NZ615694B2 - Antibodies against kidney associated antigen 1 and antigen binding fragments thereof - Google Patents
Antibodies against kidney associated antigen 1 and antigen binding fragments thereof Download PDFInfo
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- NZ615694B2 NZ615694B2 NZ615694A NZ61569412A NZ615694B2 NZ 615694 B2 NZ615694 B2 NZ 615694B2 NZ 615694 A NZ615694 A NZ 615694A NZ 61569412 A NZ61569412 A NZ 61569412A NZ 615694 B2 NZ615694 B2 NZ 615694B2
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
Abstract
Discloses antibodies and antigen binding fragments that bind to KAAG1 and associated methods and uses in treatment, detection and diagnosis of cancers comprising KAAG1 expressing cells, comprising heavy chain CDR sequences: GYTFTDDYMS; DINPYNGDTN; DPGAMDY; and light chain CDR sequences: RSSQSLLHSNGNTYLE; TVSNRFS; FQGSHVPLT. TYLE; TVSNRFS; FQGSHVPLT.
Description
ANTIBODIES AGAINST KIDNEY ASSOCIATED ANTIGEN 1 AND ANTIGEN
BINDING FRAGMENTS THEREOF
FIELD OF THE INVENTION
The present invention relates to specific antibodies or antigen binding fragments that
specifically bind to kidney associated n 1 (KAAG1) and their use for the
treatment, detection and sis of cancer. Delivery of a therapeutic agent to cells
with these antibodies or antigen binding fragments is particularly contemplated.
BACKGROUND OF THE ION
Among gynecologic malignancies, ovarian cancer accounts for the highest tumor—
related mortality in women in the United States (Jemal et al., 2005). It is the fourth
leading cause of cancer-related death in women in the US (Menon et al., 2005). The
American Cancer Society ted a total of 22,220 new cases in 2005 and
attributed 16,210 deaths to the e (Bonome et al., 2005). For the past 30 years,
the statistics have remained largely the same - the majority of women who develop
ovarian cancer will die of this disease (Chambers and Vanderhyden, 2006). The
e carries a 1:70 lifetime risk and a ity rate of >60% (Chambers and
Vanderhyden, 2006). The high mortality rate is due to the difficulties with the early
detection of ovarian cancer when the malignancy has already spread beyond the
ovary. Indeed, >80% of ts are diagnosed with advanced staged disease (stage
III or IV) (Bonome et al., 2005). These patients have a poor prognosis that is reflected
in <45% 5—year survival rate, although 80% to 90% will initially respond to
chemotherapy (Berek et al., 2000). This sed success compared to 20% 5-year
survival rate years earlier is, at least in part, due to the ability to optimally debulk
tumor tissue when it is confined to the ovaries, which is a significant prognostic factor
for ovarian cancer (Bristow R. E., 2000; Brown et al., 2004). In patients who are
sed with early disease (stage I), the 5-yr survival ranges from >90 (Chambers
and Vanderhyden, 2006).
Ovarian cancer comprises a heterogeneous group of tumors that are derived from the
surface epithelium of the ovary or from surface inclusions. They are classified into
serous, mucinous, endometrioid, clear cell, and r (transitional) types
corresponding to the different types of lia in the organs of the female
reproductive tract (Shih and Kurman, 2005). Of these, serous tumors account for
~60% of the ovarian cancer cases diagnosed. Each histologic subcategory is further
divided into three groups: benign, intermediate (borderline tumor or low ancy
potential , and malignant, reflecting their clinical behavior (Seidman et al.,
2002). LMP represents 10% to 15% of tumors diagnosed as serous and is a
conundrum as they display atypical nuclear structure and metastatic behavior, yet
they are considerably less aggressive than rade serous tumors. The 5-year
survival for patients with LMP tumors is 95% in contrast to a <45% survival for
advanced high-grade e over the same period (Berek et al., 2000).
Presently, the diagnosis of ovarian cancer is accomplished, in part, through routine
analysis of the medical history of patients and by performing physical, ultrasound and
x-ray examinations, and hematological ing. Two alternative strategies have
been reported for early hematological detection of serum biomarkers. One approach
is analysis of serum samples by mass spectrometry to find ns or protein
fragments of unknown identity that detects the presence or absence of cancer (Mor et
al., 2005; Kozak et al., 2003). However, this strategy is expensive and not broadly
available. atively, the presence or absence of known proteins/peptides in the
serum is being detected using antibody rrays, ELISA, or other r
approaches. Serum testing for a protein biomarker called CA—125 (cancer antigen-
125) has long been widely performed as a marker for n cancer. However,
although ovarian cancer cells may produce an excess of these protein les,
there are some other cancers, ing cancer of the fallopian tube or endometrial
cancer (cancer of the lining of the uterus), 60% of people with pancreatic cancer, and
%-25% of people with other malignancies with ed levels of CA-125. The CA-
125 test only returns a true positive result for about 50% of Stage l ovarian cancer
patients and has a 80% chance of returning true positive results from stage II, ill, and
iv ovarian cancer patients. The other 20% of ovarian cancer patients do not show any
increase in CA-125 concentrations. In addition, an elevated CA—125 test may indicate
other benign activity not associated with cancer, such as menstruation, pregnancy, or
endometriosis. Consequently, this test has very limited clinical ation for the
detection of early stage disease when it is still treatable, ting a positive
predictive value (PPV) of <10%. Even with the addition of ultrasound screening to CA-
125, the PPV only improves to around 20% (Kozak et al., 2003). Thus, this test is not
an effective screening test.
Despite improved knowledge of the etiology of the disease, aggressive cytoreductive
surgery, and modern combination chemotherapy, there has been only little change in
mortality. Poor outcomes have been attributed to (1) lack of adequate screening tests
for early disease detection in combination with only subtle presentation of symptoms
at this stage - diagnosis is frequently being made only after progression to later
stages, at which point the peritoneal dissemination of the cancer limits effective
treatment and (2) the frequent development of resistance to standard
chemotherapeutic strategies limiting improvement in the 5-year survival rate of
patients. The initial chemotherapy regimen for ovarian cancer includes the
combination of latin (Paraplatin) and paclitaxel ). Years of clinical trials
have proved this ation to be most ive after effective surgery - reduces
tumor volume in about 80% of the women with newly diagnosed ovarian cancer and
40% to 50% will have complete regression - but studies continue to look for ways to
improve patient response. Recent nal infusion of chemotherapeutics to target
hard-to-reach cells in combination with intravenous delivery has increased the
effectiveness. However, severe side s often lead to an lete course of
treatment. Some other chemotherapeutic agents include doxorubicin, cisplatin,
cyclophosphamide, cin, etoposide, stine, topotecan hydrochloride,
ifosfamide, 5-fluorouracil and melphalan. More recently, clinical trials have
demonstrated that intraperitoneal administration of cisplatin confers a survival
advantage compared to systemic intravenous chemotherapy stra and McGuire,
2007). The excellent al rates for women with early stage disease receiving
chemotherapy provide a strong rationale for research efforts to develop gies to
improve the detection of ovarian cancer. Furthermore, the discovery of new ovarian
cancer-related biomarkers will lead to the development of more effective therapeutic
strategies with l side effects for the future treatment of ovarian cancer.
Notwithstanding these recent advances in the understanding and the treatment for
ovarian cancer, the use of chemotherapy is invariably associated with severe adverse
reactions, which limit their use. Consequently, the need for more specific strategies
such as combining n tissue specificity with the selectivity of monoclonal
antibodies should permit a significant ion in rget-associated side effects.
The use of monoclonal antibodies for the therapy of ovarian cancer is beginning to
emerge with an increasing number of ongoing clinical trials (Oei et al., 2008;
Nicodemus and berek, 2005). Most of these trials have examined the use of
monoclonal antibodies conjugated to radioisotopes, such as yttrium-90, or antibodies
that target tumor antigens already identified in other cancer types. An example of this
is the use of bevacizumab, which targets ar endothelial growth factor (Burger,
2007). There are very few ovarian cancer specific antigens that are currently under
investigation as therapeutic targets for monoclonal antibodies. Some examples
include the use of a protein termed B7-H4 (Simon et al., 2006) and more recently
folate receptor-alpha (Ebel et al., 2007), the latter of which has ly entered
Phase ll clinical trials.
Kidney associated antigen 1 (KAAG1) was originally cloned from a cDNA library
derived from a histocompatibility leukocyte antigen-B7 renal carcinoma cell line as an
antigenic peptide presented to cytotoxic T lymphocytes (Van den Eynde et al., 1999;
Genebank accession no. Q9UBP8, SEQ ID NOs.:28; 29). The locus containing
KAAG1 was found to encode two genes transcribed on opposite DNA strands. The
sense strand was found to encode a transcript that encodes a protein termed
DCDC2. Expression studies by these authors found that the KAAG1 antisense
transcript was tumor specific and exhibited very little expression in normal tissues
s the DCDC2 sense ript was ubiquitously expressed (Van den Eynde et
al., 1999). The expression of the KAAG1 transcript in cancer, and in particular
ovarian cancer, renal cancer, lung , colon cancer, breast cancer and
melanoma was disclosed in the published patent application No.
(the entire content of which is incorporated herein by
nce). Van den Eynde et al., also observed RNA expression in renal
carcinomas, colorectal omas, melanomas, sarcomas, leukemias, brain tumors,
thyroid tumors, mammary carcinomas, tic carcinomas, oesophageal
carcinomas, bladder tumor, lung carcinomas and head and neck tumors. Recently,
strong genetic evidence obtained through linkage disequilibrium studies found that
the VMP/DCDCZ/KAAG1 locus was associated with dyslexia (Schumacher et al.,
2006; Cope et al., 2005). One of these reports pointed to the DCDCZ marker as the
culprit in dyslexic ts since the function of this n in cortical neuron
migration was in accordance with symptoms of these patients who often display
abnormal neuronal migration and maturation (Schumacher et al., 2006).
SUMMARY OF THE INVENTION
The invention relates to specific AAG1 antibodies and antigen binding
fragments and their use for the treatment, detection and diagnosis of cancer
comprising tumor cells expressing KAAG1 or a KAAG1 t. Exemplary
embodiments of such cancer includes, for e, ovarian cancer, skin cancer,
renal cancer, colorectal cancer, a, ia, brain cancer, cancer of the
thyroid, breast cancer, prostate , cancer of the oesophagus, bladder cancer,
lung cancer and head and neck cancer.
The antibodies or antigen binding fragments may be ularly effective at targeting
KAAG1 or KAAG1 variant expressed at the surface of the tumor cells.
In fact, the antibodies and n binding fragments of the present invention appear
to have improved ability to bind to KAAG1-expressing tumor cells in comparison with,
for example, the 3D3 and 3G10 antibodies disclosed in . These
antibodies and antigen binding fragments are also internalized and may ore be
useful to deliver therapeutic agents to tumor cells. Our results suggest that antibodies
and antigen binding nts having the desired characteristics (e.g., improved
binding and internalization) generally bind to a C-terminal region of KAAG1 ted
by amino acids 61 to 84. However, although both the 3A4 and 3G10 antibodies bind
to the same region, the 3A4 antibody appears to bind to the surface of tumor cells
more ently than the 3G10 antibody. In ular, cancer cells that express the
KAAG1 antigen require approximately 10-fold less 3A4 compared to 3G10 in flow
cytometry experiments, an approach that measures the direct binding of the
antibodies to the surface of the cells. In addition, in binding experiments using surface
plasmon resonance, it was discovered that the affinity constant (KD) of 3A4 for
KAAG1 is below 10 picomolar, s antibodies 3D3 and 3G10 exhibited affinity
constants (KD) greater than 200 picomolar (20-fold lower affinity). Therefore, these
increases in binding ability of 3A4 are expected to translate into ed therapeutic
activity.
The t invention provides in one aspect thereof, an isolated or substantially
purified dy or antigen binding fragment which may be capable of specific
binding to a sequence which is identical to at least 10 (e.g., 10 to 20 or more)
consecutive amino acids located between amino acids 61 to 84 of KAAG1 (SEQ ID
NO.:29)
The present invention also provides isolated antibodies or antigen binding fragments
capable of competing with the dy or antigen binding fragment described herein.
In a further aspect, the invention relates to specific antibodies or antigen binding
fragments having the amino acid sequences described herein. Such antibodies or
antigen binding nts may be in the form of monoclonal antibodies, polyclonal
antibodies, chimeric antibodies, humanized antibodies and human antibodies
(isolated) as well as antigen binding fragments having the characteristics described
herein. Antibodies or antigen binding fragments encompassing permutations of the
light and/or heavy chains between a monoclonal, ic, humanized or human
antibody are also encompassed th.
The antibodies or antigen g fragments of the present invention may thus
se amino acids of a human constant region and/or framework amino acids of a
human dy.
The term "antibody” refers to intact antibody, monoclonal or polyclonal antibodies.
The term “antibody” also encompasses multispecific antibodies such as bispecific
antibodies. Human antibodies are usually made of two light chains and two heavy
chains each comprising variable regions and constant regions. The light chain
variable region comprises 3 CDRs, identified herein as CDRL1 or L1, CDRL2 or L2
and CDRL3 or L3 flanked by framework regions. The heavy chain variable region
comprises 3 CDRs, identified herein as CDRH1 or H1, CDRH2 or H2 and CDRH3 or
H3 d by framework regions. The CDRs of the humanized antibodies of the
present invention have been identified using the Kabat and Chotia definitions (e.g.,
CDRH2 set forth in SEQ ID NO.:56). However. others (Abhinandan and Martin, 2008)
have used modified approaches based loosely on Kabat and Chotia resulting in the
delineation of shorter CDRs (e.g., CDRH2 set forth in SEQ ID NO.:6).
The term "antigen-binding fragment", as used herein, refers to one or more fragments
of an antibody that retain the ability to bind to an antigen (e.g., KAAG1, secreted form
of KAAG1 or ts thereof). It has been shown that the antigen-binding on of
an antibody can be performed by fragments of an intact antibody. Examples of
binding fragments encompassed within the term "antigen-binding fragment" of an
antibody e (i) a Fab fragment, a lent fragment ting of the VL, VH,
CL and CH1 domains; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment ting of the VL and VH
domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR), e.g., VH CDR3. Furthermore, although
the two domains of the Fv fragment, VL and VH, are coded for by separate genes,
they can be joined, using recombinant methods, by a tic linker that enables
them to be made as a single polypeptide chain in which the VL and V... regions pair to
form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 3-426; and Huston et at. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883). Such single chain antibodies are also intended to be encompassed
within the term "antigen—binding fragment" of an antibody. Furthermore, the antigen-
binding fragments include binding-domain immunoglobulin fusion proteins comprising
(i) a binding domain polypeptide (such as a heavy chain variable region, a light chain
variable region, or a heavy chain variable region fused to a light chain variable region
via a linker peptide) that is fused to an immunoglobulin hinge region polypeptide, (ii)
an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and
(iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant
region. The hinge region may be modified by ing one or more cysteine residues
with serine es so as to prevent dimerization. Such binding-domain
immunoglobulin fusion ns are further disclosed in US 2003/0118592 and US
2003/0133939. These dy fragments are ed using conventional
techniques known to those with skill in the art, and the fragments are screened for
utility in the same manner as are intact antibodies.
The term ized antibody” encompasses futiy humanized antibody (i.e.,
frameworks are 100% humanized) and partially humanized antibody (e.g., at least
one variable domain contains one or more amino acids from a human antibody, while
other amino acids are amino acids of a non—human parent antibody). Typically a
“humanized antibody” contains CDRs of a man parent antibody (e.g., mouse,
rat, rabbit, man primate, etc.) and frameworks that are cal to those of a
natural human antibody or of a human antibody consensus. In such instance, those
ized antibodies" are characterized as fuiiy humanized. A “humanized
antibody” may also contain one or more amino acid substitutions that have no
correspondence to those of the human antibody or human antibody consensus. Such
substitutions include, for example, back-mutations (e.g., roduction of non-human
amino acids) that may preserve the antibody characteristics (e.g., affinity, specificity
etc.). Such substitutions are usually in the framework region. A “humanized antibody”
optionaIiy also comprise at least a portion of a constant region (Fc) which is typically
that of a human antibody. Typically, the nt region of a “humanized antibody” is
identical to that of a human antibody.
The term “natural human antibody” refers to an antibody that is encoded (encodable)
by the human antibody repertoire, i.e., germline sequence.
The term ric antibody” refers to an antibody having non-human variable
region(s) and human constant .
The term “hybrid antibody” refers to an antibody comprising one of its heavy or light
chain variable region (its heavy or light chain) from a certain types of antibody (e.g.,
humanized) while the other of the heavy or light chain variable region (the heavy or
light chain) is from another type (e.g., murine, ic).
In some embodiments, the heavy chain and/or light chain framework region of the
humanized antibody may comprises from one to thirty amino acids from the non—
human antibody which is sought to be humanized and the remaining portion being
from a natural human dy or a human antibody consensus. In some instances,
the humanized antibody may comprise from 1 to 6 non-human CDRs and often the
six CDRs are non-human.
The natural human dy selected for zation of the non—human parent
antibody may comprise a variable region having a three-dimensional structure similar
to that of (superimposable to) a (modeled) variable region of the non—human parent
antibody. As such, the zed antibody has a greater chance of having a three-
dimensional ure similar to that of the non—human parent antibody.
The light chain variable region of the natural human antibody selected for
humanization purposes, may have, for example an overall (over the entire light chain
le region) of at least 70%, 75%, 80%, etc. identity with that of the non-human
parent antibody. Alternatively, the light chain framework region of the natural human
dy selected for humanization purposes, may have, for example, at least 70%
75%, 80%, 85% etc. sequence identity with the light chain framework region of the
non-human parent antibody. In some embodiments, the natural human antibody
selected for humanization purposes may have the same or substantially the same
number of amino acids in its light chain complementarity determining region to that of
a light chain complementarity determining region of the non-human parent antibody.
The heavy chain variable region of the l human antibody selected for
humanization purposes, may have, for example an overall (over the entire heavy
chain variable region) of at least 60%, 70%, 75%, 80%, etc. identity with that of the
non-human parent antibody. Also in accordance with the t invention, the
human framework region amino acid residues of the humanized antibody heavy
chain may be from a l human antibody heavy chain framework region having at
least 70%, 75%, 89% etc. identity with a heavy chain framework region of the non-
human parent antibody. In some embodiments, the natural human antibody selected
for humanization purposes may have the same or substantially the same number of
amino acids in its heavy chain mentarity ining region to that of a heavy
chain complementarity determining region of the non-human parent antibody.
The natural human antibody that is selected for zation of the non-human
parent antibody may comprise a variable region having a three-dimensional structure
similar to that of (superimposable to) a (modeled) variable region of the non-human
parent antibody. As such, the humanized or hybrid antibody has a greater chance of
having a three-dimensional structure similar to that of the non—human parent
antibody.
For example, the natural human dy heavy chain variable region which
may be
selected for humanization purposes may have the ing characteristics: a) a
three—dimensional structure similar to or identical imposable) to that of a heavy
chain of the non-human antibody and/or b) a framework region having an amino acid
sequence at least 70% identical to a heavy chain framework region of the non-human
antibody. Optionally, (a number of) amino acid residues in a heavy chain CDR (e.g.,
all three CDRs) is the same or substantially the same as that of the non-human
heavy chain CDR amino acid residues.
Alternatively, the l human antibody light chain variable region which may be
selected for humanization purposes may have the ing teristics: a) a
three-dimensional structure similar to or identical (superimposable) to that of a light
chain of the non—human antibody, and/or b) a framework region having an amino acid
sequence at least 70% identical to a light chain framework region of the non-human
antibody. Optionally, (a number of) amino acid residues in a light chain CDR (e.g., all
three CDRs) that is the same or substantially the same as that of the non-human light
chain CDR amino acid residues.
A typical antigen binding site is comprised of the variable regions formed by the
pairing of a light chain immunoglobulin and a heavy chain immunoglobulin. The
structure of the antibody variable regions is very consistent and exhibits very similar
structures. These variable regions are typically sed of relatively homologous
framework regions (FR) interspaced with three hypervariable regions termed
Complementarity Determining Regions (CDRs). The overall binding activity of the
antigen binding fragment is often dictated by the sequence of the CDRs. The FRs
often play a role in the proper positioning and alignment in three dimensions of the
CDRs for optimal antigen binding.
Antibodies and/or antigen binding fragments of the t invention may originate,
for example, from a mouse, a rat or any other mammal or from other s such as
through recombinant DNA technologies.
In another aspect, the invention provides an antibody or antigen binding fragment
f e of specific binding to Kidney associated n 1 (KAAG1) having a
heavy chain variable region comprising the amino acid sequence set forth in SEQ ID
NO.:5, SEQ ID NO.:6 and SEQ ID NO.:7 and a light chain variable region comprising
the amino acid ce set forth in SEQ ID NO.: 8, SEQ ID NO.:9 and SEQ ID
NO.:10.
In another aspect, the invention provides an antibody or antigen binding fragment
thereof that specifically binds to kidney associated antigen 1 (KAAG1) and has an
ty constant (KD) of less than 10 picomolar.
In another aspect, the invention provides an antibody or antigen binding fragment
thereof capable of competing with the antibody or antigen binding fragment thereof of
the invention and having an affinity constant (KD) of less than 1nM, provided that said
antibody is not the 3G10 antibody or antigen binding fragment thereof.
In r aspect, the invention provides a nucleic acid encoding a light chain
variable region and/or a heavy chain variable region of the antibody or antigen
binding fragment of the invention.
In another aspect, the invention provides a vector comprising the c acid of the
invention.
In another , the invention provides an ed cell comprising the nucleic acid
of the invention, the vector of the invention or the antibody or antigen binding
nt of the invention.
In another aspect, the ion provides a pharmaceutical composition comprising
the antibody or antigen binding fragment of the ion, and a pharmaceutically
acceptable carrier.
In r aspect, the ion provides a method for detecting KAAG1 or a KAAG1
variant, the method comprising contacting an isolated cell expressing KAAG1 or the
KAAG1 variant or a sample comprising or suspected of comprising KAAG1 or the
KAAG1 variant with the antibody or n binding fragment thereof of the invention
and measuring binding.
In another aspect, the invention provides a composition comprising the antibody or
antigen binding nt of the invention, and a carrier.
In another aspect, the invention provides a kit comprising the antibody or antigen
binding fragment of the ion.
In another , the invention provides use of the dy or antigen binding
fragment of the invention, the pharmaceutical composition of the invention or the
composition of the ion in the manufacture of a medicament for detection,
diagnosis or treatment of cancer comprising cells expressing KAAG1 or a KAAG1
variant.
In another aspect, the ion provides use of the antibody or antigen binding
fragment of the invention, the pharmaceutical composition of the invention or the
composition of the ion in the detection of a tumor ex vivo or in the diagnosis of
cancer ex vivo, wherein the tumor or cancer comprises cells expressing KAAG1 or a
KAAG1 variant.
In another aspect, the invention provides a method for obtaining an antibody or
antigen binding fragment thereof, suitable for use as an antibody-drug conjugate for
the treatment of cancer comprising cells sing KAAG1 or a KAAG1 variant, the
method comprising: a. providing an antibody or antigen binding fragment thereof
which specifically binds to an epitope comprised between amino acids 61 and 84 of
KAAG1 or a KAAG1 variant; b. testing alization of the antibody or antigen
binding fragment within an isolated cell expressing KAAG1 or a KAAG1 variant or in a
non-human animal and; c. isolating an antibody or an antigen binding nt which
is internalized.
In another aspect, the invention provides a method of making the antibody or antigen
binding fragment thereof of the invention, sing culturing an isolated cell of the
invention so that the antibody or n g fragment f is produced.
Further scope, ability and advantages of the present invention will become
apparent from the non-restrictive detailed description given after. It should be
understood, however, that this detailed description, while ting exemplary
embodiments of the invention, is given by way of example only, with reference to the
accompanying drawings.
Any discussion of documents, acts, als, devices, articles or the like which has
been included in the present specification is not to be taken as an admission that any
or all of these matters form part of the prior art base or were common general
knowledge in the field relevant to the present disclosure as it existed before the
priority date of each claim of this application.
Throughout this specification the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the ion of a stated t, integer
or step, or group of elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or steps.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results from the ELISA that compares the binding of the 3A4
chimeric anti-KAAG1 antibody with a control antibody when incubated with increasing
concentrations of recombinant human KAAG1. The binding curve of 3A4 is shown by
the lighter colored line.
Figure 2 shows a histogram that describes the results from ELISA analyses to map
the epitope specificity of the 3A4 anti-KAAG1 antibody. The results showed that 3A4
interacted with a sequence of amino acids contained in the carboxy-terminus of
KAAG1 between amino acids 61 – 84. The binding of 3A4 was compared with 3C4,
3D3, and 3G10 anti-KAAG1 antibodies that were known to interact with regions 1 –
, 36 – 60, and 61 – 84 of KAAG1, tively.
Figure 3A shows the results of flow cytometry performed on SKOV-3 and TOV-21G
ovarian cancer cells with the 3A4 anti-KAAG1 antibody (darker line) compared with a
control IgG (lighter line).
Figure 3B shows the results of flow cytometry med on 293E human kidney
cells with the 3A4 anti—KAAG1 antibody r line) compared with a control lgG
(lighter line).
Figure 4 represents the detection of the KAAG1 antigen on the surface of SKOV-3
cells by flow cytometry with the 3A4 anti-KAAG1 antibody. The fluorescence signal
decreases with time when the cells were incubated at 37 C, which suggests that the
antibody complex was internalized during the incubation when the cells were
incubated with 3A4.
Figure 5 shows the internalization of 3A4 anti-KAAG1 antibody and its co-localization
with LAMP1, a protein associated with endosomal and lysosomal membranes.
Figure 6A and BB are graphs representing FACs analysis of tumor cells exposed to
different anti—KAAG1 antibodies.
Figure 7 are schematics enting 2 likely representation of the KAAG1
orientation in the cell membrane.
Figure 8 is a molecular model n diagram) of the murine 3A4 variable domain.
CDR loops are colored in black and labelled L1. L2 and L3 in the light chain and H1,
H2 and H3 in the heavy chain. The framework region is shown in gray.
Figure 9a is a molecular models of humanized antibody Lh1Hh1 (i.e., humanized
light chain 1 and humanized heavy chain 1) of 3A4 variable domains. CDR loops are
colored in black and ed L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine framework to human framework are rendered in ball-and-stick
representation. Lh1 ated the humanized light chain of variant 1 and HM
designated the heavy chain of variant 1.
Figure 9b is a molecular models of humanized antibody Lh1Hh2 (i.e., humanized
light chain 1 and humanized heavy chain 2) of 3A4 variable domains. CDR loops are
colored in black and labelled L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine framework to human framework are rendered in nd-stick
entation. Lh1 designated the humanized light chain of variant 1 and Hh2
designated the heavy chain of variant 2.
Figure 9c is a molecular models of humanized antibody Lh1Hh3 (i.e., humanized
light chain 1 and humanized heavy chain 3) of 3A4 le domains. CDR loops are
colored in black and labelled L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side—chains of residues
mutated from murine ork to human framework are rendered in ball-and-stick
representation. Lh1 designated the humanized light chain of variant 1 and Hh3
designated the heavy chain of variant 3.
Figure 9d is a molecular models of humanized antibody Lh1Hh4 (i.e., humanized
light chain 1 and humanized heavy chain 4) of 3A4 variable domains. CDR loops are
colored in black and labelled L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine framework to human framework are rendered in ball-and-stick
entation. Lh1 designated the zed light chain of variant 1 and HM
designated the heavy chain of variant 4.
Figure 9e is a molecular models of humanized antibody Lh2Hh1 (i.e., humanized
light chain 2 and humanized heavy chain 1) of 3A4 variable domains. CDR loops are
colored in black and labelled L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine framework to human framework are ed in nd-stick
representation. Lh2 designated the humanized light chain of variant 2 and HM
designated the heavy chain of variant 1.
Figure 9f is a molecular models of humanized antibody Lh2Hh2 (i.e., humanized
light chain 2 and humanized heavy chain 2) of 3A4 variable domains. CDR loops are
colored in black and ed L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine framework to human framework are rendered in ball-and-stick
representation. Lh2 designated the humanized light chain of variant 2 and Hh2
designated the heavy chain of variant 2.
Figure 99 is a molecular models of humanized antibody Lh2Hh3 (i.e., humanized
light chain 2 and zed heavy chain 3) of 3A4 variable domains. CDR loops are
colored in black and ed L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine ork to human framework are rendered in ball-and-stick
representation. Lh2 designated the humanized light chain of variant 2 and Hh3
designated the heavy chain of variant 3.
Figure 9h is a molecular models of humanized antibody Lh2Hh4 (i.e., humanized
light chain 2 and humanized heavy chain 4) of 3A4 variable domains. CDR loops are
d in black and labelled L1, L2 and L3 in the light chain and H1, H2 and H3 in
the heavy chain. The framework region is shown in gray. The side-chains of residues
mutated from murine ork to human framework are rendered in ball-and-stick
representation. Lh2 designated the humanized light chain of variant 2 and HM
designated the heavy chain of variant 4.
Figure 10a is an amino acid sequence alignment of the 3A4 variable domains of the
murine and humanized light chains. The light chain has two humanized variants (Lh1
an Lh2). The CDRs are shown in bold and indicted by CDRL1, CDRL2 and CDRL3.
Back ons in the human framework regions that are murine amino acids are
underlined in the humanized sequences.
Figure 10b is an amino acid ce alignment of the 3A4 variable s of the
murine and humanized heavy chains. The heavy chain has four zed variants
(Hh1 to Hh4). The CDRs are shown in bold and indicted by CDRH1, CDRH2 and
CDRH3. Back mutations in the human framework regions that are murine amino
acids are underlined in the humanized sequences.
Figure 11A is an ent of murine 3A4 light chain variable region (SEQ ID NO.:4)
with a light chain variable region variant (SEQ ID NO.:33) using the ClustalW2
program (Larkin M.A., et al., (2007) ClustalW and ClustalX version 2. Bioinformatics
2007 : 2947-2948) where an “*" (asterisk) indicates positions which have a
single, fully conserved residue, wherein (colon) indicates conservation between
groups of strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix
and where (period) indicates conservation between groups of weakly similar
properties - g =< 0.5 in the Gonnet PAM 250 matrix.
Figure 113 is an alignment of murine 3A4 heavy chain variable region (SEQ ID
N02) with a light chain variable region variant (SEQ ID NO.:38) using the ClustalW2
m (Larkin M.A., et al., (2007) ClustalW and ClustalX version 2. Bioinformatics
2007 23(21): 2947-2948) where an “*” (asterisk) indicates positions which have a
single, fully ved residue, wherein (colon) indicates conservation between
groups of strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix
and where (period) indicates conservation between groups of weakly r
properties - scoring =< 0.5 in the Gonnet PAM 250 .
Figure 12a represents plasmid Map of pKCR5—3A4-HC-Variant 1. The heavy chains
of the humanized 3A4 variants were cloned in the same manner into the Hindlll site
of pK-CR5. Consequently the resulting plasmids are identical to 3A4-HC
variant 1 except for the sequence of the heavy chain immunoglobulin variable
domain.
Figure 12b represents plasmid Map of pMPG—CR5-3A4-LC-Variant 1. The light
chains of the humanized variants 1 and 2 of 3A4 antibody were cloned in the same
manner into the BamHl site of pMPG-CRS. Consequently, the resulting plasmid is
identical to pMPG-CR5-3A4-LC-Variant 1, except for the sequence of the light chain
immunoglobulin variable domain.
Figure 13 represents an analysis of antibody tion after transient transfection in
CHO cells. Supernatant (13 days post—transfection) of CHOcTA cells transfected with
the different combinations of light and heavy chains of zed 3A4 antibody were
analyzed by western blot. Quantification of antibody produced in the atants
was determined after scanning the bands of the western blot t dilution of a
known standard (human purified lgG antibody). Mr molecular weight marker (kDa).
Figure 14 is a graph of a ex GT5 gel filtration of recombinant KAAG1 sample.
KAAG1 was injected over the gel filtration and separated at 0.4 ml/min. The largest
peak between fractions 15 — 19.
Figure 15 is a Table listing the rate and affinity constants for the murine and
humanized variants of the 3A4 antibody.
Figure 16A is an histogram illustrating the association rates (Ka) of the humanized
dies.
Figure 163 is an histogram rating the dissociation rates (Kd) of the humanized
antibodies.
Figure 16C is an histogram illustrating the affinity constants (KB) of the humanized
antibodies.
Figure 17a illustrates humanized 3A4 variants binding to KAAG1 in an ELISA. This
figure shows the comparative binding of 3A4 humanized antibody variants and the
murine 3A4. Concentration-dependent binding profiles of the humanized heavy
chains (Hh1, Hh2, Hh3 and HM) assembled with the Lh1 light chain variant.
Figure 17b illustrates humanized 3A4 variants binding to KAAG1 in an ELISA. This
figure shows the comparative binding of 3A4 humanized antibody variants and the
murine 3A4. Concentration-dependent binding profiles of the humanized heavy
chains (Hh1, Hh2, Hh3 and HM) assembled with the Lh2 light chain variant.
Figure 18 illustrates humanized 3A4 ts binding to KAAG1 on the surface of
cancer cells. This illustration shows the comparative binding activity of the
humanized and the murine 3A4 antibodies on the unpermeabilized SKOV—3 ovarian
cancer cells.
DETAILED PTION OF THE INVENTION
The expression and biological activity of KAAG1 in cancer cells
The present invention relates to the use of antibodies to target tumors found in
various cancer types, in particular n cancer. In order to direct the antibodies to
the , the identification of tumor-specific antigens that are expressed at the cell
surface of the cancer cells must be carried out. There are several technologies that
are available to identify tumor-specific ns and the method that was used to
identify KAAG1 in ovarian tumors, an innovative discovery platform called Subtractive
Transcription-based ication of mRNA (STAR), is described in the published
patent application No. published under No. WO/2007/147265
on December 27, 2007.
Analysis of the ovarian cancer STAR libraries yielded many genes that encode
secreted and cell e proteins. One of these, termed AB-0447, contained an open
reading frame that d a polypeptide of 84 amino acids, ponding to SEQ
ID NO.:29 that was encoded by a cDNA of 885 base pairs with the nucleotide
sequence shown in SEQ ID NO.:28. A search of publicly available databases
revealed that the AB-0447 nucleotide ce was identical to that of a gene called
KAAG1. Bioinformatic analysis ted a membrane-anchored protein that presents
its functional domain to the extracellular tment. KAAG1 was originally cloned
from a kidney cancer library as a cell surface antigen, a result that confirms its
membrane zation. Additionally, our studies showed that the protein was
processed at its amino-terminus, a result that was consistent with ge of a
functional signal peptide at or between amino acids 30 and 34. Furthermore, transient
sion of the full-length cDNA resulted in detection of cleaved KAAG1 in the
culture medium. This last finding indicated that this membrane-anchored protein could
be shed from the cells when sed at high levels. in contrast, expression of an
amino-truncated mutant of KAAG1 resulted in intra-cellular retention of the protein.
There are currently no published reports that shed any light on its function and the
over-expression of KAAG1 in ovarian cancer, as disclosed by this invention, has
never been usly documented.
We have thus investigated whether KAAG1 could be used for antibody-based
diagnostics and therapeutics.
Several ovarian cancer cell-based models have been established, such as TOV-21G,
TOV-112D, OV-90, and others, and are ar to those skilled in the art. These cells
are part of a collection of human ovarian cancer cell lines d from patients with
ovarian tumors or ascites fluid. These cell lines have undergone an in-depth analysis,
including global gene expression patterns on rrays that make them excellent
cell-based models for human ovarian cancer. The growth properties, gene expression
patterns, and response to chemotherapeutic drugs indicated that these cell lines are
very representative of ovarian tumor behavior in vivo (Benoit et al., 2007). RT-PCR
analysis of total RNA isolated from these ovarian cancer cell lines showed that the
KAAG1 transcript was weakly expressed in the cell lines derived from primary tumors.
in contrast, cell lines d from ascitic fluid contained high levels of KAAG1
expression. The increased expression of KAAG1 in cells from the ascitic fluid
suggested that the nment of the cells influences the regulation of the KAAG1
gene. c cells are associated with advanced disease and this pattern of
expression implies that increased KAAG1 levels are associated with anchorage-
independent growth. In concordance with this latter suggestion, KAAG1 expression
was found to significantly increase in cell lines derived from primary tumors when
these cells were cultured as spheroids in 3D cultures. These spheroids have been
extensively terized and were found to y many properties associated with
tumors in vivo (Cody et al., 2008). Thus, expression of KAAG1 was found to be
significantly increased in models that mimic tumor progression, in particular during the
evolution of ovarian cancer.
With the demonstration that KAAG1 expression is ted in ovarian cancer cells,
the function of this gene in ovarian cancer cell behavior was examined in cell-based
assays. To that effect, RNA interference (RNAi) was used to knock down the
expression of the endogenous KAAGi gene in the ovarian cancer cell lines and it was
found that decreased expression of KAAG1 resulted in a significant reduction in the
migration of the cells as ined in a standard cell ty assay, as exemplified
by a wound healing (or h) assay. This type of assay measures the speed at
which cells fill a denuded area in a confluent monolayer. sed expression of
KAAG1 resulted in a reduction in the survival of ovarian cancer cell lines as ed
by a clonogenic assay, such as a colony survival assay. Those skilled in the art may
use other methods to evaluate the requirement of KAAG1 in the behavior of cancer
cells, in particular ovarian cancer cells.
Based on the expression of KAAG1 in a large proportion of ovarian tumors, its limited
expression in normal tissues, and a concordance between expression levels and
increased malignancy, and a ve biological role for KAAG1 in the behavior of
n cancer cell lines, KAAG1 was chosen as a therapeutic target for the
development of antibodies for the detection, prevention, and treatment of n
cancer. Expression of KAAG1 in cancers, other than ovarian cancer also lead the
Applicant to the evaluation of eutic or diagnostic antibodies for other cancer
indications.
The present invention therefore provides anti-KAAG1 antibodies and antigen binding
fragments thereof which specifically target KAAG1 and which may be used, for
example, as an antibody-drug conjugate.
Such antibodies and antigen binding fragments include for example, onal
antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies,
dy fragments, single chain antibodies, domain antibodies, and polypeptides
having an antigen binding region.
Antibodies and_antigen binding fragments that binds to KAAG1
Antibodies were initially ed from Fab librairies for their specificity towards the
antigen of interest.
The le regions of the antibodies or antigen binding fragments described herein
may be fused with constant regions of a desired species thereby aliowing recognition
of the dy by effector cells of the desired species. The constant region may
originate, for example, from an IgG1, IgG2, lgG3, or lgG4 subtype. Cloning or
synthesizing a constant region in frame with a variable region is well within the scope
of a person of skill in the art and may be performed, for example, by recombinant
DNA technology. Thus, antibodies comprising constant region of a human antibody
as well as antibodies or antigen binding fragments comprising framework amino acids
of a human antibody are also encompassed by the present invention.
The present invention therefore es in an exemplary embodiment, an isolated
antibody or antigen binding fragment comprising a light chain variable region having;
a. a CDRL1 sequence comprising SEQ ID NO.:8 or as set forth in
SEQID NO.:8;
b. a CDRL2 sequence comprising SEQ ID NO.:9 or as set forth in SEQ
ID NO.:9, or;
c. a CDRL3 sequence comprising SEQ ID NO.:10 or as set forth in SEQ
ID NO.:10.
The isolated antibody or antigen binding fragment may also comprise a heavy chain
variable region ;
a. a CDRH1 sequence comprising SEQ ID NO.:5 or as set forth in SEQ
ID NO.:5;
b. a CDRH2 sequence comprising SEQ ID NO.:6 or as set forth in SEQ
ID NO.:6, or;
c. a CDRH3 sequence comprising SEQ ID N02? or as set forth in SEQ
ID NO.:7.
In an exemplary embodiment, the antibody or antigen binding fragment may se
any individual CDR or a combination of CDR1, CDR2 and/or CDR3 of the light chain
variable region. The CDR3 may more particularly be selected. Combination may
include for example, CDRL1 and CDRL3; CDRL1 and CDRL2; CDRL2 and CDRL3
and; CDRL1, CDRL2 and CDRL3.
In another exemplary embodiment, the antibody or antigen g fragment may
comprise any dual CDR or a ation of CDR1, CDR2 and/or CDR3 of the
heavy chain variable region. The CDR3 may more ularly be selected.
ation may include for example, CDRH1 and CDRH3; CDRH1 and CDRH2;
CDRH2 and CDRH3 and; CDRH1, CDRH2 and CDRH3.
In accordance with the present invention, the antibody or antigen binding fragment
may comprise at least two CDRs of a CDRL1, a CDRL2 or a CDRL3.
Also in accordance with the present invention, the antibody or antigen binding
fragment may comprise one CDRL1, one CDRL2 and one CDRL3.
Further in accordance with the present invention, the antibody or n binding
nt may comprise:
a. At least two CDRs of a CDRL1, CDRL2 or CDRL3 and;
b. At least two CDRs of a CDRH1, one CDRH2 or one CDRH3.
The antibody or n binding fragment may more preferably comprise one CDRL1,
one CDRL2 and one CDRL3.
The dy or antigen g fragment may also more preferably comprise one
CDRH1, one CDRH2 and one CDRH3.
In accordance witht the present invention, the antibody or antigen binding fragment
may comprise one CDRH1, one CDRH2 or one CDRH3.
In accordance witht the t invention, the antibody or antigen binding fragment
may also comprise one CDRH1, one CDRH2 and one CDRH3.
When only one of the light chain variable region or the heavy chain variable region is
available, an antibody or antigen-binding fragment may be reconstituted by screening
a library of complementary variable regions using methods known in the art
(Portolano et al. The Journal of Immunology (1993) 150:880-887, Clarkson et al.,
Nature (1991) 352:624-628).
Also encompassed by the present invention are polypeptides or antibodies
comprising variable chains having at least one conservative amino acid substitution in
at least one of the CDRs bed herein (in comparison with the original CDR).
The present invention also encompasses polypeptides or antibodies sing
le chains having at least one conservative amino acid substitution in at least
two of the CDRs (in comparison with the original CDRs).
The present invention also encompasses polypeptides or antibodies comprising
le chains having at least one conservative amino acid substitution in the 3
CDRs (in ison with the original CDRs).
The present invention also encompasses polypeptides or antibodies comprising
variable chains having at least two conservative amino acid substitutions in at least
one of the CDRs (in comparison with the original CDRs).
The present invention also encompasses polypeptides or dies comprising
le chains having at least two conservative amino acid substitutions in at least
two of the CDRs (in comparison with the original CDRs).
The present invention also encompasses polypeptides or antibodies comprising
variable chains having at least two conservative amino acid substitutions in the 3
CDRs (in ison with the original CDRs).
In another aspect, the present invention relates to a polypeptide, antibody or antigen
binding fragment comprising (on a single polypeptide chain or on separate
ptide chains) at least one cornplementarity-determining region of a light chain
variable region and at least one complementarity—determining region of a heavy chain
variable region of one of the antibodies or antigen binding fragment bed .
The present ion relates in another aspect thereof to anti-KAAG1 antibodies that
may comprise (on a single polypeptide chain or on separate polypeptide chains) all
six complementarity—determining regions (CDRs) of the antibody or antigen binding
fragment described herein.
Variant antibody and antigen binding fragments
The present invention also encompasses variants of the antibodies or antigen binding
fragments described herein. t antibodies or antigen binding fragments included
are those having a ion in the amino acid sequence. For example, variant
antibodies or n binding nts included are those having at least one variant
CDR (two, three, four, five or six variant CDRs or even twelve variant CDRs), a
variant light chain variable region, a variant heavy chain variable region, a variant light
chain and/or a t heavy chain. Variant antibodies or antigen binding fragments
ed in the present invention are those having, for example, similar or improved
binding affinity in comparison with the original antibody or antigen binding fragment.
As used herein the term “variant” s to any of the ce described herein
and includes for example, a variant CDR (either CDRL1, CDRLZ, CDRL3, CDRH1,
CDRH2 and/or CDRH3), a variant light chain variable region, a t heavy chain
variable region, a variant light chain, a variant heavy chain, a t antibody, a
variant antigen binding fragment and a KAAG1 variant.
Variant antibodies or n binding fragments encompassed by the present
invention are those which may comprise an insertion, a deletion or an amino acid
substitution (conservative or non—conservative). These variants may have at least one
amino acid residue in its amino acid sequence removed and a different residue
inserted in its place.
The antibody or antigen g fragment of the present invention may have a light
chain variable region and/or heavy chain variable region as described above and
may further comprise amino acids of a constant region, such as, for example, amino
acids of a constant region of a human antibody.
In an exemplary embodiment, the antibody or antigen binding fragment of the present
invention may comprise, for example, a human lgG1 constant region.
In accordance with r exemplary embodiment of the invention, the antigen
binding fragment may be, for e, a scFv, a Fab, a Fab' or a (Fab');
A site of interest for substitutional mutagenesis includes the hypervariable regions
(CDRs), but cations in the framework region or even in the constant region are
also contemplated. Conservative substitutions may be made by exchanging an amino
acid (of a CDR, variable chain, antibody, etc.) from one of the groups listed below
(group 1 to 6) for another amino acid of the same group.
Other exemplary embodiments of conservative substitutions are shown in Table 1A
under the heading of “preferred substitutions“. If such tutions result in a
undesired property, then more substantial changes, denominated "exemplary
substitutions" in Table 1A, or as further bed below in reference to amino acid
classes, may be introduced and the products screened.
It is known in the art that variants may be generated by substitutional mutagenesis
and retain the biological activity of the polypeptides of the present invention. These
variants have at least one amino acid residue in the amino acid ce removed
and a different residue inserted in its place. For example, one site of interest for
substitutional mutagenesis may include a site in which particular residues obtained
from various species are identical. Examples of substitutions identified as
“conservative substitutions” are shown in Table 1A. If such substitutions result in a
change not d, then other type of substitutions, denominated “exemplary
tutions" in Table 1A, or as further described herein in reference to amino acid
classes, are introduced and the products screened.
Substantial modifications in function or immunological identity are accomplished by
selecting tutions that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the substitution, for e, as
a sheet or helical conformation. (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side chain properties:
(group 1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),
Valine (Val), e (Leu), lsoieucine (lle)
(group 2) neutral hydrophilic: ne (Cys), Serine (Ser), Threonine
(Thr)
(group 3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)
(group 4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),
Lysine (Lys), ne (Arg)
(group 5) residues that influence chain orientation: Glycine (Gly), Proline
(Pro); and
(group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)
Non-conservative substitutions will entail exchanging a member of one of these
classes for another.
Table 1A. Amino acid substitution
Original residue 7 Exemplary substitution Conservative substitution
Ala (A) lVal, Leu, lle Val
Arg (R) ‘ Lys, Gln, Asn Lys
Asn (N) lGln, His, Lys, Arg, Asp Gln
IEys (C)Asp (D) , AlaGlu, Asn Glu
JSer
Original residue Exemplary substitution Conservative substitution
lle(| Leu, Val, Met, Ala, Phe,
norieucine
Norleucine, lle, Val, Met, lle
Ala, Phe
Arg, Gln, Asn Arg
Leu, Phe, lle Leu
Leu, Val, lle, Ala, Tyr Tyr
Ser (8) Thr
Thr (T) Ser
Trp (W) Tyr, Phe Tyr
-norieucineVal (V) lle, Leu, Met, Phe, Ala,
Variant antibody or antigen binding fragment may have substantial sequence
similarity and/or ce ty in its amino acid ce in comparison with that
the original antibody or antigen binding fragment amino acid sequence. The degree of
similarity between two ces is based upon the percentage of identities (identical
amino acids) and of conservative substitution.
Generally, the degree of similarity and ty between variable chains has been
determined herein using the Blast2 ce program (Tatiana A. Tatusova, Thomas
L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and
nucleotide sequences", FEMS Microbiol Lett. 174:247-250) using default settings, i.e.,
blastp program, BLOSUMBZ matrix (open gap 11 and extension gap penalty 1; gapx
dropoff 50, expect 10.0, word size 3) and activated filters.
Percent identity will therefore be indicative of amino acids which are identical in
ison with the original peptide and which may occupy the same or similar
position.
Percent similarity will be indicative of amino acids which are identical and those which
are replaced with conservative amino acid substitution in comparison with the original
peptide at the same or similar position.
Variants of the present invention therefore comprise those which may have at least
70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an
original sequence or a portion of an original sequence.
Exemplary embodiments of variants are those having at least 81% sequence identity
to a sequence described herein and 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ce
similarity with an original sequence or a portion of an original sequence.
Other exemplary embodiments of variants are those having at least 82% sequence
identity to a sequence described herein and 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
similarity with an al ce or a portion of an original sequence.
r exemplary embodiments of variants are those having at least 85% sequence
identity to a sequence described herein and 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an
al sequence or a portion of an original sequence.
Other exemplary embodiments of variants are those having at least 90%
sequence
identity to a sequence bed herein and 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of
an original sequence.
Additional ary embodiments of ts are those having at least 95%
sequence ty to a sequence described herein and 95%, 96%, 97%, 98%, 99% or
100% sequence similarity with an original sequence or a portion of an original
sequence.
Yet additional exemplary embodiments of variants are those having at least 97%
sequence identity to a sequence described herein and 97%, 98%, 99% or 100%
sequence similarity with an original sequence or a n of an original sequence.
For a e of concision the applicant provides herein a Table IB illustrating
exemplary embodiments of individual variants encompassed by the present invention
and comprising the ied % ce identity and % sequence rity. Each
“X” is to be construed as defining a given variant.
Table 1 B
t (%) sequence identity
-m--m-----mmmmmmmmm
X X --------------
--------------—------
--------------------
g 84 X ---------------
E X X X - - -
a X X X IX - II.-
3 x x x x x
g x x x x x x x
3 x x x x x x x x -
3 x x x x x x x x x x
§ x x x x x x x x x x x '--
L; x x x x x x x x x x x
8 x x x x x x x x x x x I
a T—
x x x x x x x x x x x x a
x x x x x x x x x x x x x
x x x x x x x x x x x x x x x x
x x x x x x x x x x x x x x x x
x x x x x x x x x x x x x x x |x x x x
J x \x g x x x x x x X x x x x x x x x x
x x x x x x x x x x x x x x x x x x x x x
The present invention encompasses CDRs, light chain variable regions, heavy chain
variable regions, light , heavy chains, antibodies and/or antigen binding
fragments which comprise at least 80% identity with the sequence described .
Exemplary embodiments of the antibody or antigen binding fragment of the present
invention are those comprising a light chain variable region comprising a sequence at
least 70%, 75%, 80% identical to SEQ ID NO.:4.
These light chain variable region may comprise a CDRL1 sequence at least 80 %
identical to SEQ ID NO.:8, a CDRL2 sequence at least 80 % identical to SEQ ID
NO.:9 and a CDRL3 sequence at least 80 % identical to SEQ ID NO.:10.
In an exemplary ment of the present invention, any of the antibodies provided
herein may comprise a CDRL1 sequence which may be at least 90 % identical to
SEQ ID NO.:8.
In another exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRL1 sequence which may be 100% cal to
SEQ ID NO.:8.
In another exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRL2 ce at least 90 % cal to SEQ ID
NO.:9.
In yet another exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRL2 sequence which may be 100% identical to
SEQ ID NO.:9.
In another exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRL3 sequence which may be at least 90 %
identical to SEQ ID NO.:10.
In an additional exemplary ment of the present invention, any of the antibodies
provided herein may comprise a CDRL3 sequence which may be 100% identical to
SEQ ID NO.:10.
In an exemplary ment, the antibody or n binding fragment may comprise
a heavy chain variable region comprising a ce at least 70%, 75%, 80%
identical to SEQ ID NO.:2.
These heavy chain variable regions may comprise a CDRH1 sequence at least 80 %
cal to SEQ ID NO.:5, a CDRH2 sequence at least 80 % identical to SEQ ID
NO.:6 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.:7.
In an exemplary embodiment of the present invention, any of the antibodies provided
herein may comprise a CDRH1 sequence which may be at least 90 % identical to
SEQ ID N05.
In another exemplary embodiment of the present invention, any of the dies
provided herein may comprise a CDRH1 sequence which may be 100% identical to
SEQ ID NO.:5.
In yet another exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRH2 sequence which may be at least 90 %
identical to SEQ ID N056.
In a further exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRH2 sequence which may be 100% identical to
SEQ ID NO.:6.
In yet a further exemplary embodiment of the t invention, any of the antibodies
provided herein may comprise a CDRH3 sequence which may be at least 90 %
identical to SEQ ID NO.:7.
In an onal exemplary embodiment of the present invention, any of the antibodies
provided herein may comprise a CDRH3 sequence which may be 100% identical to
SEQ ID NO.:7.
In some ces, the variant antibody heavy chain variable region may comprise
amino acid deletions or additions (in combination or not with amino acid
substitutions). Often 1, 2, 3, 4 or 5 amino acid deletions or additions may be tolerated.
Exemplary ments of variant antibody or antigen g fragments include
those having a light chain le region as set forth in SEQ ID NO.:30:
SEQ ID NO.:30
DXVMTQTPLSLXVXXGXXASlSCRSSQSLLHSNGNTYLEWYLQKPGQSPXLLIHTVS
NRFSGVPDRFSGSGSGTDFTLKISRVEAEDXGWYCFQGSHVPLTFGXGTXLEXK,
wherein at least one of the amino acids identified by X is an amino acid substitution
(conservative or non-conservative) in ison with a corresponding amino acid in
the polypeptide set forth in SEQ ID NO.:4. The amino acid substitution may be, for
example, an amino acid found at a corresponding position of a natural human
antibody or a human antibody consensus. The amino acid substitution may be, for
example conservative.
Another ary embodiment of a variant antibody or antigen binding fragment
include those having a light chain variable region as set forth in SEQ ID NO.:31:
SEQ ID NO.:31
DXa1VMTQTPLSLXa2VX33Xa4GXa5XasASISCRSSQSLLHSNGNTYLEWYLQKPGQSP
Xa7LLlHTVSNRFSGVPDRFSGSGSGTDFTLKlSRVEAEDXasGWYCFQGSHVPLTF
GXaeGTXaioLEXanK,
Wherein X31 may be a hydrophobic amino acid;
Wherein X32 may be A or P;
Wherein X33 may be neutral hilic amino acid;
Wherein X34 may be L or P;
n X35 may be an acidic amino acid;
Wherein Xaa may be Q or P;
Wherein X37 may be a basic amino acid;
Wherein X38 may be a hydrophobic amino acid;
Wherein Xag may be A or Q;
Wherein X310 may be a basic amino acid; or
Wherein X311 may be a hydrophobic amino acid,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a ponding amino acid in
the polypeptide set forth in SEQ ID NO.:4.
An additional exemplary embodiment of a variant antibody or antigen binding
fragment include those having a light chain le region as set forth in SEQ ID
NO.:32:
SEQ ID NO.:32
DXA1VMTQTPLSLXAZVXA3XA4GXA5XA5ASISCRSSQSLLHSNGNTYLEWYLQKPGQSP
XA7LLIHTVSNRFSGVPDRFSGSGSGTDFTLKlSRVEAEDXAaGWYCFQGSHVPLTF
GXAQGTXMOLEXAHK
Wherein XA1 may be V or |
Wherein XAZ may be A or P
Wherein XA3 may be S or T
Wherein XA4 may be L or P
Wherein XA5 may be D or E
Wherein XAe may be Q or P
n XA7 may be K or Q
Wherein XAB may be L or V
Wherein XAg may be A or Q
Wherein Xmo may be R or K or
Wherein XA11 may be L or I,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in
the polypeptide set forth in SEQ ID NO.:4.
in accordance with an embodiment, the light chain variable domain variant may have
a sequence as set forth in SEQ lD NO.:33 or 34:
SEQ ID NO.:33
TPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLlYTVS
NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGWYCFQGSHVPLTFGQGTKLEIK.
SEQ ID NO.:34
DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVS
NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGWYCFQGSHVPLTFGQGTKLElK.
Exemplary ments of variant antibody or antigen binding fragments include
those having a heavy chain variable region as set forth in SEQ ID NO.:35.
SEQ ID NO.:35
QXQLVQSGXEXXKPGASVKXSCKASGYTFTDDYMSWVXQXXGXXLEWXGDINPY
NGDTNYNQKFKGXXXXTXDXSXSTAYMXLXSLXSEDXAWYCARDPGAMDYWGQ
GTXVTVSS,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or nservative) in comparison with a corresponding amino acid in
the polypeptide set forth in SEQ lD N02. The amino acid substitution may be, for
example, an amino acid found at a corresponding position of a l human
antibody or a human antibody consensus. The amino acid substitution may be, for
example conservative.
Another exemplary embodiment of a variant antibody or antigen binding fragment
include those having a heavy chain variable region as set forth in SEQ ID NO.:36:
SEQ ID NO.:36
QXmQLVQSGngEngxmKPGASVKXb5$CKASGYTFTDDYMSWVszQXwngGngXM
oLEWXm1GDINPYNGDTNYNQKFKGXMZXM3Xb14Xb15TXb15DXb1ysXmSTAYMXmgLsz
OSLXb21SEDszzAWYCARDPGAMDYWGQGTXb23VTVSS,
Wherein Xb1 may be a hydrophobic amino acid;
Wherein sz may be P or A;
Wherein Xb3 may be a hydrophobic amino acid;
n Xb4 may be V or K;
Wherein sz may be a hydrophobic amino acid;
Wherein sz may be a basic amino acid;
Wherein be may be S or A;
Wherein Xba may be H or P;
Wherein ng may be a basic amino acid;
Wherein Xbm may be S or G;
n XW may be a hydrophobic amino acid;
Wherein Xb12 may be a basic amino acid;
Wherein Xm may be a hydrophobic amino acid;
n Xb14 may be I or T;
n Xb15 may be a hydrophobic amino acid;
Wherein Xms may be a hydrophobic amino acid;
Wherein an may be K or T;
Wherein Xm may be a neutral hydrophiiic amino acid;
Wherein Xb19 may be Q or E;
Wherein szo may be N or S;
Wherein Xb21 may be T or R;
Wherein szz may be a neutral hydrophiiic amino acid; or
Wherein Xb23 may be S or L,
wherein at least one of the amino acid identified by X is an amino acid tution
(conservative or non-conservative) in comparison with a corresponding amino acid in
the polypeptide set forth in SEQ ID NO.:2.
An additional exemplary embodiment of a variant antibody or antigen binding
fragment include those having a heavy chain variable region as set forth in SEQ ID
NO.:37:
SEQ ID NO.:37
QXB1QLVQSGXBzEX33XB4KPGASVKXBssCKASGYTFTDDYMSWVXBGQXmXBaGXBgX
B10LEWXB11GDlNPYNGDTNYNQKFKGXB12X313XB14XB15TXB1BDXB17SXB188TAYMXB19
LXBZOSLXBmSEDXBZZAVYYCARDPGAMDYWGQGTX523VTVSS
Wherein X31 may be I or V;
Wherein X32 may be P or A;
Wherein X33 may be M or V;
Wherein X34 may be V or K;
Wherein X35 may be M or V;
Wherein X36 may be K or R;
Wherein X37 may be S or A;
Wherein X38 may be H or P;
Wherein X39 may be K or Q;
Wherein X510 may be S or G;
Wherein X311 may be I or M;
Wherein X312 may be K or R;
Wherein X313 may be A or V;
n X314 may be I or T;
Wherein X315 may be L or I;
Wherein X316 may be V or A;
n X317 may be K or T;
Wherein X315 may be S or T;
Wherein X319 may be Q or E;
n X320 may be N or S;
Wherein X321 may be T or R;
Wherein X522 may be S or T; or
Wherein X323 is S or L,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in
the polypeptide set forth in SEQ ID NO.:2.
In accordance with an embodiment, the heavy chain variable domain variant may
have a sequence as set forth in any one of SEQ ID No.38 to 41:
SEQ ID NO.:38
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGD| N PY
NGDTNYNQKFKGRVTITADTSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQG
TLVTVSS.
SEQ ID NO.:39
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPY
NGDTNYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQG
TLVTVSS.
SEQ ID NO.:40
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYN
GDTNYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQG
TLVTVSS.
SEQ ID NO. :41
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYN
GDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGT
LVTVSS.
Production of the antibodies in cells
The AAG1 antibodies that are disclosed herein can be made by a variety of
methods familiar to those skilled in the art, such as hybridoma methodology or by
recombinant DNA methods.
In an exemplary embodiment of the invention, an anti—KAAG1 antibodies (e.g., an
antibody which can compete with the antibodies disclosed herewith) may be
produced by the conventional hybridoma technology, where a mouse is immunized
with an antigen, spleen cells ed and fused with myeloma cells lacking HGPRT
expression and hybrid cells ed by hypoxanthine, aminopterin and thymine
(HAT) containing media.
In an additional exemplary embodiment of the invention, the anti-KAAG1 dies
may be produced by inant DNA methods.
in order to express the anti-KAAG1 dies, nucleotide sequences able to encode
any one of a light and heavy immunoglobulin chains described herein or any other
may be inserted into an expression vector, Le, a vector that contains the elements for
transcriptional and ational control of the ed coding sequence in a particular
host. These elements may include regulatory ces, such as enhancers,
constitutive and inducible promoters, and 5' and 3' un-translated regions. Methods
that are well known to those skilled in the art may be used to construct such
expression vectors. These methods include in vitro recombinant DNA techniques,
synthetic ques, and in vivo genetic recombination.
A y of expression vector/host cell systems known to those of skill in the art may
be utilized to express a polypeptide or RNA derived from nucleotide sequences able
to encode any one of a light and heavy immunoglobulin chains described .
These include, but are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression s; yeast
transformed with yeast expression vectors; insect cell systems infected with
baculovirus vectors; plant cell systems transformed with viral or bacterial sion
s; or animal cell systems. For long-term production of recombinant proteins in
mammalian systems, stable expression in cell lines may be effected. For example,
nucleotide sequences able to encode any one of a light and heavy immunoglobulin
chains described herein may be transformed into cell lines using expression vectors
that may contain viral origins of replication and/or endogenous expression ts
and a selectable or visible marker gene on the same or on a separate vector. The
invention is not to be limited by the vector or host cell employed. In n
embodiments of the present invention, the nucleotide sequences able to encode
one of a light and heavy immunoglobulin chains described herein may each be ligated
into a separate expression vector and each chain expressed separately. In another
embodiment, both the light and heavy chains able to encode any one of a light and
heavy immunoglobulin chains described herein may be ligated into a single
expression vector and sed simultaneously.
Alternatively, RNA and/or polypeptide may be expressed from a vector comprising
nucleotide sequences able to encode any one of a light and heavy immunoglobulin
chains described herein using an in vitro transcription system or a coupled in vitro
transcription/translation system respectively.
of a
in general, host cells that contain nucleotide sequences able to encode any one
light and heavy immunoglobulin chains described herein and/or that express a
polypeptide encoded by the nucleotide ces able to encode any one of a light
and heavy immunoglobulin chains described herein, or a portion thereof, may be
identified by a variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA/DNA or DNA/RNA hybridizations,
PCR amplification, and protein bioassay or immunoassay techniques that include
membrane, solution, or chip based technologies for the detection and/or quantification
of nucleic acid or amino acid sequences. Immunological methods for detecting and
measuring the expression of polypeptides using either specific polyclonal or
monoclonal antibodies are known in the art. Examples of such techniques include
enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RlAs), and
fluorescence ted cell sorting (FACS). Those of skill in the art may readily adapt
these methodologies to the present invention.
Host cells comprising nucleotide sequences able to encode any one of a light and
heavy immunoglobulin chains described herein may thus be cultured under conditions
for the transcription of the corresponding RNA (mRNA, etc.) and/or the expression of
the polypeptide from cell e. The polypeptide produced by a cell may be secreted
or may be ed intracellularly depending on the ce and/or the vector used.
In an exemplary embodiment, expression s ning tide sequences
able to encode any one of a light and heavy immunoglobulin chains described herein
secretion of the polypeptide
may be designed to contain signal ces that direct
through a prokaryotic or otic cell membrane.
Due to the inherent degeneracy of the genetic code, other DNA sequences that
encode the same, substantially the same or a onally equivalent amino acid
sequence may be produced and used, for e, to express a polypeptide
encoded by nucleotide sequences able to encode any one of a light and heavy
immunoglobulin chains described herein. The nucleotide ces of the present
invention may be engineered using methods generally known in the art in order to
alter the tide sequences for a variety of purposes including, but not limited to,
modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide ces. For
example, oligonucleotide-mediated site-directed mutagenesis may be used to
introduce mutations that create new restriction sites, alter glycosylation patterns,
change codon preference, produce splice variants, and so forth.
In addition, a host cell strain may be chosen for its ability to te expression of
the inserted ces or to process the expressed polypeptide in the desired
fashion. Such modifications of the polypeptide include, but are not d to,
acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. In
an exemplary embodiment, anti-KAAG1 antibodies that contain particular
glycosylation ures or patterns may be desired. Post-translational processing,
which cleaves a “prepro” form of the polypeptide, may also be used to specify protein
targeting, folding, and/or activity. Different host cells that have specific cellular
machinery and characteristic mechanisms for ranslational activities (e.g., CHO,
HeLa, MDCK, HEK293, and W138) are available commercially and from the
American Type e Collection (ATCC) and may be chosen to ensure the correct
modification and processing of the expressed polypeptide.
Those of skill in the art wi|l readily appreciate that natural, modified, or recombinant
nucleic acid sequences may be ligated to a heterologous sequence resulting in
translation of a fusion polypeptide containing heterologous polypeptide moieties in
any of the entioned host s. Such heterologous polypeptide moieties
may facilitate purification of fusion polypeptides using commercially available affinity
es. Such moieties include, but are not limited to, glutathione S-transferase
(GST), e g protein, thioredoxin, calmodulin binding peptide, 6-His (His),
FLAG, c-myc, utinin (HA), and antibody epitopes such as monoclonal antibody
epitopes.
In yet a further aspect, the present invention relates to a cleotide which may
comprise a nucleotide sequence encoding a fusion protein. The fusion protein may
comprise a fusion partner (e.g., HA, Fc, etc.) fused to the polypeptide (e.g., complete
light chain, complete heavy chain, variable regions, CDRs etc.) described .
Those of skill in the art will also y recognize that the nucleic acid and
polypeptide sequences may be synthesized, in whole or in part, using chemical or
enzymatic methods well known in the art. For example, peptide synthesis may be
performed using various solid-phase techniques and machines such as the ABI 431A
Peptide synthesizer (PE Biosystems) may be used to automate synthesis. If desired,
the amino acid sequence may be altered during sis and/or combined with
sequences from other proteins to produce a variant protein.
Antibody coniugates
The antibody or antigen binding fragment of the t invention may be conjugated
with a detectable moiety (i.e., for ion or diagnostic purposes) or with a
therapeutic moiety (for therapeutic purposes).
A table moiety" is a moiety detectable by spectroscopic, photochemical,
mical, immunochemical, al and/or other physical means. A detectable
moiety may be d either directly and/or indirectly (for example via a linkage,
such as, without limitation, a DOTA or NHS linkage) to antibodies and antigen binding
nts thereof of the present invention using methods well known in the art. A
wide variety of detectable moieties may be used, with the choice depending on the
sensitivity required, ease of conjugation, stability requirements and available
instrumentation. A suitable detectable moiety include, but is not limited to, a
fluorescent label, a radioactive label (for example, t limitation, 125l, Inm, T099,
l131 and including positron emitting isotopes for PET scanner etc), a nuclear magnetic
resonance active label, a luminiscent label, a uminescent label, a chromophore
label, an enzyme label (for example and without limitation horseradish peroxidase,
alkaline phosphatase, etc.), quantum dots and/or a nanoparticle. Detectable moiety
may cause and/or produce a detectable signal thereby ng for a signal from the
detectable moiety to be detected.
In r ary embodiment of the invention, the antibody or antigen binding
nt thereof may be coupled (modified) with a therapeutic moiety (e.g., drug,
cytotoxic moiety, anti-cancer agent).
In an exemplary embodiment, the anti-KAAG1 antibodies and antigen binding
fragments may comprise a chemotherapeutic, a cytotoxic agent or an anti-cancer
drug (e.g., small molecule). Such chemotherapeutic or cytotoxic agents include, but
are not limited to, Yttrium-90, Scandium-47, m-186, Iodine-131, Iodine-125,
and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lum),
bismuth (e.g., Bi213), copper (e.g., Cu67)). In other instances, the chemotherapeutic,
cytotoxic agent or anti-cancer drug may be comprised of, among others known to
those skilled in the art, 5-fluorouracil, adriamycin, irinotecan, taxanes, pseudomonas
endotoxin, ricin, auristatins (e.g., monomethyl auristatin E, monomethyl auristatin F),
maytansinoids (e.g., mertansine) and other toxins.
Alternatively, in order to carry out the methods of the present invention and as known
in the art, the antibody or antigen binding fragment of the present invention
(conjugated or not) may be used in ation with a second molecule (e.g., a
secondary antibody, etc.) which is able to specifically bind to the antibody or antigen
binding fragment of the present invention and which may carry a desirable detectable,
diagnostic or therapeutic .
Pharmaceutical compositions of the antibodies and their use
Pharmaceutical compositions of the anti-KAAG1 antibodies or antigen binding
fragments (conjugated or not) are also encompassed by the present invention. The
pharmaceutical ition may comprise an anti-KAAG1 antibody or an antigen
binding nt and may also contain a pharmaceutically acceptable carrier.
Other s of the invention relate to a ition which may comprise the
antibody or antigen binding fragment described herein and a carrier.
The present invention also relates to a pharmaceutical composition which may
comprise the antibody or antigen g fragment described herein and a
pharmaceutically acceptable carrier.
In addition to the active ingredients, a pharmaceutical composition may contain
pharmaceutically able carriers comprising water, PBS, salt solutions, gelatins,
oils, alcohols, and other excipients and auxiliaries that facilitate processing of the
active compounds into preparations that may be used pharmaceutically. In other
ces, such preparations may be sterilized.
As used herein, "pharmaceutical composition" means therapeutically effective
amounts of the agent er with pharmaceutically able diluents,
preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically
ive amount" as used herein refers to that amount which es a therapeutic
effect for a given condition and administration n. Such compositions
are liquids
or lized or otherwise dried formulations and include diluents of various buffer
content (e.g., Tris-HCI., acetate, phosphate), pH and ionic strength, additives such
albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20,
Tween 80, Pluronic F68, bile acid . Solubilizing agents (e.g., glycerol,
hylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or
tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as
polyethylene glycol to the n, xation with metal ions, or incorporation of
the material into or onto particulate preparations of ric compounds such as
ctic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions,
micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
Such compositions will influence the physical state, solubility, stability, rate of in vivo
release, and rate of in vivo clearance. Controlled or sustained release compositions
include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also
comprehended by the invention are particulate compositions coated with polymers
(e.g., poloxamers or poloxamines). Other embodiments of the compositions of the
invention incorporate particulate forms protective gs, protease inhibitors or
tion enhancers for various routes of administration, including parenteral,
pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical
composition is administered parenterally, paracancerally, transmucosally,
transdermally, intramuscularly, intravenously, intradermally, subcutaneously,
intraperitonealy, intraventricularly, intracranially and intratumorally.
Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical
carrier" are known in the art and include, but are not limited to, 0.01-O.1 M or 0.05 M
phosphate buffer or 0.8 % saline. Additionally, such pharmaceutically acceptable
carriers may be aqueous or non—aqueous solutions, suspensions, and emulsions.
Examples of ueous solvents are propylene , polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters such as ethyl .
Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, ing saline and buffered media. Parenteral es include sodium
chloride on, Ringer's dextrose, dextrose and sodium de, lactated Ringer's
d oils. intravenous es include fluid and nutrient replenishers, electrolyte
replenishers such as those based on Ringer's dextrose, and the like. Preservatives
and other additives may also be present, such as, for example, antimicrobials,
antioxidants, collating agents, inert gases and the like.
For any compound, the therapeutically effective dose may be estimated initially either
in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs.
An animal model may also be used to determine the concentration range and route of
administration. Such information may then be used to determine useful doses and
routes for administration in humans. These techniques are well known to one skilled
in the art and a therapeutically effective dose refers to that amount of active
ingredient that ameliorates the symptoms or condition. Therapeutic cy and
toxicity may be determined by standard pharmaceutical procedures in cell cultures or
with experimental animals, such as by calculating and contrasting the E050 (the dose
therapeutically effective in 50% of the population) and LD5o (the dose lethal to 50% of
the population) statistics. Any of the eutic compositions described above may
be applied to any subject in need of such therapy, including, but not limited to,
mammals such as dogs, cats, cows, horses, rabbits, monkeys, and humans.
The pharmaceutical compositions utilized in this invention may be stered by
any number of routes including, but not limited to, oral, intravenous, uscular,
intra—arterial, edullary, intrathecal, entricular, transdermal, aneous,
intraperitoneal, intranasal, l, topical, sublingual, or rectal means.
The term "treatment" for purposes of this disclosure refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the object is slow down
(lessen) the targeted pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to have the disorder or
those in whom the disorder is to be prevented. Particularly, subjects in need include
subjects with an elevated level of one or more cancer markers.
The anti-KAAG1 antibodies and antigen g fragments thereof may have
eutic uses in the treatment of various cancer types, such as ovarian cancer,
renal cancer, colon cancer, lung cancer, melanoma, etc. In an ary
embodiment, the antibodies and fragments have therapeutic uses in ovarian cancer.
In a more particular embodiment the subject may have, for example, a recurrent
ovarian cancer. in yet another embodiment, the subject may have, for example, a
metastatic cancer.
in certain ces, the AAG1 antibodies and fragments may block the
interaction of KAAG1 with its protein partners. The anti-KAAG1 antibodies of the
present invention may particularly be used to deliver a eutic moiety to a cell
expressing KAAGt.
The anti-KAAGt dies and antigen binding fragments thereof may have
therapeutic uses in the treatment of various types of ovarian cancer. Several different
cell types may give rise to ent ovarian cancer histotypes. The most common
form of ovarian cancer is comprised of tumors that originate in the epithelial cell layer
of the ovary or the fallopian tube. Such epithelial ovarian cancers include serous
tumors, endometroid tumors, mucinous tumors, clear cell tumors, and borderline
tumors. In other embodiments, the anti-KAAG1 antibodies and antigen binding
fragments thereof have uses in the ent of other types of ovarian cancer such as
germ line and sex cord ovarian cancer.
In n instances, the AAG1 antibodies and n binding nts
thereof may be administered concurrently in combination with other treatments given
for the same condition. As such, the antibodies may be administered with anti-mitotics
(eg., taxanes), platinum-based agents (eg., cisplatin), DNA damaging agents (eg.
Doxorubicin) and other anti-cancer therapies that are known to those skilled in the art.
In other instances, the anti-KAAG1 antibodies and antigen binding fragments thereof
may be administered with other therapeutic antibodies. These include, but are not
limited to, antibodies that target EGFR, CD—20, and Her2.
The present invention relates in a further aspect thereof to a method for inhibiting the
growth of a KAAG1-expressing cell, the method which may comprise contacting the
cell with an effective amount of the antibody or antigen binding fragment described
herein.
The present invention also asses method of ng cancer or inhibiting the
growth of a KAAG1 expressing cells in a mammal, the method may comprise
administering the antibody or antigen binding fragment, for example, conjugated with
a eutic moiety described herein to a subject in need.
In further aspects, the present invention provides method of treatment, stic
methods and method of detection using the antibody or antigen binding fragment of
the present invention and the use of these dies or antigen binding fragment in
the manufacture of a pharmaceutical composition or drug for such es.
The invention therefore relates to the use of the ed antibody or antigen binding
fragment described herein in the (manufacture of a pharmaceutical ition for)
treatment of cancer.
The antibody or antigen g fragment may more particularly be applicable for
malignant tumors including, for example, a malignant tumor having the ability to
metastasize and/or tumor cells characterized by age-independent growth.
The antibody or antigen binding fragment of the present invention may also be used
in the diagnosis of cancer. The sis of cancer may be performed in vivo by
administering the antibody or antigen binding fragment of the present invention to a
mammal having or suspected of having a cancer. The diagnosis may also be
performed ex vivo by contacting a sample obtained from the mammal with the
antibody or antigen binding fragment and determining the presence or absence of
cells (tumor cells) expressing KAAGl or a KAAG1 variant.
The present invention ore also encompasses method of detecting cancer or
ing a KAAGl expressing cells in a , the method may comprise
administering the antibody or antigen binding nt described herein to a subject
in need.
The present invention relates in another aspect thereof to a method for detecting a
cell sing KAAG1 or a KAAG1 variant, the method may comprise contacting the
cell with an antibody or antigen g fragment described herein and detecting a
complex formed by the antibody and the or KAAG1 variant-expressing cell.
Exemplary embodiments of antibodies or antigen binding fragments used in detection
methods are those which are capable of binding to the ellular region of KAAG1.
Other exemplary embodiments of antibodies or antigen binding fragments used in
detection methods are those which bind to KAAG1 or KAAG1 variant expressed at
the surface of a tumor cells.
Subject in need which would benefit from treatment, ion or diagnostic methods
described herein are those which have or are suspected of having cancer, e.g.,
ovarian cancer (e.g., serous, endometroid, clear cell or mucinous), skin cancer (e.g.,
mas, squamous cell carcinomas), renal cancer (e.g., papillary cell carcinomas,
clear cell carcinomas), colorectal cancer (e.g., colorectal carcinomas), sarcoma,
leukemia, brain tumor, thyroid tumor, breast cancer (e.g., mammary carcinomas),
prostate cancer (e.g., prostatic carcinomas), oesophageal tumor, bladder tumor, lung
tumor (e.g., lung carcinomas) or head and neck tumor and especially when the
cancer is characterized as being malignant and/or when the cells sing KAAG1
or a KAAG1 variant are characterized by anchorage-independent growth.
Subjects having cancer may be identified by imaging, tissue biopsy, genetic testing.
Alternatively, subjects having cancer may be identified by the presence of cancer
markers in their bodily fluids using standard assays (e.g., ELISA and the like).
Especially encompassed by the present invention are patients having or susceptible
of having ovarian cancer (e.g., serous, troid, clear cell or mucinous), skin
cancer (e.g., melanomas, squamous cell carcinomas) or renal cancer (e.g., papillary
cell carcinomas) and especially when the cancer is characterized as being malignant
and/or when the cells expressing KAAG1 or a KAAG1 variant are characterized by
anchorage-independent growth.
Another aspect of the invention s to a method for detecting KAAG1 (SEQ ID
NO.:29), a KAAG1 variant having at least 80% sequence identity with SEQ ID NO.:29
or a secreted form of circulating form of KAAG1 or KAAG1 variant, the method may
comprise ting a cell expressing KAAG1 or the KAAG1 variant or a sample
(biopsy, serum, plasma, urine etc.) comprising or suspected of comprising KAAG1 or
the KAAG1 variant with the dy or antigen binding fragments described herein
and measuring binding. The sample may originate from a mammal (e.g., a human)
which may have cancer (e.g., ovarian , a atic cancer) or may be
suspected of having cancer (e.g., ovarian , a metastatic cancer). The sample
may be a tissue sample obtained from the mammal or a cell culture supernatant.
in accordance with the invention the sample may be a serum sample, a plasma
sample, a blood sample, semen or ascitic fluid obtained from the mammal. The
antibody or antigen binding fragment described herein may advantageously detect a
secreted or circulating form (circulating in blood) of KAAG1.
The method may comprise fying the complex formed by the antibody or n
binding fragment bound to KAAG1 or to the KAAG1 variant.
The binding of an antibody to an antigen will cause an increase in the expected
molecular weight of the n. A physical change therefore occurs upon specific
binding of the dy or antigen binding fragment and the antigen.
Such changes may be detected using, for example, electrophoresis followed by
Western blot and coloration of the gel or blot, mass spectrometry, HPLC coupled with
a computer, FACS or else. Apparatus capable of computing a shift in molecular
weight are known in the art and include for example, PhosphorimagerTM.
When the antibody comprises for example a detectable label, the antigen-antibody
complex may be ed by the fluorescence emitted by the label, radiation emission
of the label, enzymatic activity of a label provided with its substrate or else.
ion and/or measurement of binding between an antibody or antigen binding
nt and an antigen may be performed by s methods known in the art.
Binding between an antibody or antigen binding fragment and an antigen may be
monitored with an apparatus capable of ing the signal emitted by the detectable
label (radiation emission, fluorescence, color change etc.). Such apparatus provides
data which indicates that binding as ed and may also provide indication as to
the amount of antibody bound to the n. The apparatus (usually coupled with a
computer) may also be capable of calculating the difference between a background
signal (e.g., signal ed in the absence of antigen-antibody binding) or
ound noise and the signal obtained upon specific antibody—antigen binding.
Such apparatuses may thus provide the user with indications and conclusions as to
whether the antigen has been detected or not.
Additional aspects of the invention relates to kits which may include one or more
ner containing one or more antibodies or antigen binding fragments bed
herein.
Nucleic acids, vectors and cells
Antibodies are usually made in cells allowing expression of the light chain and heavy
chain expressed from a vector(s) comprising a nucleic acid ce encoding the
light chain and/or heavy chain.
The present therefore encompasses nucleic acids capable of encoding any of the
CDRs, light chain variable regions, heavy chain variable regions, light chains, heavy
chains described herein.
The present invention therefore relates in a further aspect to a c acid encoding
a light chain variable region and/or a heavy chain variable region of an antibody which
is e of ic binding to KAAG1.
Exemplary embodiments of nucleic acids of the present invention include nucleic
acids encoding a light chain variable region comprising:
a. a CDRL1 as set forth in SEQ ID NO.:8 or comprising SEQ ID NO.:8;
b. a CDRL2 as set forth in SEQ ID NO.:9 or comprising SEQ ID NO.:9,
c. a CDRL3 sequence as set forth in SEQ ID NO.:10 or comprising SEQ
ID NO.:10.
In accordance with the present invention, the nucleic acid may encode a light chain
variable region which may se at least two CDRs of a CDRL1, a CDRL2 or a
CDRL3.
Also in accordance with the present invention, the nucleic acid may encode a light
chain le region which may comprise one CDRL1, one CDRL2 and one CDRL3.
The present invention also relates to a nucleic acid encoding a heavy chain variable
region comprising:
a. a CDRH1 sequence as set forth in SEQ ID NO.:5 or comprising SEQ
ID NO.:5;
b. a CDRH2 sequence as set forth in SEQ ID NO.:6 or comprising SEQ
ID NO.:6, or;
c. a CDRH3 sequence as set forth in SEQ ID NO.:7 or comprising SEQ
ID NO.:7.
In accordance with the present invention, the nucleic acid may encode a heavy chain
le region which may comprise at least two CDRs of a CDRH1, a CDRH2 or a
CDRH3.
In accordance with the present invention, the nucleic acid may encode a heavy chain
le region which may comprise one CDRH1, one CDRH2 and one CDRH3.
Also encompassed by the present invention are nucleic acids encoding antibody
variants having at least one conservative amino acid substitution.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least one conservative amino acid substitution.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least one conservative amino acid substitution in at least two of the
CDRs.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least one conservative amino acid substitution in the 3 CDRs.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least two conservative amino acid substitutions in at least one of the
CDRs.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least two conservative amino acid substitutions in at least two of the
CDRs.
In accordance with the present invention, the nucleic acid may encode a CDR
comprising at least two conservative amino acid substitutions in the 3 CDRs.
Other aspects of the invention relate to a nucleic acid encoding a light chain le
region having at least 70%. 75%, 80% sequence identity to SEQ ID NO.:4.
Yet other aspects of the invention relate to a nucleic acid encoding a heavy chain
variable region having at least 70%. 75%, 80% sequence identity to SEQ ID NO.:2.
In yet another aspect, the present ion relates to a vector comprising the nucleic
acids bed .
In accordance with the present invention, the vector may be an expression .
Vector that ns the elements for riptional and translational control of the
inserted coding sequence in a particular host are known in the art. These elements
may include regulatory sequences, such as enhancers, tutive and inducible
promoters, and 5' and 3' un-translated regions. Methods that are well known to those
skilled in the art may be used to construct such expression vectors. These methods
include in vitro inant DNA techniques, synthetic techniques, and in vivo
genetic recombination.
In r aspect the present invention relates to an isolated cell which may comprise
the nucleic acid, dies or antigen binding fragment described herein.
The ed cell may comprise a nucleic acid encoding a light chain le region
and a nucleic acid encoding a heavy chain variable region either on separate vectors
or on the same . The isolated cell may also comprise a nucleic acid encoding a
light chain and a nucleic acid encoding a heavy chain either on separate vectors or on
the same vector.
In accordance with the present invention, the cell may be capable of expressing,
assembling and/or secreting an dy or antigen g fragment thereof.
In another aspect, the present invention provides a cell which may comprise and/or
may express the antibody described herein.
In accordance with the invention, the cell may comprise a nucleic acid ng a
light chain variable region and a nucleic acid encoding a heavy chain variable region.
The cell may be capable of expressing, assembling and/or secreting an antibody or
antigen binding fragment thereof.
The examples below are presented to further outline details of the present invention.
EXAMPLES
Example 1
This example describes the binding of antibody 3A4 to KAAG1.
The antibodies that bind KAAG1 were generated using the Alere phage y
technology. A detailed ption of the technology and the methods for generating
these antibodies can be found in the US. Patent No. 6,057,098. In addition, a
detailed description of the generation of antibodies against KAAG1 can be found in
2009/001586. Briefly, the technology es stringent panning of phage
libraries that display the antigen binding nts (Fabs). After a several rounds of
panning, a library, termed the Omniclonal, was obtained that was enriched for
recombinant Fabs containing light and heavy chain variable regions that bound to
KAAGI with very high affinity and specificity. From this library, more precisely
designated Omniclonal AL0003 AZZB, 96 individual recombinant monoclonal Fabs
were prepared from E. coli and tested for KAAG1 binding. The monoclonal
designated 3A4 was derived from this 96-well plate of onal antibodies based
on its high binding activity for recombinant KAAG1 and its affinity for KAAG1 on the
surface of ovarian cancer cells.
The nucleotide sequences of the variable regions of the heavy and light chain
immunoglobulin chains are shown in SEQ ID NOS.:1 and 3, respectively and the
polypeptide sequences of the variable regions of the heavy and light chain
immunoglobulin chains are shown in SEQ ID NOS.:2 and 4, tively. The
complementarity determining regions (CDRs) of the 3A4 heavy chain immunoglobulin
are shown in SEQ ID NOS.:5, 6 and 7, respectively and the CDRs of the 3A4 light
chain immunoglobulin are shown in SEQ ID NOS.:8, 9 and 10, respectively.
Aside from the possibility of conducting interaction s between the Fab
monoclonals and the KAAG1 protein, the use of Fabs is limited with t to
conducting meaningful in vitro and in vivo studies to te the biological on of
the antigen. Thus, it was necessary to transfer the light and heavy chain variable
regions contained in the 3A4 Fabs to full dy scaffolds, to generate mouse—
human chimeric IgG1. The expression vectors for both the light and heavy
immunoglobulin chains were constructed such that i) the original bacterial signal
peptide sequences upstream of the Fab expression vectors were replaced by
mammalian signal peptides and ii) the light and heavy chain constant regions in the
mouse antibodies were replaced with human constant regions. The methods to
accomplish this er utilized standard molecular biology techniques that are
familiar to those skilled in the art. A brief overview of the methodology is described
here.
Light chain expression vector — an existing mammalian expression pIasmid, called
pTTVHBG (Durocher et al., 2002), ed to be used in the 293E transient
transfection system was modified to accommodate the mouse light chain variable
region. The resuIting mouse-human chimeric light chain contained a mouse variable
region followed by the human kappa constant . The cDNA sequence encoding
the human kappa constant domain was amplified by PCR with s OGS1773 and
OGS1774 (SEQ ID NOS:11 and 12, respectively). The nucleotide sequence and the
ponding amino acid sequence for the human kappa constant region are shown
in SEQ ID NOS:13 and 14, respectively. The resulting 321 base pair PCR product
was ligated into pTTVH8G ately downstream of the signal peptide sequence of
human VEGF A (NM_003376). This cloning step also positioned unique restriction
endonuclease sites that permitted the precise positioning of the cDNAs encoding the
mouse light chain variable regions. The sequence of the final expression plasmid,
called pTTVK1, is shown in SEQ ID NO.:15. Based on the 3A4 light chain variable
sequence shown in SEQ lD NO.:3, a PCR primer specific for the light chain variable
region was designed that incorporated, at its 5’-end, a sequence cal to the last
base pairs of the VEGF A signal peptide. The sequence of this primer is shown in
SEQ ID NO:16. A reverse primer (SEQ ID NO.:17) incorporated, at its 3’-end, a
sequence identical to the first 20 base pairs of the human kappa constant domain.
Both the PCR fragments and the digested pTTVK1 were d with the 3’ — 5’
lease activity of T4 DNA polymerase resulting in complimentary ends that
were joined by annealing. The annealing reactions were transformed into ent
E. coli and the expression plasmids were verified by sequencing to ensure that the
mouse light chain variable regions were properly inserted into the pTTVK1 expression
vector.
Heavy chain expression vector— the expression vector that produced the 3A4 heavy
chain immunoglobulin was designed in a similar manner to the pTTVK1 described
above for production of the light chain immunoglobulins. Plasmid pYD11 her et
al., 2002), which contains the human lgGK signal peptide sequence as well as the
CH2 and CH3 s of the human Fc domain of lgG1, was modified by ligating the
cDNA sequence encoding the human constant CH1 region. PCR primers OGS1769
and OGS1770 (SEQ ID NOS:18 and 19), designed to contain unique restriction
endonuclease sites, were used to amplify the human lgG1 CH1 region containing the
nucleotide sequence and corresponding amino acid sequence shown in SEQ ID
NOS:2O and 21. Following ligation of the 309 base pair fragment of human CH1
ately downstream of the lgGK signal e sequence, the modified plasmid
(SEQ ID ) was designated pYD15. When a selected heavy chain variable
region is ligated into this vector, the ing plasmid encodes a full IgG1 heavy chain
immunoglobulin with human constant regions. A PCR primers ic for the heavy
chain variable region of antibody 3A4 (SEQ ID NOS:1) was designed that
incorporated, at its 5’-end, a sequence identical to the last 20 base pairs of the lgGK
signal peptide. The sequence of this primers is shown in SEQ ID NOS:23. A reverse
primer (SEQ lD ) incorporated, at its 3’-end, a sequence identical to the first 20
base pairs of the human CH1 constant . Both the PCR fragments and the
digested pYDi5 were treated with the 3’ — 5’ exonuclease activity of T4 DNA
polymerase resulting in complimentary ends that were joined by annealing. The
annealing reactions were transformed into competent E. coli and the expression
plasmids were verified by sequencing to ensure that the mouse heavy chain variable
regions were properly inserted into the pYD15 expression vector.
Expression of human 3A4 chimeric [961 in 293E cells — The sion vectors
prepared above that encoded the light and heavy chain globulins were
sed in 293E cells using the transient transfection system (Durocher et al.,
2002). The ratio of light to heavy chain was zed in order to achieve the most
yield of antibody in the tissue culture medium and it was found to be 9:1 (L2H).
Binding of chimeric 3A4 to KAAG1 — To measure the ve binding of the 3A4
monoclonal antibody, recombinant human KAAG1 was produced in 293E cells using
the large-scale transient transfection technology (Durocher et al., 2002; Durocher,
2004). The expression and purification of human recombinant KAAG1 as an F0 fusion
protein is found in . To carry out the binding of Fc-KAAG1 to the
antibody preparation, the Fc—KAAG1 was biotinylated with NHS-biotin (Pierce,
Rockford, IL) and 10 ng/well was coated in a streptavidin 96-well plate for 1h at room
temperature. Purified chimeric 3A4 was added at increasing concentrations and
incubated at room temperature for 30 minutes. Bound dy was detected with
HRP-conjugated human anti-kappa light chain antibody in the presence of TMB liquid
substrate (Sigma-Aldrich Canada Ltd., Oakville, ON) and readings were conducted at
450 nm in microtiter plate reader. As shown in Figure 1, 3A4 interacted with the
immobilized KAAG1 protein in a dose-dependent manner. When the control unrelated
lgG was incubated with the recombinant KAAG1, no g activity was observed,
even at the very highest tration. This result demonstrated that 3A4 binds to
human KAAGl. The binding of 3A4 was compared to the binding of the chimeric 3D3
(described in Tremblay and Filion (2009)), that has different epitope city (see
Example 2). The binding ty of 3A4 is very r to 3D3 in this type of assay
(see Figure 1).
Example 2
This example describes the epitope g studies to determine which region of
KAAG1 the 3A4 antibody binds to.
To further delineate the regions of KAAG1 that are bound by the 3A4 antibody,
truncated mutants of KAAG1 were expressed and used in the ELISA. As for the full
length KAAG1, the truncated versions were amplified by PCR and ligated into
BamHI/HindIII digested pYD5. The primers that were used combined the forward
ucleotide with the sequence shown in SEQ ID NO.:25 with primers of SEQ ID
NOS:26 and 27, to produce Fc-fused fragments that ended at amino acid number 60
and 35 of KAAG1, respectively. The expression of these recombinant mutants was
conducted as was described above for the full length Fc-KAAG1 and purified with
Protein-A agarose.
Based on the teachings of Tremblay and Filion (2009), it was known that other
antibodies interacted with specific regions of recombinant KAAG1. Thus, anti-KAAG1
dy 3C4, 3D3, and 3G10 interacted with the regions 1 - 35, 36 — 60, and 61 ~ 84
of KAAG1, tively. These binding results were reproduced and are shown in
Figure 2. In order to determine the region in KAAG1 that is bound by the 3A4
antibody, the ELISA was performed using the KAAG1 truncated Fc-fusions according
to a similar protocol that was described in Example 1. The only modifications were the
use of different biotinylated Fc-KAAG1 truncated mutants. The results show that the
binding specificity of 3A4 is similar to 3G10. In KAAG1 mutants that do not have
amino acids sequences beyond amino acid 60, the binding of 3A4 to KAAG1 does not
occur. This indicates that 3A4 interacts with a region delineated by amino acids 61 —
84 of human KAAG1. The observation that 3A4 and 3D3 have lly cal
binding activity as measured by ELISA le 1) but have very different epitope
specificity suggests that the binding ties of 3A4 is quite distinct of 3D3.
Example 3
This example describes the ability of 3A4 to bind to KAAG1 on the surface of cancer
cell lines
Flow cytometry was used to detect KAAG1 on the surface of cell lines. Based on RT-
PCR expression analyses using KAAG1 mRNA specific primers, selected cancer cell
lines were expected to express KAAG1 protein. To verify this, ovarian cancer cells
3 and TOV-21G) and a control cell lines that showed very little KAAG1
expression (293E). The cells were harvested using 5 mM EDTA, counted with a
hemocytometer, and resuspended in FCM buffer (0.5% BSA, 10 ug/ml goat serum in
1x PBS) at a cell density of 2 x 106 cells/ml. Chimeric 3A4 or a l IgG were
added to 100 pl of cells at a final concentration of 5 pig/ml and incubated on ice for 2h.
The cells were washed in cold PBS to remove unbound antibodies, resuspended in
100 pl FCM buffer containing anti—human lgG conjugated to FITC (diluted 1:200) as a
secondary antibody and ted on ice for 45min on ice. Following another washing
step in cold PBS, the cells were resuspended in 250 pl FCM buffer and analyzed with
a flow cytometer. The results from this experiment are shown in Figure 3A and 38.
Incubation of the cell lines with the control antibody resulted in histograms that
corresponded to the signal that was typically obtained when the antibody was omitted
from the cells. This established the background signal of these FCM values (Figures
3A and 38). By contrast, incubation of the SKOV-3, TOV—21G with the 3A4 chimeric
antibody resulted in a strong fluorescence signal (Figures 3A). This indicated that the
dy ently detects KAAG1 on the e of these cancer cells. The 293E
cells, a human kidney cell line, was expected to show very little KAAG1 expression
and indeed, FCM histogram showed almost no shift compared to the control antibody
(see Figure 38). Therefore, 3A4 specifically ed KAAG1 on the surface of cancer
cells. The activity of 3A4 was compared to the 303, an AAG1 antibody
described in the teachings of Tremblay and Filion (2009). Based on this patent
application, it was known that 3D3 could detect KAAG1 on the surface of cancer cells
as measured by FCM. This was confirmed when the 3D3 was incubated in the
presence of SKOV-3 and TOV-21G cells (see Figure 3A). The fluorescence signal
was not as high compared to the 3A4, indicating that 3A4 has different and increased
ability to detect KAAG1 on the surface of n cancer cells. Other results obtained
in our laboratory indicate that 3A4 could detect KAAG1 on the surface of cancer cells
under conditions where 3D3 exhibited no activity in this assay ts not .
Taken together, these observations and the difference in epitope specificity of 3A4
compared to 3D3 suggests that these antibodies have distinct anti-KAAG1 properties.
Example 4
Methods for use of the 3A4 anti-KAAG1 antibody as an dy conjugate
As demonstrated above, the KAAG1 antigen was detected by 3A4 on the surface of
cancer cells using flow cytometry. There are several different molecular events that
can occur upon binding of an dy to its target on the surface of cells. These
include i) blocking accessibility to another cell-surface antigen/receptor or a ligand, ii)
formation of a relatively stable antibody-antigen complex to allow cells to be ed
via ADCC or CDC, iii) signalling events can occur as exemplified by agonistic
antibodies, iv) the complex can be internalized, or v) the x can be shed from
the cell surface. To address this question we wished to examine the behavior of the
3A4 antibody-KAAG1 complex on the surface of the cells. SKOV-3 cells were plated,
washed, and incubated with 5 ug/ml chimeric 3A4 antibody as described in Example
3. After washing, complete OSE medium was added and the cells placed at 37 C for
up to 90 minutes. The cells were removed at the indicated times (see Figure 4),
rapidly cooled, prepared for cytometry with onjugated anti-human lgG and the
results were expressed as the percentage of mean fluorescence ity (Mean
fluorescence intensity, %) remaining. As illustrated in Figure 4, the fluorescence
signal ses rapidly over a period of 30 - 45 minutes. This result indicates that
the 3A4/KAAG1 complex disappeared from the cells, which indicated that an
internalization of the complex likely occurred. Preliminary studies to elucidate the
ism responsible for this decrease in urface fluorescence have revealed
that the complex appears to be internalized.
These findings were further confirmed by conducting immunofluorescence on live
cells to see if this internalization could be copically observed. SKOV-3 cells
were seeded on cover slips in full medium (OSE medium (Wisent) containing 10%
FBS, 2 mM glutamine, 1 mM sodium-pyruvate, 1X non-essential amino acids, and
antibiotics). Once the cells were properly d, fresh medium was added
containing the 3A4 anti-KAAG1 chimeric antibody at 10 ug/ml and incubating at 37 C
for 4h. The cells were washed in PBS then fixed in 4% paraformaldehyde (in PBS)
for 20 min. After washing, the cells were permeabilized with 0.1% Triton X-100 in
PBS for 5 min. Blocking was performed with 1.5% dry milk in PBS for 1h. Lysosomal-
associated membrane protein 1 , Chang et al., 2002) was detected by
incubating with anti—LAMP1 (Santa Cruz, sc-18821, diluted 1:100) in 1.5 % milk in
PBS for 2h. After washing in PBS, the secondary antibodies were added together in
1.5% milk and incubated for 1h. For the anti—KAAG1 chimeric antibodies the
secondary antibody was a ine Red-X conjugated donkey uman lgG
(H+L) diluted 1:300. For the anti-LAMP1 antibody the secondary antibody was a
DyLight488-conjugated goat anti-mouse lgG (H+L) diluted 1:300. Both secondary
antibodies were from Jackson ImmunoResearch. The coverslips were washed in
PBS and mounted in ProLong Gold antifade t with DAPl. As seen in Figure
5A, after 4 hours of incubation at 37 C in the presence of SKOV-3 ovarian cancer
cells, the 3A4 antibody was able to be ed in complexes predominantly near the
peri-nuclear area (arrows, see red staining in the left panel in Figure 5A), which is
typical of endosomal-Iysosomal-based internalization pathways. This observation
was further confirmed when a mal marker, LAMP1 was visualized and was
found to be also expressed in these areas s, see green staining in the middle
panel in Figure 5A). Importantly, the merging of the two images resulted in the
3A4 and the anti-LAMP1
appearance of yellow-orange structures indicating that the
antibodies were present in the same structures s, see yellow ng in the
right panel in Figure 5A). The co-localization of 3A4, which binds to KAAG1 on the
surface of cancer cells, with LAMP1, a marker of late endosomes/lysosomes, shows
that the antibody/antigen complex was internalized and that it follows a pathway that
is amenable for the release of a payload that would be conjugated to the 3A4
antibody. Identical results were observed in another cancer cell line, TOV-21G (see
Figure SB).
Taken together, these studies demonstrated that dies specific for KAAG1 such
as 3A4 might have uses as an antibody-drug conjugate (ADC). Thus, the high level
of ovarian cancer specificity of KAAG1 coupled with the capacity of this target to be
internalized in cells would support the development of applications as an ADC.
Example 5
Preferential detection of KAAG1 on the surface of cancer cells.
Although l antibodies cting with different epitopes of the KAAG1 protein
were developed, the accessibility of these epitopes when KAAG1 is expressed on the
surface of intact cancer cells was not fully elucidated. ormatics analysis of the
y amino acid structure of KAAG1 (total number of amino acids in the human
protein is 84) did not reveal any s sequences that might correspond to a trans-
membrane domain and therefore how KAAG1 was anchored to the cell membrane
was not fully known.
The antibodies generated against KAAG1 were found to bind to three different
regions in the KAAG1 protein (see ). Most of the antibodies
interact with amino acids 35 — 60 in the KAAGl protein and are exemplified by
antibodies 3D3 and 3612 in this application. Antibodies that interact with the
carboxy-terminal end of KAAG1 between amino acids 61 — 84 are exemplified by
antibody 3A4. Finally, antibodies that interact with the amino-terminal region of the
protein, as exemplified by 304, showed very little binding to cells that express
KAAG1.
This ation shows that when KAAG1 is expressed in cells, the carboxy-terminal
region (amino acids 61 - 84) is exposed to the extracellular space and that
antibodies that target this region are the most efficient at detecting and potentially
treating KAAGl-positive cells. Antibodies that bind to the middle region of KAAGl
(amino acids 35 — 60) can also detect KAAG1 on the cells surface but to a lesser
extent than antibodies that interact with the carboxy-terminus.
Ovarian cancer cell lines such as SKOV-3, are positive for KAAG1 expression. These
cells were used to detect the expression KAAG1 by flow cytometry, which is a
method that allows the detection of cell surface proteins and is well known by those
skilled in the art. Briefly, for each sample 100,000 cells were incubated on ice for 1h
with the y antibody (either AAG1, or the control antibody) at a
concentration of 1 ug/ml. After several washes with ice-cold PBS, the stained cells
were incubated with the secondary antibody that was conjugated to a fluorochrome
(FlTC) which detects the presence of the primary antibody bound to the cells. After
several additional washes, the cells were analyzed with a flow cytometer. The s
expressed in Figure 6 show the Y-axis representing the number of counts (cells) and
the X-axis representing the quantity of fluorescence (fluorescence signal). When
SKOV-3 cells were incubated with the 3A4 antibody, a large shift in fluorescence was
observed indicating that there was nt KAAG1 protein on the surface of the
cells (Figure 6A) and that it was efficiently recognized by this antibody. Under
identical conditions, the antibodies that interact with the middle region of KAAG1,
3612 and 3D3 (Figure 6A) were significantly less efficacious for detecting KAAGl.
When the cells were incubated with increased concentration of 3612 or 3D3, KAAG1
could be detected on the cell surface (not shown). When the cells were ted
with either the l lgG (Figure 6A) or the 3C4, an antibody against the amino
terminus of KAAG1 (Figure 6A), no signal was observed. These results indicate that
antibodies that interact with the carboxy-terminus of KAAG1 can detect the antigen
on the surface of cancer cells more ently then antibodies directed against other
regions of KAAG1. This implied that the carboxy-terminus of KAAG1 is d to
the extracellular (outside) space of the cell. Similar results were ed for other
cancer cell lines that express KAAG1.
The experiment was also med in SKOV-3 cells that were permeabilized with
Triton X-100. Triton X-100 is typically used to permeabilize cell membranes and
release membrane proteins. When the bilized cells were incubated with 3A4
and measured in the flow cytometer (see Figure 6B), the signal was similar to that
obtained in intact cells. ngiy, when the permeabilized cells were ted with
the 3G12 antibody that binds to the middle region of KAAG1 (Figure SB), the signal
was as strong as the 3A4. These results indicate that the middle region of KAAG1 is
likely present in the cell membrane or the inside of the cell. A similar result was
obtained with the 303 antibody, another middle-region binder (Figure GB) but the
signal obtained for 303 was not as strong. As before, igG control did not show any
detectable signal in this assay (Figure 6B). interestingly, incubation of the cells with
the 304 antibody which binds to the amino region of KAAG1, did not result in any
detectable signal (Figure 6B). This last result suggested that the amino region of
KAAG1 is likely cleaved off during the transport of the n to the cell ne.
Overall, these experiments provide much insight into the structure and orientation of
the KAAG1 antigen when it is expressed on the surface of cancer cells. Based on
these data, two models for the structure of KAAG1 at the cell surface is proposed
(Figure 7). In the first model (Figure 7, Model A), the data suggests that the middle
portion is actually the transmembrane region of KAAG1 that is only lly exposed
to the extra—cellular space. This would make the carboxy-terminus of KAAG1 easily
detectable and the middle region more difficult to bind. In the second model (Figure
7, Model B), KAAG1 is anchored to the membrane by another protein that itself is
embedded in the cell membrane. Again, the carboxy-terminus would be easily
accessible by antibodies such as 3A4 but the ction between KAAG1 and the
protein partner would make access to the middle region difficult. The results g
that antibodies consisting of both the y—terminai binders (as ified by
3A4) and middle-region binders (as exemplified by 3612 and 3D3) tested in the
ce of permeabilized cells is in agreement with both models. The inability of the
304 antibody to bind to KAAG1 in intact or permeabilized cells is likely due to the
lack of amino acids contained in the amino-terminus of the mature processed
membrane form of KAAG1 and both models are in agreement with this.
These s imply that antibodies that target the carboxy-terminus of KAAG1 in
cancer cells, in particular the region spanned by amino acids 61 — 84, are the most
appropriate for the development of antibodies for uses as therapeutics for the
treatment of carcinomas that express KAAG1. In addition, other uses for the KAAG1
antibodies that bind to the carboxy-terminai region include diagnostic reagents for the
detection of carcinomas that express KAAG1.
Antibodies or antigen binding fragments having a binding specificity similar to the
3A4 antibody may be generated or isolated by immunizing an animal with the C-
terminal portion of KAAG1 according to methods known in the art, including
hybridoma technology, by screening a library of antibody or antigen binding
fragments with the C—terminal portion of KAAG1 and/or performing competition assay
of isolated antibodies or antigen binding fragment with the 3A4 antibody described
herein.
Example 6
Humanization by design of the 3A4 mouse monoclonal dy
3D modeling of the variable s of the mouse 3A4 monoclonal antibody.
This task was accomplished by homology modeling. The most similar template
structures to the murine 3A4 variable region sequences of the light and heavy chains
(SEQ ID NOs: 4 and 2) were identified by a BLAST search against PDB. To build an
initial model of the mouse 3A4 variable region the following template ures were
used (PDB codes): 2|PU (chain L) for the light chain, and 1F11 (chain B) for the
heavy chain. Other suitable templates can be found in the PDB entry 2DDQ for the
light chain, and in the PDB s 3lY3, 1KTR, 2VXT, 1A6T ad 1lGl for the heavy
chain. Required mutations were operated on these template structures according to
the murine 3A4 sequences: 7 mutations in the 2|PU light chain, and 17 mutations
plus a due deletion in the 1F11 heavy chain. The mutated structures
corresponding to the heavy and light chains of the murine 3A4 variable regions were
assembled into two—chain antibody structures by mposing the heavy and light
chains of the tive template structures. The resulting structure of the led
3A4 variable region was first refined by energy zation with the AMBER force—
field and a stepwise release of constraints, ranging from the CDR loops that were
relaxed first, to the ne heavy atoms of the framework region that were fully
d only in the last stage. The CDR-H3 loop in each antibody variable region
structure was then refined by Monte-Carlo-minimization (MCM) conformational
ng, in which dihedral angles in the CDR-H3 region were sampled in each MCM
cycle followed by energy minimization of a predefined region extending 10 A around
the initial conformation of the CDR-H3 loop. A entation of the modeled variable
region of the mouse 3A4 antibody is given in Figure 8. The structures of the human
or humanized variable sequences most similar to each of the 3A4 variable
sequences were also identified from PDB, and then superimposed onto the modeled
structures of the murine 3A4 variable s. These structures include PDB entries
3QCT, 3AAZ, 1WT5 and 3M80 for the light chain, and PDB s 1|9R, 3NFP,
1T04, 1ZA6, 3HC4, 207T and 1WT5 for the heavy chain. These ures were
used to assist in the modeling of mutations in the framework region in order to build
the humanized 3D-structures starting from the modeled murine BD-structure.
Characterization of the mouse 3A4 amino-acid sequences and modeled ure.
This step was d out to estimate the humanness index, antigen contact
propensity index, to delineate the CDRs, canonical residues, inter-chain packing
(VHNL interface residues), variable-/constant—region packing (VH/CH and VL/CL
interface residues), unusual framework residues, potential N- and O-glycosylation
sites, buried residues, Vernier zone residues, and proximity to CDRs. Internet-
available resources and local software were used to assess these properties.
ion of the best human chain and heavy-chain frameworks for the mouse
CDRs.
This was done by standard sequence gy comparison against a local copy of
human germline databases (VBASE), t other sequence libraries (Genbank and
SwissProt), as well as the set of human framework consensus sequences. BLAST
searches were conducted to retrieve sequence matches with highest homology in the
framework region only (thus excluding CDRs) while matching the length of the CDR
loops. The human frameworks identified for the light and heavy chains of the 3A4
antibody correspond to the k2 and M classes, respectively. Several human germline
framework sequences that are most r to the 3A4 framework sequences were
retained in addition to the human sus sequences for these classes.
Identification of framework residues for back-mutations and design of multiple
humanized variants.
This is an important step that flags amino-acid residues that should be mutated to the
corresponding human sequences with particular care. These residues represent
y candidates for back-mutations to the mouse sequences in case of affinity
loss. It is the most difficult and unpredictable step of humanization by design,
particularly in the absence of an experimental ure of the antibody-antigen
complex. It relies on the identification of residues in one or more of the following
categories: canonical, CDR—H3, Vernier zone, unusual, CDR-proximal (within 5 A),
inter-chain packing, and glycosylation-site residues. Such residues might affect
antigen-binding site and affinity directly or indirectly. The antigen contact sity
index as well as amino-acid occurrence in human germline databases at each
position are also extremely important in deciding whether a certain residue can be
safely mutated from the mouse sequence to the human sequence. Humanization of
the 3A4 dy light chain variable region involves 11 mutations to its proposed
humanized framework for 100% framework humanization. Humanization of the 3A4
antibody heavy chain variable region involves 23 mutations to its proposed
humanized framework for 100% framework humanization. These 100% humanized
variable region sequences are labelled Lvh1 and th1, respectively (SEQ ID NOsz33
and 38). Additional humanized sequences were also designed in which several
residues from the 3A4 mouse sequences were retained based on careful structural
and ative sequence analyses that te a high probability of altering
antigen-binding affinity if mutations are to be introduced at these positions. These
sequences of the variable regions are labelled Lvh2, th2, th3 and th4 (SEQ ID
N03: 34, 39, 40 and 41).
The two humanized light chain variants (including the constant region) are fied
herein as Lh1 (SEQ ID NO.: 43) and Lh2 (SEQ ID ). The four humanized
heavy chain ts (including the constant region_ are identified herein as Hh1
(SEQ ID NO.:46), Hh2 (SEQ ID NO.:47), Hh3 (SEQ ID NO.:48) and HM (SEQ ID
NO.:49). The two humanized light chain and 4 humanized heavy chain can be
assembled into 8 humanized antibodies (Lh1Hh1, Lh1Hh2, Lh1Hh3, Lh1Hh4,
Lh2Hh1, Lh2Hh2, Lh2Hh3, and Lh2Hh4). Molecular models for all these
combinations were constructed by homology ng ng from the 3D model of
the murine 3A4 le region, and are depicted in Figures 9a-9h.
In the case of 3A4 light-chain humanized ce Lvh2 (SEQ ID NO:34),
ork residues Val-L2 and 5 were retained from the mouse sequence
since residue L2 is semi—buried, contacts both CDR-L1 and CDR-L3, and has
n—contacting propensity, while residue L45 approaches the heavy-chain. We
note that both these murine residues may occur in human frameworks. In the case of
3A4 heavy-chain humanized sequence th2 (SEQ ID N0239), framework residues
Ile-H2 and Lys-L73 were ed from the mouse sequence since residue H2 is
semi-buried, contacts both CDR—H1 and CDR-H3, and has antigen-contacting
propensity, while residue H73 belongs to the Vernier zone supporting CDR-H2, and
both these murine residues may occur in human frameworks. In the case of 3A4
chain humanized sequence th3 (SEQ ID NO:40), lle-H2 and Lys-L73 back-
mutations were retained and in addition to these, framework residues lle-H48, Ala-
H67, 9 and Val-H71 were retained from the mouse ce since all these
additional murine residues are buried residues and belong to the Vernier zone
supporting CDR-H2, and also murine residue H71 may occur in human frameworks.
In the case of 3A4 heavy-chain humanized sequence th4 (SEQ ID NO:41), all 6
back—mutations of the th3 humanized variant were included plus additional two
mouse framework residues Lys-H38 and Lys-H66 since they represent semi—buried
residues close to . The resulting amino acid sequences of the murine and
humanized chains are listed in Table 1. The alignment of the murine and humanized
light chain variable regions is shown in Figure 10a and the alignment of the murine
and humanized heavy chain variable regions is shown in Figure 10b.
Figure 11a and 11b is an alignment of the murine light chain variable region with the
100% humanized light chain variable region and the murine heavy chain variable
region with the 100% humanized heavy chain variable region respectively. This
figure illustrates the amino acids that are preserved and those that have been chosen
for substitution.
Example 7.
Assembly and expression of 3A4 humanized variant antibodies
The purpose of these investigations is to ine the kinetics ters of anti-
clusterin antibodies. In particular, to determine whether the humanization of the 3A4
anti-KAAG1 monoclonal antibody s the kinetics parameters of its binding to
human KAAG1. To this end, a kinetic analysis method was developed using the
ProteOn XPR36 instrument from . Human KAAG1 was immobilized on a
sensor chip. Full length antibodies or Fab fragments were injected and allowed to
interact with the immobilized KAAG1.
Construction of d encoding the chimeric (murine) heavy and light chains of
The heavy and light chains of the ic antibody were amplified by PCR from the
original murine immunoglobulin chains using the following oligonucleotide primer
pairs: heavy chain, 5’—oligo encoded by SEQ ID NO: 50 and 3’-oligo encoded by SEQ
ID NO:51; light chain, 5'-oligo d by SEQ ID NO: 52 and 3’-oligo encoded by
SEQ ID NO:53. The ing PCR ts were digested by Hind Ill and cloned into
pK-CR5 (SEQ ID NO:21) previously digested with Hind Ill.
Construction of plasmids encoding the zed heavy chain 3A4 ts 1, 2, 3
and 4
The fragments coding for the humanized heavy chain region of the antibody 3A4
(Hh1, Hh2, Hh3 and HM) were ordered from GenScript (Piscataway, USA). The
DNA fragments including the kozak and stop codon sequences were digested with
Hindlll and cloned into the Hindlll site of plasmid pK-CR5 previously
dephosphorylated with calf intestinal phosphatase (NEB) to prevent recircularization.
Figure 12a shows the map of the plasmid -3A4-HC-variant1. All heavy chain
variants of the humanized 3A4 were constructed in a similar manner.
Construction of plasmids ng the zed light chain 3A4 variants 1 and 2
The fragments coding for the human light chain regions of the antibody 3A4 (Lh1 and
Lh2) were ordered from GenScript. The DNA fragments including the kozak and stop
codon sequences was digested with BamHl and cloned into the BamHl site of
plasmid pMPG-CR5 (SEQ lD NO:55) previously dephosphorylated with calf intestinal
phosphatase (NEB) to prevent recircularization. Figure 12b shows the map of the
plasmid pMPG-CR5-3A4-LC-variant1. All light chain variants of the zed 3A4
were constructed in a similar manner.
Transient transfection study
d DNA was isolated from small cultures of E. coli using the Mini-Prep kit
(Qiagen Inc, sauga, ON) according to the manufacturer’s recommendation.
Briefly, 2 ml of LB medium containing 100 ug/ml of ampicillin were inoculated with a
single colony picked after ligation and transformation. The cultures were incubated at
37°C overnight with vigorous shaking (250 RPM). The plasmid was then isolated
from 1.5 ml of culture using the protocols, buffers, and columns provided by the kit.
The DNA was eluted using 50 ul of sterile water. Plasmid DNA was isolated from
large culture of E. coli using the Plasmid Plus Maxi kit (Qiagen Inc, Mississauga, ON)
according to the manufacturer’s endation. 200 mL of LB medium containing
100 ug/mL ampicillin were inoculated with a single fresh colony of E. coli and
incubated overnight at 37°C with vigorous shaking (250 RPM). The bacteria (130 mL
of culture for the heavy chain and 180 mL of culture for the light chain) were pelleted
by centrifugation at 6000 x g, for 15 min, at 4°C and the plasmid was isolated using
the protocols, buffers and columns provided by the kit. The pure plasmids was
resuspended in e 50 mM Tris, pH8 and quantified by measuring the optical
density at 260 nm. Before transfection the purified plasmid were sterilized by
extraction with phenol/chloroform followed by ethanol precipitation. The plasmid were
resuspended in sterile 50 mM Tris, pH 8 and quantified by optical density at 260 nm.
Before ection, the cells (CHO-cTA) were washed with PBS and ended at
a concentration of 4.0 X 106 cell/ml in growth medium (CD-CHO, lnvitrogen) without
dextran sulfate for 3 h in suspension e. For each plasmid combination, 45 ml of
cells were ected by adding slowly 5 ml of CDCHO medium supplemented with
pg/ml of each plasmid and 50 pg/ml of polyethylenimine (PEI Max; Polysciences).
The final concentration was 1 pg/ml of each plasmid and 5 ug/m1 of PEI. After 2 h,
the cells were erred at 30°C. The next days, 50 ug/mL of dextran sulfate and
3.75 ml of each supplement (Efficient Feed A and B lnvitrogen) were added to the
cells and they were incubated at 30°C for 13 days. 2.5 ml of Feed A and 2.5 ml of
Feed B were added at day 4, 6, 8 and 11. On day 13, the supernatant was clarified
by centrifugation and filtered through a 0.22 pM .
CHO cells (CHOcTA) were transfected with plasmids encoding the different variants
of humanized heavy and light chains of the 3A4 antibody regulated by the CR5
promoter. Transfection with different combinations of light and heavy chains was
performed. As control, cells were also transfected with ds ng the
chimeric/murine antibody.
Purification of antibody
ml of supernatant from the CH0 cell transfections were concentrated by
centrifugation using the Amicon Ultra (Ultacell-50k) cassette at 1500 rpm. The
concentrated antibody (550 pl) was purified using the Nab spin kit Protein A Plus
(Thermo Scientific) according to the manufacture’s endations. The purified
antibodies were then desalted using PBS and the concentrating Amicon Ultra
(UltraceI-10K) cassette at 2500 rpm to a final volume of 250 pl. The purified antibody
was quantified by reading the ODzeo using the op spectrophotometer and kept
frozen at -20°C. An aliquote of the purified antibody was resuspended into an equal
volume of Laemmli 2X and heated at 95°C for 5 min and chilled on ice. A standard
curve was made using known amount of purified human lgG1 kappa from Human
Myeloma plasma (Athens Research). The samples were separated on a
polyacrylamide Novex 10% Tris-Glycine gel (lnvitrogen Canada Inc, Burlington, ON)
and transferred onto a Hybond-N nitrocellulose membrane ham ence
Corp, Baie e, QC) for 1 h at 275 mA. The membrane was blocked for 1 h in
0.15% Tween 20, 5% skimmed milk in PBS and incubated for 1 hr with an Goat anti-
Human lgG (H+L) conjugated to Cy5 (Jackson, Cat# 109—176-099). The signal was
revealed and quantified by scanning with the Typhoon Trio+ scanner (GE Healtcare).
As shown in Figure 13, all combinations of the 3A4 humanized antibody variants
were sed in CHO cells.
Example 8.
Kinetic analysis of murine and humanized 3A4 antibody
Supplies
GLM sensorchips, the Biorad ProteOn amine coupling kit (EDC, sNHS and
ethanolamine), and 10mM sodium acetate buffers were purchased from Bio-Rad
Laboratories (Mississauga, ON). HEPES buffer, EDTA, and NaCl were purchased
from from Sigma-Aldrich lle, ON). Ten percent Tween 20 solution was
purchased from Teknova (Hollister, CA). The goat anti-human lgG Fc fragment
specific antibody was purchased from Jackson ImmunoResearch. The gel filtration
column Superdex 75 10/300 GL was purchased from GE care.
Gel filtration
The KAAG1 protein at a concentration of 3.114 mg/ml and a volume of 220 uL was
injected onto the Superdex G75 column. The separation was done at 0.4ml/min in
HBST running buffer (see below) without Tween 20. The volume of the fractions
collected was 500 uL. Concentration of KAAG1 in each fractions was determined by
OD 280 using an extension coefficient of 5500 and a MW of 8969. Figure 14
represents the e of the gel filtration of KAAG1. A small peak of potential
aggregate is eluting at around 11 ml. The protein g at 13 ml was used as
e for the SPR assay (fractions 15 — 19).
SPR biosensor assays
All surface plasmon resonance assays were carried out using a BioRad ProteOn
XPR36 instrument (Bio-Rad Laboratories Ltd. ssauga, ON) with HBST running
buffer (10mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 pH 7.4)
at a ature of 25°C. The anti-mouse Fc capture surface was generated using a
GLM sensorchip activated by a 1:5 dilution of the standard BioRad sNHS/EDC
solutions injected for 300 s at 30 uL/min in the analyte (horizontal) direction.
Immediately after the activation, a 13 ug/mL solution of anti-human lgG Fc fragment
specific in 10 mM NaOAc pH 4.5 was injected in the analyte direction at a flow rate of
uL/min until approximately 8000 resonance units (RUs) were immobilized.
Remaining active groups were quenched by a 300 3 injection of 1M ethanolamine at
uL/min in the analyte ion, and this also ensures mock—activated pots
are created for blank referencing. The screening of the 3A4 variants for binding to
KAAG1 occurred in two steps: an indirect capture of 3A4 variants from cell
supernatant onto the anti-human lgG Fc fragment specific surface in the ligand
direction(vertical) followed by a KAAG1 injection in the analyte direction. Firstly, one
buffer ion for 30 s at 100 uL/min in the ligand direction was used to stabilize the
baseline. For each 3A4 capture, unpurified 3A4 ts in cell-culture media were
diluted to 4 °/o in HBST, or approximately 1.25 pg/mL of purifed 3A4 in HBST was
used. Four to five 3A4 ts along with wild-type 3A4 were simultaneously injected
in individual ligand channels for 240 s at flow 25 pL/min. This resulted in a saturating
3A4 capture of approximately 400-700 RUs onto the uman lgG Fc fragment
specific surface. The first ligand l was left empty to use as a blank control if
required. This 3A4 capture step was immediately followed by two buffer injections in
the analyte direction to stabilize the baseline, and then the gel filtration purified
KAAG1 was injected. For a typical screen, five KAAG1 concentrations (8, 2.66, 0.89,
0.29, and 0.098 nM) and buffer control were simultaneously injected in individual
analyte channels at 50 uL/min for 120 s with a 600s dissociation phase, resulting in a
set of binding sensorgrams with a buffer reference for each of the captured 3A4
variants. The anti-human lgG Fc fragment specific — 3A4 xes were
regenerated by a 18 8 pulse of 0.85% phosphoric acid for 18 s at 100 pL/min to
prepare the anti-human lgG Fc fragment specific surface for the next injection cycle.
Sensorgrams were aligned and double-referenced using the buffer blank ion
and interspots, and the ing sensorgrams were analyzed using ProteOn
Manager software v3.0. The c and affinity values were determined by fitting the
referenced sensorgrams to the 1:1 Langmuir binding model using local Rmax, and
affinity constants (KD M) were derived from the resulting rate constants (kd s'1/ k2,1 M'1s'
Determination of rate and affinity constants
Figure 15 summarizes the association (k3, 1/Ms) and dissociation (kd, 1/s) rate
constants as well as affinity (KD, M) nts for the interaction of KAAG1 with
purified murine 3A4, murine 3A4 transiently expressed as a chimeric and transiently
expressed zed variants. These constants are graphically represented in
Figure 16. The ation rate constant is very similar for the pure parental, chimeric
and zed 3A4 variants (Figure 16a). The dissociation rate constants is similar
for the transiently express chimeric as ed to the pure parental 3A4 with
suggest that the transfection procedure did not alter the parameters of the interaction
of KAAG1 with the antibody (Figure 16b). However all zed variants seem to
have a slightly altered off rate, i.e. quicker dissociation rate (Figure 16b). This is
reflected in the affinity constants (Figure 160). In summary, there is a linear
correlation between the binding affinity (logKD) of the humanized variant and the
number of back-mutations made in the parent antibody (Lch) with a decrease in the
g affinity as the number of mutations is increasing. However, the difference in
binding affinity is only 4 fold different between the worse variant (H1L1, 0.47 nM)
which has no mouse residue retained and the best variant which has 10 mouse
residues retained (H4L2, 0.1 nM). Finally, the binding affinity of all variants for
KAAG1 was found to be sub-nanomolar and the best variant (H4L2, 0.1 nM)
exhibited an affinity about 6-fold weaker than the murine (Lch, 0.057 nM). Overall,
these results indicate that humanization was successful as all of the variants
displayed very high affinity for KAAG1.
Example 9.
Binding of 3A4 humanized variants to KAAG1 in an ELISA
ELISA methods were also used to e the g activity of the zed 3A4
variants to the murine 3A4 antibody. Recombinant human KAAG1 was coated in 96-
well plates O/N, washed and incubated for 1h at RT with increasing quantities of
murine or humanized 3A4 variants. Following another round of washing steps, an
anti-human antibody conjugated to HRP was added to the wells and the bound 3A4
antibody was measured metrically at Abs450. As shown in Figure 17A, the
humanized variants (Lh1Hh1, Lh1Hh2, Lh1Hh3 and Lh1Hh4) displayed very similar
binding to KAAG1 when compared to the murine 3A4 (Lch). This result ted
that all four zed heavy chain variants were comparable to the original h3A4
heavy chain when assembled with the L1 variant of the humanized light chain. Figure
178 shows the results when the heavy chain variants were assembled with Lh2
variant of the 3A4 humanized light chain. In this instance, there was a difference in
the binding of the variants. For example, Lh2hh4 was the variant with the closest
profile compared to the murine 3A4. This was in agreement with the SPR data (see
Example 3), which showed that the variant 4 of the heavy chain had the highest
affinity for KAAG1. Taken together, these binding results show that the humanized
variants all interact with human KAAG1 in this assay. Although there were some
subtle differences, the binding in ELISA was in concordance with the SPR results.
Example 10.
g of 3A4 humanized variants on the surface of cancer cells
Flow cytometry was used to evaluate the capacity of the humanized 3A4 variants to
ct with KAAG1 expressed on the surface of cancer cells. To this end, SKOV-3
ovarian cancer cells, which we had previously showed were efficiently bound by 3A4
by flow try, were incubated with the eight zed variants and the original
murine antibody. Briefly, SKOV-3 cells were detached from the plate with EDTA and
incubated on ice with either 3.0 mg/ml, 0.3 mg/ml or 0.3 mg/ml of the antibodies for
1h. After three washing steps, the cells were incubated with the secondary antibody,
anti-human lgG-conjugated to FITC for 1h on ice. Cell surface fluorescence was
measured in a flow cytometer and the values ae shown in the histogram of Figure 18.
As depicted, all variants could detect KAAG1 on the surface on unpermeabilized and
the est s were obtained at the highest concentration of 3A4 dies (3
mg/ml) and decreased as the concentration of the antibody was decreased. Among
the different variants, the ones with the most murine back-mutations (Figure 18, see
Lh1Hh4 and Lh2Hh4) interacted with KAAG1 on the surface of cells with the highest
activity. In fact, Lh1Hh4 and Lh2hh4 appeared to be slight improved cell surface
binding to KAAG1 compared to the murine 3A4 antibody (Lch).
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mammalian cells expressing them.2004; US. patent application No. 60/662,392.
- ay GB, Filion M. Antibodies that specifically block the biological activity
of a tumor antigen. 2009; .
- Durocher Y, Kamen A, Perret S, Pham PL. Enhanced production of
recombinant proteins by transient transfection of suspension-growing mammalian
cells. 2002; Canadian patent ation No. CA 2446185.
- Durocher Y. Expression s for enhanced transient gene expression and
mammalian cells expressing them.2004; US. patent application No. 60/662,392.
- Chang MH, Karageorgos LE, Meikle PJ. CD107a (LAMP-1) and CD107b
(LAMP-2). 2002; J Biol Regul Homeost Agents. 16:147—51.
- Abhinandan, KR and Martin, ACR. Analysis and improvements to Kabat and
urally correct numbering of antibody variable domains. 2008; Mol lmmunol,
45, 3832-3839.
Sequences referred to in the description
SEQ lD NO.:1 — 3A4 heavy chain variable region nucleotide sequence
CAGATCCAGTTGGTGCAATCTGGACCTGAGATGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAG
GCTTCTGGATACACATTCACTGACGACTACATGAGCTGGGTGAAACAGAGCCATGGAAAGAGCCTTGAG
TGGATTGGAGATATTAATCCTTACAACGGTGATACTAACTACAACCAGAAGTTCAAGGGCAAGGCCATA
TTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAACAGCCTGACATCGGAAGACTCAGCA
GTCTATTACTGTGCAAGAGACCCGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC
SEQ ID NO.:2 — 3A4 heavy chain variable region polypeptide sequence
QIQLVQSGPEMVKPGASVKMSCKASGYTFTDDYMSWVKQSHGKSLEWIGDINPYNGDTNYNQKFKGKAI
LTVDKSSSTAYMQLNSLTSEDSAVYYCARDPGAMDYWGQGTSVTVSS
SEQ lD NO.:3 — 3A4 light chain variable region nucleotide sequence
GATGTTGTGATGACCCAAACTCCACTCTCCCTGGCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC
AGATCTAGTCAGAGCCTTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTTCAGAAACCAGGC
CAGTCTCCAAAGCTCCTGATCCACACAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGATTCAGTGGC
AGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTAC
CAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAGGCTGGAGCTGAAA
SEQ ID NO.:4 — 3A4 light chain variable region polypeptide sequence
DVVMTQTPLSLAVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIHTVSNRFSGVPDRFSG
FTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTRLELK
SEQ ID NO.:5 — 3A4 heavy chain CDR1 polypeptide ce
GYTFTDDYMS
SEQ ID NO.:6 — 3A4 heavy chain CDR2 polypeptide sequence
GDTN
SEQ ID NO.:7 — 3A4 heavy chain CDR3 polypeptide sequence
DPGAMDY
SEQ ID NO.:8 — 3A4 light chain CDR1 polypeptide sequence
RSSQSLLHSNGNTYLE
SEQ lD NO.:9 — 3A4 light chain CDR2 polypeptide sequence
TVSNRFS
SEQ ID NO.:10 — 3A4 light chain CDR3 polypeptide sequence
FQGSHVPLT
SEQ ID NO.:11 — OGS1773
GTAAGCAGCGCTGTGGCTGCACCATCTGTCTTC
SEQ ID NO.:12 — OGS1774
GTAAGCGCTAGCCTAACACTCTCCCCTGTTGAAGC
SEQ ID NO.:13 — human kappa constant nucleotide ce
GCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT
GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTC
CAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC
ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC
CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO.:14 — human kappa nt polypeptide sequence
AVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO.:15
CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGGGTGAGTACTCCC
TCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCA
CCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCC
ACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCATGAACTTTCTGCTGTCTTGGGTGCATTGG
AGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCAGGCTTGAGACGGAGCTTACAGCGCT
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA
TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGGTACCGCGGCCGCTTCGAATGAGATC
CCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGT
GTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTA
GAGCCCCGCCGCCGGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATG
TAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACC
AAACACAAAGGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGA
TTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTAACT
CTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGACTACGGGAG
GCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCA
ATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATAC
TATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTT
AGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGG
TCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAGGAGCGGGCA
GTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGT
TGTGAACAGTAAGGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTT
GGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACCCCTTGGGCAATA
AATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCC
GTAAAGACTGGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGA
TACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTT
GTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAA
ATTAAACGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTG
TGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAAT
AACTTGGCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCAC
CCCGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGAC
CACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGA
GAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC
TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATA
TCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAG
CTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTA
TCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATAT
CTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGC
ATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTAT
CCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTA
TATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGCTG
TCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC
ATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT
TTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA
TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT
TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT
TTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAA
GAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAG
CATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG
GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT
CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC
ACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC
CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG
GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG
GATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA
CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC
TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT
GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG
ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG
CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC
AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT
CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG
TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAA
AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA
CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC
TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCT
GTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC
GAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT
CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTG
AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATT
GTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTC
GACCAATTCTCATGTTTGACAGCTTATCATCGCAGATCCGGGCAACGTTGTTGCATTGCTGCAGGCGCA
GAACTGGTAGGTATGGCAGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTCATTGGTTA
ATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTA
TATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAAT
TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCC
TGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAAT
AGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT
GTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA
GTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT
GATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCA
CCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAA
CCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTT
AGTGAACCGTCAGATCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGCTCGCGGTT
GAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTACTC
CGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACC
AGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTGG
CGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGT
SEQ ID NO.:16 — OGS18500
ATGCCAAGTGGTCCCAGGCTGATGTTGTGATGACCCAAACTCC
SEQ ID NO:.17 — 4
GGGAAGATGAAGACAGATGGTGCAGCCACAGTCCG
SEQ ID NO.:18 — OGS1769
GTAAGCGCTAGCGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCC
SEQ ID NO.:19 — OGS1770
GTAAGCGAATTCACAAGATTTGGGCTCAACTTTCTTG
SEQ ID NO.:20 — human globulin CH1 region nucleotide sequence
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCA
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
SEQ ID NO.:21 — human immunoglobulin CH1 region ptide sequence
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC
VOJZZ
CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGGGTGAGTACTCCC
TCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCA
CCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCC
ACTCCCAGGTCCAAGTTTGCCGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGG
GTTCCAGGTTCCACTGGCGGAGACGGAGCTTACGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCAA
GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT
CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGT
GAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGAATTCACTCACAC
ATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA
GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT
CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA
GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAA
GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGATCCCCCGACCTCGACCTCTG
GCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGA
CATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGCCCCGCCGCCGGACG
AACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCTTCAGTTGGTT
GGTACAACTTGCCAACTGAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGTATATACTATCCAG
ACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAA
GGGTCCTAAGGAACAGCGATGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAGGAC
AAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGA
GAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC
TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATA
TCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAG
CATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTA
TCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATAT
CTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGC
ATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTAT
CCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTA
TATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGCTG
TCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC
ATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT
TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA
TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT
TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT
TTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAA
CTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAG
CATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCG
GCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGAT
CATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC
ACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC
CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG
GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTG
GGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA
CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC
TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT
GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG
ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG
CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGC
AGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTA
GCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT
CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCG
TGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAA
AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA
CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC
CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCT
GTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGC
GAGTCAGTGAGCGAGGAAGCGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGA
ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC
GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA
ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG
TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGAC
GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATC
TACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGG
TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAAT
CAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGG
TGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCTCACTCTCTTCCGCATCGCTGTC
TGCGAGGGCCAGCTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATC
CCGTCGGCCTCCGAACGGTACTCCGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGATC
GGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGG
CAGCGGGTGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGT
SEQ 23 — OGS1879
GGGTTCCAGGTTCCACTGGCCAGATCCAGTTGGTGCAATCTGG
EQ ID NO.:24 — OGS1810
GGGGCCAGGGGAAAGACAGATGGGCCCTTCGTTGAGGC
SEQ ID NO.:25
GTAAGCGGATCCATGGATGACGACGCGGCGCCC
SEQ ID NO.:26
GTAAGCAAGCTTAGGCCGCTGGGACAGCGGAGGTGC
SEQ ID NO.:27
GTAAGCAAGCTTGGCAGCAGCGCCAGGTCCAGC
SEQ ID NO.:28 .
GAGGGGCATCAATCACACCGAGAAGTCACAGCCCCTCAACCACTGAGGTGTGGGGGGGTAGGGATCTGC
TCATATCAACCCCACACTATAGGGCACCTAAATGGGTGGGCGGTGGGGGAGACCGACTCACTT
GAGTTTCTTGAAGGCTTCCTGGCCTCCAGCCACGTAATTGCCCCCGCTCTGGATCTGGTCTAGCTTCCG
GATTCGGTGGCCAGTCCGCGGGGTGTAGATGTTCCTGACGGCCCCAAAGGGTGCCTGAACGCCGCCGGT
CACCTCCTTCAGGAAGACTTCGAAGCTGGACACCTTCTTCTCATGGATGACGACGCGGCGCCCCGCGTA
GAAGGGGTCCCCGTTGCGGTACACAAGCACGCTCTTCACGACGGGCTGAGACAGGTGGCTGGACCTGGC
GCTGCTGCCGCTCATCTTCCCCGCTGGCCGCCGCCTCAGCTCGCTGCTTCGCGTCGGGAGGCACCTCCG
CTGTCCCAGCGGCCTCACCGCACCCAGGGCGCGGGATCGCCTCCTGAAACGAACGAGAAACTGACGAAT
CCACAGGTGAAAGAGAAGTAACGGCCGTGCGCCTAGGCGTCCACCCAGAGGAGACACTAGGAGCTTGCA
GGACTCGGAGTAGACGCTCAAGTTTTTCACCGTGGCGTGCACAGCCAATCAGGACCCGCAGTGCGCGCA
CCACACCAGGTTCACCTGCTACGGGCAGAATCAAGGTGGACAGCTTCTGAGCAGGAGCCGGAAACGCGC
GGGGCCTTCAAACAGGCACGCCTAGTGAGGGCAGGAGAGAGGAGGACGCACACACACACACACACACAA
ATATGGTGAAACCCAATTTCTTACATCATATCTGTGCTACCCTTTCCAAACAGCCTA
SEQ ID NO.:29
MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLSQRPHRTQGAGSP
PETNEKLTNPQVKEK
SEQ ID NO.:30 (variant light chain variable region)
DXVMTQTPLSLXVXXGXXASISCRSSQSLLHSNGNTYLEWYLQKPGQSPXLLIHTVSNRFSG
VPDRFSGSGSGTDFTLKlSRVEAEDXGWYCFQGSHVPLTFGXGTXLEXK
wherein at least one of the amino acids identified by X is an amino acid substitution
rvative or non-conservative) in comparison with a corresponding amino acid in the
polypeptide set forth in SEQ ID NO.:4. The amino acid substitution may be, for example
conservative.
SEQ ID NO.:31 (variant light chain variable region)
DXa1VMTQTPLSLXaZVXa3Xa4GX35XasASISCRSSQSLLHSNGNTYLEWYLQKPGQSPXa7LLIHT
VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDXaaGVYYCFQGSHVPLTFGXagGTXa1oLEXa11
n X31 may be a hobic amino acid;
Wherein Xag may be A or P;
Wherein X33 may be neutral hydrophilic amino acid;
Wherein Xa4 may be L or P;
Wherein X35 may be an acidic amino acid;
Wherein X35 may be Q or P;
n X37 may be a basic amino acid;
Wherein X38 may be a hydrophobic amino acid;
Wherein X39 may be A or Q;
Wherein X310 may be a basic amino acid; or
Wherein X311 may be a hobic amino acid,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in the
polypeptide set forth in SEQ ID NO.:4.
SEQ ID NO.:32 (variant light chain variable region)
DXA1VMTQTPLSLXAZVXA3XA4GXA5XA5ASISCRSSQSLLHSNGNTYLEWYLQKPGQSPXMLLIH
TVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDXAaGWYCFQGSHVPLTFGXAgGTXA1OLEXA
Wherein XA1 may be V or I
Wherein XA2 may be A or P
Wherein XA3 may be S or T
Wherein XA4 may be L or P
Wherein XA5 may be D or E
Wherein XAG may be Q or P
n XA7 may be K or Q
Wherein XAB may be L or V
Wherein XAg may be A or Q
Wherein XA10 may be R or K or
Wherein XA11 may be L or I,
wherein at least one of the amino acid identified by X is an amino acid substitution
rvative or non-conservative) in comparison with a ponding amino acid in the
polypeptide set forth in SEQ ID NO.:4.
SEQ ID NO.:33 (variant 1 light chain variable region: Lvh1)
DIVMTQTPLSLPVTPGEPAS|SCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGV
PDRFSGSGSGTDFTLKISRVEAEDVGWYCFQGSHVPLTFGQGTKLEIK
SEQ ID NO.:34 (variant 2 light chain variable region: Lvh2)
DWMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSG
VPDRFSGSGSGTDFTLKlSRVEAEDVGWYCFQGSHVPLTFGQGTKLEIK
SEQ ID NO.:35 (variant heavy chain variable region)
QXQLVQSGXEXXKPGASVKXSCKASGYTFTDDYMSWVXQXXGXXLEWXGDINPYNGDTNY
NQKFKGXXXXTXDXSXSTAYMXLXSLXSEDXAVYYCARDPGAMDYWGQGTXVTVSS
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in the
polypeptide set forth in SEQ ID N02. The amino acid substitution may be, for example
conservative.
SEQ ID NO.:36 (variant heavy chain le )
QXmQLVQSGszEXb3Xb4KPGASVKXb5SCKASGYTFTDDYMSWVszQXWngGngXmoLEWXb
11GDINPYNGDTNYNQKFKGXM2Xb13Xb14Xb15TXb15DXWSXD188TAYMXb19LszoSLXb21SEDsz
2AVYYCARDPGAMDYWGQGTXmVTVSS
Wherein Xm may be a hydrophobic amino acid;
Wherein sz may be P or A;
Wherein Xb3 may be a hydrophobic amino acid;
Wherein Xb4 may be V or K;
Wherein Xb5 may be a hydrophobic amino acid;
Wherein sz may be a basic amino acid;
Wherein Xb7 may be S or A;
Wherein ng may be H or P;
Wherein ng may be a basic amino acid;
Wherein Xmo may be S or G;
Wherein an may be a hydrophobic amino acid;
Wherein Xm may be a basic amino acid;
Wherein Xm may be a hydrophobic amino acid;
Wherein XW may be i or T;
Wherein Xb15 may be a hydrophobic amino acid;
Wherein Xma may be a hydrophobic amino acid;
Wherein Xb17 may be K or T;
Wherein Xm may be a neutral hydrophilic amino acid;
Wherein Xb19 may be Q or E;
Wherein ngo may be N or S;
n Xb21 may be T or R;
Wherein Xm may be a neutral hydrophilic amino acid; or
Wherein sza may be S or L,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in the
polypeptide set forth in SEQ ID NO.:2.
SEQ ID NO.:37 nt heavy chain variable region)
QXB1QLVQSGXBZEX33XB4KPGASVKXB5SCKASGYTFTDDYMSWVXBGQXB7XBBGX39XB10LEW
X311GDINPYNGDTNYNQKFKGXB12XB13XB14XB15TXB16DXB17SXB188TAYMXB19LXBZOSLX321SE
DXBzzAWYCARDPGAMDYWGQGTXmVTVSS
n X31 may be I or V;
Wherein X32 may be P or A;
Wherein X33 may be M or V;
Wherein X34 may be V or K;
Wherein X35 may be M or V;
Wherein X35 may be K or R;
Wherein X37 may be S or A;
Wherein X38 may be H or P;
Wherein X39 may be K or Q;
Wherein X510 may be S or G;
Wherein X311 may be i or M;
Wherein X312 may be K or R;
Wherein X313 may be A or V;
Wherein X314 may be I or T;
n X315 may be L or I;
Wherein X316 may be V or A;
Wherein X517 may be K or T;
Wherein X313 may be S or T;
Wherein X319 may be Q or E;
Wherein X320 may be N or S;
Wherein X321 may be T or R;
Wherein X322 may be S or T; or
Wherein X323 may be S or L,
wherein at least one of the amino acid identified by X is an amino acid substitution
(conservative or non-conservative) in comparison with a corresponding amino acid in the
polypeptide set forth in SEQ ID NO.:2.
SEQ ID NO.:38 (variant 1 heavy chain variable region: th1)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTN
YNQKFKGRVTITADTSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGTLVTVSS
SEQ ID NO.:39 (variant 2 heavy chain variable region: th2)
Q|QLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNY
RVTITADKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGTLVTVSS
SEQ ID NO.:40 (variant 3 heavy chain variable region: th3)
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYNGDTNY
NQKFKGRATLTVDKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGTLVTVSS
SEQ ID NO.:41 (variant 4 heavy chain le region: th4)
QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNY
NQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSS
SEQ ID NO: 42 3A4 murine light (kappa) chain
DWMTQTPLSLAVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIHTVSNRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGWYCFQGSHVPLTFGAGTRLELKRTVAAPSVFIFPP
SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:43 3A4 humanized light (kappa) chain variant 1; Lh1
TPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGV
PDRFSGSGSGTDFTLKISRVEAEDVGWYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPS
DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS
KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:44 3A4 humanized light (kappa) chain variant 2; Lh2
DWMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSG
VPDRFSGSGSGTDFTLKISRVEAEDVGWYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPP
SDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKWACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:45 3A4 murine heavy (lgg1) chain
QIQLVQSGPEMVKPGASVKMSCKASGYTFTDDYMSWVKQSHGKSLEWIGD!NPYNGDTNY
NQKFKGKAILTVDKSSSTAYMQLNSLTSEDSAWYCARDPGAMDYWGQGTSVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT
VLHQDWLNGKEYKCKVSNKALPAPiEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
SEQ ID NO:46 3A4 humanized heavy (Igg1) chain variant 1; HM
SGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTN
YNQKFKGRVTITADTSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
MISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
SEQ ID NO:47 3A4 humanized heavy (Igg1) chain variant 2; Hh2
QlQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWMGDINPYNGDTNY
NQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKG
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
WTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
SEQ ID NO:48 3A4 humanized heavy (lgg1) chain variant 3; Hh3
QlQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVRQAPGQGLEWIGDINPYNGDTNY
NQKFKGRATLTVDKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGTLVTVSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
SEQ ID NO:49 3A4 humanized heavy (Igg1) chain variant 4: HM
SGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNY
NQKFKGKATLTVDKSTSTAYMELSSLRSEDTAWYCARDPGAMDYWGQGTLV‘WSSASTK
GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGK
SEQ ID NO:50
ATACCCAAGCTTGCCACCATGGAGACAGACACAC
SEQ ID NO:51
ATACCCAAGCTTCATTTCCCGGGAGACAGGGAG
SEQ ID NO:52
ATACCCAAGCTTGGGCCACCATGAACTTTCTGCTGTCTTGG
SEQ ID NO:53
ATACCCAAGCTTCTAACACTCTCCCCTGTTGAAG
SEQ ID NO:54 pK-CR5
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT
TTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGA
TAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCA
ACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCC
TAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAG
ATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAA
GAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGT
AACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTC
AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC
TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCA
GTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGC
GAATTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCACATCGGCGC
GCCAAATGATTTGCCCTCCCATATGTCCTTCCGAGTGAGAGACACAAAAAATTCCAACAC
ACTATTGCAATGAAAATAAATTTCCTTTATTAGCCAGAGGTCGAGATTTAAATAAGCTTGC
TAGCAGATCTTTGGACCTGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGGATGTCA
TTGTCACTCAAGTGTATGGCCAGATCGGGCCAGGTGAATATCAAATCCTCCTCGTTTTTG
GAAACTGACAATCTTAGCGCAGAAGTAATGCCCGCTTTTGAGAGGGAGTACTCACCCCA
ACAGCTGGATCTCAAGCCTGCCACACCTCACCTCGACCATCCGCCGTCTCAAGACCGCC
TACTTTAATTACATCATCAGCAGCACCTCCGCCAGAAACAACCCCGACCGCCACCCGCT
GCCGCCCGCCACGGTGCTCAGCCTACCTTGCGACTGTGACTGGTTAGACGCCTTTCTC
GAGAGGTTTTCCGATCCGGTCGATGCGGACTCGCTCAGGTCCCTCGGTGGCGGAGTAC
CGTTCGGAGGCCGACGGGTTTCCGATCCAAGAGTACTGGAAAGACCGCGAAGAGTTTG
TCCTCAACCGCGAGCCCAACAGCTGGCCCTCGCAGACAGCGATGCGGAAGAGAGTGAC
CGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCG
TCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGGCCTCCCACCGTA
CACGCCTACCTCGACCCGGGTACCAATCTTATAATACAAACAGACCAGATTGTCTGTTTG
TTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGT
TTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTC
TGTTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTAAGGTTGTCGAGTGAAGACG
AAAGGGTTCATTAAGGCGCGCCGTCGACCTCGAGGGGGGGCCCGGTACCCAGCTTTTG
TTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT
GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA
AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG
AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG
GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCT
CGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC
CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC
CGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA
GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT
ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTT
ACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG
CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC
CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG
GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA
GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA
AGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG
TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCA
TACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTC
TGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG
GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATG
AGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCT
GTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGG
AGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGC
TCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCT
GCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT
GTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG
CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT
GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA
AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT
GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA
GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC
GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACT
CTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT
GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA
ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTT
TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATG
TATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC
SEQ ID NO:55 pMPG-CR5
GTCGACGATACCGTGCACTTAATTAAGCGCGCTCGACCAAATGATTTGCCCTCCCATATG
TCCTTCCGAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCT
TTATTAGCCAGAGGTCGAGGTCGGGGGATCCGTTTAAACTTGGACCTGGGAGTGGACAC
CTGTGGAGAGAAAGGCAAAGTGGATGTCATTGTCACTCAAGTGTATGGCCAGATCGGGC
CAGGTGAATATCAAATCCTCCTCGTTTTTGGAAACTGACAATCTTAGCGCAGAAGTAATG
CCCGCTTTTGAGAGGGAGTACTCACCCCAACAGCTGGATCTCAAGCCTGCCACACCTCA
CCTCGACCATCCGCCGTCTCAAGACCGCCTACTTTAATTACATCATCAGCAGCACCTCC
GCCAGAAACAACCCCGACCGCCACCCGCTGCCGCCCGCCACGGTGCTCAGCCTACCTT
GCGACTGTGACTGGTTAGACGCCTTTCTCGAGAGGTTTTCCGATCCGGTCGATGCGGAC
TCGCTCAGGTCCCTCGGTGGCGGAGTACCGTTCGGAGGCCGACGGGTTTCCGATCCAA
TGGAAAGACCGCGAAGAGTTTGTCCTCAACCGCGAGCCCAACAGCTGGCCCT
CAGCGATGCGGAAGAGAGTGACCGCGGAGGCTGGATCGGTCCCGGTGTCTT
CTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACG
AGCTCTGCTTATATAGGCCTCCCACCGTACACGCCTACCTCGACCCGGGTACCAATCTT
ATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTGTTT
GTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGTCTG
TTTGTTATAATACAAACAGACCAGATTGTCTGTTTGTTATAATACAAACAGACCAGATTGT
CTGTTTGTTAAGGTTGTCGAGTGAAGACGAAAGGGTTAATTAAGGCGCGCCGTCGACTA
GCTTGGCACGCCAGAAATCCGCGCGGTGGTTTTTGGGGGTCGGGGGTGTTTGGCAGCC
ACAGACGCCCGGTGTTCGTGTCGCGCCAGTACATGCGGTCCATGCCCAGGCCATCCAA
AAACCATGGGTCTGTCTGCTCAGTCCAGTCGTGGACCAGACCCCACGCAACGCCCAAAA
TAATAACCCCCACGAACCATAAACCATTCCCCATGGGGGACCCCGTCCCTAACCCACGG
TGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTT
CACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCC
AATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTT
ATGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGC
GGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCATCTCCCCTATT
CCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACA
CAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCC
CGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAA
ATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCC
GGAGCCGCGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTG
CTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAAT
CCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGG
ACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGG
CCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCAT
CACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATG
TAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGA
TCGGCCGCAGCGATCGCATCCATGGCCTCCGCGACCGGCTGCAGAACAGCGGGCAGTT
CGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGAGATGCAATAGGT
CAGGCTCTCGCTGAATTCCCCAATGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGAT
GCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGC
AGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGA
GAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACG
TCGCGGTGAGTTCAGGCTTTTTCATATCTCATTGCCCGGGATCTGCGGCACGCTGTTGA
TAAGCGGGTCGCTGCAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTT
AATATGCGAAGTGGACCTGGGACCGCGCCGCCCCGACTGCATCTGCGTGTTCGAATTC
GCCAATGACAAGACGCTGGGCGGGGTTTGTGTCATCATAGAACTAAAGACATGCAAATA
TATTTCTTCCGGGGACACCGCCAGCAAACGCGAGCAACGGGCCACGGGGATGAAGCAG
GGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGG
ATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCA
GGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTC
GCGGCTCTTACCAGCCTAACTTCGATCACTGGACCGCTGATCGTCACGGCGATTTATGC
CGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTT
GTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGG
AAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATCAATTCT
TGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCC
ATCTCCAGCAGCCGCACGCGGCGCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT
GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA
GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAG
CTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC
TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTG
GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT
GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA
CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG
AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGC
GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA
AACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAA
AAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAA
ACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT
AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTT
ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG
TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCC
AGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAA
CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC
CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGC
AACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC
ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAA
AGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTAT
CACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT
TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA
GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAA
ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTG
AGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTC
ACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAG
GGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT
CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAG
GGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCA
TGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCAT
GTTTGACAGCTTATCTCTAGCAGATCCGGAATTCCCCTCCCCAATTTAAATGAGGACCTA
ACCTGTGGAAATCTACTGATGTGGGAGGCTGTAACTGTACAAACAGAGGTTATTGGAATA
ACTAGCATGCTTAACCTTCATGCAGGGTCACAAAAAGTGCATGACGATGGTGGAGGAAA
ACCTATTCAAGGCAGTAATTTCCACTTCTTTGCTGTTGGTGGAGACCCCTTGGAAATGCA
GGGAGTGCTAATGAATTACAGGACAAAGTACCCAGATGGTACTATAACCCCTAAAAACCC
AACAGCCCAGTCCCAGGTAATGAATACTGACCATAAGGCCTATTTGGACAAAAACAATGC
TTATCCAGTTGAGTGCTGGGTTCCTGATCCTAGTAGAAATGAAAATACTAGGTATTTTGG
GACTTTCACAGGAGGGGAAAATGTTCCCCCAGTACTTCATGTGACCAACACAGCTACCA
CAGTGTTGCTAGATGAACAGGGTGTGGGGCCTCTTTGTAAAGCTGATAGCCTGTATGTTT
CAGCTGCTGATATTTGTGGCCTGTTTACTAACAGCTCTGGAACACAACAGTGGAGAGGC
CTTGCAAGATATTTTAAGATCCGCCTGAGAAAAAGATCTGTAAAGAATCCTTACCTAATTT
CCTTTTTGCTAAGTGACCTTATAAACAGGAGAACCCAGAGAGTGGATGGGCAGCCTATG
TATGGTATGGAATCCCAGGTAGAAGAGGTTAGGGTGTTTGATGGCACAGAAAGACTTCC
AGGGGACCCAGATATGATAAGATATATTGACAAACAGGGACAATTGCAAACCAAAATGCT
TTAAACAGGTGCTTTTATTGTACATATACATTTAATAAATGCTGCTTTTGTATAAGCCACTT
TTAAGCTTGTGTTATTTTGGGGGTGGTGTTTTAGGCCTTTTAAAACACTGAAAGCCTTTAC
ACAAATGCAACTCTTGACTATGGGGGTCTGACCTTTGGGAATGTTCAGCAGGGGCTGAA
GAGACTTGGGAAGAGCATTGTGATTGGGATTCAGTGCTTGATCCATGTCCAGA
GTCTTCAGTTTCTGAATCCTCTTCTCTTGTAATATCAAGAATACATTTCCCCATGCATATAT
TATATTTCATCCTTGAAAAAGTATACATACTTATCTCAGAATCCAGCCTTTCCTTCCATTCA
ACAATTCTAGAAGTTAAAACTGGGGTAGATGCTATTACAGAGGTAGAATGCTTCCTAAAC
CCAGAAATGGGGGATCTGC
SEQ ID NO.:56— 3A4 humanized heavy chain CDR2 polypeptide sequence
DINPYNGDTNYNQKFKG
Claims (40)
1. An antibody or antigen binding fragment thereof capable of specific g to Kidney associated antigen 1 (KAAG1) having a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO.:5, SEQ ID NO.:6 and SEQ ID NO.:7 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO.: 8, SEQ ID NO.:9 and SEQ ID NO.:10.
2. The antibody or antigen g fragment thereof of claim 1, wherein the heavy chain le region is as set forth in SEQ ID NO.: 35, SEQ ID NO.:36, or SEQ ID NO.:37 and wherein the light chain variable region is as set forth in SEQ ID NO.:30, SEQ ID NO.:31, or SEQ ID NO.:32.
3. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region is as set forth in SEQ ID NO.:38, SEQ ID NO.:39, SEQ ID NO.:40, SEQ ID NO.:41 or SEQ ID NO.:2 and wherein the light chain variable region is as set forth in SEQ ID NO.:33, SEQ ID NO.:34 or SEQ ID NO.:4.
4. The antibody or antigen g fragment thereof of any one of claims 1 to 3, wherein the dy comprises the heavy chain set forth in SEQ ID NO.: 49, SEQ ID NO.:46, SEQ ID NO.:47, SEQ ID NO.:48 or SEQ ID NO.:45 and/or the light chain set forth in SEQ ID NO.: 43, SEQ ID NO.:44 or SEQ ID NO.:
5. The antibody or antigen g fragment thereof of claim 3, n said antibody or antigen binding fragment thereof comprises: a. the heavy chain variable region as set forth in SEQ ID NO.:41 and the light chain variable region as set forth in SEQ ID NO.:33; b. the heavy chain as set forth in SEQ ID NO.:49 and the light chain as set forth in SEQ ID NO.:43; c. the heavy chain variable region as set forth in SEQ ID NO.:38 and the light chain le region as set forth in SEQ ID NO.:33; d. the heavy chain as set forth in SEQ ID NO.:46 and the light chain as set forth in SEQ ID NO.:43; e. the heavy chain variable region as set forth in SEQ ID NO.:39 and the light chain variable region as set forth in SEQ ID NO.:33; f. the heavy chain as set forth in SEQ ID NO.:47 and the light chain as set forth in SEQ ID NO.:43; g. the heavy chain variable region as set forth in SEQ ID NO.:40 and the light chain variable region as set forth in SEQ ID NO.:33; h. the heavy chain as set forth in SEQ ID NO.:48 and the light chain as set forth in SEQ ID NO.:43; i. the heavy chain variable region as set forth in SEQ ID NO.:41 and the light chain variable region as set forth in SEQ ID NO.:34; j. the heavy chain as set forth in SEQ ID NO.:49 and the light chain as set forth in SEQ ID ; k. the heavy chain variable region as set forth in SEQ ID NO.:38 and the light chain variable region as set forth in SEQ ID NO.:34; l. the heavy chain as set forth in SEQ ID NO.:46 and the light chain as set forth in SEQ ID NO.:44; m. the heavy chain variable region as set forth in SEQ ID NO.:39 and the light chain variable region as set forth in SEQ ID NO.:34; n. the heavy chain as set forth in SEQ ID NO.:47 and the light chain as set forth in SEQ ID NO.:44; o. the heavy chain variable region as set forth in SEQ ID NO.:40 and the light chain variable region as set forth in SEQ ID NO.:34; p. the heavy chain as set forth in SEQ ID NO.:48 and the light chain as set forth in SEQ ID NO.:44; q. the heavy chain variable region set forth in SEQ ID NO.:2 and the light chain variable region set forth in SEQ ID NO.:4; or r. the heavy chain set forth in SEQ ID NO.:45 and the light chain set forth in SEQ ID NO.:42.
6. An antibody or antigen binding fragment thereof that specifically binds to kidney associated antigen 1 (KAAG1) and has an affinity constant (KD) of less than 10 picomolar.
7. The antibody or antigen binding fragment thereof of claim 6, wherein said antibody or antigen binding fragment thereof competes with the dy or antigen binding fragment thereof of claim 1 to 5.
8. The antibody or antigen g fragment thereof of any one of claims 1 to 3, 6 or 7, wherein the antibody comprises a constant region of a human IgG1 antibody.
9. An antibody or antigen binding fragment thereof capable of competing with the antibody or n binding fragment thereof of any one of claims 1 to 5 and having an affinity constant (KD) of less than 1nM, provided that said antibody is not the 3G10 dy or antigen binding fragment thereof.
10. The dy or antigen binding fragment thereof of claim 1, having a light chain variable region at least 70% identical to SEQ ID NO.:4 and/or a heavy chain variable region at least 70% identical to SEQ ID NO.:2 and sing at least one amino acid substitution e of a complementarity determining region (CDR) in comparison with SEQ ID NO.:4 or SEQ ID NO.:2..
11. The antibody or antigen g fragment thereof of any one of claims 1 to 3 or 6 to 10, wherein the antibody is a monoclonal antibody, a chimeric dy, an hybrid antibody, a humanized antibody or a human antibody or an antigen binding fragment thereof.
12. The antibody or antigen binding fragment of any one of claims 1 to 11, wherein the antibody or antigen binding fragment thereof is conjugated with a therapeutic moiety or with a detectable moiety.
13. The antibody or antigen binding fragment of any one of claims 1 to 11, wherein the antibody or antigen binding fragment thereof is conjugated with a cytotoxic agent.
14. The antibody or antigen binding fragment thereof of claim 13, n the cytotoxic agent comprises an auristatin.
15. The antibody or antigen binding fragment thereof of claim 14, wherein the auristatin comprises monomethyl auristatin E or monomethyl auristatin F.
16. A nucleic acid encoding a light chain le region and/or a heavy chain variable region of the antibody or antigen g fragment of any one of claims 1 to 11.
17. A vector comprising the c acid of claim 16.
18. The vector of claim 17, wherein said vector is an expression vector.
19. An isolated cell comprising the nucleic acid of claim 16, the vector of claim 17 or the antibody or antigen binding fragment of any one of claims 1 to 15.
20. The isolated cell of claim 19, n said cell comprises a nucleic acid encoding a light chain variable region and a nucleic acid ng a heavy chain variable region.
21. The isolated cell of claim 20, wherein said cell is capable of expressing, ling and/or secreting an antibody or antigen binding fragment thereof.
22. A pharmaceutical composition comprising the antibody or antigen binding fragment of any one of claims 1 to 15, and a pharmaceutically acceptable
23. A composition comprising the antibody or antigen binding fragment of any one of claims 1 to 15, and a carrier.
24. A method for detecting KAAG1 or a KAAG1 variant, the method comprising contacting an isolated cell expressing KAAG1 or the KAAG1 variant or a sample comprising or suspected of comprising KAAG1 or the KAAG1 variant with the antibody or antigen binding fragment thereof of any one of claims 1 to 15 and measuring binding.
25. The method of claim 24, wherein the sample is from a subject having or suspected of having cancer.
26. The method of claim 25, wherein the cancer is metastatic.
27. The method of any one of claims 24 to 26, wherein the sample is a serum , a plasma sample or a blood sample obtained from a mammal.
28. The method of any one of claims 24 to 26, n the sample is a tissue sample obtained from a mammal.
29. The method of any one of claims 24 to 26, wherein the sample is a cell e or a supernatant.
30. The method of any one of claims 24 to 29, comprising quantifying the amount of antibody bound to KAAG1 or the KAAG1 variant.
31. A kit comprising the antibody or antigen binding nt of any one of claims 1 to 15.
32. Use of the dy or antigen binding fragment of any one of claims 1 to 15, the pharmaceutical composition of claim 22 or the composition of claim 23 in the manufacture of a medicament for detection, diagnosis or treatment of cancer comprising cells expressing KAAG1 or a KAAG1 variant.
33. Use of the antibody or antigen g fragment of any one of claims 1 to 15, the ceutical composition of claim 22 or the composition of claim 23 in the detection of a tumor ex vivo or in the diagnosis of cancer ex vivo, wherein the tumor or cancer comprises cells expressing KAAG1 or a KAAG1 variant.
34. The use as d in claim 32 or claim 33, wherein the cancer is selected from the group consisting of ovarian cancer, skin cancer, renal cancer, colorectal , sarcoma, leukemia, brain cancer, d cancer, breast cancer, prostate cancer, oesophageal cancer, bladder cancer, lung cancer and head and neck cancer.
35. The use as defined in claim 34, wherein the cancer is ovarian cancer.
36. The use as defined in claim 35, wherein the cancer is recurrent n cancer.
37. The use as defined in any one of claims 32 to 36, wherein the cancer is metastatic.
38. A method for obtaining an antibody or antigen binding fragment thereof, le for use as an antibody-drug conjugate for the treatment of cancer sing cells expressing KAAG1 or a KAAG1 variant, the method comprising: a. providing an antibody or antigen binding fragment thereof which specifically binds to an epitope comprised between amino acids 61 and 84 of KAAG1 or a KAAG1 variant; b. testing internalization of the antibody or n binding fragment within an isolated cell expressing KAAG1 or a KAAG1 variant or in a non-human animal and; c. isolating an antibody or an antigen binding fragment which is internalized.
39. A method of making the antibody or antigen binding fragment thereof of any one of claims 1 to 11, comprising culturing an isolated cell as defined in any one of claims 19 to 21 so that the antibody or antigen binding fragment thereof is produced.
40. The method of claim 39, comprising conjugating the antibody or n binding nt f with a therapeutic moiety or with a detectable moiety. ‘vn an1
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161470063P | 2011-03-31 | 2011-03-31 | |
US61/470,063 | 2011-03-31 | ||
US201161533346P | 2011-09-12 | 2011-09-12 | |
US61/533,346 | 2011-09-12 | ||
PCT/CA2012/000296 WO2012129668A1 (en) | 2011-03-31 | 2012-03-28 | Antibodies against kidney associated antigen 1 and antigen binding fragments thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ615694A NZ615694A (en) | 2015-01-30 |
NZ615694B2 true NZ615694B2 (en) | 2015-05-01 |
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