|
Cancer Patent Abstract
Described herein are methods and compositions that can be used for
diagnosis and treatment of cancer.
Cancer Patent Claims
What is claimed is:
1. An isolated antibody or antigen binding fragment thereof comprising:
the heavy chain variable region of SEQ ID NO:26 and the light chain
variable region of SEQ ID NO:34.
2. The antibody of claim 1 wherein the antibody is conjugated to
a detectable label.
3. The antibody of claim 1 wherein the antibody is conjugated to
an effector selected from the group consisting of a toxin, a chemotherapeutic
agent and a radioisotope.
4. The antibody of claim 3 wherein the toxin is auristatin.
5. The antibody of claim 1 wherein the heavy chain variable region
is encoded by SEQ ID NO:25 and the light chain variable region is
encoded by SEQ ID NO:3.
6. A pharmaceutical composition comprising the antibody of claim
1 and a pharmaceutically acceptable excipient.
7. A pharmaceutical composition comprising the antibody of claim
4 and a pharmaceutically acceptable excipient.
Cancer Patent Description
FIELD OF THE INVENTION
The invention relates to the identification and generation of antibodies
that specifically bind to TMEFF2 proteins that are involved in cancer;
and to the use of such antibodies and compositions comprising them
in the diagnosis, prognosis and therapy of cancer.
BACKGROUND OF THE INVENTION
Prostate cancer is the most frequently diagnosed cancer and the
second leading cause of male cancer death in North America and northern
Europe. Early detection of prostate cancer using a serum test for
prostate-specific antigen (PSA) has dramatically improved the treatment
of the disease (Oesterling, 1992, J. Am. Med. Assoc. 267:2236-2238
and DiVita et al. (1997) Cancer: Principles and Practices of Oncology,
5th ed. Lippincott-Raven pub.). Treatment of prostate cancer consists
largely of surgical prostatectomy, radiation therapy, androgen ablation
therapy and chemotherapy. Although many prostate cancer patients
are effectively treated, the current therapies can all induce serious
side effects which diminish quality of life. For example, patients
who present with metastatic disease are most often treated with
androgen-ablation therapy. Chemical or surgical castration has been
the primary treatment for symptomatic metastatic prostate cancer
for over 50 years. While this testicular androgen deprivation therapy
usually results in stabilization or regression of the disease (in
80% of patients), progression of metastatic prostate cancer eventually
develops (Panvichian et al., Cancer Control 3(6):493-500 (1996);
Afrin and Stuart, 1994, J. S. C. Med. Assoc. 90:231-236). Metastatic
disease is currently considered incurable. Thus, the primary goals
of treatment are to prolong survival and improve quality of life
(Rago, Cancer Control 5(6):513-521 (1998)).
Clearly, the identification of novel therapeutic targets and diagnostic
markers is essential for improving the current treatment of prostate
cancer patients. Recent advances in molecular medicine have increased
the interest in tumor-specific cell surface antigens that could
serve as targets for various immunotherapeutic or small molecule
strategies. Antigens suitable for immunotherapeutic strategies should
be highly expressed in cancer tissues and ideally not expressed
in normal adult tissues. One such antigen is TMEFF2.
The TMEFF2 protein contains 2 follistatin-like domains and a conserved
EGF-like domain. The gene encoding the protein was first characterized
from a human brain cDNA library (see Uchida, et al. (1999) Biochem.
Biophys. Res. Commun. 266:593-602), and later isolated from a human
fetal brain cDNA library (see Horie, et al. (2000) Genomics 67:146-152).
See also, e.g., Online Mendelian Inheritance in Man, number 605734;
Unigene Cluster Hs.22791; LocusLink 23671; and other linked sites.
TMEFF2 has been referred to as tomoregulin, TR, hyperplastic polyposis
gene 1, HPP 1, and TENB2. TMEFF2's nucleic acid sequence can be
identified by ATCC Accession Nos. AF264150, AB004064, AB017269,
and AF179274. TMEFF2's amino acid sequence can be identified by
ATCC Accession Nos. AAF91397, BAA90820, BAA87897, and AAD55776.
TMEFF2's UniGene Cluster identification number is hs.22791, Locuslink
identification number is 23671, and OMIM identification number is
605734.
The gene has also been implicated in certain cancerous conditions.
Young, et al. (2001) Proc. Nat'l Acad. Sci. USA 98:265-270 reported
expression in colorectal polyps. Glynne-Jones, et al. (2001) Int.
J. Cancer 94:178-184 reported it as a marker for prostate cancer.
Treatments such as surgery, radiation therapy, and cryotherapy
are potentially curative when the cancer remains localized. Therefore,
early detection of cancer is important for a positive prognosis
for treatment.
Thus, antibodies that can be used for diagnosis and prognosis and
effective treatment of cancer, and including particularly metastatic
cancer, would be desirable. Accordingly, provided herein are compositions
and methods that can be used in diagnosis, prognosis, and therapy
of certain cancers.
SUMMARY OF THE INVENTION
The present invention provides anti-TMEFF2 antibodies that are
surprisingly well internalized and are particularly useful for making
conjugated antibodies for therapeutic purposes. In some embodiments,
the antibodies of the present invention are therapeutically useful
in persons diagnosed with cancer and other proliferative conditions,
including benign proliferative conditions. In one aspect, the antibodies
of the present invention can be used to treat proliferative conditions
of the prostate including, e.g., benign prostate hyperplasia and
prostate cancer. In another aspect, the antibodies of the present
invention can be used to treat malignant and benign proliferative
conditions of the brain including, e.g., gliobastomas, oligodendrogliomas,
anablastic astrocytomas, meningiomas, medulloblastomas, and neuroblastomas.
In particular, the present invention provides anti-TMEFF2 antibodies
that are particularly useful as selective cytotoxic agents for TMEFF2
expressing cells. Without wishing to be bound by theory it is believed
that the antibodies of the invention recognize a TMEFF2 epitope
that effects an increased internalization, and thus enhanced cell
killing, when conjugated to a cytotoxic moiety.
The present invention provides antibodies that competitively inhibit
binding of TMEFF2#19 (ATCC Accession No. PTA-4127) to TMEFF2. In
some embodiments the antibodies are further conjugated to an effector
component. The effector component can be a label (e.g., a fluorescent
label) or can be cytotoxic moiety (e.g., a radioisotope or a cytotoxic
chemical) An exemplary cytotoxic chemical is auristatin.
The antibodies of the invention can be whole antibodies or can
antibody fragments. In some embodiments the immunoglobulin is a
humanized antibody. An exemplary antibody of the invention is TMEFF2#19
(ATCC Accession No. PTA-4127).
The invention also provides pharmaceutical compositions comprising
a pharmaceutically acceptable excipient and the antibody of the
invention. In these embodiments, the antibody can be further conjugated
to an effector component. The effector component can be a label
(e.g., a fluorescent label) or can be cytotoxic moiety (e.g., a
radioisotope or a cytotoxic chemical) An exemplary cytotoxic chemical
is auristatin. The antibodies in the pharmaceutical compositions
can be whole antibodies or can antibody fragments. In some embodiments
the immunoglobulin is a humanized antibody. An exemplary antibody
TMEFF2#19 (ATCC Accession No. PTA-4127).
The invention further provides immunoassays using the immunoglobulins
of the invention. These methods involve detecting a prostate cancer
cell in a biological sample from a patient by contacting the biological
sample with an antibody of the invention. The antibody is typically
conjugated to a label such as fluorescent label.
The invention provides methods of inhibiting proliferation of a
prostate cancer-associated cell. The method comprises contacting
the cell with an antibody of the invention. In most embodiments,
the cancer cell is in a patient, typically a human. The patient
may be undergoing a therapeutic regimen to treat metastatic prostate
cancer or may be suspected of having prostate cancer.
The invention also provides a method of treating prostate cancer
with an antibody to TMEFF2, wherein said prostate cancer is selected
from the group consisting of a primary prostate cancer, metastatic
prostate cancer, locally advanced prostate cancer, androgen independent
prostate cancer, prostate cancer that has been treated with neoadjuvant
therapy, and prostate cancer that is refractory to treatment with
neoadjuvant therapy.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel reagents and methods for treatment,
diagnosis and prognosis for certain cancers using antibodies against
TMEFF2. In particular, the present invention provides anti-TMEFF2
antibodies that are particularly useful as selective cytotoxic agents
for TMEFF2 expressing cells. Without wishing to be bound by theory
it is believed that the antibodies of the invention recognize a
TMEFF2 epitope that effects an increased internalization and thus
enhanced cell killing, when conjugated to a cytotoxic moiety. In
addition, antibodies of the invention are useful because they recognize
the non-glycosylated form of the protein. This is advantageous because
antibodies that recognize the glycosylated portion of the protein
may only recognize a subset of the expressed proteins. The invention
is based, in part, on analysis of approximately 100 hybridoma supernatants.
Epitope mapping of antibodies showing high affinity binding was
carried out through competitive binding analyses. Using this methodology
antibodies recognizing a number of individual epitopes were identified.
The antibodies were then assessed for TMEFF2 dependent cell death
in vitro. Using these methods antibodies that promoted significant
cell death were identified.
Definitions
"Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene, or fragments thereof, that specifically
binds and recognizes an antigen. The recognized immunoglobulin genes
include the kappa, lambda, alpha, gamma, delta, epsilon, and mu
constant region genes, as well as the myriad immunoglobulin variable
region genes. Light chains are classified as either kappa or lambda.
Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
which in turn define the immunoglobulin classes, IgG, IgM, IgA,
IgD and IgE, respectively. Typically, the antigen-binding region
of an antibody or its functional equivalent will be most critical
in specificity and affinity of binding. See Paul, Fundamental Immunology.
However, recombinant methods exist to chimerize and generate changed
classes and effector functions.
An exemplary immunoglobulin (antibody) structural unit comprises
a tetramer of four polypeptides. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD).
The N-terminus of each chain defines a variable region of about
100 to 110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number
of well-characterized fragments produced by digestion with various
peptidases. Thus, e.g., pepsin digests an antibody below the disulfide
linkages in the hinge region to produce F(ab').sub.2, a dimer of
Fab which itself is a light chain joined to VH-CH1 by a disulfide
bond. The F(ab').sub.2 may be reduced under mild conditions to break
the disulfide linkage in the hinge region, thereby converting the
F(ab').sub.2 dimer into an Fab' monomer. The Fab' monomer is essentially
Fab with part of the hinge region (see Fundamental Immunology (Paul
ed., 3d ed. 1993). While various antibody fragments are defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by using recombinant DNA methodology. Thus, the term
antibody, as used herein, also includes antibody fragments either
produced by the modification of whole antibodies, or those synthesized
de novo using recombinant DNA methodologies (e.g., single chain
Fv) or those identified using phage display libraries (see, e.g.,
McCafferty, et al. (1990) Nature 348:552-554).
For preparation of antibodies, e.g., recombinant, monoclonal, or
polyclonal antibodies, many technique known in the art can be used
(see, e.g., Kohler & Milstein (1975) Nature 256:495-497; Kozbor,
et al. (1983) Immunology Today 4:72; Cole, et al., pp. 77-96 in
Monoclonal Antibodies and Cancer Therapy (1985); Coligan (1991)
Current Protocols in Immunology; Harlow & Lane (1988) Antibodies:
A Laboratory Manual; and Goding(1986) Monoclonal Antibodies: Principles
and Practice (2d ed.). Techniques for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies
to polypeptides of this invention. Also, transgenic mice, or other
organisms such as other mammals, may be used to express humanized
antibodies. Alternatively, phage display technology can be used
to identify antibodies and heteromeric Fab fragments that specifically
bind to selected antigens (see, e.g., McCafferty, et al. (1990)
Nature 348:552-554; Marks, et al. (1992) Biotechnology 10:779-783).
A "chimeric antibody" is an antibody molecule in which
(a) the constant region, or a portion thereof, is altered, replaced
or exchanged so that the antigen binding site (variable region)
is linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable
region, or a portion thereof, is altered, replaced or exchanged
with a variable region having a different or altered antigen specificity.
"Epitope" or "antigenic determinant" refers
to a site on an antigen to which an antibody binds. Epitopes can
be formed both from contiguous amino acids or noncontiguous amino
acids juxtaposed by tertiary folding of a protein. Epitopes formed
from contiguous amino acids are typically retained on exposure to
denaturing solvents whereas epitopes formed by tertiary folding
are typically lost on treatment with denaturing solvents. An epitope
typically includes at least 3, and more usually, at least 5 or 8-10
amino acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray crystallography
and 2-dimensional nuclear magnetic resonance. See, e.g., "Epitope
Mapping Protocols" in Morris (ed. 1996) Methods in Molecular
Biology, Vol. 66.
The term "TMEFF2 protein" or "TMEFF2 polynucleotide"
refers to nucleic acid and polypeptide polymorphic variants, alleles,
mutants, and interspecies homologues that: (1) have a nucleotide
sequence that has greater than about 60% nucleotide sequence identity,
65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98% or 99% or greater nucleotide sequence identity, preferably
over a region of over a region of at least about 25, 50, 100, 200,
500, 1000, or more nucleotides, to a nucleotide sequence of SEQ
ID NO:1; (2) bind to antibodies, e.g., polyclonal antibodies, raised
against an immunogen comprising an amino acid sequence encoded by
a nucleotide sequence of SEQ ID NO: 1, and conservatively modified
variants thereof, (3) specifically hybridize under stringent hybridization
conditions to a nucleic acid sequence, or the complement thereof
of SEQ ID NO: 1 and conservatively modified variants thereof or
(4) have an amino acid sequence that has greater than about 60%
amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino sequence
identity, preferably over a region of at least about 25, 50, 100,
200, or more amino acids, to an amino acid sequence of SEQ ID NO:2.
A polynucleotide or polypeptide sequence is typically from a mammal
including, but not limited to, primate, e.g., human; rodent, e.g.,
rat, mouse, hamster; cow, pig, horse, sheep, or other mammal. A
"TMEFF2 polypeptide" and a "TMEFF2 polynucleotide,"
include both naturally occurring or recombinant forms. A number
of different variants have been identified. See, e.g., LocusLink
23671.
A "full length" TMEFF2 protein or nucleic acid refers
to a prostate cancer polypeptide or polynucleotide sequence, or
a variant thereof, that contains all of the elements normally contained
in one or more naturally occurring, wild type TMEFF2 polynucleotide
or polypeptide sequences. For example, a full length TMEFF2 nucleic
acid will typically comprise all of the exons that encode for the
full length, naturally occurring protein. The "full length"
may be prior to, or after, various stages of post-translation processing
or splicing, including alternative splicing.
"Biological sample" as used herein is a sample of biological
tissue or fluid that contains nucleic acids or polypeptides, e.g.,
of a TMEFF2 protein, polynucleotide or transcript. Such samples
include, but are not limited to, tissue isolated from primates,
e.g., humans, or rodents, e.g., mice, and rats. Biological samples
may also include sections of tissues such as biopsy and autopsy
samples, frozen sections taken for histologic purposes, blood, plasma,
serum, sputum, stool, tears, mucus, hair, skin, etc. Biological
samples also include explants and primary and/or transformed cell
cultures derived from patient tissues. A biological sample is typically
obtained from a eukaryotic organism, most preferably a mammal such
as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent,
e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
"Providing a biological sample" means to obtain a biological
sample for use in methods described in this invention. Most often,
this will be done by removing a sample of cells from an animal,
but can also be accomplished by using previously isolated cells
(e.g., isolated by another person, at another time, and/or for another
purpose), or by performing the methods of the invention in vivo.
Archival tissues, having treatment or outcome history, will be particularly
useful.
The term "prostate cancer stage" or grammatical equivalents
thereof refer to the size of a cancer and whether it has spread
beyond its original site. Prostate cancer is generally divided into
four stages, from small and localized (stage 1), to spread into
surrounding tissue (stage 3 and 4). If the cancer has spread to
other parts of the body, this is known as secondary prostate cancer
(or metastatic prostate cancer). There are two systems of prostate
cancer staging the conventional system of the American Urological
Association and a new system based on detection-of prostate cancer
by way of prostate serum antigen (PSA) tests. The new system known
as the Tumor, Nodes and Metastasis System or TNM. In the conventional
AUA system stage A corresponds to clinically unsuspected prostate
cancer. Stage B corresponds to a tumor confined to the prostate
gland (localized). Stage C corresponds to a tumor outside prostate
capsule, and stage D corresponds to metastasis into the pelvic lymph
node. Stage D2 is distant metastatic cancer into distant lymph nodes,
organs, soft tissue or bone.
In the TNM system stages include T1: The tumor is within the prostate
gland and is too small to be detected during a rectal examination,
but may be detected through tests such as PSA test. There are generally
no symptoms. T2: The tumor is still within the prostate gland but
is large enough to be felt during a digital rectal examination or
show up on ultrasound. Often there are no symptoms. T3/T4: The cancer
has spread beyond the prostate gland into the surrounding tissues.
This is known as locally advanced prostate cancer. T1 and T2 tumors
are known as early prostate cancer. T3 and T4 are known as locally
advanced prostate cancer. If the lymph nodes, bones or other parts
of the body are affected this is called secondary or metastatic
cancer. "Locally advanced prostate cancer" refers to prostate
cancer that shows some evidence of metastasis, or developing metastasis.
The term "neoadjuvant therapy" also known as "neoadjuvant
androgen depravation therapy" refers to the treatment of prostate
cancer by giving adjuvant hormone blocking drugs before surgery.
The terms "identical" or percent "identity,"
in the context of two or more nucleic acids or polypeptide sequences,
refer to two or more sequences or subsequences that are the same
or have a specified percentage of amino acid residues or nucleotides
that are the same (e.g., about 60% identity, preferably 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region, when compared and aligned for
maximum correspondence over a comparison window or designated region)
as measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment
and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/
or the like). Such sequences are then said to be "substantially
identical." This definition also refers to, or may be applied
to, the compliment of a test sequence. The definition also includes
sequences that have deletions and/or additions, as well as those
that have substitutions, as well as naturally occurring, e.g., polymorphic
or allelic variants, and man-made variants. As described below,
the preferred algorithms can account for gaps and the like. Preferably,
identity exists over a region that is at least about 25 amino acids
or nucleotides in length, or more preferably over a region that
is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into
a computer, subsequence coordinates are designated, if necessary,
and sequence algorithm program parameters are designated. Preferably,
default program parameters can be used, or alternative parameters
can be designated. The sequence comparison algorithm then calculates
the percent sequence identities for the test sequences relative
to the reference sequence, based on the program parameters.
A "comparison window", as used herein, includes reference
to a segment of one of the number of contiguous positions selected
from the group consisting typically of from about 20 to 600, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same number
of contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known
in the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443,
by the search for similarity method of Pearson & Lipman (1988)
Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel, et al. (eds. 1995 and supplements) Current
Protocols in Molecular Biology.
Preferred examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity include the BLAST
and BLAST 2.0 algorithms, which are described in Altschul, et al.
(1977) Nuc. Acids Res. 25:3389-3402 and Altschul, et al. (1990)
J. Mol. Biol. 215:403-410. BLAST and BLAST 2.0 are used, with the
parameters described herein, to determine percent sequence identity
for the nucleic acids and proteins of the invention. Software for
performing BLAST analyses is publicly available through the National
Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
This algorithm involves first identifying high scoring sequence
pairs (HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued threshold
score T when aligned with a word of the same length in a database
sequence. T is referred to as the neighborhood word score threshold
(Altschul et al., supra). These initial neighborhood word hits act
as seeds for initiating searches to find longer HSPs containing
them. The word hits are extended in both directions along each sequence
for as far as the cumulative alignment score can be increased. Cumulative
scores are calculated using, e.g., for nucleotide sequences, the
parameters M (reward score for a pair of matching residues; always
>0) and N (penalty score for mismatching residues; always <0).
For amino acid sequences, a scoring matrix is used to calculate
the cumulative score. Extension of the word hits in each direction
are halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more negative-scoring
residue alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T, and X determine the sensitivity
and speed of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, an expectation (E) of 10,
M=5, N=-4 and a comparison of both strands. For amino acid sequences,
the BLASTP program uses as defaults a wordlength of 3, and expectation
(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff
(1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50,
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which
a match between two nucleotide or amino acid sequences would occur
by chance. For example, a nucleic acid is considered similar to
a reference sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.2, more preferably less than about 0.01, and most preferably
less than about 0.001. Log values may be large negative numbers,
e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
An indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first
nucleic acid is immunologically cross reactive with the antibodies
raised against the polypeptide encoded by the second nucleic acid,
as described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, e.g., where the two peptides
differ only by conservative substitutions. Another indication that
two nucleic acid sequences are substantially identical is that the
two molecules or their complements hybridize to each other under
stringent conditions, as described below. Yet another indication
that two nucleic acid sequences are substantially identical is that
the same primers can be used to amplify the sequences.
A "host cell" is a naturally occurring cell or a transformed
cell that contains an expression vector and supports the replication
or expression of the expression vector. Host cells may be cultured
cells, explants, cells in vivo, and the like. Host cells may be
prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,
insect, amphibian, or mammalian cells such as CHO, HeLa, and the
like (see, e.g., the American Type Culture Collection catalog or
web site, www.atcc.org).
The terms "isolated," "purified," or "biologically
pure" refer to material that is substantially or essentially
free from components that normally accompany it as found in its
native state. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel electrophoresis
or high performance liquid chromatography. A protein or nucleic
acid that is the predominant species present in a preparation is
substantially purified. In particular, an isolated nucleic acid
is separated from some open reading frames that naturally flank
the gene and encode proteins other than protein encoded by the gene.
The term "purified" in some embodiments denotes that a
nucleic acid or protein gives rise to essentially one band in an
electrophoretic gel. Preferably, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure. "Purify" or "purification"
in other embodiments means removing at least one contaminant from
the composition to be purified. In this sense, purification does
not require that the purified compound be homogenous, e.g., 100%
pure.
The terms "polypeptide," "peptide" and "protein"
are used interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers, those containing modified residues, and non-naturally
occurring amino acid polymer.
The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine.
Amino acid analogs refers to compounds that have the same basic
chemical structure as a naturally occurring amino acid, e.g., an
.alpha. carbon that is bound to a hydrogen, a carboxyl group, an
amino group, and an R group, e.g., homoserine, norleucine, methionine
sulfoxide, methionine methyl sulfonium. Such analogs may have modified
R groups (e.g., norleucine) or modified peptide backbones, but retain
the same basic chemical structure as a naturally occurring amino
acid. Amino acid mimetics refers to chemical compounds that have
a structure that is different from the general chemical structure
of an amino acid, but that functions similarly to a naturally occurring
amino acid.
Amino acids may be referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted single-letter
codes.
"Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical or associated, e.g., naturally
contiguous, sequences. Because of the degeneracy of the genetic
code, a large number of functionally identical nucleic acids encode
most proteins. For instance, the codons GCA, GCC, GCG and GCU all
encode the amino acid alanine. Thus, at every position where an
alanine is specified by a codon, the codon can be altered to another
of the corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also describes
silent variations of the nucleic acid. One of skill will recognize
that in certain contexts each codon in a nucleic acid (except AUG,
which is ordinarily the only codon for methionine, and TGG, which
is ordinarily the only codon for tryptophan) can be modified to
yield a functionally identical molecule. Accordingly, often silent
variations of a nucleic acid which encodes a polypeptide is implicit
in a described sequence with respect to the expression product,
but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the encoded
sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with
a chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. Typically conservative substitutions for
one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)
(see, e.g., Crceighton (1984) Proteins).
Macromolecular structures such as polypeptide structures can be
described in terms of various levels of organization. For a general
discussion of this organization, see, e.g., Alberts, et al. (1994)
Molecular Biology of the Cell (3d ed.), and Cantor & Schimmel
(1980) Biophysical Chemistry Part I: The Conformation of Biological
Macromolecules. "Primary structure" refers to the amino
acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains. Domains
are portions of a polypeptide that often form a compact unit of
the polypeptide and are typically 25 to approximately 500 amino
acids long. Typical domains are made up of sections of lesser organization
such as stretches of .beta.-sheet and .alpha.-helices. "Tertiary
structure" refers to the complete three dimensional structure
of a polypeptide monomer. "Quaternary structure" refers
to the three dimensional structure formed, usually by the noncovalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. For example, useful labels include
fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly
used in an ELISA), biotin, digoxigenin, or haptens and proteins
or other entities which can be made detectable, e.g., by incorporating
a radiolabel into the peptide or used to detect antibodies specifically
reactive with the peptide. The radioisotope may be, for example,
.sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I. In some cases,
particularly using antibodies against the proteins of the invention,
the radioisotopes are used as toxic moieties, as described below.
The labels may be incorporated into the TMEFF2 nucleic acids, proteins
and antibodies at any position. A method known in the art for conjugating
the antibody to the label may be employed, including those methods
described by Hunter, et al. (1962) Nature 144:945; David, et al.
(1974) Biochemistry 13:1014; Pain, et al. (1981) J. Immunol. Meth.
40:219; and Nygren (1982) J. Histochem. and Cytochem. 30:407. The
lifetime of radiolabeled peptides or radiolabeled antibody compositions
may extended by the addition of substances that stabilize the radiolabeled
peptide or antibody and protect it from degradation. Any substance
or combination of substances that stabilize the radiolabeled peptide
or antibody may be used including those substances disclosed in
U.S. Pat. No. 5,961,955.
An "effector" or "effector moiety" or "effector
component" is a molecule that is bound (or linked, or conjugated),
either covalently, through a linker or a chemical bond, or noncovalently,
through ionic, van der Waals, electrostatic, or hydrogen bonds,
to an antibody. The "effector" can be a variety of molecules
including, e.g., detection moieties including radioactive compounds,
fluorescent compounds, an enzyme or substrate, tags such as epitope
tags, a toxin; activatable moieties, a chemotherapeutic agent; a
lipase; an antibiotic; or a radioisotope emitting "hard",
e.g., beta radiation.
The term "recombinant" when used with reference, e.g.,
to a cell, or nucleic acid, protein, or vector, indicates that the
cell, nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the alteration
of a native nucleic acid or protein, or that the cell is derived
from a cell so modified. Thus, e.g., recombinant cells express genes
that are not found within the native (non-recombinant) form of the
cell or express native genes that are otherwise abnormally expressed,
under expressed or not expressed at all. By the term "recombinant
nucleic acid" herein is meant nucleic acid, originally formed
in vitro, in general, by the manipulation of nucleic acid, e.g.,
using polymerases and endonucleases, in a form not normally found
in nature. In this manner, operably linkage of different sequences
is achieved. Thus an isolated nucleic acid, in a linear form, or
an expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this invention. It is understood that once a recombinant
nucleic acid is made and reintroduced into a host cell or organism,
it will replicate non-recombinantly, e.g., using the in vivo cellular
machinery of the host cell rather than in vitro manipulations; however,
such nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. Similarly, a "recombinant protein"
is a protein made using recombinant techniques, e.g., through the
expression of a recombinant nucleic acid as depicted above.
The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two
or more subsequences that are not normally found in the same relationship
to each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences, e.g., from
unrelated genes arranged to make a new functional nucleic acid,
e.g., a promoter from one source and a coding region from another
source. Similarly, a heterologous protein will often refer to two
or more subsequences that are not found in the same relationship
to each other in nature (e.g., a fusion protein).
A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used herein,
a promoter includes necessary nucleic acid sequences near the start
site of transcription, such as, in the case of a polymerase II type
promoter, a TATA element. A promoter also optionally includes distal
enhancer or repressor elements, which can be located as much as
several thousand base pairs from the start site of transcription.
A "constitutive" promoter is a promoter that is active
under most environmental and developmental conditions. An "inducible"
promoter is a promoter that is active under environmental or developmental
regulation. The term "operably linked" refers to a functional
linkage between a nucleic acid expression control sequence (such
as a promoter, or array of transcription factor binding sites) and
a second nucleic acid sequence, wherein the expression control sequence
directs transcription of the nucleic acid corresponding to the second
sequence.
An "expression vector" is a nucleic acid construct, generated
recombinantly or synthetically, with a series of specified nucleic
acid elements that permit transcription of a particular nucleic
acid in a host cell. The expression vector can be part of a plasmid,
virus, or nucleic acid fragment. Typically, the expression vector
includes a nucleic acid to be transcribed operably linked to a promoter.
The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding reaction
that is determinative of the presence of the protein, in a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a particular
protein sequences at least two times the background and more typically
more than 10 to 100 times background.
Specific binding to an antibody under such conditions requires
an antibody that is selected for its specificity for a particular
protein. For example, polyclonal antibodies raised to a particular
protein, polymorphic variants, alleles, orthologs, and conservatively
modified variants, or splice variants, or portions thereof, can
be selected to obtain only those polyclonal antibodies that are
specifically immunoreactive with TMEFF2 and not with other proteins.
This selection may be achieved by subtracting out antibodies that
cross-react with other molecules. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with
a particular protein. For example, solid-phase ELISA immunoassays
are routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane (1988) Antibodies:
A Laboratory Manual for a description of immunoassay formats and
conditions that can be used to determine specific immunoreactivity).
"Tumor cell" refers to precancerous, cancerous, and normal
cells in a tumor.
"Cancer cells," "transformed" cells or "transformation"
in tissue culture, refers to spontaneous or induced phenotypic changes
that do not necessarily involve the uptake of new genetic material.
Although transformation can arise from infection with a transforming
virus and incorporation of new genomic DNA, or uptake of exogenous
DNA, it can also arise spontaneously or following exposure to a
carcinogen, thereby mutating an endogenous gene. Transformation
is associated with phenotypic changes, such as immortalization of
cells, aberrant growth control, nonmorphological changes, and/or
malignancy (see, Freshney (1994) Culture of Animal Cells: A Manual
of Basic Technique (3d ed.).
Expression of TMEFF2 Polypeptides from Nucleic Acids
Nucleic acids of the invention can be used to make a variety of
expression vectors to express TMEFF2 polypeptides which can then
be used to raise antibodies of the invention, as described below.
Expression vectors and recombinant DNA technology are well known
to those of skill in the art (see, e.g., Ausubel, supra, and Fernandez
& Hoeffler (eds. 1999) Gene Expression Systems) and are used
to express proteins. The expression vectors may be either self-replicating
extrachromosomal vectors or vectors which integrate into a host
genome. Generally, these expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the TMEFF2 protein. The term "control
sequences" refers to DNA sequences used for the expression
of an operably linked coding sequence in a particular host organism.
Control sequences that are suitable for prokaryotes, e.g., include
a promoter, optionally an operator sequence, and a ribosome binding
site. Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into
a functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably linked
to DNA for a polypeptide if it is expressed as a preprotein that
participates in the secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is operably
linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However, enhancers
do not have to be contiguous. Linking is typically accomplished
by ligation at convenient restriction sites. If such sites do not
exist, synthetic oligonucleotide adaptors or linkers are used in
accordance with conventional practice. Transcriptional and translational
regulatory nucleic acid will generally be appropriate to the host
cell used to express the TMEFF2 protein. Numerous types of appropriate
expression vectors, and suitable regulatory sequences are known
in the art for a variety of host cells.
In general, transcriptional and translational regulatory sequences
may include, but are not limited to, promoter sequences, ribosomal
binding sites, transcriptional start and stop sequences, translational
start and stop sequences, and enhancer or activator sequences. In
a preferred embodiment, the regulatory sequences include a promoter
and transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters.
The promoters may be either naturally occurring promoters or hybrid
promoters. Hybrid promoters, which combine elements of more than
one promoter, are also known in the art, and are useful in the present
invention.
In addition, an expression vector may comprise additional elements.
For example, the expression vector may have two replication systems,
thus allowing it to be maintained in two organisms, e.g., in mammalian
or insect cells for expression and in a prokaryotic host for cloning
and amplification. Furthermore, for integrating expression vectors,
the expression vector contains at least one sequence homologous
to the host cell genome, and preferably two homologous sequences
which flank the expression construct. The integrating vector may
be directed to a specific locus in the host cell by selecting the
appropriate homologous sequence for inclusion in the vector. Constructs
for integrating vectors are well known in the art (e.g., Fernandez
& Hoeffler, supra).
In addition, in a preferred embodiment, the expression vector contains
a selectable marker gene to allow the selection of transformed host
cells. Selection genes are well known in the art and will vary with
the host cell used.
The TMEFF2 proteins of the present invention are produced by culturing
a host cell transformed with an expression vector containing nucleic
acid encoding a TMEFF2 protein, under the appropriate conditions
to-induce or cause expression of the TMEFF2 protein. Conditions
appropriate for TMEFF2 protein expression will vary with the choice
of the expression vector and the host cell, and will be easily ascertained
by one skilled in the art through routine experimentation or optimization.
For example, the use of constitutive promoters in the expression
vector will require optimizing the growth and proliferation of the
host cell, while the use of an inducible promoter requires the appropriate
growth conditions for induction. In addition, in some embodiments,
the timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria,
fungi, and insect and animal cells, including mammalian cells. Of
particular interest are Saccharomyces cerevisiae and other yeasts,
E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora,
BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial
cells), THP1 cells (a macrophage cell line) and various other human
cells and cell lines.
In a preferred embodiment, the TMEFF2 proteins are expressed in
mammalian cells. Mammalian expression systems are also known in
the art, and include retroviral and adenoviral systems. One expression
vector system is a retroviral vector system such as is generally
described in PCT/US97/01019 and PCT/US97/01048, both of which are
hereby expressly incorporated by reference. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter, and the CMV promoter (see, e.g., Fernandez
& Hoeffler, supra). Typically, transcription termination and
polyadenylation sequences recognized by mammalian cells are regulatory
regions located 3' to the translation stop codon and thus, together
with the promoter elements, flank the coding sequence. Examples
of transcription terminator and polyadenylation signals include
those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian
hosts, as well as other hosts, is well known in the art, and will
vary with the host cell used. Techniques include dextran-mediated
transfection, calcium phosphate precipitation, polybrene mediated
transfection, protoplast fusion, electroporation, viral infection,
encapsulation of the polynucleotide(s) in liposomes, and direct
microinjection of the DNA into nuclei.
In some embodiments, TMEFF2 proteins are expressed in bacterial
systems. Bacterial expression systems are well known in the art.
Promoters from bacteriophage may also be used and are known in the
art. In addition, synthetic promoters and hybrid promoters are also
useful; e.g., the tac promoter is a hybrid of the trp and lac promoter
sequences. Furthermore, a bacterial promoter can include naturally
occurring promoters of non-bacterial origin that have the ability
to bind bacterial RNA polymerase and initiate transcription. In
addition to a functioning promoter sequence, an efficient ribosome
binding site is desirable. The expression vector may also include
a signal peptide sequence that provides for secretion of the TMEFF2
protein in bacteria. The protein is either secreted into the growth
media (gram-positive bacteria) or into the periplasmic space, located
between the inner and outer membrane of the cell (gram-negative
bacteria). The bacterial expression vector may also include a selectable
marker gene to allow for the selection of bacterial strains that
have been transformed. Suitable selection genes include genes which
render the bacteria resistant to drugs such as ampicillin, chloramphenicol,
erythromycin, kanamycin, neomycin and tetracycline. Selectable markers
also include biosynthetic genes, such as those in the histidine,
tryptophan and leucine biosynthetic pathways. These components are
assembled into expression vectors. Expression vectors for bacteria
are well known in the art, and include vectors for Bacillus subtilis,
E. coli, Streptococcus cremoris, and Streptococcus lividans, among
others (e.g., Fernandez & Hoeffler, supra). The bacterial expression
vectors are transformed into bacterial host cells using techniques
well known in the art, such as calcium chloride treatment, electroporation,
and others.
In one embodiment, TMEFF2 polypeptides are produced in insect cells.
Expression vectors for the transformation of insect cells, and in
particular, baculovirus-based expression vectors, are well known
in the art.
TMEFF2 polypeptides can also be produced in yeast cells. Yeast
expression systems are well known in the art, and include expression
vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa,
Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia
guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia
lipolytica.
The TMEFF2 polypeptides may also be made as a fusion protein, using
techniques well known in the art. Thus, e.g., for the creation of
monoclonal antibodies, if the desired epitope is small, the TMEFF2
protein may be fused to a carrier protein to form an immunogen.
Alternatively, the TMEFF2 protein may be made as a fusion protein
to increase expression, or for other reasons. For example, when
the TMEFF2 protein is a TMEFF2 peptide, the nucleic acid encoding
the peptide may be linked to other nucleic acid for expression purposes.
The TMEFF2 polypeptides are typically purified or isolated after
expression. TMEFF2 proteins may be isolated or purified in a variety
of ways known to those skilled in the art depending on what other
components are present in the sample. Standard purification methods
include electrophoretic, molecular, immunological and chromatographic
techniques, including ion exchange, hydrophobic, affinity, and reverse-phase
HPLC chromatography, and chromatofocusing. For example, the TMEFF2
protein may be purified using a standard anti-TMEFF2 protein antibody
column. Ultrafiltration and diafiltration techniques, in conjunction
with protein concentration, are also useful. For general guidance
in suitable purification techniques, see Scopes, Protein Purification
(1982). The degree of purification necessary will vary depending
on the use of the TMEFF2 protein. In some instances no purification
will be necessary.
One of skill will recognize that the expressed protein need not
have the wild-type TMEFF2 sequence but may be derivative or variant
as compared to the wild-type sequence. These variants typically
fall into one or more of three classes: substitutional, insertional
or deletional variants. These variants ordinarily are prepared by
site specific mutagenesis of nucleotides in the DNA encoding the
protein, using cassette or PCR mutagenesis or other techniques well
known in the art, to produce DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture as outlined above.
However, variant protein fragments having up to about 100-150 residues
may be prepared by in vitro synthesis using established techniques.
Amino acid sequence variants are characterized by the predetermined
nature of the variation, a feature that sets them apart from naturally
occurring allelic or interspecies variation of the TMEFF2 protein
amino acid sequence. The variants typically exhibit the same qualitative
biological activity as the naturally occurring analogue, although
variants can also be selected which have modified characteristics
as will be more fully outlined below.
TMEFF2 polypeptides of the present invention may also be modified
in a way to form chimeric molecules comprising a TMEFF2 polypeptide
fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of
the TMEFF2 polypeptide with a tag polypeptide which provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino-or carboxyl-terminus
of the TMEFF2 polypeptide. The presence of such epitope-tagged forms
of a TMEFF2 polypeptide can be detected using an antibody against
the tag polypeptide. Also, provision of the epitope tag enables
the TMEFF2 polypeptide to be readily purified by affinity purification
using an anti-tag antibody or another type of affinity matrix that
binds to the epitope tag. In an alternative embodiment, the chimeric
molecule may comprise a fusion of a TMEFF2 polypeptide with an immunoglobulin
or a particular region of an immunoglobulin. For a bivalent form
of the chimeric molecule, such a fusion could be to the Fc region
of an IgG molecule.
Various tag polypeptides and their respective antibodies are well
known in the art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelation
tags, the flu HA tag polypeptide and its antibody 12CA5 (Field,
et al (1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan, et at.
(1985) Molecular and Cellular Biology 5:3610-3616); and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky,
et al. (1990) Protein Engineering 3(6):547-553). Other tag polypeptides
include the FLAG-peptide (Hopp, et al. (1988) BioTechnology 6:1204-1210);
the KT3 epitope peptide (Martin, et al. (1992) Science 255:192-194);
tubulin epitope peptide (Skinner, et al. (1991) J. Biol. Chem. 266:15163-15166);
and the T7 gene 10 protein peptide tag (Lutz-Freyermnuth, et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6393-6397).
Antibodies to Cancer Proteins
Once the TMEFF2 protein is produced, it is used to generate antibodies,
e.g., for immunotherapy or immunodiagnosis. As noted above, the
antibodies of the invention recognize the same epitope as that recognized
by TMEFF2#19 (ATCC Accession No. PTA-4127. This hybridoma was deposited
at ATCC under the Budapest Treaty on Mar. 6, 2002. The American
Type Culture Collection, ATCC, is located at 10801 University Blvd.,
Manassas, Va. 20110-2209 in the United States of America.). The
ability of a particular antibody to recognize the same epitope as
another antibody is typically determined by the ability of one antibody
to competitively inhibit binding of the second antibody to the antigen.
Many of a number of competitive binding assays can be used to measure
competition between two antibodies to the same antigen. An exemplary
assay is a Biacore assay as desrcibed in the Examples, below. Briefly
in these assays, binding sites can be mapped in structural terms
by testing the ability of interactants, e.g. different antibodies,
to inhibit the binding of another. Injecting two consecutive antibody
samples in sufficient concentration can identify pairs of competing
antibodies for the same binding epitope. The antibody samples should
have the potential to reach a significant saturation with each injection.
The net binding of the second antibody injection is indicative for
binding epitope analysis. Two response levels can be used to describe
the boundaries of perfect competition versus non-competing binding
due to distinct epitopes. The relative amount of binding response
of the second antibody injection relative to the binding of identical
and distinct binding epitopes determines the degree of epitope overlap.
Other conventional immunoassays known in the art can be used in
the present invention. For example, antibodies can be differentiated
by the epitope to which they bind using a sandwich ELISA assay.
This is carried out by using a capture antibody to coat the surface
of a well. A subsaturating concentration of tagged-antigen is then
added to the capture surface. This protein will be bound to the
antibody through a specific antibody:epitope interaction. After
washing a second antibody, which has been covalently linked to a
detectable moiety (e.g., HRP, with the labeled antibody being defined
as the detection antibody) is added to the ELISA. If this antibody
recognizes the same epitope as the capture antibody it will be unable
to bind to the target protein as that particular epitope will no
longer be available for binding. If however this second antibody
recognizes a different epitope on the target protein it will be
able to bind and this binding can be detected by quantifying the
level of activity (and hence antibody bound) using a relevant substrate.
The background is defined by using a single antibody as both capture
and detection antibody, whereas the maximal signal can be established
by capturing with an antigen specific antibody and detecting with
an antibody to the tag on the antigen. By using the background and
maximal signals as references, antibodies can be assessed in a pair-wise
manner to determine epitope specificity.
A first-antibody is considered to competitively inhibit binding
of a second antibody, if binding of the second antibody to the antigen
is reduced by at least 30%, usually at least about 40%, 50%, 60%
or 75%, and often by at least about 90%, in the presence of the
first antibody using any of the assays described above.
Methods of preparing polyclonal antibodies are known to the skilled
artisan (e.g., Coligan, supra; and Harlow & Lane, supra). Polyclonal
antibodies can be raised in a mammal, e.g., by one or more injections
of an immunizing agent and, if desired, an adjuvant. Typically,
the immunizing agent and/or adjuvant will be injected in the mammal
by multiple subcutaneous or intraperitoneal injections. The immunizing
agent may include a protein encoded by a nucleic acid of the figures
or fragment thereof or a fusion protein thereof. It may be useful
to conjugate the immunizing agent to a protein known to be immunogenic
in the mammal being immunized. Examples of such immunogenic proteins
include but are not limited to keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples
of adjuvants which may be employed include Freund's complete adjuvant
and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by
one skilled in the art without undue experimentation.
The antibodies may, alternatively, be monoclonal antibodies. Monoclonal
antibodies may be prepared using hybridoma methods, such as those
described by Kohler & Milstein (1975) Nature 256:495. In a hybridoma
method, a mouse, hamster, or other appropriate host animal, is typically
immunized with an immunizing agent to elicit lymphocytes that produce
or are capable of producing antibodies that will specifically bind
to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro. The immunizing agent will typically include a polypeptide
encoded by a nucleic acid of Tables 1-2, fragment thereof, or a
fusion protein thereof. Generally, either peripheral blood lymphocytes
("PBLs") are used if cells of human origin are desired,
or spleen cells or lymph node cells are used if non-human mammalian
sources are desired. The lymphocytes are then fused with an immortalized
cell line using a suitable fusing agent, such as polyethylene glycol,
to form a hybridoma cell (pp. 59-103 in Goding (1986) Monoclonal
Antibodies: Principles and Practice). Immortalized cell lines are
usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine, and human origin. Usually, rat or mouse myeloma
cell lines are employed. The hybridoma cells may be cultured in
a suitable culture medium that preferably contains one or more substances
that inhibit the growth or survival of the unfused, immortalized
cells. For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
In one embodiment, the antibodies are bispecific antibodies. Bispecific
antibodies are monoclonal, preferably human or humanized, antibodies
that have binding specificities for at least two different antigens
or that have binding specificities for two epitopes on the same
antigen. In one embodiment, one of the binding specificities is
for a TMEFF2 protein, the other one is for any other prostate cancer
antigen. Alternatively, tetramer-type technology may create multivalent
reagents.
In a preferred embodiment, the antibodies to TMEFF2 protein are
capable of reducing or eliminating prostate cancer cells. That is,
the addition of anti-TMEFF2 antibodies (either polyclonal or preferably
monoclonal) to prostate cancer tissue (or cells containing TMEFF2)
may reduce or eliminate the prostate cancer. Generally, at least
a 25% decrease in activity, growth, size or the like is preferred,
with at least about 50% being particularly preferred and about a
95-100% decrease being especially preferred.
In a preferred embodiment the antibodies to the TMEFF2 proteins
are humanized antibodies (e.g., Xenerex Biosciences, Medarex, Inc.,
Abgenix, Inc., Protein Design Labs, Inc.) Humanized forms of non-human
(e.g., murine) antibodies are chimeric molecules of immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
which contain minimal sequence derived from non-human immunoglobulin.
Humanized antibodies include 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, selectivity, affinity, and capacity. In some
instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies
may also comprise residues which are found neither in the recipient
antibody nor in the imported CDR or framework sequences. In general,
a humanized antibody will comprise substantially all of at least
one, and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human immunoglobulin
and all or substantially all of the framework (FR) regions are those
of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin (Jones,
et al. (1986) Nature 321:522-525; Riechmann, et al. (1988) Nature
332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596).
Humanization can be essentially performed following the method of
Winter and co-workers (Jones, et al. (1986) Nature 321:522-525;
Riechmann, et al. (1988) Nature 332:323-327; Verhoeyen, et al. (1988)
Science 239:1534-1536), by substituting rodent CDRs or CDR sequences
for the corresponding sequences of a human antibody. Accordingly,
such humanized antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the corresponding sequence from a
nonhuman species.
Human antibodies can also be produced using various techniques
known in the art, including phage display libraries (Hoogenboom
& Winter (1991) J. Mol. Biol. 227:381; Marks, et al. (1991)
J. Mol. Biol. 222:581). The techniques of Cole, et al. and Boemer,
et al. are also available for the preparation of human monoclonal
antibodies (p. 77 in Cole, et al. (1985) Monoclonal Antibodies and
Cancer Therapy; and Boerner, et al. (1991) J. Immunol. 147(1):86-95).
Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or completely
inactivated. Upon challenge, human antibody production is observed,
which closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This approach
is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks, et al. (1992) Bio/Technology 10:779-783; Lonberg,
et al. (1994) Nature 368:856-859; Morrison (1994) Nature 368:812-13;
Fishwild, et al. (1996) Nature Biotechnology 14:845-51; Neuberger
(1996) Nature Biotechnology 14:826; and Lonberg & Huszar (1995)
Intern. Rev. Immunol. 13:65-93.
By immunotherapy is meant treatment of prostate cancer with an
antibody raised against TMEFF2 proteins. As used herein, immunotherapy
can be passive or active. Passive immunotherapy as defined herein
is the passive transfer of antibody to a recipient (patient). Active
immunization is the induction of antibody and/or T-cell responses
in a recipient (patient). Induction of an immune response is the
result of providing the recipient with an antigen to which antibodies
are raised. As appreciated by one of ordinary skill in the art,
the antigen may be provided by injecting a polypeptide against which
antibodies are desired to be raised into a recipient, or contacting
the recipient with a nucleic acid capable of expressing the antigen
and under conditions for expression of the antigen, leading to an
immune response.
In some embodiments, the antibody is conjugated to an effector
moiety. The effector moiety can be any number of molecules, including
labeling moieties such as radioactive labels or fluorescent labels,
or can be a therapeutic moiety. In one aspect the therapeutic moiety
is a small molecule that modulates the activity of the TMEFF2 protein.
In another aspect the therapeutic moiety modulates the activity
of molecules associated with or in close proximity to the TMEFF2
protein.
In other embodiments, the therapeutic moiety is a cytotoxic agent.
In this method, targeting the cytotoxic agent to prostate cancer
tissue or cells, results in a reduction in the number of afflicted
cells, thereby reducing symptoms associated with prostate cancer.
Cytotoxic agents are numerous and varied and include, but are not
limited to, cytotoxic drugs or toxins or active fragments of such
toxins. Suitable toxins and their corresponding fragments include
diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,
curcin, crotin, phenomycin, enomycin, auristatin and the like. Cytotoxic
agents also include radiochemicals made by conjugating radioisotopes
to antibodies raised against prostate cancer proteins, or binding
of a radionuclide to a chelating agent that has been covalently
attached to the antibody. Targeting the therapeutic moiety to transmembrane
prostate cancer proteins not only serves to increase the local concentration
of therapeutic moiety in the prostate cancer afflicted area, but
also serves to reduce deleterious side effects that may be associated
with the therapeutic moiety.
Binding Affinity of Antibodies of the Invention
Binding affinity for a target antigen is typically measured or
determined by standard antibody-antigen assays, such as Biacore
competitive assays, saturation assays, or immunoassays such as ELISA
or RIA.
Such assays can be used to determine the dissociation constant
of the antibody. The phrase "dissociation constant" refers
to the affinity of an antibody for an antigen. Specificity of binding
between an antibody and an antigen exists if the dissociation constant
(K.sub.D=1/K, where K is the affinity constant) of the antibody
is <1 .mu.M, preferably <100 nM, and most preferably <0.1
nM. Antibody molecules will typically have a K.sub.D in the lower
ranges. K.sub.D=[Ab-Ag]/[Ab][Ag] where [Ab] is the concentration
at equilibrium of the antibody, [Ag] is the concentration at equilibrium
of the antigen and [Ab-Ag] is the concentration at equilibrium of
the antibody-antigen complex. Typically, the binding interactions
between antigen and antibody include reversible noncovalent associations
such as electrostatic attraction, Van der Waals forces and hydrogen
bonds.
The antibodies of the invention specifically bind to TMEFF2 proteins.
By "specifically bind" herein is meant that the antibodies
bind to the protein with a K.sub.D of at least about 0.1 mM, more
usually at least about 1 .mu.M, preferably at least about 0.1 .mu.M
or better, and most preferably, 0.01 .mu.M or better.
Immunoassays
The antibodies of the invention can be used to detect TMEFF2 or
TMEFF2 expressing cells using any of a number of well recognized
immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241;
4,376,110; 4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Asai (ed. 1993) Methods in Cell Biology Vol.
37, Academic Press, New York; Stites & Terr (eds. 1991) Basic
and Clinical Immunology 7th Ed.
Thus, the present invention provides methods of detecting cells
that express TMEFF2. In one method, a biopsy is performed on the
subject and the collected tissue is tested in vitro. The tissue
or cells from the tissue is then contacted, with an anti-TMEFF2
antibody of the invention. Any immune complexes which result indicate
the presence of a TMEFF2 protein in the biopsied sample. To facilitate
such detection, the antibody can be radiolabeled or coupled to an
effector molecule which is a detectable label, such as a radiolabel.
In another method, the cells can be detected in vivo using typical
imaging systems. Then, the localization of the label is determined
by any of the known methods for detecting the label. A conventional
method for visualizing diagnostic imaging can be used. For example,
paramagnetic isotopes can be used for MRI. Internalization of the
antibody may be important to extend the life within the organism
beyond that provided by extracellular binding, which will be susceptible
to clearance by the extracellular enzymatic environment coupled
with circulatory clearance.
TMEFF2 proteins can also be detected using standard immunoassay
methods and the antibodies of the invention. Standard methods include,
for example, radioimmunoassay, sandwich immunoassays (including
ELISA), immunofluorescence assays, Western blot, affinity chromatography
(affinity ligand bound to a solid phase), and in situ detection
with labeled antibodies.
Administration of Pharmaceutical and Vaccine Compositions
The antibodies of the invention can be formulated in pharmaceutical
compositions. Thus, the invention also provide methods and compositions
for administering a therapeutically effective dose of an anti-TMEFF2
antibody. The exact dose will depend on the purpose of the treatment,
and will be ascertainable by one skilled in the art using known
techniques. See, e.g., Ansel, et al. (1999) Pharmaceutical Dosage
Forms and Drug Delivery; Lieberman (1992) Pharmaceutical Dosage
Forms (vols. 1-3), Dekker, ISBN 0824770846, 082476918X, 0824712692,
0824716981; Lloyd (1999) The Art Science and Technology of Pharmaceutical
Compounding Amer. Pharm. Assn.; and Pickar (1999) Dosage Calculations
Thomson. Adjustments for cancer degradation, systemic versus localized
delivery, and rate of new protein synthesis, as well as the age,
body weight, general health, sex, diet, time of administration,
drug interaction and the severity of the condition may be necessary,
and will be ascertainable with routine experimentation by those
skilled in the art. U.S. Ser. No. 09/687,576 further discloses the
use of compositions and methods of diagnosis and treatment in prostate
cancer is hereby expressly incorporated by reference.
A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals. Thus
the methods are applicable to both human therapy and veterinary
applications. In the preferred embodiment the patient is a mammal,
preferably a primate, and in the most preferred embodiment the patient
is human.
The administration of the antibodies of the present invention can
be done in a variety of ways as discussed above, including, but
not limited to, orally, subcutaneously, intravenously, intranasally,
transdermally, intraperitoneally, intramuscularly, intrapulmonary,
vaginally, rectally, or intraocularly.
The pharmaceutical compositions of the present invention comprise
an antibody of the invention in a form suitable for administration
to a patient. In the preferred embodiment, the pharmaceutical compositions
are in a water soluble form, such as being present as pharmaceutically
acceptable salts, which is meant to include both acid and base addition
salts. "Pharmaceutically acceptable acid addition salt"
refers to those salts that retain the biological effectiveness of
the free bases and that are not biologically or otherwise undesirable,
formed with inorganic acids such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid and the like,
and organic acids such as acetic acid, propionic acid, glycolic
acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,
p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically
acceptable base addition salts" include those derived from
inorganic bases such as sodium, potassium, lithium, ammonium, calcium,
magnesium, iron, zinc, copper, manganese, aluminum salts and the
like. Particularly preferred are the ammonium, potassium, sodium,
calcium, and magnesium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary, secondary,
and tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of
the following: carrier proteins such as serum albumin; buffers;
fillers such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol.
The pharmaceutical compositions can be administered in a variety
of unit dosage forms depending upon the method of administration.
For example, unit dosage forms suitable for oral administration
include, but are not limited to, powder, tablets, pills, capsules
and lozenges. It is recognized that antibodies when administered
orally, should be protected from digestion. This is typically accomplished
either by complexing the molecules with a composition to render
them resistant to acidic and enzymatic hydrolysis, or by packaging
the molecules in an appropriately resistant carrier, such as a liposome
or a protection barrier. Means of protecting agents from digestion
are well known in the art.
The compositions for administration will commonly comprise an antibody
of the invention dissolved in a pharmaceutically acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers can
be used, e.g., buffered saline and the like. These solutions are
sterile and generally free of undesirable matter. These compositions
may be sterilized by conventional, well known sterilization techniques.
The compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions such
as pH adjusting and buffering agents, toxicity adjusting agents
and the like, e.g., sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration
of active agent in these formulations can vary widely, and will
be selected primarily based on fluid volumes, viscosities, body
weight and the like in accordance with the particular mode of administration
selected and the patient's needs (e.g., (1980) Remington's Pharmaceutical
Science (18th ed.); and Hardman, et al. (eds. 2001) Goodman &
Gilman: The Pharmacological Basis of Therapeutics).
Thus, a typical pharmaceutical composition for intravenous administration
would be about 0.1 to 10 mg per patient per day. Dosages from 0.1
up to about 100 mg per patient per day may be used, particularly
when the drug is administered to a secluded site and not into the
blood stream, such as into a body cavity or into a lumen of an organ.
Substantially higher dosages are possible in topical administration.
Actual methods for preparing parenterally administrable compositions
will be known or apparent to those skilled in the art, e.g., Remington's
Pharmaceutical Science and Goodman and Gilman: The Pharmacological
Basis of Therapeutics, supra.
The compositions containing antibodies of the invention can be
administered for therapeutic or prophylactic treatments. In therapeutic
applications, compositions are administered to a patient suffering
from a disease (e.g., a cancer) in an amount sufficient to cure
or at least partially arrest the disease and its complications.
An amount adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend
upon the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the-compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the agents of this invention to
effectively treat the patient. An amount of modulator that is capable
of preventing or slowing the development of cancer in a mammal is
referred to as a "prophylactically effective dose." The
particular dose required for a prophylactic treatment will depend
upon the medical condition and history of the mammal, the particular
cancer being prevented, as well as other factors such as age, weight,
gender, administration route, efficiency, etc. Such prophylactic
treatments may be used, e.g., in a mammal who has previously had
cancer to prevent a recurrence of the cancer, or in a mammal who
is suspected of having a significant likelihood of developing cancer.
It will be appreciated that the present prostate cancer protein-modulating
compounds can be administered alone or in combination with additional
prostate cancer modulating compounds or with other therapeutic agent,
e.g., other anti-cancer agents or treatments.
In some embodiments, the antibodies of the invention can be used
to prepare targeted liposomes for delivery of a desired therapeutic
composition (e.g., anti-cancer agents) to a target cell (e.g., a
prostate cancer cell). The preparation and use of immunoliposomes
for targeted delivery of antitumor drugs is reviewed in Mastrobattista,
et al. (1999) Advanced Drug Delivery Reviews 40:103-127.
Liposomes are vesicular structures based on lipid bilayers. They
can be as small as 20 nm and as large as 10 .mu.m in diameter. They
can be unilamellar (only one bilayer surrounds an aqueous core)
or multilamellar (several bilayers concentrically oriented around
an aqueous core). The liposomes of the present invention are formed
from standard vesicle-forming lipids, which generally include neutral
and negatively charged phospholipids and a sterol, such as cholesterol.
The selection of lipids is generally guided by consideration of,
e.g., liposome size and stability of the liposomes in the bloodstream.
Targeting of liposomes using a variety of targeting agents (e.g.,
monoclonal antibodies of the invention) is well known in the art.
See, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). Standard methods
for coupling targeting agents to liposomes can be used. Antibody
targeted liposomes can be constructed using, for instance, liposomes
which incorporate protein A. See, Renneisen, et al. (1990) J. Biol.
Chem. 265:16337-16342; and Leonetti, et al. (1990) Proc. Nati. Acad.
Sci. USA 87:2448-2451.
A variety of methods are available for preparing liposomes, as
described in, e.g., Szoka, et al. (1980) Ann. Rev. Biophys. Bioeng.
9:467; U.S. Pat. Nos. 4, 235,871; 4,501,728; and 4,837,028. One
method produces multilamellar vesicles of heterogeneous sizes. In
this method, the vesicle forming lipids are dissolved in a suitable
organic solvent or solvent system and dried under vacuum or an inert
gas to form a thin lipid film. If desired, the film may be redissolved
in a suitable solvent, such as tertiary butanol, and then lyophilized
to form a more homogeneous lipid mixture which is in a more easily
hydrated powder-like form. This film is covered with an aqueous
solution of the targeted drug and the targeting component (antibody)
and allowed to hydrate, typically over a 15-60 minute period with
agitation. The size distribution of the resulting multilamellar
vesicles can be shifted toward smaller sizes by hydrating the lipids
under more vigorous agitation conditions or by adding solubilizing
detergents such as deoxycholate.
Kits for Use in Diagnostic and/or Prognostic Applications
For use in diagnostic, research, and therapeutic applications suggested
above, kits are also provided by the invention. In the diagnostic
and research applications such kits may include any or all of the
following: assay reagents, buffers, and TMEFF2-specific antibodies
of the invention. A therapeutic product may include sterile saline
or another pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials containing
directions (e.g., protocols) for the practice of the methods of
this invention. While the instructional materials typically comprise
written or printed materials they are not limited to such. Any medium
capable of storing such instructions and communicating them to an
end user is contemplated by this invention. Such media include,
but are not limited to electronic storage media (e.g., magnetic
discs, tapes, cartridges, chips), optical media (e.g., CD ROM),
and the like. Such media may include addresses to internet sites
that provide such instructional materials.
EXAMPLES
Example 1
Approximately 12 anti-TMEFF2 hybridoma supernatants were selected
from an initial pool of roughly one hundred, based on off rates
(kd) for binding to covalently immobilized TMEFF2-FLAG protein as
measured by BIAcore.TM.. Supernatants exhibiting the lowest dissociation
rate constants were chosen for larger scale purification. The sequences
of variable regions of antibodies TMEFF2 #19, TMEFF2 #10, TMEFF2
#18, TMEFF2 #20, TMEFF2 #21 are presented in Table 1. A kinetic
evaluation was carried out on each purified antibody by measuring
binding to TMEFF2-FLAG over a range of antigen concentrations. Affinity
constants (K.sub.D) were then determined using the global fitting
procedure described in the BIAapplications Handbook Biacore AB,
BIAapplications Handbook, version AB, 1998, Application Notes, Note
101 (June 1995); Daiss, et al. (1994) Methods: A companion to Methods
in Enzymology Volume 6, p143-156. In addition, pair-wise epitope
mapping was carried out through a competitive binding analysis.
This was accomplished by exposing the TMEFF2-FLAG surface to a saturating
amount of one antibody sample and measuring the response level of
a second injected antibody. Using this methodology antibodies recognizing
a number of individual epitopes were selected for further study.
Each antibody of interest was covalently coupled to the synthetic
toxin auristatin (Int. J. Oncol. 15:367-72 (1999)) (pAE), a dolastatin
10 derivative, and assessed for TMEFF2 dependent cell death in vitro.
The cell death assay (Proc. Nat'l Acad. Sci. USA 93:8618-23(1996))
was executed by first determining a cell density that exhibits linear
cell growth over several days. Populations of dividing cells were
then incubated with multiple concentrations of toxin-conjugated
TMEFF2 antibodies (or a negative control) for one hour, followed
by removal of the antibody and gentle washing. Four days later,
cell viability was determined by using the Celltiter 96 assay (Promega).
In this manner a prostate cancer cell line stably expressing TMEFF2
(PC3-TMEFF2), was compared with the parental cell line that does
not (PC3).
Two antibodies corresponding to distinct epitopes, as determined
by BIAcore, have been assessed for their ability to interfere with
cell survival in vitro. One of these antibodies, TMEFF2 #19-pAE,
appears to promote significant cell death in PC3-TMEFF2 cells, but
not in the parental line. The other antibody, #21-pAE, also causes
cell death, but with somewhat less potency than #19-pAE. A negative
control antibody that does not recognize a cell surface marker in
PC3 cells, TIB-pAE, does not affect cell survival in either cell
line. Additionally, another prostate cancer line, LnCAP, which has
been determined to express small amounts of surface TMEFF2, also
displayed sensitivity to #19-pAE relative to TIB-pAE. These results
show that #19-pAE is a potent and selective cytotoxic agent on TMEFF2
expressing cells.
TABLE-US-00001 TABLE 1 TMEFF2#19.Heavy chain variable region. SEQ
ID NO: 1 GATGTACAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCAC-
T GGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGG-
C TTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATC-
T GAGAACCAGTTTTTCCTGAACTTGAGATCTGTGACTACTGAGGACACAGCAACATATTATTGTGCAAGAGGTTT-
A CGACGAGGGGACTATTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ
ID NO: 2 DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWSWIRQFPGNKLEWMGFISYDGSNKYNPSLKNRISITRDT-
S ENQFFLNLRSVTTEDTATYYCARGLRRGDYSMDYWGQGTSVTVSS TMEFF2#19.Light
chain variable region SEQ ID NO: 3 GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGTGTCAGCATCACCTGCAAGGC-
C AGTCAGAATGTCGTTACAGCTGTAGCCTGGTATCGACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTC-
G GCATCCAATCGGCACACTGGAGTCCCTGACCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT-
C AACAATATGCAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCGTTCACGTTCGGAGG-
G GGGACCAAGCTGGAAATAAAA SEQ ID NO: 4 DIVMTQSQKFMSTSVGDSVSITCKASQNVVTAVAWYRQKPGQSPKLLIYSASNRHTGVPDRFTGSGSGTDFTLT-
I NNMQSEDLADYFCQQYSSYPFTFGGGTKLEIK TMEFF2#10. heavy chain variable
region SEQ ID NO: 5 GAAGTGAACCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTC-
T GGATTCACTTTCAGTGACTATTACATGTTCTGGATTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATA-
C ATTAGTAATGGTGGTGGTAATACCTATTATTCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGC-
C AAGAACACCCTGTACCTCCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACGGGG-
A TTACGACGAGGGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ
ID NO: 6 EVNLVESGGGLVQPGGSLKLSCATSGFTFSDYYMFWIRQTPEKRLEWVAYISNGGGNTYYSDTVKGRFTISRDN-
A KNTLYLQMSRLKSEDTAMYYCARRGLRRGGAMDYWGQGTSVTVSS TMEFF2#10. Light
chain variable region SEQ ID NO: 7 GACATTGTTTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGC-
C AGCCAAAGTGTTGATTACGGTGGTTATGGTTATATAAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACT-
C CTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGATTT-
C ACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAGTCTATTACTGTCAACAAAGTTATGTGGATCCATT-
C ACGTTCGGCTCGGGGACAAAGTTGGAAATAATC SEQ ID NO: 8 DIVLTQSPASLAVSLGQRATISCKASQSVDYGGYGYINWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTD-
F TLNIHPVEEEDAAVYYCQQSYVDPFTFGSGTKLEII TMEFF2#18. Heavy Chain variable
region SEQ ID NO: 9 CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTC-
T GGGTATACCTTCACAAACTATGGAATGAGCTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTG-
G ATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGGCGGTTTGCCTTCTCTTTGGAAACCTC-
T GCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGGGGGTGATGC-
T TACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO: 10 QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMSWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLET-
S ASTAYLQINNLKNEDTATYFCGGDAYWGQGTLVTVSA TMEFF2#18. Light Chain variable
region SEQ ID NO: 11 GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGC-
C AGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACT-
C CTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT-
C ACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCACCACAGTAGGGAGCTTCGGAC-
G TTCGGTGGAGGCACCAAACTGGAAATCAAA SEQ ID NO: 12 DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTD-
F TLNIHPVEEEDAATYYCQHSRELRTFGGGTKLEIK TMEFF2#20. Heavy Chain variable
region SEQ ID NO: 13 GAGATCCAGCTGCAGCAGTCTGGACCTGAGCTGATGAAGCCTGGGGCTTCAGTGAAGATATCTTGCAAGGCTTC-
T ACTTACTCATTCACTAGGTACTTCATGCACTGGGTGAAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGATA-
T ATTGATCCTTTCAATGGTGGTACTGGCTACAATCAGAAATTCAAGGGCAAGGCCACATTGACTGTAGACAAATC-
T TCCAGCACAGCCTACATGCATCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAACGTATGG-
C TCCGACTACTTTCACTATTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO:
14 EIQLQQSGPELMKPGASVKISCKASTYSFTRYFMHWVKQSHGESLEWIGYIDPFNGGTGYNQKFKGKATLTVDK-
S SSTAYMHLSSLTSEDSAVYYCVTYGSDYFDYWGQGTTLTVSS TMEFF2#20. Light chain
variable region SEQ ID NO: 15 GACATTGTGATGACCCAGCCACAAAAATTCATGTCCACGTCTGTAGGCGACAGGGTCAGTGTCACCTGCAAGGC-
C AGTCAGAATGTGGAAACTGATGTAGTCTGGTATCAACAGAAACCTGGGCAACCACCTAAAGCACTGATTTACTC-
G GCATCCTACCGGCACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAAATTTCACTCTCACCAT-
C AGCACTGTACAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAACTATCCATTCACGTTCGGCTC-
G GGGACAAAGTTGGAAATAATA SEQ ID NO: 16 DIVMTQPQKFMSTSVGDRVSVTCKASQNVETDVVWYQQKPGQPPKALIYSASYRHSGVPDRFTGSGSGTNFTLT-
I STVQSEDLAEYFCQQYNNYPFTFGSGTKLEII TMEFF2#21. Heavy chain variable
region SEQ ID NO: 17 CAGATCCACTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTC-
T GGATATACCTTCACAAACTTTGCAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTCAAGTGGATGGGCTG-
G ATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTC-
T GTCAGTATTGCCTATTTGCAGATCAACAGCCTCAAAAATGAGGACACGGCTACATATTTCTGTTCAAAATTTGA-
C TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 18 QIHLVQSGPELKKPGETVKISCKASGYTFTNFAMNWVKQAPGKGFKWMGWINTYTGEPTYADDFKGRFAFSLET-
S VSIAYLQINSLKNEDTATYPCSKFDYWGQGTTLTVSS TMEFF2#21 .Light Chain variable
region SEQ ID NO: 19 GACATCCAGATGACTCAGTCTCCAGCCTCCCTATATGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGC-
A AGTGAGAATATTTACAGTTATTTAGCATGGTTTCAGCAGAAACAGGGAAAATCTCCTCACCTCCTGGTCTATAA-
T GCAAAAACCTTAGCAGCAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGAT-
C ACCAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCCACGTGGACGTTCGG-
T GGAGGCACCAAGCTGGAAATCAAA SEQ ID NO: 20 DIQMTQSPASLYASVGETVTITCRASENIYSYLAWFQQKQGKSPHLLVYNAKTLAAGVPSRFSGSGSGTQFSLK-
I TSLQPEDFGSYYCQHHYGTPTWTFGGGTKLEIK
Relatively low amounts of the TMEFF2 protein are detectable on
the cell surface of cancer cell lines, as assessed by FACS analysis
using the TMEFF2 #19 antibody. Thus, the effectiveness of the toxin-conjugated
#19 antibody at killing cells specifically expressing this target
was surprising. However, experiments designed to assess the ability
of specific antibody:target combinations to be internalized has
generated novel data that explains the efficiency of the toxin-conjugated
anti TMEFF2 antibodies at killing. It has become apparent that this
particular target protein shows an incredibly high rate of internalization.
In these internalization experiments, cells expressing TMEFF2 are
incubated at different temperatures, and for different lengths of
time, in the presence of anti-TMEFF2 antibody. After incubation
with anti-TMEFF2 antibody for 1 hour at 4.degree. C., the cells
are washed and further incubated with a fluorescently labeled anti-mouse
antibody. By fluorescent microscopy a low level of specific antibody
binding to the TMEFF2 at the cell surface is observed. In contrast,
when cells are incubated at 37.degree. C. for 1 hour, a temperature
that allows for protein trafficking and internalization, and are
then subjected to permeabilization and staining with the fluorescently
labeled anti-mouse antibody, the majority of the fluorescence is
detected within the cells. Such data indicates that the specific
antibody:target combination has been internalized--a result that
is further confirmed by subjecting the cells to an acid--stripping
step prior to the detection step. The acid stripping removes all
protein still present at the cell surface leaving behind only the
internalized antibody:target proteins. In contrast to other antibody:target
combinations such as herceptin:Her2 and anti-ephrinA3: ephrinA3,
these experiments have shown that the TMEFF2 protein, as recognized
by the specific anti-TMEFF2 antibodies, is internalized at a very
rapid rate and also that almost complete internalization of the
cell surface protein is observed within the 1 hour period. These
data, showing the surprisingly efficient internalization of TMEFF2
account for the efficiency of the toxin-conjugated anti-TMEFF2 antibodies
at killing.
Example 2
Using standard techniques as described above, humanized TMEFF2#19
antibodies were generated. The sequences of four humanized heavy
chain variable regions and three humanized light chain variable
regions are presented in Table 2. The heavy and light chain variable
regions may be used to combine into binding sites, and among the
tested combinations, retain binding affinity. These antibodies can
be used in in vivo mouse models to inhibit growth of tumor cells
in vivo.
TABLE-US-00002 TABLE 2 VH 1.0 DNA SEQ ID NO: 21 GATGTACAACTTCAGGAGTCAGGACCTGGCCTCOTCAAACCTTCTGAGACCCTGTCTCTCACCTGCGCAGTCAC-
T GGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGTTTCCAGGAAAGAAACTGGAATGGATGGG-
C TTCATAACCTACGACCCTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATC-
T GAGAACCAGTTTTTCCTGAACTTGTCTTCTGTCACTCCAGCACACACAGCAACATATTATTGTGCAAGAGGTTT-
A CGACGAGGGCACTATTCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCA VH
1.0 AMINO ACIDS SEQ ID NO: 22 DVQLQESGPGLVKPSETLSLTCAVTGYSITSGYYWSWIRQFPGKKLEWMGFISYDGSNKYNPSLKNRISITRDT-
S ENQFFLKLSSVTAADTATYYCARGLRRGDYSMDYWGQGTLVTVSS VH 2.0 DNA SEQ ID
NO: 23 GATGTACAACTTCAGGACTCAGGACCTGGCCTCCTGAAACCTTCTGAGACCCTGTCTCTCACCTGCGCAGTCAC-
T CGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGCCTCCACGAAAGGGCCTGCAATGGATGGG-
C TTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATC-
T GAGAACCAGTTTTTCCTCAAGTTGTCTTCTGTGACTGCACCAGACACACCAGTCTATTATTGTGCAAGAGGTTT-
A CGACGAGGCCACTATTCTATGCACTACTGGGGTCAAGGAACCCTCGTCACCGTCTCCTCA VH
2.0 AMINO ACIDS SEQ ID NO: 24 DVQLQESGPGLVKPSETLSLTCAVTGYSITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRISITRDT-
S ENQFFLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSS VH 3.0 DNA SEQ ID
NO: 25 GATGTACAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTGAGACCCTGTCTCTCACCTCCGCAGTCAG-
C GGCTACTCCATCACCAGTGGTTATTACTGGAGCTGGATCCGGCAGCCTCCAGGAAAGGGCCTCCAATGGATGGG-
C TTCATAAGCTACGACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCACCATCTCCCGTGACACATC-
T AAGAACCAGTTTTCCCTGAAGTTGTCTTCTGTCACTGCAGCACACACACCAGTCTATTATTGTGCAACAGGTTT-
A CGACGAGGGCACTATTCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCA VH
3.0 AMINO ACIDS SEQ ID NO: 26 DVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRDT-
S KNQFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSS VH 4.0 DNA SEQ ID
NO: 27 GATGTACAACTTCAGGAGTCAGGACCTGCCCTCGTGAAACCTTCTCACACCCTGTCTCTCACCTGCGCAGTCAG-
C GGCTACTCCATCACCAGTGCTTATTACTGGAGCTCCATCCGGCAGTTTCCAGGAAAGAPACTGGAATCGATGGG-
C TTCATAAGCTACCACGGTTCCAATAAGTATAATCCATCTCTCAAAAATCGAATCACCATCTCCCGTGACACATC-
T AAGAACCAGTTTTCCCTGAAGTTGTCTTCTGTGACTGCAGCAGACACAGCAACATATTATTGTGCAAGAGGTTT-
A CGACGAGGGCACTATTCTATCGACTACTCGCGTCAAGGAACCCTGGTCACCCTCTCCTCA VH
4.0 AMINO ACIDS SEQ ID NO: 20 DVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQFPGKKLEWMGFISYDGSNKYNPSLKNRITISRDT-
S KNQFSLKLSSVTAADTATYYCARGLRRGDYSMDYWGQGTLVTVSS VL 1.0 DNA SEQ ID
NO: 29 GACATTCAGATGACCCAGTCTCAATCTAGTATGTCCACATCAGTACGAGACCGAGTCACCATCACCTGCAAGGC-
C AGTCAGAATGTCGTTACAGCTGTACCCTGGTATCGACAGAAACCAGGAAAGTCTCCTAAACTACTGATTTACTC-
G GCATCCAATCGCCACACTGGAGTCCCTAGTCGCTTCTCTGCCAGTGCATCTCCCACAGATTTCACTCTCACCAT-
C TCTAGCATGCAGCCTGAAGACTTCGCAGATTATTTCTGCCAGCAATATACCAGCTATCCGTTCACGTTCGGAGG-
G GGGACCAAGCTCGAGATCAAACGG VL 1.0 AMINO ACIDS SEQ ID NO: 30 DIQMTQSQSSMSTSVGDRVTITCKASQNVVTAVAWYRQKPGKSPKLLIYSASNRHTGVPSRFSGSGSGTDFTLT-
I SSMQPEDFADYFCQQYSSYPFTFGGGTKLEIKR VL 2.0 DNA SEQ ID NO: 31 GACATTCAGATGACCCAGTCTCCATCTAGTCTGTCCCCTTCAGTACGAGACCGAGTCACCATCACCTGCAAGGC-
C AGTCAGAATGTGGTTACAGCTGTAGCCTGGTATCGACACAAACCAGGAAAGTCTCCTAAACTACTGATTTACTC-
G GCATCCAATCGGCACACTGCAGTCCCTAGTCCCTTCTCTGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT-
C TCTAGCCTGCAGCCTGAAGACTTCGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCGTTCACGTTCGGAGG-
G GGGACCAAGGTCGAGATCAAACGG VL 2.0 AMINO ACIDS SEQ ID NO: 32 DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYRQKPGKSPKLLIYSASNRHTGVPSRFSGSGSGTDFTLT-
I SSLQPEDFADYFCQQYSSYPFTFGGGTKVEIKR VL 3.0 DNA SEQ ID NO: 33 GACATTCAGATGACCCAGTCTCCATCTAGTCTGTCCGCTTCAGTAGGAGACCGAGTCACCATCACCTGCAAGGC-
C AGTCAGAATGTGGTTACAGCTGTAGCCTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAACTACTGATTTACTC-
G GCATCCAATCGGCACACTGGAGTCCCTAGTCGCTTCTCTGGCAGTGGATCTGGGACAGATTTCACTCTCACCAT-
C TCTAGCCTGCAGCCTGAAGACTTCGCAACCTATTATTGCCAGCAATATAGCACCTATCCGTTCACGTTCGGAGG-
G GGCACCAACGTCGAGATCAAACGG VL 3.0 AMINO ACIDS SEQ ID NO: 34 DIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYSASNRHTCVPSRFSGSGSGTDFTLT-
I SSLQPEDFATYYCQQYSSYPFTFGGGTKVEIKR
Example 3
Auristatin E Conjugated Anti-TMEFF2 Antibodies Target and Kill
Prostate Cancer Tumors in Vivo
The TMEFF2 gene is highly and specifically expressed in clinical
prostate cancer samples. To demonstrate that the protein product
of the TMEFF2 gene is a therapeutic target for the treatment of
prostate cancer, the human prostate cancer cell line LNCAP was modeled
in SCID (severe combined immunodeficient) mice. Gene expression
analysis shows that TMEFF2 is highly expressed in LNCAP cells grown
on plastic in tissue culture and also when grown as xenograft tumors
in SCID mice.
To determine the in vivo effects of toxin-conjugated anti-TMEFF2
antibodies (#19-pMMVCAE), LNCAP cells were grown as xenograft tumors
in SCID mice. After the tumors reached a certain size (average of
100 mm.sup.3), the animals were distributed into 3 groups and subjected
to treatment with either a) control vehicle, b) #19-pMMVCAE, or
c) isotype control-MMVCAE (an antibody that does not recognize molecules
on the surface of LNCAP cells). Conjugated antibodies were used
at 0.25 mg/kg of drug equivalent (.about.5 mg/kg of antibody-drug
conjugate), and were administered at 4 day intervals. Tumor size
was measured twice a week. Animal weight was monitored throughout
the experiment and serum PSA (prostate-specific antigen) levels
were measured at various time intervals during the experiment.
The results showed that treatment with #19-pMMVCAE significantly
reduced LNCAP tumor growth. In fact, established LNCAP tumors regressed
in size (to less than 100 mm.sup.3), serum PSA (a surrogate marker
for prostate tumor burden) levels significantly dropped (<10
ng/ml), while animal weight remained steady and animals appeared
healthy. This is in contrast to mice that received either control
vehicle or the isotype control-MMVCAE. The tumors in these mice
grew rapidly and had to be sacrificed at days 50-60 post tumor implantation
due to the large size of the tumors (>500 mm.sup.3). In addition,
the animals lost considerable amount of weight, appeared moribund
and had significantly higher levels of serum PSA (>350 ng/ml).
Treatment with humanized #1 |