U.S. patent number 5,565,354 [Application Number 08/259,372] was granted by the patent office on 1996-10-15 for production of human monoclonal antibodies specific for hepatitis b surface antigen.
This patent grant is currently assigned to Sandoz Ltd.. Invention is credited to Lars G. Ostberg.
United States Patent |
5,565,354 |
Ostberg |
October 15, 1996 |
Production of human monoclonal antibodies specific for hepatitis B
surface antigen
Abstract
Monoclonal antibodies effective for the diagnosis and treatment
of diseases caused by infection with hepatitis B have been prepared
from a cell line obtained by fusing a xenogeneic hybridoma
designated SPAZ 4 with blood cells of a patient immunized with
hepatitis B vaccine. The invention also provides cocktails of the
above monoclonal antibodies specific for different epitopes of
hepatitis B surface antigen.
Inventors: |
Ostberg; Lars G. (Convent
Station, NJ) |
Assignee: |
Sandoz Ltd. (Basel,
CH)
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Family
ID: |
27558835 |
Appl.
No.: |
08/259,372 |
Filed: |
June 14, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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871426 |
Apr 21, 1992 |
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676036 |
Mar 27, 1991 |
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538796 |
Jun 15, 1990 |
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192754 |
May 11, 1988 |
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925196 |
Oct 31, 1986 |
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904517 |
Sep 5, 1986 |
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Current U.S.
Class: |
435/339;
424/149.1; 435/70.21; 530/388.15; 530/388.3 |
Current CPC
Class: |
C07K
16/082 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
16/08 (20060101); A61K 38/00 (20060101); C12N
005/20 (); C07K 016/08 () |
Field of
Search: |
;530/388.15,388.3
;435/70.21,240.27 ;935/99,100 ;424/149.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0389983 |
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Oct 1990 |
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EP |
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2113715 |
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Aug 1983 |
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GB |
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9114703 |
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Oct 1991 |
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WO |
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Other References
Ogata et al (1993) Proc. Nat'l. Acad. Sci. 90: 3014-3018. .
Matsui et al. (1982) Nichi Dai Ishi (J. Nihon Univ. Med. Assoc.)
41(12): 1163-1171 (English translation, pp. 1-23). .
Matsui et al (1982) Biol. Abstr. 77(6): 4766, Abstract# 43590.
.
Goldstein et al. (1986) Transplantation 42: 507-511. .
Ehrlich et al. (1992) Hum. Antibod. Hybridomas 3: 2-7. .
Perillo et al (1990) N. Engl. J. Med. 323: 295-301. .
Janssen et al (1994) Antiviral Res. 23: 251-257. .
Hoofnagle et al "Serologic Responses in HB; In Viral Hepatitis"
(Vyas et al. eds.) Franklin Press., 1978, pp. 219-242. .
Roitt (1991) "Essential Immunology", Blackwell Scientific
Publications, Oxford, pp. 65-68 & 74. .
Burragi et al (1985) Cancer Res. 45: 3378-3387. .
Goding J. W. (1983) "Monoclonal Antibodies", Academic Press, Inc.,
Orlando, pp. 118-125. .
Davidsohn et al (1974) "Clinical Diagnosis By Laboratory Methods",
W. B. Saunders Co., Philadelphia, pp. 848-851 & 861-865. .
Roitt (1991) "Essential Immunology", Blackwell Scientific
Publications, Oxford, pp. 68-75. .
Cotran et al (1989) "Robbins Pathologic Basis of Disease", W. B.
Saunders Company, Philadelphia, pp. 927-928. .
Todo et al (1991) Hepatology 13: 619-626. .
Demetris et al (1986) Am. J. Pathol. 125: 161-172. .
Starzi et al (1989) Transplantation Proceedings 21: 2197-2200.
.
Andris et al (1992) J. Immunology 149: 4053-4059. .
Kozbor et al (1984) J. Immunol. 133: 3001-3005. .
Teng et al (1983) Proc Nat'l Acad Sci.80: 7308-7312. .
Steinitz et al (1979) J. Clin. Lab. Immunol. 2: 1-7. .
Matsui et al (1982) Nichidai Igaku Zasshi 41(12); 1163-1172 [&
Biol. Abstr. 77(6); 4766, #43590]. .
Wands et al (1981) Gastroenterology 80: 225-232. .
Imai et al (1982) J. Immunol. 128: 69-72. .
Ichimori, et al., Biochem. & Biophysical Res. Comm., vol. 129,
pp. 26-33 1985. .
Stricker, E. A. M. et al. 1985. "A Human Monoclonal IgG1.lambda.
Anti-Hepatitis B Surface Antibody " Scan. J. Immunol. 22:337-343.
.
Harada, K. et al. 1989 "Human-Human Hybridomas Secreting Hepatitis
B Virus Neutralizing Antibodies" Bio/Technology 7:374-377. .
Ichimori, Y. et al. 1987. "Establishment of Hybridoma Secreting
Human Monoclonal Antibody Against Hepatitis B Virus Surface
Antigen" Bioch. bioph. Res. Comm. 142(3):805-812..
|
Primary Examiner: Hutzell; Paula K.
Assistant Examiner: Ebert; Ray F.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Parent Case Text
This is a Continuation of application Ser. No. 07/871,426, filed
Apr. 21, 1992, now abandoned, which is a continuation-in-part of
Ser. No. 07/676,036, filed Mar. 27, 1991 (now abandoned), which is
a continuation-in-part of Ser. No. 07/538,796, filed Jun. 15, 1990
(now abandoned), which is a continuation of Ser. No. 07/192,754,
filed May 11, 1988 (now abandoned), which is a continuation-in-part
of Ser. No. 06/925,196, filed Oct. 31, 1986 (now abandoned), which
is a continuation-in-part of Ser. No. 06/904,517, filed Sep. 5,
1986 (now abandoned).
Claims
What is claimed is:
1. A cell line designated ATCC HB 9234.
2. A human monoclonal antibody produced by the cell line designated
ATCC HB 9234.
3. A Fab fragment of the human monoclonal antibody of claim 2.
4. A human monoclonal antibody comprising a heavy chain variable
region having the amino acid sequence shown in Table 8-1 (SEQ ID
NO:2) and a light chain variable region having the amino acid
sequence shown in Table 9-1 (SEQ ID NO:10).
5. A cell line producing the antibody of claim 4.
6. A Fab fragment of the antibody of claim 4.
7. A composition comprising the antibody of claim 2 and a
vehicle.
8. A composition comprising the antibody of claim 4 and a
vehicle.
9. The human monoclonal antibody of claim 4, wherein the heavy
chain variable region is encoded by the DNA sequence shown in Table
8-1 (SEQ ID NO:1) and the light chain variable domain is encoded by
the DNA sequence shown in Table 9-1 (SEQ ID NO:9).
Description
The present invention concerns hybridoma cell lines which produce
human antibodies which neutralize the hepatitis B virus, methods
for producing the cell lines, antibodies produced by the cell
lines, and uses of the antibodies, particularly
therapeutically.
The making of hybridoma cell lines for the purpose of producing
monoclonal antibodies is in general well known at this time to
researchers in this art. The present invention concerns the
obtaining of human monoclonal antibodies effective in particular
against hepatitis B surface antigen (HBsAg), such antibodies being
prepared according to a generally applicable method described by
the applicant in Hybridoma 2(4):361 (1983) and United Kingdom
Patent Application 2,113,715A, published Aug. 10, 1983. More
particularly, it has been found that a hybridoma cell line
comprising a parent rodent immortalizing cell, such as a murine
myeloma cell, e.g. SP-2, fused to a human partner cell results in
an immortalizing xenogeneic hybridoma cell. This xenogeneic
hybridoma cell may be fused to a cell capable of producing an
anti-HBsAg human antibody, resulting in a novel trioma cell line
capable of generating human antibody effective against such antigen
in the human. Alternately, when greater stability is desired, a
trioma cell line which preferably no longer has the capability of
producing its own antibody is made and this trioma is then fused
with a further cell capable of producing useful against said
antigen so as to obtain a still more stable hybridoma (quadroma)
which produces antibody against the antigen.
The applicant's publications earlier referred to describe the
preparation of a xenogeneic hybridoma referred to as SPAZ 4,
prepared from drug resistant cell line SP-2 obtainable, e.g., from
the NIGMS Human Genetic Mutant Cell Repository Ref. GM35669A (see
U.S. DHHS 1982 Catalog of Cell Lines). Preparation of SPAZ 4 is
summarized as follows. The SP-2 cell line is fused with normal
human peripheral lymphocytes by conventional techniques. A large
number of hybrids is obtained and, after approximately five weeks,
five clones are selected which show fast growth and no antibody
production. These cells are selected for resistance to
8-azaguianine and with three of these lines it is possible to
obtain mutants which are resistant to 20 .mu.g/ml of 8-azaguanine.
These cells are at the same time sensitive to
Hypoxanthine-Aminopterin-Thymidine (HAT) medium which showed that
they had lost their ability to produce hypoxanthine phosphoribosyl
transferase. One of these cell lines is SPAZ 4.
Cell line SPAZ 4 may be fused with cells obtained from the blood of
persons immunized with hepatitis B vaccine to obtain hybridoma cell
lines which provide positive cultures when standard selection
procedures are used involving binding of antibodies to relevant
viral antigens. It is preferred that said positive cultures be
placed through a second selection process in which different
subtypes of the virus are used for antigen preparation. This
provides an opportunity to pinpoint the exact antigenic determinant
recognized by the antibody.
The cell lines resulting from the fusion of a xenogeneic hybridoma
and the human monoclonal antibody producing cell (trioma) are
therefore useful in providing monoclonal antibodies capable of
effective activity in neutralizing a virus causing hepatitis, and
said antibodies can therefore prevent the spread of hepatitis
through e.g. blood transfusion. They can also be used to give
initial protection to newborn babies or exposed individuals earlier
than a vaccine could be effective. Anti-hepatitis antibodies may be
used to protect immunosuppressed patients, including
transplantation patients, from recurrent hepatitis. This is most
significant in cases of hepatitis B positive liver recipients.
Further, the antibodies can be used in diagnostic assays.
It has also been found that antibody fragments, such as Fab
fragments can also bind to hepatitis B virus surface antigen. These
fragments also make up part of this invention.
Specific antibodies which have been made according to this
invention include PE1-1, ZM1-1, ZM1-2, MD3-4 and LO3-3, each of
these antibodies being of the IgG.sub.1 class.
The cell line producing PE1-1 was deposited at the American Type
Culture Collection 12301 Parklawn Drive, Rockville, Md. 20852 on
Oct. 16, 1986 and given accession number ATCC HB 9234; the cell
line producing ZM1-1 was deposited as ATCC HB 9191 on Sep. 4, 1986
and the cell line producing ZM1-2 was deposited as ATCC HB 9192 on
Sep. 4, 1986. The address of the American Type Culture Collection
is 12301 Parklawn Drive, Rockville, Md. 20852.
The cell lines of the present invention all behave as typical
(mouse.times.human).times.human hybridomas and produce their
respective antibodies in concentrations ranging up to 25 mg/l in
standard suspension culture.
DESCRIPTION OF THE FIGURES
FIG. 1 shows the results of a direct binding enzyme linked
immunoassay comparing binding kinetics of antibody PE1-1 (shown by
the single line) and antibody ZM1-2 (double line). Details are
given in Example 4A.
FIG. 2 shows the serum levels of antibody PE1-1 in rhesus monkey
serum determined at various times post-dosage. Details are given in
Example 4C.
Throughout the specification and claims, the same designation is
given to both the cell line and the antibody it produces, i.e. cell
line PE1-1 produces monoclonal antibody PE1-1; cell line ZM1-1
produces monoclonal antibody ZM1-1, etc. It is felt that one of
ordinary skill in the art will understand whether the cell line or
the antibody is being discussed.
Monoclonal antibody and cell line PE1-1 has also been referred to
by the inventor and the inventor's assignee as OST 577 and 64-577.
Likewise, monoclonal antibody and cell line ZM1-2 have also been
referred to as 265-695, and monoclonal antibody and cell line L03-3
have been referred to as 266-215.
The antibodies and antibody fragments obtained according to this
invention have good specificity for hepatitis B surface antigen in
in vitro ELISA binding assays.
As the antibodies of the present invention are of human origin,
they are advantageously used in human therapy, as no allergenic
response develops with repeated therapy, as occurs with murine or
ovine antibodies. Thus, another aspect of this invention is a
method of treating hepatitis B through the administration of one or
more of the aforementioned antibodies. It has been found that
repeated doses of approximately 10-40 mg antibody will
substantially reduce the amount of circulating HBsAg. Additional
doses were found to decrease the amount of HBsAg levels to below
the detectable limits of antigen tests.
Another aspect of this invention is a cocktail of two or more
monoclonal antibodies. This mixture is particularly suited for
administration to patients who carry a non-wild type strain of
hepatitis B virus which does not bind well with a given single
monoclonal antibody. For example, one compassionate need patient
who suffered from hepatocellular carcinoma and chronic hepatitis B
was given antibody PE1-1 prior to liver transplantation, and
repeated doses thereafter. (Details are given below in Example 5).
After approximately four and one-half months of treatment, low
levels of serum HBsAg could be detected with a polyclonal antibody,
but not with PE1-1. Polymerase chain reaction (PCR) DNA analysis of
the 230 base pair region of the HBsAg gene corresponding to the
putative monoclonal antibody binding domain was performed. The PCR
DNA was cloned into M13 bacteriophage and the resulting DNA was
sequenced. Analysis of clones from each of the serum samples
revealed two variant sequences when compared to PCR DNA from the
original liver and pre-antibody therapy. The variant DNA codes for
two different amino acids in the S protein of HBsAg and also codes
for a stop codon (UAG) in the viral polymerase gene. Both variant
genes contain an amino acid change resulting in the substitution of
arginine for glycine in a conserved peptide domain.
Since the monoclonal antibodies PE1-1, ZM1-2, ZM1-1, MD3-4 and
L03-3 have been shown to bind to different epitopes, and at least
one of the monoclonal antibodies has been found to bind to every
variant virus tested to date to a sufficient extent to render it
clinically useful, another aspect of this invention is a cocktail
of two or more of the monoclonal antibodies selected from the group
consisting of: PE1-1, ZM1-2, ZM1-1, MD3-4 and L03-3. Particularly
preferred are cocktails of two monoclonal antibodies, especially
the mixture of PE1-1 and ZM1-2 and the mixture of PE1-1 and L03-3.
The ratio of the monoclonal antibodies present in the mixture may
vary depending on many factors apparent to one of ordinary skill in
the art and include: the genotype of the hepatitis virus or viruses
present in the patient's serum, the relative binding strengths of
the antibodies chosen, the epitopes to which the chosen antibodies
bind, and economic considerations. Generally, the antibodies will
be present in a ratio ranging from 1:99, more typically from 25:75,
and preferably in a substantially equal amount.
Sections of the PE1-1, ZM1-1, ZM1-2 and MD3-4 were sequenced using
standard techniques. The sequence obtained for the V.sub.H region
of PE1-1 is given in Table 8-1, and areas corresponding to the
CDR1, CDR2, and CDR3 (D.sub.H and J.sub.H4) are noted. As the CDR
regions are particularly important regions in determining the
binding properties of an antibody, this invention includes an
antibody that has an amino acid sequence of its CDR1 region which
is substantially similar to that of PE1-1, as set forth in Table
8-1. This invention also includes an antibody that has an amino
acid sequence in its CDR2 region which is substantially similar to
that of PE1-1, as set forth in Table 8-1. Further, this invention
also comprises an antibody that has an amino acid sequence of its
CDR3 region which is substantially similar to the CDR3 region of
PE1-1, as set forth in Table 8-1.
Likewise, the V.sub.H region of ZM1-1 was sequenced as is given in
Table 8-2. Areas corresponding to its CDR1, CDR2, and CDR3 (D.sub.H
and J.sub.H4) are also indicated. This invention includes an
antibody which has an amino acid sequence of its CDR1 region which
is substantially similar to that of ZM1-1 as set forth in Table
8-2. Also, this invention includes an antibody which has an amino
acid sequence of its CDR2 region which is substantially similar to
that of ZM1-1 as set forth in Table 8-2, and further this invention
also comprises an antibody that has an amino acid sequence of its
CDR3 region which is substantially similar to that of ZM1-1 as set
forth in Table 8-2.
The DNA sequences which code for the regions of ZM1-2 and MD3-4 are
given in Table 8-3 and 8-4 respectively. This invention also
includes any antibody which has amino acid sequences which are
substantially similar to that of the regions of ZM1-2 and MD3-4 as
set forth in Tables 8-3 and 8-4.
The DNA sequences which code for the V.sub.H regions of PE1-1,
ZM1-1, ZM1-2 and MD3-4 were determined and appear in Tables 8-1 and
8-2, 8-3 and 8-4 respectively. These sequences or appropriate
fragments may be used in cloning antibodies (or modified
antibodies) or as probes. Antibodies which are produced through
genetic engineering processes (rather than conventional harvesting
from hybridomas) can be made using cloning techniques which are
known in the art. DNA from other sources may be used to produce a
synthetic antibody molecule which retains the binding
characteristics of PE1-1, ZM1-1, ZM1-2 and MD3-4 by virtue of
having substantially similar CDR1, CDR2, and/or CDR3 regions. Such
antibodies are within the scope of this invention.
The DNA sequences which code for the V.sub.L light chain variable
regions of PE1-1, ZM1-1, ZM1-2 and MD3-4 are given in Tables 9-1,
9-2, 9-3, and 9-4, respectively. This invention also includes any
antibody which has amino acid sequences which are substantially
similar to that of the regions of PE1-1, ZM1-1, ZM1-2 and MD3-4 as
set forth in Tables 9-1, 9-2, 9-3 and 9-4.
Also within the scope of this invention are the DNA sequences which
code for the V.sub.H region, the V.sub.L region, the CDR1 regions,
the CDR2 regions and/or the CDR3 regions of PE1-1, ZM1-1, ZM1-2 and
MD3-4. Also included is DNA which would hybridize to any of the
aforementioned sequences under stringent hybridization conditions.
This DNA is substantially free from other DNA of the donor mammal,
and may contain introns or it may be cDNA.
As used throughout the specification and claims, the following
definitions are intended. An amino acid sequence is "substantially
similar" to another amino acid sequence if their amino acid
homology is at least 80%. Referring to DNA, "stringent
hybridization conditions" are those in which hybridization is
effected at 60.degree. C. in 2.5 X saline citrate buffer (SSC)
followed merely by rinsing at 37.degree. C. at a reduced buffer
concentration which will not affect the hybridizations which take
place. "Associated mammalian DNA" means DNA present in the mammal
which is the source of the V.sub.H antibody chain, but which is not
involved in coding for an antibody or antibody fragment.
The invention is more fully exemplified in the following
non-limiting examples.
EXAMPLE 1
Production of Antibody Cell Lines
Human volunteers are immunized with hepatitis B vaccine. MD3-4,
ZM1-2, ZM1-1, and PE1-1 hybridoma cell lines are derived from
lymphocytes of individuals immunized with HEPTAVAX.RTM. vaccine
(Merck & Co.). Cell line L03-3 is developed from cells of an
individual injected several times with HEPTAVAX.RTM. vaccine, and
just preceding the fusion, RECOMBIVAX.RTM. (Merck & Co.).
Peripheral blood lymphocytes are purified by density gradient
centrifugation on a cushion of Percoll (Pharmacia Inc.), density
1.085 g/ml. The isolated lymphocytes are washed three times in
Hank's Balanced Salt Solution and mixed with an equal number of
cells from (mouse.times.human) cell line SPAZ-4. The cell mixture
is pelleted at room temperature with 400 x g for 5 minutes. After
removing the medium, the cell pellet is treated with a 50% solution
of PEG-1000 in Dulbecco's Minimal Essential Medium (MEM) for 1
minute at 37.degree. C. after which the medium was slowly diluted
with Dulbecco's MEM. The cells are collected by centrifugation and
resuspended into Dulbecco's MEM containing 20% fetal bovine serums.
The cells are seeded at approximately 2.times.10.sup.6 cells per ml
into microwell plates. On the following day fresh medium containing
the components of HAT medium (hypoxanthine aminopterin thymidine)
is added in order to select against non-fused SPAZ-4 cells. On day
4 after fusion the medium is replaced with fresh medium containing
only HT as all cells sensitive to HAT-selection had been killed by
that time.
After 3 to 4 weeks, when good growth of hybridoma-like cells could
be seen microscopically, supernatants are tested for the presence
of anti-hepatitis B surface antigen antibody. An ELISA-assay using
a 1/100 dilution of HEPTAVAX.RTM. vaccine on the solid phase is
used. After incubation with the supernatants the plates are
developed with a VECTASTAIN.RTM. kit of biotinylated goat
anti-human immunoglubulin and avidin-coupled horseradish peroxidase
(Vector Laboratories Inc.). The enzyme is detected by the color
reaction with phenylenediamine. Positive cultures are picked into
new wells and a part of the cells is cloned by limiting dilution in
Dulbecco's MEM containing 20% fetal bovine serum and 10.sup.7 mouse
thymocytes per milliliter. The cloning plates are tested by the
same ELISA method as described above and positive cultures are
expanded and frozen.
All the cell lines behave as typical
(mouse.times.human).times.human hybridomas and produce their
respective antibodies in concentrations ranging up to 25 mg/l in
standard suspension culture.
EXAMPLE 2
Immunochemical Characterization
A. Antibody Class/Subclass
The immunoglobulin class of antibodies PE1-1, ZM1-1, ZM1-2, MD3-4
and L03-3 is determined using ELISA methodology. Each antibody is
captured on an antigen-coated plate and each assay is developed
with subclass specific, peroxidase-conjugated anti-human Ig (Tago).
Each of the antibodies are clearly IgG.sub.1.
B. Light Chain Type
Using ELISA methods similar to those described in A, above, each
antibody is tested with anti-.kappa. or anti-.lambda. light chain
reagents (Tago). The following results are obtained.
______________________________________ PE1-1 lambda ZM1-1 kappa
ZM1-2 kappa L03-3 lambda MD3-4 lambda
______________________________________
C. Isoelectric Focusing (IEF)
A sample of antibody L03-3 or PE1-1 is applied to gel. Each is
found to behave as a basic protein.
D. Specificity
Purified HBsAg of subtypes adw and ayr are purchased from Scripps
Laboratories, San Diego, Calif. HBsAg subtype ayw is obtained from
Connaught Laboratories (Willowdale, Ontario). ELISA assays are
performed essentially as described by Ostberg, et al. (1983)
Hybridoma 2:361-367.
PE1-1 reacts with both ayr and adw, but it reacts slightly better
with the adw subtype. L03-3 reacts substantially equally well with
ayr and adw. ZM1-1 shows higher reactivity with adw, but ZM1-2
binds slightly better to ayr. These results are confirmed for PE1-1
and L03-3 by Scatchard analysis in solid phase RIA with solid
adsorbed ayr or adw antigen. Thus, although these monoclonal
antibodies apparently do not bind to the subtypic determinant,
their reaction with HBsAg can be significantly affected by the
subtype.
G. Allotype Determination
Allotypes are determined using reagents supplied by the Central
Laboratory of the Netherlands Red Cross Transfusion Service.
Inhibition ELISA or direct binding ELISA are used. Results are
presented in Table 1, below. As can be seen, there is no apparent
restriction on high affinity anti-HBSAg antibodies with respect to
light chain or allotype.
TABLE 1 ______________________________________ Allotypes of
Anti-HBsAg Monoclonal Antibodies Allotypes Antibody a f z Km(3)
______________________________________ PE1-1 - + - * ZM1-2 + - + +
L03-3 - + - * ZM1-1 ND ND ND +
______________________________________ ND = Not determined
*Antibody has .lambda. light chain which does not have Km
allotypes
G. Affinity
The affinity for solid adsorbed HBsAg is determined for each
antibody using radiolabelled antibodies essentially as described by
Wands, et al. (1981) Gastroenterology 80:225-232, which is hereby
incorporated by reference. Antibodies are labeled with .sup.125 I
with Iodogen (Pierce). For each monoclonal except L03-3, the solid
phase absorbed HBsAg is ayw. L03-3 is assayed with both ayr and adw
with essentially the same results. Antibody-antigen incubation
occurs at room temperature.
The relative affinity is also determined using an inhibition ELISA
in which varying concentrations of soluble HBsAg (ayw subtype) are
pre-incubated with monoclonal antibody and the mixture is then
incubated at 37.degree. C. in a microtiter well coated with HBsAg.
Results are presented below in Table 2.
TABLE 2 ______________________________________ Affinity of
Monoclonal Antibodies for HBsAg Antibody Solid Phase RIA, M.sup.-1
Inhibition ELISA, M.sup.-1 ______________________________________
PE1-1 3.6 .times. 10.sup.9 .about.2 .times. 10.sup.9 ZM1-2 1.5
.times. 10.sup.9 .about.7 .times. 10.sup.8 L03-3 1.7 .times.
10.sup.9 .about.1 .times. 10.sup.8 ZM1-1 5 .times. 10.sup.9
.about.1 .times. 10.sup.8
______________________________________
As can be seen from the table above, for both PE1-1 and ZM1-2 the
ELISA results are approximately two-fold lower than the RIA
results, which is within the range of experimental error. A
Scatchard plot of the results of the RIA performed on ZM1-1
indicates that there might be a low affinity binding site. It is
thus possible that the ELISA is measuring this low affinity binding
site, as the ELISA results are some 50-fold lower than the RIA. In
addition, Scatchard plots also indicate that there are considerably
less high affinity ZM1-1 sites than ZM1-2 or PE1-1 high affinity
sites. While not wishing to be bound by theory, it appears that
ZM1-1 may have the highest affinity for HBsAg of the four
antibodies compared, but only for HBsAg in a certain spatial
arrangement. This arrangement is only manifested in a small
percentage of HBsAg molecules. It is also possible that this may be
due to bivalent binding of ZM1-1 to HBsAg while the low affinity
site is monovalent.
EXAMPLE 3
Potency of PE1-1
Antibody PE1-1 is tested for potency in the AUSAB radioimmunoassay
(Abbott). Tests are performed against the Bureau of Biologics
Reference Hepatitis B immune globin, and several commercial
hepatitis B immune globulin preparations (H-Big Immune Globin.RTM.,
Hep B Gammagee Immune Globin.RTM., and Hyper Hep Immune
Globin.RTM., all purchased from a pharmaceutical supply house).
Despite the fact that the immune globulin preparations are
polyclonal and PE1-1 is monoclonal, the binding data are within the
criteria of the Bureau of Biologics for comparing immune globulin
preparations, i.e., the lines were parallel at a probability level
less than or equal to 0.01.
Determination of potency is as follows. Preparations are compared
on a weight basis (an absorbance at 208 nm of 1.4 is assumed equal
to 1 mg/ml). Preparations of PE1-1 which have been stored at
5.degree. C. are then compared with the above polyclonal
preparations which have also been stored at 5.degree. C. The
logarithm of 1000 divided by .mu.g/ml in the preparation (i.e. the
log of a number that is inversely proportional to the concentration
of the immunoglobin, similar to the log of the dilution factor) is
then plotted vs. log counts per minute (average of triplicates).
The hypothesis that the fitted lines are parallel is tested using
analysis of variance. It is found that the lines are parallel at a
probability level of less than or equal to 0.01. Lines of all
preparations are parallel and a common slope is determined. The
x-intercepts are calculated from the common slope and the
difference in intercepts used to determine the difference in
potency. By this procedure, monoclonal antibody PE1-1 is some 435
times more potent than the Bureau of Biologics reference hepatitis
B immune globin. Since the commercial hepatitis B immune globulin
preparations were found to be two-fold (or less) more potent than
the Bureau of Biologics reference preparation, PE1-1 is at least
200 times more potent than the commercial hepatitis B immune
globulin preparations on a weight basis.
EXAMPLE 4
A. Binding Kinetics
Direct binding enzyme linked immunoassays are used to compare the
kinetics of binding to HBsAg of antibodies PE1-1 and ZM1-2. ELISA
microtiter plates are coated with HEPTAVAX.RTM. vaccine at 1
.mu.g/ml. Wells are then incubated at 37.degree. C. with 2% fetal
calf serum in phosphate buffered saline. Monoclonal antibody PE1-1
or ZM1-2 at 0.5 .mu.g/ml in 2% fetal calf serum are incubated in
the wells for various times. At the indicated times the antibody
solution is removed and the well is rinsed three times with fresh
2% fetal calf serum. The well is then incubated with 2% fetal calf
serum until the wells for the 90 minute time point contain 2% fetal
calf serum. Thus, solution is then replaced with either peroxidase
conjugated goat anti-lambda chain (PE1-1 wells) or goat anti-kappa
chain (ZM1-2 wells). Quantitation of peroxidase conjugate bound to
plastic is accomplished with the addition of O-phenylenediamine and
H.sub.2 O.sub.2. Results are presented in FIG. 1, where a single
line is PE1-1 and a double line is ZM1-2.
As can be seen in FIG. 1, at a concentration at which PE1-1 is
almost completely reacted in 5 minutes, the reaction of ZM1-2 with
solid adsorbed HBsAg is not completed in 30 minutes and may
continue to react for 90 minutes or more. Thus, PE1-1 binds
significantly faster to antigen in this assay. Assuming this also
occurs in vivo, PE1-1 is likely to be more efficient in
neutralizing viral particles before they can infect the liver.
B. Relative Position of Epitopes
The relative position of the epitopes of antibodies PE1-1, L03-3,
and ZM1-2 are determined. A simultaneous sandwich immunoassay with
a solid-adsorbed monoclonal antibody is used. The same antibody is
radiolabelled and incubated in a microtiter well with the inhibitor
and serum from a hepatitis B positive patient. Radiolabelled PE1-1
Fab fragment is used while radiolabelled L03-3 is intact IgG.
Results are presented in Table 3, below.
TABLE 3 ______________________________________ Inhibition of
Binding of Radiolabelled Monoclonal Antibody to HBsAg by Unlabelled
Monoclonal Antibodies Solid-Absorbed Iodinated Inhibitor maB maB
maB IC.sub.50 ng/ml ______________________________________ L03-3
L03-3 L03-3 10 L03-3 L03-3 PE1-1 >22,500 PE1-1 PE1-1 PE1-1 8
PE1-1 PE1-1 ZM1-2 76 PE1-1 PE1-1 LO3-3 >22,500
______________________________________
Monoclonal antibody ZM1-2 is only approximately nine times less
effective in inhibiting .sup.125 I-PE1-1's binding to HBsAg than
unlabelled PE1-1, whereas L03-3 is thousands of times less
effective. Thus, the epitopes of ZM1-2 and PE1-1 are probably near
each other on the HBsAg molecule while the L03-3 epitope is
probably on a different part of the molecule. The reciprocal
experiment, PE1-1 inhibition of radiolabelled L03-3, provides
further evidence that PE1-1 and L03-3 bind to epitopes that are not
overlapping.
The similarity of PE1-1 and ZM1-2 epitopes and their difference
from the L03-3 is confirmed by immunoassay with reduced and
alkylated HBsAg. L03-3 can bind to denatured antigen while both
ZM1-2 and PE1-1 cannot so bind. It should be noted that PE1-1 and
ZM1-2 have distinct epitopes since their reaction with different
subtypes varies.
C. Pharmokinetics of PE1-1 in Rhesus Monkeys
The pharmokinetics of PE1-1 is studied in two rhesus monkeys. Each
animal receives a single intravenous bolus injection (0.5 mg/kg) of
monoclonal antibody PE1-1. Serum levels of PE1-1 are determined at
various times post-dose using an ELISA based sandwich immunoassay
with HEPTAVAX.RTM. vaccine coated on ELISA plates and rabbit
anti-idiotypic antibodies to PE1-1. Results are shown in FIG.
2.
Serum levels of PE1-1 in the two rhesus monkeys are characterized
by a biphasic decline (t 1/2.alpha.=1 and 1.4 days; t 1/2.beta.=11
and 16 days) with the shorter half-life possibly associated with
the distribution phase of the monoclonal antibody. The volume of
distribution at steady state (Vdss) is calculated to be 114-144% of
the plasma volume, which suggests little distribution of PE1-1 to a
tissue compartment in the antigen-free monkey.
EXAMPLE 5
Clinical Trials
A. Compassionate use of PE1-1 in Two Patients with End-stage Liver
Disease Secondary to Chronic Active Hepatitis B and Hepatocellular
Carcinoma Undergoing Liver Transplantation
PE1-1 was provided on a compassionate need basis to two patients
with end-stage liver disease undergoing liver transplantation.
Patient #1 was a 56 year old male with a 20 year history of chronic
active hepatitis and a diagnosis of hepatocellular carcinoma. The
second patient was a 10 year old male thought to have been infected
with hepatitis B at birth. Patient #2 was initially evaluated for a
large mass in the right lobe of the liver, which a biopsy confirmed
was hepatocellular carcinoma.
Preoperative doses of PE1-1 were administered to these patients and
significantly reduced their circulating HBsAg levels before the
transplant procedure. Each patient also received two 20 mg doses of
PE1-1 during transplantation. Postoperative dosing then began on
the second day following surgery.
Patient #1 never became HBsAg negative, although his circulating
HBsAg levels did diminish markedly from their pretreatment level.
Patient #2 became HBsAg negative, first noted on post-transplant
day 9. Patient #1 received additional doses of PE1-1 ranging from
5-40 mg at 2-20 day intervals. Patient #2 received either 5 or 10
mg doses on average of every 21-28 days.
No adverse events were reported for either of these patients during
the period they received PE1-1. However, approximately four weeks
after Patient #1 was discharged from the hospital, it was
determined that he had metastatic malignancy. He expired on
post-transplant day 139. No evidence of recurrent hepatitis was
noted during his post-transplant course despite the presence of
detectable circulating HBsAg. Although a hepatitis B virus DNA
assay was negative preoperatively, a single positive value was
detected 60 days post-transplant.
On post transplant day 143, Patient #2 was first seen to be
positive for HBsAg. The HBsAg level fluctuated for a short time
before it then stabilized at a level signicantly below his
pretreatment levels. Isolates of this patient's hepatitis B virus
obtained before treatment with PE1-1 and at later times were
analyzed for their binding ability to PE1-1. PE1-1 was found to be
able to bind to the variant virus, but not as well as it had to the
wild-type virus.
Genetic analysis of the two viral isolates indicated single
nucleotide differences in a highly conserved region of the major
viral surface protein. Such differences, when compared to the
pre-treatment virus, could potentially encode for a single amino
acid difference which would reduce the binding ability of PE1-1 to
the hepatitis B viral binding particle.
B. Use of PE1-1 in Patients with Chronic Active Hepatitis B
Undergoing Liver Transplantation (Not Complicated by Hepatocellular
Carcinoma)
This study involved five patients who were HBsAg positive (but did
not have hepatocellular carcinoma) and who underwent liver
transplantation. Each patient was administered three daily
preoperative doses of PE1-1, (10, 20 and 40 mg, respectively) over
a three day period. The liver transplants were then performed from
a minimum of two days to a maximum of 32 days following their
preoperative dose of the study drug. An additional 40 mg dose of
PE1-1 was administered during the operation. All five transplants
were successfully completed.
The patients' HBsAg titers, liver enzymes, and other clinical
parameters were closely monitored during their hospital stays.
Follow-up evaluations and administration of PE1-1 by each patients'
private physician continued on a regular basis (approximately every
1 to 3 weeks). Dosing and other parameters varied from patient to
patient.
Two patients (#5 and #6) had similar results to Patient #2, above,
in that a variant virus appeared after a period of negative HBsAg
screening results. The sera of these patients remained active with
PE1-1. Sequence analysis indicated the presence of single
nucleotide differences between the variants in the patients' sera
and wild type virus. Two variants were detected in each patient.
Immunoassays and sequence analysis indicated that the variants in
each patient were different and they also differed from the
variants of Patient #2.
Patient #3 is a 39 year old Caucasian male who had end-stage liver
disease secondary to a 16 year history of chronic hepatitis B. The
three preoperative doses of PE1-1 that were administered to Patient
#3 caused a substantial reduction in his HBsAg titer. On
post-transplant days 2 and 3, he received 20 mg of PE1-1 and was
first noted to be HBsAg negative on post-transplant day 2. For two
months thereafter, Patient #3 received 10 mg PE1-1 on an average of
every 1 to 7 days. Since then, he has received 7.5 or 10 mg doses
of PE1-1 every 14 to 43 days. Histopathological evaluation of a
liver biopsy performed in February, 1989 was negative for both
HBsAg and HBcAg. Patient #3 remains HBsAg negative 582 days after
transplant. In addition to receiving PE1-1, he has also received
three consecutive monthly injections of RECOMBIVAX.RTM. in July,
August and September, 1989.
Patient #4 was a 40 year old Arabic female who had end-stage liver
disease secondary to a 10+ year history of chronic active Hepatitis
B. The three preoperative doses of PE1-1 given to Patient #4 caused
a substantial reduction in her HBsAg level. Patient #4 received 20
mg of PE1-1 on post-transplant days 1 and 2, and was found to be
HBsAg negative on post-transplant day 6. For two months thereafter,
she received 10 mg PE1-1 on average of every 3 to 8 days. Since
then, she received 10 mg PE1-1 every 5 to 26 days. Approximately 1
year after her transplant, the patient developed hepatic artery
thrombosis, but remained HBsAg negative, and was re-transplanted.
Three days later, due to ischemia, a third transplant was
performed. Twenty days following, a fourth transplant was performed
due to infection. The patient expired 18 days after the fourth
transplant (404 days after her initial transplant), secondary to
liver failure and bacterial sepsis. Histopathological evaluation of
a liver biopsy from her first transplanted liver showed that she
was HBsAg negative.
Patient #5 is a 38 year old Caucasian male who had end-stage liver
disease secondary to chronic active hepatitis B. The preoperative
doses of PE1-1 administered to the patient substantially lowered
his circulating HBsAg level. Patient #5 received 20 mg PE1-1 on
post-transplant days 2 and 3, and was found to be HBsAg negative on
post-transplant day 3. During the first two months post-transplant,
he received 10 mg PE1-1 on average of every 3-7 days. Later, he
received 10 mg PE1-1 every 9 to 26 days. The patient was noted to
be HBsAg positive on post-transplant day 252, although his antigen
level is substantially lower than his pre-transplant level.
Histopathological evaluation of a liver biopsy performed in January
1990 is positive for HBsAg and HBcAg.
Patient #6 is a 38 year old Caucasian male who had end-stage liver
disease secondary to chronic active hepatitis B and alcohol abuse.
This patient acquired his initial infection via a blood
transfusion. Prior to the transplant, he was positive for both
HBsAg and HBeAg. Each preoperative dose of PE1-1 caused a decrease
in the level of the patient's HBsAg titer. Patient #6 received 20
mg of PE1-1 on post-transplant days 1 and 2 and was noted to be
HBsAg negative on post-transplant day 1. For two months thereafter,
Patient #6 received 10 mg of PE1-1 on average of every 3-14 days.
Subsequently he has received 10 mg PE1-1 every 7 to 63 days on an
outpatient basis. The first HBsAg positive response was noted on
post-transplant day 251 and occurred after his longest duration (63
days) between doses of PE1-1. Although at present Patient #6 is
positive for HBsAg, his titer remains significantly lower than
pre-transplant levels.
Patient #7 is a 38 year old Caucasian female with a history of IV
drug abuse. This patient had end-stage liver disease secondary to
chronic active hepatitis B. Prior to transplantation, the patient
was positive for HBsAg and HBeAg. Each preoperative dose of PE1-1
caused a decrease in the patient's HBsAg titer. The first month
post-transplant, Patient #7 received between 10 and 40 mg of PE1-1
on the average of every 1-7 days, and was noted to be HBsAg
negative on post-transplant day 16. Subsequently, she received 10
mg PE1-1 every 15 to 29 days. Histopathological evaluation of a
liver biopsy performed in July, 1989 was negative for HBsAg and
HBcAg. Patient #7 remains HBsAg negative 464 days
post-transplant.
EXAMPLE 6
Reactivity with Variant Virusus
The reactivity of the monoclonal antibodies PE1-1, ZM1-2, and L03-3
with variant hepatitis B viruses isolated from patients described
in Example 5 is investigated. Radioimmunoassays are performed by
determining the radioactivity bound to a solid phase
adsorbed-antibody. A solution of a monoclonal antibody at a
concentration of 20 .mu.g/ml in phosphate-buffered saline
containing 0.02% NaN.sub.3 is incubated for at least 18 hours in
U-bottom wells (Falcon MicroTest III Flexible Assay Plates). The
solution is removed from the wells and the wells are then washed
three times with distilled water. Fetal calf serum at a
concentration of 2% in phosphate-buffered saline is added and
incubated overnight at room temperature with solutions of serum
HBsAg or controls and .sup.125 I-radiolabelled antibody
(approximately 4,000 cpm in 1% fetal calf serum). Wells are then
washed with distilled water three times. Individual wells are
excited and counted. Results are presented in Table 4, below.
TABLE 4 ______________________________________ Relative Reactivity
of Serum-Derived Variant HBsAg with HBsAg-Specific Monoclonal
Antibodies Sample* LO3-3:LO3-3** PE1-1:ZM1-2 ZM1-2:ZM1-2
______________________________________ Control 1.000 1.000 1.000
Patient #2 (234) 0.013 0.070 0.233 Patient #4 (251) 0.007 0.024
0.010 Patient #3 (264) 0.043 0.173 0.179
______________________________________ *Representative
HBsAgpositive serum samples derived from patients after liver
transplantation and treatment with an antiHBsAg therapeutic
monoclonal antibody PE11. Numbers in parentheses denote days after
transplantation. **LO33:LO3-3 indicates a radioimmunoassay composed
of both solidadsorbed and radiolabelled human monoclonal antibody
LO33. PE11:ZM1-2 indicates a radioimmunoassay composed of human
monoclonal antibody PE11 solidadsorbed and human monoclonal
antibody ZM12 radiolabelled. ZM12:ZM1-2 indicates a
radioimmunoassay composed of both solidadsorbed and radiolabelled
human monoclonal antibody ZM12. Control HBsAgpositive serum reacted
well with antibodies LO33, PE11 and ZM12.
EXAMPLE 7
Large Scale Production of Antibodies
To initiate a production run with cells, one or more ampule(s) of
frozen cells is removed from liquid nitrogen. After rapidly heating
in a 37.degree. C. water bath until most of the ice has melted, the
ampule is opened inside a vertical laminar flow hood. The contents
of the ampule are mixed with a 1 ml volume of Dulbecco's MEM/Ham's
F12(1:1) (DMEM/F12) to which ferric salts have been added to a
final concentration of 50 .mu.M of Fe+++. After mixing, the tube is
filled up to approximately 10 ml with the same medium and the cells
are collected by centrifugation. The cell pellet is resuspended
into 5 ml of the above mentioned medium with 20% fetal bovine serum
and seeded into 1 well of a 6-well tissue culture plate. The cells
are incubated in a 37.degree. C. incubator in a 5% CO.sub.2
-atmosphere. When the cells have established themselves in culture
and start to multiply and have an approximate cell concentration of
10.sup.6 /ml, the cells and the medium are moved into a tissue
culture flask with a surface area of 80 cm.sup.2 and diluted to 40
ml using DMEM/F12 (without serum). When the cells have once again
reached a concentration of 10.sup.6 /ml, they, and the medium, are
moved into a tissue culture flask with a surface area of 175
cm.sup.2 and further diluted to a volume of 100 ml using DMEM/F12.
When the cells have once again reached optimal concentration, the
cells and the medium are transferred into a roller bottle with a
850 cm.sup.2 surface area and diluted to a final volume of 500 ml.
When this roller bottle has reached optimal cell concentration, it
is split 1/3 into new roller bottles using the same medium as
before. This splitting process of the roller bottles is continued
until a sufficient number of bottles have been obtained in order to
give a desired number of cells to seed into the Verax System 200
reactor.
The Verax System 200
The Verax System 200 reactor is a closed cell culture system where
cells are cultivated in stainless steel weighted microspheres
(density 1.6 g/mL) composed of cross-lined type I bovine collagen.
The microspheres are loaded into a vertical transparent glass tube
through which the culture medium (same as above) is pumped,
entering at the bottom. The inlet to the tube is formed in such a
fashion that the microspheres will establish a fluidized bed
configuration when the medium is pumped through at a suitable
velocity. During operation, fresh medium is constantly added and
conditioned medium removed at a rate determined by the cell growth
as monitored by glucose consumption. Temperature is maintained at
37.degree. C.; pH is maintained at 7.1 and oxygen/nitrogen ratio is
also controlled.
After loading of the microspheres in 1% fetal bovine serum
containing medium, the reactor is run for at least three days
without cells to ascertain that the microsphere loading did not
contaminate the System. During this time the reactor is fed with
protein-free medium to reduce the priming dose of fetal bovine
serum. If all systems are operating satisfactorily, the cells from
the roller bottles are inoculated into the reactor.
The Verax System 2000
This equipment uses the same type of microspheres as the System
200, and its controls and operations are essentially the same as
for the smaller system. The System 2000 represents an approximately
15-fold scale-up compared to the System 200.
Monitoring of Yield of Antibody from Full-Scale Culture
The conditioned medium is monitored, each time the harvest tank is
emptied, for the level of human immunoglobulin in the supernatant
using an ELISA-type assay. The results are confirmed using a
Protein A HPLC method.
Harvesting of Cell Culture Media and Production of Harvest Pool
The conditioned medium is continuously being removed from the Verax
equipment into a refrigerated harvest tank. This medium is later
unloaded (using the nitrogen pressure in the verax system) into a
mobile stainless steel tank for further processing.
Cell Culture Media
The media routinely used is a 1:1 mixture of Dulbecco's MEM H21 and
Ham's F12 (Mediatech). The medium is purchased as a powder
sufficient for 50 liters of finished medium. Two such containers of
each medium powder are added into a stainless steel tank containing
approximately 190 liters of water. The powder is suspended with an
impeller until all has been dissolved. Sodium bicarbonate is added
as recommended by the manufacturer and the pH of the medium is set
to 7.4. Sodium selenite is added to a final concentration of 17.3
.mu.g/l and the volume is topped up to 2001 with water. The medium
is also supplemented with ferric ions in the form of ferric
nitrate/sodium citrate to a final concentration of 50 .mu.M Fe+++.
The medium is immediately added to the medium tank of the Verax
System S200 through the built-in sterilization filter. No protein
is added to the medium. No antibiotics of any type are ever
used.
PURIFICATION OF THE MONOCLONAL ANTIBODY
Description of Methodology of Harvesting and Purification of End
Product
The monoclonal antibody is produced in cell culture from a
hybridoma cell line in the absence of serum. This means that we
have a need to remove from the final product only components from
the cellular material. As human monoclonal antibodies are not in
themselves expected to be immunogenic, it becomes very important to
remove all potentially immunogenic components.
The goal of the purification procedures is a final product that is
more than 99.9% pure, using affinity chromatography. We depend
heavily on the biological specificity of affinity chromatography.
Each step of the purification process (summarized in Table 5) is
discussed in more detail, supra.
TABLE 5 ______________________________________ Purification Summary
Step Conditions Materials ______________________________________
Cell Removal Room Polyvinylidene difluoride Temp. filters,
0.65/0.45 .mu.m absolute. Concentration Microfiltration 4.degree.
C. Polysulfone filter nominal 30,000 daltons. Polyester (0.8 .mu.m)
and cellulose acetate (0.2 .mu.m) absolute filters. Protein A
4.degree. C. Agarose coupled Staphyloccocus chromatography aureus
Protein A. Concentration 4.degree. C. Cellulose triacetate filter,
nominal 20,000 dalton. Gel 4.degree. C. Sephacryl S-300, Ringer's
chromatography. Lactated Solution Ion exchange 4.degree. C.
Sephacryl S-300, Ringer's Lactated Solution
______________________________________
Cell Harvest and Removal of Particulate Materials from the
Conditioned Medium
Even though most of the cells are retained by the microspheres, a
sizable number of cells are present in the harvested supernatant.
To avoid gross contamination of the medium by cell components the
supernatant is filtered through a polyvinylidene difluoride 0.65
.mu.m Prostack.RTM. filter (Millipore), immediately after removal
from the Verax harvest tank. This type of filter unit works in a
tangential flow mode which allows filtration of large amount of
particulate material without clogging the filter. The cleared
medium is collected into a refrigerated stainless steel tank.
Concentration of Conditioned Medium
The conditioned medium is concentrated using a nominal 30,000
dalton polysulfone spiral wound membrane supplied by Millipore.
After concentration, the pH is set to 7.0 using 1M acetic acid. The
material is sterile filtered through a Sartobran-PH 0.8/0.2 .mu.m
(Sartorius) filter (the 0.8 .mu.m component is polyester, the 0.2
.mu.m component is cellulose acetate) before being stored at
4.degree. C. The material is microfiltrated (0.22 .mu.M Millipore)
and filled into polypropylene vessels.
Protein A Chromatography
The extremely powerful purification step utilizes the high affinity
of the human IgG1 antibody to Staphylococcus aureus Protein A.
The Protein A is purchased already coupled covalently by an amide
bond to agarose. After packing the gel in a column, the column with
its contents and attached tubing is sanitized by treatment with 70%
ethanol in water for 24 hours. The column is then equilibrated with
PBS, pH 7.0.
Performing the affinity chromatography separation on the Protein A
column involves the following sequential steps:
A) Loading.
The concentrated conditioned medium is loaded on the column with a
pump. The effluent from the column is collected and monitored for
the presence of antibody by the human immunoglobulin ELISA. The
column is loaded to such a degree that a measurable amount of
antibody-containing fluid passes through the column. The overload
fraction is separately recovered and recycled if it contains more
than 20 mg/ml antibody.
B) Washing.
To remove unbound materials the column is extensively washed with
phosphate buffered saline, pH 7 with sodium chloride added to a
final concentration of 0.5M. This wash is followed by a second
washing step using a buffer of 0.02M sodium citrate, pH 5.6,
containing 0.5M sodium chloride. This wash releases small amounts
of the human antibody.
C) Elution.
The bound monoclonal antibodies are eluted from the column using a
buffer composed of 0.02M sodium citrate, pH 3.0, containing 0.5M
sodium chloride. The eluted material is continuously diluted into a
volume of 1M Tris-HCl, pH 8.0 to rapidly restore near-neutral
conditions.
The Protein A purification is performed in a closed system
utilizing a Waters 650 Protein Purification System.
Concentration of Protein A Column Eluate
In order to make the following purification step more effective and
convenient, the eluate from the Protein A column is concentrated to
at least 5 mg/ml antibody. The concentrate is sterile filtered
through a 0.2 .mu.m filter and the sterile concentrate is stored at
4.degree. C. until sufficient materials have been collected for the
next purification step.
Size Separation by Gel Chromatography on Sephacryl S-300 High
Resolution
The antibody preparation is run on a Sephacryl S300 High Resolution
(Pharmacia) gel, packed in a Pharmacia BP113/120 column with a bed
volume of approximately 10 liters. The column is packed in Lactated
Ringer's Irrigation USP (Travenol Laboratories). The elution of the
column is monitored by a Waters 650 Protein Purification
System.
The purpose of this step is not principally additional
purification, but buffer change. After the elution of the Protein A
column the antibodies are in a complex, hypertonic buffer composed
of sodium citrate, sodium chloride and Tris-HCl. This buffer
mixture can not be used directly as a vehicle for an intravenous
injection. The buffer after this step is suitable both for
intravenous injection and for long term refrigerated storage.
Removal of Host Cell DNA by Passage over an Ion Exchange Column
Even after the Protein A chromatography, which removes the bulk of
DNA present in the concentrated supernatant, and the Sephacryl
S-300HR which removes DNA molecules that are either significantly
larger or significantly smaller than the monoclonal antibody
product, there is a small, but detectable, presence of DNA in the
antibody preparation. We have selected to remove this contaminant
by an ion exchange step on a strong anion exchanger, Q Sepharose
(Pharmacia Inc.). At the pH of Lactated Ringer's solution, antibody
proteins have a positive charge, and are repelled by the anion
exchanger. Nucleic acids, however, have a negative charge at this
pH, and will bind to the column.
The column was packed according to the manufacturer's suggestions.
After decanting the 20% ethanol solution the gel is delivered in,
100 ml of gel was suspended in 200 ml of Lactated Ringer's
solution. The slurry is poured into a Pharmacia K50/30 column, and
when the gel has packed itself to a constant volume, it is
sanitized with 1 column volume of 0.5N sodium hydroxide, followed
by 3 column volumes of Dulbecco's PBS, followed by 5 column volumes
of Lactated Ringer's solution. Immediately prior to use the column
was washed with an additional 5 column volumes of Lactated Ringer's
solution. The sample is then passed through the column and the
pass-through is collected in a sterile container.
EXAMPLE 8
Molecular Analyses of PE1-1, ZM1-1, ZM1-2 and MD3-4
The heavy variable (V.sub.H) chain of antibodies PE1-1, ZM1-1,
ZM1-2 and MD3-4 are isolated and sequenced. Total RNA is extracted
from 10.sup.7 hybridoma cells of each cell line using procedures
described in Sanz, et al. 1989 J. Immunol. 142:883, which is hereby
incorporated by reference. Single stranded DNA is synthesized using
AMV-reverse transcriptase as the enzyme and oligo-dT as the primer.
The quantity of the synthesized ss-cDNA is assessed by measuring
the incorporation of .sup.32 p-dCTP
Polymerase chain reactions (PCR) are performed essentially as
recommended by the manufacturer (Perkin Elmer Cetus, Norwalk,
Conn.). One microgram of DNA is added to a 200 .mu.m solution of
each of dATP, dCTP, dGTP and dTTP, with 100 p moles each of primer
and 5 units of Taq DNA polymerase. PCR cycles are as follows:
denaturation at 98.degree. C. for 3 minutes, annealing at
55.degree. C. for 2 minutes, and extension at 72.degree. C. for
three minutes, controlled in a DNA thermal cycler (Perkin Elmer
Cetus).
Amplified DNA is size selected on a 1.0% low melting agarose gel,
ligated into the EcoRV site of a BLUESCRIPT phagemid vector, and
transformed into CaCl.sub.2 competent BSJ72 bacteria. Single
stranded DNA for sequencing is isolated from each positive clone
after superinfection with M13K07 as described by Sanz, et al.,
supra. Sequencing is accomplished via the dideoxy chain termination
method as described by Sanger, et al. 1980 J. Mol. Biol. 143:161,
except a modified T7 DNA polymerase (Sequenase) is used as
described by Tabor, et al. 1987. PNAS (USA) 84:4767. Results are
given in Tables 8-1, 8-2, 8-3 and 8-4.
TABLE 8-1
__________________________________________________________________________
DNA sequence of the V.sub.H region of PE1-1 is shown below (SEQ ID
NO: 1). The leader, V.sub.H III, D, and J.sub.H 4 regions are
denoted by the dashed line; complementarity-determining regions
CDR1 and CDR2 are indicated by the asterisks. Amino acids appear as
single letter abbreviations below the DNA (SEQ ID NO: 2).
__________________________________________________________________________
##STR1## ATG GAG TTT GGG CTG AGC TGG GTT TTC CTC GTT GCT CTT TTA
AGA GGT GTC CAG TGT CAG GTG M E F G L S W V F L V A L L R G V Q C Q
V ##STR2## CAG CTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CTT GGG AGG
TCC CTG AGA CTC TCC TGT GCA Q L V E S G G G V V Q P G R S L R L S C
A ##STR3## GCC TCT GGA TTC ACC TTC AGT AGG TAT GGC ATG CAC TGG GTC
CGC CAG GCT CCA GGC AAG GGG A S G F T F S R Y G M H W V R Q A P G K
G ##STR4## CTG GAG TGG GTG GCA GTG ATA TCA TAT GAT GGA AGT AAT AAA
TGG TAT GCA GAC TCC GTG AAG L E W V A V I S Y D G S N K W Y A D S V
K ##STR5## GGC CGA TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACT CTG
TTT CTG CAA ATG CAC AGC CTG G R F T I S R D N S K N T L F L Q M H S
L ##STR6## AGA GCT GCG GAC ACG GGT GTA TAT TAC TGT GCG AAA GAT CAA
CTT TAC TTT GGT TCG CAG AGT R A A D T G V Y Y C A K D Q L Y F G S Q
S ##STR7## CCC GGG CAC TAC TGG GTC CAG GGA ACC CTG GTC ACC GTC TCC
TCA P G H Y W V Q G T L V T V S S
__________________________________________________________________________
TABLE 8-2
__________________________________________________________________________
DNA sequence of the V.sub.H region of ZM1-1 is shown below (SEQ ID
NO: 3). The leader, V.sub.H III, D, and J.sub.H 4 regions are
denoted by the dashed line; complementarity-determining regions
CDR1 and CDR2 are indicated by the asterisks. Amino acids appear as
single letter abbreviations below the DNA (SEQ ID NO: 4).
__________________________________________________________________________
##STR8## ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT GCT ATA TTA
GAA GGT GTC CAG TGT GAG GTG M E F G L S W V F L V A I L E G V Q C E
V ##STR9## CAG CTG GTG GAG TCT GGG GGA GGT TTG GTA CAG CCT GGG GGG
TCC CTG AGA CTC TCC TGT GCA Q L V E S G G G L V Q P G G S L R L S C
A ##STR10## GCC TCT GGA TTC ACC TTC AGT AGG TAC GAC ATG TAC TGG GTC
CGC CAA GCT ACA GGA AAA GGT A S G F T F S R Y D M Y W V R Q A T G K
G ##STR11## CTG GAG TGG GTC TCA GCT ATT GGT CCT ACT GGT GAC ACA TAC
TAT GCA GAC TCC GTG AAG GGC L E W V S A I G P T G D T Y Y A D S V K
G ##STR12## CGA TTC ACC ATC TCC AGA GAA AAT GCC AAG AAC TCC TTG TAT
CTT ACA ATG AAC GGC CTG AGA R F T I S R E N A K N S L Y L T M N G L
R ##STR13## GCC GGG GAC ACG GCT GTG TAT TAC TGT GCA AGA GAT TTA GAA
CTC TGG GGC CAG GGA ACC CTG A G D T A V Y Y C A R D L E L W G Q G T
L ##STR14## GTC ACC GTC TCC TCA V T V S S
__________________________________________________________________________
TABLE 8-3
__________________________________________________________________________
DNA sequence of the V.sub.H region of ZM1-2 is shown below (SEQ ID
NO: 5). The leader, V.sub.H IV, D, and J.sub.H 4 regions are
denoted by the dashed line; complementarity-determining regions
CDR1 and CDR2 are indicated by the asterisks. Amino acids appear as
single letter abbreviations below the DNA (SEQ ID NO: 6).
__________________________________________________________________________
##STR15## ATG AAA CAC CTG TGG TTC TTC CTC CTG CTG GTG GCS GTT CCC
AGA TGG GTC GTG TCC CAG GTG M K H L W F F L L L V A V P R W V V S Q
V ##STR16## CAG CTG CAG GAG TCG GGC CCA GGA CTG GTG AAG GCT GCG GAG
ACC CTG TCC CTC ACC TGC ACT Q L Q E S G P G L V K A S E T L S L T C
T ##STR17## GTC TCC CGT GGC TCC TTC AGT GAT TAC TTC TGG AAT TGG TTC
CGG CAG CCC GCC GGG AAG CGC V S R G S F S D Y F W N W F R Q P A G K
R ##STR18## CTG GAG TGG CTT GGG CGT GTC TAT ACC AGT GGA AGT GTC GAC
TAC AAC CCC TCC CTC AAG AGT L E W L G R V Y T S G S V D Y N P S L K
S ##STR19## CGA GTC ACC GTG TCA GTG GAC ACG TCC AAG AAG CAG TTC TCC
CTG AGG CTG AGC TCT GTG ACC R V T V S V D T S K K Q F S L R L S S V
T ##STR20## GTC GCG GAC ACG GCC GTG TAT TAT TGT GCG AGA GGA CTG TCC
GGT TTT GAC TAC TGG GGC CAG V A D T A V Y Y C A R G L S G F D Y W G
Q ##STR21## GGA GCC CTG GTC ACC GTC TCC CCA G A L V T V S P
__________________________________________________________________________
TABLE 8-4
__________________________________________________________________________
DNA sequence of the V.sub.H region of MD3-4 is shown below (SEQ ID
NO: 7). The leader, V.sub.H V, D, and J.sub.H 3 regions are denoted
by the dashed line; complementarity-determining regions CDR1 and
CDR2 are indicated by the asterisks. Amino acids appear as single
letter abbreviations below the DNA (SEQ ID NO: 8).
__________________________________________________________________________
##STR22## ATG GGG TCA ACC GCC ATC CTT GGC CTC CTC CTG GCT GTT CTC
CAA GGA GTC TGT GCC GAA GTG M G S T A I L G L L L A V L Q G V C A E
V ##STR23## CAG CTG GTG CAA TCT GGA GCA GAG GTG AAA AAG CCC GGG GAG
TCT CTG AGG ATC TCC TGT AAG Q L V Q S G A E V K K P G E S L R I S C
K ##STR24## GGT TCT GGA TAC AGC TTT ACC AGC TAC TGG ATC AGC TGG GTG
CGC CAG ATG CCC GGG AA GGC G S G Y S F T S Y W I S W V R Q M P G K
G ##STR25## CTG GAG TGG ATG GGG AGG CTT GAT CCT AGT GCC TCC TCT GCC
ATC TTC AGC CCG TCC CTC CAA L E W M G R L D P S A S S A I F S P S L
Q ##STR26## GGC CAC GTC ACC ATC TCA GTT GAC AAG TCC ATG AGG ACT GCC
TAC GTG CAG TGG AGA AGC CTG G H V T I S V D K S M R T A Y V Q W R S
L ##STR27## AAG GCC TCG GAC ACC GCC ATG TAT TAC TGT GCG AGA CAT GTC
CGC GAA AAG AGT ATG GTT CAG K A S D T A M Y Y C A R H V R E K S M V
Q ##STR28## GGA GTC ATT ATA AAG GAC GCT TTT GAT ATC TGG GGC CAA GGG
ACA ATG GTC ACC GTC TCT TCA G V I I K D A F D I W G Q G T M V T V S
S
__________________________________________________________________________
EXAMPLE 9
Following the procedures of Example 8, the light variable (V.sub.L)
chain of antibodies PE1-1, ZM1-1, ZM1-2 and MD3-4 are isolated and
sequenced. Results are given in Tables 9-1, 9-2, 9-3 and 9-4.
TABLE 9-1
__________________________________________________________________________
DNA sequence of the V.sub.L region of PE1-1 is shown below (SEQ ID
NO: 9). The leader, VV and J3 regions are denoted by the dashed
line; complementarity-determining regions CDR1, CDR2 and CDR3 are
indicated by the asterisks. Amino acids appear as single letter
abbreviations below the DNA (SEQ ID NO: 10).
__________________________________________________________________________
##STR29## CAG TCT CAG CTG ACG CAG CCG CCC TCG GTG TCA GTG GCC CCA
GGG CAG ACG GCC AGG ATT ACC Q S Q L T Q P P S V S V A P G Q T A R I
T ##STR30## TGT GGG GGA GAC ACC ATT GGG AGT AAA AGT GTG AAC TGG TTC
CAG CAG AAG CCA GGC CAG GCC C G G D N I G S K S V N W F Q Q K P G Q
A ##STR31## CCT GTC CTG GTC GTC TAT GAT GAT AAC GAA CGG CCC TCA GGC
ATT TCT GAG CGA TTC TCT GGC P V L V V Y D D N E R P S G I S E R F S
G ##STR32## TCC AAC TCT GGG AAC ACG GCC ACC CTG ACC ATC AGC AGG GTC
GAA GCC GGG GAT GAG GCC GAC S N S G N T A T L T I S R V E A G D E A
D ##STR33## TAT TAC TGT CAG GTG TGG GAT AGT AGT AGT GAT CAT GTG GTA
TTC GGC GGA GGG ACC AAG CTG Y Y C Q V W D S S S D H V V F G G G T K
L ##STR34## ACC GTC CTA T V L
__________________________________________________________________________
TABLE 9-2
__________________________________________________________________________
DNA sequence of the V.sub.L region of ZM1-1 is shown below (SEQ ID
NO: 11). The leader, VII and J5 regions are denoted by the dashed
line; complementarity-determining regions CDR1, CDR2 and CDR3 are
indicated by the asterisks. Amino acids appear as single letter
abbreviations below the DNA (SEQ ID NO: 12).
__________________________________________________________________________
##STR35## ATG GAC ACG AGG GTC CCC GCT CAG CTC CTG GGG CTG CTA ATG
CTC TGG GTC CCA GGA TCC AGT M D T R V P A Q L L G L L M L W V P G S
S ##STR36## GGG GAT GTT GTG GTG ACT CAG TCT CCA CTC TCC CTG CCC GTC
ACC CTT GGA CAG CCG GCC TCC G D V V V T Q S P L S L P V T L G Q P A
S ##STR37## ATC TCC TGC AGA TCT AGT CTA AGC CTC GTG GAC AGT GAC GGA
AAC ACC TAC TTG AAT TGG TTT I S C R S S L S L V D S D G N T Y L N W
F ##STR38## CTC CAG AGG CCA GGC CAA TCT CCA AGG CGC CTA ATT TAT CAG
CTT TCT AGC CGG GAC TCT GGG L Q R P G Q S P R R L I Y Q L S S R D S
G ##STR39## GTC CCA GAC AGA TCC AGC GGC AGT GGG TCA GGC ACT GAT TTC
ACT CTG AAA ATC AGC AGG GTG V P D R F S G S G S G T D F T L K I S R
V ##STR40## GAG GCT GAG GAT GTT GGC GTT TAT TAC TGC ATG CAA GGT ACA
CAC TGG CCG ATC ACC TTC GGC E A E D V G V Y Y C M Q G T H W P I T F
G ##STR41## CAA GGG ACA CGA CTG GAG ATT AAA CGA Q P T R L E I K R
__________________________________________________________________________
TABLE 9-3
__________________________________________________________________________
DNA sequence of the V.sub.L region of ZM1-2 is shown below (SEQ ID
NO: 13). The leader, VI and J regions are denoted by the dashed
line; complementarity-determining regions CDR1, CDR2 and CDR3 are
indicated by the asterisks. Amino acids appear as single letter
abbreviations below the DNA (SEQ ID NO: 14).
__________________________________________________________________________
##STR42## ATG AGG CCC GTC GCT CAG CTC CTG GGG CTC CTG CTG CTC TGG
TTC CCA GGT TCC AGA TGC GAC M R P V A Q L L G L L L L W F P G S R C
D ##STR43## ATC CAG ATG ACC CAG TCT CCA TCT TCC GTG TCT GCA TCT GTG
GGA GAC AGA GTC ACC GTC ACT I Q M T Q S P S S V S A S V G D R V T V
T ##STR44## TGT CGG GCG AGT CAG GGT ATT AGC AGT TGG TTA GCC TGG TAT
CAG CAG AAA CCA GGG AAA GCC C R A S Q G I S S W L A W Y Q Q K P G K
A ##STR45## CCT AAA CTC CTG ATC CAT GCT GCA TCC AGT TTG CAA AGT GGG
GTC CCA TCA AGG TTC ATC GGC P K L L I H A A S S L Q S G V P S R F I
G ##STR46## AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC ATC ACC AGC CTG
CAG GCT GAA GAT TTT GCA ACC S G S G T D F T L T I T S L Q A E D F A
T ##STR47## TAC TAT TGT CAA CAG GCT GAC AGT CTC CCT TTT ACT TTC GGC
GGA GGG ACC AAG GTG GAC TTC Y Y C Q Q A D S L P F T F G G G T K V D
F ##STR48## AAA CGA K R
__________________________________________________________________________
TABLE 9-4
__________________________________________________________________________
DNA sequence of the V.sub.L region of MD3-4 is shown below (SEQ ID
NO: 15). The VIII and J3 regions are denoted by the dashed line;
complementarity-determining regions CDR1, CDR2 and CDR3 are
indicated by the asterisks. Amino acids appear as single letter
abbreviations below the DNA (SEQ ID NO: 16).
__________________________________________________________________________
##STR49## CAG TCT CAG CTG ACG CAG CCT GCC TCA GTG TCC GTG TCC CCA
GGA CAG ACA GCC AGC ATC ACC Q S Q L T Q P A S V S V S P G Q T A S I
T ##STR50## TGC TCT GGA GAT AGA TTG GGG GAT GAA TTT GCT TCC TGG TAT
CAG CAG AAG CCA GGC CAG TCC C S G D R L G D E F A S W Y Q Q K P G Q
S ##STR51## CCT ATT CTG GTC ATC TTT GAG GAT AAC AAG AGG CCC TCA GGG
ATC CCT GAA CGA TTC TCT GGC P I L V I F E D N K R P S G I P E R F S
G ##STR52## TCC AAC TCT GGG AAC ACA GCC ACT CTG ACC ATC AGC GGG ACC
CAG GCT ATG GAT GAG GCT GAC S N S G N T A T L T I S G T Q A M D E A
D ##STR53## TAT TAC TGT CTG GCG TGG GCC AGC AGC CTT TGG GTG TTC GGC
GGA GGG ACC AAG CTG ACC GTC Y Y C L A W A S S L W V F G G G T K L T
V ##STR54## TTG
__________________________________________________________________________
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 16 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 423 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE: Hybridoma (H)
CELL LINE: PE1-1 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
1..423 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGT48
MetGluPheGlyLeuSerTrpValPheLeuValAlaLeuLeuArgGly 151015
GTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAG96
ValGlnCysGlnValGlnLeuValGluSerGlyGlyGlyValValGln 202530
CCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTC144
ProGlyArgSerLeuArgLeuSerCysAlaAlaSerGlyPheThrPhe 354045
AGTAGGTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTG192
SerArgTyrGlyMetHisTrpValArgGlnAlaProGlyLysGlyLeu 505560
GAGTGGGTGGCAGTGATATCATATGATGGAAGTAATAAATGGTATGCA240
GluTrpValAlaValIleSerTyrAspGlySerAsnLysTrpTyrAla 65707580
GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAC288
AspSerValLysGlyArgPheThrIleSerArgAspAsnSerLysAsn 859095
ACTCTGTTTCTGCAAATGCACAGCCTGAGAGCTGCGGACACGGGTGTA336
ThrLeuPheLeuGlnMetHisSerLeuArgAlaAlaAspThrGlyVal 100105110
TATTACTGTGCGAAAGATCAACTTTACTTTGGTTCGCAGAGTCCCGGG384
TyrTyrCysAlaLysAspGlnLeuTyrPheGlySerGlnSerProGly 115120125
CACTACTGGGTCCAGGGAACCCTGGTCACCGTCTCCTCA423
HisTyrTrpValGlnGlyThrLeuValThrValSerSer 130135140 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 141
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetGluPheGlyLeuSerTrpValPheLeuValAlaLeuLeuArgGly 151015
ValGlnCysGlnValGlnLeuValGluSerGlyGlyGlyValValGln 202530
ProGlyArgSerLeuArgLeuSerCysAlaAlaSerGlyPheThrPhe 354045
SerArgTyrGlyMetHisTrpValArgGlnAlaProGlyLysGlyLeu 505560
GluTrpValAlaValIleSerTyrAspGlySerAsnLysTrpTyrAla 65707580
AspSerValLysGlyArgPheThrIleSerArgAspAsnSerLysAsn 859095
ThrLeuPheLeuGlnMetHisSerLeuArgAlaAlaAspThrGlyVal 100105110
TyrTyrCysAlaLysAspGlnLeuTyrPheGlySerGlnSerProGly 115120125
HisTyrTrpValGlnGlyThrLeuValThrValSerSer 130135140 (2) INFORMATION
FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 393 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G)
CELL TYPE: Hybridoma (H) CELL LINE: ZM1-1 (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..393 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:3: ATGGAGTTTGGGCTGAGCTGGGTTTTCCTTGTTGCTATATTAGAAGGT48
MetGluPheGlyLeuSerTrpValPheLeuValAlaIleLeuGluGly 145150155
GTCCAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTTTGGTACAG96
ValGlnCysGluValGlnLeuValGluSerGlyGlyGlyLeuValGln 160165170
CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTC144
ProGlyGlySerLeuArgLeuSerCysAlaAlaSerGlyPheThrPhe 175180185
AGTAGGTACGACATGTACTGGGTCCGCCAAGCTACAGGAAAAGGTCTG192
SerArgTyrAspMetTyrTrpValArgGlnAlaThrGlyLysGlyLeu 190195200205
GAGTGGGTCTCAGCTATTGGTCCTACTGGTGACACATACTATGCAGAC240
GluTrpValSerAlaIleGlyProThrGlyAspThrTyrTyrAlaAsp 210215220
TCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCC288
SerValLysGlyArgPheThrIleSerArgGluAsnAlaLysAsnSer 225230235
TTGTATCTTACAATGAACGGCCTGAGAGCCGGGGACACGGCTGTGTAT336
LeuTyrLeuThrMetAsnGlyLeuArgAlaGlyAspThrAlaValTyr 240245250
TACTGTGCAAGAGATTTAGAACTCTGGGGCCAGGGAACCCTGGTCACC384
TyrCysAlaArgAspLeuGluLeuTrpGlyGlnGlyThrLeuValThr 255260265
GTCTCCTCA393 ValSerSer 270 (2) INFORMATION FOR SEQ ID NO:4: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 131 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetGluPheGlyLeuSerTrpValPheLeuValAlaIleLeuGluGly 151015
ValGlnCysGluValGlnLeuValGluSerGlyGlyGlyLeuValGln 202530
ProGlyGlySerLeuArgLeuSerCysAlaAlaSerGlyPheThrPhe 354045
SerArgTyrAspMetTyrTrpValArgGlnAlaThrGlyLysGlyLeu 505560
GluTrpValSerAlaIleGlyProThrGlyAspThrTyrTyrAlaAsp 65707580
SerValLysGlyArgPheThrIleSerArgGluAsnAlaLysAsnSer 859095
LeuTyrLeuThrMetAsnGlyLeuArgAlaGlyAspThrAlaValTyr 100105110
TyrCysAlaArgAspLeuGluLeuTrpGlyGlnGlyThrLeuValThr 115120125
ValSerSer 130 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 402 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE: Hybridoma (H)
CELL LINE: ZM1-2 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:
1..402 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCAGTTCCCAGATGG48
MetLysHisLeuTrpPhePheLeuLeuLeuValAlaValProArgTrp 135140145
GTCGTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAG96
ValValSerGlnValGlnLeuGlnGluSerGlyProGlyLeuValLys 150155160
GCTGCGGAGACCCTGTCCCTCACCTGCACTGTCTCCCGTGGCTCCTTC144
AlaAlaGluThrLeuSerLeuThrCysThrValSerArgGlySerPhe 165170175
AGTGATTACTTCTGGAATTGGTTCCGGCAGCCCGCCGGGAAGCGCCTG192
SerAspTyrPheTrpAsnTrpPheArgGlnProAlaGlyLysArgLeu 180185190195
GAGTGGCTTGGGCGTGTCTATACCAGTGGAAGTGTCGACTACAACCCC240
GluTrpLeuGlyArgValTyrThrSerGlySerValAspTyrAsnPro 200205210
TCCCTCAAGAGTCGAGTCACCGTGTCAGTGGACACGTCCAAGAAGCAG288
SerLeuLysSerArgValThrValSerValAspThrSerLysLysGln 215220225
TTCTCCCTGAGGCTGAGCTCTGTGACCGTCGCGGACACGGCCGTGTAT336
PheSerLeuArgLeuSerSerValThrValAlaAspThrAlaValTyr 230235240
TATTGTGCGAGAGGACTGTCCGGTTTTGACTACTGGGGCCAGGGAGCC384
TyrCysAlaArgGlyLeuSerGlyPheAspTyrTrpGlyGlnGlyAla 245250255
CTGGTCACCGTCTCCCCA402 LeuValThrValSerPro 260265 (2) INFORMATION FOR
SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 134 amino
acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetLysHisLeuTrpPhePheLeuLeuLeuValAlaValProArgTrp 151015
ValValSerGlnValGlnLeuGlnGluSerGlyProGlyLeuValLys 202530
AlaAlaGluThrLeuSerLeuThrCysThrValSerArgGlySerPhe 354045
SerAspTyrPheTrpAsnTrpPheArgGlnProAlaGlyLysArgLeu 505560
GluTrpLeuGlyArgValTyrThrSerGlySerValAspTyrAsnPro 65707580
SerLeuLysSerArgValThrValSerValAspThrSerLysLysGln 859095
PheSerLeuArgLeuSerSerValThrValAlaAspThrAlaValTyr 100105110
TyrCysAlaArgGlyLeuSerGlyPheAspTyrTrpGlyGlnGlyAla 115120125
LeuValThrValSerPro 130 (2) INFORMATION FOR SEQ ID NO:7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 441 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE:
Hybridoma (H) CELL LINE: MD3-4 (ix) FEATURE: (A) NAME/KEY: CDS (B)
LOCATION: 1..441 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGGGGTCAACCGCCATCCTTGGCCTCCTCCTGGCTGTTCTCCAAGGA48
MetGlySerThrAlaIleLeuGlyLeuLeuLeuAlaValLeuGlnGly 135140145150
GTCTGTGCCGAAGTGCAGCTGGTGCAATCTGGAGCAGAGGTGAAAAAG96
ValCysAlaGluValGlnLeuValGlnSerGlyAlaGluValLysLys 155160165
CCCGGGGAGTCTCTGAGGATCTCCTGTAAGGGTTCTGGATACAGCTTT144
ProGlyGluSerLeuArgIleSerCysLysGlySerGlyTyrSerPhe 170175180
ACCAGCTACTGGATCAGCTGGGTGCGCCAGATGCCCGGGAAGGGCCTG192
ThrSerTyrTrpIleSerTrpValArgGlnMetProGlyLysGlyLeu 185190195
GAGTGGATGGGGAGGCTTGATCCTAGTGCCTCCTCTGCCATCTTCAGC240
GluTrpMetGlyArgLeuAspProSerAlaSerSerAlaIlePheSer 200205210
CCGTCCCTCCAAGGCCACGTCACCATCTCAGTTGACAAGTCCATGAGG288
ProSerLeuGlnGlyHisValThrIleSerValAspLysSerMetArg 215220225230
ACTGCCTACGTGCAGTGGAGAAGCCTGAAGGCCTCGGACACCGCCATG336
ThrAlaTyrValGlnTrpArgSerLeuLysAlaSerAspThrAlaMet 235240245
TATTACTGTGCGAGACATGTCCGCGAAAAGAGTATGGTTCAGGGAGTC384
TyrTyrCysAlaArgHisValArgGluLysSerMetValGlnGlyVal
250255260 ATTATAAAGGACGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACC432
IleIleLysAspAlaPheAspIleTrpGlyGlnGlyThrMetValThr 265270275
GTCTCTTCA441 ValSerSer 280 (2) INFORMATION FOR SEQ ID NO:8: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 147 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetGlySerThrAlaIleLeuGlyLeuLeuLeuAlaValLeuGlnGly 151015
ValCysAlaGluValGlnLeuValGlnSerGlyAlaGluValLysLys 202530
ProGlyGluSerLeuArgIleSerCysLysGlySerGlyTyrSerPhe 354045
ThrSerTyrTrpIleSerTrpValArgGlnMetProGlyLysGlyLeu 505560
GluTrpMetGlyArgLeuAspProSerAlaSerSerAlaIlePheSer 65707580
ProSerLeuGlnGlyHisValThrIleSerValAspLysSerMetArg 859095
ThrAlaTyrValGlnTrpArgSerLeuLysAlaSerAspThrAlaMet 100105110
TyrTyrCysAlaArgHisValArgGluLysSerMetValGlnGlyVal 115120125
IleIleLysAspAlaPheAspIleTrpGlyGlnGlyThrMetValThr 130135140
ValSerSer 145 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 324 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL
SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE: Hybridoma (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..324 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:9:
CAGTCTCAGCTGACGCAGCCGCCCTCGGTGTCAGTGGCCCCAGGGCAG48
GlnSerGlnLeuThrGlnProProSerValSerValAlaProGlyGln 150155160
ACGGCCAGGATTACCTGTGGGGGAGACAACATTGGGAGTAAAAGTGTG96
ThrAlaArgIleThrCysGlyGlyAspAsnIleGlySerLysSerVal 165170175
AACTGGTTCCAGCAGAAGCCAGGCCAGGCCCCTGTCCTGGTCGTCTAT144
AsnTrpPheGlnGlnLysProGlyGlnAlaProValLeuValValTyr 180185190195
GATGATAACGAACGGCCCTCAGGCATTTCTGAGCGATTCTCTGGCTCC192
AspAspAsnGluArgProSerGlyIleSerGluArgPheSerGlySer 200205210
AACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGG240
AsnSerGlyAsnThrAlaThrLeuThrIleSerArgValGluAlaGly 215220225
GATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCAT288
AspGluAlaAspTyrTyrCysGlnValTrpAspSerSerSerAspHis 230235240
GTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA324
ValValPheGlyGlyGlyThrLysLeuThrValLeu 245250255 (2) INFORMATION FOR
SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 108 amino
acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GlnSerGlnLeuThrGlnProProSerValSerValAlaProGlyGln 151015
ThrAlaArgIleThrCysGlyGlyAspAsnIleGlySerLysSerVal 202530
AsnTrpPheGlnGlnLysProGlyGlnAlaProValLeuValValTyr 354045
AspAspAsnGluArgProSerGlyIleSerGluArgPheSerGlySer 505560
AsnSerGlyAsnThrAlaThrLeuThrIleSerArgValGluAlaGly 65707580
AspGluAlaAspTyrTyrCysGlnValTrpAspSerSerSerAspHis 859095
ValValPheGlyGlyGlyThrLysLeuThrValLeu 100105 (2) INFORMATION FOR SEQ
ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 405 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY:
unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G)
CELL TYPE: Hybridoma (H) CELL LINE: ZM1-1 (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..405 (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:11: ATGGACACGAGGGTCCCCGCTCAGCTCCTGGGGCTGCTAATGCTCTGG48
MetAspThrArgValProAlaGlnLeuLeuGlyLeuLeuMetLeuTrp 110115120
GTCCCAGGATCCAGTGGGGATGTTGTGGTGACTCAGTCTCCACTCTCC96
ValProGlySerSerGlyAspValValValThrGlnSerProLeuSer 125130135140
CTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGT144
LeuProValThrLeuGlyGlnProAlaSerIleSerCysArgSerSer 145150155
CTAAGCCTCGTGGACAGTGACGGAAACACCTACTTGAATTGGTTTCTC192
LeuSerLeuValAspSerAspGlyAsnThrTyrLeuAsnTrpPheLeu 160165170
CAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATCAGCTTTCTAGC240
GlnArgProGlyGlnSerProArgArgLeuIleTyrGlnLeuSerSer 175180185
CGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACT288
ArgAspSerGlyValProAspArgPheSerGlySerGlySerGlyThr 190195200
GATTTCACTCTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGCGTT336
AspPheThrLeuLysIleSerArgValGluAlaGluAspValGlyVal 205210215220
TATTACTGCATGCAAGGTACACACTGGCCGATCACCTTCGGCCAAGGG384
TyrTyrCysMetGlnGlyThrHisTrpProIleThrPheGlyGlnGly 225230235
ACACGACTGGAGATTAAACGA405 ThrArgLeuGluIleLysArg 240 (2) INFORMATION
FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 135
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
MetAspThrArgValProAlaGlnLeuLeuGlyLeuLeuMetLeuTrp 151015
ValProGlySerSerGlyAspValValValThrGlnSerProLeuSer 202530
LeuProValThrLeuGlyGlnProAlaSerIleSerCysArgSerSer 354045
LeuSerLeuValAspSerAspGlyAsnThrTyrLeuAsnTrpPheLeu 505560
GlnArgProGlyGlnSerProArgArgLeuIleTyrGlnLeuSerSer 65707580
ArgAspSerGlyValProAspArgPheSerGlySerGlySerGlyThr 859095
AspPheThrLeuLysIleSerArgValGluAlaGluAspValGlyVal 100105110
TyrTyrCysMetGlnGlyThrHisTrpProIleThrPheGlyGlnGly 115120125
ThrArgLeuGluIleLysArg 130135 (2) INFORMATION FOR SEQ ID NO:13: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 384 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (G) CELL TYPE:
Hybridoma (H) CELL LINE: ZM1-2 (ix) FEATURE: (A) NAME/KEY: CDS (B)
LOCATION: 1..384 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ATGAGGCCCGTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCA48
MetArgProValAlaGlnLeuLeuGlyLeuLeuLeuLeuTrpPhePro 140145150
GGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCT96
GlySerArgCysAspIleGlnMetThrGlnSerProSerSerValSer 155160165
GCATCTGTGGGAGACAGAGTCACCGTCACTTGTCGGGCGAGTCAGGGT144
AlaSerValGlyAspArgValThrValThrCysArgAlaSerGlnGly 170175180
ATTAGCAGTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCT192
IleSerSerTrpLeuAlaTrpTyrGlnGlnLysProGlyLysAlaPro 185190195
AAACTCCTGATCCATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCA240
LysLeuLeuIleHisAlaAlaSerSerLeuGlnSerGlyValProSer 200205210215
AGGTTCATCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACC288
ArgPheIleGlySerGlySerGlyThrAspPheThrLeuThrIleThr 220225230
AGCCTGCAGGCTGAAGATTTTGCAACCTACTATTGTCAACAGGCTGAC336
SerLeuGlnAlaGluAspPheAlaThrTyrTyrCysGlnGlnAlaAsp 235240245
AGTCTCCCTTTTACTTTCGGCGGAGGGACCAAGGTGGACTTCAAACGA384
SerLeuProPheThrPheGlyGlyGlyThrLysValAspPheLysArg 250255260 (2)
INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 128 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:14: MetArgProValAlaGlnLeuLeuGlyLeuLeuLeuLeuTrpPhePro 151015
GlySerArgCysAspIleGlnMetThrGlnSerProSerSerValSer 202530
AlaSerValGlyAspArgValThrValThrCysArgAlaSerGlnGly 354045
IleSerSerTrpLeuAlaTrpTyrGlnGlnLysProGlyLysAlaPro 505560
LysLeuLeuIleHisAlaAlaSerSerLeuGlnSerGlyValProSer 65707580
ArgPheIleGlySerGlySerGlyThrAspPheThrLeuThrIleThr 859095
SerLeuGlnAlaGluAspPheAlaThrTyrTyrCysGlnGlnAlaAsp 100105110
SerLeuProPheThrPheGlyGlyGlyThrLysValAspPheLysArg 115120125 (2)
INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 318 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: Homo sapiens (G) CELL TYPE: Hybridoma (H) CELL LINE:
MD3-4 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..318 (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:15:
CAGTCTCAGCTGACGCAGCCTGCCTCAGTGTCCGTGTCCCCAGGACAG48
GlnSerGlnLeuThrGlnProAlaSerValSerValSerProGlyGln 130135140
ACAGCCAGCATCACCTGCTCTGGAGATAGATTGGGGGATGAATTTGCT96
ThrAlaSerIleThrCysSerGlyAspArgLeuGlyAspGluPheAla 145150155160
TCCTGGTATCAGCAGAAGCCAGGCCAGTCCCCTATTCTGGTCATCTTT144
SerTrpTyrGlnGlnLysProGlyGlnSerProIleLeuValIlePhe 165170175
GAGGATAACAAGAGGCCCTCAGGGATCCCTGAACGATTCTCTGGCTCC192
GluAspAsnLysArgProSerGlyIleProGluArgPheSerGlySer 180185190
AACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATG240
AsnSerGlyAsnThrAlaThrLeuThrIleSerGlyThrGlnAlaMet 195200205
GATGAGGCTGACTATTACTGTCTGGCGTGGGCCAGCAGCCTTTGGGTG288
AspGluAlaAspTyrTyrCysLeuAlaTrpAlaSerSerLeuTrpVal 210215220
TTCGGCGGAGGGACCAAGCTGACCGTCTTG318 PheGlyGlyGlyThrLysLeuThrValLeu
225230 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 106 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:16:
GlnSerGlnLeuThrGlnProAlaSerValSerValSerProGlyGln 151015
ThrAlaSerIleThrCysSerGlyAspArgLeuGlyAspGluPheAla 202530
SerTrpTyrGlnGlnLysProGlyGlnSerProIleLeuValIlePhe 354045
GluAspAsnLysArgProSerGlyIleProGluArgPheSerGlySer 505560
AsnSerGlyAsnThrAlaThrLeuThrIleSerGlyThrGlnAlaMet 65707580
AspGluAlaAspTyrTyrCysLeuAlaTrpAlaSerSerLeuTrpVal 859095
PheGlyGlyGlyThrLysLeuThrValLeu 100105
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