of sequences SEQ ID Nos. 7, 2 and 3.
23. The internalizing antibody of claim 20, characterized in that it is selected
from:
a) an antibody comprising a heavy chain of sequence SEQ ID No. 37 or any
sequence exhibiting at least 80% identity with SEQ ID No. 37 and a light chain of
sequence SEQ ID No. 39 or any sequence exhibiting at least 80%> identity with SEQ ID
No. 39;
b) an antibody comprising a heavy chain of sequence SEQ ID No. 38 or any
sequence exhibiting at least 80% identity with SEQ ID No. 38 and a light chain of
sequence SEQ ID No. 40 or any sequence exhibiting at least 80% identity with SEQ ID
No. 40; and
c) an antibody comprising a heavy chain variable domain of sequence selected
from SEQ ID Nos. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80, or any sequence with at
least 80% identity with SEQ ID No. 56, 62, 64, 66, 68, 70, 72, 74, 76, 78 or 80, and a
light chain variable domain of sequence selected from SEQ ID Nos. 57 and 60 or any
sequence with at least 80%> identity with SEQ ID Nos. 57 or 60.
24. The internalizing antibody of claim 20, characterized in that it comprises:
a) a heavy chain variable domain (VH) of sequence SEQ ID No. 33 wherein said
sequence SEQ ID No. 33 comprises at least 1 back-mutation selected from the residues
20, 34, 35, 38, 48, 50, 59, 61, 62, 70, 72, 74, 76, 77, 79, 82 and 95; and
b) a light chain variable domain (VL) of sequence SEQ ID No. 35, wherein said
sequence SEQ ID No. 35 comprises at least 1 back-mutation selected from the residues
22, 53, 55, 65, 71, 72, 77 and 87.
25. The internalizing antibody according to any one of claims 4 to 24,
characterized in that it is selected from:
i) an antibody produced by the hybridoma 1-4757, 1-4773, 1-4775, 1-4736 or I-
4774 deposited at the CNCM, Institut Pasteur France on the 30 May 2013, 26 June
2013, 26 June 2013, 24 April 2013 and 26 June 2013, respectively;
ii) an antibody which competes for binding to IGF-1R with the antibody of i);
and
iii) an antibody which binds to the same epitope of IGF-1R as does the antibody
ofi).
26. A murine hybridoma selected from the hybridoma 1-4757, 1-4773, 1-4775, 1-
4736 and 1-4774 deposited at the CNCM, Institut Pasteur France on the 30 May 2013,
26 June 2013, 26 June 2013, 24 April 2013 and 26 June 2013, respectively.
27. The internalizing antibody, or an internalizing antigen binding fragment
thereof, according to any one of the claims 4 to 25, for use as an addressing vehicle for
delivering a cytotoxic agent at a host target site, said host target site consisting of an
epitope localized into the protein IGF-IR extracellular domain, preferably the human
protein IGF-IR extracellular domain (SEQ ID No. 51), more preferably the human
protein IGF-IR extracellular domain N term (SEQ ID No. 52), or any natural variant
sequence thereof.
28. An antibody-drug conjugate comprising the internalizing antibody, or an
internalizing antigen binding fragment thereof, according to any one of claims 4 to 25,
conjugated to a cytotoxic agent.
29. The antibody-drug conjugate of claim 28, for use as a medicament.
30. The antibody-drug conjugate for use according of claim 29, for the treatment
of cancer.
31. The antibody-drug conjugate for use according of claim 29 for the treatment
of an IGF-IR expressing cancer.
32. A pharmaceutical composition comprising an internalizing antibody of any
one of claims 4 to 25, or an antibody-drug conjugate of claim 28, and at least an
excipient and/or a pharmaceutical acceptable vehicle.
33. The pharmaceutical composition of claim 32 for use in the treatment of
cancer.
34. The pharmaceutical composition of claim 32 for use in the treatment of an
IGF-IR expressing cancer.
35. The use of an antibody-drug conjugate of claim 28 for the treatment of an
IGF- 1R expressing cancer.
36. A method for the treatment of an IGF-IR expressing cancer in a subject,
comprising administering to said subject an effective amount of at least the antibodydrug
conjugate of claim 28 or a pharmaceutical composition of claim 32.
37. A method of delivering a drug or a medicament to an IGF-IR expressing
cancer cell in a subject, comprising administering to said subject an effective amount of
at least the antibody-drug conjugate of claim 28 or a pharmaceutical composition of
claim 32.