U.S. patent application number 11/102000 was filed with the patent office on 2005-09-01 for expression of heterologous multi-domain proteins in yeast.
This patent application is currently assigned to APOLIFE, INC.. Invention is credited to Blackburn, Robert, Motwani, Nalini.
Application Number | 20050191726 11/102000 |
Document ID | / |
Family ID | 24296375 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191726 |
Kind Code |
A1 |
Motwani, Nalini ; et
al. |
September 1, 2005 |
Expression of heterologous multi-domain proteins in yeast
Abstract
This invention demonstrates the utility of a yeast expression
system for the expression of functional heterologous multi-domain
proteins in yeast. The yeast expression system allows for the
inclusion of a plurality of (up to three) modular expression
cassettes which may encode multiple polypeptide chains of a
heterologous multi-domain protein on a single plasmid (Twin
Cassette). Because multiple polypeptide chains may be encoded for
by the expression cassettes of the present invention in a single
vector, the system can produce equivalent amounts of the multiple
polypeptide chains, thereby enhancing the yield of a functional
heterologous multi-domain protein. For example, functional
monoclonal antibodies (MAbs) comprising a heavy chain and a light
chain of an immunoglobulin (IgG), and functional immunotoxins
comprising an antibody domain and an oxidase toxin may be produced
using the Yeast expression system of the present invention. In
addition, functional single chain antibodies, antibody fragments
and chimeric antibodies may also be produced.
Inventors: |
Motwani, Nalini; (West
Bloomfield, MI) ; Blackburn, Robert; (Warren,
MI) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Assignee: |
APOLIFE, INC.
|
Family ID: |
24296375 |
Appl. No.: |
11/102000 |
Filed: |
April 7, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11102000 |
Apr 7, 2005 |
|
|
|
10172867 |
Oct 19, 2001 |
|
|
|
10172867 |
Oct 19, 2001 |
|
|
|
09574492 |
May 19, 2000 |
|
|
|
6358733 |
|
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/254.2; 435/483; 530/388.1; 536/23.5 |
Current CPC
Class: |
C12N 15/67 20130101;
C12N 15/81 20130101; C07K 2317/55 20130101; C07K 2317/622 20130101;
C07K 16/30 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/483; 435/254.2; 530/388.1; 536/023.5 |
International
Class: |
C12P 021/06; C07H
021/04; C12N 001/18; C12N 015/74; C07K 016/46 |
Goverment Interests
[0001] The present invention involves subject matter developed
under National Institute of Health Grant Numbered 1R43AI40822-01,
so that the United States Government may have certain rights
herein.
Claims
1. A vector including one or more expression cassettes wherein one
of said expression cassettes comprises a nucleic acid encoding a
heterologous recombinant fusion protein which comprises an
immunological molecule and a toxin.
2. The vector of claim 1 wherein the expression cassettes further
comprise promoter sequences operatively linked to the nucleic acid
encoding the heterologous recombinant fusion protein.
3. The vector of claim 2 wherein the expression cassettes further
comprise translation initiation sequences and wherein said promoter
sequences are located flush with and 5' to said translation
initiation sequences.
4. The vector of claim 1 wherein the expression cassettes further
comprise a nucleic acid encoding a secretory signal sequence.
5. The vector of claim 4 wherein the secretory signal sequence is
selected from the group consisting of human secretory signal
sequences, yeast secretory sequences and secretory sequences which
are native to the heterologous fusion protein.
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. The vector of claim 1 further including an expression cassette
comprising a nucleic acid encoding an accessory molecule.
11. The vector of claim 10 wherein the accessory molecule is a
chaperone protein.
12. The vector of claim 11 wherein the chaperone protein is
selected from the group consisting of the BiP and protein disulfide
isomerase (PDI).
13. A method of producing a functional heterologous recombinant
multichain or single-chain fusion protein comprising: (a)
transforming a Saccharomyces yeast host cell with a vector, wherein
the vector comprises multiple expression cassettes, and wherein at
least one of the expression cassettes encodes the fusion protein;
(b) growing the transformed cell in culture to a stage of log phase
growth; and (c) inducing the cell culture to express the
heterologous recombinant multichain or single-chain fusion protein,
wherein the fusion protein comprises an immunotoxin, and wherein
the immunotoxin comprises an antibody domain and an oxidase toxin
domain.
14. The method of claim 13 wherein the yeast cell is Saccharomyces
cerevisiae.
15. The method of claim 14 wherein the strain of S. cerevisiae is
selected from the group consisting of Y112, Y113, Y114, Y115, Y116,
Y117, Y118, Y119, Y120, Y121, Y122, Y123, Y124 and Y125.
16. The method of claim 13 wherein the yeast host cell comprises
one or more mutations in glycosylation pathways.
17. The method of claim 16 wherein the recombinant fusion protein
is glycosylated.
18. The method of claim 13 wherein the yeast host cell comprises
supersecretory activity.
19. (canceled)
20. The method of claim 13 wherein the transformed yeast cell is
grown under fermentation parameters that improve the production of
the recombinant fusion protein.
21. The method of claim 20 wherein the fermentation parameters are
selected from the group consisting of carbon sources, buffering
systems, media formulations, vitamin levels, trace salt levels,
temperature, aeration levels, oxygen levels, pH, induction time,
and length of induction.
22. (canceled)
23. The method of claim 13 wherein the antibody domain comprises
two chains and wherein one of the chains is operably linked to the
toxin domain.
24. The method of claim 23 wherein one of the two chains is a heavy
chain, and wherein the other chain is a light chain.
25. The method of claim 23 wherein the vector comprises multiple
expression cassettes.
26. The method of claim 13 wherein the toxin is selected from the
group consisting of glucose oxidase, glucose-oxidase-related toxin,
horseradish peroxidase, and horseradish peroxidase-related
toxin.
27. A vector comprising at least two expression cassettes, wherein
a first expression cassette comprises a nucleic acid encoding a
heterologous recombinant fusion protein, wherein the fusion protein
comprises an immunological molecule and a toxin, wherein a second
expression cassette comprises a nucleic acid encoding an accessory
molecule, and wherein the accessory molecule is a chaperone protein
selected from the group consisting of a BiP and a protein disulfide
isomerase (PDI).
28. The vector of claim 27, wherein a third expression cassette
comprises a nucleic acid encoding an accessory molecule, and
wherein the accessory molecule is a chaperone protein selected from
the group consisting of a BiP and a protein disulfide isomerase
(PDI).
Description
1. INTRODUCTION
[0002] The invention is directed to a cost effective system for the
production of heterologous recombinant proteins in yeast using a
single vector to express functional multi-domain proteins,
including proteins comprising multiple polypeptide chains. The
proteins may include, but are not limited to, recombinant
monoclonal antibodies, single antibody chains, chimeric antibodies,
immunotoxins, etc. The vector of the present invention may comprise
a plurality of modular expression cassettes which facilitate the
manipulation of the expression of various subunits of a protein.
The expression cassettes may additionally comprise a hybrid,
constitutive, or inducible promoter, signal sequences for secretion
of protein, nucleic acid encoding the protein of interest (e.g. a
heavy and light chain of an antibody molecule) and a
transcriptional termination sequence. Furthermore, the invention is
directed to improved techniques to reduce development time for
production of functional heterologous recombinant multi-domain
proteins. The recombinant molecules produced by this invention are
useful for research, diagnostic and/or therapeutic
applications.
2. BACKGROUND OF THE INVENTION
2.1 MAb Expression
[0003] Monoclonal antibodies hold great promise for application in
a wide range of diagnostic and therapeutic (clinical) settings, as
evidenced by current clinical use of monoclonal antibody-derived
products for transplantation, tumor imaging, therapeutics and
diagnostics.
[0004] Currently two main methods used for commercial monoclonal
antibody ("MAb") production are generally employed; in vivo mouse
ascites fluid and in vitro cultivation of hybridoma cell lines. The
production of a MAb in vivo from mouse ascites fluid is limited in
that it produces solid tumors in mice and results in death of the
animal and low-level yields of MAbs. In vitro cultivation of
hybridoma cell lines also has limitations. For example, it has been
estimated that a minimum of 1000 clones need to be screened to find
just two MAb-producing hybridoma cell lines. Most clones are not
considered to be useful because of inappropriate specificity. In
addition, after going through several passages, hybridoma cell
lines may lose certain chromosomes and stop producing the MAb.
Recombinant production of MAbs could avoid some of the problems
associated with production of MAbs from hybridoma cell lines and
ascites fluid.
[0005] A large number of heterologous single chain polypeptides
have been produced by host cells transformed by recombinant DNA
techniques. However, very few functional multichain polypeptides
have been successfully produced by recombinant techniques.
Recombinant dimeric polypeptides have been synthesized as a single
chain polypeptide, coded for by a single DNA sequence, which is
then cleaved in the host cell subsequent to synthesis to form the
dimeric structure. In some cases the polypeptide chains are
synthesized separately and then assembled after isolation from the
host cell. Disadvantages of recombinant protein production in E.
coli include inefficient secretion, formation of insoluble protein
complexes in inclusion bodies, the presence of endotoxin, lack of
glycosylation, and lack of N-terminal methionine processing (see
Buchner, Anal. Biochem. 205:263 (1992)), which often affect the
functionality of the recombinant protein, or hinder efficient and
cost-effective production and purification.
[0006] A number of heterologous proteins have been expressed in
yeast. Examples include interferon (U.S. Pat. No. 4,775, 622,
Hitzeman, et al., Nature, 292, 717, 1991); platelet derived growth
factor (U.S. Pat. No. 4,801,542); and glyceraldehyde-3-phosphate
dehydrogenase (Holland et al., Basic Life Science, 19:291, (1981)).
Burke et al., U.S. Pat. No. 4,876,197 discloses a DNA construct
comprising a transcription regulatory region obtained from the
yeast ADH2, the regulatory region of acid phosphatase (PHO5) or
GAL4 which provides for inducible transcriptional regulation, a
transcriptional initiation region from the yeast
glyceraldehyde-3-phosphate dehydrogenase gene ("TDH3") and a
terminator region.
[0007] The structure of antibody molecules and the nature of genes
coding for them permit extensive manipulation and shuffling of
antibody genes to produce recombinant antibodies with domains from
different proteins and species. Such manipulation and shuffling can
create MAbs with desired specificity, effector functions, reduced
immunogenicity and/or binding sites for additional molecules.
Recent advances in genetic engineering have made it possible to
design and generate single chain, chimeric and humanized antibodies
with desired specificities and binding sites (Vaughan et al.,
Nature Biotech. 16:535 (1998)).
[0008] Expression of recombinant MAbs using different expression
systems such as bacteria, yeast, baculovirus and mammalian cells
have been reported (Gen. Eng. News p. 12, August (1996)). Bacterial
cells produce MAbs which accumulate as improperly folded,
non-native proteins in inclusion bodies. However, the levels of
properly folded MAbs from industrial cell cultures are generally
very low.
[0009] Humanized bispecific antibody produced from E. coli in
secreted form was found to simultaneously bind different antigens
on two different cells (Russoniello et al., Clin. Cancer Res.
4:2237 (1998)). Using Pichia pastoris, Ridder et al. have reported
production of a soluble and functional rabbit single chain antibody
fragment ("ScFv") (Biotechnology 13:255-60, (1995)). The yields of
ScFv for human leukemia inhibition factor was 100 mg/L.
Glockhushuber et al. reported production of single chain and Fab
fragments of antibodies in E. coli. (Biochem. 291362 (1990)). The
yield in this system was poor (10-100 ug/ml) with the bacterial
products being secreted in the periplasm and not glycosylated,
requiring solubilization, denaturation, reduction, and renaturation
to facilitate the formation of intramolecular disulfide bonds and
the native conformation. Glockhushuber et al., Biochem. 29:1362
(1990). Another disadvantage of E. coli derived polypeptides is
endotoxin contamination which can cause immune reactions in
patients. In addition, E. coli do not have the ability to remove
the N-formyl-methionine by post-translational modification which is
required for the production of functional antibody formation.
Glockhushuber et al., Biochem. 291362 (1990).
[0010] U.S. Pat. No. 4,816,397 ("the '397 patent") describes the
process for production of multichain polypeptides or proteins in a
single host cell, which comprises transforming the host cell with
DNA coding for each of the polypeptide chains. The invention also
describes the production of recombinant IgG heavy and light chain
or fragments thereof having an intact variable domain. While the
'397 patent describes the production of both a heavy and light
chain in a single cell, the expressed polypeptides were found in
inclusion bodies in the bacterial cells in which they were produced
and required cumbersome denaturation. Only a small fraction of the
amount expressed was retrievable in functional, soluble form.
[0011] Feasibility of expression of functional immunoglobulin (IgG)
in yeast was first reported by Wood et al. (Nature 314:446 (1985))
and Carlson (Mol. Cell Biol, 8:2638; 46, (1988)). Functional IgG
against alcohol dehydrogenase was described using a yeast inducible
promoter. Using GAL1-10 bidirectional promoter, Bowdish et al. (J.
Biol Chem., 266:11901-8 (1991)) produced properly folded Fab
fragment of a catalytic antibody, permitting the expression of low
levels of two antibody polypeptides simultaneously. However, the
expression of heavy chain gene was more efficient than that of
light chain gene from GAL110. The results of Bowdish et al.
indicate that recombinant heavy chain polypeptides are reasonably
stable in yeast cytoplasm. Typically 100-200 ug/L of Fab was
expressed which accounted for approximately 0.1% of total cellular
protein. In comparison to the prior art methods, an advantage of
the yeast expression system of the present invention is that it can
simultaneously express two proteins (or protein subunits) in
similar amounts, thereby favoring higher yields of functional
multichain molecules.
[0012] In addition, recombinant MAbs have been expressed in
hybridoma or myeloma cell lines. See David Robinson, Biotech
Bioeng. 55:783 (1997). The current methodologies are limited by a
low secretion rate of cell lines and the difficulties of selecting
human clones secreting IgG. See Bobbington et al., Biotech 10:169
(1992). The media contain as much as 50 ingredients, and can take
up to 14 days for fermentation making development of a mammalian
cells secreting MAbs slow.
[0013] In some cases the polypeptides produced by the aformentioned
techniques are not immunologically functional as they are incapable
of combining with complementary heavy or light chains to provide
functional IgG molecules.
2.2. The Role of MAbs in Therapeutics and Diagnostics
[0014] Herceptin (Genentech, San Francisco, Calif.) was the first
humanized MAb approved by the FDA for use in the treatment of human
cancer. Werner, Semin. Oncol. 26:43 (1999). However, current MAb
technology has a number of short comings. First, production is
limited by low yields, long production times and high costs of
production, as discussed above. Second, in non-chimeric form, MAbs
are immunogenic. A major drawback of MAbs produced in ascites of
mice is that these MAbs, when administered to human patients, cause
an immune response which produces neutralizing human-anti-mouse
antibodies ("HAMAs"). HAMAs limit the number of times a patient may
be treated with a mouse MAb. Several antibody variants in which
immunogenic regions have been eliminated, including chimeric and
humanized antibodies, are currently being tested in therapeutic
clinical settings.
[0015] It is believed that if the affinity and/or specificity of an
antibody (Ab) can be improved ten or twenty fold, its therapeutic
usefulness can be greatly improved. Such high affinity Abs could
target specific cells. The selective delivery of drugs to a tumor
is a major goal in cancer chemotherapy. Solid tumors are poorly
vascularized which hinders antibody penetration. Smaller molecules
such as single chain antibodies or Ab fragments may more
efficiently penetrate solid tumors. The smaller molecules have
reduced serum half life, enhanced tissue penetration, may be useful
in tumor imaging and therapy or for the treatment of acute
inflammation. However, current methods are not amenable to rapid
screening of MAbs or efficient, large-scale, cost effective
production of MAbs.
[0016] Therefore, it would be useful to have improved methods to
quickly screen for high affinity therapeutic MAbs. Therapeutic use
of MAbs may require doses ranging between several hundred
milligrams to a gram over the course of therapy. Typical expression
levels of hybridoma cell lines is between 0.2-0.5 g/L. Robbinson et
al., Biotech. Bioeng. 44:727 (1994). For a moderate market like
lung or breast cancer to achieve 30% market penetration, a company
will have to produce 60 kg purified bulk product. Using hybridoma
cell lines, this will translate to 50 runs of 14 days each for a
5000-L bioreactor which would require 2 years to produce the
required MAbs (Seaver, Genet Eng. News, Jan. 15, (1997)). Improved
methods for the quick, low cost production of MAbs would vastly
improve the introduction of therapeutic MAbs into the market.
2.3. Expression of Heterologous Proteins in Yeast
[0017] Yeast has been used in large scale fermentations for
centuries and the technology of large scale production of yeast is
well known. Yeast has several advantages as an expression system,
namely: (1) it can be grown in higher densities than bacterial and
eukaryotic cells, (2) it is capable of protein glycosylation which
is important in antibody production, (3) its post-translational
modification machinery can remove terminal methionines, and (4) it
has post-transcriptional and post-translational modification
machinery similar to that found in mammalian cells, increasing the
likelihood of expression of a soluble, functional eukaryotic
protein.
[0018] The products produced and secreted in yeast are easily
purified because of the resistance of yeast to lysis (hydrostatic
pressures), low contamination in media and low protease content in
the growth media. In addition, yeast does not have endotoxin
problems associated with bacteria or the viral contamination
problem associated with products produced by mammalian cell culture
systems. Furthermore, yeast can be grown more rapidly to high
density in simple, low cost media than other eukaryotic cells and
its genetics are well characterized and easily manipulated for the
optimization of heterologous gene expression.
2.4. Immunotoxin Expression
[0019] Molecules commonly used to construct immunotoxins have been
derived from bacteria or plant toxins. Pietersz and McKenzie,
Immunol. Rev. 129:57 (1992). First generation immunotoxins have
been constructed by linking hybridoma-generated monocolonal
antibodies to purified toxins by chemical conjugation. Pastan and
Fitzgerald, Science 254:1173 (1991); Melton and Sherwood, J. Natl
Cancer Inst. 88:153 (1996). These were found to have limited
efficacy against cancer, which led to the development of
recombinant immunotoxins, which are chimeric proteins comprising a
fusion of a truncated toxin and the variable region sequences of a
monoclonal antibody. The improved stability, tissue permeability,
and decreased immunogenicity of recombinant immunotoxins adds
greater potential for the therapeutic usage of these proteins.
However, concurrent with the advances in immunotoxin technology,
development of cost-effective production methods are essential to
provide adequate availability.
[0020] Glucose oxidase ("GO", D-glucose:oxygen 1-oxidoreductase) is
an enzyme present in several Aspergillus and Penicillium species
that utilize glucose as a substrate to generate hydrogen peroxide
and gluconolactone as byproducts of its enzymatic activity. The
functional form of the GO glycoprotein is composed of a dimer (MW
150,000) containing two bound flavin adenine dinucleotide (FAD)
cofactors. Pazur et al., Arch. Biochem. Biophys. 111:351 (1965).
The gene encoding glucose oxidase from the fungi Aspergillus niger
has been cloned, sequenced, and expressed in a functional form in
the yeast, S. cerevisiae. Whittington et al., Curr. Genet. 18:531
(1999). In addition, the A. niger glucose oxidase produced from
yeast has been shown to be more stable at higher temperatures and
at wider pH ranges than the native protein. Frederick et al., J.
Biol. Chem. 265:3793 (1990).
[0021] Exposure to glucose oxidase can induce toxicity in mammalian
cells. Salazar and Van Houten, Mutat. Res. 385:139 (1997). The
predominant toxic effect of GO has been shown to result from the
generation of hydrogen peroxide. Starke and Farber, J. Biol. Chem.
260:86-92 (1985). Preferential toxicity toward tumor cells from
GO-generated peroxide has also been demonstrated. Mavier et al.,
Hepatology 8:1673 (1988); Ben-Yoseph and Ross, Br. J. Cancer
70:1131 (1994). Purified GO induced extensive cytotoxicity (less
than 10% survival) in breast, prostate, and lung carcinoma cell
lines at a concentration of less than 0.01 units of activity/ml of
culture supernatant within three hours of exposure. In addition,
exposure of carcinoma cells to glucose oxidase may enhance
radiation-induced killing through the generation of free radical
species (hydroperoxides) induced by its enzymatic activity.
Metosh-Dickey and Winston, Free Radic. Biol. Med. 24:155 (1998);
Nutter et al., J. Biol. Chem. 267:2472 (1992); Sinha et al., Cancer
Res. 49:3844 (1989). Glucose oxidase also can function to generate
free radical products through the one-electron reduction of several
different classes of xenobiotic compounds. Metosh-Dickey and
Winston, Free Radic. Biol. Med. 24:155 (1998). Many
chemotherapeutic agents (e.g. menadione, mitomycin C, adriamycin)
are converted to active forms via single electron bioreduction.
Nutter et al., J. Biol. Chem. 267:2472 (1992); Sinha et al., Cancer
Res. 49:3844 (1989). Therefore, systemic administration of these
drugs may enhance tumoricidal activity in combination with targeted
exposure of tumor tissue to a glucose oxidase immunotoxin.
[0022] In addition to direct hydrogen peroxide-related toxicity, GO
may also serve to deplete glucose in targeted cells. A common trait
of most carcinoma cells is an extreme reliance upon glycolytic
pathways to generate phosphometabolites, e.g. energy in the form of
ATP, and to control the intracellular redox environment relative to
normal tissue. Warburg, Science 123:309 (1956). This reliance is
characterized by a downregulation of genes involved in oxidative
phoshorylation, concomitant with increased expression of glucose
uptake and transport proteins (e.g. glut1; Kozlovsky et al., J.
Biol. Chem. 272:33367 (1997)) and glycolytic enzymes (Meixensberger
et al., Neurooncol 24:153 (1995)). Exposure of cultured tumor cells
to hydrogen peroxide induces an increase in glycolytic metabolism
and glucose uptake (Kozlovsky et al., J. Biol. Chem. 272:33367
(1997)), further increasing the dependence upon available glucose
for survival. As a result of this chronic dependence upon
glycolysis, glucose-deprivation of carcinoma cells results in
significant and preferential induction of cytotoxicity (Blackburn
et al., Free Radic. Biol Med. 26:419 (1999)).
[0023] Other oxidases have also been shown to have cytotoxic
effects in mammalian cells. For example, xanthine oxidase, like
glucose oxidase, induces toxicity in mammalian cells in native and
modified forms. See Stanislawski et al., Cancer Res. 49:5497
(1989); Sawa et al., Cancer Res. 60:666 (2000).
[0024] It has also been shown that peroxidases, including
horseradish peroxidase, eosinophil peroxidase, myeloperoxidase, and
lactoperoxidase, exhibit anticancer activity when administered
alone or in combination with glucose oxidases and a source of
halide ions. See Everse et al., Br. J. Cancer 51:743 (1985);
Stanislawski et al., Cancer Res. 49:5497 (1989); Samoszuk et al.
Cancer Res. 54:2650 (1994); Odajima et al., Biol. Chem. 377:689
(1996).
[0025] Previously, several methods have been developed to deliver
glucose oxidase protein into cells via streptavidin/biotin systems
(Ohno et al., Biochem. Mol. Med. 58:227 (1996)), and liposome
vehicles (Samoszuk et al., Cancer Res. 56:87 (1996)), and through
the use of chemical conjugation to antibodies (Stanislawski et al.,
Cancer Res. 49:5497 (1989)). In each of these systems, significant
cytotoxicity could be generated in the target cells. Inefficient
conjugation, expense of manufacturing individual components,
altered protein structure and longer production times are all
disadvantages regarding the use of these procedures.
[0026] The present invention provides an improved method for
producing immunotoxins which allows for the production of large
amounts of immunotoxins at a relatively low cost in a short time
frame. In addition, the present invention facilitates the
production of various immunotoxins both analytically and in
large-scale.
[0027] There is a need for an expression system for the production
of functional multi-domain proteins which can reduce production
times and cost. The present invention demonstrates high level
expression of properly folded functional heterologous multi-domain
proteins (e.g. MAbs, single chain antibodies, chimeric antibodies,
immunotoxins, etc.) in yeast which is accomplished quickly and at
low cost. This invention describes the use of a yeast expression
system for the expression of functional multi-domain heterologous
proteins.
3. SUMMARY OF THE INVENTION
[0028] This invention demonstrates the utility of a yeast
expression system for the expression of functional heterologous
recombinant multi-domain proteins in yeast which is cost effective
and which allows for efficient production. The yeast expression
system allows for the inclusion of a plurality of (up to three)
modular expression cassettes which may encode multiple polypeptide
chains of a heterologous multi-domain protein on a single plasmid.
Because multiple polypeptide chains may be encoded by the
expression cassettes of the present invention in a single vector,
the system can produce equivalent amounts of each of the multiple
polypeptide chains, thereby enhancing the yield of a functional
heterologous multi-domain protein. For example, functional
monoclonal antibodies ("MAbs") comprising a heavy chain and a light
chain of an immunoglobulin ("IgG") may be produced using the yeast
expression system of the present invention. In addition, functional
single chain antibodies, antibody fragments and chimeric antibodies
may also be produced. This invention also relates to a system for
the cost effective production of immunotoxins in yeast.
[0029] The production of MAbs may be accomplished using the present
invention which comprises a yeast expression system including a
single plasmid comprising expression cassettes encoding both heavy
and light IgG chains. This process provides a technical and
practical advantage to other methods by providing better yields of
functional MAb (greater than 5 mg/L from 100 ml shake flask
fermentation), quicker production times, modular expression
cassettes which permit production of MAbs to any specific antigen
within a few weeks with little manipulation of the vector and lower
costs of production.
[0030] The yeast expression system can be used for expression of a
single chain, Fab fragment or a complete antibody molecule.
Cotransformation of a host yeast strain with two plasmids
containing heavy ("H") and light ("L") chains can also be used for
expressing antibodies such that the H and L chains are produced in
equivalent amounts. The yeast expression system is well suited for
commercial use by providing a low cost system in which high yield
expression is achieved (greater than 5 mg/L host cell culture) and
the proteins produced may be secreted in the medium for easy
isolation.
[0031] In addition, the present invention is directed to the
production of multi-domain recombinant proteins (e.g.
immunotoxins). For example, the production of functional
immunotoxins may be accomplished using the present invention which
comprises a yeast expression system having one or more, or a
plurality of, expression cassettes wherein each expression cassette
includes a nucleic acid comprising an antibody domain (e.g. scFv,
Fab', etc.) fused to a toxin (e.g. an oxidase toxin such as glucose
oxidase, xanthine oxidase, amino acid oxidase and peroxidases). The
present invention also offers the advantage of allowing for the
incorporation and coordinated expression of an accessory molecule
(e.g. chaperones which may improve protein folding) into a
heterologous protein production system. Co-expression of an
accessory molecule may improve the production of a functional
heterologous multi-domain protein. The recombinant proteins
generated by this system are optimized for efficient, high-yield
expression and secretion in yeast.
4. DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1. Universal cloning vector.
[0033] FIG. 2. A plasmid including an expression cassette
comprising a nucleic acid encoding IgG light chain and expression
cassette comprising a nucleic acid encoding IgG heavy chain.
[0034] FIG. 3. SDS-PAGE gel electrophoresis of supernatants of S.
cerevisiae strain Y112 transformed with the plasmid of FIG. 2.
[0035] FIG. 4. SDS-PAGE gel electrophoresis of supernatants of S.
cerevisiae strain Y112 co-transformed with a plasmid including an
expression cassette comprising a nucleic acid encoding IgG light
chain and a plasmid including an expression cassette comprising a
nucleic acid encoding IgG heavy chain.
[0036] FIG. 5. SDS-PAGE gel electrophoresis of supernatants of S.
cerevisiae strain Y113 co-transformed with a plasmid including an
expression cassette comprising a nucleic acid encoding IgG light
chain and a plasmid including an expression cassette comprising a
nucleic acid encoding IgG heavy chain.
[0037] FIG. 6. A plasmid including an expression cassette
comprising a nucleic acid encoding the fusion protein ScFv-SA.
[0038] FIGS. 7A & B. Western blot analysis of both (A) SDS-PAGE
gel electrophoresis and (B) native gel electrophoresis of
supernatants of S. cerevisiae transformed with the plasmid of FIG.
6.
[0039] FIG. 8. Western blot analysis of SDS-PAGE gel
electrophoresis of supernatants of S. cerevisiae transformed with
the plasmid of FIG. 6 as compared to purified fusion protein.
[0040] FIG. 9. Functional EGP-2 antigen binding assay for ScFv-SA
of supernatants of S. cerevisiae transformed with the plasmid of
FIG. 6.
[0041] FIG. 10. Quantitation by ELISA of ScFv-SA production of
supernatants of S. cerevisiae transformed with the plasmid of FIG.
6.
[0042] FIG. 11. A plasmid including an expression cassette
comprising a nucleic acid encoding the Fd chain of Fab-SA and an
expression cassette comprising a nucleic acid encoding the Fv_chain
of Fab-SA.
[0043] FIG. 12. Western blot analysis of SDS-PAGE gel
electrophoresis of supernatants and cell lysates of S. cerevisiae
transformed with the plasmid of FIG. 11.
[0044] FIG. 13. Comparison of Fab-SA expression in various yeast
strains.
[0045] FIG. 14. Quantitation by ELISA of Fab-SA production from
supernatants of S. cerevisiae transformed with the plasmid of FIG.
11.
[0046] FIG. 15. Expression cassette comprising an antibody gene
(ScFv) and a toxin gene (glucose oxidase).
[0047] FIG. 16. Western blot analysis of SDS-PAGE gel
electrophoresis of supernatants of S. cerevisiae transformed with a
plasmid including the expression cassette of FIG. 15.
[0048] FIG. 17. Functional EGP-2 antigen binding assay for ScFv-GO
of supernatants of S. cerevisiae transformed with a plasmid
including the expression cassette of FIG. 15.
[0049] FIGS. 18A & B. (A) Control wells measuring the glucose
activity of reagents alone, buffer (PBS), and purified glucose
oxidase, (A) functional glucose oxidase activity assay for
ScFv-GO_of supernatants of S. cerevisiae transformed with a plasmid
including the expression cassette of FIG. 15 at 0 (preculture), 24
and 48 hours post-induction, also shown is reagents alone, and
vector alone control.
[0050] FIG. 19. Western blot analysis of SDS-PAGE gel
electrophoresis showing ScFv-GO glycosylation.
[0051] FIG. 20. Comparison of ScFv-GO expression in various yeast
strains.
[0052] FIG. 21. Comparison of different media for the growth of
yeast strains transformed with the plasmid of FIG. 6.
[0053] FIG. 22. Flow diagram illustrating an antibody-fusion
protein purification scheme.
5. DETAILED DESCRIPTION OF THE INVENTION
[0054] For the purposes of clarity of description, and not by way
of limitation, the detailed description of the invention is divided
into the following subsections.
[0055] (i) the Yeast Expression System
[0056] (ii) hybrid promoters
[0057] (iii) yeast host cells
[0058] (iv) antibody expression
[0059] (v) immunotoxin expression
5.1. The Yeast Expression System
[0060] This invention describes an efficient yeast expression
system for production of functional heterologous multi-domain or
multi-chain proteins. The yeast expression system includes one or
more, or a plurality of, expression cassettes (see, e.g., FIG. 11)
comprising (a) a strong constitutive or inducible hybrid yeast
promoter, (b) a leader sequence, (c) a nucleic acid encoding a
heterologous protein, and (d) a transcription termination sequence.
Nonlimiting examples of constitutive promoters include GAPDH
(TDH3), ADH1 or the enolase promoter. As the term is used herein, a
hybrid promoter comprises an upstream activation sequence(UAS) of
an inducible promoter, such as GAL1, GAL10, ADH2 or PHO5 and the
TATA box of a strong constitutive promoter, such as, GAPDH, GAL,
ADH, enolase, etc.. The expression cassette may further comprise
secretion signals which may be natural signals from IgG molecules
or secretory signals from non-imunoglobulin molecules (e.g. human
serum albumin secretory signal, yeast secretory signal). In
preferred embodiments of the invention, the yeast expression system
may include a "high-copy" yeast vector comprising a plurality of
expression cassettes inserted at multiple cloning sites. Where the
expression cassettes encode different protein chains (e.g. heavy
and light immunoglobulin chains, see FIG. 2) the high-copy yeast
vector allows for the efficient expression of a functional
heterologous multi-domain or multi-chain protein in yeast. A
high-copy yeast vector may alternatively contain more than one
expression cassette encoding the same protein.
[0061] As used herein, a functional protein refers to a protein
which exhibits its wild-type function, e.g. a functional MAb binds
to antigen and a functional immunotoxin binds to antigen and also
retains toxin activity, although the degree of function (e.g.
binding affinity or toxin activity) may be different from
wild-type.
[0062] Recombinant protein production using the yeast expression
system of the present invention may be confirmed by techniques
known in the art, such as Western blot analysis. The system may
produce recombinant MAbs and immunotoxins in excess of 5 mg/L. This
yield can be improved (10.times.) by selection of promoters, carbon
sources and fermentation conditions. Nonlimiting examples of high
copy vectors include pPM40, YEp13, C1/1 and pSI. See, e.g. abstract
for NSBI Grant Number 1R43AI40822-01 of Motwani (1996); available
online from CRISP at
<<https://www-commons.cit.nih.gov/crisp/>>,
incorporated herein by reference.
[0063] The expression cassettes of the present invention allow for
mixing and matching of different upstream activating sequence (UAS)
elements, translation initiation sequences, promoter elements,
secretory signals and nucleic acids, using cloning techniques known
the art (see e.g., Fritsch and Manniatis, Molecular Cloning: A
Laboratory Manual, 2.sup.nd Ed. 1989; Ausubel et al. (eds.) Current
Protocols in Molecular Biology (1992); incorporated herein by
reference). Improved yields of heterologous proteins may be
achieved by such mixing and matching.
[0064] The expression system of the present invention may also
include an appropriate plasmid replication signals and/or a
selection marker to promote high copy number of the plasmid, and
thus increase expression of the polypeptides in the yeast cells.
Suitable selection markers may include, but are not limited to,
leu, ura and trp etc. In a preferred embodiment of the present
invention, the selection marker may be either ura or leu.
[0065] In one specific, non-limiting embodiment of the invention,
the vector can include three expression cassettes which can be
positioned at three restriction endonuclease sites in the high-copy
vector (e.g. XbaI, Bam HI, Eco RI, sites of the pPM40 high-copy
vector). The expression cassettes can be cloned in either direction
(e.g. two cassettes may be cloned such that transcription proceeds
in the same or opposite direction). The orientation of the
cassettes may effect the yield of recombinant heterologous
proteins. The location of these cassettes may also influence the
expression levels of the heterologous proteins. Expression yields
may be higher when a first expression cassette is cloned into the
BamHI site and a second expression cassette is cloned into the
PvuII site. In general, the distance between two expression
cassettes in the vector is at least 2500 bases. In addition, the
sequences just prior to the ATG start codon may have an affect on
the expression levels of the recombinant heterologous proteins
(e.g. the ATG right next to the first codon for the heterlogous
protein). Yields may also be improved by including two expression
cassettes comprising a nucleic acid encoding the same chain in a
single plasmid such that both expression cassettes produce the
immunoglobulin chain.
[0066] The yeast expression system of the present invention can
produce similar amounts of a first and second protein of interest
(the first and second protein may represent individual subunits of
a multichain protein). As referred to herein, similar amounts means
that the amount of the first protein produced relative to the
amount of the second protein produced is in a ratio between 0.2:1.0
to 5:1.0 and preferably 0.5:1.0 to 2:1.0. In order to produce
similar amounts of a first and second protein, a vector can be
constructed which has two expression cassettes of the present
invention, each of which comprises a nucleic acid encoding either
the first or second protein to be expressed. A yeast cell can be
transformed with the vector encoding the first and second protein,
grown in culture to log phase and induced to express the first and
second protein in similar amounts.
5.2 The Hybrid Promoter
[0067] The hybrid promoter of the present invention includes a
first nucleic acid comprising an inducible transcriptional
promoter/enhancer sequence, and a second nucleic acid comprising an
RNA polymerase binding site and transcriptional initiation site.
The use of regulatory sequences for controlling transcription of
the structural gene of interest allows for high density growth of
host cells with no or low levels of expression of the structural
gene when operatively linked thereto. Gene expression may then be
induced by changing the environmental conditions.
[0068] As non-limiting examples of sequences which may be used
according to the invention, European Patent Application No.
132,309, published Jan. 30, 1985, discloses a plasmid containing
the yeast galactose-induced promoter for galactokinase (GAL1) and
UDP-galactose epimerase (GAL10), hereinafter referred to as the
GAL-10 promoter, which is bidirectional. Broach et al.
(Manipulation of Gene Expression, ed. Inouye, 1983) disclose a
plasmid containing a GAL10 upstream activator sequence (UAS)
(herein after, GAL-UAS) which promotes transcription and an alcohol
dehydrogenase transcription terminator (ADH1) sequence derived from
YEp51 to prevent run through transcription. U.S. Pat. No. 4,615,974
discloses the use of 5' region of the yeast phosphoglycerate kinase
gene as a promoter of the transcription of interferon. When using
an expression cassette comprising GAL-UAS, transformed yeast can be
grown in rich media containing glycerol/lactic acid to high density
and then induced by switching the carbon source to galactose.
[0069] Other genes containing elements which may be used in hybrid
promoters include PHO5 and ADH2. The PHO5 gene codes for a
repressible yeast acid phosphatase which is repressed at high
concentrations of inorganic phosphate and is derepressed under
inorganic phosphate starvation. ADH2 gene is derepressed after
glucose is exhausted from the medium or when cells are shifted to a
nonfermentable carbon source. ADH2 gene regulation may have
advantages over other hybrid promoters because no exogenous inducer
is needed for derepression which reduces the cost of induction. In
addition, ADH2 derepression does not require specific host strains
or mutations, so it can be used in a variety of yeast strains with
varied genetic backgrounds. Optimal expression is obtained when
cells reach a high density making this promoter valuable for large
scale production.
[0070] In one embodiment of the present invention, hybrid promoters
were constructed by fusion of the upstream activating sequence
(UAS) from Gal1-10 promoter with the transcription initiation
sequences of the glyceraldehyde dehydrogenase (TDH3) promoter. The
expression levels of heterologous polypeptides can be increased by
strong promoter sequences. When the junction between the
translation initiation sequences of the nucleic acid encoding the
protein of interest is flush with the promoter sequences (i.e.
without extraneous sequences in between the promoter sequences and
the translation initiation sequences, i.e. the ATG start codon),
the expression levels may be improved. Constructs incorporating
such features may be prepared using a primer-directed polymerase
chain reaction by techniques known in the art. In alternative
embodiments of the present invention, expression cassettes of the
present invention are constructed so that the yeast TDH3 promoter
sequence is linked either 3' or 5' to the coding region of the
nucleic acid encoding the protein of interest.
[0071] A native yeast secretion signal sequence may be inserted
into the expression cassette of the present invention at a position
after the promoter sequences and preceding (in the correct coding
frame) the sequences encoding the protein of interest (i.e. the
secretory signal sequences are 3' to the promoter and 5' to the
sequences encoding the protein of interest) such that the secretion
signal sequence is operatively linked to the sequences encoding the
protein of interest. Nonlimiting examples of secretion signal
sequences include several yeast secretory signals (e.g. alpha
mating factor), human secretory signals (e.g. human serum albumin
secretory signal) and the secretory signal of the protein of
interest itself.
[0072] It is possible to get a flush junction between the promoter
sequences and the translation initiation sequences if a convenient
restriction site is available in the 3' end of the promoter
sequences and the 5' end of the nucleic acid. If the nucleic acid
encoding a heterologous protein does not contain an appropriate
cloning site, one can create the flush junction by making internal
primers to span sequences between the 3' end of the promoter
sequence and the 5' end of the nucleic acid (ie. flush with the
translation initiation sequences--the ATG start codon). By using
two complementary internal primers and two outside primers, one can
join any two nucleic acid molecules by polymerase chain reaction
(PCR). The hybrid promoter may be constructed in such a way that
one can replace the GAL-UAS with ADH2-UAS or the UAS of any other
regulatory promoter sequences. The 3' end of TDH3 also can be
replaced by 3' end of enolase promoter or any other strong
promoter. Thus by mixing and matching different promoter elements
one can easily construct several hybrid promoters which may improve
expression yields of heterologous recombinant proteins.
5.3 Yeast Host Cells
[0073] A yeast host for the expression of polypeptides is
advantageous for many reasons, including (1) yeast culture
constitutes a well characterized secretion system, (2) there are
many known leader sequences including host or heterologous signal
sequences for secretion, (3) some secretory proteins have been
produced in yields as great as 90% of the secreted protein, (4)
only low levels of native proteins are secreted in culture medium,
simplifying the purification of a target protein, and (5) correctly
folded proteins are produced and intracellular disulfide bonds are
formed during secretion. While a difference in glycosylation may
occur in yeast as compared to other eukaryotic cells,
hyperglycosylation can be reduced in vitro or in vivo by taking
advantage of the extensively characterized yeast glycosylation
system. For example, employing a yeast host strain containing mnn9
(Ip et al., Biochemistry 31:285 (1992)) may be useful to avoid
hyperglycosylation. In addition, a yeast strain containing two or
more genetic modifications may be used to improve the yield of
recombinant proteins. In one embodiment of the present invention,
the yeast host cell is S. cerevisiae, although other yeast may be
used, such as yeast of the genus Pichia, Kluyveromyces and
Hansenula. A yeast strain containing mutation(s) in its cell wall
can be used to increase the secretory efficiency of the expressed
recombinant protein. In preferred embodiments, the yeast host cell
is S. cerevisiae strain Y112, Y113, Y114, Y115, Y116, Y117, Y118,
Y119, Y120, Y121, Y122, Y123, Y124 or Y125.
[0074] A yeast based expression system also offers several
advantages over other systems including: (1) recombinant proteins
generated from yeast systems are acceptable for use as therapeutics
because S. cerevisiae is recognized as a "generally regarded as
safe" (GRAS) organism, (2) yeast is a eukaryotic organism and
allows for post-transcriptional and post-translational processing
and modification of proteins and is more likely to produce
functional proteins than a prokaryotic system, and (3) yeast have
been used in large scale fermentations for centuries, so the
technology for fermenting yeast is well known and a number of yeast
strains as useful hosts are commercially available. It is also
known that secretory proteins can be produced from yeast. Buckholz,
Curr. Opin. Biotechnol. 4:538 (1993). Using the yeast expression
system, N-terminal processing of both alpha and beta chains of
hemoglobin expressed in yeast has been observed (see, U.S. Pat. No.
5,827,693, incorporated herein by reference).
[0075] Furthermore, the cost of growing yeast is a fraction of the
cost of growing other eukaryotic cell cultures. By simple
manipulations of fermentation conditions, the yeast expression
system can yield high levels of recombinant multi-domain proteins
which can be increased by high density fermentations, complex
media, proper carbon and nitrogen sources, pH, temperature,
aeration (pO2), induction regimen, fed batch v.s. continuous
fermentations, use of minimal salt media, and selection of yeast
strains with supersecretory activity. These criteria can be
manipulated for optimization of MAb yields (see, e.g., FIG. 21).
Using shake flask fermentations >10 mg/L of functional
recombinant multi-domain proteins may be obtainable (Motwani et
al., Proteins Expression and Purification, 8:447, 1996). This yield
can potentially be improved 2-10.times. when 2-10 L fermenters are
used for large scale fermentations applying some of the above
criteria (Motwani, et al., Proteins Expression and Purification,
8:447, 1996). Parameters that can be altered to increase yield
include (1) carbon sources (glucose, glu+gal, raffinose,
glycerol/ethanol), (2) buffering systems (e.g. phosphate, citrate),
(3) media formulations (complex versus defined media), (4) vitamins
(e.g. biotin), (5) trace salts, (6) induction times, (7)
temperature, and (8) yeast strains (e.g. supersecretory strains,
secretory mutant strains, glycosylation pathway mutant strains,
permeable cell wall mutant strains, etc.).
5.4 Antibody Expression
[0076] Using an analogous yeast expression system, high level
expression of functional hemoglobin has been achieved (U.S. Pat.
No. 5,827,693 incorporated herein by reference). Both the alpha and
beta chains of hemoglobin were expressed at equal amounts, and the
recombinant hemoglobin ("Hb") was functional and properly folded.
In addition, the amino terminal end of the Hb was properly
post-translationally modified. Furthermore, a high yield of
hemoglobin was obtained without manipulation of fermentation
conditions. When two separate plasmids, each carrying an expression
cassette comprising a nucleic acid encoding alpha and beta globin,
were transformed in yeast, the levels of Hb production was lower
than when the twin cassette plasmid including both chains was
used.
[0077] Similarly, one may produce MAbs using a yeast expression
system. The vector of the present invention allows for modular
construction of the expression cassettes (i.e. the expression
cassettes are easily exchangeable in the vector). The expression
cassettes of the present invention may comprise a heavy and light
chain of an IgG which may be expressed simultaneously in the yeast
expression system. MAb expression yields may be greater than 5 mg/L
host cell culture and preferably greater than 20 mg/L host cell
culture. In a particularly preferred embodiment of the invention,
the MAb expression yield is greater than 100 mg/L host cell
culture. Due to the modular construction of the expression
cassettes (see, e.g., FIG. 11 and FIG. 15), one can (for example by
using PCR or restriction digestion) create MAbs comprising
heterologous heavy and light chains or humanized or single chain
antibodies with very little manipulation of the vector.
[0078] In one embodiment, a vector of the present invention allows
for the expression of a functional heterologous recombinant MAb in
a yeast host cell in excess of 5 mg/L of yeast cell culture. The
vector may include (a) a first expression cassette comprising a
nucleic acid encoding an immunoglobulin heavy chain and (b) a
second expression cassette comprising a nucleic acid encoding an
immunoglobulin light chain. The immunoglobulin heavy and light
chain may be derived, as a specific example, and not by way of
limitation, from an anti-EGP2 antibody. The functional heterologous
recombinant MAb exhibits antigen binding activity similar to a MAb
produced by other conventional methods, e.g. a MAb produced by an
ascites fluid or a hybridoma cell line.
[0079] The functional heterologous recombinant MAb may be produced
by transforming a yeast cell (e.g. S. cerevisiae) with the vector
including expression cassettes comprising nucleic acids encoding an
immunoglobulin heavy and light chain; growing the transformed yeast
cell to log phase; inducing the expression of the MAb in the yeast
cell as described in section 5.2 above; and isolating/purifying the
MAb from the yeast cell culture by standard techniques (e.g.
purification may be accomplished using a protein A-sepharose
column).
[0080] Purification of the recombinant proteins of the present
invention may be achieved by the following scheme: (1) harvesting
transformed cells from large scale fermentation and separating the
cells from the supernatant (e.g. by centrifugation), (2)
concentrating the supernatant from the large scale fermentation up
to 10 fold using a suitable molecular weight cut-off membrane
filter (e.g. 30 kDa filter) by standard techniques (e.g.
ultrafiltration), (3) subjecting the concentrated supernatant to
conventional column chromatography (e.g. cation exchange, anion
exchange, affinity chromatography, etc.), (4) purifying further by
conventional column chromatography where necessary (e.g.
hydroxyapatite (HA), DEAE, gel filtration, etc.).
[0081] FIG. 22 shows a general purification scheme for recombinant
proteins, and particularly MAbs, produced by the present invention.
The yeast are grown in a fermenter using fed batch protocols
(Hensing et al., Antoine Van Leeuwenhoek 67:261 (1995)). The
supernatant from this fermentation is then subjected to buffer
exchange as shown. The sample is then circulated over a tangential
flow filter (TFF) with a 30 kDa cut-off to remove lower molecular
weight impurities. The sample is then purified by column
chromatography using ion exchange resins (IEX, anion and cation
exchange) followed by hydroxyapatite (HA) binding and elution for
final purification.
[0082] The modular expression cassettes are easily interchanged
allowing for mixing and matching of different proteins including
mixing of domains from different species. Using this system one can
readily express MAbs to any specific antigens within a few weeks
(as compared to months by conventional methods) and with little
manipulation of the vector. This system can be used for the
production of MAbs of commercial importance (e.g. MAbs against
viruses and other agents of infectious disease, receptor molecules,
tumor associated antigens or cytochrome p450 isozymes).
[0083] The nucleic acids encoding IgG heavy and light chains,
single chain antibodies and chimeric antibodies can be derived from
naturally occurring or hybridoma (monoclonal) IgG-producing cells
with the desired specificity or selected using a phage display
library. The nucleic acids can also be obtained from a genomic RNA
or mRNA preparation using reverse transcriptase or any other
techniques known in the art.
[0084] In order to make antibodies for screening within a short
time, one can clone into the vector two expression cassettes
comprising either a heavy chain or a light chain by blunt end
ligation using techniques known in the art to obtain several
antibodies within few days. Such antibodies will not necessarily be
expressed to high levels. However, once a desired antibody is
isolated, yields may be improved by the techniques mentioned above
in sections 5.1 and 5.3. Nucleic acids encoding different heavy and
light chains may be easily interchanged by simple restriction
enzyme digestions and ligations. Prototype MAbs to several antigens
may be produced within a short time for testing.
[0085] It is also contemplated that a chimeric IgG molecule can be
produced using this technology. These molecules can be used for
immunoconjugation, immunopurification, immunoassays, cytochemical
labelling, in therapeutics as conjugated to anticancer drugs or in
diagnosis. The chimeric IgG molecules may be therapeutic
themselves. As used herein, a chimeric IgG molecule refers to any
IgG molecule comprising a variable region from one known antibody
and a constant region from another known antibody which may or may
not originate from the same species (e.g. a mouse variable region
and a human constant region). A chimeric protein, as used herein,
generally refers to a molecule comprising multiple protein domains
originating from differing naturally occurring proteins, including
chimeric antibodies and immunotoxins (e.g. a fusion protein of an
antibody chain and a toxin, e.g., ScFv-SA and ScFv-GO of Example 5
and 6 below respectively, or a fusion protein of ScFv-SA and
horseradish peroxidase).
5.5. Immunotoxin Expression
[0086] Production and therapeutic usage of glucose oxidase (GO) in
the form of a single-chain, recombinant immunotoxin has not been
demonstrated to date. GO conjugates have been described which are
produced by isolating and purifying GO and another molecule (e.g. a
peptide or monoclonal antibody) and subsequently conjugating them
together using coupling techniques (see, e.g., Casentini-Borocz and
Bringman, Antimicrobial Agents and Chemotherapy 34:875-880 (1990);
Chouchane et al., Immunology Letters 25:359-366 (1990)). While GO
is toxic to mammalian cells, it has been demonstrated that
functional GO can be efficiently expressed in the yeast S.
cerevisiae (Whittington et al., Curr. Genet. 18:531 (1990);
Frederick et al., J. Biol. Chem. 265:3793 (1990)) with a very high
cytotoxic potential per unit of activity. This enzyme is an
excellent candidate for use in the development of immunotoxins.
Recombinant immunotoxin bearing the enzymatic activity of GO may
induce specific and direct cytotoxicity toward carcinoma cells that
express the target antigen (e.g. EGP-2), and may sensitize
carcinoma cells to chemotherapeutic agents and to radiation. The
activity of GO may also inhibit tumor growth by depleting the
localized levels of glucose available for glycolytic metabolism
through bystander glucose deprivation and peroxide toxicity in
adjacent cells.
[0087] In addition to GO, other nonlimiting examples of suitable
oxidase toxins include amino acid oxidase and xanthine oxidase.
Xanthine oxidase may be particularly useful when incorporated into
the immunotoxins of the present invention since its enzymatic
activity produces both hydrogen peroxide and the highly toxic anion
radical, superoxide which may make a xanthine oxide immunotoxin
very efficient. In fact, xanthine oxidase conjugated to
polyethylene glycol has been shown to significantly reduce tumor
growth in mice following administration of the enzyme substrate,
hypoxanthine (Sawa et al., Cancer Res. 60:666 (2000). Therefore,
immunotoxins of the present invention comprising amino acid oxidase
or xanthine oxidase and antibody domains (e.g. ScFv, Fab', etc)
which may specifically bind to tumor cells (e.g. carcinoma cells)
are useful to induce tumor cell destruction and cytotoxicity of
surrounding tumor cells.
[0088] Peroxidases are also useful in the development of
immunotoxins. Peroxidases have been shown to have cytotoxic
activity when administered to tumor cells, either alone or together
with GO, Nonlimiting examples of peroxidases include horseradish
peroxidase, eosinophil peroxidase, myeloperoxidase and
lactoperoxidase.
[0089] A single-chain MAb (ScFv) directed against the human
tumor-associated antigen, epithelial glycoprotein (EGP-2) has been
produced (Brietz et al., Nucl. Med. 33:1099 (1992); Weiden et al.,
J. Nucl. Med. 34:2111 (1993)). EGP-2 expression is associated with
small-cell lung cancer, lung adenocarcinomas, renal carcinomas,
colon carcinomas, and breast carcinomas. De Leij et al., Int. J.
Cancer Supl. 8:60 (1994). Therefore, ScFv may be used to target
these cancer cells.
[0090] A nucleic acid encoding ScFv may be fused to the coding
region of a toxin gene (e.g. an oxidase or peroxidase) from
Aspergillus niger. In a preferred embodiment, a nucleic acid
encoding ScFv may be fused to the coding region of GO. The nucleic
acid encoding the fusion ScFv-GO may be cloned into an expression
cassette of the present invention (see, e.g., FIG. 15) and into the
vector of the present invention. A plurality of expression
cassettes comprising the nucleic acid encoding the fusion protein
may be cloned into the vector to improve protein yields. The
modular construction of the expression cassette can facilitate the
efficient construction of immunotoxins containing ScFv domains
specific for other cancer antigens and/or alternative toxin (e.g.
horseradish peroxidase (HRP)) molecules and thus generate series of
new immunotoxins in less time than required by current methods.
[0091] The present invention is useful for the production of
functional immunotoxins. As referred to herein, an immunotoxin is a
multi-domain, chimeric protein containing an immunological domain
(e.g. an antibody, or fragment thereof) and a toxin domain. In one
embodiment, ScFv-GO immunotoxin fusion protein is produced using
the yeast expression system of the present invention. In another
embodiment, ScFv-HRP immunotoxin fusion protein is produced using
the yeast expression system of the present invention. The
ScFv-oxidase and ScFv-peroxidase immunotoxins are useful as they
may specifically recognize cancer cells bearing the EGP-2 antigen
and demonstrate toxicity therein thus specifically eliminating
these cells. The oxidase coding sequences or peroxidase coding
sequences may be cloned in the 5' (N-terminal protein position) or
3' (C-terminal protein position) orientation to achieve optimal
functionality of the fusion protein. In a further embodiment, an
immunotoxin comprising one chain (either heavy or light) of ScFv
fused to HRP and the other chain (either heavy or light) of ScFv
fused to GO may be constructed. Such an immunotoxin can form a
multichain protein wherein the heavy and light chain of ScFv are
bound connecting the attached HRP and GO in a single multichain
molecule. Such a molecule may be administered to tumor cells for
the treatment of cancer. In addition, a single chain ScFv-GO
immunotoxin and a single chain ScFv-HRP immunotoxin may be
administered alone or_in combination to tumor cells for the
treatment of cancer.
[0092] Monoclonal antibody-based compounds (MAbs) represent the
single largest category of compounds in clinical drug development.
Large amounts of MAb based immunotoxins are required for
therapeutic use for the treatment of cancer and other diseases. The
novel functional immunotoxin fusion proteins of the present
invention offer several distinct advantages: (1) GO has
successfully been used to induce toxicity in mammalian cells in
native and modified forms, but to date, a completely recombinant
immunotoxin incorporating this toxic enzyme has not been generated
for use in mammalian cells and (2) they are produced using modular
expression cassettes of the present invention which allow for the
rapid cloning and production of secreted recombinant immunotoxins
incorporating various configurations of single-chain antibodies and
toxin genes.
[0093] In one embodiment, the vector of the present invention
allows for the expression of a functional heterologous recombinant
immunotoxin in yeast in excess of 5 mg/L of yeast cell culture. The
vector includes an expression cassette comprising a nucleic acid
encoding a fusion protein of an immunological molecule (e.g.
anti-EGP-2 antibody ScFv) and a toxin (e.g. an oxidase toxin, or a
peroxidase toxin) (see FIG. 11). The vector may also include a
plurality of expression cassettes, each comprising a nucleic acid
encoding a fusion protein of an immunological molecule and a toxin.
The vector which allows for the expression of the immunotoxin may
then be transformed into yeast. The transformed yeast may be grown
to log phase and the expression of the immunotoxin may be induced
as described in section 5.2 above. The immunotoxin may then be
isolated from the yeast cells and the media by standard techniques
known in the art. The immunotoxin thus produced is functional, i.e.
is able to bind antigen and retains toxin activity. In addition, a
vector comprising a first and second expression cassette may be
constructed wherein the first expression cassette comprises one
chain of an immunological molecule (e.g. heavy chain of ScFv) and a
toxin (e.g. glucose oxidase) and the second expression cassette
comprises another chain of an immunological molecule (e.g. light
chain of ScFv) and a different toxin (e.g. horseradish peroxidase)
wherein the two chains of the immunological molecule are capable of
binding to one another to form a multichain molecule comprising
heavy and light chain each fused to two different toxins. Such a
multichain molecule may be produced by the yeast expression system
of the present invention. Two vectors may also be constructed, one
vector comprising an expression cassette encoding a first
immunotoxin (e.g. heavy chain ScFv/GO) and a second vector
comprising an expression cassette encoding a second immunotoxin
(e.g. light chain ScFv/HRP). A yeast host cell may be transduced
with both vectors to produce the multichain molecule comprising
heavy and light chain and two toxins.
[0094] Expression yields of the immunotoxins of the present
invention may be improved using yeast strains overexpressing
catalase (for the detoxification of H.sub.2O.sub.2) which may
better tolerate a peroxide-producing immunotoxin of the present
invention. Such strains may be readily isolated by simple selection
techniques applied to yeast cells expressing the immunotoxin which
grow to higher density. For example, yeast cells that grow well
despite the presence of hydrogen peroxide in the growth medium may
be selected by simple selection techniques. Also, alternative
promoter/fermentation schemes may be used (ethanol fermentation) to
bypass glucose exposure and utilization to improve upon the yield
of recombinant immunotoxin fusion proteins. The ScFv-toxin fusion
protein expression yields may be greater than 1 mg/L host cell
culture and preferably greater than 5 mg/L host cell culture. In a
particularly preferred embodiment, the ScFv-toxin fusion protein
expression yields may be greater than 10 mg/L host cell culture and
preferably greater than 20 mg/L host cell culture. In addition,
fermentation using carbon sources other than glucose (e.g.
glycerol/ethanol, raffinose, etc) to avoid generation of hydrogen
peroxide during the course of fermentation may be used since
glucose oxidase generates hydrogen peroxide through the enzymatic
conversion of glucose.
[0095] In addition, alternative toxin and "suicide" genes (encoding
enzymes which directly indue toxicity or activate toxic forms of
prodrugs) may be used in place of oxidases and peroxidases. It may
be advantageous to enhance the immunotoxin Fv region stability and
specificity using mutagenesis to introduce disulfide-linkages
between Fv domains. Reiter and Pastan, Clin. Cancer Res. 2:245
(1996). Optimization of immunotoxin expression may be achieved via
further strain selection and manipulation of fermentation
conditions. Furthermore, it has been shown that overexpression of
either the molecular chaparone BiP or protein disulfide isomerase
(PDI) (Shusta et al., Nat. Biotech. 16:773 (1998)) can increase
secretion of single-chain antibodies 2-8 fold. Therefore, the
immunotoxin fusion proteins may be co-expressed with immunoglobin
chaperones (BiP, PDI) for the optimization of correct folding,
transport and secretion of the expressed immunotoxin fusion
protein.
[0096] The invention is further illustrated by reference to the
following examples.
6. EXAMPLES
Example 1
Hybrid Promoter Production
[0097] A. GAL-UAS:
[0098] Broach et al. (Manipulation of Gene Expression, ed. Inouye,
1983) disclose a plasmid containing a GAL10 upstream activator
sequence (UAS) (herein after, GAL-UAS). GAL-UAS was synthesized by
using YEp5 1 plasmid DNA (Bitter and Egan, Gene 69:193 (1988) and
nucleic acid primers containing appropriate cloning sites (shown in
lower case):
1 5' primer: TtgagctcCCCAGAAATAAGGC- (SEQ ID NO:1) 3' primer:
AGAAGGTTTTTTTAGcccgggCA- (SEQ ID NO:2)
[0099] B. ADH-2-UAS:
[0100] ADH2 gene is derepressed after glucose is exhausted from the
medium or when cells are shifted to a nonfermentable carbon source.
ADH2 gene regulation may have advantages over other hybrid
promoters because no exogenous inducer is needed for derepression
which reduces the cost of induction. In addition, ADH2 derepression
does not require specific host strains or mutations, so it can be
used in a variety of yeast strains with varied genetic backgrounds.
Optimal expression is obtained when cells reach a high density
making this promoter valuable for large scale production. ADH2-UAS
was synthesized by PCR using YEp51 plasmid DNA and the following
primers:
2 5' primer: (SEQ ID NO:3) 3' CGA TCG gagctc ATT AAC GCC TTT CGC
TCA TAA-5' 3' primer: (SEQ ID NO:4) 3'- G TGT CCT CTC GTA TCT TTA
CCC CAA aga tct GCG CGA-5'
[0101] C. 3' end of TDH3 Promoter:
[0102] The TDH3-3' promoter fragment, which is a transcriptional
initiation region from the yeast glyceraldehyde-3-phosphate
dehydrogenase gene was synthesized by PCR using appropriate primers
and template from plasmid gp491. The primers used were:
3 5' primer: 5' AtcccgggAAGGTTGAAACCAGTTCCCTG-3' (SEQ ID NO: 5) 3'
primer: 3'-GTGTGTATTTATTTGTTTTACCacgtgcG- C-3' (SEQ ID NO: 6)
[0103] D. 3' end of the Enolase Promoter:
[0104] The enolase promoter RNA is most abundant RNA in vegetative
yeast cells. The downstream element including the TATA box (ENO-3')
of the enolase promoter was used for construction of a hybrid
promoter, ADH2-UAS-ENO-3' (AE). The 3' end of the enolase promoter
was synthesized by using plasmid DNA from pENO8-1 (Cohen, R.,
Holland, J., Yokoi, T., and Holland, M., Mol. Cell Biol. 6, 2287,
(1986)).
4 5' primer: (SEQ ID NO: 7) 5'-CCG GCC GTC tctaga TCT GGC TTT GAT
CTT ACT ATC ATT TGG-3' 3' primer: (SEQ ID NO: 8) 3'-G TAT TGT GGT
TCG TTG ATT ATG ATA TTG ATA GTT ATT ATT AC gtgcac G GCG-5'.
[0105] The 340 bp fragment containing 3' end of enolase promoter
was synthesized by PCR.
[0106] E. Cloning the Hybrid Promoter into a Vector
[0107] All the fragments containing ADH2-UAS, GAL-1-10 UAS, TDH3-3'
and Enol-3' were cloned into pUC19 vector. A hybrid promoter can be
constructed by simple cloning of one of the UAS fragments with the
strong promoter fragments. e.g. GD contains GAL-10-UAS (G) and
TDH3-3'(D).
Example 2
Terminator Production
[0108] A. ADH-Terminator:
[0109] ADH1-transcription terminator (ADH-t) sequences were
isolated from plasmid AAH5 (Ammerer, G., 1983, Methods in
Enzymology, 101, pp. 192-201). Plasmid AAH5 was digested with BamH1
and HindIII. The resulting 450 bp fragment was isolated by gel
electrophoresis and further digested with SphI. The 320 bp fragment
of ADH-t (HindIII/SphI) was used for cloning into pUC19 vector and
the plasmid was labeled pUC19-ADHt. The restriction sites on the
terminator can be altered by PCR.
[0110] DNA ligation and transformation of E. coli was carried out
using standard procedures (Laboratory Cloning,: Sambrrok, J.,
Fritsch, E. F., and Maniatis, T. eds., Cold Spring Harbor
Laboratory Press, 1989, Second Edition). Transformed cells were
plated on LB-media with 100 mg/L ampicillin. Plates were incubated
at 37.degree. C. overnight.
Example 3
Plasmids
[0111] Plasmid pUC19-GH contains a hybrid promoter which was
constructed by the fusion of the upstream activating sequence (UAS)
of GAL1-10 promoter (G) with the downstream promoter elements of
the TDH3 promoter. The TDH3-3' promoter segment includes the
transcriptional initiation site, the "TATA" sequences, capping
sequence and RNA polymerase binding site. The hybrid promoter
fragment was cloned into pUC19 as SacI/SphI fragment and the
resulting plasmid was labeled pUC19-H.
Example 4
Antibody Production
[0112] A. Construction of Universal Plasmids Containing a GH Hybrid
Promoter, Yeast Alpha Factor Secretory Signal and ADH or
GAL-Terminator:
[0113] These plasmids were constructed as a modular unit, and each
fragment was cloned into the appropriate site into the Bluescript
vector SK.sup.+ (Invitrogen, San Diego, Calif.) (FIG. 1).
[0114] Plasmid pUC19-GH (U.S. Pat. No. 5,827,693) contains a hybrid
promoter which was constructed by the fusion of the upstream
activating sequence (UAS) of GAL1-10 promoter (G) with the
downstream promoter elements of the TDH3 promoter (referred to as
TDH3-3' and labeled as H in plasmid pUC19-GH). The TDH3-3' promoter
segment includes the transcriptional initiation site, the "TATA"
sequences, a capping sequence and a RNA polymerase binding site
(U.S. Pat. No. 5,827,693). Using the DNA from pUC19-GH as a
template and primers containing required restriction sites, a GH
promoter fragment was synthesized by PCR {KpnI (5'end) and SalI (3'
end)}.
[0115] Genomic DNA was isolated from a strain of S. cerevisiae and
a yeast signal, the .infin.-mating factor secretion signal, was
synthesized by PCR using appropriate primers. The resulting
fragment was labeled Sec1 and contains SalI (5' end) and EcoRI (3'
end) restriction sites.
[0116] The transcription termination sequences of ADH and GAL10
were cloned into the pUC19 plasmid, which resulted in plasmids
pUC-19-ADH-t and pUC-19-GAL-t. PCR was used to create appropriate
cloning sites compatible with the nucleic acids encoding antibody
heavy or light chains.
[0117] Bluescript SK.sup.+ vector (Stratagene, La Jolla, Calif.)
was used for cloning the fragments of expression unit synthesized
by PCR. The hybrid promoter GH was synthesized by PCR to contain
KpnI (5' end) and Sal I (3' end) sites. A yeast secretory signal
containing SalI (5' end) and EcoRI (3' end) was synthesized by PCR.
The SK.sup.+ DNA was cut with KpnI and EcoRI. Three way ligation
was set between the vector, GH promoter cut with KpnI/SalI, and
yeast signal sequence cut with SalI/EcoRI. The E. coli
transformants were identified by digestion with PvuII. This plasmid
is referred to as pSK.sup.+-GH-Sec1 and was cut with NotI/SacI. The
ADH terminator synthesized by PCR was cut with NotI/SacI and
ligated to the SK.sup.+GH-Sec1 cut plasmid. The positive clones
were identified by digestion with PvuII. Plasmid containing the GH
promoter, yeast .infin. factor signal and ADH-terminator was
labeled SK.sup.+-GH-Sec1-ADH-t. Similarly, a GAL terminator
synthesized by PCR was cloned as a NotI/SacI fragment into
SK.sup.+-GH-Sec1 and this plasmid is SK.sup.+-GH-Sec1-GAL-t (FIG.
1).
[0118] Sequences upstream of ATG of cDNA are critical for high
level expression of proteins (U.S. Pat. No. 5,827,693, incorporated
herein by reference). Since there is a convenient site at the 3'
end of the secretory signal, XhoI, we used this site for cloning.
This strategy can only be used for the cDNAs that do not contain an
internal XhoI site.
[0119] Nucleic acids encoding heavy (Apo-H)and light chain (Apo-L)
of NR-LU-10, hereafter referred to as NRX1 (NeoRX, Seattle, Wash.)
were cloned in universal plasmids (pBluescript SK+, Stratagene, La
Jolla, Calif.).
[0120] B. Cloning of H and L into Universal Plasmids pApo-U1 and
pApo-U2
[0121] Appropriate modification of NRX14 cDNAs were carried out
using PCR and cloned into universal vectors, p-Apo-U1 and p-Apo-U2
(ApoLife, Inc., Detroit, Mich.). The resulting clones were
identified by digestion with PvuII. Plasmids are labeled
SK.sup.+GH-Sec-Apo-L-Gt and SK.sup.+-GH-Sec-Apo-H-At.
[0122] C. Yeast Shuttle Vector Containing LEU2 d Marker--Plasmid
pPM40:
[0123] The multi-copy E. coli/S. cerevisiae shuttle vector pPM40,
used in the present invention, contains three cloning sites, BamHI,
PvuII and HpaI which allows cloning of two cDNAs in a single
plasmid. The vector also contains LEU2-d, which represents a
truncated LEU2 promoter that is expressed at very low levels. When
introduced into a leu2 auxotrophic strain, selection for leucine
prototropy results in amplification of the plasmid to a high copy
number (Erhart and Hollenberg, Bacteriol. 156:483 (1983)). Under
appropriate fermentation conditions, one can select for
transformants containing high copy plasmid (200 copies/cell) using
this vector.
[0124] D. Cloning of Expression Cassettes for L and H Chains into
Yeast Vector, pPM40
[0125] The cassettes GH-Sec-Apo-H-At and GH-Sec-Apo-L-Gt were
excised and cloned separately into pPM40 at BamH1 by blunt end
ligation (FIG. 2). The clones were identified by EcoRI digestion.
The DNA from each plasmid was transformed into yeast to check for
the expression levels of single chains. An expression vector was
obtained by cloning both cassettes on pPM40 vector (FIG. 2). There
are three cloning sites in pPM40. The expression levels may vary
depending upon the site of cloning.
[0126] Transformation of yeast strains by electroporation with DNA
from plasmids was carried out by a modification of a standard
electroporation protocol. The cells were grown in YEPD (Sherman,
Methods in Yeast Genetics (1986)) containing 2% glucose at
30.degree. C. in a shaker for 4-6 hrs to create a stock. 100 ml of
the same media was innoculated with the stock and grown to an OD600
of 1.3-1.5. Cells were harvested by centrifugation at 5,000 rpm for
5 min, washed 3' with distilled water and washed 2.times. with 1M
sterile sorbitol at 4.degree. C. The cells were resuspended in 100
ul of 1M sorbitol, and 40 ul of cell suspension was added to
Eppendorf tubes. Approximately 1000 ng of plasmid DNA were added to
the cells. The cells and plasmid DNA was incubated on ice for 5 min
and transferred to 0.2 cm electroporation cuvettes. A Bio-Rad Gene
Pulser (Richmond, Calif.) was used according to manufacturer's
directions to perform the electroperation. The transformants were
selected on YNB media (Sherman, Methods in Yeast Genetics (1986))
lacking uracil. With every experiment, a control was included of
the yeast strain with no DNA. No colonies were seen on control
plates.
[0127] Yeast strains were cotransformed with two plasmids, one
containing NRX1 (heavy chain; ApoH) and the other containing NRX1
(light chain; ApoL) to see if simultaneous expression of both
chains is possible from separate plasmids.
[0128] E. Standard Fermentation Conditions:
[0129] A preculture was grown in 100 ml YNB media (ura-,
leu.sup.+)+2% glucose for 24 hrs at 30.degree. C. This was followed
by inoculation in 200 ml fermentation media, (ura-, leu-)+2%
glucose, at 0.5 OD. The cultures were grown and harvested and
samples taken at various time points and induced with 2% galactose.
The cells were separated from the culture media by centrifugation.
The samples were then analyzed immediately as described below.
[0130] F. Analysis of Recombinant Proteins by Western Blot
Analysis:
[0131] The yeast cells and media collected at different time points
during fermentation were stored at -20.degree. C. The culture
supernatants were concentrated with Centricon 30 filters by
ultrafiltration and used for SDS-PAGE analysis. Cell pellets (10
OD) were disrupted by vortexing with autoclaved glass beads (40
mesh, BDH) in 50 mM Tris-HCL, pH 7.6, 1 mM EDTA. The mixture was
centrifuged and the supernate was stored at 4.degree. C. For
electrophoretic separation, the samples were denatured under
reducing conditions in the buffer: 1.5% w/v SDS, 2.5% v/v
bromophenol blue, 5% v/v glycerol, 2.5% .beta.-mercaptoethanol.
SDS-PAGE analysis and transfer of proteins to nitrocellulose
membrane was carried out using a Novex apparatus (Novex, San Diego,
Calif.) using precast 14% and 4-20% gradient Tris-glycine gels
according to the manufacturer's directions. Western blot analysis
was carried out using peroxidase labeled goat anti human kappa
light chain specific antibody and IgG gamma heavy chain specific
antibody (Southern Biotechnology, Inc., Burlingame, Calif.) and
detection was carried out using TMB reagent (Vector Laboratories,
Burlingame, Calif.). If protease associated degradation of proteins
was observed, 0.5 mM PMSF was included in buffers. Molecular weight
standards were obtained from Novex (San Diego, Calif.).
[0132] G. Results of Western Blot Analysis:
[0133] Production and subsequent detection of proteins in shake
flask fermentations depends upon several factors such as: yeast
strain selection, which is empirical, the growth conditions of
transformants, induction regimen, susceptibility of proteins to
proteases, and the specificity of antibodies used for protein
detection on western blots. Certain antibodies seemed to be more
susceptible to degradation and thus sample collection time was
optimized for each antibody.
[0134] The fermentation was carried out in 200 ml media using
standard conditions. The yeast strains used in the experiments
could only reach 4-5 OD in most experiments under standard
conditions. The expression of ApoH and ApoL chains of NRX-1 was
obtained by galactose induction of yeast transformants. The
presence of ApoH and ApoL chains in cell extracts and supernatant
was demonstrated by western blot analysis. When single plasmids
were used for expression, ApoL chain migrated with its expected
molecular weight (FIG. 3, lane 6) but the ApoH chain (FIG. 3, lane
9) was found to be less stable and only seen in some of the yeast
strains. When both plasmids were used for cotransformation in a
single yeast strain, this contransformant produced equivalent
amounts of both ApoH and ApoL chains (FIG. 3, lane 11). NRX-1
antibody provided by NeoRx (Seattle, Wash.) was used as a positive
control (FIG. 3, lane 1). No detectable bands were seen in the
negative control (FIG. 3, lane 7), yeast strain transformed with
pPM40 (Motwani et al., Protein Expression and Purification 8:477
(1996)). In some yeast strains (Y112 and Y113), higher expression
of antibody chains were seen in cell lysates (FIG. 4, lane 9)
compared to the one in supernatant (see Example 5 and FIG. 4, lane
4).
[0135] High levels of secretion of two bands was obtained from the
Y113 cotransformed with two plasmids for NRX-1 antibody (FIG. 5).
This supernatant was concentrated to 20.times. and unreduced sample
was run on gel with positive antibody control. Protein bands were
found which migrate at the same levels as unreduced NRX-1 antibody
molecule (FIG. 5, lane 1=purified NRX-1, lane 4=yeast expressed
NRX-1). A further reduction of this sample showed two bands which
migrated to expected mol. wt of ApoH and ApoL chains (FIG. 5, lane
4). High levels of antibody chains had to be produced to detect
fully assembled molecule migrating at 150 kd on reducing gels (FIG.
5, lane 4).
Example 5
Expression of Fusion Protein, ScFv-SA+Fab-SA
[0136] A. Construction of the ScFv-SA Expression Plasmid
[0137] The nucleic acid encoding fusion protein consisting of a
single chain Fv of NRX1 fused to the amino terminal end of
streptavidin (SA), designated ScFv-SA was obtained from NeoRx
(Seattle, Wash.). The nucleic acid encoding a single-chain
Fv/streptavidin fusion protein was cloned into the expression
cassette of the present invention. The expression cassette was
subcloned into a high copy yeast vector, pPM40 (FIG. 6), and
transformed into various S. cerevisiae strains.
[0138] B. ScFv-SA Protein Expression
[0139] The transformed yeast were grown and samples were analyzed
by SDS-PAGE electrophoresis (FIG. 7A) and native gel
electrophoresis (FIG. 7B). Samples were collected at 24 hours
(FIGS. 7A and B lanes 2 and 3), 36 hours (FIGS. 7A and B, lanes 5
and 6) and 72 hours (FIGS. 7A and B, lanes 8 and 9). In the gels,
T=tetramer and M=monomer. Western blots of reducing gel-resolved
proteins from shake flask fermentations were carried out by
standard techniques and detected anti-streptavidin antibody
reactive bands migrating in the molecular weight range of 64 kDa,
corresponding to the correct dimeric form (D) of ScFv-SA (FIG. 7A,
lanes 2, 3, 5, 6, 8 and 9). Immunoblots of the same samples run on
native gels revealed a band at 172 kDa (FIG. 7B, lanes 2, 3, 5, 6,
8 and 9). The size of this detected band corresponds to the
predicted tetrameric form (T) of the ScFv-SA protein, as the
native, functional form of streptavidin is a tetramer. Control
lanes 4, 7 and 10 were supernatants from yeast transformed with
plasmid alone. More than 90% of the recombinant protein was
secreted into the medium. The fusion protein was not detected in
the cellular lysates. The ScFv-SA produced by the Yeast expression
system of the present invention was compared to purified ScFv-SA
produced in bacteria by SDS-PAGE gel electrophoresis and western
blot analysis using antistreptavidin antibody was performed using
standard techniques. FIG. 4, lanes 2 and 3 show bacterial produced
ScFv-SA and lanes 3-7 show the ScFv-SA produced by the yeast
expression system of the present invention.
[0140] The immunoblot in FIG. 8 compares the ScFv-SA produced in
crude form from yeast (lanes 6 and 7, 2 liter fermentation) with
the purified form of the same protein that was expressed in E. coli
(lanes 2 and 3). Antistreptavidin-reactive bands were not detected
in sample from the control plasmid fermentation (lane 8). Putative
tetrameric (T) and dimeric (D) forms of ScFv-SA were detected as
shown by arrowheads. The ScFv-SA produced in the yeast expression
system of the present invention was qualitatively similar to the
corresponding protein produced in E. coli. However, the ScFv-SA
produced in E. coli contained less dimeric form due to the removal
of the dimeric form during purification.
[0141] C. Functional Antigen Binding Assay for ScFv-SA:
[0142] Antigen binding specificity was shown using crude secreted
protein from yeast fermentation to detect purified recombinant
EGP-2 protein in immunoblots, as ScFv is an anti-EGP-2 antibody.
Various concentrations of EGP-2 were analyzed by SDS-PAGE gel
electrophoresis, transferred to nitrocellulose, and subject to
western blot analysis using ScFv-SA using standard techniques. FIG.
9 shows the ability of ScFv-SA produced by the present invention to
detect EGP-2 (panel 1), (panel 2=purified E. coli produced ScFv-SA
as positive control, panel 3=negative control supernatant used in
western blot). The results indicate that the ScFv-SA fusion protein
expressed in S. cerevisiae using the yeast expression system of the
present invention maintained antigen binding activity.
[0143] D. Quantitation of Expression of ScFv-SA:
[0144] To quantitate the relative amount of ScFv-SA produced by the
yeast expression system of the present invention, ELISA plates
(VWR, Plainfield, N.J.) were coated with 1 .mu.g/ml biotinylated
ScFv-SA. Serial dilutions of either the E. coli produced ScFv-SA
(FIG. 10, rows 1-6) or the ScFv-SA produced by the yeast expression
system of the present invention (supernatant of yeast transformed
with a vector expressing ScFv-SA, FIG. 10, rows 7-11) were
incubated with the biotinylated protein. Biotin-bound streptavidin
of the ScFv-SA was detected using a horseradish
peroxidase-conjugated antibody to streptavidin and ABTS
colorimetric substrate by the protocol provided by the supplier
(Sigma, St. Louis, Mo.). Quantitation was determined using an
automatic plate reader/spectrophotometer (Dynatech, Alexandria,
Va.) at 410 nm with a 490 nm reference. FIG. 10 shows the results
of the ELISA quantitation assay. It was calculated that 10-20 mg/L
of secreted biotin reactive fusion protein was produced using the
yeast expression system of the present invention. Since only the
supernatant was analyzed, it is likely that the yeast host cells
may also contain expressed ScFv-SA. Therefore, the actual fusion
protein yields may be much higher.
Example 6
Production of the Two-Chain Fab-SA Fusion Protein
[0145] A. Construction of the Fab-SA Expression Plasmid
[0146] The nucleic acid sequences encoding the two chains the
Fab-SA fusion protein (immunoglobin light chain (V.sub.L) and the
heavy chain (V.sub.H) fused to a streptavidin gene) were obtained
from the plasmid A173B-3 (NeoRx, Seattle, Wash.). The nucleic acid
sequences were cloned separately into yeast expression cassettes in
universal plasmids (FIG. 1) wherein a first expression cassette
comprises a nucleic acid encoding the heavy chain and a second
expression cassette comprises a nucleic acid encoding the light
chain. Both expression cassettes were subsequently cloned into a
single high copy yeast vector, pPM40 (FIG. 2 and FIG. 11), and
transformed into various S. cerevisiae strains.
[0147] B. Fab-SA Protein Expression
[0148] S. cerevisiae was transformed with the Fab-SA expression
plasmid of FIG. 11 and were grown and induced as described above in
Example 4. Fermentation supernatant samples (48 hours after
induction) were analyzed by SDS-PAGE electrophoresis (FIG. 12,
lanes 2-4). Supernatant from control plasmid transformants was
included as a negative control (lane 5). In the figures,
Fd=V.sub.H/streptavidin fusion protein, and V.sub.L=light chain
variable region peptide. Western blots of reducing gel-resolved
proteins from shake flask fermentations were carried out by
standard techniques and proteins were detected using either
anti-streptavidin/horseradish peroxidase (HRP) or anti-light chain
IgG/HRP conjugated antibodies, which reacted with bands migrating
in the molecular weight range of 64 kDa and 32 kDa, corresponding
to the correct Fd chain and V.sub.L chains of Fab-SA, respectively.
More than 90% of the recombinant protein produced by the yeast
expression system of the present invention was secreted into the
medium.
[0149] In addition, the Fab-SA expression plasmid was transformed
into multiple S. cerevisiae strains and tested for optimal
expression level and functionality of the immunoglobin/fusion
chains. FIG. 13 shows immunoblots of fermentation supernatants
(panel A) and the corresponding cellular lysates (panel B) from
seven of these transformant strains that express Fab-SA. These
strains are Y124, Y125, Y112, Y116, Y114, Y113, Y120 (lanes 1-7
respectively). Strain Y116 transformed with vector alone was
included as a negative control (lane 8). Positions of the light
chain variable region (Fv) and heavy chain variable/streptavidin
fusion (Fd) bands are indicated by arrows. Differential expression
levels of the two Fab-SA chains were obtained from the various
yeast strains. All strains tested exhibited expression of the light
(Fv) chain. However, only strains Y112, Y120 (lanes 3 and 7
respectively) and Y116 produced significant levels of both the Fv
and Fd chains. Highest expression levels were obtained from strain
Y120 (lane 7).
[0150] C. Quantitation of Expression of Fab-SA
[0151] Relative quantitation of Fab-SA in various yeast strains was
determined by detection of binding to biotinylated protein in an
ELISA assay (FIG. 14) as described above in Example 5D for FIG. 10.
Serial dilutions of the E. coli produced ScFv-SA (rows 1-6) or
supernatant samples from the yeast strains Y113, Y117, Y118, Y119,
Y120 (rows 8-12 respectively) expressing Fab-SA by the yeast
expression system of the present invention were incubated with
biotinylated protein bound to ELISA plate wells. Colorimetric
detection of biotin-bound recombinant protein was used to estimate
relative yield. It was calculated that at least 10-20 mg/L of
secreted biotin reactive fusion protein was produced by all yeast
strains shown using the yeast expression system of the present
invention in small-scale fermentations. Since only the supernatant
was analyzed by this assay, it is likely that the yeast host cells
may also contain expressed ScFv-SA. Therefore, the actual fusion
protein yields may be higher.
Example 7
Production of Glucose Oxidase Immunotoxin
[0152] A Prototype Antibody Gene:
[0153] The E. coli expression vector, pSPORT-FvSA1 was obtained
from NeoRx (Seattle, Wash.). This vector contains the combined
single-chain variable region sequences of an EGP-2-specific
monoclonal antibody fused to a streptavidin gene (Breitz et al.,
Nucl. Med. 33:1099 (1992)) in a contiguous reading frame, cloned
into the pSPORT plasmid (Life Technologies, Gaithersburg, Md.).
[0154] B. Glucose Oxidase Gene:
[0155] The glucose oxidase gene sequences to be used for
immunotoxin construction have been isolated and characterized.
Whittington et al., Curr. Genet. 18:531 (1990). A genomic clone
from A. niger contains the entire GO coding region, consisting of a
single exon. It has been determined that a 22 amino acid
hydrophobic signal sequence precedes the mature peptide-coding
region.
[0156] C. Construction of the Immunotoxin ScFv-GO Fusion Gene and
Expression Cassette:
[0157] The Fv region of pSPORT-FvSA template was amplified by
polymerase chain reaction (PCR) using oligonucleotide primers which
introduced a 5' Eco RI restriction site, and a 3' Bam HI site into
the PCR product. The glucose oxidase coding region (beginning with
the first codon of the mature peptide) was obtained by PCR using a
plasmid template containing a genomic insert from A. niger and
primers which incorporated a 5' Bam HI site, and a 3' Not I site
(Life Technologies, Rockville, Md.) into the amplified sequences.
The FvSA and GO fragments were cloned by three-way ligation into a
pBluescript plasmid (Stratagene, La Jolla Calif.) containing the
yeast expression cassette described above. Eco RI and Not I
restriction sites located between the secretory and termination
sequences were utilized to insert the immunotoxin components in
frame. The resultant plasmid is(SK+[GO Fusion]). The complete
expression cassette was removed from this plasmid via restriction
digestion with KpnI and SphI, and cloned by blunt-end ligation into
either or both of the unique BamHI and HpaI sites in the pPM40
yeast shuttle vector (FIG. 15). A single expression cassette, or
multiple expression cassettes were cloned into the yeast shuttle
vector. The DNA from this plasmid was transformed into several
yeast strains and fermentation was carried out by standard
techniques known in the art.
[0158] D. Analysis of Recombinant ScFv-GO Fusion Proteins by
Western Blot Analysis:
[0159] The culture supernatants were concentrated with Centricon 30
filters by ultrafiltration and used for SDS-PAGE analysis. Cell
pellets (2 OD) were lysed directly in reducing sample buffer (1.5%
w/v SDS, 2.5% v/v bromophenol blue, 5% v/v glycerol, 2.5%
2-mercaptoethanol) and boiled at 95.degree. C. for 5 minutes prior
to electrophoretic separation. If degradation of proteins was
observed, 0.5 mM PMSF was included in the buffer. Samples were
resolved on tris-glycine gradient gels (4-20% acrylamide).
Molecular weight markers and precast gels were obtained from Novex
(San Diego, Calif.). FIG. 16 shows the results of SDS-PAGE gel
electrophoresis followed by western blot analysis with anti-glucose
oxidase antibody. Lane 1 contains the supernatant of a yeast
transformed with a negative control plasmid (no expression
cassette), Lanes 2 and 3 contain the supernatant of a yeast
transformed with a vector comprising the expression cassette
encoding ScFv-GO (FIG. 15). A band migrating at approximately 140
KD is indicated with an arrow which corresponds to a tetrameric
ScFv-GO fusion protein.
[0160] E. ScFv-GO Fusion Protein Quantitation:
[0161] Total protein from fermentation supernatants and cell
lysates was quantitated using the BCA Protein Assay System (Pierce,
Rockford Ill.). Commercially purified bovine serum albumin was used
to establish concentration standards. Detection and quantitation of
specific peptides was determined using the ECL Chemiluminescent
Western Blotting System (Amersham, Piscataway, N.J.). Detected
bands of samples and standards were recorded by densitometric scan
and relative quantitation determined using the Scion Image program
(Scion, Md.). Plasmids containing either single or multiple
expression cassettes yielded an average of 20-40 mg/L in shake
flask fermentations.
Example 8
Testing of the Immunotoxin for Functionality
[0162] A. Functional (Antigen Binding) Assay of the
GO-Immunotoxin:
[0163] Detection of functional immunotoxin binding to Western blots
of purified EGP-2 antigen was performed as described above in
Example 5 above for the ScFv-SA expressed protein. FIG. 17, panel 2
(ApoLife ScFv-GO) shows that the ScFv-GO produced by the yeast
expression system of the present system was able to bind EGP-2 as
was control ScFv-GO produced in E. coli (panel 1, positive
control). Supernatant from negative control fermentations did not
bind antigen (panel 3, negative control).
[0164] B. ELISA:
[0165] Purified glucose oxidase protein (Sigma, St. Louis, Mo.) was
added at various concentrations to generate a standard curve.
Following 5 washes (phosphate buffered saline [PBS], 0.5% Tween
20), glucose oxidase specific antibody (sheep anti-GO,
#AB1221,Chemicon, Temeula, Calif.) was incubated for 1 hour at room
temperature (RT), followed by washes and a 1 hour incubation with
horseradish peroxidase conjugated anti-sheep antibody (diluted
1:2000, Zymed, San Francisco, Calif.). After washing, the samples
were quantitated by comparison to standard curve data generated by
first order, log x/log y regression analysis.
[0166] C. Detection and Ouantitation of Glucose Oxidase
Activity:
[0167] Two methods of quantitating glucose oxidase activity were
employed. The first method has been described previously
(Wittington et al., Curr. Genet. 18:531 (1990). FIG. 18 shows an
example using this assay. Fermentation samples of transformed yeast
containing an ScFv-GO expression plasmid were harvested at various
time points post-induction and concentrated 20-fold by
ultrafiltration. A 10 i.mu.l aliquot of each sample was assayed for
glucose oxidase activity. Purified glucose oxidase protein at
various concentrations was also assayed in parallel (A3-A5).
Additional controls included reagents alone (A1), PBS (A2) and
pPM40 fermentation sample (vector alone, B5). The presence of
hydrogen peroxide, indicative of glucose oxidase activity, was
detected in B2-B4 fermentation samples expressing ScFv-GO whereas
the control fermentation (pPM40, B5) contained no detectable
hydrogen peroxide. An estimated 1000 units/L was obtained. Varying
the pH of the fermentation may improve this yield of activity since
the activity of the glucose oxidase enzyme is pH dependant.
[0168] The second method employed for detecting glucose oxidase
activity was the Amplex Red Hydrogen Peroxide Assay Kit (Cat. #
A-12212, Molecular Probes, Eugene, Oreg.) as per manufacturers
suggested protocols. This highly sensitive assay detects hydrogen
peroxide (H.sub.2O.sub.2) by its conversion of the Amplex Red
(10-acetyl-3,7-dihydroxyphenoxazine) reagent in the presence of
horseradish peroxidase to produce a highly fluorescent and stable
product, resorufin. Peroxide production is initiated by the
addition of glucose to the reaction mixture containing the
recombinant immunotoxin. The fluorescence generated by the reaction
was detected by using a fluorimeter set for excitation in the range
of 530-560 nm and emission detection at 590 nm. FIG. 18 shows the
ability of control GO (Panel A, rows 3-5, 0.25 Units/ml GO, 0.5
U/ml and 1.0 U/ml respectively) and ScFv-GO produced by the yeast
expression system of the present invention (Panel B, rows 3 and 4,
24 hour and 48 hour post-induction) to produced H.sub.2O.sub.2.
Data from peroxide generation assay is combined with specific
protein levels detection to determine specific activity (units/g
protein) and concentration (units/liter).
[0169] D. Glycosylation of ScFv-GO
[0170] FIG. 19 compares the ScFv-GO from crude fermentation
supernatants with endoglycosidase H treatment (lane 1) and without
endoglycosidase H treatment (lane 3) to detect the presence of
N-linked glycosylation residues added to the recombinant protein,
since it has been reported that N-linked glycosylation is essential
to the function of glucose oxidase. See Whittington et al., Curr.
Genet. 18:531 (1990); Frederick et al., J. Biol. Chem. 265:3793
(1990). 20 .mu.l of crude fermentation supernatant from ScFv-GO
production was treated with 10 units of endoglycosidase H
(Boehringer Mannheim, Indianapolis, Ind.) at 37.degree. C. for 16
hours prior to being resolved by SDS-PAGE electrophoresis.
Endoglycosidase H treatment (lane 2) results in a band that
migrates at a lower molecular weight and which has a more distinct
banding pattern than the untreated sample (lane 1). Therefore,
significant N-linked glycosylation is present on the yeast-secreted
form of ScFv-GO, indicative of correct post-translational
processing in the yeast expression system of the present invention.
By comparison, treatment of ScFv-SA (lanes 3, untreated and lane 4,
endoglycosidase H treated) did not result in a shift in specific
bands, indicating that N-linked glycosylation is not present in
this fusion protein.
[0171] E. Comparison of ScFv-GO Expression in Various Yeast
Strains
[0172] FIG. 20 shows the western blot analysis of ScFv-GO
immunotoxin expressed in the fermentation supernatant of various
transformed S. cerevisiae yeast strains. The yeast strains which
were employed are as follows: Y111, Y124, Y125, Y112, Y116, Y114
and Y120 (lanes 1-7, respectively). Supernatant samples were
harvested at 24 hours post-induction. These data demonstrate that
(1) Y111 did not express the GO-fusion protein, lane 1; (2) strains
Y124 and Y125, lanes 2 and 3, respectively, expressed small amounts
of the fusion protein which may be non-glycosylated; (3) Y112, lane
4, a protease deficient strain, expressed the putative
unglycosylated form of ScFv-GO; (4) Y116 and Y114, lanes 5 and 6,
respectively, expressed the glycosylated form of the ScFv-GO
protein; and (5) Y120, lane 7, expressed the glycosylated form of
the ScFv-GO protein in the highest levels.
Example 9
Comparison of Different Media for the Growth of Yeast Strains
[0173] FIG. 21 shows the relative growth, in terms of optical
density, obtained after fermentation of yeast strain Y113
(designated as RgsApoFGt (ScFv-SA) in the figure) in the various
media compositions as shown. The yeast was transformed with either
the ScFv-SA fusion protein expression vector (see Example 5) or
with vector alone (pPM40) as a control. Optimal yeast cell growth
was obtained in minimal salt medium (MSM) (Methods in Yeast
Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1986)) supplemented with additional biotin for both fusion protein
expressing and non-expressing yeast transformants.
[0174] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties.
Sequence CWU 1
1
8 1 22 DNA Artificial Sequence Synthetic Oligonucleotide 1
ttgagctccc cagaaataag gc 22 2 23 DNA Artificial Sequence Synthetic
Oligonucleotide 2 agaaggtttt tttagcccgg gca 23 3 33 DNA Artificial
Sequence Synthetic Oligonucleotide 3 cgatcggagc tcattaacgc
ctttcgctca taa 33 4 37 DNA Artificial Sequence Synthetic
Oligonucleotide 4 gtgtcctctc gtatctttac cccaaagatc tgcgcga 37 5 29
DNA Artificial Sequence Synthetic Oligonucleotide 5 atcccgggaa
ggttgaaacc agttccctg 29 6 30 DNA Artificial Sequence Synthetic
Oligonucleotide 6 gtgtgtattt atttgtttta ccacgtgcgc 30 7 42 DNA
Artificial Sequence Synthetic Oligonucleotide 7 ccggccgtct
ctagatctgg ctttgatctt actatcattt gg 42 8 52 DNA Artificial Sequence
Synthetic Oligonucleotide 8 gtattgtggt tcgttgatta tgatattgat
agttattatt acgtgcacgg cg 52
* * * * *
References