U.S. patent application number 10/901650 was filed with the patent office on 2005-03-10 for antibodies and methods for generating genetically altered antibodies with enhanced effector function.
Invention is credited to Grasso, Luigi, Nicolaides, Nicholas C., Sands, Howard, Sass, Philip M..
Application Number | 20050054048 10/901650 |
Document ID | / |
Family ID | 34115492 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054048 |
Kind Code |
A1 |
Grasso, Luigi ; et
al. |
March 10, 2005 |
Antibodies and methods for generating genetically altered
antibodies with enhanced effector function
Abstract
Dominant negative alleles of human mismatch repair genes can be
used to generate hypermutable cells and organisms. By introducing
these genes into cells and transgenic animals, new cell lines and
animal varieties with novel and useful properties can be prepared
more efficiently than by relying on the natural rate of mutation.
These methods are useful for generating genetic diversity within
immunoglobulin genes directed against an antigen of interest to
produce altered antibodies with enhanced biochemical activity.
Moreover, these methods are useful for generating
antibody-producing cells with increased level of antibody
production. The invention also provides methods for increasing the
effector function of monoclonal antibodies and monoclonal
antibodies with increased effector function.
Inventors: |
Grasso, Luigi; (Bala Cynwyd,
PA) ; Nicolaides, Nicholas C.; (Boothwyn, PA)
; Sands, Howard; (Wilmington, DE) ; Sass, Philip
M.; (Audubon, PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
34115492 |
Appl. No.: |
10/901650 |
Filed: |
July 29, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60491310 |
Jul 29, 2003 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/326; 435/455; 530/387.1; 536/23.53; 800/6 |
Current CPC
Class: |
C07K 16/4291 20130101;
C12N 15/1024 20130101; C07K 16/00 20130101; C07K 2317/92 20130101;
C07K 2317/732 20130101 |
Class at
Publication: |
435/069.1 ;
435/455; 435/326; 530/387.1; 536/023.53; 800/006 |
International
Class: |
C07H 021/04; C07K
016/18; C12N 015/87 |
Claims
What is claimed:
1. A method of producing an antibody with enhanced effector
function comprising introducing into an antibody-producing cell a
dominant negative allele of a mismatch repair gene, whereby said
antibody-producing cell becomes hypermutable; screening said
hypermutable cells for cells that produce antibodies with increased
effector function, thereby producing an antibody with enhanced
effector function.
2. The method of claim 1 wherein said dominant negative allele is a
PMS2 allele.
3. The method of claim 2 wherein said PMS2 allele comprises a
truncation mutation at codon 134.
4. The method of claim 3 wherein said truncation mutation is a
thymidine at nucleotide 424 of wild-type PMS2.
5. The method of claim 3 wherein said PMS2 allele encodes the first
133 amino acids of wild-type PMS2.
6. The method of claim 2 wherein the PMS2 allele is human PMS2.
7. The method of claim 1 wherein said effector function is
antibody-dependent cytotoxicity (ADCC) activity.
8. The method of claim 1 further comprising restoring genetic
stability to said antibody-producing cell.
9. The method of claim 1 further comprising exposing said
antibody-producing cell to a chemical mutagen.
10. The method of claim 1 wherein said antibody comprises an amino
acid substitution with proline or hydroxyproline.
11. The method of claim 1 wherein said antibody comprises an amino
acid substitution in a heavy chain variable region.
12. The method of claim 1 wherein said antibody comprises an amino
acid substitution in a light chain variable region.
13. The method of claim 1 wherein said antibody comprises an amino
acid substitution in a first framework region of a heavy chain.
14. The method of claim 13 wherein said substitution occurs at
position 6 of a first framework region comprising the amino acid
sequence of SEQ ID NO:18.
15. The method of claim 1 wherein said antibody comprises an amino
acid substitution in a second framework region of a light
chain.
16. The method of claim 14 wherein said amino acid substitution
comprises proline or hydroxyproline.
17. The method of claim 15 wherein said substitution occurs at
position 22 of a second framework region comprising the amino acid
sequence of SEQ ID NO:21.
18. The method of claim 1 wherein said antibody comprises an amino
acid substitution in a first framework region of a heavy chain and
an amino acid substitution in a second framework region of a light
chain.
19. The method of claim 17 wherein said amino acid substitution
comprises proline or hydroxyproline.
20. An antibody having enhanced effector function produced
according to the method of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This claims the benefit of U.S. Provisional Application
60/491,310, filed Jul. 29, 2003, the entire contents of which are
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention is related to the area of antibody effector
function and cellular production. In particular, it is related to
the field of mutagenesis.
BACKGROUND OF THE INVENTION
[0003] The use of antibodies to block the activity of foreign
and/or endogenous polypeptides provides an effective and selective
strategy for treating the underlying cause of disease. For example,
monoclonal antibodies (MAb), such as the FDA-approved ReoPro
(Glaser, V. (1996) "Can ReoPro repolish tarnished monoclonal
therapeutics?" Nat. Biotechnol. 14:1216-1217), an anti-platelet MAb
from Centocor; Herceptin (Weiner, L. M. (1999) "Monoclonal antibody
therapy of cancer" Semin. Oncol. 26:43-51), an anti-Her2/neu MAb
from Genentech; and Synagis (Saez-Llorens, X. E., et al. (1998)
"Safety and pharmacokinetics of an intramuscular humanized
monoclonal antibody to respiratory syncytial virus in premature
infants and infants with bronchopulmonary dysplasia" Pediat.
Infect. Dis. J. 17:787-791), an anti-respiratory syncytial virus
MAb produced by Medimmune, have been used as effective
therapeutics.
[0004] Standard methods for generating MAbs against candidate
protein targets are known by those skilled in the art. Briefly,
rodents such as mice or rats are injected with a purified antigen
in the presence of adjuvant to generate an immune response (Shield,
C. F., et al. (1996) A cost-effective analysis of OKT3 induction
therapy in cadaveric kidney transplantation. Am. J. Kidney Dis.
27:855-864). Rodents with positive immune sera are sacrificed and
splenocytes are isolated. Isolated splenocytes are fused to
melanomas to produce immortalized cell lines that are then screened
for antibody production. Positive lines are isolated and
characterized for antibody production. The direct use of rodent
MAbs as human therapeutic agents were confounded by the fact that
human anti-rodent antibody (HARA) responses occurred in a
significant number of patients treated with the rodent-derived
antibody (Khazaeli, M. B., et al., (1994) Human immune response to
monoclonal antibodies. J. Immunother. 15:42-52). In order to
circumvent the problem of HARA, the grafting of the complementarity
determining regions (CDRs), which are the critical motifs found
within the heavy and light chain variable regions of the
immunoglobulin (Ig) subunits making up the antigen binding domain,
onto a human antibody backbone found these chimeric molecules are
able to retain their binding activity to antigen while lacking the
HARA response (Emery, S. C., and Harris, W. J. "Strategies for
humanizing antibodies" In: ANTIBODY ENGINEERING C.A.K. Borrebaeck
(Ed.) Oxford University Press, N.Y. 1995. pp. 159-183). A common
problem that exists during the "humanization" of rodent-derived
MAbs (referred to hereon as HAb) is the loss of binding affinity
due to conformational changes in the 3-dimensional structure of the
CDR domain upon grafting onto the human Ig backbone (U.S. Pat. No.
5,530,101 to Queen et al.). To overcome this problem, additional
HAb vectors usually need to be engineered by inserting or deleting
additional amino acid residues within the framework region and/or
within the CDR coding region itself in order to recreate high
affinity HAbs (U.S. Pat. No. 5,530,101 to Queen et al.). This
process is a very time consuming procedure that involves the use of
expensive computer modeling programs to predict changes that may
lead to a high affinity HAb. In some instances, the affinity of the
HAb is never restored to that of the MAb, rendering them of little
therapeutic use.
[0005] Another problem that exists in antibody engineering is the
generation of stable, high yielding producer cell lines that are
required for manufacturing of the molecule for clinical materials.
Several strategies have been adopted in standard practice by those
skilled in the art to circumvent this problem. One method is the
use of Chinese Hamster Ovary (CHO) cells transfected with exogenous
Ig fusion genes containing the grafted human light and heavy chains
to produce whole antibodies or single chain antibodies, which are a
chimeric molecule containing both light and heavy chains that form
an antigen-binding polypeptide (Reff, M. E. (1993) High-level
production of recombinant immunoglobulins in mammalian cells. Curr.
Opin. Biotechnol. 4:573-576). Another method employs the use of
human lymphocytes derived from transgenic mice containing a human
grafted immune system or transgenic mice containing a human Ig gene
repertoire. Yet another method employs the use of monkeys to
produce primate MAbs, which have been reported to lack a human
anti-monkey response (Neuberger, M., and Gruggermann, M. (1997)
Monoclonal antibodies. Mice perform a human repertoire. Nature
386:25-26). In all cases, the generation of a cell line that is
capable of generating sufficient amounts of high affinity antibody
poses a major limitation for producing sufficient materials for
clinical studies. Because of these limitations, the utility of
other recombinant systems such as plants are currently being
explored as systems that will lead to the stable, high-level
production of humanized antibodies (Fiedler, U., and Conrad, U.
(1995) High-level production and long-term storage of engineered
antibodies in transgenic tobacco seeds. Bio/Technology
13:1090-1093).
[0006] Still another aspect of antibody function is the effector
mechanisms of the Mab. One of many possible ways to increase
effector function of antibodies is via changes in glycosylation.
This topic has been recently reviewed by Ruju who summarized the
proposed importance of the oligosaccharides found on human IgGs
with their degree of effector function (Raju, T S. BioProcess
International April 2003. 44-53). According to Wright and Morrison,
the microheterogeneity of human IgG oligosaccharides can affect
biological functions such as complement dependent cytotoxicty (CDC)
and antibody-dependent cytotoxicity (ADCC), binding to various Fc
receptors, and binding to C1q protein (Wright A. Morrison S L.
TIBTECH 1997, 15 26-32). It is well documented that glycosylation
patterns of antibodies can differ depending on the producing cell
and the cell culture conditions (Raju, T S. BioProcess
International April 2003. 44-53). Such differences can lead to
changes in both effector function and pharmacokinetics (Israel E J,
Wilsker D F, Hayes K C, Schoenfeld D, Simister N E. Immunology.
1996 December;89(4):573-578; Newkirk M M, Novick J, Stevenson M M,
Fournier Mi, Apostolakos P. Clin. Exp. 1996
November;106(2):259-64). Differences in effector function may be
related to the IgGs ability to bind to the Fc.gamma. receptors
(Fc.gamma.Rs) on the effector cells. Shields, et al., have shown
that IgG.sub.1 with variants in amino acid sequence that have
improved binding to Fc.gamma.R can exhibit up to 100% enhanced ADCC
using human effector cells (Shields R L, Namenuk A K, Hong K, Meng
Y G, Rae J, Briggs J, Xie D, Lai J, Stadlen A, Li B, Fox J A,
Presta L G. J Biol Chem. 2001 Mar. 2;276(9):6591-604). While these
variants include changes in amino acids not found at the binding
interface, both the nature of the sugar component as well as its
structural pattern may also contribute to the differences seen. In
addition, the presence or absence of fucose in the oligosaccharide
component of an IgG.sub.1 can improve binding and ADCC (Shields R
L, Lai J, Keck R, O'Connell L Y, Hong K, Meng Y G, Weikert S H,
Presta L G. J Biol Chem. 2002 Jul. 26;277(30):26733-40). An
IgG.sub.1 that lacked a fucosylated carbohydrate linked to
Asn.sup.297 exhibited normal receptor binding to the Fc.gamma.
receptor. In contrast, binding to the Fc.gamma.RIIA receptor was
improved 50% and accompanied by enhanced ADCC, especially at lower
antibody concentrations.
[0007] Work by Shinkawa, et al., demonstrated that an antibody to
the human IL-5 receptor produced in a rat hybridoma showed more
than 50% higher ADCC when compared to the antibody produced in
Chinese hamster ovary cells (CHO)(Shinkawa T, Nakamura K, Yaman N,
Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M,
Yamasaki M, Hanai N, Shitara K. J Biol Chem. 2003 Jan.
31;278(5):3466-73). Monosaccharide composition and oligosaccharide
profiling showed that the rat hybridoma-produced IgG.sub.1 had a
lower content of fucose than the CHO-produced protein. The authors
concluded that the lack of fucosylation of an IgG.sub.1 has a
critical role in enhancement of ADCC activity.
[0008] A different approach was taken by Umana, et al., who changed
the glycosylation pattern of chCE7, a chimeric IgG.sub.1
anti-neuroblastoma antibody (Umana P. Jean-Mairet J, Moudry R,
Amstutz H, Bailey J E. Nat Biotechnol. 1999 February; 17(2):
176-80). Using tetracycline, they regulated the activity of a
glycosyltransferase enzyme (GnTIII) which bisects oligosaccharides
that have been implicated in ADCC activity. The ADCC activity of
the parent antibody was barely above background level. Measurement
of ADCC activity of the chCE7 produced at different tetracycline
levels showed an optimal range of GnTIII expression for maximal
chCE7 in vitro ADCC activity. This activity correlated with the
level of constant region-associated, bisected complex
oligosaccharide. Newly optimized variants exhibited substantial
ADCC activity.
[0009] A method for generating diverse antibody sequences within
the variable domain that results in HAbs and MAbs with high binding
affinities to antigens would be useful for the creation of more
potent therapeutic and diagnostic reagents respectively. Moreover,
the generation of randomly altered nucleotide and polypeptide
residues throughout an entire antibody molecule will result in new
reagents that are less antigenic and/or have beneficial
pharmacokinetic properties. The invention described herein is
directed to the use of random genetic mutation throughout an
antibody structure in vivo by blocking the endogenous mismatch
repair (MMR) activity of a host cell producing immunoglobulins that
encode biochemically active antibodies. The invention also relates
to methods for repeated in vivo genetic alterations and selection
for antibodies with enhanced binding and pharmacokinetic profiles.
The methods of the invention may be used to enhance the effector
function of the antibodies.
[0010] In addition, the ability to develop genetically altered host
cells that are capable of secreting increased amounts of antibody
will also provide a valuable method for creating cell hosts for
product development. The invention described herein is directed to
the creation of genetically altered cell hosts with increased
antibody production via the blockade of MMR.
[0011] The invention facilitates the generation of antibodies with
enhanced effector function and the production of cell lines with
elevated levels of antibody production. Other advantages of the
present invention are described in the examples and figures
described herein.
SUMMARY OF THE INVENTION
[0012] The invention provides methods for generating genetically
altered antibodies (including single chain molecules) and antibody
producing cell hosts in vitro and in vivo, whereby the antibody
possesses a desired biochemical property(s), such as, but not
limited to, increased antigen binding, increased gene expression,
enhanced effector function and/or enhanced extracellular secretion
by the cell host. One method for identifying antibodies with
increased binding activity or cells with increased antibody
production is through the screening of MMR defective antibody
producing cell clones that produce molecules with enhanced binding
properties, enhanced effector function such as (but not limited to)
antibody-dependent cellular cytotoxicity (ADCC), or clones that
have been genetically altered to produce enhanced amounts of
antibody product.
[0013] The antibody producing cells suitable for use in the
invention include, but are not limited to rodent, primate, or human
hybridomas or lymphoblastoids; mammalian cells transfected with and
expressing exogenous Ig subunits or chimeric single chain
molecules; plant cells, yeast or bacteria transfected with and
expressing exogenous Ig subunits or chimeric single chain
molecules.
[0014] Thus, the invention provides methods for making hypermutable
antibody-producing cells by introducing a polynucleotide comprising
a dominant negative allele of a mismatch repair gene into cells
that are capable of producing antibodies. The cells that are
capable of producing antibodies include cells that naturally
produce antibodies, and cells that are engineered to produce
antibodies through the introduction of immunoglobulin encoding
sequences. Conveniently, the introduction of polynucleotide
sequences into cells is accomplished by transfection.
[0015] The invention also provides methods of making hypermutable
antibody producing cells by introducing a dominant negative
mismatch repair (MMR) gene such as PMS2 (preferably human PMS2),
MLH1, PMS1, MSH1, or MSH2 into cells that are capable of producing
antibodies. The dominant negative allele of a mismatch repair gene
may be a truncation mutation of a mismatch repair gene (preferably
a truncation mutation at codon 134, or a thymidine at nucleotide
424 of wild-type PMS2). The invention also provides methods in
which mismatch repair gene activity is suppressed. This may be
accomplished, for example, using antisense molecules directed
against the mismatch repair gene or transcripts.
[0016] Other embodiments of the invention provide methods for
making a hypermutable antibody producing cells by introducing a
polynucleotide comprising a dominant negative allele of a mismatch
repair gene into fertilized eggs of animals. These methods may also
include subsequently implanting the eggs into pseudo-pregnant
females whereby the fertilized eggs develop into a mature
transgenic animal. The mismatch repair genes may include, for
example, PMS2 (preferably human PMS2), MLH1, PMS1, MSH1, or MSH2.
The dominant negative allele of a mismatch repair gene may be a
truncation mutation of a mismatch repair gene (preferably a
truncation mutation at codon 134, or a thymidine at nucleotide 424
of wild-type PMS2).
[0017] The invention further provides homogeneous compositions of
cultured, hypermutable, mammalian cells that are capable of
producing antibodies and contain a dominant negative allele of a
mismatch repair gene. The mismatch repair genes may include, for
example, PMS2 (preferably human PMS2), MLHI, PMS1, MSH1, or MSH2.
The dominant negative allele of a mismatch repair gene may be a
truncation mutation of a mismatch repair gene (preferably a
truncation mutation at codon 134, or a thymidine at nucleotide 424
of wild-type PMS2). The cells of the culture may contain PMS2,
(preferably human PMS2), MLH1, or PMS1; or express a human mutL
homolog, or the first 133 amino acids of hPMS2.
[0018] The invention further provides methods for generating a
mutation in an immunoglobulin gene of interest by culturing an
immunoglobulin producing cell selected for an immunoglobulin of
interest wherein the cell contains a dominant negative allele of a
mismatch repair gene. The properties of the immunoglobulin produced
from the cells can be assayed to ascertain whether the
immunoglobulin gene harbors a mutation. The assay may be directed
to analyzing a polynucleotide encoding the immunoglobulin, or may
be directed to the immunoglobulin polypeptide itself.
[0019] The invention also provides methods for generating a
mutation in a gene affecting antibody production in an
antibody-producing cell by culturing the cell expressing a dominant
negative allele of a mismatch repair gene, and testing the cell to
determine whether the cell harbors mutations within the gene of
interest, such that a new biochemical feature (e.g.,
over-expression and/or secretion of immunoglobulin products) is
generated. The testing may include analysis of the steady state
expression of the immunoglobulin gene of interest, and/or analysis
of the amount of secreted protein encoded by the immunoglobulin
gene of interest. The invention also embraces prokaryotic and
eukaryotic transgenic cells made by this process, including cells
from rodents, non-human primates and humans.
[0020] Other aspects of the invention encompass methods of
reversibly altering the hypermutability of an antibody producing
cell, in which an inducible vector containing a dominant negative
allele of a mismatch repair gene operably linked to an inducible
promoter is introduced into an antibody-producing cell. The cell is
treated with an inducing agent to express the dominant negative
mismatch repair gene (which can be PMS2 (preferably human PMS2),
MLH1, or PMS1). Alternatively, the cell may be induced to express a
human mutL homolog or the first 133 amino acids of hPMS2. In
another embodiment, the cells may be rendered capable of producing
antibodies by co-transfecting a preselected immunoglobulin gene of
interest. The immunoglobulin genes of the hypermutable cells, or
the proteins produced by these methods may be analyzed for desired
properties, and induction may be stopped such that the genetic
stability of the host cell is restored.
[0021] The invention also embraces methods of producing genetically
altered antibodies by transfecting a polynucleotide encoding an
immunoglobulin protein into a cell containing a dominant negative
mismatch repair gene (either naturally or in which the dominant
negative mismatch repair gene was introduced into the cell),
culturing the cell to allow the immunoglobulin gene to become
mutated and produce a mutant immunoglobulin, screening for a
desirable property of said mutant immunoglobulin protein, isolating
the polynucleotide molecule encoding the selected mutant
immunoglobulin possessing the desired property, and transfecting
said mutant polynucleotide into a genetically stable cell, such
that the mutant antibody is consistently produced without further
genetic alteration. The dominant negative mismatch repair gene may
be PMS2 (preferably human PMS2), MLH1, or PMS1. Alternatively, the
cell may express a human mutL homolog or the first 133 amino acids
of hPMS2.
[0022] The invention further provides methods for generating
genetically altered cell lines that express enhanced amounts of an
antigen binding polypeptide. These antigen-binding polypeptides may
be, for example, immunoglobulins. The methods of the invention also
include methods for generating genetically altered cell lines that
secrete enhanced amounts of an antigen binding polypeptide. The
cell lines are rendered hypermutable by dominant negative mismatch
repair genes that provide an enhanced rate of genetic hypermutation
in a cell producing antigen-binding polypeptides such as
antibodies. Such cells include, but are not limited to hybridomas.
Expression of enhanced amounts of antigen binding polypeptides may
be through enhanced transcription or translation of the
polynucleotides encoding the antigen binding polypeptides, or
through the enhanced secretion of the antigen binding polypeptides,
for example.
[0023] Methods are also provided for creating genetically altered
antibodies in vivo by blocking the MMR activity of the cell host,
or by transfecting genes encoding for immunoglobulin in a MMR
defective cell host.
[0024] Antibodies with increased binding properties to an antigen
due to genetic changes within the variable domain are provided in
methods of the invention that block endogenous MMR of the cell
host. Antibodies with increased binding properties to an antigen
due to genetic changes within the CDR regions within the light
and/or heavy chains are also provided in methods of the invention
that block endogenous MMR of the cell host.
[0025] The invention provides methods of creating genetically
altered antibodies in MMR defective Ab producer cell lines with
enhanced pharmacokinetic properties in host organisms including but
not limited to rodents, primates, and man.
[0026] These and other aspects of the invention are provided by one
or more of the embodiments described below. In one embodiment of
the invention, a method for making an antibody producing cell line
hypermutable is provided. A polynucleotide encoding a dominant
negative allele of a MMR gene is introduced into an
antibody-producing cell. The cell becomes hypermutable as a result
of the introduction of the gene.
[0027] In another embodiment of the invention, a method is provided
for introducing a mutation into an endogenous gene encoding for an
immunoglobulin polypeptide or a single chain antibody. A
polynucleotide encoding a dominant negative allele of a MMR gene is
introduced into a cell. The cell becomes hypermutable as a result
of the introduction and expression of the MMR gene allele. The cell
further comprises an immunoglobulin gene of interest. The cell is
grown and tested to determine whether the gene encoding for an
immunoglobulin or a single chain antibody of interest harbors a
mutation. In another aspect of the invention, the gene encoding the
mutated immunoglobulin polypeptide or single chain antibody may be
isolated and expressed in a genetically stable cell. In a preferred
embodiment, the mutated antibody is screened for at least one
desirable property such as, but not limited to, enhanced binding
characteristics.
[0028] In another embodiment of the invention, a gene or set of
genes encoding for Ig light and heavy chains or a combination
therein are introduced into a mammalian cell host that is MMR
defective. The cell is grown, and clones are analyzed for
antibodies with enhanced binding characteristics.
[0029] In another embodiment of the invention, a method is provided
for producing new phenotypes of a cell. A polynucleotide encoding a
dominant negative allele of a MMR gene is introduced into a cell.
The cell becomes hypermutable as a result of the introduction of
the gene. The cell is grown. The cell is tested for the expression
of new phenotypes where the phenotype is enhanced secretion of a
polypeptide.
[0030] The invention also provides antibodies having increased
affinity for antigen comprising immunoglobulin molecules wherein a
substitution has been made for at least one amino acid in the
variable domain of the heavy and/or light chain. In some
embodiments, the substitution is in a position wherein the parental
amino acid in that position is an amino acid with a non-polar side
chain. In some embodiments the parental amino acid is substituted
with a different amino acid that has a non-polar side chain. In
other embodiments, the parental amino acid is replaced with a
proline or hydroxyproline. In some embodiments, the substitution(s)
are made in the framework regions of the heavy and/or light chain
variable domains. In some embodiments, the substitution(s) are made
within the first framework region of the heavy chain. In some
embodiments, the substitution(s) are made within the second
framework region of the light chain. In some embodiments, the
substitutions are made within the first framework region of the
heavy chain and the second framework region of the light chain. In
some embodiments, a substitution is made at position 6 of the first
framework region of the heavy chain as shown in SEQ ID NO: 18. In
some embodiments a substitution is made at position 22 of the
second framework region of the light chain as shown in SEQ ID
NO:21. For the specific position mutations, in some embodiments the
amino acid substitution is a proline or hydroxyproline.
[0031] The invention also provides methods for increasing the
affinity of an antibody for an antigen comprising substituting an
amino acid within the variable domain of the heavy or light chain
of the subject antibody with another amino acid having a non-polar
side chain. In some embodiments, a proline is substituted for the
original amino acid at the position. In some embodiments, proline
is used to substitute for another amino acid having a non-polar
side chain. In some embodiments alanine and/or leucine is replaced
by proline. In certain embodiments, the amino acid in position 6 of
the first framework region of the heavy chain of the antibody as
shown in SEQ ID NO: 18 is replaced with a proline. In other
embodiments, the amino acid in position 22 of the second framework
region of the light chain variable domain as shown in SEQ ID NO:21
is replaced with proline. The invention also provides antibodies
produced by these methods.
[0032] The antibodies produced in the invention may be made using
the process of the invention wherein a dominant negative allele of
a mismatch repair gene is introduced into the antibody producing
cell and the cell becomes hypermutable as described more fully
herein. Alternatively, one may disrupt mismatch repair using
chemical inhibitors of mismatch repair, such as using anthracene
and/or its derivatives as described in PCT Publication No. WO
02/054856, published Jul. 18, 2002, which is specifically
incorporated herein in its entirety. The cells treated with the
chemicals that disrupt mismatch repair or which express a dominant
negative mismatch repair gene become hypermutable. The antibodies
produced by the hypermutable cells are screened for increased
affinity, and those antibodies comprising the amino acid
substitutions described above display increased affinity for
antigen. The cells producing the antibodies which have the
increased affinity and the molecular characteristics described
herein may be rendered genetically stable again by withdrawing the
chemical inhibitor, or by rendering the cells genetically stable
through the inactivation of the expression of the dominant negative
allele. For example, a dominant negative allele that it under the
control of an inducible promoter may be inactivated by withdrawing
the inducer. Alternatively, the dominant negative allele may be
knocked out, or a CRE-LOX expression system may be used whereby the
dominant negative allele is spliced from the genome once the cells
containing a genetically diverse immunoglobulin has been
established.
[0033] In other embodiments, one of skill in the art may use any
known method of introducing mutations into proteins and selecting
for antibodies having higher affinity with the amino acid
substitutions described above. Methods of introducing mutations may
be random, such as chemical mutagenesis, or may be specific, such
as site-directed mutagenesis. Methods for random and specific
mutagenesis are well-known in the art and include, but are not
limited to, for example, chemical mutagenesis (e.g., using such
chemicals as methane sulfonate, dimethyl sulfonate, O6-methyl
benzadine, methylnitrosourea (MNU), and ethylnitrosourea (ENU));
oligonucleotide-mediated site-directed mutagenesis; alanine
scanning; and PCR mutagenesis (see, for example, Kunkel et al.
(1991) Methods Enzymol. 204:125-139), site-directed mutagenesis;
Crameri et al. (1995) BioTechniques 18(2):194-196, cassette
mutagenesis; and Haught et al. (1994) BioTechniques 16(1):47-48,
restriction selection mutagenesis).
[0034] These and other embodiments of the invention provide the art
with methods that can generate enhanced mutability in cells and
animals as well as providing cells and animals harboring
potentially useful mutations for the large-scale production of high
affinity antibodies with beneficial pharmacokinetic profiles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1. Hybridoma cells stably expressing PMS2 and PMS134
MMR genes. Shown is steady state mRNA expression of MMR genes
transfected into a murine hybridoma cell line. Stable expression
was found after 3 months of continuous growth. The (-) lanes
represent negative controls where no reverse transcriptase was
added, and the (+) lanes represent samples reverse transcribed and
PCR amplified for the MMR genes and an internal housekeeping gene
as a control.
[0036] FIG. 2. Creation of genetically hypermutable hybridoma
cells. Dominant negative MMR gene alleles were expressed in cells
expressing a MMR-sensitive reporter gene. Dominant negative alleles
such as PMS134 and the expression of MMR genes from other species
results in antibody producer cells with a hyperrnutable phenotype
that can be used to produce genetically altered immunoglobulin
genes with enhanced biochemical features as well as lines with
increased Ig expression and/or secretion. Values shown represent
the amount of converted CPRG substrate which is reflective of the
amount of function of .beta.-galactosidase contained within the
cell from genetic alterations within the pCAR-OF reporter gene.
Higher amounts of .beta.-galactosidase activity reflect a higher
mutation rate due to defective MMR.
[0037] FIG. 3. Screening method for identifying antibody-producing
cells containing antibodies with increased binding activity and/or
increased expression/secretion
[0038] FIG. 4. Generation of a genetically altered antibody with an
increased binding activity. Shown are ELISA values from 96-well
plates, screened for antibodies specific to hIgE. Two clones with a
high binding value were found in HB134 cultures.
[0039] FIG. 5. Sequence alteration within variable chain of an
antibody (a mutation within the light chain variable region in
MMR-defective HB134 antibody producer cells). Arrows indicate the
nucleotide at which a mutation occurred in a subset of cells from a
clone derived from HB 134 cells. Panel A: The change results in a
Thr to Ser change within the light chain variable region. The
coding sequence is in the antisense direction. Panel B: The change
results in a Pro to Leu change within the light chain variable
region.
[0040] FIG. 6. Generation of MMR-defective clones with enhanced
steady state Ig protein levels. A Western blot of heavy chain
immunoglobulins from HB134 clones with high levels of MAb (>500
ngs/ml) within the conditioned medium shows that a subset of clones
express higher steady state levels of immunoglobulins (Ig). The H36
cell line was used as a control to measure steady state levels in
the parental strain. Lane 1: fibroblast cells (negative control);
Lane 2: H36 cell; Lane 3: HB134 clone with elevated MAb levels;
Lane 4: HB134 clone with elevated MAb levels; Lane 5: HB 134 clone
with elevated MAb levels.
[0041] FIG. 7. MORAb-003 is able to induce cytotoxicity in human
ovarian tumor cells mediated by normal human PBMCs.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0042] Methods have been discovered for developing hypermutable
antibody-producing cells by taking advantage of the conserved
mismatch repair (MMR) process of host cells. Dominant negative
alleles of such genes, when introduced into cells or transgenic
animals, increase the rate of spontaneous mutations by reducing the
effectiveness of DNA repair and thereby render the cells or animals
hypermutable. Hypermutable cells or animals can then be utilized to
develop new mutations in a gene of interest. Blocking MMR in
antibody-producing cells such as but not limited to: hybridomas;
mammalian cells transfected with genes encoding for Ig light and
heavy chains; mammalian cells transfected with genes encoding for
single chain antibodies; eukaryotic cells transfected with Ig
genes, can enhance the rate of mutation within these cells leading
to clones that have enhanced antibody production and/or cells
containing genetically altered antibodies with enhanced biochemical
properties such as increased antigen binding. The process of MMR,
also called mismatch proofreading, is carried out by protein
complexes in cells ranging from bacteria to mammalian cells. A MMR
gene is a gene that encodes for one of the proteins of such a
mismatch repair complex. Although not wanting to be bound by any
particular theory of mechanism of action, a MMR complex is believed
to detect distortions of the DNA helix resulting from
non-complementary pairing of nucleotide bases. The
non-complementary base on the newer DNA strand is excised, and the
excised base is replaced with the appropriate base, which is
complementary to the older DNA strand. In this way, cells eliminate
many mutations that occur as a result of mistakes in DNA
replication.
[0043] Dominant negative alleles cause a MMR defective phenotype
even in the presence of a wild-type allele in the same cell. An
example of a dominant negative allele of a MMR gene is the human
gene hPMS2-134, which carries a truncating mutation at codon 134
(SEQ ID NO:15). The mutation causes the product of this gene to
abnormally terminate at the position of the 134th amino acid,
resulting in a shortened polypeptide containing the N-terminal 133
amino acids. Such a mutation causes an increase in the rate of
mutations, which accumulate in cells after DNA replication.
Expression of a dominant negative allele of a mismatch repair gene
results in impairment of mismatch repair activity, even in the
presence of the wild-type allele. Any allele which produces such
effect can be used in this invention. Dominant negative alleles of
a MMR gene can be obtained from the cells of humans, animals,
yeast, bacteria, or other organisms. Such alleles can be identified
by screening cells for defective MMR activity. Cells from animals
or humans with cancer can be screened for defective mismatch
repair. Cells from colon cancer patients may be particularly
useful. Genomic DNA, cDNA, or mRNA from any cell encoding a MMR
protein can be analyzed for variations from the wild type sequence.
Dominant negative alleles of a MMR gene can also be created
artificially, for example, by producing variants of the hPMS2-134
allele or other MMR genes. Various techniques of site-directed
mutagenesis can be used. The suitability of such alleles, whether
natural or artificial, for use in generating hypermutable cells or
animals can be evaluated by testing the mismatch repair activity
caused by the allele in the presence of one or more wild-type
alleles, to determine if it is a dominant negative allele.
[0044] A cell or an animal into which a dominant negative allele of
a mismatch repair gene has been introduced will become
hypermutable. This means that the spontaneous mutation rate of such
cells or animals is elevated compared to cells or animals without
such alleles. The degree of elevation of the spontaneous mutation
rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold,
100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell
or animal. The use of chemical mutagens such as but limited to
methane sulfonate, dimethyl sulfonate, 06-methyl benzadine, MNU,
ENU, etc. can be used in MMR defective cells to increase the rates
an additional 10 to 100 fold that of the MMR deficiency itself.
[0045] According to one aspect of the invention, a polynucleotide
encoding for a dominant negative form of a MMR protein is
introduced into a cell. The gene can be any dominant negative
allele encoding a protein, which is part of a MMR complex, for
example, PMS2, PMS1, MLH1, or MSH2. The dominant negative allele
can be naturally occurring or made in the laboratory. The
polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a
chemically synthesized polynucleotide.
[0046] The polynucleotide can be cloned into an expression vector
containing a constitutively active promoter segment (such as but
not limited to CMV, SV40, Elongation Factor or LTR sequences) or to
inducible promoter sequences such as the steroid inducible pIND
vector (Invitrogen), where the expression of the dominant negative
MMR gene can be regulated. The polynucleotide can be introduced
into the cell by transfection.
[0047] According to another aspect of the invention, an
immunoglobulin (Ig) gene, a set of Ig genes or a chimeric gene
containing whole or parts of an Ig gene can be transfected into
MMR-deficient cell hosts, the cell is grown and screened for clones
containing genetically altered Ig genes with new biochemical
features. MMR defective cells may be of human, primates, mammals,
rodent, plant, yeast or of the prokaryotic kingdom. The mutated
gene encoding the Ig with new biochemical features may be isolated
from the respective clones and introduced into genetically stable
cells (i.e., cells with normal MMR) to provide clones that
consistently produce Ig with the new biochemical features. The
method of isolating the Ig gene encoding Ig with new biochemical
features may be any method known in the art. Introduction of the
isolated polynucleotide encoding the Ig with new biochemical
features may also be performed using any method known in the art,
including, but not limited to transfection of an expression vector
containing the polynucleotide encoding the Ig with new biochemical
features. As an alternative to transfecting an Ig gene, a set of Ig
genes or a chimeric gene containing whole or parts of an Ig gene
into an MMR deficient host cell, such Ig genes may be transfected
simultaneously with a gene encoding a dominant negative mismatch
repair gene into a genetically stable cell to render the cell
hypermutable.
[0048] Transfection is any process whereby a polynucleotide is
introduced into a cell. The process of transfection can be carried
out in a living animal, e.g., using a vector for gene therapy, or
it can be carried out in vitro, e.g., using a suspension of one or
more isolated cells in culture. The cell can be any type of
eukaryotic cell, including, for example, cells isolated from humans
or other primates, mammals or other vertebrates, invertebrates, and
single celled organisms such as protozoa, yeast, or bacteria.
[0049] In general, transfection will be carried out using a
suspension of cells, or a single cell, but other methods can also
be applied as long as a sufficient fraction of the treated cells or
tissue incorporates the polynucleotide so as to allow transfected
cells to be grown and utilized. The protein product of the
polynucleotide may be transiently or stably expressed in the cell.
Techniques for transfection are well known. Available techniques
for introducing polynucleotides include but are not limited to
electroporation, transduction, cell fusion, the use of calcium
chloride, and packaging of the polynucleotide together with lipid
for fusion with the cells of interest. Once a cell has been
transfected with the MMR gene, the cell can be grown and reproduced
in culture. If the transfection is stable, such that the gene is
expressed at a consistent level for many cell generations, then a
cell line results.
[0050] An isolated cell is a cell obtained from a tissue of humans
or animals by mechanically separating out individual cells and
transferring them to a suitable cell culture medium, either with or
without pretreatment of the tissue with enzymes, e.g., collagenase
or trypsin. Such isolated cells are typically cultured in the
absence of other types of cells. Cells selected for the
introduction of a dominant negative allele of a mismatch repair
gene may be derived from a eukaryotic organism in the form of a
primary cell culture or an immortalized cell line, or may be
derived from suspensions of single-celled organisms.
[0051] A polynucleotide encoding for a dominant negative form of a
MMR protein can be introduced into the genome of an animal by
producing a transgenic animal. The animal can be any species for
which suitable techniques are available to produce transgenic
animals. For example, transgenic animals can be prepared from
domestic livestock, e.g., bovine, swine, sheep, goats, horses,
etc.; from animals used for the production of recombinant proteins,
e.g., bovine, swine, or goats that express a recombinant
polypeptide in their milk; or experimental animals for research or
product testing, e.g., mice, rats, guinea pigs, hamsters, rabbits,
etc. Cell lines that are determined to be MMR defective can then be
used as a source for producing genetically altered immunoglobulin
genes in vitro by introducing whole, intact immunoglobulin genes
and/or chimeric genes encoding for single chain antibodies into MMR
defective cells from any tissue of the MMR defective animal.
[0052] Once a transfected cell line or a colony of transgenic
animals has been produced, it can be used to generate new mutations
in one or more gene(s) of interest. A gene of interest can be any
gene naturally possessed by the cell line or transgenic animal or
introduced into the cell line or transgenic animal. An advantage of
using such cells or animals to induce mutations is that the cell or
animal need not be exposed to mutagenic chemicals or radiation,
which may have secondary harmful effects, both on the object of the
exposure and on the workers. However, chemical mutagens may be used
in combination with MMR deficiency, which renders such mutagens
less toxic due to an undetermined mechanism. Hypermutable animals
can then be bred and selected for those producing genetically
variable B-cells that may be isolated and cloned to identify new
cell lines that are useful for producing genetically variable
cells. Once a new trait is identified, the dominant negative MMR
gene allele can be removed by directly knocking out the allele by
technologies used by those skilled in the art or by breeding to
mates lacking the dominant negative allele to select for offspring
with a desired trait and a stable genome. Another alternative is to
use a CRE-LOX expression system, whereby the dominant negative
allele is spliced from the animal genome once an animal containing
a genetically diverse immunoglobulin profile has been established.
Yet another alternative is the use of inducible vectors such as the
steroid induced pIND (Invitrogen) or pMAM (Clonetech) vectors which
express exogenous genes in the presence of corticosteroids.
[0053] Mutations can be detected by analyzing for alterations in
the genotype of the cells or animals, for example by examining the
sequence of genomic DNA, cDNA, messenger RNA, or amino acids
associated with the gene of interest. Mutations can also be
detected by screening for the production of antibody titers. A
mutant polypeptide can be detected by identifying alterations in
electrophoretic mobility, spectroscopic properties, or other
physical or structural characteristics of a protein encoded by a
mutant gene. One can also screen for altered function of the
protein in situ, in isolated form, or in model systems. One can
screen for alteration of any property of the cell or animal
associated with the function of the gene of interest, such as but
not limited to Ig secretion.
[0054] Cells expressing the dominant negative alleles can be
"cured" in that the dominant negative allele can be turned off, if
inducible, eliminated from the cell, and the like such that the
cells become genetically stable once more and no longer accumulate
mutations at the abnormally high rate. The polynucleotide can be
cloned into an expression vector containing constitutively active
promoter segment (such as but not limited to CMV, SV40, Elongation
Factor or LTR sequences) or to inducible promoter sequences such as
the steroid inducible pIND vector where the expression of the
dominant negative mismatch repair gene can be regulated. The cDNA
is introduced into the cell by transfection. Upon identification of
the desired phenotype or trait the organism can then be genetically
stabilized.
[0055] Examples of mismatch repair proteins and nucleic acid
sequences include mouse PMS2 (SEQ ID NOs:5 and 6), human PMS2 (SEQ
ID NOs:7 and 8), human PMS1 (SEQ ID NOs:9 and 10), human MSH2 (SEQ
ID NOs: 11 and 12), human MLH1 (SEQ ID NOs:13 and 14), and human
PMS2-134 (SEQ ID NOs:15 and 16).
[0056] Mutant antibodies showing increased affinity for antigen
were sequenced and compared to the sequence of the wild-type (WT)
H36 parental antibody. It has been discovered that alterations of
amino acids to proline has the effect of increasing affinity for
antigen when introduced into the variable region of either the
light chain or heavy chain of the immunoglobulin molecule. While
not wishing to be bound by any particular theory of operation, it
is believed that the prolines introduce a localized area of
rigidity and lend stability to the immunoglobulin molecule,
particularly to the regions around the antigen combining sites.
[0057] Thus, the invention provides for a method to increase the
affinity of antibodies comprising replacing amino acids of the
variable domain heavy and/or light chain with proline or
hydroxyproline (collectively referred to as "proline"). In some
embodiments, the substitution of prolines is in the heavy chain
variable domain. In some embodiments, the substitution of prolines
is in the light chain variable domain. In other embodiments, the
substitution of proline is in both the heavy chain and the light
chain of the variable domain of the immunoglobulin molecule. In
some embodiments, the proline substitutes for another amino acid
having a non-polar sidechain (e.g., glycine, alanine, valine,
leucine, isoleucine, phenylalanine, methionine, tryptophan and
cysteine). In some embodiments, further exchanges of amino acids
having non-polar sidechains with other amino acids having non-polar
sidechains may also confer increased affinity of the antibody for
the antigen. In some embodiments, the amino acid substitutions are
in a framework region of the heavy chain. In other embodiments, the
amino acid substitutions are in a framework region of the light
chain.
[0058] In other embodiments, the amino acid substitutions are in a
framework region of both the heavy and light chain. In some
embodiments, the amino acid substitutions are in the first
framework region (FR1) of the heavy chain. In other embodiments,
the amino acid substitution is in the second framework region (FR2)
of the heavy chain. In other embodiments, the amino acid
substitution is in the third framework region (FR3) of the heavy
chain. In other embodiments, the amino acid substitution is in the
fourth framework region (FR4) of the heavy chain. In some
embodiments, the amino acid substitutions are in the first
framework region (FR1) of the light chain. In other embodiments,
the amino acid substitution is in the second framework region (FR2)
of the light chain. In other embodiments, the amino acid
substitution is in the third framework region (FR3) of the light
chain. In other embodiments, the amino acid substitution is in the
fourth framework region (FR4) of the light chain.
[0059] In certain embodiments of the invention, a proline
substitutes for an alanine at position 6 of SEQ ID NO:18. In other
embodiments, proline substitutes for alanine at position 6 of SEQ
ID NO: 18 and the glycine at position 9 of SEQ ID NO:18, and/or the
lysine at position 10 of SEQ ID NO:18 is substituted with an amino
acid having a non-polar side chain (preferably, valine and
arginine, respectively). In other embodiments, proline substitutes
for leucine at position 22 of SEQ ID NO:21.
[0060] The recent clinical and commercial success of anticancer
antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin)
and small molecule signal transduction inhibitors such as imatinib
mesylate, (Gleevec or STI-571), has created great interest in
"targeted" therapeutics for hematopoietic malignancies and solid
tumors. In comparison to small molecule cytotoxic agents it is
hoped that these approaches will result in lower toxicity while
maintaining or increasing the therapeutic efficacy.
[0061] Antibodies conjugated to radionuclides, drugs or toxins have
intrinsic specificity due to their specific antigen binding. The
degree of specificity is dependent on the relative specificity of
the antigen on the targeted tumor. The conjugated toxic component
complicates the approach since the radionuclide is irradiating
normal tissues during the duration of its circulation (prolonged
for a humanized antibody) and drugs and toxins can be detached from
the antibody by enzymatic and non-enzymatic mechanism, thus
delivering the toxin to normal tissue. In addition, the presence of
the conjugate can result in the body recognizing the complex as
foreign with the resulting uptake into organs of clearance such as
the liver.
[0062] Previous attempts at maximizing the therapeutic potential of
monoclonal antibodies have mostly focused on improving the affinity
and avidity of binding to the targeted antigen. The method of the
invention allows for the maximization of efficacy of an
unconjugated antibody. It is an object of the invention to generate
improved monoclonal antibodies (e.g., humanized antibodies) by
producing and assaying molecules with increases in the effector
function (Fc) of the protein, regardless of the mechanisms behind
the increases. These new molecules could then target human tumors
and have enhanced potency for tumor cell killing. The resulting
product would be expected to function at a lower dose, without an
increase in toxicity, thus increasing its therapeutic window. In
addition, many previous studies have shown that the accretion of an
antibody into a tumor is relatively low (Sands, H. Cancer Research
(Suppl) 1990, 50: 809s-813s). An increase in effector ftunction
could result in an increased therapeutic efficacy thus allowing a
humanized monoclonal antibody to have a therapeutic effect at the
accretion rates found in human tumors.
[0063] The method of the invention can enhance the effector
function of monoclonal antibodies, including, but not limited to
those currently in development for the treatment of cancer.
Glycosylation is only one of many ways in which antibody effector
function can be manipulated. The technology is ideally suited for
this study since it can yield a more potent antibody that has
minimal changes in amino acid sequence, in the glycosylation
pattern and/or in other known and unknown mechanisms. These changes
may arise due to genetic changes in the DNA resulting in the amino
acid sequence of the immunoglobulin molecule itself, or in the
cellular machinery that controls the sequence or nature of the post
translational pattern.
[0064] The method of the invention may be used to enhance
properties of antibodies, including, but not limited to, rodent
antibodies against therapeutic targets, and chimerized and
humanized versions thereof. One such antibody, referred to as
MORAb-03, binds to a cell surface adult-type, high-affinity
folate-binding glycoprotein antigen (designated MORAb-03 antigen)
of normal placenta and gestational choriocarcinomas. Expression
profiles show that MORAb-03 antigen has a restricted distribution
in normal tissues, being expressed primarily in a subset of simple
epithelia (Rettig W J, Cordon-Cardo C, Koulos J P, Lewis J L Jr,
Oettgen H F, Old U. Int J Cancer. 1985 Apr. 15;35(4):469-75; Coney
L R, Tomassetti A, Carayannopoulos L, Frasca V, Kamen B A, Colnaghi
M I, Zurawski V R Jr. Cancer Res. 1991 Nov. 15;51(22):6125-32) and
fresh frozen sections of human pancreas, proximal kidney tubules,
and bronchi. The distribution of MORAb-03 antigen was further
determined by immunohistochemical analysis of 150 tumor cell lines
and normal cell cultures as well as on primary tumor tissues using
a MORAb-03-antigen specific mouse derived monoclonal antibody
(L-26). MORAb-03 antigen was found expressed on all cultured
choriocarcinomas and teratocarcinomas. Immunohistochemistry of
primary tumors found MORAb-03 antigen expression in a significant
number of ovarian tumors and over 400 tumors of other histological
types (Garin-Chesa P, Campbell I, Saigo P E, Lewis J L Jr, Old U,
Rettig W J. Am J Pathol. 1993 February; 142(2) :557-67). Ovarian
carcinomas derived from coelomic epithelium showed the most
consistent and strongest immunostaining with the MORAb-03 antibody,
with 52 of 56 cases being MORAb-03 positive. MORAb-03 antigen was
not detected in normal fetal or adult ovary; however, it was found
present in the lining epithelia in a subset of benign ovarian
cysts.
[0065] The method of the invention may be used to develop a
production line that produces an antibody that can meet the
therapeutic and manufacturing requirements (i.e., high affinity and
production) specified by the current industry standards. The method
of the invention is suitable for application to mAbs generated
using standard murine hybridoma techniques. In some embodiments,
murine complementary determining regions (CDRs) are grafted into a
human IgG1k backbone, and the light and heavy chain cDNAs are
transfected into NS0 cells, resulting in the generation of an
antibody production system known in the industry as "transfectoma."
The process of grafting the CDRs into a human immunoglobulin
sequence is called "humanization." Unfortunately, the transfectoma
line produced the MORAb-03 at a rate of less than 1 pg/cell/day and
the humanization process had led to a reduction of the affinity,
now in the micromolar range. The method of the invention enabled
successful production of an optimized, humanized MORAb-03 antibody
with acceptable antigen binding activity (low nanomolar
dissociation constant) and production rates (>10 pg/cell/day).
ADCC assays using human ovarian cancer cells as target and
peripheral blood mononuclear cells (PBMCs) as effector cells showed
that 200 ng/ml of MORAb-03 produced in NS0 cells was able to
mediate the lysis of 44% of target cells, whereas lysis mediated by
the control IgG1 antibody was only 6% (FIG. 7). In contrast, the
same concentration of MORAb-03 produced in CHO cells mediated the
lysis of 32% of target cells, a reduction of 27% (paired T
test=0.0008) (FIG. 4). Multiple independent ADCC assays have shown
a similar trend, where MORAb-03 CHO produced in cells showed a
reduction in activity as high as 50% compared to MORAb-03 produced
in NSO cells. CHO is a standard host cell line recognized by the
FDA and is well characterized by contract manufacturing
organizations. Among its strengths are the robustness and stability
of its growth, its adaptability to different manufacturing schemes
and serum-free media, and its high efficiency and reproducibility
of antibody production. A CHO line producing MORAb-03 with ADCC
activity similar to or higher than NSO cell-produced MORAb-03 will
be an extremely valuable manufacturing asset for the production of
a therapeutic anti-cancer biologic. The method of the invention may
be applied to the cell lines producing MORAb-03 in order to
identify variants producing antibodies with enhanced ADCC
activity.
[0066] In summary, the MORAb-03 antigen is a glycoprotein whose
expression is highly restricted in normal tissues and highly
expressed in a large portion of ovarian tumors. The antibody is
capable of inducing ADCC thus making it an excellent drug candidate
for the treatment of ovarian of cancer.
[0067] For further information on the background of the invention
the following references may be consulted, each of which is
incorporated herein by reference in its entirety:
[0068] 1. Glaser, V. (1996) Can ReoPro repolish tarnished mono
clonal therapeutics? Nat. Biotechol. 14:1216-1217.
[0069] 2. Weiner, L. M. (1999) Monoclonal antibody therapy of
cancer. Semin. Oncol. 26:43-51.
[0070] 3. Saez-Llorens, X. E. et al. (1998) Safety and
pharmacokinetics of an intramuscular humanized monoclonal antibody
to respiratory syncytial virus in premature infants and infants
with bronchopulmonary dysplasia. Pediat. Infect. Dis. J.
17:787-791.
[0071] 4. Shield, C. F. et al. (1996) A cost-effective analysis of
OKT3 induction therapy in cadaveric kidney transplantation. Am. J.
Kidney Dis. 27:855-864.
[0072] 5. Kbazaeli, M. B. et al. (1994) Human immune response to
monoclonal antibodies. J. Immunother. 15:42-52.
[0073] 6. Emery, S. C. and W. J. Harris "Strategies for humanizing
antibodies" In: ANTIBODY ENGINEERING C.A.K. Borrebaeck (Ed.) Oxford
University Press, N.Y. 1995, pp. 159-183.
[0074] 7. U.S. Pat. No. 5,530,101 to Queen and Selick.
[0075] 8. Reff, M. E. (1993) High-level production of recombinant
immunoglobulins in mammalian cells. Curr. Opin. Biotechnol.
4:573-576.
[0076] 9. Neuberger, M. and M. Gruggermann, (1997) Monoclonal
antibodies. Mice perform a human repertoire. Nature 386:25-26.
[0077] 10. Fiedler, U. and U. Conrad (1995) High-level production
and long-term storage of engineered antibodies in transgenic
tobacco seeds. Bio/Technology 13:1090-1093.
[0078] 11. Baker S. M. et al. (1995) Male defective in the DNA
mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in
meiosis. Cell 82:309-319.
[0079] 12. Bronner, C. E. et al. (1994) Mutation in the DNA
mismatch repair gene homologue hMLH1 is associated with hereditary
non-polyposis colon cancer. Nature 368:258-261.
[0080] 13. de Wind N. et al. (1995) Inactivation of the mouse Msh2
gene results in mismatch repair deficiency, methylation tolerance,
hyperrecombination, and predisposition to cancer. Cell
82:321-330.
[0081] 14. Drummond, J. T. et al. (1995) Isolation of an hMSH2-p160
heterodimer that restores mismatch repair to tumor cells. Science
268:1909-1912.
[0082] 15. Modrich, P. (1994) Mismatch repair, genetic stability,
and cancer. Science 266:1959-1960.
[0083] 16. Nicolaides, N. C. et al. (1998) A Naturally Occurring
hPMS2 Mutation Can Confer a Dominant Negative Mutator Phenotype.
Mol. Cell. Biol. 18:1635-1641.
[0084] 17. Prolla, T. A. et al. (1994) MLH1, PMS1, and MSH2
Interaction during the initiation of DNA mismatch repair in yeast.
Science 264:1091-1093.
[0085] 18. Strand, M. et al. (1993) Destabilization of tracts of
simple repetitive DNA in yeast by mutations affecting DNA mismatch
repair. Nature 365:274-276.
[0086] 19. Su, S. S., R. S. Lahue, K. G. Au, and P. Modrich (1988)
Mispair specificity of methyl directed DNA mismatch corrections in
vitro. J. Biol. Chem. 263 :6829-6835.
[0087] 20. Parsons, R. et al. (1993) Hypermutability and mismatch
repair deficiency in RER+ tumor cells. Cell 75:1227-1236.
[0088] 21. Papadopoulos, N. et al. (1993) Mutation of a mutL
homolog is associated with hereditary colon cancer. Science
263:1625-1629.
[0089] 22. Perucho, M. (1996) Cancer of the microsatellite mutator
phenotype. Biol. Chem. 377:675-684.
[0090] 23. Nicolaides N. C., K. W. Kinzier, and B. Vogelstein
(1995) Analysis of the 5' region of PMS2 reveals heterogenous
transcripts and a novel overlapping gene. Genomics 29:329-334.
[0091] 24. Nicolaides, N. C. et al. (1995) Genomic organization of
the human PMS2 gene family. Genomics 30:195-206.
[0092] 25. Palombo, F. et al. (1994) Mismatch repair and cancer.
Nature 36:417.
[0093] 26. Eshleman J. R. and S. D. Markowitz (1996) Mismatch
repair defects in human carcinogenesis. Hum. Mol. Genet.
5:1489-494.
[0094] 27. Liu, T. et al. (2000) Microsatellite instability as a
predictor of a mutation in a DNA mismatch repair gene in familial
colorectal cancer. Genes Chromosomes Cancer 27:17-25.
[0095] 28. Nicolaides, N. C. et al. (1992) The Jun family members,
c-JUN and JUND, transactivate the human c-myb promoter via an Ap1
like element. J. Biol. Chem. 267:19665-19672.
[0096] 29. Shields, R. L. et al. (1995) Anti-IgE monoclonal
antibodies that inhibit allergen-specific histamine release. Int.
Arch. Allergy Immunol. 107:412-413.
[0097] 30. Frigerio L. et al. (2000) Assembly, secretion, and
vacuolar delivery of a hybrid immunoglobulin in plants. Plant
Physiol. 123:1483-1494.
[0098] 31. Bignami M, (2000) Unmasking a killer: DNA
O(6)-methylguanine and the cytotoxicity of methylating agents.
Mutat. Res. 462:71-82.
[0099] 32. Drummond, J. T. et al. (1996) Cisplatin and adriamycin
resistance are associated with MutLa and mismatch repair deficiency
in an ovarian tumor cell line. J. Biol. Chem. 271:19645-19648.
[0100] 33. Galio, L. et al. (1999) ATP hydrolysis-dependent
formation of a dynamic ternary nucleoprotein complex with MutS and
MutL. Nucl. Acids Res. 27:2325-2331.
[0101] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLE I
Stable Expression of Dominant Negative MMR Genes in Hybridoma
Cells
[0102] It has been previously shown by Nicolaides et al.
(Nicolaides et al. (1998) A Naturally Occurring hPMS2 Mutation Can
Confer a Dominant Negative Mutator Phenotype Mol. Cell. Biol.
18:1635-1641) that the expression of a dominant negative allele in
an otherwise MMR proficient cell could render these host cells MMR
deficient. The creation of MMR deficient cells can lead to the
generation of genetic alterations throughout the entire genome of a
host organisms offspring, yielding a population of genetically
altered offspring or siblings that may produce biochemicals with
altered properties. This patent application teaches of the use of
dominant negative MMR genes in antibody-producing cells, including
but not limited to rodent hybridomas, human hybridomas, chimeric
rodent cells producing human immunoglobulin gene products, human
cells expressing immunoglobulin genes, mammalian cells producing
single chain antibodies, and prokaryotic cells producing mammalian
immunoglobulin genes or chimeric immunoglobulin molecules such as
those contained within single-chain antibodies. The cell expression
systems described above that are used to produce antibodies are
well known by those skilled in the art of antibody
therapeutics.
[0103] To demonstrate the ability to create MMR defective
hybridomas using dominant negative alleles of MMR genes, we first
transfected a mouse hybridoma cell line that is known to produce an
antibody directed against the human IgE protein with an expression
vector containing the human PMS2 (cell line referred to as HBPMS2),
the previously published dominant negative PMS2 mutant referred
herein as PMS134 (cell line referred to as HB134), or with no
insert (cell line referred to as HBvec). The results showed that
the PMS 134 mutant could indeed exert a robust dominant negative
effect, resulting in biochemical and genetic manifestations of MMR
deficiency. Unexpected was the finding that the full length PMS2
also resulted in a lower MMR activity while no effect was seen in
cells containing the empty vector. A brief description of the
methods is provided below.
[0104] The MMR proficient mouse H36 hybridoma cell line was
transfected with various hPMS2 expression plasmids plus reporter
constructs for assessing MMR activity. The MMR genes were cloned
into the pEF expression vector, which contains the elongation
factor promoter upstream of the cloning site followed by a
mammalian polyadenylation signal. This vector also contains the
NEOr gene that allows for selection of cells retaining this
plasmid. Briefly, cells were transfected with 1 .mu.g of each
vector using polyliposomes following the manufacturer's protocol
(Life Technologies). Cells were then selected in 0.5 mg/ml of G418
for 10 days and G418 resistant cells were pooled together to
analyze for gene expression. The pEF construct contains an intron
that separates the exon 1 of the EF gene from exon 2, which is
juxtaposed to the 5' end of the polylinker cloning site. This
allows for a rapid reverse transcriptase polymerase chain reaction
(RT-PCR) screen for cells expressing the spliced products. At day
17, 100,000 cells were isolated and their RNA extracted using the
trizol method as previously described (Nicolaides N. C., Kinzler,
K. W., and Vogelstein, 8. (1995) Analysis of the 5' region of PMS2
reveals heterogeneous transcripts and a novel overlapping gene.
Genomics 29:329-334). RNAs were reverse transcribed using
Superscript II (Life Technologies) and PCR amplified using a sense
primer located in exon 1 of the EF gene (5'-ttt cgc aac ggg ttt gcc
g-3') (SEQ ID NO:23) and an antisense primer (5'-gtt tca gag tta
agc ctt cg-3') (SEQ ID NO:24) centered at nt 283 of the published
human PMS2 cDNA, which will detect both the full length as well as
the PMS 134 gene expression. Reactions were carried out using
buffers and conditions as previously described (Nicolaides, N. C.,
et al. (1995) Genomic organization of the human PMS2 gene family.
Genomics 30:195-206), using the following amplification parameters:
94.degree. C. for 30 sec, 52.degree. C. for 2 min, 72.degree. C.
for 2 min, for 30 cycles. Reactions were analyzed on agarose gels.
FIG. 1 shows a representative example of PMS expression in stably
transduced H36 cells.
[0105] Expression of the protein encoded by these genes were
confirmed via western blot using a polyclonal antibody directed to
the first 20 amino acids located in the N-terminus of the protein
following the procedures previously described (data not shown)
(Nicolaides et al. (1998) A Naturally Occurring hPMS2 Mutation Can
Confer a Dominant Negative Mutator Phenotype. Mol. Cell. Biol.
18:1635-1641).
EXAMPLE 2
hPMS134 Causes a Defect in MMR Activity and Hypermutability in
Hybridoma Cells
[0106] A hallmark of MMR deficiency is the generation of unstable
microsatellite repeats in the genome of host cells. This phenotype
is referred to as microsatellite instability (MI) (Modrich, P.
(1994) Mismatch repair, genetic stability, and cancer. Science
266:1959-1960; Palombo, F., et al. (1994) Mismatch repair and
cancer. Nature 36:4 17). MI consists of deletions and/or insertions
within repetitive mono-, di-, and/or tri-nucleotide repetitive
sequences throughout the entire genome of a host cell. Extensive
genetic analyses of eukaryotic cells have found that the only
biochemical defect that is capable of producing MI is defective MMR
(Strand, M., et al. (1993) Destabilization of tracts of simple
repetitive DNA in yeast by mutations affecting DNA mismatch repair.
Nature 365:274-276; Perucho, M. (1996) Cancer of the microsatellite
mutator phenotype. Biol Chem. 377:675-684; Eshleman J. R., and
Markowitz, S. D. (1996) Mismatch repair defects in human
carcinogenesis. Hum. Mol. Genet. 5:1489-494). In light of this
unique feature that defective MMR has on promoting MI, it is now
used as a biochemical marker to survey for lack of MMR activity
within host cells (Perucho, M. (1996) Cancer of the microsatellite
mutator phenotype. Biol Chem. 377:675-684; Eshleman J. R., and
Markowitz, S. D. (1996) Mismatch repair defects in human
carcinogenesis. Hum. Mol. Genet. 5:1489-494; Liu, T., et al. (2000)
Microsatellite instability as a predictor of a mutation in a DNA
mismatch repair gene in familial colorectal cancer. Genes
Chromosomes Cancer 27:17-25).
[0107] A method used to detect MMR deficiency in eukaryotic cells
is to employ a reporter gene that has a polynucleotide repeat
inserted within the coding region that disrupts its reading frame
due to a frameshift. In the case where MMR is defective, the
reporter gene will acquire random mutations (i.e., insertions
and/or deletions) within the polynucleotide repeat yielding clones
that contain a reporter with an open reading frame. We have
employed the use of an MMR-sensitive reporter gene to measure for
MMR activity in HBvec, HBPMS2, and HBPMS 134 cells. The reporter
construct used the pCAR-OF, which contains a hygromycin resistance
(HYG) gene plus a .beta.-galactosidase gene containing a 29 bp
out-of-frame poly-CA tract at the 5' end of its coding region. The
pCAR-OF reporter would not generate .beta.-galactosidase activity
unless a frame-restoring mutation (i.e., insertion or deletion)
arose following transfection. HBvec, HBPMS2, and HB 134 cells were
each transfected with pCAR-OF vector in duplicate reactions
following the protocol described in Example 1. Cells were selected
in 0.5 mg/ml G418 and 0.5 mg/ml HYG to select for cells retaining
both the MMR effector and the pCAR-OF reporter plasmids. All
cultures transfected with the pCAR vector resulted in a similar
number of HYG/G418 resistant cells. Cultures were then expanded and
tested for .beta.-galactosidase activity in situ as well as by
biochemical analysis of cell extracts. For in situ analysis,
100,000 cells were harvested and fixed in 1% gluteraldehyde, washed
in phosphate buffered saline solution and incubated in 1 ml of
X-gal substrate solution [0.15 M NaCl, 1 mM MgCl.sub.2, 3.3 mM
K.sub.4Fe(CN).sub.6, 3.3 mM K.sub.3Fe(CN).sub.6, 0.2% X-Gal] in 24
well plates for 2 hours at 37.degree. C. Reactions were stopped in
500 mM sodium bicarbonate solution and transferred to microscope
slides for analysis. Three fields of 200 cells each were counted
for blue (.beta.-galactosidase positive cells) or white
(.beta.-galactosidase negative cells) to assess for MMR
inactivation. Table 1 shows the results from these studies. While
no .beta.-galactosidase positive cells were observed in HBvec
cells, 10% of the cells per field were .beta.-galactosidase
positive in HB 134 cultures and 2% of the cells per field were
.beta.-galactosidase positive in HBPMS2 cultures.
[0108] Cell extracts were prepared from the above cultures to
measure .beta.-galactosidase using a quantitative biochemical assay
as previously described (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype Mol. Cell. Biol. 18:1635-1641; Nicolaides, N. C., et al.
(1992) The Jun family members, c-JUN and JUND, transactivate the
human c-myb promoter via an Ap1 like element. J. Biol. Chem.
267:19665-19672). Briefly, 100,000 cells were collected,
centrifuged and resuspended in 200 uls of 0.25M Tris, pH 8.0. Cells
were lysed by freeze/thawing three times and supernatants collected
after microfugation at 14,000 rpms to remove cell debris. Protein
content was determined by spectrophotometric analysis at
OD.sup.280. For biochemical assays, 20 .mu.g of protein was added
to buffer containing 45 mM 2-mercaptoethanol, 1 mM MgCl.sub.2, 0.1
M NaPO.sub.4 and 0.6 mg/ml Chlorophenol
red-.beta.-D-gaIactopyranoside (CPRG, Boehringer Mannheim).
Reactions were incubated for 1 hour, terminated by the addition of
0.5 M Na.sub.2CO.sub.3, and analyzed by spectrophotometry at 576
nm. H36 cell lysates were used to subtract out background. FIG. 2
shows the .beta.-galactosidase activity in extracts from the
various cell lines. As shown, the HB 134 cells produced the highest
amount of .beta.-galactosidase, while no activity was found in the
HBvec cells containing the pCAR-OF. These data demonstrate the
ability to generate MMR defective hybridoma cells using dominant
negative MMR gene alleles.
1TABLE 1 .beta.-galactosidase expression of HBvec, HBPMS2 and HB134
cells transfected with pCAR-OF reporter vectors. Cells were
transfected with the pCAR-OF .beta.-galactosidase reporter plasmid.
Transfected cells were selected in hygromycin and G418, expanded
and stained with X-gal solution to measure for .beta.-galactosidase
activity (blue colored cells). 3 fields of 200 cells each were
analyzed by microscopy. The results below represent the mean +/-
standard deviation of these experiments. Cell line Number Blue
Cells HBvec 0 +/- 0 HBPMS2 4 +/- 1 HB134 20 +/- 3
EXAMPLE 3
Screening Strategy to Identify Hybridoma Clones Producing
Antibodies with Higher Binding Affinities and/or Increased
Immunoglobulin Production.
[0109] An application of the methods presented within this document
is the use of MMR deficient hybridomas or other immunoglobulin
producing cells to create genetic alterations within an
immunoglobulin gene that will yield antibodies with altered
biochemical properties. An illustration of this application is
demonstrated within this example whereby the HB134 hybridoma
(Example 1), which is a MMR-defective cell line that produces an
anti-human immunoglobulin type E (hIgE) MAb, is grown for 20
generations and clones are isolated in 96-well plates and screened
for hIgE binding. FIG. 3 outlines the screening procedure to
identify clones that produce high affinity MAbs, which is presumed
to be due to an alteration within the light or heavy chain variable
region of the protein. The assay employs the use of a plate Enzyme
Linked Immunosorbant Assay (ELISA) to screen for clones that
produce high-affinity MAbs. 96-well plates containing single cells
from HBvec or HB134 pools are grown for 9 days in growth medium
(RPMI 1640 plus 10% fetal bovine serum) plus 0.5 mg/ml G418 to
ensure clones retain the expression vector. After 9 days, plates
are screened using an hIgE plate ELISA, whereby a 96 well plate is
coated with 50 uls of a 1 .mu.g/ml hIgE solution for 4 hours at
4.degree. C. Plates are washed 3 times in calcium and magnesium
free phosphate buffered saline solution (PBS-/-) and blocked in 100
.mu.ls of PBS-/- with 5% dry milk for 1 hour at room temperature.
Wells are rinsed and incubated with 100 .mu.ls of a PBS solution
containing a 1:5 dilution of conditioned medium from each cell
clone for 2 hours. Plates are then washed 3 times with PBS.sup.-/-
and incubated for 1 hour at room temperature with 50 .mu.ls of a
PBS.sup.-/- solution containing 1:3000 dilution of a sheep
anti-mouse horse radish peroxidase (HRP) conjugated secondary
antibody. Plates are then washed 3 times with PBS.sup.-/- and
incubated with 50 .mu.ls of TMB-HRP substrate (BioRad) for 15
minutes at room temperature to detect amount of antibody produced
by each clone. Reactions are stopped by adding 50 .mu.ls of 500 mM
sodium bicarbonate and analyzed by OD at 415 nm using a BioRad
plate reader. Clones exhibiting an enhanced signal over background
cells (H36 control cells) are then isolated and expanded into 10 ml
cultures for additional characterization and confirmation of ELISA
data in triplicate experiments. ELISAs are also performed on
conditioned (CM) from the same clones to measure total Ig
production within the conditioned medium of each well. Clones that
produce an increased ELISA signal and have increased antibody
levels are then further analyzed for variants that over-express
and/or over-secrete antibodies as described in Example 4. Analysis
of five 96-well plates each from HBvec or HB 134 cells have found
that a significant number of clones with a higher Optical Density
(OD) value is observed in the MMR-defective HB134 cells as compared
to the HBvec controls. FIG. 4 shows a representative example of
HB134 clones producing antibodies that bind to specific antigen (in
this case IgE) with a higher affinity. FIG. 4 provides raw data
from the analysis of 96 wells of HBvec (left graph) or HB134 (right
graph) which shows 2 clones from the HB134 plate to have a higher
OD reading due to 1) genetic alteration of the antibody variable
domain that leads to an increased binding to IgE antigen, or 2)
genetic alteration of a cell host that leads to
over-production/secretion of the antibody molecule. Anti-Ig ELISA
found that the two clones, shown in FIG. 4 have Ig levels within
their CM similar to the surrounding wells exhibiting lower OD
values. These data suggest that a genetic alteration occurred
within the antigen binding domain of the antibody which in turn
allows for higher binding to antigen.
[0110] Clones that produced higher OD values as determined by ELISA
were further analyzed at the genetic level to confirm that
mutations within the light or heavy chain variable region have
occurred that lead to a higher binding affinity hence yielding a
stronger ELISA signal. Briefly, 100,000 cells are harvested and
extracted for RNA using the Triazol method as described above. RNAs
are reverse transcribed using Superscript II as suggested by the
manufacturer (Life Technology) and PCR amplified for the antigen
binding sites contained within the variable light and heavy chains.
Because of the heterogeneous nature of these genes, the following
degenerate primers are used to amplify light and heavy chain
alleles from the parent H36 strain.
2 Light chain sense: 5'-GGA TTT TCA GGT GCA GAT TTT CAG-3' (SEQ ID
NO:1) Light chain antisense: 5'-ACT GGA TGG TGG GAA GAT GGA-3' (SEQ
ID NO:2) Heavy chain sense: 5'-A(G/T) GTN (A/C)AG CTN CAG (C/G)AG
TC-3' (SEQ ID NO:3) Heavy chain antisense: 5'-TNC CTT G(A/G)C CCC
AGT A(G/A)(A/T)C-3' (SEQ ID NO:4)
[0111] PCR reactions using degenerate oligonucleotides are carried
out at 94.degree. C. for 30 sec, 52.degree. C. for 1 mm, and
72.degree. C. for 1 min for 35 cycles. Products are analyzed on
agarose gels. Products of the expected molecular weights are
purified from the gels by Gene Clean (Bio 101), cloned into
T-tailed vectors, and sequenced to identify the wild type sequence
of the variable light and heavy chains. Once the wild type sequence
has been determined, nondegenerate primers were made for RT-PCR
amplification of positive HB134 clones. Both the light and heavy
chains were amplified, gel purified and sequenced using the
corresponding sense and antisense primers. The sequencing of RT-PCR
products gives representative sequence data of the endogenous
immunoglobulin gene and not due to PCR-induced mutations. Sequences
from clones were then compared to the wild type sequence for
sequence comparison. An example of the ability to create in vivo
mutations within an immunoglobulin light or heavy chain is shown in
FIG. 5, where HB134 clone92 was identified by ELISA to have an
increased signal for hIgE. The light chain was amplified using
specific sense and antisense primers. The light chain was RT-PCR
amplified and the resulting product was purified and analyzed on an
automated ABI377 sequencer. As shown in clone A, a residue -4
upstream of the CDR region 3 had a genetic change from ACT to TCT,
which results in a Thr to Ser change within the framework region
just preceding the CDR#3. In clone B, a residue -6 upstream of the
CDR region had a genetic change from CCC to CTC, which results in a
Pro to Leu change within framework region preceding CDR#2.
[0112] The ability to generate random mutations in immunoglobulin
genes or chimeric immunoglobulin genes is not limited to
hybridomas. Nicolaides et al. (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype Mol. Cell. Biol. 18:1635-1641) has previously shown the
ability to generate hypermutable hamster cells and produce
mutations within an endogenous gene. A common method for producing
humanized antibodies is to graft CDR sequences from a MAb (produced
by immunizing a rodent host) onto a human Ig backbone, and
transfection of the chimeric genes into Chinese Hamster Ovary (CHO)
cells which in turn produce a functional Ab that is secreted by the
CHO cells (Shields, R. L., et al. (1995) Anti-IgE monoclonal
antibodies that inhibit allergen-specific histamine release. Int
Arch. Allergy Immunol. 107:412-413). The methods described within
this application are also useful for generating genetic alterations
within Ig genes or chimeric Igs transfected within host cells such
as rodent cell lines, plants, yeast and prokaryotes (Frigerio L, et
al. (2000) Assembly, secretion, and vacuolar delivery of a hybrid
immunoglobulin in plants. Plant Physiol. 123:1483-1494).
[0113] These data demonstrate the ability to generate hypermutable
hybridomas, or other Ig producing host cells that can be grown and
selected, to identify structurally altered immunoglobulins yielding
antibodies with enhanced biochemical properties, including but not
limited to increased antigen binding affinity. Moreover,
hypermutable clones that contain missense mutations within the
immunoglobulin gene that result in an amino acid change or changes
can be then further characterized for in vivo stability, antigen
clearance, on-off binding to antigens, etc. Clones can also be
further expanded for subsequent rounds of in vivo mutations and can
be screened using the strategy listed above.
[0114] The use of chemical mutagens to produce genetic mutations in
cells or whole organisms is limited due to the toxic effects that
these agents have on "normal" cells. The use of chemical mutagens
such as MNU in MMR defective organisms is much more tolerable
yielding to a 10 to 100 fold increase in genetic mutation over MMR
deficiency alone (Bignami M, (2000) Unmasking a killer: DNA
O(6)-methylguanine and the cytotoxicity of methylating agents.
Mutat. Res. 462:71-82). This strategy allows for the use of
chemical mutagens to be used in MMR-defective Ab producing cells as
a method for increasing additional mutations within immunoglobulin
genes or chimeras that may yield functional Abs with altered
biochemical properties such as enhanced binding affinity to
antigen, etc.
EXAMPLE 4
Generation of Antibody Producing Cells with Enhanced Antibody
Production
[0115] Analysis of clones from H36 and HB134 following the
screening strategy listed above has identified a significant number
of clones that produce enhanced amounts of antibody into the
medium. While a subset of these clones gave higher Ig binding data
as determined by ELISA as a consequence of mutations within the
antigen binding domains contained in the variable regions, others
were found to contain "enhanced" antibody production. A summary of
the clones producing enhanced amounts of secreted MAb is shown in
TABLE 2, where a significant number of clones from HB134 cells were
found to produce enhanced Ab production within the conditioned
medium as compared to H36 control cells.
3TABLE 2 Generation of hybridoma cells producing high levels of
antibody. HB134 clones were assayed by ELISA for elevated Ig
levels. Analysis of 480 clones showed that a significant number of
clones had elevated MAb product levels in their CM. Quantification
showed that several of these clones produced greater than 500
ngs/ml of MAb due to either enhanced expression and/or secretion as
compared to clones from the H36 cell line. Cell Line % clones >
400 ng/ml % clones > 500 ng/ml H36 1/480 = 0.2% 0/480 = 0% HB134
50/480 = 10% 8/480 = 1.7%
[0116] Cellular analysis of HB134 clones with higher MAb levels
within the conditioned medium (CM) were analyzed to determine if
the increased production was simply due to genetic alterations at
the Ig locus that may lead to over-expression of the polypeptides
forming the antibody, or due to enhanced secretion due to a genetic
alteration affecting secretory pathway mechanisms. To address this
issue, three HB134 clones that had increased levels of antibody
within their CM were expanded. 10,000 cells were prepared for
western blot analysis to assay for intracellular steady state Ig
protein levels (FIG. 6). In addition, H36 cells were used as a
standard reference (Lane 2) and a rodent fibroblast (Lane 1) was
used as an Ig negative control. Briefly, cells were pelleted by
centrifugation and lysed directly in 300 .mu.l of SDS lysis buffer
(60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2-mercaptoethanol,
0.001% bromophenol blue) and boiled for 5 minutes. Lysate proteins
were separated by electrophoresis on 4-12% NuPAGE gels (for
analysis of Ig heavy chain). Gels were electroblotted onto
Immobilon-P (Millipore) in 48 mM Tris base, 40 mM glycine, 0.0375%
SDS, 20% methanol and blocked at room temperature for 1 hour in
Tris-buffered saline (TBS) plus 0.05% Tween-20 and 5% condensed
milk. Filters were probed with a 1:10,000 dilution of sheep
anti-mouse horseradish peroxidase conjugated monoclonal antibody in
TBS buffer and detected by chemiluminescence using Supersignal
substrate (Pierce). Experiments were repeated in duplicates to
ensure reproducibility. FIG. 6 shows a representative analysis
where a subset of clones had enhanced Ig production which accounted
for increased Ab production (Lane 5) while others had a similar
steady state level as the control sample, yet had higher levels of
Ab within the CM. These data suggest a mechanism whereby a subset
of HB134 clones contained a genetic alteration that in turn
produces elevated secretion of antibody.
[0117] The use of chemical mutagens to produce genetic mutations in
cells or whole organisms is limited due to the toxic effects that
these agents have on "normal" cells. The use of chemical mutagens
such as MNU in MMR defective organisms is much more tolerable,
yielding a 10 to 100 fold increase in genetic mutation over MMR
deficiency alone (Bignanii M, (2000) Unmasking a killer: DNA
O(6)-methylguanine and the cytotoxicity of methylating agents.
Mutat. Res. 462:71-82). This strategy allows for the use of
chemical mutagens to be used in MMR-defective Ab producing cells as
a method for increasing additional mutations within immunoglobulin
genes or chimeras that may yield functional Abs with altered
biochemical properties such as enhanced binding affinity to
antigen, etc.
EXAMPLE 5
Establishment of Genetic Stability in Hybridoma Cells with New
Output Trait.
[0118] The initial steps of MMR are dependent on two protein
complexes, called MutS.alpha. and MutL.alpha. (Nicolaides et al.
(1998) A Naturally Occurring hPMS2 Mutation Can Confer a Dominant
Negative Mutator Phenotype. Mol. Cell. Biol. 18:1635-1641).
Dominant negative MMR alleles are able to perturb the formation of
these complexes with downstream biochemicals involved in the
excision and polymerization of nucleotides comprising the
"corrected" nucleotides. Examples from this application show the
ability of a truncated MMR allele (PMS134) as well as a full length
human PMS2 when expressed in a hybridoma cell line to block MMR
resulting in a hypermutable cell line that gains genetic
alterations throughout its entire genome per cell division. Once a
cell line is produced that contains genetic alterations within
genes encoding for an antibody, a single chain antibody,
overexpression of immunoglobulin genes and/or enhanced secretion of
antibody, it is desirable to restore the genomic integrity of the
cell host. This can be achieved by the use of inducible vectors
whereby dominant negative MMR genes are cloned into such vectors
and introduced into Ab producing cells. The cells are cultured in
the presence of inducer molecules and/or conditions. Inducible
vectors include but are not limited to chemical regulated promoters
such as the steroid inducible MMTV, tetracycline regulated
promoters, temperature sensitive MMR gene alleles, and temperature
sensitive promoters.
[0119] The results described above lead to several conclusions.
First, expression of hPMS2 and PMS 134 results in an increase in
microsatellite instability in hybridoma cells. That this elevated
microsatellite instability is due to MMR deficiency was proven by
evaluation of extracts from stably transduced cells. The expression
of PMS134 results in a polar defect in MMR, which was only observed
using heteroduplexes designed to test repair from the 5' direction
(no significant defect in repair from the 3' direction was observed
in the same extracts) (Nicolaides et al. (1998) A Naturally
Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator
Phenotype. Mol. Cell. Biol. 18:1635-1641). Interestingly, cells
deficient in hMLH1 also have a polar defect in MMR, but in this
case preferentially affecting repair from the 3' direction
(Drummond, J. T, et al. (1996) Cisplatin and adriamycin resistance
are associated with MutLa and mismatch repair deficiency in an
ovarian tumor cell line. J. Biol. Chem. 271:9645-19648). It is
known from previous studies in both prokaryotes and eukaryotes that
the separate enzymatic components mediate repair from the two
different directions. Our results, in combination with those of
Drummond et al. (Shields, R. L., et al. (1995) Anti-IgE monoclonal
antibodies that inhibit allergen-specific histamine release. Int.
Arch Allergy Immunol. 107:412-413), strongly suggest a model in
which 5' repair is primarily dependent on hPMS2 while 3' repair is
primarily dependent on hMLH1. The dimeric complex between PMS2 and
MLH1 sets up this directionality. The combined results also
demonstrate that a defect in directional MMR is sufficient to
produce a MMR defective phenotype and suggests that any MMR gene
allele is useful to produce genetically altered hybridoma cells, or
a cell line that is producing Ig gene products. Moreover, the use
of such MMR alleles will be useful for generating genetically
altered Ig polypeptides with altered biochemical properties as well
as cell hosts that produce enhanced amounts of antibody
molecules.
[0120] Another method that is taught in this application is that
any method used to block MMR can be performed to generate
hypermutability in an antibody-producing cell that can lead to
genetically altered antibodies with enhanced biochemical features
such as but not limited to increased antigen binding, enhanced
pharmacokinetic profiles, etc. These processes can also to be used
to generate antibody producer cells that have increased Ig
expression as shown in Example 4, FIG. 6 and/or increased antibody
secretion as shown in Table 2.
[0121] In addition, we demonstrate the utility of blocking MMR in
antibody producing cells to increase genetic alterations within Ig
genes that may lead to altered biochemical features such as, but
not limited to, increased antigen binding affinities (FIG. 5A and
SB). The blockade of MMR in such cells can be through the use of
dominant negative MMR gene alleles from any species including
bacteria, yeast, protozoa, insects, rodents, primates, mammalian
cells, and man. Blockade of MMR can also be generated through the
use of antisense RNA or deoxynucleotides directed to any of the
genes involved in the MMR biochemical pathway. Blockade of MMR can
be through the use of polypeptides that interfere with subunits of
the MMR complex including but not limited to antibodies. Finally,
the blockade of MMR may be through the use of chemicals such as but
not limited to nonhydrolyzable ATP analogs, which have been shown
to block MMR (Galio, L, et al. (1999) ATP hydrolysis-dependent
formation of a dynamic ternary nucleoprotein complex with MutS and
MutL. Nucl. Acids Res. 27:2325-2331).
EXAMPLE 6
Analysis of Genetic Sequence of Mutant H36 Cell Lines Producing
High Affinity Antibodies
[0122] The nucleic acid sequence of the light and heavy chains of
the antibodies produced by the H36 mutant cell lines were examined
for mutations within the immunoglobulin coding sequence that
contribute to the increased affinity of the antibodies as compared
to the parent clone. The results are shown in Table 3. The data
show that proline substitutions in both the heavy and light chain
variable domains contribute to increased affinity of the antibodies
to antigen. A particular hot spot appears to be amino acid position
6 of SEQ ID NO:6 in which an amino acid substitution occurred
changing the parental alanine to proline for HB91-47, HB134DRMA13,
and HB134DRMA55. These three clones also had mutations at positions
9 and 10. In position 9, the parental valine was changed to glycine
or arginine, while at position 10 of SEQ ID NO:6, the parental
arginine was changed to lysine in both cases.
4TABLE 3 Sequence Amino acid Mean Clones Chain Change Change ELISA
Affinity H36 WT None 0.542 4.80E-08 HB-134a1 L A > T Thr >
Ser 1.632 nd HB91-34 H C Frameshift 0 0 insertion HB91-37 L T >
C Leu > Pro 1.743 1.40E-09 HB91-38 H T > A Ser > Ser 1.641
nd HB91-40 H A > G Ala > Thr 1.333 nd HB91-47 H Multiple Ala
> Pro, 1.979 3.12E-09 Val > Gly, Arg > Lys HB91-53 H TT
> AA Phe > Lys 1.144 nd HB91-62 H A > G Met > Gly 0.218
6.60E-07 HB91-71 H T > G Met > Gly 0.186 nd HB134DRMA13 H
Multiple Ala > Pro, 2.041 nd Val > Gly, Arg > Lys, Thr
> Ala HB134DRMA14 H G > A, Arg > Lys, 1.211 nd A > G
Thr > Ala HB134DRMA55 H Multiple Ala > Pro, 2.012 nd Val >
Arg, Arg > Lys, Thr > Glu, Ser > Thr
[0123] The genetically altered antibodies show the following
sequence differences and consensus sequence:
[0124] Amino acid alignment of morphogenic HB91-47 heavy chain (SEQ
ID NO:17), parental H36 heavy chain (SEQ ID NO:18), and consensus
heavy chain sequence (SEQ ID NO:19)
5 1 35 Morphogenic (1) LQQSGPELGKPGTSVKISCKASGYTFTNYGMNWVK H36
parental (1) LQQSGAELVRPGTSVKISCKASGYTFTNYGMNWVK Consensus (1)
LQQSG-EL--PGTSVKISCKASGYTFTNYGMNWVK .vertline. FR1 .vertline.CDR1
.vertline. 36 70 Morphogenic (36)
QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLE H36 parental (36)
QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLE Consensus (36)
QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLE FR2 .vertline. CDR2 .vertline.
FR3
[0125] Amino acid alignment of morphogenic HB91-37 light chain (SEQ
ID NO:20), parental H36 light chain (SEQ ID NO:21), and consensus
light chain sequence (SEQ ID NO:22)
6 1 35 Morphogenic (1) SASSSVSSSYFHWYQQKSGASPKPLIHRTSNLASG H36
parental (1) SASSSVSSSYFHWYQQKSGASLKPLIHRTSNLASG Consensus (1)
SASSSVSSSYFHWYQQKSGAS-KPLIHRTSNLASG CDR1 .vertline. FR2
.vertline.CDR2 .vertline. 36 45 Morphogenic (36) VPARFSGSGS H36
parental (36) VPARFSGSGS Consensus (36) VPARFSGSGS FR3
[0126] The data shows that, for the light chain, a substitution in
the second framework region (FR2) of the light chain at position 22
of SEQ ID NO:21 to a proline increased the binding affinity of the
antibody.
EXAMPLE 7
An Efficient Screening System for Determining the Effector
Function
[0127] Increased antibody dependent cellular cytotoxicity (ADCC)
elicited by antibody clone variants generated using the method of
the invention maybe detected as follows: In one embodiment, human
peripheral blood mononuclear cells (PBMCs), isolated from healthy
donors are used as effector cells. Briefly, 400 ml of whole blood
is diluted 1:1 (volume:volume) with phosphate buffer saline (PBS),
laid onto Ficoll-Paque (Amersham) solution, and centrifuged at
2,000 RPM, 18.degree. C., for 30 minutes. The interphase containing
the mononuclear cells is recovered and transferred into a fresh
tube and cells are washed with PBS. Red blood cells are then lysed
using ACK lysing buffer (150 mM NH.sub.4Cl, 10 mM KHCO.sub.3, and
0.1 mM Na.sub.2EDTA) for 5 minutes at room temperature. PBMCs are
washed again and their number and viability determined via trypan
blue exclusion. Typically, greater than 2.times.10.sup.8 cells are
recovered using this method, of which 60% endure the
cryo-preservation and subsequent culturing (see below). PBMCs are
then suspended in complete high glucose RMPI-1640 (Invitrogen),
containing 10% fetal bovine serum (FBS) (Invitrogen), 2 mM
L-glutamine (Invitrogen), 5% DMSO (Sigma), at a cell density of
20.times.10.sup.6/ml. Cells are transferred into cryovials, 1
ml/vial, and stored at -80.degree. C. until use. Cells are quickly
brought to 37.degree. C., washed once using pre-warmed compete
RPMI, re-suspended at a cell density of 2.5.times.10.sup.6/ml in
complete RPMI containing 10 ng/ml human recombinant interleukin 2
(hIL-2) (R&D Systems), and grown for 3 days at 37.degree. C.,
5% CO.sub.2. At the end of this incubation, PBMC viability is
typically>85%, and the expected yield will allow the screening
of greater than 600 antibody producing clones, assuming an
effector:target cell ratio of 5:1. Before the assay, cells are
washed once with PBS buffer, counted via trypan blue exclusion,
suspended in CD-CHO serum-free medium (Invitrogen) and used for
ADCC assay. The isolation of PBMCs from about ten donors will be
required to screen 5,000 clones. In the past this number has been
sufficient for the isolation of clones with desired
characteristics. PBMCs from each donor will be used for separate
runs of screening and never mixed with PBMCs of other donors.
[0128] In another embodiments, the use of human stable lines are
used as an alternate source of effector cells. It has been reported
that U937 and HL-60 cells (ATCC CRL-1593.2 and CCL-2, respectively)
are capable of effector function (Sarmay G, Lund J, Rozsnyay Z,
Gergely J, Jefferis R.). Mol. Immunol. 1992 May;29(5):633-9). This
approach tests whether these cells elicit a cytolytic response
against the target cells opsonized by the test antibody (e.g., an
antibody against a tumor antigen applied to a tumor cell). Briefly,
U937 or HL-60 cells will be cultured at 37.degree. C., 5% CO.sub.2,
in complete RPMI and stimulated with either 10 ng/ml recombinant
human interferon gamma (INF, R&D Systems) or with 100 ng/ml of
phorbol 12-myristate 13-acetate (PMA, Sigma). After a 2-day
incubation, cells are washed once with PBS buffer, counted via
trypan blue exclusion, suspended in CD-CHO serum-free medium
(Invitrogen) and used for ADCC assay.
EXAMPLE 8
Production of Cell Lines that Produce Improved Antibodies
[0129] To generate phenotypically diversified cells for the
selection of clones producing antibodies with increased ADCC
activity, mAb-producing cells will be transfected with the vector
p0124 (pEF1-hPMS2-134-IRES-TK) for the expression of the hPMS-134
gene, to inhibit mismatched DNA repair, using the Fugene reagent as
described above. After selection using G41 8 (0.75 mg/ml), cells
will be subcloned to isolate antibody producing clones
concomitantly expressing hPMS-134 protein, as assessed by ELISA and
western blotting, respectively. Cells will be allowed to expand for
greater than 20 generations and then frozen and stored in liquid
nitrogen until use.
EXAMPLE 9
Screening Cells for the Production of Antibodies with Enhanced
Effector Function
[0130] The mAb-producing cells expressing the hPMS-134 will be
subcloned by liming dilution and seeded in a flat-bottom 96-well
plate. Seeding density will be determined empirically in order to
obtain 40 single-cell colonies per plate to approximate
monoclonality.
[0131] The clones will be allowed to grow for a number of days,
which will be empirically determined, after which a sufficient
amount of antibody, capable of mediating ADCC activity, is
produced. Because the parental antibody can elicit ADCC activity at
concentrations as low as 100 ng/ml, we predict that incubating the
single cell-derived clones for 10-15 days should result in the
production of sufficient antibodies to support effector function.
At the end of this incubation period, 50 ul of conditioned medium
from each clone/well will be used to assess concentration of
antibodies by ELISA, while another 50 ul of conditioned medium from
the same well/clone will be utilized in the ADCC assay. Briefly,
for example, an anti-ovarian cancer mAb are used in conjunction
with the target cells, SKOV3 (passage 1 to 20, obtained from ATCC),
which are seeded the day before the assay in a flat-bottom 96-well
microplate at a density of 30,000 cell/well in complete growth
medium (RPMI-1640 containing 10% FBS, 2 mM L-glutamine). The
following day, the complete medium is replaced with 100 ul of
CHO-CD serum-free medium and 50 ul of antibody-containing
conditioned medium will be added to target cells and incubated for
20 minutes at 37.degree. C. Subsequently, 100 ul of serum-free
medium containing 2.times.10.sup.5 of effector cells are added to
each well and cells are incubated for 5-6 hours at 37.degree. C.,
5% CO2. Plates are then briefly centrifuged and 100 ul of
supernatant is collected from each well and transferred into ELISA
plates (Nunc). One hundred ul of LDH substrate (Roche) is added to
supernatants and incubated for 10 minutes at ambient temperature.
LDH activity will be proportional to the extent of the LDH enzyme
released from lysed target cells. Optical density at 490 um
(OD.sub.490) is obtained spectrophotometrically and percent of
cytotoxicity is determined with the formula: (sample
OD.sub.490-spontaneous OD.sub.490)/(max OD.sub.490-spontaneous
OD.sub.490).times.100%, where `spontaneous`=target cells lysis in
absence of effector cells or antibody, and `max`=target cells lysis
in the presence of 2% Triton. Cytotoxicity elicited by 100 ng/ml of
a reference antibody (protein A purified, parental antibody) will
be used as positive control. Non-specific cytotoxicity will be
monitored using 100 mg/ml of normal human IgG1. The ratio obtained
by dividing the % cytotoxicity by the concentration of the antibody
for each well/clone (i.e., ratio=50(%)/100(ng/ml)=0.5) will be set
as the criterion for selecting lead clones with potentially
enhanced effector function. Lead clones will be expanded to 50 ml
cultures and antibody will be purified from their conditioned media
by protein-A affinity column as described. ADCC activities of the
antibodies produced by the lead clones will be compared to the
parental antibody using concentrations ranging from 10-1000
ng/ml.
EXAMPLE 10
Correlating Effector Function and Receptor Binding Activity
[0132] One of the major modes of action of unconjugated therapeutic
monoclonal antibodies directed against tumor antigens is through
recruitment of immune effector populations to the tumor cells
(Clynes R, Takechi Y, Moroi Y, Houghton A, Ravetch J V. Proc. Natl.
Acad. Sci. U.S.A. 1998 Jan. 20;95(2):652-6; Clynes R A, Towers T L,
Presta L G, Ravetch J V. Nat. Med. 2000 April;6(4):443-6). It is
presumed that the efficiency with which a given antibody can
recruit immune effector cells to a tumor cell is influenced by the
affinity of the antibody for its cognate antigen on the tumor cell
surface, such that a high affinity antibody will display more
efficient recruitment of immune effectors to the tumor cell than a
lower affinity counterpart recognizing the same antigen. Limited
reports have attempted to demonstrate this relation in vitro
(Alsmadi, 0. and Tilley, S A. J. Virol. 1998 January;72(1):286-293;
McCall, A M., Shahied, L., Amoroso, A R., Horak, E M., Simmons, R
H., Nielson, U., Adams, G P., Schier, R., Marks, J D., Weiner, L M.
J. Immunol. 2001 May 15;166(10):6112-7, as well as in vivo
(Velders, M P, van Rhijn, C M., Oskam, G J., Warnaar, S O. and
Litvinov, S V. J. Cancer 1998;78(4):476-483). In order to determine
if such a correlation exists, in vitro ADCC activity of enhanced
mAbs, and the affinity of these antibodies may be compared for
their relevant antigen by surface plasmon resonance
spectroscopy.
[0133] Surface plasmon resonance spectroscopy relies on the short
range (.about.150 nm) interaction of the electrical field
(evanescent wave) generated by photons under conditions of total
internal reflection (TIR) with electrons (surface plasmons) in a
conductive film at the boundary between two media of differing
refractive indices, whereby one of the media is a thin gold layer
(conductive film) coated with an alkane linker coupled to
CM-dextran. The CM-dextran surface, which forms an extended
hydrogel in solution, projecting roughly 100-150 nm into the
flowcell, may be derivatized further with a ligand of choice by
covalent immobilization to the carboxyl groups present on the
CM-dextran layer. The angle necessary to allow the evanescent wave
to interact with the gold layer will depend on the angle necessary
to observe TIR, which in turn depends on the thickness or mass at
the surface of the chip. The instrument thus allows for observation
of the change in mass at the surface of the chip over time, as
would be observed when an analyte which interacts with the
immobilized ligand is injected into the flowcell. If injection of
analyte is followed by injection of buffer, one can follow both the
association (during injection of the analyte) and dissociation
phases (during buffer injection) of the binding. Kinetic on-rates
(k.sub.a) and off-rates (k.sub.d), as well as steady-state
equilibrium constants (K.sub.a and K.sub.d) can thus be
extrapolated.
[0134] The soluble, secreted form of the antigen will be purified
from the serum-free culture supernatant of target cells by
chromatography through Phenyl Sepharose (high sub), followed by ion
exchange on S Sepharose Fast Flow. Briefly, culture supernatant
containing secreted antigen will be loaded onto the Phenyl
Sepharose (high sub) column in the absence of additional salts.
Unbound proteins will be removed by extensive washing in HIC A (20
mM K phosphate pH 7.2), followed by elution of bound antigen using
a linear gradient of 0-20 mM CHAPS in HIC buffer. Peak
MORAb-03-containing fractions will be pooled, acidified (pH 5.5)
with 1 M citrate, then applied to a S Sepharose cation exchange
column. After washing with IEX buffer (20 mM K phosphate, pH 5.5),
bound antigen will be eluted using a linear gradient of 0-1 M NaCl
in IEX buffer. Peak fractions will be pooled, concentrated using a
Centricon centrifugal concentration device (Millipore), and
dialyzed against PBS. Based on the purity of the antigen
preparation, an additional affinity chromatography step on
covalently coupled folate Sepharose resin may be necessary
(Sadasivan, E., da Costa, M., Rothenberg, S P. and Brink, L.
Biochim. Biophys. Acta 1987;(925):36-47).
[0135] The mAb to be assayed will be purified in one step by
affinity chromatography on recombinant protein A Sepharose resin
(RPA-Sepharose, Amersham Biosciences). Immunoglobulin (Ig)
containing tissue culture supernatants will be loaded onto
RPASepharose columns by gravity, at a Ig/ml resin value of 10 mg/mL
of resin. Unbound proteins will be removed by extensive washing
with PBS, followed by elution using 0.1 M glycine-HCl pH 2.6.
Fractions will be neutralized with 1 M Tris. Peak fractions will be
pooled, and dialyzed against 1000 volumes of PBS. Ig concentration
will be determined by BCA protein assay (Pierce Chemical Co.) and
Ig-specific ELISA.
[0136] Purified antigen will be diluted into coupling buffer (10 mM
NaOAc pH 5.0), and immobilized onto the flowcell of a CM5 sensor
chip (Biacore) by amine coupling, using a mixture of
N-hydroxysuccinimide (NHS) and 1-ethyl-3-[dimethylaminopropyl]
carbodiimide hydrochloride (EDC) to activate carboxyl groups in the
CM-Dextran hydrogel attached to the surface of the CM5 sensor chip.
Activated, underivatized carboxyl groups will be quenched with 1 M
ethanolamine. A reference flowcell, consisting of the quenched
CMDextran surface, activated in the absence of antigen, will be
used to normalize all measurements. Crude, mAb-containing culture
supernatants, or purified mAb preparations will be injected at flow
rates of 30 ul/min for kinetic assays, and 5 ul/mm for steady-state
affinity ranking experiments, using HBS-EP (20 mM HEPES-OH, 150 mM
NaCl, 3 mM EDTA, 0.005% Surfactant P-20, pH 7.4) as running buffer.
Purified mAb preparations will be dialyzed against HBS-EP, using
10K MWCO Slide-A-Lyzer dialysis cassettes (Pierce) prior to their
use in Biacore analysis. For samples containing tissue culture
supernatant, BSA and soluble CM-Dextran will be added to final
concentrations of 1% and 1 mg/ml, respectively. Regeneration of the
surface will be accomplished by 30 second injection of 50 mM NaOH,
at a flow rate of 100 ul/min. Data analysis will be performed using
Bia Evaluation software (Biacore). Kinetic data will be fitted to a
simple 1:1 (Langmuir) binding model. For ranking experiments, rank
will be determined by K.sub.D values obtained from plots of Req
versus C at different concentrations of sample.
Sequence CWU 1
1
24 1 24 DNA Artificial Sequence Primer 1 ggattttcag gtgcagattt tcag
24 2 21 DNA Artificial Sequence Primer 2 actggatggt gggaagatgg a 21
3 19 DNA Artificial Sequence Primer 3 angtnnagct ncagnagtc 19 4 19
DNA Artificial Sequence Primer 4 tnccttgncc ccagtannc 19 5 859 PRT
Mus musculus 5 Met Glu Gln Thr Glu Gly Val Ser Thr Glu Cys Ala Lys
Ala Ile Lys 1 5 10 15 Pro Ile Asp Gly Lys Ser Val His Gln Ile Cys
Ser Gly Gln Val Ile 20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu
Leu Ile Glu Asn Ser Val Asp 35 40 45 Ala Gly Ala Thr Thr Ile Asp
Leu Arg Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser
Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu
Ala Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp
Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105
110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Gly Ser
115 120 125 Ala Ser Val Gly Thr Arg Leu Val Phe Asp His Asn Gly Lys
Ile Thr 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Lys Gly Thr Thr
Val Ser Val Gln 145 150 155 160 His Leu Phe Tyr Thr Leu Pro Val Arg
Tyr Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr Ser Lys
Met Val Gln Val Leu Gln Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly
Val Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg
His Ala Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 210 215 220 Glu
Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230
235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Val Cys Glu Glu Tyr
Gly 245 250 255 Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr Phe
Arg Ala Ser 260 265 270 Phe His Ser Ala Arg Thr Ala Pro Gly Gly Val
Gln Gln Thr Gly Ser 275 280 285 Phe Ser Ser Ser Ile Arg Gly Pro Val
Thr Gln Gln Arg Ser Leu Ser 290 295 300 Leu Ser Met Arg Phe Tyr His
Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320 Val Val Leu Asn
Val Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330 335 Thr Pro
Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350
Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Ala Asn 355
360 365 Lys Leu Asn Val Asn Gln Gln Pro Leu Leu Asp Val Glu Gly Asn
Leu 370 375 380 Val Lys Leu His Thr Ala Glu Leu Glu Lys Pro Val Pro
Gly Lys Gln 385 390 395 400 Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala
Asp Glu Lys Arg Val Ala 405 410 415 Ser Ile Ser Arg Leu Arg Glu Ala
Phe Ser Leu His Pro Thr Lys Glu 420 425 430 Ile Lys Ser Arg Gly Pro
Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 435 440 445 Ser Glu Lys Arg
Gly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Ser 450 455 460 Tyr Arg
Gly Leu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp 465 470 475
480 Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly
485 490 495 Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe Ser Thr
Pro Glu 500 505 510 Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser
Ser Leu Glu Asp 515 520 525 Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly
Asp Leu Asp Cys Arg Pro 530 535 540 Pro Gly Thr Gly Gln Ser Leu Lys
Pro Glu Asp His Gly Tyr Gln Cys 545 550 555 560 Lys Ala Leu Pro Leu
Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg Phe 565 570 575 Lys Thr Glu
Glu Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro 580 585 590 Gly
Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met 595 600
605 Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys
610 615 620 Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys His
Glu Leu 625 630 635 640 Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro
Gly Glu Asn Gln Ala 645 650 655 Ala Glu Asp Glu Leu Arg Lys Glu Ile
Ser Lys Ser Met Phe Ala Glu 660 665 670 Met Glu Ile Leu Gly Gln Phe
Asn Leu Gly Phe Ile Val Thr Lys Leu 675 680 685 Lys Glu Asp Leu Phe
Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 690 695 700 Asn Phe Glu
Met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu 705 710 715 720
Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu 725
730 735 Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val
Ile 740 745 750 Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys Leu Ile
Ser Leu Pro 755 760 765 Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp
Ile Asp Glu Leu Ile 770 775 780 Phe Met Leu Ser Asp Ser Pro Gly Val
Met Cys Arg Pro Ser Arg Val 785 790 795 800 Arg Gln Met Phe Ala Ser
Arg Ala Cys Arg Lys Ser Val Met Ile Gly 805 810 815 Thr Ala Leu Asn
Ala Ser Glu Met Lys Lys Leu Ile Thr His Met Gly 820 825 830 Glu Met
Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg 835 840 845
His Val Ala Asn Leu Asp Val Ile Ser Gln Asn 850 855 6 3056 DNA Mus
musculus 6 gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg
aggagacaga 60 taacctgtcg tcaggtaacg atggtgtata tgcaacagaa
atgggtgttc ctggagacgc 120 gtcttttccc gagagcggca ccgcaactct
cccgcggtga ctgtgactgg aggagtcctg 180 catccatgga gcaaaccgaa
ggcgtgagta cagaatgtgc taaggccatc aagcctattg 240 atgggaagtc
agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg 300
tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta
360 aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta
gaagaagaaa 420 actttgaagg tctagctctg aaacatcaca catctaagat
tcaagagttt gccgacctca 480 cgcaggttga aactttcggc tttcgggggg
aagctctgag ctctctgtgt gcactaagtg 540 atgtcactat atctacctgc
cacgggtctg caagcgttgg gactcgactg gtgtttgacc 600 ataatgggaa
aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg 660
tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa
720 aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca
gcaggcgtcc 780 gtgtaagctg cactaatcag ctcggacagg ggaagcggca
cgctgtggtg tgcacaagcg 840 gcacgtctgg catgaaggaa aatatcgggt
ctgtgtttgg ccagaagcag ttgcaaagcc 900 tcattccttt tgttcagctg
ccccctagtg acgctgtgtg tgaagagtac ggcctgagca 960 cttcaggacg
ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg 1020
cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc
1080 agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg
catcagtacc 1140 catttgtcgt ccttaacgtt tccgttgact cagaatgtgt
ggatattaat gtaactccag 1200 ataaaaggca aattctacta caagaagaga
agctattgct ggccgtttta aagacctcct 1260 tgataggaat gtttgacagt
gatgcaaaca agcttaatgt caaccagcag ccactgctag 1320 atgttgaagg
taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa 1380
agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct
1440 ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct
aggggtccag 1500 agactgctga actgacacgg agttttccaa gtgagaaaag
gggcgtgtta tcctcttatc 1560 cttcagacgt catctcttac agaggcctcc
gtggctcgca ggacaaattg gtgagtccca 1620 cggacagccc tggtgactgt
atggacagag agaaaataga aaaagactca gggctcagca 1680 gcacctcagc
tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca 1740
gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg
1800 acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac
catggatatc 1860 aatgcaaagc tctacctcta gctcgtctgt cacccacaaa
tgccaagcgc ttcaagacag 1920 aggaaagacc ctcaaatgtc aacatttctc
aaagattgcc tggtcctcag agcacctcag 1980 cagctgaggt cgatgtagcc
ataaaaatga ataagagaat cgtgctcctc gagttctctc 2040 tgagttctct
agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg 2100
aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag
2160 atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc
ttgggtcagt 2220 ttaacctggg atttatagta accaaactga aagaggacct
cttcctggtg gaccagcatg 2280 ctgcggatga gaagtacaac tttgagatgc
tgcagcagca cacggtgctc caggcgcaga 2340 ggctcatcac accccagact
ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa 2400 atctggaaat
attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca 2460
ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag
2520 atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc
cggccctcac 2580 gagtcagaca gatgtttgct tccagagcct gtcggaagtc
agtgatgatt ggaacggcgc 2640 tcaatgcgag cgagatgaag aagctcatca
cccacatggg tgagatggac cacccctgga 2700 actgccccca cggcaggcca
accatgaggc acgttgccaa tctggatgtc atctctcaga 2760 actgacacac
cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg 2820
ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc
2880 catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca
gctacttggg 2940 tgatccggtg ggagctcatg tgagcccagg actttgagac
cactccgagc cacattcatg 3000 agactcaatt caaggacaaa aaaaaaaaga
tatttttgaa gccttttaaa aaaaaa 3056 7 862 PRT Homo sapiens 7 Met Glu
Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10 15
Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20
25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu
Asp 35 40 45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr
Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val
Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His His Thr
Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr
Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu
Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala Lys Val
Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys Ile Ile 130 135 140 Gln
Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gln 145 150
155 160 Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe Gln Arg
Asn 165 170 175 Ile Lys Lys Glu Tyr Ala Lys Met Val Gln Val Leu His
Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly Ile Arg Val Ser Cys Thr
Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg Gln Pro Val Val Cys Thr
Gly Gly Ser Pro Ser Ile Lys 210 215 220 Glu Asn Ile Gly Ser Val Phe
Gly Gln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe Val Gln
Leu Pro Pro Ser Asp Ser Val Cys Glu Glu Tyr Gly 245 250 255 Leu Ser
Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 260 265 270
Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln 275
280 285 Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys
Arg 290 295 300 Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln
Tyr Pro Phe 305 310 315 320 Val Val Leu Asn Ile Ser Val Asp Ser Glu
Cys Val Asp Ile Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln Ile Leu
Leu Gln Glu Glu Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr Ser
Leu Ile Gly Met Phe Asp Ser Asp Val Asn 355 360 365 Lys Leu Asn Val
Ser Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 370 375 380 Ile Lys
Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gln 385 390 395
400 Asp Gln Ser Pro Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp Val Ser
405 410 415 Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr
Glu Asn 420 425 430 Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg
Ser Pro Leu Gly 435 440 445 Gln Lys Arg Gly Met Leu Ser Ser Ser Thr
Ser Gly Ala Ile Ser Asp 450 455 460 Lys Gly Val Leu Arg Pro Gln Lys
Glu Ala Val Ser Ser Ser His Gly 465 470 475 480 Pro Ser Asp Pro Thr
Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His 485 490 495 Gly Ser Thr
Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 500 505 510 Ser
His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly 515 520
525 Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp
530 535 540 Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr
Gly Cys 545 550 555 560 Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu
Ala Thr Pro Asn Thr 565 570 575 Lys Arg Phe Lys Lys Glu Glu Ile Leu
Ser Ser Ser Asp Ile Cys Gln 580 585 590 Lys Leu Val Asn Thr Gln Asp
Met Ser Ala Ser Gln Val Asp Val Ala 595 600 605 Val Lys Ile Asn Lys
Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser 610 615 620 Leu Ala Lys
Arg Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu 625 630 635 640
Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu 645
650 655 Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr
Met 660 665 670 Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn Leu Gly
Phe Ile Ile 675 680 685 Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp
Gln His Ala Thr Asp 690 695 700 Glu Lys Tyr Asn Phe Glu Met Leu Gln
Gln His Thr Val Leu Gln Gly 705 710 715 720 Gln Arg Leu Ile Ala Pro
Gln Thr Leu Asn Leu Thr Ala Val Asn Glu 725 730 735 Ala Val Leu Ile
Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 740 745 750 Phe Val
Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 755 760 765
Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val Asp 770
775 780 Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg
Pro 785 790 795 800 Ser Arg Val Lys Gln Met Phe Ala Ser Arg Ala Cys
Arg Lys Ser Val 805 810 815 Met Ile Gly Thr Ala Leu Asn Thr Ser Glu
Met Lys Lys Leu Ile Thr 820 825 830 His Met Gly Glu Met Asp His Pro
Trp Asn Cys Pro His Gly Arg Pro 835 840 845 Thr Met Arg His Ile Ala
Asn Leu Gly Val Ile Ser Gln Asn 850 855 860 8 2771 DNA Homo sapiens
8 cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct
60 aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg
gcaggtggta 120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca
gtctggatgc tggtgccact 180 aatattgatc taaagcttaa ggactatgga
gtggatctta ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa
cttcgaaggc ttaactctga aacatcacac atctaagatt 300 caagagtttg
ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc 360
tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga
420 actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta
cccccgcccc 480 agagggacca cagtcagcgt gcagcagtta ttttccacac
tacctgtgcg ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc
aaaatggtcc aggtcttaca tgcatactgt 600 atcatttcag caggcatccg
tgtaagttgc accaatcagc ttggacaagg aaaacgacag 660 cctgtggtat
gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720
cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt
780 gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat
ctcaggtttc 840 atttcacaat gcacgcatgg agttggaagg agttcaacag
acagacagtt tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc
tgcagactcg tgaatgaggt ctaccacatg 960
tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt
1020 gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa
gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtg
atgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt
aacttaataa aaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa
gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgt
ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320
aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt
1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag
acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacgg
acagagcgga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat
tctgaggggt tcagcatccc agacacgggc 1560 agtcactgca gcagcgagta
tgcggccagc tccccagggg acaggggctc gcaggaacat 1620 gtggactctc
aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680
tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca
1740 accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga
catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttg
atgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg
agttctttag ctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag
tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggag
aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040
tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat
2100 gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt
cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcac
ctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat
ctggaaatat ttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc
tccagtcact gaaagggcta aactgatttc cttgccaact 2340 agtaaaaact
ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400
agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc
2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa
actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatg
gaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac
tgaccgtagt cactgtatgg aataattggt 2640 tttatcgcag atttttatgt
tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctg
ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760
cttttcaaac c 2771 9 932 PRT Homo sapiens 9 Met Lys Gln Leu Pro Ala
Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr Ser
Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp
Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45
Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50
55 60 Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn
Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg
Gly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu
Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln Tyr
Val Leu Asp Gly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys Pro Ser
His Leu Gly Gln Gly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu Phe
Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145 150 155 160 Thr Ala
Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170 175
Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 180
185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys
Met 195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn
Met Glu Ser 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr
Leu Ser Gly Phe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His Ser
Phe Thr Ser Leu Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile
Asn Ser Arg Pro Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile
Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr Arg
Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295 300
Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 305
310 315 320 Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr
Thr Cys 325 330 335 Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn
Asn Lys Thr Asp 340 345 350 Val Ser Ala Ala Asp Ile Val Leu Ser Lys
Thr Ala Glu Thr Asp Val 355 360 365 Leu Phe Asn Lys Val Glu Ser Ser
Gly Lys Asn Tyr Ser Asn Val Asp 370 375 380 Thr Ser Val Ile Pro Phe
Gln Asn Asp Met His Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn Thr
Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 405 410 415 Gly
Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420 425
430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn
435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val
Lys His 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp
Glu Ser Gly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu Asn
Ser Ser Glu Ile Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn Ile
Leu Lys Asn Ser Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile
Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn
Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540 Ser
Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545 550
555 560 Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met
Ser 565 570 575 Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe
Leu Ile Glu 580 585 590 Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu
Gln Ile Glu Glu Leu 595 600 605 Trp Lys Thr Leu Ser Glu Glu Glu Lys
Leu Lys Tyr Glu Glu Lys Ala 610 615 620 Thr Lys Asp Leu Glu Arg Tyr
Asn Ser Gln Met Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu Ser Gln
Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr Ser
Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660 665 670
Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 675
680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met
Lys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp
Leu Glu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn Leu
Arg Phe Pro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu Val
Met Leu Leu Asn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu Phe
Lys Arg Leu Leu Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu
Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser
His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790 795
800 Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn
805 810 815 Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu
Asn Tyr 820 825 830 Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe
Tyr Gly Val Ala 835 840 845 Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu
Asn Arg Asn Ala Lys Glu 850 855 860 Val Tyr Glu Cys Arg Pro Arg Lys
Val Ile Ser Tyr Leu Glu Gly Glu 865 870 875 880 Ala Val Arg Leu Ser
Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 885 890 895 Ile Gln Asp
Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 900 905 910 Lys
Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 915 920
925 Pro Glu Thr Thr 930 10 3063 DNA Homo sapiens 10 ggcacgagtg
gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60
ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa
120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa
aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta
tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg
ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt
catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt
ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420
ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac
480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag
aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga
tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac
ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa
agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc
tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780
tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa
840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa
gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga
atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag
ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa
aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta
tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140
ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg
1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc
caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt
aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg
aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca
atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg
ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500
atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt
1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag
aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa
agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag
attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt
acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc
aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860
ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg
1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga
tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact
aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc
agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa
ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt
acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220
acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg
2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat
agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact
tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta
atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa
agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg
tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580
aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc
2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct
cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca
attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa
tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca
ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt
taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940
tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca
3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa
ataaactaat 3060 aac 3063 11 934 PRT Homo sapiens 11 Met Ala Val Gln
Pro Lys Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu 1 5 10 15 Val Gly
Phe Val Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr 20 25 30
Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu 35
40 45 Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val
Ile 50 55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser
Val Val Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp
Leu Leu Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn
Arg Ala Gly Asn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu
Ala Tyr Lys Ala Ser Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp
Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val
Val Gly Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln 145 150 155 160
Val Gly Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 165
170 175 Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu
Ile 180 185 190 Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu
Thr Ala Gly 195 200 205 Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg
Gly Gly Ile Leu Ile 210 215 220 Thr Glu Arg Lys Lys Ala Asp Phe Ser
Thr Lys Asp Ile Tyr Gln Asp 225 230 235 240 Leu Asn Arg Leu Leu Lys
Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 245 250 255 Val Leu Pro Glu
Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala 260 265 270 Val Ile
Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 275 280 285
Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile 290
295 300 Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp
Thr 305 310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys
Cys Lys Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp Ile
Lys Gln Pro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu
Asn Leu Val Glu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln
Thr Leu Gln Glu Asp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn
Arg Leu Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu
Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410
415 Val Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu
420 425 430 Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp
Phe Ser 435 440 445 Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met
Asp Gln Val Glu 450 455 460 Asn His Glu Phe Leu Val Lys Pro Ser Phe
Asp Pro Asn Leu Ser Glu 465 470 475 480 Leu Arg Glu Ile Met Asn Asp
Leu Glu Lys Lys Met Gln Ser Thr Leu 485 490 495 Ile Ser Ala Ala Arg
Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys 500 505 510 Leu Asp Ser
Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 515 520 525 Glu
Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile 530 535
540 Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn
545 550 555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala
Gln Asp Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly
Tyr Val Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala Gln
Leu Asp Ala Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala
Pro Val Pro Tyr Val Arg Pro Ala Ile 610 615 620 Leu Glu Lys Gly Gln
Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala 625 630 635 640 Cys Val
Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr 645 650 655
Phe Glu Lys Asp Lys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met 660
665 670 Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu
Met 675 680 685 Ala Gln Ile Gly Cys Phe Val Pro Cys Glu Ser Ala Glu
Val Ser
Ile 690 695 700 Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser
Gln Leu Lys 705 710 715 720 Gly Val Ser Thr Phe Met Ala Glu Met Leu
Glu Thr Ala Ser Ile Leu 725 730 735 Arg Ser Ala Thr Lys Asp Ser Leu
Ile Ile Ile Asp Glu Leu Gly Arg 740 745 750 Gly Thr Ser Thr Tyr Asp
Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu 755 760 765 Tyr Ile Ala Thr
Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe 770 775 780 His Glu
Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr Val Asn Asn Leu 785 790 795
800 His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gln
805 810 815 Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gly Ile His Val
Ala Glu 820 825 830 Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala
Lys Gln Lys Ala 835 840 845 Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly
Glu Ser Gln Gly Tyr Asp 850 855 860 Ile Met Glu Pro Ala Ala Lys Lys
Cys Tyr Leu Glu Arg Glu Gln Gly 865 870 875 880 Glu Lys Ile Ile Gln
Glu Phe Leu Ser Lys Val Lys Gln Met Pro Phe 885 890 895 Thr Glu Met
Ser Glu Glu Asn Ile Thr Ile Lys Leu Lys Gln Leu Lys 900 905 910 Ala
Glu Val Ile Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 915 920
925 Arg Ile Lys Val Thr Thr 930 12 3145 DNA Homo sapiens 12
ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag
60 gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg
gccgaggtcg 120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgac
caccacagtg cgccttttcg 180 accggggcga cttctatacg gcgcacggcg
aggacgcgct gctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatc
aagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag
taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360
atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt
420 atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt
ctctttggta 480 acaatgatat gtcagcttcc attggtgttg tgggtgttaa
aatgtccgca gttgatggcc 540 agagacaggt tggagttggg tatgtggatt
ccatacagag gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttc
tccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt
acccggagga gagactgctg gagacatggg gaaactgaga cagataattc 720
aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt
780 atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat
agtgctgtat 840 tgccagaaat ggagaatcag gttgcagttt catcactgtc
tgcggtaatc aagtttttag 900 aactcttatc agatgattcc aactttggac
agtttgaact gactactttt gacttcagcc 960 agtatatgaa attggatatt
gcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac
tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080
gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg
1140 agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag
actttacaag 1200 aagatttact tcgtcgattc ccagatctta accgacttgc
caagaagttt caaagacaag 1260 cagcaaactt acaagattgt taccgactct
atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat
gaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac
tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440
tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc
1500 tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca
acattaataa 1560 gtgcagccag agatcttggc ttggaccctg gcaaacagat
taaactggat tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta
aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatc
cagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga
agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg 1800
ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg
1860 tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga
gcacctgttc 1920 catatgtacg accagccatt ttggagaaag gacaaggaag
aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa
ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc
cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca
aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160
agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc
2220 aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct
atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattggg
aagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag
aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt
catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca
tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520
agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta
2580 agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt
cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa
gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc
tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc
acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag
ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880
cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt
2940 atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca
acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagat
ttttattttg aaaaatgaga 3060 gctgtaactg aggactgttt gcaattgaca
taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg
3145 13 756 PRT Homo sapiens 13 Met Ser Phe Val Ala Gly Val Ile Arg
Arg Leu Asp Glu Thr Val Val 1 5 10 15 Asn Arg Ile Ala Ala Gly Glu
Val Ile Gln Arg Pro Ala Asn Ala Ile 20 25 30 Lys Glu Met Ile Glu
Asn Cys Leu Asp Ala Lys Ser Thr Ser Ile Gln 35 40 45 Val Ile Val
Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile Gln Asp Asn 50 55 60 Gly
Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg Phe 65 70
75 80 Thr Thr Ser Lys Leu Gln Ser Phe Glu Asp Leu Ala Ser Ile Ser
Thr 85 90 95 Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser His
Val Ala His 100 105 110 Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys
Cys Ala Tyr Arg Ala 115 120 125 Ser Tyr Ser Asp Gly Lys Leu Lys Ala
Pro Pro Lys Pro Cys Ala Gly 130 135 140 Asn Gln Gly Thr Gln Ile Thr
Val Glu Asp Leu Phe Tyr Asn Ile Ala 145 150 155 160 Thr Arg Arg Lys
Ala Leu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile 165 170 175 Leu Glu
Val Val Gly Arg Tyr Ser Val His Asn Ala Gly Ile Ser Phe 180 185 190
Ser Val Lys Lys Gln Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro 195
200 205 Asn Ala Ser Thr Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala
Val 210 215 220 Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr
Leu Ala Phe 225 230 235 240 Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn
Tyr Ser Val Lys Lys Cys 245 250 255 Ile Phe Leu Leu Phe Ile Asn His
Arg Leu Val Glu Ser Thr Ser Leu 260 265 270 Arg Lys Ala Ile Glu Thr
Val Tyr Ala Ala Tyr Leu Pro Lys Asn Thr 275 280 285 His Pro Phe Leu
Tyr Leu Ser Leu Glu Ile Ser Pro Gln Asn Val Asp 290 295 300 Val Asn
Val His Pro Thr Lys His Glu Val His Phe Leu His Glu Glu 305 310 315
320 Ser Ile Leu Glu Arg Val Gln Gln His Ile Glu Ser Lys Leu Leu Gly
325 330 335 Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Thr Leu Leu Pro
Gly Leu 340 345 350 Ala Gly Pro Ser Gly Glu Met Val Lys Ser Thr Thr
Ser Leu Thr Ser 355 360 365 Ser Ser Thr Ser Gly Ser Ser Asp Lys Val
Tyr Ala His Gln Met Val 370 375 380 Arg Thr Asp Ser Arg Glu Gln Lys
Leu Asp Ala Phe Leu Gln Pro Leu 385 390 395 400 Ser Lys Pro Leu Ser
Ser Gln Pro Gln Ala Ile Val Thr Glu Asp Lys 405 410 415 Thr Asp Ile
Ser Ser Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu 420 425 430 Glu
Leu Pro Ala Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu 435 440
445 Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro
450 455 460 Thr Ser Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp
Val Glu 465 470 475 480 Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr
Ala Ala Cys Thr Pro 485 490 495 Arg Arg Arg Ile Ile Asn Leu Thr Ser
Val Leu Ser Leu Gln Glu Glu 500 505 510 Ile Asn Glu Gln Gly His Glu
Val Leu Arg Glu Met Leu His Asn His 515 520 525 Ser Phe Val Gly Cys
Val Asn Pro Gln Trp Ala Leu Ala Gln His Gln 530 535 540 Thr Lys Leu
Tyr Leu Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe 545 550 555 560
Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn Phe Gly Val Leu Arg Leu 565
570 575 Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Met Leu Ala Leu Asp
Ser 580 585 590 Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pro Lys Glu
Gly Leu Ala 595 600 605 Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Ala
Glu Met Leu Ala Asp 610 615 620 Tyr Phe Ser Leu Glu Ile Asp Glu Glu
Gly Asn Leu Ile Gly Leu Pro 625 630 635 640 Leu Leu Ile Asp Asn Tyr
Val Pro Pro Leu Glu Gly Leu Pro Ile Phe 645 650 655 Ile Leu Arg Leu
Ala Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys 660 665 670 Phe Glu
Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys 675 680 685
Gln Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 690
695 700 Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile
Val 705 710 715 720 Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys
His Phe Thr Glu 725 730 735 Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu
Pro Asp Leu Tyr Lys Val 740 745 750 Phe Glu Arg Cys 755 14 2484 DNA
Homo sapiens 14 cttggctctt ctggcgccaa aatgtcgttc gtggcagggg
ttattcggcg gctggacgag 60 acagtggtga accgcatcgc ggcgggggaa
gttatccagc ggccagctaa tgctatcaaa 120 gagatgattg agaactgttt
agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180 ggaggcctga
agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg 240
gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt
300 atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt
ggctcatgtt 360 actattacaa cgaaaacagc tgatggaaag tgtgcataca
gagcaagtta ctcagatgga 420 aaactgaaag cccctcctaa accatgtgct
ggcaatcaag ggacccagat cacggtggag 480 gacctttttt acaacatagc
cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540 gggaaaattt
tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca 600
gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg
660 gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga
aattggatgt 720 gaggataaaa ccctagcctt caaaatgaat ggttacatat
ccaatgcaaa ctactcagtg 780 aagaagtgca tcttcttact cttcatcaac
catcgtctgg tagaatcaac ttccttgaga 840 aaagccatag aaacagtgta
tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900 ctcagtttag
aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa 960
gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag
1020 ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc
aggacttgct 1080 ggcccctctg gggagatggt taaatccaca acaagtctga
cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc ccaccagatg
gttcgtacag attcccggga acagaagctt 1200 gatgcatttc tgcagcctct
gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260 gaggataaga
cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa 1320
ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag
1380 gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag
aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaa gatgattccc
gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat cattaacctc
actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg gacatgaggt
tctccgggag atgttgcata accactcctt cgtgggctgt 1620 gtgaatcctc
agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc 1680
aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt
1740 ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt
agatagtcca 1800 gagagtggct ggacagagga agatggtccc aaagaaggac
ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga gatgcttgca
gactatttct ctttggaaat tgatgaggaa 1920 gggaacctga ttggattacc
ccttctgatt gacaactatg tgcccccttt ggagggactg 1980 cctatcttca
ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt 2040
gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag
2100 gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa
ctcctggaag 2160 tggactgtgg aacacattgt ctataaagcc ttgcgctcac
acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat cctgcagctt
gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt aaatatggtt
atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340 cgatacaaag
tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag 2400
cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata
2460 aataaataga tgtgtcttaa cata 2484 15 133 PRT Homo sapiens 15 Met
Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10
15 Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val
20 25 30 Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser
Leu Asp 35 40 45 Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp
Tyr Gly Val Asp 50 55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly
Val Glu Glu Glu Asn Phe 65 70 75 80 Glu Gly Leu Thr Leu Lys His His
Thr Ser Lys Ile Gln Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu
Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala
Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 115 120 125 Ala Lys
Val Gly Thr 130 16 426 DNA Homo sapiens 16 cgaggcggat cgggtgttgc
atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca
aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120
ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact
180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc
agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga
aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa
acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga
tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 acttga 426 17 70
PRT Artificial Sequence Clone HB 91-47 immunoglobulin heavy chain
17 Leu Gln Gln Ser Gly Pro Glu Leu Gly Lys Pro Gly Thr Ser Val Lys
1 5 10 15 Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly
Met Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp
Met Gly Trp Ile 35 40 45 Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu 65 70 18
70 PRT Artificial Sequence Parental H36 immunoglobulin heavy chain
18 Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys
1 5 10 15 Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly
Met Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp
Met Gly Trp Ile 35 40 45 Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala
Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu 65 70 19
70 PRT Artificial Sequence Consensus immunoglobulin heavy chain
sequence 19 Leu Gln Gln Ser Gly Xaa Glu Leu Xaa Xaa Pro Gly Thr Ser
Val Lys 1 5 10 15 Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr Gly Met Asn 20 25 30 Trp Val Lys Gln Ala Pro Gly Lys Gly Leu
Lys Trp Met Gly Trp Ile 35 40 45 Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Asp Asp Phe Lys Gly Arg 50 55 60 Phe Ala Phe Ser Leu Glu 65
70 20 45 PRT Artificial Sequence Clone HB 91-37 immunoglobulin
light chain 20 Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Phe His Trp
Tyr Gln Gln 1 5 10 15 Lys Ser Gly Ala Ser Pro Lys Pro Leu Ile His
Arg Thr Ser Asn Leu 20 25 30 Ala Ser Gly Val Pro Ala Arg Phe Ser
Gly Ser Gly Ser 35 40 45 21 45 PRT
Artificial Sequence Parental immunoglobulin light chain 21 Ser Ala
Ser Ser Ser Val Ser Ser Ser Tyr Phe His Trp Tyr Gln Gln 1 5 10 15
Lys Ser Gly Ala Ser Leu Lys Pro Leu Ile His Arg Thr Ser Asn Leu 20
25 30 Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 35 40 45
22 44 PRT Artificial Sequence Consensus immunoglobulin light chain
sequence 22 Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Phe Trp Tyr Gln
Gln Lys 1 5 10 15 Ser Gly Ala Ser Xaa Lys Pro Leu Ile His Arg Thr
Ser Asn Leu Ala 20 25 30 Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
Gly Ser 35 40 23 19 DNA Artificial Sequence Sense primer 23
tttcgcaacg ggtttgccg 19 24 20 DNA Artificial Sequence Antisense
primer 24 gtttcagagt taagccttcg 20
* * * * *