ANTIBODY-DRUG CONJUGATES
Field
The present invention generally relates to anti-5T4 antibody-drug conjugates for the
treatment of cancer.
Background
Antibody-drug conjugates (ADCs) combine the binding specificity of monoclonal
antibodies with the potency of chemotherapeutic agents. The technology associated with
the development of monoclonal antibodies to tumor associated target molecules, the use
of more effective cytotoxic agents, and the design of chemical linkers to covalently bind
these components, has progressed rapidly in recent years (Ducry L , et al. Bioconjugate
Chemistry, 2 1:5-13, 2010).
Promising ADCs such as SGN-75 (US2009/1 48942) and trastuzumab-DM1
(US2009/0226465) are currently in clinical trials. However, as other tumor associated
antigens are considered for targets, numerous challenges remain. Each monoclonal
antibody must be characterized separately, an appropriate linker designed, and a suitable
cytotoxic agent identified that retains its potency upon delivery to tumor cells. One must
consider the antigen density on the cancer target and whether normal tissues express the
target antigen. Other considerations include whether the entire ADC is internalized upon
binding the target; whether a cytostatic or cytotoxic drug is preferable when considering
possible normal tissue exposure and/or the type and stage of the cancer being treated;
and, whether the linker connecting the antibody to the drug payload is a cleavable or a
non-cleavable linkage. Furthermore, the antibody to drug moiety conjugation ratio must be
sufficient without compromising the binding activity of the antibody and/or the potency of
the drug. It is evident that ADCs are complex biologies and the challenges to develop an
effective ADC remain significant.
The human 5T4 tumor associated antigen is the target antigen of the present
invention. It has recently been shown that the 5T4 antigen is expressed in high levels on
certain highly tumorigenic cells, also called tumor-initiating cells (WO201 0/1 11659).
Tumor-initiating cells show resistance to standard therapies and are believed to be
responsible for tumor recurrence and metastasis and therefore present yet another
obstacle for ADC development.
The novel anti-5T4 ADCs of the present invention overcome the challenges
associated with ADC technology and provide highly specific and potent ADCs that bind to
tumor cells expressing the 5T4 antigen and deliver sufficient cytotoxic drug to the cells,
thus providing an innovative and effective treatment for cancer.
Summary
In one embodiment, an antibody-drug conjugate of the present invention has the
formula: Ab-(LU-D)p or a pharmaceutically acceptable salt thereof wherein, Ab is an anti-
5T4 antibody or antigen binding portion thereof, comprising a heavy chain variable region
having a VH CDR1 region as shown in SEQ ID NO: 5, a VH CDR2 region as shown in
SEQ ID NO: 6, and a VH CDR3 region as shown in SEQ ID NO: 7; LU is a linker unit
selected from the group consisting of maleimidocaproyl and maleimidocaproyl-Val-Cit-
PABA ; p is an integer from about 1 to about 8; and D is a Drug unit selected from the
group consisting of MMAE, MMAF, and MMAD.
The present invention further provides anti-5T4 antibody-drug conjugates wherein
said anti-5T4 antibody or antigen binding portion thereof, comprises a heavy chain variable
region having (a) a VH CDR1 region as shown in SEQ ID NO: 5, (b) a VH CDR2 region as
shown in SEQ ID NO: 6, and (c) a VH CDR3 region as shown in SEQ ID NO: 7.
The present invention further provides an anti-5T4 antibody-drug conjugate wherein
said anti-5T4 antibody or antigen binding portion thereof, comprises a light chain variable
region having (a) a VL CDR1 region as shown in SEQ ID NO: 8, (b) a VL CDR2 region as
shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown in SEQ ID NO: 10.
The present invention further provides an anti-5T4 antibody-drug conjugate wherein
said anti-5T4 antibody or antigen binding portion thereof, further comprises a heavy chain
variable region having (a) a VH CDR1 region as shown in SEQ ID NO: 5, (b) a VH CDR2
region as shown in SEQ ID NO: 6, and (c) a VH CDR3 region as shown in SEQ ID NO: 7
and a light chain variable region having (a) a VL CDR1 region as shown in SEQ ID NO: 8,
(b) a VL CDR2 region as shown in SEQ ID NO: 9, and (c) a VL CDR3 region as shown in
SEQ ID NO: 10.
The present invention further provides an anti-5T4 antibody-drug conjugate wherein
said anti-5T4 antibody or antigen binding portion thereof, comprises the VH region of SEQ
ID NO: 3 and the VL region of SEQ ID NO: 4.
The present invention further provides an anti-5T4 antibody-drug conjugate wherein
said anti-5T4 antibody consists of a heavy chain having SEQ ID NO: 1 and a light chain
having SEQ ID NO: 2.
The present invention further provides an anti-5T4 antibody-drug conjugate
wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID NO:1 and a light chain
having SEQ ID NO: 2, (b) said LU is maleimidocaproyl, (c) said Drug is MMAF, and (d) p
is an integer of about 4.
The present invention further provides an anti-5T4 antibody-drug conjugate
wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID NO:1 and a light chain
having SEQ ID NO: 2, (b) said LU is maleimidocaproyl-Val-Cit-PABA, (c) said Drug is
MMAE, and (d) p is an integer of about 4.
The present invention further provides an anti-5T4 antibody-drug conjugate
wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID NO:1 and a light chain
having SEQ ID NO: 2, (b) said LU is maleimidocaproyl-Val-Cit-PABA, (c) said Drug is
MMAD, and (d) p is an integer from about 1 to about 8.
The present invention further provides an anti-5T4 antibody-drug conjugate
wherein: (a) said anti-5T4 antibody consists of a heavy chain having SEQ ID NO:15 and a
light chain having SEQ ID NO: 2, (b) said LU is maleimidocaproyl-Val-Cit-PABA, (c) said
Drug is MMAE, and (d) p is an integer of about 1 to about 8.
The present invention provides an anti-5T4 antibody-drug conjugate wherein said
antibody recognizes an epitope on human 5T4 antigen wherein said epitope comprises
amino acid residues 173-258 and 282-361 of the amino acid sequence of SEQ ID NO: 11.
The present invention provides a pharmaceutical composition comprising an
antibody-drug conjugate indicated above and a pharmaceutically acceptable carrier.
The present invention further provides a method of treating a 5T4-positive cancer in
a patient in need thereof, comprising administering to said patient an antibody-drug
conjugate indicated above.
The present invention further provides a method of treating a 5T4-positive cancer
wherein said cancer is selected from the group consisting of carcinomas of the bladder,
breast, cervix, colon, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas,
liver, skin, stomach, and testes.
More preferably, the present invention provides a method of treating a 5T4-positive
cancer wherein said cancer is selected from the group consisting of colorectal, breast,
pancreatic, and non-small cell lung carcinomas.
The invention further provides an antibody-drug conjugate indicated above for use
in therapy.
The invention further provides the use of an antibody-drug conjugate indicated
above for the manufacture of a medicament.
The invention further provides the use indicated above, wherein said use is for the
treatment of a 5T4-positive cancer and wherein said cancer is selected from the group
consisting of carcinomas of the bladder, breast, cervix, endometrium, kidney, lung,
esophagus, ovary, prostate, pancreas, skin, stomach, and testes.
More preferably, the invention further provides the use indicated above, wherein
said use is for the treatment of a 5T4-positive cancer wherein said cancer is selected from
the group consisting of colorectal, breast, pancreatic, and non-small cell lung carcinomas.
The invention further provides a nucleic acid that encodes an anti-5T4 antibody, a
vector comprising said nucleic acid, and a host cell comprising said vector.
The invention further provides a process for producing an anti-5T4 antibody
comprising cultivating the host cell comprising the above mentioned vector and recovering
the antibody from the cell culture.
The invention further provides a process for producing an anti-5T4 antibody-drug
conjugate comprising: (a) taking the antibody recovered from the cell culture, (b)
chemically linking said antibody via a linker unit selected from the group consisting of
maleimidocaproyl or maleimidocaproyl-Val-Cit to a Drug unit selected from the group
consisting of MMAE, MMAD, or MMAF, and (c) purifying the antibody-drug conjugate.
Detailed Description
The present invention provides anti-5T4 antibody-drug conjugates for the treatment
of cancer. In order that the present invention is more readily understood, certain terms are
first defined.
All amino acid abbreviations used in this disclosure are those accepted by the
United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 B) S).
5T4 refers to the 5T4 oncofetal antigen, a 72 kDa highly glycosylated
transmenbrance glycoprotein comprising a 42 kDa non-glycosylated core
(see US 5,869,053). Human 5T4 is expressed in numerous cancer types, including
carcinomas of the bladder, breast, cervix, colon, endometrium, kidney, lung, esophagus,
ovary, prostate, pancreas, liver, skin, stomach, and testes. Highly tumorigenic cells, also
called cancer stem cells or tumor-initiating cells have been shown to have high levels of
5T4 expression (WO2010/1 11659). Anti-5T4 antibodies of the invention include antibodies
that specifically bind the human 5T4 antigen (see US 2007/0231333).
An "antibody" is an immunoglobulin molecule capable of specific binding to a target,
such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the immunoglobulin molecule. As
used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal
antibodies, but also any antigen binding fragment (i.e., "antigen-binding portion") or single
chain thereof, fusion proteins comprising an antibody, and any other modified configuration
of the immunoglobulin molecule that comprises an antigen recognition site including, for
example without limitation, Fab, Fab', F(ab')2, an Fd fragment consisting of the VH and
CH1 domains, an Fv fragment consisting of the VL and VH domains of a single arm of an
antibody, an isolated complementarity determining region (CDR), scFv, single domain
antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies,
diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.
An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub
class thereof), and the antibody need not be of any particular class. Depending on the
antibody amino acid sequence of the constant region of its heavy chains, 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 regions
that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon,
gamma, and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well known.
A "variable region" of an antibody refers to the variable region of the antibody light
chain or the variable region of the antibody heavy chain, either alone or in combination. As
known in the art, the variable regions of the heavy and light chain each consist of four
framework regions (FRs) connected by three complementarity determining regions (CDRs)
also known as hypervariable regions, contribute to the formation of the antigen binding site
of antibodies. If variants of a subject variable region are desired, particularly with
substitution in amino acid residues outside of a CDR region (i.e., in the framework region),
appropriate amino acid substitution, preferably, conservative amino acid substitution, can
be identified by comparing the subject variable region to the variable regions of other
antibodies which contain CDR1 and CDR2 sequences in the same canonincal class as the
subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901 -91 7, 1987). When
choosing FR to flank subject CDRs, e.g., when humanizing or optimizing an antibody, FRs
from antibodies which contain CDR1 and CDR2 sequences in the same canonical class
are preferred.
A "CDR" of a variable domain are amino acid residues within the variable region
that are identified in accordance with the definitions of the Kabat, Chothia, the cumulation
of both Kabat and Chothia, AbM, contact, and/or conformational definitions or any method
of CDR determination well known in the art. Antibody CDRs may be identified as the
hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992,
Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH,
Washington, D.C. The positions of the CDRs may also be identified as the structural loop
structures originally described by Chothia and others. See, e.g., Chothia et al., 1989,
Nature 342:877-883. Other approaches to CDR identification include the "AbM definition,"
which is a compromise between Kabat and Chothia and is derived using Oxford
Molecular's AbM antibody modeling software (now Accel rys®), or the "contact definition" of
CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol.
Biol., 262:732-745. In another approach, referred to herein as the "conformational
definition" of CDRs, the positions of the CDRs may be identified as the residues that make
enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of
Biological Chemistry, 283: 1156-1 166. Still other CDR boundary definitions may not strictly
follow one of the above approaches, but will nonetheless overlap with at least a portion of
the Kabat CDRs, although they may be shortened or lengthened in light of prediction or
experimental findings that particular residues or groups of residues or even entire CDRs
do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs
defined by any approach known in the art, including combinations of approaches. The
methods used herein may utilize CDRs defined according to any of these approaches. For
any given embodiment containing more than one CDR, the CDRs may be defined in
accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational
definitions.
The term "monoclonal antibody" ( ab) refers to an antibody that s derived from a
single copy or clone, including e.g., any eukaryotic, prokaryotic, or phage clone, and not
the method by which it is produced. Preferably, a monoclonal antibody of the invention
exists in a homogeneous or substantially homogeneous population.
"Humanized" antibody refers to forms of non-human (e.g. murine) antibodies that
are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv,
Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain
minimal sequence derived from non-human immunoglobulin. Preferably, humanized
antibodies are human immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are replaced by residues from a
CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the
desired specificity, affinity, and capacity.
The term "chimeric antibody" is intended to refer to antibodies in which the variable
region sequences are derived from one species and the constant region sequences are
derived from another species, such as an antibody in which the variable region sequences
are derived from a mouse antibody and the constant region sequences are derived from a
human antibody.
Antibodies of the invention can be produced using techniques well known in the art,
e.g., recombinant technologies, phage display technologies, synthetic technologies or
combinations of such technologies or other technologies readily known in the art (see, for
example, Jayasena, S.D., Clin. Cbe . 45: 1628-50 (1999) and Feliouse, FA, et a J.
o Biol., 373(4):924-40 (2007))
Tables 1 and 2 below depict preferred CDRs for the antibodies of the present
invention.
Table 1
Table 2
The present invention includes an antibody or antigen binding portion thereof, that
comprises:
a) a light chain variable region comprising:
i) a LCDR1 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 8 and 17;
ii) a LCDR2 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 9 and 18; and
iii) a LCDR3 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 10 and 19; and
b) a heavy chain variable region comprising:
i) a HCDR1 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 5 and 22;
ii) a HCDR2 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6 and 23; and
iii) a LCDR1 having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7 and 24.
A preferred antibody or antigen binding portion thereof, of the invention comprises:
a) a LCVR comprising: a LCDR1 of SEQ ID NO: 8, a LCDR2 of SEQ ID NO: 9, and
a LCDR3 of SEQ ID NO: 10; and
b) a HCVR comprising: a HCDR1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 6,
and a HCDR3 of SEQ ID NO: 7.
Preferred monoclonal antibodies of the invention are referred to herein as A 1 (a
humanized anti-5T4 lgG1 antibody); A1-lgG4 (a humanized anti-5T4 lgG4 antibody); A3 (a
mouse/human chimeric antibody); and A3hu (a humanized anti-5T4 lgG1 antibody). The
SEQ ID NOs of the amino acid sequences encoding Mabs A 1,A1-lgG4 and A3 are
provided in Table 3 below:
Table 3
The phrases "an antibody recognizing an antigen" and "an antibody specific for an
antigen" are used interchangeably herein with the term "an antibody which binds
specifically to an antigen."
Anti-5T4 Antibody-Drug conjugate refers to an anti-5T4 antibody or antigen binding
portion thereof, as described herein linked to a cytotoxic drug moiety (D) via a linker unit
molecule (LU).
Linker Unit (LU): LU describes the direct or indirect linkage of the antibody to the
drug. Attachment of a linker to a mAb can be accomplished in a variety of ways, such as
through surface lysines, reductive-coupling to oxidized carbohydrates, and through
cysteine residues liberated by reducing interchain disulfide linkages. A variety of ADC
linkage systems are known in the art, including hydrazone-, disulfide- and peptide-based
linkages.
Drug (D): A drug is any substance having biological or detectable activity, for
example, therapeutic agents, detectable labels, binding agents, etc., and prodrugs, which
are metabolized to an active agent in vivo. The terms drug and payload are used
interchangeably. In some embodiments, the Drug is an auristatin, such as auristatin E
(also known in the art as a derivative of dolastatin-10) or a derivative thereof. The
auristatin can be, for example, an ester formed between auristatin E and a keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid or benzoylvaleric acid to
produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and
MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent
Nos. 6,884,869, 7,098,308, 7,256,257, 7,423,1 16, 7,498,298 and 7,745,394, each of which
is incorporated by reference herein in its entirety and for all purposes.
Auristatins have been shown to interfere with microtubule dynamics and nuclear
and cellular division and have anticancer activity. Auristatins of the present invention bind
tubulin and can exert a cytotoxic or cytostatic effect on a 5T4 expressing cell or cell line.
There are a number of different assays, known in the art, that can be used for determining
whether an auristatin or resultant antibody-drug conjugate exerts a cytostatic or cytotoxic
effect on a desired cell or cell line. Methods for determining whether a compound binds
tubulin are known in the art. See, for example, Muller et al., Anal. Chem 2006, 78, 4390-
4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; and Hamel et al., The
Journal of Biological Chemistry, 1990 265:28, 17141-17149.
Examples of drugs or payloads are selected from the group consisting of DM1
(maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-
1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin-D or Nmethyl-
L-valyl-N-[(1 S,2R)-2-methoxy-4-[(2S)-2-[(1 R,2R)-1 -methoxy-2-methyl-3-oxo-3-
[[(1 S)-2-phenyl-1 -(2-thiazolyl)ethyl]amino]propyl]-1 -pyrrolidinyl]-1 -[(1 S)-1 -methylpropyl]-4-
oxobutyl]-N-methyl- (9CI)- L-valinamide), mc-MMAF (maleimidocaproylmonomethylauristatin
F or N-[6-(2,5-dihydro-2,5-dioxo-1 H-pyrrol-1 -yl)-1 -oxohexyl]-Nmethyl-
L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-
(aR,pR,2S)-p-methoxy-a-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-
PABA-MMAE (6-maleimidocaproyl -ValcCit-(p-aminobenzyloxycarbonyl)-
monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1 H-pyrrol-1 -yl)-1 -oxohexyl]-
L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-
[(1 S,2R)-4-[(2S)-2-[(1 R,2R)-3-[[(1 R,2S)-2-hydroxy-1 -methyl-2-phenylethyl]amino]-1 -
methoxy-2-methyl-3-oxopropyl]-1 -pyrrolidinyl]-2-methoxy-1 -[(1 S)-1 -methylpropyl]-4-
oxobutyl]-N-methyl-L-valinamide). DM1 is a derivative of the tubulin inhibitor maytansine
while MMAD, MMAE, and MMAF are auristatin derivatives. The preferred payloads of the
present invention are selected from the group consisting of mc-MMAF and mc-Val-Cit-
PABA-MMAE.
The term "epitope ** refers to tha portion of a moiecuto capable of being recognized
by and bound by an antibody at one or more of the antibody's antigen-binding regions.
Epitopes often consist of a chemically active surface grouping of molecules such as amino
acids or sugar side chains and have specific three-dimensional structural characteristics as
well as specific charge characteristics. The term "antigenic epitope" as used herein, is
defined as a portion of a polypeptide to which an antibody can specifically bind as
determined by any method well known in the art, for example, by conventional
immunoassays. A "nonlinear epitope" or "conformational epitope" comprises
noncontiguous polypeptides (or amino acids) within the antigenic protein to which an
antibody specific to the epitope binds.
The term "binding affinity (KD)" as used herein, is intended to refer to the
dissociation rate of a particular antigen-antibody interaction. The K is the ratio of the rate
of dissociation, also caS!ed the "off-rate {k f)", to the association rate, or "on- rate ( )".
Thus, K equals k f / k and is expressed as a molar concentration ( ). It follows that the
smaller the , the stronger the affinity of binding. Therefore, a K of 1 m indicates weak
binding affinity compared to a K of 1 n . KD values for antibodies can be determined
using methods well established in the art. One method for determining the K D of an
antibody is by using surface plasmon resonance (SPR), typically using a biosensor system
such as a Biacore® system.
The term "specifically binds" as used herein in reference to the binding between an
antibody and a 5T4 antigen and the antibody binds the 5T4 antigen with a K D less than
about 30 nM as determined by SPR at 25°C.
Pharmaceutically acceptable salt as used herein refers to pharmaceutically
acceptable organic or inorganic salts of a molecule or macromolecule.
The term "potency" is a measurement of biological activity and may be designated
as IC50, or effective concentration of antibody needed to inhibit 50% of growth of a 5T4
positive cell line as described in Example 3. Alternatively, potency may refer to anti-tumor
activity as determined in an in vivo tumor xenograph model as shown in Example 4.
The terms "polynucleotide" or "nucleic acid molecule", as used herein, are intended
to include DNA molecules and RNA molecules. A nucleic acid molecule may be singlestranded
or double-stranded, but preferably is double-stranded DNA.
The polynucleotides that encode the antibodies of the present invention may include
the following: only the coding sequence for the variant, the coding sequence for the
variant and additional coding sequences such as a functional polypeptide, or a signal or
secretory sequence or a pro-protein sequence; the coding sequence for the antibody and
non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding
sequence for the antibody. The term 'polynucleotide encoding an antibody" encompasses
a polynucleotide which includes additional coding sequence for the variant but also a
polynucleotide which includes additional coding and/or non-coding sequence. It is known
in the art that a polynucleotide sequence that is optimized for a specific host
cell/expression system can readily be obtained from the amino acid sequence of the
desired protein (see GENEART ® AG, Regensburg, Germany).
The polynucleotides encoding the antibodies of the present invention will typically
include an expression control polynucleotide sequence operably linked to the antibody
coding sequences, including naturally-associated or heterologous promoter regions known
in the art. Preferably, the expression control sequences will be eukaryotic promoter
systems in vectors capable of transforming or transfecting eukaryotic host cells, but control
sequences for prokaryotic hosts may also be used. Once the vector has been incorporated
into the appropriate host cell line, the host cell is propagated under conditions suitable for
expressing the nucleotide sequences, and, as desired, for the collection and purification of
the antibodies. Preferred eukaryotic cell lines include the CHO cell lines, various COS cell
lines, HeLa cells, myeloma cell lines, transformed B-cells, or human embryonic kidney cell
lines. The most preferred host cell is a CHO cell line.
The present invention encompasses antibodies or antigen-binding portions thereof
that bind to a specific epitope on the 5T4 antigen. The epitope identified is a nonlinear or
conformational epitope comprising a first contact with the human 5T4 antigen (SEQ ID NO:
) between amino acid residues 173 and 252 and comprising a second contact between
amino acid residues 276 and 355 (see Example 7). Thus, the CDRs and heavy and light
chain variable regions described herein are used to make full-length antibodies as well as
functional fragments and analogs that maintain the binding affinity of the protein employing
the CDRs specific for the above mentioned epitope of the 5T4 antigen.
The binding affinity of antibodies of the present invention is determined using SPR
(Example 6). In these experiments the 5T4 antigens are immobilized at low densities onto
a BIAcore® chip and antibodies are flowed past. Build up of mass at the surface of the
chip is measured. This analytical method allows the determination in real time of both on
and off rates to obtain affinity (KD) for binding. The humanized antibodies of the present
invention have a KD of between about 0.30 and about 30 nM; about 0.30 and about 20 nM;
about 0.30 and about 10 nM; about 0.5 and about 7 nM; about 1.0 and about 5 nM; and
about 1.0 and about 3 nM.
Conjugation of Drugs to an Antibody
The drug has, or is modified to include, a group reactive with a conjugation point on
the antibody. For example, a drug can be attached by alkylation (e.g., at the epsilon-amino
group lysines or the N-terminus of antibodies), reductive amination of oxidized
carbohydrate, transesterification between hydroxyl and carboxyl groups, amidation at
amino groups or carboxyl groups, and conjugation to thiols. In some embodiments, the
number of drug moieties, p, conjugated per antibody molecule ranges from an average of
1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from
an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is an
average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p ranges from an average of
about 1 to about 8; about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to
about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from
about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to
about 4 or about 2 to about 3. For examples of chemistries that can be used for
conjugation, see, e.g., Current Protocols in Protein Science (John Wiley & Sons, Inc.),
Chapter 15 (Chemical Modifications of Proteins) (the disclosure of which is incorporated by
reference herein in its entirety.)
For example, when chemical activation of the protein results in formation of free
thiol groups, the protein may be conjugated with a sulfhydryl reactive agent. In one
aspect, the agent is one which is substantially specific for free thiol groups. Such agents
include, for example, malemide, haloacetamides (e.g., iodo, bromo or chloro), haloesters
(e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzylic
halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio.
Linkers
The drug can be linked to an antibody by a linker. Suitable linkers include, for
example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to
cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a
peptide linker cleavable by an intracellular protease, such as lysosomal protease or an
endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker,
such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or
maleimidocapronic -valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker.
Another linker is Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate
(smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with
sulfhydryls (thiols, -SH), while its Sulfo-NHS ester is reactive toward primary amines (as
found in Lysine and the protein or peptide N-terminus). Yet another linker is
maleimidocaproyi (mc). Other suitable linkers include linkers hydrolyzable at a specific pH
or a pH range, such as a hydrazone linker. Additional suitable cleavable linkers include
disulfide linkers. The linker may be covalently bound to the antibody to such an extent that
the antibody must be degraded intracellularly in order for the drug to be released e.g. the
mc linker and the like.
A linker can include a group for linkage to the antibody. For example, linker can
include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide,
haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro),
halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or
chloride), vinyl sulfone and pyridylthio). See generally Wong, Chemistry of Protein
Conjugation and Cross-linking; CRC Press, Inc., Boca Raton, 1991 .
Immunotherapy
For immunotherapy, an antibody can be conjugated to a suitable drug, such as a
cytotoxic or cytostatic agent, an immunosuppressive agent, a radioisotope, a toxin, or the
like. The conjugate can be used for inhibiting the multiplication of a tumor cell or cancer
cell, causing apoptosis in a tumor or cancer cell, or for treating cancer in a patient. The
conjugate can be used accordingly in a variety of settings for the treatment of animal
cancers. The conjugate can be used to deliver a drug to a tumor cell or cancer cell.
Without being bound by theory, in some embodiments, the conjugate binds to or
associates with a cancer-cell or a tumor-associated antigen, and the conjugate and/or drug
can be taken up inside a tumor cell or cancer cell through receptor-mediated endocytosis.
The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix
protein associated with the tumor cell or cancer cell. Once inside the cell, one or more
specific peptide sequences within the conjugate (e.g., in a linker) are hydrolytically cleaved
by one or more tumor-cell or cancer-cell-associated proteases, resulting in release of the
drug. The released drug is then free to migrate within the cell and induce cytotoxic or
cytostatic or other activities. In some embodiments, the drug is cleaved from the antibody
outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell, or acts
at the cell surface.
Therapy for Cancer
As discussed above, cancers, including, but not limited to, a tumor, metastasis, or
other disease or disorder characterized by uncontrolled cell growth, can be treated or
prevented by administration of a protein-drug conjugate.
In other embodiments, methods for treating or preventing cancer are provided,
including administering to a patient in need thereof an effective amount of a conjugate and
a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is that with
which treatment of the cancer has not been found to be refractory. In some embodiments,
the chemotherapeutic agent is that with which the treatment of cancer has been found to
be refractory. The conjugate can be administered to a patient that has also undergone a
treatment, such as surgery for treatment for the cancer. In another embodiment, the
additional method of treatment is radiation therapy.
Multi-Drug Therapy for Cancer
Methods for treating cancer include administering to a patient in need thereof an
effective amount of an antibody-drug conjugate and another therapeutic agent that is an
anti-cancer agent. Suitable anticancer agents include, but are not limited to, methotrexate,
taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine,
cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,
dacarbazine, procarbizine, topotecan, nitrogen mustards, Cytoxan, etoposide, 5-
fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin,
daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.
The ADCs of the present invention can be in the form of a pharmaceutical
composition for administration that are formulated to be appropriate for the selected mode
of administration, and pharmaceutically acceptable diluent or excipients, such as buffers,
surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents,
carriers, and the like. Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton Pa., 18th ed., 1995, incorporated herein by reference, provides a compendium of
formulation techniques as are generally known to practitioners.
These pharmaceutical compositions may be administered by any means known in
the art that achieve the generally intended purpose to treat cancer. The preferred route of
administration is parenteral, defined herein as referring to modes of administration that
include but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and
intraarticular injection and infusion. The dosage administered will be dependent upon the
age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired.
Compositions within the scope of the invention include all compositions wherein an
ADC is present in an amount that is effective to achieve the desired medical effect for
treating cancer. While individual needs may vary from one patient to another, the
determination of the optimal ranges of effective amounts of all of the components is within
the ability of the clinician of ordinary skill.
Example 1
Preparation of an anti-5T4 ADC
5T4-A1 antibody drug conjugate (ADC) is prepared via partial reduction of the mAb
with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced Cys residues
with the desired maleimide terminated linker-payload. In particular, 5T4-A1 mAb is
partially reduced via addition of 2.8 molar excess of tris(2-carboxyethyl)phosphine (TCEP)
in 100 mM HEPES (4-(2-hydroxyethy!)-1-piperazineeihanesulfonic a d buffer), pH 7.0 and
1 mM diethylenetriaminepentaacetic acid (DTPA) for 2 h at 37 °C. The desired linkerpayload
is then added to the reaction mixture at a linker-payload/mAb-thiol molar ratio of
5.5 (maleimidocapronic-monomethylauristatin F [mc-MMAF]) or 8 (maleimidocapronic -
valine-citruline-p-aminobenzyloxycarbonyl- monomethylauristatin E [mc-Val-Cit-PABAMMAE])
and reacted for an additional 1 h at 25°C in the presence of 15% v/v of
dimethylacetamide (DMA). After the 1 h incubation period, N-ethylmaleimide (4.5 fold
excess for mc-MMAF and 2 fold excess for mc-Val-Cit-PABA-MMAE) is added to cap the
unreacted thiols and is allowed to react for 15 minutes followed by addition of 6 fold excess
L-Cys to quench any unreacted linker-payload. The reaction mixture is dialyzed overnight
at 4 °C in phosphate buffered saline (PBS), pH 7.4, and purified via SEC (AKTA explorer,
Superdex 200 10/30 GL column). The ADC is further characterized via size exclusion
chromatography (SEC ) for purity, hydrophobic interaction chromatography (HIC), and
liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI MS) to
calculate loading, and the concentration is determined via UV spectrophotometer.
Example 2
Binding Studies
Cells expressing the 5T4 antigen, and the negative control Raji cells, are plated at a
density of 500,000 cells/well on non-tissue culture treated 96 well plates and kept on ice.
Dilutions of the A 1 and A 1-lgG4 antibodies or A 1-mcMMAF ADC are made in 3% bovine
serum albumin BSA in Dulbecco's phosphate buffered saline (DPBS) and added to the
plate at a final concentration of 10mg/mL. The plates are then incubated on ice for 1 hour
followed by 2 washes. The secondary antibody, PE (phycoerythrin) conjugated Goat Anti-
Human IgG Fc is added to the wells. After 30 minutes of incubation at 4°C, the mean
fluorescence intensity is then measured using a flow cytometer.
The data in Table 4 indicates that the A 1 antibody binds a diverse panel of 5T4
positive cell lines. The data in Table 5 indicates that similar binding on several different
cell lines is observed with the A 1 and A 1-lgG4 antibodies as well as the A1-mcMMAF
ADC.
Table 4
Table 5
Example 3
Cytotoxicity Assay
Cell lines expressing 5T4, and the negative control Raji cell line, are cultured with
increasing concentrations of ADC. After four days, viability of each culture is assessed.
IC50 values are calculated by logistic non-linear regression and are presented as ng
Ab/mL. A 1-mcMMAF, A1-vcMMAE, A3-mcMMAF and A3-mcMMAE are shown to inhibit
the growth of 5T4 expressing cell lines (MDAMB435/5T4, MDAMB468, and
MDAMB361 DYT2), while being inactive on 5T4 negative cells (Raji), Table 6.
Table 6
Additionally, 5T4+ primary lung tumor 37622a cells are isolated and grown in
culture. Cells are cultured with increasing concentrations of ADC. Ten days later, viability
of each culture is assessed using the MTS method. IC50 values were calculated by logistic
non-linear regression and are presented as ng Ab/ml. A1-mcMMAF, A 1-vcMMAE, A3-
mcMMAF, and A3-vcMMAE inhibit the growth of the primary lung tumor cells, Table 7.
Table 7
Example 4
Subcutaneous Xenograft Model
Female, athymic (nude) mice (or another strain of immunosupressed mice) are
injected s.c. with MDAMB435/5T4, MDAMB361 DYT2, or H 975 tumor cells. Mice with
staged tumors, approximately 0.1 to 0.3 g (n=6 to 10 mice/treatment group) are
administered intravenously Q4Dx4 with normal saline (vehicle), A 1-mcMMAF, A1-
vcMMAE, A1-mcMMAD, A1-smccDM1 , A3-mcMMAF, A3-vcMMAE, or a nonbinding
control antibody conjugated to either mcMMAF or vcMMAE, at the dose of 3 mg Ab/kg. All
ADCs are dosed based on Ab content. Tumors are measured at least once a week and
their size (mm2 ± SEM) is calculated as mm2 = 0.5 x (tumor width2) x (tumor length).
The data in Table 8 indicates that A 1-mcMMAF, A 1-vcMMAE, A1-vcMMAD, A3-
mcMMAF, and A3-vcMMAE inhibit the growth of MDAMB435/5T4 xenografts while A 1-
mcMMAD and A1-smccDM1 were not active in this model.
The data in Table 9 indicates that A 1-mcMMAF, A 1-vcMMAE, A1-vcMMAD, A1-
smccDMI , A3-mcMMAF, and A3-vcMMAE inhibit the growth of MDAMB361 DYT2
xenografts while A 1-mcMMAD was not active in this model.
The data in Table 10 indicates that A 1-mcMMAF, A 1-vcMMAE, A 1-vcMMAD, A3-
mcMMAF, and A3-vcMMAE inhibit the growth of H 1975 xenografts while A 1-mcMMAD and
A1-smccDM1 were not active in this model.
Table 8
GT= group terminated due to large tumor size
Table 9
GT= group terminated due to large tumor size
Table 10
GT= group terminated due to arge tumor size
Alternatively, nude mice with 37622a primary tumor cell xenografts established
subcutaneously are treated iv Q4Dx4 with A -mcMMAF, A1-mcMMAD, A 1-vcMMAD, or
A3-mcMMAF at the dose of 3 mg Ab/kg and the tumor growth is monitored over the period
of 96 days. Table 11 demonstrates that A 1-mcMMAF, A 1-vcMMAD and A3-mcMMAF
inhibit the growth of 37622a primary tumor xenografts compared to vehicle control treated
animals while A1-mcMMAD was not active in this model.
Table 11
GT= group terminated due to large tumor size
Unexpectedly, the data in Tables 8- show that ADCs with the same antibody and
drug payload but with different linkers had a dissimilar efficacy profile i.e. A1-mcMMAD vs
AI-vcMMAD in all four xenograft models. In addition, the data show that ADCs with the
same antibody and linker but with different drug payloads also had a different efficacy
profile i.e. A1-mcMMAF vs A1-mcMMAD, in all four xenograft models. Thus, the drug
MMAD is effective in all four xenograft models when linked to the A 1 antibody by the vc
linker but has no activity in any of the xenograft models tested when linked by the mc
linker. In contrast, the drug MMAF is highly effective in all 4 xenograft models when linked
to the A 1 antibody with the mc linker while the chemically related drug MMAD has no
activity in all 4 xenograft models when linked to the same antibody by the same linker.
Yet another unexpected observation is seen with the ADC A1-smccDM1 (Tables 8-
10). This ADC was very effective against the MDAMB361 DYT2 xenograft but had
essentially no effect against the MDAMB435/5T4 and the H1975 xenografts even though
all the xenografts have a high expression of the 5T4 target antigen. This data illustrates
that the effectiveness of the linker-payload could not be predicted even when the same
high affinity antibody is utilized or even when the same ADC is used.
Example 5
Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)
ADCC assay:
Blood from a healthy volunteer is collected into a BD Vacutainer CPT cell
preparation tube with sodium heparin. Human peripheral blood mononucleocytes (PBMC)
are harvested and resuspended in assay buffer (RPMI 1640 supplemented with 10 mM
HEPES) at 2.5 x 107 cells/ml. Target cells (MDAMB435/5T4 or MDAMB435/neo) are
seeded at a density of 1 x 104 cells/well in a 96 well assay plate. A 1 antibody or A 1-
mcMMAF are added, then human PBMC effector cells (5 x 105) are dispensed into the
wells for an effector:target cell ratio (E:T) of 50:1 . The assay plate is incubated at 37 °C for
4 hours for ADCC activity. The plate is harvested by adding equal volume of CytoTox-One
reagent (Promega). Stop solution (Promega; 50 ul) is added to each well and lactate
dehydrogenase release was quantified by measuring fluorescence intensity. As a positive
control, 2 m I of lysis buffer per well is added to generate a maximum LDH release (100%
cytotoxicity) in control wells. Percent cytotoxicity is calculated using the following equation:
experimental - effector spontaneous - target spontaneous
%Specific Cytotoxicity = X 100
target maximum - target spontaneous
Where "experimental" corresponds to the signal measured in one of the
experimental conditions, "effector spontaneous" corresponds to the signal measured in the
presence of PBMC alone, "target spontaneous" corresponds to the signal measured in the
presence of target cells alone, and "target maximum" corresponds to the signal measured
in the presence of detergent-lysed target cells alone.
The ADCC activity of A1-lgG1 Ab and A1-mcMMAF compared to A1-lgG4 Ab is
shown in Table 12. Both the A 1 antibody and A1-mcMMAF demonstate comparable
ADCC activity indicating that the ADCC activity of A1-mcMMAF may contribute to its anti
tumor activity.
Table 12
Example 6
Binding Affinity
Surface plasmon resonance (SPR) analysis is performed utilizing the BIAcore ® to
determine the affinity constants for A 1-lgG1 and A1-lgG4 binding to either human or
cynomolgus 5T4 at pH 6.0 and pH 7.4. BIAcore ® technology utilizes changes in the
refractive index at the surface layer upon binding of the huA1 antibody variants to the
human 5T4 protein immobilized on the surface layer. Binding is detected by SPR of laser
light refracting from the surface. Analysis of the signal kinetics on-rate and off-rate allows
the discrimination between non-specific and specific interactions. The 5T4 proteins used
for this analysis consisted of the human or cynomolgus 5T4 ectodomain fused to the
human lgG1-Fc domain and low densities (45.1 and 45.4 RU for human and cynomolgus
respectively) are immobilized onto a CM5 chip to accurately measure affinity constants.
The measurement of specific binding to the 5T4 ectodomain is attained by
subtracting binding to a reference surface that had only human lgG1-Fc protein
immobilized onto the CM5 chip at the same density to that on the 5T4-Fc surfaces. Next,
various concentrations of A 1, A1-lgG4, or A3 antibodies in either HBS-EP pH 7.4 or MESEP
pH 6.0 buffer are injected over the surface. The surface is regenerated two times with
Glycine pH 1.7 + 0.05% Surfactant P20 (GE Healthcare, BR-1 000-54) between injection
cycles.
Results show that the A 1 has a slightly higher affinity for human 5T4 using the lowdensity
5T4 surface at both pH 6.0 and pH 7.4 relative to A1-lgG4 ( 1 .5-fold and 1.2-fold
respectively, Table 13). Additionally, A 1 exhibited slightly better binding to cynomolgus
5T4 at both pH 6.0 and pH 7.4 compared to A 1-lgG4 ( 1 .7-fold and 1.2-fold respectively)
and both A 1 and A 1-lgG4 bound human 5T4, 3 - 4 fold better than cynomolgus 5T4 (Table
12).
Table 13
Comparing the A 1 and A3 antibodies, it is apparent that the A 1 antibody binds
human and cynomolgus 5T4 better at pH 7.4 relative to pH 6.0 while the A3 antibody
exhibits enhanced binding at pH 6.0 compared to pH 7.4, Table 14.
Table 14
Example 7
Epitope Mapping Using 5T4 Chimeras
To identify the epitopes to which each of the A 1 and A3 antibodies bind, an enzyme
linked immunosorbent assay (ELISA) is performed using ( 1) 5T4 ectodomain Fc construct
and (2) human/mouse 5T4 chimera constructs transiently expressed in COS-1 cells. The
ectodomain includes the amino-terminal region, two leucine-rich repeats, and the
intervening hydrophilic region. Mouse and rat 5T4 ectodomains contain a 6 amino acid
direct repeat within their hydrophilic region.
Fusion proteins containing a 5T4 ectodomain and a Fc constant region from human
lgG1 are prepared using human 5T4 (amino acids 1-355), mouse 5T4 (amino acids 1-
361 ) , rat 5T4 (amino acids 1-361 ) , cynomologus monkey 5T4 (amino acids 1-355),
chimpanzee 5T4 (amino acids 1-355), and black-tailed marmoset (amino acids 1-355).
The binding results with human/mouse 5T4 chimera constructs are summarized in Table
14, which indicates specific binding, partial binding, or lack of binding, by the A 1 and A3
antibodies.
Table 15 refers to binding ability of the antibodies to the various human/mouse
chimeras and the nomenclature is designated by mouse 5T4 content. When no binding is
observed, this indicates where the antibody binds human 5T4 since these antibodies do
not bind mouse 5T4. For example, the A3 antibody has the most N-terminal binding
epitope (between 83 - 163) and this is shown by lack of binding to the 5T4 chimera that
has residues 83 - 163 replaced by mouse 5T4, hence A3 can no longer bind. Based upon
these results, it is determined that humanized A 1 antibody has a first contact with human
5T4 between amino acid residues 173 and 252 and a second contact with human 5T4
between amino acid residues 276 and 355. The A3 antibody binds the first leucine-rich
repeat region of human 5T4 between amino acid residues 83 through 163. The number of
amino acid residues corresponds to the human 5T4 antigen amino acid sequence of SEQ
ID NO: 11.
Table 15
Example 8
Comparison of A1-mcMMAF ADC with A1-lgG4-CM ADC
A1-mcMMAF is compared to A1-lgG4-AcBut calicheamicin (A1-IGG4-CM) for both
safety and efficacy. A1-4-CM is comprised of the A1-lgG4 antibody conjugated with the
linker, AcBut [-(4' acetylphenoxy) butanoic acid], to a calicheamicin payload. The
calicheamicins are potent antitumor agents of a class of enediyne antibiotics derived from
the bacterium Micromonospora echinospora.
The cell binding activity of A 1 Ab, A 1-lgG4 Ab, A 1-mcMMAF ADC and A 1-lgG4-CM
ADC are compared using several 5T4 positive cell lines (see Example 2, Table 5). The
data indicates that similar binding is observed with the A 1 and A1-lgG4 antibodies as well
as the A1-mcMMAF ADC, all of which have a higher mean fluorescent intensity than A 1-
lgG4-CM for all the 5T4 positive cell lines tested.
A1-mcMMAF and A1-lgG4-CM are tested side-by-side in the MDAMB435/5T4
subcutaneous xenograft model. Both ADCs are given iv (Q4dx2) when the tumors reach
approximately 200 mm2 in size. The anti-tumor activity of A 1-lgG4-CM at a dose of 3
mg/kg is similar to the anti-tumor activity of A1-mcMMAF administered at dose of 10 mg/kg
(Table 16). Based upon these results, the anti-tumor activity of A1-lgG4-CM is
approximately 3.3 fold more potent than A1-mcMMAF.
Table 16
It could be expected that the 3.3 fold enhanced potency of A 1-lgG4-CM over that of
A1-mcMMAF would translate into a 3.3 fold enhanced safety margin of A1-mcMMAF over
that of A 1-lgG4-CM in an animal toxicity study. However, when the safety profile of A 1-
lgG4-CM in cynomolgus macques is reviewed, it is determined that A 1-lgG4-CM is at least
100 fold more toxic than A1-mcMMAF in the cynomolgus macque. When A1-lgG4-CM is
administered at 0.032, 0.095 and 0.32 mg Ab/kg/cycle (2, 6, 20 mg calicheamicin/kg/cycle)
to male (n=3) and female (n=3) cynomolgus macques, toxicity is observed at each dose
level. After 2 cycles (2 doses), 4 out of 6 animals in the 0.095 treatment group are either
euthanized or found dead. On the other hand, no deaths are observed at dosages up to
10 mg/kg with A 1-mcMMAF (247 mg mcMMAF/kg/cycle), after 2 cycles (2 doses), over the
same 4 week time period. In summary, the 10 mg/kg dosage group of A 1-mcMMAF is
safe while the 0.096 mg/kg dosage group of A1-lgG4-CM is deemed toxic when both are
administered twice to cynomolgus macques in a 4 week observation period.
Unexpectedly, these results demonstrate a 105 fold (10/0.095=105) safety margin
of A 1-mcMMAF over that of A 1-lgG4-CM, rather than the expected 3.3 fold safety margin
based on the relative anti-tumor potency of each ADC. This data reveals the unpredictable
nature of antibody-drug conjugates that utilize antibodies to the same antigen target but
are conjugated to a different drug payload.
Example 9
A1-mcMMAF Mouse PK/PD Modeling and Clinical Dose Predictions
PK/PD modeling has been used to quantify the tumor response of A 1-mcMMAF in
mouse xenograft studies, in order to determine efficacious concentration across cell lines.
The transit compartment tumor kill PK/PD model used was previously described by
Simeoni et al. (Simeoni et al, Cancer Res, 64:1094, (2004)). The model has been
modified to account for linear, exponential and logistic growth of tumor, and saturative
killing by the drug. PK/PD model parameters include:
g ex exponential growth
kg logistic growth
wo initial tumor volume
tau transduction rate
kmax maximum kill rate
kCeo concentration at half max kill rate
The PK PD modeling results are used to calculate the Tumor Static Concentration
(TSC, Equation 1) . This is the drug concentration where tumor growth is equal to tumor
death rates and tumor volume remains unchanged. TSC can be defined as the minimal
concentration required for efficacy. TSC is used to give guidance on clinical dose
selection, with concentrations of >TSC required for efficacy in the clinic.
For A1-mcMMAF, mouse PK was determined in a separate study (3mg/kg IV,
female athymic nu/nu mice). Mouse xenograft studies were completed using 3 different
5T4 cell lines with A 1-mcMMAF administered at dose levels between 1 and 30 mg/kg
every 4 days: cell line MDAMB435/5T4 (dosed at 1, 3, 10, and 30 mg/kg), cell line H1975
(dosed at 1, 3, and 10 mg/kg) and cell line 37622A (dosed at 1 and 10 mg/kg). PK/PD
modeling was performed as described and TSCs are reported in Table 17.
Mouse PK/PD parameters for each xenograft cell line were combined with predicted
human PK of A 1-mcMMAF to simulate doses required for efficacy in the clinic. Using this
methodology, A 1-mcMMAF has a predicted minimally efficacious clinical dose of about
0.22 to about 2.3 mg/kg Q3 weeks [every three weeks] (Table 17).
In an embodiment of the present invention, dose ranges can be in the range from
about 0.18 mg/kg to about 2.7 mg/kg, from about 0.22 mg/kg to about 2.6 mg/kg, from
about 0.27 mg/kg to about 2.5 mg/kg, from about 0.32 mg/kg to about 2.3 mg/kg, from
about 0.37 mg/kg to about 2.15 mg/kg, from about 0.42 mg/kg to about 2.10 mg/kg, from
about 0.47 mg/kg to about 2.05 mg/kg, from about 0.52 mg/kg to about 2.00 mg/kg, from
about 0.57 mg/kg to about 1.95 mg/kg, from about 0.62 mg/kg to about 1.90 mg/kg, from
about 0.67 mg/kg to about 1.85 mg/kg, from about 0.72 mg/kg to about 1.80 mg/kg, from
about 0.82 mg/kg to about 1.70 mg/kg, from about 0.92 mg/kg to about 1.60 mg/kg, from
about 1.02 mg/kg to about 1.50 mg/kg, from about 1.12 mg/kg to about 1.40 mg/kg, or
from about 1.20 mg/kg to about 1.30 mg/kg, with dosing at Q3 weeks. Preferably, dose
ranges can be in the range from about 0.22 mg/kg to about 2.3 mg/kg.
Equation 1
1.
g u w -k.max
Table 17
SEQUENCE LISTING
SEQ ID NO:l Humanized Al human IgGl heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 2 Humanized Al human Kappa light chain:
DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSG
YGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC
SEQ ID NO: 3 Al-VH
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSS
SEQ ID NO: 4 Al-VL
DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSG
YGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIK
SEQ ID NO: 5 Al-HC CDR1
NFGMN
SEQ ID NO: 6 Al-HC CDR2
WINTNTGEPRYAEEFKG
SEQ ID NO: 7 Al-HC CDR3
DWDGAYFFDY
SEQ ID NO: 8 A1-LC-CDR1
KASQSVSNDVA
SEQ ID NO: 9 A1-LC-CDR2
FATNRYT
SEQ ID NO: 10 A1-LC-CDR3
QQDYSSPWT
SEQ ID NO: 11 Human 5T4 antigen
MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASS
FSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNR
NLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAEL
AALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPF
AFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMW
AALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLD
LSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAE
LQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDR
LTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLG
IVLAL IGAI FLL LYLNRKG IKKWMH IRDACRDHME GYH
YRYEINADPRLTNLSSNSDV
SEQ I D NO : 12 Humanized A l human IgG4m heavy chain:
EVQLVESGGGLVQPGGSLRLSCAASGYTFT NFGMNWVRQAPGKGLEWV AWINTNTGEPRYAEEFKG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DWDGAYFFDY WGQGTLVTVSSASTKGPSVFPLAP
CSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS
IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ I D NO : 13 Al human IgG4m VH (A1-IGG4-VH)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSS
SEQ I D NO : 14 Al-IgG4-VH-CDRl
NFGMN
SEQ I D NO : 15 Chimeric A3 heavy chain (muA3-huIgGl )
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSV
KDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ I D NO : 16 Chimeric A3 VH
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSV
KDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSS
SEQ ID NO: 17 Chimeric A3 VH-CDR1
TYAMN
SEQ ID NO: 18 Chimeric A3 VH-CDR2
RIRSKSNNYATYYADSVKD
SEQ ID NO: 19 Chimeric A3 VH-CDR3
QWDYDVRAMNY
SEQ ID NO: 20 Chimeric A3 light chain (muA3-huKappa)
DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSG
SGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC
SEQ ID NO: 21 Chimeric A3 VL
DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSG
SGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQGTKLEIK
SEQ ID NO: 22 Chimeric A3 VL-CDR1
KASQDVDTAVA
SEQ ID NO: 23 Chimeric A3 VL-CDR2
WASTRLT
SEQ ID NO: 24 Chimeric A3 VL-CDR3
QQYSSYPYT
SEQ ID NO: 25 Humanized A 3 human IgGl heavy chain:
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSV
KDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFP
LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSL
GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 26 Humanized A 3 VH :
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSV
KDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSS
SEQ ID NO: 27 Humanized A 3 VH-CDR1 :
TYAMN
SEQ ID NO: 28 Humanized A 3 VH-CDR2 :
RIRSKSNNYATYYADSVKD
SEQ ID NO: 29 Humanized A 3 VH-CDR3 :
QWDYDVRAMNY
SEQ ID NO: 30 Humanized A 3 human Kappa light chain:
DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH
QGLSSPVTKSFNRGEC
SEQ ID NO: 31 Humanized A 3 VL :
DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIK
SEQ ID NO: 32 Humanized A 3 VL-CDR1 :
KASQDVDTAVA
SEQ ID NO: 33 Humanized A 3 VL-CDR2 :
WASTRLT
SEQ ID NO: 34 Humanized A 3 VL-CDR3 :
QQYSSYPYT

We Claim:
1. An antibody-drug conjugate of the formula:
Ab-(LU-D)p
or a pharmaceutically acceptable salt thereof wherein;
(a) Ab is an anti-5T4 antibody or antigen binding portion thereof, comprising a
heavy chain variable region having:
(i) a VH CDR1 region as shown in SEQ ID NO: 5,
(ii) a VH CDR2 region as shown in SEQ ID NO: 6, and
(iii) a VH CDR3 region as shown in SEQ ID NO: 7;
(b) LU is a linker unit selected from the group consisting of maleimidocaproyi and
maleimidocaproyl-Val-Cit,
(c) p is an integer from about 1 to about 8, and
(d) D is a Drug unit selected from the group consisting of MMAE, MMAD, and
MMAF.
2. The antibody-drug conjugate of Claim 1 wherein said anti-5T4 antibody or antigen
binding portion thereof, comprises a light chain variable region having:
(a) a VL CDR1 region as shown in SEQ ID NO: 8,
(b) a VL CDR2 region as shown in SEQ ID NO: 9, and
(c) a VL CDR3 region as shown in SEQ ID NO: 10.
3. The antibody-drug conjugate of Claims 1 or 2 wherein said anti-5T4 antibody or antigen
binding portion thereof, further comprises:
1) a heavy chain variable region having
(a) a VH CDR1 region as shown in SEQ ID NO: 5,
(b) a VH CDR2 region as shown in SEQ ID NO: 6, and
(c) a VH CDR3 region as shown in SEQ ID NO: 7.
2) a light chain variable region having
(a) a VL CDR1 region as shown in SEQ ID NO: 8,
(b) a VL CDR2 region as shown in SEQ ID NO: 9, and
(c) a VL CDR3 region as shown in SEQ ID NO: 10.
4. The antibody-drug conjugate of any one of Claims 1-3, wherein said anti-5T4 antibody
or antigen binding portion thereof, comprises the VH region of SEQ ID NO: 3 and the VL
region of SEQ ID NO: 4 .
5. The antibody-drug conjugate of any one of Claims 1-4 wherein said anti-5T4 antibody
or antigen binding portion thereof, consists of a heavy chain having SEQ ID NO:1 and a
light chain having SEQ ID NO: 2.
6. The antibody-drug conjugate of any one of Claims 1-5, wherein said antibody or antigen
binding portion thereof, recognizes an epitope on human 5T4 antigen, wherein said
epitope comprises amino acid residues 173-258 and 282-361 of the amino acid sequence
of SEQ ID NO:1 1.
7. The antibody-drug conjugate of any one of Claims 1-6, wherein said LU is
maleimidocaproyl.
8. The antibody-drug conjugate of any one of Claims 1-7, wherein said Drug is MMAF.
9. The antibody-drug conjugate of any one of Claims 1-8, wherein p is an integer from
about 1 to about 4.
10. The antibody-drug conjugate of Claim 1 wherein:
(a) said anti-5T4 antibody consists of a heavy chain having SEQ ID NO:1 and a
light chain having SEQ ID NO: 2,
(b) said LU is maleimidocaproyl,
(c) said Drug is MMAF, and
(d) p is 4.
11. A pharmaceutical composition comprising the antibody-drug conjugate of any one of
Claims 1-10 and a pharmaceutically acceptable carrier.
12. A method of treating 5T4-positive cancer in a patient in need thereof, comprising
administering to said patient the antibody-drug conjugate according to any one of Claims
1-1 1.
13. The method of Claim 12 wherein said cancer is selected from the group consisting of
colorectal, breast, pancreatic, and non-small cell lung carcinomas.
14. The antibody-drug conjugate of any one of Claims 1-1 1 for use in therapy.
15. The use of the antibody-drug conjugate of any one of Claims 1- 11 for the manufacture
of a medicament.
16. The use according to Claims 14 or 15, wherein said use is for the treatment of 5T4-
positive cancer.
17. A nucleic acid that encodes the anti-5T4 antibody of any one of Claims 1-6.
18. A vector comprising the nucleic acid of Claim 17.
19. A host cell comprising the vector according to Claim 18.
20. A process for producing an antibody comprising cultivating the host cell of Claim 19
and recovering the antibody from the culture.
A process for producing an anti-5T4 antibody-drug conjugate comprising:
(a) chemically linking a linker unit selected from the group consisting of
maleimidocaproyi or maleimidocaproyl-Val-Cit to a Drug unit selected from the
group consisting of MMAE, MMAD, or MMAF;
(b) conjugating said linker-drug to the antibody recovered from the cell culture of
Claim 20 , and;
(c) purifying the antibody-drug conjugate.