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Cancer Patent Abstract
The present application describes a method for making antibodies
which can be used for cancer diagnosis or therapy. The application
also discloses a method for identifying an antigen which is differentially
expressed on the surface of two or more distinct cell populations.
The application additionally describes human antibodies directed
against decay accelerating factor (DAF), as well as therapeutic
compositions comprising such antibodies. Moreover, the application
discloses a method of treating lung cancer with antibodies directed
against DAF.
Cancer Patent Claims
What is claimed is:
1. An isolated human antibody directed against human decay accelerating
factor (DAF) which has a binding affinity for human DAF of about
10 nM or better, and which binds an epitope on DAF bound by an antibody
selected from the group consisting of LU30 having variable light
chain and heavy chain sequences of SEQ ID NOs: 1 and 4, respectively,
LU13 having variable light chain and heavy chain sequences of SEQ
ID NOs: 2 and 5, respectively, and LU20 having variable light chain
and heavy chain sequences of SEQ ID NOs: 3 and 6, respectively.
2. The antibody of claim 1 comprising antigen-binding amino acid
residues of an antibody selected from the group consisting of LU30
having variable light chain and heavy chain sequences of SEQ ID
NOs: 1 and 4, respectively, LU13 having variable light chain and
heavy chain sequences of SEQ ID NOs: 2 and 5, respectively, and
LU20 having variable light chain and heavy chain sequences of SEQ
ID NOs: 3 and 6, respectively.
3. The antibody of claim 1 which is selected from the group consisting
of LU30 having variable light chain and heavy chain sequences fo
SEQ ID NOs: 1 and 4, respectively, LU13, having variable light chain
and heavy chain sequences of SEQ ID NOs: 2 and 5, respectively and
LU20, having variable light chain and heavy chain sequences of SEQ
ID NOs: 3 and 6, respectively.
4. A pharmaceutical composition comprising the antibody of claim
1 and a pharmaceutically acceptable carrier.
5. An article of manufacture comprising the pharmaceutical composition
of claim 4 and a package insert instructing the user of the composition
to treat a patient having lung cancer, or predisposed to lung cancer,
with said composition, wherein said patient having lung cancer or
predisposed to lung cancer exhibits overexpression of Decay Accelerating
Factor bound by the antibody of the composition.
6. The article of manufacture of claim 5 wherein the lung cancer
is selected from the group eonsisting of small-cell lung cancer,
non-small cell lung cancer, large cell lung carcinoma, lung adenocarcinoma,
and squamous cell lung carcinoma.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns a method for making antibodies which
can, for example, be used for cancer diagnosis or therapy. The invention
further provides a method for identifying an antigen which is differentially
expressed on the surface of distinct cell populations. The present
invention additionally provides human antibodies directed against
decay accelerating factor (DAF), as well as therapeutic compositions
comprising such antibodies. Moreover, the invention pertains to
a method of treating lung cancer with antibodies directed against
DAF.
2. Description of Related Art
The demonstration of significant anti-tumor efficacy of antibodies
has long been sought-after in the clinic and recently obtained using
"naked" chimeric/humanized antibodies (Riethmuller et
al., Lancet, 343: 1177-1183 (1994); Riethmuller et al., J. Clin.
Oncol., 16: 1788-1794 (1998); Maloney et al., Blood, 90: 2188-2195
(1997); McLaughlin et al., J. Clin. Oncol., 16: 2825-2833 (1998);
and Baselga et al., J. Clin. Oncol., 14: 697-699 (1996)) antibodies
as well as with radiolabeled murine antibodies (Press et al., N.
Engl. J. Med., 329: 1219-1224 (1993); Press et al., Lancet, 346:
336-340, (1995); Kaminski et al., N. Engl. J. Med., 329: 459-495
(1993); Kaminski et al., J. Clin. Oncol., 14: 1974-1981 (1996)).
Indeed a chimeric anti-CD20 antibody (Reff et al., Blood, 83: 435-445
(1994)) and a chimeric/humanized anti-HER2 antibody (Carter et al.
PNAS (USA) 89:4285-4289 (1992)) have recently been approved by US
Federal Drug Administration for the treatment of non-Hodgkin's lymphoma
and metastatic breast cancer, respectively. These successes with
anti-tumor antibodies in patients has led to renewed interest in
the identification of novel tumor-associated antigens suitable for
antibody targeting.
The traditional approach to obtaining tumor-specific antibodies
has been to immunize mice with tumor cells and to screen the resultant
monoclonal antibodies for their binding specificity. Unfortunately
tumor-binding antibodies obtained in this way often cross-react
with many normal cells, which may interfere with their clinical
utility. Ideally one would like to select rather than screen for
antibodies that bind selectively to tumor. The advent of antibody
fragment display on phage (McCafferty et al., Nature, 348: 552-554
(1990)) and the development of large (>10.sup.10 clone) phage
display libraries (Griffiths et al., EMBO J., 13:3245-3260 (1994),
Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996)) offers a potential
way of making antibodies. With antibody phage screening, unlike
hybridoma technology, it is readily possible to obtain antibodies
binding antigens that are highly conserved between mouse and man
(Nissim et al., EMBO J., 13:692-698 (1994)).
Naive antibody phage libraries have proved to be a rapid and general
method for identifying antibodies binding to purified antigens (Griffiths
et al., EMBO J., 13:3245-3260 (1994); Vaughan et al. Nat. Biotechnol.
14: 309-314 (1996); Nissim et al., EMBO J., 13:692-698 (1994)).
In contrast, panning cellular targets with antibody phage has proved
much more difficult because of the much lower effective antigen
concentration, greater antigen complexity and the tendency of phage
to bind non-specifically to cells. Nevertheless, antibodies against
cell surface antigens have been identified (Marks et al, Bio/Technol.,
11: 1145-1149 (1992); Portolano et al., J. Immunol., 151:2839-2851
(1993); de Kruif et al, Proc. Natl. Acad. Sci. USA, 92:3938-3942
(1995); Van Ewijk et al., Proc. Natl. Acad. Sci. USA, 94:3903-3908
(1997); Cai et al, Proc. Natl. Acad. Sci. USA, 92:6537-6541 (1995);
Cai et al Proc. Natl. Acad. Sci. USA, 93:6280-6285 (1996); Cai et
al, Proc. Natl. Acad. Sci. USA, 94:9261-9266 (1997)). Melanoma specific
antibodies have been identified by selecting for antibody phage
that bind to melanoma cells but not melanocytes using antibody phage
libraries constructed from human donors immunized with their own
tumor cells (Cai et al, Proc. Natl. Acad. Sci. USA, 92:6537-6541
(1995); Cai et al Proc. Natl. Acad. Sci. USA, 93:6280-6285 (1996);
Cai et al., Proc. Natl. Acad. Sci. USA, 94:9261-9266 (1997)).
Decay Accelerating Factor (DAF), is a GPI-anchored protein that
acts together with two other GPI-anchored proteins, CD46 and CD59,
in protecting host cells from complement-mediated cell lysis (Nicholson-Weller
et al. J. Lab. Clin. Med., 123:485-491 (1994)). DAF is expressed
at widely varying levels on tumor cell lines and its overexpression
correlates with enhanced resistance to complement-mediated cell
lysis in vitro (Cheung et al., J. Clin. Invest., 81:1122-1128 (1988)).
DAF overexpression has been observed on a variety of human tumor
tissues including 6/9 lung adenocarcinomas and 2/7 lung squamous
cell carcinomas (Niehans et al., Am. J. Path., 149:129-142 (1996)).
Regarding normal lung tissue, DAF has been detected by immunohistochemistry
on the alveolar epithelium, interstitium and endothelium as well
as the bronchial epithelium, glands and ducts plus blood vessels
(Niehans et al., Am. J. Path., 149:129-142 (1996)).
Other publications relating to DAF include Hara et al. Immunology
Letters 37:145-152 (1993); Nicholson-Weller and Wang J. Lab. Clin.
Med. 123(4):485491 (1994); Lublin et al. J. Immunol. 137:1629-1635
(1986); WO99/43800; WO98/39659; U.S. Pat. No. 5,695,945; U.S. Pat.
No. 5,763,224; and WO 86/07062.
Vollmers et al. Cancer Research 49: 2471-2476 (1989); and Vollmers
et al. Cancer 76(4): 550-558 (1995) describe the human IgM monoclonal
antibody "SC-1" which is said inhibit growth of stomach
adenocarcinoma cells in vitro and in vivo by inducing apoptosis.
Vollmers et al. Oncology Reports 5:549-552 (1998) reports the results
of a clinical trial in which patients with poorly differentiated
stomach adenocarcinoma were treated with the SC-1 antibody. The
later publication, Hensel et al. Cancer Research 59:5299-5306 (1999),
identifies DAF as the antigen bound by SC-1.
SUMMARY OF THE INVENTION
In the present application, a large naive antibody phage library
was used to search for cancer-associated antigens, thus obviating
the need for creating custom libraries from immunized donors. In
addition, antibodies were selected using live rather than fixed
cells, to obtain antibodies primarily against native rather than
denatured antigens. This was done to facilitate subsequent expression
cloning of corresponding antigen as well as enhance the therapeutic
potential of antibodies obtained. Indeed an antigen corresponding
to a scFv fragment identified with significant tumor selectivity
was cloned according to the present methods.
Accordingly, the invention provides a method for making an antibody
comprising the following steps: (a) binding antibody phage from
a naive antibody phage library to a live cancer cell; (b) selecting
an antibody phage or antibody which binds selectively to the live
cancer cell; and (c) identifying an antigen to which the antibody
phage or antibody binds.
The invention further provides an antibody derived according to
the method of the preceding paragraph and optionally including amino
acid sequence alterations (e.g. additions, deletions and/or substitutions)
compared to the antibody selected in step (b)). Moreover, the invention
provides a method for detecting the antigen comprising exposing
a sample suspected of containing the antigen to the antibody or
altered antibody and determining binding of the antibody or altered
antibody to the sample. The invention further provides a method
for treating a mammal having a disease or disorder comprising administering
the above antibody or altered antibody to the mammal in an amount
effective to treat the disease or disorder.
The invention further provides a method for identifying an antigen
which is differentially expressed on the surface of two or more
distinct cell populations, comprising the following steps: (a) binding
antibody phage from a naive antibody phage library to a first cell
population; (b) binding the antibody phage to a second cell population
which is distinct from the first cell population; (c) selecting
an antibody phage or antibody which binds selectively to the first
cell population; and (d) identifying an antigen to which the antibody
phage or antibody in (c) binds.
The invention further provides an antagonist, such as an antibody,
directed against an antigen, wherein the antigen has been identified
according to the method of the previous paragraph.
The invention additionally relates to an isolated human antibody
which is directed against, or specifically binds to, human decay
accelerating factor (DAF), obtainable by the methods herein. The
invention further provides a human antibody which has better binding
affinity for DAF than the human IgM SC-1 antibody has for DAF, e.g.
about 10 nM or better binding affinity for human DAF (for instance,
in the range from about 10 nM to about 1 pM). An example of an antibody
with such strong binding affinity for DAF is the LU30 antibody herein
which has a binding affinity (K.sub.d) for DAF of about 13 nM as
determined using a BIACORE.TM. instrument. The antibody optionally
binds an epitope on DAF bound by the LU30, LU13 or LU20 antibodies
herein disclosed. The human antibody may, for instance, comprise
antigen-binding amino acid residues of the LU30, LU13 or LU20 antibodies.
The application additionally provides the human antibodies designated
LU30, LU13 and LU20 herein as well as variants of any one of those
antibodies. Preferred amino acid sequence variants comprise VH and
VL domains which together share about 90-100%, and preferably about
95-100%, and most preferably 98-100%, amino acid sequence identity
with the VH and VL amino acid sequences of the LU30, LU13 or LU20
antibodies as depicted in FIGS. 5A and 5B herein. One preferred
amino acid sequence variant is an affinity matured variant, which
comprises one or more amino acid sequence modifications (e.g. about
1-20, and most preferably about 3-10 amino acid substitutions) in
one or more hypervariable regions of the LU30, LU13 or LU20 VH and/or
VL amino acid sequences disclosed herein. Another type of variant
is a glycosylation variant which has altered glycosylation compared
to a parent antibody and thus may have altered effector function(s).
While Fv fragment forms (e.g. single chain Fv fragments, scFv) of
the LU30, LU13 or LU20 antibodies may be used, the variable regions
of these antibodies are optionally fused to heterologous polypeptide(s)
such as (1) a toxin polypeptide(s) to generate an immunotoxin or
(2) antibody constant region sequences to make larger antibody molecules,
such as Fab fragments, F(ab').sub.2 fragments or intact antibodies.
Such intact antibodies generally have human heavy and light chain
constant regions and, therefore, have antibody effector functions,
such as antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement dependent cytotoxicity (CDC).
In another embodiment, the invention pertains to a pharmaceutical
composition comprising a human antibody directed against DAF and
a pharmaceutically acceptable carrier. In addition, the invention
provides an article of manufacture comprising the pharmaceutical
composition and a package insert instructing the user of the composition
to treat a patient having, or predisposed to, lung cancer with the
composition. The lung cancer to be treated includes small-cell lung
cancer, non-small cell lung cancer, large cell lung carcinoma, lung
adenocarcinoma, and squamous cell lung carcinoma.
In yet a further embodiment, the invention relates to method of
treating lung cancer comprising administering a therapeutically
effective amount of an antibody directed against decay accelerating
factor (DAF) to a human patient. Candidates for treatment with the
anti-DAF antibody are optionally screened to determine DAF expression
by tumor cells. For instance, DAF overexpression, and/or expression
of a DAF glycoform, by the tumor may be assessed using diagnostic
procedures available in the art, such as immunohistochemistry (IHC)
or a DNA-based assay (e.g. fluorescent in situ hybridization, FISH).
This way, a subpopulation of cancer patients (e.g. DAF-overexpressing
patients or patients expressing a cancer-related variant of DAF)
may be identified and those patients can be treated as described
herein. The antibody may be administered in the neoadjuvant, adjuvant
or metastatic settings. Moreover, the antibody used for such therapy
may be conjugated with a cytotoxic agent (examples of which are
provided below) in order to generate an immunotoxin. Preferably,
the antibody is a human antibody (e.g. one which has a binding affinity
for DAF of about 10 nM or better). The antibody for such therapy
optionally binds an epitope on DAF bound by any one of the LU30,
LU13, LU20, 791T36 or SC-1 antibodies. The antibody for therapy
may, therefore, comprise antigen-binding amino acid residues of
the LU30, LU13, LU20, 791T36 or SC-1 antibodies. The patient may
optionally be treated with a second different cytotoxic agent, wherein
the second cytotoxic agent is therapeutically effective against
lung cancer. Examples of such second cytotoxic agents include, but
are not limited to, navelbine, gemcitabine, a taxoid, carboplatin,
cisplatin, etoposide, cyclosphosphamide, mitomycin, vinblastine,
an anti-ErbB2 antibody (e.g. HERCEPTIN.RTM., sold by Genentech,
Inc., South San Francisco), an anti-angiogenic factor antibody (e.g.
an anti-VEGF antibody), an anti-mucin antibody, or a second antibody
directed against a different epitope on DAF. Such therapy with the
combination of the antibody and the second cytotoxic agent may result
in a synergistic therapeutic effect against lung cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts flow cytometric analysis of phage populations from
rounds 1, 2 and 3 binding to tumor cell line 1264 (dark) used for
selection and non-tumor cell line BEAS-2B (light) used for counter-selection.
Also shown is a negative control phage population.
FIGS. 2A-C depict dendrograms for tumor-selective scFv satisfying
primary and secondary selection criteria (Table 1). Comparisons
were made between scFv amino acid sequences (FIG. 2A) as well as
their component V.sub.H domains (FIG. 2B), and V.sub.L domains (FIG.
2C).
FIG. 3 shows flow cytometric analysis of scFv fragments with tumor
(1264, A549, CALU6 and SKLU1) and non-tumor (BEAS-2B and NHEK) cell
lines.
FIG. 4 shows binding of LU30 scFv (3 .mu.g/ml) to 1264 cells in
the absence and presence of recombinant human DAF (30 .mu.g/ml).
FIGS. 5A and 5B depict the amino acid sequences of the variable
light (VL) (FIG. 5A; SEQ ID NOS: 1-3, respectively) and variable
heavy (VH) (FIG. 5B; SEQ ID NOS:4-6, respectively) domains of human
antibodies LU30, LU13 and LU20 identified in Example 1. Complementarity
Determining Region (CDR) residues are those residues in bold and
hypervariable loop residues are within brackets.
TABLE-US-00001 TABLE 1 Primary screening of scFv phage clones BstNI
fingerprint # tumor selective clone identity type clones.sup.a (#
clones sequenced) 1 110 LU4 (8) 2 49 LU1 (7) 3 10 LU20 (9) 4 7 LU13
(3), LU34 (4).sup.b 5 4 LU22 (4) 6 3 LU36 (3) 7 3 LU41 (3) 8 3 LU57
(2) 9 3 LU3 (1), LU77 (2).sup.b 10 2 LU30 (2) 11 1 LU7 (1) 12 1
LU71 (1) 13 1 LU100 (1) 14 1 LU60 (1).sup.c 15 1 LU78 (1) .sup.aTumor
selective clones by phage ELISA: robust binding to 1264 cells (A.sub.450-A.sub.650
.gtoreq.0.3) and much weaker binding to BEAS-2B cells (.gtoreq.10-fold
lower signal), as judged by phage ELISA. .sup.bClones LU13 and LU34
are predicted from their nucleotide sequences to generate identical
fingerprint patterns, whereas clones LU3 and LU77 share closely
related fingerprints that were not distinguishable by our electrophoretic
analysis. .sup.cCodon 3 in V.sub.H is amber (TAG) that will be read
through as glutamine in the supE E. coli strain, TG1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
The term "antibody" is used in the broadest sense and
specifically covers intact monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
"Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; "linear antibodies"
(U.S. Pat. No. 5,641,870); single-chain antibody molecules such
as single chain Fv fragments (scFv); and multispecific antibodies
formed from antibody fragments.
An "intact" antibody is one which comprises an antigen-binding
variable region as well as a tight chain constant domain (C.sub.L)
and heavy chain constant domains, C.sub.H1, C.sub.H2 and C.sub.H3.
The constant domains may be native sequence constant domains (e.g.
human native sequence constant domains) or amino acid sequence variants
thereof. Preferably, the intact antibody has one or more effector
functions.
Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc region
or amino acid sequence variant Fc region) of an antibody. Examples
of antibody effector functions include C1q binding; complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g. B cell receptor, BCR), etc.
Depending on the amino acid sequence of the constant domain of
their heavy chains, intact antibodies can be assigned to different
"classes". There are five major classes of intact antibodies:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into "subclasses" (isotypes), e.g., IgG1 (including
human A and non-A allotypes), IgG2, IgG3, IgG4, IgA, and IgA2. The
subunit structures and three-dimensional configurations of different
classes of immunoglobulins are well known.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on
a target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express FcgammaRIII
only, whereas monocytes express FcgammaRI, FcgammaRII and FcgammaRIII.
FcR expression on hematopoietic cells is summarized in Table 3 on
page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
To assess ADCC activity of a molecule of interest, an in vitro ADCC
assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337
may be performed. Useful effector cells for such assays include
peripheral blood mononuclear cells (PBMC) and Natural Killer (NK)
cells. Alternatively, or additionally, ADCC activity of the molecule
of interest may be assessed in vivo, e.g., in a animal model such
as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
"Complement dependent cytotoxicity" or "CDC"
refer to the ability of a molecule to lyse a target in the presence
of complement. The complement activation pathway is initiated by
the binding of the first component of the complement system (C1q)
to a molecule (e.g. an antibody) complexed with a cognate antigen.
To assess complement activation, a CDC assay, e.g. as described
in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may
be performed.
The "antigen-binding" amino acid residues of an antibody
are those residues which contact antigen and result in specific
binding of the antibody to that antigen. Generally, the antigen-binding
residues coincide with the hypervariable region residues of an antibody.
The hypervariable regions generally comprise amino acid residues
from a "complementarity determining region" or "CDR"
(e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3)
in the heavy chain variable domain; Kabat et al., Sequences of Proteins
of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework
Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
The term "monoclonal antibody" as used herein refers
to an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population
are identical except for possible naturally occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody preparations
which typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against
a single determinant on the antigen. In addition to their specificity,
the monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous population
of antibodies, and is not to be construed as requiring production
of the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries.
The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding sequences
in antibodies derived from a particular species or belonging to
a particular antibody class or subclass, while the remainder of
the chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such antibodies,
so long as they exhibit the desired biological activity (U.S. Pat.
No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855
(1984)).
A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising non-human
antigen-binding residues. Human antibodies can be produced using
various techniques known in the art. In the preferred embodiment,
the human antibody is selected from a phage library, where that
phage library expresses human antibodies (Vaughan et al. Nature
Biotechnology 14:309-314 (1996): Sheets et al PNAS (USA) 95:6157-6162
(1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks
et al, J. Mol. Biol., 222:581 (1991); and Example 1 herein). Human
antibodies can also be made by introducing 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, for
example, 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, Bio/Technology 10: 779-783 (1992); Lonberg et al.,
Nature 368: 856-859 (1994); Morrison, Nature 368:812-13 (1994);
Fishwild et al., Nature Biotechnology 14: 845-51 (1996); Neuberger,
Nature Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern.
Rev. Immunol. 13:65-93 (1995). Alternatively, the human antibody
may be prepared via immortalization of human B lymphocytes producing
an antibody directed against a target antigen (such B lymphocytes
may be recovered from an individual or may have been immunized in
vitro); see, e.g., Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147 (1):86-95 (1991); U.S. Pat. No. 5,750,373.
"Humanized" forms of non-human (e.g., murine) antibodies
are chimeric 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. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a complementarity determining region (CDR) of the recipient
are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired specificity,
affinity, and capacity. In some instances, Fv framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies may comprise
residues which are found neither in the recipient antibody nor in
the imported CDR or framework sequences. These modifications are
made to further refine and maximize antibody performance. In general,
the humanized antibody will comprise substantially all of at least
one, and typically two, variable domains, in which all or substantially
all of the CDRs correspond to those of a non-human immunoglobulin
and all or substantially all of the FRs are those of a human immunoglobulin
sequence. The humanized antibody optimally also will comprise at
least a portion of an immunoglobulin constant region (Fc), typically
that of a human immunoglobulin. For further details, see Jones et
al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The
humanized antibody includes a PRIMATIZED.TM. antibody wherein the
antigen-binding region of the antibody is derived from an antibody
produced by immunizing macaque monkeys with the antigen of interest.
"Single chain Fv" or "scFv" antibody fragments
comprise the V.sub.H and V.sub.L domains of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the scFv to form the desired
structure for antigen binding. For a review of scFv see Pluckthun
in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments
with two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable domain
(V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using
a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites. Diabodies
are described more fully in, for example, EP 404,097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred affinity
matured antibodies will have nanomolar or even picomolar affinities
for the target antigen. Affinity matured antibodies are produced
by procedures known in the art. Marks et al. Rio/Technology 10:779-783
(1992) describes affinity maturation by V.sub.H and V.sub.L domain
shuffling. Random mutagenesis of CDR and/or framework residues is
described by: Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813
(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.
Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9
(1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
An antibody which is "directed against" or which "specifically
binds to" an antigen of interest, e.g. DAF antigen, is one
capable of binding that antigen with sufficient affinity such that
the antibody is useful as a therapeutic agent in targeting the antigen.
The antigen here is normally DAF as it exists in a patient to be
treated with the antibody (especially the antigen expressed by tumor
cells in the patient). Notwithstanding this, various forms of DAF
(e.g. native, recombinant, and synthetic DAF, including DAF variants
and fragments) may be used to generate or raise the antibody.
The "binding affinity" of an antibody for a target antigen,
such as DAF, may be determined by equilibrium methods (e.g. enzyme-linked
immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics
(e.g. BIACORE.TM. analysis; see Example 1 below), for example.
To determine whether an antibody binds to an "epitope"
on an antigen, such as DAF, bound by another antibody, a routine
cross-blocking assay such as that described in Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane
(1988), can be performed.
The term "antibody phage" refers to a bacteriophage with
an antibody (particularly an antibody fragment such as a scFv, diabody,
linear antibody or Fab) displayed on the surface thereof.
A "naive antibody phage library" comprises a plurality
of antibody phages which have not been derived from an immunized
host, i.e. a "non-immunized" phage display library (see,
e.g. Vaughan et al. Nat. Biotechnol. 14: 309-314 (1996); and Sheets
et al. PNAS (USA) 95:6157-6162 (1988)). Exemplary methods for generating
such "naive" or "non-immunized" phage libraries
are elaborated herein.
The act of "binding" antibody phage to a cell or cell
population entails exposing or contacting the antibody phage to/with
the cell or cell population under appropriate conditions and for
a sufficient period of time such that the antibody displayed on
the surface of the phage noncovalently binds to one or more antigens
on the cell or cell population. Generally, those antigen(s) to which
the antibody bind(s) are present at the surface of a cell (i.e.
are "cell surface antigen(s)"). The "antigen"
is generally a protein, but may be a non-protein molecule such as
a lipid, carbohydrate, glycolipid, nucleic acid etc.
A "live" cell is one which has not been histologically
fixed with a fixative such as glutaraldehyde. The live cell may
be a "primary" cell which has, e.g., been surgically removed
from a mammal or a "cell line" capable of being continuously
cultivated in cell culture. A "live cancer cell" is a
cancer or tumor cell which has not been histologically fixed and
a "live non-cancer cell" is a noncancerous cell (i.e.
one which has not been derived from a cancer or tumor) which has
not been histologically fixed.
A "distinct" cell or cell population is one which is
genotypically and/or phenotypically different from another cell
or cell population to which it is being compared. The "distinct"
cells or cell populations may however, be of the same tissue-type;
for example, a cancer cell and a non-cancer cell of the same tissue
type. In the Example below, lung cancer cell lines (1264, SKLU1,
A549 and CALU6) and non-cancer lung cell lines (BEAS-2B, CCD19LU
and NHBE 4683) were utilized as distinct cell populations.
By "selecting" an antibody phage or antibody is meant
choosing for further analysis, or for employment in further method(s),
an antibody phage or antibody derived therefrom.
An antibody phage or antibody which "binds selectively"
to a cell or cell population is one which binds preferentially to
that cell or cell population compared to a distinct cell or cell
population. The antibody phage or antibody preferably binds selectively
to a cancer cell compared to a non-cancer cell of the same tissue-type.
Such selective binding can be determined by a number of methods
known in the art including ELISA (with scFv, Fab or antibody phage);
flow cytometry (with scFv, Fab or antibody phage); and immunohistochemistry
(with scFv, Fab or antibody phage).
The act of "counter-selecting" herein refers to binding
antibody phage from an antibody phage library to a first cell or
first cell population (e.g. a non-cancer cell or cell population)
which is distinct from a second cell or second cell population of
interest (e.g. a cancer cell or cancer cell population) and substracting
or removing those antibody phage which bind to the first cell or
cell population (e.g. the antibody phage which bind to the first
cell or first cell population are not subjected to subsequent analyses
or screening(s)). This may, for example, be achieved by centrifuging
antibody phage bound to the first cell(s) and using the supernatant
thereby obtained for further analysis or screening.
"Expression cloning" refers to the act of characterizing
a nucleic acid encoding a protein (e.g. a protein antigen) of interest,
wherein the method involves detecting that protein expressed by
the nucleic acid. Detection is possible using an antibody directed
against the protein, e.g., an antibody phage or antibody derived
from a naive phage library as described herein.
"Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented. The patient to be treated herein
may have, or be predisposed to, cancer (e.g. lung cancer). The patient
who is "predisposed" to cancer, may display risk factor(s),
such as DAF overexpression and/or expression of a DAF glycoform
thought to be associated with cancer.
"Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm animals,
and zoo, sports, or pet animals, such as dogs, horses, cats, cows,
etc. Preferably, the mammal is human.
The term "therapeutically effective amount" refers to
an amount of a drug effective to treat a disease or disorder in
a mammal. In the case of cancer, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop) cancer
cell infiltration into peripheral organs; inhibit (i.e., slow to
some extent and preferably stop) tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve to some extent one or more
of the symptoms associated with the disorder. To the extent the
drug may prevent growth and/or kill existing cancer cells, it may
be cytostatic and/or cytotoxic. For cancer therapy, efficacy can,
for example, be measured by assessing the time to disease progression
(TTP) and/or determining the response rate (RR).
The terms "cancer" and "cancerous" refer to
or describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer include,
but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,
and leukemia. More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various
types of head and neck cancer.
The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include radioactive
isotopes (e.g. At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes
of Lu), chemotherapeutic agents, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof.
A "chemotherapeutic agent" is a chemical compound useful
in the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide
and trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including
the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin;
a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
prednimustine, trofosfamide, uracil mustard; nitrosureas such as
carmustine, chlorozotocin, fotemustine, lomustine, nirnustine, ranimustine;
antibiotics such as the enediyne antibiotics (e.g. calicheamicin,
especially calicheamicin .gamma..sub.1.sup.I and calicheamicin .theta..sup.I.sub.1,
see, e.g., Agnew Chem Intl. Ed. Engl. 33:183-186 (1994); dynemicin,
including dynemicin A; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antiobiotic chromomophores),
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,
dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,
idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimeterxate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine;
elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine
and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; rhizoxin; sizofiran; spirogermanium;
tenuazonic acid; triaziquone; 2, 2',2''-trichlorotriethylamine;
trichothecenes (especially T-2 toxin, verracurin A, roridin A and
anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE.RTM.,
Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda;
ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine
(DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable
salts, acids or derivatives of any of the above. Also included in
this definition are anti-hormonal agents that act to regulate or
inhibit hormone action on tumors such as anti-estrogens including
for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene (Fareston); and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), and luteinizing hormone (LH); hepatic growth factor;
fibroblast growth factor; prolactin; placental lactogen; tumor necrosis
factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor; integrin;
thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet-growth
factor; transforming growth factors (TGFs) such as TGF-alpha and
TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide
factors including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native sequence
cytokines.
The term "prodrug" as used in this application refers
to a precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in
Cancer Chemotherapy" Biochemical Society Transactions, 14,
pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs:
A Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al, (ed.), pp. 247-267, Humana Press (1985).
The prodrugs of this invention include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing
prodrugs, optionally substituted phenoxyacetamide-containing prodrugs
or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine
and other 5-fluorouridine prodrugs which can be converted into the
more active cytotoxic free drug. Examples of cytotoxic drugs that
can be derivatized into a prodrug form for use in this invention
include, but are not limited to, those chemotherapeutic agents described
above.
The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic products,
that contain information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such therapeutic
products.
Drug combinations that are "synergistic" are those in
which the combined action (e.g. the ability to treat cancer) of
the drugs is clinically superior to that of each acting separately.
"Decay Accelerating Factor (DAF)" and "CD55"
are used interchangeably herein and refer to DAF protein as disclosed
in U.S. Pat. No. 5,763,224 and expressly incorporated herein by
reference, including variants and isoforms thereof (see U.S. Pat.
No. 5,763,224; Caras et al. Nature 325: 545-549 (1987); Lublin et
al. J. Immunol. 137:1629-1635 (1986); Hara et al. Immunol. Lett.
37:145-152 (1993); and WO99/43800). Preferred DAF is native sequence
human DAF, including native sequence human secreted DAF (DAF-A)
and membrane-bound DAF (DAF-B) (Caras et al. Nature 325: 545-549
(1987)). This definition specifically includes glycosylation variants
of DAF, particularly where those variants are preferentially expressed
by tumor cells (such as gastric tumor cells, Hensel et al. Cancer
Research 59:5399-5306 (1999), or lung tumor cells) compared to normal
cells of the same tissue type. An example of a glycosylation variant
is the "791Tgp72 antigen" described in WO99/43800, expressly
incorporated herein by reference.
Examples of antibodies directed against DAF (or antibodies which
specifically bind to DAF) include the murine monoclonal antibodies
IA10, IIH6 and VIIIA7 as described in WO86/07062 published Dec.
4, 1986 and expressly incorporated herein by reference; the human
antibodies herein designated LU30, LU13 and LU20; the murine 110
and BRIC 216 monoclonal antibodies directed against DAF as described
in WO99/43800; the murine 791T36 antibody directed against the 791Tgp72
antigen (ATCC HB9173; WO99/43800); the D17 murine antibody described
in Hara et al. Immunol. Lett. 37:145-152 (1993) which binds DAF
on blood cells, but not in semen or on testis; the human SC-1 antibody
(Vollmers et al. Cancer 76(4): 550-558 (1995); Vollmers et al. Cancer
Research 49: 2471-2476 (1989); Vollmers et al. Oncology Reports
5:549-522 (1998); and Hensel et al. Cancer Research 59:5299-5306
(1999)), as well as variants of any one of the above antibodies.
Antibody variants including amino acid sequence variants (e.g. affinity
matured antibodies and humanized variants of murine antibodies),
glycosylation variants with altered effector function, etc.
A "native sequence" protein comprises the amino acid
sequence of a protein as found in nature, e.g. in a human. The native
sequence protein can be made by recombinant or other synthetic means,
or may be isolated from a native source.
"Percent (%) amino acid sequence identity" herein is
defined as the percentage of amino acid residues in a candidate
sequence that are identical with the amino acid residues in a selected
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that are within
the skill in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate parameters
for measuring alignment, including any algorithms needed to achieve
maximal alignment over the full-length of the sequences being compared.
For purposes herein, however, % amino acid sequence identity values
are obtained as described below by using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc. has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559,
where it is registered under U.S. Copyright Registration No. TXU510087,
and is publicly available through Genentech, Inc., South San Francisco,
Calif. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid sequence
B (which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with, or against a given amino acid sequence B) is calculated
as follows: 100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of amino
acid sequence A is not equal to the length of amino acid sequence
B, the % amino acid sequence identity of A to B will not equal the
% amino acid sequence identity of B to A.
II. Modes for Carrying out the Invention
The present application provides a method for making an antibody
useful, for example, for cancer diagnosis or therapy, and a method
for identifying an antigen which is differentially expressed on
the surface of two or more distinct cell populations. These methods
employ a naive antibody phage library that can be prepared according
to known techniques, including those discussed below.
Antibody Phage Library Preparation
The antigen-binding domain of an antibody is formed from two variable
(V) regions of about 110 amino acids, one each from the light (VL)
and heavy (VH) chains, that both present three hypervariable regions.
Variable domains can be displayed functionally on phage, for example
as single-chain Fv (scFv) fragments, in which VH and VL are covalently
linked through a short, flexible peptide, or as Fab fragments, in
which they are each fused to a constant domain and interact non-covalently,
as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
The naive repertoire of an animal (the repertoire before antigen
challenge) provides it with antibodies that can bind with moderate
affinity (Kd of about 10.sup.6 to 10.sup.7 M.sup.-1) to essentially
any non-self molecule. The sequence diversity of antibody binding
sites is not encoded directly in the germline but is assembled in
a combinatorial manner from V gene segments. Each combinatorial
rearrangement of V-gene segments in stem cells gives rise to a B
cell that expresses a single VH-VL combination. Immunization triggers
any B cells making a combination that binds the immunogen to proliferate
(clonal expansion) and to secrete the corresponding antibody. These
naive antibodies are then matured to high affinity (Kd better than
10.sup.9 M.sup.-1) by a process of mutagenesis and selection known
as affinity maturation. It is after this point that cells are normally
removed to prepare hybridomas and generate high-affinity monoclonal
antibodies.
At three stages of this process, repertoires of VH and VL genes
can be separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be searched
for antigen-binding clones as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Unlike libraries from immunized sources,
a naive repertoire can be cloned to provide a single source of human
antibodies to a wide range of non-self and also self antigens without
any immunization as described by Vaughan et al. Nat. Biotechnol.
14: 309-314 (1996); and Sheets et al. PNAS (USA) 95:6157-6162 (1988).
Finally, naive libraries can also be made synthetically by cloning
the unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992); and
Griffiths et al., EMBO J., 13:3245-3260 (1994).
Phage display mimics the B cell. Filamentous phage is used to display
antibody fragments by fusion to the minor coat protein pIII. The
antibody fragments can for example be displayed as single chain
Fv fragments, in which VH and VL domains are connected on the same
polypeptide chain by a flexible polypeptide spacer, e.g. as described
by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments,
in which one chain is fused to pIII and the other is secreted into
the bacterial host cell periplasm where assembly of a Fab-coat protein
structure which becomes displayed on the phage surface by displacing
some of the wild type coat proteins, e.g. as described in Hoogenboom
et al., Nucl. Acids Res., 19: 4133-4137 (1991). When antibody fragments
are fused to the N-terminus of pill, the phage is infective. However,
if the N-terminal domain of pIII is excised and fusions made to
the second domain, the phage is not infective, and wild type pIII
must be provided by helper phage.
The pIII fusion and other proteins of the phage can be encoded
entirely within the same phage replicon, or on different replicons.
When two replicons are used, the pIII fusion is encoded on a phagemid,
a plasmid containing a phage origin of replication. Phagemids can
be packaged into phage particles by "rescue" with a helper
phage such as M13K07 that provides all the phage proteins, including
pIII, but due to a defective origin is itself poorly packaged in
competitions with the phagemids as described in Vieira and Messing,
Meth. Enzymol., 153: 3-11 (1987). In a preferred method, the phage
display system is designed such that the recombinant phage can be
grown in host cells under conditions permitting no more than a minor
amount of phage particles to display more than one copy of the Fv-coat
protein fusion on the surface of the particle as described in Bass
et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, published
Jun. 11, 1992.
In general, nucleic acids encoding antibody gene fragments are
obtained from immune cells harvested from humans or animals. The
use of spleen cells and/or B cells or other PBLs from an unimmunized
donor provides a better representation of the possible antibody
repertoire, and also permits the construction of an antibody library
using any animal (human or non-human) species. For libraries incorporating
in vitro antibody gene construction, stem cells are harvested from
the subject to provide nucleic acids encoding unrearranged antibody
gene segments. The immune cells of interest can be obtained from
a variety of animal species, such as human, mouse, rat, lagomorpha,
luprine, canine, feline, porcine, bovine, equine, and avian species,
etc.
Nucleic acid encoding antibody variable gene segments (including
VH and VL segments) are recovered from the cells of interest and
amplified. In the case of rearranged VH and VL gene libraries, the
desired DNA can be obtained by isolating genomic DNA or mRNA from
lymphocytes followed by polymerase chain reaction (PCR) with primers
matching the 5' and 3' ends of rearranged VH and VL genes as described
in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837 (1989),
thereby making diverse V gene repertoires for expression. The V
genes can be amplified from cDNA and genomic DNA, with back primers
at the 5' end of the exon encoding the mature V-domain and forward
primers based within the J-segment as described in Orlandi et al.,
supra and in Ward et al, Nature, 341: 544-546 (1989). However, for
amplifying from cDNA, back primers can also be based in the leader
exon as described in Jones et al., Biotechnol., 9: 88-89 (1991),
and forward primers within the constant region as described in Sastry
et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al., supra or Sastry et al., supra. Preferably,
the library diversity is maximized by using PCR primers targeted
to each V-gene family in order to amplify all available VH and VL
arrangements present in the immune cell nucleic acid sample, e.g.
as described in the method of Marks et al., J. Mol. Biol., 222:
581-597 (1991) or as described in the method of Orum et al., Nucleic
Acids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA
into expression vectors, rare restriction sites can be introduced
within the PCR primer as a tag at one end as described in Orlandi
et al., supra, or by further PCR amplification with a tagged primer
as described in Clackson et al., Nature, 352: 624-628 (1991).
Repertoires of synthetically rearranged V genes can be derived
in vitro from V gene segments. Most of the human VH-gene segments
have been cloned and sequenced (reported in Tomlinson et al., J.
Mol. Biol., 227: 776-798 (1992)), and mapped (reported in Matsuda
et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including
all the major comformations of the H1 and H2 loop) can be used to
generate diverse VH gene repertoires with PCR primers encoding H3
loops of diverse sequence and length as described in Hoogenboom
and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can
also be made with all the sequence diversity focussed in a long
H3 loop of a single length as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 89: 4457-4461 (1992). One can also make synthetic
light chain repertoires (Williams and Winter, Eur. J. Immunol.,
23: 1456-1461 (1993)). Synthetic V gene repertoires, based on a
range of VH and VL folds, and L3 and H3 lengths, will encode antibodies
of considerable structural diversity. Following amplification of
V-gene encoding DNAs, germline V-gene segments can be rearranged
in vitro according to the methods of Hoogenboom and Winter, J. Mol.
Biol., 227: 381-388 (1992).
Repertoires of antibody fragments can be constructed by combining
VH and VL gene repertoires together in several ways. Each repertoire
can be created in different vectors, and the vectors recombined
in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-126
(1993), or in vivo by combinatorial infection, e.g., the loxP system
described in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266
(1993); and Griffiths et al., EMBO J., 13:3245-3260 (1994). The
in vivo recombination approach exploits the two-chain nature of
Fab fragments to overcome the limit on library size imposed by E.
coli transformation efficiency. Naive VH and VL repertoires are
cloned separately, one into a phagemid and the other into a phage
vector. The two libraries are then combined by phage infection of
phagemid-containing bacteria so that each cell contains a different
combination and the library size is limited only by the number of
cells present (about 10.sup.12 clones). Both vectors contain in
vivo recombination signals so that the VH and VL genes are recombined
onto a single replicon and are co-packaged into phage virions. These
huge libraries provide large numbers of diverse antibodies of good
affinity (Kd of about 10.sup.-8 M).
Alternatively, the repertoires may be cloned sequentially into
the same vector, e.g. as described in Barbas et al., Proc. Natl.
Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR
and then cloned, e.g. as described in Clackson et al., Nature, 352:
624-628 (1991). PCR assembly can also be used to join VH and VL
DNAs with DNA encoding a flexible peptide spacer to form single
chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).
The antibodies produced by naive libraries (either natural or synthetic)
can be of moderate affinity (Kd of about 10.sup.6 to 10.sup.7 M.sup.-1),
but affinity maturation can also be mimicked in vitro by constructing
and reselecting from secondary libraries as described in Winter
et al. (1994), supra. For example, mutation can be introduced at
random in vitro by using error-prone polymerase (reported in Leung
et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et
al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram
et al., Proc Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally,
affinity maturation can be performed by randomly mutating one or
more CDRs, e.g. using PCR with primers carrying random sequence
spanning the CDR of interest, in selected individual Fv clones and
screening for higher affinity clones. WO 96/07754 (published Mar.
14, 1996) describes a method for inducing mutagenesis in a complementarity
determining region of an immunoglobulin light chain to create a
library of light chain genes. Another effective approach is to recombine
the VH or VL domains selected by phage display with repertoires
of naturally occurring V domain variants obtained from unimmunized
donors and screen for higher affinity in several rounds of chain
reshuffling as described in Marks et al., Biotechnol., 10: 779-783
(1992). This technique allows the production of antibodies and antibody
fragments with affinities in the 10.sup.-9 M range.
The antibody phage library of particular interest herein is one
which comprises from about 10.sup.9 to about 10.sup.15 antibody
phage.
Screening for Useful Antibodies/Antigens
The naive antibody phage library is panned with or screened against
live cancer cells. The cancer cells may, for example, be surgically
removed from a cancer patient or may be derived from a cancer cell
line. Various cancer cell lines are publicly available, e.g. from
the American Type Culture Collection (ATCC). Exemplary cancer cell
lines include breast cancer cell lines such as SK-BR-3, BT-483,
MCF-7, BT-20, ZR-751, MDA-MB-231, CAMA1, BT-474; lung adenocarcinoma
cell lines such as SKLU1, A549, and 1264; glioma cancer cell lines
such as Hs683; ovarian carcinoma lines such as SK-OV-3 and Hey;
colorectal carcinoma cell lines including HT-29 and Ls180; prostate
carcinoma cell lines such as DU145; gastric carcinoma cell lines
exemplified by MS; and renal carcinoma cell lines such as Caki-1.
The cancer from which the cancer cell is derived may be a carcinoma,
lymphoma, blastoma, sarcoma, or leukemia. Exemplary cancer types
from which the cancer cell may be procured include lung cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer,
colon cancer, colorectal cancer, salivary gland carcinoma, kidney
cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,
or hepatic carcinoma. Preferably, the cancer cell is a lung cancer
cell.
In a preferred embodiment of the invention, the method includes
a counter-selection step using a live non-cancer cell which is preferably
of the same tissue type as the cancer cell. Counter-selection can
be carried out at any time including before, during and after (or
combinations thereof) screening the antibody phage library with
the live cancer cells. In the preferred embodiment however, counter-selection
precedes at least one step involving panning against the live cancer
cells of interest. According to this counter-selection step, the
"subtracted" antibody phage (e.g. those present in a supernatant)
are then exposed to the live cancer cells. It was surprisingly discovered
herein that antibodies against an antigen shared by the cancer cell
and non-cancer cell could be identified, in spite of this counter-selection
step being performed prior to screening the cancer cell of interest.
It was anticipated that such a counter-selection step may have depleted
the phage library of antibodies capable of binding the shared antigen.
The non-cancer cells may, for example, have been surgically removed
from a patient or may be obtained from some other in vivo source
of the cells, or may be derived from a non-cancer cell line, such
cell lines being publicly available, e.g., from the ATCC.
The cells to be screened will oftentimes be "adherent"
to the extent they adhere to the surface of a cell culture plate
or other solid phase in which they are cultured. The present application
provides an improved method for detaching the cells from the surface
to which they are adhered comprising the use of a solution which
does not include any protease and preferably comprises EDTA for
detaching the cancer cells. This avoids proteolytic degradation
of cell surface antigens resulting from the commonly used trypsin
release step.
The cancer and non-cancer cells are not fixed prior to exposure
to antibody phage in the antibody phage library. Use of such live
cells serves to preserve surface antigens in their native state.
Hence, the antibodies prepared according to the present method are
more likely to bind the antigen in its endogenous state in a mammal
and hence serve as superior diagnostic (e.g. in vivo diagnostic)
and therapeutic antibodies.
Antibody phage from the naive antibody phage library are contacted
with, or bound to, the cancer cells (and optionally the non-cancer
cells). Prior to this binding step, an aliquot of antibody phage
may be blocked to reduce non-specific binding to cell surfaces.
Such blocked antibody phage may be added to the cells. Alternatively,
the cells, which are optionally blocked, may be added to the antibody
phage. The cells and antibody phage are contacted for a sufficient
period of time and under suitable conditions such that binding of
the phage to cell surface antigen(s) occurs. Such conditions can
be determined without undue experimentation. Moreover, panning steps
may be repeated as desired to achieve the desired binding between
cell surface antigens and antibody phage. Cells may be pelleted
in-between panning steps via centrifugation or other means as desired.
Binding of antibody phage or antibody derived from the phage to
cells may, for example, be determined by established methodologies
such as ELISA, flow cytometry and immunohistochemistry.
Hence, an antibody phage or antibody is selected which binds selectively
to the cancer cell of interest Such "cancer-selective"
antibodies may be subjected to one or more further analyses. For
example, clone analysis (e.g. restriction enzyme cleaving and finger
printing and/or DNA sequencing) may be carried out according to
known procedures. Alternatively, or additionally, cancer-selectivity
of selected antibodies or antibody phage may be determined by comparing
binding of the antibodies or antibody phage to cancer cells and
non-cancer cells, e.g. of the same tissue type. Such screening may
be performed using the cancer and non-cancer cells used to screen
the phage library, or other cancer and non-cancer cells.
The selected antibody or antibodies may be altered or modified
as desired to generate an antibody particularly adapted for in vivo
therapy or diagnosis. Such alteration may involve one or more amino
acid substitutions in one or more hypervariable regions of the antibody
to increase its affinity for antigen; i.e. the selected antibody
may be "affinity matured". Moreover, the antibody or affinity
matured antibody may be fused to, or conjugated with, a cytotoxic
agent, enzyme (e.g. for ADEPT, see below), detectable label, or
other antibody (to generate a bispecific antibody). Such alterations
are discussed in more detail below in the Section entitled "Other
Methods for Making Antibodies". The variable domain sequences
of the antibody or affinity matured antibody may be fused to human
constant region sequences so as to generate a larger antibody molecule,
such as a Fab, F(ab').sub.2 or intact antibody, depending, for example,
on the intended use of the antibody.
Nucleic acid encoding the antibody (which has optionally been altered
as explained in the previous paragraph) may be isolated and inserted
into a recombinant expression vector and used to transform a suitable
host cell for expression of the antibody. Exemplary host cells include
prokaryotic host cells (e.g. E. coli), yeast cells (such as Saccharomyces
cerevisiae and Pichia pastoris), mammalian cells such as lymphoid
cells and Chinese Hamster Ovary (CHO) cells, or plant cells. The
expressed antibody recovered from the host cell, may be used for
various diagnostic and therapeutic applications such as those discussed
hereinbelow.
The present method facilitates identification of an antigen expressed
at higher levels on a first cell population (generally a cancer
cell) compared to a second cell population (e.g. a non-cancer cell
of the same tissue type as the first cell). For example, the level
of expression of the antigen on the first cell population or cancer
cell may be about two fold or about five fold to about 100 fold
or about 1000 fold greater than the level of expression of the antigen
on the second cell population or non-cancer cell. Such antigens
can be targeted in therapy or diagnosis using antagonists, such
as antibodies, or small molecule drugs directed thereagainst. Antibodies
directed against such "over-expressed" antigens can be
prepared by screening antibody phage libraries as discussed above,
or according to other methods for making antibodies available in
the art, including those discussed below.
Another advantage of the present invention is the ability to easily
expression clone nucleic acid encoding the antigen. To expression
clone the antigen, a cDNA library may be prepared, e.g., from the
cancer cell used to screen the phage library. The cDNA's thus prepared
are expressed in a suitable host cell and expression of the desired
protein can be screened for using one or more antibodies selected
from the phage library. This way, cDNA encoding the antigen can
be identified and sequenced.
In the present Example, an anti-penta-histidine antibody was used
to cross-link, via their penta-histidine epitope tags, scFv fragments
used to screen for expression of desired antigen. This cross-linking
increased the avidity of the interaction between the scFv and antigen.
In addition, an anti-mouse antibody was coated on an assay plate
and bound the antibody-linked cells to the assay plate.
Other Methods for Making Antibodies
As disclosed above, the present methods provide means for identifying
antigens expressed at higher levels on one cell compared to another.
Such cells may, for example, be cancer cells and the antigen of
interest thereon may be one which is useful for targeting with an
antibody for therapy or diagnosis.
Once an antigen is identified as described herein, one can generate
further antibodies thereagainst by screening antibody phage libraries
as discussed above, or an antibody can be made by other techniques
such as those disclosed below.
In one embodiment, a polyclonal antibody is raised against the
antigen of interest. Polyclonal antibodies are preferably raised
in animals by multiple subcutaneous (sc) or intraperitoneal (ip)
injections of the relevant antigen and an adjuvant. It may be useful
to conjugate the relevant antigen to a protein that is immunogenic
in the species to be immunized, e.g., keyhole limpet hemocyanin,
serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
using a bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where
R and R.sup.1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates,
or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g of the protein
or conjugate (for rabbits or mice, respectively) with 3 volumes
of Freund's complete adjuvant and injecting the solution intradermally
at multiple sites. One month later the animals are boosted with
1/5 to 1/10 the original amount of peptide or conjugate in Freund's
complete adjuvant by subcutaneous injection at multiple sites. Seven
to 14 days later the animals are bled and the serum is assayed for
antibody titer. Animals are boosted until the titer plateaus. Preferably,
the animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different cross-linking
reagent. Conjugates also can be made in recombinant cell culture
as protein fusions. Also, aggregating agents such as alum are suitably
used to enhance the immune response.
Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Thus, the modifier
"monoclonal" indicates the character of the antibody as
not being a mixture of discrete antibodies.
For example, the monoclonal antibodies may be made using the hybridoma
method first described by Kohler et al., Nature, 256:495 (1975),
or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal,
such as a hamster, is immunized as hereinabove described to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the protein used for immunization.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp.59-103 Academic Press (1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, parental myeloma
cells. For example, if the parental myeloma cells lack the enzyme
hypoxanthine guanine pliosphoribosyl 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.
Preferred myeloma cells are those that fuse efficiently, support
stable high-level production of antibody by the selected antibody-producing
cells, and are sensitive to a medium such as HAT medium. Among these,
preferred myeloma cell lines are murine myeloma lines, such as those
derived from MOPC-21 and MPC-11 mouse tumors available from the
Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 or X63-Ag8-653 cells available from the American Type Culture
Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma
cell lines also have been described for the production of human
monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur
et al., Monoclonal Antibody Production Techniques and Applications,
pp.51-63 Marcel Dekker, Inc., New York, (1987)).
Culture medium in which hybridoma cells are growing is assayed
for production of monoclonal antibodies directed against the antigen.
Preferably, the binding specificity of monoclonal antibodies produced
by hybridoma cells is determined by immunoprecipitation or by an
in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example,
be determined by the Scatchard analysis of Munson et al., Anal.
Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of
the desired specificity, affinity, and/or activity, the clones may
be subcloned by limiting dilution procedures and grown by standard
methods (Goding, Monoclonal Antibodies: Principles and Practice,
pp.59-103 Academic Press (1986)). Suitable culture media for this
purpose include, for example, D-MEM or RPMI-1640 medium. In addition,
the hybridoma cells may be grown in vivo as ascites tumors in an
animal.
The monoclonal antibodies secreted by the subclones are suitably
separated from the culture medium, ascites fluid, or serum by conventional
immunoglobulin purification procedures such as, for example, protein
A-Sepharose, hydroxylapatite chromatography, gel electrophoresis,
dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of murine antibodies). The hybridoma
cells serve as a preferred source of such DNA. Once isolated, the
DNA may be placed into expression vectors, which are then transfected
into host cells such as E. coli cells, simian COS cells, Chinese
Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. Review articles on recombinant
expression in bacteria of DNA encoding the antibody include Skerra
et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun,
Immunol. Revs., 130:151-188 (1992).
The DNA also may be modified, for example, by substituting the
coding sequence for human heavy- and light-chain constant domains
in place of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or
by covalently joining to the immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted
for the constant domains of an antibody, or they are substituted
for the variable domains of one antigen-combining site of an antibody
to create a chimeric bivalent antibody comprising one antigen-combining
site having specificity for an antigen and another antigen-combining
site having specificity for a different antigen.
Methods for humanizing non-human antibodies are well known in the
art. Preferably, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen
et al., Science, 239:1534-1536 (1988)), 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 non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR residues
and possibly some FR residues are substituted by residues from analogous
sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to
be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit"
method, the sequence of the variable domain of a rodent antibody
is screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the rodent
is then accepted as the human framework region (FR) for the humanized
antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et
al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular
framework region derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized antibodies
(Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta
et al., J. Immunol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role
of the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially involved
in influencing antigen binding.
Alternatively, it is now possible to produce transgenic animals
(e.g., mice) that are capable, upon immunization, of producing a
full repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that
the homozygous deletion of the antibody heavy-chain joining region
(J.sub.H) gene in chimeric and germ-line mutant mice results in
complete inhibition of endogenous antibody production. Transfer
of the human germ-line immunoglobulin gene array in such germ-line
mutant mice will result in the production of human antibodies upon
antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad.
Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258
(1993); Bruggermann et al., Year in Immuno., 7:33 (1993). Human
antibodies can also be derived from phage-display libraries (Hoogenboom
et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,
222:581-597 (1991)).
Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al., Journal
of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan
et al., Science, 229:81 (1985)). However, these fragments can now
be produced directly by recombinant host cells. For example, the
antibody fragments can be isolated from the antibody phage libraries
discussed above. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab').sub.2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According
to another approach, F(ab').sub.2 fragments can be isolated directly
from recombinant host cell culture. Other techniques for the production
of antibody fragments will be apparent to the skilled practitioner.
In other embodiments, the antibody of choice is a single chain Fv
fragment (scFv). See WO 93/16185.
Bispecific antibodies are antibodies that have binding specificities
for at least two different epitopes. Exemplary bispecific antibodies
may bind to two different epitopes of the antigen of interest. Alternatively,
an arm which binds antigen of interest may be combined with an arm
which binds to a triggering molecule on a leukocyte such as a T-ell
receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (Fc.gamma.R),
such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII
(CD16) so as to focus cellular defense mechanisms to the cell expressing
the antigen of interest. Bispecific antibodies may also be used
to localize cytotoxic agents to cells which express the antigen.
These antibodies possess an antigen-binding arm and an arm which
binds the cytotoxic agent (e.g. saporin, anti-interferon-.gamma.,
vinca alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length antibodies
or antibody fragments (e.g. F(ab').sub.2 bispecific antibodies).
Methods for making bispecific antibodies are known in the art.
Traditional production of full length bispecific antibodies is based
on the coexpression of two immunoglobulin heavy chain-light chain
pairs, where the two chains have different specificities (Milstein
et al., Nature 305:537-539 (1983)). Because of the random assortment
of immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules,
of which only one has the correct bispecific structure. Purification
of the correct molecule, which is usually done by affinity chromatography
steps, is rather cumbersome, and the product yields are low. Similar
procedures are disclosed in WO 93/08829, and in Traunecker et al.,
EMBO J., 10:3655-3659 (1991).
According to a different approach, antibody variable domains with
the desired binding specificities (antibody-antigen combining sites)
are fused to immunoglobulin constant domain sequences. The fusion
preferably is with an immunoglobulin heavy chain constant domain,
comprising at least part of the hinge, CH2, and CH3 regions. It
is preferred to have the first heavy-chain constant region (CH1)
containing the site necessary for light chain binding, present in
at least one of the fusions. DNAs encoding the immunoglobulin heavy
chain fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide fragments
in embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is, however,
possible to insert the coding sequences for two or all three polypeptide
chains in one expression vector when the expression of at least
two polypeptide chains in equal ratios results in high yields or
when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies
are composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity)
in the other arm. It was found that this asymmetric structure facilitates
the separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin
light chain in only one half of the bispecific molecule provides
for a facile way of separation. This approach is disclosed in WO
94/04690. For further details of generating bispecific antibodies
see, for example, Suresh et al., Methods in Enzymology, 121:210
(1986).
According to another approach described in WO96/27011, the interface
between a pair of antibody molecules can be engineered to maximize
the percentage of heterodimers which are recovered from recombinant
cell culture. The preferred interface comprises at least a part
of the C.sub.H.sup.3 domain of an antibody constant domain. In this
method, one or more small amino acid side chains from the interface
of the first antibody molecule are replaced with larger side chains
(e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the heterodimer
over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For example, one of the antibodies in the heteroconjugate
can be coupled to avidin, the other to biotin. Such antibodies have,
for example, been proposed to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies
may be made using any convenient cross-linking methods. Suitable
cross-linking agents are well known in the art, and are disclosed
in U.S. Pat. No. 4,676,980, along with a number of cross-linking
techniques.
Techniques for generating bispecific antibodies from antibody fragments
have also been described in the literature. For example, bispecific
antibodies can be prepared using chemical linkage. Brennan et al.,
Science, 229: 81 (1985) describe a procedure wherein intact antibodies
are proteolytically cleaved to generate F(ab').sub.2 fragments.
These fragments are reduced in the presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent
intermolecular disulfide formation. The Fab' fragments generated
are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to form the bispecific antibody.
The bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab'-SH
fragments from E. coli, which can be chemically coupled to form
bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225
(1992) describe the production of a fully humanized bispecific antibody
F(ab').sub.2 molecule. Each Fab' fragment was separately secreted
from E. coli and subjected to directed chemical coupling in vitro
to form the bispecific antibody. The bispecific antibody thus formed
was able to bind to cells overexpressing the ErbB2 receptor and
normal human T cells, as well as trigger the lytic activity of human
cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced
using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins
were linked to the Fab' portions of two different antibodies by
gene fusion. The antibody homodimers were reduced at the hinge region
to form monomers and then re-oxidized to form the antibody heterodimers.
This method can also be utilized for the production of antibody
homodimers. The "diabody" technology described by Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided
an alternative mechanism for making bispecific antibody fragments.
The fragments comprise a heavy-chain variable domain (V.sub.H) connected
to a light-chain variable domain (V.sub.L) by a tinker which is
too short to allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be prepared. Tutt et al. J. Immunol.
147: 60 (1991).
It may be desirable to modify the antibody of the invention with
respect to effector function, so as to enhance the effectiveness
of the antibody in treating cancer, for example. For example cysteine
residue(s) may be introduced in the Fc region, thereby allowing
interchain disulfide bond formation in this region. The homodimeric
antibody thus generated may have improved internalization capability
and/or increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195
(1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric
antibodies with enhanced anti-tumor activity may also be prepared
using heterobifunctional cross-linkers as described in Wolff et
al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody
can be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent,
toxin (e.g. a small molecule toxin or an enzymatically active toxin
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates
have been described above.
Conjugates of an antibody and one or more small molecule toxins,
such as a calicheamicin, a maytansine (U.S. Pat. No. 5,208,020),
a trichothene, and CC 1065 are also contemplated herein.
In one preferred embodiment of the invention, the antibody is conjugated
to one or more maytansine molecules (e.g. about 1 to about 10 maytansine
molecules per antibody molecule). Maytansine may, for example, be
converted to May-SS-Me which may be reduced to May-SH3 and reacted
with modified antibody (Chari et al. Cancer Research 52: 127-131
(1992)) to generate a maytansinoid-antibody immunoconjugate.
Another immunoconjugate of interest comprises an antibody conjugated
to one or more calicheamicin molecules. The calicheamicin family
of antibiotics are capable of producing double-stranded DNA breaks
at sub-picomolar concentrations. Structural analogues of calicheamicin
which may be used include, but are not limited to, .gamma..sub.1.sup.I,
.alpha..sub.2.sup.I, .alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I,
PSAG and .theta..sup.I.sub.1 (Hinman et al. Cancer Research 53:
3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)).
See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and 5,773,001
expressly incorporated herein by reference.
Enzymatically active toxins and fragments thereof which can be
used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. See, for example, WO
93/21232 published Oct. 28, 1993.
The present invention further contemplates an immunoconjugate formed
between an antibody and a compound with nucleolytic activity (e.g.
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
A variety of radioactive isotopes are available for the production
of radioconjugated anti-DAF antibodies. Examples include At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32 and radioactive isotopes of Lu.
Conjugates of the antibody and cytotoxic agent may be made using
a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disucciimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives
(such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates
(such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds
(such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al. Science
238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody. See WO94/11026.
The linker may be a "cleavable linker" facilitating release
of the cytotoxic drug in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, dimethyl linker or disulfide-containing
linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be
used.
Alternatively, a fusion protein comprising the antibody and cytotoxic
agent may be made, e.g. by recombinant techniques or peptide synthesis.
In another embodiment, the antibody may be conjugated to a "receptor"
(such streptavidin) for utilization in tumor pretargeting wherein
the antibody-receptor conjugate is administered to the patient,
followed by removal of unbound conjugate from the circulation using
a clearing agent and then administration of a "ligand"
(e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide).
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known
in the art, such as described in Epstein et al., Proc. Natl. Acad.
Sci, USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA,
77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes
with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising phosphatidylcholine,
cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter. Fab' fragments of the antibody
of the present invention can be conjugated to the liposomes as described
in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide
interchange reaction. A chemotherapeutic agent is optionally contained
within the liposome. See Gabizon et al. J. National Cancer Inst.
81(19)1484 (1989).
The antibodies of the present invention may also be used in Antibody
Dependent Enzyme Mediated Prodrug Therapy (ADEPT) by conjugating
the antibody to a prodrug-activating enzyme which converts a prodrug
(e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an active
anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.
4,975,278.
The enzyme component of the immunoconjugate useful for ADEPT includes
any enzyme capable of acting on a prodrug in such a way so as to
covert it into its more active, cytotoxic form.
Enzymes that are useful in the method of this invention include,
but are not limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs; cytosine
deaminase useful for converting non-toxic 5-fluoro |