U.S. patent application number 10/974678 was filed with the patent office on 2005-05-19 for immunoconjugates and humanized antibodies specific for b-cell lymphoma and leukemia cells.
This patent application is currently assigned to Immunomedics, Inc.. Invention is credited to Hansen, Hans, Leung, Shui-on.
Application Number | 20050106108 10/974678 |
Document ID | / |
Family ID | 23112129 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106108 |
Kind Code |
A1 |
Leung, Shui-on ; et
al. |
May 19, 2005 |
Immunoconjugates and humanized antibodies specific for B-cell
lymphoma and leukemia cells
Abstract
A chimeric LL2 monoclonal antibody is described in which the
complementarity determining regions (CDRs) of the light and heavy
chains of the murine LL2 anti-B-lymphoma, anti-leukemia cell
monoclona lantibody has been recombinantly joined to the human
kappa and IgG.sub.1 constant region domains, respectively, which
retains the immunospecificity and B-cell lymphoma and leukemia cell
internalization capacity of the parental murine LL2 monoclonal
antibody, and which has the potential of exhibiting reduced human
anti-mouse antibody production activity. A humanized LL2 monoclonal
antibody is described in which the CDRs of the light and heavy
chains have been recombinantly joined to a framework sequence of
human light and heavy chains variable regions, respectively, and
subsequently linked to human kappa and IgG.sub.1 constant region
domains, respectively, which retains the immunospecificity and
B-lymphoma and leukemia cell internalization capacities of the
parental murine and chimeric LL2 monoclonal antibodies, and which
has the potential for exhibiting reduced human anti-mouse antibody
production activity. Vectors for producing recombinant chimeric and
humanized chimeric monoclonal antibodies are provided. Isolated
DNAs encoding the amino acid sequences of the LL2 variable light
and heavy chain and CDR framework regions are described. Conjugates
of chimeric and humanized chimeric LL2 antibodies with cytotoxic
agents or labels find use in therapy and diagnosis of B-cell
lymphomas and leukemias.
Inventors: |
Leung, Shui-on; (Madison,
NJ) ; Hansen, Hans; (Picayune, MS) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1717 RHODE ISLAND AVE, NW
WASHINGTON
DC
20036-3001
US
|
Assignee: |
Immunomedics, Inc.
Morris Plains
NJ
|
Family ID: |
23112129 |
Appl. No.: |
10/974678 |
Filed: |
October 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10974678 |
Oct 28, 2004 |
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10446689 |
May 29, 2003 |
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10446689 |
May 29, 2003 |
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09741843 |
Dec 22, 2000 |
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09741843 |
Dec 22, 2000 |
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09127902 |
Aug 3, 1998 |
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6187287 |
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09127902 |
Aug 3, 1998 |
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08690102 |
Jul 31, 1996 |
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5789554 |
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08690102 |
Jul 31, 1996 |
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08289576 |
Aug 12, 1994 |
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Current U.S.
Class: |
424/9.34 ;
424/155.1; 424/178.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61K 47/6877 20170801; C07K 2317/41 20130101; C07K 2317/77
20130101; A61K 47/6867 20170801; A61P 35/02 20180101; C07K 2319/00
20130101; C07K 16/3061 20130101; A61K 38/00 20130101; C07K 2317/24
20130101; A61K 47/6851 20170801 |
Class at
Publication: |
424/009.34 ;
424/155.1; 424/178.1 |
International
Class: |
A61K 049/00; A61K
039/395 |
Claims
1. An immunoconjugate comprising an antibody or a fragment thereof
specific for B-cells which is conjugated to Y-90 through a DTPA
chelating agent.
2. The immunoconjugate of claim 1, wherein the DPTA chelating agent
is aminohexyl DTPA.
3. The immunoconjugate of claim 1, wherein at least 47.3% of Y-90
is chelated.
4. The immunoconjugate of claim 1, wherein the immunoconjugate is
internalizing.
5. The immunoconjugate of claim 4, wherein conjugation through the
DPTA chelating agent to Y-90 does not reduce binding to B-cells or
internalization function relative to the unconjugated antibody.
6-10. (canceled)
Description
[0001] CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0002] This application is a continuation of U.S. Ser. No.
09/741,843 filed on Dec. 22, 2000, which is a continuation of U.S.
Ser. No. 09/127,902 filed on Aug. 3, 1998, now U.S. Pat. No.
6,187,287, which is a continuation of U.S. Ser. No. 08/690,102
filed on Jul. 31, 1996, now U.S. Pat. No. 5,789,554, which is a
continuation of U.S. Ser. No. 08/289,576 filed on Aug. 12, 1994,
now abandoned. This application claims only subject matter
disclosed in the parent applications and therefore presents no new
matter.
BACKGROUND OF THE INVENTION
[0003] The invention relates generally to immunoconjugates for
diagnostic and therapeutic uses in cancer. In particular, the
invention relates to recombinantly produced chimeric and humanized
monoclonal antibodies directed against B-cell lymphoma and leukemia
cells, which antibodies can be covalently conjugated to a
diagnostic or therapeutic reagent without loss of antibody binding
and internalization function and with reduced production of human
anti-mouse antibodies.
[0004] Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia
are B-cell malignancies that remain important contributors to
cancer mortality. The response of these malignancies to various
forms of treatment is mixed. They respond reasonably well to
chemotherapy, and, in cases where adequate clinical staging of NHL
is possible, as for patients with localized disease, satisfactory
treatment may be provided using field radiation therapy (Hall et
al., Radiology for the Radiologist, Lippincott, Philadelphia, 1989,
pp 365-376). However, the toxic side effects associated with
chemotherapy and the toxicity to the hematopoietic system from
local, as well as whole body, radiotherapy, limits the use of these
therapeutic methods. About one-half of the patients die from the
disease (Posner et al., Blood, 61: 705 (1983)).
[0005] The use of targeting monoclonal antibodies conjugated to
radionuclides or other cytotoxic agents offers the possibility of
delivering such agents directly to the tumor site, thereby limiting
the exposure of normal tissues to toxic agents (Goldenberg, Semin.
Nucl. Med., 19: 332 (1989)). In recent years, the potential of
antibody-based therapy and its accuracy in the localization of
tumor-associated antigens have been demonstrated both in the
laboratory and clinical studies (see., e.g., Thorpe, TIBTECH, 11:
42 (1993); Goldenberg, Scientific American, Science & Medicine,
1: 64 (1994); Baldwin et al., U.S. Pat. Nos. 4,925,922 and
4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095;
Irie et al., U.S. Pat. No. 5, 196,337; Hellstrom et al., U.S. Pat.
Nos. 5,134,075 and 5,171,665). In general, the use of radio-labeled
antibodies or antibody fragments against tumor-associated markers
for localization of tumors has been more successful than for
therapy, in part because antibody uptake by the tumor is generally
low, ranging from only 0.01% to 0.001% of the total dose injected
(Vaughan et al., Brit, J. Radiol., 60: 567 (1987)). Increasing the
concentration of the radiolabel to increase the dosage to the tumor
is counterproductive generally as this also increases exposure of
healthy tissue to radioactivity.
[0006] LL-2 (EPB2) is a highly specific anti-B-cell lymphoma and
anti-lymphocytic leukemia cell murine monoclonal antibody (mAb)
that is rapidly internalized by such cells and that can overcome
some of the aforementioned difficulties (Shih et al,. Int, J.
Cancer, 56: 538 (1994)). LL2, which is of the IgG2a antibody type,
was developed using the Raji B-lymphoma cell line as the source af
antigen (Pawlak-Byczkowska et al., Cancer Res., 49: 4568 (1989)).
Murine LL2 (mLL2) is known to react with an epitope of CD22
(Belisle et al., Proc Amer. Assn. Clin. Res., 34: A2873 (1993)).
CD22 molecules are expressed in the cytoplasm of progenitor and
early pre-B cells, and appear in the cell surface of mature
B-cells.
[0007] By immunostaining of tissue sections, mLL2 was shown to
react with 50 of 51 B-cell lymhomas tested. mLL2 is a highly
sensitive means of detecting B-cell lymphoma cell in vivo, as
determined by a radioimmunodetection method (Murthy et al., Eur. J.
Nucl. Med., 19: 394 (1992)). The Fab' fragment of mLL2 labeled with
.sup.99mTc localized to 63 of 65 known lesions in Phase II trial
patients with B-cell lymphoma (Mills et al., Proc. Amer. Assn.
Cancer Res., 14: A2857 (1993)). In addition, .sup.131I-labeled mLL2
was therapeutically effective in B-cell lymphoma patients
(Goldenberg et al., J. Clin. Oncol., 9: 548 (1991)). mLL2 Fab'
conjugated to the exotoxin PE38KDEL induced complete remissions of
measurable human lymphoma xenografts (CA-46) growing in nude mice
(Kreitman et al., Cancer Res., 53: 819 (1993)).
[0008] The clinical use of mLL2, just as with most other promising
murine antibodies, has been limited by the development in humans of
a HAMA response. While a HAMA response is not invariably abserved
following injection of mLL2, in a significant number of cases
patients developed HAMA following a single treatment with mLL2.
This can limit the diagnostic and therapeutic usefulness of such
antibody conjugates, not only because of the potential anaphylactic
problem, but also as a major portion of the circulating conjugate
may be complexed to and sequestered by the circulating anti-mouse
antibodies. This is exemplified by one study in which about 30% of
the patients developed low level HAMA response following a single
injection of about 6 mg of mLL2 .sup.131I-IgG and nearly all
developed a strong HAMA response with additional injections. On the
other hand, with mLL2 Fab' labeled with .sup.99mTc, no HAMA
response was observed. Such HAMA responses in general pose a
potential obstacle to realizing the full diagnostic and therapeutic
potential of the mLL2 antibody.
[0009] Although, as noted above, the use of fragments of mLL2, such
as F(ab').sub.2 and Fab', partially alleviate/circumvent these
problems of immunogenicity, there are circumstances in which whole
IgG is more desirable, such as when induction of cellular immunity
is intended for therapy, or where an antibody with enhanced
survival time is required.
[0010] In order to maximize the value of the mLL2 IgG antibody as a
therapeutic or diagnostic modality and increase its utility in
multiple and continuous administration modalities, it is an object
of this invention to produce a mouse/human chimeric mAb (cLL2) and
humanized mAb (hLL2) related to mLL2 that retain the
antigen-binding specificity of mLL2, but that elicit reduced HAMA
in a subject receiving same.
[0011] It is another object of this invention to provide DNA
sequences encoding the amino acid sequences of the variable regions
of the light and heavy chains of the cLL2 and hLL2 mAbs, including
the complementarity determining regions (CDR).
[0012] It is also an object of this invention provide conjugates of
the hLL2 and cLL2 mAbs containing therapeutic or diagnostic
modalities.
[0013] It is a further object of this invention to provide methods
of therapy and diagnosis that utilize the humanized and chimeric
mAbs of the invention.
[0014] These objects have been achieved by the invention described
below in the specification and appended claims.
SUMMARY OF THE INVENTION
[0015] In one aspect of the invention, there is provided a cLL2 mAb
related to mLL2 mAb, in which the murine light (VK) and heavy (VH)
chain variable regions are joined to the human constant light
(kappa) and heavy (IgG.sub.1) chains.
[0016] This chimeric mAb retains the B-lymphoma and leukemia cell
targeting and internalization properties of the parental mLL2.
[0017] In another aspect of the invention, there is provided a hLL2
mAb related to mLL2 mAb, in which the complementarity-determining
regions (CDRs) of the light and heavy chains of the mLL2 mAb are
joined to the framework (FR) sequence of human VK and VH regions,
respectively, and subsequently to the human kappa and IgG.sub.1
constant region domains, respectively. This humanized antibody
retains the B-lymphoma and leukemia cell targeting and
internalizaiton characteristics of the parental mLL2 mAb, and can
exhibit a lowered HAMA reaction.
[0018] In still another aspect, there is provided isolated
polynucleotides comprising DNA sequences encoding the amino acid
sequences of the variable light and heavy chains, respectively, of
the hLL2 and cLL2 mAbs.
[0019] In an additional aspect, there is provided the amino acid
sequences of the CDRs of the VK and VH chains.
[0020] In yet another aspect, there are provided conjugates in
which the hLL2 or cLL2 mAb is covalently bonded to a diagnostic or
therapeutic reagent.
[0021] In still another aspect, there are provided methods whereby
the aforementioned mAb conjugates can be used to diagnose or treat
B-cell lymphomas and lymphocytic leukemias.
[0022] These and other aspects and embodiments of the invention
will become apparent by reference to the following specification
and appended claims.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 is a comparison of the murine with the humanized LL2
VK (FIG. 1A) and VH (FIG. 1B) domains. Only hFR sequences
(designated as REIHuVK and EUHuVH) different than mFR sequences
(designated as murine) are shown, and designated by asterisks. More
residues in these positions were retained in the humanized
structure. CDRs are boxed. FR residues showing CDR contacts by
computer modeling are underlined.
[0024] FIG. 2 shows vicinal relationships of the LL2 CDRs to their
framework regions (FRs). Separate energy-minimized models for the
VL and VH domains of mLL2 were constructed, and all FR residues
within a radius of 4.5 .ANG. or any CDR atom were identified as
potential CDR-FR contacts. CDRs of the light (L1,L2, and L3, FIG.
2A) and heavy (H1, H2, and H3, FIG. 2B)) chains are shown as "ball
and stick" representations superimposed on their respective,
space-filling FRs.
[0025] FIG. 3 shows the light chain (FIG 3A) staging (VKpBR) and
mammalian expression (pKH) vectors, and the heavy chain (FIG. 3B)
staging (VHpBS) and mammalian expression (pG1g) vectors.
[0026] FIG. 4 shows the double-stranded DNA and amino acid
sequences of the LL2 VK domain (FIG. 4A) and the LL2 VH domain
(FIG. 4B). Amino acid sequences encoded by the corresponding DNA
sequences are given as one letter codes. CDR amino acid sequences
are boxed. The Asn-glycosylation site located in FR1 of LL2VK (FIG.
4A) is shown as the underlined NVT sequence.
[0027] FIG. 5A shows the double stranded DNA and corresponding
amino acid residues of the hLL2 VK domain. CDR amino acid sequences
are boxed. The corresponding data for the VH domain is shown in
FIG. 5B.
[0028] FIG. 6 is a schematic diagram representation of the PCR/gene
synthesis of the humanized VH region and the subcloning into the
staging vector, VHpBS.
[0029] FIG. 7 shows SDS-PAGE analysis of mLL2 and cLL2 antibodies
under non-reducing (lanes 6-8) and reducing (lanes 3-5, light and
heavy chains) conditions. Lanes 3 and 6 include a control
antibody.
[0030] FIG. 8 shows SDS-PAGE analysis of different versions of cLL2
and hLL2 antibodies under reducing (lanes 3-5) and non-reducing
(lanes 6-8) conditions.
[0031] FIG. 9 shows SDS-PAGE anaylsis on mix-and-match cLL2 and
hLL2 antibodies under reducing (lanes 3-6) and non-reducing (lanes
7-10) conditions, cLL2 serves as the control.
[0032] FIG. 10. shows the results of a comparative Raji cell
competitive antibody binding assay involving mLL2 and cLL2
antibodies competing for binding to cells against tracer
radiolabeled mLL2.
[0033] FIG. 11 shows the results of a comparative Raji cell
competitive antibody binding assay in which mixed
humanized/chimeric LL2s were compared to cLL2 (FIG. 11A), and two
versions of hLL2 compared to cLL2 (FIG. 11B).
[0034] FIG. 12 shows a comparison of antibody
internalization:surface binding ratios as a function of time for
cLL2, cLL2 (Q to V mutagenesis), hLL2 and mLL2 antibodies.
[0035] FIG. 13 shows an SDS-PAGE analysis of mLL2 and cLL2 after
deglycosylation by endoglycosidase F.
[0036] FIG. 14 shows the effect of deglycosylation of mLL2 on its
binding affinity to Raji cells.
DETAILED DESCRIPTION OF THE INVENTION
[0037] cDNAs encoding the VL and VH regions of the mLL2 mAb have
been isolated and separately recombinantly subcloned into mammalian
expression vectors containing the genes encoding kappa and
IgG.sub.1 constant regions, respectively, of human antibodies.
Cotransfection of mammalian cells with these two recombinant DNAs
expressed a cLL2 mAb that, like the parent mLL2 mAb, bound avidly
to, and was rapidly internalized by, B-lymphoma cells.
[0038] The CDRs of the VK and VH DNAs have been similarly
recombinantly linked to the framework (FR) sequences of the human
VK and VH regions, respectively, which are subsequently linked,
respectively, to the human kappa and IgG.sub.1 constant regions, so
as to express in mammalian cells as described above hLL2.
[0039] In this specification, the expressions "cLL2" or "cLL2 mAb"
are intended to refer to the chimeric monoclonal antibody
constructed by joining or subcloning the murine VK and VH regions
to the human constant light and heavy chains, respectively. The
expressions "hLL2" or "hLL2 mAB" are intended to refer to the
humanization of the chimeric monoclonal antibody by replacing the
murine FR sequences in cLL2 with that of human framework
regions.
[0040] Covalent conjugates between cLL2 and hLL2 mAbs and a
diagnostic or chemotherapeutic reagent, formulated in
pharmaceutically acceptable vehicles (see, e.g., Remington's
Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton,
Pa., 1990) can be prepared that have the advantages, compared to
prior art antibody conjugates, of B-cell lymphoma-specific and
leukemia cell-specific targeting, rapid internalization into target
cells, rapid liberation of the diagnostic or chemotherapeutic
reagent intracellularly (thereby increasing effectiveness of the
reagent), and a potential reduction of the HAMA response in the
human patient.
[0041] As the VK-appended carbohydrate moiety of the cLL2 mAb is
shown herein not to be involved in binding to B-lymphoma cells, it
is preferred to use conjugates in which the reagent is bound to the
antibody through such carbohydrate moieties, such as through oxided
carbohydrate derivatives. Methods for the production of such
conjugates and their use in diagnostics and therapeutics are
provided, for expample, in Shih et al., U.S. Pat. No. 5,057,313,
Shih et al., Int. J. Cancer 41: 832 (1988), and copending, commonly
owned Hansen et al., U.S. Ser. No. 08/162,912, the contents of
which are incorporated herein by reference. Direct linkage of the
reagent to oxidized carbohydrate without the use of a polymeric
carrier is described in McKearn et al., U.S. Pat. No. 5,156,840,
which is also incorporated by reference.
[0042] A wide variety of diagnostic and therapeutic reagents can be
advantageously conjugated to the antibodies of the invention. These
include: chemotherapeutic drugs such as doxorubicin, methotrexate,
taxol, and the like; chelators, such as DTPA, to which detectable
labels such as fluorescent molecules or cytotoxic agents such as
heavy metals or radionuclides can be complexed; and toxins such as
Pseudomonas exotoxin, and the like. Several embodiments of these
conjugates are described in the examples below.
[0043] Cell lines and culture media used in the present invention
include LL2 (EPB-2) hybridoma cells (Pawlak-Byczkowska et al. 1989
above), Sp2/0-Ag14 myeloma cells (ATCC, Rockville, Md.) and Raji
cells. These cells are preferably cultured in Dulbecco's modified
Eagle's Medium (DMEM) supplemented with 10% FCS (Gibco/BRL,
Gaithersburg, Mass.), 2mM L-glutamine and 75 .mu.g/ml gentamicin,
(complete DMEM). Transfectomas are grown in Hybridoma Serum Free
Medium, HSFM, (Gibco/BRL, Gaithersburg, Mass.) containing 10% of
FCS and 75 .mu.g/ml gentamicin (complete HSFM) or, where indicated,
in HSFM containing only antibiotics. Selection of the transfectomas
may be carried out in complete HSFM containing 500 .mu.g/ml of
hygromycin (Calbiochem, San Diego, Calif.). All cell lines are
preferably maintained at 37.degree. C. in 5%CO.sub.2.
[0044] An important aspect of this invention is that antibody
variable domains can be modeled by computer modeling (see, for
example, Dion, in Goldenberg et al. eds., Cancer Therapy With
Radiolabeled Antibodies, CRC Press, Boca Raton, Fla., 1994) which
is incorporated by reference. In general, the 3-D structure for
both the mLL22 and hLL2 mAbs are best modeled by homology. The high
frequency of residue identities (75.0 to 92.3%) between the deduced
primary sequences of mLL2 light chain FR regions and human REI (VK)
facilitates this approach because of the availability of
crystallographic data from the Protein Data Bank (PDR Code 1REI,
Bernstein et al., J. Mol. Biol. 112: 535 (1977)), which is
incorporated by reference. Similarly, antibody EU (VH) sequences
can be selected as the computer counterparts for FR1 to FR3 of the
mLL2 heavy chain; FR4 was based on NEWM. As X-ray coordinate data
is currently lacking for the EU sequence. NEWM structural data (PDR
Code 3FAB) for FRs 1 to 4 can be used, and amino acid side groups
can be replaced to correspond to mLL2 or EU (hLL2) as needed. The
CDR of the light chain can be modeled from the corresponding
sequence of 1MCP (L1 and L2) and 1REI (L3). For heavy chain CDRs,
H1 and H2 can be based on 2HFL and 1MCP, respectively, while H3 can
be modeled de novo. Wherever possible, side group replacements
should be performed so as to maintain the torsion angle between
C.alpha. and C.beta.. Energy minimization may be accomplished by
the AMBER forcefield (Weiner et al, J. Amer. Chem. Soc. 106: 765
(1984) using the convergent method. Potentially critical FR-CDR
interactions can be determined by initially modeling the light and
heavy variable chains of mLL2. All FR residues within a 4.5 .ANG.
radius of all atoms within each CDR can thereby be identified and
retained in the final design model of hLL2.
[0045] Once the sequences for the hLL2 VK and VH domains are
designed, CDR engrafting can be accomplished by gene synthesis
using long synthetic DNA oligonucleotides as templates and short
oligonucleotides as primers in a PCR reaction. In most cases, the
DNA encloding the VK or VH domain will be approximately 350 bp
long. By taking advantage of codon degeneracy, a unique restriction
site may easily be introduced, without changing the encoded amino
acids, at regions close to the middle of the V gene DNA sequence.
For example, at DNA nucleotide positions 157-162 (amino acid
positions 53 and 54) for the hLL2 VH domain, a unique AvrII site
can be introduced while maintaining the originally designed amino
acid sequence (FIG. 4B). Two long non-overlapping single-stranded
DNA oligonucleotides (.about.150 bp) upstream and downstream of the
AvrII site (see, for example, oligo A and oligo B, Example 3 below)
can be generated by automated DNA oligonucleotide synthesizer
(Cyclone Plus DNA Synthesizer, Milligen-Biosearch). As the yields
of full length DNA oligonucleotides such as oligos A and B may be
expected to be low, they can be amplified by two pairs of flanking
oligonucleotides (oligo Seq. ID Nos. 7 and 8 for oligo A; oligo
Seq. ID Nos. 9 and 10 for oligo B, Example 3) in a PCR reaction.
The primers can be designed with the necessary restriction sites to
facilitate subsequent subcloning. Primers for oligo A and for oligo
B should contain overlapping sequence at the AvrII site so that the
resultant PCR product for oligo A and B, respectively, can be
joined in-frame at the AvrII site to form a full length DNA
sequence (ca 350 bp) encoding the hLL2 VH domain. The ligation of
the PCR products for oligo A (restriction-digested with PstI and
AvrII) and B (restriction-digested with AvrII and BstEII) at the
AvrII site and their subcloning into the PstII/BstEII sites of the
staging vector, VHpBs, can be completed in a single
three-fragment-ligation step (See, for example, Example 3). The
subcloning of the correct sequence into VHpBS can be first analyzed
by restriction digestion analysis and subsequently confirmed by
sequencing reaction according to Sanger et al., Proc. Natl. Acad.
Sci. USA 74: 5463 (1997).
[0046] The HinkIII/BamHI fragment containing the Ig promoter,
leader sequence and the hLL2 VH sequence can be excised from the
staging vector and subcloned to the corresponding sites in a
pSVgpt-based vector, pG1g, which contains the genomic sequence of
the human IgG constant region, an Ig enhancer and gpt selection
marker, forming the final expression vector, hLL2pG1g. Similar
strategies can be employed for the construction of the hLL2 VK
sequence. The restriction site chosen for the ligation of the PCR
products for the long oligonucloetides (oligos C and D, see
examples below) can be NruI in this case.
[0047] The DNA sequence containing the Ig promoter, leader sequence
and the hLL2 VK sequence can be excised from the staging vector
VKpBR by treatment with BamH1/HindIII, and can be subcloned into
the corresponding sites of a pSVhyg-based vector, pKh, which
contains the genomic sequence of human kappa chain constant
regions, a hygromycin selection marker, an Ig and a kappa enhancer,
forming the final expression vector, hLL2pKh.
[0048] As humanization sometimes results in a reduction or even
loss of antibody affinity, additional modification might be
required in order to restore the original affinity (See, for
example, Tempest et al., Bio/Technology 9: 266 (1991); Verhoeyen et
al., Science 239: 1534 (1988)), which are incorporated by
reference. Knowing that cLL2 exhibits a binding affinity comparable
to that of its murine counterpart (see Example 5 below), defective
designs, if any, in the original version of hLL2 can be identified
by mixing and matching the light and heavy chains of cLL2 to those
of the humanized version. SDS-PAGE analysis of the different
mix-and-match humanized chimeric LL2 under non-reducing (the
disulfide L-H chain connections remain intact) and reducing
condition (the chains separate, permitting analyses of the
relationships of the different types of light and heavy chains on
the properties of the molecule). For example, migrtation as
multiple bands or as a higher apparent molecular size can be due to
the presence of a glycan group at the N-linked glycosylation site
found at the FR1 region of the murine VK domain of LL2. For another
example, a discrete band migrating at about 25 kDa is the expected
molecular size for a non-glycosylated light chain.
[0049] In general, to prepare cLL2 mAb, VH and VK chains of mLL2
can be obtained by PCR cloning using DNA products and primers.
Orlandi et al., infra, and Leung et al., infra. The VK PCR primers
may be subcloned into a pBR327 based staging vector (VKpBR) as
described above. The VH PCR products may be subcloned into a
similar pBluescript-based staging vector (VHpBS) as described
above. The fragments containing the VK and VH sequences, along with
the promoter and signal peptide sequences, can be excised from the
staging vectors using HindIII and BamHI restriction endonucleases.
The VK fragments (about 600 bp) can be subcloned into a mammalian
expression vector (for example, pKh) conventionally. pKh is a
pSVhyg-based expression vector containing the genomic sequence of
the human kappa constant region. an Ig enhancer, a kappa enhancer
and the hygromucin-resistant gene. Similarly, the about 800 bp VH
fragments can be subcloned into pG1g, a pSVgpt-based expression
vector carrying the genomic sequence of the human IgG1 constant
region, an Ig enhancer and the xanthine-guanine phosphoribosyl
transferase (gpt) gene. The two plasmids may be transfected into
mammalian expression cells, such as Sp2/0-Ag14 cells, by
electroporation and selected for hygromycin resistance. Colonies
surviving selection are expanded, and supernatant fluids monitored
for production of cLL2 mAb by an ELISA method. A transfection
efficiency of about 1-10.times.10.sup.6 cells is desirable. An
antibody expression level of between 0.10 and 2.5 .mu.g/ml can be
expected with this system.
[0050] RNA isolation, cDNA synthesis, and amplification can be
carried out as follows. Total cell RNA can be prepared from a LL2
hybridoma cell line, using a total of about 10.sup.7 cells,
according to Sambrook et al., (Molecular Cloning: A Laboratory
Manual, Second ed., Cold Spring Harbor Press, 1989), which is
incorporated by reference. First strand cDNA can be reverse
transcribed from total RNA conventionally, such as by using the
SuperScript preamplification system (Gibco/BRL., Gaithersburg,
Md.). Briefly, in a reaction volume of 20 .mu.l, 50 ng of random
primers can be annealed to 5 .mu.g of RNAs in the presence of 2
.mu.l of 10.times.synthesis buffer [200 mM Tris-HCl (pH 8.4), 500
mM KCl, 25 mM MgCl.sub.2, 1 mg/ml BSA], 1 .mu.l of 10 mM dNTP mix,
2 .mu.l of 0.1 M DTT, and 200 units of SuperScript reverse
transcriptase. The elongation step is initially allowed to proceed
at room temperature for 10 min followed by incubation at 42.degree.
C. for 50 min. The reaction can be terminated by heating the
reaction mixture at 90.degree. C. for 5 min.
[0051] The VK and VH sequences of cLL2 or hLL2 can amplified by PCR
as described by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86:
3833 (1989)) which is incorporated by reference. VK sequences may
be amplified using the primers CK3BH and VK5-3 (Leung et al.,
BioTechniques, 15: 286 (1993), which is incorporated by reference),
while VH sequences can be amplified using the primer CH1B which
anneals to the CH1 region of murine 1gG, and VHIBACK (Orlandi et
al., 1989 above). The PCR reaction mixtures containing 10 .mu.l of
the first strand cDNA product, 9 .mu.l of 10.times. PCR buffer [500
mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgC12, and 0.01% (w/v)
gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), can be subjected to
30 cycles of PCR. Each PCR cycle preferably consists of
denaturation at 94.degree. C. for 1 min, annealing at 50.degree. C.
for 1.5 min, and polymerization at 72.degree. C. for 1.5 min.
Amplified VK and VH fragments can be purified on 2% agarosse
(BioRad, Richmond, Calif.). See Example 3 for a method for the
synthesis of an oligo A (149-mer) and an oligo B (140-mer) on an
automated Cyclone Plus DNA synthesizer (Milligan-Biosearch) for use
in constructing humanized V genes.
[0052] PCR products for VK can be subcloned into a staging vector,
such as a pBR327-based staging vector VKpBR that contains an Ig
promoter, a signal peptide sequence and convenient restriction
sites to facilitate in-frame ligation of the VK PCR products. PCR
products for VH can be subcloned into a similar staging vector,
such as the pBluescript-based VHpBS. Individual clones containing
the respective PCR products may be sequenced by, for example, the
method of Sanger et al., Proc. Natl. Acad. Sci., USA, 74: 5463
(1977) which is incorporated by reference.
[0053] The DNA sequences described herein are to be taken as
including all alleles, mutants and variants thereof, whether
occurring naturally or induced.
[0054] The two plasmids can be co-transfected into an appropriate
cell, e.g., myeloma Sp2/0-Ag14, colonies selected for hygromycin
resistance, and supernatant fluids monitored for produciton of cLL2
or hLL2 antibodies by, for example, an ELISA assay, as described
below.
[0055] Transfection, and assay for antibody secreting clones by
ELISA, can be carried out as follows. About 10 .mu.g of hLL2pKh
(light chain expression vector) and 20 .mu.g of hLL2pG1g (heavy
chain expression vector) can be used for the transfection of
5.times.10.sup.6 SP2/0 myeloma cells by electroporation (BioRad,
Richmond, Calif.) according to Co et al., J. Immunol., 148: 1149
(1992) which is incorporated by reference. Following transfection,
cells may be grown in 96-well microtiter plates in complete HSFM
medium (GIBCO, Gaithersburg, Md.) at 37.degree. C., 5% CO.sub.2.
The selection process can be initiated after two days by the
addition of hygromycin selection medium (Calbiochem, San Diego,
Calif.) at a final concentration of 500 .mu.g/ml of hygromycin.
Colonies typically emerge 2-3 weeks post-electroporation. The
cultures can then be expanded for further analysis.
[0056] Transfectoma clones that are positive for the secretion of
chimeric or humanized heavy chain can be identified by ELISA assay.
Briefly, supernatant samples (100 .mu.l) from transfectoma cultures
are added in triplicate to ELISA microtiter plates precoated with
goat anti-human (GAH)-IgG, F(ab').sub.2 fragment-specific antibody
(Jackson ImmunoResearch, West Grove, Pa.). Plates are incubated for
1 h at room temperature. Unbound proteins are removed by washing
three times with wash buffer (PBS containing 0.05% polysorbate 20).
Horseradish peroxidase (HRP) conjugated GAH-IgG, Fc
fragment-specific anitbodies (Jackson ImmunoResearch, West Grove,
Pa.) are added to the wells, (100 .mu.l of antibody stock diluted
.times.10.sup.4, supplemented with the unconjugated antibody to a
final concentration of 1.0 .mu.g/ml). Following an incubation of 1
h, the plates are washed, typically three times. A reaction
solution, [100 .mu.l, containing 167 .mu.g of
orthophenylene-diamine (OPD) (Sigma, St. Louis, Mo.), 0.025%
hydrogen peroxide in PBS], is added to the wells. Color is allowed
to develop in the dark for 30 minutes. The reaction is stopped by
the addition of 50 .mu.l of 4 N HCl solution into each well before
measuring absorbance at 490 nm in an automated ELISA reader
(Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodies are
than determined relative to an irrelevant chimeric antibody
standard (obtainable from Scotgen, Ltd., Edinburg, Scotland).
[0057] Antibodies can be isolated from cell culture media as
follows. Transfectoma cultures are adapted to serum-free medium.
For production of chimeric antibody, cells are grown as a 500 ml
culture in roller bottles using HSFM. Cultures are centrifuged and
the supernatant filtered through a 0.2 micron membrane. The
filtered medium is passed through a protein A column (1.times.3 cm)
at a flow rate of 1 ml/min. The resin is then washed with about 10
column volumes of PBS and protein A-bound antibody is eluted from
the column with 0.1 M glycine buffer (pH 3.5) containing 10 mM
EDTA. Fractions of 1.0 ml are collected in tubes containing 10
.mu.l of 3 M Tris (pH 8.6), and protein concentrations determined
from the absorbancies at 280/260 nm. Peak fractions are pooled,
dialyzed against PBS, and the antibody concentrated, for example,
with the Centricon 30 (Amicon, Beverly, Mass.). The antibody
concentration is determined by ELISA, as before, and its
concentration adjusted to about 1 mg/ml using PBS. Sodium azide,
0.01% (w/v), is conveniently added to the sample as
preservative.
[0058] Comparative binding affinities of the mLL2, cLL2 and hcLL2
antibodies thus isolated may be determined by direct
radioimmunoassay. mLL2 can be labeled with .sup.131I or .sup.125I
using the chloramine T method (see, for example, Greenwood et al.,
Biochem. J., 89: 123 (1963) which is incorporated by reference).
The specific activity of the iodinated antibody is typically
adjusted to about 10 .mu.Ci/.mu.g. Unlabeled and labeled antibodies
are diluted to the appropriate concentrations using reaction medium
(HSFM supplemented with 1% horse serum and 100 .mu.g/ml
gentamicin). The appropriate concentrations of both labeled and
unlabeled antibodies are added together to the reaction tubes in a
total volume of 100 .mu.l. A culture of Raji cells is sampled and
the cell concentration determined. The culture is centrifuged and
the collected cells washed once in reaction medium followed by
resuspension in reaction medium to a final concentration of about
10.sup.7 cells/ml. All procedures are carried out in the cold at
4.degree. C. The cell suspension, 100 .mu.l, is added to the
reaction tubes. The reaction is carried out at 4.degree. C. for 2 h
with periodic gentle shaking of the reaction tubes to resuspend the
cells. Following the reaction period, 5 ml of wash buffer (PBS
containing 1% BSA) is added to each tube. The suspension is
centrifuged and the cell pellet wahed a second time with another 5
ml of wash buffer. Following centrifugation, the amount of
remaining radioactivity remaining in the cell pellet is determined
in a gamma counter (Minaxi, Packard Instruments, Sterling,
Va.).
[0059] The Raji cell surface antigen binding affinities of
mix-and-match and fully humanized antibodies can be compared to
that of cLL2 using various concentrations of mLL2 F(ab').sub.2
fragments devoid of the Fc portion as competitors, as evaluated by
flow cytometry assay. Residual surface-bound LL2 antibodies
carrying the human Fc portions (cLL2 and mix-and-match LL2) can be
detected by a FITC-labeled anti-human Fc specific antibody in a
flow cytometry assay. Where mix-and-match LL2 antibodies exhibit
antigen-binding affinities similar to that of cLL2, it can be
concluded that the original designs for the humanization of both
the light and heavy chains reatin the mLL2 immunoreactivity.
[0060] The internalization of mLL2, cLL2 and hLL2 antibodies into
target cells can be followed by fluorescence labeling, essentially
according to the procedure of Pirker et al., J. Clin. Invest., 76:
1261 (1985), which is incorporated by reference. Cultured Raji
cells are centrifuged and the cells resuspended in fresh medium to
a concentration of about 5.times.10.sup.6 cells/ml. To each well of
a 96-well microtiter plate, 100 .mu.l of the cell suspension is
added. The antibodies, 40 .mu.g/ml, in a volume of 100 .mu.l are
added to the reaction wells at timed intervals so as to terminate
all reations simultaneously. The plate is incubated at 37.degree.
C. in a CO.sub.2 cell culture incubator. Unbound antibodies are
removed by washing the cells three times with cold 1% FCS/PBS at
the end of the incubation. The cells are then treated with 1 ml of
Formaid-Fresh [10% formalin solution (Fisher, Fair Lawn, N.J.)] for
15 min at 4.degree. C. After washing, antibodies present either on
the cell surface or inside the cells are detected by treatment with
FITC-labeled goat anti-mouse antibody (Tago, Burlingame, Calif.),
or FITC-labeled goat anti-human antibody (Jackson ImmunoResearch,
West Grove, Pa.), depending on whether the antibody being assayed
for is murine, chimeric, or humanized, respectively. Fluorescence
distribution are evaluated using a BH-2 flourescence microscope
(Olympus, Lake Success, N.Y.).
[0061] The rate of antibody internalization can be determined
according to Opresko et al., (J. Biol. Chem., 262: 4116 (1987)),
using radioiodinated antibody as tracer. Briefly, radiolabeled
antibodies (1.times.10.sup.4 cpm) are incubated with Raji cells
(1.times.10.sup.6 cells/ml) at 4.degree. C. for 2 h in 0.5 ml of
DMEM medium containing 1% human serum. Following the reaction
interval, non-specifically bound antibodies are removed by washing
three times with 0.5 ml of DMEM medium. To each of the reaction
tubes 0.5 ml of DMEM medium is added and the suspension incubated
at 37.degree. C. for the determination of internalization. At timed
intervals, triplicates of cells are removed and chilled immediately
in an ice bath to stop further internalization. Cells are
centrifuged at 1000.times.g for 5 min at 4.degree. C. The
supernatant is removed and counted for radioactivity. The
surface-bound radioactivity is removed by treatment with 1 ml 0.1 M
acetate/0.1 M glycine buffer at pH 3.0 for 8 min. inh the cold.
Radioactivity removed by the acid treatment, and that remaining
associated with the cells, are determined. The ratio of the
CPM.sub.internalization/CPM.sub.surface is plotted versus time to
determine the rate of internalization from the slope.
[0062] Detailed protocols for oligonucleotide-directed mutagenesis
and related techniques for mutagenesis of cloned DNA are
well-known. For example, see Sambrook et al. and Ausubel et al.
above.
[0063] Asn-linked glycosylation sites may be introduced into
antibodies using conventional site-directed oligonucleotide
mutagenesis reactions. For example, to introduce an Asn in position
18 of a kappa protein, one may alter codon 18 from AGG to AAC. To
accomplish this, a single stranded DNA templated containing the
antibody light chain sequence is prepared from a suitable strain of
E. coli (e.g., dut.sup.31ung-) in order to obtain a DNA molecule
containing a small number of uracils in place of thymidine. Such a
DNA template can be obtained by M13 cloning or by in vitro
transcription using a SP6 promoter. See, for example, Ausubel et
al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, NY, 1987. An oligonucleotide containing the mutated sequence
is synthesized conventionally, annealed to the single-stranded
template and the product treated with T4 DNA polymerase and T4 DNA
ligase to produce a double-stranded DNA molecule. Transformation of
wild type E. coli (dut.sup.30 ung.sup.30 ) cells with the
double-stranded DNA provides an efficient recovery of mutated
DNA.
[0064] Alternatively, an Asn-linked glycosylation site can be
introduced into an antibody light chain using an oligonucleotide
containing the desired mutation as the primer and DNA clones of the
variable regions for the VL chain, or by using RNA from cells that
produce the antibody of interest as a template. Also see, Huse, in
ANTIBODY ENGINEERING: A PRACTICAL GUIDE, Boerrebaeck, ed., W. H.
Freeman & Co., pp 103-120, 1992. Site-directed mutagenesis can
be performed, for example, using the TRANSFORMER.TM. kit (Clontech,
Palo Alto, Calif.) according to the manufacturer's
instructions.
[0065] Alternatively, a glycosylation site can be introduced by
synthesizing an antibody chain with mutually priming
oligonucleotides, one such containing the desired mutation. See,
for example, Uhlmann, Gene 71: 29 (1988); Wosnick et al., Gene 60:
115 (1988); Ausubel et al., above, which are incorporated by
reference.
[0066] Although the general description above referred to the
introduction of an Asn glycosylation site in position 18 of the
light chain of an antibody, it will occur to the skilled artisan
that it is possible to introduce Asn-linked glycosylation sites
elsewhere in the light chain, or even in the heavy chain variable
region.
[0067] The representative embodiments described below are simply
used to illustrate the invention. Those skilled in these arts will
recognize that variations of the present materials fall within the
broad generic scope of the claimed invention. The contents of all
references mentioned herein are incorporated by reference.
EXAMPLE 1
Choice of Human Frameworks and Sequence Design for the Humanization
of LL2 Monoclonal Antibody
[0068] By comparing the murine variable (V) region framework (FR)
sequences of LL2 to that of human antibodies in the Kabat data base
(Kabat et al., Sequences of Proteins of Immunological Interest, 5th
ed., U.S. Department of Health and Human Services, U.S. Government
Printing Office, Washington, D.C.), which is incorporated by
reference, the human REI (FIG. 1A, Sequence ID No. 1) and EU (FIG.
1B, Sequence ID No. 2) sequences were found to exhibit the highest
degree of sequence homology to the FRs of VK and VH domains of LL2,
respectively. Therefore, the REI and EU FRs were selected as the
human frameworks onto which the CDRs for LL2 VK and VH were
grafted, respectively. The FR4 sequence of NEWM, however, rather
than that of EU, was used to replace the EU FR4 sequence for the
humanization of LL2 heavy chain. Based on the results of computer
modeling studies (FIGS. 2A and 2B), murine FR residues having
potential CDR contacts, which might affect the affinity and
specificity of the resultant antibody, were retained in the design
of the humanized FR sequences (FIG. 1).
[0069] Two versions of humanized heavy chain were constructed. In
the first version (hLL2-1), the glutamine (Q) at amino acid
position 5 (Kabat numbering) was introduced to include a PstI
restriction site to facilitate its subcloning into the staging
vector (FIG. 3). This murine residue was converted, by
oligo-directed mutagenesis, to the human EU residue valine (V) in
hLL2-2. It should be noted that in the original murine kappa chain
variable sequence, a potential N-linked glycosylation site was
identified at positions 18-20 (FIG. 1) and was used for
carbohydrate addition. This glycosylation site was not included in
the REI FR sequence used for LL2 light chain humanization.
[0070] See Example 3 for more oligonucleotide detail.
EXAMPLE 2
PCR Cloning and Sequence Elucidation for LL2 Heavy and Light Chain
Variable Regions
[0071] The variable regions for both heavy (VH) and light (VK)
chains of mLL2 (IgG2a) were obtained by PCR cloning using DNA
primers as described in general above and in greater detail in
Example 3, below. As PCR is prone to mutation, the variable region
sequence of multiple individual clones for either the heavy or
light chains was determined for six clones and confirmed to be
identical prior to use for the construction of the chimeric
antibody.
[0072] The PCR products for VK were subcloned into a pBR327-based
staging vector, VKpBR, which contained an Ig promoter, a signal
peptide sequence and convenient restriction sites to facilitate
in-frame ligation of the VK PCR products (FIG. 3A). The PCR
products for VH were subcloned into a similar pBluescript-based
staging vector, VHpBS (FIG. 3B).
[0073] As noted above, at least six individual clones containing
the respective PCR products were sequenced according to the method
of Sanger et al., 1977, above. All were shown to bear identical
sequences and their respective sequences were elucidated, as shown
in FIG. 4A for LL2 VK (Sequence ID NO. 3) and FIG. 4B for LL2 VH
(Sequence ID NO. 4). No defective mutations were identified within
the sequences encoding the VK and VH regions. Comparison of the
PCR-amplified variable region sequences of LL2 with the Kabat
database (Kabat et al., above) suggested that the VK and VH
sequences of LL2 belong to subgroup 5 and 2B, respectively.
Important residues such as Cys for intra-domain disulfide linkage
were retained at appropriate positions.
[0074] In the FR1 framework region of VK, an N-linked carbohydrate
attachment site, Asn-Val-Ser, was identified at position 18-20
(FIG. 4A), suggesting that the VK of LL2 might be glycosylated. As
will be detailed below, SDS-PAGE analysis under reducing conditions
demonstrated that this Asn glycosylation site is indeed utilized
for carbohydrate addition. The presence of the glycosylation site
in the variable region does not, however, appear to affect the
immunoreactivity of the antibody. A comparison of the
immunoreactivity of mLL2 with that of cLL2 in a competitive RIA
showed that the two antibodies have nearly identical
activities.
EXAMPLE 3
PCR/Gene Synthesis of the Humanized V Genes
[0075] The designed sequence for the hLL2 VH domain, the
construction of the hLL2 VH domain by long oligonucleotides and
PCR, and the staging vector VHpBS containing the hLL2 VH domain are
summarized in the sketch shown in FIG. 6.
[0076] For the construction of the hLL2 VH domain, oligo A
(149-mer) and oligo B(140-mer) were synthesized on an automated
CYCLONE PLUS.TM. DNA synthesizer (Milligen Bioresearch).
[0077] Oligo A (Sequence ID No. 7 below) represents the minus
strand of the hLL2 VH domain complementary to nt 24 to 172.
1 Sequence ID No. 7 5'-TAT AAT CAT TCC TAG GAT TAA TGT ATC CAA TCC
ATT CCA GAC CCT GTC CAG GTG CCT GCC TGA CCC AGT GCA GCC AGT AGC TAG
TAA AGG TGT AGC CAG AAG CCT TGC AGG AGA CCT TCA CTG ATG ACC CAG GTT
TCT TGA CTT CAG CC-3'
[0078] Oligo B (Sequence ID No. 8 below) represents the minus
strand of the hLL2 VH domain complementary to nt 180 to 320.
2 Sequence ID No. 8 5'-CCC CAG TAG AAC GTA ATA TCC CTT GCA CAA AAA
TAA AAT GCC GTG TCC TCA GAC CTC AGG CTG CTC AGC TCC ATG TAG GCT GTA
TTG GTG GAT TCG TCT GCA GTT ATT GTG GCC TTG TCC TTG AAG TTC TGA
TT-3'
[0079] Oligos A and B were cleaved from the support and deprotected
by treatment with concentrated ammonium hydroxide. After the sample
were vacuum-dried (SpeedVac, Savant, Farmingdale, N.Y.) and
resuspended in 100 .mu.l of water, incomplete oligomers (less than
100-mer) were removed by centrifugation through a
CHROMOSPIN-100.TM. column (Clonetech, Palo Alto, Calif.) before the
DNA oligomers were amplified by PCR. All flanking primers for the
seperate amplifications and PCR cloning of oligos A and B were
purified by SDS-PAGE essentially according to the methods of
Sambrook et al., 1989, above. From the CHROMASPIN-purified oligo A,
1 .mu.l of sample stock was PCR-amplified in a reaction volume of
100 .mu.l by adding 5 .mu.l of 5 .mu.M of oligo Sequence ID No.
9:
[0080] 5'-CCA GCT GCA GCA ATC AGG GGC TGA AGT CAA GAA ACC TG-3'
[0081] and oligo Sequence ID No. 10:
[0082] 5'-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA ATG-3'
[0083] in the presence of 10 .mu.l of 10.times.PCR Buffer (500 mM
KCl, 100 mM Tris.HCL buffer, pH 8.3, 15 mM MgCl.sub.2) and 5 units
of AMPLITAQ.TM. DNA polymerase (Perkin Elmer Cetus, Norwalk,
Conn.). This reaction mixture was subjected to 30 cycles of PCR
reaction consisting of denaturation at 94.degree. C. for 1 minute,
annealing at 50.degree. C. for 1.5 minutes, and polymerization at
72.degree. C. for 1.5 minutes.
[0084] Oligo B was PCR-amplified by the primer pairs Sequence ID
No. 11:
3 5'-TAA TCC TAG GAA TGA TTA TAC TGA GTA CAA TCA GAA CTT CAA GGA
CCA G-3'
[0085] and Sequence ID No. 12:
[0086] 5'-GGA GAC GGT GAC CGT GGT GCC TTG GCC CCA GTA GAA CGT AGT
AA-3'under similar conditions.
[0087] Double-stranded PCR-amplified products for oligos A and B
were gel-purified, restriction-digested with PstI/AvrII (PCR
product of oligo A) and BstEII/AvrII (PCR product of oligo B), and
sucloned into the complementary PstI/BstEII sites of the heavy
chain staging vector, VHpBS. The humanized VH sequence was
subcloned into the pG1g vector, resulting in the final human IgG1
heavy chain expression vector, hLL2pG1g.
[0088] For constructing the full length DNA of the humanized VK
sequence, oligo E (150-mer) and oligo F (121-mer) were synthesized
as described above.
[0089] Oligo E Sequence ID No. 13:
4 5'-CCT AGT GGA TGC CCA GTA GAT CAG CAG TTT AGG TGC TTT CCC TGG
TTT CTG GTG GTA CCA GGC CAA GTA GTT CTT GTG ATT TGC ACT GTA TAA AAC
ACT TTG ACT GGA CTT ACA GCT CAT AGT GAC CCT ATC TCC AAC AGA TGC GCT
CAG-3'
[0090] represents the minus strand of the humanized VK domain
complementary to nt 31 to 180, and this sequence was PCR-amplified
by oligo Sequence ID No. 14:
5 5'-GAC AAG CTT CAG CTG ACC CAG TCT CCA TCA TCT CTG AGC GCA TCT
GTT GGA G-3'
[0091] and oligo Sequence ID No. 15
6 5'-AGA GAA TCG CGA AGG GAC ACC AGA TTC CCT AGT GGA TGC CCA
GTA-3'.
[0092] Oligo F Sequence ID No. 16:
7 5'-GCA CCT TGG TCC CTC CAC CGA ACG TCC ACG AGG AGA GGT ATT GGT
GAC AAT AAT ATG TTG CAA TGT CTT CTG GTT GAA GAG AGC TGA TGG TGA AAG
TAA AAT CTG TCC CAG ATC CGC TGC C-3'
[0093] represents the minus strand of the humanized LL2 VK domain
complementary to nt 208 to 327, and was PCR amplified by oligo
Sequence ID No. 17:
[0094] 5'-GAC AAG CTT TCG CGA TTC TCT GGC AGC GGA TCT GGG ACA
G-3'
[0095] and oligo Sequence ID No. 18:
[0096] 5'-GAC CGG CAG ATC TGC ACC TTG GTC CCT CCA CCG-3'.
[0097] Gel-purified PCR products for oligos E and F were
restriction-digested with PvuII/NruI and NruI/BglIII, respectively.
The two PCR fragments E and F were then joined at the NruI site and
ligated to the complementary PvuI/BcII sites of the light chain
staging vector, VKpBR. The humanized VK sequence was subcloned into
vector pKh to form the final human kappa chain expression vector,
hLL2pKh.
[0098] To express the humanized antibodies, about 10 .mu.g of
linearized hLL2pKh and 20 .mu.g of linearized hLL2pG1g were used to
transfect 5.times.10.sup.6 SP2/0 cells by electroporation. The
transfectomas were selected with hygromycin at 500 .mu.g/ml and
secreted antibody was purified on a 1.times.3 cm column of protein
A. After concentrating the purified antibody by Centricon 30
centifugation, antibody concentration was determined by ELISA. The
final concentration of the antibody was adjusted to 1 mg/ml in PBS
buffer containing 0.01% (w/v) sodium azide as a preservative.
[0099] FIG. 1 (Sequence ID Nos. 1 and 2), there is compared the
amino acid sequence between murine and humanized LL2 VK domains
(FIG. 1A) and between murine and humanized LL2 VH domains (FIG.
1B). In the VK chain, human REI framework sequences were used for
all FRs. In the VH chain, human EU framework sequences were used
for FR 1-3, and NEWM sequences were used for FR-4. Only human FR
sequences that are different from that of the mouse are shown.
Asterisks indicate murine FR sequences that are different form that
of the human FR at corresponding positions. Murine residues at
these positions were retained in the humanized structure. CDRs are
boxed.
[0100] In FIG. 4A (Sequence ID No. 3) there are shown the double
stranded DNA and corresponding amino acid sequences (shown by
single letter code) of the humanized LL2 VK domain. CDR 1-3 amino
acid sequences are boxed. The corresponding display for VH is shown
in FIG. 4B (Sequence ID No. 4).
[0101] In FIG. 5A (Sequence ID No. 5) and FIG. 5B (Sequence ID No.
6) there are shown double-stranded DNA sequences and amino acid
sequences of humanized LL2 VK and LL2 VH, respectively. Amino acid
sequences are shown by the single-letter code, and CDR amino acid
sequences are boxed.
EXAMPLE 4
Construction, Expression and Purification of Chimeric LL2
Antibodies
[0102] The fragments containing the VK and VH sequences of LL2,
together with the promoter and signal peptide sequences, were
excised from LL2VKpBR and LL2VHpBS, respectively, by double
restriction digestion with HindIII and BamHI. The about 600 bp VK
fragments were then subcloned into the HindIII/BamHI site of a
mammalian expression vector, pKh (FIG. 3A). pKh is a pSVhyg-based
expression vector containing the genomic sequence of the human
kappa constant region, an Ig enhancer, a kappa enhancer and the
hygromycin-resistant gene. Similarly, the ca. 800 bp VH fragments
were subcloned into the corresponding HindIII/BamHI site of pG1g
(FIG. 3B), a pSVgpt-based expression vector carrying the genomic
sequence of the human IgG1 constant region, an Ig enhancer and the
xanthine-guanine phosphoribosyltransferase (gpt) gene. The final
expression vectors are designated as LL2pKh and LL2pG1g,
respectively.
[0103] The two plasmids were co-transfected into Sp2/0-Ag14 cells
by electroporation and selected for hygromycin resistance.
Supernatants from colonies surviving selection were monitored for
chimeric antibody secretion by ELISA assay (see above). The
transfection efficiency was approximately 1-10.times.10.sup.6
cells. The antibody expression level, in a terminal culture, was
found to vary in the range between <0.10 and 2.5/.mu.g/ml.
[0104] FIG. 7 shows the results of analyzing protein A-purified
mLL2 (lanes 4 and 7) and cLL2 (lanes 5 and 8) by SDS-PAGE under
reducing and non-reducing conditions, respectively. HMW stands for
high molecular weight protein markers, and LMW for light molecular
weight markers. The light chains of both mLL2 and cLL2 (lanes 4 and
5) migrated primarily as a doublet band, with a higher than
expected apparent molecular weight. As the human kappa constant
region of cLL2 is know to contain no potential glycosylation site,
it can be inferred that the potential glycosylation site identified
in the FR1 region of LL2 VK domain was utilized.
[0105] FIG. 8 shows the results of analyzing different versions of
hLL2 and cLL2 antibodies by SDS-PAGE under reducing and
non-reducing conditions. As before, LMW and HMW are molecular
weight markers. Lanes 3 and 6 are cLL2 antibodies. Lanes 4 and 7
are hLL2 with seven murine FR residues in the VH domain (hLL2-1).
Lanes 5 and 8 are hLL2 with 6 murine FR residues in the VH domain
(hLL2-2). The humanized light chains migrated more rapidly and as
more discrete bands compared to chimeric light chains.
[0106] FIG. 9 shows the results of SDS-PAGE analysis on
mix-and-match and cLL2 and hLL2 antibodies under both reducing and
non-reducing conditions. Lanes 1 and 2 are molecular weight
markers. Lanes 3 and 7 are cLL2. Lanes 4 and 8 are mix-and-match
with a humanized light and chimeric heavy chain [(hL/cH)LL2]. Lanes
5 and 9 are chimeric light and humanized heavy (Version 1) chains
[(cL/hH)LL2-1]. Lanes 6 and 10 are chimeric light and a humanized
heavy (version 2) chains [(cL/hH)LL2-2]. The humanized LL2 version
1 contains 7 murine FR residues in the VH domain, while version 2
contains 6 murine FR residues in the VH domain. It is noteworthy
that the position of the light chain of (hL/cH)LL2 (lane 4) is
different from that of the others, suggesting that there is no
carbohydrate attachment to the humanized LL2 light chain.
EXAMPLE 5
Binding of cLL2 Antibody to Raji Cell Surface Antigens
[0107] A competition cell binding assay was carried out to assess
the immunoreactivity of cLL2 relative to the parent mLL2. Using
.sup.131I-labeled mLL2 (0.025 .mu.g/ml) as a probe, Raji cells were
incubated with the antibodies and the relative binding to the cells
determined from the amount of cell-bound labeled mLL2 (see above).
As shown by the competition assays described in FIG. 10, both mLL2
and cLL2 antibodies exhibited similar binding activities.
[0108] The results were confirmed by a second competition assay
based on flow cytometry. Briefly, using Raji cells as before and
varying the concentration of one antibody relative to other, as
before, the amount of bound mLL2 or cLL2 was determined with
FITC-labeled anti-mouse Fc or anti-human Fc antibodies followed by
analysis using flow cytometry.
EXAMPLE 6
Binding of hLL2 Antibodies to Raji Cells
[0109] In experiments similar to those of Example 5, the antigen
binding affinities of the three different combinations of
mix-and-match or humanized LL2 were compared with that of cLL2 in
the flow cytometry assay.
[0110] Briefly, 1 .mu.g of cLL2, mix-and-match LL2, hLL2-1 or
hLL2-2 antibodies were incubated with 10.sup.8 Raji cells in the
presence of varying concentrations of mLL2 F(ab').sub.2 fragments
(as competitor) in a final volume of 100 .mu.l of PBS buffer
supplemented with 1% FCS and 0.01% sodium azide. The mixture was
incubated for 30 minutes at 4.degree. C., and washed three times
with PBS to remove unbound antibodies. By taking advantage of the
presence of human Fc portions in the antibodies, the binding levels
of the antibodies were assessed by adding a 20.times.diluted
FITC-labeled goat anti-human IgG1, Fc fragment-specific antibodies
(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed
three times with PBS, and fluorescence intensities measured by a
FACSCAN fluorescence activated cell sorter (Becton-Dickinson,
Bedford, Mass.). The results are shown in FIG. 11A.
[0111] Using the same methods, cLL2 was compared to two versions of
hLL2 (FIG. 11B).
[0112] The results shown in FIGS. 11A and B demonstrate that the
immunoreactivity of cLL2 is similar or identical to that of
humanized or mix-and-match antibodies. Taken together with the
comparison of cLL2 with mLL2 (FIG. 10), the authenticity of the
sequences for chimeric and humanized VK and VH obtained is
established, and the functionality of cLL2 and hLL2 confirmed.
EXAMPLE 7
Internalization of mLL2 and cLL2 by Raji Cells
[0113] One of the unique characteristics of the LL2 antibody is its
rapid internalization upon binding to Raji cells (Shih et al., 1994
above). Murine LL2 after internalization is likely to be rapidly
transferred to the Golgi apparatus and from there to the lysosomes,
the organelle responsible for the degradation of a wide variety of
biochemicals (Keisari et al., Immunochem., 10: 565 (1973)).
[0114] Rates of antibody internalization were determined according
to Opresko et al., 1987 above. The ratio of
CPM.sub.intracellular/CPM.sub.su- rface was determined as a
function of time.
[0115] Rates of LL2 antibody internalization were determined by
incubating radiolabeled LL2 antibody (1.times.10.sup.6 cpm) with
0.5.times.10.sup.6 Raji cells in 0.5 ml of DMEM buffer containing
1% human serum for 2 hrs. at 4.degree. C. Excess human serum was
included to saturate Raji cell surface Fc receptors in order to
exclude or minimize non-antigen-specific internalization mediated
through the Fc receptors. Unbound radiolabeled LL2 antibodies were
removed from the cells by washing three times with 0.5 ml portions
of DMEM at 4.degree. C. Cells were then incubated at 37.degree. C.,
and, at timed intervals, aliquots of the cell suspension were
transferred to ice in order to stop internalization. The cells in
these aliquots were isolated by centrifugation at 1,000.times.g for
5 mins. at 4.degree. C., and surface bound radiolabeled LL2
stripped off cells with 1 ml of 0.1 M glycine acetate buffer, pH 3,
for 8 mins. at 4.degree. C. Radioactivity thus obtained (CPM
surface) and radioactivity remaining in the cells (CPM
intracellular) were determined. Rates of internalization were
calculated from the slope of the plot of intracellular:surface
radioactivity ratios as a function of time.
[0116] As shown in FIG. 12, mLL2, cLL2, cLL2Q and hLL2 antibodies
were internalized at a similar rate (Ke=0.107 (mLL2) to 0.1221
(cLL2Q, NVT to QVT mutation). Those numbers suggested that
approximately 50% of the surface-bound antibody could be
internalized in 10 min. The results show that neither chimerization
nor humanization nor deglycosylation by mutagenesis of mLL2
antibodies impair rates of internalization.
[0117] The pattern of internalization for mLL2, cLL2 and hLL2 was
also monitored by fluorescence microscopy on a time-course basis
using a FITC-labeled second antibody probe as described in the
specification. Internalization of both antibodies was observed in
at the earliest time point measurable. At 5 minutes, antibodies
were seen both on the cell surface and internalized in areas
immediately adjacent to the membrane as cytoplasmic micro-vesicles.
At 15 min. post-incubation, the fine dots dispersed around the
intramembrane began to merge into a group of granules, at locations
believed to be the Golgi apparatus. As more antibodies were being
internalized after 30 min. of incubation, redistribution of the
grouped antibodies to scattered locations, probably the lysosomes
in which the antibodies were degraded, was observed. At 2 hrs
post-incubation, most of the antibodies were found inside the cell.
Only strong surface staining was observed when LL2 was incubated
for 20 min on ice. Both mLL2 and cLL2 were internalized with a
similar pattern. The internalization of LL2 was associated
specifically with antigent-antibody binding, as the irrelevant
control humanized antibody demonstrated only dull surface
staining.
[0118] A103 antibody (an IgG2a antibody that binds to the surface
of all human epithelial cells but does not internalize efficiently
(Mattes et al., Hybridoma, 2: 253 (1983)) showed strong membrane
staining at up to 2 h, while the anti-transferrin receptor antibody
(5F9) internalized rapidly, just as did LL2.
EXAMPLE 8
Role of Glycosylation Site in FR1 Region of LL2 VK Sequence
[0119] Of particular inventive interest is the identification of an
Asn-glycosylation site at position 18-20 within the FR1 region of
the LL2 NVT light chain sequence (FIG. 4A). As shown above,
SDS-PAGE analysis under reducing condition suggests that the Asn
glycosylation site is utilized for carbohydrate addition.
[0120] In this example, the influence of the carbohydrate moiety at
position 18-20 on the functional acitivities of the light chains
was examined.
[0121] Murine and chimeric LL2 light chains were treated with (+)
or without (-) endoglycosidase F conventionally, and the antibody
products examined by SDS-PAGE under reducing and non-reducing
conditions (FIG. 13). There was no distinction between the antibody
types as to electrophoretic behavior. In both cases,
deglycosylation reduced the rate of migration of the light
chain.
[0122] The effect of deglycosylation on the binding affinity to
Raji cells of the mLL2 antibody is shown in FIG. 14. Removing
carbohydrate by endoglycosidase F was without influence on the
binding activity.
[0123] A mutation was introduced at position 18 of the light chain
so that the Asn was replaced with Gln to produce LL2Q VK FR1.
SDS-PAGE analyses demonstrated that the NVT to QVT mutation
abolished glycosylation of the antibody. Comparison of the Raji
cell binding affinity for cLL2 with and without light chain VK
glycosylation demonstrated that the carbohydrate moiety was without
influence on binding of the antibody to these cells.
[0124] It can be concluded that the presence of the carbohydrate
site in the variable region does not affect the immunoreactivity of
the antibody. Computer modeling studies suggested that the VK
carbohydrate moiety in LL2 is remotely positioned from the CDRs and
forms a "cap" over the bottom loops of the FR-associated
.beta.-barrels supporting the CDRs.
[0125] Humanization without inclusion of the original glycosylation
site resulted in a CDR-grafted LL2 antibody with immunoreactivity
comparable to that of its murine counterpart.
[0126] These characteristics indicate that the glycosylation site
can be used for conjugating therapeutic or diagnostic agents to LL2
without compromising the ability of the antibody to bind and
internalize in B-lymphoma or leukemia cells.
EXAMPLE 9
Conjugation of LL2 at its Carbohydrate-bearing Site
[0127] The apparent lack of involvement of the variable region
carbohydrate moiety in the functional activities of mLL2, cLL2and
hLL2 mAbs indicates that this moiety could profitably be used as
the site of attachment of cytotoxic or detection agents such as
radionuclides or toxins, and thereby avoid potential interference
with the binding of the conjugate to a cell surface.
[0128] Using procedures described in Shih et al., U.S. Pat. No.
5,057,313 (which is incorporated by reference) for preparing
antibody conjugates through an oxidized carbohydrate moiety of the
antibody and a primary alkylamino group of a polymeric carrier to
which are covalently one or more of a variety of drugs, toxins,
chelators and detectable labels, a doxorubicin-dextran-LL2 antibody
fragment devoid of appended glycans was produced containing
multiple copies of the drug. The carbohydrate moieties of the cLL2
VK FR1 region involved were those covalently bound to the Asn
glycosylation site.
[0129] In one synthesis, dextran (18-40 kDa) was converted to an
amino dextran by oxidation of the dextran by NaIO.sub.4, Schiff
base formation with NH.sub.2--CH.sub.2--CHOH--CH.sub.2--NH.sub.2,
and reduction with NaBH.sub.4. The amino dextran was then condensed
with doxorubicin (DOX) in the presence of succinic anhydride and
1-ethyl-3-(3-dimethylaminopropy- l)carbodiimide to produce
DOX-aminodextran. The latter was then condensed with an aldehydic
group on LL2 VK FR-1 produced by oxidizing the carbohydrate moiety
of the antibody fragment with NaIO.sub.4.
[0130] In one preparation of DOX-LL2, the number of moles of DOX
attached to dextran was 14 moles per mole dextran, and the number
of moles of doxorubicin per mole F(ab')2 was 8.9. The
immunoreactivity in the Raji cell binding assay above was about 80%
of control values.
[0131] This conjugation system is not limited to the mLL2 antibody.
In a comparative study, 15-19 moles of DOX/mole of cLL2 were
bound.
[0132] The conjugation possibilities are not limited to the use of
a carrier dextran as in the example above. For example, the
carbohydrate moiety of the LL2 VK FR1 region can be oxidized to
produce aldehydic groups. These in turn can be reacted with an
amino group on any drug to produce a Schiff base which, upon
reduction, produces multiple copies of the drug stably linked to
the antibody via alkyamine groups.
[0133] For example, where the drug is aminohexyl DTPA (a chelating
agent), there is produced a LL2 covalently bound to a chelator. The
chelator can be used to deliver to target tissues, for example, a
radionuclide or paramagnetic metal ion, with a potential for
diagnostic and therapeutic uses. DTPA-LL2 conjugates were produced
containing 5.5 moles of the chelator/mole of antibody which, in
turn, chelated 47.3% of Y-90 and 97.4% In-111.
[0134] It should be emphasized that the above-described examples
merely describe several specific embodiments of the invention, and
applicants do not intend to be limited as to scope of claims by
these specific examples.
[0135] Applicants also incorporate by reference all publications
and patents cited in the specification.
Sequence CWU 1
1
25 1 339 DNA Murinae gen. sp. CDS (1)..(339) 1 gac att cag ctg acc
cag tct cca tca tct ctg gct gtg tct gca gga 48 Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 gaa aac gtc
act atg agc tgt aag tcc agt caa agt gtt tta tac agt 96 Glu Asn Val
Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 gca
aat cac aag aac tac ttg gcc tgg tac cag cag aaa cca ggg cag 144 Ala
Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45 tct cct aaa ctg ctg atc tac tgg gca tcc act agg gaa tct ggt gtc
192 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60 cct gat cgc ttc aca ggc agc gga tct ggg aca gat ttt act
ctt acc 240 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr 65 70 75 80 atc agc aga gta caa gtt gaa gac ctg gca att tat
tat tgt cac caa 288 Ile Ser Arg Val Gln Val Glu Asp Leu Ala Ile Tyr
Tyr Cys His Gln 85 90 95 tac ctc tcc tcg tgg acg ttc ggt gga ggg
acc aag ctg gag atc aaa 336 Tyr Leu Ser Ser Trp Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105 110 cgt 339 Arg 2 113 PRT Murinae
gen. sp. 2 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser
Ala Gly 1 5 10 15 Glu Asn Val Thr Met Ser Cys Lys Ser Ser Gln Ser
Val Leu Tyr Ser 20 25 30 Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Arg Val
Gln Val Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln 85 90 95 Tyr Leu
Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110
Arg 3 348 DNA Murinae gen. sp. CDS (1)..(348) 3 cag gtc cag ctg cag
gag tca ggg gct gaa ctg tca aaa cct ggg gcc 48 Gln Val Gln Leu Gln
Glu Ser Gly Ala Glu Leu Ser Lys Pro Gly Ala 1 5 10 15 tca gtg aag
atg tcc tgc aag gct tct ggc tac acc ttt act agc tac 96 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 tgg
ctg cac tgg ata aaa cag agg cct gga cag ggt ctg gaa tgg att 144 Trp
Leu His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 gga tac att aat cct agg aat gat tat act gag tac aat cag aac ttc
192 Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe
50 55 60 aag gac aag gcc aca ttg act gca gac aaa tcc tcc agc aca
gcc tac 240 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 atg caa ctg agc agc ctg aca tct gag gac tct gca
gtc tat tac tgt 288 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 gca aga agg gat att act acg ttc tac tgg
ggc caa ggc acc act ctc 336 Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp
Gly Gln Gly Thr Thr Leu 100 105 110 aca gtc tcc tcg 348 Thr Val Ser
Ser 115 4 116 PRT Murinae gen. sp. 4 Gln Val Gln Leu Gln Glu Ser
Gly Ala Glu Leu Ser Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His
Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly
Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55
60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln
Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 5 339 DNA Homo
sapiens CDS (1)..(339) 5 gac att cag ctg acc cag tct cca tca tct
ctg agc gca tct gtt gga 48 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 gat agg gtc act atg agc tgt aag
tcc agt caa agt gtt tta tac agt 96 Asp Arg Val Thr Met Ser Cys Lys
Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 gca aat cac aag aac tac
ttg gcc tgg tac cag cag aaa cca ggg aaa 144 Ala Asn His Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 gca cct aaa ctg
ctg atc tac tgg gca tcc act agg gaa tct ggt gtc 192 Ala Pro Lys Leu
Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 cct tcg
cga ttc tct ggc agc gga tct ggg aca gat ttt act ttc acc 240 Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 65 70 75 80
atc agc tct ctt caa cca gaa gac att gca aca tat tat tgt cac caa 288
Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys His Gln 85
90 95 tac ctc tcc tcg tgg acg ttc ggt gga ggg acc aag gtg cag atc
aaa 336 Tyr Leu Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys Val Gln Ile
Lys 100 105 110 cgt 339 Arg 6 113 PRT Homo sapiens 6 Asp Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25
30 Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
Thr Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Trp Thr Phe Gly
Gly Gly Thr Lys Val Gln Ile Lys 100 105 110 Arg 7 348 DNA Homo
sapiens CDS (1)..(348) 7 cag gtc cag ctg gtc caa tca ggg gct gaa
gtc aag aaa cct ggg tca 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 tca gtg aag gtc tcc tgc aag gct
tct ggc tac acc ttt act agc tac 96 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 tgg ctg cac tgg gtc agg
cag gca cct gga cag ggt ctg gaa tgg att 144 Trp Leu His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 gga tac att aat
cct agg aat gat tat act gag tac aat cag aac ttc 192 Gly Tyr Ile Asn
Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55 60 aag gac
aag gcc aca ata act gca gac gaa tcc acc aat aca gcc tac 240 Lys Asp
Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80
atg gag ctg agc agc ctg agg tct gag gac acg gca ttt tat ttt tgt 288
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85
90 95 gca aga agg gat att act acg ttc tac tgg ggc caa ggc acc acg
gtc 336 Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr
Val 100 105 110 acc gtc tcc tcg 348 Thr Val Ser Ser 115 8 116 PRT
Homo sapiens 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Arg Asn
Asp Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55 60 Lys Asp Lys Ala Thr
Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90 95 Ala
Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr Val 100 105
110 Thr Val Ser Ser 115 9 116 PRT Homo sapiens 9 Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn
Phe 50 55 60 Lys Asp Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Phe Tyr Phe Cys 85 90 95 Ala Arg Arg Asp Ile Thr Thr Phe Tyr
Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 10 149
DNA Artificial Sequence Synthetic sequence 10 tataatcatt cctaggatta
atgtatccaa tccattccag accctgtcca ggtgcctgcc 60 tgacccagtg
cagccagtag ctagtaaagg tgtagccaga agccttgcag gagaccttca 120
ctgatgaccc aggtttcttg acttcagcc 149 11 134 DNA Artificial Sequence
Synthetic sequence 11 ccccagtaga acgtaatatc ccttgcacaa aaataaaatg
ccgtgtcctc agacctcagg 60 ctgctcagct ccatgtaggc tgtattggtg
gattcgtctg cagttattgt ggccttgtcc 120 ttgaagttct gatt 134 12 38 DNA
Unknown Primer 12 ccagctggtc caatcagggg ctgaagtcaa gaaacctg 38 13
33 DNA Unknown Primer 13 aagtggatcc tataatcatt cctaggatta atg 33 14
49 DNA Unknown Primer 14 taatcctagg aatgattata ctgagtacaa
tcagaacttc aaggacaag 49 15 44 DNA Unknown Primer 15 ggagacggtg
accgtggtgc cttggcccca gtagaacgta gtaa 44 16 150 DNA Artificial
Sequence Synthetic sequence 16 cctagtggat gcccagtaga tcagcagttt
aggtgctttc cctggtttct gctggtacca 60 ggccaagtag ttcttgtgat
ttgcactgta taaaacactt tgactggact tacagctcat 120 agtgacccta
tctccaacag atgcgctcag 150 17 52 DNA Unknown Primer 17 gacaagcttc
agctgaccca gtctccatca tctctgagcg catctgttgg ag 52 18 45 DNA Unknown
Primer 18 agagaatcgc gaagggacac cagattccct agtggatgcc cagta 45 19
121 DNA Artificial Sequence Synthetic sequence 19 gcaccttggt
ccctccaccg aacgtccacg aggagaggta ttggtgacaa taatatgttg 60
caatgtcttc tggttgaaga gagctgatgg tgaaagtaaa atctgtccca gatccgctgc
120 c 121 20 40 DNA Unknown Primer 20 gacaagcttt cgcgattctc
tggcagcgga tctgggacag 40 21 33 DNA Unknown Primer 21 gaccggcaga
tctgcacctt ggtccctcca ccg 33 22 339 DNA Murinae gen. sp. 22
acgtttgatc tccagcttgg tccctccacc gaacgtccac gaggagaggt attggtgaca
60 ataataaatt gccaggtctt caacttgtac tctgctgatg gtaagagtaa
aatctgtccc 120 agatccgctg cctgtgaagc gatcagggac accagattcc
ctagtggatg cccagtagat 180 cagcagttta ggagactgcc ctggtttctg
ctggtaccag gccaagtagt tcttgtgatt 240 tgcactgtat aaaacacttt
gactggactt acagctcata gtgacgtttt ctcctgcaga 300 cacagccaga
gatgatggag actgggtcag ctgaatgtc 339 23 348 DNA Murinae gen. sp. 23
cgaggagact gtgagagtgg tgccttggcc ccagtagaac gtagtaatat cccttcttgc
60 acagtaatag actgcagagt cctcagatgt caggctgctc agttgcatgt
aggctgtgct 120 ggaggatttg tctgcagtca atgtggcctt gtccttgaag
ttctgattgt actcagtata 180 atcattccta ggattaatgt atccaatcca
ttccagaccc tgtccaggcc tctgttttat 240 ccagtgcagc cagtagctag
taaaggtgta gccagaagcc ttgcaggaca tcttcactga 300 ggccccaggt
tttgacagtt cagcccctga ctcctgcagc tggacctg 348 24 339 DNA Homo
sapiens 24 acgtttgatc tgcaccttgg tccctccacc gaacgtccac gaggagaggt
attggtgaca 60 ataatatgtt gcaatgtctt ctggttgaag agagctgatg
gtgaaagtaa aatctgtccc 120 agatccgctg ccagagaatc gcgaagggac
accagattcc ctagtggatg cccagtagat 180 cagcagttta ggtgctttcc
ctggtttctg ctggtaccag gccaagtagt tcttgtgatt 240 tgcactgtat
aaaacacttt gactggactt acagctcata gtgaccctat ctccaacaga 300
tgcgctcaga gatgatggag actgggtcag ctgaatgtc 339 25 348 DNA Homo
sapiens 25 cgaggagacg gtgaccgtgg tgccttggcc ccagtagaac gtagtaatat
cccttcttgc 60 acaaaaataa aatgccgtgt cctcagacct caggctgctc
agctccatgt aggctgtatt 120 ggtggattcg tctgcagtta ttgtggcctt
gtccttgaag ttctgattgt actcagtata 180 atcattccta ggattaatgt
atccaatcca ttccagaccc tgtccaggtg cctgcctgac 240 ccagtgcagc
cagtagctag taaaggtgta gccagaagcc ttgcaggaga ccttcactga 300
tgacccaggt ttcttgactt cagcccctga ttggaccagc tggacctg 348
* * * * *