ANTIBODIES THAT SPECIFICALLY BIND TO THE EPHA2 RECEPTOR
The present invention relates to an antibody or an epitope-binding fragment thereof
that specifically binds to an EphA2 receptor.
It further relates to a conjugate comprising a cytotoxic agent that is covaiently bound to
the antibody and a method for preparing such a conjugate.
BACKGROUND OF THE INVENTION
Cancer is a disease characterized by uncontrolled proliferation, resulting from aberrant
signal transduction. The most dangerous forms of cancer are malignant cells which
have the ability to spread, either by direct growth into adjacent tissue through invasion,
or by implantation into distant sites by metastasis. Metastatic cells have acquired the
ability to break away from the primary tumor, translocate to distant sites through the
bloodstream or lymphatic system, and colonize distant and foreign microenvironments.
It is now clear that the Eph molecules are involved in disease states such as cancer.
Eph receptors are a unique family of receptor tyrosine kinases (RTK), the largest in the
genome, consisting of fourteen receptors, divided into two groups A and B, that interact
with eight membrane-bound ephrin ligands (Pasquale, E. B. et al., 2005, Nature
Reviews Mol. Cell Biol., 6: 462-475). Binding of Eph receptors to their ligands induces
receptor clustering, activation of kinase activity, and subsequent trans-phosphorylation
of the cytoplasmic domains on tyrosine residues, creating docking sites for a number of
signaling proteins (Kullander, K. and Klein, R., 2002, Nature Reviews Mol. Cell Biol., 3:
475-486; Noren, N. K. and Pasquale, E. B., 2004, Cell signal., 16: 655-666 ).
Overexpression of the EphA2 receptor has been reported in cancers of the ovary,
breast, prostate, lung, colon, oesophagus, renal cell, cervix, and melanoma. EphA2
was suggested to be a positive regulator of cell growth and survival in malignant cells
(Landen, C. N. et al., 2005, Expert. Opin. Ther. Targets, 9 (6): 1179-1187). A role for
EphA2 in cancer has also been described, since EphA2 overexpression alone is
sufficient to transform mammary epithelial cells into a malignant phenotype (Zelinski et
al, 2001, Cancer Res., 61: 2301-2306), and increases spontaneous metastasis to
distant sites (Landen, C. N. et al., 2005, Expert. Opin. Ther. Targets, 9 (6): 1179-1187).
Furthermore, increasing evidence suggests that EphA2 is involved in tumor
angiogenesis (Ogawa et al., 2000, Oncogene, 19: 6043-6052; Cheng et al. 2002, Mol.
Cancer Res., 1: 2-11; Cheng et al, 2003, Neoplasia, 5 (5): 445-456; Dobrzanski et al.,
2004, Cancer Res., 64:910-919).

Phosphorylation of EphA2 has been shown to be linked to its abundance. Tyrosine
phosphorylated EphA2 is rapidly internalized and fated for degradation, whereas
unphosphorylated EphA2 demonstrates reduced turnover and therefore accumulates at
the cell surface. It is currently thought that this kind of model might contribute to the
high frequency of EphA2 overexpression in cancer (Landen, C. N. et a/., 2005, Expert.
Opin. Ther. Targets, 9 (6): 1179-1187). However, reality may be more complex, since
recent data seem to indicate a role for EphA2 kinase-dependent and -independent
functions in tumor progression (Fang W. B., 2005, Oncogene, 24: 7859-7868).
Agonistic antibodies have been developed which promote EphA2 tyrosine
phosphorylation and internalization, ultimately resulting in inhibition of tumor cell growth
(Dodge-Zantek et a/., 1999, Cell Growth & Differ., 10: 629-638; WO 01/12172, WO
03/094859, WO 2004/014292, WO 2004/101764, WO 2006/023403, WO 2006/047637,
WO 2007/030642).
Application WO 2006/084226 discloses antibodies which neither increase nor decrease
EphA2 kinase activity but are capable of impeding tumor cell proliferation. However,
there is no indication therein that these antibodies prevent ephrinA1 binding to the
receptor and inhibit ephrinA1-induced EphA2 phosphorylation. The use of antagonistic
antibodies has been proposed in WO 2004/092343, but no actual antibody was
disclosed therein. Antibodies recognizing EphA2 which are genuine antagonists have
been described in WO 2008/010101, as well as humanized variants and conjugates
thereof. These antibodies and derivatives thereof inhibit EphA2 kinase-dependent
tumor cell growth.
Nevertheless, there is still a need for novel and efficacious medicaments which can be
used in cancer therapy.
SUMMARY OF THE INVENTION
It is an object of the invention to provide new agents that specifically bind to class A
Eph receptor family members, such as EphA2, and inhibit the cellular activity of the
receptor by antagonizing the receptor. Thus, the present invention includes antibodies
or fragments thereof that recognize the EphA2 receptor, preferably human, and
function as antagonists of said receptor. These antibodies are devoid of any agonist
activity.
In one embodiment, the antibody or an epitope-binding fragment thereof of the present
invention comprises at least one heavy chain and at least one light chain, said heavy
chain comprising three sequential complementarity-determining regions having amino

acid sequences represented by SEQ ID NOS: 1, 2, and 3, and said light chain
comprising three sequential complementarity-determining regions having amino acid
sequences represented by SEQ ID NOS: 4, 5, and 6. In a preferred embodiment, said
antibody or epitope-binding fragment thereof is a humanized or resurfaced. In an even
more preferred embodiment, the heavy chain of said antibody or epitope-binding
fragment thereof comprises an amino acid sequence consisting of SEQ ID NO: 12, and
the light chain of said antibody or epitope-binding fragment thereof comprises an amino
acid sequence consisting of SEQ ID NO: 14.
In a further embodiment the heavy chain of said antibody has an amino acid sequence
SEQ ID NO: 18, and the light chain of said antibody has an amino acid sequence SEQ
ID NO: 16.
In another embodiment, the said antibody or epitope-binding fragment thereof is
conjugated to a cytotoxic agent. It is therefore an aspect of this invention to provide a
conjugate of an antibody of the present invention or an epitope-binding fragment
thereof, wherein said conjugate comprises a bound cytotoxic agent chosen between:
the compound of formula (XIV):
the compound of formula (XIII):

In a preferred embodiment, the conjugate comprises the compound of formula (XIII) as
the cytotoxic agent. In a further preferred embodiment, the number of maytansinoid
molecules bound per antibody molecule (DAR) in said conjugate is comprised between
4 and 7 maytansinoid molecules/antibody molecule, said DAR being determined by
measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm.
In another aspect, it is provided a method for preparing a conjugate which comprises
the steps of:
(i) bringing into contact an optionally-buffered aqueous solution of a cell-binding agent
with a solution of a cytotoxic compound;
(ii) then optionally separating the conjugate which was formed in (i) from the unreacted
reagents and any aggregate which may be present in the solution;
wherein the cell-binding agent is an antibody according to claims 1-3, and a cytotoxic
agent chosen between:
the compound of formula (XVII):


wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3 -carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom;
and
the compound of formula (XVIII):

wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
suifophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom.

Conjugates obtainable by said method are comprised within the scope of this invention.
In particular, such conjugate have a structure chosen between the structures of the
formula (XV):

wherein Ab is an antibody according to the present invention and n is an integer
comprised between 1 and 15. In a preferred embodiment, n is comprised between 4
and 7. In another preferred embodiment, the conjugate of the invention has the
structure of the formula (XV).

The affinity of the antibody of the invention or epitope-binding fragment thereof for the
antigen is not affected by the conjugation process; on the other hand, conjugation of a
cytotoxic agent to antibodies of the prior art results in a decreased affinity for EphA2.
The said antibody or epitope-binding fragment thereof of the present invention, when
conjugated to a cytotoxic agent, shows more potency and is more selective at killing
tumor cells expressing EphA2 than the conjugates of the prior art. In addition, the
conjugates of the present invention display advantageous pharmacokinetic properties
over the conjugates of the prior art, such as a slower clearance, a better exposure, and
an increased stability of the conjugate in vivo. Such properties would be particularly
useful, along with the high cytotoxic efficacy and selectivity, for developing a
medicament which is both safe and efficacious.
Therefore, the invention encompasses a pharmaceutical composition containing an
antibody of the invention or epitope-binding fragment thereof, or a conjugate thereof,
and a pharmaceutically acceptable carrier or excipient. The antibodies of the invention
or epitope-binding fragments thereof, or conjugates thereof, can be used as a
medicament. In particular, they can be used to make a medicament to treat cancer. In
a preferred embodiment, the said cancer is chosen between carcinoma, including that
of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix,
thyroid and skin; including squamous cell carcinoma ; hematopoietic tumors of
lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic
leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma ; hematopoietic
tumors of myeloid lineage, including acute and chronic myelogenous leukemias and
promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyosarcoma and osteosarcoma; other tumors, including melanoma, seminoma,
teratocarcinoma,, thyroid follicular cancer, xeroderma pigmentosum,
keratoactanthoma; tumors of the central and peripheral nervous system, including
astrocytoma, neuroblastoma, glioma, and schwannomas.
FIGURE LEGENDS
Figure 1: Sequences of the CDR, the VH and the VL of mu2H 11R35R74, and of the VH
and the VL of hu2H11R35R74.
Figure 2: HRMS spectrum of hu2H11R35R74-PEG4-NHAc-DM4 using method C
(example 1a.1.)
Figure 3: HRMS spectrum of hu2H11R35R74-PEG4-Mal-DM4 using method C
(example 1 b. 1.)

Figure 4: Kinetics analysis of the phosphorylation of EphA2 in MDA-MB-231 cells. WT:
wild type untreated cells; MW: molecular weight marker; P-EphA2: phosphorylated
Epha2
Figures 5A and 5B: Inhibition of the phosphorylation of EphA2 after induction by
EphrinA1/Fc respectively on NCI-H1299 cells (5A) and MDA-MB-231 cells (5B); WT:
wild type untreated cells; MW: molecular weight marker; P-EphA2: phosphorylated
EphA2.
Figure 6: Cytotoxic activity of hu2H11 R35R74-PEG4-Mal-DM4 (small filled circles:
DAR = 6.7; large filled circles: DAR = 7.0) as compared to hu2H11-PEG4-Mal-DM4.
Figure 7: Time plot (semi-logarithmic scale) representation of the mean plasma
concentrations (n=2) of hu2H11-SPDB-DM4 after a single dose intravenous
administration of 20 mg/kg of immunoconjugate in HGS to male CD-1 mice. The total
concentration of human IgG (filled diamonds) and the conjugate fraction (open
squares) are shown.
Figure 8: Time plot (semi-logarithmic scale) representation of the mean plasma
concentrations (n=2) of hu2H11R35R74-PEG4-NHAc-DM4 after a single dose
intravenous administration of 20 mg/kg of immunoconjugate in HGS to male CD-1
mice. The total concentration of human IgG (filled diamonds) and the conjugate fraction
(open squares) are shown.
Figures 9A and 9B: PK parameters for hu2H11R35R74-PEG4-NHAc-DM4 at various
DARs, Bar graph representation of the exposure to (AUC(O-inf); figure 9A) and
clearance (CI; figure 9B) of several ADCs as a function of the DAR after a single dose
intravenous administration of 20 mg/kg of immunoconjugate in HGS to male CD-1 mice
(n=4).
Figure 10: illustrates the mapping of EphA2 epitope for Fab2H11.
Figure 11: is the sequence of EphA2 wherein residues in dark grey are part of the
epitope; residues in light grey are not visible in the crystal structures.
Figure 12 A: represents the overall structure of the complex and figure 12B is a
magnification of the part with the two Fab mutations.
Figure 13: is a represents the structure of the paratope.
Figure 14: HRMS spectrum of hu2H11 R35R74-PEG4-NMeAc-DM4.

Figure 15: HRMS spectrum of hu2H11R35R74-PEG8-NHAc-DM4.
Figure 16: HRMS spectrum of hu2H11R35R74-PEG4-AIIyl-DM4.
Figure 17: HRMS spectrum of hu2H11-PEG4-NHAc-DM4.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined herein, scientific and technical terms used in connection with
the present invention shall have the meanings that are commonly understood by those
of ordinary skill in the art. Further, unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the singular. Generally,
nomenclatures used in connection with, and techniques of, cell and tissue culture,
molecular biology, immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known and commonly used
in the art. The practice of the present invention employs, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant techniques),
microbiology, cell biology, biochemistry, and immunology, which are within the skill of
the art. Such techniques are explained fully in the literature, such as Molecular Cloning:
A Laboratory Manual, second edition (Sambrook et al, 1989); Oligonucleotide
Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987);
Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology
(F. M. Ausubel et ah, eds., 1987, and periodic updates); PCR: The Polymerase Chain
Reaction, (Mullis et al, ed., 1994); A Practical Guide to Molecular Cloning (Perbal
Bernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al., 2001).
Enzymatic reactions and purification techniques are performed according to
manufacturer's specifications, as commonly accomplished in the art or as described
herein. The nomenclatures used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses, chemical analyses,
pharmaceutical preparation, formulation, and delivery, and treatment of patients.
New antibodies and fragments thereof, capable of specifically binding the EphA2
receptor and antagonizing said receptor are herein provided. In particular, the novel
antibodies or fragments of the invention specifically bind Eph receptors on the cell
surface, but are preferentially devoid of any agonist activity. On the other hand, they
are capable of inhibiting the cellular functions of the receptor even in the presence of its

ligands.
As used herein, the term "EphA2 receptor" refers to a tyrosine kinase belonging to the
Eph receptors family (reviewed in Pasquale, E. B. et a/., 2005, Nature Reviews Mol.
Ceil Biol., 6, 462-475), and comprising, for example, an amino sequence as in
Genbank accession Nos NM_004431 (human EphA2), NM_010139 (murine EphA2), or
NXM_345596 (rat EphA2). Human EphA2 is a preferred EphA2 receptor. The term
"EphA2 ligand" as used herein refers to a protein that binds to, and optionally activates
(e.g. stimulates the autophosphorylation of), an EphA2 receptor. A preferred EphA2
ligand herein is "ephrinA1, which binds to the EphA2 receptor and comprises, for
example, an amino sequence as in Genbank accession NM_004428 (human
ephrinA1).
The term "antagonist" as used herein refers to a molecule which is capable of inhibiting
one or more of the biological activities of a target molecule, such as an EphA2
receptor. Antagonists may act by interfering with the binding of a receptor to a ligand
and vice versa, by decreasing EphA2 phosphorylation that could be induced by a
ligand, and/or by inhibiting the intracellular pathways that are induced by the binding of
such ligand, and/or by inhibiting the homo/hetero-oligomerization of EphA receptors.
The antagonist may completely block receptor-ligand interactions or may substantially
reduce such interactions. All such points of intervention by an antagonist shall be
considered equivalent for purposes of this invention. Thus, included within the scope of
the invention are antagonists (e.g. neutralizing antibodies) that bind to EphA2 receptor,
EphA2 ligand or a complex of an EphA2 receptor and EphA2 ligand; amino acid
sequence variants or derivatives of an EphA2 receptor or EphA2 ligand which
antagonize the interaction between an EphA2 receptor and EphA2 ligand; soluble
EphA2 receptor or soluble EphA2 ligand, optionally fused to a heterologous molecule
such as an immunoglobulin region (e.g. an immunoadhesin); a complex comprising an
EphA2 receptor in association with EphA2 ligand; synthetic or native sequence
peptides which bind to EphA2 receptor or EphA2 ligand. In a preferred embodiment,
the antagonist is an antibody.
The term "agonist" as used herein refers to any compound, including a protein, a
polypeptide, a peptide, an antibody, an antibody fragment, a conjugate, a large
molecule, a small molecule, capable of activating one or more of the biological
activities of the target molecule. EphA2 agonists act by stimulating phosphorylation of
the protein, thereby triggering degradation of said protein.

Thus in a preferred embodiment the present invention provides, among other features,
anti-EphA2 monoclonal antibodies, anti-EphA2 humanized antibodies, and fragments
of the anti-EphA2 antibodies. Each of the antibodies and antibody fragments of the
present invention is designed to specifically recognize and bind the EphA2 receptor,
and acts as an EphA2 receptor antagonist, inhibiting the phosphorylation induced by
EphA2 ligands.
The EphA2 receptor belongs to a family of receptor whose cytoplasmic tail
phosphorylation is increased after ligand binding to interact with a variety of adapter
and signaling proteins, leading to the activation of different downstream cellular
signaling pathways (Kullander, K. and Klein, R., 2002, Nature Reviews Mol. Cell Biol.,
3: 475-486; Noren, N. K. and Pasquale, E. B., 2004, Cell signal., 16: 655-666 ). As
used herein, the term "EphA2-mediated signaling" refers to all the cellular events which
occur in response to ligand binding by EphA2. Whereas antibodies disclosed in the
prior art agonize the EphA2 receptor, and, in particular, increase the tyrosine
phosphorylation of the EphA2 protein, the antibodies and antibody fragments of the
invention are preferentially devoid of any such agonistic properties. In particular, they
are unable to stimulate EphA2 phoshorylation by themselves. Like the antibodies
described in WO 2008/010101, the antagonistic antibodies and antibody fragments of
the invention are devoid of any agonist activity. In a specific embodiment, they are
unable to promote tyrosine phosphorylation of EphA2, unlike other antibodies
described in the prior art (Dodge-Zantek et a/., 1999, Cell Growth & Differ., 10: 629-
638; WO 01/12172, WO 03/094859, WO 2004/014292, WO 2004/101764, WO
2006/023403, WO 2006/047637, WO 2007/030642).
This invention provides actual antagonistic anti-EphA2 antibodies. The antibodies and
antibody fragments of the invention have the ability of inhibiting the cellular functions of
the EphA2 receptor, even in the presence of the ligands of said EphA2 receptor, e.g.
ephrinAI. In one embodiment, the antibodies and antibody fragments of the invention
can inhibit the binding of a ligand to an EphA2 receptor. In a preferred embodiment, the
binding of ephrinAI to EphA2 is prevented by the antibodies and fragments thereof
provided by this invention. Remarkably, in another embodiment, the antibodies and
antibody fragments of the invention are capable of inhibiting tyrosine phosphorylation of
the EphA2 receptor, even in the presence of ephrinA1.
Antibodies
The term "antibody" is used herein in the broadest sense and specifically covers

monoclonal antibodies (including full length monoclonal antibodies) of any isotype such
as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies, chimeric
antibodies, and antibody fragments. An antibody reactive with a specific antigen can be
generated by recombinant methods such as selection of libraries of recombinant
antibodies in phage or similar vectors, or.by immunizing an animal with the antigen or
an antigen-encoding nucleic acid.
A typical antibody is comprised of two identical heavy chains and two identical light
chains that are joined by disulfide bonds. Each heavy and light chain contains a
constant region and a variable region. As used herein, "VH" or "VH" refers to the
variable region of an immunoglobulin heavy chain of an antibody, including the heavy
chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment. Reference to "VL" or "VL"
refers to the variable region of the immunoglobulin light chain of an antibody, including
the light chain of an Fv, scFv, dsFv, Fab, Fab', or F(ab')2 fragment. Each variable
region contains three segments called "complementarity-determining regions" ("CDRs")
or "hypervariable regions", which are primarily responsible for binding an epitope of an
antigen. They are usually referred to as CDR1, CDR2, and CDR3, numbered
sequentially from the N-terminus. The more highly conserved portions of the variable
regions are called the "framework regions" ("FR"). The variable domains of native
heavy and light chains each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three CDRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The CDRs in each chain are held
together in close proximity by the FR regions and, with the CDRs from the other chain,
contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th edition, National Institute of
Health, Bethesda, MD, 1991).
The constant domains are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity (ADCC), phagocytosis via binding to Fcγ receptor,
half/life clearance rate via neonatal Fc receptor (FcRn) and complement dependent
cytotoxicity (CDC) via the C1q component of the complement cascade.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be
assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on
the amino acid sequences of their constant domains.
Depending on the amino acid sequences of the constant domains of their heavy

chains, antibodies (immunoglobulins) can be assigned to different classes. There are
five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of
these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4,
lgA1, and lgA2 . The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called Α,Δ,Ε,Γ, and μ, respectively. Within light and
heavy chains, the variable and constant regions are joined by a "J" region of about 12
or more amino acids, with the heavy chain also including a "D" region of about 10 more
amino acids (see e.g., Fundamental Immunology Ch. 7, Paul, W., ed., 2nd edition,
Raven Press, N. Y., 1989 ). The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well known and described
generally in, for example, Abbas et al.(Cellular and Mol. Immunology, 4th edition, W. B.
Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by
covalent or non-covalent association of the antibody with one or more other proteins or
peptides.
A "polyclonal antibody" is an antibody which was produced among or in the presence of
one or more other, non-identical antibodies. In general, polyclonal antibodies are
produced from a B-lymphocyte in the presence of several other B-lymphocytes
producing non-identical antibodies. Usually, polyclonal antibodies are obtained directly
from an immunized animal.
A "monoclonal antibody", as used herein, is an antibody obtained from a population of
substantially homogeneous antibodies, i.e. the antibodies forming this population are
essentially identical except for possible naturally occurring mutations which might be
present in minor amounts. These antibodies are directed against a single epitope and
are therefore highly specific.
A "naked antibody" for the purposes herein is an antibody which is not conjugated to a
cytotoxic moiety or radiolabel.
An "epitope" is the site on the antigen to which an antibody binds. It can be formed by
contiguous residues or by non-contiguous residues brought into close proximity by the
folding of an antigenic protein. Epitopes formed by contiguous amino acids are typically
retained on exposure to denaturing solvents, whereas epitopes formed by non-
contiguous amino acids are typically lost under said exposure.
As used herein, the term "KD" refers to the dissociation constant of a particular
antibody/antigen interaction. "Binding affinity" generally refers to the strength of the
sum total of non-covalent interactions between a single binding site of a molecule {e.g.,

an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as
used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1 : 1
interaction between members of a binding pair (e.g., antibody and antigen). The affinity
of a molecule X for its partner Y can generally be represented by the KD. Affinity can be
measured by common methods known in the art, including those described herein.
Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily,
whereas high- affinity antibodies generally bind antigen faster and tend to remain
bound longer. A variety of methods of measuring binding affinity are known in the art,
any of which can be used for purposes of the present invention.
The present invention proceeds from a murine anti-EphA2 antibody, herein
mu2H11R35R74, which is fully characterized with respect to the amino acid sequences
of both light and heavy chains, the identification of the CDRs, the identification of
surface amino acids, and means for its expression in recombinant form.
The murine antibody of the invention can be obtained for example by site-directed
mutagenesis of the antibody 53.2H11. The 53.2H11 antibody is produced by a
hybridoma deposited under the Budapest Treaty on June 16, at the American Type
Culture Collection, under the accession number PTA-7662, and is described in PCT
application WO 2008/010101. Thus, the amino acid sequences of the both light and
heavy chains of 53.2H11, the identification of the CDRs, the identification of surface
amino acids, as well as polynucleotide sequences encoding said light and heavy
chains are all disclosed in WO 2008/010101.
The primary amino acid and DNA sequences of antibody mu2H11R35R74 light and
heavy chains, and of humanized versions thereof, are disclosed herein. In one
embodiment, this invention provides antibodies or epitope-binding fragment thereof
comprising one or more CDRs having an amino acid sequence selected from the group
consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6.
In a preferred embodiment, the antibodies of the invention comprise at least one heavy
chain and at least one light chain, and said heavy chain comprises three sequential
CDRs having amino acid sequences selected from the group consisting of SEQ ID
NOS: 1, 2, 3, and said light chain comprises three sequential CDRs having amino acid
sequences selected from the group consisting of SEQ ID NOS: 4, 5, 6.
In a preferred embodiment the paratope of said monoclonal antibodies or epitope-
binding fragments thereof comprises in the light chain: Arg35 of Loop L1, Tyr54, Arg58
and Asp60 of L2.

In a preferred embodiment the paratope of said monoclonal antibodies or epitope-
binding fragments thereof comprises heavy chain: Thr30, Ala31, Tyr32 and Tyr33 of
Loop H1, Asn52, Tyr54, Asn55 and Phe57 of H2 and Glu99, Phe100, Tyr101, Gly102,
Tyr103 and Tyr105 of H3.
Said antibodies or an epitope-binding fragments thereof can comprise mutations:
at the position: Thr H28
at one of few of the following positions on the light chain: 35, 26 to 31, 34 to 37, 55,
56, 57, 59 and 94 to 102, and/or
at one of few of the following positions on the heavy chain: 28, 54 and 57.
In another embodiment the antibodies of the invention specifically bind to an epitope of
human EphA2 receptor comprising residues Gly49, Lys50, Gly51, Asp53, Cys70,
Asn71, Val72, Met73, Ser74, Gly75, Gln77, Phe 108, Pro109, Gly110, Gly111, Ser113
and Ser114 of the LBD from the extra-cellular domain of EphA2 receptor, or a
conservatively substituted form thereof.
In another embodiment, the antibodies of the invention comprise a VH having an amino
acid sequence consisting of SEQ ID NO 8. In another preferred embodiment, the
antibodies of the invention comprise a VL having an amino acid sequence consisting of
SEQ ID NO 10.
Humanized or Resurfaced 2H11R35R74 Antibody
As used herein, the term "humanized antibody" refers to a chimeric antibody which
contains minimal sequence derived from non-human immunoglobulin. A "chimeric
antibody", as used herein, is an antibody in which the constant region, or a portion
thereof, is altered, replaced, or exchanged, so that the variable region is linked to a
constant region of a different species, or belonging to another antibody class or
subclass. "Chimeric antibody" also refers to an antibody in which the variable region, or
a portion thereof, is altered, replaced, or exchanged, so that the constant region is
linked to a variable region of a different species, or belonging to another antibody class
or subclass.
The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody,
such as a murine antibody, for introduction into a human, while maintaining the full
antigen binding affinity and specificity of the antibody. Humanized antibodies, or
antibodies adapted for non-rejection by other mammals, may be produced using

several technologies such as resurfacing and CDR grafting. As used herein, the
resurfacing technology uses a combination of molecular modeling, statistical analysis
and mutagenesis to alter the non-CDR surfaces of antibody variable regions to
resemble the surfaces of known antibodies of the target host.
Strategies and methods for the resurfacing of antibodies, and other methods for
reducing immunogenicity of antibodies within a different host, are disclosed in US
Patent 5,639,641, which is hereby incorporated in its entirety by reference. Briefly, in a
preferred method, (1) position alignments of a pool of antibody heavy and light chain
variable regions is generated to give a set of heavy and light chain variable region
framework surface exposed positions wherein the position alignments for all variable
regions are at least about 98% identical; (2) a set of heavy and light chain variable
region framework surface exposed amino acid residues is defined for a rodent antibody
(or fragment thereof); (3) a set of heavy and light chain variable region framework
surface exposed amino acid residues that is most closely identical to the set of rodent
surface exposed amino acid residues is identified; (4) the set of heavy and light chain
variable region framework surface exposed amino acid residues defined in step (2) is
substituted with the set of heavy and light chain variable region framework surface
exposed amino acid residues identified in step (3), except for those amino acid
residues that are within 5 A of any atom of any residue of the complementarity-
determining regions of the rodent antibody; and (5) the humanized rodent antibody
having binding specificity is produced.
Another preferred method of humanization of antibodies, based on the identification of
flexible residues, has been described in PCT application WO 2009/032661. Said
method comprises the following steps: (1) building a homology model of the parent
mAb and running a molecular dynamics simulation; (2) analyzing the flexible residues
and identification of the most flexible residues of a non-human antibody molecule, as
well as identifying residues or motifs likely to be a source of heterogeneity or of
degradation reaction; (3) identifying a human antibody which displays the most similar
ensemble of recognition areas as the parent antibody; (4) determining the flexible
residues to be mutated, residues or motifs likely to be a source of heterogeneity and
degradation are also mutated; and (5) checking for the presence of known T cell or B
cell epitopes. The flexible residues can be found using a molecular dynamics
calculation using an implicit solvent model, which accounts for the interaction of the
water solvent with the protein atoms over the period of time of the simulation.
Antibodies can be humanized using a variety of other techniques including CDR-

grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089),
veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991, Molecular
Immunology 28(4/5): 489-498; Studnicka G. M. et al., 1994, Protein Engineering 7(6):
805-814; Roguska MA et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91:969-973), and
chain shuffling (U.S. Pat. No. 5,565,332).
In certain embodiments both the variable and constant regions of the antibodies, or
antigen-binding fragments, variants, or derivatives thereof are fully human. Fully human
antibodies can be made using techniques that are known in the art. For example, fully
human antibodies against a specific antigen can be prepared by administering the
antigen to a transgenic animal which has been modified to produce such antibodies in
response to antigenic challenge, but whose endogenous loci have been disabled.
Exemplary techniques that can be used to make such antibodies are described in US
patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully
.. human antibodies can likewise be produced by various display technologies, e.g.,
phage display or other viral display systems. See also U.S. Pat. Nos. 4,444,887,
4,716,111, 5,545,806, and 5,814,318; and international patent application publication
numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096,
WO 96/33735, and WO 91/10741 (said references incorporated by reference in their
entireties).
The present invention provides humanized antibodies or fragments thereof, which
recognize EphA2 receptor and act as antagonists. In a preferred embodiment, the
humanized antibodies or epitope-binding fragments thereof have the additional ability
to inhibit growth of a cancer cell expressing the EphA2 receptor. In a further
embodiment, the humanized antibody or epitope-binding fragment thereof have the
additional ability to inhibit the migration of a metastatic cancer cell expressing the
EphA2 receptor.
A preferred embodiment of such a humanized antibody is a humanized 2H11R35R74
antibody, or an epitope-binding fragment thereof.
In another preferred embodiment, the humanized antibodies of the invention are
obtained by site-directed mutagenesis of the polynucleotide sequences encoding
hu53.2H11 (WO 2008/010101) referred herein as hu2H11.
In more preferred embodiments, there are provided resurfaced or humanized versions
of the 2H11R35R74 antibody wherein surface-exposed residues of the antibody or its
fragments are replaced in both light and heavy chains to more closely resemble known

human antibody surfaces. The humanized 2H11R35R74 antibody or epitope-binding
fragments thereof of the present invention have improved properties. For example,
humanized 2H11R35R74 antibodies or epitope-binding fragments thereof specifically
recognize EphA2 receptor. More preferably, the humanized 2H11R35R74 antibody or
epitope-binding fragments thereof have the additional ability to inhibit the growth of an
EphA2 receptor-expressing cell.
The humanized versions of the 2H11R35R74 antibody are also fully characterized
herein with respect to their respective amino acid sequences of both light and heavy
chain variable regions, the DNA sequences of the genes for the light and heavy chain
variable regions, the identification of the CDRs, the identification of their surface amino
acids, and disclosure of a means for their expression in recombinant form. However,
the scope of the present invention is not limited to antibodies and fragments comprising
these sequences. Instead, all antibodies and fragments that specifically bind to EphA2
receptor are included in the present invention. Preferably, the antibodies and fragments
that specifically bind to EphA2 receptor antagonize the biological activity of the
receptor. More preferably, such antibodies further are substantially devoid of agonist
activity. Thus, antibodies and epitope-binding antibody fragments of the present
invention may differ from the 2H11R35R74 antibody or the humanized derivatives
thereof, in the amino acid sequences of their scaffold, CDRs, and/or light chain and
heavy chain, and still fall within the scope of the present invention.
The CDRs of the 2H11R35R74 antibody have been determined by solving the crystal
structure of the Fab fragment of 2H11R35R74 in complex with the extra-cellular
domain of the EphA2 receptor. The residues from the 2H11R35R74 which interact with
the extra-cellular domain of EphA2 have been identified. Accordingly, antibodies and
fragments are provided that have improved properties produced by, for example,
affinity maturation of an antibody of the present invention.
The mouse light chain IgVK and JK germline genes and heavy chain IgVh and Jh
germline genes from which 53.2H11 was likely derived have been identified, and were
disclosed in WO 2008/010101. The accession numbers of said germline sequences
are respectively MMU231196 and AF303833. Such germline gene sequences are
useful to identify somatic mutations in the antibodies, including in the CDRs.
The sequences of the heavy chain and light chain variable regions of the 2H11R35R74
antibody, and the sequences of their CDRs were not previously known and are set
forth in this application. Such information can be used to produce humanized versions

of the 2H11R35R74 antibody. It is also possible to obtain the humanized 2H11R35R74
antibodies of the invention by site-directed mutagenesis of hu2H11. These humanized
anti-EphA2 antibodies or their derivatives may also be used as the cell binding agent of
the conjugates of the present invention.
Thus, in one embodiment, this invention provides humanized antibodies or epitope-
binding fragment thereof comprising one or more CDRs having an amino acid
sequence selected from the group consisting of SEQ ID NOS: 1,2, 3, 4, 5, 6. In a
preferred embodiment, the humanized antibodies of the invention comprise at least one
heavy chain and at least one light chain, wherein said heavy chain comprises three
sequential CDRs having amino acid sequences represented by SEQ ID NOS: 1, 2, and
3, and wherein said light chain comprises three sequential CDRs having amino acid
sequences represented by SEQ ID NOS: 4, 5, and 6.
In one embodiment, this invention provides a humanized 2H11R35R74 antibody or
fragments thereof which comprises a VH having an amino acid sequence consisting of
SEQ ID NO. 12. In another embodiment, this invention provides a humanized
2H11R35R74 antibody or fragments thereof which comprises a VL having an amino
acid sequence consisting of SEQ ID NO 14.
In a preferred embodiment, a humanized 2H11R35R74 antibody is provided, which
comprises at least one heavy chain and at least one light chain, wherein said heavy
chain comprises three sequential CDRs having amino acid sequences represented by
SEQ ID NOS: 1, 2, and 3, wherein said light chain comprises three sequential CDRs
having amino acid sequences represented by SEQ ID NOS: 4, 5, and 6, wherein said
heavy chain has an amino acid sequence consisting of SEQ ID NO. 12, and wherein
said light chain has an amino acid sequence consisting of SEQ ID NO. 14.
Polynucleotides, vectors, and host cells.
Nucleic acids encoding anti-EphA2 antibodies of the invention are provided. In one
embodiment, the nucleic acid molecule encodes a heavy and/or a light chain of an anti-
EphA2 immunoglobulin. In a preferred embodiment, a single nucleic acid encodes a
heavy chain of an anti-EphA2 immunoglobulin and another nucleic acid molecule
encodes the light chain of an anti-EphA2 immunoglobulin.
In another aspect of this invention, there are provided polynucleotides encoding
polypeptides having an amino acid sequence selected from the group of SEQ ID NOS:
1, 2, 3, 4, 5, 6, 8, 10, 12, 14,16 and 18. In a preferred embodiment, the polynucleotide
of the invention is selected from the group consisting of SEQ ID NOs: 7, 9,11,13,15

and 17. The invention is not limited to said polynucleotides per se but also includes all
polynucleotides displaying at least 80 % identity with said polynucleotides.
The term "polynucleotide" as referred to herein means a polymeric form of nucleotides
of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified
form of either type of nucleotide. The term includes single and double stranded forms
of DNA.
The term "isolated polynucleotide" as used herein shall mean a polynucleotide of
genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its
origin the "isolated polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the"isolated polynucleotide'' found in nature, (2) is operably
linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in
nature as part of a larger sequence.
The invention provides vectors comprising the polynucleotides of the invention. In one
embodiment, the vector contains a polynucleotide encoding a heavy chain of an anti-
EphA2 immunoglobulin. In another embodiment, said polynucleotide encodes the light
chain of an anti-EphA2 immunoglobulin. The invention also provides vectors
comprising polynucleotide molecules encoding fusion proteins, modified antibodies,
antibody fragments, and probes thereof.
In order to express the heavy and/or light chain of the anti-EphA2 antibodies of the
invention, the polynucleotides encoding said heavy and/or light chains are inserted into
expression vectors such that the genes are operatively linked to transcriptional and
translational sequences.
"Operably linked" sequences include both expression control sequences that are
contiguous with the gene of interest and expression control sequences that act in trans
or at a distance to control the gene of interest. The term "expression control sequence"
as used herein refers to polynucleotide sequences which are necessary to effect the
expression and processing of coding sequences to which they are ligated. Expression
control sequences include appropriate transcription initiation, termination, promoter and
enhancer sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that
enhance translation efficiency (i. e., Kozak consensus sequence); sequences that
enhance protein stability; and when desired, sequences that enhance protein secretion.
The nature of such control sequences differs depending upon the host organism; in
prokaryotes, such control sequences generally include promoter, ribosomal binding

site, and transcription termination sequence; in eukaryotes, generally, such control
sequences include promoters and transcription termination sequence. The term
"control sequences" is intended to include, at a minimum, all components whose
presence is essential for expression and processing, and can also include additional
components whose presence is advantageous, for example, leader sequences and
fusion partner sequences.
The term "vector", as used herein, is intended to refer to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been linked. One type of
vector is a "plasmid", which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral genome. Certain
vectors are capable of autonomous replication in a host cell into which they are
introduced (e. g., bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e. g., non- episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome.
Certain vectors are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as "recombinant expression
vectors" (or simply, "expression vectors"). In general, expression vectors of utility in
recombinant DNA techniques are in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to include such
forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retrovirus,
EBV-derived episomes, and all the other vectors that the skilled man will know to be
convenient for ensuring the expression of the heavy and/or light chains of the
antibodies of the invention. The skilled man will realize that the polynucleotides
encoding the heavy and the light chains can be cloned into different vectors or in the
same vector. In a preferred embodiment, said polynucleotides are cloned in the same
vector.
Polynucleotides of the invention and vectors comprising these molecules can be used
for the transformation of a suitable mammalian host cell, or any other type of host cell
known to the skilled person. The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a recombinant expression vector
has been introduced. It should be understood that such terms are intended to refer not
only to the particular subject cell but also to the progeny of such a cell. Because certain

modifications may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be identical to the parent cell,
but are still included within the scope of the term "host cell" as used herein.
Transformation can be by any known method for introducing polynucleotides into a
host cell. Such methods are well known of the man skilled in the art and include
dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated
transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into
liposomes, biolistic injection and direct microinjection of DNA into nuclei.
Antibody Fragments
The antibodies of the present invention include both the full length antibodies
discussed above, as well as epitope-binding fragments. As used herein, "antibody
fragments" include any portion of an antibody that retains the ability to bind to the
epitope recognized by the full length antibody, generally termed "epitope-binding
fragments." Examples of antibody fragments include, but are not limited to, Fab, Fab'
and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs
(dsFv) and fragments comprising either a VL or VH region. Epitope-binding fragments,
including single-chain antibodies, may comprise the variable region(s) alone or in
combination with the entirety or a portion of the following: hinge region, CH1, CH2, and
CH3 domains.
Such fragments may contain one or both Fab fragments or the F(ab')2 fragment.
Preferably, the antibody fragments contain all six CDRs of the whole antibody, although
fragments containing fewer than all of such regions, such as three, four or five CDRs,
are also functional. Further, the fragments may be or may combine members of any
one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the
subclasses thereof.
Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes
such as papain (Fab fragments) or pepsin (F(ab')2 fragments).
The "single-chain FVs" ("scFvs") fragments are epitope-binding fragments that contain
at least one fragment of an antibody heavy chain variable region (VH) linked to at least
one fragment of an antibody light chain variable region (VL). The linker may be a short,
flexible peptide selected to ensure that the proper three-dimensional folding of the (VL)
and (VH) regions occurs once they are linked so as to maintain the target molecule
binding-specificity of the whole antibody from which the single-chain antibody fragment
is derived. The carboxyl terminus of the (VL) or (VH) sequence may be covalently linked

by a linker to the amino acid terminus of a complementary (VL) or (VH) sequence.
Single-chain antibody fragments of the present invention contain amino acid
sequences having at least one of the variable or complementarity determining regions
(CDRs) of the whole antibodies described in this specification, but are lacking some or
all of the constant domains of those antibodies. These constant domains are not
necessary for antigen binding, but constitute a major portion of the structure of whole
antibodies. Single-chain antibody fragments may therefore overcome some of the
problems associated with the use of antibodies containing a part or all of a constant
domain. For example, single-chain antibody fragments tend to be free of undesired
interactions between biological molecules and the heavy-chain constant region, or
other unwanted biological activity. Additionally, single-chain antibody fragments are
considerably smaller than whole antibodies and may therefore have greater capillary
permeability than whole antibodies, allowing single-chain antibody fragments to localize
and bind to target antigen-binding sites more efficiently. Also, antibody fragments can
be produced on a relatively large scale in prokaryotic cells, thus facilitating their
production. Furthermore, the relatively small size of single-chain antibody fragments
makes them less likely to provoke an immune response in a recipient than whole
antibodies.
Single-chain antibody fragments may be generated by molecular cloning, antibody
phage display library or similar techniques well known to the skilled artisan. These
proteins may be produced, for example, in eukaryotic cells or prokaryotic cells,
including bacteria. The epitope-binding fragments of the present invention can also be
generated using various phage display methods known in the art. In phage display
methods, functional antibody domains are displayed on the surface of phage particles
which carry the polynucleotide sequences encoding them. In particular, such phage
can be utilized to display epitope-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage expressing an epitope-
binding domain that binds the antigen of interest can be selected or identified with
antigen, e.g., using labelled antigen bound or captured to a solid surface or bead.
Phage used in these methods are typically filamentous phage including fd and M13
binding domains expressed from phage with Fab, Fv or disulfide-stabilized Fv antibody
domains recombinantly fused to either the phage gene III or gene VIII protein.
Examples of phage display methods that can be used to make the epitope-binding
fragments of the present invention include those disclosed in Brinkman ef a/., 1995, J.
Immunol. Methods, 182:41-50; Ames ef a/., 1995, J. Immunol. Methods, 184:177-186;

Kettleborough et al., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene
187: 9-18; Burton et al., 1994, Advances in Immunology, 57: 191-280; PCT application
No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;
WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;
each of which is incorporated herein by reference in its entirety.
After phage selection, the regions of the phage encoding the fragments can be isolated
and used to generate the epitope-binding fragments through expression in a chosen
host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, using
recombinant DNA technology, e.g., as described in detail below. For example,
techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in PCT publication
WO 92/22324; Mullinax et al., 1992, BioTechniques, 12(6): 864-869; Sawai et al.,
1995, AJRI, 34: 26-34; and Better et al., 1988, Science, 240: 1041-1043; said
references incorporated by reference in their entireties. Examples of techniques which
can be used to produce single-chain Fvs and antibodies include those described in
U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology
203: 46-88; Shu era/., 1993, Proc. Natl. Acad. Sci. U.S.A., 90: 7995-7999; Skerra et
al., 1988, Science, 240:1038-1040.
Functional Equivalents
Also included within the scope of the invention are functional equivalents of the anti-
EphA antibody and the humanized anti-EphA2 receptor antibody. The term "functional
equivalents" includes antibodies with homologous sequences, chimeric antibodies,
artificial antibodies and modified antibodies, for example, wherein each functional
equivalent is defined by its ability to bind to EphA2 receptor. The skilled artisan will
understand that there is an overlap in the group of molecules termed "antibody
fragments" and the group termed "functional equivalents." Methods of producing
functional equivalents are known to the person skilled in the art and are disclosed, for
example, in PCT Application WO 93/21319, European Patent No. EP 0239400; PCT
Application WO 89/09622; European Patent No. EP 0338745; and European Patent
Application EP 0332424, which are incorporated in their respective entireties by
reference.
Antibodies with homologous sequences are those antibodies with amino acid

sequences that have sequence homology with amino acid sequence of an anti-EphA
antibody and a humanized anti-EphA antibody of the present invention. Preferably
homology is with the amino acid sequence of the variable regions of the anti-EphA
antibody and humanized anti-EphA antibody of the present invention. "Sequence
homology" as applied to an amino acid sequence herein is defined as a sequence with
at least about 90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably
at least about 95%, 96%, 97%, 98%, or 99% sequence homology to another amino
acid sequence, as determined, for example, by the FASTA search method in
accordance with Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. U.S.A., 85: 2444-
2448.
A chimeric antibody is one in which different portions of an antibody are derived from
different animal species. For example, an antibody having a variable region derived
from a murine monoclonal antibody paired with a human immunoglobulin constant
region. Methods for producing chimeric antibodies are known in the art. See, e.g.,
Morrison, 1985, Science, 229:1202; Oi etal., 1986, BioTechniques, 4: 214; Gillies et
a/., 1989, J. Immunol. Methods, 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816,397, which are incorporated herein by reference in their entireties.
Humanized forms of chimeric antibodies are made by substituting the complementarity
determining regions of, for example, a mouse antibody, into a human framework
domain, e.g., see PCT Pub. No. WO 92/22653. Humanized chimeric antibodies
preferably have constant regions and variable regions other than the complementarity
determining regions (CDRs) derived substantially or exclusively from the corresponding
human antibody regions and GDRs derived substantially or exclusively from a mammal
other than a human.
Artificial antibodies include scFv fragments, diabodies, triabodies, tetrabodies and mru
(see reviews by Winter, G. and Milstein, C, 1991, Nature, 349: 293-299; Hudson, P.J.,
1999, Current Opinion in Immunology, 11: 548-557), each of which has antigen-binding
ability. In the single chain Fv fragment (scFv), the VH and VL domains of an antibody
are linked by a flexible peptide. Typically, this linker peptide is about 15 amino acid
residues long. If the linker is much smaller, for example 5 amino acids, diabodies are
formed, which are bivalent scFv dimers. If the linker is reduced to less than three amino
acid residues, trimeric and tetrameric structures are formed that are called triabodies
and tetrabodies. The smallest binding unit of an antibody is a CDR, typically the CDR2
of the heavy chain which has sufficient specific recognition and binding that it can be
used separately. Such a fragment is called a molecular recognition unit or mru. Several

such mrus can be linked together with short linker peptides, therefore forming an
artificial binding protein with higher avidity than a single mm.
The functional equivalents of the present application also include modified antibodies,
e.g., antibodies modified by the covalent attachment of any type of molecule to the
antibody. For example, modified antibodies include antibodies that have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. The covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. These modifications may be
carried out by known techniques, including, but not limited to, specific chemical
cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the modified antibodies may contain one or more non-classical amino acids.
Functional equivalents may be produced by interchanging different CDRs on different
chains within different frameworks. Thus, for example, different classes of antibody are
possible for a given set of CDRs by substitution of different heavy chains, whereby, for
example, lgG1-4, IgM, lgA1-2, IgD, IgE antibody types and isotypes may be produced.
Similarly, artificial antibodies within the scope of the invention may be produced by
embedding a given set of CDRs within an entirely synthetic framework.
Functional equivalents may be readily produced by mutation, deletion and/or insertion
within the variable and/or constant region sequences that flank a particular set of
CDRs, using a wide variety of methods known in the art.
The antibody fragments and functional equivalents of the present invention encompass
those molecules with a detectable degree of binding to EphA2, when compared to the
2H11R35R74 antibody. A detectable degree of binding includes all values in the range
of at least 10-100%, preferably at least 50%, 60% or 70%, more preferably at least
75%, 80%, 85%, 90%, 95% or 99% the binding ability of the murine 2H11R35R74
antibody to EphA2.
Improved Antibodies
The CDRs are of primary importance for epitope recognition and antibody binding.
However, changes may be made to the residues that comprise the CDRs without
interfering with the ability of the antibody to recognize and bind its cognate epitope. For
example, changes that do not affect epitope recognition, yet increase the binding
affinity of the antibody for the epitope may be made.

Thus, also included in the scope of the present invention are improved versions of both
the murine and humanized antibodies, which also specifically recognize and bind
EphA2, preferably with increased affinity.
Several studies have surveyed the effects of introducing one or more amino acid
changes at various positions in the sequence of an antibody, based on the knowledge
of the primary antibody sequence, on its properties such as binding and level of
expression (Yang, W. P. et al., 1995, J. Mol. Biol., 254: 392-403; Rader, C. et al., 1998,
Proc. Natl. Acad. Sci. U.S.A., 95: 8910-8915; Vaughan, T. J. et al., 1998, Nature
Biotechnology, 16: 535-539).
In these studies, equivalents of the primary antibody have been generated by changing
the sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or
framework regions, using methods such as oligonucleotide-mediated site-directed
mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-
strains of E. coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16: 535-539; Adey,
N. B. et al., 1996, Chapter 16, pp. 277-291, in "Phage Display of Peptides and
Proteins", Eds. Kay, B. K. et al., Academic Press). These methods of changing the
sequence of the primary antibody have resulted in improved affinities of the secondary
antibodies (Gram, H. et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89: 3576-3580; Boder,
E. T. et al., 2000, Proc. Natl. Acad. Sci. U.S.A., 97: 10701-10705; Davies, J. and
Riechmann, L, 1996, Immunotechnolgy, 2: 169-179; Thompson, J. et al., 1996, J. Mol.
Biol., 256: 77-88; Short, M. K. etal., 2002, J. Biol. Chem., 277: 16365-16370;
Furukawa, K. et al., 2001, J. Biol. Chem., 276: 27622-27628).
By a similar directed strategy of changing one or more amino acid residues of the
antibody, the antibody sequences described in this invention can be used to develop
anti-EphA2 antibodies with improved functions, including improved affinity for EphA2.
Preferred amino acid substitutions are those which: (1) reduce susceptibility to
proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming
protein complexes, and (4) confer or modify other physico-chemical or functional
properties of such analogs. Analogs can include various muteins of a sequence other
than the naturally-occurring peptide sequence. For example, single or multiple amino
acid substitutions (preferably conservative amino acid substitutions) may be made in
the naturally-occurring sequence (preferably in the portion of the polypeptide outside
the domain (s) forming intermolecular contacts). A conservative amino acid substitution
should not substantially change the structural characteristics of the parent sequence

(e.g., a replacement amino acid should not tend to break a helix that occurs in the
parent sequence, or disrupt other types of secondary structure that characterizes the
parent sequence). Examples of art-recognized polypeptide secondary and tertiary
structures are described in Proteins, Structures and Molecular Principles (Creighton,
Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure
(C. Branden and J. Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and
Thornton et a/., 1991, Nature, 354: 105, which are each incorporated herein by
reference.
Improved antibodies also include those antibodies having improved characteristics that
are prepared by the standard techniques of animal immunization, hybridoma formation
and selection for antibodies with specific characteristics.
The interaction between the constant region of an antibody and various Fc receptors
(FcyR) is believed to mediate the effector functions of the antibody which include
antibody-dependent cellular cytotoxicity (ADCC), fixation of complement, phagocytosis
and half-life/clearance of the antibody. Various modifications to the constant region of
antibodies of the invention may be carried out depending on the desired property. For
example, specific mutations in the constant region to render an otherwise lytic
antibody, non-lytic is detailed in EP 0629 240B1 and EP 0307 434B2 or one may
incorporate a salvage receptor binding epitope into the antibody to increase serum half
life (see US 5,739,277). There are five currently recognised human Fey receptors, FcyR
(I), FcyRlla, FcyRllb, FcyRllla and neonatal FcRn. Shields et al. (J.Biol.Chem., 27: 6591-
6604, 2001) demonstrated that a common set of IgGI residues is involved in binding all
FcyRs, while FcyRII and FcyRIII utilize distinct sites outside of this common set. One
group of IgGI residues reduced binding to all FcyRs when altered to alanine: Pro-238,
Asp-265, Asp-270, Asn-297 and Pro-239. All are in the IgG CH2 domain and clustered
near the hinge joining CH1 and CH2. While FcγRI utilizes only the common set of IgGI
residues for binding, FcγRII and FcγRIII interact with distinct residues in addition to the
common set.
Alteration of some residues reduced binding only to FcyRII (e.g. Arg-292) or FcyRIII
(e.g. Glu-293). Some variants showed improved binding to FcyRII or FcyRIII but did not
affect binding to the other receptor (e.g. Ser-267Ala improved binding to FcgRII but
binding to FcyRIII was unaffected). Other variants exhibited improved binding to FcyRII
or FcγRIII with reduction in binding to the other receptor (e.g. Ser-298Ala improved
binding to FcyRIII and reduced binding to FcyRII). For FcγRIlla, the best binding IgGI

variants had combined alanine substitutions at Ser-298, Glu-333 and Lys-334. The
neonatal FcRn receptor is believed to be involved in both antibody clearance and the
transcytosis across tissues (see Junghans R.P, 1997, Immunol. Res., 16: 29-57 and
Ghetie et al., 2000, Annu.Rev.lmmunol. 18: 739-766). Human IgGI residues
determined to interact directly with human FcRn includes Ne253, Ser254, Lys288,
Thr307, Gln311, Asn434 and His435. Switches at any of these positions described in
this section may enable increased serum half-life and/or altered effector properties of
antibodies of the invention.
Other modifications include glycosylation variants of the antibodies of the invention.
Glycosylation of antibodies at conserved positions in their constant regions is known to
have a profound effect on antibody function, particularly effector functioning such as
those described above, see for example, Boyd et al (Mol. Immunol,. 32: 1311-1318,
1996). Glycosylation variants of the antibodies or antigen binding fragments thereof of
the present invention wherein one or more carbohydrate moiety is added, substituted,
deleted or modified are contemplated. Introduction of an asparagine-X- serine or
asparagine-X-threonine motif creates a potential site for enzymatic attachment of
carbohydrate moieties and may therefore be used to manipulate the glycosylate of an
antibody. In Raju et al. (Biochemistry 40: 8868-8876, 2001 ) the terminal sialylation of a
TNFR-IgG immunoadhesin was increased through a process of regalactosylation
and/or resialylation using β-1,4-galactosyltransferace and/or alpha, 2,3
sialyltransferase. Increasing the terminal sialylation is believed to increase the half-life
of the immunoglobulin. Antibodies, in common with most glycoproteins, are typically
produced as a mixture of glycoforms. This mixture is particularly apparent when
antibodies are produced in eukaryotic, particularly mammalian cells. A variety of
methods have been developed to manufacture defined glycoforms (see Zhang et al.
2004, Science 303: 371; Sears et al, 2001, Science 291: 2344; Wacker et al., 2002,
Science 298: 1790; Davis et al. 2002, Chem.Rev. 102: 579; Hang et al., 2001,
Acc.Chem. Res. 34: 727). Thus the invention contemplates a plurality of (monoclonal)
antibodies (which may be of the IgG isotype, e.g. IgGI) as herein described comprising
a defined number (e.g. 7 or less, for example 5 or less such as two or a single)
glycoform(s) of said antibodies or antigen binding fragments thereof.
Therefore, improved antibodies according to the invention include in particular
antibodies with enhanced functional properties. Of special interest are those antibodies
with enhanced ability to mediate cellular cytotoxic effector functions such as ADCC.
Such antibodies may be obtained by making single or multiple substitutions in the

constant framework of the antibody, thus altering its interaction with the Fc receptors.
Methods for designing such mutants can be found for example in Lazar et al. (2006,
Proc. Natl. Acad. Sci. U.S.A. 103(11): 4005-4010) and Okazaki et al. (2004, J. Mol.
Biol. 336(5): 1239-49). See also WO 03/074679, WO 2004/029207, WO 2004/099249,
WO2006/047350, WO 2006/019447, WO 2006/105338, WO 2007/041635. It is also
possible to use cell lines specifically engineered for production of improved antibodies.
In particular, these lines have altered regulation of the glycosylation pathway, for
example resulting in antibodies which are poorly fucosylated or even totally
defucosylated. Such cell lines and methods for engineering them are disclosed in e.g.
Shinkawa et al. (2003, J. Biol. Chem. 278(5): 3466-3473), Ferrara et al. (2006, J. Biol.
Chem. 281(8): 5032-5036; 2006, Biotechnol. Bioeng. 93(5): 851-61), EP 1 272 527 B1,
EP 1 331 266, EP 1 498 490, EP 1 498 491, EP 1 676 910, EP 1 792 987, and WO
99/54342.
Further embodiments of the invention include antibodies of the invention or antigen
binding fragments thereof coupled to a non-proteinaeous polymer such as polyethylene
glycol (PEG), polypropylene glycol or polyoxyalkylene. Conjugation of proteins to PEG
is an established technique for increasing half-life of proteins, as well as reducing
antigenicity and immunogenicity of proteins. The use of PEGylation with different
molecular weights and styles (linear or branched) has been investigated with intact
antibodies as well as Fab' fragments (Koumenis I. L et al., 2000, Int. J. Pharmaceut.
198:83-95).
The present invention also includes cytotoxic conjugates, or antibody-drug conjugates,
or conjugates. As used herein, all these terms have the same meaning and are
interchangeable.
These cytotoxic conjugates comprise two primary components, a cell-binding agent
and a cytotoxic agent.
As used herein, the term "cell binding agent" refers to an agent that specifically
recognizes and binds the EphA2 receptor on the cell surface. In one embodiment, the
cell binding agent specifically recognizes the EphA2 receptor such that it allows the
conjugate to act in a targeted fashion with little side-effects resulting from non-specific
binding.
In another embodiment, the cell binding agent of the present invention also specifically
recognizes the EphA2 receptor so that the conjugate will be in contact with the target
cell for a sufficient period of time to allow the cytotoxic drug portion of the conjugate to

act on the cell, and/or to allow the conjugates to be internalized by the cell.
In a preferred embodiment, the cytotoxic conjugates comprise an anti-EphA2 antibody
as the cell binding agent, more preferably the murine 2H11R35R74 monoclonal
antibody. In a more preferred embodiment, the cytotoxic conjugate comprises a
humanized 2H11R35R74 antibody or an epitope-binding fragment thereof. The
2H11R35R74 antibody is able to specifically recognize an EphA receptor, such as
EphA2, and directs the cytotoxic agent to an abnormal cell or a tissue, such as cancer
cells, in a targeted fashion.
The second component of the cytotoxic conjugates of the present invention is a
cytotoxic agent. The term "cytotoxic agent", as used herein, refers to a substance that
reduces or blocks the function, or growth, of cells and/or causes destruction of cells.
In preferred embodiments, the cytotoxic agent is a taxoid, a maytansinoid such as DM1
or DM4, a small drug, a tomaymycin derivative, a prodrug, CC-1065 or a CC-1065
analog. In preferred embodiments, the cell binding agents of the present invention are
covalently attached, directly or via a cleavable or non-cleavable linker, to the cytotoxic
agent. "Linker", as used herein, means a chemical moiety comprising a covalent bond
or a chain of atoms that covalently attaches an antibody to a drug moiety.
Thus, this invention contemplates the use of conjugates between (1) a cell binding
agent that recognizes and binds the EphA2 receptor, and (2) a cytotoxic agent. In the
cytotoxic conjugates, the cell binding agent has a high affinity for the EphA2 receptor
and the cytotoxic agent has a high degree of cytotoxicity for cells expressing the EphA2
receptor, such that the cytotoxic conjugates of the present invention form effective
killing agents.
Conjugates of antagonistic EphA2 antibodies have been previously described. For
example, WO 2008/010101 disclosed humanized 37.3D7 and humanized 53.2H11
antibodies conjugated to L-DM4, N2'deacetyl-N2'(4-methyl-4-mercapto-1-oxopentyl)-
maytansine, using SPDB (4-[2-pyridyldithio]butanoic acid N-hydroxysuccinimide ester)
linker (see Example 10 of WO 2008/010101; hu37.3D7-SPDB-DM4 and hu2H11-
SPDB-DM4).
When conjugated to a cytotoxic agent, the antibodies of the invention show a number
of advantageous properties over the antibodies of the prior art. In particular,
conjugation does not affect the affinity of the antibodies of the invention for the EphA2
receptor, whereas the binding of 53.2H11 to EphA2 is negatively affected by the
attachment of a cytotoxic agent onto said 53.2H11 antibody.

The cell binding agents, cytotoxic agents, and linkers are discussed in more detail
below.
Cell Binding Agents
The effectiveness of the compounds of the present invention as therapeutic agents
depends on the careful selection of an appropriate cell binding agent. Cell binding
agents may be of any kind presently known, or that become known, and includes
peptides and non-peptides. The cell binding agent may be any compound that can bind
a cell, either in a specific or non-specific manner. Generally, these can be antibodies
(especially monoclonal antibodies), lymphokines, hormones, growth factors, vitamins,
nutrient-transport molecules (such as transferrin), or any other cell binding molecule or
substance.
More specific examples of cell binding agents that can be used include:
polyclonal antibodies;
monoclonal antibodies;
fragments of antibodies such as Fab, Fab', and F(ab')2, Fv (Parham, 1983, J. Immunol.,
131:2895-2902; Spring et al., 1974, J. Immunol., 113: 470-478; Nisonoff et al., 1960,
Arch. Biochem. Biophys., 89: 230-244).
Preferably, a humanized anti-EphA2 antibody is used as the cell binding agent of the
present invention. More preferably the humanized anti-EphA2 antibody is a humanized
2H11R35R74 antibody.
Cytotoxic Agents
In another embodiment, the humanized antibody or an epitope-binding fragment
thereof can be conjugated to a drug, such as a maytansinoid, a tomaymycin derivative
or a duocarmycin derivative to form a prodrug having specific cytotoxicity towards
antigen-expressing cells by targeting the drug to the EphA2 receptor. Cytotoxic
conjugates comprising such antibodies and a small, highly toxic drug (e.g.,
maytansinoids, tomaymycin derivatives, and CC-1065 and CC-1065 analogs) can be
used as a therapeutic for treatment of tumors, such as, for example, breast and ovarian
tumors.
The cytotoxic agent used in the cytotoxic conjugate of the present invention may be
any compound that results in the death of a cell, or induces cell death, or in some
manner decreases cell viability. Preferred cytotoxic agents include, for example,

maytansinoids and maytansinoid analogs, tomaymycin derivatives, and CC-1065 and
CC-1065 analogs, defined below. These cytotoxic agents are conjugated to the
antibodies, antibody fragments, functional equivalents, improved antibodies and their
analogs as disclosed herein.
The cytotoxic conjugates may be prepared by in vitro methods. In order to link a drug
or prodrug to the antibody, a linking group is used. Suitable linking groups are well
known in the art and include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups and esterase labile groups.
Examplary linking groups are disulfide groups and thioether groups. For example,
conjugates can be constructed using a disulfide exchange reaction or by forming a
thioether bond between the antibody and the drug or prodrug. Examples of linkers
carrying such linking groups include N-succinimidyl pyridyldithiopropionate (SPDP) and
N-succinimidyl pyridyldithiobutyrate (SPDB), whose dithiopyridyl reactive group (see
Bourdon M.A. et al., Biochem. J., 173: 723-737, 1978; US 5208020) reacts with a cytotoxic
chemical reactive group such as -SH to form a new bond -S-S-. The N-succinimidyloxy
group then preferentially reacts with the amino groups present on the antibody in order to
form amide bonds.
In another preferred embodiment, the cytotoxic agent is linked to the cell binding agent
using polyethylene glycol (PEG) linking groups, as set forth in US 6,716,821.
Exemplary PEG linking groups include heterobifunctional PEG linkers that bind to
cytotoxic agents and cell binding agents through a functional sulfhydryl or disulfide
group at one end, and an active ester at the other end (US 6,716,821). It is also
possible to use PEG linkers which do not bind to cytotoxic agents through a functional
sulfhydryl or disulfide group.
Specifically contemplated is a cytotoxic agent bearing a polyethylene glycol (PEG)
linking group having a terminal active ester, of formula (I):

wherein Z is said cytotoxic agent, said cytotoxic agent being selected from the group of
maytansinoids and maytansinoid analogs, tomaymycin derivatives, and CC-1065 and

CC-1065 analogs, and
wherein Y is Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom.
In another preferred embodiment, a cytotoxic agent is provided, said cytotoxic agent
bearing a polyethylene glycol (PEG) linking group having a terminal active ester, and of
formula (II):

wherein Z is said cytotoxic agent, said cytotoxic agent being selected from the group of
maytansinoids and maytansinoid analogs, tomaymycin derivative, and CC-1065 and
CC-1065 analogs, and
wherein Y is Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom.
Preparation of the conjugate
In general, the conjugate can be obtained by a process comprising the steps of:
(i) bringing into contact an optionally-buffered aqueous solution of a cell-binding agent
with a solution of a cytotoxic compound;
(ii) then optionally separating the conjugate which was formed in (i) from the unreacted
reagents and any aggregate which may be present in the solution.
In one aspect, the cell-binding agent is an antibody; more specifically, the cell-binding
agent is the mu2H11R35R74 antibody or a humanized version thereof. In another
aspect, the cytotoxic agent is a compound of either formula (I) or (II), wherein Z is a
maytansinoid; in particular, Z is DM4.
It is understood that conjugates obtainable by this process are comprised within the
scope of the invention.

The aqueous solution of cell-binding agent can be buffered with buffers such as, e.g.
potassium phosphate or N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes
buffer). The buffer depends upon the nature of the cell-binding agent. The cytotoxic
compound is in solution in an organic polar solvent, e.g. dimethyl sulfoxide (DMSO) or
dimethylacetamide (DMA).
The reaction temperature is usually comprised between 20 and 40°C. The reaction
time can vary from 1 to 24 hours. The reaction between the cell-binding agent and the
cytotoxic agent can be monitored by size exclusion chromatography (SEC) with a
refractometric and/or UV detector. If the conjugate yield is too low,, the reaction time
can be extended.
A number of different chromatography methods can be used by the person skilled in
the art in order to perform the separation of step (ii): the conjugate can be purified e.g.
by SEC, adsorption chromatography (such as ion exchange chromatography, I EC),
hydrophobic interaction chromatograhy (HIC), affinity chromatography, mixed-support
chromatography such as hydroxyapatite chromatography, or high performance liquid
chromatography (HPLC). Purification by dialysis or diafiltration can also be used.
An example of a process which can be used is described in the Example l.b.1.
As used herein, the term "aggregates" means the associations which can be formed
between two or more cell-binding agents, said agents being modified or not by
conjugation. The aggregates can be formed under the influence of a great number of
parameters, such as a high concentration of cell-binding agent in the solution, the pH of
the solution, high shearing forces, the number of bonded dimers and their hydrophobic
character, the temperature (see Wang & Gosh, 2008, J. Membrane Sci., 318: 311-316,
and references cited therein); note that the relative influence of some of these
parameters is not clearly established. In the case of proteins and antibodies, the
person skilled in the art will refer to Cromwell et al. (2006, AAPS Jounal, 8(3): E572-
E579). The content in aggregates can be determined with techniques well known to the
skilled person, such as SEC (see Walter et al., 1993, Anal. Biochem., 212(2): 469-480).
After step (i) or (ii), the conjugate-containing solution can be submitted to an additional
step (iii) of ultrafiltration and/or diafiltration.
The conjugate is recovered at the end of these steps in an aqueous solution.
Maytansinoids
Among the cytotoxic agents that may be used in the present invention to form a

cytotoxic conjugate, are maytansinoids and maytansinoid analogs. Examples of
suitable maytansinoids include maytansinol and maytansinol analogs. Maytansinoids
are drugs that inhibit microtubule formation and that are highly toxic to mammalian
cells.
Examples of suitable maytansinol analogues include those having a modified aromatic
ring and those having modifications at other positions. Such suitable maytansinoids are
disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946;
4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;
4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.
Specific examples of suitable analogues of maytansinol having a modified aromatic
ring include:
(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reduction of
ansamytocin P2);
(2) C-20-hydroxy (or C-20-demethyl) +/-C-19-dechioro (U.S. Pat. Nos. 4,361,650 and
4,307,016) (prepared by demethylation using Streptomyces or Actinomyces or
dechlorination using LAH); and
(3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechloro (U.S. Pat. No 4,294,757)
(prepared by acylation using acyl chlorides).
Specific examples of suitable analogues of maytansinol having modifications of other
positions include:
(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction of maytansinol with H2S
or P2S5);
(2) C-14-alkoxymethyl (demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);
(3) C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.
4,450,254) (prepared from Nocardia);
(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the conversion of
maytansinol by Streptomyces);
(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated from Trewia
nudiflora);
(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by the
demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No 4,371,533) (prepared by the titanium trichloride/LAH
reduction of maytansinol).
In a preferred embodiment, the cytotoxic conjugates of the present invention utilize the
thiol-containing maytansinoid (DM1), formally termed N2-deacetyl-N2-(3-mercapto-1-
oxopropyl)-maytansine, as the cytotoxic agent. DM1 is represented by the following
structural formula (III):

In another preferred embodiment, the cytotoxic conjugates of the present invention
utilize the thiol-containing maytansinoid DM4, formally termed N2-deacetyl-N-2 (4-
methyl-4-mercapto-1-oxopentyl)-maytansine, as the cytotoxic agent. DM4 is
represented by the following structural formula (IV):

In further embodiments of the invention, other maytansines, including thiol and
disulfide-containing maytansinoids bearing a mono or di-alkyl substitution on the

carbon atom bearing the sulfur atom, may be used. These include a maytansinoid
having, at C-3, C-14 hydroxymethyl, C-15 hydroxy, or C-20 desmethyi, an acylated
amino acid side chain with an acyl group bearing a hindered sulfhydryl group, wherein
the carbon atom of the acyl group bearing the thiol functionality has one or two
substituents, said substituents being CH3, C2H5, linear or branched alky! or alkenyl
having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical,
and further wherein one of the substituents can be H, and wherein the acyl group has a
linear chain length of at least three carbon atoms between the carbonyl functionality
and the sulfur atom.
Such additional maytansines include compounds represented by formula (V):

wherein:
Y' represents
(CR7R8)1(CR9=CR10)p(CEC)qAr(CR5R6)mDu(CR11=CR12)r(CEC)sBt(CR3R4)nCR1R2SZ,
wherein:
R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from
1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical,
and in addition R2 can be H;
A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical;

R3,. R4, R5, R6,. R7, R8, R9, R10, R11, and R12 are each independently H, CH3, C2H5, linear
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic
or heterocycloalkyl radical;
I, m, n, o, p, q, r, s, and t are each independently 0 or an integer of from 1 to 5,
provided that at least two of I, m, n, o, p, q, r, s and t are not zero at any one time; and
Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple
or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.
Preferred embodiments of formula (V) include compounds of formula (V) wherein:
R1 is methyl, R2 is H and Z is H.
R1 and R2 are methyl and Z is H.
R1 is methyl, R2 is H, and Z is -SCH3.
R1 and R2 are methyl, and Z is -SCH3.
Such additional maytansines also include compounds represented by formula (Vl-L),
(Vl-D), or (VI-D.L):

wherein:

Y represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ,
wherein:
R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from
1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl, or heterocyclic aromatic or heterocycloalkyl radical,
and in addition R2 can be H;
R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having
from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or
heterocycloalkyl radical;
I, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0;
Z is H, SR or -COR wherein R is linear or branched alkyl or alkenyl having from
1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or
simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical; and
May represents a maytansinoid which bears the side chain at C-3, C-14
hydroxymethyl, C-15 hydroxy or C-20 desmethyl.
Preferred embodiments of formulas (Vl-L), (Vl-D) and (VI-D.L) include compounds of
formulas (Vl-L), (Vl-D) and (VI-D,L) wherein:
R1 is methyl, R2 is H, R5, R6, R7, and R8 are each H, I and m are each 1, n is 0, and Z
is H.
R1 and R2 are methyl, R5, R6, R7, R8 are each H, I and m are 1, n is 0, and Z is H.
R1 is methyl, R2 is H, R5, R6, R7, and R8 are each H, I and m are each 1, n is 0, and Z is
-SCH3.
R1 and R2 are methyl, R5, R6, R7, R8 are each H, I and m are 1, n is 0, and Z is -SCH3.
Preferably the cytotoxic agent is represented by formula (Vl-L).
Such additional maytansines also include compounds represented by formula (VII):


wherein:
Y represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ,
wherein:
R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from
1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical,
and in addition R2 can be H;
R3, R4, R5, R6, R7 and R8 are each independently H, CH3, C2H5, linear alkyl or
alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having
from 3 to 10 carbon atoms, phenyl, substituted phenyl, or heterocyclic aromatic or
heterocycloalkyl radical;
I, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0; and
Z is H, SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or
simple or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.
Preferred embodiments of formula (VII) include compounds of formula (VII) wherein:
R1 is methyl, R2 is H, R5, R6, R7, and R8 are each H; I and m are each 1; n is 0; and Z
is H.
R1 and R2 are methyl; R5, Re, R7, R8 are each H, I and m are 1; n is 0; and Z is H.

, R1 is methyl, R2 is H, R5, R6, R7, and R8 are each H, I and m are each 1, n is 0, and Z
is -SCH3.
R1 and R2 are methyl, R5, R6, R7, R8 are each H, I and m are 1, n is 0, and Z is -SCH3.
Such additional maytansines further include compounds represented by formula (VIIII-
L), (Vlll-D), or (VIII-D.L):

wherein:
Y2 represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2SZ2,
wherein:
R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from
1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical,
and in addition R2 can be H;
R3, R4, R5. R6, R7 and R8 are each independently H, CH3, C2H5, linear cyclic
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic
or heterocycloalkyl radical;
I, m and n are each independently an integer of from 1 to 5, and in addition n
can be 0;
Z2 is SR or COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, or simple
or substituted aryl or heterocyclic aromatic or heterocycloalkyl radical; and

May is a maytansinoid. .
Such additional maytansines also include compounds represented by formula (IX):

wherein:
Y2' represents
(CR7R8),(CR9=CR10)p(CEC)qAr(CR5R6)mDu(CR11=CR12)r(CEC)sBt(CR3R4)nCR1R2SZ2!
wherein:
R1 and R2 are each independently CH3, C2H5, linear branched or alkyl or alkenyl
having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having from 3 to 10 carbon
atoms, phenyl, substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical,
and in addition R2 can be H;
A, B, and D each independently is cycloalkyl or cycloalkenyl having 3 to 10
carbon atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl
radical;
R3, R4, R5, R6, R7, R8, R9. R10, R11, and R12 are each independently H, CH3,
C2H5, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl
or alkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic
aromatic or heterocycloalkyl radical;
I, m, n, 0, p, q, r, s, and t are each independently 0 or an integer of from 1 to 5,
provided that at least two of I, m, n, o, p, q, r, s and t are not zero at any one time; and

Z2 is SR or -COR, wherein R is linear alkyl or alkenyl having from 1 to 10 carbon
atoms, branched or cyclic alkyl or alkenyl having from 3-10 carbon atoms, or simple or
substituted aryl or heterocyclic aromatic or heterocycloalkyl radical.
Preferred embodiments of formula (IX) include compounds of formula (IX) wherein: R1
is methyl, R2 is H.
The above-mentioned maytansinoids can be conjugated to anti-EphA antibody
2H11R35R74 or a homologue or fragment thereof, wherein the antibody is linked to the
maytansinoid using the thiol or disulfide functionality that is present on the acyl group of
an acylated amino acid side chain found at C-3, C-14 hydroxymethyl, C-15 hydroxy or
C-20 desmethyl of the maytansinoid, and wherein the acyl group of the acylated amino
acid side chain has its thiol or disulfide functionality located at a carbon atom that has
one or two substituents, said substituents being CH3, C2H5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to
10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl radical, and in addition one of the substituents can be H, and wherein
the acyl group has a linear chain length of at least three carbon atoms between the
carbonyl functionality and the sulfur atom.
A preferred conjugate of the present invention is the one that comprises the anti-EphA
antibody 2H11R35R74 or a homologue or fragment thereof, conjugated to, or
obtainable by conjugation with, a maytansinoid of formula (X):

wherein:
Y1' represents

(CR7R8)i(CR9=CR1o)p(CEC)qAr(CR5R6)mDu(CR11=CR12)r(CEC)sBt(CR3R4)nCRiR2S-I
wherein:
A, B, and D, each independently is cycloalkyl or cycloalkenyl having 3-10 carbon
atoms, simple or substituted aryl, or heterocyclic aromatic or heterocycloalkyl radical;
R3, R4, R5, R6, R7, R8, R9, R10, R11, and R12 are each independently H, CH3, C2H5, linear
alkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl
having from 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic
or heterocycloalkyl radical; and
I, m, n, o, p, q, r, s, and t are each independently 0 or an integer of from 1 to 5,
provided that at least two of I, m, n, o, p, q, r, s and t are non-not zero at any one time.
Preferably, R1, is methyl, R2 is H, or R1 and R2 are methyl.
An even more preferred conjugate of the present invention is the one that comprises
the anti-EphA antibody 2H11R35R74 or a homologue or fragment thereof, conjugated
to a maytansinoid of formula (Xl-L), (Xl-D), or (XI-D,L):

wherein:
Y1 represents (CR7R8)l(CR5R6)m(CR3R4)nCR1R2S-,
wherein:
R1 and R2 are each independently CH3, C2H5, linear alkyl or alkenyl having from 1 to 10
carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to 10 carbon atoms,

. phenyl, substituted phenyl, heterocyclic aromatic or heterocycloalkyl radical, and in
addition R2can be H;
R3, R4, R5, R5, R7, and R8 are each independently H, CH3, C2H5, linear alkyl or alkenyl
having from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to
10 carbon atoms, phenyl, substituted phenyl or heterocyclic aromatic or
heterocycloalkyl radical;
I, m and n are each independently an integer of from 1 to 5, and in addition n can be 0;
and
May represents a maytansinol which bears the side chain at C-3, C-14 hydroxymethyl,
C-15 hydroxy or C-20 desmethyl.
Preferred embodiments of formulas (Xl-L), (Xl-D) and (XI-D.L) include compounds of
formulas (Xl-L), (Xl-D) and (XI-D.L) wherein:
R1 is methyl, R2 is H, or R1 and R2 are methyl,
R1i is methyl, R2 is H, R5, R6, R7 and R8 are each H; I and m are each 1; n is 0,
R1 and R2 are methyl; R5, R6, R7 and R8 are each H; I and m are 1; n is 0.
Preferably the cytotoxic agent is represented by formula (Xl-L).
A further preferred conjugate of the present invention is the one that comprises the
anti-EphA antibody 2H11R35R74 or a homologue or fragment thereof, conjugated to a
maytansinoid of formula (XII):


wherein the substituents are as defined for formula (XI) above.
Especially preferred are any of the above-described compounds, wherein R-, is H, R2 is
methyl, R5, R6, R7 and R8 are each H, I and m are each 1, and n is 0.
Further especially preferred are any of the above-described compounds, wherein R-,
and R2 are methyl, R5, R6, R7, R8 are each H, I and m are 1, and n is 0
Further, the L-aminoacyl stereoisomer is preferred.
Each of the maytansinoids taught in pending U.S. patent application number
10/849,136, filed May 20, 2004, may also be used in the cytotoxic conjugate of the
present invention. The entire disclosure of U.S. patent application number 10/849,136
is incorporated herein by reference.
Disulfide-containing linking groups
In order to link the maytansinoid to a cell binding agent, such as the 2H11R35R74
antibody, the maytansinoid comprises a linking moiety. The linking moiety contains a
chemical bond that allows for the release of fully active maytansinoids at a particular
site. Suitable chemical bonds are well known in the art and include disulfide bonds,
acid labile bonds, photolabile bonds, peptidase labile bonds and esterase labile bonds.
The linking moiety also comprises a reactive chemical group. In a preferred
embodiment, the reactive chemical group is used to covalently bind to the
maytansinoid via a disulfide bond linking moiety.
Particularly preferred reactive chemical groups are N-succinimidyl esters and N-
sulfosuccinimidyl esters.
Particularly preferred maytansinoids comprising a linking moiety that contains a
reactive chemical group are C-3 esters of maytansinol and its analogs where the
linking moiety contains a disulfide bond and the chemical reactive group comprises a
N-succinimidyl or N-sulfosuccinimidyl ester.
Many positions on maytansinoids can serve as the position to chemically link the
linking moiety. For example, the C-3 position having a hydroxyl group, the C-14
position modified with hydroxymethyl, the C-15 position modified with hydroxy and the
C-20 position having a hydroxy group are all expected to be useful. However the C-3
position is preferred and the C-3 position of maytansinol is especially preferred.
While the synthesis of esters of maytansinol having a linking moiety is described in
terms of disulfide bond-containing linking moieties, one of skill in the art will understand

that linking moieties with other chemical bonds (as described above) can also be used ,
with the present invention, as can other maytansinoids. Specific examples of other
chemical bonds include acid labile bonds, photolabile bonds, peptidase labile bonds
and esterase labile bonds. The disclosure of U.S. Patent No. 5,208,020, incorporated
herein, teaches the production of maytansinoids bearing such bonds.
The synthesis of maytansinoids and maytansinoid derivatives having a disulfide moiety
that bears a reactive group is described in U.S. Patent Nos. 6, 441,163 and 6,333,410,
and U.S. Application No. 10/161,651, each of which is herein incorporated by
reference.
PEG-containing linking groups
Maytansinoids may also be linked to cell binding agents using PEG linking groups, as
set forth in U.S. Patent No. 6,716,821. These PEG linking groups are soluble both in
water and in non-aqueous solvents, and can be used to join one or more cytotoxic
agents to a cell binding agent. Exemplary PEG linking groups include hetero-
bifunctional PEG linkers that bind to cytotoxic agents and cell binding agents at
opposite ends of the linkers through a functional sulfhydryl or disulfide group at one
end, and an active ester at the other end.
As a general example of the synthesis of a cytotoxic conjugate using a PEG linking
group, reference is again made to U.S. Patent No. 6,716,821 for specific details.
Synthesis begins with the reaction of one or more cytotoxic agents bearing a reactive
PEG moiety with a cell-binding agent, resulting in displacement of the terminal active
ester of each reactive PEG moiety by an amino acid residue of the cell binding agent,
such as the 2H11R35R74 antibody, to yield a cytotoxic conjugate comprising one or
more cytotoxic agents covalently bonded to a cell binding agent through a PEG linking
group. It is also possible to use PEG linking groups which do not bind to cytotoxic
agents through a functional sulfhydryl or disulfide group.
Thus comprised within the scope of the invention is the maytansinoid of formula (XIII),
herein referred to as PEG4-NHAc-DM4:


Also comprised within the meaning of the invention is the maytansinoid of formula
(XIV), herein referred to as PEG4-Mal-DM4:

Also comprised within the meaning of the invention is the maytansinoid of formula
(XXIV), herein referred to as SPDB-DM4:


Also comprised within the meaning of the invention is the maytansinoid of formula
(XXV), herein referred to as:_PEG4-NMeAc-DM4

Also comprised within the meaning of the invention is the maytansinoid of formula
(XXVI), herein referred to as: PEG8-NHAc-DM4


Also comprised within the meaning of the invention is the maytansinoid of formula
(XXVII), herein referred to as:.PEG4-AIIyl-DM4

The reactive group-containing maytansinoids, such as DM4, are reacted with an
antibody, such as the 2H11R35R74 antibody, to produce cytotoxic conjugates, wherein
the cytotoxic is covalently attached to the antibody. These conjugates may be purified
by HPLC or by gel-filtration.
A preferred embodiment of the invention is a conjugate of the 2H11R35R74 antibody,
or of a humanized version thereof, said conjugate comprising a cytotoxic agent
attached covalently to said 2H11R35R74 antibody, said cytotoxic agent being chosen
between the maytansinoid of formula (XIII) and the maytansinoid of formula (XIV). In a
more preferred embodiment, the conjugation of the maytansinoid of formula (XIII) to the
2H11R35R74 antibody of the invention will result in a 2H11 R35R74-PEG4-NHAc-DM4
conjugate. In another further preferred embodiment, the maytansinoid of formula (XIV)
is conjugated to the 2H11R35R74 antibody of the invention to yield a 2H11R35R74-
PEG4-Mal-DM4 conjugate.
Thus, a preferred embodiment is drawn to an antibody-drug conjugate having a
structure consisting of the structure of the formula (XV):


wherein Ab is an antibody of the invention and n is an integer comprised between 1
and 15. Preferentially, n is comprised between 1 and 10. Even more preferentially, n is
comprised between 5 and 7. In another further preferred embodiment, the antibody of
the invention is the 2H11R35R74 antibody or a humanized version thereof, and the
conjugate is a 2H11 R35R74-PEG4-NHAc-DM4 conjugate.
Thus, another preferred embodiment is drawn to an antibody-drug conjugate having a
structure consisting of the structure of the formula (XVI):

wherein Ab is an antibody of the invention and n is an integer comprised between 1
and 15. Preferentially, n is comprised between 1 and 10. Even more preferentially, n is
comprised between 5 and 7. In another further preferred embodiment, the antibody of

the invention is the 2H11R35R74 antibody or a humanized version thereof, and the
conjugate is a 2H11 R35R74-PEG4-MaI-DM4 conjugate.
Several excellent schemes for producing such antibody-maytansinoid conjugates are
provided in U.S. Patent No. 6,333,410, and U.S. Application Nos. 09/867,598,
10/161,651 and 10/024,290, each of which is incorporated herein in its entirety.
As explained above, in general, the conjugate can be obtained by a process
comprising the steps of:
(i) bringing into contact an optionally-buffered aqueous solution of an antibody with a
solution of a maytansinoid;
(ii) then optionnally separating the conjugate which was formed in (i) from the
unreacted reagents and any aggregate which may be present in the solution.
More specifically, a solution of an antibody in aqueous buffer may be incubated with a
molar excess of maytansinoid having a disulfide moiety that bears a reactive group.
The reaction mixture can be quenched by addition of excess amine (such as
ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate may then be purified
by gel-filtration.
In one aspect of the process, the antibody is the mu2H11R35R74 antibody or a
humanized version thereof. In another aspect of that process, the cytotoxic agent is a
cytotoxic agent chosen between:
the compound of formula (XVII):

wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-

. sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyIoxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenylpxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom; and
the compound of formula (XVIII):

wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom.
An example of a process which can be used to with an antibody and a compound of
either formula (XVII) or (XVIII) is given in Example I.
The number of maytansinoid molecules bound per antibody molecule ("drug-to-
antibody ratio" or "DAR") can be determined spectrophotometrically by measuring the
ratio of the absorbance at 252 nm and 280 nm of a solution of the substantially purified
conjugate (that is after step (ii)). In particular, said DAR can be determined
spectrophotometrically using the measured extinction coefficients at respectively 280
and 252 nm for the antibody: s^so = 224,000 M"1 cm"1 and 8^52 = 82,880 M"1cnr1;
assuming an average 160,000 molecular weight for the antibody, and for the
maytansinoid, sD280 = 5,180 M"1cm"1 and ED252 = 26,159 M~1cm"1). The method of
calculation is derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies
and Protocols, vol 525, 445, Springer Science and is described in more details below:
The absorbances for the conjugate at 252 nm (A262) and at 280 nm (A28o) are measured
either on the monomeric peak of the SEC analysis (allowing to calculate the
"DAR(SEC)" parameter) or using a classic spectrophotometer apparatus (allowing to

calculate the "DAR(UV)" parameter). The absorbances can be expressed as follows:

wherein:
CD and CA are respectively the concentrations in the solution of the maytansinoid and of
the antibody
εD252and εD280are respectively the molar extinction coefficients of the maytansinoid at
252 nm and 280 nm
εA252 and εA280 are respectively the molar extinction coefficients of the antibody at 252
nm and 280 nm.
Resolution of these two equations with two unknowns leads to the following equations:

The average DAR is then calculated from the ratio of the drug concentration to that of
the antibody:
DAR = cD / cA
The average DAR measured with a UV spectrophometer (DAR(UV)) is more
particularly above 4, more particularly between 4 and 10, even more particularly
between 4 and 7, even more particularly between 5.5 and 8 and even more particularly
between 5.9 and 7.5
In yet a further preferred embodiment, the invention comprises a conjugate of the
2H11R35R74 antibody, or of a humanized version thereof, and a compound of either
formula (XVII) or (XVIII), wherein the DAR is comprised between 4 and 7 maytansinoid
molecules/antibody molecule, said DAR being determined by measuring
spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm of a solution
of the substantially purified conjugate.
The conjugates obtainable by the above-process are comprised within the scope of this

invention. In a particular aspect, such conjugates have a structure chosen between
formula (XV) and formula (XVI), wherein Ab is an antibody of the invention, and
wherein n is comprised between 4 and 10. In a preferred embodiment, n is comprised
between 4 and 7. In another preferred embodiment, said conjugates have the structure
of formula (XV).
Conjugates of antibodies with maytansinoid drugs can be evaluated for their ability to
suppress proliferation of various unwanted cell lines in vitro. For example, cell lines
such as the human epidermoid carcinoma line A-431, the human small cell lung cancer
cell line SW2, the human breast tumor line SKBR3 and the Burkitt's lymphoma cell line
Namalwa can easily be used for the assessment of cytotoxicity of these compounds.
Cells to be evaluated can be exposed to the compounds for 24 hours and the surviving
fractions of cells measured in direct assays by known methods. IC50 values can then be
calculated from the results of the assays.
Tomaymycin derivatives
The cytotoxic according to the present invention may also be a tomaymycin derivative.
Tomaymycin derivatives are pyrrolo[1,4]benzodiazepines (PBDs), a known class of
compounds exerting their biological properties by covalently binding to the N2 of
guanine in the minor groove of DNA. PBDs include a number of minor groove binders
such as anthramycin, neothramycin and DC-81.
Novel tomaymycin derivatives that retain high cytotoxicity and that can be effectively
linked to cell binding agents are described in the International Application No.
PCT/IB2007/000142, whose content is herein incorporated by reference. The cell
binding agent-tomaymycin derivative complexes permit the full measure of the
cytotoxic action of the tomaymycin derivatives to be applied in a targeted fashion
against unwanted cells only, therefore avoiding side effects due to damage to non-
targeted healthy cells.
The cytotoxic agent according to the present invention comprises one or more
tomaymycin derivatives, linked to a cell binding agent, such as the 2H11R35R74
antibody, via a linking group. The linking group is part of a chemical moiety that is
covalently bound to a tomaymycin derivative through conventional methods. In a
preferred embodiment, the chemical moiety can be covalently bound to the
tomaymycin derivative via a disulfide bond.
The tomaymycin derivatives useful in the present invention have the formula (XX)
shown below:


wherein
— represents an optional single bond;
represents either a single bond or a double bond ;
provided that when represents a single bond, U and IT, the same or different,
independently represent H, and W and W, the same or different, are independently
selected from the group consisting of OH, an ether such as -OR, an ester (e.g. an
acetate), such as -OCOR, a carbonate such as -OCOOR, a carbamate such as -
OCONRR', a cyclic carbamate, such that N10 and C11 are a part of the cycle, a urea
such as -NRCONRR', a thiocarbamate such as -OCSNHR, a cyclic thiocarbamate
such that N10 and C11 are a part of the cycle, -SH, a sulfide such as -SR, a sulfoxide
such as -SOR, a sulfone such as -SOOR, a sulfonate such as -S03 -, a sulfonamide
such as -NRSOOR, an amine such as —NRR', optionally cyclic amine such that N10
and C11 are a part of the cycle, a hydroxylamine derivative such as -NROR', an amide
such as -NRCOR, an azido such as -N3, a cyano, a halo, a trialkyl or
triarylphosphonium, an aminoacid-derived group; Preferably W and W are the same or
different and are OH, OMe, OEt, NHCONH2, SMe;
and when represents a double bond, U and U' are absent and W and W
represent H;
R1, R2, R1', R2' are the same or different and independently chosen from Halide or
Alkyl optionally substituted by one or more Hal, CN, NRR', CF3, OR, Aryl, Het, S(O)qR,
or R1 and R2 and R1' and R2' form together a double bond containing group =B and
=B' respectively.
Preferably, R1 and R2 and R1' and R2' form together a double bond containing group

=B and =B' respectively.
■ B and B' are the same or different and independently chosen from Alkenyl being
optionally substituted by one or more Hal, CN, NRR', CF3, OR, Aryl, Het, S(O)qR or B
and B' represent an oxygen atom.
Preferably, B=B'.
More preferably, B=B'= =CH2 or =CH-CH3,
■ X, X' are the same or different and independently chosen from one or more
-O-, -NR-, -(C=O)-, -S(O)q-.
Preferably, X=X'.
More preferably, X=X'=O.
■ A, A' are the same or different and independently chosen from Alkyl or Alkenyl
optionally containing an oxygen, a nitrogen or a sulfur atom, each being optionally
substituted by one or more Hal, CN, NRR', CF3, OR, S(O)qR, Aryl, Het, Alkyl, Alkenyl.
Preferably, A=A'.
More preferably, A=A'=linearunsubstituted alkyl.
■ Y, Y' are the same or different and independently chosen from H, OR;
Preferably, Y=YMore preferably, Y=Y'=OAIkyl, more preferably OMethyl.
■ T is -NR-, -O-, -S(O)q-, or a 4 to 10-membered aryl, cycloalkyl, heterocyclic or
heteroaryl, each being optionally substituted by one or more Hal, CN, NRR', CF3, R,
OR, S(O)qR, and/or linker(s), or a branched Alkyl, optionally substituted by one or more
Hal, CN, NRR', CF3, OR, S(O)qR and/or linker(s), or a linear Alkyl substituted by one or
more Hal, CN, NRR', CF3, OR, S(O)qR and/or linker(s).
Preferably, T is a 4 to 10-membered aryl or heteroaryl, more preferably phenyl or
pyridyl, optionally substituted by one or more linker(s).
Said linker comprises a linking group. Suitable linking groups are well known in the art
and include thiol, sulfide, disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups and esterase labile groups. Preferred are
disulfide groups and thioether groups.
When the linking group is a thiol-, sulfide (or so-called thioether -S-) or disulfide (-S-S-)

-containing group, the side chain carrying the thiol, the sulfide or disulfide group can be
linear or branched, aromatic or heterocyclic. One of ordinary skill in the art can readily
identify suitable side chains.
Preferably, said linker is of formula:
-G-D-(Z)p-S-Z'
where
G is a single or double bond, -O-, -S- or-NR-;
D is a single bond or-E-, -E-NR-, -E-NR-F-, -E-O-, -E-O-F-, -E-NR-CO-, -E-NR-CO-F-,
-E-CO-, -CO-E-, -E-CO-F, -E-S-, -E-S-F-, -E-NR-C-S-, -E-NR-CS-F- ;
where E and F are the same or different and are independently chosen from linear or
branched -(OCH2CH2)iAIkyl(OCH2CH2)j-, -Alkyl(OCH2CH2)i-Alkyl-, -(OCH2CH2)i-, -
(OCH2CH2)iCycloaIkyl(OCH2CH2)j-,-(OCH2CH2)iHeterocyclic(OCH2CH2)j-,-
(OCH2CH2)iAryl(OCH2CH2)j-, -(OCH2CH2)iHeteroaryl(OCH2CH2)j-,-Alkyl-
(OCH2CH2)iAlkyl(OCH2CH2)j-, -Alkyl-(OCH2CH2)i-, -Alkyl-
(OCH2CH2)iCycloalkyl(OCH2CH2)j-,-Alkyi(OCH2CH2)iHeterocyclic(OCH2CH2)j-,-
Alkyl-(OCH2CH2)iAryl(OCH2CH2)j-,-Alkyl(OCH2CH2)iHeteroaryl(OCH2CH2)j-, -
Cycloalkyl-Alkyl-, -Alkyl-Cycloalkyl-, -Heterocyclic-Alkyl-, -Alkyl-Heterocyclic-, -Alkyl-
Aryl-, -Aryl-Alkyl-, -Alkyl-Heteroaryl- , -Heteroaryl-Alkyl-;
where i and j, identical or different are integers and independently chosen from 0, 1 to
2000;
Z is linear or branched -Alkyl-;
pisO or 1;
Z represents H, a thiol protecting group such as COR, R20 or SR20, wherein R20
represents H, methyl, Alkyl, optionally substituted Cycloalkyl, aryl, heteroaryl or
heterocyclic, provided that when Z' is H, said compound is in equilibrium with the
corresponding compound formed by intramolecular cyclisation resulting from addition
of the thiol group -SH on the imine bond -NH= of one of the PBD moieties.
■ n, n', equal or different are 0 or 1.
■ q is 0, 1 or 2.
■ R, R' are equal or different and independently chosen from H, Alkyl, Aryl, each being
optionally substituted by Hal, CN, NRR', CF3, R, OR, S(O)qR, Aryl, Het;

or their pharmaceutically acceptable salts, hydrates, or hydrated salts, or the
polymorphic crystalline structures of these compounds or their optical isomers,
racemates, diastereomers or enantiomers.
The compounds of the general formula (XX) having geometrical and stereoisomers are
also a part of the invention.
The N-10, C-11 double bond of tomaymycin derivatives of formula (XX) is known to be
readily convertible in a reversible manner to corresponding imine adducts in the
presence of water, an alcohol, a thiol, a primary or secondary amine, urea and other
nucleophiles. This process is reversible and can easily regenerate the corresponding
tomaymycin derivatives in the presence of a dehydrating agent, in a non-protic organic
solvant, in vacuum or at high temperatures (Z. Tozuka, 1983, J. Antibiotics, 36: 276).
Thus, reversible derivatives of tomaymycin derivatives of general formula (XXI) can
also be used in the present invention:

where A, X, Y, n, T, A', X', Y', n\ R1, R2, R1\ R2' are defined as in formula (XX) and
W, W are the same or different and are selected from the group consisting of OH, an
ether such as -OR, an ester (e.g. an acetate), such as -OCOR, -COOR, a carbonate
such as -OCOOR, a carbamate such as -OCONRR', a cyclic carbamate, such that
N10 and C11 are a part of the cycle, a urea such as -NRCONRR', a thiocarbamate
such as -OCSNHR, a cyclic thiocarbamate such that N10 and C11 are a part of the
cycle, -SH, a sulfide such as -SR, a sulphoxide such as -SOR, a sulfone such as -
SOOR, a sulphonate such as -S03-, a sulfonamide such as -NRSOOR, an amine
such as -NRR', optionally cyclic amine such that N10 and C11 are a part of the cycle,
a hydroxylamine derivative such as -NROR', an amide such as -NRCOR, -
NRCONRR', an azido such as -N3, a cyano, a halo, a trialkyl ortriarylphosphonium, an
aminoacid-derived group. Preferably, W and W are the same or different and are OH,
Ome, Oet, NHCONH2, SMe.

Compounds of formula (XXI) may thus be considered as solvates, including water
when the solvent is water; these solvates can be particularly useful.
Preferred compounds are those of formula (XXII) or (XXIII):

where X, X', A, A', Y, Y', T, n, n' are defined as above.
The compounds of formula (XX) may be prepared in a number of ways well known to
those skilled in the art. The compounds can be synthesized, for example, by
application or adaptation of the methods described below, or variations thereon as
appreciated by the skilled artisan. The appropriate modifications and substitutions will
be readily apparent and well known or readily obtainable from the scientific literature to
those skilled in the art. In particular, such methods can be found in R.C. Larock,
Comprehensive Organic Transformations, Wiley-VCH Publishers, 1999.
Methods for synthesizing the tpmaymycin derivatives which may be used in the
invention are described in the International Application No. PCT/IB2007/000142.
Compounds of the present invention may be prepared by a variety of synthetic routes.
The reagents and starting materials are commercially available, or readily synthesized
by well-known techniques by one of ordinary skill in the arts (see, for example, WO
00/12508, WO 00/12507, W0 2005/040170, WO 2005/085260, FR1516743, M. Mori et
a/., 1986, Tetrahedron, 42:3793-3806).

The conjugate molecules of the invention may be formed using any techniques. The
tomaymycin derivatives of the invention may be linked to an antibody or other cell
binding agent via an acid labile linker, or by a photolabile linker. The derivatives can be
condensed with a peptide having a suitable sequence and subsequently linked to a cell
binding agent to produce a peptidase labile linker. The conjugates can be prepared to
contain a primary hydroxyl group, which can be acylated and then linked to a cell
binding agent to produce a conjugate that can be cleaved by intracellular esterases to
liberate free derivative. Preferably, the derivatives are synthesized to contain a free or
protected thiol group, and then one or more disulfide or thiol-containing derivatives are
each covalently linked to the cell binding agent via a disulfide bond or a thioether link.
Numerous methods of conjugation are taught in USP 5,416,064 and USP 5,475,092.
The tomaymycin derivatives can be modified to yield a free amino group and then
linked to an antibody or other cell binding agent via an acid labile linker or a photolabile
linker. The tomaymycin derivatives with a free amino or carboxyl group can be
condensed with a peptide and subsequently linked to a cell binding agent to produce a
peptidase labile linker. The tomaymycin derivatives with a free hydroxyl group on the
linker can be acylated and then linked to a cell binding agent to produce a conjugate
that can be cleaved by intracellular esterases to liberate free drug. Most preferably, the
tomaymycin derivatives are treated to create a free or protected thiol group, and then
the disulfide- or thiol containing tomaymycin dimers are linked to the cell binding agent
via disulfide bonds.
Preferably, monoclonal antibody- or cell binding agent-tomaymycin derivative
conjugates are those that are joined via a disulfide bond, as discussed above, that are
capable of delivering tomaymycin derivatives. Such cell binding conjugates are
prepared by known methods such as by modifying monoclonal antibodies with
succinimidyl pyridyl-dithiopropionate (SPDP) (Carlsson era/., 1978, Biochem. J., 173:
723-737). The resulting thiopyridyl group is then displaced by treatment with thiol-
containing tomaymycin derivatives to produce disulfide linked conjugates. Alternatively,
in the case of the aryldithio- tomaymycin derivatives, the formation of the cell binding
conjugate is effected by direct displacement of the aryl-thiol of the tomaymycin
derivative by sulfhydryl groups previously introduced into antibody molecules.
Conjugates containing 1 to 10 tomaymycin derivative drugs linked via a disulfide bridge
are readily prepared by either method.
More specifically, a solution of the dithio-nitropyridyl modified antibody at a
concentration of 2.5 mg/ml in 0.05 M potassium phosphate buffer, at pH 7.5 containing

2 mM EDTA is treated with the thiol-containing tomaymycin derivative (1.3 molar
eq./dithiopyridyl group). The release of nitropyridinethione from the modified antibody is
monitored spectrophotometrically at 325 nm and is complete in about 16 hours. The
antibody-tomaymycin derivative conjugate is purified and freed of unreacted drug and
other low molecular weight material by gel filtration through a column of Sephadex G-
25 or Sephacryl S300. The number of tomaymycin derivative moieties bound per
antibody molecule can be determined by measuring the ratio of the absorbance at 230
nm and 275 nm. An average of 1-10 tomaymycin derivative molecules/antibody
molecule can be linked via disulfide bonds by this method.
The effect of conjugation on binding affinity towards the antigen-expressing cells can
be determined using the methods previously described by Liu et al., 1996, Proc. Natl.
Acad. Sci. U.S.A., 93: 8618-8623. Cytotoxicity of the tomaymycin derivatives and their
antibody conjugates to cell lines can be measured by back-extrapolation of cell
proliferation curves as described in Goldmacher ef a/., 1985, J. Immunol., 135: 3648-
3651. Cytotoxicity of these compounds to adherent cell lines can be determined by
clonogenic assays as described in Goldmacher ef a/., 1986, J. Cell Biol., 102:1312-
1319.
CC-1065 Analogues
The cytotoxic agent used in the cytotoxic conjugates according to the present invention
may also be CC-1065 or a derivative thereof.
CC-1065 is a potent anti-tumor antibiotic isolated from the culture broth of
Streptomyces zelensis. CC-1065 is about 1000-fold more potent in vitro than are
commonly used anti-cancer drugs, such as doxorubicin, methotrexate and vincristine
(B.K. Bhuyan ef al., 1982, Cancer Res., 42, 3532-3537). CC-1065 and its analogs are
disclosed in U.S. Patent Nos. 6,372,738, 6,340,701, 5,846,545 and 5,585,499.
The cytotoxic potency of CC-1065 has been correlated with its alkylating activity and its
DNA-binding or DNA-intercalating activity. These two activities reside in separate parts
of the molecule. Thus, the alkylating activity is contained in the cyclopropapyrroloindole
(CPI) subunit and the DNA-binding activity resides in the two pyrroloindole subunits.
Although CC-1065 has certain attractive features as a cytotoxic agent, it has limitations
in therapeutic use. Administration of CC-1065 to mice caused a delayed hepatotoxicity
leading to mortality on day 50 after a single intravenous dose of 12.5 ug/kg (V. L
Reynolds ef al., 1986, J. Antibiotics, XXIX: 319-334). This has spurred efforts to
develop analogs that do not cause delayed toxicity, and the synthesis of simpler

analogs modeled on CC-1065 has been described (M.A. Warpehoski et a!., 1988, J.
Med. Chem., 31: 590-603).
In another series of analogs, the CPI moiety was replaced by a
cyclopropa[c]benz[e]indole (CBI) moiety (D.L. Bogeref a/., 1990, J. Org. Chem., 55:
5823-5833; D.L. Bogeref a/., 1991, BioOrg. Med. Chem. Lett., 1: 115-120). These
compounds maintain the high in vitro potency of the parental drug, without causing
delayed toxicity in mice. Like CC-1065, these compounds are alkylating agents that
bind to the minor groove of DNA in a covalent manner to cause cell death. However,
clinical evaluation of the most promising analogs, Adozelesin and Carzelesin, has led
to disappointing results (B.F. Foster et a/., 1996, Investigational New Drugs, 13: 321-
326; I. Wolff era/., 1996, Clin. Cancer Res., 2:1717-1723). These drugs display poor
therapeutic effects because of their high systemic toxicity.
The therapeutic efficacy of CC-1065 analogs can be greatly improved by changing the
in vivo distribution through targeted delivery to the tumor site, resulting in lower toxicity
to non-targeted tissues, and thus, lower systemic toxicity. In order to achieve this goal,
conjugates of analogs and derivatives of CC-1065 with cell-binding agents that
specifically target tumor cells have been described (US Patents; 5,475,092; 5,585,499;
5,846,545). These conjugates typically display high target-specific cytotoxicity in vitro,
and exceptional anti-tumor activity in human tumor xenograft models in mice (R.V. J.
Chari et a/., 1995, Cancer Res., 55: 4079-4084).
Recently, prodrugs of CC-1065 analogs with enhanced solubility in aqueous medium
have been described (European Patent Application No. 06290379.4). In these
prodrugs, the phenolic group of the alkylating portion of the molecule is protected with
a functionality that renders the drug stable upon storage in acidic aqueous solution,
and confers increased water solubility to the drug compared to an unprotected analog.
The protecting group is readily cleaved in vivo at physiological pH to give the
corresponding active drug. In the prodrugs described in EP 06290379.4, the phenolic
substituent is protected as a sulfonic acid containing phenyl carbamate which
possesses a charge at physiological pH, and thus has enhanced water solubility. In
order to further enhance water solubility, an optional polyethylene glycol spacer can be
introduced into the linker between the indolyl subunit and the cleavable linkage such as
a disulfide group. The introduction of this spacer does not alter the potency of the drug.
Methods for synthesizing CC-1065 analogs that may be used in the cytotoxic
conjugates of the present invention, along with methods for conjugating the analogs to

cell binding agents such as antibodies, are described in detail in ER 06290379.4
(whose content is incorporated herein by reference) and U.S. Patent Nos. 5,475,092,
5,846,545, 5,585,499, 6,534,660 and 6,586,618 and in U.S. Application Nos.
10/116,053 and 10/265,452.
Other Drugs
Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine,
melphalan, leptomycin derivatives, mitomycin C, chlorambucil, calicheamicin, tubulysin
and tubulysin analogs, duocarmycin and duocarmycin analogs, dolastatin and
dolastatin analogs such as auristatins are also suitable for the preparation of
conjugates of the present invention. The drug molecules can also be linked to the
antibody molecules through an intermediary carrier molecule such as serum albumin.
Doxorubicin and Daunorubicin compounds, as described, for example, in U.S. Patent
No. 6,630,579, may also be useful cytotoxic agents.
Therapeutic Composition
The invention also relates to a therapeutic composition for the treatment of a
hyperproliferative disorder in a mammal which comprises a therapeutically effective
amount of a compound of the invention and a pharmaceutically acceptable carrier. In
one embodiment said pharmaceutical composition is for the treatment of cancer,
including (but not limited to) the following: carcinoma, including that of the bladder,
breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;
including squamous cell carcinoma ; hematopoietic tumors of lymphoid lineage,
including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell
lymphoma, T-cell lymphoma, Burkitt's lymphoma ; hematopoietic tumors of myeloid
lineage, including acute and chronic myelogenous leukemias and promyelocytic
leukemia; tumors of mesenchymal origin, including fibrosarcoma and
rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma,
neuroblastoma and glioma; tumors of the central and peripheral nervous system,
including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of
mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma;
and other tumors, including melanoma, xeroderma pigmentosum, keratoactanthoma,
seminoma, thyroid follicular cancer and teratocarcinoma, and other cancers yet to be
determined in which EphA2 is expressed predominantly. In a preferred embodiment,
the pharmaceutical compositions of the invention are used for treatment of cancer of
the lung, breast, colon, prostate, kidney, pancreas, ovary, cervix and lymphatic organs,

osteosarcoma, synovial carcinoma, a sarcoma, head and neck, a glioma, gastric, liver
or other carcinomas in which EphA2 is expressed. In particular, the cancer is a
metastatic cancer. In another embodiment, said pharmaceutical composition relates to
other disorders such as, for example, autoimmune diseases, such as systemic lupus,
rheumatoid arthritis, and multiple sclerosis; graft rejections, such as renal transplant
rejection, liver transplant rejection, lung transplant rejection, cardiac transplant
rejection, and bone marrow transplant rejection; graft versus host disease; viral
infections, such as mV infection, HIV infection, AIDS, etc.; and parasite infections, such
as giardiasis, amoebiasis, schistosomiasis, and others as determined by one of
ordinary skill in the art.
The instant invention provides pharmaceutical compositions comprising:
an effective amount of an antibody, antibody fragment or antibody conjugate of the
present invention, and
a pharmaceutically acceptable carrier, which may be inert or physiologically active.
As used herein, "pharmaceutically-acceptable carriers" includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, and the like that are
physiologically compatible. Examples of suitable carriers, diluents and/or excipients
include one or more of water, saline, phosphate buffered saline, dextrose, glycerol,
ethanol, and the like, as well as combination thereof. In many cases, it will be
preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride
in the composition. In particular, relevant examples of suitable carrier include: (1)
Dulbecco's phosphate buffered saline, pH'~ 7.4, containing or not containing about
1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride
(NaCI)), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as
tryptamine and a stabilizing agent such as Tween 20.
The compositions herein may also contain a further therapeutic agent, as necessary for
the particular disorder being treated. Preferably, the antibody, antibody fragment or
antibody conjugate of the present invention, and the supplementary active compound
will have complementary activities, that do not adversely affect each other. In a
preferred embodiment, the further therapeutic agent is an antagonist of fibroblast-
growth factor (FGF), hepatocyte growth factor (HGF), tissue factor (TF), protein C,
protein S, platelet-derived growth factor (PDGF), or HER2 receptor.
The compositions of the invention may be in a variety of forms. These include for
example liquid, semi-solid, and solid dosage forms, but the preferred form depends on

the intended mode of administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions. The preferred mode of
administration is parenteral (e.g. intravenous, intramuscular, intraperinoneal,
subcutaneous). In a preferred embodiment, the compositions of the invention are
administered intravenously as a bolus or by continuous infusion over a period of time.
In another preferred embodiment, they are injected by intramuscular, subcutaneous,
intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional
routes, to exert local as well as systemic therapeutic effects. They can be also
administered by nebulisation.
Sterile compositions for parenteral administration can be prepared by incorporating the
antibody, antibody fragment or antibody conjugate of the present invention in the
required amount in the appropriate solvent, followed by sterilization by microfiltration.
As solvent or vehicle, there may be used water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, and the like, as well as a combination thereof. In many
cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or
sodium chloride in the composition. These compositions may also contain adjuvants, in
particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile
compositions for parenteral administration may also be prepared in the form of sterile
solid compositions which may be dissolved at the time of use in sterile water or any
other injectable sterile medium.
The doses depend on the desired effect, the duration of the treatment and the route of
administration used; they are generally between 5 mg and 1000 mg per day for an
adult with unit doses ranging from 1 mg to 250 mg of active substance.
In general, the doctor will determine the appropriate dosage depending on the age,
weight and any other factors specific to the subject to be treated.
Therapeutic methods of use
In another embodiment, the present invention provides a method for inhibiting the
EphA2 receptor activity by administering an antibody which antagonizes said EphA2
receptor, to a patient in need thereof. Any of the type of antibodies, antibody fragments,
or cytotoxic conjugates of the invention, may be used therapeutically. The invention
thus includes the use of antagonistic anti-EphA2 antibodies, fragments thereof, or
cytotoxic conjugates thereof as medicaments. In a preferred embodiment, the
antagonistic anti-EphA2 antibody is the 2H11R35R74 antibody or a humanized variant
thereof.

In a preferred embodiment, antibodies, antibody fragments, or cytotoxic conjugates of
the invention are used for the treatment of a hyperproliferative disorder in a mammal. In
a more preferred embodiment, one of the pharmaceutical compositions disclosed
above, and which contains an antibody, antibody fragment, or cytotoxic conjugate of
the invention, is used for the treatment of a hyperproliferative disorder in a mammal. It
is also an embodiment of the invention that the antibodies, antibody fragments, and
cytotoxic conjugates of the invention can also be used to make a medicament to treat
said hyperproliferative disorder in a mammal. In one embodiment, the disorder is a
cancer. In particular, the cancer is a metastatic cancer. The antibodies, antibody
fragments, and cytotoxic conjugates of the invention can also be used to treat the
neovascularization of said cancer tumor.
Accordingly, the pharmaceutical compositions of the invention are useful in the
treatment or prevention of a variety of cancers, including (but not limited to) the
following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung,
ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma;
hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic
leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's
lymphoma ; hematopoietic tumors of myeloid lineage, including acute and chronic
myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin,
including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma,
seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and
peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and
schwannomas; tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma,
xeroderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma, and other cancers yet to be determined in which EphA is expressed
predominantly. In a preferred embodiment, the cancer is a cancer of the lung, breast,
colon, prostate, kidney, pancreas, uterus, ovary, cervix and lymphatic organs,
osteosarcoma, synovial carcinoma, a sarcoma, head and neck, a glioma, gastric, liver
or other carcinomas in which EphA is expressed. In another embodiment, said
pharmaceutical composition relates to other disorders such as, for example,
autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and multiple
sclerosis; graft rejections, such as renal transplant rejection, liver transplant rejection,
lung transplant rejection, cardiac transplant rejection, and bone marrow transplant
rejection; graft versus host disease; viral infections, such as mV infection, HIV infection,

AIDS, etc.; and parasite infections, such as giardiasis, amoebiasis, schistosomiasis,
and others as determined by one of ordinary skill in the art.
Similarly, the present invention provides a method for inhibiting the growth of selected
cell populations comprising contacting target cells, or tissue containing target cells, with
an effective amount of an antibody, antibody fragment or antibody conjugate of the
present invention, or an antibody, antibody fragment or a therapeutic agent comprising
a cytotoxic conjugate, either alone or in combination with other cytotoxic or therapeutic
agents.
The method for inhibiting the growth of selected cell populations can be practiced in
vitro, in vivo, or ex vivo. As used herein, "inhibiting growth" means slowing the growth
of a cell, decreasing cell viability, causing the death of a cell, lysing a cell and inducing
cell death, whether over a short or long period of time.
Examples of in vitro uses include treatments of autologous bone marrow prior to their
transplant into the same patient in order to kill diseased or malignant cells; treatments
of bone marrow prior to its transplantation in order to kill competent T cells and prevent
graft-versus-host-disease (GVHD); treatments of cell cultures in order to kill all cells
except for desired variants that do not express the target antigen; or to kill variants that
express undesired antigen.
The conditions of non-clinical in vitro use are readily determined by one of ordinary skill
in the art.
Examples of clinical ex vivo use are to remove tumor cells or lymphoid cells from bone
marrow prior to autologous transplantation in cancer treatment or in treatment of
autoimmune disease, or to remove T cells and other lymphoid cells from autologous or
allogeneic bone marrow or tissue prior to transplant in order to prevent graft versus
host disease (GVHD). Treatment can be earned out as follows. Bone marrow is
harvested from the patient or other individual and then incubated in medium containing
serum to which is added the cytotoxic agent of the invention. Concentrations range
from about 10 uM to 1 pM, for about 30 minutes to about 48 hours at about 37°C. The
exact conditions of concentration and time of incubation, i.e., the dose, are readily
determined by one of ordinary skill in the art. After incubation the bone marrow cells
are washed with medium containing serum and returned to the patient by i.v. infusion
according to known methods. In circumstances where the patient receives other
treatment such as a course of ablative chemotherapy or total-body irradiation between
the time of harvest of the marrow and reinfusion of the treated cells, the treated marrow

cells are stored frozen in liquid nitrogen using standard medical equipment.
For clinical in vivo use, the antibody, the epitope-binding antibody fragment, or the
cytotoxic conjugate of the invention will be supplied as solutions that are tested for
sterility and for endotoxin levels. Examples of suitable protocols of cytotoxic conjugate
administration are as follows. Conjugates are given weekly for 4 weeks as an i.v. bolus
each week. Bolus doses are given in 50 to 100 ml of normal saline to which 5 to 10 ml
of human serum albumin can be added. Dosages will be 10 ug to 100 mg per
administration, i.v. (range of 100 ng to 1 mg/kg per day). More preferably, dosages will
range from 50 pg to 30 mg. Most preferably, dosages will range from 1 mg to 20 mg.
After four weeks of treatment, the patient can continue to receive treatment on a
weekly basis. Specific clinical protocols with regard to route of administration,
excipients, diluents, dosages, times, etc., can be determined by one of ordinary skill in
the art as the clinical situation warrants.
Diagnostic
The antibodies or antibody fragments of the invention can also be used to detect
EphA2 in a biological sample in vitro or in vivo. In one embodiment, the anti-EphA2
antibodies of the invention are used to determine the level of EphA2 in a tissue or in
cells derived from the tissue. In a preferred embodiment, the tissue is a diseased
tissue. In a preferred embodiment of the method, the tissue is a tumor or a biopsy
thereof. In a preferred embodiment of the method, a tissue or a biopsy thereof is first
excised from a patient, and the levels of EphA2 in the tissue or biopsy can then be
determined in an immunoassay with the antibodies or antibody fragments of the
invention. The tissue or biopsy thereof can be frozen or fixed. The same method can
be used to determine other properties of the EphA2 protein, such as its level of tyrosine
phosphorylation, cell surface levels, or cellular localization.
The above-described method can be used to diagnose a cancer in a subject known to
or suspected to have a cancer, wherein the level of EphA2 measured in said patient is
compared with that of a normal reference subject or standard. Said method can then
be used to determine whether a tumor expresses EphA2, which may suggest that the
tumor will respond well to treatment with the antibodies, antibody fragments or antibody
conjugates of the present invention. Preferably, the tumor is a cancer of the lung,
breast, colon, prostate, kidney, pancreas, uterus, ovary, cervix and lymphatic organs,
osteosarcoma, synovial carcinoma, a sarcoma, a glioma, gastric, liver, head and neck
or other carcinomas in which EphA2 is expressed, and other cancers yet to be

determined in which EphA2 is expressed predominantly.
The present invention further provides for monoclonal antibodies, humanized
antibodies and epitope-binding fragments thereof that are further labeled for use in
research or diagnostic applications. In preferred embodiments, the label is a radiolabel,
a fluorophore, a chromophore, an imaging agent or a metal ion.
A method for diagnosis is also provided in which said labeled antibodies or epitope-
binding fragments thereof are administered to a subject suspected of having a cancer,
and the distribution of the label within the body of the subject is measured or
monitored.
Kit
The present invention also includes kits, e.g., comprising a described cytotoxic
conjugate and instructions for the use of the cytotoxic conjugate for killing of particular
cell types. The instructions may include directions for using the cytotoxic conjugates in
vitro, in vivo or ex vivo.
Typically, the kit will have a compartment containing the cytotoxic conjugate. The
cytotoxic conjugate may be in a lyophilized form, liquid form, or other form amendable
to being included in a kit. The kit may also contain additional elements needed to
practice the method described on the instructions in the kit, such a sterilized solution
for reconstituting a lyophilized powder, additional agents for combining with the
cytotoxic conjugate prior to administering to a patient, and tools that aid in
administering the conjugate to a patient.
The present invention also relates to an article of manufacture comprising:
- a) a packaging material
- b) an antibody or epitope-binding fragment thereof or a conjugate, and
c) a label or package insert contained within said packaging material indicting
that said antibody or epitope-binding fragment thereof is effective for treating cancer.
EXAMPLES
Example 1
Preparation of conjugates
Conjugation of 2H11R35R74 to DM4
General synthetic schemes

Method A: High Pressure Liquid Chromatography- Mass Spectrometry (LCMS)
Spectra have been obtained on a Waters UPLC-SQD system in positive and/or
negative eiectrospray mode (ES+/-). Chromatographic conditions are the following:
column: ACQUITY BEH C18, 1,7 pm - 2,1x30 mm ; solvents: A: H20 (0,1% formic acid)

B: CH3CN (0,1 % formic acid); column temperature: 45°C ; flow rate: 0,6 ml/min ;
gradient (2 min): from 5 to 50% of B in 1 min ; 1,3 min: 100% of B ; 1,45 min: 100% of
B ; 1,75 min: 5% of B.
Method B: High Pressure Liquid Chromatography- Mass Spectrometry (LCMS)
Spectra have been obtained on a Waters ZQ system in positive and/or negative
electrospray mode (ES+/-). Chromatographic conditions are the following: column:
XBridge C18 2,5 urn 3x50 mm ; solvents: A: H20 (0,1 % formic acid) B: CH3CN (0,1 %
formic acid ; column temperature: 70°C ; flow rate: 0,9 ml/min ; gradient (7 min): from 5
to 100 % of B in 5,3 min ; 5,5 min: 100 % of B ; 6,3 min: 5 % of B.
Method C: deqlvcosvlation and High Resolution Mass Spectrometry of
immunoconiuqate (HRMS)
Deglycosylation is a technique of enzymatic digestion by means of glycosidase. The
deglycosylation is made from 500 ul of conjugated +100 ul of Tris buffer HCI 50 mM +
10 ul of glycanase-F enzyme (100 units of freeze-dried enzyme/100 Ml of water). The
medium is vortexed and maintained one night at 37°C. The deglycosylated sample is
then ready to be analyzed in HRMS. Mass spectra were obtained on a Waters Q-Tof-2
system in electrospray positive mode (ES+). Chromatographic conditions are the
following: column: 4 urn BioSuite 250 URH SEC 4,6x300 mm (Waters); solvents: A:
ammonium formate 25 mM +1% formic acid: B: CH3CN ; column temperature: 30°C ;
flow rate 0,4 ml/min ; isocratic elution 70% A + 30% B (15 min).
Method D: Analytical Size Exclusion Chromatography (SEC)
> Column: TSKgel G3000 SWXL 5pm column, 7.8 mm x 30 cm, TOSOH
BIOSCIENCE, LLC Part#: 08541
> Mobile Phase: KCI (0.2M), KH2P04 (0.052M) K2HP04 (0.107M), iPrOH (20% in
volume)
> Analysis Conditions: isocratic elution at 0.5 ml/min for 30 minutes

Method E : mass spectrometry (MS)
Spectra have been obtained through chemical ionisation (reactant gas : ammoniac) on
a WATERS GCTof system (direct introduction without LC).
Method F : High Pressure Liquid Chromatography - Mass Spectrometry (LCMS)
Spectra have been obtained on a Waters UPLC-SQD system in positive and/or
negative electrospray mode (ES+/-). Chromatographic conditions are the following :
column : ACQUITY BEH C18 1,7 urn - 2,1x50 mm ; solvents : A : H20 (0,1% formic
acid) B : CH3CN (0,1% formic acid); column temperature : 50°C ; flow rate : 1 ml/min ;
gradient (2 min): from 5 to 50% of B in 0.8 min ; 1,2 min : 100% of B ; 1,85 min : 100%
of B; 1,95 : 5% of B.
Abbreviations:
AcOEt: ethyl acetate ; ALK: (Ci-Ci2)alkylene group, particularly (d-C6)alkylene ; DAR:
Drug Antibody Ratio ; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene ; DCC: N,N-
dicyclohexylcarbodiimide ; DCM: dichloromethane ; DEAD: diethylazodicarboxylate ;
DIC: N,N'-diisopropylcarbodiimide ; DIPEA: N,N-diisopropylethylamine ; DMA:
dimethylacetamide ; DMAP: 4-dimethylaminopyridine ; DME: dimethoxyethane ; DMF:
dimethylformamide ; DMSO: dimethylsulfoxyde ; s: molar extinction coefficient; EEDQ:
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; EDCI: N-(3-dimethylaminopropyl)-N'-
ethylcarbodiimide ; EDTA: ethylene-diamine-tetraacetic acid ; Fmoc:
fluorenylmethoxycarbonyl; Hal: halogen atom ; HOBt: 1-hydroxybenzotriazole ;
HEPES: 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid ; NHS: N-
hydroxysuccinimide ; iPrOH: iso-propyl alcool; NMP: N-methylpyrrolidinone ; Rf:
retention factor; RP: reduced pressure ; RT: room temperature ; SEC: Size Exclusion
Chromatography ; TBDMS: ferf-butyldimethylsilyl ; TEA: triethylamine ; TFA:
trifluoroacetic acid ; TFAA: trifluoroacetic anhydride ;TFF: Tangential Flow Filtration ;
THF: tetrahydrofurane ; TIPS: triisopropylsilyl ;TLC: Thin Layer Chromatography ;tR:
retention time.
Buffers contents:
> Buffer A (pH 6.5): NaCI (50mM), KPi (50mM), EDTA (2mM)
> Buffer HGS (pH 5.5): Histidine (10mM), Glycine (130 mM), sucrose 5% (w/v), HCI
(8mM)

Parameters for Ab and L-DM4 concentration calculations (reference for the method
of calculation: Therapeutic Antibodies and Protocols, vol 525, 445):
> Molar extinction coefficients for hu2H11R35R74 (224000 at 280nM ; 82880 at
252nM) and L-DM4 (5180 at 280nM ; 26159 at 252nM), assuming an average
160000 molecular weight for the antibody.
Example 1a:
1a.1. Preparation of conjugate linked with PEG4-acetamido:
hu2H11R35R74-PEG4-NHAC-DM4

Under magnetic stirring, at room temperature, 9 ml of hu2H11R35R74 (14.36 mg/ml in
buffer A) are added, then 16.85 ml of buffer A, 3.23 ml of HEPES 1M, 1.59 ml of DMA,
followed by 1.64 ml of a 10mM DMA solution of L-DM4-AcNH-PEG4-CONHS activated
ester. After 1 hours 30 minutes at room temperature, an extra 0.085 ml of 10mM DMA
solution of L-DM4-AcNH-PEG4-CONHS activated ester is added. After 1 hours 45

minutes at room temperature, the crude conjugation medium is diluted with 60 ml of
HGS buffer and purified by TFF on Pellicon 3 cassettes. The sample is diafiltered
against ~ 10 sample volumes of HGS buffer and then collected. The TFF tank and lines
are washed with an extra 10 ml of HGS buffer. The two solutions are mixed, filter-
sterilized through 0.22 urn PVDF, concentrated on Amicon 15 and filter-sterilized
through 0.22 pm PVDF. 17 ml of hu2H11R35R74-PEG4-NHAc-DM4 immunoconjugate
(c = 5.76 mg/ml) was thus obtained. The immunoconjugate is then analyzed for final
drug load and monomeric purity.
SEC analysis (method D): DAR (SEC) = 5.4 ; RT = 16.757 ; monomeric purity = 99.5%
HRMS data (method C): see fig.2
1a.2. Preparation of L-DM4-AcNH-PEG4-CONHS activated ester:

Under magnetic stirring, at room temperature, 154.3 mg of L-DM4 are introduced in a
glass vial. A solution of 90 mg of 3-[2-(2-{2-[2-(2-bromo-acetylamino)-ethoxy]-ethoxy]-
ethoxy)-ethoxy]-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester in 0.94 ml of DMA is then
added, followed by 36 ul of DIEA. After 23 hours at room temperature, the reaction
medium is diluted with 5 ml of AcOEt and washed with 7 ml of water. The aqueous
phase is extracted with 5 ml of AcOEt. The combined organic phases are dried over
magnesium sulphate, concentrated to dryness under reduced pressure. 228 mg of pale
yellow viscous oil are obtained, which product is diluted with a minimum amount of
DMA and purified by flash-chromatography on 30g of C18-grafted silica gel (gradient of

elution water.acetonitrile 95:5 to 5:95 by volume). After concentration of fractions 2 and
3 under reduce pressure, a colourless viscous oil is obtained, which product is diluted
with a minimum amount of DMA and purified by flash-chromatography on 30g of CIS-
grafted silica gel (gradient of elution wateracetonitrile 95:5 to 5:95 by volume). After
concentration of fractions 33 to 35 under reduce pressure, 41 mg of L-DM4-AcNH-
PEG4-CONHS activated ester are obtained in the form of a white meringue-like
product, the characteristics of which are as follows:
Mass spectra: method B
Retention time (min) = 4,06
[M+H-H20]+: m/z 1164; [M+H]+: m/z 1182 ;
[M-H+HC02H]-: m/z 1226
NMR analysis 1H (500 MHz. 5 in ppm. chloroform-d): 0,80 (s, 3 H); 1,21 (s, 3 H); 1,22
(s, 3 H); 1,25 (m, 1 H); 1,29 (d, J=6,7 Hz, 6 H); 1,46 (m, 1 H); 1,57 (d, J=13,4 Hz, 1 H);
1,64 (s, 3 H ); 1,76 to 1,83 (m, 1 H); 1,88 to 1,96 (m, 1 H); 2,18 (dd, J=2,5 et 14,3 Hz,
1 H); 2,36 (m, 1 H); 2,53 (m, 1 H); 2,61 (dd, J=12,5 et 14,3 Hz, 1 H); 2,82 to 2,92 (m,
10 H); 2,98 (d, J=16,7 Hz, 1 H); 3,03 (d, J=9,6 Hz, 1 H); 3,15 (d, J=12,9 Hz, 1 H); 3,22
(s, 3 H); 3,32 (s large, 1 H); 3,36 (s, 3 H); 3,42 (m, 2 H); 3,50 (d, J=9,1 Hz, 1 H); 3,53 (t,
J=5,2 Hz, 2 H); 3,58 to 3,67 (m, 13 H); 3,84 (t, J=6,4 Hz, 2 H); 3,99 (s, 3 H); 4,27
(m,1 H); 4,77 (dd, J=2,9 et 11,9 Hz, 1 H); 5,42 (q, J=6,7 Hz, 1 H); 5,66 (dd, J=9,1 et
15,4 Hz, 1 H ); 6,23 (s,1 H); 6,43 (dd, J=11,3 et 15,4 Hz, 1 H); 6,64 (d, J=1,1 Hz, 1 H);
6,74 (d, J=11,3 Hz, 1 H); 6,85 (d, J=1,1 Hz, 1 H); 7,08 (t, J=5,2 Hz, 1 H).
1a.3. Preparation of 3-r2-(2-|2-f2-(2-bromo-acetvlamino)-ethoxv1-ethoxv>-ethoxv)-
ethoxvl-propionic acid 2,5-dioxo-pyrrolidin-1-vl ester:

Under magnetic stirring, at room temperature, 671.4 mg of 3-(2-{2-[2-(2-amino-ethoxy)-
ethoxy]-ethoxy}-ethoxy)-propionic acid (CA(PEG)4, Pierce) are introduced in a glass
vial. A solution of 597.4 mg of bromo-acetic acid 2,5-dioxo-pyrrolidin-1-yl ester in 14 ml

of dichloromethane is then added. After 15 minutes at room temperature, 0.396 ml of
DIC (N,N'-diisopropylcarbodiimide) is added. After 1 hour 30 minutes, the crude
reaction medium is filtered on sintered glass, and the filtrate is purified by flash-
chromatography on 100g of CN-grafted silica gel (gradient of elution
n.heptane/iPrOH/AcOEt with increasing iPrOH portion). After concentration of fractions
30 to 45 under reduce pressure, 761 mg of 3-[2-(2-{2-[2-(2-bromo-acetylamino)-
ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester are
obtained in the form of a colourless oil, the characteristics of which are as follows:
Mass spectra: method A
Retention time (min) = 0,74
[M+H]+:m/z 483/485
[M-H+HC02H]-: m/z 527/529
Bromo-acetic acid 2,5-dioxo-pyrrolidin-1-yl ester could be prepared following published
protocol (Biochemistry, 1974, 481).
Example 1b:
1b.1. Preparation of conjugate linked with PEG4-Mal:
hu2H11R3!iR74-PEG4-Mal-DM4

Under magnetic stirring, at room :emperature, 4 ml of hu2H11R35R74 (14.36 mg/ml in
buffer A) are added, then 7.5 ml of buffer A, 1.45 ml of HEPES 1M, 1.15 ml of DMA,
followed by 0.305 ml of a 10mM DMA solution of L-DM4-Mal-PEG4-CONHS activated

ester. After 7 hour at room temperature, the crude conjugation medium is diluted with
70 ml of HGS buffer and purified by TFF on Pellicon 3 cassette. The sample is
diafiltered against ~ 10 sample volumes of HGS buffer and then collected. The TFF
tank and lines are washed with an extra 10 ml of HGS buffer. The two solutions are
mixed, concentrated on Amicon 15 and filter-sterilized through 0.22 (xm PVDF. 8.0 ml
of hu2H11R35R74-PEG4-Mal-DM4 immunoconjugate (c = 5.09 mg/ml) was thus
obtained. The immunoconjugate is then analyzed for final drug load and monomeric
purity.
SEC analysis (method D): DAR (SEC) = 5.3 ; RT = 16.680 ; monomeric purity = 99.5%
HRMS data (method C): see fiq.3
1b.2. Preparation of L-DM4-Mal-PEG4-CONHS activated ester:

Under magnetic stirring, at room temperature, 50 mg of L-DM4,17.2 mg of supported
DIEA (3.72 mmol/g), and a solution of 36.2 mg of 3-{2-[2-(2-{2-[3-(2,5-dioxo-2!5-
dihydro-pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionicacid
2,5-dioxo-pyrrolidin-1-yl ester (commercially available, SM(PEG)4, Pierce) in 360 ul of
DMA are successively added. After 1 hours 30 minutes at room temperature the
reaction medium is filtered, the solids are washed with AcOEt, and the combined
filtrates are directly purified by flash-chromatography on 14 g of CN grafted silica gel
(gradient of elution heptane:AcOEt:iPrOH with increasing contribution of iPrOH). After
concentration of fractions containing the expected product under reduce pressure, 29.8
mg of L-DM4-Ma!-PEG4-CONHS activated ester are obtained in the form of a
colourless glass, the characteristics of which are as follows:

Mass spectra: method A
Retention time (min) = 1,24 /1,25 (2 diastereoisomers)
[M+H]+:m/z1293
[M-H+HC02H]-: m/z 1337
Example 1c: Preparation of conjugate linked with SPDB:
hu2H11R35R74-SPDB-DM4

Humanized 2H11R35R74 antibodies were conjugated to L-DM4 N2'deacetyl-N2'(4-
methyl-4-mercapto-1-oxopentyl)-maytansine using SPDB (4-[2-pyridyldithio]butanoic
acid N-hydroxsuccinimde ester) linker using the same protocol as previously described
in WO 2008010101A9 with other hu2H11 antibodies. Briefly, the antibody was modified
at 8 mg/mL with 5.5 or 6.5 folds molar excess of SPDB for hu2H11 and hu37.3D7
respectively. The reaction was carried out in Buffer A (50 mM KPi/50 mM NaCI/2 mM
EDTA, pH 6.5, 95% v/v) with EtOH (5% v/v) for 90 minutes at room temperature. The
modified antibody was then purified by SephadexG25 desalting column with Buffer A.
Next, the modified antibody was reacted with a 1.7-fold molar excess of DM4 over
SPDB linker. The reaction was carried out at 2.5 mg/mL antibody in Buffer A (97% v/v)
and DMA (dimethylacetamide, 3% v/v) at room temperature for 20 hours. The
conjugate was purified by SephadexG25 desalting column with 10 mM Histidine, 130
mM Glycine, 5% sucrose, pH5.5. The drug to antibody ratio was 4.0 for hu37.3D7-


1d.1 Preparation of conjugate hu2H11R35R74-PEG4-NMeAc-DM4
Under magnetic stirring at RT, 4 ml of hu2H11R35R74 (14.36 mg/ml in buffer A) are
added, then 7.5 ml of buffer A, 1.45 ml of HEPES 1M, 1.05 ml of DMA, followed by
0.39 ml of a 10mM DMA solution of L-DM4-AcNMe-PEG4-CONHS activated ester.
After 30 min at RT, an extra 0.19 ml of 10 mM DMA solution of L-DM4-AcNMe-PEG4-
CONHS activated ester is added. After 3 hrs at RT, the crude conjugation medium is
diluted with 65 ml of HGS buffer and purified by TFF on Pellicon 3 cassette. The
sample is diafiltered against ~10 sample volumes of HGS buffer and then collected.
The TFF tank and lines are washed with an extra 10 ml of HGS buffer. The two
solutions are mixed, concentrated on Amicon 15 and filter-sterilized through 0.22 |im
PVDF. 8.5 ml of hu2H11R35R74-PEG4-NMeAc-DM4 conjugate (c=6.01 mg/ml) was
thus obtained. The conjugate is then analyzed for final drug load and monomeric purity.
SEC analysis (D): DAR (SEC)= 5.5 ; RT= 16.7 min ; monomeric purity= 99.4% ; HRMS
data : see Fig-H-
id^ Preparation of L-DM4-AcNMe-PEG4-CONHS activated ester
Under magnetic stirring at RT, 133.4 mg of L-DM4 are introduced in a glass vial. A
solution of 85 mg of 3-{2-[2-(2-{2-[(2-bromo-acetyl)-methyl-amino]-ethoxy}-ethoxy)-

ethoxy]-ethoxy]-propionic.acid 2,5-dioxo-pyrrolidin-.1-yl ester in 0.2 ml of DMA is then
added, followed by 32.9 ul of DIEA. After 1 hours at RT, the reaction medium is
purified by flash-chromatography on 30 g of C18-grafted silica gel (gradient of elution
water:acetonitrile 95:5 to 5:95 by volume). After concentration of fractions containing
the desired product under RP, 71.3 mg of L-DM4-AcNMe-PEG4-CONHS activated
ester are obtained in the form of a colourless glass-like product. Mass spectra (D) :
RT=O.98 min ; [M+H-H20]+ : m/z 1178 (main signal) ; [M+Na]+ : m/z 1218 ; [M-
H+HC02H]- : m/z 1240 ; 1H NMR (500 MHz. 8 in ppm . chloroform-d'): 0,81 (s, 3 H);
1,20 to 1,33 (m, 13 H) ; 1,42 to 1,52 (m, 1 H) ; 1,56 to 1,61 (m, 1 H) ; 1,65 (s, 3 H);
1,73 to 1,83 (m, 1 H); 1,96 to 2,04 (m, 1 H); 2,19 (dd, J=2,8 and 14,4 Hz, 1 H) ; 2,29
to 2,41 (m, 1 H); 2,55 to 2,66 (m, 2 H); 2,83 to 2,93 (m, 12 H); 3,04 (d, J=9,8 Hz, 1 H)
; 3,12 (d, J=12,7 Hz, 1 H); 3,18 to 3,25 (m, 5 H); 3,37 (s, 3 H); 3,47 to 3,54 (m, 3 H);
3,57 to 3,68 (m, 15 H); 3,85 (t, J=6,6 Hz, 2 H); 3,99 (s, 3 H); 4,29 (m, 1 H); 4,79 (dd,
J=2,8 and 12,2 Hz, 1 H); 5,41 (q, J=6,7 Hz, 1 H) ; 5,68 (dd, J=9,3 et 15,2 Hz, 1 H);
6,23 (s, 1 H); 6,43 (dd, J=11,0 and 15,2 Hz, 1 H); 6,66 (s, 1 H); 6,74 (d, J=11,0 Hz, 1
H); 6,83 (s, 1 H).
1d.3 Preparation of 3-l2-r2-(242-r(2-bromo-acetvl)-methvl-amino1-ethoxvV
ethoxv)-ethoxv1-ethoxv}-propionic acid 2.5-dioxo-pyrrolidin-1-vl ester

Under magnetic stirring, at RT, in a round bottom flask, 115.1 mg of 3-(2-{2-[2-(2-
methylamino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid, 1.5 ml of DCM, 97.3 mg
of bromo-acetic acid 2,5-dioxo-pyrrolidin-1-yl ester are successively introduced. After 2
h, 72 ul of DIEA are added, and after a further 1 hour at RT, 70.2 ul of DIC are added.
The crude reaction medium is kept 4 hrs at RT, 16 hrs at -20°C, and then purified by
flash-chromatography on 30 g of silica gel (gradient of elution DCM:methanol from
0:100 to 3:97 by volume). After concentration of fractions containing the desired
product under RP, 85.8 mg of 3-{2-[2-(2-{2-[(2-bromo-acetyl)-methyl-amino]-ethoxy}-
ethoxy)-ethoxy]-ethoxy}-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester are obtained in
the form of a white solid. Mass spectra (A): RT= 0,84 min ; [M+H]+ : m/z 497/499

1d.4 Preparation of 3-(2-(2-r2-(2-methvlamino-ethoxv)-ethoxv1-ethoxv>-ethoxv)-
propionic acid

Under an inert atmosphere of argon, in a round bottom flask, with magnetic stirring,
120.1 mg of 3-[2-(2-{2-[2-(2,2,2-trifluoro-acetylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-
propionic acid methyl ester, 1 ml of anhydrous THF and 59.8 pi of CH3I and
successively introduced. The reaction medium is cooled with a ice/water bath at about
0°C, and 16.1 mg of NaH (50% pure in oil) is slowly added by small portions. After 15
min at 0°C, and 1 hr at RT, the crude reaction medium is concentrated to dryness
under RP, and diluted with 0.5 ml of THF and 0.8 ml of water. At RT, 30.6 mg of LiOH
is then added to the reaction medium. The crude reaction medium is kept 2 hrs at RT,
16 hrs at -20°C, and then purified by flash-chromatography on 30 g of C18-grafted
silica gel (gradient of elution water:acetonitrile from 95:5 to 5:95 by volume). After
concentration of fractions containing the desired product under RP, 115.3 mg of 3-(2-
{2-[2-(2-methylamino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid are obtained in
the form of a yellow oil.
1d.5 Preparation of 3-f2-(2-(2-f2-(2,2,2-trifluoro-acetvlamino)-ethoxv1-ethoxv>-
ethoxv^-ethoxvl-propionic acid methyl ester

Under an inert atmosphere of argon, in a round bottom flask, with magnetic stirring,
230 mg of 3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid
(CA(PEG)4, Pierce) 2 ml of DCM and 1 ml of methanol are successively introduced. At
RT, 1 ml of trimethylsilyldiazomethane (2M solution in hexane) is slowiy added to the
reaction medium. After 2 hrs at RT, the excess of trimethylsilyldiazomethane is
neutralized by addition of acetic acid. The crude reaction medium is then evaporated to
dryness under RP. The residue obtained is diluted with 2 ml of DCM, cooled to 0°C
with a water-ice bath, then 363 pi of TEA and 300 pi of TFAA are successively added.
After 2 hrs 30 min at RT and 19 hrs at -20°C, 363 pi of TEA and 300 pi of TFAA are
successively added. After 4 hrs 30 min at RT and the crude medium is stocked at -

20°C and then purified by flash-chromatography on 30 g of C18-grafted silica gel
(gradient of elution watenacetonitrile from 95:5 to 5:95 by volume). After concentration
of fractions containing the desired product under RP, 123 mg of 3-[2-(2-{2-[2-(2,2,2-
trifluoro-acetylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionic acid methyl ester are
obtained in the form of a pale-yellow oil. Mass spectra (A): RT= 0,90 min ; [M+H]+ :
m/z 376 ; [M-H]- : m/z 374.
Example 1e: Preparation of conjugate hu2H11R35R74-PEG8-NHAc-DM4

1e.1 Preparation of conjugate hu2H11R35R74-PEG8-NHAc-DM4
Under magnetic stirring at RT, 4 ml of hu2H11R35R74 (14.36 mg/ml in buffer A) are
added, then 7.5 ml of buffer A, 1.45 ml of HEPES 1M, 1.05 ml of DMA, followed by
0.405 ml of a 10 mM DMA solution of L-DM4-AcNMe-PEG8-CONHS activated ester.
After 30 min at RT, an extra 0.1 ml of 10 mM DMA solution of L-DM4-AcNMe-PEG8-
CONHS activated ester is added. After 1 hr 45 min at RT, the crude conjugation
medium is diluted with 60 ml of HGS buffer and purified by TFF on Pellicon 3 cassette.
The sample is diafiltered against -10 sample volumes of HGS buffer and then
collected. The TFF tank and lines are washed with an extra 10 ml of HGS buffer. The
two solutions are mixed, concentrated on Amicon 15 and filter-sterilized through 0.22
\im PVDF. 7.0 ml of hu2H11R35R74-PEG8-AcNMe-DM4 conjugate (c= 6.95 mg/ml)
was thus obtained. The conjugate is then analyzed for final drug load and monomeric
purity. SEC analysis (D) : DAR (SEC)= 5.0 ; RT= 16.593 min ; monomeric purity =
99.5% ; HRMS data : see Fig. 15.

1e.f Preparation of L-DM4-AcNH-PEG8-CONHS activated ester
Under magnetic stirring at RT, 65 mg of 3-{2-[2-(2-{2-[2-(2-{2-[2-(3-bromo-
propionylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy>ethoxy]-ethoxy}-
propionic acid 2,5-dioxo-pyrrolidin-1-yl ester are introduced in a glass vial, followed by
a solution of 67.7 mg of L-DM4 in 0.85 ml of DMA and 16,5 pi of DIEA. After 48 hrs at
RT, the reaction medium is purified by flash-chromatography on 10 g of silica gel
(gradient of elution DCM:MeOH 100:0 to 90:10 by volume). After concentration of
fractions 18 to 26 under RP, 17 mg of L-DM4-AcNH-PEG8-CONHS activated ester are
obtained in the form of a colourless glass. Mass spectra (B): : RT= 4,08 min ; [M+H-
H20]+ : m/z 1340 (main signal); [M+Na]+ : m/z 1380 ; [M-H+HC02H]- : m/z 1402 ; 1H
NMR (400 MHz. 5 in ppm . chloroform-d^: 0.81 (s, 3 H); 1,22 (s, 3 H) ; 1,23 (s, 3 H);
1,26 (m, 1 H); 1,30 (d, J=6,8 Hz, 6 H); 1,41 to 1,52 (m, 1 H); 1,65 (s, 3 H); 1,80 (m, 1
H); 1,89 to 1,99 (m, 1 H);2,19(m, 1 H); 2,37 (m, 1 H); 2,47 a 2,67 (m, 2 H); 2,81 a
2,93 (m, 10 H) ; 2,99 (d, J=16,6 Hz, 1 H) ; 3,04 (d, J=9,8 Hz, 1 H) ; 3,16 (d broad,
J=13,7 Hz, 1 H); 3,23 (s, 3 H); 3,32 (s broad, 1 H); 3,37 (s, 3 H); 3,44 (m, 2 H); 3,51
(d, J=9,1 Hz, 1 H); 3,54 (t, 7=5,4 Hz, 2 H); 3,59 a 3,73 (m, 29 H); 3,86 (t, J=6,6 Hz, 2
H) ; 4,00 (s, 3 H) ; 4,22 to 4,33 (m, 1 H) ; 4,78 (dd, J=2,9 and 12,2 Hz, 1 H) ; 5,43 (q,
J=6,8 Hz, 1 H) ; 5,67 (dd, J=9,0 et 15,2 Hz, 1 H) ; 6,23 (s, 1 H) ; 6,44 (dd, J=11,2 et
15,2 Hz, 1 H); 6,65 (d, J=1,5 Hz, 1 H); 6,75 (d, J=11,2 Hz, 1 H); 6,86 (d, J=1,5 Hz, 1
H); 7,02 a 7,13 (m, 1 H).
1e.g Preparation of 3-l2-r2-(2-l2-r2-(2-f2-r2-(3-bromo-propionviamino)-ethoxv1-
ethoxvl-ethoxv)-ethoxv1-ethoxv>-ethoxv)-ethoxv1-ethoxvVpropionic acid 2.5-
dioxo-pvrrolidin-1-vl ester:

Under magnetic stirring at RT, 100 mg of 3-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-
ethoxy)-propionic acid (CA(PEG)4, Pierce), 2 ml of DCM and 53.5 mg of bromo-acetic
acid 2,5-dioxo-pyrrolidin-1-yl ester are successively introduced in a glass vial. After 1 hr
at RT, 35.1 pi of DIC are added. After 1 hr, the crude reaction medium is filtered on

sintered glass, concentrated to dryness under RP, dilute with 10 ml of AcOEt, filtered
on sintered glass and concentrated to dryness under RP. 76.5 mg of 3-{2-[2-(2-{2-[2-(2-
{2-[2-(3-bromo-propionylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-
ethoxy]-ethoxy}-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester are obtained in the form of
a colourless oil. Mass spectra (A) : RT= 0,80 min ; [M+H]+ : m/z 659/661 ; [M-
H+HC02H]-: m/z 703/705
Example 1f: Preparation of conjugate hu2H11R35R74-PEG4-Allyl-DM4
StmnnrWi TiCCt
1f.1 Preparation of conjugate hu2H11R35R74-PEG4-Allvl-DM4
Under magnetic stirring at RT, 4 ml of hu2H11R35R74 (14.36 mg/ml in buffer A) are
added, then 7.5 ml of buffer A, 1.45 ml of HEPES 1M, 1.14 ml of DMA, followed by 0.3
ml of a 10 mM DMA solution of L-DM4-Allyl-PEG4-CONHS activated ester. After 30
min at RT, an extra 0.125 ml of 10 mM DMA solution of L-DM4-Allyl-PEG4-CONHS
activated ester is added. After 1 hr 25 min at RT, the crude conjugation medium is
diluted with 65 ml of HGS buffer and purified by TFF on Pellicon 3 cassette. The
sample is diafiltered against -10 sample volumes of HGS buffer and then collected.
The TFF tank and lines are washed with an extra 10 ml of HGS buffer. The two
solutions are mixed, concentrated on Amicon 15 and filter-sterilized through 0.22 yum
PVDF. 8.0 ml of hu2H11R35R74-PEG4-Allyl-DM4 conjugate (c=5.22 mg/ml) was thus
obtained. The conjugate is then analyzed for final drug load and monomeric purity.
SEC analysis (H) : DAR (SEC)= 5.3 ; RT= 16.767 min ; monomeric purity= 99.4% ;
HRMS data : see Fig.16.

1f.2 Preparation of L-DM4-Allvl-PEG4-CONHS activated ester
Under magnetic stirring at RT, 70 mg of L-DM4, 45 mg of 3-(2-{2-[2-(4-bromo-but-2-
eny1oxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Bromo-
Allyl-PEG4-C0NHS), 0.5 ml of DMA and 23.5 pl of DIEA are successively introduced in
a glass vial. After 2 hrs at RT and 17 hrs at -20°C, 50 pi of DIEA is added. After 24 firs
at RT, the reaction medium is purified by flash-chromatography on 30 g of C-18 grafted
silica gel (gradient of elution water.acetonitrile 95:5 to 5:95 by volume). After
concentration of fractions containing the expected product under RP, 47.1 mg of L-
DM4-Allyl-PEG4-CONHS activated ester are obtained in the form of a white solid.
Mass spectra (D1: RT= 1,06 min ; [M+Na]+ : m/z 1173 ; 1H NMR (500 MHz. 5 in ppm .
chloroform-d) : 0,81 (s, 3 H); 1,18 a 1,39 (m, 13 H); 1,42 to 1,52 (m, 1 H) ; 1,58 (d,
J=13,4 Hz, 1 H); 1,65 (s, 3 H); 1,73 to 1,82 (m, 1 H) ; 1,86 a 1,95 (m, 1 H); 2,19 (d,
J=14,3 Hz, 1 H); 2,40 (m, 1 H) ; 2,51 to 2,65 (m, 2 H); 2,82 to 2,95 (m, 9 H) ; 2,98 to
3,07 (m, 2 H); 3,12 (d, J=12,6 Hz, 1 H); 3,18 to 3,27 (m, 1 H); 3,23 (s, 3 H) ;3,36 (s, 3
H) ; 3,51 (d, .7=9,1 Hz, 1 H) ; 3,54 a 3,82 (m, 13 H) ; 3,86 (t, J=6,4 Hz, 2 H) ; 3,91 a
3,95 (m, 2 H); 3,99 (s, 3 H); 4,28 (t, J=11,0 Hz, 1 H); 4,78 (dd, J=2,6 et 11,9 Hz, 1 H)
; 5,44 (q, J=6,7 Hz, 1 H); 5,49 to 5,63 (m, 2 H) ; 5,68 (dd, J=9,1 and 15,0 Hz, 1 H) ;
6,24 (s, 1 H); 6,43 (dd, J=11,1 and 15,0 Hz, 1 H) ; 6,66 (s, 1 H); 6,77 (d, J=11,1 Hz,1
H); 6,83 (s, 1 H).
1f.3 Preparation of 3-(2-f2-f2-(4-bromo-but-2-envloxv>-ethoxv1-ethoxv\-ethoxv)-
propionic acid 2,5-dioxo-pyrrolidin-1 -yl ester

At RT, 200 mg of 3-(2-{2-[2-(4-bromo-but-2-enyloxy)-ethoxy]-ethoxy>ethoxy)-propionic
acid, 4 ml of DCM and 232.3 mg of supported DCC (2 equivalents) are successively
introduced in a glass vial. After 1 hr at RT, 64.8 mg of NHS are added. After 5 hrs at
RT, the crude reaction medium is filtered on sintered glass, solids are washed with
DCM, and the combined filtrates are concentrated to dryness under RP. Purification by
flash-chromatography on 15 g of silica gel (gradient of elution MeOH:DCM 0:100 to
10:90 by volume), and concentration of fractions containing the expected product under

RP, afforded 46 mg of 3-(2-{2-[2-(4-bromo-but-2-enyloxy)-ethoxy]-ethoxy}-ethoxy)- ,
propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Bromo-Allyl-PEG4-CONHS) are obtained
in the form of a pale yellow oil. Mass spectra (A): RT= 1,02 min ; [M+H]+ : m/z 454/456
; [M+Na]+ : m/z 476/478 ; [M-H+HC02H]- : m/z 498/500.
1f.4 Preparation of 3-(2-{2-T2-(4-brorno-but-2-envloxy)-ethoxvl-ethoxv}-ethoxv)-
propionic acid

At RT, a solution of 1 g of 3-(2-{2-[2-(4-bromo-but-2-enyloxy)-ethoxy]-ethoxy}-ethoxy)-
propionic acid tert-butyl ester (commercially available), 6 ml of TFA and 3 ml of DCM is
stirred during 3 hrs, and then concentrated to dryness under RP. The oily residue is
diluted with toluene and concentrated to dryness under RP affording 853 mg of 3-(2-{2-
[2-(4-bromo-but-2-enyloxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid in the form of a
brown oil.
Example 1q: Preparation of conjugate hu2H11-PEG4-NHAc-DM4
Conjugate hu2H11-PEG4-NHAc-DM4 could be prepared in a manner similar to
example 1 : under stirring, at RT, 1 ml of hu2H11 (8.52 mg/ml in buffer A) is added,
then 0.7 ml of buffer A, 0.213 ml of HEPES 1M, 0.7 ml of DMA, followed by 0.085 ml of
a 10 mM DMA solution of L-DM4-AcNH-PEG4-CONHS activated ester diluted with
0.128 ml of DMA. After 2 hrs at RT, the crude medium is concentrated on Amicon 4 at
7000 G, buffer exchanged with HGS buffer on Nap-10 column, and finally purified on a
5 ml Zeba column. 1.15 ml of hu2H11-PEG4-NHAc-DM4 conjugate (c=3.78 mg/ml)
was thus obtained. The conjugate is then analyzed for final drug load and monomeric
purity. SEC analysis (method D): DAR (UV)= 6.6 ; DAR (SEC)= 5.6 ; RT= 15.387 min ;
monomeric purity= 99.7% ; HRMS data : see Fig.17.
Example 2
Inhibition of EphA2 autophosphorylation activity by hu2H11R35R74
Materials and Methods
Cell lines and Antibodies
The breast adenocarcinoma cell line MDA-MB-231 was obtained from ECAAC (ref. #
92020424). The non-small cell lung carcinoma cell line NCI-H1299 was obtained from

ATCC (ref. # CRL-5803). Both cell lines were maintained in Dulbecco!s modified Eagle
medium (DMEM) supplemented with 10 % heat inactivated foetal calf serum and 2 mM
L-Gln. The recombinant mouse Ephrin-A1 extracellular domain/Fc chimera
(EphrinA1/Fc) was obtained from Sigma (ref. # E 9902) or from R&D Systems (ref. #
602-A1). The anti-Eck/EphA2 clone D7 antibody (Ab) and the anti-phosphotyrosine Ab
4G10 were obtained from Millipore (ref. # 05-480 and 05-321 respectively). The chKTI
isotype control Ab was generated at Immunogen Inc. It corresponds to the chimeric
version of the anti-Kunitz Soybean Trypsin Inhibitor Ab (KTI, ATCC ref. # HB9515)
where the Ig heavy and light chain constant regions were replaced by the human K light
chain and human y1 heavy chain. The recombinant Ab was purified at ImmunoGen
(chKTI lot #2539-91) from culture supernatants of HEK293T cells transiently
transfected with an expression plasmid for the heavy and light chains. The humanized
anti-EphA2 Abs hu2H11 (lot #LP08191) and hu2H11R35R74 (lot # LP09077) were
produced at Sanofi-Aventis from stably transfected Lonza Chinese hamster ovary
(CHO)/GS cell lines. Both Abs were purified from culture supernatants by affinity
chromatography on protein A-sepharose followed by anion exchange chromatography
according to standard procedures. An additional chromatography step was performed
on ceramic hydroxyapatite. All preparations were stored in 1xPBS at 4°C and tested
for low endotoxin levels by using the kinetic LAL method. The anti-actin antibody
clone C4 was from Millipore (ref. # MAB1501). The peroxydase (PO) conjugated goat
anti-mouse IgG Ab was from Jackson Immunoresearch (ref. # 115-035-003).
Induction of phosphorylation
MDA-IVIB-231 cells were plated in complete medium in P1Q0 Petri-dishes (5 plates per
sample) at 3 x 106 cells per plate and incubated for 48 hrs at 37°C in a C02 incubator.
Cells were serum starved for 18 hrs and subsequently treated with hu2H11 or hu2H11-
R35/R74 10ug/mLorwith EphrinAI/Fc at 2 ug/mL 10minto2 hrs at 37°C.
Inhibition of phosphorylation
Serum starved MDA-MB-231 or NCI-H1299 cells were incubated with chKTI, hu2H11
or hu2H11R35R74 (10 ug/mL) 1 hrs at 37°C. EphrinA1/Fc was then added at 1 ug/mL
and cells were further incubated 10-30 min at 37°C.
Preparation of cell extracts

Cell samples were harvested on ice by scraping, transferred into 15 mL conical tubes
and centrifuged 5 min at 1300 rpm. After one wash with 1 x phosphate buffered saline
(PBS; Invitrogen # 14190) cells were resuspended in 300 uL of lysis buffer (Biosource
ref.# FNN0011) extemporaneously supplemented with 1 mM phenylmethylsulphonyl
fluoride (PMSF), 1 x protease inhibitor cocktail (Sigma ref. # P2714) and 1 x Halt
phosphatase inhibitor (Pierce ref. # 78420). Cell extracts were kept on ice 45 min with
occasionnal vortexing, centrifuged 10 min at 15000 rpm and kept at -80°C until further
use. Protein concentration was determined by the bicinchoninic acid (BCA) method
using a kit from Pierce (ref. # 23227).
Immunoprecipitation
A pre-cleaning step of cell extracts (0.3-0.5 mg per sample) was performed by adding
protein G Sepharose 4 fast flow (GE Healthcare Life Sciences ref. # 17-0618-01)
previously equilibrated in lysis buffer (1 hr at 4°C on a rotator). Incubation was
performed 30 min at 4°C on a rotator. Samples were centrifuged 2 min at 1500 rpm,
supernatant were collected and incubated over night on a rotator with the anti-EphA2
Ab clone D7 (4 pL per sample). Immunoprecipitation was performed with Protein G
Sepharose 4 hrs at 4°C on a rotator. Samples were centrifuged 2 min at 1500 rpm at
4°C and washed 3 times 5 min with 100 uL of lysis buffer. Immunoprecipitated beads
were resuspended in 50 uL of 4 x NuPAGE LDS sample buffer (Invitrogen ref. #
NP0007) supplemented with NuPAGE reducing agent (Invitrogen ref. # NP0009),
heated 5 min at 95°C centrifuged 5 min 1500 rpm and kept at -20°C or subjected to
electrophoresis.
Immunoblotting
Immunoprecipitates or cell extracts were loaded on a 4-12% Bis-Tris Midi gel
(Invitrogen # NP0322BOX) with reference molecular weight markers (GE healthcare
Life Sciences ref. # RPN800) and electrophoresis was performed 3 hrs at 150 V in
MOPS-SDS buffer 1 x (Invitrogen ref. # NP0001). Electroblotting was performed on
PVDF membranes (Invitrogen ref. # LC2007) with an i-blot™ apparatus (Invitrogen)
using programme 3. Blocking of the membranes was performed in TBST 1x (i.e. Tris-
Buffered Saline Sigma ref. # T 5912, 0.1% Tween 20 Sigma ref. # P1379)
supplemented with 5% Bovine serum albumin. Labeling with the anti-EphA2 Ab clone
D7 or the anti-Phosphotyrosine 4G10 Ab were performed over night at 4°C in the same
buffer. Labeling with anti-Actin Ab clone C4 was performed 1 h at room temperature.

After.subsequent washing of the membranes with 1 x TBST development of the
Immunoblots was performed using the PO-conjugated goat anti-mouse Ab and the
ECL kit from Perkin Elmer (ref. # NEL 104001EA). Luminescence was read on a
Fuji4000 apparatus.
Results
Induction of the phosphorylation of EphA2 by hu2H11 or hu2H11R35R74 Abs was
investigated in MDA-MB231 cells using recombinant EphrinA1/Fc as a positive control.
Results are presented in figure 4. No induction of phosphorylation could be detected
with any of the two Abs from 10 min to 2 hrs while recombinant EphrinA1/Fc induced a
strong phosphorylation of the EphA2 receptor at 10 min with a decline of the signal
starting at 1 hr and a degradation of the receptor beginning to be visible at 2 hrs.
Inhibition of the phosphorylation of EphA2 after induction by EphrinA1/Fc was
investigated in NCI-H1299 and MDA-MB-231 cells. Results are presented in figures 5
A and 5B. In both cases, pre-incubation of the cells with any of the two Abs inhibited
phosphorylation of the EphA2 receptor by EphrinA1/Fc.
We can conclude that hu2H 11 and hu2H11R35R74 have similar inhibitory activity on
the EphA2 receptor.
Example 3
Binding Characterization of Conjugated Anti-EphA2 Antibodies, hu2H11R35R74 and
hu2H11.
The interaction of the anti-EphA2 antibodies hu2H11 and hu2H11R35R74, either
naked or conjugated to DM4 with the PEG4-NHAc linker, with immobilized EphA2-Fc
was monitored by surface plasmon resonance detection using a BIAcore 3000
instrument (GE healthcare, N°CH321). EphA2-Fc (10 ug/ml; Acetate buffer
pH = 4.5) was coupled to the matrix of a C1 sensor chip (GE healthcare BR-1005-40 )
at 10 ul/min using a standard amine coupling protocol with EDC (N-ethyl-N'-[dimethyi-
aminopropyl]carbodiimide)/NHS (N-hydroxysuccinimide). The density was controlled at
an increased response level of-100 response units (RU) in kinetic binding
experiments. IgGs were diluted in 0.01 M Hepes, pH 7.4 containing 0.15 M NaCI, 3mM
EDTA, and 0.005% P20. All subsequent dilutions were made in the same buffer. All

binding experiments were performed at 25 °C with IgG concentrations typically ranging
from 50 to 0.2 nM at a flow rate of 50 pl/min.
Data were collected for approximately 12 min (2 min association time and 10 min
dissociation time) and 1 min pulse at 30 pl/min of 1M NaCI, 50 mM NaOH was used to
regenerate the surfaces. IgGs were also flowed over an uncoated cell and the
sensorgrams from the blank runs were substracted from those obtained with EphA2-Fc
coupled chips. Data were fitted to a 1:1 Langmuir binding model with drifting baseline.
This algorithm calculates both the kon and the kon, from which the apparent equilibrium
dissociation constant KDl is deduced as the ratio of the two rate constants (koff/kon). The
values obtained are indicated in Table I.
The affinity constant was first measured with both naked hu2H11 and hu2H11R35R74.
Analysis of the binding data of hu2H11 to EphA2-Fc give a KD of 0.30 nM; this value
was similar to the KD of hu2H11R35R74 (0.22 nM). However, when conjugated
antibodies were used in the assay, the results were dramatically different: conjugated
hu2H11 with a high drug-to-antibody-ratio such as 7.5 displayed a KD increased by 5.4
fold, when compared to the naked antibody (1.62 nM vs. 0.30 nM). On the other hand,
the KD the hu2H11R35R74 conjugated at a drug-to-antibody ratio of 7.4 was not
different from the one of the naked antibody (0.27nM vs. 0.22 nM). Moreover, the KD
was not significantly different for drug-to-antibody ratios ranging from 5.6 to 8.4.
We conclude that the affinity of the 2H11R35R74 antibody is not affected by
conjugation, even at high-drug-to-antibody ratios.
Example 4
Inhibition of growth of EphA2 expressing tumor cells by humanized 2H11R35R74-
PEG4-Mal-DM4
Inhibition of Lovo tumor cells
Lovo tumor ceils (2000 per well) were plated in 96-well tissue culture plates in complete
serum-containing media. Conjugates were serially diluted and added to triplicate wells
at concentrations ranging between 10"7 and 10"12 M. Cells were cultured at 37°C/5%
C02 in the presence of the antibody-cytotoxic compound conjugates for 5 days, after
which time a 4 h WST8 assay was performed according to the manufacturer's
instructions (Dojindo Cell Counting Kit-8, Cat.#CK04) to evaluate cell survival and
growth. Cell-free reagent blanks were subtracted from the test well readings and the

data were plotted as surviving fractions obtained by dividing readings of the conjugate-
treated cells by the average of readings from control wells of vehicle-treated cells.
The cytotoxic potency of two lots of hu2H11 R35R74-PEG4-Mal-DM4 at high
maytansine/antibody ratios (6,70 D/A and 7.00 D/A) was compared with that of a wild-
type hu2H11-PEG4-Mal-DM4 conjugate having a comparable maytansine/antibody
ratio (6.99 D/A). As may be seen in Figure 7, the two hu2H11R35R74-PEG4-Mal-DM4
conjugates showed higher potency than the corresponding conjugate of the wild-type
hu2H11 against the EphA2-positive Lovo cell line (Table II).
Example 5
Inhibition of growth of EphA2 expressing tumor cells by humanized 2H11R35R74-
PEG4-NHAC-DM4
Inhibition of growth of MDA-MB231 and SKMEL28
Cells in exponential phase of growth were trypsinized and resuspended in their
respective culture medium (DMEM/F12 Gibco #21331; 10% SVF Gibco # 10500-056;
2nM Glutamine Gibco #25030 for MDA-MB231 cells; DMEM (Gibco # 11960) 10% SVF
Gibco # 10500-056; 2nM Glutamine Gibco # 25030 for SKMEL-28 cells). Cell
suspension was distributed in 96-well Cytostar culture plates (GE Healthcare Europe, #
RPNQ0163 ) in complete serum-containing media at a density of 5000 cells/well (MDA-
MB231, SKMEL-28). After coating for 4 hours, serial dilutions of conjugates were
added to triplicate wells at concentrations ranging between 10-7 and 10"12 M. Cells
were cultured at 37°C/5% C02 in the presence of the antibody-cytotoxic compound
conjugates for 3 days. The 4th day, 10 ul of a solution of 14C thymidine (0.1 uCi/well
(Perkin Elmer # NEC56825000) was added to each well. The uptake of 14C thymidine
was measured 96 hours after the experiment has been started with a microbeta
radioactive counter (Perkin Elmer). Cell-free reagent blanks were substracted from the
test well readings and the data were plotted as surviving fractions obtained by dividing
readings of the conjugate-treated cells by the average of readings from control wells of
vehicle-treated cells. In some experiments, the naked antibody (2H11 or 2H11R35R74)
was added to the wells at a concentration of 1 uM at the beginning of the experiment,
and inhibition of proliferation was measured as previously described.
Results
Results reported in table III suggest that the 2H11R35R74-PEG4-NHAc-DM4 as well
as the 2H11R35R74-PEG4-Mal-DM4 conjugates are better than former conjugates in

terms of in vitro proliferation inhibition on MDA-MB231 cells and in vitro selectivity
against antigen minus cells (SKMEL-28)
Example 6
Pharmacokinetics Study
The present study was designed to evaluate the pharmacokinetic behavior of the
hu2H11R35R74-PEG4-NHAC-DM4 conjugate (DAR = 5.5) in comparison to the
hu2H11-SPDB-DM4 conjugate (DAR = 3.9) in CD-1 mice. Animals received 20 mg/kg
by IV route of each conjugate and blood was collected at 0, 8, 24, 48, 72, 120, 168,
240, 336, 504, 672 h post-injection. Plasma levels of antibody drug conjugates were
measured to establish basic single dose pharmacokinetic parameters under standard
conditions. The plasma concentrations of conjugates and their antibody component
(total antibody, a sum of conjugated antibody and any de-conjugated antibody) were
measured by specific ELISA techniques.
The clearance-related pharmacokinetic parameters for the antibody component of
hu2H11-SPDB-DM4 (total) were calculated to be CI (clearance) of 0.00043 L/h/kg, T1/2
(terminal half life) of 160 h, AUC 0-inf (area under the concentration time-curves from
time zero to infinity) of 47,000,000 ng.h/mL, Vdss (steady-state volume of distribution)
of 0.095 L/kg and CO (concentration at time 0) of 270,000 ng/mL.
The pharmacokinetic parameters for hu2H11-SPDB-DM4 conjugate were calculated to
be CI of 0.00070 L/h/kg, T1/2 of 87 h, AUC 0-inf of 28,000,000 ng.h/mL, Vdss of 0.080
L/kg and CO of 360,000 ng/mL
The PK parameters for the antibody component of hu2H11 R35R74-PEG4-NHAc-DM4
(total) were CI of 0.00027 L/h/kg, T1/2 of 190 h, AUC 0-inf of 73,000,000 ng.h/mL, Vdss
of 0.068 L/kg and CO of 530,000 ng/mL.
Finally, the hu2H11 R35R74-PEG4-NHAc-DM4 conjugate showed values of: 0.00036
L/h/kg for clearance, T1/2 of 150 h, AUC 0-inf of 56,000,000 ng.h/mL, Vdss of 0.069
L/kg and CO of 600,000 ng/mL.
In conclusion, hu2H11-SPDB-DM4 conjugate is cleared faster than hu2H11R35R74-
PEG4-NHAc-DM4. Furthermore, compared with hu2H11-SPDB-DM4, hu2H11R35R74-
PEG4-NHAc-DM4 showed a better exposure (AUC 0-inf) and a narrower separation
between the total conjugated antibody and total antibody curves (compare figs 7 and
8).

Example 7
Antitumor effect of hu2H11 R35R74-PEG4-NHAc-DM4 and hu2H11-PEG4-NHAc-DM4
conjugate against a primary colon tumor, CR-LRB-004P implanted in female SCID
mice
Materials and Methods
For the evaluation of anti-tumor activity of conjugates, animals were weighed daily and
tumors were measured 2 times weekly by caliper. Tumor weights were calculated using
the formula mass (mg) = [length (mm) * width (mm)2]^. Antitumor activity evaluation
was done at the highest non toxic dose (HNTD).
A dosage producing a 20% body weight loss (bwl) at nadir (mean of group) or 10% or
more drug deaths, was considered an excessively toxic dosage. Animal body weights
included the tumor weights. The primary efficacy end points are AT/AC, percent
median regression, partial and complete regressions (PR and CR) and Tumor free
survivors (TFS).
Changes in tumor volume for each treated (T) and control (C) are calculated for each
tumor by subtracting the tumor volume on the day of first treatment (staging day) from
the tumor volume on the specified observation day. The median AT is calculated for the
treated group and the median AC is calculated for the control group. Then the ratio
AT/AC is calculated and expressed as a percentage:

The dose is considered as therapeutically active when AT/AC is lower than 40% and
very active when AT/AC is lower than 10%. If AT/AC is lower than 0, the dose is
considered as highly active and the percentage of regression is dated (ref 1):
% tumor regression: is defined as the % of tumor volume decrease in the treated group
at a specified observation day compared to its volume on the first day of first treatment.
At a specific time point and for each animal, % regression is calculated. The median %
regression is then calculated for the group.


Partial regression (PR): Regressions are defined as partial if the tumor volume
decreases to 50 % of the tumor volume at the start of treatment.
Complete regression (CR): Complete regression is achieved when tumor volume = 0
mm3 (CR is considered when tumor volume cannot be recorded).
TFS: Tumor free is defined as the animals with undetectable tumors at the end of the
study (>100 days post last treatment)
Results
The antitumor effect of hu2H11-PEG4-NHAc-DM4 -conjugate and hu2H11R35R74-
PEG4-NHAc-DM4 was evaluated at 2 dose levels against a measurable primary colon
tumor, CR-LRB-004P, strongly expressing target, S.C. implanted in female SCID mice.
Control group was left untreated. Doses were expressed in milligram of protein per
kilogram. hu2H11R35R74-PEG4-NHAC-DM4 was administered at 40 and 10 mg/kg, by
an intravenous (IV) bolus injection, on day 15. To give equivalent dose of DM4,
hu2H11-PEG4-NHAc-DM4 was administered at 44 and 11 mg/kg.
As shown on Table V, using a single administration schedule in CR-LRB-004P tumor,
hu2H11R35R74-PEG4-NHAC-DM4 was active at 40 and 10 mg/kg with a AT/AC of 28
and 39% respectively while hu2H11-PEG4-NHAc-DM4 was active only at 44 mg/kg
with a AT/AC of 26%. At 10 mg/kg, hu2H11-PEG4-NHAc-DM4 was not active in this
model.
From these results, hu2H11R35R74-PEG4-NHAc-DM4-conjugate at lower dose
exhibited a better activity than hu2H11-PEG4-NHAc-DM4 conjugate.
Example 8
Impact of the PAR on the anti-tumor activity of hu2H11R35R74-PEG4-NHAc-DM4
against prostatic adenocarcinoma PC-3 in SCID female mice
The effect of the DAR on the antitumor activity of antibody drug conjugate
hu2H11R35R74-PEG4-NHAc-DM4 was evaluated comparing two low effective doses
at six different Drug antibody ratios (DAR) on Prostatic PC-3 tumors S.C. implanted in

female SCID. Control group was left untreated. Doses were expressed in milligram of
protein per kilogram. DAR was determined by an UV method. hu2H11R35R74-PEG4-
NHAc-DM4 was administered at 10 and 5 mg/kg with DARs at 3.4, 4.4, 5.9, 6.2, 7.4
and 8.4, respectively, by an intravenous (IV) bolus injection, on day 16.
Materials and Methods
For the evaluation of anti-tumor activity of conjugates, animals were weighed daily and
tumors were measured 2 times weekly by caliper. Tumor weights were calculated using
the formula mass (mg) = [length (mm) * width (mmf|/2. Antitumor activity evaluation
was done at the highest non toxic dose (HNTD).
A dosage producing a 20% body weight loss (bwl) at nadir (mean of group) or 10% or
more drug deaths, was considered an excessively toxic dosage. Animal body weights
included the tumor weights. The primary efficacy end points are AT/AC, percent
median regression, partial and complete regressions (PR and CR) and Tumor free
survivors (TFS).
Changes in tumor volume for each treated (T) and control (C) are calculated for each
tumor by subtracting the tumor volume on the day of first treatment (staging day) from
the tumor volume on the specified observation day. The median AT is calculated for the
treated group and the median AC is calculated for the control group. Then the ratio
AT/AC is calculated and expressed as a percentage:

The dose is considered as therapeutically active when AT/AC is lower than 40% and
very active when AT/AC is lower than 10%. If AT/AC is lower than 0, the dose is
considered as highly active and the percentage of regression is dated (ref 1):
% tumor regression: is defined as the % of tumor volume decrease in the treated group
at a specified observation day compared to its volume on the first day of first treatment.
At a specific time point and for each animal, % regression is calculated. The median %
regression is then calculated for the group.


Partial regression (PR): Regressions are defined as partial if the tumor volume
decreases to 50 % of the tumor volume at the start of treatment.
Complete regression (CR): Complete regression is achieved when tumor volume = 0
mm3 (CR is considered when tumor volume cannot be recorded).
TFS: Tumor free is defined as the animals with undetectable tumors at the end of the
study (>100 days post last treatment)
Results
As illustrated in table VI using a single administration schedule, hu2H11R35R74-
PEG4-NHAc-DM4 at 10 mg/kg showed an activity from a DAR of 4.4 to the higher DAR
of 8.4.
At 5 mg/kg, hu2H11 R35R74-PEG4-NHAc-DM4 showed an activity from a DAR of 5.9
to the higher DAR of 8.4.
In conclusion, the DAR has an effect on the tumor activity of hu2H11R35R74-PEG4-
NHAc-DM4 and one can deduce from these results that the optimal DAR of
hu2H 11 R35R74-PEG4-NHAc-DM4 is at least equal to 5.9.
Example 9
Impact of the DAR on the PK parameters of hu2H11R35R74-PEG4-NHAc-DM4.
The pharmacokinetic properties of hu2H11 R35R74-PEG4-NHAc-DM4 at different drug-
antibody ratio (DAR) were evaluated in male CD-1 mice after a single intravenous
administration of 20 mg/kg of conjugate. Plasma levels of conjugates were measured
to establish basic single dose pharmacokinetic parameters under standard conditions.
PK parameters were compared to those of the naked parental antibody. The plasma
concentrations of conjugates and their antibody component (total antibody, a sum of
conjugated antibody and any de-conjugated antibody) were measured by specific
ELISA techniques.
Results (see figures 9A and 9B) show a reverse correlation between the DAR values
and the exposure to the total antibody components with AUC 0-inf values of

. 83,000,000, 61,000,000,48,000,000, 46,000,000,41,000,000 and 27,000,000 ng-h/mL
for DAR of 0, 3.4, 4.3, 5.9, 6.6 and 7.4, respectively.
Similarly there is a reverse correlation between the DAR values and the exposure to
the conjugate with AUC 0-inf values of 39,000,000, 30,000,000,27,000,000,
29,000,000 and 20,000,000 ng-h/mL for DAR of 3.4, 4.3, 5.9, 6.6 and 7.4, respectively.
There is a perfect correlation between the DAR values and the elimination of the
antibody component with CI values of 0.00024, 0.00033, 0.00042, 0.00043, 0.00049
and 0.00074 L/h/kg for DAR 0, 3.4, 4.3, 5.9, 6.6 and 7.4, respectively.
Similarly there is almost a perfect correlation between the DAR values and the
elimination of the conjugate with CI values of 0.00051, 0.00066, 0.00075, 0.00069,
0.00099 L/h/kg for DAR 3.4, 4.3, 5.9, 6.6 and 7.4, respectively.
In conclusion, the DAR has an impact on the PK parameters with a decreased
exposure and an increased elimination when the DAR increases.
According to results from efficacy and PK evaluation, the optimal DAR will be included
between 5.9 and 7.4.
Example 10
Evaluation of hu2H11R35R74-PEG4-NHAc-PM4 against prostatic
adenocarcinoma PC-3 in SCID female mice
The antitumor effect of antibody drug conjugate hu2H11 R35R74-PEG4-NHAc-DM4
was evaluated at 8 dose levels against measurable prostatic PC-3 tumor, strongly
expressing target, S.C. implanted in female SCID mice. Control group was left
untreated. Doses are expressed in milligram of protein per kilogram.
They were administered at 160,120, 80, 40, 20,10, 5 and 2.5 mg/kg, by an
intravenous (IV) bolus injection, on day 17.
Materials and Methods
For the evaluation of anti-tumor activity of conjugates, animals were weighed daily and
tumors were measured 2 times weekly by caliper. Tumor weights were calculated using
the formula mass (mg) = [length (mm) * width (mm)2]^. Antitumor activity evaluation
was done at the highest non toxic dose (HNTD).

A dosage producing a 20% body weight loss (bwl) at nadir (mean of group) or 10% or
more drug deaths, was considered an excessively toxic dosage. Animal body weights
included the tumor weights. The primary efficacy end points are AT/AC, percent
median regression, partial and complete regressions (PR and CR) and Tumor free
survivors (TFS).
Changes in tumor volume for each treated (T) and control (C) are calculated for each
tumor by subtracting the tumor volume on the day of first treatment (staging day) from
the tumor volume on the specified observation day. The median AT is calculated for the
treated group and the median AC is calculated for the control group. Then the ratio
AT/AC is calculated and expressed as a percentage:

The dose is considered as therapeutically active when AT/AC is lower than 40% and
very active when AT/AC is lower than 10%. If AT/AC is lower than 0, the dose is
considered as highly active and the percentage of regression is dated (ref 1):
% tumor regression: is defined as the % of tumor volume decrease in the treated group
at a specified observation day compared to its volume on the first day of first treatment.
At a specific time point and for each animal, % regression is calculated. The median %
regression is then calculated for the group.

Partial regression (PR): Regressions are defined as partial if the tumor volume
decreases to 50 % of the tumor volume at the start of treatment.
Complete regression fCR): Complete regression is achieved when tumor volume = 0
mm3 (CR is considered when tumor volume cannot be recorded).
TFS: Tumor free is defined as the animals with undetectable tumors at the end of the
study (>100 days post last treatment)

Results
Using a single administration schedule, the highest dose of conjugate tested
(160mg/kg) was found to be toxic, inducing body weight loss and drug-related deaths.
As illustrated on table VIM at the HNTD (120 mg/kg) and other lowest doses, the
compound was highly active. For all doses except for 2.5 mg/kg, hu2H11R35R74-
PEG4-NHAc-DM4 induced partial regressions and for 120, 80 and 20 mg/kg, it induced
complete regressions. In addition, the tumor model was cachexic, and the
administration of the compound reduced the body weight loss at nadir in comparison
with Control
In conclusion, hu2H11R35R74-PEG4-NHAc-DM4 showed a high activity with a good
dose-effect on Prostatic PC-3 tumor model.
Example 11
Mapping of the epitope and identification of the paratope by structure
determination of the crystal structure of the extra-cellular domain of EphA2
receptor in complex with the Fab fragment from hu2H11-R35-R74 at 2.1 A
resolution.
Material and Methods
Initial crystallization trials were made on glycosylated extra-cellular domain of EphA2
receptor-Fc in complex with the Fab of hu2H11R35-74. Crystals were obtained only in
presence of trypsin. These crystals were analyzed and peptide mapping showed that
they contained the Fab, some portion of the N-terminal domain of extra-cellular domain
of EphA2 and of the Fc. Another batch of complex was produced, this time using
aglycosylated extra-cellular domain of EphA2 receptor with terminal His-tag, in
complex with recombinant Fab fragment from hu2H11R34-R74-His. Both constructs of
the EphA2 receptor provide the same structure of the complex between EphA2
receptor and hu2H11R34-R75.
Extra-cellular region of EphA2 receptor is made of 4 domains and has been shown to
be very flexible: the LBD (Ligand Binding Domain), the CRD (Cystein-Rich Domain)
and two Fibronectin Repeats, nFN3 and cFN3. The different domains were used as
search models for molecular replacement calculation either alone or in combination;

models of the variable and constant domains of the Fab were also produced and used,
as well as a model of the Fc (pdb code 1IGT) to solve the crystal structure.
Various crystallization conditions were tested and a 2.1A dataset collected at the ESRF
was used to solve the structure of the complex. It has enabled us to analyze the
interface between the extra-cellular domain of EphA2 receptor and hu2H11R34-R74.
Even though the full length EphA2 extracellular region (25-534) was present initially,
the crystals contain only the LBD and CRD domains of the protein (residues 25-325 or
327 depending on the crystal (here a sequential numbering is used).
Results
Epitope mapping
Figure 10: illustrates the mapping of the extra-cellular domain of EphA2 receptor
epitope for hu2H11R34-R75 (epitope residues are defined as residues which contain
atoms that lie within 4A from any atom of the CDR residues of hu2H11R34-R74 Fab
fragment).
The epitope of extra-cellular domain of EphA2 receptor when bound to Fab fragment of
hu2H11R34-R74 is a conformational epitope that includes residues of the LBD domain
Gly49, Lys50, Gly51, Asp53, Cys70, Asn71, Val72, Met73, Ser74, Gly75, Gln77,
Phe108, Pro109, Gly110, Gly111, Ser113 and Ser114.
Figure 11: shows residues from the extra-cellular domain of EphA2 receptor that are
part of the epitope (represented in dark grey); residues in light grey are not visible in
the crystal structures.
Figure 12 A: represents the overall structure of the complex and figure 11B is a
magnification of the part with the two mutations introduced in position 35 and 74..
Conjugation of hu2H11 occurs on surface lysine residues. Two of these were mutated
into arginine. These two residues, heavy chain R74 and light chain R35 are depicted in
figure 12B:
R74 lies in proximity of the interface, facing away from the extra-cellular domain of
EphA2 receptor and about 10A from the nearest extra-cellular domain

R35 is one of the paratope residues and it makes an H-bond with Asp53 from the
extra-cellular domain of EphA2 receptor. A lysine reside would very likely make the
same interactions with the antigen and its conjugation would clearly be detrimental for
antibody binding.
Paratope analysis
The interface between the extra-celiuiar domain of EphA2 receptor and hu2H11R35-
R74 does not involve all CDR loop. As can be seen from Fig 12, mainly the heavy
chain CDRs are involved in binding. This suggests that changes in the CDRs of
hu2H11, particularly in the light chain but not exclusively, can be introduced without
adversely affect binding to the EphA2 receptor. Loop L3 of the light chain is not
involved at all in the interface, while only one residue at the end of L1 (Arg35) interacts
with the extra-cellular domain of EphA2. This implies that changes in loop L3 should
not impact binding to EphA2 and that loop L1 should tolerate mutations/insertions of
amino acid residues as long as they don't destabilize the conformation and orientation
of Arg35 residue.
The paratope of hu2H11-R35-R74 for EphA2 receptor involves the following residues
of the light chain: Arg35 of Loop L1, Tyr54, Arg58 and Asp60 of L2. It involves in the
heavy chain the following residues: Thr30, Ala31, Tyr32 and Tyr33 of Loop H1, Asn52,
Tyr54, Asn55 and Phe57 of H2 and Glu99, PhelOO, Tyr101, Gly102, Tyr103 and
Tyr105 of H3. A sequential numbering scheme is used for the light and heavy chains.
It might be possible to improve the affinity of the hu2H11-R35-R74 antibody for EphA2
receptor using the structural data and for instance by the approach described Clark et
al (Protein Science (2006), 15:949-960) or Lippow et al (Nature Biotech (2007),
10:1171-76).
Loop H1: it might be possible to create additional interactions, for instance with Asp76,
by judicious mutation of Thr H28.
Interactions between the light chain and Epha2 receptor do not involve many residues.
Nevertheless, creating new interactions would probably require significant insertions in
either loop L1 or L3.
Interactions between these loops and EphA2 receptor occur via rather large or long
residues: any change in this environment might result in loss of binding affinity.

This X-ray structure highlights residues from the CDRs that can be mutated, that
should not impact binding to EphA2.
In the following descriptions, residues from the paratope should not be modified, unless
so specified to preserve the binding to the EphA2 receptor. It also understood that
mutations in the CDRs should not affect the conformation and orientation of the
residues from the paratope to preserve binding to the EphA2 receptor. Residue
numbering is sequential and does not follow Kabbat conventions as used in Al-Lazikani
((1997) J. Mol. Biol. 273, 927-948). "X" represents "any residue", while "-" indicates that
no modification should be made at this position.
In the CDR L1 (SEQ ID °4) except D 33 and R35 there is no restriction on sequence
provided length of the loop is conserved and it adopts canonical structure L1-kappa4
as described in J. Mol. Biol. (1992) 227: 799-817, J. Mol. Biol. (1992) 227: 776-798 and
in Al-Lazikani et al (cited supra). This means that torsion angles of peptide bonds fall
within the accepted range defined in Fig 5 of Al-Lazikani et al (cited supra).
Arg35 has been mutated to prevent conjugation on this residue. A Lys would
nevertheless make the same interactions with EphA2 and hence it is predicted that
parent hu2H11 antibody will make the same interaction with EphA2 receptor and
engage the same epitope of EphA2 receptor.
In the CDR L2 (SEQ ID N°5) both Leu could be replaced by the conservative
substitution such as lle. Val might be replaced by lle (less favorable). The first Ser of
An Asp is the preferred residue at position 33.


the loop can be replaced by any residue, while the second might be replaced by
another hydrophilic/charged reside. The Tyr residue (54) should not be changed.

In the CDR l_3 (SEQ ID N°6) there is no restriction on sequence nor length of the loop
as long as it adopts canonical structure L3-kappa1 as defined in Al-Lazikani et al (cited
supra).

Around the CDR H1 (SEQ ID N°1) GYTFTAYY) the first Thr of the loop (Thr 28) is a
potential position for affinity maturation.

In the CDR H2 (SEQ ID N°2) the Phe and Tyr can be replaced by any aromatic residue
(F/Y/W).


As for the CDR H3 (EFYGYRYFDV) no change should be made to this loop.

Ephrin binding site
The hu2H11 antibody shows functional activity: it inhibits ephrin-A1 binding and ephrin-
A1 induced phosphorylation of EphA2. The structure of the LBD and CRD domains of
the EphA2 receptor in complex with ephrinAI has been published (PDB code 3MBW).
When superimposing this structure on that of EphA2 in complex with the Fab fragment
of hu2H11R35R74, it clearly appears that the light chain of the Fab fragment of
2H11R35R74 overlaps with the binding area of ephrin-A1 on EphA2 and hence
confirms the competitive nature of the hu2H11 antibody.
Example 12
Inhibition of growth of EphA2 expressing tumor cells by humanized
2H11R35R74-DM4 conjugates
MDA-MB231 cells in exponential phase of growth were trypsinized and resuspended in
culture medium (DMEM/F12 Gibco #21331; 10% SVF Gibco# 10500-056; 2nM
Glutamine Gibco #). Cell suspension was distributed in 96-well Cytostar culture plates
(GE Healthcare Europe, # RPNQ0163 ) in complete serum-containing media at a
density of 5000 cells/well After coating for 4 hours, serial dilutions of conjugates were
added to triplicate wells at concentrations ranging between 10-7 and 10-12 M. Cells

were cultured at 37°C/5% C02 in the presence of the antibody-cytotoxic compound
conjugates for 3 days. The 4th day, 10 ul of a solution of 14C thymidine (0.1 uCi/well
(Perkin Elmer # NEC56825000) was added to each well. The uptake of 14C thymidine
was measured 96 hours after the experiment has been started with a microbeta
radioactive counter (Perkin Elmer). Cell-free reagent blanks were substracted from the
test well readings and the data were plotted as surviving fractions obtained by dividing
readings of the conjugate-treated cells by the average of readings from control wells of
vehicle-treated cells. In some experiments, the naked antibody (2H11 or 2H11R35R74)
was added to the wells at a concentration of 1 uM at the beginning of the experiment,
and inhibition of proliferation was measured as previously described.
Results
Results reported in table IX suggest that all 2H11R35R74 DM4 conjugates tested are
as potent as the hu2H11 R35R74-Peg4-AcNH-DM4 in inhibiting the growth of MDA-
MB231 cells.
Example 13
Evaluation of different linkers on the anti-tumor activity of 2h11-DM4 conjugates
against colon adenocarcinoma Lovo in SCID female mice
Materials and Methods
For the evaluation of anti-tumor activity of conjugates, animals were weighed daily and
tumors were measured 2 times weekly by caliper. Tumor weights were calculated using
the formula mass (mg) = [length (mm) x width (mm)2]/2. Antitumor activity evaluation
was done at the highest non toxic dose (HNTD).
A dosage producing a 20% body weight loss (bwl) at nadir (mean of group) or 10% or
more drug deaths, was considered an excessively toxic dosage. Animal body weights
included the tumor weights. The primary efficacy end points are AT/AC, percent
median regression, partial and complete regressions (PR and CR) and Tumor free
survivors (TFS).
Changes in tumor volume for each treated (T) and control (C) are calculated for each
tumor by subtracting the tumor volume on the day of first treatment (staging day) from
the tumor volume on the specified observation day. The median AT is calculated for the
treated group and the median AC is calculated for the control group. Then the ratio

ΔT/ΔC is calculated and expressed as a percentage: ,

The dose is considered as therapeutically active when ΔT/ΔC is lower than 40% and
very active when ΔT/ΔC is lower than 10%. If ΔT/ΔC is lower than 0, the dose is
considered as highly active and the percentage of regression is dated (ref 1):
% tumor regression: is defined as the % of tumor volume decrease in the treated group
at a specified observation day compared to its volume on the first day of first treatment.
At a specific time point and for each animal, % regression is calculated. The median %
regression is then calculated for the group.

Partial regression (PR): Regressions are defined as partial if the tumor volume
decreases to 50 % of the tumor volume at the start of treatment.
Complete regression (CR): Complete regression is achieved when tumor volume = 0
mm3 (CR is considered when tumor volume cannot be recorded).
TFS: Tumor free is defined as the animals with undetectable tumors at the end of the
study (>100 days post last treatment)
Results
The antitumor activity of antibody drug 2M1-DM4 conjugates with different non
cleavable linkers hu2H11-R35R74-PEG4-AcNH-DM4, hu2H11-R35R74-PEG8-AcNH-
DM4, hu2H11-R35R74-PEG4-AcNMe-DM4, hu2H11-R35R74-PEG4-Allyl-DM4 and
hu2H11-R35R74-Acetyl-DM4 was evaluated comparing the same dose of 600 ug of
DM4/kg on Colon Lovo tumors S.C. implanted in female SCID. Control group was left
untreated. Doses were expressed in microgram of DM4 per kilogram. The conjugates
were administered by an intravenous (IV) bolus injection, on day 14.
As illustrated in Table X using a single administration schedule, ail five conjugate
exhibited the same high activity on Lovo tumor model with a ΔT/ΔC < 0 and the same
impact on the body weight loss (-13.3 % in the control group versus -9.5% to 11.2% for
treated groups). The hu2H11-R35R74-PEG4-AcNH-DM4 exhibited the best efficacy
with a tumor regression of 82% and 1CR compared to tumor regressions of 72%, 69%,

41% and 33% without CR for hu2H11-R35R74-PEG4-AcNMe-DM4, hu2H11-R35R74-
Acetyl-DM4, hu2H11-R35R74-PEG4-Ally]-DM4 and hu2H11-R35R74-PEG8-AcNH-
DM4, respectively.
TABLES
Table I: Biacore analysis of the binding of hu2H11R35R74 and conjugates thereof to
EphA2


Table II: Cytotoxic activity of hu2H11R35R74-PEG4-Mal-DM4 on Lovo cells

Table III: Cytotoxicity of hu2H11R35R74 and conjugates thereof on MDA MB231 cells
and SKMEL-28 cells

CLAIMS
1. An antibody or an epitope-binding fragment thereof that specifically binds to an
EphA2 receptor and comprising at least one heavy chain and at least one light
chain, wherein said heavy chain comprises three sequential complementarity-
determining regions having amino acid sequences represented by SEQ ID NOS: 1,
2, and 3, and wherein said light chain comprises three sequential complementarity-
determining regions having amino acid sequences represented by SEQ ID NOS: 4,
5, and 6.
2. An antibody or an epitope-binding fragment thereof according to claim 1, wherein
said monoclonal antibody or epitope-binding fragment thereof is a humanized or
resurfaced antibody or epitope-binding fragment thereof.
3. An antibody or an epitope-binding fragment thereof according to claim 1, wherein
said heavy chain comprises an amino acid sequence consisting of SEQ ID NO: 12
and wherein said light chain comprises an amino acid sequence consisting of SEQ
ID NO: 14.
4. An antibody or an epitope-binding fragment thereof according to claim 1, wherein
said heavy chain consists in an amino acid sequence SEQ ID NO: 18, and wherein
said light chain consists in an amino acid sequence SEQ ID NO: 16.
5. A conjugate of an antibody or an epitope-binding fragment according to claims 1 -4,
wherein said conjugate comprises a cytotoxic agent chosen between:
a) the maytansinoid of formula (XIII):


b) the maytansinoid of formula (XIV):

c) the maytansinoid of formula (XXIV):


d) the maytansinoid of formula (XXV):

e) the maytansinoid of formula (XXVI):


f) the maytansinoid of formula (XXVII):

6. A conjugate according to claim 5, wherein said cytotoxic agent is covalently bound
to the antibody.
7. A conjugate according to any of claims 5 or 6, wherein the cytotoxic agent is the
maytansinoid of formula (XIII).
8. A method for preparing a conjugate comprising the steps of:
(i) bringing into contact an optionally-buffered aqueous solution of a cell-binding agent
with a solution of a cytotoxic compound;
(ii) then optionally separating the conjugate which was formed in (i) from the unreacted
reagents and any aggregate which may be present in the solution;
wherein the cell-binding agent is an antibody according to claims 1-4, and a cytotoxic
agent chosen between:
the compound of formula (XVII):


wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-
sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom; and
the compound of formula (XVIII):

wherein Y is N-succinimidyloxy, N-sulfosuccinimidyloxy, N-phthalimidyloxy, N-
sulfophthalimidyloxy, 2-nitrophenyloxy, 4-nitrophenyloxy, 2,4-dinitrophenyloxy, 3-

sulfonyl-4-nitrophenyloxy, 3-carboxy-4-nitrophenyloxy, imidazolyl, or halogen atom; and
and of the formula (XVI):
9. A conjugate obtainable by the method of claim 8.
10. A conjugate according to claim 9, said conjugate having a structure chosen
between the structures of the formula (XV):



wherein Ab is an antibody according to claims 1-3 and n is an integer comprised
between 1 and 15.
11. The antibody-drug conjugate of claim 10 wherein n is comprised between 4 and 7.
12. The antibody-drug conjugate of claim 11 having the structure of formula (XV).
13. A pharmaceutical composition containing an antibody or epitope-binding fragment
thereof according to any of claims 1-4, or a conjugate according to any of claims 6,
7 and 8, and a pharmaceutically acceptable carrier or excipient.
14. An antibody or epitope-binding fragment thereof according to any of claims 1-4, or
a conjugate according to any of claims 5, 6, and 7, for use as a medicament.
15. The use of an antibody or epitope-binding fragment thereof according to any of
claims 1-4, or a conjugate according to any of claims 5, 6, and 7, to make a
medicament to treat cancer.
16. The use of claim 15 wherein the cancer is chosen between carcinoma, including
that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach,
cervix, thyroid and skin; including squamous cell carcinoma ; hematopoietic tumors
of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute
lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma ;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous
leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including
fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma,
seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and
peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and
schwannomas; tumors of mesenchymal origin, including fibrosarcoma,
rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma,
xeroderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer
and teratocarcinoma.
17. An antibody or an epitope-binding fragment thereof according to claims 1-4,
wherein the paratope of said monoclonal antibody or epitope-binding fragment
thereof comprises in the light chain: Arg35 of Loop L1, Tyr54, Arg58 and Asp60 of
L2.
18. An antibody or an epitope-binding fragment thereof according to claims 1-4,
wherein the paratope of said monoclonal antibody or epitope-binding fragment
thereof comprises in the heavy chain: Thr30, Ala31, Tyr32 and Tyr33 of Loop H1,

Asn52, Tyr54, Asn55 and Phe57 of H2 and Glu99, Phe100, Tyr101, Gly102,
Tyr103 and Tyr 105 of H3.
19. An antibody or an epitope-binding fragment thereof according to claims 1-4,
wherein said monoclonal antibody or epitope-binding fragment thereof comprises
mutation at the position: Thr H28.
20. An antibody or an epitope-binding fragment thereof according to claims 1-4,
wherein said monoclonal antibody or epitope-binding fragment thereof comprises
mutation at one of few of the following positions on the light chain: 35, 26 to 31, 34
to 37, 55, 56, 57, 59 and 94 to 102.
21. An antibody or an epitope-binding fragment thereof according to claims 1-4,
wherein said monoclonal antibody or epitope-binding fragment thereof comprises
mutation at one of few of the following positions on the heavy chain: 28, 54 and 57.
22. An antibody or an epitope-binding fragment thereof according to claims 1-4, which
specifically binds to an epitope of human EphA2 receptor comprising residues
Gly49, Lys50, Gly51, Asp53, Cys70, Asn71, Val72, Met73, Ser74, Gly75, Gln77,
Phe108, Pro109, Gly110, Gly111, Ser113 and Ser114.of the LBD from the extra-
cellular domain of EphA2 receptor, or a conservatively substituted form thereof.
23. A conjugate according to any of claims 6, 7 and 8 having an average DAR above
4, the DAR being measured with a UV spectrophotometer and determined by the
following equation DAR = CD / CA
with :

A252 and A280 being the absorbances of the conjugate measured on the
UV spectrophotometer at respectively 252 and 280 nm

24. , A conjugate according to claim 23 having an average DAR comprised between. 4
and 10 or 5 and 8.
25. A conjugate according to claim 23 having an average DAR comprised between 5.9
and 7.5.
26. An article of manufacture comprising:

- a) a packaging material
- b) an antibody or epitope-binding fragment thereof according to any of claims 1 -
4, or a conjugate according to any of claims 6 to 8 and 23 to 25, and
- c) a label or package insert contained within said packaging material indicting
that said antibody or epitope-binding fragment thereof is effective for treating
cancer.

Abstract:

The present disclosure
relates to an antibody or an epitope-binding fragment thereof that specifically binds to an EphA2 receptor. It
further relates to a conjugate comprising a cytotoxic agent which is covalently bound to the antibody and a
method for preparing such a conjugate.