Title: Chimeric cell-targeting pathogenic organism and method of therapeutic use
Abstract: The invention chimeric organism comprises a chimeric surface integrin-like fusion protein in which the I domain has been replaced by an antibody fragment that binds a disease-associated antigen on a cell. Binding of the antibody fragment to the disease-associated antigen triggers virulent transformation of the chimeric pathogenic organism so as to cause the organism to infiltrate the target cell with specificity. Preferably, the chimeric organism is a chimeric pathogenic C. albicans having an INT1 fusion protein in which the I domain is replaced by an antibody fragment, preferably a single chain antibody, and in which expression of an iron transporter gene necessary for infiltration of a target cell is triggered under the control of a EFG1p response element. Binding of the antibody to the disease-associated antigen causes filamentous transformation in the chimeric pathogenic C. albicans and specific infiltration of target cells. The invention chimeric pathogenic organisms are used in therapeutic methods to specifically infiltrate and destroy diseased cells to which the antibody fragment binds while remaining non-pathogenic to normal cells.
Patent Number: 6,929,941 Issued on 08/16/2005 to Odom
| Inventors:
|
Odom; Duncan (Cambridge, MA)
|
| Assignee:
|
California Institute of Technology (Pasadena, CA)
|
| Appl. No.:
|
672408 |
| Filed:
|
September 26, 2003 |
| Current U.S. Class: |
435/252.3; 435/254.1; 435/254.22 |
| Intern'l Class: |
C12N 001/14; C12N 001//16; C12N 001//18; C12N 001//20 |
| Field of Search: |
435/2523,254.1,254.22
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Lo et al (Mol. Biol. Cell vol. 9, pp 161-171, 1998).
Gale et al., "Linkage of Adhesion, Filamentous Growth, and Virulence in Candida
albicans to Single Gene, INT1," Science, 279:1355-1358 (1998).
Moritz et al., "Cytotoxic T lymphocytes with a grafted recognition specificity
for ERBB2-expressing tumor cells," Proc. Natl. Acad. Sci. USA, 91:4318-4322 (1994).
Wels et al., "Biotechnological and gene therapeutic strategies in cancer treatment,"
Gene 159:73-80 (1995).
Calderone et al., "Adherence and Receptor Relationships of Candida albicans",
Microbiol. Rev., 55(1):1-20 (1991).
Gale et al., "Linkage of Adhesion, Filamentous Growth, and Virulence in Candida
albicans to a Single Gene, INT1," Science, 279:1355-1358 (1998).
Moritz et al., "Cytotoxic T lymphocytes with a grafted recognition specificity
for ERBB2-expressing tumor cells," Proc. Natl. Acad. Sci. USA, 91:4318-4322 (1994).
Wels et al., "Biotechnological and gene therapeutic strategies in cancer treatment,"
Gene 159:73-80 (1995).
Sonneborn et al., "Control of White-Opaque Phenotypic Switching in Candida
albicans by the Efg1p Morphogenetic Regulator", Infect. Immun., 67(9):4655-4660 (1999).
|
Primary Examiner: Navarro; Mark
Attorney, Agent or Firm: DLA Piper Rudnick Gray Cary US LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. application Ser. No. 10/002,389
filed Nov. 30, 2001, now U.S. Pat. No. 6,638,756; which claims priority under 35
USC §119(e) to U.S. Application Ser. Nos. 60/297,995 filed Jun. 13, 2001,
now abandoned and 60/251,253 filed Dec. 5, 2000, now abandoned. The disclosure
of each of the prior applications is considered part of and is incorporated by
reference in the disclosure of the application.
Claims
1. A method for generating a chimeric
Candida organism from a pathogenic
organism that possesses in the wild-type an INT1 protein with an I domain, said
method comprising:
replacing the I domain in the INT1 protein of the pathogenic organism with an
antibody fragment that binds to a disease-associated antigen on a diseased cell;
wherein the wild-type pathogenic organism undergoes virulent transformation by
binding of the I domain of the surface INT1 protein to a cell, and wherein the
chimeric
Candida organism undergoes virulent transformation by binding of
the antibody fragment to the disease-associated antigen on the cell.
2. The method of claim 1, wherein the pathogenic organism is
C. albicans and
wherein the method further comprises disabling the wild-type high-affinity iron
transporter gene in the
C. albicans, and
introducing a DNA construct comprising a wild-type high-affinity iron transporter
gene under the control of a EFG1p response element,
wherein binding of the antibody fragment to the disease-associated antigen triggers
expression of the high-affinity iron transporter gene in the DNA construct and
filamentous transformation in the chimeric pathogenic
C. albicans.
3. The method of claim 2, wherein the antibody fragment is a single chain antibody.
4. The method of claim 2, wherein the antibody fragment binds to an antigen on
a tumor cell.
5. The method of claim 4, wherein the disease-associated antigen is contained
in an abnormal surface protein of the tumor cell.
Description
FIELD OF THE INVENTION
This invention relates to treatment of diseases characterized by production
of cell surface markers using antibody-targeted compositions. More particularly,
this invention relates to chimeric organisms that express an antibody fragment
and to the use of such chimeric organisms in treatment of diseases characterized
by production of cell surface markers.
BACKGROUND OF THE INVENTION
Many recent gene therapy approaches have exploited the specificity of antibody
binding to target cancer cell lines in order to deliver either drugs or immune
responses to an actual tumor location. Most cancer cell lines misregulate cell
surface proteins and polysaccharides, and are thus easily distinguished from normal
somal cells by antibodies (R. E. Hawkins et al.,
Gene Therapy (1998), 5:1581-1583).
It is apparent that established carcinomas have successfully avoided activating
the immune response within their hosts. Direct attempts to rectify this by recruiting
the body's humoral immune response to tumors by injection of murine derived antibodies
can unfortunately cause serious and even life threatening human anti-mouse responses
(R. K. Jain et al.,
J Natl. Cancer Inst. (1989) 81:570-576 and D. Colcher
et al.,
J. Natl. Cancer Inst. (1990) 82:1191-1197). In addition, the overall
penetration of antibodies into tumors is limited due to the high molecular weights
of these molecules (K. A. Chester et al.,
Adv. Drug Delivery Rev. (1996) 22:303-313).
In an attempt to limit both the size of the antibody and the mouse-character
of
the antibody, single chain antibodies (scFvs) that encapsulate the binding features
of the Fv region of the antibody without the bulk of the native antibody sequence
in the c1, c2, and c3 domains have been developed. One methodology to generate
scFvs involves tethering the antigen binding domains of V
H and V
L
together using a short flexible peptide linker (R. E. Bird et al.,
Science
(1988) 242:423-426). Another approach involves the generation de novo of molecular
diversity, instead of generating monoclonal antibodies in mice. By using combinatorial
antibody libraries on the surface of filamentous bacteriophage screened against
immobilized antigen, a single polypeptide chain that is amenable to fusion with
other proteins can be generated (J. S. Huston et al.,
Proc. Natl. Acad. Sci.
USA (1988) 85:5879-5883; J. McCafferty,
Nature (1990) 348:552-554; R.
H. J. Begent et al.,
Nature Med. (1996) 2:979-984, reviewed in K. A. Chester
et al.,
Adv. Drug Delivery Rev. (1996) 22, 303-313). The scFvs obtained
by either methodology above show better tumor penetration, but therapeutic application
is still in early stages (G. Reitmuller et al.,
Lancet (1994) 343:1177-1183).
However, fusions between imaging agents and scFvs have found wide acceptance and
extensive application in tumor imaging and radiochemotherapeutic delivery (see
J. Bhatia et al.,
Cancer (1999) 85:571-577 and A. M. Wu et al.,
Tumor
Targeting (1999) 4:47-58 and references therein).
Antibody recognition has also been used to target cancer cells by incorporation
of an scFv into the envelope protein of a retrovirus (S. J. Russell et al.,
Nuc.
Acids Res. (1993) 21:1081-1085 and F. Martin et al.,
Human Gene Therapy
(1998) 9:737-746). This targeting is modest, but offers some promise, as has
been demonstrated for certain types of melanoma (Martin 1998). In addition, adenovirus
infection has been used to allow the transient expression of tumor-targeting scFv
fusion proteins in whole organisms with moderate success (H. A. Whittington et
al.,
Gene Therapy (1998) 5:770-777). Unfortunately, low survivability of
adenoviruses carrying antibody generating expression vectors limits their impact.
The most promising therapeutic techniques relying on the specificity of antibody
binding focus on engineering T-cells that express antibody fragments fused to surface
proteins, and are thus directed to tumor surfaces (recent work reviewed in F. Paillard,
Human Gene Therapy (1999) 10:151-153). Some of these T-cells are at present
in clinical trials. Strategies used to date, however, have drawbacks, including
limited efficacy against established tumors, though demonstrating some slowing
of tumor metastasis (R. P. McGuinness et al,
Human Gene Therapy (1999) 10:165-173).
Limited effectiveness against established tumors may be due to the inability of
the T-cells to penetrate solid cell masses (Paillard 1999). True protection against
establishment of invasive carcinoma was obtained only by coinjection of modified
T-cells with the tumorogenic line. In clinical applications, this may permit stabilization
and localization of established tumors, but not reductive treatment. Another potential
problem is that suicide signals T-cells use to induce apoptosis, like tumor necrosis
factor I, are often not functional against carcinomas. Even when they are effective,
successful cancer cell lines will rapidly adapt to apoptotic signals, and have
even been known to induce apoptosis in attacking T-cells (K. Shiraki,
Proc.
Natl. Acad. Sci. USA (1996) 94:6420-6425). In addition, T-cells bearing these
chimeras are assembled separately for each patient ex vivo due to possible MHC
incompatibilities that could result in serious allergic reactions were T-cells
from other humans introduced therapeutically.
Candida albicans is the most commonly isolated invasive fungal pathogen
in humans. This organism is representative of several that switch between two major
classes of morphology. The first morphology is the ellipsoid blastospore. Like
most yeast,
C. albicans assumes this architecture when growing non-pathogenically.
Upon binding of
C. albicans to mammalian tissues (i.e. via the I domain
of the INT-1 protein), the cell morphology switches to various filamentous forms,
including germ tubes and hyphae, that are capable of aggressively invading host
tissue (reviewed by R. A. Calderone,
Microbol. Rev. (1991) 55, 1-20). Systemic
infection of a vulnerable host by
C. albicans results in high levels of
mortality. For example, more than 30% of immunocompromised HIV patients are systemically
infected despite appropriate treatment regimes. In addition,
C. albicans infection
commonly leads to death in premature infants, diabetics, and surgical patients.
To date, the ability of this pathogenic organism to infect cells when the cell
morphology switches to a filamentous form has not been utilized for therapeutic
purposes, such as in cancer therapy.
Thus, the need exists in the art for new and better compositions and methods
of their use for treating various types of cancers and other diseases associated
with production of an abnormal protein.
SUMMARY OF THE INVENTION
The present invention overcomes these and other problems in the art by providing
chimeric organisms having a chimeric surface integrin-like protein in which the
I domain has been replaced by an antibody fragment that binds a disease-associated
antigen on a cell. Binding of the antibody fragment to the disease-associated antigen
on the cell triggers virulent transformation of the chimeric pathogenic organism
and allows the organism to infect the cell.
In one embodiment according to the present invention, there are provided chimeric
pathogenic
C. albicans modified to contain an integrin1 (INT1) fusion protein
in which the I domain is replaced by an antibody fragment that binds to a disease-associated
antigen on a diseased cell. The chimeric
C.albicans further contains a disabled
wild-type high affininity iron transporter (CAFTR) gene, and a DNA construct comprising
a wild-type CAFTR gene under the control of an enhanced filamentous growth protein
(EFG1p) response element, wherein binding of the antibody to the disease-associated
antigen triggers expression of the CAFTR gene in the DNA construct and filamentous
transformation in the chimeric pathogenic
C. albicans.
In another embodiment according to the present invention, there are provided
methods
for treating a disease associated with the presence of cells having a disease-associated
surface antigen in a subject in need thereof by administering to the subject a
therapeutically effective amount of an invention chimeric pathogenic organism so
as to cause binding of the antibody fragment to the disease-associated antigen
on the cells, thereby treating the disease by triggering infiltration of the chimeric
pathogenic
C. Albicans into the cells without substantial damage to healthy cells.
In yet another embodiment, the present invention provides methods for generating
a chimeric therapeutic organism from a pathogenic organism that possesses in the
wild-type an integrin-like protein with an I domain. In the invention methods,
the I domain in the integrin-like protein of the pathogenic organism is replaced
with an antibody fragment that binds to a disease-associated antigen on a diseased
cell. In the chimeric therapeutic organism, virulent transformation occurs upon
binding of the antibody fragment to the disease-associated antigen on the cell.
A BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-1 to 1-23 show the nucleotide sequence
of the gene that encodes the integrin-like INT1 protein
C. Albicans (GenBank
Accession #U35070) (SEQ ID NO:1).
FIG. 2 shows the nucleotide sequences of seven primers used in construction
of the chimeric
C. albicans of Example 1 (SEQ ID NOS:2 through 8, respectively).
FIG. 3 is a schematic drawing showing human integrin structure (adapted from
M. J. Humphries,
Biochem. Soc. Trans. (2000) 28:311-340).
FIG. 4 is a schematic drawing showing two pathways by which hyphal development
in yeast is regulated.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides chimeric pathogenic organisms derived from wild
type organisms wherein virulent transformation of the organism is controlled in
the wild-type organism by binding of the I domain of a surface integrin-like protein
to a cell. The invention chimeric organism comprises a chimeric surface integrin-like
protein in which the I domain is replaced by an antibody fragment that binds a
disease-associated antigen on a cell. Binding of the antibody fragment to the disease-associated
antigen triggers virulent transformation of the chimeric pathogenic organism so
as to cause the organism to infiltrate the cell. Virulent invasion of the cell
by the chimeric pathogen inhibits growth of the diseased cell.
The invention pathogenic chimeric organism represents a new approach to employing
otherwise pathogenic organisms to assist in disease treatment. Although the present
invention is described for illustrative purposes with reference to a reingeneered
C albicans, suitable pathogenic organisms in addition to
C. albicans
that can be engineered according to the methods disclosed herein are pathogenic
organisms that become virulent (e.g., switch to a filamentous invasive form) upon
binding of its integrin-like surface protein (i.e., a cell-cell communication protein)
to a target on another cell and in which the binding domain of the surface protein
can be replaced with a antibody fragment that binds to a desired target cell associated
with a disease state. Preferably the chimeric pathogen also is relatively harmless
to mammalian cells until binding of the antibody fragment contained in its surface protein.
In higher eukaryotes, integrins are one of the most important classes of surface
proteins responsible for intercellular communication (reviewed in F. G. Giancotti
Science (1999) 285:1028-1032 and M. J. Humphries,
Biochem. Soc. Trans.
(2000) 28:311-340). Generally, integrins are heterodimers, each subunit of
which consists of a cytosolic domain with one tyrosine used as a kinase regulatory
site, a transmembrane domain, and four EFG-like repeats. As used herein, the term
"integrin-like protein" refers to a cell-cell communication transmembrane protein
that contains one or more of the above features.
There are various other domains on the integrin proteins, including metal binding
MIDAS loops and β propeller domains. Notably, in nine of the fifteen human
integrin I subunits, there is a protruding region known alternately as the IA,
or the I domain, which appears to regulate integrin targeting. This suggests that
the absence or presence of the I domain has little, if any, effect on the integrin's
ability to transduce signals, but instead regulates which signals are transduced.
The I domain is the only region whose structure has been solved crystallographically
(both bound to its target proteins and unbound). Based on these studies, it is
believed that the I domain alone is indeed sufficient for binding to collagen (J.
Emsley
Cell (2000) 101:47-56).
In the invention chimeric organism, the endogenous binding region of the surface
integrin-like protein, which nonspecifically targets cells (e.g., those containing
fibrinogen), is replaced with an antibody fragment, such as a single chain antibody.
As a result, rather than nonspecifically binding to any cell containing a binding
site for the endogenous binding region, the invention chimeric pathogen binds with
specificity to cells that express the target antigen. Binding of the chimeric pathogen
to a cell containing an epitope for the antibody fragment triggers virulent invasion
of the disease-associated cell. Other cells (e.g., healthy cells) are not bound
by the chimeric organism. As a result, pathogenic infiltration of non-targeted
cells does not take place.
In one embodiment, the invention provides a chimeric pathogenic
C. albicans
comprising an INT1 fusion protein in which the I domain is replaced by an antibody
fragment that binds to a disease-associated antigen on a cell. Preferably, the
INT1 protein in the invention pathogenic organism is a fusion protein in which
a single chain antibody replaces the native I domain. The nucleotide sequence encoding
INT1 is shown in FIGS. 1A-W (SEQ ID NO:1 herein). Construction of such a chimeric
INT1 is described in Example 1 below.
As used herein, the term "disease-associated antigen" means either that the antigen
is not expressed in normal, healthy cells, or that the antigen is expressed in
abnormal quantity in diseased cells. Existence of disease-associated antigen on
cells greatly increases the amount of the chimeric pathogen that attacks such disease-associated cells.
Preferably the antibody fragment is a single chain antibody (scFv), ),
but Fv and CDR fragments can also be used. The antibody fragment is preferably
incorporated into the surface integrin-like protein to form a fusion protein.
In a preferred embodiment, the chimeric pathogen is a chimeric
Candida albicans,
the most common fungal pathogen of humans. In immunocompromised individuals,
C. albicans is a dangerous, and sometimes lethal pathogen. The primary protein
responsible for
C. albicans targeting is an integrin-like transmembrane
protein known as INT1. INT1 contains an integrin-like domain (known as the I domain),
which is the putative targeting region of this protein. FIGS. 1A-W show the nucleotide
sequence of the gene that encodes the integrin-like INT1 protein of
C. Albicans
(GenBank Accession # U35070) (SEQ ID NO:1).
The preferred antibody fragment for incorporation into the INT1 protein as a
fusion protein is a single chain antibody (scFv), but Fv and CDR fragments can
also be used. Thus, in this embodiment, the power and specificity of scFv antibody
fragments, which now exist against a considerable number of cell surface targets
in cancer cell lines, is combined with the ability to not only bind to the cancer
cell mass, but to invade and destroy tumors aggressively and selectively in a manner
independent and complementary to the body's own defenses.
The present invention exploits the mechanism involved in the filamentous transformation
of
C. albicans and similar pathogenic organisms, which is controlled by
a regulatory system similar to that used by
Saccharomyces cerevisiae to
alter its own morphology under nitrogen starvation conditions and agar invasive
conditions. The evolutionary conservation of this pathway has greatly facilitated
deconvoluting the biochemistry involved, and recent research has resulted in a
better understanding of proteins responsible for
C. albicans intercellular
binding and pathogenicity. In particular, INT1 is the previously known but unidentified
surface protein that is strongly crossreactive with antibodies for certain leukocyte
integrins, and appears to be the primary protein responsible for attaching to target
cells (C. A. Gale et al.,
Science (1998) 279:1355-1358). INT1 is a transmembrane
surface protein isolated by cDNA screening of the
C. albicans genome by
oligonucleotide probes derived from the conserved region of human integrins (C.
Gale et al.,
Proc. Natl. Acad. Sci. USA (1996) 93:357-361). Over the last
fifteen years, many reports have appeared in the literature describing surface
proteins that are related to I-subunits of the leukocyte integrins IM/l2 (Mac-1;
CD11b/CD18) and IX/θ2 (p150,95; CD11c/CD18) (C. M. Bendel,
J. Clin Invest.
(1993) 92:1840-1849 and references therein). Many monoclonal antibodies that
recognize epitopes of these leukocyte surface proteins cross react to blastospores
and germ tubes of
C. albicans, sometimes with the same affinity as to the
original human targets.
Upon ligand binding, INT1 signals cell morphology changes that induce hyphae
growth. This signaling pathway appears to be largely independent of the mating
factor MAPK pathway, which is most commonly associated with morphologic changes.
Unlike the pathway that is triggered by ligand binding, the MAPK pathway is triggered
by environmental stimulation, such as changes in pH, temperature, mating signaling
or nutrient availability, and terminates in transcription factor STE12 in
S.
cerevisiae (Gale 1996; C. J. Gimeno et al.,
Cell (1992) 68, 1077-1090
1992 and R. L. Roberts et al.,
Genes Dev. (1994) 8, 2974-2985) (FIG.
4).
The second pathway to hyphal morphology, which is less well characterized, depends
on direct stimulation by serum. Addition of mammalian serum to an otherwise spheroplast
culture of
C. albicans induces hyphae growth, even when the signaling cascade
terminating in STE12 (briefly described above) is completely knocked out (Lo 1997).
INT1 instead communicates morphology changes via a second, STE12 independent, pathway
that appears to have as an intermediary protein Ash1. Ash 1 is a daughter cell-specific
protein that helps regulate filamentous growth, and may interact with STE20 (S.
Chandarlapaty et al.,
Mol. Cell Biol. (1998) 18, 2884-2891). This pathway
terminates at the transcriptional level in a bHLH class protein known as PHD1 recently
isolated in
S. cerevisiae. The homologous protein in
C. albicans is
known as EFG1. The sequence for EFG1 is fully described in W. R. Stoldt et al.,
EMBO Journal (1997) 16, 1982-1991, which is incorporated herein by reference
in its entirety.
For both
S. cerevisiae and
C albicans, overexpression of their
respective analog is sufficient to induce hyphae growth (Stoldt 1997). Importantly,
elimination of filamentous growth can only be achieved after disabling both of
these two pathways. This observation also rules out a third pathway for signaling.
In construction of the illustrative
C. albicans chimeric pathogen, the
integrin homolog INT1 was isolated using conserved transmembrane sequences from
mammalian integrins to clone the cDNA copy of the gene in
C. albicans. Removal
of the INT1 protein reduces specific adhesion of
C. albicans to HeLa cells
by 39%. Therefore, though INT1 is a critical protein for binding to a target, other
proteins as well must serve to help mediate this interaction (vide infra), though
these proteins have yet to be identified. Still, previous research appears to be
consistent with cell surface binding by a single, integrin-like protein of approximate
MW of 165 kDa. Interestingly, transgenic experiments where INT1 was overexpressed
on the surface of
Saccharomyces cerevisiae, a nonadhesive and nonpathogenic
species, caused strong adhesion to mammalian cells. Thus, INT1 alone is sufficient
for target binding.
Deletion of the INT1 gene cripples filamentous growth of
Candida, though
not entirely eliminating it. It has been shown that invasive growth of this type
is necessary for parasitic microorganisms to successfully invade host tissues (W-S
Lo et al.,
Mol. Biol. Cell (1998) 9:161-171). In vivo testing of the pathogenicity
of an INT1 of pathogenicity of the INT1 deletion strain of
C. albicans on
mice was conducted and showed a dramatic reduction in mouse lethality compared
to wild-type strains (Gale 1996).
The similarity of INT1 in
C. albicans to mammalian integrins is not limited
to antibody cross reactivity and sequence similarity in the transmembrane region.
Notably, INT1 also appears to contain numerous motifs similar by homology to mammalian
integrin motifs. These include (1) two FE-hand divalent cation binding sites that
likely mediate target binding; (2) a single cytosolic tyrosine for kinase signaling;
and, most importantly, (3) a region that appears to be homologous to the I domain
of integrins. Similar to the higher mammalian IM and IX that recognize iC3b and
fibrinogen, the I domain like region in
C. albicans INT1 is generally thought
to be the binding site that targets iC3b. This is further supported by its ˜25%
sequence identity with the fibrogen binding domain of
Staphylococcus aureus.
In the illustrative preferred embodiment of the invention chimeric pathogen,
the
I domain of the wild-type INT1 protein, which nonspecifically targets fibrinogen,
is replaced with an scFv that targets cancer cells. Many scFvs already have been
developed that bind to a wide variety of tumor cells for therapeutic applications.
Such studies take advantage of the severe misregulation of surface protein populations
in tumors by utilizing scFvs that bind epitopes found in such surface proteins.
For example, therapeutic applications involving T-cell, viral, and/or drug targeting
has already been proven in vivo using scFvs shown in Table 1 below.
| TABLE 1 |
| |
| |
|
CANCER LINE |
|
|
| ANTIBODY |
ANTIGEN |
AND LOCATION |
REFERENCES |
OTHER |
| |
| CC49 |
TAG-72 |
Adenocarcinoma |
McGuiness 1999 |
|
| |
|
(colon, |
Shu 1993 |
| |
|
ovarian, breast) |
Kashmiri 1995 |
| FRP5 |
ERBB2 |
Breast, ovarian |
Moritz 1994 |
Previously used to |
| |
|
|
Harwerth 1992 |
construct cytotoxic |
| |
|
|
Hynes 1993 |
C-lymphocytes. Also |
| |
|
|
|
used to direct virus |
| |
|
|
|
targeting (Galmiche |
| |
|
|
|
1997) |
| GA733.2 |
EGP-2 |
Various |
Ren-Heidenreich |
| |
|
|
2000 |
| HMN-14 |
CEA |
Colorectal, breast, |
Nolan 1999 |
Previously used to |
| |
|
pancreas, other |
|
construct killer |
| |
|
|
|
T-cells |
| VFF17 |
CD44 |
Cervical cancer, |
Dall 1997 |
| |
|
lymph metastases |
Hekele 1996 |
| MOV19 |
I-FR |
Nonmucinous ovarian |
Melani 1998 |
| |
|
carcinoma |
| 7.16.4 |
Neu |
Breast |
Katsumata 1995 |
Antigen (neu) is same |
| |
|
|
Stankovski 1993 |
as ERBB2, and is |
| |
|
|
Disis 1997 |
protein bound by |
| |
|
|
|
Herceptin. |
| MLuCl |
L(Y) TAA |
Various |
Mezzanzanica |
Targets misregulated |
| |
|
|
1998 |
carbohydrates. Lewis |
| |
|
|
|
(Y) tumor associated |
| |
|
|
|
antigen |
| |
By replacing the I domain in the integrin-like surface protein with a scFv that
binds to a disease-associated tumor cell, the invention chimeric pathogenic organisms
are engineered to take advantage of the severely misregulated production of surface
protein populations in tumors. In the present invention, the antibody fragment,
preferably as a scFv, is incorporated into the position of the native binding domain
of an integrin-like protein (i.e., the creation of a fusion protein that contains
the scFv incorporated in the place of the I domain in the wild-type pathogenic
organism). Many antibody fragments have already been tested for selective binding
to a known tumor-associated antigen, for example, as shown in Table 1. Representative
non-limiting examples of tumor associated antigens to which scFvs of the invention
chimeric pathogens bind include GAG-72, ERBB2, EGP-2, CEA, CD44, I-FR, neu, the
Lewis (Y) tumor associated antigen, and the like.
As used herein, the terms "disease- or tumor-associated antigen" and "disease-
or tumor-associated epitope" encompass antigens and epitopes, respectively, found
in surface proteins produced in large amounts in various types of tumors as well
as various types of marker proteins (and the epitopes contained therein) that are
found associated with tumor cells and not found associated with normal cells. Representative
non-limiting examples of tumors having associated antigens to which antibody fragments
(e.g., scFvs) of the invention chimeric pathogens bind includes adenocarcinoma
of colon, ovary or breast; cervical cancer, nonmucinous ovarian carcinoma; breast,
ovarian, colorectal, and pancreatic cancer, and the like. Invention chimeric pathogenic
organisms are incapable of infiltrating a cell in the subject until the antibody
fragment in the chimeric integrin-like protein binds to its target epitope, triggering
a virulent transformation of the chimeric pathogenic organism. Therefore, the invention
chimeric pathogenic organisms are substantially incapable of pathogenic activity,
such as infiltration, of cells other than their target cells (e.g., cancer cells).
Preferably, the antibody fragment is a scFv and is introduced in the
place of the I domain of INT-1 in
C. albicans. Once engineered to replace
the wild-type binding domain of the INT1 protein with an antigen binding region
(e.g. scFv) from cancer-specific antibodies, the invention mutant
C. albicans
strain will specifically bind to a cancer line dictated by the targeting of
the scFv-INT1 fusion protein.
Optionally, in order to direct pathogenicity specifically to the target
cell (e.g., a carcinoma cell) a gene in the pathogenic organism from which the
chimeric organism is derived that is required for invasive growth is disabled or
removed and a DNA construct comprising a reengineered copy of the gene necessary
for invasive growth is introduced into the chimeric organism under the regulatory
control of a transcription factor that regulates filamentous transformation of
the organism. However, the gene removed should be one that does not significantly
affect vegetative growth of the organism so that large quantities of this chimeric
organism can be produced using standard culture techniques.
For example, in
C albicans, the wild-type gene is placed under the control
of a EFG1p response element. While the CaFTR1 gene is currently preferred for reengineering
in
C. albicans, those of skill in the art can readily substitute for reengineering
(i.e., in the place of the CaFTR1 gene) another gene from the pathogenic organism
that is essential or preferred for pathogenic invasion.
Preferably, in the invention chimeric
C. albicans, the wild-type
CAFTR gene is either disabled or removed and a DNA construct comprising a wild-type
CAFTR gene under the control of a EFG1p response element is introduced. Overexpression
of EFG1 in
C. albicans leads to enhanced filamentous growth in liquid and
on solid media. Overexpression of EFG1 by a PCK1p-EFG1 fusion is described by A.
Sonneborn,
Infect Immun (1999) 67:9:4655-60, which is incorporated herein
by reference in its entirety (See also, V. R. Stoldt et al.,
EMBO J (1997)
16:8 1982-91). The nucleotide sequence for the CaFTR1 gene of
C. albicans is
found at NCBI GenBank Number AF195775.
CaFTR1 extracts iron from mammalian tissues that withhold metals from microbial
predators as a defense mechanism (D. M. DeSilva et al.,
Physiol. Rev. (1996)
76, 31-47 and H. Gunshin et al.,
Nature (1997) 388, 482-488). Removal of
the native CaFTR1 completely abrogates pathogenicity. Mice injected with a mutant
C. albicans having a disabled CaFTR1 gene survive entirely; while those
injected with an equal amount of wild-type
C. albicans do not. Under circumstances
of normal unicellular growth in an abundance of iron, though, CaFTR1 is not an
essential gene. In conditions where iron is in limited quantities, for instance
during circulation through a host designed to have limiting nutrient levels, this
gene is highly upregulated. Removal of the CaFTR1 gene only causes a growth (and
thus invasion) deficiency when pathogenesis is initiated. This protein is normally
regulated entirely independently from the morphology signaling pathway, and its
concentration is dependent only on the heavy metals detected in the environment.
By placing this protein under the transcriptional control of the cell morphology
pathway initiated by INT1, as described herein, the pathogenicity of the overall
assembly can be tightly restricted to scFv-INT1 targeted cells.
In this preferred embodiment of the invention chimeric pathogenic organism, binding
of the antibody to the disease-associated antigen triggers both expression of the
CAFTR gene in the DNA construct and filamentous transformation of the chimeric
pathogenic
C. albicans. Since expression of INT1 in wild-type
C. albicans
is activated by INT1 binding to other cells, placing expression of the
C.
albicans iron transporter under control of the Efg1 expression system ensures
that both the pathogenicity and the binding of the invention chimeric organism
is directed specifically to target cells. If the antibody fragment incorporated
in the place of the INT1 binding domains is specific for a tumor cell, the pathogenicity
of the
C. albicans cell line is directed specifically towards target cancerous
cells, and nonspecific toxicity is inhibited. In other words, the virulence of
the engineered strain of
C. albicans will only be activated once scFv-INT1
binds to its target antigen on the surface of the carcinoma.
The gene that triggers filamentous growth can be disabled in the invention chimeric
organism using any method known in the art, for example, by disruption of the gene
at both diploid loci using standard techniques. The native gene can be reintroduced
under the control of a response element (e.g., a transcription factor) that regulates
filamentous transformation of the organism using known techniques, such as by use
of homologous recombination (as described in Example 1 herein). This regulatory
reassignment of the gene that triggers transformation tightly limits the pathogenicity
dependent on this protein to the specified target.
In yet another embodiment, the present invention provides methods for treating
a disease associated with the presence of cells having a disease-associated surface
antigen in a subject in need thereof. The invention method includes administering
to the subject, a therapeutically effective amount of an invention chimeric pathogenic
organism so as to cause binding of the antibody fragment to the disease-associated
antigen on the cell, thereby specifically treating the disease by triggering infiltration
of the chimeric pathogenic organism into the cells without substantial damage to
healthy cells. The invention method may further include administering to the subject
an immunosuppressive agent to inhibit the subject's immune system from destroying
the chimeric pathogen prior to achieving a therapeutic effect. Representative immunosuppressive
agents useful in the practice of the invention methods include such agents as cyclosporin
A, OKT3, FK506, mycophenolate mofetil (MMF), azathioprine, corticosteroids (such
as prednisone), antilymphocyte globulin, antithymocyte globulin, and the like.
In a preferred embodiment of the invention methods, the invention chimeric pathogen
is used to target and attack tumor cells.
In invention therapeutic methods, the chimeric pathogenic organisms are used
to
infiltrate and destroy both ex vivo cell lines (e.g., tumor cell lines), as well
as in vivo murine models of human carcinomas, and the like. The invention pathogenic
organisms also have specific utility as research reagents for the testing of therapeutic
compositions. For example, the invention chimeric organisms can be used to compare
the therapeutic effect against a particular cell line of various antibody fragments
engineered into the surface integrin-like protein. Binding of invention organisms
to these ex vivo and in vivo models can be tested for efficacy using known assays
(e.g., mouse tumor models) to determine binding of the antibody fragment (e.g.,
single chain antibody) to the target antigen on a disease-associated cell.
The chimeric pathogens used in practice of the invention method can be administered
for therapeutic purposes, such as treatment of tumor, by any route known to those
of skill in the art, such as intraarticularly, intracisternally, intraocularly,
intraventricularly, intrathecally, intravenously, intramuscularly, intraperitoneally,
intradermally, intratracheally, intracavitarily, and the like, as well as by any
combination of any two or more thereof.
The most suitable route for administration will vary depending upon the disease
state to be treated, or the location of the suspected condition or tumor to be
treated. For example, for treatment of inflammatory conditions and various tumors,
local administration, including administration by injection directly into the body
part containing the tumor provides the advantage that the chimeric pathogen can
be administered in a high concentration without risk of the complications that
may accompany systemic administration thereof.
The chimeric pathogen is administered in "a therapeutically effective amount."
An effective amount is the quantity of a chimeric pathogen necessary to aid in
treatment, inhibition or destruction of diseased tissue (e.g. tumor) under treatment
in a subject. A "subject" as the term is used herein is contemplated to include
any mammal, such as a domesticated pet, farm animal, or zoo animal, but preferably
is a human. Amounts effective for therapeutic use will, of course, depend on such
factors as the size and location of the body part to be treated, the affinity of
the antibody fragment for the target antigen, the type of target tissue, as well
as the route of administration. Local administration of the targeting construct
will typically require a smaller dosage than any mode of systemic administration,
although the local concentration of the chimeric pathogen may, in some cases, be
higher following local administration than can be achieved with safety upon systemic administration.
The invention composition can also be formulated as a sterile injectable suspension
according to known methods using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example,
as a solution in 1-4, butanediol. Sterile, fixed oils are conventionally employed
as a solvent or suspending medium. For this purpose any bland fixed oil may be
employed, including synthetic mono- or diglycerides, fatty acids (including oleic
acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut
oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate, or the
like. Buffers, preservatives, antioxidants, and the like, can be incorporated as
required, or, alternatively, can comprise the formulation.
Preferably the antibody fragment is a scFv incorporated into the chimeric
surface protein of the pathogen as a targeting device and is not relied upon as
the toxic agent. Rather, it is the pathogenic organism itself that invades and
destroys the target cells in accordance with the present invention. A single chain
antibody (scFv) is a genetically engineered molecule containing the variable region
of the light chain and the variable region of the heavy chain, linked by a suitable
polypeptide linker as a genetically fused single chain molecule. Methods of making
these fragments are known in the art. (See for example, Harlow & Lane,
Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated
herein by reference). As used in this invention, the term "epitope" means any antigenic
determinant on an antigen to which the paratope of an antibody binds. Epitopic
determinants usually consist of chemically active surface groupings of molecules
such as amino acids or sugar side chains and usually have specific three-dimensional
structural characteristics, as well as specific charge characteristics.
Fv fragments comprise an association of V
H and V
L chains.
This association may be noncovalent, as described in Inbar et al.,
Proc. Nat'l
Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be linked
by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
See, e.g., Sandhu,
Crit. Rev. Biotech. 12:437, 1992; and Singer et al.,
J. Immunol. 150:2844, 1993. Preferably, the Fv fragments comprise V
H
and V
L chains connected by a peptide linker. These single-chain
antigen binding proteins (scFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the V
H and V
L domains connected
by an oligonucleotide. The structural gene is inserted into an expression vector,
which is subsequently introduced into a host cell such as
E. coli. The recombinant
host cells synthesize a single polypeptide chain with a linker peptide bridging
the two V domains. Methods for producing scFvs are described, for example, by Whitlow
et al.,
Methods: a Companion to Methods in Enzymology, 2: 97, 1991; Bird
et al.,
Science 242:423-426, 1988; Pack et al.,
Bio/Technology 11:1271-77,
1993; Sandhu, supra, and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby
incorporated by reference in its entirety.
Another form of an antibody fragment suitable for incorporation as a fusion
protein in invention chimeric pathogenic organisms is a peptide coding for a single
complementarity-determining region (CDR). CDR peptides ("minimal recognition units")
can be obtained by constructing genes encoding the CDR of an antibody of interest.
Such genes are prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells. See, for example,
Larrick et al.,
Methods: a Companion to Methods in Enzymology, 2: 106, 1991.
Antibodies that bind to a tumor cell or other disease-associated antigen
can be prepared using an intact polypeptide or biologically functional fragment
containing small peptides of interest as the immunizing antigen. The polypeptide
or a peptide used to immunize an animal (derived, for example, from translated
cDNA or chemical synthesis) can be conjugated to a carrier protein, if desired.
Commonly used carriers that are chemically coupled to the peptide include keyhole
limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus
toxoid, and the like. The coupled peptide is then used to immunize the animal (e.g.,
a mouse, a rat, or a rabbit).
The preparation of such monoclonal antibodies is conventional. See, for example,
Kohler & Milstein,
Nature 256:495, 1975; Coligan et al., sections 2.5.1-2.6.7;
and Harlow et al., in:
Antibodies: a Laboratory Manual, page 726 (Cold Spring
Harbor Pub., 1988), which are hereby incorporated by reference. Briefly, monoclonal
antibodies can be obtained by injecting mice with a composition comprising an antigen,
verifying the presence of antibody production by removing a serum sample, removing
the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells
to produce hybridomas, cloning the hybridomas, selecting positive clones that produce
antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
Monoclonal antibodies can be isolated and purified from hybridoma cultures by a
variety of well-established techniques. Such isolation techniques include affinity
chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG), in:
Methods
in Molecular Biology, Vol. 10, pages 79-104 (Humana Press, 1992).
Antibodies of the present invention may also be derived from subhuman
primate antibodies. General techniques for raising therapeutically useful antibodies
in baboons can be found, for example, in Goldenberg et al., International Patent
Publication WO 91/11465 (1991) and Losman et al., 1990,
Int. J. Cancer 46:310,
which are hereby incorporated by reference. Alternatively, a therapeutically useful
antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal
antibodies are produced by transferring mouse complementarity determining regions
from heavy and light variable chains of the mouse immunoglobulin into a human variable
domain, and then substituting human residues in the framework regions of the murine
counterparts. The use of antibody components derived from humanized monoclonal
antibodies obviates potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al.,
Proc. Nat'l Acad. Sci.
USA 86:3833,1989, which is hereby incorporated in its entirety by reference.
Techniques for producing humanized monoclonal antibodies are described, for example,
by Jones et al.,
Nature 321:522, 1986; Riechmann et al.,
Nature 332:323,
1988; Verhoeyen et al.,
Science 239:1534, 1988; Carter et al.,
Proc.
Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu,
Crit. Rev. Biotech. 12:437,
1992; and Singer et al.,
J. Immunol. 150:2844, 1993, which are hereby incorporated
by reference.
It is also possible to use anti-idiotype technology to produce monoclonal antibodies,
which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made
to a first monoclonal antibody will have a binding domain in the hyper variable
region that is the "image" of the epitope bound by the first monoclonal antibody.
The assembly, selection, and integration of these chimeric scFv-INT1 products
are conducted using standard molecular biology, for example as is described in
Example 1 herein. The proper assembly of the invention chimeric scFv-INT-1 protein
and adhesion to the epitopes in target cell lines, e.g., tumor cell lines, can
be tested by introduction of the chimeric assembly into
S. cerevisiae, preferably
under the control of a promoter, such as the actin promoter, that is constantly
activated in such yeast cell lines. Yeast cells (e.g.,
Saccharomyces cerevisiae)
possess an efficient and precise system for genetic recombination. The natural
process of homologous recombination depends on a system of enzymes that search
for regions of sequence homology between two DNA molecules (which may be entire
chromosomes). Once homology is found, an exchange of information is possible.
Plasmids or other vectors carrying recombinant-DNA (r-DNA) clones which
contain naturally-occurring yeast sequences and which are introduced into cells
by standard transformation methods are capable of stably integrating into the yeast
genome at sites of homology. The efficiency of this process can be increased by
up to a thousand-fold by introducing a double-strand break within a DNA sequence
on the incoming DNA molecule that is homologous to a sequence resident in the yeast
cell. The cloned yeast DNA on the transforming vector is referred to herein as
the targeting sequence, and the site of integration is referred to herein as the
target site.
In one process described in U.S. Pat. No. 5,783,385 to Treco , et al., which
is
incorporated herein by reference in its entirety, a targeting DNA molecule, e.g.,
a bacterial plasmid, which is non-replicating in yeast is introduced into the population
of host yeast cells containing the r-DNA. The bacterial plasmid has a selectable
marker gene that functions in yeast and a first targeting DNA sequence which is
homologous in part to a second target r-DNA clone sequence. Preferentially, the
targeting plasmid is cut with a restriction endonuclease that introduces a double-strand
break within the targeting sequence, thereby linearizing the bacterial plasmid
and providing DNA ends which are recombinogenic to stimulate the process of homologous
recombination with host yeast sequences. Because the plasmid is non-replicating
in yeast, stable transformation with the selectable marker can only proceed by
homologous recombination. The efficiency of transformation by homologous recombination
is increased when the plasmid is cut by restriction enzyme digestion within the
targeting DNA sequence homologous in part to the target r-DNA sequence.
The host yeast cells are grown under conditions such that only those yeast cells
that have been stably transformed, i.e., have had the plasmid and selectable marker
stably integrated in the host cell by homologous recombination will be able to
grow. In a correctly targeted event, the entire plasmid is stably incorporated
contained in the host yeast cell by homologous recombination of the targeting DNA
sequence of the plasmid and the homologous target r-DNA clone sequence. Only those
few host yeast cells that contain the desired, target r-DNA clone sequence (and
have thereby undergone homologous recombination with the targeting plasmid) are
able to grow under the new growth conditions, due to the introduction of the yeast-selectable
marker gene contained on the targeting plasmid.
The vast majority of the population of the host yeast cells containing r-DNA
clone sequences that are not homologous to the targeting DNA sequence contained
on the plasmid, do not have the plasmid incorporated by homologous recombination
and, therefore, do not acquire the marker gene that is essential for growth under
the selection conditions. Therefore, it is preferable that any yeast-selectable
marker gene that is contained on the incoming targeting plasmid has been deleted
entirely or almost entirely from the genome of the host yeast strain that is used
for the vector. This prevents any spurious homologous recombination events between
the incoming yeast-selectable marker gene and any other natural yeast genetic loci.
If a yeast-selectable marker gene on the incoming targeting plasmid is not deleted
from the yeast genome, but is retained as a mutated, non-functional portion of
the yeast chromosome, more positive scores for homologous recombination will have
to be screened to ensure that the homologous recombination event has taken place
between the targeting DNA sequence on the bacterial plasmid and the desired, target
r-DNA clone sequence. Cells with the integrated marker can grow into colonies when
plated on appropriate selective media.
Alternatively, a yeast-selectable marker gene on the incoming targeting
DNA molecule can be a bacterial gene that confers drug resistance to yeast cells,
e.g., the CAT or neo genes from Tn9 and Tn903, or bacterial amino acid or amino
acid nucleoside prototrophy genes, e.g., the
E. coli argH, trpC, and pyrF genes.
Methods for plasmid purification, restriction enzyme digestion of plasmid
DNA and gel electrophoresis, use of DNA modifying enzymes, ligation, transformation
of bacteria, transformation of yeast by the lithium acetate method, preparation
and Southern blot analysis of yeast DNA, tetrad analysis of yeast, preparation
of liquid and solid media for the growth of
E. coli and yeast, and all standard
molecular biological and microbiological techniques can be carried out essentially
as described in Ausubel et al. (Ausubel, F. M. et al.,
Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New
York, 1987).
Once the proper assembly of the invention chimeric scFv-INT-1 protein and adhesion
to the epitopes in target cell lines, e.g., tumor cell lines, has been tested in
a non-pathogenic yeast cell (e.g.,
Saccharomyces cerevisiae) homologous
recombination can be used to insert a polynucleotide sequence encoding the chimeric
scFv-INT1 into
Candida albicans, and similar ex vivo experiments as those
performed for
S. cerevisiae will be performed to assure that replacement
of the I domain does not seriously impair the proper folding and targeting of scFv-INT1.
At this point, ex vivo experiments verifying adhesion of this mutant
C. albicans
strain to cancer cells are performed, in addition to preliminary in vivo mice
experiments to ascertain that this targeting alone is adequate in mice to restrict
pathogenicity and targeting to tumors.
The most common model for human cancers is a murine subject that has been transfected
with human carcinomas. After an incubation period varying from weeks to months
after carcinoma introduction to allow growth of test tumors, transfected mice will
be treated with the genetically modified
C. albicans. Survival of the mice
and tumor spreading are monitored over time. Biopsies of the tumorous tissues can
also be taken to investigate
C. albicans invasion. By using large groups
of genetically identical mice, aggregate data can be collected.
Evolution has optimized certain organisms to invade mammalian tissue. The
present invention harnesses this powerful and highly pathogenic trait to generate
a new weapon against cancer and other diseases characterized by the presence of
cells with a disease-associated antigen. In contrast to more indirect methodologies
previously applied that recruit the natural immune system responses, fusion scFv-INT1
proteins targeted to disease-associated tissues will direct aggressive invasion
of the naturally invasive pathogen to diseased host tissue. The method of the present
invention is a novel approach to cancer treatment that recruits the previously
untapped resource of pathogenic organisms (e.g. fungi) as potent and specific therapy
to eliminate diseased tissue characterized by disease-associated antigens.
The invention will now be described by reference to the following non-limiting
illustrative example:
EXAMPLE 1
Construction of the scFv-INT1 Fusion Gene
Using bulk genomic DNA from
C. albicans, primers 1 and 2 (shown in FIGS.
2A-B) (SEQ ID NOS:2 and 3) are used for PCR amplification of the INT1 gene (available
from GenBank under accession number U35070) (SEQ ID NO:1) as previously described
(Gale 1996). These primers insert SacI and ApaI restriction sites at the 5′
and 3′ ends of the coding region of INT1, respectively. These restriction
sites are both nonexistent in the ORF of the gene (see gene sequence in FIGS. 1A-W).
The 5 kB product of this PCR reaction is isolated using a standard Qiagen desalting
kit, digested with the appropriate enzymes SacI and ApaI, and ligated into predigested
and dephosphorylated pBluescript II SK (+) phagemid plasmid according to the manufacturer's
instructions (Product #212205, Stratagene, LaJolla, Calif.). ssDNA incorporating
the INT1 gene is then generated using standard techniques with helper phage and
uridine in dut
-; ung
-; strains of
E. Coli according
to the manufacturer's instructions.
To introduce multiple cloning sites in the ssDNA PCR product in the place of
the
I domain of INT1, primer 7 (shown in FIGS. 2A-B) (SEQ ID NO:8) is used in a standard
polymerase/ligase reaction; also thus eliminating the I domain. Isolation of the
generated plasmids is performed using standard techniques.
Single chain antibodies (scFvs) having the target antigen binding region of
a desired antigen are generated using reverse transcriptase PCR of the bulk RNA
from antibody-generating cell lines using primers 4 to 6 (shown in FIGS. 2A-B)
(SEQ ID NOS:5, 6, and 7). The binding regions are subcloned into the cut and dephosphorylated
plasmid prepared as described above, and then a fusion gene is isolated and characterized
using techniques described in Z. Eshhar et al.,
Methods in
