Monoclonal Antibodies That Specifically Block Biological Activity Of A Tumor Antigen

54 Pages·2013·3.92 MB·English

US 20130189272A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2013/0189272 A1
Grasso et al. (43) Pub. Date: Jul. 25, 2013
(54) MONOCLONAL ANTIBODIES THAT Publication Classi?cation
SPECIFICALLY BLOCK BIOLOGICAL
ACTIVITY OF A TUMOR ANTIGEN (51) Int. Cl.
C07K 16/28 (2006.01)
(71) Applicant: MORPHOTEK, INC.; Exton; PA (US) (52) US, Cl,
_ _ CPC .................................... .. C07K16/28 (2013.01)
(72) Inventorsl Lulgl Gram, Bryn MaWr, PA (US); USPC 424/1431; 424/178.1; 435/334; 435/696;
Nicholas C. Nicolaides; Garnett Valley; 43 5 @5421
PA (US); Philip M. Sass; Audubon; PA
US
( ) (57) ABSTRACT
(73) Assignee: MORPHOTEK, INC.; Exton; PA (U S) _ _ _ _ _
This mvent1on relates to novel monoclonal ant1bod1es that
(21) App1_ NO; 13/826,964 speci?cally bind to the alpha-folate receptor. In some
embodiments; the antibodies inhibit a biological activity of
(22) Filed; Mar, 14, 2013 folate receptor-0t (FR-0t). The antibodies are useful in the
treatment of certain cancers; particularly cancers that have
Related US. Application Data increased cell surface expression of the alpha-folate receptor
(60) Continuation of application No. 12/500,144; ?led on ( FR“ )3 Such as .ovanan’ breast’. renal.’ Colorectal’ lung’
Jul 9 2009 which is a division of a lication NO endometnal; or bram cancer. The mvent1on also relates to
11/'05’6 776 ’?1ed on Feb 11 2005 novgpabandoned ' cells expressing the monoclonal antibodies; antibody deriva
’ ’ ' ’ ’ ' tives; such as chimeric and humanized monoclonal antibod
(60) Provisional application No. 60/544,364; ?led on Feb. ies; antibody fragments; and methods of detecting and treat
12, 2004. ing cancer using the antibodies; derivatives; and fragments.
Patent Application Publication Jul. 25, 2013 Sheet 1 0f 5 US 2013/0189272 A1
Figure 1
-tetramer
471071077161"
Patent Application Publication Jul. 25, 2013 Sheet 2 0f 5 US 2013/0189272 A1
Figure 2
Patent Application Publication Jul. 25, 2013 Sheet 3 0f 5 US 2013/0189272 A1
Figure 3
Patent Application Publication Jul. 25, 2013 Sheet 4 of 5 US 2013/0189272 A1
Figure 4
I
hybridoma clones expressing dominant
negative MMR gene
Antigen-speci?c ELISA Anti-lg ELISA
I Isolate and expand positive clones I
i
Con?rmatory ELISAs in triplicate experiments
i
Sequence analysis; binding affinity assay and/or ADCC assay

US 2013/0189272 A1 Jul. 25, 2013
MONOCLONAL ANTIBODIES THAT MOv18 Was a GPI-linked protein. This Was subsequently
SPECIFICALLY BLOCK BIOLOGICAL identi?ed as the human folate binding protein (Coney et al.
ACTIVITY OF A TUMOR ANTIGEN (1991) Cancer Res. 5 1 (22): 6125-6 1 32). Tomassetti et al.
shoWed that MOv18 recogniZes a soluble form and a GPI
CROSS-REFERENCE TO RELATED anchored form of the folate binding protein in IGROVl cells
APPLICATION (Tomassetti et al. (1993)FEBSLeZZ. 3 17(1 -2): 143-146). Sub
sequent Work combined the variable regions of the mouse
[0001] This application is a continuation of Us. applica
MOv18 With human IgG1 (kappa) constant region to create a
tion Ser. No. 12/500,144, ?led Jul. 9, 2009, Which is a con
chimeriZed MOvl 8 antibody. The chimeriZed antibody medi
tinuation of Us. application Ser. No. 11/056,776, ?led Feb.
ated higher and more speci?c lysis of IGROV 1 cells at
11, 2005, Which claims bene?t ofU.S. Appl. No. 60/544,364,
10-100-fold loWer antibody concentrations (Coney et al.
?led Feb. 12, 2004. The content of each of these applications
(1994) Cancer Res. 54(9):2448-2455). The 38 kDa antigen
is incorporated herein by reference in its entirety.
appears to be the monomeric form of FR-ot.
FIELD OF THE INVENTION [0005] Us. Pat. No. 5,952,484 describes a humanized anti
body that binds to a 38 kDa protein (FR-0t). The antibody Was
[0002] This invention relates to puri?ed novel monoclonal named LK26. The original mouse monoclonal antibody Was
antibodies that speci?cally bind to the alpha-folate receptor described by Rettig in European Patent Application No.
(“FR-0t”) and compositions thereof. In some embodiments, 861041705 (published as EP0197435 and issued in the Us.
the antibodies of the invention block the biological activity of
as U.S. Pat. No. 4,851,332).
FR-ot. The antibodies and compositions of the invention are
[0006] Ovarian cancer is a major cause of death due to
useful in the treatment of certain cancers, particularly cancers gynecological malignancy. Although chemotherapy is the
that have increased cell surface expression of the alpha-folate
recommended treatment and has enjoyed some success, the
receptor, such as ovarian, breast, renal, colorectal, lung,
5-year survival rate is still less than 40%.
endometrial, or brain cancer. The invention also relates to
[0007] A dif?cult problem in antibody therapy in cancer is
hybridoma cells expressing the monoclonal antibodies, anti
that often the target of the antibody is expressed by normal
body derivatives, such as chimeric and humaniZed mono
clonal antibodies, antibody fragments, mammalian cells tissues as Well as cancerous tissues. Thus, the antibodies that
expressing the monoclonal antibodies, derivatives and frag are used to kill cancer cells also have a deleterious effect on
normal cells. Finding unique targets or targets that are pref
ments, compositions of puri?ed antibodies of the invention,
erentially expressed in cancer tissues has proven dif?cult in
and methods of detecting and treating cancer using the anti
many cancers. Identi?cation of preferentially expressed tar
bodies, derivatives, fragments, and compositions of the
gets and the ability to block the biological activity of such
invention.
targets may be an effective treatment for cancer. As such,
more effective antibody therapies for ovarian and other FR-ot
BACKGROUND OF THE INVENTION
bearing cancers that avoids or minimiZes reactivity With nor
[0003] There are three major isoforms of the human mem mal tissues are needed.
brane folate binding protein, 0t, [3, and y. The 0t and [3 isoforms
have about 70% amino acid sequence homology, and differ SUMMARY OF THE INVENTION
dramatically in their stereospeci?city for some folates. Both
isoforms are expressed in fetal and adult tissue, although [0008] In some embodiments, the invention provides anti
normal tissue generally expresses loW to moderate amounts bodies that speci?cally bind to FR-ot. The antibodies of the
of FR-[3. FR-ot, hoWever, is expressed in normal epithelial invention preferably block a biological activity of FR-ot. In
cells, and is frequently strikingly elevated in a variety of some embodiments, the invention provides antibody-produc
carcinomas (Ross etal. (1994) Cancer 73(9):2432-2443; Ret ing cells and compositions of antibodies that speci?cally bind
tig et al. (1988) Proc. Natl. Acad. Sci. USA 85:3110-3114; to FR-ot Wherein the cells and compositions are substantially
Campbell et al. (1991) Cancer Res. 51:5329-5338; Coney et free of FR-ot binding competitors. In some embodiments,
al. (1991) Cancer Res. 51 :6125-6132; Weitman et al. (1992) antibody-producing cells that produce antibodies comprising
Cancer Res. 52:3396-3401; Garin-Chesa et al. (1993) Am. J. substantially only antibody of the invention are provided. In
Pathol. 142:557-567; Holm et al. (1994) APMIS 1021413 preferred embodiments, the antibodies of the invention bind
419; Franklin et al. (1994) Int. J. Cancer 8 (Suppl.):89-95; FR-ot With a binding af?nity of at least about 1><10_7 M, at
Miotti et al. (1987)Inl. J. Cancer 39:297-303; andVegglan et least about 1><10_8 M, at least about 1><10_9 M, and most
al. (1989) Tumori 75:510-513). FR-ot is overexpressed in preferably at least about 1><10_l0 M.
greater than 90% of ovarian carcinomas (Sudimack and Lee [0009] It has been discovered that tumors that overexpress
(2000) Adv. Drug Deliv. Rev. 41(2):147-62). FR-ot generally FR-ot tend to favor the formation of multimeric forms of
attaches to the cell surface membrane via a GPI anchor. GPI FR-ot, for example tetramers. Without Wishing to be bound by
anchors contain oligosaccharides and inositol phospholipids. any particular theory, it is believed that the formation of the
[0004] In 1987, Miotti et al. described three neW mono multimeric form of FR-ot is driven by a mass effect due to the
clonal antibodies that recogniZed antigens on human ovarian accumulation of larger amounts of FR-ot on the surface of
carcinoma cells (Miotti et al. (1987)Inl. J. Cancer 39(3):297 tumor cells. Previously, other researchers only found higher
303). One of these Was designated MOv18, Which recogniZes molecular Weight species of FR-ot in gel ?ltration assays
a 38 kDa protein on the surface of choriocarcinoma cells. Which represented FR-ot inserted into Triton X-100 micelles
MOv18 is a murine, IgG1, kappa antibody and mediates via their hydrophobic tails (Holm et al. (1997)Bi0sci. Reports
speci?c cell lysis of the ovarian carcinoma cell line, IGROVl . 17(4):415-427). In some embodiments, the invention pro
Alberti et al. ((1990) Biochem. Biophys. Res. Commun. 171 vides antibodies that speci?cally bind to the multimeric form
(3)11051-1055) shoWed that the antigen recogniZed by of FR-ot and not the monomeric form.
US 2013/0189272 A1 Jul. 25, 2013
[0010] In some embodiments, the antibodies of the inven antifolate agent and antibody of the invention may be admin
tion (a) bind to an epitope of FR-Ot other than the epitope istered at the same time or simultaneously (that is, together),
bound by antibody LK26; (b) bind FR-Ot With greater a?inity or in any order.
than antibody LK26; (c) out-compete antibody LK26 for [0018] The invention also provides methods for decreasing
binding to the multimeric form of FR-Ot and thereby block the the groWth of cancer cells using monoclonal antibodies that
biological activity of FR-Ot; and/or (d) are puri?ed relative to speci?cally bind to FR-Ot, preferably mammalian FR-Ot. The
LK26. methods of the invention may be used to modulate the groWth
[0011] In some embodiments, the antibodies of the inven of cancer cells and the progression of cancer in mammals,
tion recognize a disul?de-dependent epitope. including humans. The cancer cells that may be inhibited
include all cancer cells that have an increased expression of
[0012] Some embodiments of the invention relate to anti
FR-Ot in relation to normal human tissues, such as but not
bodies comprising a heavy chain comprising an amino acid
limited to ovarian, breast, renal, colorectal, lung, endometrial,
sequence of SEQ ID N015. In some embodiments, the heavy
or brain cancer cells.
chain comprises an amino acid sequence of SEQ ID N016.
[0019] Also provided by the invention are compositions of
[0013] In some embodiments, the antibodies of the inven antibodies of the invention. In preferred embodiments, the
tion comprise a light chain comprising the amino acid compositions are substantially pure. Substantially pure com
sequence of SEQ ID N012. In some embodiments of the positions of antibodies of the invention preferably comprise
invention, the antibodies comprise a light chain comprising at least about 90%, more preferably at least about 95%, even
the amino acid sequence of SEQ ID N013. more preferably at least about 99%, and most preferably
[0014] The invention further provides antibodies compris about 100% by Weight of antibodies of the invention.
ing a heavy chain comprising an amino acid of SEQ ID N015
BRIEF DESCRIPTION OF THE DRAWINGS
or SEQ ID N016 and a light chain comprising an amino acid
sequence of SEQ ID N012 or SEQ ID N013. The antibodies [0020] FIG. 1 shoWs a Western blot of tumor cells shoWing
of the invention preferably comprise a heavy chain compris the tetrameric and monomeric forms of FR-Ot.
ing an amino acid sequence of SEQ ID N015 and a light chain [0021] FIG. 2 shoWs a Western blot of Escherichia coli
comprising an amino acid sequence of SEQ ID N012 and expressed FR-Ot.
more preferably comprise a heavy chain comprising an amino
[0022] FIG. 3 shoWs a Western blot of FR-Ot solubiliZed in
acid sequence of SEQ ID N016 and a light chain comprising
the presence or absence of Triton X-lOO.
an amino acid sequence of SEQ ID N013. In some embodi
[0023] FIG. 4 illustrates a screening method for identifying
ments of the invention, the heavy chain of the antibody is
antibody-producing cells of the invention.
encoded by a nucleic acid comprising the nucleotide
[0024] FIG. 5A illustrates a sequence alignment of light
sequence of SEQ ID N017. In some embodiments of the
chain of an anti-FR-Ot antibody of the invention having an
invention, the light chain of the antibody is encoded by a
amino acid sequence of SEQ ID N013 and the light chain of
nucleic acid comprising the nucleotide sequence of SEQ ID
an aberrant translation product having an amino acid
N018.
sequence of SEQ ID N01 24. FIG. 5B illustrates a sequence
[0015] The antibodies of the invention may be chimeric alignment of the nucleic acid sequence of a light chain of an
antibodies, including, but not limited to human-mouse chi anti-FR-Ot antibody of the invention having a sequence of
meric antibodies. The antibodies of the invention may also be SEQ ID N018 and a nucleic acid sequence encoding the
humaniZed antibodies. The invention also provides: cells, aberrant translation product having a sequence of SEQ ID
including hybridoma cells, that express the antibodies of the N0125.
invention; polynucleotides that encode the antibodies of the
invention; vectors comprising the polynucleotides that DETAILED DESCRIPTION OF ILLUSTRATIVE
encode the antibodies of the invention; and expression cells EMBODIMENTS
comprising the vectors of the invention.
[0025] The reference Works, patents, patent applications,
[0016] The invention also provides methods of producing
and scienti?c literature, including accession numbers to Gen
an antibody that speci?cally binds to FR-Ot. In some embodi
Bank database sequences that are referred to herein establish
ments, the method comprises the step of culturing the anti
the knowledge of those With skill in the art and are hereby
body-producing cells of the invention. The cells of the inven
incorporated by reference in their entirety to the same extent
tion may be insect cells or animal cells, preferably,
as if each Was speci?cally and individually indicated to be
mammalian cells. incorporated by reference. Any con?ict betWeen any refer
[0017] The invention further provides methods of inhibit ence cited herein and the speci?c teachings of this speci?ca
ing the groWth of dysplastic cells associated With increased tion shall be resolved in favor of the latter. Likewise, any
expression of FR-Ot comprising administering to a patient con?ict betWeen an art-understood de?nition of a Word or
With such dysplastic cells a composition comprising an anti phrase and a de?nition of the Word or phrase as speci?cally
body of the invention. The antibody preferably blocks a bio taught in this speci?cation shall be resolved in favor of the
logical activity of FR-Ot. The methods may be used for various latter.
dysplastic conditions, such as, but not limited to ovarian, [0026] Standard reference Works setting forth the general
breast, renal, colorectal, lung, endometrial, or brain cancer. In principles of recombinant DNA technology knoWn to those of
preferred embodiments, the patients are human patients. In skill in the art include Ausubel et al. CURRENT PROTOCOLs IN
some embodiments, the antibodies are conjugated to cyto MOLECULAR BIOLOGY, John Wiley & Sons, NeW York (1998);
toxic agents such as, but not limited to radionuclides, toxins, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL,
and chemotherapeutic agents. In some embodiments, the 2D ED., Cold Spring Harbor Laboratory Press, Plainview, NY.
antibodies are co-administered With an antifolate agent. The (1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND
US 2013/0189272 A1 Jul. 25, 2013
CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca sloWing or stopping) of groWth of tumor cells in vivo (c)
Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS: A promotion of cell death; (d) inhibition of degeneration; (e)
PRACTICAL APPROACH, IRL Press, Oxford (1991). relieving to some extent one or more of the symptoms asso
[0027] As used herein, the term “epitope” refers to the ciated With the abnormal condition; and (f) enhancing the
portion of an antigen to Which a monoclonal antibody spe function of a population of cells. The monoclonal antibodies
ci?cally binds. and derivatives thereof described herein effectuate the thera
[0028] As used herein, the term “conformational epitope” peutic effect alone or in combination With conjugates or addi
refers to a discontinuous epitope formed by a spatial relation tional components of the compositions of the invention.
ship betWeen amino acids of an antigen other than an unbro [0037] As used herein, the term “inhibits the progression of
ken series of amino acids. cancer” refers to an activity of a treatment that sloWs the
[0029] As used herein, the term “multimeric” refers to a modulation of neoplastic disease toWard end-stage cancer in
grouping of tWo or more identical or nearly identical units. As relation to the modulation toWard end-stage disease of
used herein, the term “tetrameric” refers to a grouping of four, untreated cancer cells.
identical or nearly identical units. [0038] As used herein “blocks a biological activity of
[0030] As used herein, the term “monomeric” refers to a FR-Ot” refers to the ability of the antibodies (or fragments
single unit of a mature protein that assembles in groups With thereof) of the invention to prevent folate binding to FR-Ot, to
other units. prevent the uptake of folate by cells, or to inhibit signal
[0031] As used herein, the term “inhibition of groWth of transduction in the cell triggered by folate.
dysplastic cells in vitro” means a decrease in the number of [0039] As used herein, the term “about” refers to an
tumor cells, in culture, by at least about 5%, preferably about approximation of a stated value Within an acceptable range.
10%, more preferably about 20%, more preferably about Preferably the range is +/—5% of the stated value.
30%, more preferably about 40%, more preferably about [0040] As used herein, the term “neoplastic disease” refers
50%, more preferably about 60%, more preferably about to a condition marked by abnormal proliferation of cells of a
70%, more preferably about 80%, more preferably about tissue.
90%, more preferably about 95%, more preferably about [0041] As used herein, the term “Wild-type” refers to a
99%, and most preferably 100%. In vitro inhibition of tumor native sequence, for example, a native nucleic acid sequence
cell groWth may be measured by assays knoWn in the art, such encoding or amino acid sequence of a heavy or light chain of
as the GEO cell soft agar assay. the antibodies of the invention. Examples of Wild-type
[0032] As used herein, the term “inhibition of groWth of sequences of the invention include the sequences of SEQ ID
dysplastic cells in vivo” means a decrease in the number of NOs: 1 -8.
tumor cells, in an animal, by at least about 5%, preferably [0042] As used herein, the term “FR-0t binding competi
about 10%, more preferably about 20%, more preferably tors” refers to aberrant transcripts of the nucleic acids encod
about 30%, more preferably about 40%, more preferably ing antibodies of the invention and aberrant translation prod
about 50%, more preferably about 60%, more preferably ucts of the antibodies of the invention that do not have the
about 70%, more preferably about 80%, more preferably biological properties of the anti-FR-Ot antibodies of the inven
about 90%, more preferably about 95%, more preferably tion (e.g., antigenbinding a?inity, ability to block a biological
about 99%, and most preferably 100%. In vivo modulation of activity of FR-Ot). For example, an aberrant transcript may
tumor cell groWth may be measured by assays knoWn in the contain a deletion, a frameshift, a nonsense mutation, or a
art, for example but not limited to using the Response Evalu missense mutation. An example of an aberrant translation
ation Criteria in Solid Tumors (RECIST) parameters (avail product is an alternative splice variant. An example of a FR-Ot
able online through the National Cancer Institute Cancer binding competitor is an antibody comprising a light chain
Therapy Evaluation Program). having an amino acid sequence of SEQ ID NO:24:
[0033] As used herein, “dysplastic cells” refer to cells that
exhibit abnormal groWth properties, such as but not limited to
groWth in soft agar, lack of contact inhibition, failure to MGWSCIILFLVATATGVHSDIQLTQSPSSLSASVGDRVTIT
undergo cell cycle arrest in the absence of serum, and forma
CSVSSSISSNNLHWYQQKPAASSQRTSPPTTANSGVVTRTC
tion of tumors When injected into immune-compromised
mice. Dysplastic cells include, but are not limited to tumors,
TRSAKGPRWKSNELWLHHLSSSSRHLMSS .
hyperplasia, and the like.
[0043] The light chain of such an FR-Ot binding competitor
[0034] The term “preventing” refers to decreasing the prob
may be encoded by a nucleic acid having a nucleic acid
ability that an organism contracts or develops an abnormal
sequence of SEQ ID NO:25:
condition.
[0035] The term “treating” refers to having a therapeutic
effect and at least partially alleviating or abrogating an abnor
ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA
mal condition in the organism. Treating includes inhibition of
tumor groWth, maintenance of inhibited tumor groWth, and GGTGTCCACTCCGACATCCAGCTGACCCAGAGCCCAAGCAGCCTG
induction of remission.
AGCGCCAGCGTGGGTGACAGAGTGACCATCACCTGTAGTGTCAGC
[0036] The term “therapeutic effect” refers to the inhibition
of an abnormal condition. A therapeutic effect relieves to TCAAGTATAAGTTCCAACAACTTGCACTGGTACCAGCAGAAGCCC
some extent one or more of the symptoms of the abnormal
condition. In reference to the treatment of abnormal condi GCAGCCTCCAGCCAGAGGACATCGCCACCTACTACTGCCAACAGT
tions, a therapeutic effect can refer to one or more of the
GGAGTAGTTACCCGTACATGTACACGTTCGGCCAAGGGACCAAGG
folloWing: (a) an increase or decrease in the proliferation,
groWth, and/or differentiation of cells; (b) inhibition (i.e.,
US 2013/0189272 A1 Jul. 25, 2013
and monomers or dimers of antibody heavy or light chains or
— c ont inued
TGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCC mixtures thereof. Antibodies of the invention are preferably
monoclonal antibodies.
CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT
[0049] The antibodies of the invention may include intact
immunoglobulins of any isotype including types IgA, IgG,
GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA
IgE, IgD, IgM (as Well as subtypes thereof). The antibodies
AGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA preferably include intact I gG and more preferably I gG1. The
light chains of the immunoglobulin may be kappa or lambda.
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCC
The light chains are preferably kappa.
TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT [0050] The antibodies of the invention include portions of
intact antibodies that retain antigen-binding speci?city, for
GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
example, Fab fragments, Fab‘ fragments, F(ab')2 fragments,
TCAACAGGGGAGAGTGTTAA. F(v) fragments, heavy chain monomers or dimers, light chain
monomers or dimers, dimers consisting of one heavy and one
[0044] As used herein, the term “puri?ed” means a condi light chain, and the like. Thus, antigen binding fragments, as
tion of being suf?ciently separated from other proteins or Well as full-length dimeric or trimeric polypeptides derived
nucleic acids With Which it Would naturally be associated, so from the above-described antibodies are themselves useful.
as to exist in “substantially pure” form. “Puri?ed” is not [0051] A “chimeric antibody” is an antibody produced by
meant to exclude arti?cial or synthetic mixtures With other recombinant DNA technology in Which all or part of the hinge
compounds or materials, or the presence of impurities that do and constant regions of an immuno globulin light chain, heavy
not interfere With the fundamental activity, and that may be chain, or both, have been substituted for the corresponding
present, for example, due to incomplete puri?cation, addition regions from another animal’ s immuno globulin light chain or
heavy chain. In this Way, the antigen-binding portion of the
of stabilizers, or compounding into, for example, immuno
genic preparations or pharmaceutically acceptable prepara parent monoclonal antibody is grafted onto the backbone of
another species’ antibody. One approach, described in EP
tions. A “puri?ed” antibody preferably means an antibody
substantially free of FR-ot binding competitors. The term 0239400 to Winter et al. describes the substitution of one
species’ complementarity determining regions (CDRs) for
“substantially pure” means comprising at least about 50-60%
by Weight of a given material (e. g., nucleic acid, protein, etc.). those of another species, such as substituting the CDRs from
human heavy and light chain immuno globulin variable region
More preferably, the preparation comprises at least about
75% by Weight, and most preferably about 90-95% by Weight domains With CDRs from mouse variable region domains.
of the given compound. Purity is measured by methods appro These altered antibodies may subsequently be combined With
priate for the given material (e. g., chromatographic methods, human immunoglobulin constant regions to form antibodies
agarose or polyacrylamide gel electrophoresis, HPLC analy that are human except for the substituted murine CDRs Which
are speci?c for the antigen. Methods for grafting CDR
sis, and the like).
regions of antibodies may be found, for example in Riech
[0045] As used herein, the phrase “substantially free of mann et al. (1988) Nature 332:323-327 and Verhoeyen et al.
FR-ot binding competitors” refers to a condition of having (1988) Science 239:1534-1536.
less than about 50%, more preferably less than about 40%,
[0052] The direct use of rodent monoclonal antibodies
more preferably less than about 30%, more preferably less (MAbs) as human therapeutic agents led to human anti-ro
than about 20%, more preferably less than about 10%, more dent antibody (“HARA”) (for example, human anti-mouse
preferably less than about 5%, more preferably less than
antibody (“HAMA”)) responses Which occurred in a signi?
about 1%, more preferably less than about 0.5%, and most cant number of patients treated With the rodent-derived anti
preferably about 0% by Weight of FR-ot binding competitors.
body (KhaZaeli, et al., (1994) Immunolhen 15:42-52). Chi
[0046] Antibodies meric antibodies containing feWer murine amino acid
sequences are believed to circumvent the problem of eliciting
[0047] The antibodies of the invention speci?cally bind
an immune response in humans.
folate receptor-alpha (FR-0t). In some embodiments, the anti
[0053] Re?nement of antibodies to avoid the problem of
bodies of the invention speci?cally bind a monomeric form of
HARA responses led to the development of “humanized anti
FR-ot. In some embodiments, the antibodies of the invention
bodies.” HumaniZed antibodies are produced by recombinant
speci?cally bind a multimeric form of FR-ot (e.g., a tetrameric
DNA technology, in Which at least one of the amino acids of
form) and not the monomeric form of FR-ot. Preferred anti
a human immunoglobulin light or heavy chain that is not
bodies of the invention block a biological activity of FR-ot. In
required for antigen binding has been substituted for the
preferred embodiments, the antibodies block a biological
corresponding amino acid from a nonhuman mammalian
activity of FR-ot on FR-ot-bearing cells. Antibodies of the
immunoglobulin light or heavy chain. For example, if the
invention preferably induce antibody-dependent cellular
immuno globulin is a mouse monoclonal antibody, at least one
cytotoxicity (ADCC) of FR-ot-bearing cells. Examples of
amino acid that is not required for antigen binding is substi
FR-ot-bearing cells include but are not limited to ovarian,
tuted using the amino acid that is present on a corresponding
lung, breast, brain, renal, colorectal, and endometrial cancer
human antibody in that position. Without Wishing to be bound
cells.
by any particular theory of operation, it is believed that the
[0048] Preferred antibodies, and antibodies suitable for use “humaniZation” of the monoclonal antibody inhibits human
in the method of the invention, include, for example, fully immunological reactivity against the foreign immunoglobu
human antibodies, human antibody homologs, humaniZed lin molecule.
antibody homologs, chimeric antibody homologs, Fab, Fab‘, [0054] As a non-limiting example, a method of performing
F(ab')2 and F(v) antibody fragments, single chain antibodies, complementarity determining region (CDR) grafting may be